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Sample records for human anti-met monoclonal

  1. Discovery of Fully Human Anti-MET Monoclonal Antibodies with Antitumor Activity against Colon Cancer Tumor Models In Vivo

    Directory of Open Access Journals (Sweden)

    Edward Htun van der Horst

    2009-04-01

    Full Text Available The receptor tyrosine kinase MET is a major component controlling the invasive growth program in embryonic development and in invasive malignancies. The discovery of therapeutic antibodies against MET has been difficult, and antibodies that compete with hepatocyte growth factor (HGF act as agonists. By applying phage technology and cell-based panning strategies, we discovered two fully human antibodies against MET (R13 and R28, which synergistically inhibit HGF binding to MET and elicit antibody-dependent cellular cytotoxicity. Cell-based phosphorylation assays demonstrate that R13 and R28 abrogate HGF-induced activation of MET, AKT1, ERK1/2, and HGF-induced migration and proliferation. FACS experiments suggest that the inhibitory effect is mediated by “locking” MET receptor in a state with R13, which then increases avidity of R28 for the extracellular domain of MET, thus blocking HGF binding without activating the receptor. In vivo studies demonstrate that the combination of R13/28 significantly inhibited tumor growth in various colon tumor xenograft models. Inhibition of tumor growth was associated with induction of hypoxia. Global gene expression analysis shows that inhibition of HGF/MET pathway significantly upregulated the tumor suppressors KLF6, CEACAM1, and BMP2, the negative regulator of phosphatidylinositol-3-OH-kinase PIK3IP1, and significantly suppressed SCF and SERPINE2, both enhancers of proliferation and invasiveness. Moreover, in an experimental metastasis model, R13/28 increased survival by preventing the recurrence of otherwise lethal lung metastases. Taken together, these results underscore the utility of a dual-antibody approach for targeting MET and possibly other receptor tyrosine kinases. Our approach could be expanded to drug discovery efforts against other cell surface proteins.

  2. Reshaped Human Monoclonal Antibodies for Therapy and Passive Immunization

    Science.gov (United States)

    1991-11-01

    Monoclonal Antibodies for Therapy and Passive Immunisation by Reshaping Rodent Monoclonal Antibodies". Two mouse monoclonal antibody producing cell...could be simply extended to make human monoclonals, but this has proved not to be the case. There are difficulties in finding appropriately immunised ...human donors and suitable fusion partners for the antibody producing cells. In vitro immunisation techniques have been tried, but only low affinity 1gM

  3. Production of Monoclonal Antibody against Human Nestin

    OpenAIRE

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad Mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2010-01-01

    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140?250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such a...

  4. Crossreactivity of boar sperm monoclonal antibodies with human ...

    African Journals Online (AJOL)

    Monoclonal antibodies against the head (H mabs) and tail (Tmabs) of boar spermatozoa were produced. Spermatozoa from boar, stallion, bull, human, ram, goat and rabbit were independently incubated with the monoclonal antibodies and later stained by immunofluorescence method. There were positive reactions of the ...

  5. Analysis of human chorionic gonadotropin-monoclonal antibody ...

    Indian Academy of Sciences (India)

    However we observed that in a high affinity antigen-antibody system [human chorionic gonadotropin-monoclonal antibody (hCG-mAb)] dissociation is insignificant and the sensogram data cannot be used to measure the equilibrium and kinetic parameters. At low concentrations of mAb the complete sensogram could be ...

  6. Generation of monoclonal antibodies to native active human glycosyltransferases

    DEFF Research Database (Denmark)

    Vester-Christensen, Malene Bech; Bennett, Eric Paul; Clausen, Henrik

    2013-01-01

    using monoclonal antibodies therefore provides an excellent strategy to analyze the glycosylation process in cells. A major drawback has been difficulties in generating antibodies to glycosyltransferases and validating their specificities. Here we describe a simple strategy for generating...... and characterizing monoclonal antibodies to human glycosyltransferases. This strategy includes a process for recombinant production and purification of enzymes for immunization, a simple selection strategy for isolation of antibodies with optimal properties for in situ detection of enzyme expression......, and a comprehensive strategy for characterizing the fine specificity of such antibodies....

  7. Development of Human Monoclonal Antibodies Against Respiratory Syncytial Virus Using a High Efficiency Human Hybridoma Technique.

    Science.gov (United States)

    Alvarado, Gabriela; Crowe, James E

    2016-01-01

    Human monoclonal antibodies against RSV have high potential for use as prophylaxis or therapeutic molecules, and they also can be used to define the structure of protective epitopes for rational vaccine design. In the past, however, isolation of human monoclonal antibodies was difficult and inefficient. Here, we describe contemporary methods for activation and proliferation of primary human memory B cells followed by cytofusion to non-secreting myeloma cells by dielectrophoresis to generate human hybridomas secreting RSV-specific monoclonal antibodies. We also provide experimental methods for screening human B cell lines to obtain RSV-specific lines, especially lines secreting neutralizing antibodies.

  8. 78 FR 7438 - Prospective Grant of Exclusive License: Development of Human Monoclonal Antibodies Against DR4

    Science.gov (United States)

    2013-02-01

    ... Human Monoclonal Antibodies Against DR4 AGENCY: National Institutes of Health, Public Health Service... Monoclonal Antibodies Against DR4'' (HHS Ref. No. E-158-2010/0) to Customized Biosciences, Inc., which is... relates to the development of two human monoclonal antibodies (mAbs) that bind to death receptor 4 (``DR4...

  9. Human monoclonal antibodies: the residual challenge of antibody immunogenicity.

    Science.gov (United States)

    Waldmann, Herman

    2014-01-01

    One of the major reasons for seeking human monoclonal antibodies has been to eliminate immunogenicity seen with rodent antibodies. Thus far, there has yet been no approach which absolutely abolishes that risk for cell-binding antibodies. In this short article, I draw attention to classical work which shows that monomeric immunoglobulins are intrinsically tolerogenic if they can be prevented from creating aggregates or immune complexes. Based on these classical studies two approaches for active tolerization to therapeutic antibodies are described.

  10. Humanization and simultaneous optimization of monoclonal antibody.

    Science.gov (United States)

    Kuramochi, T; Igawa, T; Tsunoda, H; Hattori, K

    2014-01-01

    Antibody humanization is an essential technology for reducing the potential risk of immunogenicity associated with animal-derived antibodies and has been applied to a majority of the therapeutic antibodies on the market. For developing an antibody molecule as a pharmaceutical at the current biotechnology level, however, other properties also have to be considered in parallel with humanization in antibody generation and optimization. This section describes the critical properties of therapeutic antibodies that should be sufficiently qualified, including immunogenicity, binding affinity, physiochemical stability, expression in host cells and pharmacokinetics, and the basic methodologies of antibody engineering involved. By simultaneously optimizing the antibody molecule in the light of these properties, it should prove possible to shorten the research and development period necessary to identify a highly qualified clinical candidate and consequently accelerate the start of the clinical trial.

  11. [Neutralizing Monoclonal and Chimeric Antibodies to Human IFN-γ].

    Science.gov (United States)

    Larina, M V; Aliev, T K; Solopova, O N; Pozdnyakova, L P; Korobova, S V; Yakimov, S A; Sveshnikov, P G; Dolgikh, D A; Kirpichnikov, M P

    2015-01-01

    Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

  12. 77 FR 9678 - Prospective Grant of Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies...

    Science.gov (United States)

    2012-02-17

    ... Human Anti-CD22 Monoclonal Antibodies for the Treatment of Human Cancers and Autoimmune Disease AGENCY... Monoclonal Antibodies Against CD22'' , U.S. patent application 12/934,214 entitled ``Human Monoclonal... and m972 (SMB-002) monoclonal antibodies as therapies for the treatment of B cell cancers and...

  13. Antigen-specific human monoclonal antibodies from transgenic mice.

    Science.gov (United States)

    Mompó, Susana Magadán; González-Fernández, Africa

    2014-01-01

    Due to the difficulties found when generating fully human monoclonal antibodies (mAbs) by the traditional method, several efforts have attempted to overcome these problems, with varying levels of success. One approach has been the development of transgenic mice carrying immunoglobulin (Ig) genes in germ line configuration. The engineered mouse genome can undergo productive rearrangement in the B cell population, with the generation of mouse B lymphocytes expressing human Ig (hIg) chains. To avoid the expression of mouse heavy or light chains, the endogenous mouse Ig (mIg) loci must be silenced by gene-targeting techniques. Subsequently, to obtain antigen-specific mAbs, conventional immunization protocols can be followed and the mAb technique used (fusion of activated B cells with mouse myeloma cells, screening, cloning, freezing, and testing) with these animals expressing human Ig genes. This chapter describes the type of transgenic knockout mice generated for various research groups, provides examples of human mAbs developed by research groups and companies, and includes protocols of immunization, generation, production, and purification of human mAbs from such mice. In addition, it also addresses the problems detected, and includes some of the methods that can be used to analyze functional activities with human mAbs.

  14. Two monoclonal anti-CD3 antibodies can induce different events in human T lymphocyte activation

    NARCIS (Netherlands)

    Roosnek, E. E.; van Lier, R. A.; Aarden, L. A.

    1987-01-01

    Two monoclonal antibodies, WT32 and CLB-T3/4.2a, directed against the CD3 complex were used to study the mechanism of activation of human peripheral T lymphocytes. WT32, a mouse monoclonal IgG2a antibody with a low avidity (much less than OKT3) for the CD3 complex, effectively induces mitogenesis of

  15. Molecular Insights into Fully Human and Humanized Monoclonal Antibodies: What are the Differences and Should Dermatologists Care?

    Science.gov (United States)

    Mallbris, Lotus; Davies, Julian; Glasebrook, Andrew; Tang, Ying; Glaesner, Wolfgang; Nickoloff, Brian J

    2016-07-01

    In recent years, a large number of therapeutic monoclonal antibodies have come to market to treat a variety of conditions including patients with immune-mediated chronic inflammation. Distinguishing the relative clinical efficacy and safety profiles of one monoclonal antibody relative to another can be difficult and complex due to different clinical designs and paucity of head-to-head comparator studies. One distinguishing feature in interpreting clinical trial data by dermatologists may begin by determining whether a monoclonal antibody is fully human or humanized, which can be discerned by the generic name of the drug. Herein, this commentary highlights the distinctions and similarities of fully human and humanized monoclonal antibodies in their nomenclature, engineering, and clinical profiles. While there are a number of differences between these types of monoclonal antibodies, current evidence indicates that this designation does not impart any measurable impact on overall clinical efficacy and safety profiles of a given drug. Based on molecular insights provided in this commentary, it is clear that each monoclonal antibody, irrespective of being fully human or humanized, should be individually assessed for its clinical impact regarding safety and efficacy. Going beyond the type of generic name ascribed to a monoclonal antibody will be an ever-increasing theme for dermatologists as more therapeutic monoclonal antibodies emerge to potentially treat a wider scope of diseases with cutaneous manifestations.

  16. Specific MR imaging of human-lymphocytes by monoclonal antibody-guided dextran-magnetite particles

    NARCIS (Netherlands)

    Bulte, J. W. M.; Hoekstra, Y; Kamman, R. L.; Magin, R. L.; Webb, A. G.; Briggs, R. W.; Go, K. G.; Hulstaert, C. E.; Miltenyi, S.; The, T. Hauw; de Leij, L

    Human lymphocytes were labeled with biotinylated anti-lymphocyte-directed monoclonal antibodies, to which streptavidin and subsequently biotinylated dextran-magnetite particles were coupled. This labeling resulted in a strong and selective negative contrast enhancement of lymphocyte suspensions at

  17. Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets

    NARCIS (Netherlands)

    ter Meulen, Jan; Bakker, Alexander B. H.; van den Brink, Edward N.; Weverling, Gerrit J.; Martina, Byron E. E.; Haagmans, Bart L.; Kuiken, Thijs; de Kruif, John; Preiser, Wolfgang; Spaan, Willy; Gelderblom, Hans R.; Goudsmit, Jaap; Osterhaus, Albert D. M. E.

    2004-01-01

    SARS coronavirus continues to cause sporadic cases of severe acute respiratory syndrome (SARS) in China. No active or passive immunoprophylaxis for disease induced by SARS coronavirus is available. We investigated prophylaxis of SARS coronavirus infection with a neutralising human monoclonal

  18. Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma.

    Science.gov (United States)

    Peng, Hong; Brimijoin, Stephen; Hrabovska, Anna; Krejci, Eric; Blake, Thomas A; Johnson, Rudolph C; Masson, Patrick; Lockridge, Oksana

    2016-01-05

    Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immuno extract BChE from the plasma of humans, monkey and other animals. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  19. Human monoclonal antibodies broadly neutralizing against influenza B virus.

    Directory of Open Access Journals (Sweden)

    Mayo Yasugi

    2013-02-01

    Full Text Available Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus.

  20. The Use of Humanized Monoclonal Antibodies for the Prevention of Respiratory Syncytial Virus Infection

    Directory of Open Access Journals (Sweden)

    Marcello Lanari

    2013-01-01

    Full Text Available Monoclonal antibodies are widely used both in infants and in adults for several indications. Humanized monoclonal antibodies (palivizumab have been used for many years for the prevention of respiratory syncytial virus infection in pediatric populations (preterm infants, infants with chronic lung disease or congenital heart disease at high risk of severe and potentially lethal course of the infection. This drug was reported to be safe, well tolerated and effective to decrease the hospitalization rate and mortality in these groups of infants by several clinical trials. In the present paper we report the development and the current use of monoclonal antibodies for prophylaxis against respiratory syncytial virus.

  1. Quantitative PET imaging of Met-expressing human cancer xenografts with {sup 89}Zr-labelled monoclonal antibody DN30

    Energy Technology Data Exchange (ETDEWEB)

    Perk, Lars R.; Stigter-van Walsum, Marijke; Vosjan, Maria J.W.D.; Leemans, C.R. [VU University Medical Centre, Department of Otolaryngology-Head and Neck Surgery, De Boelelaan 1117, P.O. Box 7057, Amsterdam (Netherlands); Visser, Gerard W.M.; Kloet, Reina W. [VU University Medical Centre, Nuclear Medicine and PET Research, Amsterdam (Netherlands); Giaccone, Giuseppe [National Cancer Institute, Medical Oncology Branch CCR, Bethesda, MD (United States); Albano, Raffaella; Comoglio, Paolo M. [University of Turin Medical School, Division of Molecular Oncology, Institute for Cancer Research and Treatment (IRCC), Turin (Italy); Dongen, Guus A.M.S. van [VU University Medical Centre, Department of Otolaryngology-Head and Neck Surgery, De Boelelaan 1117, P.O. Box 7057, Amsterdam (Netherlands); VU University Medical Centre, Nuclear Medicine and PET Research, Amsterdam (Netherlands)

    2008-10-15

    Targeting the c-Met receptor with monoclonal antibodies (MAbs) is an appealing approach for cancer diagnosis and treatment because this receptor plays a prominent role in tumour invasion and metastasis. Positron emission tomography (PET) might be a powerful tool for guidance of therapy with anti-Met MAbs like the recently described MAb DN30 because it allows accurate quantitative imaging of tumour targeting (immuno-PET). We considered the potential of PET with either {sup 89}Zr-labelled (residualising radionuclide) or {sup 124}I-labelled (non-residualising radionuclide) DN30 for imaging of Met-expressing tumours. The biodistribution of co-injected {sup 89}Zr-DN30 and iodine-labelled DN30 was compared in nude mice bearing either the human gastric cancer line GLT-16 (high Met expression) or the head-and-neck cancer line FaDu (low Met expression). PET images were acquired in both xenograft models up to 4 days post-injection (p.i.) and used for quantification of tumour uptake. Biodistribution studies in GTL-16-tumour-bearing mice revealed that {sup 89}Zr-DN30 achieved much higher tumour uptake levels than iodine-labelled DN30 (e.g. 19.6%ID/g vs 5.3%ID/g, 5 days p.i.), while blood levels were similar, indicating internalisation of DN30. Therefore, {sup 89}Zr-DN30 was selected for PET imaging of GLT-16-bearing mice. Tumours as small as 11 mg were readily visualised with immuno-PET. A distinctive lower {sup 89}Zr uptake was observed in FaDu compared to GTL-16 xenografts (e.g. 7.8%ID/g vs 18.1%ID/g, 3 days p.i.). Nevertheless, FaDu xenografts were also clearly visualised with {sup 89}Zr-DN30 immuno-PET. An excellent correlation was found between PET-image-derived {sup 89}Zr tumour uptake and ex-vivo-assessed {sup 89}Zr tumour uptake (R {sup 2} = 0.98). The long-lived positron emitter {sup 89}Zr seems attractive for PET-guided development of therapeutic anti-c-Met MAbs. (orig.)

  2. Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants

    NARCIS (Netherlands)

    ter Meulen, Jan; van den Brink, Edward N.; Poon, Leo L. M.; Marissen, Wilfred E.; Leung, Cynthia S. W.; Cox, Freek; Cheung, Chung Y.; Bakker, Arjen Q.; Bogaards, Johannes A.; van Deventer, Els; Preiser, Wolfgang; Doerr, Hans Wilhelm; Chow, Vincent T.; de Kruif, John; Peiris, Joseph S. M.; Goudsmit, Jaap

    2006-01-01

    BACKGROUND: Experimental animal data show that protection against severe acute respiratory syndrome coronavirus (SARS-CoV) infection with human monoclonal antibodies (mAbs) is feasible. For an effective immune prophylaxis in humans, broad coverage of different strains of SARS-CoV and control of

  3. 77 FR 5036 - Prospective Grant of Exclusive License: The Development of Human Anti-Mesothelin Monoclonal...

    Science.gov (United States)

    2012-02-01

    ... the treatment of pancreatic cancer, ovarian cancer, lung cancer, mesothelioma, and stomach/gastric... the treatment of mesothelin-expressing cancers, including mesothelioma, lung cancer, stomach/gastric... Human Anti-Mesothelin Monoclonal Antibodies for the Treatment of Human Cancers AGENCY: National...

  4. Immunoreactivity of skate electrocytes towards monoclonal antibodies against human dystrophin and dystrophin-related (DMDL) protein.

    Science.gov (United States)

    Dowdall, M J; Ellis, J M; Nguyen thi Man; Morris, G E

    1992-04-13

    Monoclonal antibodies against human dystrophin have been used to demonstrate the existence of a dystrophin-like protein in the electrocytes of skate electric organ. This protein is also present in skate muscle and resembles that found in Torpedo electric organ. Monoclonal antibodies against a human autosomal homologue of dystrophin (DMDL protein) did not detect a similar protein in skate or Torpedo. Immunocytochemical staining of the innervated and non-innervated faces of the electrocyte membrane was obtained using the anti-dystrophin antibodies only.

  5. Characterization of pathogenic human monoclonal autoantibodies against GM-CSF.

    Science.gov (United States)

    Wang, Yanni; Thomson, Christy A; Allan, Lenka L; Jackson, Linda M; Olson, Melanie; Hercus, Timothy R; Nero, Tracy L; Turner, Amanda; Parker, Michael W; Lopez, Angel L; Waddell, Thomas K; Anderson, Gary P; Hamilton, John A; Schrader, John W

    2013-05-07

    The origin of pathogenic autoantibodies remains unknown. Idiopathic pulmonary alveolar proteinosis is caused by autoantibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF). We generated 19 monoclonal autoantibodies against GM-CSF from six patients with idiopathic pulmonary alveolar proteinosis. The autoantibodies used multiple V genes, excluding preferred V-gene use as an etiology, and targeted at least four nonoverlapping epitopes on GM-CSF, suggesting that GM-CSF is driving the autoantibodies and not a B-cell epitope on a pathogen cross-reacting with GM-CSF. The number of somatic mutations in the autoantibodies suggests that the memory B cells have been helped by T cells and re-entered germinal centers. All autoantibodies neutralized GM-CSF bioactivity, with general correlations to affinity and off-rate. The binding of certain autoantibodies was changed by point mutations in GM-CSF that reduced binding to the GM-CSF receptor. Those monoclonal autoantibodies that potently neutralize GM-CSF may be useful in treating inflammatory disease, such as rheumatoid arthritis and multiple sclerosis, cancer, and pain.

  6. Naturalizing activity and safety of human monoclonal antibodies against of hepatitis C virus.

    Science.gov (United States)

    Abelhafez, Tawfeek H; Tabll, Ashraf A; El-Awady, Mostafa K; Mashaly, Mohammad M; El Shenawy, Reem; El-Abd, Yasmine S; Shaker, Maysa H; Abdel Malak, Camelia A

    2017-09-29

    Assessment of neutralizing activity of the human monoclonal antibodies against HCV and also study its safety in experimental small animals (Swiss mice). Assessment of neutralizing activity of the human monoclonal antibodies against HCV envelope regions (E1, E2) by two methods (by HCV cc infectious system and by using positive HCV positive serum as source of HCV particles (neutralizing assay 2). Dot ELISA were used to study the activity of the generated antibodies. We tested the safety and toxicity of the generated human antibodies by assessment the changes in biochemistry of liver function tests and changes in kidney function test, Complete blood counts (CBC) and study the pathological changes with different concentration of purified human antibodies. Human Abs # 5 & 11 showed neutralizing activity by (neutralizing assay 2) but were not neutralizing by HCV cc assay. Human Abs # 12 & 15 showed neutralizing activity by two methods i.e our generated human antibodies Abs# 5 &11 & 12 & 15 were neutralizing for HCV genotype 4a and Abs # 12 & 15 were neutralizing for HCV genotypes 4a and 2a. Liver and kidney functions and CBC results indicated that doses of 10 μg, 100 μg were safe. The histopathological results indicated that the dose of 10 μg of purified human monoclonal antibodies per mouse body weight was safe. The generated human monoclonal antibodies can be used to develop a potent immunotherapy that can be administrated for the post-transplantation patients to prevent the recurrence of HCV infection. Also, the monoclonal antibodies can be used to develop a vaccine against HCV.

  7. SPAM-8, a mouse-human heteromyeloma fusion partner in the production of human monoclonal antibodies. Establishment of a human monoclonal antibody against cytomegalovirus.

    Science.gov (United States)

    Gustafsson, B; Jondal, M; Sundqvist, V A

    1991-01-01

    A heteromyeloma (mouse x human) cell line (SPAM-8) was produced by fusing mouse myeloma cells (SP2/0) with human peripheral blood lymphocytes. The cells were sensitive to aminopterin and resistant to ouabain. The cells showed a doubling time of about 19 hours and a cloning efficiency of 0.8 cells/well (to obtain growth in 50% of wells seeded) using mouse thymocytes as feeder cells. The number of chromosomes was about 86 and 1% of the total DNA was of human origin. Fusion of SPAM-8 cells with lymphocytes prepared from human spleens resulted in approximately one hybridoma per 10(5) seeded lymphocytes. A trioma (human x [mouse x human]) cell line was established by fusing cells of an Epstein-Barr virus-transformed B cell line with SPAM-8 cells. The trioma cells produced antibodies (IgG1, K) against cytomegalovirus, in a concentration of 7 micrograms/ml in spent medium, over a period of six months of continuous culture. The results obtained indicate that the heteromyeloma SPAM-8 may be used as a fusion partner in the production of human monoclonal antibodies.

  8. Human Monoclonal Antibodies Targeting Glypican-2 in Neuroblastoma | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    Researchers at the National Cancer Institute’s Laboratory of Molecular Biology (NCI LMB) have developed and isolated several single domain monoclonal human antibodies against GPC2. NCI seeks parties interested in licensing or co-developing GPC2 antibodies and/or conjugates.

  9. A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

    NARCIS (Netherlands)

    Huls, GA; Heijnen, IAFM; Cuomo, ME; Koningsberger, JC; Boel, E; de Vries, ARV; Loyson, SAJ; Helfrich, W; Henegouwen, GPV; van Meijer, M; de Kruif, J; Logtenberg, T

    A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab), The purified huMab had an affinity of 5 nM and effectively mediated tumor cell

  10. A human monoclonal antibody cocktail as a novel component of rabies postexposure prophylaxis

    NARCIS (Netherlands)

    de Kruif, John; Bakker, Alexander B. H.; Marissen, Wilfred E.; Kramer, R. Arjen; Throsby, Mark; Rupprecht, Charles E.; Goudsmit, Jaap

    2007-01-01

    The currently recommended treatment for individuals exposed to rabies virus is the combined administration of rabies vaccine and rabies immune globulin (RIG). This review sets out the criteria used to guide development of a cocktail of human monoclonal antibodies as a replacement for RIG. Using this

  11. Production and characterisation of monoclonal antibodies against native and disassembled human catalase

    NARCIS (Netherlands)

    Wiemer, E. A.; Ofman, R.; Middelkoop, E.; de Boer, M.; Wanders, R. J.; Tager, J. M.

    1992-01-01

    Catalase isolated from human erythrocytes was used to immunise mice, in order to generate hybridomas producing specific monoclonal antibodies to the enzyme. Hybridomas secreting anti-(catalase) antibodies were identified by a modified enzyme-linked immunosorbent assay (ELISA) using either

  12. 76 FR 63317 - Prospective Grant of Exclusive License: The Development of Human Anti-Mesothelin Monoclonal...

    Science.gov (United States)

    2011-10-12

    ... therapy for the treatment of pancreatic cancer, ovarian cancer, lung cancer, mesothelioma, and stomach... Treatment of Human Cancers AGENCY: National Institutes of Health, Public Health Service, HHS. ACTION: Notice... concerns a monoclonal antibody and methods of using the antibody for the treatment of mesothelin-expressing...

  13. Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies

    DEFF Research Database (Denmark)

    Hansen, J E; Clausen, H; Nielsen, C

    1990-01-01

    Carbohydrate structures are often involved in the initial adhesion of pathogens to target cells. In the present study, a panel of anticarbohydrate monoclonal antibodies (MAbs) was tested for their ability to inhibit in vitro human immunodeficiency virus infectivity. MAbs against three different N...

  14. Use of a monoclonal antibody to distinguish between precursor and mature forms of human lysosomal alpha-glucosidase

    NARCIS (Netherlands)

    Oude Elferink, R. P.; Strijland, A.; Surya, I.; Brouwer-Kelder, E. M.; Kroos, M.; Hilkens, J.; Hilgers, J.; Reuser, A. J.; Tager, J. M.

    1984-01-01

    The maturation of lysosomal alpha-glucosidase in cultured human skin fibroblasts was studied using a monoclonal antibody that distinguishes between the precursor and mature forms of the enzyme. Monoclonal antibodies against alpha-glucosidase isolated from placenta were produced by the hybridoma

  15. Monoclonal antibodies reacting with multiple epitopes on the human insulin receptor.

    OpenAIRE

    Soos, M A; Siddle, K; Baron, M D; Heward, J M; Luzio, J P; Bellatin, J; Lennox, E S

    1986-01-01

    Monoclonal antibodies for the human insulin receptor were produced following immunization of mice with IM-9 lymphocytes and/or purified placental receptor. Four separate fusions yielded 28 antibodies, all of which reacted with receptor from human placenta, liver and IM-9 cells. Some antibodies cross-reacted to varying degrees with receptor from rabbit, cow, pig and sheep, but none reacted with rat receptor. At least 10 distinct epitopes were recognized as indicated by species specificity and ...

  16. Humanization and Characterization of an Anti-Human TNF-α Murine Monoclonal Antibody

    Science.gov (United States)

    Chiu, Wei-Chun; Lai, Ya-Ping; Chou, Min-Yuan

    2011-01-01

    A murine monoclonal antibody, m357, showing the highly neutralizing activities for human tumor necrosis factor (TNF-α) was chosen to be humanized by a variable domain resurfacing approach. The non-conserved surface residues in the framework regions of both the heavy and light chain variable regions were identified via a molecular modeling of m357 built by computer-assisted homology modeling. By replacing these critical surface residues with the human counterparts, a humanized version, h357, was generated. The humanized h357 IgG1 was then stably expressed in a mammalian cell line and the purified antibody maintained the high antigen binding affinity as compared with the parental m357 based on a soluble TNF-α neutralization bioassay. Furthermore, h357 IgG1 possesses the ability to mediate antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity upon binding to cells bearing the transmembrane form of TNF-α. In a mouse model of collagen antibody-induced arthritis, h357 IgG significantly inhibited disease progression by intra-peritoneal injection of 50 µg/mouse once-daily for 9 consecutive days. These results provided a basis for the development of h357 IgG as therapeutic use. PMID:21305012

  17. Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

    Energy Technology Data Exchange (ETDEWEB)

    Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

    2000-02-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG{sub 1}), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of {sup 99m}Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14{+-}2.50 %ID/g, 5.06{+-}2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy.

  18. A monoclonal antibody defining human B cell differentiation antigen (HLB-1 antigen).

    Science.gov (United States)

    Kasai, K; Koshiba, H; Ishii, Y; Kikuchi, K

    1983-01-01

    A new monoclonal antibody specific for human B cell differentiation antigen (HLB-1) is produced by a hybridoma established by fusion of splenocytes of mice immunized with the Epstein-Barr virus (EBV)-transformed peripheral B cell line, RPMI-8057. This monoclonal, antibody designated anti-HLB-1 monoclonal antibody (anti-HLB-1), reacted with surface immunoglobulin (sIg)-positive B cells of normal peripheral blood and lymphoid tissues and sIg-positive leukemic cells. The cells of T cell leukemia, non-T non-B acute lymphoblastic leukemia (ALL) and nonlymphoid leukemia were HLB-1 negative. These data were further confirmed by studying a panel of cultured human hematopoietic cell lines. Anti-HLB-1 reacted with B cell lines derived from pre-B, Burkitt's lymphoma, B cell type ALL and EBV-transformed peripheral B cells. Anti-HLB-1 was reactive with only one of three human myeloma cell lines, and with none of the T cell, myeloid and non-T non-B ALL cell lines. This newly defined HLB-1 antigen is different from other conventional human B cell markers such as sIg, Ia antigens, and receptors for the Fc portion of Ig and complement C3.

  19. Generation of human antigen-specific monoclonal IgM antibodies using vaccinated "human immune system" mice

    NARCIS (Netherlands)

    Becker, P.D.; Legrand, N.; van Geelen, C.M.M.; Noerder, M.; Huntington, N.D.; Lim, A.; Yasuda, E.; Diehl, S.A.; Scheeren, F.A.; Ott, M.; Weijer, K.; Wedemeyer, H.; Di Santo, J.P.; Beaumont, T.; Guzman, C.A.; Spits, H.

    2010-01-01

    Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the 'humanization' of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of

  20. Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

    Science.gov (United States)

    Jensen, Ronald H.; Vanderlaan, Martin; Bigbee, William L.; Stanker, Larry H.; Branscomb, Elbert W.; Grabske, Robert J.

    1988-01-01

    The present invention provides monoclonal antibodies specific to and distinguish between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype.

  1. The monoclonal antibody GRC1 produced against human cornea recognizes a common determinant of collagen.

    Science.gov (United States)

    Lopez Nevot, M A; Cardona, L; Doblaré, E; Muñoz, C; Ruiz-Cabello, F; Garrido, F

    1990-02-01

    The monoclonal antibody GRC1 was obtained by immunizing BALB/c mice with human cornea. Screening was performed by indirect immunofluorescence in cryostatic sections of several tissues: cornea, skin, placenta, hyaline cartilage, blood vessels, and nerves. GRC1 was seen to recognize fibrillar structures in all of these tissues. The pattern of reaction was interstitial and membranous. On cornea, GRC1 reacts definitely with Bowman's membrane and diffusely with the stroma, while on skin it shows strongly positive reactivity with the papillary dermis and with the basement membrane. It also reacts on hyaline cartilage at the periphery of the condrocytic lacunae. These immunohistologic results suggest that GRC1 recognized human collagen. In order to investigate further the subtype of collagen defined by GRC1, an ELISA was performed with purified collagens of several types: I, II, III, IV, and V. The monoclonal antibody GRC1 defines a common determinant in types III, IV, and V.

  2. Human-monoclonal-antibody therapy protects nonhuman primates against advanced Lassa fever.

    Science.gov (United States)

    Mire, Chad E; Cross, Robert W; Geisbert, Joan B; Borisevich, Viktoriya; Agans, Krystle N; Deer, Daniel J; Heinrich, Megan L; Rowland, Megan M; Goba, Augustine; Momoh, Mambu; Boisen, Mathew L; Grant, Donald S; Fullah, Mohamed; Khan, Sheik Humarr; Fenton, Karla A; Robinson, James E; Branco, Luis M; Garry, Robert F; Geisbert, Thomas W

    2017-10-01

    There are no approved treatments for Lassa fever, which is endemic to the same regions of West Africa that were recently devastated by Ebola. Here we show that a combination of human monoclonal antibodies that cross-react with the glycoproteins of all four clades of Lassa virus is able to rescue 100% of cynomolgus macaques when treatment is initiated at advanced stages of disease, including up to 8 d after challenge.

  3. Analysis of human chorionic gonadotropin-monoclonal antibody ...

    Indian Academy of Sciences (India)

    Unknown

    used in high affinity systems to measure the association rate constant of the reaction and the functional capacity of the ligand (hCG) immobilized on the chip. We provide a rational explanation for the discrepancies generally observed in most of the BIAcore sensograms. [Ashish B and Murthy G S 2004 Analysis of human ...

  4. Human Neutralizing Monoclonal Antibody Inhibition of Middle East Respiratory Syndrome Coronavirus Replication in the Common Marmoset.

    Science.gov (United States)

    Chen, Zhe; Bao, Linlin; Chen, Cong; Zou, Tingting; Xue, Ying; Li, Fengdi; Lv, Qi; Gu, Songzhi; Gao, Xiaopan; Cui, Sheng; Wang, Jianmin; Qin, Chuan; Jin, Qi

    2017-06-15

    Middle East respiratory syndrome coronavirus (MERS-CoV) infection in humans is highly lethal, with a fatality rate of 35%. New prophylactic and therapeutic strategies to combat human infections are urgently needed. We isolated a fully human neutralizing antibody, MCA1, from a human survivor. The antibody recognizes the receptor-binding domain of MERS-CoV S glycoprotein and interferes with the interaction between viral S and the human cellular receptor human dipeptidyl peptidase 4 (DPP4). To our knowledge, this study is the first to report a human neutralizing monoclonal antibody that completely inhibits MERS-CoV replication in common marmosets. Monotherapy with MCA1 represents a potential alternative treatment for human infections with MERS-CoV worthy of evaluation in clinical settings. © Crown copyright 2017.

  5. Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

    DEFF Research Database (Denmark)

    Aasted, Bent; Blixenkrone-Møller, Merete; Larsen, Else Bang

    1988-01-01

    Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four...... antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen......-reactive antibody reacted with lymphocytes from mink. The anti-C3b-R antibody reacted with lymphocytes from horse, swine, dog, and cat, and the anti-HLA-DR reacted with lymphocytes from cow, goat, sheep, horse, dog, cat, and mink....

  6. The Effects of Anti-Hcg Monoclonal Antibodies on Human Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Mirshahi M

    2011-12-01

    Full Text Available Background: Human cancer cell lines express human choriogonadotropin (hCG, its subunits and derivatives, regardless of their origin and type. It appears that hCG is a common phenotype in human cancer cell lines. In this research, the effects of hCG targeting monoclonal antibodies (7D9, T18H7 and T8B12 on human cancer cell lines were evaluated. Methods: Monoclonal antibody secreting hybridomas were proliferated and injected intraperitoneally to Balb/C mice after treatment with pristine. Two weeks later, ascites fluid was collected. Purification of aforementioned antibodies from ascites fluid was performed using G-protein affinity followed by ion exchange chromatography. SDS-PAGE and ELISA confirmed the structure and functional integrity of the purified antibodies, respectively. Two human cancer cell lines "Hela" and "MDA" were treated by the purified antibodies. Three days later, different wells were imaged and the cells counted. Results: SDS-PAGE gel (None-reducing indicated consistency of band migration patterns with control antibodies. ELISA test using hCG antigens indicated that the produced antibodies could detect hCG antigens. Cell lines were cultured and treated with different concentrations of each antibody. Counting and imaging different wells of treated plates, indicated that 7D9 antibody had a more significant (P<0.01 cytotoxic effect on cancer cell lines than the control cells. Conclusion: HCG targeting monoclonal antibodies can be used for targeted cancer therapy, as human cancer cells express hCG gene. 7D9 antibody that exhibits protease activity is a proper candidate for this purpose, as it possesses both antagonistic and enzymatic properties.

  7. Fully-human Monoclonal Antibodies Against Human EphrinB2 and EphB4 | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Cancer Institute's Cancer and Inflammation Program is seeking statements of capability or interest from parties interested in licensing fully-human monoclonal antibodies against human EphrinB2 and EphB4.

  8. Production and Characterization of a Murine Monoclonal Antibody Against Human Ferritin

    Science.gov (United States)

    Bayat, Ali Ahmad; Yeganeh, Omid; Ghods, Roya; Zarnani, Amir Hassan; Ardekani, Reza Bahjati; Mahmoudi, Ahmad Reza; Mahmoudian, Jafar; Haghighat-Noutash, Farzaneh; Jeddi-Tehrani, Mahmood

    2013-01-01

    Background Ferritin is an iron storage protein, which plays a key role in iron metabolism. Measurement of ferritin level in serum is one of the most useful indicators of iron status and also a sensitive measurement of iron deficiency. Monoclonal antibodies may be useful as a tool in various aspects of ferritin investigations. In this paper, the production of a murine monoclonal antibody (mAb) against human ferritin was reported. Methods Balb/c mice were immunized with purified human ferritin and splenocytes of hyper immunized mice were fused with Sp2/0 myeloma cells. After four times of cloning by limiting dilution, a positive hybridoma (clone: 2F9-C9) was selected by ELISA using human ferritin. Anti-ferritin mAb was purified from culture supernatants by affinity chromatography. Results Determination of the antibody affinity for ferritin by ELISA revealed a relatively high affinity (2.34×109 M -1) and the isotype was determined to be IgG2a. The anti-ferritin mAb 2F9-C9 reacted with 79.4% of Hela cells in flow cytometry. The antibody detected a band of 20 kDa in K562 cells, murine and human liver lysates, purified ferritin in Western blot and also ferritin in human serum. Conclusion This mAb can specifically recognize ferritin and may serve as a component of ferritin diagnostic kit if other requirements of the kit are met. PMID:24285995

  9. Development of broad-spectrum human monoclonal antibodies for rabies post-exposure prophylaxis.

    Science.gov (United States)

    De Benedictis, Paola; Minola, Andrea; Rota Nodari, Elena; Aiello, Roberta; Zecchin, Barbara; Salomoni, Angela; Foglierini, Mathilde; Agatic, Gloria; Vanzetta, Fabrizia; Lavenir, Rachel; Lepelletier, Anthony; Bentley, Emma; Weiss, Robin; Cattoli, Giovanni; Capua, Ilaria; Sallusto, Federica; Wright, Edward; Lanzavecchia, Antonio; Bourhy, Hervé; Corti, Davide

    2016-04-01

    Currently available rabies post-exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 (binding to antigenic site I and III, respectively), were selected for their potency and broad-spectrum reactivity. In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non-RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development (namely CR57, CR4098, and RAB1) and commercially available human RIG. In vivo, the RVC20-RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post-vaccination antibody response. © 2016 Humabs BioMed SA Published under the terms of the CC BY 4.0 license.

  10. Bacterial expression of a human monoclonal antibody-alkaline phosphatase conjugate specific for Entamoeba histolytica.

    Science.gov (United States)

    Tachibana, Hiroshi; Takekoshi, Masataka; Cheng, Xun-Jia; Nakata, Yuta; Takeuchi, Tsutomu; Ihara, Seiji

    2004-01-01

    We previously produced human monoclonal antibody Fab fragments specific to Entamoeba histolytica in Escherichia coli. In order to use these Fab fragments for diagnostic purposes, an expression vector to produce a fusion protein of Fab and alkaline phosphatase (PhoA) in E. coli was designed and constructed. The E. coli PhoA gene was fused to the 3' terminus of the gene encoding the heavy-chain Fd region. The kappa and Fd genes from a previously prepared antibody clone, CP33, which is specific for the 260-kDa lectin of E. histolytica, were used as human antibody genes. When the fusion protein of CP33 and PhoA was incubated with paraformaldehyde-fixed trophozoites of E. histolytica and developed with a substrate, the trophozoites appeared to be stained. These results demonstrate the feasibility of bacterial expression of a human monoclonal antibody-PhoA conjugate specific for E. histolytica and that the antibody can be used to detect E. histolytica antigen without the use of chemically conjugated secondary antibodies.

  11. Prophylactic and therapeutic activity of fully human monoclonal antibodies directed against Influenza A M2 protein

    Directory of Open Access Journals (Sweden)

    Gwerder Myriam

    2009-12-01

    Full Text Available Abstract Influenza virus infection is a prevalent disease in humans. Antibodies against hemagglutinin have been shown to prevent infection and hence hemagglutinin is the major constituent of current vaccines. Antibodies directed against the highly conserved extracellular domain of M2 have also been shown to mediate protection against Influenza A infection in various animal models. Active vaccination is generally considered the best approach to combat viral diseases. However, passive immunization is an attractive alternative, particularly in acutely exposed or immune compromized individuals, young children and the elderly. We recently described a novel method for the rapid isolation of natural human antibodies by mammalian cell display. Here we used this approach to isolate human monoclonal antibodies directed against the highly conserved extracellular domain of the Influenza A M2 protein. The identified antibodies bound M2 peptide with high affinities, recognized native cell-surface expressed M2 and protected mice from a lethal influenza virus challenge. Moreover, therapeutic treatment up to 2 days after infection was effective, suggesting that M2-specific monoclonals have a great potential as immunotherapeutic agents against Influenza infection.

  12. Domain-Specific Monoclonal Antibodies Against Human Rev-erbβ.

    Science.gov (United States)

    Chen, Fang; Li, Yanqing; Zhao, Junli; Mao, Qinwen; Xia, Haibin

    2017-07-01

    The nuclear receptor Rev-erbβ is a potent transcriptional factor whose functional study has been limited by the lack of suitable antibodies against it. To better understand Rev-erbβ's biological roles, we generated five hybridoma cell lines secreting antibodies against human Rev-erbβ in mice immunized with the purified, prokaryotically expressed recombinant Rev-erbβ-6His fusion protein. Using Western blotting and immunofluorescence analyses, all the five monoclonal antibodies (MAbs) showed strong immunoreactivity to both prokaryotically and eukaryotically expressed recombinant Rev-erbβ. An immunoprecipitation study showed that all five monoclonal antibodies against Rev-erbβ were able to pull down the recombinant Rev-erbβ-Flag protein, but only one of the MAbs against Rev-erbβ, 37H8, could pull down the endogenous Rev-erbβ protein. Furthermore, domain specificity of these MAbs was characterized. Due to the high similarities between Rev-erbα and Rev-erbβ in the C and E domains, those C and E domain-specific anti-Rev-erbβ antibodies can react with human Rev-erbα as well. The MAbs produced in the study will provide a valuable tool for investigating the function of Rev-erbβ.

  13. [Effect of monoclonal antibodies against LI-cadherin on the proliferation of human hepatocellular carcinoma cells].

    Science.gov (United States)

    Chen, Xiao-ting; DU, Hong-yan; Yuan, Shao-fei; Wang, Sen-ming; Li, Ming

    2009-05-01

    To obtain monoclonal antibodies (mAb) against LI-cadherin and investigate their effects on the proliferation of human hepatocellular carcinoma cells. Balb/c mice were immunized with recombinant LI-cadherin, and hybridoma cell lines secreting monoclonal antibodies against LI-cadherin were established with routine cell fusion and subcloning approach. The specificity of these mAbs was determined by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The effect of the mAbs obtained on the growth of HepG2 cells was assessed using inverted microscope and MTT assay. Two hybridoma cell lines (F001 and F002) stably secreting specific mAbs were obtained. Western blot analysis showed that the two antibodies specifically recognized LI-cadherin antigen derived from human eucaryotic cells or tissue. Treatment of the HepG2 cells with the mAbs resulted in reduced viable cell number and changes in the cell morphologies, and the two mAbs inhibited the proliferation of HepG2 cells in a concentration-dependent manner (Pcells in vitro, which facilitates further study of the relationship between LI-cadherin and tumors.

  14. Potent Human Monoclonal Antibodies against SARS CoV, Nipah and Hendra Viruses

    Science.gov (United States)

    Prabakaran, Ponraj; Zhongyu, Zhu; Xiao, Xiaodong; Biragyn, Arya; Dimitrov, Antony S.; Broder, Christopher C.; Dimitrov, Dimiter S.

    2009-01-01

    Polyclonal antibodies have a century-old history of being effective against some viruses; recently, monoclonal antibodies (mAbs) have also shown success. The humanized mAb Synagis (palivizumab) remains still the only mAb against respiratory syncytial virus (RSV) infections approved by the U.S. Food and Drug Administration (FDA). Recently, several potent human monoclonal antibodies (hmAbs) targeting the Severe Acute Respiratory Syndrome-Associated coronavirus (SARS CoV) S glycoproteins were developed quickly after the virus was identified in 2003. Among these antibodies, m396 and S230.15 exhibit exceptional potency and cross-reactivity as they neutralize isolates from the first and second outbreaks and from palm civets both in vitroand in mice. Similarly, the first fully hmAbs against two other paramyxoviruses, Hendra virus (HeV) and Nipah virus (NiV), which can cause up to 75% mortality, were recently developed; one of them, m102.4, shows exceptional cross-reactive potency against both NiV and HeV. Three-dimensional molecular structures of envelope glycoproteins from these viruses in complexes with antibodies and/or receptors were recently determined. Structural analyses along with other experiments have provided insights into the molecular mechanisms of receptor recognition and antibody neutralization, and suggested that these antibodies alone or in combination could successfully fight the viruses’ heterogeneity and mutability which is a major problem in the development of effective therapeutic agents against viruses, including therapeutic antibodies. PMID:19216624

  15. Elastolytic activity of human blood monocytes characterized by a new monoclonal antibody against human leucocyte elastase. Relationship to rheumatoid arthritis

    DEFF Research Database (Denmark)

    Jensen, H S; Christensen, L D

    1990-01-01

    The leucocyte elastase of human blood monocytes was investigated by applying a new monoclonal antibody which did not block the enzyme activity against elastin. In a fixed population of mononuclear cells (MNC) and using fluorescence activated cell sorting (FACS), the human leucocyte elastase (HLE......) antibody identified a subgroup of CD14+ cells which contained all the elastase activity and which could be blocked by a specific chloromethylketone elastase inhibitor. By anti-CD14 labelling the HLE positive cells were identified as monocytes and amounted to 88% of this cell type (median: range 72...

  16. Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition

    DEFF Research Database (Denmark)

    Green, M R; Couchman, J R

    1985-01-01

    Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map...

  17. Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: Fine mapping and escape mutant analysis

    NARCIS (Netherlands)

    Marissen, W.E.; Kramer, R.A.; Rice, A.; Weldon, W.C.; Niezgoda, M.; Faber, M.; Slootstra, J.W.; Meloen, R.H.; Clijsters-van der Horst, M.; Visser, T.J.; Jongeneelen, M.; Thijsse, S.; Throsby, M.; Kruif, de J.; Rupprecht, C.E.; Dietzschold, B.; Goudsmit, J.; Bakker, A.B.H.

    2005-01-01

    Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We

  18. Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: fine mapping and escape mutant analysis

    NARCIS (Netherlands)

    Marissen, Wilfred E.; Kramer, R. Arjen; Rice, Amy; Weldon, William C.; Niezgoda, Michael; Faber, Milosz; Slootstra, Jerry W.; Meloen, Rob H.; Clijsters-van der Horst, Marieke; Visser, Therese J.; Jongeneelen, Mandy; Thijsse, Sandra; Throsby, Mark; de Kruif, John; Rupprecht, Charles E.; Dietzschold, Bernhard; Goudsmit, Jaap; Bakker, Alexander B. H.

    2005-01-01

    Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We

  19. The preparation, characterization, and application of environment-friendly monoclonal antibodies for human blood cell.

    Science.gov (United States)

    Zhou, Chenjie; Gao, Xuechao; He, Shixiang; Gao, Xiaoling; Zhuang, Jialin; Huang, Lirong; Guo, Hengchang

    2017-03-01

    Monoclonal anti-human blood group A (51A8) and B (63B6) antibody reagents were prepared using the serum-free technique. The aims of this research were to characterize the serum-free reagents and prove their reliabilities in routine use. Experiments including antigen-antibody agglutination testing, stability testing, SDS-PAGE, protein and IgM quantification, flow cytometry, and variable domain sequencing were performed to characterize the anti-A (51A8) and anti-B (63B6) reagents. Over 12 000 samples were tested using these reagents as routine blood grouping reagents. Serum-free anti-A (51A8) and anti-B (63B6) reagents were stable in longitudinal and accelerated testing, and their high purity was shown in SDS-PAGE and IgM quantification. These reagents have high specificity to red blood cells in serologic agglutination testing and flow cytometric analysis. A1 and A2 subgroup antigens can be distinguished clearly by patterns of flow cytometric histograms. No discrepancy was found in clinical trials of 12 000 samples. To reduce the risk of being affected by any animal additives, a serum-free culture system was applied to get mass-production of monoclonal anti-A/B antibodies. The high specificity and the high purity of the reagents were verified by the lab experiments. Lab research and clinical trial showed that serum-free monoclonal anti-A (51A8) and anti-B (63B6) reagents meet the requirements of routine blood grouping reagents. Moreover, these reagents featured ultra-high purity that is missing in other commercial counterparts, and therefore are recommended as more environment-friendly reagents.

  20. Hersintuzumab: A novel humanized anti-HER2 monoclonal antibody induces potent tumor growth inhibition.

    Science.gov (United States)

    Amiri, Mohammad Mehdi; Golsaz-Shirazi, Forough; Soltantoyeh, Tahereh; Hosseini-Ghatar, Reza; Bahadori, Tannaz; Khoshnoodi, Jalal; Navabi, Shadi Sadat; Farid, Samira; Karimi-Jafari, Mohammad Hossein; Jeddi-Tehrani, Mahmood; Shokri, Fazel

    2017-10-06

    Humanized monoclonal antibodies (mAbs) against HER2 including trastuzumab and pertuzumab are widely used to treat HER2 overexpressing metastatic breast cancers. These two mAbs recognize distinct epitopes on HER2 and their combination induces a more potent blockade of HER2 signaling than trastuzumab alone. Recently, we have reported characterization of a new chimeric mAb (c-1T0) which binds to an epitope different from that recognized by trastuzumab and significantly inhibits proliferation of HER2 overexpressing tumor cells. Here, we describe humanization of this mAb by grafting all six complementarity determining regions (CDRs) onto human variable germline genes. Humanized VH and VL sequences were synthesized and ligated to human γ1 and κ constant region genes using splice overlap extension (SOE) PCR. Subsequently, the humanized antibody designated hersintuzumab was expressed and characterized by ELISA, Western blot and flow cytometry. The purified humanized mAb binds to recombinant HER2 and HER2-overexpressing tumor cells with an affinity comparable with the chimeric and parental mouse mAbs. It recognizes an epitope distinct from those recognized by trastuzumab and pertuzumab. Binding of hersintuzumab to HER2 overexpressing tumor cells induces G1 cell cycle arrest, inhibition of ERK and AKT signaling pathways and growth inhibition. Moreover, hersintuzumab could induce antibody-dependent cell cytotoxicity (ADCC) on BT-474 cells. This new humanized mAb is a potentially valuable tool for single or combination breast cancer therapy.

  1. Monoclonal antibodies reacting with multiple epitopes on the human insulin receptor.

    Science.gov (United States)

    Soos, M A; Siddle, K; Baron, M D; Heward, J M; Luzio, J P; Bellatin, J; Lennox, E S

    1986-04-01

    Monoclonal antibodies for the human insulin receptor were produced following immunization of mice with IM-9 lymphocytes and/or purified placental receptor. Four separate fusions yielded 28 antibodies, all of which reacted with receptor from human placenta, liver and IM-9 cells. Some antibodies cross-reacted to varying degrees with receptor from rabbit, cow, pig and sheep, but none reacted with rat receptor. At least 10 distinct epitopes were recognized as indicated by species specificity and binding competition experiments. All of these epitopes appeared to be on extracellular domains of the receptor as shown by binding of antibodies to intact cells. In some cases the epitopes were further localized to alpha or beta subunits by immunoblotting. Several antibodies inhibited binding of 125I-insulin to the receptor, some had no effect on binding, and others enhanced the binding of 125I-insulin. It is concluded that these antibodies will be valuable probes of receptor structure and function.

  2. Stoichiometry of monoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1

    DEFF Research Database (Denmark)

    Schønning, Kristian; Lund, O; Lund, O S

    1999-01-01

    In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes are coexpr......In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes...

  3. Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model.

    Directory of Open Access Journals (Sweden)

    Xiaodong Xiao

    2010-10-01

    Full Text Available Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252 and two anti-V-specific human mAb (m253, m254 by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.

  4. Development of novel mouse hybridomas producing monoclonal antibodies specific to human and mouse nucleolar protein SURF-6.

    Science.gov (United States)

    Polzikov, Mikhail A; Kordyukova, Maria Yu; Zavalishina, Larisa E; Magoulas, Charalambos; Zatsepina, Olga V

    2012-02-01

    SURF-6 is an evolutionarily conserved nucleolar protein that is important for cell viability; however, its function in mammals still remains uncertain. The aim of this study is to generate monoclonal antibodies to human SURF-6 protein suitable for fundamental and biomedical research. The full-size human SURF-6 was expressed as a recombinant GST-fusion protein and used as an antigen to generate monoclonal antibodies, S79 and S148, specific for SURF-6. The monoclonal antibody produced by hybridoma clone S79 specifically recognizes endogenous SURF-6 by Western and immunofluorescence analyses in various cultured human cells, and by immunohistochemistry in paraffin-embedded sections of human breast cancer samples. Moreover, S79 immunoprecipitates protein complexes containing SURF-6 from HeLa cells extracts. The antibody S79 recognizes SURF-6 only in human cells; however, the antibody produced by hybridoma clone S148 can detect SURF-6 of human and mouse origin. Monoclonal antibodies to the nucleolar protein SURF-6 described in this work can be a useful tool for studies of ribosome biogenesis in normal and cancer cells.

  5. Application of a human monoclonal antibody in a rapid competitive anti-HIV ELISA.

    Science.gov (United States)

    Döpel, S H; Porstmann, T; Grunow, R; Jungbauer, A; Von Baehr, R

    1989-01-17

    The ELISA is the established screening technique for the detection of antibodies directed against HIV. The first generation assays, mostly based on the sandwich principle, employed purified virus from cell culture and gave both false-positive and false-negative results. Sandwich-type assays preferentially detect IgG antibodies, require a high serum dilution and are two-step procedures. In order to detect an immune response as early as possible after infection anti-HIV antibodies of the IgM class should also be measured. To this end a competitive ELISA has been developed using a solid phase-adsorbed recombinant HIV envelope protein and an enzyme-labelled human monoclonal antibody. This detects both IgM and IgG antibodies, the results are available within 1 h and a serum predilution is not necessary.

  6. Characterisation of new monoclonal antibodies reacting with prions from both human and animal brain tissues

    DEFF Research Database (Denmark)

    Hvass, Henriette Cordes; Bergström, Ann-Louise; Ohm, Jakob

    2008-01-01

    Post-mortem diagnosis of transmissible spongiform encephalopaties (prion diseases) is primarily based on the detection of a protease resistant, misfolded disease associated isoform (PrPSc) of the prion protein (PrPc) on neuronal cells. These methods depend on antibodies directed aganinst Pr......Pc and capable of reacting with PrpSc in situ (immunohistochemistry on nervous tissue sections) or with the unfolded form of the protein (western and paraffin embedded tissue (PET) blotting). Here, high-affinity monoclonal antibodies (mAbs 1.5D7, 1.6F4) were produced against synthetic PrP peptides in wild......-type mice and used for western blotting and immunohistochemistry to detect several types of human prion-disease associated PrPSc, including sporadic Creutzfeldt-Jakob Disease (CJD) (subtypes MM1 and V"), familial CJD and Gerstmann-Sträussler-Scheinker (GSS) disease PrPSc as well as PrPSc of bovine...

  7. Anion exchange chromatographic distribution of human monoclonal immunoglobulin G is determined by heavy chain subclass and level of sialic acid expression

    Directory of Open Access Journals (Sweden)

    Ilić Vesna

    2012-01-01

    Full Text Available Anion exchange chromatography is widely accepted method for purification of immunoglobulins. In this work, we used human monoclonal immunoglobulin G (IgG with structure and solubility of normal human IgG as a model for studying chromatographic behavior of particular molecular forms of IgG. Human sera with monoclonal IgG were fractionated on strong anion exchanger, Q Sepharose Fast Flow. With 20 mM Tris pH 7.5 as a start buffer, 42% of human monoclonal IgG passed through column, and 58% of them remained adsorbed. Bound monoclonal IgG were eluted from the exchanger by linear increasing of concentration of NaCl from 0 to 0.5 M. Chromatographic distribution of human monoclonal IgG correlated with their electrophoretic mobility in agarose gels, and it was dependent on γ heavy chain isotype. Light chain type, as well as serum concentration of monoclonal IgG did not influence their chromatographic behavior. The level of heavy chain sialic acid expression, but not of galactose and N-acetylglucosamine, significantly determined chromatographic distribution of serum monoclonal IgG. Additionally to the information on the chromatographic behavior of human monoclonal IgG, we believe that presented data could provide useful information about the possible use of Q Sepharose Fast Flow matrix for the isolation of specific molecular forms of human IgG.

  8. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    Directory of Open Access Journals (Sweden)

    Yongjun Yin

    2016-05-01

    Full Text Available Activating mutations in fibroblast growth factor receptor 3 (FGFR3 have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9, a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11 with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3.

  9. Novel human monoclonal antibody combination effectively neutralizing natural rabies virus variants and individual in vitro escape mutants

    NARCIS (Netherlands)

    Bakker, Alexander B. H.; Marissen, Wilfred E.; Kramer, R. Arjen; Rice, Amy B.; Weldon, William C.; Niezgoda, Michael; Hanlon, Cathleen A.; Thijsse, Sandra; Backus, Harold H. J.; de Kruif, John; Dietzschold, Bernhard; Rupprecht, Charles E.; Goudsmit, Jaap

    2005-01-01

    The need to replace rabies immune globulin (RIG) as an essential component of rabies postexposure prophylaxis is widely acknowledged. We set out to discover a unique combination of human monoclonal antibodies (MAbs) able to replace RIG. Stringent criteria concerning neutralizing potency, affinity,

  10. Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies.

    Science.gov (United States)

    Schwelberger, Hubert G; Feurle, Johannes; Houen, Gunnar

    2017-01-01

    The lack of suitable antibodies for the histamine inactivating enzyme histamine N-methyltransferase (HMT) has so far prevented the direct analysis of HMT proteins in man and other mammals. A series of monoclonal antibodies was produced by immunizing mice with human and porcine HMT expressed in vitro. Antibodies were characterized by immunoblotting and immunohistochemical staining. Six different monoclonal antibodies specific for human HMT and four different monoclonal antibodies specific for porcine HMT were obtained that can detect HMT with up to tenfold greater sensitivity than the most sensitive enzymatic assays currently available. Using these antibodies allowed us to confirm the expression and cellular localization of HMT in various human and porcine tissues, where the presence of the enzyme had previously been deduced from activity measurement and HMT mRNA analysis. Immunohistochemical staining of human and porcine tissue sections clearly showed that HMT is a cytosolic protein, which is localized in specific cells of most mammalian tissues. The new monoclonal antibodies not only allow a comprehensive quantitative evaluation of the expression of HMT at the cellular level in man and other mammals but will also facilitate sensitive analyses of disease-associated alterations of this protein.

  11. First administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity

    NARCIS (Netherlands)

    Bakker, A. B. H.; Python, C.; Kissling, C. J.; Pandya, P.; Marissen, W. E.; Brink, M. F.; Lagerwerf, F.; Worst, S.; van Corven, E.; Kostense, S.; Hartmann, K.; Weverling, G. J.; Uytdehaag, F.; Herzog, C.; Briggs, D. J.; Rupprecht, C. E.; Grimaldi, R.; Goudsmit, J.

    2008-01-01

    Immediate passive immune prophylaxis as part of rabies post-exposure prophylaxis (PEP) often cannot be provided due to limited availability of human or equine rabies immunoglobulin (HRIG and ERIG, respectively). We report first clinical data from two phase I studies evaluating a monoclonal antibody

  12. Monoclonal antibody GB3, a new probe for the study of human basement membranes and hemidesmosomes

    Energy Technology Data Exchange (ETDEWEB)

    Verrando, P.; Pisani, A.; Serieys, N.; Ortonne, J.P. (UER Medecine, Nice (France)); Hsi, Baeli; Yeh, Changjing (INSERM U210, Nice (France))

    1987-05-01

    A monoclonal antibody, GB3, has been raised against human amnion. Not only does GB3 bind to amniotic basement membrane, but it also recognizes an antigenic structure expressed by epidermal as well as by some other human basement membranes. This antigen is synthesized (and excreted) by cultured normal human epidermal keratinocytes. It is expressed to a lesser extent by the A431 epidermoid carcinoma cell line, but is not expressed by the SV40 virus-transformed SVK14 keratinocyte cell line. In ultrastructural studies, this antigen was located in the epidermal basement membrane, both in the lamina densa and in the lamina lucida, associated with hemidesmosomes. It was identified as a protein by in vitro proteolytic cleavage studies. The radio-immunoprecipitates from cultured human keratinocytes, analyzed by SDS-PAGE, showed that GB3 recognized five polypeptides of 93.5, 125, 130, 146 and 150 kD under reducing conditions. The tissue distribution of the antigen and the molecular weights (MWs) of its constitutive polypeptides suggest that it is different from other known components of basement membranes. It may provide a biochemical marker for hemidesmosomes. Furthermore, GB3 represents an interesting and original clinical probe, since the antigenic structure recognized by GB3 is lacking in Junctional Epidermolysis Bullosa, a lethal genodermatosis in which a dermo-epidermal splitting occurs at the level of lamina lucida.

  13. A novel human anti-interleukin-1β neutralizing monoclonal antibody showing in vivo efficacy.

    Science.gov (United States)

    Goh, Angeline X H; Bertin-Maghit, Sebastien; Ping Yeo, Siok; Ho, Adrian W S; Derks, Heidi; Mortellaro, Alessandra; Wang, Cheng-I

    2014-01-01

    The pro-inflammatory cytokine interleukin (IL)-1β is a clinical target in many conditions involving dysregulation of the immune system; therapeutics that block IL-1β have been approved to treat diseases such as rheumatoid arthritis (RA), neonatal onset multisystem inflammatory diseases, cryopyrin-associated periodic syndromes, active systemic juvenile idiopathic arthritis. Here, we report the generation and engineering of a new fully human antibody that binds tightly to IL-1β with a neutralization potency more than 10 times higher than that of the marketed antibody canakinumab. After affinity maturation, the derived antibody shows a>30-fold increased affinity to human IL-1β compared with its parent antibody. This anti-human IL-1β IgG also cross-reacts with mouse and monkey IL-1β, hence facilitating preclinical development. In a number of mouse models, this antibody efficiently reduced or abolished signs of disease associated with IL-1β pathology. Due to its high affinity for the cytokine and its potency both in vitro and in vivo, we propose that this novel fully human anti-IL-1β monoclonal antibody is a promising therapeutic candidate and a potential alternative to the current therapeutic arsenal.

  14. Epitope location for two monoclonal antibodies against human cystatin C, representing opposite aggregation inhibitory properties.

    Science.gov (United States)

    Behrendt, Izabela; Prądzińska, Martyna; Spodzieja, Marta; Kołodziejczyk, Aleksandra S; Rodziewicz-Motowidło, Sylwia; Szymańska, Aneta; Czaplewska, Paulina

    2016-07-01

    Human cystatin C (hCC), like many other amyloidogenic proteins, dimerizes and possibly makes aggregates by subdomain swapping. Inhibition of the process should suppress the fibrillogenesis leading to a specific amyloidosis (hereditary cystatin C amyloid angiopathy, HCCAA). It has been reported that exogenous agents like monoclonal antibodies against cystatin C are able to suppress formation of cystatin C dimers and presumably control the neurodegenerative disease. We have studied in detail two monoclonal antibodies (mAbs) representing very different aggregation inhibitory potency, Cyst10 and Cyst28, to find binding sites in hCC sequence responsible for the immunocomplex formation and pave the way for possible immunotherapy of HCCAA. We used the epitope extraction/excision mass spectrometry approach with the use of different enzymes complemented by affinity studies with synthetic hCC fragments as a basic technique for epitope identification. The results were analyzed in the context of hCC structure allowing us to discuss the binding sites for both antibodies. Epitopic sequences for clone Cyst28 which is a highly potent dimerization inhibitor were found in N-terminus, loop 1 and 2 (L1, L2) and fragments of β2 and β3 strands. The crucial difference between conformational epitope sequences found for both mAbs seems to be the lack of interactions with hCC via N-terminus and the loop 1 in the case of mAb Cyst10. Presumably the interactions of mAbs with hCC via L1 and β sheet fragments make the hCC structure rigid and unable to undergo the swapping process.

  15. New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

    Science.gov (United States)

    O'Brien, Carmel M; Chy, Hun S; Zhou, Qi; Blumenfeld, Shiri; Lambshead, Jack W; Liu, Xiaodong; Kie, Joshua; Capaldo, Bianca D; Chung, Tung-Liang; Adams, Timothy E; Phan, Tram; Bentley, John D; McKinstry, William J; Oliva, Karen; McMurrick, Paul J; Wang, Yu-Chieh; Rossello, Fernando J; Lindeman, Geoffrey J; Chen, Di; Jarde, Thierry; Clark, Amander T; Abud, Helen E; Visvader, Jane E; Nefzger, Christian M; Polo, Jose M; Loring, Jeanne F; Laslett, Andrew L

    2017-03-01

    The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640. © 2016 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  16. Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

    Energy Technology Data Exchange (ETDEWEB)

    Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Oshita, Masatoshi; Ideno, Shoji [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Yunoki, Mikihiro [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Kuhara, Motoki [Ina Laboratory, Medical and Biological Laboratories Corporation, Ltd., Ina, Nagano 396-0002 (Japan); Yamamoto, Naomasa [Department of Biochemistry, School of Pharmaceutical Sciences, Ohu University, Koriyama, Fukushima 963-8611 (Japan); Okuno, Yoshinobu [Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa 768-0061 (Japan); Ikuta, Kazuyoshi, E-mail: ikuta@biken.osaka-u.ac.jp [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan)

    2009-09-11

    Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

  17. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

    Science.gov (United States)

    Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  18. Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

    Directory of Open Access Journals (Sweden)

    Koushan Sineh sepehr

    2013-02-01

    Full Text Available Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells.

  19. Protection of rabbits and immunodeficient mice against lethal poxvirus infections by human monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Lindsay Crickard

    Full Text Available Smallpox (variola virus is a bioweapon concern. Monkeypox is a growing zoonotic poxvirus threat. These problems have resulted in extensive efforts to develop potential therapeutics that can prevent or treat potentially lethal poxvirus infections in humans. Monoclonal antibodies (mAbs against smallpox are a conservative approach to this problem, as the licensed human smallpox vaccine (vaccinia virus, VACV primarily works on the basis of protective antibody responses against smallpox. Fully human mAbs (hmAbs against vaccinia H3 (H3L and B5 (B5R, targeting both the mature virion (MV and extracellular enveloped virion (EV forms, have been developed as potential therapeutics for use in humans. Post-exposure prophylaxis was assessed in both murine and rabbit animal models. Therapeutic efficacy of the mAbs was assessed in three good laboratory practices (GLP studies examining severe combined immunodeficiency mice (SCID given a lethal VACV infection. Pre-exposure combination hmAb therapy provided significantly better protection against disease and death than either single hmAb or vaccinia immune globulin (VIG. Post-exposure combination mAb therapy provided significant protection against disease and death, and appeared to fully cure the VACV infection in ≥50% of SCID mice. Therapeutic efficacy was then assessed in two rabbit studies examining post-exposure hmAb prophylaxis against rabbitpox (RPXV. In the first study, rabbits were infected with RPVX and then provided hmAbs at 48 hrs post-infection, or 1 hr and 72 hrs post-infection. Rabbits in both groups receiving hmAbs were 100% protected from death. In the second rabbitpox study, 100% of animal treated with combination hmAb therapy and 100% of animals treated with anti-B5 hmAb were protected. These findings suggest that combination hmAb treatment may be effective at controlling smallpox disease in immunocompetent or immunodeficient humans.

  20. Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile Virus.

    Science.gov (United States)

    Throsby, Mark; Geuijen, Cecile; Goudsmit, Jaap; Bakker, Arjen Q; Korimbocus, Jehanara; Kramer, R Arjen; Clijsters-van der Horst, Marieke; de Jong, Maureen; Jongeneelen, Mandy; Thijsse, Sandra; Smit, Renate; Visser, Therese J; Bijl, Nora; Marissen, Wilfred E; Loeb, Mark; Kelvin, David J; Preiser, Wolfgang; ter Meulen, Jan; de Kruif, John

    2006-07-01

    Monoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred twenty-one MAbs specifically bound to the viral envelope (E) protein and four MAbs to the premembrane (prM) protein. Enzyme-linked immunosorbent assay-based competitive-binding assays with representative E protein-specific MAbs demonstrated that 24/51 (47%) bound to domain II while only 4/51 (8%) targeted domain III. In vitro neutralizing activity was demonstrated for 12 MAbs, and two of these, CR4374 and CR4353, protected mice from lethal WNV challenge at 50% protective doses of 12.9 and 357 mug/kg of body weight, respectively. Our data analyzing three infected individuals suggest that the human anti-WNV repertoire after natural infection is dominated by nonneutralizing or weakly neutralizing MAbs binding to domain II of the E protein, while domain III-binding MAbs able to potently neutralize WNV in vitro and in vivo are rare.

  1. Strategies to Obtain Diverse and Specific Human Monoclonal Antibodies From Transgenic Animals.

    Science.gov (United States)

    Brüggemann, Marianne; Osborn, Michael J; Ma, Biao; Buelow, Roland

    2017-08-01

    Techniques to obtain large quantities of antigen-specific monoclonal antibodies (mAbs) were first established in the 1970s when Georges Köhler and César Milstein immortalized antibody-producing mouse B-lymphocytes by fusion with myeloma cells (http://www.whatisbiotechnology.org/exhibitions/milstein). Combined with the expression of human antibodies in transgenic animals, this technique allowed upon immunization the generation of highly specific fully human mAbs for therapeutic applications. Apart from being extremely beneficial, mAbs are a huge success commercially. However, despite cell fusion generating many useful mAbs questions have been asked about which types of cells are prone to fuse and whether other methods may identify a wider range of binders. The discovery that expression libraries, using Escherichia coli or yeast, produced different specificities was intriguing and more recently Next-Generation Sequencing has identified wide-ranging usage with highly diverse and unique repertoires. Another strategy is the combination of flow cytometry sorting of antigen-binding B lymphocytes and single-cell reverse transcription polymerase chain reaction followed by reexpression, which has identified many high-affinity mAbs.

  2. Monoclonal Antibodies to the Human Insulin Receptor that Activate Glucose Transport but not Insulin Receptor Kinase Activity

    Science.gov (United States)

    Forsayeth, John R.; Caro, Jose F.; Sinha, Madhur K.; Maddux, Betty A.; Goldfine, Ira D.

    1987-05-01

    Three mouse monoclonal antibodies were produced that reacted with the α subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

  3. Monoclonal Antibodies.

    Science.gov (United States)

    Killington, R. A.; Powell, K. L.

