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Sample records for human anti-streptococcal monoclonal

  1. New monoclonal antibody to human apolipoprotein J

    Czech Academy of Sciences Publication Activity Database

    Čapková, Jana; Geussová, Gizela; Pěknicová, Jana

    2002-01-01

    Roč. 2002, č. 48 (2002), s. 40-42 ISSN 0015-5500 R&D Projects: GA ČR GV524/96/K162 Grant - others:NFDK-MAOB(XE) 1985-NFDK-MAOB Institutional research plan: CEZ:AV0Z5052915 Keywords : apo J * human spermatoza * monoclonal antibody Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.615, year: 2002

  2. Radioimmunodetection of human melanoma tumor xenografts with human monoclonal antibodies

    International Nuclear Information System (INIS)

    Gomibuchi, Makoto; Saxton, R.E.; Lake, R.R.; Katano, Mitsuo; Irie, R.F.

    1986-01-01

    A human IgM monoclonal antibody has been established that defines a tumor-associated membrane antigen expressed on human melanoma cells. The antigen has been identified as the ganglioside GD2. In this paper, the authors describe the potential usefulness of the human monoclonal antibody for radioimaging. Nude mice bearing tumors derived from a human melanoma cell line were used as a model. Antibody activity was degradated significantly after labeling with 131 I by the use of a modified chloramine-T method. After testing various concentrations, labeled antibody of a specific activity of 2.8μCi/μg produced the best results. Balb/c nude mice bearing a GD2-positive M14 melanoma cell line were injected with 10-30μg of labeled antibody, and its radiolocalization in different organs and in the whole body were evaluated. The best tumor image was obtained on Day 6. The labeled antibody uptake ratio between tumor and muscle was 9.2:1; the ratio between tumor and liver was 1.4:1. These studies represent the first report of experimental tumor imaging with human monoclonal antibody. Human monoclonals will probably prove to be superior reagents for tumor imaging in melanoma patients if the problem of anti-body radiolysis is resolved. (author)

  3. 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 ...

  4. Sensitive chain specific radioimmunoassay for human immunoglobulins using monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Sikora, K; Alderson, T St.J.; Ellis, J [Ludwig Institute for Cancer Research, Cambridge (UK)

    1983-02-25

    A sensitive radioimmunoassay is described for human immunoglobulins. This solid-phase assay uses commercially available monoclonal antibodies and is specific for different Ig chain types. Levels of less than 20 ng/ml Ig are detectable. The assay is suitable for the analysis of human hybridoma supernatants.

  5. Human monoclonal antibodies reactive with human myelomonocytic leukemia cells.

    Science.gov (United States)

    Posner, M R; Santos, D J; Elboim, H S; Tumber, M B; Frackelton, A R

    1989-04-01

    Peripheral blood mononuclear cells from a patient with chronic myelogenous leukemia (CML), in remission, were depleted of CD8-positive T-cells and cultured with Epstein-Barr virus. Four of 20 cultures (20%) secreted human IgG antibodies selectively reactive with the cell surfaces of certain human leukemia cell lines. Three polyclonal, Epstein-Barr virus-transformed, B-cell lines were expanded and fused with the human-mouse myeloma analogue HMMA2.11TG/O. Antibody from secreting clones HL 1.2 (IgG1), HL 2.1 (IgG3), and HL 3.1 (IgG1) have been characterized. All three react with HL-60 (promyelocytic), RWLeu4 (CML promyelocytic), and U937 (monocytic), but not with KG-1 (myeloblastic) or K562 (CML erythroid). There is no reactivity with T-cell lines, Burkitt's cell lines, pre-B-leukemia cell lines, or an undifferentiated CML cell line, BV173. Leukemic cells from two of seven patients with acute myelogenous leukemia and one of five with acute lymphocytic leukemia react with all three antibodies. Normal lymphocytes, monocytes, polymorphonuclear cells, red blood cells, bone marrow cells, and platelets do not react. Samples from patients with other diverse hematopoietic malignancies showed no reactivity. Immunoprecipitations suggest that the reactive antigen(s) is a lactoperoxidase iodinatable series of cell surface proteins with molecular weights of 42,000-54,000 and a noniodinatable protein with a molecular weight of 82,000. Based on these data these human monoclonal antibodies appear to react with myelomonocytic leukemic cells and may detect a leukemia-specific antigen or a highly restricted differentiation antigen.

  6. Generation and Characterization of Novel Human IRAS Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Bo Wang

    2009-01-01

    Full Text Available Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS, has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10–120aa through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10–120aa. Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.

  7. 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.

  8. Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

    Directory of Open Access Journals (Sweden)

    M Jedi-Tehrani

    2005-10-01

    Full Text Available Introduction: As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. Material and Methods: To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund’s adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Results: Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. Conclusion: The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization.