    1984-01-01

    Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)

  4. Humanization and characterization of an anti-ricin neutralization monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Wei-Gang Hu

    Full Text Available Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9 variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with K(D of 1.63 nM, comparable to its parental murine D9 (2.55 nM. In a mouse model, intraperitoneal (i.p. administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.

  5. Screening of anti-human leukocyte monoclonal antibodies for reactivity with equine leukocytes.

    Science.gov (United States)

    Ibrahim, Sherif; Saunders, Kelly; Kydd, Julia H; Lunn, D Paul; Steinbach, Falko

    2007-09-15

    Three hundred and seventy-nine monoclonal antibodies (mAbs) against various human CD molecules supplied to the HLDA8 animal homologues section (including four isotype controls) were analysed for cross-reactivity with equine leukocytes. First, flow cytometric identification of positively reacting mAbs was performed in one laboratory. Thereafter, a second round of flow cytometric evaluation was performed, involving three laboratories participating in the study. The first test-round indicated 17 mAbs as potentially positive. After the second round of flow cytometric analysis, 14 mAbs remained (directed against CD2, CD11a, CD18, CD44, CD45, CD49d, CD91, CD163 and CD172) where cross-reactivity was anticipated based on similarities between the human and equine staining pattern. Additionally, there was 1 mAb with weak likely positive reactivity, 12 mAbs with positive staining, which likely do not reflect valuable data, 5 mAbs with clear alternate expression pattern from that expected from humans, 5 mAbs with a questionable staining pattern itself, i.e. that was variable between the three labs, 32 mAbs with weak-positive expression and alternate staining pattern, and 279 negative mAbs (including the four isotype controls) were detected. In 31 cases, more appropriate target cells, such as thymocytes or stem cells, were not available for the screening. The results underline the value of this "cross-reactivity" approach for equine immunology. However, as only a few mAbs against leukocyte surface antigens reacted positively (approximately 4% of the mAbs submitted), the analysis of further anti-human mAbs and directed efforts to develop species-specific anti-CD mAb are still required.

  6. Humanization and characterization of an anti-ricin neutralization monoclonal antibody.

    Science.gov (United States)

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Negrych, Laurel M; Cherwonogrodzky, John W

    2012-01-01

    Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9) variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with K(D) of 1.63 nM, comparable to its parental murine D9 (2.55 nM). In a mouse model, intraperitoneal (i.p.) administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.

  7. A neutralizing human monoclonal antibody protects African Green monkeys from Hendra virus challenge

    Science.gov (United States)

    Bossart, Katharine N.; Geisbert, Thomas W.; Feldmann, Heinz; Zhu, Zhongyu; Feldmann, Friederike; Geisbert, Joan B.; Yan, Lianying; Feng, Yan-Ru; Brining, Doug; Scott, Dana; Wang, Yanping; Dimitrov, Antony S.; Callison, Julie; Chan, Yee-Peng; Hickey, Andrew C.; Dimitrov, Dimiter S.; Broder, Christopher C.; Rockx, Barry

    2012-01-01

    Hendra virus (HeV) is a recently emerged zoonotic paramyxovirus that can cause a severe and often fatal disease in horses and humans. HeV is categorized as a biosafety level 4 agent, which has made the development of animal models and testing of potential therapeutics and vaccines challenging. Infection of African Green monkeys (AGMs) with HeV was recently demonstrated and disease mirrored fatal HeV infection in humans, manifesting as a multisystemic vasculitis with widespread virus replication in vascular tissues and severe pathologic manifestations in the lung, spleen and brain. Here, we demonstrate that m102.4, a potent HeV neutralizing human monoclonal antibody (hmAb), can protect AGMs from disease post infection (p.i.) with HeV. Fourteen AGMs were challenged intratracheally with a lethal dose of HeV and twelve subjects were infused twice with a 100 mg dose of m102.4 beginning at either 10 hr, 24 hr or 72 hr p.i. and again approximately 48 hrs later. The presence of viral RNA, infectious virus and HeV-specific immune responses demonstrated that all subjects were infected following challenge. All twelve AGMs that received m102.4 survived infection; whereas the untreated control subjects succumbed to disease on day 8 p.i.. Animals in the 72 hr treatment group exhibited neurological signs of disease but all animals started to recover by day 16 p.i.. These results represent successful post-exposure in vivo efficacy by an investigational drug against HeV and highlight the potential impact a hmAb can have on human disease. PMID:22013123

  8. Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: KD values, binding pairs, and amino acid sequences.

    Science.gov (United States)

    Peng, Hong; Brimijoin, Stephen; Hrabovska, Anna; Targosova, Katarina; Krejci, Eric; Blake, Thomas A; Johnson, Rudolph C; Masson, Patrick; Lockridge, Oksana

    2015-10-05

    Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at -80 °C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10(-9) M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. Novel Monoclonal Antibodies Recognizing Human Prostate-Specific Membrane Antigen (PSMA) as Research and Theranostic Tools.

    Science.gov (United States)

    Nováková, Zora; Foss, Catherine A; Copeland, Benjamin T; Morath, Volker; Baranová, Petra; Havlínová, Barbora; Skerra, Arne; Pomper, Martin G; Barinka, Cyril

    2017-05-01

    Prostate-specific membrane antigen (PSMA) is a validated target for the imaging and therapy of prostate cancer. Here, we report the detailed characterization of four novel murine monoclonal antibodies (mAbs) recognizing human PSMA as well as PSMA orthologs from different species. Performance of purified mAbs was assayed using a comprehensive panel of in vitro experimental setups including Western blotting, immunofluorescence, immunohistochemistry, ELISA, flow cytometry, and surface-plasmon resonance. Furthermore, a mouse xenograft model of prostate cancer was used to compare the suitability of the mAbs for in vivo applications. All mAbs demonstrate high specificity for PSMA as documented by the lack of cross-reactivity to unrelated human proteins. The 3F11 and 1A11 mAbs bind linear epitopes spanning residues 226-243 and 271-288 of human PSMA, respectively. 3F11 is also suitable for the detection of PSMA orthologs from mouse, pig, dog, and rat in experimental setups where the denatured form of PSMA is used. 5D3 and 5B1 mAbs recognize distinct surface-exposed conformational epitopes and are useful for targeting PSMA in its native conformation. Most importantly, using a mouse xenograft model of prostate cancer we show that both the intact 5D3 and its Fab fragment are suitable for in vivo imaging. With apparent affinities of 0.14 and 1.2 nM as determined by ELISA and flow cytometry, respectively, 5D3 has approximately 10-fold higher affinity for PSMA than the clinically validated mAb J591 and, therefore, is a prime candidate for the development of next-generation theranostics to target PSMA. Prostate 77:749-764, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  10. Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Felix Hart

    2017-05-01

    Full Text Available IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich and hematological (CD20 cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.

  11. Identification of a human monoclonal antibody to replace equine diphtheria antitoxin for treatment of diphtheria intoxication.

    Science.gov (United States)

    Sevigny, Leila M; Booth, Brian J; Rowley, Kirk J; Leav, Brett A; Cheslock, Peter S; Garrity, Kerry A; Sloan, Susan E; Thomas, William; Babcock, Gregory J; Wang, Yang

    2013-11-01

    Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 μg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic.

  12. Characterization of a human monoclonal antibody against Shiga toxin 2 expressed in Chinese hamster ovary cells.

    Science.gov (United States)

    Akiyoshi, D E; Rich, C M; O'Sullivan-Murphy, S; Richard, L; Dilo, J; Donohue-Rolfe, A; Sheoran, A S; Chapman-Bonofiglio, S; Tzipori, S

    2005-07-01

    Shiga toxin-producing Escherichia coli infections can often lead to the development of hemolytic-uremic syndrome (HUS) in a small percentage of infected humans. Patients with HUS receive only supportive treatment as the benefit of antibiotic therapy remains uncertain. We have previously reported the generation and preclinical evaluation of neutralizing human monoclonal antibodies (HuMAbs) against the Shiga toxins (Stx). In this paper, we describe the expression in Chinese hamster ovary (CHO) cells of 5C12 HuMAb, which is directed against the A subunit of Stx2. The cDNAs of the light and heavy chain immunoglobulin (Ig) variable regions of 5C12 HuMAb were isolated and cloned into an expression vector containing human IgG1 constant regions. The vector was transfected into CHO cells, and transfectants secreting Stx2-specific antibody were screened by an Stx2-specific enzyme-linked immunosorbent assay. The CHO-produced recombinant 5C12 (r5C12) showed similar specificity and binding affinity to Stx2 as the parent hybridoma-produced 5C12. More significantly, the r5C12 displayed the same neutralizing activity as the parent 5C12 in vitro and in vivo. In the mouse toxicity model, both antibodies significantly and equally prolonged survival at a dose of 0.312 microg/mouse. The data showed that since r5C12, produced in CHO cells, was equally effective as the parent 5C12, it is our choice candidate as a potential prophylactic or therapeutic agent against hemolytic-uremic syndrome.

  13. Radioimmunoassay of bovine, ovine and porcine luteinizing hormone with a monoclonal antibody and a human tracer

    Energy Technology Data Exchange (ETDEWEB)

    Fosberg, M.; Tagle, R.; Madej, A.; Molina, J.R.; Carlsson, M.-A.

    1993-01-01

    A radioimmunoassay for bovine (bLH), ovine (oLH) and porcine (pLH) luteinizing hormone was developed using a human [sup 125]ILH tracer from a commercial kit and a monoclonal antibody (518B7) specific for LH but with low species specificity. Standard curves demonstrated similar binding kinetics when bLH, oLH and pLH were incubated with tracer and antibody for 2 h at room temperature. A 30-min delay in the addition of the tracer gave sufficient sensitivity when analysing pLH. Separation of antibody-bound LH from free hormone was achieved by using second antibody-coated micro Sepharose beads. The assay was validated and the performance compared with that of an RIA currently in use for determination of bLH (coefficient of correlation: 0.99 and 0.98). Regardless of the standards used, intra-assay coefficients of variation were <10% for LH concentrations exceeding 1 [mu]g/L. The inter-assay coefficients of variation were <15%. The assay was used for clinical evaluation demonstrating the pre-ovulatory LH surge in two cyclic cows, LH pulsatility in an oophorectomized ewe and LH response to GnRH injection in a boar. (au) (7 refs.).

  14. Preparative purification of human monoclonal antibody isoforms in a multi-compartment electrolyser with immobiline membranes.

    Science.gov (United States)

    Righetti, P G; Wenisch, E; Jungbauer, A; Katinger, H; Faupel, M

    1990-02-02

    The performance of a multi-compartment electrolyser with isoelectric Immobiline membranes for large-scale protein purification is evaluated. Owing to the presence of isoelectric membranes possessing a high buffering capacity and ionic strength, isoelectric protein precipitation inside the membranes, one of the major drawbacks of present membrane uses, is fully avoided. In addition, owing to this novel membrane technology, pH gradient decay, typical of isoelectric focusing in carrier ampholytes, is fully eliminated and pH and conductivity constancy is guaranteed in all flow chambers for running periods of more than 11 days (160,000 V h). The membranes described possess a unique selectivity, in that they act by modulating the surface charge (i.e., the mobility) of macroions crossing or tangential to them. The concept of isoelectric Immobiline membranes acting like a pH-stat unit is introduced. Protein homogeneity in each chamber of the electrolyser can be achieved even when purifying human monoclonal antibodies against HIV-1, which possess high pI values (9.0-9.6), are large molecules (Mr 150,000) and are fractionated in the presence of large micelles of neutral detergents.

  15. Pharmacology and placental transfer of a human alphav integrin monoclonal antibody in rabbits.

    Science.gov (United States)

    Martin, Pauline L; Zhou, Zhao; Van den Bulck, Kathleen; Kwok, Deborah; Powers, Gordon; Jiao, Qun; Schantz, Allen; Treacy, George

    2010-04-01

    Intetumumab is a human IgG1 anti-alphav-integrin monoclonal antibody that inhibits angiogenesis. Integrin binding and angiogenesis are important in reproduction including fertilization, implantation, and embryofetal development. These studies were designed to determine the pharmacological relevance of the rabbit for the evaluation of potential effects on embryofetal development and to evaluate the placental transfer of intetumumab in rabbits. In vitro pharmacology studies evaluated the binding of intetumumab to rabbit cells and the inhibition of vessel sprouting from rabbit aorta. For the evaluation of placental transfer, pregnant rabbits (8/group) were injected intravenously with intetumumab 50 or 100 mg/kg every 2 days from Gestation Day (GD)7 to GD19. Maternal sera, fetal homogenates/sera, and amniotic fluid were collected at necropsy on GD19 or GD28 for evaluation of intetumumab concentrations. Clinical condition of the dams was monitored and fetuses were screened for abnormalities. Intetumumab (5-40 microg/mL) inhibited aortic cell adhesion to vitronectin and vessel sprouting from rabbit aortic rings. Immunohistochemical staining of rabbit tissues demonstrated binding of intetumumab to placenta. Administration of intetumumab to pregnant rabbits was well tolerated by the dams and the fetuses did not show major abnormalities. Fetal exposure to intetumumab relative to maternal exposure was rabbit is a pharmacologically relevant species for evaluation of potential developmental effects of intetumumab. Intetumumab crosses the rabbit placenta during the fetal period (GD 19-28). 2010 Wiley-Liss, Inc.

  16. Utilisation of tracer monoclonal antibodies for the immunoscintigraphic detection of human colorectal cancers

    Energy Technology Data Exchange (ETDEWEB)

    Chatal, J.F.; Douillard, J.Y.; Kremer, M.; Curtet, C.; Le Mevel, B. (INSERM, Faculte de Medecine, Nantes (France)); Fumoleau, P. (Centre Rene Gauducheau, Nantes (France)); Bourdoiseau, M. (Office des Rayonnements Ionisants, CEA Saclay, Gif-sur-Yvette (France))

    1983-01-01

    Two monoclonal antibodies, 17-1A and 19-9, with recognized human gastrointestinal cancers in cell cultures, were labeled with iodine 131 for immunoscintigraphic application. With the intact /sup 131/I-17-1A antibody, 21 out of 35 (60%) primary or secondary colorectal cancer sites were visualized, whereas all 21 nonepitheliomatous colic cancer sites or noncolic cancer sites were negative. With F(ab')/sub 2/ fragments of the 19-9 antibody, 18 out of 27 (67%) colorectal cancer sites were positive. With both radioantibodies, the bestly contrasted tumor images were late, 4 to 5 days after injection. A study with paired-label technique, associating a specific iodine-131-labeled antibody with a non-specific iodine-125-labeled immunoglobulin, demonstrated, that tumor uptake was indeed specific for the 17-1A or 19-9 antibody in tumor and normal colon fragments obtained during operations on 4 patients. A preliminary prospective study showed that only immunoscintigraphy was able to confirm and localize a recurrence of rectal cancer in one patient. A larger series will be necessary to validate the clinical benefit of the technique, as compared with the results of other diagnostic techniques, before immunoscintigraphy can be proposed for routine clinical use.

  17. Production of a monoclonal antibody in plants with a humanized N-glycosylation pattern.

    Science.gov (United States)

    Schähs, Matthias; Strasser, Richard; Stadlmann, Johannes; Kunert, Renate; Rademacher, Thomas; Steinkellner, Herta

    2007-09-01

    In recent years, plants have become an attractive alternative for the production of recombinant proteins. However, their inability to perform authentic mammalian N-glycosylation may cause limitations for the production of therapeutics. A major concern is the presence of beta1,2-xylose and core alpha1,3-fucose residues on complex N-linked glycans, as these N-glycan epitopes are immunogenic in mammals. In our attempts towards the humanization of plant N-glycans, we have generated an Arabidopsis thaliana knockout line that synthesizes complex N-glycans lacking immunogenic xylose and fucose epitopes. Here, we report the expression of a monoclonal antibody in these glycan-engineered plants that carry a homogeneous mammalian-like complex N-glycan pattern without beta1,2-xylose and core alpha1,3-fucose. Plant and Chinese hamster ovary (CHO)-derived immunoglobulins (IgGs) exhibited no differences in electrophoretic mobility and enzyme-linked immunosorbent specificity assays. Our results demonstrate the feasibility of a knockout strategy for N-glycan engineering of plants towards mammalian-like structures, thus providing a significant improvement in the use of plants as an expression platform.

  18. Applications of monoclonal antibodies and recombinant cytokines for the treatment of human colorectal and other carcinomas

    Energy Technology Data Exchange (ETDEWEB)

    Greiner, J.W.; Smalley, R.V.; Borden, E.C.; Martin, E.W.; Guadagni, F.; Roselli, M.; Schlom, J. (Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD (United States))

    1991-01-01

    Monoclonal antibodies (MAbs) which recognize a human tumor antigen, termed tumor-associated glycoprotein-72 (TAG-72), have successfully been used to localize primary as well as metastatic colorectal tumor lesions in patients. The localization of the anti-TAG-72 MAbs has also been exploited intraoperatively using a hand-held gamma probe. That procedure, termed radioimmunoguided surgery (RIGS), has identified occult tumors which were not detected using standard external imaging techniques. In another clinical trial, interferon-gamma (IFN-gamma) was administered intraperitoneally to patients diagnosed with either gastrointestinal or ovarian carcinoma with secondary ascites. Analysis of the tumor cells isolated from the malignant ascites revealed a substantial increase in TAG-72 expression on the surface of tumor cells isolated from seven of eight patients. The results provide evidence that the combination of an anti-carcinoma MAb with the administration of a cytokine, such as IFN-gamma, may be an effective approach for the detection and subsequent treatment, of colorectal carcinoma. 15 references.

  19. Monoclonal antibody binding to the macrophage-specific receptor sialoadhesin alters the phagocytic properties of human and mouse macrophages.

    Science.gov (United States)

    De Schryver, Marjorie; Cappoen, Davie; Elewaut, Dirk; Nauwynck, Hans J; Maes, Louis; Caljon, Guy; Cos, Paul; Delputte, Peter L

    2017-02-01

    Sialoadhesin (Sn) is a surface receptor expressed on macrophages in steady state conditions, but during inflammation, Sn can be upregulated both on macrophages and on circulating monocytes. It was shown for different species that Sn becomes internalized after binding with monoclonal antibodies. These features suggest that Sn is a potential target for immunotherapies. In this study, human and mouse macrophages were treated with anti-Sn monoclonal antibodies or F(ab')2 fragments and the effect of their binding to Sn on phagocytosis was analyzed. Binding of antibodies to Sn resulted in delayed and reduced phagocytosis of fluorescent beads. No effect was observed on Fc-mediated phagocytosis or phagocytosis of bacteria by human macrophages. In contrast, an enhanced phagocytosis of bacteria by mouse macrophages was detected. These results showed that stimulation of Sn could have different effects on macrophage phagocytosis, depending both on the type of phagocytosis and cellular background. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Characterisation of new monoclonal antibodies reacting with prions from both human and animal brain tissues.

    Science.gov (United States)

    Cordes, Henriette; Bergström, Ann-Louise; Ohm, Jakob; Laursen, Henning; Heegaard, Peter M H

    2008-09-15

    Post-mortem diagnosis of transmissible spongiform encephalopathies (prion diseases) is primarily based on the detection of a protease resistant, misfolded disease associated isoform (PrP(Sc)) of the prion protein (PrP(C)) on neuronal cells. These methods depend on antibodies directed against PrP(C) and capable of reacting with PrP(Sc)in situ (immunohistochemistry on nervous tissue sections) or with the unfolded form of the protein (western and paraffin embedded tissue (PET) blotting). Here, high-affinity monoclonal antibodies (mAbs 1.5D7, 1.6F4) were produced against synthetic PrP peptides in wild-type mice and used for western blotting and immunohistochemistry to detect several types of human prion-disease associated PrP(Sc), including sporadic Creutzfeldt-Jakob Disease (CJD) (subtypes MM1 and VV2), familial CJD and Gerstmann-Sträussler-Scheinker (GSS) disease PrP(Sc) as well as PrP(Sc) of bovine spongiform encephalopathy (bovine brain), scrapie (ovine brain) and experimental scrapie in hamster and in mice. The antibodies were also used for PET-blotting in which PrP(Sc) blotted from brain tissue sections onto a nitrocellulose membrane is visualized with antibodies after protease and denaturant treatment allowing the detection of protease resistant PrP forms (PrP(RES)) in situ. Monoclonal antibodies 1.5D7 and 1.6F4 were raised against the reported epitope (PrP153-165) of the commercial antibody 6H4. While 1.5D7 and 1.6F4 were completely inhibitable by PrP153-165, 6H4 was not, indicating that the specificity of 6H4 is not defined completely by PrP153-165. The two antibodies performed similarly to 6H4 in western blotting with human samples, but showed less reactivity and enhanced background staining with animal samples in this method. In immunohistochemistry 1.5D7 and 1.6F4 performed better than 6H4 suggesting that the binding affinity of 1.5D7 and 1.6F4 with native (aggregated) PrP(Sc)in situ was higher than that of 6H4. On the other hand in PET-blotting, 6H4

  1. Neutralization of Zika virus by germline-like human monoclonal antibodies targeting cryptic epitopes on envelope domain III

    OpenAIRE

    Wu, Yanling; Li, Shun; Du, Lanying; Wang, Chunyu; Zou, Peng; Hong, Binbin; Yuan, Mengjiao; Ren, Xiaonan; Tai, Wanbo; Kong, Yu; Zhou, Chen; Lu, Lu; Zhou, Xiaohui; Jiang, Shibo; Ying, Tianlei

    2017-01-01

    The Zika virus (ZIKV), a flavivirus transmitted by Aedes mosquitoes, has emerged as a global public health concern. Pre-existing cross-reactive antibodies against other flaviviruses could modulate immune responses to ZIKV infection by antibody-dependent enhancement, highlighting the importance of understanding the immunogenicity of the ZIKV envelope protein. In this study, we identified a panel of human monoclonal antibodies (mAbs) that target domain III (DIII) of the ZIKV envelope protein fr...

  2. Myostatin blockade with a fully human monoclonal antibody induces muscle hypertrophy and reverses muscle atrophy in young and aged mice

    OpenAIRE

    Latres, Esther; Pangilinan, Jeffrey; Miloscio, Lawrence; Bauerlein, Roy; Na, Erqian; Potocky, Terra B.; Huang, Ying; Eckersdorff, Mark; Rafique, Ashique; Mastaitis, Jason; Lin, Calvin; Murphy, Andrew J.; Yancopoulos, George D.; Gromada, Jesper; Stitt, Trevor

    2015-01-01

    Background Loss of skeletal muscle mass and function in humans is associated with significant morbidity and mortality. The role of myostatin as a key negative regulator of skeletal muscle mass and function has supported the concept that inactivation of myostatin could be a useful approach for treating muscle wasting diseases. Methods We generated a myostatin monoclonal blocking antibody (REGN1033) and characterized its effects in vitro using surface plasmon resonance biacore and cell-based Sm...

  3. Characterization of a Type-Common Human Recombinant Monoclonal Antibody to Herpes Simplex Virus with High Therapeutic Potential

    Science.gov (United States)

    De Logu, Alessandro; Williamson, R. Anthony; Rozenshteyn, Roman; Ramiro-Ibañez, Fernando; Simpson, Cindy D.; Burton, Dennis R.; Paolo Sanna, Pietro

    1998-01-01

    We report the characterization of a type-common human recombinant monoclonal antibody previously isolated by antigen selection from a phage-displayed combinatorial antibody library established from a herpes simplex virus (HSV)-seropositive individual. Competition with well-characterized murine monoclonal antibodies and immunodetection of gD truncations revealed that this antibody recognizes the group Ib antigenic site of glycoprotein D, a highly conserved and protective type-common determinant. To our knowledge, this is the first human group Ib monoclonal antibody ever described. The antibody also displayed first-order neutralization kinetics and a high neutralization rate constant, was capable of completely inhibiting syncytium formation by a fusogenic strain of HSV type 1, and efficiently neutralized low-passage clinical isolates of both HSV serotypes. Taken together with our earlier observations of the in vivo antiviral activities of this human recombinant antibody in animal models of HSV infection, the present results support the high therapeutic potential of this antibody. PMID:9774565

  4. Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies.

    Science.gov (United States)

    Pietsch, E C; Dong, J; Cardoso, R; Zhang, X; Chin, D; Hawkins, R; Dinh, T; Zhou, M; Strake, B; Feng, P-H; Rocca, M; Santos, C Dos; Shan, X; Danet-Desnoyers, G; Shi, F; Kaiser, E; Millar, H J; Fenton, S; Swanson, R; Nemeth, J A; Attar, R M

    2017-02-24

    CD47, a broadly expressed cell surface protein, inhibits cell phagocytosis via interaction with phagocyte-expressed SIRPα. A variety of hematological malignancies demonstrate elevated CD47 expression, suggesting that CD47 may mediate immune escape. We discovered three unique CD47-SIRPα blocking anti-CD47 monoclonal antibodies (mAbs) with low nano-molar affinity to human and cynomolgus monkey CD47, and no hemagglutination and platelet aggregation activity. To characterize the anti-cancer activity elicited by blocking CD47, the mAbs were cloned into effector function silent and competent Fc backbones. Effector function competent mAbs demonstrated potent activity in vitro and in vivo, while effector function silent mAbs demonstrated minimal activity, indicating that blocking CD47 only leads to a therapeutic effect in the presence of Fc effector function. A non-human primate study revealed that the effector function competent mAb IgG1 C47B222-(CHO) decreased red blood cells (RBC), hematocrit and hemoglobin by >40% at 1 mg/kg, whereas the effector function silent mAb IgG2σ C47B222-(CHO) had minimal impact on RBC indices at 1 and 10 mg/kg. Taken together, our findings suggest that targeting CD47 is an attractive therapeutic anti-cancer approach. However, the anti-cancer activity observed with anti-CD47 mAbs is Fc effector dependent as are the side effects observed on RBC indices.

  5. Biochemical characterization of AMG 102: a neutralizing, fully human monoclonal antibody to human and nonhuman primate hepatocyte growth factor.

    Science.gov (United States)

    Burgess, Teresa L; Sun, Jan; Meyer, Susanne; Tsuruda, Trace S; Sun, Jilin; Elliott, Gary; Chen, Qing; Haniu, Mitsuru; Barron, Will F; Juan, Todd; Zhang, Ke; Coxon, Angela; Kendall, Richard L

    2010-02-01

    AMG 102 is a fully human monoclonal antibody that selectively targets and neutralizes hepatocyte growth factor/scatter factor (HGF/SF). A detailed biochemical and functional characterization of AMG 102 was done to support its clinical development for the treatment of cancers dependent on signaling through the HGF/SF:c-Met pathway. In competitive equilibrium binding experiments, AMG 102 bound to human and cynomolgus monkey HGF with affinities of approximately 19 pmol/L and 41 pmol/L, respectively. However, AMG 102 did not detect mouse or rabbit HGF on immunoblots. Immunoprecipitation experiments showed that AMG 102 preferentially bound to the mature, active form of HGF, and incubation of AMG 102/HGF complexes with kallikrein protease indicated that AMG 102 had no apparent effect on proteolytic processing of the inactive HGF precursor. AMG 102 inhibited human and cynomolgus monkey HGF-induced c-Met autophosphorylation in PC3 cells with IC(50) values of 0.12 nmol/L and 0.24 nmol/L, respectively. AMG 102 also inhibited cynomolgus monkey HGF-induced migration of human MDA-MB-435 cells but not rat HGF-induced migration of mouse 4T1 cells. Epitope-mapping studies of recombinant HGF molecules comprising human/mouse chimeras and human-to-mouse amino acid substitutions showed that amino acid residues near the NH(2)-terminus of the beta-chain are critical for AMG 102 binding. Bound AMG 102 protected one trypsin protease cleavage site near the NH(2)-terminus of the beta-chain of human HGF, further substantiating the importance of this region for AMG 102 binding. Currently, AMG 102 is in phase II clinical trials in a variety of solid tumor indications. Mol Cancer Ther; 9(2); 400-9.

  6. Human Monoclonal antibodies - A dual advantaged weapon to tackle cancer and viruses

    Directory of Open Access Journals (Sweden)

    Kurosawa G

    2014-11-01

    Full Text Available Human monoclonal antibodies (mAbs are powerful tools as pharmaceutical agents to tackle cancer and infectious diseases. Antibodies (Abs are present in blood at the concentration of 10 mg/ml and play a vital role in humoral immunity. Many therapeutic Abs have been reported since early 1980s. Human mAb technology was not available at that time and only the hybridoma technology for making mouse mAbs had been well established. In order to avoid various potential problems associated with use of mouse proteins, two different technologies to make human/mouse chimeric Ab as well as humanized Ab were developed crossing the various hurdles for almost twenty years and mAb based drugs such as rituximab, anti-CD20 Ab, and trastuzumab, anti-HER2 Ab, have been approved by the US Food and Drug Administration (FDA for treatment of non-Hodgkin's lymphoma and breast cancer in 1997 and 1998, respectively. These drugs are well recognized and accepted by clinicians for treatment of patients. The clinical outcome of the treatment with mAb has strongly encouraged the researchers to develop much more refined mAbs. In addition to chimeric Ab and humanized Ab, now human mAbs can be produced by two technologies. The first is transgenic mice that produce human Abs and the second is human Ab libraries using phage-display system. Until now, several hundreds of mAbs against several tens of antigens (Ags have been developed and subjected to clinical examinations. While many Abs have been approved as therapeutic agents against hematological malignancies, the successful mAbs against solid tumors are still limited. However, many researchers have suggested that developing potential mAbs agents should be possible and incurable cancers may become curable within another decade. Though it is hard to say explicitly that this prediction is correct, a passion for this development should be worth supporting to lead to a successful outcome which will lead to patient benefits. Our institute

  7. Detection of Signal Regulatory Protein α in Saimiri sciureus (Squirrel Monkey) by Anti-Human Monoclonal Antibody

    Science.gov (United States)

    de Souza, Hugo Amorim dos Santos; Costa-Correa, Edmar Henrique; Bianco-Junior, Cesare; Andrade, Márcia Cristina Ribeiro; Lima-Junior, Josué da Costa; Pratt-Riccio, Lilian Rose; Daniel-Ribeiro, Cláudio Tadeu; Totino, Paulo Renato Rivas

    2017-01-01

    Non-human primates (NHP) are suitable models for studying different aspects of the human system, including pathogenesis and protective immunity to many diseases. However, the lack of specific immunological reagents for neo-tropical monkeys, such as Saimiri sciureus, is still a major factor limiting studies in these models. An alternative strategy to circumvent this obstacle has been the selection of immunological reagents directed to humans, which present cross-reactivity with NHP molecules. In this context and considering the key role of inhibitory immunoreceptors—such as the signal regulatory protein α (SIRPα)—in the regulation of immune responses, in the present study, we attempted to evaluate the ability of anti-human SIRPα monoclonal antibodies to recognize SIRPα in antigen-presenting S. sciureus peripheral blood mononuclear cells (PBMC). As shown by flow cytometry analysis, the profile of anti-SIRPα staining as well as the levels of SIRPα-positive cells in PBMC from S. sciureus were similar to those observed in human PBMC. Furthermore, using anti-SIRPα monoclonal antibody, it was possible to detect a decrease of the SIRPα levels on surface of S. sciureus cells after in vitro stimulation with lipopolysaccharides. Finally, using computed-based analysis, we observed a high degree of conservation of SIRPα across six species of primates and the presence of shared epitopes in the extracellular domain between humans and Saimiri genus that could be targeted by antibodies. In conclusion, we have identified a commercially available anti-human monoclonal antibody that is able to detect SIRPα of S. sciureus monkeys and that, therefore, can facilitate the study of the immunomodulatory role of SIRPα when S. sciureus is used as a model. PMID:29312325

  8. Detection of Signal Regulatory Protein α in Saimiri sciureus (Squirrel Monkey by Anti-Human Monoclonal Antibody

    Directory of Open Access Journals (Sweden)

    Hugo Amorim dos Santos de Souza

    2017-12-01

    Full Text Available Non-human primates (NHP are suitable models for studying different aspects of the human system, including pathogenesis and protective immunity to many diseases. However, the lack of specific immunological reagents for neo-tropical monkeys, such as Saimiri sciureus, is still a major factor limiting studies in these models. An alternative strategy to circumvent this obstacle has been the selection of immunological reagents directed to humans, which present cross-reactivity with NHP molecules. In this context and considering the key role of inhibitory immunoreceptors—such as the signal regulatory protein α (SIRPα—in the regulation of immune responses, in the present study, we attempted to evaluate the ability of anti-human SIRPα monoclonal antibodies to recognize SIRPα in antigen-presenting S. sciureus peripheral blood mononuclear cells (PBMC. As shown by flow cytometry analysis, the profile of anti-SIRPα staining as well as the levels of SIRPα-positive cells in PBMC from S. sciureus were similar to those observed in human PBMC. Furthermore, using anti-SIRPα monoclonal antibody, it was possible to detect a decrease of the SIRPα levels on surface of S. sciureus cells after in vitro stimulation with lipopolysaccharides. Finally, using computed-based analysis, we observed a high degree of conservation of SIRPα across six species of primates and the presence of shared epitopes in the extracellular domain between humans and Saimiri genus that could be targeted by antibodies. In conclusion, we have identified a commercially available anti-human monoclonal antibody that is able to detect SIRPα of S. sciureus monkeys and that, therefore, can facilitate the study of the immunomodulatory role of SIRPα when S. sciureus is used as a model.

  9. Monoclonal antibodies to the reactive centre loop (RCL) of human corticosteroid-binding globulin (CBG) can protect against proteolytic cleavage.

    Science.gov (United States)

    Lewis, John G; Elder, Peter A

    2017-07-01

    Corticosteroid-binding globulin (CBG) binds most of the cortisol in circulation and is a non-functional member of the family of serine protease inhibitors (serpins) with an exposed elastase sensitive reactive centre loop (RCL). The RCL can be cleaved by human neutrophil elastase, released from activated neutrophils, and can also be cleaved at nearby site(s) by elastase released by Pseudomonas aeruginosa, and at two further sites, also within the RCL, by bovine chymotrypsin. Cleavage of the RCL results in a conformational change accompanied by a marked decrease in affinity for cortisol and hence its release at the site of proteolysis. These cleavages are irreversible and the similar half-lives of cleaved and intact CBG could mean that there may be some advantage in slowing the rate of CBG cleavage in acute inflammation thereby increasing the proportion of intact CBG in circulation. Here we show, for the first time, that pre-incubation of tethered human CBG with two monoclonal antibodies to the RCL of CBG protects against cleavage by all three enzymes. Furthermore, in plasma, pre-incubation with both RCL monoclonal antibodies delays neutrophil elastase cleavage of the RCL and one of these RCL monoclonal antibodies also delays bovine chymotrypsin cleavage of the RCL. These findings may provide a basis and rationale for the concept of the use of RCL antibodies as therapeutic agents to effectively increase the proportion of intact CBG in circulation which may be of benefit in acute inflammation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Generation and characterization of ixekizumab, a humanized monoclonal antibody that neutralizes interleukin-17A

    Directory of Open Access Journals (Sweden)

    Liu L

    2016-04-01

    Full Text Available Ling Liu,1 Jirong Lu,1 Barrett W Allan,2 Ying Tang,2 Jonathan Tetreault,1 Chi-kin Chow,1 Barbra Barmettler,2 James Nelson,2 Holly Bina,1 Lihua Huang,3 Victor J Wroblewski,4 Kristine Kikly1 1Biotechnology Discovery Research, Indianapolis, IN, 2Applied Molecular Evolution, Lilly Biotechnology Center, San Diego, CA, 3Bioproduct Research and Development, 4Drug Disposition, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA Abstract: Interleukin (IL-17A exists as a homodimer (A/A or as a heterodimer (A/F with IL-17F. IL-17A is expressed by a subset of T-cells, called Th17 cells, at inflammatory sites. Most cell types can respond to the local production of IL-17A because of the near ubiquitous expression of IL-17A receptors, IL-17RA and IL-17RC. IL-17A stimulates the release of cytokines and chemokines designed to recruit and activate both neutrophils and memory T-cells to the site of injury or inflammation and maintain a proinflammatory state. IL-17A-producing pathogenic T-cells contribute to the pathogenesis of autoimmune diseases, including psoriasis, psoriatic arthritis, rheumatoid arthritis, and ankylosing spondylitis. This study describes the generation and characterization of ixekizumab, a humanized IgG4 variant IL-17A-neutralizing antibody. Ixekizumab binds human and cynomolgus monkey IL-17A with high affinity and binds rabbit IL-17A weakly but does not bind to rodent IL-17A or other IL-17 family members. Ixekizumab effectively inhibits the interaction between IL-17A and its receptor in binding assays and potently blocks IL-17A-induced GRO or KC secretion in cell-based assays. In an in vivo mouse pharmcodynamic model, ixekizumab blocks human IL-17A-induced mouse KC secretion. These data provide a comprehensive preclinical characterization of ixekizumab, for which the efficacy and safety have been demonstrated in human clinical trials in psoriasis and psoriatic arthritis.Keywords: ixekizumab, IL-17A monoclonal antibody

  11. Epitope Mapping of Human HER2 Specific Mouse Monoclonal Antibodies Using Recombinant Extracellular Subdomains

    Science.gov (United States)

    Hosseini Ghatar, Reza; Soltantoyeh, Tahereh; Bahadori, Tannaz; Golara, Maryam; Hassannia, Hadi; Khosravi Eghbal, Roya; Khoshnoodi, Jalal; Judaki, Mohammad Ali; Golsaz-Shirazi, Forough; Jeddi-Tehrani, Mahmood; Amiri, Mohammad Mehdi; Shokri, Fazel

    2017-11-26

    Background: Human epidermal growth factor receptor 2 (HER2) is overexpressed in several human malignancies and numerous studies have indicated that it plays important roles in the development and maintenance of the malignant phenotype. Targeting of HER2 molecules with monoclonal antibodies (mAbs) is a promising therapeutic approach. However, anti-HER2 mAbs affect cancer cells differently, depending on the distinct epitopes which are the targets. Methods: Reactivity of a panel of 8 mouse anti-HER2 mAbs was investigated by ELISA and Western blotting using different subdomains of the extracellular domain (ECD) of HER2. All subdomains, including I, II, III, IV, I+II, III+IV and full HER2-ECD were constructed and expressed in CHO cells. Cross-reactivity of the mAbs with other members of the human HER family and Cynomolgus HER2 was also studied by ELISA. The mAbs were also tested by immunohistochemistry (IHC) using HER2 positive breast cancer tissues. Results: Our results demonstrated that 3 out of 8 mAbs detected conformational epitopes (1T0, 2A8 and 1B5), while 5 mAbs identified linear epitopes (1F2, 1H9, 4C7, 1H6 and 2A9). Three of the mAbs recognized subdomain I, one reacted with subdomain I+II, 2 recognized either subdomain III or IV and 2 recognized subdomain III+IV. However, none of our mAbs recognized the subdomain II alone. The mAbs displayed either inhibitory or stimulatory effects on HER2-overexpressing tumor cells and did not react with other members of the human HER family. The pattern of IHC results implied better reactivity of the mAbs recognizing linear epitopes. Conclusions: Our findings suggest that paired subdomains of HER2 are essential for mapping of mAbs recognizing conformational epitopes. Moreover, there seems to be no association between subdomain specificity and antitumor activity of our anti-HER2 mAbs. 10.22034/APJCP.2017.18.11.3103

  12. Affinity maturation to improve human monoclonal antibody neutralization potency and breadth against hepatitis C virus.

    Science.gov (United States)

    Wang, Yong; Keck, Zhen-yong; Saha, Anasuya; Xia, Jinming; Conrad, Fraser; Lou, Jianlong; Eckart, Michael; Marks, James D; Foung, Steven K H

    2011-12-23

    A potent neutralizing antibody to a conserved hepatitis C virus (HCV) epitope might overcome its extreme variability, allowing immunotherapy. The human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 glycoprotein. Previous studies showed that HC-1 neutralizes most HCV genotypes but has modest potency. To improve neutralization, we affinity-matured HC-1 by constructing a library of yeast-displayed HC-1 single chain Fv (scFv) mutants, using for selection an E2 antigen from one of the poorly neutralized HCVpp. We developed an approach by parallel mutagenesis of the heavy chain variable (VH) and κ-chain variable (Vk) genes separately, then combining the optimized VH and Vk mutants. This resulted in the generation of HC-1-related scFv variants exhibiting improved affinities. The best scFv variant had a 92-fold improved affinity. After conversion to IgG1, some of the antibodies exhibited a 30-fold improvement in neutralization activity. Both surface plasmon resonance and solution kinetic exclusion analysis showed that the increase in affinity was largely due to a lowering of the dissociation rate constant, Koff. Neutralization against a panel of HCV pseudoparticles and infectious 2a HCV virus improved with the affinity-matured IgG1 antibodies. Interestingly, some of these antibodies neutralized a viral isolate that was not neutralized by wild-type HC-1. Moreover, propagating 2a HCVcc under the selective pressure of WT HC-1 or affinity-matured HC-1 antibodies yielded no viral escape mutants and, with the affinity-matured IgG1, needed 100-fold less antibody to achieve complete virus elimination. Taken together, these findings suggest that affinity-matured HC-1 antibodies are excellent candidates for therapeutic development.

  13. Microcalorimetric study of adsorption of human monoclonal antibodies on cation exchange chromatographic materials.

    Science.gov (United States)

    Dieterle, Michael; Blaschke, Tim; Hasse, Hans

    2008-09-26

    Adsorption of two human monoclonal antibodies on two different strong cation exchange resins is studied by isothermal titration microcalorimetry and independent adsorption isotherm measurements. The pH value is varied between 4.5 and 7.0, using different buffer systems, the temperature is always 25 degrees C. The adsorption isotherm data is fitted using two different Langmuir type models. Combining the calorimetric and the adsorption data, the specific enthalpy of adsorption of the protein Deltah(p)(ads) is determined. At pH values near 7.0, where the antibodies are only weakly charged, the adsorption is exothermal. At small loadings the absolute number of Deltah(p)(ads) is then large and almost constant but it significantly decreases at higher loadings. This shows that the arrangement of antibody molecules on the absorber material depends on the loading and is less favourable at higher loadings. Despite the high positive charge of the antibody at pH values of about 5 the value of Deltah(p)(ads) is almost zero along the entire isotherm. Furthermore, at pH 4.5 even endothermal effects are observed, although high binding capacities are found. At these conditions the adsorption process seems to be strongly influenced by the ions bound to the antibody. Their release upon absorption explains the endothermal caloric effect. The adsorption equilibrium constant K(eq) is calculated from the isotherms. From Deltag(p)(ads) and the calorimetric results for Deltah(p)(ads), Deltas(p)(ads), the entropy change upon adsorption of the protein is found for the different studied conditions.

  14. N-terminal or signal peptide sequence engineering prevents truncation of human monoclonal antibody light chains.

    Science.gov (United States)

    Gibson, S J; Bond, N J; Milne, S; Lewis, A; Sheriff, A; Pettman, G; Pradhan, R; Higazi, D R; Hatton, D

    2017-09-01

    Monoclonal antibodies (mAbs) contain short N-terminal signal peptides on each individual polypeptide that comprises the mature antibody, targeting them for export from the cell in which they are produced. The signal peptide is cleaved from each heavy chain (Hc) and light chain (Lc) polypeptide after translocation to the ER and prior to secretion. This process is generally highly efficient, producing a high proportion of correctly cleaved Hc and Lc polypeptides. However, mis-cleavage of the signal peptide can occur, resulting in truncation or elongation at the N-terminus of the Hc or Lc. This is undesirable for antibody manufacturing as it can impact efficacy and can result in product heterogeneity. Here, we describe a truncated variant of the Lc that was detected during a routine developability assessment of the recombinant human IgG1 MEDI8490 in Chinese hamster ovary cells. We found that the truncation of the Lc was caused due to the use of the murine Hc signal peptide together with a lambda Lc containing an SYE amino acid motif at the N-terminus. This truncation was not caused by mis-processing of the mRNA encoding the Lc and was not dependent on expression platform (transient or stable), the scale of the fed-batch culture or clonal lineage. We further show that using alternative signal peptides or engineering the Lc SYE N-terminal motif prevented the truncation and that this strategy will improve Lc homogeneity of other SYE lambda Lc-containing mAbs. Biotechnol. Bioeng. 2017;114: 1970-1977. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. The effect of anti-human plasminogen monoclonal antibodies on Glu-plasminogen activation by plasminogen activators

    Directory of Open Access Journals (Sweden)

    M. Akrami

    2006-07-01

    Full Text Available Background: Human plasminogen is a plasma glycoprotein synthesized mainly in the liver. Conversion of plasminogen to plasmin by plasminogen activators is a key event in the fibrinolytic system. In this study, we investigated the effects of two anti-human plasminogen monoclonal antibodies, A1D12 and MC2B8 on Glu-plasminogen activation in presence of u-PA, t-PA and streptokinase. Methods: Producing of Hybridoma antibodies was performed by fusion of spleen cells from BALB/C mice immunized with Glu-plasminogen and NS1 myeloma cells. Antibody binding to Human Glu-plasminogen was assessed using an ELISA assay. Activation of plasminogen was determined by measuring plasmin generation using the chromogenic substrate S-2251 and the effect of monoclonal antibodies, A1D12 and MC2B8 on plasminogen activation in solution was then evaluated. Initial rates and kinetic parameters of plasminogen activation in the presence of monoclonal antibodies were calculated. The effect of the monoclonal antibody MC2B8 on the rate of plasmin hydrolysis was measured. The effect of F(ab'2 fragment of A1D12 on u-PA catalyzed-plasminogen activation also compared with the effect of the whole antibody in this reaction. Results: ELISA assay showed that the antibodies reacted well with antigens. A1D12 increased the maximum velocity (Vmax of plasminogen activation by each of the three plasminogen activators and MC2B8 decreased it. In all activation reactions, the KM value of plasminogen activation did not significantly change in the presence of antibody A1D12 whereas antibody MC2B8 increased the KM value of plasminogen activation by u-PA, fibrin monomer dependent t-PA and streptokinase. Monoclonal antibody MC2B8 had no significant effect on plasmin hydrolysis rate of synthetic substrate S-2251. Activation rate of plasminogen by u-PA in the lower concentration of F (ab2 fragment of A1D12 was identical to activation in the presence of the whole antibody. Conclusion: The binding of

  16. Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-γ Using Human-Bovine Interferon-γ Chimeras

    Science.gov (United States)

    Zuber, Bartek; Rudström, Karin; Ehrnfelt, Cecilia

    2016-01-01

    Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-γ. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-γ in ELISA, 2 epitope clusters with 5 mAbs in each were defined; 2 mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-γ, epitopes were identified using 7 h/bIFN-γ chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-γ residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2 mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN-γ-mediated activation of human cells, in line with the involvement of region A in the IFN-γ receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes. PMID:27336613

  17. Monoclonal antibodies reveal multiple forms of expression of human microsomal epoxide hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Hongying; Takagi, Akira [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kayano, Hidekazu [Department of Pathology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Koyama, Isamu [Department of Digestive and General Surgery, Saitama International Medical Center, Faculty of Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2012-04-01

    In a previous study, we developed five kinds of monoclonal antibodies against different portions of human mEH: three, anti-N-terminal; one, anti-C-terminal; one, anti-conformational epitope. Using them, we stained the intact and the permeabilized human cells of various kinds and performed flow cytometric analysis. Primary hepatocytes and peripheral blood mononuclear cells (PBMC) showed remarkable differences. On the surface, hepatocytes exhibited 4 out of 5 epitopes whereas PBMC did not show any of the epitopes. mEH was detected inside both cell types, but the most prominent expression was observed for the conformational epitope in the hepatocytes and the two N-terminal epitopes in PBMC. These differences were also observed between hepatocyte-derived cell lines and mononuclear cell-derived cell lines. In addition, among each group, there were several differences which may be related to the cultivation, the degree of differentiation, or the original cell subsets. We also noted that two glioblastoma cell lines reveal marked expression of the conformational epitope on the surface which seemed to correlate with the brain tumor-associated antigen reported elsewhere. Several cell lines also underwent selective permeabilization before flow cytometric analysis, and we noticed that the topological orientation of mEH on the ER membrane in those cells was in accordance with the previous report. However, the orientation on the cell surface was inconsistent with the report and had a great variation between the cells. These findings show the multiple mode of expression of mEH which may be possibly related to the multiple roles that mEH plays in different cells. -- Highlights: ► We examine expression of five mEH epitopes in human cells. ► Remarkable differences exist between hepatocytes and PBMC. ► mEH expression in cell lines differs depending on several factors. ► Some glioblastoma cell lines reveal marked surface expression of mEH. ► Topology of mEH on the cell

  18. Monoclonal antibody "gold rush".

    Science.gov (United States)

    Maggon, Krishan

    2007-01-01

    The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush.

  19. Monoclonal antibodies against a synthetic peptide from human immunodeficiency virus type 1 Nef protein

    DEFF Research Database (Denmark)

    Steinaa, L; Wulff, A M; Saermark, T

    1994-01-01

    Monoclonal antibodies against a synthetic peptide (aa 138-152) from HIV-1 Nef protein were produced and characterized. Three hybridoma lines producing monoclonal antibodies (MAbs) against the synthetic peptide were generated by fusion between P3-X63 Ag8.653 myeloma cells and BALB/c splenocytes from...... mice immunized with the synthetic peptide coupled to keyhole limpet hemocyanin (KLH). The hybridomas were screened and selected by ELISA with the peptide coupled to bovine serum albumin (BSA) immobilized to the polystyrene surface and specificity for the peptide was confirmed by competitive ELISA...

  20. Molecular profile of a human monoclonal antibody Fab fragment specific for Epstein-Barr virus gp350/220 antigen.

    Science.gov (United States)

    Bugli, F; Bastidas, R; Burton, D R; Williamson, R A; Clementi, M; Burioni, R

    2001-04-01

    Experimental evidence indicates Epstein Barr virus (EBV) envelope glycoprotein gp350/220 elicits a potent virus neutralizing response in the infected human host that may play an important role in restricting viral pathogenesis. In this study, we report the molecular cloning in combinatorial phage display vectors, of the IgG1 repertoire of an individual naturally infected with EBV, and describe the recovery and characterization of a monoclonal antibody recognizing gp350/220. A detailed understanding of the human antibody response in EBV infection will identify antibodies of potential use in anti-viral prophylaxis and will advance the production of more effective vaccine candidates.

  1. Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use.

    Science.gov (United States)

    Stevens, Misty W; Tawney, Rachel L; West, C Michael; Kight, Alicia D; Henry, Ralph L; Owens, S Michael; Gentry, W Brooks

    2014-01-01

    Ch-mAb7F9, a human-mouse chimeric monoclonal antibody (mAb) designed to bind (+)-methamphetamine (METH) with high affinity and specificity, was produced as a treatment medication for METH abuse. In these studies, we present the preclinical characterization that provided predictive evidence that ch-mAb7F9 may be safe and effective in humans. In vitro ligand binding studies showed that ch-mAb7F9 is specific for and only binds its target ligands (METH, (+)-amphetamine, and 3,4-methylenedioxy-N-methylamphetamine) with high affinity. It did not bind endogenous neurotransmitters or other medications and was not bound by protein C1q, thus it is unlikely to stimulate in vivo complement-dependent cytotoxicity. Isothermal titration calorimetry potency studies showed that METH binding by ch-mAb7F9 is efficient. Pharmacokinetic studies of METH given after ch-mAb7F9 doses in rats demonstrated the in vivo application of these in vitro METH-binding characteristics. While METH had little effect on ch-mAb7F9 disposition, ch-mAb7F9 substantially altered METH disposition, dramatically reducing the volume of distribution and clearance of METH. The elimination half-life of METH was increased by ch-mAb7F9, but it was still very fast compared with the elimination of ch-mAb7F9. Importantly, the rapid elimination of unbound METH combined with previous knowledge of mAb:target ligand binding dynamics suggested that ch-mAb7F9 binding capacity regenerates over time. This finding has substantial therapeutic implications regarding the METH doses against which ch-mAb7F9 will be effective, on the duration of ch-mAb7F9 effects, and on the safety of ch-mAb7F9 in METH users who use METH while taking ch-mAb7F9. These results helped to support initiation of a Phase 1a study of ch-mAb7F9.

  2. Seroepidemiology of Human Papillomavirus 16 (HPV16) L2 and Generation of L2-Specific Human Chimeric Monoclonal Antibodies.

    Science.gov (United States)

    Wang, Joshua W; Jagu, Subhashini; Wu, Wai-Hong; Viscidi, Raphael P; Macgregor-Das, Anne; Fogel, Jessica M; Kwak, Kihyuck; Daayana, Sai; Kitchener, Henry; Stern, Peter L; Gravitt, Patti E; Trimble, Cornelia L; Roden, Richard B S

    2015-07-01

    Presently, the seroprevalence of human papillomavirus (HPV) minor capsid antigen L2-reactive antibody is not well understood, and no serologic standard exists for L2-specific neutralizing antibodies. Therefore, we screened a total of 1,078 serum samples for HPV16 L2 reactivity, and these were obtained from four prior clinical studies: a population-based (n = 880) surveillance study with a high-risk HPV DNA prevalence of 10.8%, a cohort study of women (n = 160) with high-grade cervical intraepithelial neoplasia (CIN), and two phase II trials in women with high-grade vulvar intraepithelial neoplasia (VIN) receiving imiquimod therapy combined with either photodynamic therapy (PDT) (n = 19) or vaccination with a fusion protein comprising HPV16 L2, E7, and E6 (TA-CIN) (n = 19). Sera were screened sequentially by HPV16 L2 enzyme-linked immunosorbent assay (ELISA) and then Western blot. Seven of the 1,078 serum samples tested had L2-specific antibodies, but none were detectably neutralizing for HPV16. To develop a standard, we substituted human IgG1 sequences into conserved regions of two rodent monoclonal antibodies (MAbs) specific for neutralizing epitopes at HPV16 L2 residues 17 to 36 and 58 to 64, creating JWW-1 and JWW-2, respectively. These chimeric MAbs retained neutralizing activity and together reacted with 33/34 clinically relevant HPV types tested. In conclusion, our inability to identify an HPV16 L2-specific neutralizing antibody response even in the sera of patients with active genital HPV disease suggests the subdominance of L2 protective epitopes and the value of the chimeric MAbs JWW-1 and JWW-2 as standards for immunoassays to measure L2-specific human antibodies. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Rational methods for predicting human monoclonal antibodies retention in protein A affinity chromatography and cation exchange chromatography. Structure-based chromatography design for monoclonal antibodies.

    Science.gov (United States)

    Ishihara, Takashi; Kadoya, Toshihiko; Yoshida, Hideaki; Tamada, Taro; Yamamoto, Shuichi

    2005-11-04

    Rational methods for predicting the chromatographic behavior of human monoclonal antibodies (hMabs) in protein A affinity chromatography and cation exchange chromatography from the amino acid sequences information were proposed. We investigated the relation between the structures of 28 hMabs and their chromatographic behavior in protein A affinity chromatography and cation exchange chromatography using linear gradient elution experiments. In protein A affinity chromatography, the elution pH of the hMabs was correlated with not only the structure of the Fc region (subclass), but also that of the variable region. The elution pH of hMabs that have LYLQMNSL sequences in between the CDR2 and CDR3 regions of the heavy chain became lower among the same subclass of hMabs. In cation exchange chromatography, the peak salt concentrations IR of hMabs that have the same sequences of variable regions (or that have a structural difference in their Fc region, which puts them into a subclass) were similar. The IR values of hMabs were well correlated with the equilibrium association constant Ke, and also with the surface positive charge distribution of the variable region of the heavy chain (corrected surface net positive charge (cN) of the VH region). Based on these findings, we developed rational methods for predicting the retention behavior, which were also tested with eight additional hMabs. By considering the information on the number of binding sites associated with protein adsorption as determined experimentally, and the surface positive charge distribution from the three-dimensional structure of Mab A, we hypothesized that hMabs is separated by cation exchange chromatography as the surface positive charge distribution of the VH region is recognized.

  4. Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS

    Science.gov (United States)

    He, Lidong; Anderson, Lissa C.; Barnidge, David R.; Murray, David L.; Hendrickson, Christopher L.; Marshall, Alan G.

    2017-05-01

    With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error 4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background.

  5. Integrin alphaIIbbeta3-specific synthetic human monoclonal antibodies and HCDR3 peptides that potently inhibit platelet aggregation.

    Science.gov (United States)

    Chung, Junho; Rader, Christoph; Popkov, Mikhail; Hur, Young-Mi; Kim, Hyun-Kyung; Lee, Young-Joon; Barbas, Carlos F

    2004-02-01

    The interaction of fibrinogen with integrin alphaIIbbeta3 (GPIIb/IIIa), in part mediated by an RGD tripeptide motif, is an essential step in platelet aggregation. Based on their inhibition of platelet aggregation, three integrin alphaIIbbeta3 inhibitors are clinically approved. The clinically most widely used integrin alphaIIbbeta3 inhibitor abciximab is a chimeric mouse/human antibody that induces thrombocytopenia, often severe, in 1-2% of patients due to a human anti-mouse antibody (HAMA) response. In addition, unlike other ligands mimicking small molecular drugs, abciximab cross-reacts with integrin alphavbeta3 and alphaMbeta2. Here we used phage display to select monoclonal antibodies specific to integrin alphaIIbbeta3 from a synthetic human antibody library based on the randomized HCDR3 sequence VGXXXRADXXXYAMDV. The selected antibodies revealed a strong consensus in HCDR3 (V(V/W)CRAD(K/R)RC) and high specificity toward integrin alphaIIbbeta3 but not to other RGD binding integrins such as alphavbeta3, alphavbeta5, and alpha5beta1. The selected antibodies as well as three synthetic peptides (VWCRADRRC, VWCRADKRC, and VVCRADRRC) whose sequences were derived from the HCDR3 sequences of the selected antibodies strongly inhibited the interaction between integrin alphaIIbbeta3 and fibrinogen and platelet aggregation ex vivo. To our knowledge, these are the first fully human monoclonal antibodies that are specific to integrin alphaIIbbeta3 and can potently inhibit platelet aggregation.

  6. Critical epitopes in the nucleocapsid protein of SFTS virus recognized by a panel of SFTS patients derived human monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Li Yu

    Full Text Available BACKGROUND: SFTS virus (SFTSV is a newly discovered pathogen to cause severe fever with thrombocytopenia syndrome (SFTS in human. Successful control of SFTSV epidemic requires better understanding of the antigen target in humoral immune responses to the new bunyavirus infection. METHODOLOGY/PRINCIPAL FINDINGS: We have generated a combinatorial Fab antibody phage library from two SFTS patients recovered from SFTSV infection. To date, 94 unique human antibodies have been generated and characterized from over 1200 Fab antibody clones obtained by screening the library with SFTS purified virions. All those monoclonal antibodies (MAbs recognized the nucleocapsid (N protein of SFTSV while none of them were reactive to the viral glycoproteins Gn or Gc. Furthermore, over screening 1000 mouse monoclonal antibody clones derived from SFTSV virions immunization, 462 clones reacted with N protein, while only 16 clones were reactive to glycoprotein. Furthermore, epitope mapping of SFTSV N protein was performed through molecular simulation, site mutation and competitive ELISA, and we found that at least 4 distinct antigenic epitopes within N protein were recognized by those human and mouse MAbs, in particular mutation of Glu10 to Ala10 abolished or significantly reduced the binding activity of nearly most SFTS patients derived MAbs. CONCLUSIONS/SIGNIFICANCE: The large number of human recombinant MAbs derived from SFTS patients recognized the viral N protein indicated the important role of the N protein in humoral responses to SFTSV infection, and the critical epitopes we defined in this study provided molecular basis for detection and diagnosis of SFTSV infection.

  7. Epratuzumab, a humanized monoclonal antibody targeting CD22: characterization of in vitro properties.

    Science.gov (United States)

    Carnahan, Josette; Wang, Paul; Kendall, Richard; Chen, Ching; Hu, Sylvia; Boone, Tom; Juan, Todd; Talvenheimo, Jane; Montestruque, Silvia; Sun, Jilin; Elliott, Gary; Thomas, John; Ferbas, John; Kern, Brent; Briddell, Robert; Leonard, John P; Cesano, Alessandra

    2003-09-01

    Epratuzumab is a novel humanized antihuman CD22 IgG1 antibody that has recently shown promising clinical activity, both as a single agent and in combination with rituximab, in patients with non-Hodgkin's lymphomas (NHL). In an attempt to better understand the mode of action of epratuzumab, the antibody was tested in vitro in a variety of cell-based assays similar to those used to evaluate the biological activity of other therapeutic monoclonal antibodies, including rituximab. In this report, we present epratuzumab activities as they relate to binding, signaling, and internalization of the receptor CD22. Chinese hamster ovary-expressed CD22 extracellular domain was used to measure epratuzumab affinity on Biacore. CD22 receptor density and internalization rate were measured indirectly using a monovalently labeled, noncompeting (with epratuzumab) anti-CD22 antibody on Burkitt lymphoma cell lines, primary B cells derived from fresh tonsils, and B cells separated from peripheral blood samples obtained from patients with chronic lymphocytic leukemia or healthy volunteers. Epratuzumab-induced CD22 phosphorylation was measured by immunoprecipitation/Western blot and compared with that induced by anti-IgM stimulation. Epratuzumab binds to CD22-extracellular domain, with an affinity of K(D) = 0.7 nM. Binding of epratuzumab to B cell lines, or primary B cells from healthy individuals and patients with NHL, results in rapid internalization of the CD22/antibody complex. Internalization appears to be faster at early time points in cell lines than in primary B cells and NHL patient-derived B cells, but the maximum internalization reached is comparable for all B cell populations after several hours of treatment and appears to reach saturation at antibody concentrations of 1-5 micro g/ml. Finally, epratuzumab binding results in modest but significant CD22 phosphorylation. Epratuzumab represents an excellent anti-CD22 ligating agent, highly efficacious in inducing CD22

  8. Adsorption behavior of a human monoclonal antibody at hydrophilic and hydrophobic surfaces.

    Science.gov (United States)

    Couston, Ruairidh G; Skoda, Maximilian W; Uddin, Shahid; van der Walle, Christopher F

    2013-01-01

    One aspiration for the formulation of human monoclonal antibodies (mAb) is to reach high solution concentrations without compromising stability. Protein surface activity leading to instability is well known, but our understanding of mAb adsorption to the solid-liquid interface in relevant pH and surfactant conditions is incomplete. To investigate these conditions, we used total internal reflection fluorescence (TIRF) and neutron reflectometry (NR). The mAb tested ("mAb-1") showed highest surface loading to silica at pH 7.4 (~12 mg/m(2)), with lower surface loading at pH 5.5 (~5.5 mg/m(2), further from its pI of 8.99) and to hydrophobized silica (~2 mg/m(2)). The extent of desorption of mAb-1 from silica or hydrophobized silica was related to the relative affinity of polysorbate 20 or 80 for the same surface. mAb-1 adsorbed to silica on co-injection with polysorbate (above its critical micelle concentration) and also to silica pre-coated with polysorbate. A bilayer model was developed from NR data for mAb-1 at concentrations of 50-5000 mg/L, pH 5.5, and 50-2000 mg/L, pH 7.4. The inner mAb-1 layer was adsorbed to the SiO₂ surface at near saturation with an end-on" orientation, while the outer mAb-1 layer was sparse and molecules had a "side-on" orientation. A non-uniform triple layer was observed at 5000 mg/L, pH 7.4, suggesting mAb-1 adsorbed to the SiO₂ surface as oligomers at this concentration and pH. mAb-1 adsorbed as a sparse monolayer to hydrophobized silica, with a layer thickness increasing with bulk concentration - suggesting a near end-on orientation without observable relaxation-unfolding.

  9. Adsorption behavior of a human monoclonal antibody at hydrophilic and hydrophobic surfaces

    Science.gov (United States)

    Couston, Ruairidh G.; Skoda, Maximilian W.; Uddin, Shahid; van der Walle, Christopher F.

    2013-01-01

    One aspiration for the formulation of human monoclonal antibodies (mAb) is to reach high solution concentrations without compromising stability. Protein surface activity leading to instability is well known, but our understanding of mAb adsorption to the solid-liquid interface in relevant pH and surfactant conditions is incomplete. To investigate these conditions, we used total internal reflection fluorescence (TIRF) and neutron reflectometry (NR). The mAb tested (“mAb-1”) showed highest surface loading to silica at pH 7.4 (~12 mg/m2), with lower surface loading at pH 5.5 (~5.5 mg/m2, further from its pI of 8.99) and to hydrophobized silica (~2 mg/m2). The extent of desorption of mAb-1 from silica or hydrophobized silica was related to the relative affinity of polysorbate 20 or 80 for the same surface. mAb-1 adsorbed to silica on co-injection with polysorbate (above its critical micelle concentration) and also to silica pre-coated with polysorbate. A bilayer model was developed from NR data for mAb-1 at concentrations of 50–5000 mg/L, pH 5.5, and 50–2000 mg/L, pH 7.4. The inner mAb-1 layer was adsorbed to the SiO2 surface at near saturation with an end-on” orientation, while the outer mAb-1 layer was sparse and molecules had a “side-on” orientation. A non-uniform triple layer was observed at 5000 mg/L, pH 7.4, suggesting mAb-1 adsorbed to the SiO2 surface as oligomers at this concentration and pH. mAb-1 adsorbed as a sparse monolayer to hydrophobized silica, with a layer thickness increasing with bulk concentration - suggesting a near end-on orientation without observable relaxation-unfolding. PMID:23196810

  10. Isolation, production and characterization of fully human monoclonal antibodies directed to Plasmodium falciparum MSP10.

    Science.gov (United States)

    Maskus, Dominika J; Bethke, Susanne; Seidel, Melanie; Kapelski, Stephanie; Addai-Mensah, Otchere; Boes, Alexander; Edgü, Güven; Spiegel, Holger; Reimann, Andreas; Fischer, Rainer; Barth, Stefan; Klockenbring, Torsten; Fendel, Rolf

    2015-07-16

    Semi-immunity against the malaria parasite is defined by a protection against clinical episodes of malaria and is partially mediated by a repertoire of inhibitory antibodies directed against the blood stage of Plasmodium falciparum, in particular against surface proteins of merozoites, the invasive form of the parasite. Such antibodies may be used for preventive or therapeutic treatment of P. falciparum malaria. Here, the isolation and characterization of novel human monoclonal antibodies (humAbs) for such applications is described. B lymphocytes had been selected by flow cytometry for specificity against merozoite surface proteins, including the merozoite surface protein 10 (MSP10). After Epstein-Barr virus (EBV) transformation and identification of promising resulting lymphoblastoid cell lines (LCLs), human immunoglobulin heavy and light chain variable regions (Vh or Vl regions) were secured, cloned into plant expression vectors and transiently produced in Nicotiana benthamiana in the context of human full-size IgG1:κ. The specificity and the affinity of the generated antibodies were assessed by ELISA, dotblot and surface plasmon resonance (SPR) spectroscopy. The growth inhibitory activity was evaluated based on growth inhibition assays (GIAs) using the parasite strain 3D7A. Supernatants from two LCLs, 5E8 and 5F6, showed reactivity against the second (5E8) or first (5F6) epidermal growth factor (EGF)-like domain of MSP10. The isolated V regions were recombinantly expressed in their natural pairing as well as in combination with each other. The resulting recombinant humAbs showed affinities of 9.27 × 10(-7) M [humAb10.1 (H5F6:κ5E8)], 5.46 × 10(-9) M [humAb10.2 (H5F6:κ5F6)] and 4.34 × 10(-9) M [humAb10.3 (H5E8:κ5E8)]. In GIAs, these antibodies exhibited EC50 values of 4.1 mg/ml [95% confidence interval (CI) 2.6-6.6 mg/ml], 6.9 mg/ml (CI 5.5-8.6 mg/ml) and 9.5 mg/ml (CI 5.5-16.4 mg/ml), respectively. This report describes a platform for the isolation of

  11. A high-affinity human monoclonal IgM antibody reacting with multiple strains of Mycoplasma hominis

    DEFF Research Database (Denmark)

    Moller, SA; Birkelund, Svend; Borrebaeck, CA

    1990-01-01

    Human monoclonal antibodies were produced against Mycoplasma hominis by in vitro immunization of peripheral blood lymphocytes from a healthy seropositive donor using low amounts of antigen (5 ng/ml). The immune B lymphocytes were subsequently immortalized by Epstein-Barr virus transformation...... (24 hr x 10(6) cells)-1. The specificity of one antibody, 129.1, was further characterized by enzyme-linked immunosorbent assay against several different species of Mycoplasma and by Western blot analysis. The antibody specifically bound to a protein of Mr 100,000 present in 10 of 12 different strains...

  12. Stoichiometry of monoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1

    DEFF Research Database (Denmark)

    Schønning, Kristian; Lund, O; Lund, O S

    1999-01-01

    In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes...... neutralization gradually increased. Virus neutralization by virion aggregation was minimal, as MAb binding to HIV-1 Env did not interfere with an AMLV Env-mediated infection by HIV-1(AMLV/HIV-1) pseudotypes of CD4(-) HEK293 cells. MAb neutralization of chimeric virions could be described as a third...

  13. De novo protein sequencing, humanization and in vitro effects of an antihuman CD34 mouse monoclonal antibody.

    Science.gov (United States)

    Fan, Chia-Yu; Huang, Sheng-Yu; Chou, Min-Yuan; Lyu, Ping-Chiang

    2017-03-01

    QBEND/10 is a mouse immunoglobulin lambda-chain monoclonal antibody with strict specificity against human hematopoietic progenitor cell antigen CD34. Our in vitro study showed that QBEND/10 impairs the tube formation of human umbilical vein endothelial cells (HUVECs), suggesting that the antibody may be of potential benefit in blocking tumor angiogenesis. We provided a de novo protein sequencing method through tandem mass spectrometry to identify the amino acid sequences in the variable heavy and light chains of QBEND/10. To reduce immunogenicity for clinical applications, QBEND/10 was further humanized using the resurfacing approach. We demonstrate that the de novo sequenced and humanized QBEND/10 retains the biological functions of the parental mouse counterpart, including the binding kinetics to CD34 and blockage of the tube formation of the HUVECs.

  14. B-cell display-based one-step method to generate chimeric human IgG monoclonal antibodies.

    Science.gov (United States)

    Lin, Waka; Kurosawa, Kohei; Murayama, Akiho; Kagaya, Eri; Ohta, Kunihiro

    2011-02-01

    The recent development of screening strategies based on the generation and display of large libraries of antibody fragments has allowed considerable advances for the in vitro isolation of monoclonal antibodies (mAbs). We previously developed a technology referred to as the 'ADLib (Autonomously Diversifying Library) system', which allows the rapid screening and isolation in vitro of antigen-specific monoclonal antibodies (mAbs) from libraries of immunoglobulin M (IgM) displayed by the chicken B-cell line DT40. Here, we report a novel application of the ADLib system to the production of chimeric human mAbs. We have designed gene knock-in constructs to generate DT40 strains that coexpress chimeric human IgG and chicken IgM via B-cell-specific RNA alternative splicing. We demonstrate that the application of the ADLib system to these strains allows the one-step selection of antigen-specific human chimeric IgG. In addition, the production of chimeric IgG can be selectively increased when we modulate RNA processing by overexpressing the polyadenylation factor CstF-64. This method provides a new way to efficiently design mAbs suitable for a wide range of purposes including antibody therapy.

  15. The human leucocyte-common (LC) molecule: dissection of leukaemias using monoclonal antibodies directed against framework and restricted antigenic determinants.

    Science.gov (United States)

    Ritter, M A; Sauvage, C A; Pegram, S M; Myers, C D; Dalchau, R; Fabre, J W

    1985-01-01

    Monoclonal antibodies have previously been raised against two separate antigenic determinants on the human LC molecule. One, F10.89.4, recognizes a 'framework' epitope on all LC molecules; these are found on the majority of leucocytes. The other, F8.11.13, recognizes only a 'restricted' epitope present on a subset of these molecules; this subset is found on B lymphocytes and a subpopulation of T lymphocytes. LC molecules on myeloid cells do not carry the 'restricted' antigenic determinant. We have investigated the differential expression of these LC epitopes on human leukaemias, using immunofluorescence on fresh leukaemic blasts and established cell lines. Our study shows that, as on normal haemopoietic cells, LC molecules on B leukaemias bear both 'framework' and 'restricted' epitopes, while the majority of T leukaemias bear only the 'framework' determinant. The small proportion of T cells that are F8.11.13+ ('restricted' epitope) are relatively mature, being of either OKT4+ or OKT8+ phenotype, and may be in an activated state (HLA-DR+). However, in contrast to normal haemopoietic cells, some myeloid leukaemias carry both 'framework' and 'restricted' epitopes (30% AML and AMML samples are F10.89.4+, F8.11.13+), and it is within this group that all TdT+ AML and AMML cases lie. Thus, these monoclonal antibodies should be useful for studying haemopoiesis in man and for analyzing human haemopoietic malignancies.

  16. From Monoclonal Antibodies to Chimeric Antigen Receptors for the Treatment of Human Malignancies

    OpenAIRE

    Caruana, Ignazio; Diaconu, Iulia; Dotti, Gianpietro

    2014-01-01

    Monoclonal antibodies (mAbs) and their directly derived cell-based application known as chimeric antigen receptors (CARs) ensue from the need to develop novel therapeutic strategies that retain high anti-tumor activity, but carry reduced toxicity compared to conventional chemo- and radio-therapies. In this concise review article we will summarize the application of antibodies designed to target antigens expressed by tumor cells, and the transition from these antibodies to the generation of CARs.

  17. A novel monoclonal antibody for detection of galectin-9 in tissue sections: application to human tissues infected by oncogenic viruses

    Directory of Open Access Journals (Sweden)

    Barjon Clément

    2012-07-01

    Full Text Available Abstract Background Galectin-9 is a mammalian lectin which possesses immunosuppressive properties. Excessive production of galectin-9 has been reported in two types of human virus-associated diseases chronic hepatitis C and nasopharyngeal carcinoma associated to the Epstein-Barr virus. The objective of this study was to produce new monoclonal antibodies targeting galectin-9 in order to improve its detection in clinical samples, especially on tissue sections analysed by immunohistochemistry. Methods Hybridomas were produced through immunization of mice with the recombinant c-terminus part of galectin-9 (residues 191 to 355 of the long isoform and semi-solid fusion of spleen cells with Sp2/0 cells. Monoclonal antibodies were characterized using ELISA, epitope mapping, western blot and immunohistochemistry. Results We selected seven hybridomas producing antibodies reacting with our recombinant c-terminus galectin-9 in ELISA. Five of them reacted with the epitope “TPAIPPMMYPHPA” (common to all isoforms, residues 210 to 222 of the long isoform and stained all three isoforms of galectin-9 analysed by western blot. One of them, 1G3,demonstrated very good sensitivity and specificity when used for immunohistochemistry. Using 1G3, we could confirm the intense and constant expression of galectin-9 by Epstein-Barr virus positive malignant cells from nasopharyngeal carcinomas. In most samples, specific staining was detected in both cytoplasm and nuclei. Galectin-9 was also detected in liver biopsies from patients infected by the human hepatitis C or B viruses with expression not only in inflammatory leucocytes and Kupffer cells, but also in hepatocytes. In contrast, galectin-9 was virtually absent in non-infected liver specimens. Conclusion The 1G3 monoclonal antibody will be a powerful tool to assess galectin-9 expression and distribution especially in diseases related to oncogenic viruses.

  18. Characterization of Entamoeba histolytica intermediate subunit lectin-specific human monoclonal antibodies generated in transgenic mice expressing human immunoglobulin loci.

    Science.gov (United States)

    Tachibana, Hiroshi; Cheng, Xun-Jia; Tsukamoto, Hideo; Itoh, Johbu

    2009-01-01

    Four fully human monoclonal antibodies (MAbs) to Entamoeba histolytica intermediate subunit lectin (Igl) were prepared in XenoMouse mice, which are transgenic mice expressing human immunoglobulin loci. Examination of the reactivities of these MAbs to recombinant Igl1 and Igl2 of E. histolytica showed that XEhI-20 {immunoglobulin G2(kappa) [IgG2(kappa)]} and XEhI-28 [IgG2(kappa)] were specific to Igl1, XEhI-B5 [IgG2(kappa)] was specific to Igl2, and XEhI-H2 [IgM(kappa)] was reactive with both Igls. Gene analyses revealed that the V(H) and V(L) germ lines were VH3-48 and L2 for XEhI-20, VH3-21 and L2 for XEhI-28, VH3-33 and B3 for XEhI-B5, and VH4-4 and A19 for XEhI-H2, respectively. Flow cytometry analyses showed that the epitopes recognized by all of these MAbs were located on the surfaces of living trophozoites. Confocal microscopy demonstrated that most Igl1 and Igl2 proteins were colocalized on the surface and in the cytoplasm, but different localization patterns in intracellular vacuoles were also present. The preincubation of trophozoites with XEhI-20, XEhI-B5, and XEhI-H2 caused significant inhibition of the adherence of trophozoites to Chinese hamster ovary cells, whereas preincubation with XEhI-28 did not do so. XEhI-20, XEhI-B5, and XEhI-H2 were injected intraperitoneally into hamsters 24 h prior to intrahepatic challenge with E. histolytica trophozoites. One week later, the mean abscess size in groups injected with one of the three MAbs was significantly smaller than that in controls injected with polyclonal IgG or IgM isolated from healthy humans. These results demonstrate that human MAbs to Igls may be applicable for immunoprophylaxis of amebiasis.

  19. Reactivity of commercially available monoclonal antibodies to human CD antigens with peripheral blood leucocytes of dromedary camels (Camelus dromedarius

    Directory of Open Access Journals (Sweden)

    Jamal Hussen

    2017-05-01

    Full Text Available Monoclonal antibodies (mAbs to cell surface molecules have been proven as a key tool for phenotypic and functional characterization of the cellular immune response. One of the major difficulties in studying camel cellular immunity consists in the lack of mAbs that dtect their leukocyte differentiation antigens. In the present study two-parameter flow cytometry was used to screen existing commercially available mAbs to human leukocyte antigens and major histocompatibility molecules (MHC for their reactivity with camel leukocytes. The comparison of patterns of reactivity obtained after labelling human and camel leukocytes have shown that mAbs specific to human cluster of differentiation (CD 18, CD11a, CD11b and CD14 are predicted to be cross-reactive with homologous camel antigens.

  20. Treatment of Lassa virus infection in outbred guinea pigs with first-in-class human monoclonal antibodies.

    Science.gov (United States)

    Cross, Robert W; Mire, Chad E; Branco, Luis M; Geisbert, Joan B; Rowland, Megan M; Heinrich, Megan L; Goba, Augustine; Momoh, Mambu; Grant, Donald S; Fullah, Mohamed; Khan, Sheik Humarr; Robinson, James E; Geisbert, Thomas W; Garry, Robert F

    2016-09-01

    Lassa fever is a significant health threat to West African human populations with hundreds of thousands of annual cases. There are no approved medical countermeasures currently available. Compassionate use of the antiviral drug ribavirin or transfusion of convalescent serum has resulted in mixed success depending on when administered or the donor source, respectively. We previously identified several recombinant human monoclonal antibodies targeting the glycoprotein of Lassa virus with strong neutralization profiles in vitro. Here, we demonstrate remarkable therapeutic efficacy using first-in-class human antibodies in a guinea pig model of Lassa infection thereby presenting a promising treatment alternative. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Monoclonal antibodies that define canine homologues of human CD antigens: summary of the First International Canine Leukocyte Antigen Workshop (CLAW).

    Science.gov (United States)

    Cobbold, S; Metcalfe, S

    1994-03-01

    A panel of 127 monoclonal antibodies against canine leukocyte antigens, including controls, was distributed to 29 laboratories that performed a variety of experiments to identify groups of antibodies against the canine equivalents of some of the human CD antigens. Cluster analysis was performed centrally, using the submitted antibody binding data from immunofluorescence, ELISA and immuno-histology experiments. Immunoprecipitation for molecular weight determination was also performed centrally with T-cell blasts and a B-cell line as the sources of antigen. Clusters of three or more antibodies were found that defined the canine equivalents of the CD5, CD4, CD8 and Thy-1 antigens, and these could be used to label T-cell subsets from the peripheral blood. Other groups of monoclonal antibodies recognized the canine homologues of the CD11/18 group of antigens, CD44 and the CD45/CD45R antigen family: these should be useful in isolating functional subsets of CD4+ helper T cells. There was a cluster of four antibodies that bound strongly to platelets (probably CD41 antigen), three antibodies that were specific to B cells (including CD21) and two antibodies against a granulocyte antigen (possibly CD15). A number of reagents were found against canine MHC-II and immunoglobulin, with some of the latter able to distinguish between Ig subclasses. Properties of each of the canine antigens defined by these monoclonal antibodies are discussed and compared with other species. The availability of such a panel of reagents should allow rapid improvements in the immunological diagnosis of canine disease, and there might now be a potential for testing novel therapeutic strategies in a clinical veterinary setting.

  2. From monoclonal antibodies to chimeric antigen receptors for the treatment of human malignancies.

    Science.gov (United States)

    Caruana, Ignazio; Diaconu, Iulia; Dotti, Gianpietro

    2014-10-01

    Monoclonal antibodies (mAbs) and their directly derived cell-based application known as chimeric antigen receptors (CARs) ensue from the need to develop novel therapeutic strategies that retain high anti-tumor activity, but carry reduced toxicity compared to conventional chemo- and radiotherapies. In this concise review article, we will summarize the application of antibodies designed to target antigens expressed by tumor cells, and the transition from these antibodies to the generation of CARs. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Cross-protection of newly emerging HPAI H5 viruses by neutralizing human monoclonal antibodies: A viable alternative to oseltamivir.

    Science.gov (United States)

    Ren, Huanhuan; Wang, Guiqin; Wang, Shuangshuang; Chen, Honglin; Chen, Zhiwei; Hu, Hongxing; Cheng, Genhong; Zhou, Paul

    2016-01-01

    Newly emerging highly pathogenic avian influenza (HPAI) H5N2, H5N3, H5N5, H5N6, H5N8 and H5N9 viruses have been spreading in poultry and wild birds. The H5N6 viruses have also caused 10 human infections with 4 fatal cases in China. Here, we assessed the cross-neutralization and cross-protection of human and mouse monoclonal antibodies against 2 viruses: a HPAI H5N8 virus, A/chicken/Netherlands/14015526/2014 (NE14) and a HPAI H5N6 virus, A/Sichuan/26221/2014 (SC14). The former was isolated from an infected chicken in Netherlands in 2014 and the latter was isolated from an infected human patient in Sichuan, China. We show that antibodies FLA5.10, FLD21.140, 100F4 and 65C6, but not AVFluIgG01, AVFluIgG03, S139/1 and the VRC01 control, potently cross-neutralize the H5N8 NE14 and H5N6 SC14 viruses. Furthermore, we show that a single injection of >1 mg/kg of antibody 100F4 at 4 hours before, or 20 mg/kg antibody 100F4 at 72 hours after, a lethal dose of H5N8 NE14 enables mice to withstand the infection. Finally, we show that a single injection of 0.5 or 1 mg/kg antibody 100F4 prophylactically or 10 mg/kg 100F4 therapeutically outperforms a 5-day course of 10 mg/kg/day oseltamivir treatment against lethal H5N8 NE14 or H5N6 SC14 infection in mice. Our results suggest that further preclinical evaluation of human monoclonal antibodies against newly emerging H5 viruses is warranted.

  4. Immunologic analysis of human breast cancer progesterone receptors. 1. Immunonaffinity purification of transformed receptors and production of monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Estes, P.A.; Suba, E.J.; Lawler-Heavner, J.; Elashry-Stowers, D.; Wei, L.L.; Toft, D.O.; Sullivan, W.P.; Horwitz, K.B.; Edwards, D.P.

    1987-09-22

    A monoclonal antibody (MAb), designated PR-6, produced against chick oviduct progesterone receptors cross-reacts with the M/sub r/ 120,000 human B receptors. An immunomatrix prepared with PR-6 was used to purify progesterone receptors (PR) from T47D human breast cancer cells. Single-step immunoaffinity chromatography results in enrichment of B receptors (identified by immunoblot with PR-6 and by photoaffinity labeling with (/sup 3/H)promegestone) to a specific activity of 1915 pmol/mg of protein (or 23% purity) and with 27% yield. Purity and yields as judged by gel electrophoresis and densitometric scanning of the B protein were approximately 1.7-fold higher due to partial loss in hormone binding activity at the elution step. B receptors purified under these conditions are transformed and biologically active. They were maintained as undergraded 120-kDa doublets and retained both hormone and DNA binding activities. These purified B receptors were used as immunogen for production of four monoclonal antibodies against human PR. Three of the MAbs, designated as B-30 (IgG/sub 1/), B-64 (IgG/sub 1/), and B-11 (IgM), are specific for B receptors. The fourth MAb, A/B-52 (IgG/sub 1/), reacts with both A and B receptors. The IgG MAbs are monospecific for human PR since they recognize and absorb native receptor-hormone complexes, displace the sedimentation of 4S receptors on salt containing sucrose gradients, and, by immunoblot assay of crude T47D cytosol, react only with receptor polypeptides. Although mice were injected with B receptors only, production of A/B-52 which recognized both A and B receptors provides evidence that these two proteins share regions of structural homology.

  5. Development of a novel anti-human aspartyl-(asparaginyl) β-hydroxylase monoclonal antibody with diagnostic and therapeutic potential.

    Science.gov (United States)

    Huyan, Ting; Li, Qi; Dong, Dan-Dan; Yang, Hui; Xue, Xiao-Ping; Huang, Qing-Sheng

    2017-03-01

    Human aspartyl-(asparaginyl)-β-hydroxylase (HAAH) has recently been the subject of several studies, as it was previously observed to be overexpressed in numerous types of carcinoma cells and tissues in patient tumor samples. HAAH has been implicated in tumor invasion and metastasis, indicating that it may be an important target and biomarker for tumor diagnosis and treatment. However, the immunological tools currently available for the study of this protein, including monoclonal antibodies, are limited, as is the present knowledge regarding the role of HAAH in tumor therapy and diagnosis. In the present study, a recombinant C-terminal domain of HAAH was expressed in Pichia pastoris and a novel monoclonal antibody (mAb) targeting HAAH (HAAH-C) was constructed. Immunofluorescence and antibody-dependent cellular cytotoxicity (ADCC) assays were used to demonstrate the specificity and ADCC activity of this antibody. The results demonstrated that this anti-C-terminal HAAH mAB, in combination with an existing anti-N terminal HAAH mAb, exhibited a high response to native HAAH from carcinoma cell culture supernatant, as measured with a double antibody sandwich enzyme-linked immunosorbent assay. This validated novel mAB-HAAH-C may prompt further studies into the underlying mechanisms of HAAH, and the exploration of its potential in tumor diagnosis and therapy.

  6. An ultra-sensitive monoclonal antibody-based enzyme-linked immunosobent assay for dibutyl phthalate in human urinary

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Lifang [Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Science, Zhejiang University, Hangzhou 310058 (China); Lei, Yajing [Hangzhou EPIE Bio-detection Technology Limited, Hangzhou 310051 (China); Zhang, Dai; Ahmed, Shabbir [Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Science, Zhejiang University, Hangzhou 310058 (China); Chen, Shuqing, E-mail: chenshuqing@zju.edu.cn [Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Science, Zhejiang University, Hangzhou 310058 (China)

    2016-01-15

    Dibutyl phthalate (DBP) has been extensively used as a plasticizer in many daily products, which is highly toxic to human, notably affecting the reproductive and developmental function. As the previous method is expensive, time-consuming, low sensitivity and just focused on the environment. Present study was aimed to establish an ultra-sensitive and simple method based on good quality monoclonal antibody, applying to evaluate excretion level of DBP in urine samples of Chinese population directly. A monoclonal antibody was generated and characterized after fusion of myeloma cells with spleen cells isolated from BALB/c mouse. The mouse was previously immunized using a specially designed amino derivative of DBP conjugated with bovine serum albumin (BSA) as immunogen. Cross-reactivity values of the monoclonal antibody against DBP, di-isobutyl phthalate (DIBP) were observed 100% and 1.25%, while for dimethyl phthalate (DMP), butyl benzyl phthalate (BBP) and didecyl phthalate (DDP) the values were < 0.06%. The standard curve was constructed at 0–50 ng mL{sup −1} and good linearity (R{sup 2} = 0.994) was achieved. The observed IC{sub 50} (7.34 ng mL{sup −1}) and LOD (0.06 ng mL{sup −1}) values was improved 1000-fold to polyclonal antibody and 5-fold to other monoclonal antibodies. A total 1246 urine samples were analyzed and the detection frequency of DBP was observed 72.87% by ic-ELISA. The 95th percentile and mean concentration of DBP were 12.07 and 3.00 ng mL{sup −1}. Acceptable recovery rates of DBP were 97.8–114.3% and coefficients variation 5.93–11.09%. The concentrations of DBP in females were found significantly higher (p < 0.05) than males. Similarly, the DBP in middle aged and low educated individuals was found higher (p < 0.001) than the others. Considering the adverse health effects, DBP internal exposure in the Chinese population should be reduced. The ic-ELISA method has been proved as a cost effective, specific, and highly sensitive screening

  7. Idiotype vaccines against human T cell acute lymphoblastic leukemia. I. Generation and characterization of biologically active monoclonal anti-idiotopes.

    Science.gov (United States)

    Bhattacharya-Chatterjee, M; Pride, M W; Seon, B K; Kohler, H

    1987-08-15

    A murine monoclonal anti-tumor antibody termed SN2 (Ab1), isotype IgG1-kappa, that defines a unique human T cell leukemia-associated cell-surface glycoprotein, gp37 (m.w. 37,000), was used to generate monoclonal anti-idiotype antibodies (Ab2) in syngeneic BALB/c mice. The Ab2 were screened on the basis of their binding to the F(ab')2 fragments of SN2 and not to the F(ab')2 of pooled normal BALB/c mice sera IgG1 or to an unrelated BALB/c monoclonal antibody of the same isotype. Fifteen Ab2, obtained from two fusions, were specific for the SN2 idiotope and not against isotype or allotype determinants. To find out whether these Ab2 are directed against the paratope of SN2, the binding of radiolabeled SN2 to leukemic MOLT-4 and JM cells which contain gp37 as a surface constituent was studied in the presence of these anti-idiotopes. Clone 4EA2 inhibited the binding 100% at a concentration of 50 ng and 4DC6 inhibited 90% at a concentration of 250 ng. A third clone 4DD6 gave about 50% inhibition. Similar was the inhibition of SN2 binding to insolubilized MOLT-4 antigen or cell membrane preparation. The binding of SN2 (Ab1) to 4EA2 and 4DC6 was also inhibited by semipurified preparation of gp37 antigen. These results demonstrate that at least two of the anti-idiotope antibodies are binding either at or near the binding site idiotope of SN2. Next, the purified Ab2 was used to immunize syngeneic mice to induce antibody binding to MOLT-4 cells or gp37. Sera from mice immunized with 4EA2 and 4DC6 coupled to keyhole limpet hemocyanin contained antibodies which bind to semipurified gp37 antigen and MOLT-4 cells. Immune sera inhibited the binding of iodinated Ab2 and Ab1 indicating that an anti-anti-idiotopic antibody (Ab3) in mice shares idiotopes with Ab1 (SN2). Also, the binding of iodinated Ab2 to Ab1 was inhibited by rabbit antisera specific for gp37. Collectively, these data suggest that anti-idiotype antibodies 4EA2 and 4DC6 may be useful in the generation of idiotype

  8. Humanization of rabbit monoclonal antibodies via grafting combined Kabat/IMGT/Paratome complementarity-determining regions: Rationale and examples.

    Science.gov (United States)

    Zhang, Yi-Fan; Ho, Mitchell

    2017-04-01

    Rabbit monoclonal antibodies (RabMAbs) can recognize diverse epitopes, including those poorly immunogenic in mice and humans. However, there have been only a few reports on RabMAb humanization, an important antibody engineering step usually done before clinical applications are investigated. To pursue a general method for humanization of RabMAbs, we analyzed the complex structures of 5 RabMAbs with their antigens currently available in the Protein Data Bank, and identified antigen-contacting residues on the rabbit Fv within the 6 Angstrom distance to its antigen. We also analyzed the supporting residues for antigen-contacting residues on the same heavy or light chain. We identified "HV4" and "LV4" in rabbit Fvs, non-complementarity-determining region (CDR) loops that are structurally close to the antigen and located in framework 3 of the heavy chain and light chain, respectively. Based on our structural and sequence analysis, we designed a humanization strategy by grafting the combined Kabat/IMGT/Paratome CDRs, which cover most antigen-contacting residues, into a human germline framework sequence. Using this strategy, we humanized 4 RabMAbs that recognize poorly immunogenic epitopes in the cancer target mesothelin. Three of the 4 humanized rabbit Fvs have similar or improved functional binding affinity for mesothelin-expressing cells. Interestingly, 4 immunotoxins composed of the humanized scFvs fused to a clinically used fragment of Pseudomonas exotoxin (PE38) showed stronger cytotoxicity against tumor cells than the immunotoxins derived from their original rabbit scFvs. Our data suggest that grafting the combined Kabat/IMGT/Paratome CDRs to a stable human germline framework can be a general approach to humanize RabMAbs.

  9. Generation of human monoclonal antibodies against ganglioside antigens and their applications in the diagnosis and therapy of cancer

    Energy Technology Data Exchange (ETDEWEB)

    Alfonso, M. [Dept. of Tumor Cell Biology, Div. of Cancer Biology, Danish Cancer Society, Copenhagen (Denmark)]|[Dept. of Research and Development, Center of Molecular Immunology, Havana (Cuba); Zeuthen, J. [Dept. of Tumor Cell Biology, Div. of Cancer Biology, Danish Cancer Society, Copenhagen (Denmark)

    1996-10-01

    Different approaches to generating human monoclonal antibodies (MAbs) against tumor-associated ganglioside antigens have been carried out in several laboratories. A specific goal addressed by our laboratory is to produce human MAbs to several ganglioside antigens of relevance as therapeutic targets, such as the GM2, GD2, GD3 and GM3 gangliosides in melanoma. In vitro immunization of human B lymphocytes from normal donors was performed using liposomes containing gangliosides as the immunizing antigen combined with either complete tetanus toxoid or a synthetic peptide corresponding to a T helper epitope to stimulate in vitro immunization. Specific human anti-ganglioside antibodies were obtained, indicating that the antibdoy response found in vitro was antigen-driven. To overcome the widely reported problems concerning stability of immunoglobulin production by the antibody-secreting cell lines, a method of positive selection using GM3-coated magnetic beads has been developed in order to rescue unstable clones. Development of new methods to reproducibly generate ganglioside-specific human MAbs will amplify the possibilities for diagnostic and therapeutic applications. (orig.).

  10. [Evolution of monoclonal antibodies in cancer treatment].

    Science.gov (United States)

    Kubczak, Małgorzata; Rogalińska, Małgorzata

    Since late 90s of last century the new age of directed therapy began using mainly biological constructs produced in rodents called monoclonal antibodies. The side effects of monoclonal antibodies were a challenge for pharmaceutical companies to improve the biological properties of these biological drugs. The humanization of monoclonal constructs was an idea to improve monoclonal antibodies next generation activity cancer cell reduction in humans. Moreover for some other patients sensitive for monoclonal antibodies therapy could also potentially induce immunological differences that might imply on human health. The new idea related to monoclonal antibodies was to design a small molecule constructs of nanoantibodies with ability to enter into cells. Such small molecules could find their targets inside human cells, even in nuclei leading to differences in cancer cells expression. The existing knowledge on monoclonal antibodies as well as directed activity of nanoantibodies could improve anticancer treatment efficancy of diseases.

  11. 3-hydroxykynurenine-mediated modification of human lens proteins: structure determination of a major modification using a monoclonal antibody.

    Science.gov (United States)

    Staniszewska, Magdalena M; Nagaraj, Ram H

    2005-06-10

    Tryptophan can be oxidized in the eye lens by both enzymatic and non-enzymatic mechanisms. Oxidation products, such as kynurenines, react with proteins to form yellow-brown pigments and cause covalent cross-linking. We generated a monoclonal antibody against 3-hydroxykynurenine (3OHKYN)-modified keyhole limpet hemocyanin and characterized it using 3OHKYN-modified amino acids and proteins. This monoclonal antibody reacted with 3OHKYN-modified N(alpha)-acetyl lysine, N(alpha)-acetyl histidine, N(alpha)-acetyl arginine, and N(alpha)-acetyl cysteine. Among the several tryptophan oxidation products tested, 3OHKYN produced the highest concentration of antigen when reacted with human lens proteins. A major antigen from the reaction of 3OHKYN and N(alpha)-acetyl lysine was purified by reversed phase high pressure liquid chromatography, which was characterized by spectroscopy and identified as 2-amino-3-hydroxyl-alpha-((5S)-5-acetamino-5-carboxypentyl amino)-gamma-oxo-benzene butanoic acid. Enzyme-digested cataractous lens proteins displayed 3OHKYN-derived modifications. Immunohistochemistry revealed 3OHKYN modifications in proteins associated with the lens fiber cell plasma membrane. The low molecular products (<10,000 Da) isolated from normal lenses after reaction with glucosidase followed by incubation with proteins generated 3OHKYN-derived products. Human lens epithelial cells incubated with 3OHKYN showed intense immunoreactivity. We also investigated the effect of glycation on tryptophan oxidation and kynurenine-mediated modification of lens proteins. The results showed that glycation products failed to oxidize tryptophan or generate kynurenine modifications in proteins. Our studies indicate that 3OHKYN modifies lens proteins independent of glycation to form products that may contribute to protein aggregation and browning during cataract formation.

  12. Binding Affinity, Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200

    DEFF Research Database (Denmark)

    Lindegren, Sture; Andrade, Luciana N S; Bäck, Tom

    2015-01-01

    The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovar...

  13. Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins

    NARCIS (Netherlands)

    Kummer, J. Alain; Kamp, Angela M.; van Katwijk, Marcel; Brakenhoff, Just P.J.; Radosevic, Katarina; Radosevic, K.; van Leeuwen, Anne Marie; Borst, Jannie; Verweij, Cornelis L.; Hack, C. Erik

    1993-01-01

    The human serine proteases granzymes A and B are expressed in cytotoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies. Seven

  14. Intracerebral and subcutaneous xenografts of human sclc in the nude rat - comparison of monoclonal-antibody localization and tumor-infiltrating lymphocytes

    NARCIS (Netherlands)

    Bulte, J. W. M.; Go, K. Gwan; Zuiderveen, F.; The, T. Hauw; de Leij, Lou

    In the WAG/Rij nude rat, subcutaneous (s.c.) and intracerebral (i.c.) xenografts of the human SCLC cell line GLC-28 were evaluated for their growth behavior, in vivo monoclonal antibody binding and presence of tumor infiltrating lymphocytes. For the i.c. xenografts, two models of cerebral tumor

  15. In vivo activity of a mixture of two human monoclonal antibodies (anti-HBs) in a chronic hepatitis B virus carrier chimpanzee

    NARCIS (Netherlands)

    R. Heijtink; W. Paulij; P.A.C. van Bergen (Patrick); M.H. van Roosmalen (Mark); D. Rohm; B. Eichentopf; E. Muchmore; A.D.M.E. Osterhaus (Albert); R.A. de Man (Robert)

    1999-01-01

    textabstractA 35-year-old female hepatitis B virus carrier chimpanzee was infused with one dose of a mixture of human monoclonal antibodies 9H9 and 4-7B (antibodies against hepatitis B virus surface antigen; HBsAg). Blood samples were taken before and up to 3 weeks

  16. A panel of monoclonal antibodies against the prion protein proves that there is no prion protein in human pancreatic ductal epithelial cells.

    Science.gov (United States)

    Yang, Liheng; Zhang, Yan; Hu, Lipeng; Zhu, Ying; Sy, Man-Sun; Li, Chaoyang

    2014-08-01

    Prion diseases are a group of neurodegenerative diseases that are fatal. The study of these unique diseases in China is hampered by a lack of resources. Amongst the most important resources for biological study are monoclonal antibodies. Here, we characterize a panel of monoclonal antibodies specific for cellular prion protein by enzyme-linked immunosorbent assay (ELISA), immunofluorescent staining, flow cytometry, and western blotting. We identify several antibodies that can be used for specific applications and we demonstrate that there is no prion protein expression in human pancreatic ductal epithelial cells (HPDC).

  17. Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcγR Engagement.

    Science.gov (United States)

    Dahan, Rony; Barnhart, Bryan C; Li, Fubin; Yamniuk, Aaron P; Korman, Alan J; Ravetch, Jeffrey V

    2016-06-13

    While engagement of the inhibitory Fcγ-receptor (FcγR) IIB is an absolute requirement for in vivo antitumor activity of agonistic mouse anti-CD40 monoclonal antibodies (mAbs), a similar requirement for human mAbs has been disputed. By using a mouse model humanized for its FcγRs and CD40, we revealed that FcγRIIB engagement is essential for the activity of human CD40 mAbs, while engagement of the activating FcγRIIA inhibits this activity. By engineering Fc variants with selective enhanced binding to FcγRIIB, but not to FcγRIIA, significantly improved antitumor immunity was observed. These findings highlight the necessity of optimizing the Fc domain for this class of therapeutic antibodies by using appropriate preclinical models that accurately reflect the unique affinities and cellular expression of human FcγR. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Therapeutic administration of a recombinant human monoclonal antibody reduces the severity of chikungunya virus disease in rhesus macaques.

    Directory of Open Access Journals (Sweden)

    Rebecca Broeckel

    2017-06-01

    Full Text Available Chikungunya virus (CHIKV is a mosquito-borne virus that causes a febrile syndrome in humans associated with acute and chronic debilitating joint and muscle pain. Currently no licensed vaccines or therapeutics are available to prevent or treat CHIKV infections. We recently isolated a panel of potently neutralizing human monoclonal antibodies (mAbs, one (4N12 of which exhibited prophylactic and post-exposure therapeutic activity against CHIKV in immunocompromised mice. Here, we describe the development of an engineered CHIKV mAb, designated SVIR001, that has similar antigen binding and neutralization profiles to its parent, 4N12. Because therapeutic administration of SVIR001 in immunocompetent mice significantly reduced viral load in joint tissues, we evaluated its efficacy in a rhesus macaque model of CHIKV infection. Rhesus macaques that were treated after infection with SVIR001 showed rapid elimination of viremia and less severe joint infiltration and disease compared to animals treated with SVIR002, an isotype control mAb. SVIR001 reduced viral burden at the site of infection and at distant sites and also diminished the numbers of activated innate immune cells and levels of pro-inflammatory cytokines and chemokines. SVIR001 therapy; however, did not substantively reduce the induction of CHIKV-specific B or T cell responses. Collectively, these results show promising therapeutic activity of a human anti-CHIKV mAb in rhesus macaques and provide proof-of-principle for its possible use in humans to treat active CHIKV infections.

  19. The transcriptional profiling of human in vivo-generated plasma cells identifies selective imbalances in monoclonal gammopathies.

    Directory of Open Access Journals (Sweden)

    Luis M Valor

    Full Text Available Plasma cells (PC represent the heterogeneous final stage of the B cells (BC differentiation process. To characterize the transition of BC into PC, transcriptomes from human naïve BC were compared to those of three functionally-different subsets of human in vivo-generated PC: i tonsil PC, mainly consisting of early PC; ii PC released to the blood after a potent booster-immunization (mostly cycling plasmablasts; and, iii bone marrow CD138+ PC that represent highly mature PC and include the long-lived PC compartment. This transcriptional transition involves subsets of genes related to key processes for PC maturation: the already known protein processing, apoptosis and homeostasis, and of new discovery including histones, macromolecule assembly, zinc-finger transcription factors and neuromodulation. This human PC signature is partially reproduced in vitro and is conserved in mouse. Moreover, the present study identifies genes that define PC subtypes (e.g., proliferation-associated genes for circulating PC and transcriptional-related genes for tonsil and bone marrow PC and proposes some putative transcriptional regulators of the human PC signatures (e.g., OCT/POU, XBP1/CREB, E2F, among others. Finally, we also identified a restricted imbalance of the present PC transcriptional program in monoclonal gammopathies that correlated with PC malignancy.

  20. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Kai Xu

    Full Text Available The henipaviruses, represented by Hendra (HeV and Nipah (NiV viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4 was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.

  1. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

    Science.gov (United States)

    Xu, Kai; Rockx, Barry; Xie, Yihu; DeBuysscher, Blair L; Fusco, Deborah L; Zhu, Zhongyu; Chan, Yee-Peng; Xu, Yan; Luu, Truong; Cer, Regina Z; Feldmann, Heinz; Mokashi, Vishwesh; Dimitrov, Dimiter S; Bishop-Lilly, Kimberly A; Broder, Christopher C; Nikolov, Dimitar B

    2013-01-01

    The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.

  2. Prophylactic and therapeutic testing of Nicotiana-derived RSV-neutralizing human monoclonal antibodies in the cotton rat model.

    Science.gov (United States)

    Zeitlin, Larry; Bohorov, Ognian; Bohorova, Natasha; Hiatt, Andrew; Kim, Do H; Pauly, Michael H; Velasco, Jesus; Whaley, Kevin J; Barnard, Dale L; Bates, John T; Crowe, James E; Piedra, Pedro A; Gilbert, Brian E

    2013-01-01

    Severe lower respiratory tract infection in infants and small children is commonly caused by respiratory syncytial virus (RSV). Palivizumab (Synagis(®)), a humanized IgG1 monoclonal antibody (mAb) approved for RSV immunoprophylaxis in at-risk neonates, is highly effective, but pharmacoeconomic analyses suggest its use may not be cost-effective. Previously described potent RSV neutralizers (human Fab R19 and F2-5; human IgG RF-1 and RF-2) were produced in IgG format in a rapid and inexpensive Nicotiana-based manufacturing system for comparison with palivizumab. Both plant-derived (palivizumab-N) and commercial palivizumab, which is produced in a mouse myeloma cell line, showed protection in prophylactic (p plant-derived human mAbs directed against alternative epitopes displayed neutralizing activity, but conferred less protection in vivo than palivizumab-N or palivizumab. Palivizumab remains one of the most efficacious RSV mAbs described to date. Production in plants may reduce manufacturing costs and improve the pharmacoeconomics of RSV immunoprophylaxis and therapy.

  3. Monoclonal antibody to human cartilage cells and its reactivities to chondrocytic tumors.

    Science.gov (United States)

    Eisenbrey, A B; Chen, J C; Dyer, C A; Bernstein, J; Poulik, M D

    1991-01-01

    A murine monoclonal antibody (E10) was made against cultured cartilage cells. The E10 antibody binding is localized to the surface of cultured cartilage cells in suspension and is present in the cytoplasm in paraffin embedded sections. There is no reactivity with cartilage matrix, or with the matrix of cartilaginous tumors. Reactivity is removed by treatment with trypsin and hyaluronidase, but not by treatment with heparinase, neuraminidase, and chondroitinase. Regeneration of E10 antigen after trypsinization takes 48 hours in chondrocytes in tissue culture. SDS-polyacrylamide gel electrophoresis of an E10 immune precipitate of cultured chondrocytes results in two peaks: one at a very high molecular weight and a small fragment at approximately 250 kd. Specificity has been demonstrated by cytofluorometry, immunofluorescence, and immunohistochemistry, in both frozen and paraffin-embedded tissues. Positive reactivity was seen in cultured cartilage cells, chondrocytes in fetal and adult cartilage, chondrosarcomas, and chordomas. Minimal reactivity was found in a chondromyxoid liposarcoma. Acinar cells of salivary and sweat glands and mast cells in various tissues and tumors were also positive. There was no reactivity with other tissues and tumors, including myxoid and mucinous tumors and epithelial tissues.

  4. Broadly neutralizing human monoclonal JC polyomavirus VP1–specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy

    Science.gov (United States)

    Jelcic, Ivan; Combaluzier, Benoit; Jelcic, Ilijas; Faigle, Wolfgang; Senn, Luzia; Reinhart, Brenda J.; Ströh, Luisa; Nitsch, Roger M.; Stehle, Thilo; Sospedra, Mireia; Grimm, Jan; Martin, Roland

    2016-01-01

    In immunocompromised individuals, JC polyomavirus (JCPyV) may mutate and gain access to the central nervous system resulting in progressive multifocal leukoencephalopathy (PML), an often fatal opportunistic infection for which no treatments are currently available. Despite recent progress, the contribution of JCPyV-specific humoral immunity to controlling asymptomatic infection throughout life and to eliminating JCPyV from the brain is poorly understood. We examined antibody responses against JCPyV major capsid protein VP1 (viral protein 1) variants in the serum and cerebrospinal fluid (CSF) of healthy donors (HDs), JCPyV-positive multiple sclerosis patients treated with the anti-VLA-4 monoclonal antibody natalizumab (NAT), and patients with NAT-associated PML. Before and during PML, CSF antibody responses against JCPyV VP1 variants show “recognition holes”; however, upon immune reconstitution, CSF antibody titers rise, then recognize PML-associated JCPyV VP1 variants, and may be involved in elimination of the virus. We therefore reasoned that the memory B cell repertoire of individuals who recovered from PML could be a source for the molecular cloning of broadly neutralizing antibodies for passive immunization. We generated a series of memory B cell-derived JCPyV VP1-specific human monoclonal antibodies from HDs and a patient with NAT-associated PML-immune reconstitution inflammatory syndrome (IRIS). These antibodies exhibited diverse binding affinity, cross-reactivity with the closely related BK polyomavirus, recognition of PML-causing VP1 variants, and JCPyV neutralization. Almost all antibodies with exquisite specificity for JCPyV, neutralizing activity, recognition of all tested JCPyV PML variants, and high affinity were derived from one patient who had recovered from PML. These antibodies are promising drug candidates for the development of a treatment of PML. PMID:26400911

  5. High-yield production of a human monoclonal IgG by rhizosecretion in hydroponic tobacco cultures.

    Science.gov (United States)

    Madeira, Luisa M; Szeto, Tim H; Henquet, Maurice; Raven, Nicole; Runions, John; Huddleston, Jon; Garrard, Ian; Drake, Pascal M W; Ma, Julian K-C

    2016-02-01

    Rhizosecretion of recombinant pharmaceuticals from in vitro hydroponic transgenic plant cultures is a simple, low cost, reproducible and controllable production method. Here, we demonstrate the application and adaptation of this manufacturing platform to a human antivitronectin IgG1 monoclonal antibody (mAb) called M12. The rationale for specific growth medium additives was established by phenotypic analysis of root structure and by LC-ESI-MS/MS profiling of the total protein content profile of the hydroponic medium. Through a combination of optimization approaches, mAb yields in hydroponic medium reached 46 μg/mL in 1 week, the highest figure reported for a recombinant mAb in a plant secretion-based system to date. The rhizosecretome was determined to contain 104 proteins, with the mAb heavy and light chains the most abundant. This enabled evaluation of a simple, scalable extraction and purification protocol and demonstration that only minimal processing was necessary prior to protein A affinity chromatography. MALDI-TOF MS revealed that purified mAb contained predominantly complex-type plant N-glycans, in three major glycoforms. The binding of M12 purified from hydroponic medium to vitronectin was comparable to its Chinese hamster ovary (CHO)-derived counterpart. This study demonstrates that in vitro hydroponic cultivation coupled with recombinant protein rhizosecretion can be a practical, low-cost production platform for monoclonal antibodies. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  6. Establishing in vitro in vivo correlations to screen monoclonal antibodies for physicochemical properties related to favorable human pharmacokinetics.

    Science.gov (United States)

    Avery, Lindsay B; Wade, Jason; Wang, Mengmeng; Tam, Amy; King, Amy; Piche-Nicholas, Nicole; Kavosi, Mania S; Penn, Steve; Cirelli, David; Kurz, Jeffrey C; Zhang, Minlei; Cunningham, Orla; Jones, Rhys; Fennell, Brian J; McDonnell, Barry; Sakorafas, Paul; Apgar, James; Finlay, William J; Lin, Laura; Bloom, Laird; O'Hara, Denise M

    2017-12-22

    Implementation of in vitro assays that correlate with in vivo human pharmacokinetics (PK) would provide desirable preclinical tools for the early selection of therapeutic monoclonal antibody (mAb) candidates with minimal non-target-related PK risk. Use of these tools minimizes the likelihood that mAbs with unfavorable PK would be advanced into costly preclinical and clinical development. In total, 42 mAbs varying in isotype and soluble versus membrane targets were tested in in vitro and in vivo studies. MAb physicochemical properties were assessed by measuring non-specific interactions (DNA- and insulin-binding ELISA), self-association (affinity-capture self-interaction nanoparticle spectroscopy) and binding to matrix-immobilized human FcRn (surface plasmon resonance and column chromatography). The range of scores obtained from each in vitro assay trended well with in vivo clearance (CL) using both human FcRn transgenic (Tg32) mouse allometrically projected human CL and observed human CL, where mAbs with high in vitro scores resulted in rapid CL in vivo. Establishing a threshold value for mAb CL in human of 0.32 mL/hr/kg enabled refinement of thresholds for each in vitro assay parameter, and using a combinatorial triage approach enabled the successful differentiation of mAbs at high risk for rapid CL (unfavorable PK) from those with low risk (favorable PK), which allowed mAbs requiring further characterization to be identified. Correlating in vitro parameters with in vivo human CL resulted in a set of in vitro tools for use in early testing that would enable selection of mAbs with the greatest likelihood of success in the clinic, allowing costly late-stage failures related to an inadequate exposure profile, toxicity or lack of efficacy to be avoided.

  7. Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders

    Directory of Open Access Journals (Sweden)

    Liang Xu

    2017-09-01

    Full Text Available Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26, a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity.Methods: By screening a combinatorial library of human single-chain fragment variable (scFv antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells.Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action

  8. A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words

    Energy Technology Data Exchange (ETDEWEB)

    Mirick, G. R.; Bradt, B. M.; Denardo, S. J.; Denardo, G. L. [Calfornia Univ., Sacramento (United States). Davis Medical Center

    2004-12-01

    The United States Food and Drugs Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ({sup 9}0yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ({sup 1}31I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) antiCD20 MAns for use in radioimmunotherapy (RIT) of non-Hodgikin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, essays were developed to determine HAGE (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to humanize MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades.

  9. Detection of HLA-G on human extravillous cytotrophoblast and skeletal muscle with a new monoclonal antibody MEM-G/1.

    Science.gov (United States)

    Lodererová, A; Honsová, E; Viklický, O

    2003-01-01

    Using immunohistochemistry with the newly available monoclonal antibody MEM-G/1 the reaction patterns on frozen and formaldehyde-fixed paraffin-embedded sections on human placentas, lymph nodes, skeletal muscles, and kidney and liver allografts were compared. HLA-G (a nonclassical major histocompatibility complex class I molecule that is assumed to influence the immune response during pregnancy and some pathological conditions) was found within human extravillous cytotrophoblast but not within villous cytotrophoblast and placental mesenchymal tissue. No HLA-G expression on human lymph nodes, tonsils, and kidney and liver allografts was demonstrated. However, HLA-G expression was observed in all samples of skeletal muscle. The binding capacity of monoclonal antibody MEM-G/1 provides new possibilities to study physiological and pathophysiological roles of HLA-G in humans.

  10. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.

    Directory of Open Access Journals (Sweden)

    Xiaohua Ye

    2016-03-01

    Full Text Available Enterovirus 71 (EV71 is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3 of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2, the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.

  11. Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum.

    Directory of Open Access Journals (Sweden)

    Gaëtan Vanier

    Full Text Available Monoclonal antibodies (mAbs represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans.

  12. Isolation of HIV-1-neutralizing mucosal monoclonal antibodies from human colostrum.

    Directory of Open Access Journals (Sweden)

    James Friedman

    Full Text Available Generation of potent anti-HIV antibody responses in mucosal compartments is a potential requirement of a transmission-blocking HIV vaccine. HIV-specific, functional antibody responses are present in breast milk, and these mucosal antibody responses may play a role in protection of the majority of HIV-exposed, breastfeeding infants. Therefore, characterization of HIV-specific antibodies produced by B cells in milk could guide the development of vaccines that elicit protective mucosal antibody responses.We isolated B cells from colostrum of an HIV-infected lactating woman with a detectable neutralization response in milk and recombinantly produced and characterized the resulting HIV-1 Envelope (Env-specific monoclonal antibodies (mAbs.The identified HIV-1 Env-specific colostrum mAbs, CH07 and CH08, represent two of the first mucosally-derived anti-HIV antibodies yet to be reported. Colostrum mAb CH07 is a highly-autoreactive, weakly-neutralizing gp140-specific mAb that binds to linear epitopes in the gp120 C5 region and gp41 fusion domain. In contrast, colostrum mAb CH08 is a nonpolyreactive CD4-inducible (CD4i gp120-specific mAb with moderate breadth of neutralization.These novel HIV-neutralizing mAbs isolated from a mucosal compartment provide insight into the ability of mucosal B cell populations to produce functional anti-HIV antibodies that may contribute to protection against virus acquisition at mucosal surfaces.

  13. MINOR HUMAN-ANTIBODY RESPONSE TO A MOUSE AND CHIMERIC MONOCLONAL-ANTIBODY AFTER A SINGLE IV INFUSION IN OVARIAN-CARCINOMA PATIENTS - A COMPARISON OF 5 ASSAYS

    NARCIS (Netherlands)

    BUIST, MR; KENEMANS, P; VANKAMP, GJ; Haisma, Hidde

    The human anti-(mouse Ig) antibody (HAMA) response was measured in serum of 52 patients suspected of having ovarian carcinoma who had received an i.v. injection of either the murine monoclonal antibody (mAb) OV-TL 3 F(ab')(2) (n = 28, 1 mg) or the chimeric mouse/human mAb MOv18 (cMOv18; n = 24, 3

  14. Method and cell lines for the production of monoclonal antibodies to human glycophorin A

    Science.gov (United States)

    Bigbee, W.L.; Fong, S.S.N.; Jensen, R.H.; Vanderlaan, M.

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that differentiate between the M and N forms of human glycophorin A. These antibodies have potential application as human blood group reagents, as markers for terminally differentiated erythroid cells and as immunofluorescent labels of somatically variant human erythrocytes.

  15. Further phenotypic characterization and isolation of human hematopoietic progenitor cells using a monoclonal antibody to the c-kit receptor.

    Science.gov (United States)

    Briddell, R A; Broudy, V C; Bruno, E; Brandt, J E; Srour, E F; Hoffman, R

    1992-06-15

    A mouse antihuman monoclonal IgG2a antibody, termed stem cell receptor-1 (SR-1), specific for a determinant of the c-kit ligand receptor (KR), was used as an immunologic probe to analyze KR expression by human bone marrow hematopoietic progenitor cells. Monoclonal antibodies to CD34 and HLA-DR were used in a multicolor staining protocol in conjunction with SR-1 to further define the phenotypes of various classes of hematopoietic progenitor cells. Expression of KR (SR-1+) on hematopoietic progenitor cells identified subpopulations of cells expressing CD34 (CD34+). While one-half of the CD34- and HLA-DR-expressing cells (CD34+ HLA-DR+) expressed the KR (SR-1+), one-third of the CD34+ cells that lacked HLA-DR expression (CD34+ HLA-DR-) were SR-1+. The CD34+ HLA-DR+ SR-1+ cell population contained the vast majority of the more differentiated progenitor cells, including the colony-forming unit (CFU) granulocyte-macrophage; burst-forming unit-erythrocyte; CFU-granulocyte, erythrocyte, macrophage, megakaryocyte; and the CFU-megakaryocyte. The overall progenitor cell cloning efficiency of this subpopulation was greater than 31%. By contrast, the CD34+ HLA-DR- SR-1+ cell population contained fewer of these more differentiated progenitor cells but exclusively contained the more primitive progenitor cells, the BFU-megakaryocyte, high proliferative potential-colony-forming cell, and long-term bone marrow culture-initiating cell. The overall progenitor cell cloning efficiency of this subpopulation was greater than 7%. Both the CD34+ HLA-DR- and CD34+ HLA-DR+ cell subpopulations lacking KR expression contained few assayable hematopoietic progenitor cells. Long-term bone marrow cultures initiated with CD34+ HLA-DR- SR-1+ but not CD34+ HLA-DR- SR-1- cells, which were repeatedly supplemented with c-kit ligand (KL) and interleukin-3, generated assayable progenitor cells of at least 2 lineages for 10 weeks. These experiments demonstrate the expression of the KR throughout the

  16. High prevalence of human anti-bovine IgG antibodies as the major cause of false positive reactions in two-site immunoassays based on monoclonal antibodies

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Koch, Claus; Jensen, Charlotte H

    2004-01-01

    A sandwich ELISA for quantification of the endometrial protein PP14 revealed false positive reactions in 81% of male sera (n = 54). The PP14 ELISA was based on two monoclonal antibodies (Mabs) with different epitope specificities--a catcher and a biotinylated indicator. The monoclonal antibodies...... were purified by protein G affinity chromatography from culture supernatant containing 10% (v/v) fetal calf serum (FCS). Human anti-animal IgG (bovine, mouse, horse, and swine) antibodies and human anti-bovine serum albumin antibodies were measured using an ELISA design, with direct bridging...... of the solid phase and biotinylated antigens. The false positive reactions were abolished by addition of 1% (v/v) bovine serum to the dilution buffer (DB). Human anti-bovine IgG antibodies (HABIA) were detected in 99 out of 104 sera from blood donors (50 females; 54 males). HABIA levels in male sera (n = 54...

  17. A murine monoclonal anti-idiotypic antibody detects a common idiotope on human, mouse and rabbit antibodies to allergen Lol p IV.

    Science.gov (United States)

    Zhou, E M; Dzuba-Fischer, J M; Rector, E S; Sehon, A H; Kisil, F T

    1991-09-01

    A syngeneic mouse monoclonal anti-idiotypic antibody (anti-Id), designated as B1/1, was generated against a monoclonal antibody (MoAb 91) specific for Ryegrass pollen allergen Lol p IV. This anti-Id recognized an idiotope (Id) that was also present on other monoclonal antibodies with the same specificity as MoAb 91. Observations that (i) the anti-Id inhibited the binding of MoAb 91 to Lol p IV and (ii) the Id-anti-Id interaction could be inhibited by Lol p IV indicated that the Id was located within or near the antigen combining site. These properties served to characterize B1/1 as an internal image anti-Id. Evidence that an immune response in different species to Lol p IV elicits the formation of antibodies which express a common Id was provided by the observations that (i) the Id-anti-Id interactions could be inhibited by mouse, human and rabbit antisera to Lol p IV and (ii) the binding of these antisera to Lol p IV could be inhibited by the anti-Id. Interestingly, the internal image anti-Id B1/1 also recognized an Id on a monoclonal antibody which was directed to an epitope of Lol p IV, different from that recognized by MoAb 91.

  18. Monoclonal Antibodies production technology

    Directory of Open Access Journals (Sweden)

    Flávia Rocha

    2011-02-01

    Full Text Available Since the first cells were capable of maintain a continuous antibody supply, developed by Köhler and Milstein in 1975, its use in medicine and industry showed a great potential. New researches were developed to enhance the use of such cells, including immunizations, mieloma cells, fusion methodology, screening techniques, cloning, culture media, among several details which enable and optimizes its use. Nowadays, monoclonal antibodies are a well-established tool for proteomics research and it have countless applications on several knowledge areas, mainly human and/or animal disease diagnostic, identification and tracking of allergenic compounds in food and residues in the environment. This review can be used by professionals, researches and students searching for a compiled papers contributing to the improvement of the monoclonal antibodies technology, used at different knowledge areas such as human diseases and diseases and disorders in agriculture and livestock chain.

  19. Monoclonal Antibodies.

    Science.gov (United States)

    Geskin, Larisa J

    2015-10-01

    Use of monoclonal antibodies (mAbs) has revolutionized cancer therapy. Approaches targeting specific cellular targets on the malignant cells and in tumor microenvironment have been proved to be successful in hematologic malignancies, including cutaneous lymphomas. mAb-based therapy for cutaneous T-cell lymphoma has demonstrated high response rates and a favorable toxicity profile in clinical trials. Several antibodies and antibody-based conjugates are approved for use in clinical practice, and many more are in ongoing and planned clinical trials. In addition, these safe and effective drugs can be used as pillars for sequential therapies in a rational stepwise manner. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Monoclonal antibodies to human glycophorin A and cell lines for the production thereof

    Science.gov (United States)

    Vanderlaan, Martin; Bigbee, William L.; Jensen, Ronald H.; Fong, Stella S. N.; Langlois, Richard G.

    1988-01-01

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that are highly specific to and exhibit high affinity for glycophorin A.sup.N and differentiate between the M and N forms of human glycophorin A.

  1. First-in-Human Phase I Study of Lumretuzumab, a Glycoengineered Humanized Anti-HER3 Monoclonal Antibody, in Patients with Metastatic or Advanced HER3-Positive Solid Tumors

    DEFF Research Database (Denmark)

    Meulendijks, Didier; Jacob, Wolfgang; Martinez-Garcia, Maria

    2016-01-01

    PURPOSE: A first-in-human phase I study was conducted to characterize safety, efficacy, and pharmacokinetic (PK) and pharmacodynamic (PD) properties of lumretuzumab, a humanized and glycoengineered anti-HER3 monoclonal antibody, in patients with advanced cancer. EXPERIMENTAL DESIGN: Twenty-five p...

  2. First-in-Human Phase I Study of Lumretuzumab, a Glycoengineered Humanized Anti-HER3 Monoclonal Antibody, in Patients with Metastatic or Advanced HER3-Positive Solid Tumors

    NARCIS (Netherlands)

    Meulendijks, Didier; Jacob, Wolfgang; Martinez-Garcia, Maria; Taus, Alvaro; Lolkema, Martijn P; Voest, Emile E; Langenberg, Marlies H G; Fleitas Kanonnikoff, Tania; Cervantes, Andres; De Jonge, Maja J; Sleijfer, Stefan; Soerensen, Morten Mau; Thomas, Marlene; Ceppi, Maurizio; Meneses-Lorente, Georgina; James, Ian; Adessi, Celine; Michielin, Francesca; Abiraj, Keelara; Bossenmaier, Birgit; Schellens, Jan H M; Weisser, Martin; Lassen, Ulrik N

    2016-01-01

    PURPOSE: A first-in-human phase I study was conducted to characterize safety, efficacy, and pharmacokinetic (PK) and pharmacodynamic (PD) properties of lumretuzumab, a humanized and glycoengineered anti-HER3 monoclonal antibody, in patients with advanced cancer. EXPERIMENTAL DESIGN: Twenty-five

  3. Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins

    Science.gov (United States)

    Luo, Shen; Zhang, Baolin

    2015-01-01

    Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5–6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5–6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development. PMID:26338058

  4. A Recombinant Humanized Anti-Cocaine Monoclonal Antibody Inhibits the Distribution of Cocaine to the Brain in Rats

    Science.gov (United States)

    Gooden, Felicia C. T.; Tabet, Michael R.; Ball, William J.

    2014-01-01

    The monoclonal antibody (mAb), h2E2, is a humanized version of the chimeric human/murine anti-cocaine mAb 2E2. The recombinant h2E2 protein was produced in vitro from a transfected mammalian cell line and retained high affinity (4 nM Kd) and specificity for cocaine over its inactive metabolites benzoylecgonine (BE) and ecgonine methyl ester. In rats, pharmacokinetic studies of h2E2 (120 mg/kg i.v.) showed a long terminal elimination half-life of 9.0 days and a low volume of distribution at steady state (Vdss) of 0.3 l/kg. Pretreatment with h2E2 produced a dramatic 8.8-fold increase in the area under the plasma cocaine concentration-time curve (AUC) and in brain a concomitant decrease of 68% of cocaine’s AUC following an i.v. injection of an equimolar cocaine dose. Sequestration of cocaine in plasma by h2E2, shown via reduction of cocaine’s Vdss, indicates potential clinical efficacy. Although the binding of cocaine to h2E2 in plasma should inhibit distribution and metabolism, the elimination of cocaine remained multicompartmental and was still rapidly eliminated from plasma despite the presence of h2E2. BE was the major cocaine metabolite, and brain BE concentrations were sixfold higher than in plasma, indicating that cocaine is normally metabolized in the brain. In the presence of h2E2, brain BE concentrations were decreased and plasma BE was increased, consistent with the observed h2E2-induced changes in cocaine disposition. The inhibition of cocaine distribution to the brain confirms the humanized mAb, h2E2, as a lead candidate for development as an immunotherapy for cocaine abuse. PMID:24733787

  5. Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins.

    Science.gov (United States)

    Luo, Shen; Zhang, Baolin

    2015-01-01

    Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5-6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5-6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.

  6. High-Avidity and Potently Neutralizing Cross-Reactive Human Monoclonal Antibodies Derived from Secondary Dengue Virus Infection

    Science.gov (United States)

    Tsai, Wen-Yang; Lai, Chih-Yun; Wu, Yi-Chieh; Lin, Hong-En; Edwards, Carolyn; Jumnainsong, Amonrat; Kliks, Srisakul; Halstead, Scott; Mongkolsapaya, Juthathip; Screaton, Gavin R.

    2013-01-01

    The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies (Abs) and vaccine development. Previous studies of human dengue-immune sera reported that a significant proportion of anti-E Abs, known as group-reactive (GR) Abs, were cross-reactive to all four DENV serotypes and to one or more other flaviviruses. Based on studies of mouse anti-E monoclonal antibodies (MAbs), GR MAbs were nonneutralizing or weakly neutralizing compared with type-specific MAbs; a GR response was thus not regarded as important for vaccine strategy. We investigated the epitopes, binding avidities, and neutralization potencies of 32 human GR anti-E MAbs. In addition to fusion loop (FL) residues in E protein domain II, human GR MAbs recognized an epitope involving both FL and bc loop residues in domain II. The neutralization potencies and binding avidities of GR MAbs derived from secondary DENV infection were stronger than those derived from primary infection. GR MAbs derived from primary DENV infection primarily blocked attachment, whereas those derived from secondary infection blocked DENV postattachment. Analysis of the repertoire of anti-E MAbs derived from patients with primary DENV infection revealed that the majority were GR, low-avidity, and weakly neutralizing MAbs, whereas those from secondary infection were primarily GR, high-avidity, and potently neutralizing MAbs. Our findings suggest that the weakly neutralizing GR anti-E Abs generated from primary DENV infection become potently neutralizing MAbs against the four serotypes after secondary infection. The observation that the dengue immune status of the host affects the quality of the cross-reactive Abs generated has implications for new strategies for DENV vaccination. PMID:24027331

  7. Monoclonal antibody to a subset of human monocytes found only in the peripheral blood and inflammatory tissues

    Energy Technology Data Exchange (ETDEWEB)

    Zwadlo, G.; Schlegel, R.; Sorg, C.

    1986-07-15

    A monoclonal antibody is described that was generated by immunizing mice with cultured human blood monocytes. The antibody (27E10) belongs to the IgG1 subclass and detects a surface antigen at M/sub r/ 17,000 that is found on 20% of peripheral blood monocytes. The antigen is increasingly expressed upon culture of monocytes, reaching a maximum between days 2 and 3. Stimulation of monocytes with interferon-..gamma.. (IFN-..gamma..), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and lipopolysaccharide (LPS) Ylalanine (fMLP) increased the 27E10 antigen density. The amount of 27E10-positive cells is not or is only weakly affected. The antigen is absent from platelets, lymphotyces, and all tested human cell lines, yet it cross-reacts with 15% of freshly isolated granulocytes. By using the indirect immunoperoxidase technique, the antibody is found to be negative on cryostat sections of normal human tissue (skin, lung, and colon) and positive on only a few monocyte-like cells in liver and on part of the cells of the splenic red pulp. In inflammatory tissue, however, the antibody is positive on monocytes/macrophages and sometimes on endothelial cells and epidermal cells, depending on the stage and type of inflammation, e.g., BCG ranulomas are negative, whereas psoriasis vulgaris, atopic dermatitis, erythrodermia, pressure urticaria, and periodontitis contain positively staining cells. In contact eczemas at different times after elicitation (6 hr, 24 hr, and 72 hr), the 27E10 antigen is seen first after 24 hr on a few infiltrating monocytes/macrophages, which increase in numbers after 72 hr.

  8. Ligation of human Fc receptor like-2 by monoclonal antibodies down-regulates B-cell receptor-mediated signalling

    Science.gov (United States)

    Shabani, Mahdi; Bayat, Ali Ahmad; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Hojjat-Farsangi, Mohammad; Ulivieri, Cristina; Amirghofran, Zahra; Baldari, Cosima Tatiana; Shokri, Fazel

    2014-01-01

    B-cell antigen receptor (BCR) signalling and its regulation through negative and positive regulators are critical for balancing B-cell response and function. Human Fc receptor like-2 (FCRL2), a member of the newly identified FCRL family, could influence B-cell signalling due to possession of both immunoreceptor tyrosine-based activation and inhibitory motifs (ITAM and ITIM). Since the natural ligand of FCRL2 has not been identified, we generated FCRL2-specific monoclonal antibodies (mAbs) and employed them to investigate the influence of FCRL2 stimulation on BCR signalling in an FCRL2-expressing B-cell line. Two anti-FCRL2 mAb-producing hybridoma clones (5A7-E7 and 3D8-G8) were selected. None of the mAbs displayed any cross-reactivity with the other members of the FCRL family including recombinant FCRL1, -3, -4 and -5, as tested by FACS and ELISA techniques. Engagement of the FCRL2 by these mAbs resulted in significant inhibition of BCR signalling mediators such as calcium mobilization and phosphorylation of the mitogen-activated protein kinases Erk, p38 and Jnk. These findings indicate that the FCRL2 ITIM motifs are functional and the anti-FCRL2 mAbs may mimic the natural ligand of FCRL2 by induction of inhibitory signals in B cells. PMID:24797767

  9. Homotypic aggregation of human cell lines by HLA class II-, class Ia- and HLA-G-specific monoclonal antibodies

    DEFF Research Database (Denmark)

    Odum, Niels; Ledbetter, J A; Martin, P

    1991-01-01

    adhesion between T and B cells by activating the CD18/CD11a (LFA-1) adhesion pathway. Here we report that monoclonal antibodies (mAb) against HLA-DR (L243, p4.1, HB10a, VI15) and certain broad class II reacting mAb (TU35, TU39), but not anti-DQ (TU22, Leu-10) mAb, induced homotypic aggregation of human......, but not the class I-negative parental line, 221, showed homotypic aggregation in response to an HLA-G specific mAb (87G) and a broad reacting class I-specific mAb (IOT2). Both cell lines responded with aggregation to anti-class II mAb (TU35). The anti-class I mAb, W6/32, had no effect on all cell lines tested......Major histocompatibility complex (MHC) class II molecules have been implicated in cell adhesion in two ways. In addition to the well-established role of class II antigens in low-affinity adhesion provided by interactions between class II and CD4, recent data indicated that class II may also induce...

  10. An epitope tag derived from human transcription factor IIB that reacts with a polyol-responsive monoclonal antibody.

    Science.gov (United States)

    Duellman, Sarah J; Thompson, Nancy E; Burgess, Richard R

    2004-05-01

    Polyol-responsive monoclonal antibodies (PR-mAbs) provide a strategy to purify active, nondenatured proteins by a single-step immunoaffinity chromatography procedure. The high affinity interaction between these antibodies and the antigen can be dissociated in the presence of a nonchaotropic salt and a low molecular weight polyhydroxylated compound (polyol). The epitope for PR-mAb IIB8 is located near the N-terminus of the human transcription factor IIB (TFIIB). The epitope is an eight amino acid sequence, TKDPSRVG, that can be fused to a desired protein for use as a purification tag. This epitope tag (termed hIIB) was fused to the C-terminus of green fluorescent protein (GFP). An additional GFP fusion protein utilized another version of hIIB containing a point mutation at position two. These fusion proteins, expressed in Escherichia coli, allowed successful separation of the desired protein in a single chromatographic step. This strategy extends PR-mAb gentle-release purification to numerous expressed proteins.

  11. Neutralization of Zika virus by germline-like human monoclonal antibodies targeting cryptic epitopes on envelope domain III.

    Science.gov (United States)

    Wu, Yanling; Li, Shun; Du, Lanying; Wang, Chunyu; Zou, Peng; Hong, Binbin; Yuan, Mengjiao; Ren, Xiaonan; Tai, Wanbo; Kong, Yu; Zhou, Chen; Lu, Lu; Zhou, Xiaohui; Jiang, Shibo; Ying, Tianlei

    2017-10-11

    The Zika virus (ZIKV), a flavivirus transmitted by Aedes mosquitoes, has emerged as a global public health concern. Pre-existing cross-reactive antibodies against other flaviviruses could modulate immune responses to ZIKV infection by antibody-dependent enhancement, highlighting the importance of understanding the immunogenicity of the ZIKV envelope protein. In this study, we identified a panel of human monoclonal antibodies (mAbs) that target domain III (DIII) of the ZIKV envelope protein from a very large phage-display naive antibody library. These germline-like antibodies, sharing 98%-100% hoLogy with their corresponding germline IGHV genes, bound ZIKV DIII specifically with high affinities. One mAb, m301, broadly neutralized the currently circulating ZIKV strains and showed a synergistic effect with another mAb, m302, in neutralizing ZIKV in vitro and in a mouse model of ZIKV infection. Interestingly, epitope mapping and competitive binding studies suggest that m301 and m302 bind adjacent regions of the DIII C-C' loop, which represents a recently identified cryptic epitope that is intermittently exposed in an uncharacterized virus conformation. This study extended our understanding of antigenic epitopes of ZIKV antibodies and has direct implications for the design of ZIKV vaccines.

  12. Generation and characterization of new monoclonal antibodies targeting the PHF1 and AT8 epitopes on human tau.

    Science.gov (United States)

    Strang, Kevin H; Goodwin, Marshall S; Riffe, Cara; Moore, Brenda D; Chakrabarty, Paramita; Levites, Yona; Golde, Todd E; Giasson, Benoit I

    2017-07-31

    Tauopathies are a group of neurodegenerative disorders, including Alzheimer's disease, defined by the presence of brain pathological inclusions comprised of abnormally aggregated and highly phosphorylated tau protein. The abundance of brain tau aggregates correlates with disease severity and select phospho-tau epitopes increase at early stages of disease. We generated and characterized a series of novel monoclonal antibodies directed to tau phosphorylated at several of these phospho-epitopes, including Ser396/Ser404, Ser404 and Thr205. We also generated phosphorylation independent antibodies against amino acid residues 193-211. We show that most of these antibodies are highly specific for tau and strongly recognize pathological inclusions in human brains and in a transgenic mouse model of tauopathy. They also reveal epitope-specific differences in the biochemical properties of Alzheimer's disease sarkosyl-insoluble tau. These new reagents will be useful for investigating the progression of tau pathology and further as tools to target the cellular transmission of tau pathology.

  13. Absolute quantitation of host cell proteins in recombinant human monoclonal antibodies with an automated CZE-ESI-MS/MS system.

    Science.gov (United States)

    Zhu, Guijie; Sun, Liangliang; Linkous, Travis; Kernaghan, Dawn; McGivney, James B; Dovichi, Norman J

    2014-05-01

    We report the first use of CZE for absolute characterization of host cell proteins (HCPs) in recombinant human monoclonal antibodies. An electrokinetically pumped nanoelectrospray interface was used to couple CZE with a tandem mass spectrometer. Three isotopic-labeled peptides (LSFDKDAMVAR, VDIVENQAMDTR, and LVSDEMVVELIEK) were synthesized by direct incorporation of an isotope-labeled lysine or arginine. The heavy-labeled peptides were spiked in the HCP digests at known concentrations. After CZE-ESI-MS/MS analysis, the peaks of native and isotopic-labeled peptides were extracted with mass tolerance ≤ 5 ppm from the electropherograms, and the ratios of peak area between native and isotopic-labeled peptides pairs were calculated. Calibration curves (the ratios of peak area versus spiked peptide amount) with R(2) values of 0.999, 0.997, and 0.999 were obtained for the three HCP peptides, and the absolute amounts of the three proteins present were determined to be at the picomole level in a 20 μg sample of digested HCPs. The target proteins were present at the 7-30 ppt level in the purified HCP samples. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of “preneoplastic antigen”-like molecules

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Hongying [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Yoshimura, Kazunori [Department of Physiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kobayashi, Nobuharu; Sugiyama, Kazuo [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Sawada, Jun-ichi; Saito, Yoshiro [Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2012-04-01

    Microsomal epoxide hydrolase (mEH) is a drug metabolizing enzyme which resides on the endoplasmic reticulum (ER) membrane and catalyzes the hydration of reactive epoxide intermediates that are formed by cytochrome P450s. mEH is also thought to have a role in bile acid transport on the plasma membrane of hepatocytes. It is speculated that efficient execution of such multiple functions is secured by its orientation and association with cytochrome P450 enzymes on the ER membrane and formation of a multiple transport system on the plasma membrane. In certain disease status, mEH loses its association with the membrane and can be detected as distinct antigens in the cytosol of preneoplastic foci of liver (preneoplastic antigen), in the serum in association with hepatitis C virus infection (AN antigen), or in some brain tumors. To analyze the antigenic structures of mEH in physiological and pathological conditions, we developed monoclonal antibodies against different portions of mEH. Five different kinds of antibodies were obtained: three, anti-N-terminal portions; one anti-C-terminal; and one, anti-conformational epitope. By combining these antibodies, we developed antigen detection methods which are specific to either the membrane-bound form or the linearized form of mEH. These methods detected mEH in the culture medium released from a hepatocellular carcinoma cell line and a glioblastoma cell line, which was found to be a multimolecular complex with a unique antigenic structure different from that of the membrane-bound form of mEH. These antibodies and antigen detection methods may be useful to study pathological changes of mEH in various human diseases. -- Highlights: ► Monoclonal antibodies against different portions of mEH were developed. ► They discriminate between the membrane-bound and the linearized forms of mEH. ► We analyze the antigenic structure of the altered form of mEH in tumor cells. ► Preneoplastic antigen is a multimolecular complex of mEH with

  15. Treatment with a human recombinant monoclonal IgG antibody against oxidized LDL in atherosclerosis-prone pigs reduces cathepsin S in coronary lesions

    DEFF Research Database (Denmark)

    Poulsen, Christian Bo; Al-Mashhadi, Ahmed Ludvigsen; Von Wachenfeldt, Karin

    2016-01-01

    Background Immunization with oxidized LDL (oxLDL) reduces atherosclerosis in rodents. We tested the hypothesis that treatment with a human recombinant monoclonal antibody against oxLDL will reduce the burden or composition of atherosclerotic lesions in hypercholesterolemic minipigs. Methods...... and results Thirty-eight hypercholesterolemic minipigs with defective LDL receptors were injected with an oxLDL antibody or placebo weekly for 12 weeks. An 18F-fluorodeoxyglucose positron emission tomography (FDG PET) scan (n = 9) was performed before inclusion and after 3 months of treatment. Blood samples.......03) with no difference in CD68 or CD163 positivity. Conclusions In hypercholesterolemic minipigs, treatment with a human recombinant monoclonal antibody against oxLDL reduced cathepsin S in coronary lesions without any effect on the burden of atherosclerosis or aortic FDG-PET signal....

  16. Affinity isolation of antigen-specific circulating B cells for generation of phage display-derived human monoclonal antibodies

    DEFF Research Database (Denmark)

    Ditzel, Henrik

    2009-01-01

    A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries generated from...... the frequency of antibody phage particles of interest in the library and allow for efficient isolation monoclonal antibodies with the predefined specificity....

  17. B-cell display-based one-step method to generate chimeric human IgG monoclonal antibodies

    OpenAIRE

    Lin, Waka; Kurosawa, Kohei; Murayama, Akiho; Kagaya, Eri; Ohta, Kunihiro

    2010-01-01

    The recent development of screening strategies based on the generation and display of large libraries of antibody fragments has allowed considerable advances for the in vitro isolation of monoclonal antibodies (mAbs). We previously developed a technology referred to as the ‘ADLib (Autonomously Diversifying Library) system’, which allows the rapid screening and isolation in vitro of antigen-specific monoclonal antibodies (mAbs) from libraries of immunoglobulin M (IgM) displayed by the chicken ...

  18. Transgenic mouse strains as platforms for the successful discovery and development of human therapeutic monoclonal antibodies.

    Science.gov (United States)

    Green, Larry L

    2014-03-01

    Transgenic mice have yielded seven of the ten currently-approved human antibody drugs, making them the most successful platform for the discovery of fully human antibody therapeutics. The use of the in vivo immune system helps drive this success by taking advantage of the natural selection process that produces antibodies with desirable characteristics. Appropriately genetically-engineered mice act as robust engines for the generation of diverse repertoires of affinity- matured fully human variable regions with intrinsic properties necessary for successful antibody drug development including high potency, specificity, manufacturability, solubility and low risk of immunogenicity. A broad range of mAb drug targets are addressable in these mice, comprising both secreted and transmembrane targets, including membrane multi-spanning targets, as well as human target antigens that share high sequence identity with their mouse orthologue. Transgenic mice can routinely yield antibodies with sub-nanomolar binding affinity for their antigen, with lead candidate mAbs frequently possessing affinities for binding to their target of less than 100 picomolar, without requiring any ex vivo affinity optimization. While the originator transgenic mice platforms are no longer broadly available, a new generation of transgenic platforms is in development for discovery of the next wave of human therapeutic antibodies.

  19. A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection Test for the Diagnosis of Human Brucellosis

    Science.gov (United States)

    Patra, Kailash P.; Saito, Mayuko; Atluri, Vidya L.; Rolán, Hortensia G.; Young, Briana; Kerrinnes, Tobias; Smits, Henk; Ricaldi, Jessica N.; Gotuzzo, Eduardo; Gilman, Robert H.; Tsolis, Renee M.; Vinetz, Joseph M.

    2014-01-01

    Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases. PMID:24901521

  20. Prophylactic and postexposure efficacy of a potent human monoclonal antibody against MERS coronavirus

    Science.gov (United States)

    Corti, Davide; Zhao, Jincun; Pedotti, Mattia; Simonelli, Luca; Agnihothram, Sudhakar; Fett, Craig; Fernandez-Rodriguez, Blanca; Foglierini, Mathilde; Agatic, Gloria; Vanzetta, Fabrizia; Gopal, Robin; Langrish, Christopher J.; Barrett, Nicholas A; Sallusto, Federica; Baric, Ralph S.; Varani, Luca; Zambon, Maria; Perlman, Stanley; Lanzavecchia, Antonio

    2015-01-01

    Middle East Respiratory Syndrome (MERS) is a highly lethal pulmonary infection caused by a previously unidentified coronavirus (CoV), likely transmitted to humans by infected camels. There is no licensed vaccine or antiviral for MERS, therefore new prophylactic and therapeutic strategies to combat human infections are needed. In this study, we describe, for the first time, to our knowledge, the isolation of a potent MERS-CoV–neutralizing antibody from memory B cells of an infected individual. The antibody, named LCA60, binds to a novel site on the spike protein and potently neutralizes infection of multiple MERS-CoV isolates by interfering with the binding to the cellular receptor CD26. Importantly, using mice transduced with adenovirus expressing human CD26 and infected with MERS-CoV, we show that LCA60 can effectively protect in both prophylactic and postexposure settings. This antibody can be used for prophylaxis, for postexposure prophylaxis of individuals at risk, or for the treatment of human cases of MERS-CoV infection. The fact that it took only 4 mo from the initial screening of B cells derived from a convalescent patient for the development of a stable chinese hamster ovary (CHO) cell line producing neutralizing antibodies at more than 5 g/L provides an example of a rapid pathway toward the generation of effective antiviral therapies against emerging viruses. PMID:26216974

  1. The value of non-human primates in the development of therapeutic monoclonal antibodies

    NARCIS (Netherlands)

    Van Meer, P.J.K.|info:eu-repo/dai/nl/34153790X; Kooijman, M.|info:eu-repo/dai/nl/322905788; Van Der Laan, J.W.|info:eu-repo/dai/nl/374879966; Moors, E.H.M.|info:eu-repo/dai/nl/20241664X; Schellekens, H.|info:eu-repo/dai/nl/068406762

    2011-01-01

    The pharmaceutical industry is increasingly focusing on the development of biological therapeutics. These molecules generally cause no off-target toxicity and are highly species specific. Therefore, non-human primates (NHPs) are often the only relevant species in which to conduct regulatory safety

  2. Monoclonal antibodies to Pneumocystis carinii

    DEFF Research Database (Denmark)

    Kovacs, J A; Halpern, J L; Lundgren, B

    1989-01-01

    To increase understanding of the antigenic structure of Pneumocystis carinii, we developed monoclonal antibodies to rat and human P. carinii. The specificity of the antibodies was demonstrated by immunofluorescence and immunoblot studies. Only one of five monoclonal antibodies to rat P. carinii...... reacted with human P. carinii, and none of four monoclonal antibodies to human P. carinii reacted with rat P. carinii. Two antibodies to human P. carinii reacted by immunofluorescence with only one human P. carinii isolate. Immunoblot studies identified major antigens of rat P. carinii with molecular...... antigenically different. Further studies with these antibodies should increase understanding of the antigenic nature of P. carinii and of the interaction of P. carinii with its host....

  3. Efficacy and safety of treatment with an anti-m2e monoclonal antibody in experimental human influenza.

    Science.gov (United States)

    Ramos, Eleanor L; Mitcham, Jennifer L; Koller, Teri D; Bonavia, Aurelio; Usner, Dale W; Balaratnam, Ganesh; Fredlund, Paul; Swiderek, Kristine M

    2015-04-01

    The efficacy of TCN-032, a human monoclonal antibody targeting a conserved epitope on M2e, was explored in experimental human influenza. Healthy volunteers were inoculated with influenza A/Wisconsin/67/2005 (H3N2) and received a single dose of the study drug, TCN-032, or placebo 24 hours later. Subjects were monitored for symptoms, viral shedding, and safety, including cytokine measurements. Oseltamivir was administered 7 days after inoculation. Although the primary objective of reducing the proportion of subjects developing any grade ≥2 influenza symptom or pyrexia, was not achieved, TCN-032-treated subjects showed 35% reduction (P = .047) in median total symptom area under the curve (days 1-7) and 2.2 log reduction in median viral load area under the curve (days 2-7) by quantitative polymerase chain reaction (P = .09) compared with placebo-treated subjects. TCN-032 was safe and well tolerated with no additional safety signals after administration of oseltamivir. Serum cytokine levels (interferon γ, tumor necrosis factor α, and interleukin 8 and 10) were similar in both groups. Genotypic and phenotypic analyses showed no difference between virus derived from subjects after TCN-032 treatment and parental strain. These data indicate that TCN-032 may provide immediate immunity and therapeutic benefit in influenza A infection, with no apparent emergence of resistant virus. TCN-032 was safe with no evidence of immune exacerbation based on serum cytokine expression. Clinicaltrials.gov registry number. NCT01719874. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  4. High affinity anti-TIM-3 and anti-KIR monoclonal antibodies cloned from healthy human individuals.

    Directory of Open Access Journals (Sweden)

    Stefan Ryser

    Full Text Available We report here the cloning of native high affinity anti-TIM-3 and anti-KIR IgG monoclonal antibodies (mAbs from peripheral blood mononuclear cells (PBMC of healthy human donors. The cells that express these mAbs are rare, present at a frequency of less than one per 105 memory B-cells. Using our proprietary multiplexed screening and cloning technology CellSpot™ we assessed the presence of memory B-cells reactive to foreign and endogenous disease-associated antigens within the same individual. When comparing the frequencies of antigen-specific memory B-cells analyzed in over 20 screening campaigns, we found a strong correlation of the presence of anti-TIM-3 memory B-cells with memory B-cells expressing mAbs against three disease-associated antigens: (i bacterial DNABII proteins that are a marker for Gram negative and Gram positive bacterial infections, (ii hemagglutinin (HA of influenza virus and (iii the extracellular domain of anaplastic lymphoma kinase (ALK. One of the native anti-KIR mAbs has similar characteristics as lirilumab, an anti-KIR mAb derived from immunization of humanized transgenic mice that is in ongoing clinical trials. It is interesting to speculate that these native anti-TIM-3 and anti-KIR antibodies may function as natural regulatory antibodies, analogous to the pharmacological use in cancer treatment of engineered antibodies against the same targets. Further characterization studies are needed to define the mechanisms through which these native antibodies may function in healthy and disease conditions.

  5. Developmental Toxicity and Fertility Assessment in Rabbits with Tabalumab: A Human IgG4 Monoclonal Antibody.

    Science.gov (United States)

    Breslin, William J; Hilbish, Kim G; Martin, Jennifer A; Halstead, Carolyn A; Edwards, Tammy L

    2015-06-01

    Tabalumab is a human immunoglobulin G subclass 4 monoclonal antibody that has been under development for autoimmune disorders. Tabalumab has full neutralizing activity against both soluble and membrane B-cell activating factor, a B-cell survival factor. The objectives of these studies were to assess the effects of tabalumab on embryo-fetal development and on male (M) and female (F) fertility in rabbits, a pharmacologically relevant species. Doses were administered at 0 (vehicle control), 0.3 (embryo-fetal study only), 1.0, and 30 mg/kg. In the embryo-fetal study, pregnant rabbits does were given a single dose by intravenous injection on gestation day (GD) 7. In the fertility studies, tabalumab was administered by intravenous injection every 7 days starting 2 (F) or 4 (M) weeks before mating, during cohabitation, and until necropsy (M) or through GD 18 (F). Treated animals were mated with untreated partners. Parental clinical signs, body weight, food consumption, blood lymphocyte phenotyping, organ weights, morphologic pathology, ovarian and uterine observations, sperm parameters, and fertility indices were evaluated along with conceptus viability, weight, and morphology. Exposure assessments were made in all main study animals and satellite animals. No adverse parental, reproductive, or developmental effects were observed in any study at any dose. A pharmacodynamic response consisting of dose-dependent decreases in the percent and number of total B lymphocytes and increases in the percent and/or number of total T lymphocytes was observed in parental rabbits at 1.0 and 30 mg/kg. In conclusion, no adverse reproductive or developmental effects were observed in rabbits following exposure to tabalumab at doses as high as 30 mg/kg and exposures at least 14-fold greater than human exposure levels. © 2015 Wiley Periodicals, Inc.

  6. Clearance Kinetics and External Dosimetry of Iodine-131-labeled Murine and Humanized Monoclonal Antibody A33 in Patients with Colon Cancer: Radiation Safety Implications

    OpenAIRE

    Dauer, Lawrence T.; Boylan, Daniel C; Williamson, Matthew J.; Germain, Jean St.; Steven M. Larson

    2009-01-01

    The monoclonal antibody (mAb) A33 detects a membrane antigen that is expressed on greater than 95% of metastatic human colorectal cancers. Previous studies have shown excellent tumor-targeting of iodine-131 labeled murine and humanized forms of the mAb. A retrospective analysis of whole body clearance in the murine form was performed for comparison to the humanized form. Serial whole-body dose rate measurements were obtained for 55 treatments on 30 patients participating in phase I/II dose es...

  7. Generation and characterization of ixekizumab, a humanized monoclonal antibody that neutralizes interleukin-17A.

    Science.gov (United States)

    Liu, Ling; Lu, Jirong; Allan, Barrett W; Tang, Ying; Tetreault, Jonathan; Chow, Chi-Kin; Barmettler, Barbra; Nelson, James; Bina, Holly; Huang, Lihua; Wroblewski, Victor J; Kikly, Kristine

    2016-01-01

    Interleukin (IL)-17A exists as a homodimer (A/A) or as a heterodimer (A/F) with IL-17F. IL-17A is expressed by a subset of T-cells, called Th17 cells, at inflammatory sites. Most cell types can respond to the local production of IL-17A because of the near ubiquitous expression of IL-17A receptors, IL-17RA and IL-17RC. IL-17A stimulates the release of cytokines and chemokines designed to recruit and activate both neutrophils and memory T-cells to the site of injury or inflammation and maintain a proinflammatory state. IL-17A-producing pathogenic T-cells contribute to the pathogenesis of autoimmune diseases, including psoriasis, psoriatic arthritis, rheumatoid arthritis, and ankylosing spondylitis. This study describes the generation and characterization of ixekizumab, a humanized IgG4 variant IL-17A-neutralizing antibody. Ixekizumab binds human and cynomolgus monkey IL-17A with high affinity and binds rabbit IL-17A weakly but does not bind to rodent IL-17A or other IL-17 family members. Ixekizumab effectively inhibits the interaction between IL-17A and its receptor in binding assays and potently blocks IL-17A-induced GRO or KC secretion in cell-based assays. In an in vivo mouse pharmcodynamic model, ixekizumab blocks human IL-17A-induced mouse KC secretion. These data provide a comprehensive preclinical characterization of ixekizumab, for which the efficacy and safety have been demonstrated in human clinical trials in psoriasis and psoriatic arthritis.

  8. The Fab Fragment of a Humanized Anti-Toll Like Receptor 4 (TLR4 Monoclonal Antibody Reduces the Lipopolysaccharide Response via TLR4 in Mouse Macrophage

    Directory of Open Access Journals (Sweden)

    Binggang Cai

    2015-10-01

    Full Text Available Lipopolysaccharides (LPS can induce acute inflammation, sepsis, or chronic inflammatory disorders through the Toll receptor 4 (TLR4 signaling pathway. The TLR4/MD2 (myeloid differentiation protein 2 complex plays a major role in the immune response to LPS. However, there is not a good method to suppress the immune response induced by LPS via this complex in macrophages. In this article, we aimed to evaluate the effects of humanized anti-TLR4 monoclonal antibodies on LPS-induced responses in mouse macrophages. The peritoneal macrophages of mice were incubated with anti-TLR4 monoclonal antibodies and stimulated with LPS. The expression levels of cytokines were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. Additionally, activation of various signaling pathways was evaluated by Western blotting. The results showed that the humanized anti-TLR4 monoclonal antibody blocked the inflammatory cytokines expression at both the mRNA and protein level. We also found that the Fab fragment significantly inhibited the nuclear factor kappaB signaling pathway by reducing the phosphorylation of the inhibitor of kappaBalpha and decreasing the translocation of p65, resulting in the suppression of p38, extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, and IFN-β regulatory factor 3 phosphorylation. Therefore, our study showed that this humanized anti-TLR4 monoclonal antibody could effectively protect against LPS-induced responses by blocking the TLR4 signaling pathway in mouse peritoneal macrophages.

  9. Cross-reactive dengue human monoclonal antibody prevents severe pathologies and death from Zika virus infections

    OpenAIRE

    Kam, Yiu-Wing; Lee, Cheryl Yi-Pin; Teo, Teck-Hui; Howland, Shanshan W.; Amrun, Siti Naqiah; Lum, Fok-Moon; See, Peter; Kng, Nicholas Qing-Rong; Roland G. Huber; Xu, Mei-Hui; Tan, Heng-Liang; Choo, Andre; Maurer-Stroh, Sebastian; Ginhoux, Florent; Fink, Katja

    2017-01-01

    Zika virus (ZIKV) infections have been linked with neurological complications and congenital Zika syndrome. Given the high level of homology between ZIKV and the related flavivirus dengue virus (DENV), we investigated the level of cross-reactivity with ZIKV using a panel of DENV human mAbs. A majority of the mAbs showed binding to ZIKV virions, with several exhibiting neutralizing capacities against ZIKV in vitro. Three of the best ZIKV-neutralizing mAbs were found to recognize diverse epitop...

  10. Amphipathic variable region heavy chain peptides derived from monoclonal human Wegener's anti-PR3 antibodies stimulate lymphocytes from patients with Wegener's granulomatosis and microscopic polyangiitis

    Science.gov (United States)

    Peen, E; Malone, C; Myers, C; Williams, R C; Peck, A B; Csernok, E; Gross, W L; Staud, R

    2001-01-01

    Amphipathic variable-region heavy chain 11-mer peptides from monoclonal human IgM antiproteinase-3 antibodies were studied for peripheral blood lymphocyte stimulation in 21 patients with Wegener's granulomatosis (WG) or microscopic polyangiitis (MPA), connective tissue disease controls and normal control subjects. Positive T-cell activation was observed in most experiments with WG patients' lymphocytes using amphipathic VH-region peptides from four different human monoclonal anti-PR3 antibodies. Control peptides of the same length but without amphipathic characteristics along with other amphipathic peptides not derived from monoclonal anti-PR3 sequence were employed as controls. No significant lymphocyte stimulation was observed with normal controls, but positive stimulation with amphipathic VH peptides was also recorded in other connective tissue disease controls mainly patients with rheumatoid arthritis. Amphipathic peptides not derived from anti-PR3 sequence did not stimulate WG lymphocytes. Our findings indicate that lymphocyte reactivity as an element of cell-mediated immunity may be activated by amphipathic VH-region amino acid sequences of autoantibodies which are themselves associated with diseases such as WG. PMID:11529926

  11. Monoclonal antibodies recognizing the non-tandem repeat regions of the human mucin MUC4 in pancreatic cancer.

    Directory of Open Access Journals (Sweden)

    Maneesh Jain

    Full Text Available The MUC4 mucin is a high molecular weight, membrane-bound, and highly glycosylated protein. It is a multi-domain protein that is putatively cleaved into a large mucin-like subunit (MUC4α and a C-terminal growth-factor like subunit (MUC4β. MUC4 plays critical roles in physiological and pathological conditions and is aberrantly overexpressed in several cancers, including those of the pancreas, cervix, breast and lung. It is also a potential biomarker for the diagnosis, prognosis and progression of several malignancies. Further, MUC4 plays diverse functional roles in cancer initiation and progression as evident from its involvement in oncogenic transformation, proliferation, inhibition of apoptosis, motility and invasion, and resistance to chemotherapy in human cancer cells. We have previously generated a monoclonal antibody 8G7, which is directed against the TR region of MUC4, and has been extensively used to study the expression of MUC4 in several malignancies. Here, we describe the generation of anti-MUC4 antibodies directed against the non-TR regions of MUC4. Recombinant glutathione-S-transferase (GST-fused MUC4α fragments, both upstream (MUC4α-N-Ter and downstream (MUC4α-C-Ter of the TR domain, were used as immunogens to immunize BALB/c mice. Following cell fusion, hybridomas were screened using the aforementioned recombinant proteins ad lysates from human pancreatic cell lines. Three anti MUC4α-N-Ter and one anti-MUC4α-C-Ter antibodies were characterized by several inmmunoassays including enzyme-linked immunosorbent assay (ELISA, immunoblotting, immunofluorescene, flow cytometry and immunoprecipitation using MUC4 expressing human pancreatic cancer cell lines. The antibodies also reacted with the MUC4 in human pancreatic tumor sections in immunohistochemical analysis. The new domain-specific anti-MUC4 antibodies will serve as important reagents to study the structure-function relationship of MUC4 domains and for the development of MUC4

  12. Characterisation of new monoclonal antibodies reacting with prions from both human and animal brain tissues

    DEFF Research Database (Denmark)

    Cordes, H.; Bergstrom, A.L.; Ohm, J.

    2008-01-01

    Post-mortem diagnosis of transmissible spongiform encephalopathies (prion diseases) is primarily based on the detection of a protease resistant, misfolded disease associated isoform (PrP(Sc)) of the prion protein (PrP(C)) on neuronal cells. These methods depend on antibodies directed against Pr......-type mice and used for western blotting and immunohistochemistry to detect several types of human prion-disease associated PrP(Sc), including sporadic Creutzfeldt-Jakob Disease (CJD) (subtypes MM1 and VV2), familial CJD and Gerstmann-Straussler-Scheinker (GSS) disease PrP(Sc) as well as PrP(Sc) of bovine...... spongiform encephalopathy (bovine brain), scrapie (ovine brain) and experimental scrapie in hamster and in mice. The antibodies were also used for PET-blotting in which PrP(Sc) blotted from brain tissue sections onto a nitrocellulose membrane is visualized with antibodies after protease and denaturant...

  13. Generation and characterization of cross neutralizing human monoclonal antibody against 4 serotypes of dengue virus without enhancing activity

    Directory of Open Access Journals (Sweden)

    Subenya Injampa

    2017-11-01

    Full Text Available Background Dengue disease is a leading cause of illness and death in the tropics and subtropics. Most severe cases occur among patients secondarily infected with a different dengue virus (DENV serotype compared with that from the first infection, resulting in antibody-dependent enhancement activity (ADE. Our previous study generated the neutralizing human monoclonal antibody, D23-1B3B9 (B3B9, targeting the first domain II of E protein, which showed strong neutralizing activity (NT against all four DENV serotypes. However, at sub-neutralizing concentrations, it showed ADE activity in vitro. Methods In this study, we constructed a new expression plasmid using the existing IgG heavy chain plasmid as a template for Fc modification at position N297Q by site-directed mutagenesis. The resulting plasmid was then co-transfected with a light chain plasmid to produce full recombinant IgG (rIgG in mammalian cells (N297Q-B3B9. This rIgG was characterized for neutralizing and enhancing activity by using different FcγR bearing cells. To produce sufficient quantities of B3B9 rIgG for further characterization, CHO-K1 cells stably secreting N297Q-B3B9 rIgG were then established. Results The generated N297Q-B3B9 rIgG which targets the conserved N-terminal fusion loop of DENV envelope protein showed the same cross-neutralizing activity to all four DENV serotypes as those of wild type rIgG. In both FcγRI- and RII-bearing THP-1 cells and FcγRII-bearing K562 cells, N297Q-B3B9 rIgG lacked ADE activity against all DENV serotypes at sub-neutralizing concentrations. Fortunately, the N297Q-B3B9 rIgG secreted from stable cells showed the same patterns of NT and ADE activities as those of the N297Q-B3B9 rIgG obtained from transient expression against DENV2. Thus, the CHO-K1 stably expressing N297Q-B3B9 HuMAb can be developed as high producer stable cells and used to produce sufficient amounts of antibody for further characterization as a promising dengue therapeutic

  14. Cross-reactivity of human monoclonal antibodies generated with peripheral blood lymphocytes from dengue patients with Japanese encephalitis virus

    Directory of Open Access Journals (Sweden)

    Pipattanaboon C

    2013-08-01

    Full Text Available Chonlatip Pipattanaboon,1,3,8,* Tadahiro Sasaki,2,8,* Mitsuhiro Nishimura,2,8 Chayanee Setthapramote,1,8 Pannamthip Pitaksajjakul,1,4,8 Pornsawan Leaungwutiwong,1,3,8 Kriengsak Limkittikul,5,8 Orapim Puiprom,6 Mikiko Sasayama,6 Panjaporn Chaichana,6 Tamaki Okabayashi,6 Takeshi Kurosu,2,8 Ken-ichiro Ono,7,8 Pongrama Ramasoota,1,4,8 Kazuyoshi Ikuta2,8 1Center of Excellence for Antibody Research, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 2Department of Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; 3Department of Microbiology and Immunology, 4Department of Social and Environmental Medicine, 5Department of Tropical Pediatrics, Faculty of Tropical Medicine, Mahidol University, 6Mahidol-Osaka Center for Infectious Diseases, Bangkok, Thailand; 7Medical and Biological Laboratories Corporation Ltd, Nagano, Japan; 8JST/JICA, Science and Technology Research Partnership for Sustainable Development, Tokyo, Japan *These authors made an equal contribution to this study Background: Hybridomas that produce human monoclonal antibodies (HuMAbs against Dengue virus (DV had been prepared previously using peripheral blood lymphocytes from patients with DV during the acute and convalescent phases of a secondary infection. Anti-DV envelope glycoprotein (E 99 clones, anti-DV premembrane protein (prM 8 clones, and anti-DV nonstructural protein 1 (NS1 4 clones were derived from four acute-phase patients, and anti-DV E 2 clones, anti-DV prM 2 clones, and anti-DV NS1 8 clones were derived from five convalescent-phase patients. Methods and results: In the present study, we examined whether these clones cross-reacted with Japanese encephalitis virus (JEV, which belongs to the same virus family. Forty-six of the above-described 99 (46/99 anti-E, 0/8 anti-prM, and 2/4 anti-NS1 HuMAbs from acute-phase, and 0/2 anti-E, 0/2 anti-prM, and 5/8 anti-NS1 HuMAbs from convalescent-phase showed neutralizing activity against

  15. Characterization of a monoclonal antibody against the 3D polymerase of enterovirus 71 and its use for the detection of human enterovirus A infection.

    Science.gov (United States)

    Kiener, Tanja K; Lim, Xiao Fang; Jia, Qiang; Meng, Tao; Chow, Vincent Tak Kwong; Kwang, Jimmy

    2012-03-01

    Over the last decade, frequent epidemic outbreaks of hand, foot and mouth disease have been observed in the Asia-Pacific region. Hand, foot and mouth disease is caused by different viruses from the enterovirus family, mainly coxsackievirus A16 and enterovirus 71 (EV71) from the human enterovirus A family. Severe disease and neurological complications are associated more often with EV71 infection, and can lead occasionally to fatal brain stem encephalitis in young children. The rapid progression and high mortality of severe hand, foot and mouth disease makes the direct detection of antigens early in infection essential. The best method for virus detection is the use of specific monoclonal antibodies. The generation and characterization of a monoclonal antibody specific for the 3D polymerase of human enterovirus A and the development of a virus detection dot blot assay are described. A recombinant 3CD protein from EV71 C4 strain was used as an immunogen to generate monoclonal antibodies (MAbs). Screening of hybridoma cells led to the isolation of monoclonal antibody 4B12 of the immunoglobulin IgG1 isotype. MAb 4B12 recognizes the linear epitope DFEQALFS close to the active site of the 3D polymerase, corresponding to amino acid positions 53-60 of 3D and 1784-1791 of enterovirus 71 polyprotein. The presence of 3D polymerase and its precursor 3CD proteinase in purified virus particles was confirmed. MAb 4B12 was used successfully to detect all enterovirus 71 subgenotypes in a denaturing dot blot assay with a sensitivity of 10 pg of 3D protein and 10(4) tissue culture infective dose of virus particles. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants.

    Science.gov (United States)

    Ma, Julian K-C; Drossard, Jürgen; Lewis, David; Altmann, Friedrich; Boyle, Julia; Christou, Paul; Cole, Tom; Dale, Philip; van Dolleweerd, Craig J; Isitt, Valerie; Katinger, Dietmar; Lobedan, Martin; Mertens, Hubert; Paul, Mathew J; Rademacher, Thomas; Sack, Markus; Hundleby, Penelope A C; Stiegler, Gabriela; Stoger, Eva; Twyman, Richard M; Vcelar, Brigitta; Fischer, Rainer

    2015-10-01

    Although plant biotechnology has been widely investigated for the production of clinical-grade monoclonal antibodies, no antibody products derived from transgenic plants have yet been approved by pharmaceutical regulators for clinical testing. In the Pharma-Planta project, the HIV-neutralizing human monoclonal antibody 2G12 was expressed in transgenic tobacco (Nicotiana tabacum). The scientific, technical and regulatory demands of good manufacturing practice (GMP) were addressed by comprehensive molecular characterization of the transgene locus, confirmation of genetic and phenotypic stability over several generations of transgenic plants, and by establishing standard operating procedures for the creation of a master seed bank, plant cultivation, harvest, initial processing, downstream processing and purification. The project developed specifications for the plant-derived antibody (P2G12) as an active pharmaceutical ingredient (API) based on (i) the guidelines for the manufacture of monoclonal antibodies in cell culture systems; (ii) the draft European Medicines Agency Points to Consider document on quality requirements for APIs produced in transgenic plants; and (iii) de novo guidelines developed with European national regulators. From the resulting process, a GMP manufacturing authorization was issued by the competent authority in Germany for transgenic plant-derived monoclonal antibodies for use in a phase I clinical evaluation. Following preclinical evaluation and ethical approval, a clinical trial application was accepted by the UK national pharmaceutical regulator. A first-in-human, double-blind, placebo-controlled, randomized, dose-escalation phase I safety study of a single vaginal administration of P2G12 was carried out in healthy female subjects. The successful completion of the clinical trial marks a significant milestone in the commercial development of plant-derived pharmaceutical proteins. © 2015 Society for Experimental Biology, Association of

  17. Passive immunization against dental caries and periodontal disease: development of recombinant and human monoclonal antibodies.

    Science.gov (United States)

    Abiko, Y

    2000-01-01

    and periodontal diseases are summarized, and the biotechnological approaches for developing recombinant and human-type antibodies are introduced. Furthermore, our own attempts to construct single-chain variable fragments (ScFv) and human-type antibodies capable of neutralizing virulence factors are discussed.

  18. A mathematical model of a recombinant humanized anti-cocaine monoclonal antibody's effects on cocaine pharmacokinetics in mice.

    Science.gov (United States)

    Wetzel, Hanna N; Zhang, Tongli; Norman, Andrew B

    2017-09-01

    A recombinant humanized anti-cocaine monoclonal antibody (mAb), h2E2, is at an advanced stage of pre-clinical development as an immunotherapy for cocaine abuse. It is hypothesized that h2E2 binds to and sequesters cocaine in the blood. A three-compartment model of the effects of h2E2 on cocaine's distribution was constructed. The model assumes that h2E2 binds to cocaine and that the h2E2-cocaine complex does not enter the brain but distributes between the central and peripheral compartments. Free cocaine is eliminated from both the central and peripheral compartments, and h2E2 and the h2E2-cocaine complex are eliminated from the central compartment only. This model was tested against a new dataset measuring cocaine concentrations in the brain and plasma over 1h in the presence and absence of h2E2. The mAb significantly increased plasma cocaine concentrations with a concomitant significant decrease in brain concentration. Plasma concentrations declined over the 1-hour sampling period in both groups. With a set of parameters within reasonable physiological ranges, the three-compartment model was able to qualitatively and quantitatively simulate the increased plasma concentration in the presence of the antibody and the decreased peak brain concentration in the presence of antibody. Importantly, the model explained the decline in plasma concentrations over time as distribution of the cocaine-h2E2 complex into a peripheral compartment. This model will facilitate the targeting of ideal mAb PK/PD properties thus accelerating the identification of lead candidate anti-drug mAbs. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Development of a multi-product leached protein A assay for bioprocess samples containing recombinant human monoclonal antibodies.

    Science.gov (United States)

    Ren, Diya; Darlucio, Maria R; Chou, Judy H

    2011-03-07

    The detection of low level of protein A leached from monoclonal antibody downstream purification process is often interfered by the presence of excess amount of product antibody. In order to prevent this interference, we developed a new multi-product leached protein A assay that used acidification to completely dissociate the IgG-ProteinA complex, followed by neutralization under selected condition to prevent re-formation of the IgG-ProteinA complex. The amount of protein A was then determined by a sandwich immunoassay using Meso Scale Discovery technology. The assay takes approximately 3h to complete for one 96-well plate of samples, and this has been successfully applied to samples containing different monoclonal antibody products examined so far. The data demonstrates that this assay is robust and efficient in determining leached protein A contamination during purification of recombinant monoclonal antibodies. Copyright © 2010 Elsevier B.V. All rights reserved.

  20. A broadly neutralizing human monoclonal antibody is effective against H7N9.

    Science.gov (United States)

    Tharakaraman, Kannan; Subramanian, Vidya; Viswanathan, Karthik; Sloan, Susan; Yen, Hui-Ling; Barnard, Dale L; Leung, Y H Connie; Szretter, Kristy J; Koch, Tyree J; Delaney, James C; Babcock, Gregory J; Wogan, Gerald N; Sasisekharan, Ram; Shriver, Zachary

    2015-09-01

    Emerging strains of influenza represent a significant public health threat with potential pandemic consequences. Of particular concern are the recently emerged H7N9 strains which cause pneumonia with acute respiratory distress syndrome. Estimates are that nearly 80% of hospitalized patients with H7N9 have received intensive care unit support. VIS410, a human antibody, targets a unique conserved epitope on influenza A. We evaluated the efficacy of VIS410 for neutralization of group 2 influenza strains, including H3N2 and H7N9 strains in vitro and in vivo. VIS410, administered at 50 mg/kg, protected DBA mice infected with A/Anhui/2013 (H7N9), resulting in significant survival benefit upon single-dose (-24 h) or double-dose (-12 h, +48 h) administration (P cytokine responses for nine of the 11 cytokines measured. Based on these results, we find that VIS410 may be effective either as monotherapy or combined with antivirals in treating H7N9 disease, as well as disease from other influenza strains.

  1. Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain.

    Directory of Open Access Journals (Sweden)

    Sharad P Adekar

    2008-08-01

    Full Text Available Botulinum neurotoxins (BoNT are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC, a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored.We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A. The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro.An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.

  2. In Vivo Efficacy of a Cocktail of Human Monoclonal Antibodies (CL184 Against Diverse North American Bat Rabies Virus Variants

    Directory of Open Access Journals (Sweden)

    Richard Franka

    2017-09-01

    Full Text Available Following rabies virus (RABV exposure, a combination of thorough wound washing, multiple-dose vaccine administration and the local infiltration of rabies immune globulin (RIG are essential components of modern post-exposure prophylaxis (PEP. Although modern cell-culture-based rabies vaccines are increasingly used in many countries, RIG is much less available. The prohibitive cost of polyclonal serum RIG products has prompted a search for alternatives and design of anti-RABV monoclonal antibodies (MAbs that can be manufactured on a large scale with a consistent potency and lower production costs. Robust in vitro neutralization activity has been demonstrated for the CL184 MAb cocktail, a 1:1 protein mixture of two human anti-RABV MAbs (CR57/CR4098, against a large panel of RABV isolates. In this study, we used a hamster model to evaluate the efficacy of experimental PEP against a lethal challenge. Various doses of CL184 and commercial rabies vaccine were assessed for the ability to protect against lethal infection with representatives of four distinct bat RABV lineages of public health relevance: silver-haired bat (Ln RABV; western canyon bat (Ph RABV; big brown bat (Ef-w1 RABV and Mexican free-tailed bat RABV (Tb RABV. 42–100% of animals survived bat RABV infection when CL184 (in combination with the vaccine was administered. A dose-response relationship was observed with decreasing doses of CL184 resulting in increasing mortality. Importantly, CL184 was highly effective in neutralizing and clearing Ph RABV in vivo, even though CR4098 does not neutralize this virus in vitro. By comparison, 19–95% survivorship was observed if human RIG (20 IU/kg and vaccine were used following challenge with different bat viruses. Based on our results, CL184 represents an efficacious alternative for RIG. Both large-scale and lower cost production could ensure better availability and affordability of this critical life-saving biologic in rabies enzootic

  3. Characterization of ABT-806, a Humanized Tumor-Specific Anti-EGFR Monoclonal Antibody.

    Science.gov (United States)

    Reilly, Edward B; Phillips, Andrew C; Buchanan, Fritz G; Kingsbury, Gillian; Zhang, Yumin; Meulbroek, Jonathan A; Cole, Todd B; DeVries, Peter J; Falls, Hugh D; Beam, Christine; Gu, Jinming; Digiammarino, Enrico L; Palma, Joann P; Donawho, Cherrie K; Goodwin, Neal C; Scott, Andrew M

    2015-05-01

    Despite clinical efficacy, current approved agents targeting EGFR are associated with on-target toxicities as a consequence of disrupting normal EGFR function. MAb 806 is a novel EGFR antibody that selectively targets a tumor-selective epitope suggesting that a mAb 806-based therapeutic would retain antitumor activity without the on-target toxicities associated with EGFR inhibition. To enable clinical development, a humanized variant of mAb 806 designated ABT-806 was generated and is currently in phase 1 trials. We describe the characterization of binding and functional properties of ABT-806 compared with the clinically validated anti-EGFR antibody cetuximab. ABT-806 binds the mutant EGFRvIII with high affinity and, relative to cetuximab, exhibits increased potency against glioblastoma multiforme cell line and patient-derived xenografts expressing this form of the receptor. ABT-806 also inhibits the growth of squamous cell carcinoma xenograft models expressing high levels of wild-type EGFR, associated with inhibition of EGFR signaling, although higher doses of ABT-806 than cetuximab are required for similar activity. ABT-806 enhances in vivo potency of standard-of-care therapies used to treat glioblastoma multiforme and head and neck squamous cell carcinoma. An indium-labeled version of ABT-806, [(111)In]-ABT-806, used to investigate the relationship between dose and receptor occupancy, revealed greater receptor occupancy at lowers doses in an EGFRvIII-expressing model and significant uptake in an orthotopic model. Collectively, these results suggest that ABT-806 may have antitumor activity superior to cetuximab in EGFRvIII-expressing tumors, and similar activity to cetuximab in tumors highly overexpressing wild-type EGFR with reduced toxicity. ©2015 American Association for Cancer Research.

  4. Cross-reactive dengue human monoclonal antibody prevents severe pathologies and death from Zika virus infections

    Science.gov (United States)

    Kam, Yiu-Wing; Lee, Cheryl Yi-Pin; Teo, Teck-Hui; Howland, Shanshan W.; Amrun, Siti Naqiah; See, Peter; Kng, Nicholas Qing-Rong; Huber, Roland G.; Xu, Mei-Hui; Tan, Heng-Liang; Choo, Andre; Ginhoux, Florent; Fink, Katja; Wang, Cheng-I; Ng, Lisa F.P.

    2017-01-01

    Zika virus (ZIKV) infections have been linked with neurological complications and congenital Zika syndrome. Given the high level of homology between ZIKV and the related flavivirus dengue virus (DENV), we investigated the level of cross-reactivity with ZIKV using a panel of DENV human mAbs. A majority of the mAbs showed binding to ZIKV virions, with several exhibiting neutralizing capacities against ZIKV in vitro. Three of the best ZIKV-neutralizing mAbs were found to recognize diverse epitopes on the envelope (E) glycoprotein: the highly conserved fusion-loop peptide, a conformation-specific epitope on the E monomer, and a quaternary epitope on the virion surface. The most potent ZIKV-neutralizing mAb (SIgN-3C) was assessed in 2 type I interferon receptor–deficient (IFNAR–/–) mouse models of ZIKV infection. Treatment of adult nonpregnant mice with SIgN-3C rescued mice from virus-induced weight loss and mortality. The SIgN-3C variant with Leu-to-Ala mutations in the Fc region (SIgN-3C-LALA) did not induce antibody-dependent enhancement (ADE) in vitro but provided similar levels of protection in vivo. In pregnant ZIKV-infected IFNAR–/– mice, treatment with SIgN-3C or SIgN-3C-LALA significantly reduced viral load in the fetal organs and placenta and abrogated virus-induced fetal growth retardation. Therefore, SIgN-3C-LALA holds promise as a ZIKV prophylactic and therapeutic agent. PMID:28422757

  5. Cross-reactive dengue human monoclonal antibody prevents severe pathologies and death from Zika virus infections.

    Science.gov (United States)

    Kam, Yiu-Wing; Lee, Cheryl Yi-Pin; Teo, Teck-Hui; Howland, Shanshan W; Amrun, Siti Naqiah; Lum, Fok-Moon; See, Peter; Kng, Nicholas Qing-Rong; Huber, Roland G; Xu, Mei-Hui; Tan, Heng-Liang; Choo, Andre; Maurer-Stroh, Sebastian; Ginhoux, Florent; Fink, Katja; Wang, Cheng-I; Ng, Lisa F P; Rénia, Laurent

    2017-04-20

    Zika virus (ZIKV) infections have been linked with neurological complications and congenital Zika syndrome. Given the high level of homology between ZIKV and the related flavivirus dengue virus (DENV), we investigated the level of cross-reactivity with ZIKV using a panel of DENV human mAbs. A majority of the mAbs showed binding to ZIKV virions, with several exhibiting neutralizing capacities against ZIKV in vitro. Three of the best ZIKV-neutralizing mAbs were found to recognize diverse epitopes on the envelope (E) glycoprotein: the highly conserved fusion-loop peptide, a conformation-specific epitope on the E monomer, and a quaternary epitope on the virion surface. The most potent ZIKV-neutralizing mAb (SIgN-3C) was assessed in 2 type I interferon receptor-deficient (IFNAR-/-) mouse models of ZIKV infection. Treatment of adult nonpregnant mice with SIgN-3C rescued mice from virus-induced weight loss and mortality. The SIgN-3C variant with Leu-to-Ala mutations in the Fc region (SIgN-3C-LALA) did not induce antibody-dependent enhancement (ADE) in vitro but provided similar levels of protection in vivo. In pregnant ZIKV-infected IFNAR-/- mice, treatment with SIgN-3C or SIgN-3C-LALA significantly reduced viral load in the fetal organs and placenta and abrogated virus-induced fetal growth retardation. Therefore, SIgN-3C-LALA holds promise as a ZIKV prophylactic and therapeutic agent.

  6. Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia

    Directory of Open Access Journals (Sweden)

    Kosinski Marek

    2011-04-01

    Full Text Available Abstract Background The increasing availability of different monoclonal antibodies (mAbs opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells. Results The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to

  7. Characterization and expression of the human T cell receptor-T3 complex by monoclonal antibody F101.01

    DEFF Research Database (Denmark)

    Geisler, C; Plesner, T; Pallesen, G

    1988-01-01

    A murine monoclonal antibody (MoAb) F101.01 reacting with the T cell receptor (TCR)-T3 complex is presented. Immunohistological studies showed that F101.01 specifically stains T-zone lymphocytes in lymph nodes, tonsils, and splenic tissue. Two-colour immunofluorescence and flow cytometry demonstr...

  8. The relationship between human T-lymphocyte subsets defined by monoclonal antibodies and by avidity differences to sheep erythrocytes

    DEFF Research Database (Denmark)

    Hokland, P; Hokland, M; Heron, I

    1982-01-01

    differences to sheep erythrocytes. Through a correlation was demonstrated between the T4+ (inducer) cells and the high avidity ("active") T cells and between the T8+ (suppressor) cells and low avidity T cells, these subsets were far from identical, and it is concluded that the application of monoclonal...

  9. Biological Validation of Plant-derived Anti-human Colorectal Cancer Monoclonal Antibody CO17-1A

    NARCIS (Netherlands)

    Jamal, Arshad; Ahn, Mi-Hyun; Song, Mira; Oh, Eun-Yi; Hong, Juyeon; Choo, Young-Kug; Ko, Kinarm; Han, Yeon Soo; Oh, Seung Han; Van Der Linden, Joke; Leusen, Jeanette H. W.; Ko, Kisung

    We validated expression and biological activities of plant-derived monoclonal antibody (MAb(P)) CO17-1A for its efficacy in cancer immunotherapy. PCR and immunoblot analyses demonstrated insertion and expression of heavy and light chains of MAb CO17-1A in transgenic plants, respectively. Confocal

  10. Pharmacokinetics, biodistribution and dosimetry of {sup 99m}Tc-labeled anti-human epidermal growth factor receptor humanized monoclonal antibody R3 in rats

    Energy Technology Data Exchange (ETDEWEB)

    Escobar, Normando Iznaga; Morales, Alejo Morales; Duconge, Jorge; Torres, Idania Caballero; Fernandez, Eduardo; Gomez, Jose A

    1998-01-01

    The pharmacokinetics, biodistribution and dosimetry of {sup 99m}Tc-labeled anti-human epidermal growth factor receptor (anti-hEGF-r) humanized monoclonal antibody (MAb) R3 was investigated following intravenous injection in normal Wistar rats. Serum disappearance curves were best fit by a two-compartment model having a mean distribution half-life (t{sub (1(2{alpha}}{sub ))}) of 0.250 h and a mean elimination (t{sub (1(2{beta}}{sub ))}) of 13.89 h. Among the various organs, a little accumulation of the radiolabeled antibody was found only in kidneys. Biodistribution and dosimetry studies in humans were performed by extrapolation of the animal data to humans. Absorbed dose to normal organs and the remainder of the whole body were estimated using the medical internal radiation dose formula, and dose contributions from radioactivity in transit through the gastrointestinal tract were estimated using a compartment model. Extrapolated values of radiation absorbed dose to normal organs in rads per millicurie administered were whole body, 0.0085; lower large intestine wall, 0.0898; small intestine, 0.0530; upper large intestine wall, 0.0731; and kidneys, 0.0455. The effective dose equivalent predicted was 0.0162 rem/mCi and the effective dose was found to be 0.015 rem/mCi. On the basis of the pharmacokinetics, biodistribution and internal radiation dosimetry information obtained in this study, a diagnostic phase I clinical trial with {sup 99m}Tc-labeled humanized MAb R3 conjugate in patients should be supported.

  11. Nonclinical safety of mavrilimumab, an anti-GMCSF receptor alpha monoclonal antibody, in cynomolgus monkeys: Relevance for human safety

    Energy Technology Data Exchange (ETDEWEB)

    Ryan, Patricia C., E-mail: ryanp@medimmune.com [MedImmune, LLC, Gaithersburg, MD (United States); Sleeman, Matthew A. [MedImmune, LLC, Cambridge (United Kingdom); Rebelatto, Marlon [MedImmune, LLC, Gaithersburg, MD (United States); Wang, Bing; Lu, Hong [MedImmune, LLC, Moutain View, CA (United States); Chen, Xiaomin [Novartis, East Hanover, NJ (United States); Wu, Chi-Yuan [MedImmune, LLC, Moutain View, CA (United States); Hinrichs, Mary Jane; Roskos, Lorin [MedImmune, LLC, Gaithersburg, MD (United States); Towers, Heidi [MedImmune, LLC, Cambridge (United Kingdom); McKeever, Kathleen; Dixit, Rakesh [MedImmune, LLC, Gaithersburg, MD (United States)

    2014-09-01

    Mavrilimumab (CAM-3001) is an investigational human IgG4 monoclonal antibody (MAb) targeting GM-CSF receptor alpha which is currently being developed for the treatment of RA. GM-CSF plays a central role in the pathogenesis of rheumatoid arthritis (RA) through the activation, differentiation, and survival of macrophages and neutrophils. To support clinical development, the nonclinical safety of mavrilimumab was evaluated in several studies with cynomolgus monkeys as the pharmacologically relevant species. Comprehensive toxicity parameters were assessed in each study, and treatment duration ranged from 4 to 26 weeks. Mavrilimumab has an acceptable safety profile in monkeys with no changes in any parameters other than microscopic findings in lung. In several studies, minimal accumulation of foamy alveolar macrophages was observed. This finding was only seen in studies of at least 11 weeks duration, was reversible following a dose-free recovery period and was considered non-adverse. At higher dose levels (≥ 30 mg/kg/week), in a 26-week repeat-IV dose study, the presence of lung foreign material, cholesterol clefts, and granulomatous inflammation was also observed in a few animals and was considered adverse. The dose- and time-related accumulation of foamy macrophages in lung following exposure to mavrilimumab observed in several NHP studies was expected based upon the known role of GM-CSFRα signaling in the function of alveolar macrophages. Overall, a clean no-observed-adverse-effect-level (NOAEL) without any effects in lung was established and provided adequate clinical safety margins. In clinical studies in RA patients, mavrilimumab has demonstrated good clinical activity with adequate safety to support further clinical development. A Phase 2b study of mavrilimumab in subjects with RA is in progress. - Highlights: • Mavrilimumab is a MAB targeting GM-CSFRα being developed for RA therapy. • Mavrilimumab has an acceptable safety profile in cynomolgus monkeys.

  12. Comparison of an anti-rabies human monoclonal antibody combination with human polyclonal anti-rabies immune globulin

    NARCIS (Netherlands)

    Goudsmit, Jaap; Marissen, Wilfred E.; Weldon, William C.; Niezgoda, Michael; Hanlon, Cathleen A.; Rice, Amy B.; Kruif, John de; Dietzschold, Bernhard; Bakker, Alexander B. H.; Rupprecht, Charles E.

    2006-01-01

    The World Health Organization estimates human mortality from endemic canine rabies to be 55,000 deaths/year. Limited supply hampers the accessibility of appropriate lifesaving treatment, particularly in areas where rabies is endemic. Anti-rabies antibodies are key to protection against lethal

  13. Modified cytokeratins expressed on the surface of carcinoma cells undergo endocytosis upon binding of human monoclonal antibody and its recombinant Fab fragment

    DEFF Research Database (Denmark)

    Ditzel, H J; Garrigues, U; Andersen, C B

    1997-01-01

    display selection and the human Fab fragment was expressed in bacteria. Analysis by confocal laser scanning microscopy demonstrated that COU-1 bound in a uniform punctate pattern to the surface of viable carcinoma cells stained at 4 degrees C, and binding increased significantly when cells were cultured...... was significantly reduced. Similar results were obtained using intact IgM COU-1 and the recombinant Fab fragment. Immunohistological studies indicated that COU-1, in contrast to murine monoclonal antibodies against normal cytokeratin 8 and 18, could differentiate between malignant and normal colon epithelia...... represent a new target for immunoconjugates and may explain the promising results of the phase I/II clinical study....

  14. An IgE epitope of Bet v 1 and fagales PR10 proteins as defined by a human monoclonal IgE

    DEFF Research Database (Denmark)

    Hecker, J.; Diethers, A.; Schulz, D.

    2012-01-01

    BACKGROUND: Analyses of the molecular basis underlying allergenicity and allergen cross-reactivity, as well as improvement of allergy diagnostics and therapeutics, are hampered by the lack of human monoclonal IgE antibodies and knowledge about their epitopes. Here, we addressed the consecutive...... formats including IgE. Using variants of Bet v 1, the epitope of the antibody was mapped and extrapolated to other PR10 proteins. RESULTS: The obtained antibodies exhibited pronounced reactivity with Bet v 1, but were not reactive with the homologous PR10 protein Mal d 1. The epitope as defined by the Ig...

  15. Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies: peripheral glycosylation of HIV envelope glycoprotein gp120 may be a target for virus neutralization

    DEFF Research Database (Denmark)

    Hansen, J E; Clausen, H; Nielsen, C

    1990-01-01

    Carbohydrate structures are often involved in the initial adhesion of pathogens to target cells. In the present study, a panel of anticarbohydrate monoclonal antibodies (MAbs) was tested for their ability to inhibit in vitro human immunodeficiency virus infectivity. MAbs against three different N......- and O-linked carbohydrate epitopes (LeY, A1, and sialyl-Tn) were able to block infection by cell-free virus as well as inhibit syncytium formation. Inhibition of virus infectivity was independent of virus strain (HTLVIIIB or patient isolate SSI-002), the cell line used for virus propagation (H9 or MT4...

  16. Phage display derived human monoclonal antibodies isolated by binding to the surface of live primary breast cancer cells recognize GRP78

    DEFF Research Database (Denmark)

    Jakobsen, Charlotte G; Rasmussen, Nicolaj; Laenkholm, Anne-Vibeke

    2007-01-01

    of novel targets and human antibodies against them. We have isolated single-chain human mAbs from a large naïve antibody phage display library by panning on a single-cell suspension of freshly isolated live cancer cells from a human breast cancer specimen, and these antibodies were shown to specifically......Clinical trials using monoclonal antibodies (mAb) against cell-surface markers have yielded encouraging therapeutic results in several cancer types. Generally, however, anticancer antibodies are only efficient against a subpopulation of cancers, and there is a strong need for identification...... bound strongly to several cancers, including 45% breast carcinomas, 35% lung cancers, and 86% melanomas, but showed no or weak binding to normal tissues. A yeast two-hybrid screen of a large human testis cDNA library identified the glucose-regulated protein of 78 kDa (GRP78) as the antigen recognized...

  17. Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved protein.

    Science.gov (United States)

    Baerga-Ortiz, Abel; Hughes, Carrie A; Mandell, Jeffrey G; Komives, Elizabeth A

    2002-06-01

    The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen.

  18. Monoclonal antibody Po66 uptake by human lung tumours implanted in nude mice: effect of co-administration with doxorubicin.

    OpenAIRE

    Desrues, B.; L?na, H.; Brichory, F.; Ram?e, M. P.; Toujas, L; Delaval, P.; Dazord, L

    1995-01-01

    The efficacy of radioimmunotherapy of tumours with radiolabelled monoclonal antibodies (MAbs) depends on the amount of antibody taken up by the tumour and on its intratumoral distribution. In the case of MAbs directed against intracellular antigens, increasing the permeability of the cytoplasmic membrane may augment the bioavailability of the antigen for the antibody. This raises the question whether the induction of tumour necrosis by chemotherapy can enhance the tumour uptake of radiolabell...

  19. Development and characterization of a pre-treatment procedure to eliminate human monoclonal antibody therapeutic drug and matrix interference in cell-based functional neutralizing antibody assays.

    Science.gov (United States)

    Xu, Weifeng; Jiang, Hao; Titsch, Craig; Haulenbeek, Jonathan R; Pillutla, Renuka C; Aubry, Anne-Françoise; DeSilva, Binodh S; Arnold, Mark E; Zeng, Jianing; Dodge, Robert W

    2015-01-01

    Biological therapeutics can induce an undesirable immune response resulting in the formation of anti-drug antibodies (ADA), including neutralizing antibodies (NAbs). Functional (usually cell-based) NAb assays are preferred to determine NAb presence in patient serum, but are often subject to interferences from numerous serum factors, such as growth factors and disease-related cytokines. Many functional cell-based NAb assays are essentially drug concentration assays that imply the presence of NAbs by the detection of small changes in functional drug concentration. Any drug contained in the test sample will increase the total amount of drug in the assay, thus reducing the sensitivity of NAb detection. Biotin-drug Extraction with Acid Dissociation (BEAD) has been successfully applied to extract ADA, thereby removing drug and other interfering factors from human serum samples. However, to date there has been no report to estimate the residual drug level after BEAD treatment when the drug itself is a human monoclonal antibody; mainly due to the limitation of traditional ligand-binding assays. Here we describe a universal BEAD optimization procedure for human monoclonal antibody (mAb) drugs by using a LC-MS/MS method to simultaneously measure drug (a mutant human IgG4), NAb positive control (a mouse IgG), and endogenous human IgGs as an indicator of nonspecific carry-over in the BEAD eluate. This is the first report demonstrating that residual human mAb drug level in clinical sample can be measured after BEAD pre-treatment, which is critical for further BEAD procedure optimization and downstream immunogenicity testing. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Clearance kinetics and external dosimetry of 131I-labeled murine and humanized monoclonal antibody A33 in patients with colon cancer: radiation safety implications.

    Science.gov (United States)

    Dauer, Lawrence T; Boylan, Daniel C; Williamson, Matthew J; St Germain, Jean; Larson, Steven M

    2009-05-01

    The monoclonal antibody (mAb) A33 detects a membrane antigen that is expressed on greater than 95% of metastatic human colorectal cancers. Previous studies have shown excellent tumor-targeting of (131)I-labeled murine and humanized forms of the mAb. A retrospective analysis of whole-body clearance in the murine form was performed for comparison to the humanized form. Serial whole-body dose rate measurements were obtained for 55 treatments on 30 patients participating in phase I/II dose escalation studies of therapeutic (131)I-murine A33 mAb. Whole-body retention fractions over time were derived. Each treatment was fit with exponential curves to determine the effective half-lives and corresponding clearance fractions. There was a large variability in the calculated mono-exponential clearance effective half-life time, with a mean value of 36.5 h +/- 8.5 h. A bi-exponential fit of all combined data shows that 60% of the administered dose rapidly clears with a biological half-time of 23.9 h and 40% clears with a slower biological half-time of 101.2 h. The whole-body clearance proved to be more rapid in the murine form when compared with recent studies on the humanized form of radiolabeled A33 mAb. The variability in whole-body clearance reinforces the need for patient-specific tracer dosimetry for clinical care and radiation safety precautions. In addition, the slower clearance of the humanized form of the A33 mAb requires longer term radiation safety precautions than the earlier murine form. As other monoclonal antibodies progress from murine to humanized forms, radiopharmacokinetics should be evaluated for clinical and radiation safety implications.

  1. IMC-A12, a human IgG1 monoclonal antibody to the insulin-like growth factor I receptor.

    Science.gov (United States)

    Rowinsky, Eric K; Youssoufian, Hagop; Tonra, James R; Solomon, Phillip; Burtrum, Douglas; Ludwig, Dale L

    2007-09-15

    Targeted monoclonal antibody therapy is an important strategy in cancer therapeutics. Among the most promising characteristics of therapeutic targets are those that modulate the growth and survival of malignant neoplasms and their sensitivity to anticancer therapies. The insulin-like growth factor-I receptor (IGF-IR) is overexpressed in many types of solid and hematopoietic malignancies, and has been implicated as a principal cause of heightened proliferative and survival signaling. IGF-IR has also been shown to confer resistance to cytotoxic, hormonal, and targeted therapies, suggesting that therapeutics targeting IGF-IR may be effective against a broad range of malignancies. IMC-A12 (ImClone Systems Incorporated), a fully human monoclonal IgG1 antibody that binds with high affinity to the IGF-IR, inhibits ligand-dependent receptor activation and downstream signaling. IMC-A12 also mediates robust internalization and degradation of the IGF-IR. In human tumor xenograft models, IGF-IR blockade by IMC-A12 results in rapid and profound growth inhibition of cancers of the breast, lung, colon, and pancreas, and many other neoplasms. Although promising single-agent activity has been observed, the most impressive effects of targeting the IGF-IR with IMC-A12 have been noted when this agent was combined with cytotoxic agents or other targeted therapeutics. The results with IMC-A12 to date suggest that it may be an effective therapeutic in a diverse array of oncologic indications.

  2. Phage Display Approaches for the Isolation of Monoclonal Antibodies Against Dengue Virus Envelope Domain III from Human and Mouse Derived Libraries

    Directory of Open Access Journals (Sweden)

    Subhash G. Vasudevan

    2012-02-01

    Full Text Available Domain III of the dengue virus envelope protein (EDIII, aa295-395 has an immunoglobulin fold and is the proposed receptor-binding domain of the virus. Previous studies have shown that monoclonal antibodies against EDIII can be neutralizing and have therapeutic potential. Here, cloned Fab-phage libraries of human and mouse origin were screened for DENV specific antibodies. Firstly, bacterially expressed EDIII or whole virus particles were used as bait in biopanning against a large naïve human Fab-phage library ( > 10 billion independent clones. Multiple panning strategies were employed, and in excess of 1000 clones were screened, but all of the antibodies identified bound the envelope in regions outside EDIII suggesting EDIII antibodies are virtually absent from the naïve human repertoire. Next, a chimeric Fab-phage library was constructed from a panel of EDIII specific mouse hybridomas by pooling the VH and VL chain sequences from the hybridomas and cloning these into the pComb3X phagemid vector with human CH and CL encoding sequences. Biopanning against EDIII identified a unique antibody (C9 that cross-reacts with EDIII from DENV1-3 and, in the IgG format, binds and neutralizes DENV2 in cell-based assays. Sequence analysis and saturation mutagenesis of complementary determining regions (CDR in the C9 light chain suggest an antigen recognition model in which the LCDR3 is a key determinant of EDIII specificity, while modifications in LCDR1 and LCDR2 affect DENV serotype cross-reactivity. Overall, this study supports the current prevailing opinion that neutralizing anti-EDIII monoclonal antibodies can be readily generated in murine systems, but in humans the anti-DENV immune response is directed away from domain III.

  3. A novel monoclonal antibody of human stem cell factor inhibits umbilical cord blood stem cell ex vivo expansion

    Directory of Open Access Journals (Sweden)

    Fan Jie

    2012-12-01

    Full Text Available Abstract Stem cell factor (SCF activates hematopoietic stem cell (HSC self-renewal and is being used to stimulate the ex vivo expansion of HSCs. The mechanism by which SCF supports expansion of HSCs remains poorly understood. In cord blood ex vivo expansion assays, a newly produced anti-SCF monoclonal antibody (clone 23C8 was found to significantly inhibit the expansion of CD34+ cells. This antibody appears to bind directly to a part of SCF that is critical for biological activity toward expansion of CD34+ cells, which is located in the first 104 amino acids from the NH2-terminus.

  4. Characterization of Entamoeba histolytica Intermediate Subunit Lectin-Specific Human Monoclonal Antibodies Generated in Transgenic Mice Expressing Human Immunoglobulin Loci ▿

    Science.gov (United States)

    Tachibana, Hiroshi; Cheng, Xun-Jia; Tsukamoto, Hideo; Itoh, Johbu

    2009-01-01

    Four fully human monoclonal antibodies (MAbs) to Entamoeba histolytica intermediate subunit lectin (Igl) were prepared in XenoMouse mice, which are transgenic mice expressing human immunoglobulin loci. Examination of the reactivities of these MAbs to recombinant Igl1 and Igl2 of E. histolytica showed that XEhI-20 {immunoglobulin G2(κ) [IgG2(κ)]} and XEhI-28 [IgG2(κ)] were specific to Igl1, XEhI-B5 [IgG2(κ)] was specific to Igl2, and XEhI-H2 [IgM(κ)] was reactive with both Igls. Gene analyses revealed that the VH and VL germ lines were VH3-48 and L2 for XEhI-20, VH3-21 and L2 for XEhI-28, VH3-33 and B3 for XEhI-B5, and VH4-4 and A19 for XEhI-H2, respectively. Flow cytometry analyses showed that the epitopes recognized by all of these MAbs were located on the surfaces of living trophozoites. Confocal microscopy demonstrated that most Igl1 and Igl2 proteins were colocalized on the surface and in the cytoplasm, but different localization patterns in intracellular vacuoles were also present. The preincubation of trophozoites with XEhI-20, XEhI-B5, and XEhI-H2 caused significant inhibition of the adherence of trophozoites to Chinese hamster ovary cells, whereas preincubation with XEhI-28 did not do so. XEhI-20, XEhI-B5, and XEhI-H2 were injected intraperitoneally into hamsters 24 h prior to intrahepatic challenge with E. histolytica trophozoites. One week later, the mean abscess size in groups injected with one of the three MAbs was significantly smaller than that in controls injected with polyclonal IgG or IgM isolated from healthy humans. These results demonstrate that human MAbs to Igls may be applicable for immunoprophylaxis of amebiasis. PMID:19001071

  5. Biological function analysis of monoclonal antibodies against human granulins in vitro using U251 cells as a model.

    Science.gov (United States)

    Li, Yanqing; Li, Ya; Ye, Mingfu; Wang, Dongyang; Zhao, Junli; Sun, Xiaohong; Mao, Qinwen; Xia, Haibin

    2017-02-01

    Progranulin (PGRN), a highly glycosylated, secreted 593 amino acid precursor protein, is a multifunctional molecule that is critical for early embryogenesis, wound repair, inflammatory and tumorigenesis. PGRN can be proteolytically cleaved into seven cysteine-rich granulin (Grn) peptides: G, F, B, A, C, D and E. Both PGRN and its constituent Grn peptides have been implicated in a wide variety of biological activities. However, their functions are far from clear, and the lack of granulin domain-specific antibodies has hindered the progress of the functional study of PGRN and Grns. Monoclonal antibodies against GrnB, GrnA, GrnC and GrnF have been previously developed by our laboratory. In this study, we generated monoclonal antibodies (MAbs) against GrnD, GrnG and GrnE by using recombinant proteins HSA-GrnG, HSA-GrnD and HSA-GrnE as immunogens, and characterized them by indirect ELISA, Western blot and immunocytochemistry. Furthermore, the neutralizing activities of the MAbs against seven Grns were tested in vitro using the U251 cell line. This full antibody panel of MAbs against seven Grns will be a valuable tool for elucidating the biological roles of PGRN and Grns in different physiopathological processes, which will further promote the development of PGRN-based clinical diagnosis and therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Itolizumab – a humanized anti-CD6 monoclonal antibody with better side effects profile for the treatment of psoriasis [Corrigendum

    Directory of Open Access Journals (Sweden)

    Menon R

    2015-06-01

    Full Text Available Menon R, David BG. Clinical, Cosmetic and Investigational Dermatology. 2015;8:215–22.On page 215, please note correspondence should have been listed as:   Roshni Menon, D II/17,JIPMER Campus, Dhanvanthri Nagar,Pondicherry, India 605006Tel +91 944 320 8140Email roshnijagdish@gmail.com.On page 215, the first sentence of the Introduction was “Psoriasis is a chronic inflammatory disease of the skin characterized by exacerbations and remissions affecting 1%–3% of the world’s population, and approximately 20% of patients have moderate to severe disease.1,2” however should have been “Psoriasis is a chronic inflammatory disease of the skin characterized by exacerbations and remissions affecting 1%–3% of the world’s population. Approximately 20% of patients have moderate to severe disease.1,2”On page 217, 219, and 221 the running header was “Itolizumab – aCD6 monoclonal antibody for the treatment of psoriasis” however should have been “Itolizumab – a humanized anti CD6 monoclonal antibody for the treatment of psoriasis”.On page 218, Table 1, the second column heading was listed as “Anand et al25 n=40 (moderate–severe psoriasis” however should have been “Anand et al25 n=40/32 weeks (moderate–severe psoriasis”.Read the original article 

  7. Fully human broadly neutralizing monoclonal antibodies against influenza A viruses generated from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Weibin [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Chen, Aizhong [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Miao, Yi [Shanghai Xuhui Central Hospital, Shanghai 200031 (China); Xia, Shengli [Center for Disease Control and Prevention of Henan Province, Zhengzhou 450016 (China); Ling, Zhiyang; Xu, Ke; Wang, Tongyan [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Xu, Ying; Cui, Jun; Wu, Hongqiang; Hu, Guiyu; Tian, Lin; Wang, Lingling [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Shu, Yuelong [Chinese Center for Disease Control and Prevention, Beijing 102206 (China); Ma, Xiaowei [Hualan Biological Bacterin Company, Xinxiang 453003 (China); Xu, Bianli; Zhang, Jin [Center for Disease Control and Prevention of Henan Province, Zhengzhou 450016 (China); Lin, Xiaojun, E-mail: linxiaojun@hualan.com [Hualan Biological Bacterin Company, Xinxiang 453003 (China); Bian, Chao, E-mail: cbian@sibs.ac.cn [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Sun, Bing, E-mail: bsun@sibs.ac.cn [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)

    2013-01-20

    Whether the 2009 pandemic H1N1 influenza vaccine can induce heterosubtypic cross-protective anti-hemagglutinin (HA) neutralizing antibodies is an important issue. We obtained a panel of fully human monoclonal antibodies from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient. Most of the monoclonal antibodies targeted the HA protein but not the HA1 fragment. Among the analyzed antibodies, seven mAbs exhibited neutralizing activity against several influenza A viruses of different subtypes. The conserved linear epitope targeted by the neutralizing mAbs (FIEGGWTGMVDGWYGYHH) is part of the fusion peptide on HA2. Our work suggests that a heterosubtypic neutralizing antibody response primarily targeting the HA stem region exists in recipients of the 2009 pandemic H1N1 influenza vaccine. The HA stem region contains various conserved neutralizing epitopes with the fusion peptide as an important one. This work may aid in the design of a universal influenza A virus vaccine.

  8. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    Energy Technology Data Exchange (ETDEWEB)

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Luque, Daniel; Terrón, María C. [Unidad de Microscopía Electrónica y Confocal, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Calder, Lesley J. [National Institute for Medical Research, MRC, Mill Hill, London NW7 1AA (United Kingdom); Melero, José A., E-mail: jmelero@isciii.es [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain)

    2014-07-15

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.

  9. Two new monoclonal antibodies to human monocytes and granulocytes: isolation of membrane antigens and lack of effects of antibodies on leukocyte functions in vitro.

    Science.gov (United States)

    Stevenson, H C; Kimball, E; Schroff, R W; Buescher, S; Clarke, G; Gregorio, T; Wilburn, S; Foon, K A

    1984-01-01

    Mice were immunized with purified human monocytes or granulocytes obtained by leukapheresis and isolated on dextran gradients or by countercurrent centrifugation-elutriation. A monoclonal antibody, Mo95, was generated in response to monocytes and was found to react strongly with monocytes, large granular lymphocytes (LGL), granulocytes, eosinophils, and some myelomonocytic leukemia cells, but not with normal T or B lymphocytes, platelets, red cells, or leukemic cell lines. Mo95 is an IgG1 antibody, which precipitated a 95 kD molecular weight antigen. Addition of the Mo95 antibody to monocytes in the absence of complement did not inhibit lysozyme secretion nor did it affect superoxide production, C3b-rosetting, nitrotetrazolium blue reduction, phagocytosis, or chemotactic responses. A second antibody, PMN70, was found to react exclusively with granulocytes and not with monocytes, lymphocytes, LGL, platelets, red cells, or any of the myelomonocytic, T-cell-derived or B-cell-derived leukemic cell lines tested. The PMN70 antibody immunoprecipitated a 70 kD molecular weight antigen found only on mature granulocytes. Mo95 and PMN70 appear to be distinct from five other tested monoclonal antibodies reactive to monocytes and/or granulocytes on the basis of the fluorescent cell sorter and immunoprecipitation studies performed.

  10. Wheat germ cell-free system-based production of hemagglutinin-neuraminidase protein of human parainfluenza virus type 3: generation and characterization of monoclonal antibody

    Directory of Open Access Journals (Sweden)

    Satoko eMatsunaga

    2014-05-01

    Full Text Available Human parainfluenza virus 3 (HPIV3 commonly causes respiratory disorders in infants and young children. Monoclonal antibodies (MAbs have been produced to several components of HPIV3 and commercially available. However, the utility of these antibodies for several immunological and proteomic assays for understanding the nature of HPIV3 infection remain to be characterized. Herein, we report the development and characterization of monoclonal antibodies against hemagglutinin-neuraminidase (HN of HPIV3. A recombinant full-length HPIV3-HN was successfully synthesized using the wheat-germ cell-free protein production system. After immunization and cell fusion, 36 mouse hybridomas producing MAbs to HPIV3-HN were established. The MAbs obtained were fully characterized using ELISA, immunoblotting and immunofluorescent analyses. Of the MAbs tested, single clone was found to be applicable in both flow cytometry and immunoprecipitation procedures. By utilizing the antibody, we newly identified HPIV3-HN binding host proteins via immunoprecipitation-based mass spectrometry analysis. This study provides the availability of our newly-developed MAbs as a valuable tool for the study of HPIV3 infection as well as the several diagnostic tests of this virus.

  11. Radiobromination of humanized anti-HER2 monoclonal antibody trastuzumab using N-succinimidyl 5-bromo-3-pyridinecarboxylate, a potential label for immunoPET

    Energy Technology Data Exchange (ETDEWEB)

    Mume, Eskender [Organic Chemistry, Department of Chemistry, Uppsala University, S-751 24 Uppsala (Sweden); Orlova, Anna [Affibody AB, S-161 02 Bromma (Sweden); Malmstroem, Per-Uno [Division of Urology, Department of Surgical Sciences, Uppsala University, S-751 85 Uppsala (Sweden); Lundqvist, Hans [Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, S-751 85 Uppsala (Sweden); Sjoeberg, Stefan [Organic Chemistry, Department of Chemistry, Uppsala University, S-751 24 Uppsala (Sweden); Tolmachev, Vladimir [Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, S-751 85 Uppsala (Sweden)]. E-mail: vladimir.tolmachev@bms.uu.se

    2005-08-01

    Combining the specificity of radioimmunoscintigraphy and the high sensitivity of PET in an in vivo detection technique could improve the quality of nuclear diagnostics. Positron-emitting nuclide {sup 76}Br (T {sub 1/2}=16.2 h) might be a possible candidate for labeling monoclonal antibodies (mAbs) and their fragments, provided that the appropriate labeling chemistry has been established. For internalizing antibodies, such as the humanized anti-HER2 monoclonal antibody, trastuzumab, radiobromine label should be residualizing, i.e., ensuring that radiocatabolites are trapped intracellularly after the proteolytic degradation of antibody. This study evaluated the chemistry of indirect radiobromination of trastuzumab using N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate. Literature data indicated that the use of this method provided residualizing properties for iodine and astatine labels on some antibodies. An optimized 'one-pot' procedure produced an overall labeling efficiency of 45.5{+-}1.2% over 15 min. The bromine label was stable under physiological and denaturing conditions. The labeled trastuzumab retained its capacity to bind specifically to HER2-expressing SKOV-3 ovarian carcinoma cells in vitro (immunoreactivity more than 75%). However, in vitro cell test did not demonstrate that the radiobromination of trastuzumab using N-succinimidyl 5-bromo-3-pyridinecarboxylate improves cellular retention of radioactivity in comparison with the use of N-succinimidyl 4-bromobenzoate.

  12. Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Yang [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Sasaki, Tadahiro [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Kubota-Koketsu, Ritsuko [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Inoue, Yuji [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Yasugi, Mayo [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Yamashita, Akifumi; Ramadhany, Ririn; Arai, Yasuha [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Du, Anariwa [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Boonsathorn, Naphatsawan [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Ibrahim, Madiha S. [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Damanhour University, Damanhour (Egypt); and others

    2014-07-18

    Highlights: • Influenza infection can elicit heterosubtypic antibodies to group 1 influenza virus. • Three human monoclonal antibodies were generated from an H1N1-infected patient. • The antibodies predominantly recognized α-helical stem of viral hemagglutinin (HA). • The antibodies inhibited HA structural activation during the fusion process. • The antibodies are potential candidates for future antibody therapy to influenza. - Abstract: Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.

  13. Human monoclonal antibodies against glucagon receptor improve glucose homeostasis by suppression of hepatic glucose output in diet-induced obese mice.

    Directory of Open Access Journals (Sweden)

    Wook-Dong Kim

    Full Text Available AIM: Glucagon is an essential regulator of hepatic glucose production (HGP, which provides an alternative therapeutic target for managing type 2 diabetes with glucagon antagonists. We studied the effect of a novel human monoclonal antibody against glucagon receptor (GCGR, NPB112, on glucose homeostasis in diet-induced obese (DIO mice. METHODS: The glucose-lowering efficacy and safety of NPB112 were investigated in DIO mice with human GCGR for 11 weeks, and a hyperinsulinemic-euglycemic clamp study was conducted to measure HGP. RESULTS: Single intraperitoneal injection of NPB112 with 5 mg/kg effectively decreased blood glucose levels in DIO mice for 5 days. A significant reduction in blood glucose was observed in DIO mice treated with NPB112 at a dose ≥5 mg/kg for 6 weeks, and its glucose-lowering effect was dose-dependent. Long-term administration of NPB112 also caused a mild 29% elevation in glucagon level, which was returned to the normal range after discontinuation of treatment. The clamp study showed that DIO mice injected with NPB112 at 5 mg/kg were more insulin sensitive than control mice, indicating amelioration of insulin resistance by treatment with NPB112. DIO mice treated with NPB112 showed a significant improvement in the ability of insulin to suppress HGP, showing a 33% suppression (from 8.3 mg/kg/min to 5.6 mg/kg/min compared to the 2% suppression (from 9.8 mg/kg/min to 9.6 mg/kg/min in control mice. In addition, no hypoglycemia or adverse effect was observed during the treatment. CONCLUSIONS: A novel human monoclonal GCGR antibody, NPB112, effectively lowered the glucose level in diabetic animal models with mild and reversible hyperglucagonemia. Suppression of excess HGP with NPB112 may be a promising therapeutic modality for the treatment of type 2 diabetes.

  14. Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies: peripheral glycosylation of HIV envelope glycoprotein gp120 may be a target for virus neutralization

    DEFF Research Database (Denmark)

    Hansen, J E; Clausen, H; Nielsen, C

    1990-01-01

    Carbohydrate structures are often involved in the initial adhesion of pathogens to target cells. In the present study, a panel of anticarbohydrate monoclonal antibodies (MAbs) was tested for their ability to inhibit in vitro human immunodeficiency virus infectivity. MAbs against three different N......- and O-linked carbohydrate epitopes (LeY, A1, and sialyl-Tn) were able to block infection by cell-free virus as well as inhibit syncytium formation. Inhibition of virus infectivity was independent of virus strain (HTLVIIIB or patient isolate SSI-002), the cell line used for virus propagation (H9 or MT4......), and the cell type used as the infection target (MT4, PMC, or selected T4 lymphocytes). Inhibition was observed when viruses were preincubated with MAbs but not when cells were preincubated with MAbs before inoculation, and the MAbs were shown to precipitate 125I-labeled gp120. The MAbs therefore define...

  15. Non-reducing terminal fucose within N-linked glycan plays a significant role in the recognition of human milk lactoferrin by the 1CF11 monoclonal antibody.

    Science.gov (United States)

    Fukaya, Shinichi; Yabe, Tomio; Kanamaru, Yoshihiro

    2010-01-01

    We have recently demonstrated that the 1CF11 monoclonal antibody bound human milk lactoferrin (hLf) through the recognition of two distinct portions of the molecule, namely the N-glycan-relevant and -irrelevant structural elements. In this present study, we prepared four immunoreactive peptide fractions containing N-linked glycan from tryptic digests of reduced and alkylated hLf by using a concanavalin A lectin column and reverse-phase HPLC. Deglycosylation of these fractions and a competitive binding assay using fucosylated oligosaccharides revealed that the non-reducing terminal fucose residue in N-linked glycan(s) played a significant role in recognizing the N-glycan-relevant element in hLf by 1CF11.

  16. A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses

    Energy Technology Data Exchange (ETDEWEB)

    Boonsathorn, Naphatsawan; Panthong, Sumolrat [Medical Life Sciences Institute, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Koksunan, Sarawut [Medical Life Sciences Institute, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Chittaganpitch, Malinee; Phuygun, Siripaporn; Waicharoen, Sunthareeya [National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Prachasupap, Apichai [Medical Life Sciences Institute, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Sasaki, Tadahiro [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Kubota-Koketsu, Ritsuko [Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa (Japan); Yasugi, Mayo [Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka (Japan); Ono, Ken-ichiro [Ina Laboratory, Medical and Biological Laboratories Corporation, Ltd., Ina, Nagano (Japan); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Arai, Yasuha [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); and others

    2014-09-26

    Highlights: • A human monoclonal antibody against influenza virus was produced from a volunteer. • The antibody was generated from the PBMCs of the volunteer using the fusion method. • The antibody neutralized heterosubtypically group 1 influenza A viruses (H1 and H9). • The antibody targeted a novel epitope in globular head region of the hemagglutinin. • Sequences of the identified epitope are highly conserved among H1 and H9 subtypes. - Abstract: Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.

  17. Effects of treatment with a fully human anti-tumour necrosis factor alpha monoclonal antibody on the local and systemic homeostasis of interleukin 1 and TNFalpha in patients with rheumatoid arthritis.

    NARCIS (Netherlands)

    Barrera Rico, P.; Joosten, L.A.B.; Broeder, A. den; Putte, L.B.A. van de; Riel, P.L.C.M. van; Berg, W.B. van den

    2001-01-01

    OBJECTIVES: To study the short term effects of a single dose of D2E7, a fully human anti-tumour necrosis factor (TNFalpha) monoclonal antibody (mAb), on the local and systemic homeostasis of interleukin 1beta (IL1beta) and TNFalpha in patients with rheumatoid arthritis (RA). METHODS: All patients

  18. Comparison of the monoclonal antibodies 17-1A and 323/A3 : The influence of the affinity on tumour uptake and efficacy of radioimmunotherapy in human ovarian cancer xenografts

    NARCIS (Netherlands)

    Kievit, E; Pinedo, HM; Schluper, HMM; Haisma, HJ; Boven, E

    The low-affinity monoclonal antibody (MAb) chimeric 17-1A(c-17-1A) and the high-affinity MAb mouse 323/A3 (m-323/A3) were used to study the effect of the MAb affinity on the tumour uptake and efficacy of radioimmunotherapy in nude mice bearing subcutaneously the human ovarian cancer xenografts FMa,

  19. Pharmacokinetics and pharmacokinetic/pharmacodynamic associations of ofatumumab, a human monoclonal CD20 antibody, in patients with relapsed or refractory chronic lymphocytic leukaemia: a phase 1-2 study

    DEFF Research Database (Denmark)

    Coiffier, Bertrand; Losic, Nedjad; Rønn, Birgitte Biilmann

    2010-01-01

    The purpose of this phase 1-2 study was to investigate the association between the pharmacokinetic properties of ofatumumab, a human monoclonal CD20 antibody, and outcomes in 33 patients with relapsed/refractory chronic lymphocytic leukaemia receiving 4 weekly infusions of ofatumumab. The ofatumu...

  20. Safety and efficacy of ofatumumab, a fully human monoclonal anti-CD20 antibody, in patients with relapsed or refractory B-cell chronic lymphocytic leukemia: a phase 1-2 study

    DEFF Research Database (Denmark)

    Coiffier, B.; Lepretre, S.; Pedersen, L.M.

    2008-01-01

    Safety and efficacy of the fully human anti-CD20 monoclonal antibody, ofatumumab, was analyzed in a multicenter dose-escalating study including 33 patients with relapsed or refractory chronic lymphocytic leukemia. Three cohorts of 3 (A), 3 (B), and 27 (C) patients received 4, once weekly, infusio...

  1. Increased half-life and enhanced potency of Fc-modified human PCSK9 monoclonal antibodies in primates.

    Directory of Open Access Journals (Sweden)

    Yijun Shen

    Full Text Available Blocking proprotein convertase subtilisin kexin type 9 (PCSK9 binding to low-density lipoprotein receptor (LDLR can profoundly lower plasma LDL levels. Two anti-PCKS9 monoclonal antibodies (mAbs, alirocumab and evolocumab, were approved by the FDA in 2015. The recommended dose is 75 mg to 150 mg every two weeks for alirocumab and 140mg every two weeks or 420 mg once a month for evolocumab. This study attempted to improve the pharmacokinetic properties of F0016A, an IgG1 anti-PCKS9 mAb, to generate biologically superior molecules. We engineered several variants with two or three amino acid substitutions in the Fc fragment based on prior knowledge. The Fc-modified mAbs exhibited increased binding to FcRn, resulting in prolonged serum half-life and enhanced efficacy in vivo. These results demonstrate that Fc-modified anti-PCKS9 antibodies may enable less frequent or lower dosing of antibodies by improved recycling into the blood.

  2. Itolizumab – a humanized anti-CD6 monoclonal antibody with a better side effects profile for the treatment of psoriasis

    Directory of Open Access Journals (Sweden)

    Menon R

    2015-04-01

    Full Text Available Roshni Menon, Brinda G David Department of Dermatology, Venereology and Leprosy, Sri Venkateshwaraa Medical College Hospital and Research Centre, Ariyur, Pondicherry, India Abstract: Management of psoriasis is a challenge to the treating physician. The chronic inflammatory state of psoriasis with exacerbations and remissions necessitate “on-and-off” treatment schedules. The safety profiles of drugs and tolerability issues for patients are important factors to be considered during treatment. Various biological agents targeting T-cells and the inflammatory cytokines are available for systemic treatment of psoriasis. However, major causes of concern while using these drugs are risk of susceptibility to infection and development of anti-drug antibodies, which will affect the pharmacokinetic properties, efficacy, and safety profile of the drug. Itolizumab, a humanized anti-CD6 monoclonal antibody, is a new molecule that acts by immunomodulating the CD6 molecule. CD6 is a co-stimulatory molecule required for optimal T-cell stimulation by the antigen-presenting cells. This step is crucial in T-cell proliferation to form Th1 and Th17 cells, which play a major role in the pathogenesis of psoriasis. This article deals with the properties of Itolizumab and its role in the treatment of psoriasis. Based on the available published data, Itolizumab seems to have a better adverse effects profile and at the same time comparatively less efficacy when compared to other biological agents available for treating psoriasis. Larger studies with longer duration are required to clearly depict the long-term side effects profile. Keywords: Itolizumab, CD6, psoriasis, monoclonal antibody, biologicals 

  3. A humanized monoclonal antibody neutralizes yellow fever virus strain 17D-204 in vitro but does not protect a mouse model from disease.

    Science.gov (United States)

    Calvert, Amanda E; Dixon, Kandice L; Piper, Joseph; Bennett, Susan L; Thibodeaux, Brett A; Barrett, Alan D T; Roehrig, John T; Blair, Carol D

    2016-07-01

    The yellow fever virus (YFV) vaccine 17D-204 is considered safe and effective, yet rare severe adverse events (SAEs), some resulting in death, have been documented following vaccination. Individuals exhibiting post-vaccinal SAEs are ideal candidates for antiviral monoclonal antibody (MAb) therapy; the time until appearance of clinical signs post-exposure is usually short and patients are quickly hospitalized. We previously developed a murine-human chimeric monoclonal antibody (cMAb), 2C9-cIgG, reactive with both virulent YFV and 17D-204, and demonstrated its ability to prevent and treat YF disease in both AG129 mouse and hamster models of infection. To counteract possible selection of 17D-204 variants that escape neutralization by treatment with a single MAb (2C9-cIgG), we developed a second cMAb, 864-cIgG, for use in combination with 2C9-cIgG in post-vaccinal therapy. MAb 864-cIgG recognizes/neutralizes only YFV 17D-204 vaccine substrain and binds to domain III (DIII) of the viral envelope protein, which is different from the YFV type-specific binding site of 2C9-cIgG in DII. Although it neutralized 17D-204 in vitro, administration of 864-cIgG had no protective capacity in the interferon receptor-deficient AG129 mouse model of 17D-204 infection. The data presented here show that although DIII-specific 864-cIgG neutralizes virus infectivity in vitro, it does not have the ability to abrogate disease in vivo. Therefore, combination of 864-cIgG with 2C9-cIgG for treatment of YF vaccination SAEs does not appear to provide an improvement on 2C9-cIgG therapy alone. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  5. Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.

    Science.gov (United States)

    Iwamoto, Ryo; Takagi, Mika; Akatsuka, Jun-Ichi; Ono, Ken-Ichiro; Kishi, Yoshiro; Mekada, Eisuke

    2016-04-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

  6. Pre-exposure Prophylaxis With OspA-Specific Human Monoclonal Antibodies Protects Mice Against Tick Transmission of Lyme Disease Spirochetes.

    Science.gov (United States)

    Wang, Yang; Kern, Aurélie; Boatright, Naomi K; Schiller, Zachary A; Sadowski, Andrew; Ejemel, Monir; Souders, Colby A; Reimann, Keith A; Hu, Linden; Thomas, William D; Klempner, Mark S

    2016-07-15

    Tick transmission of Borrelia spirochetes to humans results in significant morbidity from Lyme disease worldwide. Serum concentrations of antibodies against outer surface protein A (OspA) were shown to correlate with protection from infection with Borrelia burgdorferi, the primary cause of Lyme disease in the United States. Mice transgenic for human immunoglobulin genes were immunized with OspA from B. burgdorferi to generate human monoclonal antibodies (HuMabs) against OspA. HuMabs were generated and tested in in vitro borreliacidal assays and animal protection assays. Nearly 100 unique OspA-specific HuMabs were generated, and 4 HuMabs (221-7, 857-2, 319-44, and 212-55) were selected as lead candidates on the basis of borreliacidal activity. HuMabs 319-44, 857-2, and 212-55 were borreliacidal against 1 or 2 Borrelia genospecies, whereas 221-7 was borreliacidal (half maximal inhibitory concentration, Lyme disease. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  7. Use of Monoclonal Antibodies for the Identification of Leishmania spp. Isolated from Humans and Wild Rodents in the State of Campeche, Mexico

    Directory of Open Access Journals (Sweden)

    Silvia B CantoLara

    1999-05-01

    Full Text Available The genus Leishmania includes 30 described species which infect a wide variety of mammalian hosts. The precise identification of leishmanial parasites at the species level is very important in order to determine whether an organism, causing the disease in a given area, is of the same biotype as that found in suspected mammalian reservoirs. The objectives of the present study were (1 to identify leishmanial parasites isolated from humans and wild rodents from the State of Campeche, an endemic focus of localized cutaneous leishmaniasis (LCL in southern Mexico, using an indirect immunofluorescent assay (IFA with monoclonal antibodies (Mabs; and (2 to determine if the parasites of the two types of hosts were of the same biotype. All the wild rodents (six Ototylomys phyllotis, eight Oryzomys melanotis, five Peromyscus yucatanicus and two Sigmodon hispidus and 96% (24/25 of the human isolates were identified as Leishmania (L. mexicana confirming that this specific LCL focus is a wild zoonosis. The presence of one human isolate of L. (Viannia braziliensis in the State of Campeche, confirmed the importance of an accurate taxonomic identification at species level.

  8. Use of monoclonal antibodies for the identification of Leishmania spp. isolated from humans and wild rodents in the State of Campeche, Mexico.

    Science.gov (United States)

    Canto-Lara, S B; Van Wynsberghe, N R; Vargas-González, A; Ojeda-Farfán, F F; Andrade-Narváez, F J

    1999-01-01

    The genus Leishmania includes 30 described species which infect a wide variety of mammalian hosts. The precise identification of leishmanial parasites at the species level is very important in order to determine whether an organism, causing the disease in a given area, is of the same biotype as that found in suspected mammalian reservoirs. The objectives of the present study were (1) to identify leishmanial parasites isolated from humans and wild rodents from the State of Campeche, an endemic focus of localized cutaneous leishmaniasis (LCL) in southern Mexico, using an indirect immunofluorescent assay (IFA) with monoclonal antibodies (Mabs); and (2) to determine if the parasites of the two types of hosts were of the same biotype. All the wild rodents (six Ototylomys phyllotis, eight Oryzomys melanotis, five Peromyscus yucatanicus and two Sigmodon hispidus) and 96% (24/25) of the human isolates were identified as Leishmania (L.) mexicana confirming that this specific LCL focus is a wild zoonosis. The presence of one human isolate of L. (Viannia) braziliensis in the State of Campeche, confirmed the importance of an accurate taxonomic identification at species level.

  9. Biodistribution of {sup 99m}Tc-labeled anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody h-R3 in a xenograft model of human lung adenocarcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Morales-Morales, Alejo; Duconge, Jorge; Caballero-Torres, Idania; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Iznaga-Escobar, Normando E-mail: normando@ict.cim.sld.cu

    1999-04-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody (MAb) h-R3 is an (IgG{sub 1}), which binds to an extracellular domain of EGF-R. It was used to evaluate the biodistribution on nude mice xenografted with H-125 human lung adenocarcinoma cell line. Results were compared with its murine version of the MAb ior-egf/r3. Twenty-one athymic female 4NMRI nu/nu mice were injected intraperitoneally with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled MAbs. Immunoreactivity of {sup 99m}Tc-labeled MAbs were measured by enzyme-linked immunosorbent assay (ELISA) on H-125 cell line and the immunoreactive fractions was determined by the Lindmo method. Among all organs, significant accumulation was found in serum (27.05 {+-} 2.08 %ID/g) and tumor (3.903 {+-} 0.89 %ID/g) at 4 h after injection. These values decreased to 5.03 {+-} 0.50 %ID/g and 2.19 {+-} 0.56 %ID/g for serum and tumor, respectively. The immunoreactive fraction was found to be 0.70, with a correlation coefficient r=0.9984. With the good biodistribution and tumor uptake of the {sup 99m}Tc-labeled humanized antibody h-R3, a phase I diagnostic clinical trial of tumor with epithelial origin should be pursued.

  10. Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor

    Directory of Open Access Journals (Sweden)

    Manetta J

    2014-08-01

    Full Text Available Joseph Manetta, Holly Bina, Paul Ryan, Niles Fox, Derrick R Witcher, Kristine Kikly Biotechnology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA Abstract: B-cell activating factor (BAFF is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect. Keywords: autoimmunity, B-cell malignancies, B-cell survival factor, BAFF

  11. A human monoclonal antibody targeting the stem cell factor receptor (c-Kit) blocks tumor cell signaling and inhibits tumor growth

    Science.gov (United States)

    Lebron, Maria B; Brennan, Laura; Damoci, Christopher B; Prewett, Marie C; O’Mahony, Marguerita; Duignan, Inga J; Credille, Kelly M; DeLigio, James T; Starodubtseva, Marina; Amatulli, Michael; Zhang, Yiwei; Schwartz, Kaben D; Burtrum, Douglas; Balderes, Paul; Persaud, Kris; Surguladze, David; Loizos, Nick; Paz, Keren; Kotanides, Helen

    2014-01-01

    Stem cell factor receptor (c-Kit) exerts multiple biological effects on target cells upon binding its ligand stem cell factor (SCF). Aberrant activation of c-Kit results in dysregulated signaling and is implicated in the pathogenesis of numerous cancers. The development of more specific and effective c-Kit therapies is warranted given its essential role in tumorigenesis. In this study, we describe the biological properties of CK6, a fully human IgG1 monoclonal antibody against the extracellular region of human c-Kit. CK6 specifically binds c-Kit receptor with high affinity (EC50 = 0.06 nM) and strongly blocks its interaction with SCF (IC50 = 0.41 nM) in solid phase assays. Flow cytometry shows CK6 binding to c-Kit on the cell surface of human small cell lung carcinoma (SCLC), melanoma, and leukemia tumor cell lines. Furthermore, exposure to CK6 inhibits SCF stimulation of c-Kit tyrosine kinase activity and downstream signaling pathways such as mitogen-activated protein kinase (MAPK) and protein kinase B (AKT), in addition to reducing tumor cell line growth in vitro. CK6 treatment significantly decreases human xenograft tumor growth in NCI-H526 SCLC (T/C% = 57) and Malme-3M melanoma (T/C% = 58) models in vivo. The combination of CK6 with standard of care chemotherapy agents, cisplatin and etoposide for SCLC or dacarbazine for melanoma, more potently reduces tumor growth (SCLC T/C% = 24, melanoma T/C% = 38) compared with CK6 or chemotherapy alone. In summary, our results demonstrate that CK6 is a c-Kit antagonist antibody with tumor growth neutralizing properties and are highly suggestive of potential therapeutic application in treating human malignancies harboring c-Kit receptor. PMID:24921944

  12. In vitro and in vivo properties of a fully human IgG1 monoclonal antibody that combats multidrug resistant Pseudomonas aeruginosa

    Science.gov (United States)

    ADAWI, AZMI; BISIGNANO, CARLO; GENOVESE, TIZIANA; FILOCAMO, ANGELA; KHOURI-ASSI, CAMELLIA; NEVILLE, ANAT; FEUERSTEIN, GIORA Z.; CUZZOCREA, SALVATORE; NEVILLE, LEWIS F.

    2012-01-01

    The development of an anti-bacterial drug in the form of a monoclonal antibody (mAb) targeting an exposed virulence factor, represents an innovative therapeutic strategy. Consequently, a fully human IgG1 mAb (LST-007) targeting Pseudomonas aeruginosa (PA) flagellin type b was recombinantly expressed and characterized in vitro and in an infection model driven by a multidrug resistant (MDR) PA strain. LST-007 demonstrated a highly specific binding towards whole PA bacteria harboring flagellin type b and its recombinant counterpart, with a KD of 7.4×10−10 M. In bioactivity assays, LST-007 or titers of Cmax sera derived from pharmacokinetic studies, markedly attenuated PA motility in an equipotent manner. In vivo, parenteral LST-007 (20 mg/kg) given as a single or double-dosing paradigm post-infection, afforded survival (up to 75% at Day 7) in a lethal model of pneumonia driven by the intratracheal (i.t.) instillation of an LD80 of the MDR PA isolate. This protective effect was markedly superior to that of imipenem (30% survival at Day 7) and totally devoid with an irrelevant, human isotype mAb. These data lay credence that LST-007 may be a valuable adjunct to the limited list of anti-bacterials that can tackle MDR PA strains, thereby warranting its continued development for eventual clinical evaluation. PMID:22735858

  13. Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V.; Xia, Di

    2016-07-27

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space groupP1), with unit-cell parametersa= 40.67,b= 44.91,c= 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.

  14. Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

    Energy Technology Data Exchange (ETDEWEB)

    Kaufmann, Bärbel; Vogt, Matthew R.; Goudsmit, Jaap; Holdaway, Heather A.; Aksyuk, Anastasia A.; Chipman, Paul R.; Kuhn, Richard J.; Diamond, Michael S.; Rossmann, Michael G. (Purdue); (WU-MED); (Crucell)

    2010-11-15

    Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.

  15. Rationale for the development of IMC-3G3, a fully human immunoglobulin G subclass 1 monoclonal antibody targeting the platelet-derived growth factor receptor alpha.

    Science.gov (United States)

    Shah, Gaurav D; Loizos, Nick; Youssoufian, Hagop; Schwartz, Jonathan D; Rowinsky, Eric K

    2010-02-15

    A large body of evidence suggests that the platelet-derived growth factor (PDGF) family and associated receptors are potential targets in oncology therapeutic development because of their critical roles in the proliferation and survival of various cancers and in the regulation and growth of the tumor stroma and blood vessels. Several small molecules that nonspecifically target the PDGF signaling axis are in current use or development as anticancer therapies. However, for the majority of these agents, PDGF and its receptors are neither the primary targets nor the principal mediators of anticancer activity. IMC-3G3, a fully human monoclonal antibody of the immunoglobulin G subclass 1, specifically binds to the human PDGF receptor alpha (PDGFRalpha) with high affinity and blocks PDGF ligand binding and PDGFRalpha activation. The results of preclinical studies and the frequent expression of PDGFRalpha in many types of cancer and in cancer-associated stroma support a rationale for the clinical development of IMC-3G3. Currently, IMC-3G3 is being evaluated in early clinical development for patients with several types of solid malignancies. (c) 2010 American Cancer Society.

  16. Generation and testing anti-influenza human monoclonal antibodies in a new humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO. IL-2Rγc KO. NOD).

    Science.gov (United States)

    Mendoza, Mirian; Ballesteros, Angela; Qi, Qiu; Sang, Luis Pow; Shashikumar, Soumya; Casares, Sofia; Brumeanu, Teodor-D

    2017-11-14

    Pandemic outbreaks of influenza type A viruses have resulted in numerous fatalities around the globe. Since the conventional influenza vaccines (CIV) provide less than 20% protection for individuals with weak immune system, it has been considered that broadly cross-neutralizing antibodies may provide a better protection. Herein, we showed that a recently generated humanized mouse (DRAGA mouse; HLA-A2. HLA-DR4. Rag1KO. IL-2Rγc KO. NOD) that lacks the murine immune system and expresses a functional human immune system can be used to generate cross-reactive, human anti-influenza monoclonal antibodies (hu-mAb). DRAGA mouse was also found to be suitable for influenza virus infection, as it can clear a sub-lethal infection and sustain a lethal infection with PR8/A/34 influenza virus. The hu-mAbs were designed for targeting a human B-cell epitope (180WGIHHPPNSKEQ QNLY195) of hemagglutinin (HA) envelope protein of PR8/A/34 (H1N1) virus with high homology among seven influenza type A viruses. A single administration of HA180-195 specific hu-mAb in PR8-infected DRAGA mice significantly delayed the lethality by reducing the lung damage. The results demonstrated that DRAGA mouse is a suitable tool to (i) generate heterotype cross-reactive, anti-influenza human monoclonal antibodies, (ii) serve as a humanized mouse model for influenza infection, and (iii) assess the efficacy of anti-influenza antibody-based therapeutics for human use.

  17. 75 FR 3244 - Prospective Grant of Exclusive License: Monoclonal Antibodies Against Smallpox/Orthopoxviruses

    Science.gov (United States)

    2010-01-20

    ... in the following patent applications: E-145-2004/0,1,2,3,4, Purcell et al., ``Monoclonal Antibodies... limited to monoclonal antibodies against orthopoxviruses (smallpox) for use in humans. Properly filed... HUMAN SERVICES National Institutes of Health Prospective Grant of Exclusive License: Monoclonal...

  18. Differentiation of the emerging human pathogens Trichosporon asahii and Trichosporon asteroides from other pathogenic yeasts and moulds by using species-specific monoclonal antibodies.

    Science.gov (United States)

    Davies, Genna E; Thornton, Christopher R

    2014-01-01

    The fungal genus Trichosporon contains emerging opportunistic pathogens of humans, and is the third most commonly isolated non-candidal yeast from humans. Trichosporon asahii and T. asteroides are the most important species causing disseminated disease in immunocompromised patients, while inhalation of T. asahii spores is the most important cause of summer-type hypersensitivity pneumonitis in healthy individuals. Trichosporonosis is misdiagnosed as candidiasis or cryptococcosis due to a lack of awareness and the ambiguity of diagnostic tests for these pathogens. In this study, hybridoma technology was used to produce two murine monoclonal antibodies (MAbs), CA7 and TH1, for detection and differentiation of Trichosporon from other human pathogenic yeasts and moulds. The MAbs react with extracellular antigens from T. asahii and T. asteroides, but do not recognise other related Trichosporon spp., or unrelated pathogenic yeasts and moulds including Candida, Cryptococcus, Aspergillus, Fusarium, and Scedosporium spp., or the etiologic agents of mucormycosis. Immunofluorescence and Western blotting studies show that MAb CA7, an immunoglobulin G1 (IgG1), binds to a major 60 kDa glycoprotein antigen produced on the surface of hyphae, while TH1, an immunoglobulin M (IgM), binds to an antigen produced on the surface of conidia. The MAbs were used in combination with a standard mycological growth medium (Sabouraud Dextrose Agar) to develop an enzyme-linked immunosorbent assay (ELISA) for differentiation of T. asahii from Candida albicans and Cryptococcus neoformans in single and mixed species cultures. The MAbs represent a major advance in the identification of T. asahii and T. asteroides using standard mycological identification methods.

  19. Generation of novel monoclonal antibodies for the enrichment and characterization of human corneal endothelial cells (hCENC) necessary for the treatment of corneal endothelial blindness.

    Science.gov (United States)

    Ding, Vanessa; Chin, Angela; Peh, Gary; Mehta, Jodhbir S; Choo, Andre

    2014-01-01

    Corneal transplantation is the primary treatment option to restore vision for patients with corneal endothelial blindness. Although the success rate of treatment is high, limited availability of transplant grade corneas is a major obstacle. Tissue-engineered corneal endothelial grafts constructed using cultivated human corneal endothelial cells (hCENC) isolated from cadaveric corneas may serve as a potential graft source. Currently, tools for the characterization of cultured hCENC and enrichment of hCENC from potential contaminating cells such as stromal fibroblasts are lacking. In this study, we describe the generation and characterization of novel cell surface monoclonal antibodies (mAbs) specific for hCENC. These mAbs could be used for enrichment and characterization of hCENC. Out of a total of 389 hybridomas, TAG-1A3 and TAG-2A12 were found to be specific to the corneal endothelial monolayer by immunostaining of frozen tissue sections. Both mAbs were able to clearly identify hCENC with good 'cobblestone-like' morphology from multiple donors. The antigen targets for TAG-1A3 and TAG-2A12 were found to be CD166/ALCAM and Peroxiredoxin-6 (Prdx-6), respectively, both of which have not been previously described as markers of hCENC. Additionally, unlike other Prdx-6 mAbs, TAG-2A12 was found to specifically bind cell surface Prdx-6, which was only expressed on hCENC and not on other cell types screened such as human corneal stromal fibroblasts (hCSF) and human pluripotent stem cells (hPSC). From our studies, we conclude that TAG-1A3 and TAG-2A12 are promising tools to quantitatively assess hCENC quality. It is also noteworthy that the binding specificity of TAG-2A12 could be used for the enrichment of hCENC from cell mixtures of hCSF and hPSC.

  20. Identifying the emerging human pathogen Scedosporium prolificans by using a species-specific monoclonal antibody that binds to the melanin biosynthetic enzyme tetrahydroxynaphthalene reductase.

    Science.gov (United States)

    Thornton, Christopher R; Ryder, Lauren S; Le Cocq, Kate; Soanes, Darren M

    2015-04-01

    The dematiaceous (melanized) fungus Scedosporium prolificans is an emerging and frequently fatal pathogen of immunocompromised humans and which, along with the closely related fungi Pseudallescheria boydii, Scedosporium apiospermum and S. aurantiacum in the Pseudallescheria-Scedosporium complex, is a contributing aetiology to tsunami lung and central nervous system infections in near-drowning victims who have aspirated water laden with spores. At present, the natural habitat of the fungus is largely unknown, and accurate detection methods are needed to identify environmental reservoirs of infectious propagules. In this study, we report the development of a monoclonal antibody (mAb) (CA4) specific to S. prolificans, which does not cross-react with closely related fungi in the Pseudallescheria-Scedosporium complex or with a wide range of mould and yeast species pathogenic to humans. Using genome sequencing of a soil isolate and targeted gene disruption of the CA4 antigen-encoding gene, we show that mAb CA4 binds to the melanin-biosynthetic enzyme tetrahydroxynaphthalene reductase. Enzyme-deficient mutants produce orange-brown or green-brown spore suspensions compared with the black spore suspension of the wild-type strain. Using mAb CA4 and a mAb (HG12) specific to the related fungi P. boydii, P. apiosperma, S. apiospermum and S. aurantiacum, we demonstrate how the mAbs can be used in combination with a semiselective isolation procedure to track these opportunistic pathogens in environmental samples containing mixed populations of human pathogenic fungi. Specificity of mAb CA4 was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of fungi isolated from estuarine muds. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  1. Five birds, one stone: neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody.

    Science.gov (United States)

    Rouha, Harald; Badarau, Adriana; Visram, Zehra C; Battles, Michael B; Prinz, Bianka; Magyarics, Zoltán; Nagy, Gábor; Mirkina, Irina; Stulik, Lukas; Zerbs, Manuel; Jägerhofer, Michaela; Maierhofer, Barbara; Teubenbacher, Astrid; Dolezilkova, Ivana; Gross, Karin; Banerjee, Srijib; Zauner, Gerhild; Malafa, Stefan; Zmajkovic, Jakub; Maier, Sabine; Mabry, Robert; Krauland, Eric; Wittrup, K Dane; Gerngross, Tillman U; Nagy, Eszter

    2015-01-01

    Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.

  2. Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure.

    Science.gov (United States)

    Strasser, Richard; Stadlmann, Johannes; Schähs, Matthias; Stiegler, Gabriela; Quendler, Heribert; Mach, Lukas; Glössl, Josef; Weterings, Koen; Pabst, Martin; Steinkellner, Herta

    2008-05-01

    A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic human N-glycosylation (i.e. the presence of beta1,2-xylosylation and core alpha1,3-fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down-regulation of the endogenous beta1,2-xylosyltransferase (XylT) and alpha1,3-fucosyltransferase (FucT) genes in Nicotiana benthamiana, a tobacco-related plant species widely used for recombinant protein expression. Three glyco-engineered lines with significantly reduced xylosylated and/or core alpha1,3-fucosylated glycan structures were generated. The human anti HIV monoclonal antibody 2G12 was transiently expressed in these glycosylation mutants as well as in wild-type plants. Four glycoforms of 2G12 differing in the presence/absence of xylose and core alpha1,3-fucose residues in their N-glycans were produced. Notably, 2G12 produced in XylT/FucT-RNAi plants was found to contain an almost homogeneous N-glycan species without detectable xylose and alpha1,3-fucose residues. Plant-derived glycoforms were indistinguishable from Chinese hamster ovary (CHO)-derived 2G12 with respect to electrophoretic properties, and exhibited functional properties (i.e. antigen binding and HIV neutralization activity) at least equivalent to those of the CHO counterpart. The generated RNAi lines were stable, viable and did not show any obvious phenotype, thus providing a robust tool for the production of therapeutically relevant glycoproteins in plants with a humanized N-glycan structure.

  3. Frequent Use of the IgA Isotype in Human B Cells Encoding Potent Norovirus-Specific Monoclonal Antibodies That Block HBGA Binding.

    Directory of Open Access Journals (Sweden)

    Gopal Sapparapu

    2016-06-01

    Full Text Available Noroviruses (NoV are the most common cause of non-bacterial acute gastroenteritis and cause local outbreaks of illness, especially in confined situations. Despite being identified four decades ago, the correlates of protection against norovirus gastroenteritis are still being elucidated. Recent studies have shown an association of protection with NoV-specific serum histo-blood group antigen-blocking antibody and with serum IgA in patients vaccinated with NoV VLPs. Here, we describe the isolation and characterization of human monoclonal IgG and IgA antibodies against a GI.I NoV, Norwalk virus (NV. A higher proportion of the IgA antibodies blocked NV VLP binding to glycans than did IgG antibodies. We generated isotype-switched variants of IgG and IgA antibodies to study the effects of the constant domain on blocking and binding activities. The IgA form of antibodies appears to be more potent than the IgG form in blocking norovirus binding to histo-blood group antigens. These studies suggest a unique role for IgA antibodies in protection from NoV infections by blocking attachment to cell receptors.

  4. Generation of specific monoclonal antibodies against the extracellular loops of human claudin-3 by immunizing mice with target-expressing cells.

    Science.gov (United States)

    Ando, Hiroshi; Suzuki, Masayo; Kato-Nakano, Mariko; Kawamoto, Shinobu; Misaka, Hirofumi; Kimoto, Naoya; Furuya, Akiko; Nakamura, Kazuyasu

    2015-01-01

    Human claudin-3 (CLDN3) is a tetraspanin transmembrane protein of tight junction structures and is known to be over-expressed in some malignant tumors. Although a specific monoclonal antibody (MAb) against the extracellular domains of CLDN3 would be a valuable tool, generation of such MAbs has been regarded as difficult using traditional hybridoma techniques, because of the conserved sequence homology of CLDN3s among various species. In addition, high sequence similarity is shared among claudin family members, and potential cross-reactivity of MAb should be evaluated carefully. To overcome these difficulties, we generated CLDN3-expressing Chinese hamster ovary and Sf9 cells to use an immunogens and performed cell-based screening to eliminate cross-reactive antibodies. As a result, we generated MAbs that recognized the extracellular loops of CLDN3 but not those of CLDN4, 5, 6, or 9. Further in vitro studies suggested that the isolated MAbs possessed the desired binding properties for the detection or targeting of CLDN3.

  5. A novel two-component Tobacco mosaic virus-based vector system for high-level expression of multiple therapeutic proteins including a human monoclonal antibody in plants.

    Science.gov (United States)

    Roy, Gourgopal; Weisburg, Sangeetha; Rabindran, Shailaja; Yusibov, Vidadi

    2010-09-15

    Expression of multiple therapeutic proteins from Tobacco mosaic virus (TMV)-based vectors was not successful when plants were coinoculated with a mixture of two TMV vectors engineered to express two foreign genes individually. Here, we have engineered and developed a defective RNA (dRNA)-based TMV vector (dRT-V) that utilizes two components of the same virus, with the dRNA component depending on the helper virus for replication. Agrobacterium-mediated coinoculation of Nicotiana benthamiana plants with both components of the dRT-V resulted in high-level expression of a human growth hormone and a lichenase-fused lethal factor protein of Bacillus anthracis. Furthermore, both heavy and light chains were expressed and assembled into a monoclonal antibody (mAb) specific to the protective antigen of B. anthracis, and the average yield of the purified antibody obtained was 120 mg/kg of fresh tissue. Our data suggest that dRT-V has a potential for rapid, cost-effective, large-scale manufacturing of multiple therapeutic proteins including mAbs in response to any biological emergencies. Copyright 2010 Elsevier Inc. All rights reserved.

  6. Applying antibody-sensitive hypervariable region 1-deleted hepatitis C virus to the study of escape pathways of neutralizing human monoclonal antibody AR5A

    DEFF Research Database (Denmark)

    Velazquez-Moctezuma, Rodrigo; Law, Mansun; Bukh, Jens

    2017-01-01

    isolates with high antibody resistance, or antibodies with moderate potency, it remains challenging to induce escape mutations in vitro. Here, as proof-of-concept, we used antibody-sensitive HVR1-deleted (ΔHVR1) viruses to generate escape mutants for a human monoclonal antibody, AR5A, targeting a rare....... The mutation did not induce viral fitness loss, but abrogated AR5A binding to HCV particles and intracellular E1/E2 complexes. Culturing J6/JFH1ΔHVR1 (genotype 2a), for which fitness was decreased by L665W, with AR5A generated AR5A-resistant viruses with the substitutions I345V, L665S, and S680T, which we...... effect but sensitized the virus to AR5A. Of note, H77/JFH1L665S was non-viable. The resistance mutations did not affect cell-to-cell spread or E1/E2 interactions. Finally, introducing L665W, identified in genotype 1, into genotypes 2–6 parental and HVR1-deleted variants (not available for genotype 4a) we...

  7. Effect of an anti-human Co-029/tspan8/Tspan8 mouse monoclonal antibody on tumour growth in a nude mouse model

    Directory of Open Access Journals (Sweden)

    Naouel eAilane

    2014-09-01

    Full Text Available New therapeutic agents are needed in digestive tract tumours. Co-029/tspan8 is a tetraspanin frequently expressed on human colorectal tumours, In this work, we report the effects of the monoclonal antibody Ts29.2, targeting Co-029/tspan8, on colorectal tumor cells in vitro and after implantation in nude mice. HT29, Isreco1 and SW480 colorectal tumor cell lines were used for this study. HT29 has a strong endogenous expression of Co-029/tspan8, whereas Isreco1 cells don’t express Co-029/tspan8 and SW480 has only a weak expression. Isreco1 and SW480 were transduced to express Co-029/tspan8 at the same level as HT29. In order to check the specificity of the effect of monoclonal antibody Ts29.2, low Co029/tspan8 expressing SW480 cells were injected simultaneously with transduced cells in the back, on the left and right sides of the mice. With an early treatment, Ts29.2 mAb inhibited growth of tumors expressing Co-029/tspan8 up to 70%, whereas a delayed treatment was less efficient. No effect of the antibody on cell proliferation or apoptosis induction was detected in vitro. No increase of activated caspase 3 labeling was observed in vivo and areas occupied by vessels were not significantly different between treated mice and controls. This suggests that the action of Ts29.2 is linked neither to cellular toxicity nor to the inhibition of the previously reported angiogenic properties of Co-029/tspan8. An inhibition of cell proliferation in vivo is demonstrated by a reduction of the mitotic index in HT29 tumors of Ts29.2 treated mice. The discrepancy between in vitro and in vivo data on cell proliferation suggests that the binding of Ts29.2 to tumour cells may modify their response to signals issued from the microenvironment. Given the restricted pattern of tissue expression of the tetraspanin Co-029/tspan8, these preliminary results put forth for consideration the antibody targeting of this tetraspanin in further investigations for therapeutic

  8. Generation and functional characterization of anti-human and anti-mouse IL-36R antagonist monoclonal antibodies.

    Science.gov (United States)

    Ganesan, Rajkumar; Raymond, Ernest L; Mennerich, Detlev; Woska, Joseph R; Caviness, Gary; Grimaldi, Christine; Ahlberg, Jennifer; Perez, Rocio; Roberts, Simon; Yang, Danlin; Jerath, Kavita; Truncali, Kristopher; Frego, Lee; Sepulveda, Eliud; Gupta, Priyanka; Brown, Su-Ellen; Howell, Michael D; Canada, Keith A; Kroe-Barrett, Rachel; Fine, Jay S; Singh, Sanjaya; Mbow, M Lamine

    2017-10-01

    Deficiency of interleukin (IL)-36 receptor antagonist (DITRA) syndrome is a rare autosomal recessive disease caused by mutations in IL36RN. IL-36R is a cell surface receptor and a member of the IL1R family that is involved in inflammatory responses triggered in skin and other epithelial tissues. Accumulating evidence suggests that IL-36R signaling may play a role in the pathogenesis of psoriasis. Therapeutic intervention of IL-36R signaling offers an innovative treatment paradigm for targeting epithelial cell-mediated inflammatory diseases such as the life-threatening psoriasis variant called generalized pustular psoriasis (GPP). We report the discovery and characterization of MAB92, a potent, high affinity anti-human IL-36 receptor antagonistic antibody that blocks human IL-36 ligand (α, β and γ)-mediated signaling. In vitro treatment with MAB92 directly inhibits human IL-36R-mediated signaling and inflammatory cytokine production in primary human keratinocytes and dermal fibroblasts. MAB92 shows exquisite species specificity toward human IL-36R and does not cross react to murine IL-36R. To enable in vivo pharmacology studies, we developed a mouse cross-reactive antibody, MAB04, which exhibits overlapping binding and pharmacological activity as MAB92. Epitope mapping indicates that MAB92 and MAB04 bind primarily to domain-2 of the human and mouse IL-36R proteins, respectively. Treatment with MAB04 abrogates imiquimod and IL-36-mediated skin inflammation in the mouse, further supporting an important role for IL-36R signaling in epithelial cell-mediated inflammation.

  9. The human CD38 monoclonal antibody daratumumab shows antitumor activity and hampers leukemia-microenvironment interactions in chronic lymphocytic leukemia

    DEFF Research Database (Denmark)

    Matas-Céspedes, Alba; Vidal-Crespo, Anna; Rodriguez, Vanina

    2017-01-01

    mononuclear cells to analyze antibodydependent cell cytotoxicity (ADCC), murine and human macrophages to study antibody-dependent cell phagocytosis (ADCP), or human serum to analyze complement-dependent cytotoxicity (CDC). The effect of daratumumab on CLL cell migration and adhesion to extracellular matrix...... was characterized. Daratumumab activity was validated in two in vivo models. Results: Daratumumab demonstrated efficient lysis of patientderived CLL cells and cell lines by ADCC in vitro and ADCP both in vitro and in vivo whereas exhibited negligible CDC in these cells. To demonstrate the therapeutic effect...

  10. Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus

    DEFF Research Database (Denmark)

    Meunier, Jean-Christophe; Russell, Rodney S.; Goossens, Vera

    2008-01-01

    The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human...

  11. High-yield production of a human monoclonal IgG by rhizosecretion in hydroponic tobacco cultures

    NARCIS (Netherlands)

    Madeira, L.M.; Szeto, T.H.; Henquet, Maurice; Raven, Nicole; Runions, John; Huddleston, Jon; Garrard, Ian; Drake, P.M.W.; Ma, Julian K.C.

    2016-01-01

    Rhizosecretion of recombinant pharmaceuticals from in vitro hydroponic transgenic plant cultures is a simple, low cost, reproducible and controllable production method. Here, we demonstrate the application and adaptation of this manufacturing platform to a human antivitronectin IgG1

  12. Uptake of 111In-labeled fully human monoclonal antibody TSP-A18 reflects transferrin receptor expression in normal organs and tissues of mice.

    Science.gov (United States)

    Sugyo, Aya; Tsuji, Atsushi B; Sudo, Hitomi; Nomura, Fumiko; Satoh, Hirokazu; Koizumi, Mitsuru; Kurosawa, Gene; Kurosawa, Yoshikazu; Saga, Tsuneo

    2017-03-01

    Transferrin receptor (TfR) is an attractive molecule for targeted therapy of cancer. Various TfR-targeted therapeutic agents such as anti-TfR antibodies conjugated with anticancer agents have been developed. An antibody that recognizes both human and murine TfR is needed to predict the toxicity of antibody-based agents before clinical trials, there is no such antibody to date. In this study, a new fully human monoclonal antibody TSP-A18 that recognizes both human and murine TfR was developed and the correlation analysis of the radiolabeled antibody uptake and TfR expression in two murine strains was conducted. TSP-A18 was selected using extracellular portions of human and murine TfR from a human antibody library. The cross-reactivity of TSP-A18 with human and murine cells was confirmed by flow cytometry. Cell binding and competitive inhibition assays with [111In]TSP-A18 showed that TSP-A18 bound highly to TfR-expressing MIAPaCa-2 cells with high affinity. Biodistribution studies of [111In]TSP-A18 and [67Ga]citrate (a transferrin-mediated imaging probe) were conducted in C57BL/6J and BALB/c-nu/nu mice. [111In]TSP-A18 was accumulated highly in the spleen and bone containing marrow component of both strains, whereas high [67Ga]citrate uptake was only observed in bone containing marrow component and not in the spleen. Western blotting indicated the spleen showed the strongest TfR expression compared with other organs in both strains. There was significant correlation between [111In]TSP-A18 uptake and TfR protein expression in both strains, whereas there was significant correlation of [67Ga]citrate uptake with TfR expression only in C57BL/6J. These findings suggest that the difference in TfR expression between murine strains should be carefully considered when testing for the toxicity of anti-TfR antibody in mice and the uptake of anti-TfR antibody could reflect tissue TfR expression more accurately compared with that of transferrin-mediated imaging probe such as [67Ga]citrate.

  13. Ofatumumab, a human anti-CD20 monoclonal antibody, for treatment of rheumatoid arthritis with an inadequate response to one or more disease-modifying antirheumatic drugs: results of a randomized, double-blind, placebo-controlled, phase I/II study

    DEFF Research Database (Denmark)

    Østergaard, Mikkel; Baslund, Bo; Rigby, William

    2010-01-01

    To investigate the safety and efficacy of ofatumumab, a novel human anti-CD20 monoclonal antibody (mAb), in patients with active rheumatoid arthritis (RA) whose disease did not respond to > or = 1 disease-modifying antirheumatic drug....

  14. Inhibition of human airway sensitization by a novel monoclonal anti-IgE antibody, 17-9.

    Science.gov (United States)

    Rabe, K F; Watson, N; Dent, G; Morton, B E; Wagner, K; Magnussen, H; Heusser, C H

    1998-05-01

    We investigated the effect of a novel mouse IgG2b nonanaphylactogenic anti-human IgE antibody, 17-9, on allergen and histamine responses in passively sensitized human airways in vitro to determine the specific contribution of IgE to the sensitization process. Bronchial rings were sensitized with serum containing high levels of allergen-specific IgE (Dermatophagoides farinae), or with a hapten-specific chimeric humanized IgE (JW8). There was a concentration-dependent contraction of serum-sensitized bronchial rings to D. farinae (517 +/- 188 mg tension at 10 U/ml, n = 8) that was not observed in nonsensitized controls. This response was practically abolished when tissues were sensitized in the presence of 100 microg/ml anti-IgE antibody 17-9 (54 +/- 20 mg). In tissues sensitized with the anti-NIP IgE, JW8, there was a concentration-dependent contraction to the specific antigen NIP-BSA (560 +/- 154 mg at 0.3 microg/ml, n = 5) that was not observed in nonsensitized control subjects and that was substantially inhibited when 17-9 was present in the sensitization buffer (124 +/- 109 mg). The inhibition with 17-9 was specific, as pretreatment with a non-IgE-specific IgG2b antibody did not affect allergen responses. Potency and maximal contractions to histamine in serum-sensitized tissues were significantly elevated compared with nonsensitized controls; this was not affected by the presence of 17-9 during sensitization (pEC50 = 5.1 +/- 0.2 versus 5.0 +/- 0.3 in tissues sensitized in the absence of 17-9). In tissues sensitized with JW8 there was no significant increase in responsiveness to histamine. We conclude that allergen responses in sensitized human airways are dependent on IgE levels in the sensitizing serum while nonspecific (hyper)responsiveness depends on serum factors other than IgE. Nonanaphylactogenic anti-human IgE antibodies effectively inhibit allergen responses of human airways in vitro but may not affect other factors inducing hyperresponsiveness.

  15. Development of a complete human IgG monoclonal antibody to transferrin receptor 1 targeted for adult T-cell leukemia/lymphoma.

    Science.gov (United States)

    Shimosaki, Shunsuke; Nakahata, Shingo; Ichikawa, Tomonaga; Kitanaka, Akira; Kameda, Takuro; Hidaka, Tomonori; Kubuki, Yoko; Kurosawa, Gene; Zhang, Lilin; Sudo, Yukio; Shimoda, Kazuya; Morishita, Kazuhiro

    2017-03-25

    Iron is an essential nutrient for normal cell growth, and reprogramming of iron metabolism is essential to tumor cell survival and progression. HTLV-1-associated adult T-cell leukemia/lymphoma (ATLL) has no effective therapy and high levels of cell surface transferrin receptor 1 (TFR1) expression have been reported in ATLL by us and other groups. In this study, to develop a novel molecular-targeted therapy against TFR1 to modulate iron metabolism, we initially determined the expression pattern of several iron-related genes along with TFR1 and found that ATLL cells presented characteristic of an iron-deficiency state such as high expression of iron-regulatory protein 2 (IRP2) and low expression of its E3 ubiquitin-ligase, FBXL5. Therefore, we developed human IgG monoclonal antibodies to human TFR1 using a phage display method (ICOS method) to block the incorporation of the transferrin (TF)-iron complex into ATLL cells for inhibiting cell growth. One of the mAbs, JST-TFR09, presented its greater affinity to TFR1 on ATLL cells in flow cytometry (FCM) analysis than those of commercially available anti-TFR1 antibodies and identified high expression of TFR1 in most of the acute-type ATLL cells. Moreover, JST-TFR09 could interfere with binding between TFR1 and TF, which resulted in effective blockade of TFR1 internalization and induction of cell apoptosis by the treatment of ATLL cells with JST-TFR09. JST-TFR09 showed dual activities through direct cell cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), and the treatment of JST-TFR09 significantly suppressed cell growth of ATLL cells with induction of apoptosis in in vitro and in vivo experiments. Thus, JST-TFR09 described here may become a promising therapeutic antibody for the treatment of ATLL. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Accelerating Influenza Research: Vaccines, Antivirals, Immunomodulators and Monoclonal Antibodies. The Manufacture of a New Wild-Type H3N2 Virus for the Human Viral Challenge Model.

    Science.gov (United States)

    Fullen, Daniel J; Noulin, Nicolas; Catchpole, Andrew; Fathi, Hosnieh; Murray, Edward J; Mann, Alex; Eze, Kingsley; Balaratnam, Ganesh; Borley, Daryl W; Gilbert, Anthony; Lambkin-Williams, Rob

    2016-01-01

    Influenza and its associated diseases are a major cause of morbidity and mortality. The United States Advisory Committee on Immunization Practices recommends influenza vaccination for everyone over 6 months of age. The failure of the flu vaccine in 2014-2015 demonstrates the need for a model that allows the rapid development of novel antivirals, universal/intra-seasonal vaccines, immunomodulators, monoclonal antibodies and other novel treatments. To this end we manufactured a new H3N2 influenza virus in compliance with Good Manufacturing Practice for use in the Human Viral Challenge Model. We chose an H3N2 influenza subtype, rather than H1N1, given that this strain has the most substantial impact in terms of morbidity or mortality annually as described by the Centre for Disease Control. We first subjected the virus batch to rigorous adventitious agent testing, confirmed the virus to be wild-type by Sanger sequencing and determined the virus titres appropriate for human use via the established ferret model. We built on our previous experience with other H3N2 and H1N1 viruses to develop this unique model. We conducted an initial safety and characterisation study in healthy adult volunteers, utilising our unique clinical quarantine facility in London, UK. In this study we demonstrated this new influenza (H3N2) challenge virus to be both safe and pathogenic with an appropriate level of disease in volunteers. Furthermore, by inoculating volunteers with a range of different inoculum titres, we established the minimum infectious titre required to achieve reproducible disease whilst ensuring a sensitive model that can be translated to design of subsequent field based studies. ClinicalTrials.gov NCT02525055.

  17. High-avidity monoclonal antibodies against the human scavenger class B type I receptor efficiently block hepatitis C virus infection in the presence of high-density lipoprotein.

    Science.gov (United States)

    Catanese, Maria Teresa; Graziani, Rita; von Hahn, Thomas; Moreau, Martine; Huby, Thierry; Paonessa, Giacomo; Santini, Claudia; Luzzago, Alessandra; Rice, Charles M; Cortese, Riccardo; Vitelli, Alessandra; Nicosia, Alfredo

    2007-08-01

    The human scavenger class B type 1 receptor (SR-B1/Cla1) was identified as a putative receptor for hepatitis C virus (HCV) because it binds to soluble recombinant HCV envelope glycoprotein E2 (sE2). High-density lipoprotein (HDL), a natural SR-B1 ligand, was shown to increase the in vitro infectivity of retroviral pseudoparticles bearing HCV envelope glycoproteins and of cell culture-derived HCV (HCVcc), suggesting that SR-B1 promotes viral entry in an HDL-dependent manner. To determine whether SR-B1 participates directly in HCV infection or facilitates HCV entry through lipoprotein uptake, we generated a panel of monoclonal antibodies (MAbs) against native human SR-B1. Two of them, 3D5 and C167, bound to conformation-dependent SR-B1 determinants and inhibited the interaction of sE2 with SR-B1. These antibodies efficiently blocked HCVcc infection of Huh-7.5 hepatoma cells in a dose-dependent manner. To examine the role of HDL in SR-B1-mediated HCVcc infection, we set up conditions for HCVcc production and infection in serum-free medium. HCVcc efficiently infected Huh-7.5 cells in the absence of serum lipoproteins, and addition of HDL led to a twofold increase in infectivity. However, the HDL-induced enhancement of infection had no impact on the neutralization potency of MAb C167, despite its ability to inhibit both HDL binding to cells and SR-B1-mediated lipid transfer. Of note, MAb C167 also potently blocked Huh-7.5 infection by an HCV strain recovered from HCVcc-infected chimpanzees. These results demonstrate that SR-B1 is essential for infection with HCV produced in vitro and in vivo and suggest the possible use of anti-SR-B1 antibodies as therapeutic agents.

  18. Potency of a human monoclonal antibody to diphtheria toxin relative to equine diphtheria anti-toxin in a guinea pig intoxication model.

    Science.gov (United States)

    Smith, Heidi L; Cheslock, Peter; Leney, Mark; Barton, Bruce; Molrine, Deborah C

    2016-08-17

    Prompt administration of anti-toxin reduces mortality following Corynebacterium diphtheriae infection. Current treatment relies upon equine diphtheria anti-toxin (DAT), with a 10% risk of serum sickness and rarely anaphylaxis. The global DAT supply is extremely limited; most manufacturers have ceased production. S315 is a neutralizing human IgG1 monoclonal antibody to diphtheria toxin that may provide a safe and effective alternative to equine DAT and address critical supply issues. To guide dose selection for IND-enabling pharmacology and toxicology studies, we dose-ranged S315 and DAT in a guinea pig model of diphtheria intoxication based on the NIH Minimum Requirements potency assay. Animals received a single injection of antibody premixed with toxin, were monitored for 30 days, and assigned a numeric score for clinical signs of disease. Animals receiving ≥ 27.5 µg of S315 or ≥ 1.75 IU of DAT survived whereas animals receiving ≤ 22.5 µg of S315 or ≤ 1.25 IU of DAT died, yielding a potency estimate of 17 µg S315/IU DAT (95% CI 16-21) for an endpoint of survival. Because some surviving animals exhibited transient limb weakness, likely a systemic sign of toxicity, DAT and S315 doses required to prevent hind limb paralysis were also determined, yielding a relative potency of 48 µg/IU (95% CI 38-59) for this alternate endpoint. To support advancement of S315 into clinical trials, potency estimates will be used to evaluate the efficacy of S315 versus DAT in an animal model with antibody administration after toxin exposure, more closely modeling anti-toxin therapy in humans.

  19. Ofatumumab, a human anti-CD20 monoclonal antibody, for treatment of rheumatoid arthritis with an inadequate response to one or more disease-modifying antirheumatic drugs: results of a randomized, double-blind, placebo-controlled, phase I/II study

    DEFF Research Database (Denmark)

    Østergaard, Mikkel; Baslund, Bo; Rigby, William

    2010-01-01

    To investigate the safety and efficacy of ofatumumab, a novel human anti-CD20 monoclonal antibody (mAb), in patients with active rheumatoid arthritis (RA) whose disease did not respond to > or = 1 disease-modifying antirheumatic drug.......To investigate the safety and efficacy of ofatumumab, a novel human anti-CD20 monoclonal antibody (mAb), in patients with active rheumatoid arthritis (RA) whose disease did not respond to > or = 1 disease-modifying antirheumatic drug....

  20. Heterologous radioimmunoassay for llama and alpaca luteinizing hormone with a monoclonal antibody, and equine standard and a human tracer

    Energy Technology Data Exchange (ETDEWEB)

    Aba, M.A.; Forsberg, M. [Swedish Univ. of Agricultural Sciences, Dept. of Clinical Chemistry, Faculty of Veterinary medicine, Uppsala (Sweden)

    1995-12-31

    A radioimmunoassay for llama and alpaca LH was developed using a human I{sup 125}LH tracer from a commercial kit, equine LH diluted in human LH free serum as standard, and amonoclonal antibody (518B7) specific for LH but with low species specificity. A 60-min delay in the addition of the tracer and overnight incubation gave a sensitivity of 0.8 {mu}3g L{sup -1}. The intra-assay coefficient of variation was 37% at 1 {mu}g L{sup -1}, declined to 15% at 4 {mu}g L{sup -1} and was below 6% for concentrations up to 32 {mu}g L{sup -1}. The inter-assay coefficients of variation for 3 control samples were 20% (2.8 {mu}g L{sup -1}), 16% (7.1 {mu}g L{sup -1}) and 9.8% (19 {mu}g L{sup -1}). In an attempt to increase sensitivity, all tubes were preincubated for 4 h at room temperature before adding the tracer, and the sample volume was increased from 50 {mu}L to 100 {mu}L (in the standard curve the increased volume was compensated for by human LH free serum). With this protocol, the assay sensitivity was 0,5 {mu}g L{sup -1}. The assay was validated clinically and demonstrated increased concentrations of LH after mating in llamas and alpacas. Furthermore, the assay was used to monitor LH responses to a single dose of GnRH in llamas (adult males and females at different ages). (au) 9 refs.

  1. Mining the human autoantibody repertoire: isolation of potent IL17A-neutralizing monoclonal antibodies from a patient with thymoma.

    Science.gov (United States)

    Beerli, Roger R; Bauer, Monika; Fritzer, Andrea; Rosen, Lindsey B; Buser, Regula B; Hanner, Markus; Maudrich, Melanie; Nebenfuehr, Mario; Toepfer, Jorge Alejandro Sepulveda; Mangold, Susanne; Bauer, Anton; Holland, Steven M; Browne, Sarah K; Meinke, Andreas

    2014-01-01

    Anti-cytokine autoantibodies have been widely reported to be present in human plasma, both in healthy subjects and in patients with underlying autoimmune conditions, such as autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) or thymic epithelial neoplasms. While often asymptomatic, they can cause or facilitate a wide range of diseases including opportunistic infections. The potential therapeutic value of specific neutralizing anti-cytokine autoantibodies has not been thoroughly investigated. Here we used mammalian cell display to isolate IL17A-specific antibodies from a thymoma patient with proven high-titer autoantibodies against the same. We identified 3 distinct clonotypes that efficiently neutralized IL17A in a cell-based in vitro assay. Their potencies were comparable to those of known neutralizing antibodies, including 2, AIN457 (secukinumab) and ixekizumab that are currently in clinical development for the treatment of various inflammatory disorders. These data clearly demonstrate that the human autoantibody repertoire can be mined for antibodies with high therapeutic potential for clinical development.

  2. Targeting FMS-related tyrosine kinase receptor 3 with the human immunoglobulin G1 monoclonal antibody IMC-EB10.

    Science.gov (United States)

    Youssoufian, Hagop; Rowinsky, Eric K; Tonra, James; Li, Yiwen

    2010-02-15

    FMS-related tyrosine kinase receptor 3 (FLT3) is a class III receptor tyrosine kinase that holds considerable promise as a therapeutic target in hematologic malignancies. Current efforts directed toward the development of small-molecule tyrosine kinase inhibitors of FLT3 may be limited by off-target toxicities and the development of drug resistance. Target-specific antibodies could overcome these hurdles and provide additional mechanisms to enhance the antitumor efficacy of FLT3 inhibitors. IMC-EB10 is a novel antibody directed against FLT3. The binding of IMC-EB10 to FLT3 results in antiproliferative effects in vitro and in mouse models engrafted with human leukemia cells that harbor wild-type or constitutively activated FLT3. Future clinical trials will test these notions formally and will identify the most appropriate opportunities for this member of a new generation of antileukemic therapies. (c) 2010 American Cancer Society.

  3. First‐in‐human assessment of PRX002, an anti–α‐synuclein monoclonal antibody, in healthy volunteers

    Science.gov (United States)

    Schenk, Dale B.; Koller, Martin; Ness, Daniel K.; Griffith, Sue G.; Grundman, Michael; Zago, Wagner; Soto, Jay; Atiee, George; Ostrowitzki, Susanne

    2016-01-01

    ABSTRACT Background: α‐Synuclein is a major component of pathologic inclusions that characterize Parkinson's disease. PRX002 is an antibody that targets α‐synuclein, and its murine parent antibody 9E4 has been shown in preclinical studies to reduce α‐synuclein pathology and to protect against cognitive and motor deteriorations and progressive neurodegeneration in human α‐synuclein transgenic mice. Methods: This first‐in‐human, randomized, double‐blind, placebo‐controlled, phase 1 study assessed the impact of PRX002 administered to 40 healthy participants in 5 ascending‐dose cohorts (n = 8/cohort) in which participants were randomly assigned to receive a single intravenous infusion of study drug (0.3, 1, 3, 10, or 30 mg/kg; n = 6/cohort) or placebo (n = 2/cohort). Results: PRX002 demonstrated favorable safety, tolerability, and pharmacokinetic profiles at all doses tested, with no immunogenicity. No serious adverse events, discontinuations as a result of adverse events, or dose‐limiting toxicities were reported. Serum PRX002 exposure was dose proportional; the average terminal half‐life across all doses was 18.2 days. A significant dose‐dependent reduction in free serum α‐synuclein (unbound to PRX002) was apparent within 1 hour after PRX002 administration, whereas total α‐synuclein (free plus bound) increased dose‐dependently, presumably because of the expected change in kinetics following antibody binding. Conclusions: This study demonstrates that serum α‐synuclein can be safely modulated in a dose‐dependent manner after single intravenous infusions of an anti–α‐synuclein antibody. These findings support continued development of PRX002, including further characterization of its safety, tolerability, pharmacokinetics, and pharmacodynamic effects in the central nervous system in patients with Parkinson's disease. © 2016 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of

  4. High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies

    Science.gov (United States)

    Liao, Hua-Xin; Levesque, Marc C.; Nagel, Ashleigh; Dixon, Ashlyn; Zhang, Ruijun; Walter, Emmanuel; Parks, Robert; Whitesides, John; Marshall, Dawn J.; Hwang, Kwan-Ki; Yang, Yi; Chen, Xi; Gao, Feng; Munshaw, Supriya; Kepler, Thomas B.; Denny, Thomas; Moody, M. Anthony; Haynes, Barton F.

    2009-01-01

    Defining human B cell repertoires to viral pathogens is critical for design of vaccines that induce broadly protective antibodies to infections such as HIV-1 and influenza. Single B cell sorting and cloning of immunoglobulin (Ig) heavy- and light-chain variable regions (VH and VL) is a powerful technology for defining anti-viral B cell repertoires. However, the Ig-cloning step is time-consuming and prevents high-throughput analysis of the B cell repertoire. Novel linear Ig heavy- and light-chain gene expression cassettes were designed to express Ig VH and VL genes isolated from sorted single B cells as IgG1 antibody without a cloning step. The cassettes contain all essential elements for transcriptional and translational regulation, including CMV promoter, Ig leader sequences, constant region of IgG1 heavy- or Ig light-chain, poly(A) tail and substitutable VH or VL genes. The utility of these Ig gene expression cassettes was established using synthetic VH or VL genes from an anti-HIV-1 gp41 mAb 2F5 as a model system, and validated further using VH and VL genes isolated from cloned EBV-transformed antibody-producing cell lines. Finally, this strategy was successfully used for rapid production of recombinant influenza mAbs from sorted single human plasmablasts after influenza vaccination. These Ig gene expression cassettes constitute a highly efficient strategy for rapid expression of Ig genes for high-throughput screening and analysis without cloning. PMID:19428587

  5. The Human CD38 Monoclonal Antibody Daratumumab Shows Antitumor Activity and Hampers Leukemia-Microenvironment Interactions in Chronic Lymphocytic Leukemia.

    Science.gov (United States)

    Matas-Céspedes, Alba; Vidal-Crespo, Anna; Rodriguez, Vanina; Villamor, Neus; Delgado, Julio; Giné, Eva; Roca-Ho, Heleia; Menéndez, Pablo; Campo, Elías; López-Guillermo, Armando; Colomer, Dolors; Roué, Gaël; Wiestner, Adrian; Parren, Paul W H I; Doshi, Parul; van Bueren, Jeroen Lammerts; Pérez-Galán, Patricia

    2017-03-15

    Purpose: To establish a proof-of-concept for the efficacy of the anti-CD38 antibody daratumumab in the poor prognosis CD38 + chronic lymphocytic leukemia (CLL) subtype. Experimental Design: The mechanism of action of daratumumab was assessed in CLL primary cells and cell lines using peripheral blood mononuclear cells to analyze antibody-dependent cell cytotoxicity (ADCC), murine and human macrophages to study antibody-dependent cell phagocytosis (ADCP), or human serum to analyze complement-dependent cytotoxicity (CDC). The effect of daratumumab on CLL cell migration and adhesion to extracellular matrix was characterized. Daratumumab activity was validated in two in vivo models. Results: Daratumumab demonstrated efficient lysis of patient-derived CLL cells and cell lines by ADCC in vitro and ADCP both in vitro and in vivo whereas exhibited negligible CDC in these cells. To demonstrate the therapeutic effect of daratumumab in CLL, we generated a disseminated CLL mouse model with the CD38 + MEC2 cell line and CLL patient-derived xenografts (CLL-PDX). Daratumumab significantly prolonged overall survival of MEC2 mice, completely eliminated cells from the infiltrated organs, and significantly reduced disease burden in the spleen of CLL-PDX. The effect of daratumumab on patient-derived CLL cell dissemination was demonstrated in vitro by its effect on CXCL12-induced migration and in vivo by interfering with CLL cell homing to spleen in NSG mice. Daratumumab also reduced adhesion of CLL cells to VCAM-1, accompanied by downregulation of the matrix metalloproteinase MMP9. Conclusions: These unique and substantial effects of daratumumab on CLL viability and dissemination support the investigation of its use in a clinical setting of CLL. Clin Cancer Res; 23(6); 1493-505. ©2016 AACR . ©2016 American Association for Cancer Research.

  6. Identification and characterization of a broadly cross-reactive HIV-1 human monoclonal antibody that binds to both gp120 and gp41.

    Directory of Open Access Journals (Sweden)

    Mei-Yun Zhang

    Full Text Available Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb, designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env. M43 bound to recombinant gp140 s from various primary isolates, to membrane-associated Envs on transfected cells and HIV-1 infected cells, as well as to recombinant gp120 s and gp41 fusion intermediate structures containing N-trimer structure, but did not bind to denatured recombinant gp140 s and the CD4 binding site (CD4bs mutant, gp120 D368R, suggesting that the m43 epitope is conformational and overlaps the CD4bs on gp120 and the N-trimer structure on gp41. M43 neutralized 34% of the HIV-1 primary isolates from different clades and all the SHIVs tested in assays based on infection of peripheral blood mononuclear cells (PBMCs by replication-competent virus, but was less potent in cell line-based pseudovirus assays. In contrast to CD4, m43 did not induce Env conformational changes upon binding leading to exposure of the coreceptor binding site, enhanced binding of mAbs 2F5 and 4E10 specific for the membrane proximal external region (MPER of gp41 Envs, or increased gp120 shedding. The overall modest neutralization activity of m43 is likely due to the limited binding of m43 to functional Envs which could be increased by antibody engineering if needed. M43 may represent a new class of bnAbs targeting conformational epitopes overlapping structures on both gp120 and gp41. Its novel epitope and possibly new mechanism(s of neutralization could helpdesign improved vaccine immunogens and candidate therapeutics.

  7. Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay

    Science.gov (United States)

    Lin, Hong-En; Tsai, Wen-Yang; Liu, I-Ju; Li, Pi-Chun; Liao, Mei-Ying; Tsai, Jih-Jin; Wu, Yi-Chieh; Lai, Chih-Yun; Lu, Chih-Hsuan; Huang, Jyh-Hsiung; Chang, Gwong-Jen; Wu, Han-Chung; Wang, Wei-Kung

    2012-01-01

    Background The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. Methodology/Principal Findings We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. Conclusions/Significance Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level. PMID:22235356

  8. AMG 479, a fully human anti-insulin-like growth factor receptor type I monoclonal antibody, inhibits the growth and survival of pancreatic carcinoma cells.

    Science.gov (United States)

    Beltran, Pedro J; Mitchell, Petia; Chung, Young-A; Cajulis, Elaina; Lu, John; Belmontes, Brian; Ho, Joanne; Tsai, Mei Mei; Zhu, Min; Vonderfecht, Steven; Baserga, Renato; Kendall, Richard; Radinsky, Robert; Calzone, Frank J

    2009-05-01

    Pancreatic carcinoma is a leading cause of cancer deaths, and recent clinical trials of a number of oncology therapeutics have not substantially improved clinical outcomes. We have evaluated the therapeutic potential of AMG 479, a fully human monoclonal antibody against insulin-like growth factor (IGF) type I receptor (IGF-IR), in two IGF-IR-expressing pancreatic carcinoma cell lines, BxPC-3 and MiaPaCa2, which also differentially express insulin receptor (INSR). AMG 479 bound to IGF-IR (K(D) 0.33 nmol/L) and blocked IGF-I and IGF-II binding (IC(50) AMG 479 completely inhibited ligand-induced (IGF-I, IGF-II, and insulin) activation of IGF-IR homodimers and IGF-IR/INSR hybrids (but not INSR homodimers) leading to reduced cellular viability in serum-deprived cultures. AMG 479 inhibited >80% of basal IGF-IR activity in BxPC-3 and MiaPaCa2 xenografts and prevented IGF-IR and IGF-IR/INSR hybrid activation following challenge with supraphysiologic concentrations of IGF-I. As a single agent, AMG 479 inhibited (∼ 80%) the growth of pancreatic carcinoma xenografts, and long-term treatment was associated with reduced IGF-IR signaling activity and expression. Efficacy seemed to be the result of two distinct biological effects: proapoptotic in BxPC-3 and antimitogenic in MiaPaCa2. The combination of AMG 479 with gemcitabine resulted in additive inhibitory activity both in vitro and in vivo. These results indicate that AMG 479 is a clinical candidate, both as a single agent and in combination with gemcitabine, for the treatment of patients with pancreatic carcinoma

  9. HIV-1 envelope glycoprotein resistance to monoclonal antibody 2G12 is subject-specific and context-dependent in macaques and humans.

    Science.gov (United States)

    Malherbe, Delphine C; Sanders, Rogier W; van Gils, Marit J; Park, Byung; Gomes, Michelle M; Schuitemaker, Hanneke; Barnett, Susan; Haigwood, Nancy L

    2013-01-01

    HIV-1 Envelope (Env) protein is the sole target of neutralizing antibodies (NAbs) that arise during infection to neutralize autologous variants. Under this immune pressure, HIV escape variants are continuously selected and over the course of infection Env becomes more neutralization resistant. Many common alterations are known to affect sensitivity to NAbs, including residues encoding potential N-linked glycosylation sites (PNGS). Knowledge of Env motifs associated with neutralization resistance is valuable for the design of an effective Env-based vaccine so we characterized Envs isolated longitudinally from a SHIV(SF162P4) infected macaque for sensitivity to neutralizing monoclonal antibodies (MAbs) B12, 2G12, 4E10 and 2F5. The early Env, isolated from plasma at day 56 after infection, was the most sensitive and the late Env, from day 670, was the most resistant to MAbs. We identified four PNGS in these Envs that accumulated over time at positions 130, 139, 160 and 397. We determined that removal of these PNGS significantly increased neutralization sensitivity to 2G12, and conversely, we identified mutations by in silico analyses that contributed resistance to 2G12 neutralization. In order to expand our understanding of these PNGS, we analyzed Envs from clade B HIV-infected human subjects and identified additional glycan and amino acid changes that could affect neutralization by 2G12 in a context-dependent manner. Taken together, these in vitro and in silico analyses of clade B Envs revealed that 2G12 resistance is achieved by previously unrecognized PNGS substitutions in a context-dependent manner and by subject-specific pathways.

  10. Characterization of a recombinant humanized anti-cocaine monoclonal antibody produced from multiple clones for the selection of a master cell bank candidate.

    Science.gov (United States)

    Wetzel, Hanna N; Webster, Rose P; Saeed, Fatima O; Kirley, Terence L; Ball, William J; Norman, Andrew B

    2017-06-03

    We have generated a humanized anti-cocaine monoclonal antibody (mAb), which is at an advanced stage of pre-clinical development. We report here in vitro binding affinity studies, and in vivo pharmacokinetic and efficacy studies of the recombinant mAb. The overall aim was to characterize the recombinant antibody from each of the three highest producing transfected clones and to select one to establish a master cell bank. In mAb pharmacokinetic studies, after injection with h2E2 (120 mg/kg iv) blood was collected from the tail tip of mice over 28 days. Antibody concentrations were quantified using ELISA. The h2E2 concentration as a function of time was fit using a two-compartment pharmacokinetic model. To test in vivo efficacy, mice were injected with h2E2 (120 mg/kg iv), then one hour later injected with an equimolar dose of cocaine. Blood and brain were collected 5 min after cocaine administration. Cocaine concentrations were quantified using LC/MS. The affinity of the antibody for cocaine was determined using a [3H] cocaine binding assay. All three antibodies had long elimination half-lives, 2-5 nM Kd for cocaine, and prevented cocaine's entry into the brain by sequestering it in the plasma. Pharmacokinetic and radioligand binding assays supported designation of the highest producing clone 85 as the master cell bank candidate. Overall, the recombinant h2E2 showed favorable binding properties, pharmacokinetics, and in vivo efficacy. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. HIV-1 envelope glycoprotein resistance to monoclonal antibody 2G12 is subject-specific and context-dependent in macaques and humans.

    Directory of Open Access Journals (Sweden)

    Delphine C Malherbe

    Full Text Available HIV-1 Envelope (Env protein is the sole target of neutralizing antibodies (NAbs that arise during infection to neutralize autologous variants. Under this immune pressure, HIV escape variants are continuously selected and over the course of infection Env becomes more neutralization resistant. Many common alterations are known to affect sensitivity to NAbs, including residues encoding potential N-linked glycosylation sites (PNGS. Knowledge of Env motifs associated with neutralization resistance is valuable for the design of an effective Env-based vaccine so we characterized Envs isolated longitudinally from a SHIV(SF162P4 infected macaque for sensitivity to neutralizing monoclonal antibodies (MAbs B12, 2G12, 4E10 and 2F5. The early Env, isolated from plasma at day 56 after infection, was the most sensitive and the late Env, from day 670, was the most resistant to MAbs. We identified four PNGS in these Envs that accumulated over time at positions 130, 139, 160 and 397. We determined that removal of these PNGS significantly increased neutralization sensitivity to 2G12, and conversely, we identified mutations by in silico analyses that contributed resistance to 2G12 neutralization. In order to expand our understanding of these PNGS, we analyzed Envs from clade B HIV-infected human subjects and identified additional glycan and amino acid changes that could affect neutralization by 2G12 in a context-dependent manner. Taken together, these in vitro and in silico analyses of clade B Envs revealed that 2G12 resistance is achieved by previously unrecognized PNGS substitutions in a context-dependent manner and by subject-specific pathways.

  12. [Cloning, ligation and expression of the variable region genes of the monoclonal antibody against human HnRNPA2/B1].

    Science.gov (United States)

    Wang, Xia; Peng, Xiao-dong; Li, Guang; Hu, Li-juan; Bi, Jian-hong

    2004-12-01

    To clone the variable region genes of the monoclonal antibody (McAb) against human heterogeneous nuclear ribonucleoprotein A2/B1 (HnRNPA2/B1), ligate them to assemble single chain Fv (ScFv) gene and express in Escherichia coli. The specificity of the anti-HnRNPA2/B1 McAb 3E8 to synthetic HnRNPA2/B1 peptide, HnRNPA2/B1 protein in lung cancer cells were examined by dot-immunobinding assay, Western blot and immunohistochemistry. The variable region genes of heavy chain (VH) and light chain (VL) were amplified from hybridoma cell by reverse transcription-polymerase chain reaction(RT-PCR), and then were linked by a linker peptide using SOE-PCR (splicing by overlap extension-PCR) to construct recombination ScFv gene. The latter was cloned into the expression vector pET28 (a+) and expressed in E coli BL21. The expressed product was identified by SDS-PAGE and competitive ELISA inhibition test. It was shown that the McAb combined specifically with synthetic HnRNPA2/B1 peptide and HnRNPA2/B1 protein in three lung cancer cells. The cloned VH gene and VL gene were 345 bp and 309 bp respectively and were linked successfully to obtain ScFv gene. The ScFv protein was expressed in the form of inclusion body, with molecular weight of 28,000 and immunoreactivity to HnRNPA2/B1. VH gene, VL gene and ScFv gene of anti-HnRNPA2/B1 antibody were cloned, constructed and functionally expressed in E coli. These results provide the experimental basis for elucidating the role of HnRNPA2/B1 in lung cancer.

  13. Phase I pharmacologic and biologic study of ramucirumab (IMC-1121B), a fully human immunoglobulin G1 monoclonal antibody targeting the vascular endothelial growth factor receptor-2.

    Science.gov (United States)

    Spratlin, Jennifer L; Cohen, Roger B; Eadens, Matthew; Gore, Lia; Camidge, D Ross; Diab, Sami; Leong, Stephen; O'Bryant, Cindy; Chow, Laura Q M; Serkova, Natalie J; Meropol, Neal J; Lewis, Nancy L; Chiorean, E Gabriela; Fox, Floyd; Youssoufian, Hagop; Rowinsky, Eric K; Eckhardt, S Gail

    2010-02-10

    PURPOSE To evaluate the safety, maximum-tolerated dose (MTD), pharmacokinetics (PKs), pharmacodynamics, and preliminary anticancer activity of ramucirumab (IMC-1121B), a fully human immunoglobulin G(1) monoclonal antibody targeting the vascular endothelial growth factor receptor (VEGFR)-2. PATIENTS AND METHODS Patients with advanced solid malignancies were treated once weekly with escalating doses of ramucirumab. Blood was sampled for PK studies throughout treatment. The effects of ramucirumab on circulating vascular endothelial growth factor-A (VEGF-A), soluble VEGFR-1 and VEGFR-2, tumor perfusion, and vascularity using dynamic contrast-enhanced magnetic resonance imaging were assessed. Results Thirty-seven patients were treated with 2 to 16 mg/kg of ramucirumab. After one patient each developed dose-limiting hypertension and deep venous thrombosis at 16 mg/kg, the next lower dose (13 mg/kg) was considered the MTD. Nausea, vomiting, headache, fatigue, and proteinuria were also noted. Four (15%) of 27 patients with measurable disease had a partial response (PR), and 11 (30%) of 37 patients had either a PR or stable disease lasting at least 6 months. PKs were characterized by dose-dependent elimination and nonlinear exposure consistent with saturable clearance. Mean trough concentrations exceeded biologically relevant target levels throughout treatment at all dose levels. Serum VEGF-A increased 1.5 to 3.5 times above pretreatment values and remained in this range throughout treatment at all dose levels. Tumor perfusion and vascularity decreased in 69% of evaluable patients. CONCLUSION Objective antitumor activity and antiangiogenic effects were observed over a wide range of dose levels, suggesting that ramucirumab may have a favorable therapeutic index in treating malignancies amenable to VEGFR-2 inhibition.

  14. A novel monoclonal antibody to human laminin α5 chain strongly inhibits integrin-mediated cell adhesion and migration on laminins 511 and 521.

    Directory of Open Access Journals (Sweden)

    Zenebech Wondimu

    Full Text Available Laminins, a large family of αβγ heterotrimeric proteins mainly found in basement membranes, are strong promoters of adhesion and migration of multiple cell types, such as tumor and immune cells, via several integrin receptors. Among laminin α (LMα chains, α5 displays the widest tissue distribution in adult life and is synthesized by most cell types. Here, we have generated and characterized five novel monoclonal antibodies (mAbs to the human LMα5 chain to further study the biological relevance of α5 laminins, such as laminins 511 (α5β1γ1 and 521 (α5β2γ1. As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMα5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited α3β1/α6β1 integrin-mediated adhesion and migration of glioma, melanoma, and carcinoma cells on laminin-511 and, together with mAb 4C7, on laminin-521. Accordingly, mAb 8G9 abolished the interaction of soluble α3β1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMα5 chain but largely hindered by mAb 4E10 to a LMβ1 chain epitope near the globular domain of laminin-511. Thus, mAb 8G9 defines a novel epitope localized at or near the integrin-binding globular domain of the LMα5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.

  15. Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL lambda genes define a conserved antigenic structure.

    Science.gov (United States)

    Gorny, Miroslaw K; Sampson, Jared; Li, Huiguang; Jiang, Xunqing; Totrov, Maxim; Wang, Xiao-Hong; Williams, Constance; O'Neal, Timothy; Volsky, Barbara; Li, Liuzhe; Cardozo, Timothy; Nyambi, Phillipe; Zolla-Pazner, Susan; Kong, Xiang-Peng

    2011-01-01

    Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.

  16. Sonoporation delivery of monoclonal antibodies against human papillomavirus 16 E6 restores p53 expression in transformed cervical keratinocytes.

    Directory of Open Access Journals (Sweden)

    Melissa Togtema

    Full Text Available High-risk types of human papillomavirus (HPV, such as HPV16, have been found in nearly all cases of cervical cancer. Therapies targeted at blocking the HPV16 E6 protein and its deleterious effects on the tumour suppressor pathways of the cell can reverse the malignant phenotype of affected keratinocytes while sparing uninfected cells. Through a strong interdisciplinary collaboration between engineering and biology, a novel, non-invasive intracellular delivery method for the HPV16 E6 antibody, F127-6G6, was developed. The method employs high intensity focused ultrasound (HIFU in combination with microbubbles, in a process known as sonoporation. In this proof of principle study, it was first demonstrated that sonoporation antibody delivery into the HPV16 positive cervical carcinoma derived cell lines CaSki and SiHa was possible, using chemical transfection as a baseline for comparison. Delivery of the E6 antibody using sonoporation significantly restored p53 expression in these cells, indicating the antibody is able to enter the cells and remains active. This delivery method is targeted, non-cytotoxic, and non-invasive, making it more easily translatable for in vivo experiments than other transfection methods.

  17. Anti-L-NGFR and -CD34 monoclonal antibodies identify multipotent mesenchymal stem cells in human adipose tissue.

    Science.gov (United States)

    Quirici, Nadia; Scavullo, Cinzia; de Girolamo, Laura; Lopa, Silvia; Arrigoni, Elena; Deliliers, Giorgio Lambertenghi; Brini, Anna T

    2010-06-01

    Stem cells hold great promise in tissue engineering for repairing tissues damaged by disease or injury. Mesenchymal stem cells (MSCs) are multipotent cells able to proliferate and differentiate into multiple mesodermal tissues such as bone, cartilage, muscle, tendon, and fat. We have previously reported that the low-affinity nerve growth factor receptor (L-NGFR or CD271) defines a subset of cells with high proliferative, clonogenic, and multipotential differentiation ability in adult bone marrow (BM). It has been recently shown that adipose tissue is an alternative source of adult multipotent stem cells and human adipose-derived stem cells, selected by plastic adherence (PA hASCs), have been extensively characterized for their functional potentials in vitro. In this study, immunoselected L-NGFR(+) and CD34(+) subpopulations have been analyzed and compared with the PA hASCs. Phenotypic profile of freshly purified subpopulations showed an enrichment in the expression of some stem cell markers; indeed, a great percentage of L-NGFR(+) cells co-expressed CD34 and CD117 antigens, whereas the endothelial-committed progenitor markers KDR and P1H12 were mainly expressed on CD34(+) cells. Differently from PA hASCs, the immunoseparated fractions showed high increments in cell proliferation, and the fibroblast colony-forming activity (CFU-F) was maintained throughout the time of culture. Furthermore, the immunoselected populations showed a greater differentiative potential toward adipocytes, osteoblasts, and chondrocyte-like cells, compared to PA hASCs. Our data suggest that both CD34(+) and L-NGFR(+) hASCs can be considered alternative candidates for tissue engineering and regenerative medicine applications.

  18. A single dose of neuron-binding human monoclonal antibody improves spontaneous activity in a murine model of demyelination.

    Directory of Open Access Journals (Sweden)

    Aleksandar Denic

    Full Text Available Our laboratory demonstrated that a natural human serum antibody, sHIgM12, binds to neurons in vitro and promotes neurite outgrowth. We generated a recombinant form, rHIgM12, with identical properties. Intracerebral infection with Theiler's Murine Encephalomyelitis Virus (TMEV of susceptible mouse strains results in chronic demyelinating disease with progressive axonal loss and neurologic dysfunction similar to progressive forms of multiple sclerosis. To study the effects of rHIgM12 on the motor function of TMEV-infected mice, we monitored spontaneous nocturnal activity over many weeks. Nocturnal behavior is a sensitive measure of rodent neurologic function because maximal activity changes are expected to occur during the normally active night time monitoring period. Mice were placed in activity boxes eight days prior to treatment to collect baseline spontaneous activity. After treatment, activity in each group was continuously recorded over 8 weeks. We chose a long 8-week monitoring period for two reasons: (1 we previously demonstrated that IgM induced remyelination is present by 5 weeks post treatment, and (2 TMEV-induced demyelinating disease in this strain progresses very slowly. Due to the long observation periods and large data sets, differences among treatment groups may be difficult to appreciate studying the original unfiltered recordings. To clearly delineate changes in the highly fluctuating original data we applied three different methods: (1 binning, (2 application of Gaussian low-pass filters (GF and (3 polynomial fitting. Using each of the three methods we showed that compared to control IgM and saline, early treatment with rHIgM12 induced improvement in both horizontal and vertical motor function, whereas later treatment improved only horizontal activity. rHIgM12 did not alter activity of normal, uninfected mice. This study supports the hypothesis that treatment with a neuron-binding IgM not only protects neurons in vitro, but

  19. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

    Sondergeld, P.; van de Donk, N. W. C. J.; Richardson, P. G.

    2015-01-01

    The development of monoclonal antibodies (mAbs) for the treatment of disease goes back to the vision of Paul Ehrlich in the late 19th century; however, the first successful treatment with a mAb was not until 1982, in a lymphoma patient. In multiple myeloma, mAbs are a very recent and exciting add...

  20. Human Monoclonal Antibodies to a Novel Cluster of Conformational Epitopes on HCV E2 with Resistance to Neutralization Escape in a Genotype 2a Isolate

    Science.gov (United States)

    Keck, Zhen-yong; Xia, Jinming; Wang, Yong; Wang, Wenyan; Krey, Thomas; Prentoe, Jannick; Carlsen, Thomas; Li, Angela Ying-Jian; Patel, Arvind H.; Lemon, Stanley M.; Bukh, Jens; Rey, Felix A.; Foung, Steven K. H.

    2012-01-01

    The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other regions that will be relevant for vaccine design, we employed yeast surface display of antibodies that bound to genotype 1a H77C E2 mutant proteins containing a substitution either at Y632A (to avoid selecting non-neutralizing antibodies) or D535A. A panel of nine human monoclonal antibodies (HMAbs) was isolated and designated as HC-84-related antibodies. Each HMAb neutralized cell culture infectious HCV (HCVcc) with genotypes 1–6 envelope proteins with varying profiles, and each inhibited E2 binding to the viral receptor CD81. Five of these antibodies neutralized representative genotypes 1–6 HCVcc. Epitope mapping identified a cluster of overlapping epitopes that included nine contact residues in two E2 regions encompassing aa418–446 and aa611–616. Effect on virus entry was measured using H77C HCV retroviral pseudoparticles, HCVpp, bearing an alanine substitution at each of the contact residues. Seven of ten mutant HCVpp showed over 90% reduction compared to wild-type HCVpp and two others showed approximately 80% reduction. Interestingly, four of these antibodies bound to a linear E2 synthetic peptide encompassing aa434–446. This region on E2 has been proposed to elicit non-neutralizing antibodies in humans that interfere with neutralizing antibodies directed at an adjacent E2 region from aa410–425. The isolation of four HC-84 HMAbs binding to the peptide, aa434–446, proves that some antibodies to this region are to highly conserved epitopes mediating broad virus neutralization. Indeed, when HCVcc were passaged in the presence of each of these antibodies, virus escape was not observed. Thus, the cluster of HC-84 epitopes, designated as antigenic domain D, is

  1. A randomized, double-blind study of AMG 108 (a fully human monoclonal antibody to IL-1R1) in patients with osteoarthritis of the knee.

    Science.gov (United States)

    Cohen, Stanley B; Proudman, Susanna; Kivitz, Alan J; Burch, Francis X; Donohue, John P; Burstein, Deborah; Sun, Yu-Nien; Banfield, Christopher; Vincent, Michael S; Ni, Liyun; Zack, Debra J

    2011-07-29

    AMG 108 is a fully human, immunoglobulin subclass G2 (IgG2) monoclonal antibody that binds the human interleukin-1 (IL-1) receptor type 1, inhibiting the activity of IL-1a and IL-1b. In preclinical studies, IL-1 inhibition was shown to be beneficial in models of osteoarthritis (OA). The purpose of this two-part study was to evaluate the safety and pharmacokinetics (PK; Part A) and clinical effect (Part B) of AMG 108 in a double-blind, placebo-controlled, multiple-dose study in patients with OA of the knee. In Part A, patients received placebo or AMG 108 subcutaneously (SC; 75 mg or 300 mg) or intravenously (IV; 100 mg or 300 mg) once every 4 weeks for 12 weeks; in Part B, patients received placebo or 300 mg AMG 108 SC, once every 4 weeks for 12 weeks. The clinical effect of AMG 108 was measured in Part B by using the Western Ontario and McMaster Universities (WOMAC) osteoarthritis index pain score. In Part A, 68 patients were randomized, and 64 received investigational product. In Part B, 160 patients were randomized, and 159 received investigational product. AMG 108 was well tolerated. Most adverse events (AEs), infectious AEs, serious AEs and infections, as well as withdrawals from the study due to AEs occurred at similar rates in both active and placebo groups. One death was reported in an 80-year-old patient (Part A, 300 mg IV AMG 108; due to complications of lobar pneumonia). AMG 108 serum concentration-time profiles exhibited nonlinear PK. The AMG 108 group in Part B had statistically insignificant but numerically greater improvement in pain compared with the placebo group, as shown by the WOMAC pain scores (median change, -63.0 versus -37.0, respectively). The safety profile of AMG 108 SC and IV was comparable with placebo in patients with OA of the knee. Patients who received AMG 108 showed statistically insignificant but numerically greater improvements in pain; however, minimal, if any, clinical benefit was observed. This study is registered with Clinical

  2. Endothelin A receptor antagonism enhances inhibitory effects of anti-ganglioside GD2 monoclonal antibody on invasiveness and viability of human osteosarcoma cells.

    Directory of Open Access Journals (Sweden)

    Bo Liu

    Full Text Available Endothelin-1 (ET-1/endothelin A receptor (ETAR signaling is important for osteosarcoma (OS progression. Monoclonal antibodies (mAbs targeting ganglioside GD2 reportedly inhibit tumor cell viability independent of the immune system. A recent study suggests that ganglioside GD2 may play an important role in OS progression. In the present study, we for the first time explored the effects of anti-GD2 mAb alone or in combination with ETAR antagonist on OS cell invasiveness and viability. Human OS cell lines Saos-2, MG-63 and SJSA-1 were treated with control IgG (PK136 mAb, 50 µg/mL, anti-GD2 14G2a mAb (50 µg/mL, selective ETAR antagonist BQ123 (5 µM, or 14G2a (50 µg/mL+BQ123 (5 µM. Cells with knockdown of ETAR (ETAR-shRNA with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K inhibitor BKM120 (50 µM were used as a positive control. Our results showed that BQ123, ETAR-shRNA and 14G2a mAb individually decreased cell invasion and viability, matrix metalloproteinase-2 (MMP-2 expression and activity, PI3k activity, and phosphorylation at serine 473 (ser473 of Akt in OS cells. 14G2a mAb in combination with BQ123 or ETAR-shRNA showed significantly stronger inhibitory effects compared with each individual treatment. In all three cell lines tested, 14G2a mAb in combination with BQ123 showed the strongest inhibitory effects. In conclusion, we provide the first in vitro evidence that anti-ganglioside GD2 14G2a mAb effectively inhibits cell invasiveness, MMP-2 expression and activity, and cell viability in human OS cells. ETAR antagonist BQ123 significantly enhances the inhibitory effects of 14G2a mAb, likely mainly through inhibiting the PI3K/Akt pathway. This study adds novel insights into OS treatment, which will serve as a solid basis for future in vivo studies on the effects of combined treatment of OS with anti-ganglioside GD2 mAbs and ETAR antagonists.

  3. Heterogeneity in both cytokine production and responsiveness of a panel of monoclonal human Epstein-Barr virus-transformed B-cell lines

    NARCIS (Netherlands)

    Jochems, G. J.; Klein, M. R.; Jordens, R.; Pascual-Salcedo, D.; van Boxtel-Oosterhof, F.; van Lier, R. A.; Zeijlemaker, W. P.

    1991-01-01

    To optimize growth and Ig production of in vitro-cultured Epstein-Barr virus (EBV)-transformed B cells, a panel of six monoclonal EBV B-cell lines was analyzed for autocrine growth factor production and responsiveness to various cytokines. Three cell lines produced Il-I and four produced Il-6,

  4. Enhancement of retroviral infection in vitro by anti-Le(y) IgG: reversal by humanization of monoclonal mouse antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Sørensen, A M; Arendrup, M

    1993-01-01

    Monoclonal mouse IgG3 antibody (ABL 364) against the carbohydrate Le(y) antigen enhanced infection in vitro with HTLV-1 and with HIV-1 when propagated in both transformed and normal lymphocytes. Enhancement was independent of complement, occurred with both lymphocytes and monocytes as target cells...

  5. First clinical use of ofatumumab, a novel fully human anti-CD20 monoclonal antibody in relapsed or refractory follicular lymphoma

    DEFF Research Database (Denmark)

    Hagenbeek, Anton; Gadeberg, Ole Vestergaard; Johnson, Peter

    2008-01-01

    Ofatumumab is a unique monoclonal antibody that targets a distinct small loop epitope on the CD20 molecule. Preclinical data show that ofatumumab is active against B-cell lymphoma/chronic lymphocytic leukemia cells with low CD20-antigen density and high expression of complement inhibitory molecul...

  6. REFINEMENT OF AN INDIRECT IMMUNOTOXIN ASSAY OF MONOCLONAL-ANTIBODIES RECOGNIZING THE HUMAN SMALL-CELL LUNG-CANCER CLUSTER-2 ANTIGEN

    NARCIS (Netherlands)

    DERBYSHIRE, EJ; DELEIJ, L; WAWRZYNCZAK, EJ

    Monoclonal antibodies (Mabs) from the Second International Workshop on Small Cell Lung Cancer (SCLC) Antigens that recognise the cluster 2 SCLC-associated antigen mediated potent and selective cytotoxic effects in an indirect assay of immunotoxin cytotoxicity. In this assay, the NCI-H69 cell line

  7. Antitumor efficacy of TRA-8 anti-DR5 monoclonal antibody alone or in combination with chemotherapy and/or radiation therapy in a human breast cancer model.

    Science.gov (United States)

    Buchsbaum, Donald J; Zhou, Tong; Grizzle, William E; Oliver, Patsy G; Hammond, Charlotte J; Zhang, Sijian; Carpenter, Mark; LoBuglio, Albert F

    2003-09-01

    A monoclonal antibody (TRA-8) has been developed that binds to death receptor 5 (DR5), one of two death receptors bound by tumor necrosis factor-related apoptosis-inducing ligand. The purpose of this study was to evaluate in vitro the binding and cytotoxicity of TRA-8 to human breast cancer cell lines. The antitumor efficacy of TRA-8 was evaluated in a xenograft human breast cancer murine model, as a single agent and in combination with chemotherapy or radiation therapy. Anti: The binding of TRA-8 to a panel of nine human breast cancer cell lines was evaluated by indirect immunofluorescence and flow cytometry. Cytotoxicity of TRA-8 alone and in the presence of Adriamycin or paclitaxel was measured in vitro using the ATP-lite assay. Antitumor efficacy was determined by treatment of nude mice bearing well-established s.c. DR5-positive 2LMP human breast cancer xenografts with TRA-8 alone or in combination with Adriamycin or paclitaxel. Tumor size and regression rates were determined. In addition, a study was carried out with TRA-8 and Adriamycin in combination with 3 Gy (60)Co irradiation of 2LMP xenografts on days 9 and 17. All nine human breast cancer cell lines expressed DR5 with TRA-8 reactivity varying from strongly to weakly positive. Four cell lines were sensitive to TRA-8 cytotoxicity with IC(50) of 17-299 ng/ml, whereas other cell lines had weak cytotoxicity or were resistant. In vivo studies demonstrated significant inhibition of growth of 2LMP xenografts by TRA-8 treatment alone. The combination of TRA-8 + Adriamycin or paclitaxel produced significant inhibition of tumor growth as compared with controls or either agent alone. An aggregate analysis of all 166 animals studied demonstrated that TRA-8 alone or in combination with Adriamycin, paclitaxel, or radiation produced a significant increase in tumor doubling time compared with any modality alone with mean doubling time in days of 12 (untreated), 14 (radiation), 17 (Adriamycin), 25 (paclitaxel), 39

  8. Anti-CD20 Monoclonal Antibody Treatment of Human Herpesvirus 8-Associated, Body Cavity-Based Lymphoma with an Unusual Phenotype in a Human Immunodeficiency Virus-Negative Patient

    Science.gov (United States)

    Pérez, Celeste L.; Rudoy, Silvia

    2001-01-01

    Human herpesvirus 8 (HHV-8), or Kaposi's sarcoma-associated herpesvirus, is a gammaherpesvirus first detected in Kaposi's sarcoma tumor cells and subsequently in primary effusion lymphoma (PEL) tumor cells and peripheral blood mononuclear cells from PEL patients. PEL has been recognized as an individual nosologic entity based on its distinctive features and consistent association with HHV-8 infection. PEL is an unusual form of body cavity-based B-cell lymphoma (BCBL). It occurs predominantly in human immunodeficiency virus (HIV)-positive patients but occasionally also in elderly HIV-negative patients. We describe a case of PEL, with ascites, bilateral pleural effusions, and a small axillary lymphadenopathy, in a 72-year-old HIV-negative man. PCR performed on a lymph node specimen and in liquid effusion was positive for HHV-8 and negative for Epstein-Barr virus. The immunophenotype of the neoplastic cells was B CD19+ CD20+ CD22+ with coexpression of CD10 and CD23 and with clonal kappa light chain rearrangement. The patient was treated with Rituximab, a chimeric (human-mouse) anti-CD20 monoclonal antibody. Thirteen months later, the patient continued in clinical remission. This is the first report of an HHV-8-associated BCBL in an HIV-negative patient in Argentina. PMID:11527816

  9. A versatile method for protein-based antigen bioanalysis in non-clinical pharmacokinetics studies of a human monoclonal antibody drug by an immunoaffinity liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Onami, Ichio; Ayabe, Miho; Murao, Naoaki; Ishigai, Masaki

    2014-03-21

    A versatile immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify the total concentration of a protein-based antigen in non-clinical pharmacokinetics (PK) studies of a human monoclonal antibody drug. The method combines using magnetic beads that have been coated with a commercial anti-human Fc region antibody to capture an immune complex of the antigen and antibody drug, with subsequent digestion and quantification of the antigen-derived tryptic peptide via LC-MS/MS. Although a typical immunoassay or an immunoaffinity LC-MS/MS assay requires an antigen-specific antibody that uses a different epitope from the antibody drug, this method requires only a commercial anti-human Fc region antibody. The method was applied to quantify total receptor activator of nuclear factor-κB ligand (RANKL) in the presence of denosumab, a humanized monoclonal antibody (mAb) specific to RANKL. The assay was validated as fit-for-purpose and found to be accurate (based antigen assays in the early drug stage. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Applying antibody-sensitive hypervariable region 1-deleted hepatitis C virus to the study of escape pathways of neutralizing human monoclonal antibody AR5A

    Science.gov (United States)

    Velázquez-Moctezuma, Rodrigo; Bukh, Jens

    2017-01-01

    Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. With 3–4 million new HCV infections yearly, a vaccine is urgently needed. A better understanding of virus escape from neutralizing antibodies and their corresponding epitopes are important for this effort. However, for viral isolates with high antibody resistance, or antibodies with moderate potency, it remains challenging to induce escape mutations in vitro. Here, as proof-of-concept, we used antibody-sensitive HVR1-deleted (ΔHVR1) viruses to generate escape mutants for a human monoclonal antibody, AR5A, targeting a rare cross-genotype conserved epitope. By analyzing the genotype 1a envelope proteins (E1/E2) of recovered Core-NS2 recombinant H77/JFH1ΔHVR1 and performing reverse genetic studies we found that resistance to AR5A was caused by substitution L665W, also conferring resistance to the parental H77/JFH1. The mutation did not induce viral fitness loss, but abrogated AR5A binding to HCV particles and intracellular E1/E2 complexes. Culturing J6/JFH1ΔHVR1 (genotype 2a), for which fitness was decreased by L665W, with AR5A generated AR5A-resistant viruses with the substitutions I345V, L665S, and S680T, which we introduced into J6/JFH1 and J6/JFH1ΔHVR1. I345V increased fitness but had no effect on AR5A resistance. L665S impaired fitness and decreased AR5A sensitivity, while S680T combined with L665S compensated for fitness loss and decreased AR5A sensitivity even further. Interestingly, S680T alone had no fitness effect but sensitized the virus to AR5A. Of note, H77/JFH1L665S was non-viable. The resistance mutations did not affect cell-to-cell spread or E1/E2 interactions. Finally, introducing L665W, identified in genotype 1, into genotypes 2–6 parental and HVR1-deleted variants (not available for genotype 4a) we observed diverse effects on viral fitness and a universally pronounced reduction in AR5A sensitivity. Thus, we were able to take advantage of the neutralization-sensitive HVR1

  11. Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques.

    Science.gov (United States)

    Santra, Sampa; Tomaras, Georgia D; Warrier, Ranjit; Nicely, Nathan I; Liao, Hua-Xin; Pollara, Justin; Liu, Pinghuang; Alam, S Munir; Zhang, Ruijun; Cocklin, Sarah L; Shen, Xiaoying; Duffy, Ryan; Xia, Shi-Mao; Schutte, Robert J; Pemble Iv, Charles W; Dennison, S Moses; Li, Hui; Chao, Andrew; Vidnovic, Kora; Evans, Abbey; Klein, Katja; Kumar, Amit; Robinson, James; Landucci, Gary; Forthal, Donald N; Montefiori, David C; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Robb, Merlin L; Michael, Nelson L; Kim, Jerome H; Soderberg, Kelly A; Giorgi, Elena E; Blair, Lily; Korber, Bette T; Moog, Christiane; Shattock, Robin J; Letvin, Norman L; Schmitz, Joern E; Moody, M A; Gao, Feng; Ferrari, Guido; Shaw, George M; Haynes, Barton F

    2015-08-01

    HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.

  12. Enrichment and purging of human embryonic stem cells by detection of cell surface antigens using the monoclonal antibodies TG30 and GCTM-2.

    Science.gov (United States)

    Polanco, Juan Carlos; Wang, Bei; Zhou, Qi; Chy, Hun; O'Brien, Carmel; Laslett, Andrew L

    2013-12-06

    Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all somatic cell lineages. Differentiated hESC derivatives can potentially be used in transplantation therapies to treat a variety of cell-degenerative diseases. However, hESC differentiation protocols usually yield a mixture of differentiated target and off-target cell types as well as residual undifferentiated cells. For the translation of differentiated hESC-derivatives from the laboratory to the clinic, it is important to be able to discriminate between undifferentiated (pluripotent) and differentiated cells, and generate methods to separate these populations. Safe application of hESC-derived somatic cell types can only be accomplished with pluripotent stem cell-free populations, as residual hESCs could induce tumors known as teratomas following transplantation. Towards this end, here we describe a methodology to detect pluripotency associated cell surface antigens with the monoclonal antibodies TG30 (CD9) and GCTM-2 via fluorescence activated cell sorting (FACS) for the identification of pluripotent TG30(Hi)-GCTM-2(Hi) hESCs using positive selection. Using negative selection with our TG30/GCTM-2 FACS methodology, we were able to detect and purge undifferentiated hESCs in populations undergoing very early-stage differentiation (TG30(Neg)-GCTM-2(Neg)). In a further study, pluripotent stem cell-free samples of differentiated TG30(Neg)-GCTM-2(Neg) cells selected using our TG30/GCTM-2 FACS protocol did not form teratomas once transplanted into immune-compromised mice, supporting the robustness of our protocol. On the other hand, TG30/GCTM-2 FACS-mediated consecutive passaging of enriched pluripotent TG30(Hi)-GCTM-2(Hi) hESCs did not affect their ability to self-renew in vitro or their intrinsic pluripotency. Therefore, the characteristics of our TG30/GCTM-2 FACS methodology provide a sensitive assay to obtain highly

  13. Recombinant humanized monoclonal antibody against CD18 (rhuMAb CD18) in traumatic hemorrhagic shock: results of a phase II clinical trial. Traumatic Shock Group.

    Science.gov (United States)

    Rhee, P; Morris, J; Durham, R; Hauser, C; Cipolle, M; Wilson, R; Luchette, F; McSwain, N; Miller, R

    2000-10-01

    Activated neutrophils have been shown to play a pivotal role in resuscitation injury after traumatic hemorrhagic shock. Blocking the adhesion of neutrophils with a recombinant humanized monoclonal antibody against CD18 (rhuMAb CD18) may reduce resuscitation injury but increase the risk of infection. This was a dose-finding phase II study to determine safety, pharmacokinetics, pharmacodynamics, and clinical outcome parameters for additional studies. This was a prospective, placebo-controlled, randomized (3:1), double-blind phase II trial enrolling 116 blunt and penetrating trauma patients from 14 trauma centers over a 9-month period. Patients with hypotension (blood pressure history of cardiopulmonary resuscitation in the trauma center. An independent Drug Safety and Monitoring Review Board periodically reviewed unblinded data for safety issues and to give approval for dose escalation. Minor and major infection rates in rhuMAb CD18 groups were comparable to placebo. There was no evidence of antibody formation against rhuMAb CD18. Linear PK was observed within the dose range studied. Duration of neutrophil binding was dose-dependent, with 2 mg/kg resulting in greater than 90% neutrophil CD18 receptor saturation for approximately 48 hours. The mortality was 6.7% (2 of 30) in the placebo group, 4.8% (1 of 21) in the 0.5-mg/kg group, 8.5% (4 of 47) in the 1-mg/kg group, and 0% (0 of 18) in the 2-mg/kg group. The study was not powered for efficacy, and none of the efficacy variables demonstrated statistical significance. Favorable trends were seen in the 2-mg/kg group as compared with placebo in median intensive care unit length of stay (5 vs. 9 days) and median time on ventilator (34 vs. 72 hours). A single 2-mg/kg dose of rhuMAb CD18 maintains greater than 90% saturation of neutrophil CD18 receptors for approximately 48 hours in patients with traumatic hemorrhagic shock undergoing resuscitation. There was no trend toward increased infection. A larger trial is needed to

  14. Biodistribution and elimination kinetics of systemic Stx2 by the Stx2A and Stx2B subunit-specific human monoclonal antibodies in mice

    Directory of Open Access Journals (Sweden)

    Sheoran Abhineet

    2012-06-01

    Full Text Available Abstract Background Hemolytic uremic syndrome (HUS leading to acute kidney failure, is a condition linked to the production of primarily Shiga toxin 2 (Stx2 by some E. coli serotypes. We have previously shown that Stx2 A subunit-specific human monoclonal antibody (HuMAb 5C12, and B subunit-specific HuMAb 5H8 inhibit cultured cell death, and protect mice and piglets from fatal Stx2-intoxication. We have also shown that 5H8 blocks binding of Stx2 to its cell-surface receptor globotriaosyl ceramide (Gb3, whereas Stx2 when complexed with 5C12 binds Gb3 with higher affinity than Stx2. The mechanism by which 5C12 neutralizes Stx2 in vitro involves trapping of Stx2 in the recycling endosomes and releasing it into the extracellular environment. Because of the clinical implications associated with the formation of Stx2/antibody complexes and the potential for trapping and clearance through a severely damaged kidney associated with HUS, we investigated the likely site(s of Stx2/antibody localization and clearance in intoxicated mice treated with antibody or placebo. Results Mice were injected with radiolabeled Stx2 (125I-Stx2 4 hours after administration of 5C12, 5H8, or phosphate buffered saline (PBS and the sites of localization of labeled Stx2, were investigated 3, 24 and 48 hours later. The liver recorded statistically much higher concentrations of labeled Stx2 for groups receiving 5C12 and 5H8 antibodies after 3, 24 and 48 hours, as compared with the PBS group. In contrast, highest levels of labeled Stx2 were detected in the kidneys of the PBS group at all 3 sampling times. Mice receiving either of the two HuMAbs were fully protected against the lethal effect of Stx2, as compared with the fatal outcome of the control group. Conclusions The results suggest that HuMAbs 5C12 and 5H8 promoted hepatic accumulation and presumably clearance of toxin/antibody complexes, significantly diverting Stx2 localization in the kidneys, the target of Stx2 and the

  15. Evaluation of (89Zr-labeled human anti-CD147 monoclonal antibody as a positron emission tomography probe in a mouse model of pancreatic cancer.

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    Aya Sugyo

    Full Text Available INTRODUCTION: Pancreatic cancer is an aggressive cancer and its prognosis remains poor. Therefore, additional effective therapy is required to augment and/or complement current therapy. CD147, high expression in pancreatic cancer, is involved in the metastatic process and is considered a good candidate for targeted therapy. CD147-specfic imaging could be useful for selection of appropriate patients. Therefore, we evaluated the potential of a fully human anti-CD147 monoclonal antibody 059-053 as a new positron emission tomography (PET probe for pancreatic cancer. METHODS: CD147 expression was evaluated in four pancreatic cancer cell lines (MIA Paca-2, PANC-1, BxPC-3, and AsPC-1 and a mouse cell line A4 as a negative control. Cell binding, competitive inhibition and internalization assays were conducted with (125I-, (67Ga-, or (89Zr-labeled 059-053. In vivo biodistribution of (125I- or (89Zr-labeled 059-053 was conducted in mice bearing MIA Paca-2 and A4 tumors. PET imaging with [(89Zr]059-053 was conducted in subcutaneous and orthotopic tumor mouse models. RESULTS: Among four pancreatic cancer cell lines, MIA Paca-2 cells showed the highest expression of CD147, while A4 cells had no expression. Immunohistochemical staining showed that MIA Paca-2 xenografts also highly expressed CD147 in vivo. Radiolabeled 059-053 specifically bound to MIA Paca-2 cells with high affinity, but not to A4. [(89Zr]059-053 uptake in MIA Paca-2 tumors increased with time from 11.0±1.3% injected dose per gram (ID/g at day 1 to 16.9±3.2% ID/g at day 6, while [(125I]059-053 uptake was relatively low and decreased with time, suggesting that 059-053 was internalized into tumor cells in vivo and (125I was released from the cells. PET with [(89Zr]059-053 clearly visualized subcutaneous and orthotopic tumors. CONCLUSION: [(89Zr]059-053 is a promising PET probe for imaging CD147 expression in pancreatic cancer and has the potential to select appropriate patients with CD147

  16. Localization of a membrane glycoprotein in benign fibrocystic disease and infiltrating duct carcinomas of the human breast with the use of a monoclonal antibody to guinea pig milk fat globule membrane.

    Science.gov (United States)

    Greenwalt, D. E.; Johnson, V. G.; Kuhajda, F. P.; Eggleston, J. C.; Mather, I. H.

    1985-01-01

    With monoclonal antibody D-274, raised against guinea pig milk fat globule membrane, the distribution of mucinlike glycoproteins of Mrs greater than or equal to 400,000 was determined in benign fibrocystic disease and infiltrating duct carcinoma of the human breast. These glycoproteins, called collectively PAS-I, were detected in 19 out of 20 cases of benign fibrocystic disease and in at least 26 out of 47 cases of infiltrating duct carcinoma. PAS-I was concentrated on luminal surfaces of ducts and alveoli in morphologically differentiated regions of the tumors. In areas where the glandular nature of the tissue was less evident in infiltrating duct carcinoma, the PAS-I determinant recognized by antibody D-274 was present on irregular luminal surfaces and in the cytoplasm. There was a negative correlation between the short-term recurrence (less than 2 years) of infiltrating duct carcinoma and the detection of strong positive staining with antibody D-274. The results are discussed with reference to recent studies on PAS-I in human breast tissue using monoclonal antibodies raised against human milk fat globule membrane. Images Figure 1 Figure 2 Figure 3 PMID:2579563

  17. The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum.

    OpenAIRE

    Bailyes, E M; Seabrook, R N; Calvin, J; Maguire, G A; Price, C P; Siddle, K; Luzio, J P

    1987-01-01

    1. Liver and bone alkaline phosphatase isoenzymes were solubilized with the zwitterionic detergent sulphobetaine 14, and purified to homogeneity by using a monoclonal antibody previously raised against a partially-purified preparation of the liver isoenzyme. Both purified isoenzymes had a specific activity in the range 1100-1400 mumol/min per mg of protein with a subunit Mr of 80,000 determined by SDS/polyacrylamide gel electrophoresis. Butanol extraction instead of detergent solubilization, ...

  18. Monoclonal antibodies in pediatric allergy

    Directory of Open Access Journals (Sweden)

    Amelia Licari

    2015-10-01

    Full Text Available Production of monoclonal antibodies (mAbs involving human-mouse hybrid cells was first described in 1970s, but these biologics are now used for a variety of diseases including cancers, autoimmune disorders and allergic diseases. The aim of this article is to review current and future applications of mAbs, in particular focusing on anti-IgE therapy, in the field of pediatric allergy. Proceedings of the 11th International Workshop on Neonatology and Satellite Meetings · Cagliari (Italy · October 26th-31st, 2015 · From the womb to the adultGuest Editors: Vassilios Fanos (Cagliari, Italy, Michele Mussap (Genoa, Italy, Antonio Del Vecchio (Bari, Italy, Bo Sun (Shanghai, China, Dorret I. Boomsma (Amsterdam, the Netherlands, Gavino Faa (Cagliari, Italy, Antonio Giordano (Philadelphia, USA

  19. Anticuerpos monoclonales contra la gonadotropina coriónica humana (hCG para su uso en la detección de embarazo Monoclonal antibodies against human chorionic gonadotropin (HCG for their use in pregnancy detection

    Directory of Open Access Journals (Sweden)

    Bertha V. Rodríguez Pendás

    2004-12-01

    Full Text Available Se reporta la generación de 2 anticuerpos monoclonales (AcM de ratón dirigidos contra la hormona gonadotropina coriónica humana (hCG, a partir de la inmunización de ratones BALB/c con hCG humana, purificada en el Instituto Nacional de Endocrinología (INEN. Los AcM obtenidos son de la clase IgG y fueron purificados a partir de líquido ascítico, mediante cromatografía de afinidad en proteína G Sepharosa. El estudio de afinidad y especificidad demostró que estos anticuerpos podían ser útiles en ensayos inmunoenzimáticos, con el uso de uno de ellos en el sistema microELISA, de nuestra institución, para la detección cualitativa de embarazo en orina.The generation of 2 mouse monoclonal antibodies directed against the human chorionic gonadotropin hormone (CGh, starting from the immunization of BALB/c mice with human CGh purified at the National Institute of Endocrinology (NIEN is reported. IgG monoclonal antibodies were obtained. They were purified starting from the ascitic fluid by affinity chromatography in protein G Sepharose. The affinity and specificity study showed that these antibodies could be useful in immunoenzimatic assays, using one of them in the microELISA system of our institution for the qualitative detection of pregnancy in urine.

  20. Development and characterization of a monoclonal antibody specific for human basophils and the identification of a unique secretory product of basophil activation.

    Science.gov (United States)

    McEuen, A R; Buckley, M G; Compton, S J; Walls, A F

    1999-01-01

    Despite increasing evidence that basophils can infiltrate into inflamed tissues during allergic reactions, determination of the extent of infiltration and elucidation of their role in allergic disease has been frustrated by the lack of reliable means for detecting this cell type in tissues. In the present study, we report on a new monoclonal antibody specific for basophils and on the initial characterization of the antigen it recognizes. Basophils were isolated from peripheral blood by Percoll density gradient centrifugation and a positive-selection immunomagnetic procedure and injected into mice to produce monoclonal antibodies. A hybridoma clone, designated BB1, secreted antibody of the IgG2a isotype; this antibody bound selectively to basophils on immunocytochemistry but did not react with any other cell type or tissue structure, although it did stain a proportion of cells from the basophilic cell line KU812F. In sections of mixed populations of peripheral blood cells, similar numbers of cells stained with Alcian blue dye and BB1 over a wide range of basophil purity. BB1 antibody was effective in identifying basophils in sections of mixed cells or in tissues after fixation with ethanol, Carnoy's solution, or formalin. Staining of basophils with BB1 gave a granular appearance, although flow cytometry indicated that some antigen was also present on the surface of the cell. Activation of these cells with anti-IgE antibody or with the calcium ionophore A23187 provoked release of the antigen in parallel with that of histamine. BB1 antibody did not, by itself, stimulate histamine release. The molecular mass of the antigen was determined on Hedrick-Smith gels to be 124+/-11 kd. This new monoclonal antibody will be a valuable experimental tool in future studies, allowing the reliable detection of basophils in tissues of patients with allergic and chronic inflammatory disease; in addition, the antigen it identifies has potential as a unique marker of basophil activation.

  1. HuEP5C7 as a humanized monoclonal anti-E/P-selectin neurovascular protective strategy in a blinded placebo-controlled trial of nonhuman primate stroke.

    Science.gov (United States)

    Mocco, J; Choudhri, Tanvir; Huang, Judy; Harfeldt, Elisabeth; Efros, Lyubov; Klingbeil, Corine; Vexler, Vladimir; Hall, William; Zhang, Yuan; Mack, William; Popilskis, Sulli; Pinsky, David J; Connolly, E Sander

    2002-11-15

    Although inhibiting interaction of beta(2) integrins with cognate immunoglobulin class adhesion receptor ligands is an effective neuroprotective strategy in small mammal models of stroke, the strategy has failed in human trials. A completely different antiadhesion receptor strategy was therefore rigorously tested in a model that may more closely approximate human reperfused stroke. Early leukoadhesive events in postischemic cerebral microvessels are mediated by upregulated selectin-class adhesion receptors on endothelial cells. Therefore, a blocking antibody prepared against common P- and E-selectin epitopes was humanized to suppress complement activation and tested in a reperfused hemispheric stroke model in Papio anubis (baboon). Histological examination of postischemic cerebral microvessels revealed a strong upregulation of E-and P-selectin expression. Placebo-blinded administration of the humanized anti-human E- and P-selectin monoclonal antibody (HuEP5C7, 20 mg/kg IV, n=9; placebo, n=9) immediately after the onset of 1 hour of temporary ischemia resulted in trends showing reduced polymorphonuclear leukocyte (PMN) infiltration into ischemic cortex, reduced infarct volumes (by 41%), improved neurological score (by 35%), and improved ability to self-care (by 39%). Importantly, there was no evidence of systemic complement activation, immune suppression, or pathological coagulopathy associated with this therapy. These data suggest that a humanized anti-E/P-selectin antibody approach is safe and may be effective as a clinical treatment for human stroke.

  2. Novel Clostridium difficile Anti-Toxin (TcdA and TcdB) Humanized Monoclonal Antibodies Demonstrate In Vitro Neutralization across a Broad Spectrum of Clinical Strains and In Vivo Potency in a Hamster Spore Challenge Model.

    Science.gov (United States)

    Qiu, Hongyu; Cassan, Robyn; Johnstone, Darrell; Han, Xiaobing; Joyee, Antony George; McQuoid, Monica; Masi, Andrea; Merluza, John; Hrehorak, Bryce; Reid, Ross; Kennedy, Kieron; Tighe, Bonnie; Rak, Carla; Leonhardt, Melanie; Dupas, Brian; Saward, Laura; Berry, Jody D; Nykiforuk, Cory L

    2016-01-01

    Clostridium difficile (C. difficile) infection (CDI) is the main cause of nosocomial antibiotic-associated colitis and increased incidence of community-associated diarrhea in industrialized countries. At present, the primary treatment of CDI is antibiotic administration, which is effective but often associated with recurrence, especially in the elderly. Pathogenic strains produce enterotoxin, toxin A (TcdA), and cytotoxin, toxin B (TcdB), which are necessary for C. difficile induced diarrhea and gut pathological changes. Administration of anti-toxin antibodies provides an alternative approach to treat CDI, and has shown promising results in preclinical and clinical studies. In the current study, several humanized anti-TcdA and anti-TcdB monoclonal antibodies were generated and their protective potency was characterized in a hamster infection model. The humanized anti-TcdA (CANmAbA4) and anti-TcdB (CANmAbB4 and CANmAbB1) antibodies showed broad spectrum in vitro neutralization of toxins from clinical strains and neutralization in a mouse toxin challenge model. Moreover, co-administration of humanized antibodies (CANmAbA4 and CANmAbB4 cocktail) provided a high level of protection in a dose dependent manner (85% versus 57% survival at day 22 for 50 mg/kg and 20 mg/kg doses, respectively) in a hamster gastrointestinal infection (GI) model. This study describes the protective effects conferred by novel neutralizing anti-toxin monoclonal antibodies against C. difficile toxins and their potential as therapeutic agents in treating CDI.

  3. Novel Clostridium difficile Anti-Toxin (TcdA and TcdB Humanized Monoclonal Antibodies Demonstrate In Vitro Neutralization across a Broad Spectrum of Clinical Strains and In Vivo Potency in a Hamster Spore Challenge Model.

    Directory of Open Access Journals (Sweden)

    Hongyu Qiu

    Full Text Available Clostridium difficile (C. difficile infection (CDI is the main cause of nosocomial antibiotic-associated colitis and increased incidence of community-associated diarrhea in industrialized countries. At present, the primary treatment of CDI is antibiotic administration, which is effective but often associated with recurrence, especially in the elderly. Pathogenic strains produce enterotoxin, toxin A (TcdA, and cytotoxin, toxin B (TcdB, which are necessary for C. difficile induced diarrhea and gut pathological changes. Administration of anti-toxin antibodies provides an alternative approach to treat CDI, and has shown promising results in preclinical and clinical studies. In the current study, several humanized anti-TcdA and anti-TcdB monoclonal antibodies were generated and their protective potency was characterized in a hamster infection model. The humanized anti-TcdA (CANmAbA4 and anti-TcdB (CANmAbB4 and CANmAbB1 antibodies showed broad spectrum in vitro neutralization of toxins from clinical strains and neutralization in a mouse toxin challenge model. Moreover, co-administration of humanized antibodies (CANmAbA4 and CANmAbB4 cocktail provided a high level of protection in a dose dependent manner (85% versus 57% survival at day 22 for 50 mg/kg and 20 mg/kg doses, respectively in a hamster gastrointestinal infection (GI model. This study describes the protective effects conferred by novel neutralizing anti-toxin monoclonal antibodies against C. difficile toxins and their potential as therapeutic agents in treating CDI.

  4. Safety, pharmacokinetics, and pharmacodynamics of AMG 102, a fully human hepatocyte growth factor-neutralizing monoclonal antibody, in a first-in-human study of patients with advanced solid tumors.

    Science.gov (United States)

    Gordon, Michael S; Sweeney, Christopher S; Mendelson, David S; Eckhardt, S Gail; Anderson, Abraham; Beaupre, Darrin M; Branstetter, Daniel; Burgess, Teresa L; Coxon, Angela; Deng, Hongjie; Kaplan-Lefko, Paula; Leitch, Ian M; Oliner, Kelly S; Yan, Lucy; Zhu, Min; Gore, Lia

    2010-01-15

    The aims were to assess the safety, pharmacokinetics, maximum tolerated dose, and antitumor activity of AMG 102, a fully human hepatocyte growth factor/scatter factor (HGF/SF)-neutralizing monoclonal antibody, in patients with solid tumors. Patients (N = 40) with refractory advanced solid tumors were enrolled into six sequential dose-escalation cohorts (0.5, 1, 3, 5, 10, or 20 mg/kg AMG 102 i.v. every 2 weeks) and a dose-expansion cohort (20 mg/kg AMG 102 every 2 weeks). Safety, anti-AMG 102 antibody formation, pharmacokinetics, tumor response, and exploratory biomarkers were assessed. AMG 102 was well tolerated up to the planned maximum dose of 20 mg/kg, and the maximum tolerated dose was not reached. Treatment-related adverse events were generally mild and included fatigue (13%), constipation (8%), nausea (8%), vomiting (5%), anorexia (5%), myalgia (5%), and hypertension (5%). Two patients experienced dose-limiting toxicities: one patient (0.5 mg/kg cohort) experienced grade 3 hypoxia and grade 3 dyspnea and one patient (1 mg/kg cohort) experienced grade 3 upper gastrointestinal hemorrhage. No anti-AMG 102 antibodies were detected, and AMG 102 had linear pharmacokinetics within the dose range investigated. Sixteen of 23 (70%) evaluable patients had a best response of stable disease with progression-free survival ranging from 7.9 to 40 weeks. Circulating levels of the biomarker HGF/SF (bound and unbound) increased in a dose-dependent manner, whereas soluble c-Met concentrations were generally similar across doses. AMG 102 is safe and well tolerated, has a favorable pharmacokinetic profile, and will be further investigated as a monotherapy and in combination with other agents.

  5. Nuclear oncology with monoclonal antibodies and peptides

    Energy Technology Data Exchange (ETDEWEB)

    Hosono, Makoto [Saitama Medical School, Kawagoe (Japan). Saitama Medical Center

    1998-10-01

    Imaging and therapy using radiolabeled monoclonal antibodies have proved useful in many clinical studies. However, immunogenicity of mouse antibodies to human and insufficient tumor-to-normal tissue ratios remained to be solved. Chimerization and humanization by genetic engineering, and multistep targeting techniques have enabled lower immunogenicity and higher tumor-to-normal tissue contrast. Peptides like somatostatin-analogs have been reportedly useful in imaging tumors, which are either somatostatin receptor positive or negative. Elevated normal tissue accumulation of radiolabeled peptides is a drawback in aiming internal radiation therapy. (author). 51 refs.

  6. Stromal cells from human long-term marrow cultures, but not cultured marrow fibroblasts, phagocytose horse serum constituents: studies with a monoclonal antibody that reacts with a species-specific epitope common to multiple horse serum proteins.

    Science.gov (United States)

    Charbord, P; Tippens, D; Wight, T S; Gown, A M; Singer, J W

    1987-01-01

    This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These data indicate that marrow stromal cells specifically accumulate horse serum proteins which might partially explain the horse serum requirement for long-term marrow culture maintenance. The data also suggest further similarities between marrow stromal and smooth muscle cells and additional differences between marrow fibroblasts and marrow stromal cells.

  7. A fundamental study of immunoscintigraphy with sup 131 I-labeled anti-CA 19-9 and anti-CEA monoclonal antibodies; Imaging of tumor-bearing mice by IMACIS-1 and cell ELISA with human tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Nogami, Toshihiko; Miura, Hiroshi; Ohmi, Shoichi; Kazahaya, Yasuhiro (CIS DIAGNOSTIC K.K., Chiba (Japan))

    1990-05-01

    A study was made on 2 types of {sup 131}I-labeled anti-CA 19-9 and anti-CEA mouse monoclonal antibodies (IMACIS-1) against human cancer related antigen as to their usefulness in radioimmunoimaging. Tumor-bearing nude mice were used for comparison. The transplanted tumors (SW948, COLO 201) were clearly visualized 48-72 hours after administration of IMACIS-1. Tumor/blood ratio 72 hours after administration: 8.69 in COLO 201 and 5.70 in SW948, showing ca. 10-15 times as high as those in PC-3 and HEp-2. IMACIS-1 therefore is considered useful in radioimmunoimaging of cancer. Analysis was made by in vitro cell ELISA. As a result, both of the cells specifically reacted with anti-CA 19-9 but not anti-CEA. (author).

  8. Site-directed in vitro immunization leads to a complete human monoclonal IgG4λ that binds specifically to the CDR2 region of CTLA-4 (CD152 without interfering the engagement of natural ligands

    Directory of Open Access Journals (Sweden)

    Hsu Shu-Ching

    2007-08-01

    Full Text Available Abstract Background The ability to acquire fully human monoclonal antibodies (mAbs with pre-defined specificities is critical to the development of molecular tags for the analysis of receptor function in addition to promising immunotherapeutics. Yet most of the arriving affinity maturated and complete human immunoglobulin G (IgG molecules, which are actually derived from single human B cells, have not widely been used to study the conserved self antigens (Ags such as CD152 (cytotoxic T lymphocyte antigen-4, CTLA-4 because proper hosts are lacking. Results Here we developed an optimized protocol for site-directed in vitro immunizing peripheral blood mononuclear cells (PBMC by using a selected epitope of human CD152, an essential receptor involved in down-regulation of T cell activation. The resultant stable trioma cell lines constantly produce anti-CD152 mAb (γ4λhuCD152, which contains variable (V regions of the heavy chain and the light chain derived from the VH3 and Vλ human germline genes, respectively, and yet displays an unusual IgG4 isotype. Interestingly, γ4λhuCD152 has a basic pI not commonly found in myeloid monoclonal IgG4λs as revealed by the isoelectric focusing (IEF analysis. Furthermore, γ4λhuCD152 binds specifically, with nanomolar affinity, to an extracellular constituency encompassing the putative second complementarity determining region (CDR2 of CD152, whereby it can react to activated CD3+ cells. Conclusion In a context of specific cell depletion and conditioned medium,in vitro induction of human Abs against a conserved self Ag was successfully acquired and a relatively basic mAb, γ4λhuCD152, with high affinity to CDR2 of CD152 was thus obtained. Application of such a human IgG4λ mAb with designated CDR2 specificity may impact upon and prefer for CD152 labeling both in situ and ex situ, as it does not affect the binding of endogenous B7 ligands and can localize into the confined immunological synapse which may

  9. Polyclonal and monoclonal antibodies in clinic.

    Science.gov (United States)

    Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

    2014-01-01

    Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic.

  10. Monoclonal antibodies against genetically manipulated hepatitis B core antigen.

    Science.gov (United States)

    Hlozánek, I; Korec, E; Dostálová, V; Stará, J; König, J; Bichko, V V; Seichertová, A; Gren, E J

    1987-01-01

    Four different hybridoma clones secreting anti-HBcAg antibodies were constructed by fusing cells of the mouse myeloma line SP2/0 with lymphocytes from mice immunized with bacterially produced HBcAg. The monoclonal antibodies were immunologically characterized and used for HBcAg detection by ELISA. This monoclonal-antibody-based assay was compared with ELISA based on polyclonal human anti-HBcAg IgG for sensitivity and specificity. The monoclonal antibody reacted specifically both with the bacterially produced HBcAg and HBcAg isolated from human liver, but did not react with HBeAg. The human polyclonal antibody reacted with HBcAg, but also with HBeAg.

  11. Efficacy of Urtoxazumab (TMA-15 Humanized Monoclonal Antibody Specific for Shiga Toxin 2 Against Post-Diarrheal Neurological Sequelae Caused by Escherichia coli O157:H7 Infection in the Neonatal Gnotobiotic Piglet Model

    Directory of Open Access Journals (Sweden)

    Rodney A. Moxley

    2017-01-01

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC is the most common cause of hemorrhagic colitis and hemolytic uremic syndrome in human patients, with brain damage and dysfunction the main cause of acute death. We evaluated the efficacy of urtoxazumab (TMA-15, Teijin Pharma Limited, a humanized monoclonal antibody against Shiga toxin (Stx 2 for the prevention of brain damage, dysfunction, and death in a piglet EHEC infection model. Forty-five neonatal gnotobiotic piglets were inoculated orally with 3 × 109 colony-forming units of EHEC O157:H7 strain EDL933 (Stx1+, Stx2+ when 22–24 h old. At 24 h post-inoculation, piglets were intraperitoneally administered placebo or TMA-15 (0.3, 1.0 or 3.0 mg/kg body weight. Compared to placebo (n = 10, TMA-15 (n = 35 yielded a significantly greater probability of survival, length of survival, and weight gain (p <0.05. The efficacy of TMA-15 against brain lesions and death was 62.9% (p = 0.0004 and 71.4% (p = 0.0004, respectively. These results suggest that TMA-15 may potentially prevent or reduce vascular necrosis and infarction of the brain attributable to Stx2 in human patients acutely infected with EHEC. However, we do not infer that TMA-15 treatment will completely protect human patients infected with EHEC O157:H7 strains that produce both Stx1 and Stx2.

  12. A specific assay for quantification of human C4c by use of an anti-C4c monoclonal antibody

    DEFF Research Database (Denmark)

    Pilely, Katrine; Skjoedt, Mikkel-Ole; Nielsen, Christian

    2014-01-01

    a mouse monoclonal antibody (mAb) that is able to detect fluid phase C4c without interference from other products generated from the complement component C4. The C4c specific mAb was tested in different enzyme-linked immunosorbent assay (ELISA) combinations with various types of in vitro activated sera......, with a mean of 0.85mg/L and a range of 0.19-2.21mg/L. We believe that the C4c specific antibody and the ELISA might be important tools in the future assessment of in vivo activation in situations where the classical or the lectin complement pathways are involved in the pathogenesis....... and samples from factor I deficient patients. The specificity of the mAb was further evaluated by immunoprecipitation techniques and by analysis of eluted fragments of C4 after immunoaffinity chromatography. The anti-C4c mAb was confirmed to be C4c specific, as it showed no cross-reactivity with native (un...

  13. Inhibition of P-Selectin and PSGL-1 Using Humanized Monoclonal Antibodies Increases the Sensitivity of Multiple Myeloma Cells to Bortezomib

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    Barbara Muz

    2015-01-01

    Full Text Available Multiple myeloma (MM is a plasma cell malignancy localized in the bone marrow. Despite the introduction of novel therapies majority of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1 play a key role in proliferation of MM and using small-molecule inhibitors of P-selectin/PSGL-1 sensitized MM cells to therapy. However, these small-molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. Therefore, we tested blocking of P-selectin and PSGL-1 using functional monoclonal antibodies in order to sensitize MM cells to therapy. We have demonstrated that inhibiting the interaction between MM cells and endothelial and stromal cells decreased proliferation in MM cells and in parallel induced loose-adhesion to the primary tumor site to facilitate egress. At the same time, blocking this interaction in vivo led to MM cells retention in the circulation and delayed homing to the bone marrow, thus exposing MM cells to bortezomib which contributed to reduced tumor growth and better mice survival. This study provides a better understanding of the biology of P-selectin and PSGL-1 and their roles in dissemination and resensitization of MM to treatment.

  14. Targeting the Human T-Cell Inducible COStimulator Molecule with a Monoclonal Antibody Prevents Graft-vs-Host Disease and Preserves Graft vs Leukemia in a Xenograft Murine Model

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    Aude Burlion

    2017-06-01

    Full Text Available BackgroundGraft-vs-host disease (GVHD is a major complication of allogenic bone marrow transplantation (BMT. Targeting costimulatory molecules with antagonist antibodies could dampen the excessive immune response that occurs, while preserving the beneficial graft vs leukemia (GVL of the allogeneic response. Previous studies using a mouse model of GVHD have shown that targeting the T-cell Inducible COStimulator (ICOS, CD278 molecule is beneficial, but it is unclear whether the same applies to human cells.MethodsHere, we assessed whether a monoclonal antibody (mAb to human ICOS was able to antagonize the costimulatory signal delivered in vivo to human T cells. To test this hypothesis, we used a xenogeneic model of GVHD where human peripheral blood mononuclear cells were adoptively transferred in immunocompromised NOD.SCID.gc-null mice (NSG.ResultsIn this model, control mice invariably lost weight and died by day 50. In contrast, 65% of the mice receiving a single injection of the anti-hICOS mAb survived beyond 100 days. Moreover, a significant improvement in survival was obtained in a curative xeno-GVHD setting. Mechanistically, administration of the anti-hICOS mAb was associated with a strong reduction in perivascular infiltrates in liver and lungs and reduction in frequencies and numbers of human T cells in the spleen. In addition, the mAb prevented T-cell expansion in the blood during xeno-GVHD. Importantly, GVHD-protected mice retained the ability to control the P815 mastocytoma cell line, mimicking GVL in humans.ConclusionA mAb-targeting human ICOS alleviated GVHD without impairing GVL in a xenograft murine model. Thus, ICOS represents a promising target in the management of BMT, preventing GVHD while preserving GVL.

  15. Fragmentation of monoclonal antibodies

    Science.gov (United States)

    Vlasak, Josef

    2011-01-01

    Fragmentation is a degradation pathway ubiquitously observed in proteins despite the remarkable stability of peptide bond; proteins differ only by how much and where cleavage occurs. The goal of this review is to summarize reports regarding the non-enzymatic fragmentation of the peptide backbone of monoclonal antibodies (mAbs). The sites in the polypeptide chain susceptible to fragmentation are determined by a multitude of factors. Insights are provided on the intimate chemical mechanisms that can make some bonds prone to cleavage due to the presence of specific side-chains. In addition to primary structure, the secondary, tertiary and quaternary structures have a significant impact in modulating the distribution of cleavage sites by altering local flexibility, accessibility to solvent or bringing in close proximity side chains that are remote in sequence. This review focuses on cleavage sites observed in the constant regions of mAbs, with special emphasis on hinge fragmentation. The mechanisms responsible for backbone cleavage are strongly dependent on pH and can be catalyzed by metals or radicals. The distribution of cleavage sites are different under acidic compared to basic conditions, with fragmentation rates exhibiting a minimum in the pH range 5–6; therefore, the overall fragmentation pattern observed for a mAb is a complex result of structural and solvent conditions. A critical review of the techniques used to monitor fragmentation is also presented; usually a compromise has to be made between a highly sensitive method with good fragment separation and the capability to identify the cleavage site. The effect of fragmentation on the function of a mAb must be evaluated on a case-by-case basis depending on whether cleavage sites are observed in the variable or constant regions, and on the mechanism of action of the molecule. PMID:21487244

  16. The classification of Sejroe group serovars of Leptospira interrogans with monoclonal antibodies

    NARCIS (Netherlands)

    Terpstra, W. J.; Korver, H.; van Leeuwen, J.; Klatser, P. R.; Kolk, A. H.

    1985-01-01

    Using the hybridoma technique we produced monoclonal antibodies to serovars of Leptospira interrogans. We focussed on serovar hardjo which is an important pathogen for humans and animals, and on other serovars of the Sejroe group. With combinations of monoclonals, characteristic patterns of

  17. An ELISA-inhibition test using monoclonal antibody for the serology of leprosy

    NARCIS (Netherlands)

    Klatser, P. R.; de Wit, M. Y.; Kolk, A. H.

    1985-01-01

    In this study a mouse monoclonal antibody (47-9) is described, which recognized an epitope on the 36 kD protein antigen of M. leprae. The monoclonal antibody showed specificity for M. leprae. An ELISA-inhibition test based on the competitive inhibition by antibodies from human test sera of the

  18. Monoclonal gammopathies of renal significance.

    Science.gov (United States)

    Caravaca-Fontán, Fernando; Gutiérrez, Eduardo; Delgado Lillo, Ramón; Praga, Manuel

    The term monoclonal gammopathy of renal significance (MGRS) comprises a group of diseases pathogenetically characterised by proliferation of a B-cell or plasma cell clone that synthesises and secretes a monoclonal immunoglobulin or its components (light and/or heavy chains), that may deposit and cause glomerular, tubular, interstitial and/or vascular damage. The importance of differentiating the term MGRS from other monoclonal gammopathies lies in the fact that diagnostic and therapeutic procedures aimed at controlling monoclonal protein synthesis and secretion can be indicated, irrespective of the classic criteria based on malignant tumour expansion. Renal pathology associated with MGRS is highly heterogeneous, and therefore renal biopsy should be considered a key diagnostic tool. A precise diagnostic approach, however, must also identify the monoclonal protein in plasma and/or in urine, together with a complete haematological study in order to determine the nature and extension of cell clones. Recent advances in the understanding of these entities have resulted in significant improvements in clinical course and survival in several forms of MGRS, although more studies and clinical experience are needed in order to delineate more effective therapeutic strategies. In this review, we summarise the main clinical and pathological features of MGRS, highlighting the most appropriate diagnostic approach and current therapeutic options. Copyright © 2017 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.

  19. Epitope of titin A-band-specific monoclonal antibody Tit1 5 H1.1 is highly conserved in several Fn3 domains of the titin molecule. Centriole staining in human, mouse and zebrafish cells

    Directory of Open Access Journals (Sweden)

    Mikelsaar Aavo-Valdur

    2012-09-01

    Full Text Available Abstract Background Previously we have reported on the development of a new mouse anti-titin monoclonal antibody, named MAb Titl 5 H1.1, using the synthetic peptide N-AVNKYGIGEPLESDSVVAK-C which corresponds to an amino acid sequence in the A-region of the titin molecule as immunogen. In the human skeletal muscles, MAb Titl 5 H1.1 reacts specifically with titin in the A-band of the sarcomere and in different non-muscle cell types with nucleus and cytoplasm, including centrioles. In this report we have studied the evolutionary aspects of the binding of MAb Tit1 5 H1.1 with its target antigen (titin. Results We have specified the epitope area of MAb Tit1 5 H1.1 by subpeptide mapping to the hexapeptide N-AVNKYG-C. According to protein databases this amino acid sequence is located in the COOH-terminus of several different Fn3 domains of the A-region of titin molecule in many organisms, such as human being, mouse, rabbit, zebrafish (Danio rerio, and even in sea squirt (Ciona intestinalis. Our immunohisto- and cytochemical studies with MAb Tit1 5 H1.1 in human, mouse and zebrafish tissues and cell cultures showed a striated staining pattern in muscle cells and also staining of centrioles, cytoplasm and nuclei in non-muscle cells. Conclusions The data confirm that titin can play, in addition to the known roles in striated muscle cells also an important role in non-muscle cells as a centriole associated protein. This phenomenon is highly conserved in the evolution and is related to Fn3 domains of the titin molecule. Using titin A-band-specific monoclonal antibody MAb Tit1 5 H1.1 it was possible to locate titin in the sarcomeres of skeletal muscle cells and in the centrioles, cytoplasm and nuclei of non-muscle cells in phylogenetically so distant organisms as Homo sapiens, Mus musculus and zebrafish (Danio rerio.

  20. A phase II study of a human anti-PDGFRα monoclonal antibody (olaratumab, IMC-3G3) in previously treated patients with metastatic gastrointestinal stromal tumors.

    Science.gov (United States)

    Wagner, A J; Kindler, H; Gelderblom, H; Schöffski, P; Bauer, S; Hohenberger, P; Kopp, H-G; Lopez-Martin, J A; Peeters, M; Reichardt, P; Qin, A; Nippgen, J; Ilaria, R L; Rutkowski, P

    2017-03-01

    This study evaluated tumor response to olaratumab (an anti-PDGFRα monoclonal antibody) in previously treated patients with metastatic gastrointestinal stromal tumor (GIST) with or without PDGFRα mutations (cohorts 1 and 2, respectively). Patients received olaratumab 20 mg/kg intravenously every 14 days until disease progression, death, or intolerable toxicity occurred. Outcome measures were 12-week tumor response, progression-free survival (PFS), overall survival (OS), and safety. Of 30 patients enrolled, 21 patients received ≥1 dose of olaratumab. In the evaluable population (cohort 1, n = 6; cohort 2, n = 14), no complete response (CR) or partial response (PR) was observed. Stable disease (SD) was observed in 3 patients (50.0%) in cohort 1 and 2 patients (14.3%) in cohort 2. Progressive disease (PD) was observed in 3 patients (50.0%) in cohort 1 and 12 patients (85.7%) in cohort 2. The 12-week clinical benefit rate (CR + PR + SD) (90% CI) was 50.0% (15.3-84.7%) in cohort 1 and 14.3% (2.6-38.5%) in cohort 2. SD lasted beyond 12 weeks in 5 patients (cohort 1, n = 3; cohort 2, n = 2). Median PFS (90% CI) was 32.1 (5.0-35.9) weeks in cohort 1 and 6.1 (5.7-6.3) weeks in cohort 2. Median OS was not reached in cohort 1 and was 24.9 (14.4-49.1) weeks in cohort 2. All patients in cohort 1 and 9 (64.3%) in cohort 2 experienced an olaratumab-related adverse event (AE), most commonly fatigue (38.1%), nausea (19.0%), and peripheral edema (14.3%). Two grade ≥3 olaratumab-related events were reported (cohort 1, syncope; cohort 2, hypertension). Olaratumab had an acceptable AE profile in patients with GIST. While there was no apparent effect on PFS in patients without PDGFRα mutations, patients with PDGFRα-mutant GIST (all with D842V mutations) treated with olaratumab had longer disease control compared with historical data for this genotype. NCT01316263.

  1. Detection of Campylobacter species using monoclonal antibodies

    Science.gov (United States)

    Young, Colin R.; Lee, Alice; Stanker, Larry H.

    1999-01-01

    A panel of species specific monoclonal antibodies were raised to Campylobacter coli, Campylobacter jejuni and Campylobacter lari. The isotypes, and cross-reactivity profiles of each monoclonal antibody against an extensive panel of micro- organisms, were determined.

  2. Uses of monoclonal antibody 8H9

    Science.gov (United States)

    Cheung, Nai-Kong V.

    2013-04-09

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  3. Generation and characterization of a monoclonal antibody to ...

    African Journals Online (AJOL)

    Penicillic acid is one of the main mycotoxins in moldy feedstuff and has toxic effect on livestock and poultry and probably humans due to food chain transmission. The objective of this study was to generate and characterize a monoclonal antibody to penicillic acid for the efficient detection of penicillic acid from Penicillium ...

  4. Prediction of clinical pharmacokinetics of AMG 181, a human anti-α 4 β 7 monoclonal antibody for treating inflammatory bowel diseases.

    Science.gov (United States)

    Li, Hong; Köck, Kathleen; Wisler, John A; Rees, William A; Prince, Peter J; Reynhardt, Kai O; Hsu, Hailing; Yu, Zhigang; Borie, Dominic C; Salinger, David H; Pan, Wei-Jian

    2015-02-01

    The purpose of this study was to predict a safe starting dose of AMG 181, a human anti-α 4 β 7 antibody for treating inflammatory bowel diseases, based on cynomolgus monkey pharmacokinetic (PK) and pharmacodynamic (PD) data. A two-compartment model with parallel linear and target-mediated drug disposition for AMG 181 PK in cynomolgus monkey was developed. The estimated parameters were allometrically scaled to predict human PK. An E max PD model was used to relate AMG 181 concentration and free α 4 β 7 receptor data in cynomolgus monkey. AMG 181 clinical doses were selected based on observed exposures at the no adverse effect level of 80 mg·kg(-1) in monkeys, the predicted human exposures, and AMG 181 concentration expected to produce greater than 50% α 4 β 7 receptor occupancy in humans. The predicted human AMG 181 clearance and central volume of distribution were 144 mL·day(-1) and 2900 mL, respectively. The estimated EC50 for free α 4 β 7 receptor was 14 ng·mL(-1). At the 0.7 mg starting dose in humans, the predicted exposure margins were greater than 490,000 and AMG 181 concentrations were predicted to only briefly cover the free α 4 β 7 receptor EC10. Predictions for both C max and AUC matched with those observed in the first-in-human study within the 7 mg subcutaneous to 420 mg intravenous dose range. The developed model aided in selection of a safe starting dose and a pharmacological relevant dose escalation strategy for testing of AMG 181 in humans. The clinically observed human AMG 181 PK data validated the modeling approach based on cynomolgus monkey data alone.

  5. Enhancement of retroviral infection in vitro by anti-Le(y) IgG: reversal by humanization of monoclonal mouse antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Sørensen, A M; Arendrup, M

    1993-01-01

    also enhanced infection, a human/mouse chimeric antibody and a fully humanized antibody had no enhancing effect on free virus infection. We suggest that binding of anti-Le(y) ABL 364 or its F(ab)2 fragment induced a conformational change in the gp120 oligomers facilitating the process of infection......, and that this function was abrogated by the IgG1 Fc of the chimeric and the humanized antibodies. The observations indicate that the non-paratope domains of antiviral antibodies can influence their function as neutralizing or enhancing for infection....

  6. Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq.

    Science.gov (United States)

    Busby, Michele; Xue, Catherine; Li, Catherine; Farjoun, Yossi; Gienger, Elizabeth; Yofe, Ido; Gladden, Adrianne; Epstein, Charles B; Cornett, Evan M; Rothbart, Scott B; Nusbaum, Chad; Goren, Alon

    2016-01-01

    The robustness of ChIP-seq datasets is highly dependent upon the antibodies used. Currently, polyclonal antibodies are the standard despite several limitations: They are non-renewable, vary in performance between lots and need to be validated with each new lot. In contrast, monoclonal antibody lots are renewable and provide consistent performance. To increase ChIP-seq standardization, we investigated whether monoclonal antibodies could replace polyclonal antibodies. We compared monoclonal antibodies that target five key histone modifications (H3K4me1, H3K4me3, H3K9me3, H3K27ac and H3K27me3) to their polyclonal counterparts in both human and mouse cells. Overall performance was highly similar for four monoclonal/polyclonal pairs, including when we used two distinct lots of the same monoclonal antibody. In contrast, the binding patterns for H3K27ac differed substantially between polyclonal and monoclonal antibodies. However, this was most likely due to the distinct immunogen used rather than the clonality of the antibody. Altogether, we found that monoclonal antibodies as a class perform equivalently to polyclonal antibodies for the detection of histone post-translational modifications in both human and mouse. Accordingly, we recommend the use of monoclonal antibodies in ChIP-seq experiments.

  7. Monoclonal antibodies: application in radiopharmacy.

    Science.gov (United States)

    Ligiero, Thais Braga; de Souza Albernaz, Marta; de Carvalho, Samira Marques; de Oliveira, Silvia Maria Velasques; Santos-Oliveira, Ralph

    2013-12-01

    In this study was carried on a systematic review of the data was carried out in the topic of monoclonal antibodies in the last 40 years. All the data collected and summarized revealed that this new class of medicine may bring great advance in the field of radiopharmacy, oncology and imaging.

  8. Mapping of rat brain using the Synuclein-1 monoclonal antibody reveals somatodendritic expression of alpha-synuclein in populations of neurons homologous to those vulnerable to Lewy body formation in human synucleopathies.

    Science.gov (United States)

    Andringa, Gerda; Du, Fu; Chase, Thomas N; Bennett, M Catherine

    2003-10-01

    The neuronal protein alpha-synuclein has been implicated in the pathogenesis of Parkinson disease and other neurodegenerative diseases. Although many studies report that alpha-synuclein expression is restricted to neuronal presynaptic terminals, this protein aggregates in Lewy bodies in somata that are typically distant from their axon terminals. Few studies have addressed this paradox and there has been no compelling explanation proposed for the apparent discrepancy between the locus of neuronal alpha-synuclein expression and the loci of Lewy bodies in the majority of Parkinson disease cases. We explored this issue by extensively characterizing the monoclonal antibody Synuclein-1 (Syn-1) and using this highly selective antibody to map the distribution of alpha-synuclein throughout rat brain and in human substantia nigra (SN). Additionally, alpha-synuclein expression in rat SN detected by 2 polyclonal antibodies against alpha-synuclein was compared with that detected by the Syn-1 antibody. In contrast with many previous reports, alpha-synuclein was detected by Syn-1 in neuronal somata and dendrites in restricted brain regions, as well as more ubiquitously in axons and terminals. The strongest alpha-synuclein neuronal expression in rat was found in brainstem and cortical regions that are homologous to regions prone to Lewy body formation in humans. The Syn-1 antibody labeled abundant somatodendritic alpha-synuclein in both rat and human SN, a major locus of Lewy body formation and neurodegeneration in Parkinson disease. By contrast, very few immunoreactive somata in the rat SN were labeled by the 2 polyclonal antibodies. We explore possible explanations for the differences in conflicting reports of patterns of alpha-synuclein expression in brain, including differences among antibodies.

  9. A mixture of functionally oligoclonal humanized monoclonal antibodies that neutralize Clostridium difficile TcdA and TcdB with high levels of in vitro potency shows in vivo protection in a hamster infection model.

    Science.gov (United States)

    Davies, Nicola L; Compson, Joanne E; Mackenzie, Brendon; O'Dowd, Victoria L; Oxbrow, Amanda K F; Heads, James T; Turner, Alison; Sarkar, Kaushik; Dugdale, Sarah L; Jairaj, Mark; Christodoulou, Louis; Knight, David E O; Cross, Amanda S; Hervé, Karine J M; Tyson, Kerry L; Hailu, Hanna; Doyle, Carl B; Ellis, Mark; Kriek, Marco; Cox, Matthew; Page, Matthew J T; Moore, Adrian R; Lightwood, Daniel J; Humphreys, David P

    2013-03-01

    Clostridium difficile infections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments, C. difficile infection (CDI) is still a major cause of patient suffering, death, and substantial health care costs. Clostridium difficile exerts its major pathological effects through the actions of two protein exotoxins, TcdA and TcdB, which bind to and disrupt gut tissue. Antibiotics target the infecting bacteria but not the exotoxins. Administering neutralizing antibodies against TcdA and TcdB to patients receiving antibiotic treatment might modulate the effects of the exotoxins directly. We have developed a mixture of three humanized IgG1 monoclonal antibodies (MAbs) which neutralize TcdA and TcdB to address three clinical needs: reduction of the severity and duration of diarrhea, reduction of death rates, and reduction of the rate of recurrence. The UCB MAb mixture showed higher potency in a variety of in vitro binding and neutralization assays (∼10-fold improvements), higher levels of protection in a hamster model of CDI (82% versus 18% at 28 days), and higher valencies of toxin binding (12 versus 2 for TcdA and 3 versus 2 for TcdB) than other agents in clinical development. Comparisons of the MAb properties also offered some insight into the potential relative importance of TcdA and TcdB in the disease process.

  10. Renal involvement in secondary amyloidosis of Muckle-Wells syndrome: marked improvement of renal function and reduction of proteinuria after therapy with human anti-interleukin-1β monoclonal antibody canakinumab.

    Science.gov (United States)

    Scarpioni, Roberto; Rigante, Donato; Cantarini, Luca; Ricardi, Marco; Albertazzi, Vittorio; Melfa, Luigi; Lazzaro, Antonio

    2015-07-01

    Muckle-Wells syndrome (MWS) is a rare hereditary autoinflammatory disorder characterized by recurrent urticaria-like skin rashes, arthralgias, conjunctivitis, hypoacusia, and risk of reactive AA amyloidosis due to the progressive accumulation of amyloid fibrils in different organs. Its genetic defect lies in mutations in the NLRP3 gene, encoding the cryopyrin protein, and resulting in interleukin (IL)-1β oversecretion. Renal involvement, in terms of proteinuria or renal insufficiency, can be observed in up to 25% of patients. Herein, we describe our experience with two Caucasian patients, father and son, aged 52 and 26 years, respectively, heterozygous for both V198M and R260W NLRP3 mutations who had AA amyloid deposits on renal biopsy. The fully human monoclonal antibody canakinumab, providing selective and prolonged IL-1β blockade, was administered in both patients every 60 days over a period of 18 months. This treatment allowed to obtain amazing results: a rapid disappearance of any clinical symptoms, the stable normalization of serum amyloid-A and, furthermore, a marked improvement of glomerular filtration rate and proteinuria with no adverse events. Our data, though limited to only two patients, emphasize that therapeutic intervention with canakinumab, suppressing both inflammation and IL-1β-mediated manifestations, can contribute to improve kidney function in MWS with overt renal amyloidosis.

  11. Phase 1/2 study of ocaratuzumab, an Fc-engineered humanized anti-CD20 monoclonal antibody, in low-affinity FcγRIIIa patients with previously treated follicular lymphoma.

    Science.gov (United States)

    Ganjoo, Kristen N; de Vos, Sven; Pohlman, Brad L; Flinn, Ian W; Forero-Torres, Andres; Enas, Nathan H; Cronier, Damien M; Dang, Nam H; Foon, Kenneth A; Carpenter, Susan P; Slapak, Christopher A; Link, Brian K; Smith, Mitchell R; Mapara, Markus Y; Wooldridge, James E

    2015-01-01

    This phase 2 study assessed the safety and efficacy of ocaratuzumab, a humanized anti-CD20 monoclonal antibody. Fifty patients with previously treated follicular lymphoma (FL) and a low-affinity genotype of FcγRIIIa received ocaratuzumab 375 mg/m(2) weekly for 4 weeks. Grade 3/4/5 adverse events (AEs) were reported in 11/1/1 patients, respectively. Serious AEs were reported by 11/50 patients, and three discontinued due to AEs. One patient died from aspiration pneumonia due to possibly drug-related nausea and vomiting. Investigator-assessed response rate was 30% (15/50), including four complete responses (CR), three CR unconfirmed (CRu) and eight partial responses (PR). Investigator-assessed median Progression-free survivial (PFS) was 38.3 weeks. Ocaratuzumab's pharmacokinetic profile was similar to that reported for rituximab. Lymphocyte subset analysis showed significant, selective reduction of B-cells during and after ocaratuzumab treatment. Ocaratuzumab at this dose and schedule is active and well tolerated in patients with previously treated FL with low affinity FcγRIIIa genotypes. ClinTrials registry number: NCT00354926.

  12. Phase Separation in Solutions of Monoclonal Antibodies

    Science.gov (United States)

    Benedek, George; Wang, Ying; Lomakin, Aleksey; Latypov, Ramil

    2012-02-01

    We report the observation of liquid-liquid phase separation (LLPS) in a solution of humanized monoclonal antibodies, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective inter-protein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable protein condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.

  13. Generation and epitope analysis of human monoclonal antibody isotypes with specificity for the timothy grass major allergen Phl p 5a

    DEFF Research Database (Denmark)

    Hecker, J.; Diethers, A.; Seismann, H.

    2011-01-01

    and cross-reactivity, standardisation of allergens as well as improvement of allergy diagnostics and therapeutics. Here we report the generation and application of the first set of authentic human IgG, IgE and IgA antibodies. On the basis of a Phl p 5a specific antibody fragment, a lambda light chain...... of the antibodies with the allergen was assessed. Applicability in allergy diagnostics was confirmed by establishment of artificial human sera. Functionality of both antibodies was further demonstrated in receptor binding studies and mediator release assays using humanised rat basophil leukaemia cells (RBL-SX38...

  14. Single-domain monoclonal antibodies for the treatment of hepatocellular carcinoma | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Cancer Institute seeks parties to license human monoclonal antibodies and immunoconjugates and co-develop, evaluate, and/or commercialize large-scale antibody production and hepatocellular carcinoma (HCC) xenograft mouse models.

  15. Antigen-Specific Monoclonal Antibodies Isolated from B Cells Expressing Constitutively Active STAT5

    NARCIS (Netherlands)

    Scheeren, F.A.; van Geelen, C.M.M.; Yasuda, E.; Spits, H.; Beaumont, T.

    2011-01-01

    Background: Fully human monoclonal antibodies directed against specific pathogens have a high therapeutic potential, but are difficult to generate. Methodology/Principal Findings: Memory B cells were immortalized by expressing an inducible active mutant of the transcription factor Signal Transducer

  16. Monoclonal Antibodies 13A4 and AC133 Do Not Recognize the Canine Ortholog of Mouse and Human Stem Cell Antigen Prominin-1 (CD133.

    Directory of Open Access Journals (Sweden)

    Kristina Thamm

    Full Text Available The pentaspan membrane glycoprotein prominin-1 (CD133 is widely used in medicine as a cell surface marker of stem and cancer stem cells. It has opened new avenues in stem cell-based regenerative therapy and oncology. This molecule is largely used with human samples or the mouse model, and consequently most biological tools including antibodies are directed against human and murine prominin-1. Although the general structure of prominin-1 including its membrane topology is conserved throughout the animal kingdom, its primary sequence is poorly conserved. Thus, it is unclear if anti-human and -mouse prominin-1 antibodies cross-react with their orthologs in other species, especially dog. Answering this issue is imperative in light of the growing number of studies using canine prominin-1 as an antigenic marker. Here, we address this issue by cloning the canine prominin-1 and use its overexpression as a green fluorescent protein fusion protein in Madin-Darby canine kidney cells to determine its immunoreactivity with antibodies against human or mouse prominin-1. We used immunocytochemistry, flow cytometry and immunoblotting techniques and surprisingly found no cross-species immunoreactivity. These results raise some caution in data interpretation when anti-prominin-1 antibodies are used in interspecies studies.

  17. Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter

    DEFF Research Database (Denmark)

    Özvegy-Laczka, Csilla; Laczkó, Rozália; Hegedűs, Csilla

    2008-01-01

    Human ABCG2 is a plasma membrane glycoprotein working as a homodimer or homo-oligomer. The protein plays an important role in the protection/detoxification of various tissues and may also be responsible for the multidrug-resistant phenotype of cancer cells. In our previous study we found that the...

  18. Development of β-lactoglobulin-specific chimeric human IgEκ monoclonal antibodies for in vitro safety assessment of whey hydrolysates

    NARCIS (Netherlands)

    Knipping, Karen; Simons, Peter; Buelens-Sleumer, Laura S; Cox, Linda; den Hartog, Marcel; de Jong, Niels; Teshima, Reiko; Garssen, Johan; Boon, Louis; Knippels, Léon M J

    BACKGROUND: Cow's milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3),

  19. The pathogenic human monoclonal anti-DNA that induces experimental systemic lupus erythematosus in mice is encoded by a VH4 gene segment.

    Science.gov (United States)

    Waisman, A; Shoenfeld, Y; Blank, M; Ruiz, P J; Mozes, E

    1995-04-01

    Systemic lupus erythematosus (SLE) can be induced in mice by immunization with a human anti-DNA IgM mAb that was derived from a patient with cold agglutinin disease. The latter anti-DNA mAb expresses the common idiotype (Id) designated 16/6 Id. The original human hybridoma 16/6 that secreted an IgM antibody that bound ssDNA and carried the 16/6 Id had switched in culture to secrete an IgG molecule. Herein we show that the IgG 16/6 antibody contains the previously reported characteristics of the original IgM 16/6 mAb: it expresses the 16/6 Id and is capable of inducing experimental SLE in susceptible mouse strains. The identify of the IgG 16/6 anti-DNA mAb to the original IgM mAb was shown both by serological techniques and at the T cell level. The human IgG 16/6 mAb was found to be encoded by a germline gene from the human VH4 gene family, with high similarity to the germline gene VH4.21 that was previously shown to code for anti-DNA antibodies isolated from SLE patients. The VH4.21 germline gene was found to also code for most antibodies with cold agglutinin activity that were isolated from patients with cold agglutinin disease.

  20. Emergence of monoclonal antibody b12-resistant human immunodeficiency virus type 1 variants during natural infection in the absence of humoral or cellular immune pressure

    NARCIS (Netherlands)

    Bunnik, Evelien M.; van Gils, Marit J.; Lobbrecht, Marilie S. D.; Pisas, Linaida; Nanlohy, Nening M.; van Baarle, Debbie; van Nuenen, Ad C.; Hessell, Ann J.; Schuitemaker, Hanneke

    2010-01-01

    Human immunodeficiency virus type 1 (HIV-1) resistance to broadly neutralizing antibodies such as b12, which targets the highly conserved CD4-binding site, raises a significant hurdle for the development of a neutralizing antibody-based vaccine. Here, 15 individuals were studied of whom seven

  1. Differentiation between Human Coronaviruses NL63 and 229E Using a Novel Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay Based on Specific Monoclonal Antibodies

    NARCIS (Netherlands)

    Sastre, Patricia; Dijkman, Ronald; Camuñas, Ana; Ruiz, Tamara; Jebbink, Maarten F.; van der Hoek, Lia; Vela, Carmen; Rueda, Paloma

    2011-01-01

    Human coronaviruses (HCoVs) are responsible for respiratory tract infections ranging from common colds to severe acute respiratory syndrome. HCoV-NL63 and HCoV-229E are two of the four HCoVs that circulate worldwide and are close phylogenetic relatives. HCoV infections can lead to hospitalization of

  2. Reactivity of cloned, expressed human Fc gamma RIII isoforms with monoclonal antibodies which distinguish cell-type-specific and allelic forms of Fc gamma RIII

    NARCIS (Netherlands)

    Trounstine, M. L.; Peltz, G. A.; Yssel, H.; Huizinga, T. W.; von dem Borne, A. E.; Spits, H.; Moore, K. W.

    1990-01-01

    We have isolated and expressed a cDNA encoding human NK cell Fc gamma RIII. The NK cell cDNA differs from the neutrophil Fc gamma RIII cDNA by a number of point mutations and encodes an additional 21 amino acids at its C-terminus. When transiently expressed neutrophil and NK cell Fc gamma RIII were

  3. Emerging monoclonal antibodies against Clostridium difficile infection.

    Science.gov (United States)

    Péchiné, Séverine; Janoir, Claire; Collignon, Anne

    2017-04-01

    Clostridium difficile infections are characterized by a high recurrence rate despite antibiotic treatments and there is an urgent need to develop new treatments such as fecal transplantation and immonotherapy. Besides active immunotherapy with vaccines, passive immunotherapy has shown promise, especially with monoclonal antibodies. Areas covered: Herein, the authors review the different assays performed with monoclonal antibodies against C. difficile toxins and surface proteins to treat or prevent primary or recurrent episodes of C. difficile infection in animal models and in clinical trials as well. Notably, the authors lay emphasis on the phase III clinical trial (MODIFY II), which allowed bezlotoxumab to be approved by the Food and Drug Administration and the European Medicines Agency. They also review new strategies for producing single domain antibodies and nanobodies against C. difficile and new approaches to deliver them in the digestive tract. Expert opinion: Only two human Mabs against TcdA and TcdB have been tested alone or in combination in clinical trials. However, many animal model studies have provided rationale for the use of Mabs and nanobodies in C. difficile infection and pave the way for further clinical investigation.

  4. Impact of V2 mutations on escape from a potent neutralizing anti-V3 monoclonal antibody during in vitro selection of a primary human immunodeficiency virus type 1 isolate.

    Science.gov (United States)

    Shibata, Junji; Yoshimura, Kazuhisa; Honda, Akiko; Koito, Atsushi; Murakami, Toshio; Matsushita, Shuzo

    2007-04-01

    KD-247, a humanized monoclonal antibody to an epitope of gp120-V3 tip, has potent cross-neutralizing activity against subtype B primary human immunodeficiency virus type 1 (HIV-1) isolates. To assess how KD-247 escape mutants can be generated, we induced escape variants by exposing bulked primary R5 virus, MOKW, to increasing concentrations of KD-247 in vitro. In the presence of relatively low concentrations of KD-247, viruses with two amino acid mutations (R166K/D167N) in V2 expanded, and under high KD-247 pressure, a V3 tip substitution (P313L) emerged in addition to the V2 mutations. However, a virus with a V2 175P mutation dominated during passaging in the absence of KD-247. Using domain swapping analysis, we demonstrated that the V2 mutations and the P313L mutation in V3 contribute to partial and complete resistance phenotypes against KD-247, respectively. To identify the V2 mutation responsible for the resistance to KD-247, we constructed pseudoviruses with single or double amino acid mutations in V2 and measured their sensitivity to neutralization. Interestingly, the neutralization phenotypes were switched, so that amino acid residue 175 (Pro or Leu) located in the center of V2 was exchanged, indicating that the amino acid at position 175 has a crucial role, dramatically changing the Env oligomeric state on the membrane surface and affecting the neutralization phenotype against not only anti-V3 antibody but also recombinant soluble CD4. These data suggested that HIV-1 can escape from anti-V3 antibody attack by changing the conformation of the functional envelope oligomer by acquiring mutations in the V2 region in environments with relatively low antibody concentrations.

  5. Use of Monoclonal Antibodies for the Identification of Leishmania spp. Isolated from Humans and Wild Rodents in the State of Campeche, Mexico

    OpenAIRE

    CantoLara Silvia B; Van Wynsberghe Nicole R; Vargas-González Alberto; Ojeda-Farfán Fanny F; Andrade-Narváez Fernando J

    1999-01-01

    The genus Leishmania includes 30 described species which infect a wide variety of mammalian hosts. The precise identification of leishmanial parasites at the species level is very important in order to determine whether an organism, causing the disease in a given area, is of the same biotype as that found in suspected mammalian reservoirs. The objectives of the present study were (1) to identify leishmanial parasites isolated from humans and wild rodents from the State of Campeche, an endemic...

  6. High-Avidity Monoclonal Antibodies against the Human Scavenger Class B Type I Receptor Efficiently Block Hepatitis C Virus Infection in the Presence of High-Density Lipoprotein▿

    OpenAIRE

    Catanese, Maria Teresa; Graziani, Rita; von Hahn, Thomas; Moreau, Martine; Huby, Thierry; Paonessa, Giacomo; Santini, Claudia; Luzzago, Alessandra; Rice, Charles M.; Cortese, Riccardo; Vitelli, Alessandra; Nicosia, Alfredo

    2007-01-01

    The human scavenger class B type 1 receptor (SR-B1/Cla1) was identified as a putative receptor for hepatitis C virus (HCV) because it binds to soluble recombinant HCV envelope glycoprotein E2 (sE2). High-density lipoprotein (HDL), a natural SR-B1 ligand, was shown to increase the in vitro infectivity of retroviral pseudoparticles bearing HCV envelope glycoproteins and of cell culture-derived HCV (HCVcc), suggesting that SR-B1 promotes viral entry in an HDL-dependent manner. To determine wheth...

  7. Development of β-lactoglobulin-specific chimeric human IgEκ monoclonal antibodies for in vitro safety assessment of whey hydrolysates.

    Science.gov (United States)

    Knipping, Karen; Simons, Peter J; Buelens-Sleumer, Laura S; Cox, Linda; den Hartog, Marcel; de Jong, Niels; Teshima, Reiko; Garssen, Johan; Boon, Louis; Knippels, Léon M J

    2014-01-01

    Cow's milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow's milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays. An oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays. Mice were immunized with bovine β-lactoglobulin, and subsequently the variable domains of dissimilar anti-β-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains. After sensitization with this pool of anti-β-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa β-lactoglobulin, and 5-10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow's milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-β-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on β-lactoglobulin, which were also recognized by serum IgEs from cow's milk allergic children. Usage of our 'unlimited' source and well-defined pool of β-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow's milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula.

  8. Development of β-lactoglobulin-specific chimeric human IgEκ monoclonal antibodies for in vitro safety assessment of whey hydrolysates.

    Directory of Open Access Journals (Sweden)

    Karen Knipping

    Full Text Available Cow's milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3, sensitized with serum IgE from cow's milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays.An oligoclonal pool of chimeric human (chuIgE antibodies against bovine β-lactoglobulin (a major allergen in whey was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays.Mice were immunized with bovine β-lactoglobulin, and subsequently the variable domains of dissimilar anti-β-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains.After sensitization with this pool of anti-β-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa β-lactoglobulin, and 5-10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow's milk powder (mainly casein showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-β-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on β-lactoglobulin, which were also recognized by serum IgEs from cow's milk allergic children.Usage of our 'unlimited' source and well-defined pool of β-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow's milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula.

  9. Matched rabbit monoclonal antibodies against αv-series integrins reveal a novel αvβ3-LIBS epitope, and permit routine staining of archival paraffin samples of human tumors

    Directory of Open Access Journals (Sweden)

    Simon L. Goodman

    2012-02-01

    The relationship between integrin expression and function in pathologies is often contentious as comparisons between human pathological expression and expression in cell lines is difficult. In addition, the expression of even integrins αvβ6 and αvβ8 in tumor cell lines is not comprehensively documented. Here, we describe rabbit monoclonal antibodies (RabMabs against the extracellular domains of αv integrins that react with both native integrins and formalin fixed, paraffin embedded (FFPE human tissues. These RabMabs, against αvβ3 (EM22703, αvβ5 (EM09902, αvβ6 (EM05201, αvβ8 (EM13309, and pan-αv (EM01309, recognize individual integrin chains in Western blots and in flow cytometry. EM22703 detected a ligand-induced binding site (LIBS, reporting an epitope enhanced by the binding of an RGD-peptide to αvβ3. αvβ8 was rarely expressed in human tumor specimens, and weakly expressed in non-small-cell lung carcinoma (NSCLC. However, ovarian carcinoma cell lines expressed αvβ8, as did some melanoma cells, whereas U87MG glioma lacked αvβ8 expression. We observed an unexpected strong expression of αvβ6 in tumor samples of invasive ductal breast adenoma, colorectal carcinoma (CRC, and NSCLC. αvβ3 was strongly expressed in some invasive NSCLC cohorts. Interestingly, PC3 prostate cell and human prostate tumors did not express αvβ3. The RabMabs stained plasma membranes in FFPE-immunohistochemistry (IHC samples of tumor cell lines from lung, ovary, colon, prostate, squamous cell carcinoma of head and neck (SCCHN, breast, and pancreas carcinomas. The RabMabs are unique tools for probing αv integrin biology, and suggest that especially αvβ6 and αvβ8 biologies still have much to reveal.

  10. Preclinical in vitro and in vivo characterization of the fully human monoclonal IgM antibody KBPA101 specific for Pseudomonas aeruginosa serotype IATS-O11

    DEFF Research Database (Denmark)

    Horn, Michael P; Zuercher, Adrian W; Imboden, Martin A

    2010-01-01

    Antigenic Typing System (IATS) serotype O11 with high avidity (5.81 x 10(7) M(-1) +/- 2.8 x 10(7) M(-1)) without cross-reacting with other serotypes. KBPA101 specifically opsonized the P. aeruginosa of IATS O11 serotype and mediated complement-dependent phagocytosis in vitro by the human monocyte-like cell...... line HL-60 at a very low concentration (half-maximal phagocytosis at 0.16 ng/ml). In vivo evaluation of KBPA101 demonstrated a dose-response relationship for protection against systemic infections in a murine burn wound sepsis model, where 70 to 100% of animals were protected against lethal challenges...

  11. First-in-Human Study of AMG 820, a Monoclonal Anti-Colony-Stimulating Factor 1 Receptor Antibody, in Patients with Advanced Solid Tumors.

    Science.gov (United States)

    Papadopoulos, Kyriakos P; Gluck, Larry; Martin, Lainie P; Olszanski, Anthony J; Tolcher, Anthony W; Ngarmchamnanrith, Gataree; Rasmussen, Erik; Amore, Benny M; Nagorsen, Dirk; Hill, John S; Stephenson, Joe

    2017-10-01

    Purpose: Binding of colony-stimulating factor 1 (CSF1) ligand to the CSF1 receptor (CSF1R) regulates survival of tumor-associated macrophages, which generally promote an immunosuppressive tumor microenvironment. AMG 820 is an investigational, fully human CSF1R antibody that inhibits binding of the ligands CSF1 and IL34 and subsequent ligand-mediated receptor activation. This first-in-human phase I study evaluated the safety, pharmacokinetics, pharmacodynamics, and antitumor activity of AMG 820.Experimental Design: Adult patients with relapsed or refractory advanced solid tumors received intravenous AMG 820 0.5 mg/kg once weekly or 1.5 to 20 mg/kg every 2 weeks until disease progression, adverse event (AE), or consent withdrawal.Results: Twenty-five patients received ≥1 dose of AMG 820. AMG 820 was tolerated up to 20 mg/kg; the MTD was not reached. One dose-limiting toxicity was observed (20 mg/kg; nonreversible grade 3 deafness). Most patients (76%) had treatment-related AEs; the most common were periorbital edema (44%), increased aspartate aminotransferase (AST; 28%), fatigue (24%), nausea (16%), increased blood alkaline phosphatase (12%), and blurred vision (12%). No patients had serious or fatal treatment-related AEs; 28% had grade ≥3 treatment-related AEs. Grade 3 AST elevations resolved when treatment was withheld. AMG 820 showed linear pharmacokinetics, with minimal accumulation (AMG 820 was tolerated with manageable toxicities up to 20 mg/kg every 2 weeks. Pharmacodynamic response was demonstrated, and limited antitumor activity was observed. Clin Cancer Res; 23(19); 5703-10. ©2017 AACR. ©2017 American Association for Cancer Research.

  12. Flow cytometric assessment of the reactivity of a panel of monoclonal antibodies (mAb) against two populations of human dendritic cells (DC)

    Science.gov (United States)

    Nunez, Rafael; Filgueira, Luis

    2001-01-01

    Background The identification of antigens on human DC has been a very difficult and elusive task because of the lack of appropriate reagents. Therefore, we evaluated by flow cytometry a panel of mAb that recognize antigens on human DC, aiming to determine the kinetics of DC antigen expression at 7, 14, 21 and 28 days in (i) Dermal DC like cells (Mo-DC) and (ii) Langerhans cell like DC (Mo-LC). In addition we aimed to identify markers for DC subpopulations. Results It was found at day 7, that mAb BG6, HP-F1, BU10, RFD-1, CMRF-44 recognized 50% of Mo-DC. Moreover, 7H5, ZM3.8, CMRF-56, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 showed increased MFI reactivity against Mo-DC. mAb BG6, BU10 and CMRF-44 recognized 50% of Mo-LC. The increase in % of positive cells was paralleled by MFI increases. At day 14, fourteen mAb recognized >50% of the Mo-DC, while five recognized 20-50% of Mo-DC. BG6 reacted with 7% of the Mo-DC. Nineteen mAb recognized >48% of Mo-LC while BG6 had negative reactivity. At day 21 and 28, all mAb reacted with >20% of Mo-DC and yielded a significant MFI with Mo-DC. Also nineteen mAb yielded significant MFI with Mo-LC while RFD-7 did not. Conclusions The immunophenotyping assays demonstrated differences between the two DC populations as well as variations in the reactivity of the mAb at diverse time points, suggesting the existence of subpopulations within the Mo-DC and Mo-LC. PMID:11504561

  13. Flow cytometric assessment of the reactivity of a panel of monoclonal antibodies (mAb against two populations of human dendritic cells (DC

    Directory of Open Access Journals (Sweden)

    Nunez Rafael

    2001-07-01

    Full Text Available Abstract Background The identification of antigens on human DC has been a very difficult and elusive task because of the lack of appropriate reagents. Therefore, we evaluated by flow cytometry a panel of mAb that recognize antigens on human DC, aiming to determine the kinetics of DC antigen expression at 7, 14, 21 and 28 days in (i Dermal DC like cells (Mo-DC and (ii Langerhans cell like DC (Mo-LC. In addition we aimed to identify markers for DC subpopulations. Results It was found at day 7, that mAb BG6, HP-F1, BU10, RFD-1, CMRF-44 recognized 50% of Mo-DC. Moreover, 7H5, ZM3.8, CMRF-56, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 showed increased MFI reactivity against Mo-DC. mAb BG6, BU10 and CMRF-44 recognized 50% of Mo-LC. The increase in % of positive cells was paralleled by MFI increases. At day 14, fourteen mAb recognized >50% of the Mo-DC, while five recognized 20-50% of Mo-DC. BG6 reacted with 7% of the Mo-DC. Nineteen mAb recognized >48% of Mo-LC while BG6 had negative reactivity. At day 21 and 28, all mAb reacted with >20% of Mo-DC and yielded a significant MFI with Mo-DC. Also nineteen mAb yielded significant MFI with Mo-LC while RFD-7 did not. Conclusions The immunophenotyping assays demonstrated differences between the two DC populations as well as variations in the reactivity of the mAb at diverse time points, suggesting the existence of subpopulations within the Mo-DC and Mo-LC.

  14. Cultivation and Immortalization of Human B-Cells Producing a Human Monoclonal IgM Antibody Binding to MDA-LDL: Further Evidence for Formation of Atherogenic MDA-LDL Adducts in Humans In Vivo

    Directory of Open Access Journals (Sweden)

    Franz Tatzber

    2017-01-01

    Full Text Available Oxidatively modified low-density lipoprotein (oLDL is firmly believed to play an important role in the initiation and development of atherosclerosis, and malonic dialdehyde (MDA is one of the major lipid peroxidation breakdown products involved in this process. In recent decades, antibodies against MDA-LDL have been detected in human and animal sera. In our study, human B-cells from the peripheral blood of a healthy female donor were fused with the SP2/0 mouse myeloma cell line. Antibody-producing hybridomas were detected by MDA-LDL-IgG/IgM enzyme-linked immunosorbent assays (ELISA and Cu++-oxidized LDL IgG/IgM (oLAb ELISA. Cells with supernatants emitting positive signals for antibodies were then cloned and after sufficient multiplication frozen and stored under liquid nitrogen. Due to the loss of antibody-producing ability, we established an MDA-LDL-IgM-producing cell line by recloning. This allowed isolation and immortalization of several human B-cells. The human donor had not been immunized with MDA-modified proteins, thus obviously producing MDA-LDL antibodies in vivo. Furthermore, using these antibodies for in vitro experiments, we were able to demonstrate that MDA epitopes are among the epitopes generated during Cu++-LDL oxidation as well. Finally, these antibodies compete in ELISA and cell culture experiments with MDA as a challenging toxin or ligand.

  15. Aggregation of Human Recombinant Monoclonal Antibodies Influences the Capacity of Dendritic Cells to Stimulate Adaptive T-Cell Responses In Vitro

    Science.gov (United States)

    Sordé, Laetitia; Koepke, Stephan; Gottlieb, Sascha; Krieg, Jennifer; Djidja, Marie-Claude; Baban, Aida; Spindeldreher, Sebastian; Koulov, Atanas V.; Kiessling, Andrea

    2014-01-01

    Subvisible proteinaceous particles which are present in all therapeutic protein formulations are in the focus of intense discussions between health authorities, academics and biopharmaceutical companies in the context of concerns that such particles could promote unwanted immunogenicity via anti-drug antibody formation. In order to provide further understanding of the subject, this study closely examines the specific biological effects proteinaceous particles may exert on dendritic cells (DCs) as the most efficient antigen-presenting cell population crucial for the initiation of the adaptive immune response. Two different model IgG antibodies were subjected to three different types of exaggerated physical stress to generate subvisible particles in far greater concentrations than the ones typical for the currently marketed biotherapeutical antibodies. The aggregated samples were used in in vitro biological assays in order to interrogate the early DC-driven events that initiate CD4 T-cell dependent humoral adaptive immune responses – peptide presentation capacity and co-stimulatory activity of DCs. Most importantly, antigen presentation was addressed with a unique approach called MHC-associated Peptide Proteomics (MAPPs), which allows for identifying the sequences of HLA-DR associated peptides directly from human dendritic cells. The experiments demonstrated that highly aggregated solutions of two model mAbs generated under controlled conditions can induce activation of human monocyte-derived DCs as indicated by upregulation of typical maturation markers including co-stimulatory molecules necessary for CD4 T-cell activation. Additional data suggest that highly aggregated proteins could induce in vitro T-cell responses. Intriguingly, strong aggregation-mediated changes in the pattern and quantity of antigen-derived HLA-DR associated peptides presented on DCs were observed, indicating a change in protein processing and presentation. Increasing the amounts of

  16. Pharmacokinetics and pharmacodynamics of a human monoclonal anti‐FGF23 antibody (KRN23) in the first multiple ascending‐dose trial treating adults with X‐linked hypophosphatemia

    Science.gov (United States)

    Imel, Erik A.; Ruppe, Mary D.; Weber, Thomas J.; Klausner, Mark A.; Ito, Takahiro; Vergeire, Maria; Humphrey, Jeffrey; Glorieux, Francis H.; Portale, Anthony A.; Insogna, Karl; Carpenter, Thomas O.; Peacock, Munro

    2015-01-01

    Abstract In X‐linked hypophosphatemia (XLH), serum fibroblast growth factor 23 (FGF23) is increased and results in reduced renal maximum threshold for phosphate reabsorption (TmP), reduced serum inorganic phosphorus (Pi), and inappropriately low normal serum 1,25 dihydroxyvitamin D (1,25[OH]2D) concentration, with subsequent development of rickets or osteomalacia. KRN23 is a recombinant human IgG1 monoclonal antibody that binds to FGF23 and blocks its activity. Up to 4 doses of KRN23 were administered subcutaneously every 28 days to 28 adults with XLH. Mean ± standard deviation KRN23 doses administered were 0.05, 0.10 ± 0.01, 0.28 ± 0.06, and 0.48 ± 0.16 mg/kg. The mean time to reach maximum serum KRN23 levels was 7.0 to 8.5 days. The mean KRN23 half‐life was 16.4 days. The mean area under the concentration–time curve (AUCn) for each dosing interval increased proportionally with increases in KRN23 dose. The mean intersubject variability in AUCn ranged from 30% to 37%. The area under the effect concentration–time curve (AUECn) for change from baseline in TmP per glomerular filtration rate, serum Pi, 1,25(OH)2D, and bone markers for each dosing interval increased linearly with increases in KRN23 AUCn. Linear correlation between serum KRN23 concentrations and increase in serum Pi support KRN23 dose adjustments based on predose serum Pi concentration. © 2015 The Authors. The Journal of Clinical Pharmacology Published by Wiley Periodicals, Inc. on behalf of American College of Clinical Pharmacology PMID:26073451

  17. Efficacy and safety of alirocumab, a fully human PCSK9 monoclonal antibody, in high cardiovascular risk patients with poorly controlled hypercholesterolemia on maximally tolerated doses of statins: rationale and design of the ODYSSEY COMBO I and II trials.

    Science.gov (United States)

    Colhoun, Helen M; Robinson, Jennifer G; Farnier, Michel; Cariou, Bertrand; Blom, Dirk; Kereiakes, Dean J; Lorenzato, Christelle; Pordy, Robert; Chaudhari, Umesh

    2014-09-20

    Alirocumab is a fully human monoclonal antibody to proprotein convertase subtilisin kexin type 9 (PCSK9) under investigation for treatment of hypercholesterolemia and reduction of cardiovascular events. The COMBO studies, part of the Phase 3 ODYSSEY clinical trial program, are designed to evaluate the efficacy and safety of alirocumab as add-on therapy to stable, maximally tolerated daily statin, with or without other lipid-lowering therapy (LLT), in a planned 966 patients with hypercholesterolemia at high cardiovascular risk. COMBO I ( http://clinicaltrials.gov/show/NCT01644175) is placebo-controlled, with a double-blind treatment period of 52 weeks, and 306 planned patients who may receive other LLTs in addition to statin therapy. COMBO II ( http://clinicaltrials.gov/show/NCT01644188) has a double-blind treatment period of 104 weeks, comparing alirocumab with ezetimibe in 660 planned patients receiving statin therapy (but no other LLTs). The primary efficacy endpoint is the difference between treatment arms in percent change in low-density lipoprotein cholesterol (LDL-C) from baseline to week 24. Both studies utilized a starting dose of alirocumab 75 mg every 2 weeks (Q2W; administered as 1 mL solution via auto-injector). Patients with LDL-C levels ≥70 mg/dL after 8 weeks of treatment were up-titrated in a blinded manner at week 12 to alirocumab 150 mg Q2W (also 1 mL auto-injector). In conclusion, the COMBO studies will provide information on the long-term efficacy and safety of alirocumab in high-risk patients when administered in addition to maximally tolerated statin therapy, with a flexible dosing strategy which allows for individualized therapy based on the degree of LDL-C lowering needed to achieve the desired treatment response. NCT01644175 ( NCT01644175). NCT01644188 ( NCT01644188).

  18. Phase I study of GC1008 (fresolimumab: a human anti-transforming growth factor-beta (TGFβ monoclonal antibody in patients with advanced malignant melanoma or renal cell carcinoma.

    Directory of Open Access Journals (Sweden)

    John C Morris

    Full Text Available In advanced cancers, transforming growth factor-beta (TGFβ promotes tumor growth and metastases and suppresses host antitumor immunity. GC1008 is a human anti-TGFβ monoclonal antibody that neutralizes all isoforms of TGFβ. Here, the safety and activity of GC1008 was evaluated in patients with advanced malignant melanoma and renal cell carcinoma.In this multi-center phase I trial, cohorts of patients with previously treated malignant melanoma or renal cell carcinoma received intravenous GC1008 at 0.1, 0.3, 1, 3, 10, or 15 mg/kg on days 0, 28, 42, and 56. Patients achieving at least stable disease were eligible to receive Extended Treatment consisting of 4 doses of GC1008 every 2 weeks for up to 2 additional courses. Pharmacokinetic and exploratory biomarker assessments were performed.Twenty-nine patients, 28 with malignant melanoma and 1 with renal cell carcinoma, were enrolled and treated, 22 in the dose-escalation part and 7 in a safety cohort expansion. No dose-limiting toxicity was observed, and the maximum dose, 15 mg/kg, was determined to be safe. The development of reversible cutaneous keratoacanthomas/squamous-cell carcinomas (4 patients and hyperkeratosis was the major adverse event observed. One malignant melanoma patient achieved a partial response, and six had stable disease with a median progression-free survival of 24 weeks for these 7 patients (range, 16.4-44.4 weeks.GC1008 had no dose-limiting toxicity up to 15 mg/kg. In patients with advanced malignant melanoma and renal cell carcinoma, multiple doses of GC1008 demonstrated acceptable safety and preliminary evidence of antitumor activity, warranting further studies of single agent and combination treatments.Clinicaltrials.gov NCT00356460.

  19. A phase I pharmacologic study of necitumumab (IMC-11F8), a fully human IgG1 monoclonal antibody directed against EGFR in patients with advanced solid malignancies.

    Science.gov (United States)

    Kuenen, Bart; Witteveen, Petronella O; Ruijter, Rita; Giaccone, Giuseppe; Dontabhaktuni, Aruna; Fox, Floyd; Katz, Terry; Youssoufian, Hagop; Zhu, Junming; Rowinsky, Eric K; Voest, Emile E

    2010-03-15

    This study aimed to determine a maximum tolerated dose (MTD) and recommended dose for disease-directed studies of necitumumab (IMC-11F8), a fully human IgG(1) monoclonal antibody directed at the epidermal growth factor receptor, and to characterize the safety profile, pharmacokinetics, preliminary antitumor activity, and immunogenicity of necitumumab. Patients with advanced solid malignancies were treated with 100 to 1,000 mg (flat dosing) necitumumab followed by a 2-week pharmacokinetics sampling period, before beginning 6-week cycles of therapy. Sixty patients received necitumumab weekly (29 patients) or every other week (31 patients). Two patients receiving 1,000 mg every 2 weeks experienced dose-limiting toxicities (DLT; grade 3 headache), accompanied by grade 3 nausea and vomiting in one patient. Occurring hours after the initial dose, these DLTs established 800 mg as the MTD. Mild dose-related skin toxicity was the most common drug-related toxicity (80%). One patient in each arm experienced grade 3 acneform rash, which responded to oral antibiotics and topical therapy. Toxicity was similar on both schedules. Necitumumab exhibited saturable elimination and nonlinear pharmacokinetics. At 800 mg (both arms), its half-life was approximately 7 days. All patients treated with >or=600 mg necitumumab achieved target trough concentrations (>or=40 microg/mL). Antibodies against necitumumab were not detected. Partial response and stable disease were experienced by 2 and 16 patients, respectively. Well tolerated, necitumumab is associated with preliminary evidence of antitumor activity, and achieves biologically relevant concentrations throughout the dosing period. The recommended dose of necitumumab for further clinical development is 800 mg (flat dose) weekly or every 2 weeks based on the clinical setting.

  20. Computational identification of epitopes in the glycoproteins of novel bunyavirus (SFTS virus) recognized by a human monoclonal antibody (MAb 4-5)

    Science.gov (United States)

    Zhang, Wenshuai; Zeng, Xiaoyan; Zhang, Li; Peng, Haiyan; Jiao, Yongjun; Zeng, Jun; Treutlein, Herbert R.

    2013-06-01

    In this work, we have developed a new approach to predict the epitopes of antigens that are recognized by a specific antibody. Our method is based on the "multiple copy simultaneous search" (MCSS) approach which identifies optimal locations of small chemical functional groups on the surfaces of the antibody, and identifying sequence patterns of peptides that can bind to the surface of the antibody. The identified sequence patterns are then used to search the amino-acid sequence of the antigen protein. The approach was validated by reproducing the binding epitope of HIV gp120 envelop glycoprotein for the human neutralizing antibody as revealed in the available crystal structure. Our method was then applied to predict the epitopes of two glycoproteins of a newly discovered bunyavirus recognized by an antibody named MAb 4-5. These predicted epitopes can be verified by experimental methods. We also discuss the involvement of different amino acids in the antigen-antibody recognition based on the distributions of MCSS minima of different functional groups.

  1. Confirmation of the immunoreactivity of monoclonal anti-human C-terminal EGFR antibodies in bronze Corydoras Corydoras aeneus (Callichthyidae Teleostei) by Western Blot method.

    Science.gov (United States)

    Mytych, Jennifer; Satora, Leszek; Kozioł, Katarzyna

    2017-12-12

    Bronze corydoras (Corydoras aeneus) uses the distal part of the intestine as accessory respiratory organ. Our previous study showed the presence of epidermal growth factor receptor (EGFR) cytoplasmic domain in the digestive tract of the bronze corydoras. In this study, using Western Blot method, we validated the results presented in the previous research. In detail, results of Western Blot analysis on digestive and respiratory part of bronze corydoras intestine homogenates confirmed the immunoreactivity of anti-cytoplasmic domain (C-terminal) human EGFR antibodies with protein band of approximately 180kDa (EGFR molecular weight). This indicates a high homology of EGFR domain between these species and the possibility of such antibody use in bronze corydoras. Statistically significantly higher EGFR expression was observed in the respiratory part of intestine when compared to the digestive part. This implies higher proliferation activity and angiogenesis of epithelium in this part of intestine, creating conditions for air respiration. Therefore, Corydoras aeneus may be considered as a model organism for the molecular studies of the mechanisms of epithelial proliferation initiation and inhibition depending on hypoxia and normoxia. Copyright © 2017. Published by Elsevier GmbH.

  2. Emergence of monoclonal antibody b12-resistant human immunodeficiency virus type 1 variants during natural infection in the absence of humoral or cellular immune pressure.

    Science.gov (United States)

    Bunnik, Evelien M; van Gils, Marit J; Lobbrecht, Marilie S D; Pisas, Linaida; Nanlohy, Nening M; van Baarle, Debbie; van Nuenen, Ad C; Hessell, Ann J; Schuitemaker, Hanneke

    2010-05-01

    Human immunodeficiency virus type 1 (HIV-1) resistance to broadly neutralizing antibodies such as b12, which targets the highly conserved CD4-binding site, raises a significant hurdle for the development of a neutralizing antibody-based vaccine. Here, 15 individuals were studied of whom seven developed b12-resistant viruses late in infection. The study investigated whether immune pressure may be involved in the selection of these viruses in vivo. Although four out of seven patients showed HIV-1-specific broadly neutralizing activity in serum, none of these patients had CD4-binding site-directed antibodies, indicating that strong humoral immunity is not a prerequisite for the outgrowth of b12-resistant viruses. In virus variants from one patient, who showed extremely weak heterologous and autologous neutralizing activity in serum, mutations were identified in the envelope that coincided with changes in b12 neutralization sensitivity. Lack of cytotoxic T-cell activity against epitopes with and without these mutations excluded a role for host cellular immunity in the selection of b12-resistant mutant viruses in this patient. However, b12 resistance correlated well with increased virus replication kinetics, indicating that selection for enhanced infectivity, possibly driven by the low availability of target cells in the later stages of disease, may coincide with increased resistance to CD4-binding site-directed agents, such as b12. These results showed that b12-resistant HIV-1 variants can emerge during the course of natural infection in the absence of both humoral and cellular immune pressure, suggestive of other mechanisms playing a role in the selective outgrowth of b12-resistant viruses.

  3. Human pharmacokinetics, biodistribution and dosimetry of the kit of monoclonal antibody IOR EGF/R3 labelled with {sup 99m} Tc

    Energy Technology Data Exchange (ETDEWEB)

    Torres, L.A.; Ramos, M.; Perera, A.; Hernandez, A.; Iznaga, M.E. N. [Solano, Ivette Alvarez, Jose L. Rodriguez. Centro de InvestigacionesClinicas. 34 no.4501 e/45 y 47 Kohly, Playa, C. Habana (Cuba)

    1998-12-31

    The aim of this work was to assess the human pharmacokinetics, biodistribution and dosimetry of the {sup 99m} Tc-labeled MAb ior egf/r3. Five patients were included in the biodistribution and dosimetric studies and three in the pharmacokinetic analysis. Multiple blood and urine samples we recollected and sequential anterior and posterior whole-body scintigraphies u pto 24 hr post-injection were performed to all patients . The internal radiation dosimetry was estimated from gamma camera imaging data using the methods developed by the Medical Internal radiation dosimetry (MIRD)committee. Raw data were computed from operations between gamma graphic images and regions of interest (ROI) using the Bio-Dose software and time-activity curves were calculated in order to determine the residence times of the source organs. The Pharmacokinetics and Biodistribution results showed that this compound have a bio exponential plasmatic and blood clearance with a rapid biodistribution phase of 9.1 {+-} 8.4 min and 12.2{+-}4.4 min, respectively, and a slower elimination phase of 6.6 {+-} 1.6 hr and 10.8 {+-} 6.8 hr. respectively. The urinary and hepatobiliary excretion showed 4.7 {+-} 0.4 % and 9.9 {+-} 1.8 % of the total administered dose,eliminated by these ways. Liver was the target organ of this product and had an uptake peak at 1 hr post-injection (61.2%) and a great retention of the MAb(T 1/2 eff = 5.3 hr, T 1/2 Biol. = 45.0 hr). The dosimetric results showed that liver, gallbladder and spleen received the higher absorbed. The effective dose and the effective equivalent dose were 1,2E-01 mSv/MBq and 9,2E-02 mSv/MBq respectively. These results allow to see the i or egf/r3 kit in a safe and controlled way. (Author)

  4. A randomized, double-blind, placebo-controlled assessment of BMS-936558, a fully human monoclonal antibody to programmed death-1 (PD-1, in patients with chronic hepatitis C virus infection.

    Directory of Open Access Journals (Sweden)

    David Gardiner

    Full Text Available Expression of the programmed death 1 (PD-1 receptor and its ligands are implicated in the T cell exhaustion phenotype which contributes to the persistence of several chronic viral infections, including human hepatitis C virus (HCV. The antiviral potential of BMS-936558 (MDX-1106 - a fully human anti-PD-1 monoclonal immunoglobulin-G4 that blocks ligand binding - was explored in a proof-of-concept, placebo-controlled single-ascending-dose study in patients (N = 54 with chronic HCV infection. Interferon-alfa treatment-experienced patients (n = 42 were randomized 5∶1 to receive a single infusion of BMS-936558 (0.03, 0.1, 0.3, 1.0, 3.0 mg/kg [n = 5 each] or 10 mg/kg [n = 10] or of placebo (n = 7. An additional 12 HCV treatment-naïve patients were randomized to receive 10 mg/kg BMS-936558 (n = 10 or placebo (n = 2. Patients were followed for 85 days post-dose. Five patients who received BMS-936558 (0.1 [n = 1] or 10 mg/kg and one placebo patient achieved the primary study endpoint of a reduction in HCV RNA ≥0.5 log10 IU/mL on at least 2 consecutive visits; 3 (10 mg/kg achieved a >4 log10 reduction. Two patients (10 mg/kg achieved HCV RNA below the lower limit of quantitation (25 IU/mL, one of whom (a prior null-responder remained RNA-undetectable 1 year post-study. Transient reductions in CD4(+, CD8(+ and CD19(+ cells, including both naïve and memory CD4(+ and CD8(+ subsets, were observed at Day 2 without evidence of immune deficit. No clinically relevant changes in immunoglobulin subsets or treatment-related trends in circulating cytokines were noted. BMS-936558 exhibited dose-related exposure increases, with a half-life of 20-24 days. BMS-936558 was mostly well tolerated. One patient (10 mg/kg experienced an asymptomatic grade 4 ALT elevation coincident with the onset of a 4-log viral load reduction. Six patients exhibited immune-related adverse events of mild-to-moderate intensity, including two cases of

  5. Novel O-linked glycans containing 6'-sulfo-Gal/GalNAc of MUC1 secreted from human breast cancer YMB-S cells: possible carbohydrate epitopes of KL-6(MUC1) monoclonal antibody.

    Science.gov (United States)

    Seko, Akira; Ohkura, Takashi; Ideo, Hiroko; Yamashita, Katsuko

    2012-02-01

    Human serum Krebs von den Lugen-6 (KL-6) antigen is a MUC1 glycoprotein (KL-6/MUC1) recognized by anti-KL-6 monoclonal antibody (KL-6/mAb) and has been utilized as a diagnostic marker for interstitial pneumonia. KL-6/mAb is thought to recognize the specific glycopeptides sequence of MUC1, but the precise glycan structure of the epitope is unclear. In this study, we determined the carbohydrate structures of KL-6/MUC1 to search the carbohydrate epitopes for KL-6/mAb. KL-6/MUC1 was purified from the culture medium of human breast cancer YMB-S cells by KL-6/mAb-affinity chromatography; the O-linked glycan structures were determined in combination with paper electrophoresis, several lectin column chromatographies, sialidase digestion and methanolysis. KL-6/MUC1 contained core 1 and extended core 1 glycans modified with one or two sialic acid/sulfate residues. Based on these structures, several synthetic glycans binding to anti-KL-6/mAb were compared with one another by surface plasmon resonance. Sequentially, related radiolabeled oligosaccharides were enzymatically synthesized and analyzed for binding to a KL-6/mAb-conjugated affinity column. 3'-sialylated, 6'-sulfated LNnT [Neu5Acα2-3(SO(3)(-)-6)Galβ1-4GlcNAcβ1-3Galβ1-4Glc], 3'-sialylated, 6-sulfated core 1 [Neu5Acα2-3Galβ1-3(SO(3)(-)-6)GalNAc] and disulfated core 1 SO(3)(-)-3Galβ1-3(SO(3)(-)-6)GalNAc exhibited substantial affinity for KL-6/mAb, and 3'-sulfated core 1 derivatives [SO(3)(-)-3Galβ1-3(±Neu5Acα2-6)GalNAc] and 3'-sialylated core 1 weakly interacted with KL-6/mAb. These results indicated that the possible carbohydrate epitopes of KL-6/mAb involve not only 3'-sialylated core 1 but also novel core 1 and extended core 1 with sulfate and sialic acid residues. Epitope expressing changes with suppression or over-expression of the Gal6ST (Gal 6-O-sulfotransferase) gene, suggesting that Gal6ST is involved in the biosynthesis of the unique epitopes of KL-6/mAb.

  6. A randomized, double-blind, placebo-controlled assessment of BMS-936558, a fully human monoclonal antibody to programmed death-1 (PD-1), in patients with chronic hepatitis C virus infection.

    Science.gov (United States)

    Gardiner, David; Lalezari, Jay; Lawitz, Eric; DiMicco, Michael; Ghalib, Rheem; Reddy, K Rajender; Chang, Kyong-Mi; Sulkowski, Mark; Marro, Steven O'; Anderson, Jeffrey; He, Bing; Kansra, Vikram; McPhee, Fiona; Wind-Rotolo, Megan; Grasela, Dennis; Selby, Mark; Korman, Alan J; Lowy, Israel

    2013-01-01

    Expression of the programmed death 1 (PD-1) receptor and its ligands are implicated in the T cell exhaustion phenotype which contributes to the persistence of several chronic viral infections, including human hepatitis C virus (HCV). The antiviral potential of BMS-936558 (MDX-1106) - a fully human anti-PD-1 monoclonal immunoglobulin-G4 that blocks ligand binding - was explored in a proof-of-concept, placebo-controlled single-ascending-dose study in patients (N = 54) with chronic HCV infection. Interferon-alfa treatment-experienced patients (n = 42) were randomized 5∶1 to receive a single infusion of BMS-936558 (0.03, 0.1, 0.3, 1.0, 3.0 mg/kg [n = 5 each] or 10 mg/kg [n = 10]) or of placebo (n = 7). An additional 12 HCV treatment-naïve patients were randomized to receive 10 mg/kg BMS-936558 (n = 10) or placebo (n = 2). Patients were followed for 85 days post-dose. Five patients who received BMS-936558 (0.1 [n = 1] or 10 mg/kg) and one placebo patient achieved the primary study endpoint of a reduction in HCV RNA ≥0.5 log10 IU/mL on at least 2 consecutive visits; 3 (10 mg/kg) achieved a >4 log10 reduction. Two patients (10 mg/kg) achieved HCV RNA below the lower limit of quantitation (25 IU/mL), one of whom (a prior null-responder) remained RNA-undetectable 1 year post-study. Transient reductions in CD4(+), CD8(+) and CD19(+) cells, including both naïve and memory CD4(+) and CD8(+) subsets, were observed at Day 2 without evidence of immune deficit. No clinically relevant changes in immunoglobulin subsets or treatment-related trends in circulating cytokines were noted. BMS-936558 exhibited dose-related exposure increases, with a half-life of 20-24 days. BMS-936558 was mostly well tolerated. One patient (10 mg/kg) experienced an asymptomatic grade 4 ALT elevation coincident with the onset of a 4-log viral load reduction. Six patients exhibited immune-related adverse events of mild-to-moderate intensity, including two cases of

  7. First clinical use of ofatumumab, a novel fully human anti-CD20 monoclonal antibody in relapsed or refractory follicular lymphoma: Results of a phase 1/2 trial

    NARCIS (Netherlands)

    A. Hagenbeek (Anton); O. Gadeberg (Ole); P. Johnson (Jürgen); L.M. Pedersen; J. Walewski (Jan); A. Hellmann (Andrzej); B.K. Link (Brian); T. Robak (Tadeusz); M. Wojtukiewicz (Marek); M. Pfreundschuh (Michael); M. Kneba (Michael); A. Engert (Andreas); P. Sonneveld (Pieter); M. Flensburg (Mimi); J. Petersen (Jørgen); N. Losic (Nedjad); J. Radford (John)

    2008-01-01

    textabstractOfatumumab is a unique monoclonal antibody that targets a distinct small loop epitope on the CD20 molecule. Preclinical data show that ofatumumab is active against B-cell lymphoma/chronic lymphocytic leukemia cells with low CD20-antigen density and high expression of complement

  8. Production and immunohistochemical characterization of monoclonal antibodies directed against renal basement membranes of rats

    OpenAIRE

    Hinsch, Klaus-Dieter; Hansen, Diethelm; Zimmermann, Astrid; Bruchhausen, Franz V.

    1988-01-01

    Basement membranes were separated from rat glomeruli and purified by mild procedures, which led to a highly enriched basement membrane fraction. Here, the production and characterization of five monoclonal antibodies against tubular and glomerular basement membranes are described. These antibodies were analyzed immunohistochemically on frozen sections of rat, bovine, and human kidneys as well as on rat embryos. One monoclonal antibody (BM 0 11) exclusively ...

  9. MONOCLONAL ANTIBODIES AND BREAST CANCER. CURRENT THERAPEUTIC PROGRESS

    OpenAIRE

    Collignon, Joëlle; Gennigens, Christine; Rorive, Andrée; Coucke, Philippe; Lifrange, Eric; Maweja, Sylvie; Fillet, Georges; Jerusalem, Guy

    2009-01-01

    About 9,500 new breast cancers are diagnosed in Belgium every year. Improvement of our knowledge of altered molecular events leading to the proliferation of tumor cells has resulted in the development of targeted therapies in subgroups of cancers. One of the first validation of targeted therapy is the anti-HER-2 monoclonal antibody trastuzumab (Herceptin) in patients with overexpression of human epidermal growth factor receptor type 2 (HER2) occurring in 20 to 25% of invasive breast carcinoma...

  10. Monoclonal Antibody Shows Promise as Potential Therapeutic for MERS | Poster

    Science.gov (United States)

    A monoclonal antibody has proven effective in preventing Middle Eastern Respiratory Syndrome (MERS) in lab animals, suggesting further development as a potential intervention for the deadly disease in humans, according to new research. MERS is a newly emerged coronavirus first detected in humans in 2012. Most cases have occurred in the Middle East, but the disease has appeared elsewhere. In all, MERS has infected more than 1,700 individuals and killed more than 600, according to the World Health Organization. No vaccines or antiviral therapies currently exist. Several candidate vaccines are being developed, and some have been tested in animal models, a prerequisite to human clinical trials.

  11. Monoclonal antibodies in chronic lymphocytic leukemia.

    Science.gov (United States)

    Ferrajoli, Alessandra; Faderl, Stefan; Keating, Michael J

    2006-09-01

    Multiple options are now available for the treatment of chronic lymphocytic leukemia. Over the last 10 years, monoclonal antibodies have become an integral part of the management of this disease. Alemtuzumab has received approval for use in patients with fludarabine-refractory chronic lymphocytic leukemia. Rituximab has been investigated extensively in chronic lymphocytic leukemia both as a single agent and in combination with chemotherapy and other monoclonal antibodies. Epratuzumab and lumiliximab are newer monoclonal antibodies in the early phase of clinical development. This article will review the monoclonal antibodies more commonly used to treat chronic lymphocytic leukemia, the results obtained with monoclonal antibodies as single agents and in combination with chemotherapy, and other biological agents and newer compounds undergoing clinical trials.

  12. Safety profile and practical considerations of monoclonal antibody treatment.

    Science.gov (United States)

    Casanova Estruch, B

    2013-04-01

    Monoclonal antibodies are immunoglobulins specially designed to act against specific targets, in such a way that their administration stops a specific pathogenic process, stimulates a particular cellular action, or changes a cell mechanism to another pathway of interest. Their production is based on the establishment of modified immortal B lymphocytes to produce a specific immunoglobulin. Depending on the level of purity, this immunoglobulin may be murine complement (ending in "o", for example muromonab); chimeric, in which all the immunoglobulin is human, except in the variable region which is murine (ending in "xi", for example, rituximab); humanised, in which all the immunoglobulin is human, except in the variable complement region which remains murine (ending in "zu", for example, natalizumab); and human complement (ending in "u", for example, adalimumab). Therefore, there will be two types of secondary effects: those arising from the action of the antibody, such as opportunistic infections due to immunosuppression, and those arising from the administration of a protein, such as anaphylactic reactions. The sources used for the present articles were articles published in PubMed, located by searching for "Monoclonal antibodies and Secondary effects", and the web pages of the European Medicines Agency (EMEA) and the US Food and Drus Administration (FDA). The secondary effects arising from the mechanisms of action were opportunistic infections, common infections, development of tumours and autoimmune phenomena, and those arising from the administration of proteins: anaphylactic reaction, cytokine release syndrome, and the development of neutralising antibodies. Finally, the management of monoclonal antibodies in clinical practice and in special situations is discussed, including administering vaccines, pregnancy and paediatric use. Reference will be made to immune recovery syndrome. Monoclonal antibodies are highly effective drugs when specifically indicated, but

  13. Suppression of blastogenesis and proliferation of activated CD4(+) T cells: intravenous immunoglobulin (IVIg) versus novel anti-human leucocyte antigen (HLA)-E monoclonal antibodies mimicking HLA-I reactivity of IVIg.

    Science.gov (United States)

    Ravindranath, M H; Terasaki, P I; Pham, T; Jucaud, V; Kawakita, S

    2014-10-01

    Activated CD4(+) T cells undergo blastogenesis and proliferation and they express several surface receptors, including β2-microglobulin-free human leucocyte antigen (HLA) heavy chains (open conformers). Intravenous immunoglobulin (IVIg) suppresses activated T cells, but the mechanism is unclear. IVIg reacts with HLA-Ia/Ib antigens but its reactivity is lost when the anti-HLA-E Ab is adsorbed out. Anti-HLA-E antibodies may bind to the peptides shared by HLA-E and the HLA-I alleles. These shared peptides are cryptic in intact HLA, but exposed in open conformers. The hypothesis that anti-HLA-E monoclonal antibodies (mAbs) that mimic HLA-I reactivity of IVIg may suppress activated T cells by binding to the shared peptides of the open conformers on the T cell surface was tested by examining the relative binding affinity of those mAbs for open conformers coated on regular beads and for intact HLA coated on iBeads, and by comparing the effects on the suppression of phytohaemagglutinin (PHA)-activated T cells of three entities: IVIg, anti-HLA-E mAbs that mimic IVIg [Terasaki Foundation Laboratory (TFL)-006 and (TFL)-007]; and anti-HLA-E antibodies that do not mimic IVIg (TFL-033 and TFL-037). Suppression of blastogenesis and proliferation of those T cells by both IVIg and the anti-HLA-E mAbs was dose-dependent, the dose required with mAbs 50-150-fold lower than with IVIg. TFL-006 and TFL-007 significantly suppressed blastogenesis and proliferation of activated CD4(+) T cells, but neither the non-IVIg-mimicking mAbs nor control antibodies did so. The suppression may be mediated by Fab-binding of TFL-006/TFL-007 to the exposed shared peptides. The mAb binding to the open conformer may signal T cell deactivation because the open conformers have an elongated cytoplasmic tail with phosphorylation sites (tryosine(320)/serine(335)). © 2014 British Society for Immunology.

  14. Safety, tolerability, pharmacokinetics, and efficacy of AMG 403, a human anti-nerve growth factor monoclonal antibody, in two phase I studies with healthy volunteers and knee osteoarthritis subjects.

    Science.gov (United States)

    Gow, Jason M; Tsuji, Wayne H; Williams, Gary J; Mytych, Daniel; Sciberras, David; Searle, Shawn L; Mant, Tim; Gibbs, John P

    2015-10-08

    Nerve growth factor plays a key role in the pathology of osteoarthritis (OA) related chronic pain. The aim of these studies was to evaluate the safety, tolerability, pharmacokinetics, and clinical response of AMG 403, a human anti-nerve growth factor monoclonal antibody, in healthy volunteers and subjects with knee OA. Two phase I, randomized, placebo-controlled, double-blind studies were conducted. The single-ascending dose study randomized healthy volunteers (n = 48) 3:1 to receive AMG 403 (1, 3, 10, or 30 mg intravenously; or 10 or 30 mg subcutaneously; n = 8 per group) or placebo. The multiple-ascending dose study randomized knee OA subjects (n = 18) 3:1 to receive AMG 403 (3, 10, or 20 mg subcutaneously once monthly for four doses) or placebo. Safety, tolerability, and pharmacokinetics (PK) were assessed for both studies. Patient's and physician's disease assessments and total WOMAC score were determined in knee OA subjects. AMG 403 appeared to be well-tolerated after single and multiple doses, except for subject-reported hyperesthesia, pain, and paresthesia (mild to moderate severity). These treatment-emergent neurosensory events showed evidence of reversibility and a possible dose-dependence. Three serious adverse events were reported in AMG 403 treated subjects, but were not considered treatment related. AMG 403 PK was linear with an estimated half-life of 19.6 to 25.8 days. After multiple doses, AMG 403 PK showed modest accumulation (≤2.4-fold increase) in systemic exposure. Knee OA diagnosis, body weight, and anti-drug antibody development did not appear to affect AMG 403 PK. Patient's and physician's disease assessments and total WOMAC score showed improvement in AMG 403 treated knee OA subjects compared with placebo. AMG 403 was generally safe and well-tolerated in both healthy volunteers and knee OA patients, and exhibited linear pharmacokinetics. Preliminary clinical efficacy was observed in knee OA subjects. ClinicalTrials.gov NCT02348879

  15. Non-random escape pathways from a broadly neutralizing human monoclonal antibody map to a highly conserved region on the hepatitis C virus E2 glycoprotein encompassing amino acids 412-423.

    Science.gov (United States)

    Keck, Zhen-yong; Angus, Allan G N; Wang, Wenyan; Lau, Patrick; Wang, Yong; Gatherer, Derek; Patel, Arvind H; Foung, Steven K H

    2014-08-01

    A challenge for hepatitis C virus (HCV) vaccine development is to define epitopes that are able to elicit protective antibodies against this highly diverse virus. The E2 glycoprotein region located at residues 412-423 is conserved and antibodies to 412-423 have broadly neutralizing activities. However, an adaptive mutation, N417S, is associated with a glycan shift in a variant that cannot be neutralized by a murine but by human monoclonal antibodies (HMAbs) against 412-423. To determine whether HCV escapes from these antibodies, we analyzed variants that emerged when cell culture infectious HCV virions (HCVcc) were passaged under increasing concentrations of a specific HMAb, HC33.1. Multiple nonrandom escape pathways were identified. Two pathways occurred in the context of an N-glycan shift mutation at N417T. At low antibody concentrations, substitutions of two residues outside of the epitope, N434D and K610R, led to variants having improved in vitro viral fitness and reduced sensitivity to HC33.1 binding and neutralization. At moderate concentrations, a S419N mutation occurred within 412-423 in escape variants that have greatly reduced sensitivity to HC33.1 but compromised viral fitness. Importantly, the variants generated from these pathways differed in their stability. N434D and K610R-associated variants were stable and became dominant as the virions were passaged. The S419N mutation reverted back to N419S when immune pressure was reduced by removing HC33.1. At high antibody concentrations, a mutation at L413I was observed in variants that were resistant to HC33.1 neutralization. Collectively, the combination of multiple escape pathways enabled the virus to persist under a wide range of antibody concentrations. Moreover, these findings pose a different challenge to vaccine development beyond the identification of highly conserved epitopes. It will be necessary for a vaccine to induce high potency antibodies that prevent the formation of escape variants, which

  16. Humanized anti-CD2 monoclonal antibody treatment of plaque psoriasis: efficacy and pharmacodynamic results of two randomized, double-blind, placebo-controlled studies of intravenous and subcutaneous siplizumab.

    Science.gov (United States)

    Bissonnette, Robert; Langley, Richard G; Papp, Kim; Matheson, Robert; Toth, Darryl; Hultquist, Micki; Geba, Gregory P; White, Barbara

    2009-07-01

    New biologic therapies focused primarily on cytokine pathways, some targeting T cell-mediated immune responses, are being developed for the treatment of psoriasis. Siplizumab is a humanized anti-CD2 monoclonal antibody that interferes with costimulation necessary for T cell activation and proliferation. We assessed the biological activity, serum concentrations, and pharmacodynamic effects of siplizumab in patients with plaque psoriasis. Two multicenter, phase II randomized, double-blind, placebo-controlled studies were conducted: one study randomized 124 patients to one of two intravenous (IV) doses (0.012 and 0.04 mg/kg) of siplizumab, given every 2 weeks x 8 doses; the other study randomized 420 patients to one of three subcutaneous (SC) dose regimens of siplizumab given weekly (5 mg for 12 weeks, 5 mg for 6 weeks, and 7 mg for 4 weeks) or placebo for 12 weeks. Adults with plaque psoriasis involving > or =10% of the body surface area and who were not receiving psoriasis therapy were eligible. Treatment with siplizumab resulted in reductions in psoriasis severity, but most of the effects were not statistically significant compared with placebo. Statistically significant differences among IV siplizumab-treated and placebo groups were observed at study day 28, with greater psoriasis area and severity index (PASI) score reductions from baseline in the siplizumab groups. The difference in PASI50 response rates between the 0.04 mg/kg siplizumab and placebo groups was also statistically significant at day 28. A trend toward clinical improvement was observed in SC siplizumab-treated groups. Significant reductions in circulating absolute lymphocyte counts and CD2+ (CD3+, CD8+, and CD16+/56+), but not CD2- (CD19+ and CD14+), lymphocyte populations were observed. These changes were not accompanied by concomitant reductions in infiltrating CD3+ lymphocytes in psoriatic lesions, epidermal thickness, or keratin 16 (K16) and intercellular adhesion molecule (ICAM) expression

  17. Monoclonal antibodies for the treatment of Ebola virus disease.

    Science.gov (United States)

    Moekotte, A L; Huson, M A M; van der Ende, A J; Agnandji, S T; Huizenga, E; Goorhuis, A; Grobusch, M P

    2016-11-01

    To date, the management of patients with suspected or confirmed Ebolavirus disease (EVD) depends on quarantine, symptomatic management and supportive care, as there are no approved vaccines or treatments available for human use. However, accelerated by the recent large outbreak in West Africa, significant progress has been made towards vaccine development but also towards specific treatment with convalescent plasma and monoclonal antibodies. Areas covered: We describe recent developments in monoclonal antibody treatment for EVD, encompassing mAb114 and the MB-003, ZMAb, ZMapp™ and MIL-77E cocktails. Expert opinion: Preventive measures, are, and will remain essential to curb EVD outbreaks; even more so with vaccine development progressing. However, research for treatment options must not be neglected. Small-scale animal and individual human case studies show that monoclonal antibodies (mAbs) can be effective for EVD treatment; thus justifying exploration in clinical trials. Potential limitations are that high doses may be needed to yield clinical efficacy; epitope mutations might reduce efficacy; and constant evolution of (outbreak-specific) mAb mixtures might be required. Interim advice based on the clinical experience to date is that treatment of patients with mAbs is sensible, provided those could be made available in the necessary amounts in time.

  18. Choriocarcinoma: blocking factor and monoclonal antibody iodine 131 imaging

    Energy Technology Data Exchange (ETDEWEB)

    Pattillo, R.A.; Khazaeli, M.B.; Ruckert, A.C.; Hussa, R.O.; Collier, B.D.; Beierwaltes, W.; Mattingly, R.F.

    1984-04-01

    Postoperative iodine 131 monoclonal antibody localization in metastatic choriocarcinoma was accomplished in this study. The monoclonal antibody was prepared to male choriocarcinoma which cross reacted with gestational choriocarcinoma. The antibody was raised against whole choriocarcinoma cells and human chorionic gonadotropin (hCG) cross reactivity was excluded. The purified antibody was iodinated with /sup 131/I and successfully imaged BeWo choriocarcinoma transplanted in nude mice; however, imaging of choriocarcinoma in a patient was verified only after resection. It is our belief that failure to sufficiently concentrate the antibody in the tumor before operation was due to blocking factor in the serum of the patient. Blocking factor and hCG dropped postoperatively. Blocking factor activity in 15 patients with metastatic trophoblastic disease was monitored and, like hCG, was found to be a sensitive indicator of the presence of disease. Its efficacy may be in the small number of patients without hCG but with persistent disease.

  19. Monoclonal Antibodies for Systemic Lupus Erythematosus (SLE

    Directory of Open Access Journals (Sweden)

    Gabriella Moroni

    2010-01-01

    Full Text Available A number of monoclonal antibodies (mAb are now under investigation in clinical trials to assess their potential role in Systemic Lupus Erythematosus (SLE. The most frequently used mAb is rituximab, which is directed against CD20, a membrane protein expressed on B lymphocytes. Uncontrolled trials reported an improvement of SLE activity in non-renal patients and other studies even reported an improvement of severe lupus nephritis unresponsive to conventional treatments. However two randomized trials failed to show the superiority of rituximab over conventional treatment in non renal SLE and in lupus nephritis. Preliminary trials reported promising results with epratuzumab, a humanized mAb directed against CD22, and with belimumab, a human mAb that specifically recognizes and inhibits the biological activity of BLyS a cytokine of the tumornecrosis-factor (TNF ligand superfamily. Other clinical trials with mAb directed against TNF-alpha, interleukin-10 (Il-10, Il-6, CD154, CD40 ligand, IL-18 or complement component C5 are under way. At present, however, in spite of good results reported by some studies, no firm conclusion on the risk-benefit profile of these mAbs in patients with SLE can be drawn from the available studies.

  20. Therapeutic monoclonal antibodies and derivatives: Historical perspectives and future directions.

    Science.gov (United States)

    Rodgers, Kyla R; Chou, Richard C

    2016-11-01

    Biologics, both monoclonal antibodies (mAbs) and fusion proteins, have revolutionized the practice of medicine. This year marks the 30th anniversary of the Food and Drug Administration approval of the first mAb for human use. In this review, we examine the biotechnological breakthroughs that spurred the explosive development of the biopharmaceutical mAb industry, as well as how critical lessons learned about human immunology informed the development of improved biologics. We also discuss the most common mechanisms of action of currently approved biologics and the indications for which they have been approved to date. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. The safety of monoclonal antibodies for treatment of colorectal cancer.

    Science.gov (United States)

    Berger, Martin D; Lenz, Heinz-Josef

    2016-06-01

    Monoclonal antibodies such as bevacizumab, ramucirumab, cetuximab and panitumumab play an important role in the treatment of metastatic colorectal cancer (mCRC). With the introduction of these drugs considerable improvements in both progression-free survival (PFS) and overall survival (OS) were achieved. However these antibodies are associated with a unique side effect profile. This review provides an overview about drug efficacy of bevacizumab, cetuximab, panitumumab and ramucirumab in the treatment algorithm of mCRC. Additionally, we discuss the most common toxicites of these monoclonal antibodies. The most common toxicities associated with the VEGF-A directed antibody bevacizumab are hypertension, proteinuria, thromboembolism, bleeding, gastrointestinal perforation and prolonged wound healing. Similarly, the rate of hypertension and proteinuria is increased during treatment with the VEGFR2 antibody ramucirumab. On the other hand the most frequent side effects of EGFR targeted antibodies are skin rash, hypersensitivity reactions and hypomagnesemia. Due to the murine portions of cetuximab the incidence of infusion reactions is more frequent compared to panitumumab which is a pure human monoclonal antibody.

  2. Recent Advances in Monoclonal Antibody Therapies for Multiple Sclerosis.

    Science.gov (United States)

    Wootla, Bharath; Watzlawik, Jens O; Stavropoulos, Nikolaos; Wittenberg, Nathan J; Dasari, Harika; Abdelrahim, Murtada A; Henley, John R; Oh, Sang-Hyun; Warrington, Arthur E; Rodriguez, Moses

    2016-06-01

    Multiple sclerosis (MS) is the most common chronic inflammatory, demyelinating disease of the CNS and results in neurological disability. Existing immunomodulatory and immunosuppressive approaches lower the number of relapses but do not cure or reverse existing deficits nor improve long-term disability in MS patients. Monogenic antibodies were described as treatment options for MS, however the immunogenicity of mouse antibodies hampered the efficacy of potential therapeutics in humans. Availability of improved antibody production technologies resulted in a paradigm shift in MS treatment strategies. In this review, an overview of immunotherapies for MS that use conventional monoclonal antibodies reactive to immune system and their properties and mechanisms of action will be discussed, including recent advances in MS therapeutics and highlight natural autoantibodies (NAbs) that directly target CNS cells. Recent challenges for MS therapy are the identification of relevant molecular and cellular targets, time frame of treatment, and antibody toxicity profiles to identify safe treatment options for MS patients. The application of monoclonal antibody therapies with better biological efficacy associated with minimum side effects possesses huge clinical potential. Advances in monoclonal antibody technologies that directly target cells of nervous system may promote the CNS regeneration field from bench to bedside.

  3. Recent Advances in Monoclonal Antibody Therapies for Multiple Sclerosis

    Science.gov (United States)

    Stavropoulos, Nikolaos; Wittenberg, Nathan J.; Dasari, Harika; Abdelrahim, Murtada A.; Henley, John R.; Oh, Sang-Hyun; Warrington, Arthur E.; Rodriguez, Moses

    2016-01-01

    Introduction Multiple sclerosis (MS) is the most common chronic inflammatory, demyelinating disease of the CNS and results in neurological disability. Existing immunomodulatory and immunosuppressive approaches lower the number of relapses but do not cure or reverse existing deficits nor improve long-term disability in MS patients. Areas Covered Monogenic antibodies were described as treatment options for MS, however the immunogenicity of mouse antibodies hampered the efficacy of potential therapeutics in humans. Availability of improved antibody production technologies resulted in a paradigm shift in MS treatment strategies. In this review, an overview of immunotherapies for MS that use conventional monoclonal antibodies reactive to immune system and their properties and mechanisms of action will be discussed, including recent advances in MS therapeutics and highlight natural autoantibodies (NAbs) that directly target CNS cells. Expert Opinion Recent challenges for MS therapy are the identification of relevant molecular and cellular targets, time frame of treatment, and antibody toxicity profiles to identify safe treatment options for MS patients. The application of monoclonal antibody therapies with better biological efficacy associated with minimum side effects possesses huge clinical potential. Advances in monoclonal antibody technologies that directly target cells of nervous system may promote the CNS regeneration field from bench to bedside. PMID:26914737

  4. Application of Monoclonal Antibodies in Veterinary Parasitology

    Directory of Open Access Journals (Sweden)

    Gupta A.

    2011-08-01

    Full Text Available The discovery of hybridoma technology by Kohler and Milstein in 1975, heralded a new era in antibody research. Mouse hybridomas were the first reliable source of monoclonal antibodies. The generation of monoclonal antibodies from species other than rats and mice, has developed slowly over the last 30 years. The advent of antibody engineering and realization of the advantages of non murine antibodies has increased their relevance recently. However, in the area of veterinary parasitology, monoclonal antibodies are just beginning to fulfill the promises inherent in their great specificity for recognizing and selectively binding to antigens. This review describes the recent advances in the application of monoclonal antibodies for immunodiagnosis / prophylaxis and immunotherapy of parasitic diseases. [Vet. World 2011; 4(4.000: 183-188

  5. Monoclonal antibodies to immunodeterminants of lipoteichoic acids.

    Science.gov (United States)

    Jackson, D E; Wong, W; Largen, M T; Shockman, G D

    1984-03-01

    Murine hybrid cell lines producing monoclonal antibodies directed against determinants present on lipoteichoic acids were generated. Hapten inhibition studies showed that one group of monoclonal antibodies was inhibited by deacylated cardiolipin, and the second group was inhibited by kojibiose. Thus, antibodies directed against the polyglycerophosphate chain, which is common to the lipoteichoic acids of many gram-positive species, and against the streptococcal group D antigen were obtained.

  6. Monoclonal antibodies to immunodeterminants of lipoteichoic acids.

    OpenAIRE

    Jackson, D E; Wong, W; Largen, M T; Shockman, G. D.

    1984-01-01

    Murine hybrid cell lines producing monoclonal antibodies directed against determinants present on lipoteichoic acids were generated. Hapten inhibition studies showed that one group of monoclonal antibodies was inhibited by deacylated cardiolipin, and the second group was inhibited by kojibiose. Thus, antibodies directed against the polyglycerophosphate chain, which is common to the lipoteichoic acids of many gram-positive species, and against the streptococcal group D antigen were obtained.

  7. Monoclonal gammopathy missed by capillary zone electrophoresis

    OpenAIRE

    Schild, Christof; Egger, Florence; Kaelin-Lang, Alain; Nuoffer, Jean-Marc

    2017-01-01

    Background: Serum protein electrophoresis is used as a screening test for monoclonal gammopathies. Here, we present a case of a high-concentration monoclonal immunoglobulin (M-protein) that was missed by serum protein electrophoresis on a Capillarys 2 capillary zone electrophoresis system. The aim of our study was to identify the reason for the failure of the system to detect the M-protein. Methods: M-protein solubility was examined in response to temperature, pH, ionic strength, the chaotrop...

  8. Antibodies and Selection of Monoclonal Antibodies.

    Science.gov (United States)

    Hanack, Katja; Messerschmidt, Katrin; Listek, Martin

    Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology.

  9. Improved monoclonal antibodies to halodeoxyuridine

    Science.gov (United States)

    Vanderlaan, M.; Dolbeare, F.A.; Gray, J.W.; Thomas, C.B.

    1983-10-18

    The development, method of production, characterization and methods of use of two hybridomas, CIdU-1 (ATCC Accession No. HB-8321) and CIdU-2 (ATCC Accession No. HB-8320), are described. These secrete IgG/sub 1/(K) immunoglobulins that react with halodeoxyuridine (HdU or halodU) such as bromo, chloro, fluoro and iodo deoxyuridine (BrdU, CldU, FdU and IdU), whether these are free in solution or incorporated into single stranded DNA in whole cells. The antibodies do not react with naturally occurring free nucleic acids or with deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) polymers. These antibodies are suitable for use in enzyme immunoassays for free CldU, FdU, IdU and BrdU and for detecting cells with these nucleotides incorporated into them. The monoclonal antibodies are useful in the detection of the sensitivity of tumor cells to specific chemotherapeutic agents, in the measurement of the rate of cellular DNA synthesis, in the measurement of the rate of proliferation of normal and malignant cells and in the detection of HPRT deficiency in cells. 1 tab.

  10. A single-arm, open-label, phase 2 clinical trial evaluating disease response following treatment with BI-505, a human anti-intercellular adhesion molecule-1 monoclonal antibody, in patients with smoldering multiple myeloma.

    Directory of Open Access Journals (Sweden)

    Stina Wichert

    Full Text Available Smoldering multiple myeloma (SMM is an indolent disease stage, considered to represent the transition phase from the premalignant MGUS (Monoclonal Gammopathy of Undetermined Significance state towards symptomatic multiple myeloma (MM. Even though this diagnosis provides an opportunity for early intervention, few treatment studies have been done and the current standard of care is observation until progression. BI-505, a monoclonal antibody directed against intercellular adhesion molecule 1 (ICAM-1 with promising anti-myeloma activity in preclinical trials, is a possible treatment approach for this patient category with potential to eliminate tumor cells with minimal long-term side effects. BI-505 was well tolerated in an earlier phase 1 trial.In this phase 2 trial the effects of BI-505 in patients with SMM were studied. Four patients were enrolled and three of them completed the first cycle of treatment defined as 5 doses of BI-505, a total of 43 mg/kg BW, over a 7-week period. In the three evaluable patients, BI-505 showed a benign safety profile. None of the patients achieved a response as defined per protocol. EudraCT number: 2012-004884-29.The study was conducted to assess the efficacy, safety and pharmacodynamics of BI-505 in patients with SMM. BI-505 showed no clinically relevant efficacy on disease activity in these patients with SMM, even if well tolerated.ClinicalTrials.gov Identifier: NCT01838369.

  11. A single-arm, open-label, phase 2 clinical trial evaluating disease response following treatment with BI-505, a human anti-intercellular adhesion molecule-1 monoclonal antibody, in patients with smoldering multiple myeloma.

    Science.gov (United States)

    Wichert, Stina; Juliusson, Gunnar; Johansson, Åsa; Sonesson, Elisabeth; Teige, Ingrid; Wickenberg, Anna Teige; Frendeus, Björn; Korsgren, Magnus; Hansson, Markus

    2017-01-01

    Smoldering multiple myeloma (SMM) is an indolent disease stage, considered to represent the transition phase from the premalignant MGUS (Monoclonal Gammopathy of Undetermined Significance) state towards symptomatic multiple myeloma (MM). Even though this diagnosis provides an opportunity for early intervention, few treatment studies have been done and the current standard of care is observation until progression. BI-505, a monoclonal antibody directed against intercellular adhesion molecule 1 (ICAM-1) with promising anti-myeloma activity in preclinical trials, is a possible treatment approach for this patient category with potential to eliminate tumor cells with minimal long-term side effects. BI-505 was well tolerated in an earlier phase 1 trial. In this phase 2 trial the effects of BI-505 in patients with SMM were studied. Four patients were enrolled and three of them completed the first cycle of treatment defined as 5 doses of BI-505, a total of 43 mg/kg BW, over a 7-week period. In the three evaluable patients, BI-505 showed a benign safety profile. None of the patients achieved a response as defined per protocol. EudraCT number: 2012-004884-29. The study was conducted to assess the efficacy, safety and pharmacodynamics of BI-505 in patients with SMM. BI-505 showed no clinically relevant efficacy on disease activity in these patients with SMM, even if well tolerated. ClinicalTrials.gov Identifier: NCT01838369.

  12. Cold denaturation of monoclonal antibodies

    Science.gov (United States)

    Lazar, Kristi L; Patapoff, Thomas W

    2010-01-01

    The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from −5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ΔGu, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. the Gibbs-Helmholtz expression fit to the ΔGu versus temperature data from fluorescence gave a ΔCp of 8.0 kcal mol−1 K−1, a maximum apparent stability of 23.7 kcal mol−1 at 18°C, and an apparent cold denaturation temperature (TCD) of −23°C. ΔGu values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative TCD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. thus, mAbs have a potential to undergo cold denaturation at storage temperatures near −20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs. PMID:20093856

  13. Mammalian tissue distribution of a large heparan sulfate proteoglycan detected by monoclonal antibodies

    DEFF Research Database (Denmark)

    Couchman, J R; Ljubimov, A V

    1989-01-01

    A panel of nine monoclonal antibodies has been characterized, all of which have reactivity with the core protein of a large heparan sulfate proteoglycan derived from the murine EHS tumor matrix. These rat monoclonal antibodies stained mouse basement membranes intensely, including those of all...... muscle, endothelia, peripheral nerve fibers and epithelia so far examined. In addition, two of the monoclonal antibodies show cross-species reactivity, staining bovine and human basement membranes, and immunoprecipitating proteoglycans from human endothelial cell cultures. These antibodies do not......, however, cross-react with avian tissues. These results show the ubiquitous distribution of a heparan sulfate proteoglycan in mammalian tissues, which will be useful in vitro and in vivo for studies on the biology of basement membrane proteoglycans and investigations of possible roles of these molecules...

  14. Monoclonal antibodies to hepatitis B surface antigen: production and characterization.

    Science.gov (United States)

    Hlozánek, I; Dostálová, V; Korec, E; Zelený, V; König, J; Nĕmecek, V

    1986-01-01

    Hybridomas secreting anti-HBsAg antibodies were produced by fusion of the mouse myeloma cell line SP2/0 with lymphocytes from mice immunized with purified HBsAg. All clones produced antibodies of the IgG1 idiotype that react with the subtype a determinant of HBsAg. An enzyme immunoassay for detection of HBsAg in human sera using monoclonal antibodies was developed and compared with commercial Sevatest ELISA HBsAg/micro I kit for detection of HBsAg in clinical serum samples.

  15. IgM monoclonal gammopathy and neuropathy in two siblings

    DEFF Research Database (Denmark)

    Jensen, T S; Schrøder, H D; Jønsson, V

    1988-01-01

    A sister and a brother with a progressive mixed axonal and demyelinating polyneuropathy were found to have a monoclonal IgM gammopathy of kappa and lambda type, respectively. Sural nerve and cutaneous nerve specimens obtained by biopsy showed deposits of IgM on myelin sheets. Sera from both...... patients contained antibodies directed to bovine peripheral nerve myelin as determined by ELISA technique and to normal human peripheral nerve myelin as demonstrated by indirect immunofluorescence histochemistry. These siblings may have a genetic predisposition to the formation of autoantibodies...... with peripheral nerve myelin as the target for the immune attack....

  16. Production and characterization of monoclonal antibodies against mink leukocytes

    DEFF Research Database (Denmark)

    Chen, W.S.; Pedersen, Mikael; Gram-Nielsen, S.

    1997-01-01

    Three monoclonal antibodies (mAbs) were generated against mink leukocytes. One antibody reacted with all T lymphocytes, one with all monocytes and one had platelet reactivity. Under reducing conditions, the T lymphocyte reactive antibody immunoprecipitated 18 kDa, 23 kDa, 25 kDa and 32-40 k...... leukocytes of human and various animal species have been analysed for cross-reactivity to mink leukocytes. We found eight to cross-react with mink. Of particular importance was an anticanine CD11a mAb, an antihuman CD79a mAb and an antihuman bcl-2 mAb....

  17. Fixed Dosing of Monoclonal Antibodies in Oncology.

    Science.gov (United States)

    Hendrikx, Jeroen J M A; Haanen, John B A G; Voest, Emile E; Schellens, Jan H M; Huitema, Alwin D R; Beijnen, Jos H

    2017-10-01

    Most monoclonal antibodies in oncology are administered in body-size-based dosing schedules. This is believed to correct for variability in both drug distribution and elimination between patients. However, monoclonal antibodies typically distribute to the blood plasma and extracellular fluids only, which increase less than proportionally with the increase in body weight. Elimination takes place via proteolytic catabolism, a nonspecific immunoglobulin G elimination pathway, and intracellular degradation after binding to the target. The latter is the primary route of elimination and is related to target expression levels rather than body size. Taken together, the minor effects of body size on distribution and elimination of monoclonal antibodies and their usually wide therapeutic window do not support body-size-based dosing. We evaluated effects of body weight on volume of distribution and clearance of monoclonal antibodies in oncology and show that a fixed dose for most of these drugs is justified based on pharmacokinetics. A survey of the savings after fixed dosing of monoclonal antibodies at our hospital showed that fixed dosing can reduce costs of health care, especially when pooling of preparations is not possible (which is often the case in smaller hospitals). In conclusion, based on pharmacokinetic parameters of monoclonal antibodies, there is a rationale for fixed dosing of these drugs in oncology. Therefore, we believe that fixed dosing is justified and can improve efficiency of the compounding. Moreover, drug spillage can be reduced and medication errors may become less likely. The currently available knowledge of elimination of monoclonal antibodies combined with the publicly available data from clinical trials and extensive population pharmacokinetic (PopPK) modeling justifies fixed dosing. Interpatient variation in exposure is comparable after body weight and fixed dosing and most monoclonal antibodies show relatively flat dose-response relationships

  18. Monoclonal Antibodies, Derived from Humans Vaccinated with the RV144 HIV Vaccine Containing the HVEM Binding Domain of Herpes Simplex Virus (HSV) Glycoprotein D, Neutralize HSV Infection, Mediate Antibody-Dependent Cellular Cytotoxicity, and Protect Mice from Ocular Challenge with HSV-1.

    Science.gov (United States)

    Wang, Kening; Tomaras, Georgia D; Jegaskanda, Sinthujan; Moody, M Anthony; Liao, Hua-Xin; Goodman, Kyle N; Berman, Phillip W; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Nitayapan, Sorachai; Kaewkungwal, Jaranit; Haynes, Barton F; Cohen, Jeffrey I

    2017-10-01

    The RV144 HIV vaccine trial included a recombinant HIV glycoprotein 120 (gp120) construct fused to a small portion of herpes simplex virus 1 (HSV-1) glycoprotein D (gD) so that the first 40 amino acids of gp120 were replaced by the signal sequence and the first 27 amino acids of the mature form of gD. This region of gD contains most of the binding site for HVEM, an HSV receptor important for virus infection of epithelial cells and lymphocytes. RV144 induced antibodies to HIV that were partially protective against infection, as well as antibodies to HSV. We derived monoclonal antibodies (MAbs) from peripheral blood B cells of recipients of the RV144 HIV vaccine and showed that these antibodies neutralized HSV-1 infection in cells expressing HVEM, but not the other major virus receptor, nectin-1. The MAbs mediated antibody-dependent cellular cytotoxicity (ADCC), and mice that received the MAbs and were then challenged by corneal inoculation with HSV-1 had reduced eye disease, shedding, and latent infection. To our knowledge, this is the first description of MAbs derived from human recipients of a vaccine that specifically target the HVEM binding site of gD. In summary, we found that monoclonal antibodies derived from humans vaccinated with the HVEM binding domain of HSV-1 gD (i) neutralized HSV-1 infection in a cell receptor-specific manner, (ii) mediated ADCC, and (iii) reduced ocular disease in virus-infected mice. IMPORTANCE Herpes simplex virus 1 (HSV-1) causes cold sores and neonatal herpes and is a leading cause of blindness. Despite many trials, no HSV vaccine has been approved. Nectin-1 and HVEM are the two major cellular receptors for HSV. These receptors are expressed at different levels in various tissues, and the role of each receptor in HSV pathogenesis is not well understood. We derived human monoclonal antibodies from persons who received the HIV RV144 vaccine that contained the HVEM binding domain of HSV-1 gD fused to HIV gp120. These antibodies were

  19. Engineering, Expression in Transgenic Plants and Characterisation of E559, a Rabies Virus-Neutralising Monoclonal Antibody

    OpenAIRE

    van Dolleweerd, CJ; Teh, AY; Banyard, AC; Both, L; Lotter-Stark, HC; Tsekoa, T; Phahladira, B; Shumba, W; Chakauya, E.; Sabeta, CT; Gruber, C.; Fooks, AR; Chikwamba, RK; Ma, JK

    2014-01-01

    Rabies post-exposure prophylaxis (PEP) currently comprises administration of rabies vaccine together with rabies immunoglobulin (RIG) of either equine or human origin. In the developing world, RIG preparations are expensive, often in short supply, and of variable efficacy. Therefore, we are seeking to develop a monoclonal antibody cocktail to replace RIG. Here, we describe the cloning, engineering and production in plants of a candidate monoclonal antibody (E559) for inclusion in such a cockt...

  20. Antibody-mediated immune suppression is improved when blends of anti-RBC monoclonal antibodies are used in mice.

    Science.gov (United States)

    Bernardo, Lidice; Amash, Alaa; Marjoram, Danielle; Lazarus, Alan H

    2016-08-25

    Although the prevention of hemolytic disease of the fetus and newborn is highly effective using polyclonal anti-D, a recombinant alternative is long overdue. Unfortunately, anti-D monoclonal antibodies have been, at best, disappointing. To determine the primary attribute defining an optimal antibody, we assessed suppression of murine red blood cell (RBC) immunization by single-monoclonal antibodies vs defined blends of subtype-matched antibodies. Allogeneic RBCs expressing the HOD antigen (hen egg lysozyme [HEL]-ovalbumin-human transmembrane Duffy(b)) were transfused into naïve mice alone or together with selected combinations of HEL-specific antibodies, and the resulting suppressive effect was assessed by evaluating the antibody response. Polyclonal HEL antibodies dramatically inhibited the antibody response to the HOD antigen, whereas single-monoclonal HEL antibodies were less effective despite the use of saturating doses. A blend of monoclonal HEL-specific antibodies reactive with different HEL epitopes significantly increased the suppressive effect, whereas a blend of monoclonal antibodies that block each other's binding to the HEL protein did not increase suppression. In conclusion, these data show that polyclonal antibodies are superior to monoclonal antibodies at suppressing the immune response to the HOD cells, a feature that can be completely recapitulated using monoclonal antibodies to different epitopes. © 2016 by The American Society of Hematology.

  1. The effect of artificial antigen-presenting cells with preclustered anti-CD28/-CD3/-LFA-1 monoclonal antibodies on the induction of ex vivo expansion of functional human antitumor T cells.

    Science.gov (United States)

    Zappasodi, Roberta; Di Nicola, Massimo; Carlo-Stella, Carmelo; Mortarini, Roberta; Molla, Alessandra; Vegetti, Claudia; Albani, Salvatore; Anichini, Andrea; Gianni, Alessandro M

    2008-10-01

    Adoptive cell therapy with ex vivo expanded autologous antitumor cytotoxic T lymphocytes represents an important therapeutic option as an anticancer strategy. In order to identify a reliable method for producing adequate amounts of functional antitumor cytotoxic T lymphocytes with a potentially long in vivo lifespan, we tested the T-cell expansion efficiency of a new artificial antigen-presenting cell-based system. Our artificial antigen-presenting cells were generated with activating (anti-CD3), co-stimulating (anti-CD28) and adhesion (anti-LFA-1) biotinylated monoclonal antibodies preclustered in microdomains held on a liposome scaffold by neutravidin rafts. The co-localization of T-cell ligands in microdomains and the targeting of an adhesion protein, increasing the efficiency of immunological synapse formation, represent the novelties of our system. The activity of our artificial antigen-presenting cells was compared with that of anti-CD3/-CD28 coated immunomagnetic microbeads and immobilized anti-CD3 monoclonal antibody (OKT3 clone), the only two commercially available artificial systems. Our artificial antigen-presenting cells expanded both polyclonal T cells and MART-1-specific CD8(+) T cells in a more efficient manner than the other systems. Stimulation with artificial antigen-presenting cells allows for the generation of viable T cells displaying an immunophenotype consistent with in vivo potential for persistence, without increasing the frequency of regulatory T cells. The starting specificity of anti MART-1 CD8(+) T cells was preserved after stimulation with artificial antigen-presenting cells and it was statistically greater when compared to the activity of the same cells expanded with the other systems. Finally, our artificial antigen-presenting cells proved to be suitable for large-scale application, minimizing the volume and the costs of T-cell expansion. Our artificial antigen-presenting cells might represent an efficient tool to rapidly obtain a

  2. INITIAL CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST THE FUNGAL HEMOLYSIN STACHYLYSIN FROM STACHYBOTRYS CHARTARUM

    Science.gov (United States)

    Stachybotrys chartarum is known to produce the hemolysin stachylysin and its detection in human serum has been proposed as a biomarker for exposure to the fungus. In this study we report the initial characterization of monoclonal antibodies (mAbs) against stachylysin and the dev...

  3. Purification process monitoring in monoclonal antibody preparation: contamination with viruses, DNA and peptide growth factors.

    NARCIS (Netherlands)

    A.R. ter Avest (Anja); E.J.J. van Zoelen (Everardus); A.D.M.E. Osterhaus (Albert); C.F. van Kreyl; G. van Steenis (Bert); H.E.M. Spijkers (Ine)

    1992-01-01

    textabstractAdministration in vivo of monoclonal antibodies to humans is challenged by considerations regarding their safety. Contamination with viruses, potentially oncogenic nucleic acids and biologically active components like growth factors and hormones forms a serious point of concern in this

  4. Comprehensive analysis of varicella-zoster virus proteins using a new monoclonal antibody collection

    NARCIS (Netherlands)

    T.L. Roviš (Tihana Lenac); S.M. Bailer (Susanne); V.R. Pothineni (Venkata R); W.J.D. Ouwendijk (Werner ); H. Šimić (Hrvoje); M. Babić (Marina); K. Miklić (Karmela); S. Malić (Suzana); M.C. Verweij; M. Baiker (Martin); O. Gonzalez (Orland); A. Brunn (Albrecht von); R. Zimmer; K. Früh (Klaus); G.M.G.M. Verjans (George); S. Jonjic (Stipan); J. Haasb (Jürgeni)

    2013-01-01

    textabstractVaricella-zoster virus (VZV) is the etiological agent of chickenpox and shingles. Due to the virus's restricted host and cell typetropism and the lack of tools for VZV proteomics, it is one of the least-characterized human herpesviruses. We generated 251monoclonal antibodies (MAbs)

  5. Anti-interleukin-17 monoclonal antibody ixekizumab in chronic plaque psoriasis

    DEFF Research Database (Denmark)

    Leonardi, Craig; Matheson, Robert; Zachariae, Claus

    2012-01-01

    Type 17 helper T cells have been suggested to play a pathological role in psoriasis. They secrete several proinflammatory cytokines, including interleukin-17A (also known as interleukin-17). We evaluated the safety and efficacy of ixekizumab (LY2439821), a humanized anti-interleukin-17 monoclonal...

  6. Anti-CD45RB monoclonal antibody prolongs renal allograft survival in cynomolgus monkeys

    NARCIS (Netherlands)

    Chen, G.; Luke, P. P. W.; Yang, H.; Visser, L.; Sun, H.; Garcia, B.; Qian, H.; Xiang, Y.; Huang, X.; Liu, W.; Senaldi, G.; Schneider, A.; Poppema, S.; Wang, H.; Jevnikar, A. M.; Zhong, R.

    Previously, an anti-CD45RB monoclonal antibody (mAb) has been shown to induce murine allograft tolerance. The present study was performed to assess the ability of an anti-human CD45RB mAb to prevent rejection in a monkey MHC-mismatched kidney transplant model. The recipients were allocated into the

  7. Intravenous cidofovir for resistant cutaneous warts in a patient with psoriasis treated with monoclonal antibodies.

    LENUS (Irish Health Repository)

    McAleer, M A

    2012-02-01

    Human papilloma virus is a common and often distressing cutaneous disease. It can be therapeutically challenging, especially in immunocompromised patients. We report a case of recalcitrant cutaneous warts that resolved with intravenous cidofovir treatment. The patient was immunocompromised secondary to monoclonal antibody therapy for psoriasis.

  8. Daratumumab: a first-in-class CD38 monoclonal antibody for the treatment of multiple myeloma

    Directory of Open Access Journals (Sweden)

    Larysa Sanchez

    2016-06-01

    Full Text Available Abstract Daratumumab is a human monoclonal antibody that targets CD38, a cell surface protein that is overexpressed on multiple myeloma (MM cells. Preclinical studies have shown that daratumumab induces MM cell death through several mechanisms, including complement-dependent cytotoxicity (CDC, antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cellular phagocytosis (ADCP, and apoptosis. Given the encouraging efficacy and acceptable safety profile of daratumumab demonstrated in clinical trials, daratumumab has emerged as a novel treatment option for myeloma and became the first monoclonal antibody approved by the FDA for the treatment of MM.

  9. Generation of monoclonal antibodies against highly conserved antigens.

    Directory of Open Access Journals (Sweden)

    Hongzhe Zhou

    Full Text Available BACKGROUND: Therapeutic antibody development is one of the fastest growing areas of the pharmaceutical industry. Generating high-quality monoclonal antibodies against a given therapeutic target is very crucial for the success of the drug development. However, due to immune tolerance, some proteins that are highly conserved between mice and humans are not very immunogenic in mice, making it difficult to generate antibodies using a conventional approach. METHODOLOGY/PRINCIPAL FINDINGS: In this report, the impaired immune tolerance of NZB/W mice was exploited to generate monoclonal antibodies against highly conserved or self-antigens. Using two highly conserved human antigens (MIF and HMGB1 and one mouse self-antigen (TNF-alpha as examples, we demonstrate here that multiple clones of high affinity, highly specific antibodies with desired biological activities can be generated, using the NZB/W mouse as the immunization host and a T cell-specific tag fused to a recombinant antigen to stimulate the immune system. CONCLUSIONS/SIGNIFICANCE: We developed an efficient and universal method for generating surrogate or therapeutic antibodies against "difficult antigens" to facilitate the development of therapeutic antibodies.

  10. Selection of Ceratitis capitata (Diptera: Tephritidae) Specific Recombinant Monoclonal Phage Display Antibodies for Prey Detection Analysis

    Science.gov (United States)

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators. PMID:23272105

  11. Monoclonal Antibody Therapy for Advanced Neuroblastoma

    Science.gov (United States)

    NCI is sponsoring two clinical trials of a monoclonal antibody called ch14.18, in combination with other drugs, to see if the antibody may be helpful for children or young adults (up to age 21) with relapsed or refractory neuroblastoma.

  12. Monoclonal antibodies reactive with hairy cell leukemia

    NARCIS (Netherlands)

    Visser, L; Shaw, A; Slupsky, J; Vos, H; Poppema, S

    Monoclonal antibodies reactive with hairy cell leukemia were developed to aid in the diagnosis of this subtype of B cell chronic lymphocytic leukemia and to gain better insight into the origin of hairy cells. Three antibodies were found to be of value in the diagnosis of hairy cell leukemia.

  13. Strain differentiation of polioviruses with monoclonal antibodies.

    NARCIS (Netherlands)

    A.D.M.E. Osterhaus (Albert); A.L. van Wezel; A.J.H. Stegmann; J.A.A.M. van Asten (Jack)

    1984-01-01

    textabstractPanels of monoclonal antibodies raised against different poliovirus type 1, 2 and 3 strains, were tested in a micro-neutralization test and in a micro-enzyme linked immunosorbent assay against a large number of poliovirus strains. The results were compared with those obtained with the

  14. Reactivity of commercially available monoclonal antibodies to ...

    African Journals Online (AJOL)

    Monoclonal antibodies (mAbs) to cell surface molecules have been proven as a key tool for phenotypic and functional characterization of the cellular immune response. One of the major difficulties in studying camel cellular immunity consists in the lack of mAbs that dtect their leukocyte differentiation antigens. In the present ...

  15. Mass-Production and Characterization of Anti-CD20 Monoclonal Antibody in Peritoneum of Balb/c Mice

    Directory of Open Access Journals (Sweden)

    Leili Aghebati

    2013-02-01

    Full Text Available Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Methods: Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After twelve days, approximately 7 ml ascetic fluid was harvested from the peritoneum of each mouse. Evaluation of mAb titration was assessed by ELISA method. In the present study, we describe a protocol for large scale production of MAbs. Results: We prepared monoclonal antibodies (mAbs with high specificity and sensitivity against human CD20 by hybridoma method and characterized them by ELISA. The subclass of antibody was IgG2a and its light chain was kappa. Ascetic fluid was purified by Protein-A Sepharose affinity chromatography and the purified monoclonal antibody was conjugated with FITC and Immunofluorescence was done for confirming the specific binding. Conclusion: The conjugated monoclonal antibody could have application in diagnosis B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells.

  16. [Preparation of clenbuterol monoclonal antibody with subtractive immunization method].

    Science.gov (United States)

    Li, Xiao-Li; Li, Xiao-Fang; Ning, Bao-An; Wu, Da-Cheng; Wang, Hong-Yong; Chen, Xiang; Ma, Xin-Hua; Ou, Guo-Rong; Gau, Zhi-Xian

    2009-11-01

    To obtain Clenbuterol monoclonal antibodies. Clenbuterol complete antigen was prepared with diazotization method. BALB/c mice was immunized with subtractive immunization, Clenbuterol monoclonal antibody was prepared with rule hybridoma technique. The mice obtained tolerance to BSA by subtractive immunization. The rate of the hybridoma cell with positive reaction which had obtained was 8.2%, and the specific clenbuterol monoclonal antibody was obtained at last. Monoclonal antibodies to micromolecule contaminant be prepared by subtractive immunization, could decrease the workload in the bolting of monoclonal antibodies, and increase the chance to obtain the antibody of expected.

  17. Monoclonal antibodies against Haemophilus lipopolysaccharides

    OpenAIRE

    Borrelli, Silvia

    1996-01-01

    The genus Haemophilus comprises a group of gram-negative bacteria with fastidious growthrequirements. Among the Haemophilus species, H. influenzae and H. ducreyi are the mostimportant in human medicine. H. influenzae is a common agent in respiratory tract infections and causes severe diseaseslike bacterial meningitidis, pneumonia, otitis and pericarditis among others. Some strains possess acapsular polysaccharide. Six capsular polysaccharides, designated types a-f, have been defined.Most...

  18. Isolation of highly active monoclonal antibodies against multiresistant gram-positive bacteria.

    Directory of Open Access Journals (Sweden)

    Friederike S Rossmann

    Full Text Available Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA. At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium, a mouse peritonitis model (using S. aureus Newman and LAC and a rat endocarditis model (using E. faecalis 12030 and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials.

  19. Ofatumumab: a novel monoclonal anti-CD20 antibody

    Directory of Open Access Journals (Sweden)

    Thomas S Lin

    2010-05-01

    Full Text Available Thomas S LinGlaxoSmithKline Oncology R&D, Collegeville, PA, USAAbstract: Ofatumumab, a novel humanized monoclonal anti-CD20 antibody, was recently approved by the FDA for the treatment of fludarabine and alemtuzumab refractory chronic lymphocytic leukemia (CLL. Ofatumumab effectively induces complement-dependent cytotoxicity (CDC in vitro, and recent studies demonstrated that ofatumumab also effectively mediates antibody-dependent cellular cytotoxicity (ADCC. Pharmacokinetic studies indicated that increased exposure to the antibody correlated with improved clinical outcome in CLL. Thus, pharmacogenomics may be important in identifying which patients are more likely to respond to ofatumumab therapy, although such studies have not yet been performed. Patients with the high-affinity FCGR3a 158 V/V polymorphism may be more likely to respond to therapy, if ADCC is the primary in vivo mechanism of action of ofatumumab. Patients with increased expression of the complement defense proteins CD55 and CD59 may be less likely to respond if ofatumumab works in vivo primarily via CDC. Patients with increased metabolism and clearance of ofatumumab may have lower exposure and be less likely to respond clinically. Thus, pharmacogenomics may determine the responsiveness of patients to ofatumumab therapy.Keywords: monoclonal antibody, CD20, CLL, NHL, lymphoma

  20. Plant Factories for the Production of Monoclonal Antibodies.

    Science.gov (United States)

    Sheshukova, E V; Komarova, T V; Dorokhov, Y L

    2016-10-01

    Like animal cells, plant cells bear mechanisms for protein synthesis and posttranslational modification (glycosylation and phosphorylation) that allow them to be seriously considered as factories for therapeutic proteins, including antibodies, with the development of biotechnology. The plant platform for monoclonal antibody production is an attractive approach due to its flexibility, speed, scalability, low cost of production, and lack of contamination risk from animal-derived pathogens. Contemporary production approaches for therapeutic proteins rely on transgenic plants that are obtained via the stable transformation of plant cells as well as the transient (temporary) expression of foreign proteins. In this review, we discuss present-day approaches for monoclonal antibody production in plants (MAPP), features of carbohydrate composition, and methods for the humanization of the MAPP carbohydrate profile. MAPPs that have successfully passed preclinical studies and may be promising for use in clinical practice are presented here. Perspectives on using MAPPs are determined by analyzing their economic benefits and production rates, which are especially important in personalized cancer therapy as well as in cases of bioterrorism and pandemics.

  1. Recent developments in monoclonal antibody radiolabeling techniques

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.; Mease, R.C.

    1989-01-01

    Monoclonal antibodies (MAbs) have shown the potential to serve as selective carriers of radionuclides to specific in vivo antigens. Accordingly, there has been an intense surge of research activity in an effort to develop and evaluate MAb-based radiopharmaceuticals for tumor imaging (radioimmunoscintigraphy) and therapy (radioimmunotherapy), as well as for diagnosing nonmalignant diseases. A number of problems have recently been identified, related to the MAbs themselves and to radiolabeling techniques, that comprise both the selectivity and the specificity of the in vivo distribution of radiolabeled MAbs. This paper will address some of these issues and primarily discuss recent developments in the techniques for radiolabeling monoclonal antibodies that may help resolve problems related to the poor in vivo stability of the radiolabel and may thus produce improved biodistribution. Even though many issues are identical with therapeutic radionuclides, the discussion will focus mainly on radioimmunoscintigraphic labels. 78 refs., 6 tabs.

  2. The future of monoclonal antibody technology

    OpenAIRE

    Zider, Alexander; Drakeman, Donald L

    2010-01-01

    With the rapid growth of monoclonal antibody-based products, new technologies have emerged for creating modified forms of antibodies, including fragments, conjugates and multi-specific antibodies. We created a database of 450 therapeutic antibodies in development to determine which technologies and indications will constitute the “next generation” of antibody products. We conclude that the antibodies of the future will closely resemble the antibodies that have already been approved for commer...

  3. Monoclonal Antibodies as Diagnostics; an Appraisal

    OpenAIRE

    Siddiqui M

    2010-01-01

    Ever since the development of Hybridoma Technology in 1975 by Kohler and Milstein, our vision for antibodies as tools for research for prevention, detection and treatment of diseases, vaccine production, antigenic characterization of pathogens and in the study of genetic regulation of immune responses and disease susceptibility has been revolutionized. The monoclonal antibodies being directed against single epitopes are homogeneous, highly specific and can be produced in unlimited quantities....

  4. SPECT assay of radiolabeled monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Jaszczak, R.J.

    1992-02-01

    The accurate determination of the biodistribution of radiolabeled monoclonal antibodies (MoAbs) is important for calculation of dosimetry and evaluation of pharmacokinetic variables such as antibody dose and route of administration. The hypothesis of this application is that the biodistribution of radiolabeled monoclonal antibodies (MoAbs) can be quantitatively determined using single photon emission computed tomography (SPECT). The major thrusts during the third year include the continued development and evaluation of improved 3D SPECT acquisition and reconstruction approaches to improve quantitative imaging of radiolabeled monoclonal antibodies (MoAbs), and the implementation and evaluation of algorithms to register serial SPECT image data sets, or to register 3D SPECT images with 3D image data sets acquired from positron emission tomography (PEI) and magnetic resonance images (MRI). The research has involved the investigation of statistical models and iterative reconstruction algorithms that accurately account for the physical characteristics of the SPECT acquisition system. It is our belief that SPECT quantification can be improved by accurately modeling the physical processes such as attenuation, scatter, geometric collimator response, and other factors that affect the measured projection data.

  5. The use of monoclonal antibodies to treat Castleman's disease.

    Science.gov (United States)

    Robey, Rebecca C; Mletzko, Salvinia; Colley, Charlotte; Balachandran, Kirsty; Bower, Mark

    2014-01-01

    Multicentric Castleman's disease (MCD) is a rare lymphoproliferative disorder presenting with heterogeneous clinical features and with a complex etiology. MCD incidence is increased in people living with HIV/AIDS when it is causally associated with Kaposi's sarcoma-associated herpes virus (KSHV). HIV-seronegative individuals present with either idiopathic or KSHV-associated MCD. Central to MCD pathology is altered expression and signaling of IL-6, which promotes B-cell proliferation and causes systemic manifestations. KSHV encodes a viral homolog of human IL-6, accounting for its role in MCD, while recent evidence shows an association between IL-6 receptor polymorphisms and idiopathic MCD. The increased understanding of mechanisms underlying the pathogenesis of MCD has guided the use of new monoclonal antibody therapies for treating this complex disorder.

  6. Self-Assembly of the Recombinant Capsid Protein of a Swine Norovirus into Virus-Like Particles and Evaluation of Monoclonal Antibodies Cross-Reactive with a Human Strain from Genogroup II

    NARCIS (Netherlands)

    Almanza, H.; Cubillos, C.; Angulo, I.; Mateos, F.; Castön, J.R.; Poel, van der W.H.M.; Vinje, J.; Bárcena, J.; Mena, I.

    2008-01-01

    Noroviruses (NoVs) are responsible for the majority of gastroenteritis outbreaks in humans. Recently, NoV strains which are genetically closely related to human genogroup II (GII) NoVs have been detected in fecal specimens from swine. These findings have raised concern about the possible role of

  7. Radioimmunotherapy with [sup 90]Y-labeled monoclonal antibodies in a nude mouse ovarian cancer model

    Energy Technology Data Exchange (ETDEWEB)

    Kairemo, K. (Dept. of Clinical Chemistry, Helsinki Univ. Central Hospital (Finland)); Ljunggren, K. (Dept. of Radiation Physics, Lund Univ. (Sweden)); Strand, S.E. (Dept. of Radiation Physics, Lund Univ. (Sweden)); Hiltunen, J. (MAP Medical Technologies, Jyvaeskylae (Finland)); Penttilae, P. (MAP Medical Technologies, Jyvaeskylae (Finland)); Nikula, T. (MAP Medical Technologies, Jyvaeskylae (Finland)); Laine, A. (Cancer Research Lab., Orion/Farmos-Group Ltd., Turku (Finland)); Wahlstroem, T. (Dept. of Obstetrics and Gynecology, Helsinki Univ. Central Hospital (Finland))

    1993-01-01

    Tumor stroma contains much fibrin, and so monoclonal antifibrin antibody can accumulate in tumors. We treated nude mice bearing human ovarian carcinoma xenografts with [sup 90]Y-labeled monoclonal antifibrin antibody Fab fragments administered intratumorally. The survival time vs. a control group was significantly prolonged and tumor growth rate was decreased. Another group of animals was treated with [sup 90]Y-labeled OC 125-monoclonal antibody; these mice received the antibodies intratumorally, intraperitoneally or intravenously. The survival time was longest in the intratumorally treated group. There was no significant difference in survival between [sup 90]Y-labeled OC 125 and antifibrin in the intratumorally treated animal groups. The tissue activity distribution studies revealed that bone marrow is the critical organ. Intratumorally injected monoclonal [sup 90]Y-antifibrin antibodies were retained at least 36 h (up to 50% of injected activity per gram tumor tissue) in the xenograft after one treatment, causing cell death. Beta-camera imaging and immunohistochemistry were performed for studies of the correlation between [sup 90]Y activity and fibrin distribution in tumor specimens. These results were in concordance. In conclusion, intratumoral administration seems suitable for radioimmunotherapy, with an antibody that targets stromal structures. The accumulation can be successfully monitored by a beta-camera. (orig.).

  8. Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model.

    Science.gov (United States)

    Duehr, James; Wohlbold, Teddy John; Oestereich, Lisa; Chromikova, Veronika; Amanat, Fatima; Rajendran, Madhusudan; Gomez-Medina, Sergio; Mena, Ignacio; tenOever, Benjamin R; García-Sastre, Adolfo; Basler, Christopher F; Munoz-Fontela, Cesar; Krammer, Florian

    2017-08-15

    Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2-/- mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro, suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity.IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two

  9. Recognition of Coccidioides immitis Antigens with Monoclonal Antibodies.

    Science.gov (United States)

    1986-09-30

    AD-A114 322 RECOGNITION OF COCCIDIOIDES IMMITIS ANTIGENS NITH 1/1 MONOCLONAL ANT ISODIESMU CALIFORNIA UNIV OAKLAND NAVAL SIOSCIENCES LAB S J KRAEGER...031 NR204-123_ 11 (1lEWnld SeuiyCasification) ([L ECOGNITIB’N OF COCCIDIQIDES IMMITIS ANTIGENS WITH MONOCLONAL ANTIBODIES 12 PERSONAL A THOR(S...specificity and suitability for diagnostic use of seven 1gM monoclonal antibodies (MAbs) prepared in 1984 with c. immitis Silveira spherules and

  10. The Role of Monoclonal Antibodies in the Management of Leukemia

    Directory of Open Access Journals (Sweden)

    Mohamad Cherry

    2010-10-01

    Full Text Available This article will review the monoclonal antibodies more commonly used in leukemias. In the last three decades, scientists have made considerable progress understanding the structure and the functions of various surface antigens, such as CD20, CD33. The introduction of rituximab, an anti CD20 monoclonal antibody, had a great impact in the treatment of lymphoproliferative disorders. Gemtuzumab, an anti CD 33 conjugated monoclonal antibody has activity in acute mylegenous leukemia (AML. As this field is undergoing a rapid growth, the years will see an increasing use of monoclonal antibodies in hematological malignancies.

  11. The Role of Monoclonal Antibodies in the Management of Leukemia

    Science.gov (United States)

    Al-Ameri, Ali; Cherry, Mohamad; Al-Kali, Aref; Ferrajoli, Alessandra

    2010-01-01

    This article will review the monoclonal antibodies more commonly used in leukemias. In the last three decades, scientists have made considerable progress understanding the structure and the functions of various surface antigens, such as CD20, CD33. The introduction of rituximab, an anti CD20 monoclonal antibody, had a great impact in the treatment of lymphoproliferative disorders. Gemtuzumab, an anti CD 33 conjugated monoclonal antibody has activity in acute mylegenous leukemia (AML). As this field is undergoing a rapid growth, the years will see an increasing use of monoclonal antibodies in hematological malignancies.

  12. Monoclonal gammopathy of undetermined significance and smoldering multiple myeloma.

    Science.gov (United States)

    Kyle, Robert A; San-Miguel, Jesus F; Mateos, Maria-Victoria; Rajkumar, S Vincent

    2014-10-01

    Monoclonal gammopathy of undetermined significance (MGUS) is characterized by an M spike less than 3 g/dL and a bone marrow containing fewer than 10% plasma cells without evidence of CRAB (hypercalcemia, renal insufficiency, anemia, or bone lesions). Light chain MGUS has an abnormal free light chain (FLC) ratio, increased level of the involved FLC, no monoclonal heavy chain, and fewer than 10% monoclonal plasma cells in the bone marrow. Smoldering multiple myeloma has an M protein of at least 3 g/dL and/or at least 10% monoclonal plasma cells in the bone marrow without CRAB features. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. [Monoclonal antibodies against PCSK9: from bench to clinic].

    Science.gov (United States)

    Guijarro Herraiz, Carlos

    2016-05-01

    Antibodies are glycoproteins with high specificity binding to multiple antigens due to the large number of structural conformations of the variable chains. Hybridoma technology (fusion of myeloma cells with immunoglobulin-producing lymphocytes) has allowed the synthesis of large quantities of unique antibodies (monoclonal [mAb]). mAbs were initially murine. Subsequently, chimeric mAbs were developed, followed by humanized mAbs and finally human mAbs. The high selectivity and good tolerance of human mAbs allows their therapeutic administration to block specific exogenous or endogenous molecules. Selective human mAbs to the catalytic domain of PCSK9 have recently been developed. These antibodies block PCSK9, favour low-density lipoprotein receptor recycling and markedly reduce circulating cholesterol. Preliminary studies indicate that lowering cholesterol through anti-PCSK9 antibodies may significantly reduce the cardiovascular complications of arteriosclerosis. Copyright © 2016 Elsevier España, S.L.U. y Sociedad Española de Arteriosclerosis. All rights reserved.

  14. A randomized, double-blind, placebo-controlled assessment of BMS-936558, a fully human monoclonal antibody to programmed death-1 (PD-1), in patients with chronic hepatitis C virus infection

    National Research Council Canada - National Science Library

    Gardiner, David; Lalezari, Jay; Lawitz, Eric; DiMicco, Michael; Ghalib, Rheem; Reddy, K Rajender; Chang, Kyong-Mi; Sulkowski, Mark; Marro, Steven O'; Anderson, Jeffrey; He, Bing; Kansra, Vikram; McPhee, Fiona; Wind-Rotolo, Megan; Grasela, Dennis; Selby, Mark; Korman, Alan J; Lowy, Israel

    2013-01-01

    Expression of the programmed death 1 (PD-1) receptor and its ligands are implicated in the T cell exhaustion phenotype which contributes to the persistence of several chronic viral infections, including human hepatitis C virus (HCV...

  15. Laboratory testing for monoclonal gammopathies: Focus on monoclonal gammopathy of undetermined significance and smoldering multiple myeloma.

    Science.gov (United States)

    Willrich, Maria A V; Murray, David L; Kyle, Robert A

    2018-01-01

    Monoclonal gammopathies (MG) are defined by increased proliferation of clonal plasma cells, resulting in a detectable abnormality called monoclonal component or M-protein. Detection of the M-protein as either narrow peaks on protein electrophoresis and discrete bands on immunofixation is the defining feature of MG. MG are classified as low-tumor burden disorders, pre-malignancies and malignancies. Since significant disease can be present at any level, several different tests are employed in order to encompass the inherent diverse nature of the M-proteins. In this review, we discuss the main characteristics and limitations of clinical assays to detect M-proteins: protein electrophoresis, immunofixation, immunoglobulin quantitation, serum free light chains and heavy-light chain assays, as well as the newly developed MALDI-TOF mass spectrometric methods. In addition, the definitions of the pre-malignancies monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), as well as monoclonal gammopathy of renal significance (MGRS) are presented in the context of the 2014 international guidelines for definition of myeloma requiring treatment, and the role of the laboratory in test selection for screening and monitoring these conditions is highlighted. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  16. Monoclonal Antibodies for Relapsing Multiple Sclerosis

    DEFF Research Database (Denmark)

    Blinkenberg, Morten; Soelberg Sørensen, Per

    2017-01-01

    leading to chronic central nervous system (CNS) demyelination, neural loss, and, finally, neurological disability. Although a number of disease-modifying treatments are available for the treatment of the inflammatory phase of MS, there is still a need for highly efficacious therapies with an acceptable...... safety profile in order to gain therapeutic control early in the disease course. Monoclonal antibodies have proven to be some of the most efficacious disease-modifying therapies in the field of MS, and recent developments in clinical research hold promise for new compounds fulfilling the need...

  17. Defining Influenza A Virus Hemagglutinin Antigenic Drift by Sequential Monoclonal Antibody Selection

    OpenAIRE

    Das, Suman R.; Hensley, Scott E.; Ince, William L.; Brooke, Christopher B.; Subba, Anju; Delboy, Mark G.; Russ, Gustav; Gibbs, James S.; Bennink, Jack R.; Yewdell, Jonathan W.

    2013-01-01

    Human influenza A virus (IAV) vaccination is limited by “antigenic drift,” rapid antibody-driven escape reflecting amino acid substitutions in the globular domain of hemagglutinin (HA), the viral attachment protein. To better understand drift, we used anti-hemagglutinin monoclonal Abs (mAbs) to sequentially select IAV escape mutants. Twelve selection steps, each resulting in a single amino acid substitution in the hemagglutinin globular domain, were required to eliminate antigenicity defined ...

  18. Detection and characterization of subvisible aggregates of monoclonal lgG in serum

    OpenAIRE

    Filipe, Vasco; Poole, Robert; Oyetayo, Olubukayo; Braeckmans, Kevin; Jiskoot, Wim

    2012-01-01

    Purpose To detect and characterize the aggregation of therapeutic monoclonal antibodies in undiluted biological fluids. Methods Fluorescently labeled subvisible IgG aggregates formed by applying either heat stress or by pH-shift were investigated immediately after addition to human serum, and after 24 h. Unstressed and stressed IgG formulations were analyzed by fluorescence single particle tracking, confocal laser scanning microscopy and flow cytometry. Results Unstressed formulations remaine...

  19. Purification of chimeric heavy chain monoclonal antibody EG2-hFc using hydrophobic interaction membrane chromatography: an alternative to protein-A affinity chromatography.

    Science.gov (United States)

    Sadavarte, Rahul; Spearman, Maureen; Okun, Natalie; Butler, Michael; Ghosh, Raja

    2014-06-01

    Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc. © 2014 Wiley Periodicals, Inc.

  20. Monoclonal gammopathy missed by capillary zone electrophoresis.

    Science.gov (United States)

    Schild, Christof; Egger, Florence; Kaelin-Lang, Alain; Nuoffer, Jean-Marc

    2011-07-01

    Serum protein electrophoresis is used as a screening test for monoclonal gammopathies. Here, we present a case of a high-concentration monoclonal immunoglobulin (M-protein) that was missed by serum protein electrophoresis on a Capillarys 2 capillary zone electrophoresis system. The aim of our study was to identify the reason for the failure of the system to detect the M-protein. M-protein solubility was examined in response to temperature, pH, ionic strength, the chaotropic agent urea and the reducing agent 2-mercaptoethanol. Precipitation of the M-protein was not cold-induced, but solubility decreased at pH 8.5 or higher, when the pH approached the apparent isoelectric point. The M-protein also precipitated in alkaline Capillarys 2 electrophoresis buffer (pH 10), which was the reason for the false-negative electrophoresis result. Precipitation of the M-protein was not related to the ionic strength of the buffer. Solubility improved in presence of urea. Pre-treatment of serum with 2-mercaptoethanol revealed the missing M-protein peak of 36 g/L on the electropherogram. This case shows that insolubility of M-proteins in alkaline buffer is one possible cause of false-negative results on capillary zone electrophoresis systems. False-negative results should be considered, especially when accompanying laboratory results are inconsistent with the electropherogram.

  1. Aggregates in monoclonal antibody manufacturing processes.

    Science.gov (United States)

    Vázquez-Rey, María; Lang, Dietmar A

    2011-07-01

    Monoclonal antibodies have proved to be a highly successful class of therapeutic products. Large-scale manufacturing of pharmaceutical antibodies is a complex activity that requires considerable effort in both process and analytical development. If a therapeutic protein cannot be stabilized adequately, it will lose partially or totally its therapeutic properties or even cause immunogenic reactions thus potentially further endangering the patients' health. The phenomenon of protein aggregation is a common issue that compromises the quality, safety, and efficacy of antibodies and can happen at different steps of the manufacturing process, including fermentation, purification, final formulation, and storage. Aggregate levels in drug substance and final drug product are a key factor when assessing quality attributes of the molecule, since aggregation might impact biological activity of the biopharmaceutical. In this review it is analyzed how aggregates are formed during monoclonal antibody industrial production, why they have to be removed and the manufacturing process steps that are designed to either minimize or remove aggregates in the final product. Copyright © 2011 Wiley Periodicals, Inc.

  2. Generation and Characterization of Anti-CD34 Monoclonal Antibodies that React with Hematopoietic Stem Cells.

    Science.gov (United States)

    Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Movassaghpour, Aliakbar; Abdolalizadeh, Jalal

    2014-01-01

    CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have generated and characterized monoclonal antibodies (mAbs) directed against a CD34 marker. Mice were immunized with two keyhole lympet hemocyanin (KLH)-conjugated CD34 peptides. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by the limiting dilution (L.D) method. Several monoclones were isolated by three rounds of limited dilutions. From these, we chose stable clones that presented sustained antibody production for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the CD34 peptides and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the mAbs (3D5) was strongly reactive against the CD34 peptide and with native CD34 from human umbilical cord blood cells (UCB) in ELISA and Western blotting analyses. The results have shown that this antibody is highly specific and functional in biomedical applications such as ELISA and Western blot assays. This monoclonal antibodies (mAb) can be a useful tool for isolation and purification of human hematopoietic stem cells (HSCs).

  3. Anti-alpha-hemolysin monoclonal antibodies mediate protection against Staphylococcus aureus pneumonia.