  9. Monoclonal antibodies to human chorionic gonadotropin and their application to two-site sandwich radioimmunoassay

    International Nuclear Information System (INIS)

    Mizuchi, A.; Iio, M.; Miyachi, Y.

    1984-01-01

    Monoclonal antibodies were prepared against human chorionic gonadotropin (HCG). One monoclonal antibody recognized a conformational determinant expressed only on native HCG molecule and another monoclonal antibody had the specificity for the epitopes located on the β-subunit of HCG. Monoclonal antibodies reacting with different antigenic determinants on the HCG molecule were used to develop a simplified 2-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized and another labeled with 125 iodine. This assay was highly specific for HCG and there was no cross-reactivity with α,β-subunit of HCG, luteinizing hormone and follicle stimulating hormone. (Auth.)

  10. Current status of cancer immunodetection with radiolabeled human monoclonal antibodies.

    Science.gov (United States)

    De Jager, R; Abdel-Nabi, H; Serafini, A; Pecking, A; Klein, J L; Hanna, M G

    1993-04-01

    The use of radiolabeled murine monoclonal antibodies (MoAbs) for cancer immunodetection has been limited by the development of human antimouse antibodies (HAMA). Human monoclonal antibodies do not elicit a significant human antihuman (HAHA) response. The generation and production of human monoclonal antibodies met with technical difficulties that resulted in delaying their clinical testing. Human monoclonal antibodies of all isotypes have been obtained. Most were immunoglobulin (Ig) M directed against intracellular antigens. Two antibodies, 16.88 (IgM) and 88BV59 (IgG3k), recognize different epitopes on a tumor-associated antigen, CTA 16.88, homologous to cytokeratins 8, 18, and 19. CTA 16.88 is expressed by most epithelial-derived tumors including carcinomas of the colon, pancreas, breast, ovary, and lung. The in vivo targeting by these antibodies is related to their localization in nonnecrotic areas of tumors. Repeated administration of 16.88 over 5 weeks to a cumulative dose of 1,000 mg did not elicit a HAHA response. Two of 53 patients developed a low titer of HAHA 1 to 3 months after a single administration of 88BV59. Planar imaging of colorectal cancer with Iodine-131 (131I)-16.88 was positive in two studies in 9 of 12 and 16 of 20 patients preselected by immunohistochemistry. Tumors less than 2 cm in diameter are usually not detected. The lack of immunogenicity and long tumor residence time (average = 17 days) makes 16.88 a good candidate for therapy. Radioimmunlymphoscintigraphy with indium-111 (111In)-LiLo-16.88 administered by an intramammary route was used in the presurgical staging of primary breast cancer. The negative predictive value of lymph node metastases for tumors less than 3 cm was 90.5%. Planar and single photon emission computed tomography imaging of colorectal carcinoma with technetium-99m (99mTc) 88BV59 was compared with computed tomography (CT) scan in 36 surgical patients. The antibody scan was more sensitive than the CT scan in detecting

  11. [Study of anti-idiotype antibodies to human monoclonal antibody].

    Science.gov (United States)

    Harada, R; Takahashi, N; Owaki, I; Kannagi, R; Endo, N; Morita, N; Inoue, M

    1992-02-01

    A human monoclonal antibody, ll-50 (IgM, lambda), was generated, which reacted specifically with a major of glycolipid present in LS174T colon cancer cells. The glycolipid antigen which reacted with the ll-50 antibody was expected to four sugar residues from its TLC mobility, and it was ascertained that the glycolipid antigen which reacted with ll-50 antibody might be Lc4 antigen [Gal beta 1----3 GLcNAc beta 1----3 Gal beta 1----4 Glc beta 1----1 Cer] judging from TLC immunostaining and ELISA when the reactivity of ll-50 antibody was tested using various pure glycolipids in 3-5 sugar residues as an antigen. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated ll-50 antibody. The serum of the Lc4 antigen recognized by ll-50 antibody was significantly higher in patients with malignant disorders than that in healthy individuals (p less than 0.05). Three mouse monoclonal anti-idiotype antibodies, G3, B3 and C5 (all IgG1), were generated by the immunization of BALB/c mice with ll-50 antibody. These anti-idiotype antibodies specifically bound to to human monoclonal antibody, ll-50 and had a significant inhibitory activity towards the binding of ll-50 antibody to the Lc4 antigen. This indicated that these anti-idiotype antibodies, G3, B3, and C5, were paratope-related anti-idiotype antibodies. G3, B3, and C5 were expected to define the nearest idiotope because they could mutually inhibit ll-50 antibody. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated anti-idiotype antibodies, G3, B3, and C5. As to the ll-50 like antibodies defined by C5 (Id-C5+), the mean serum level in patients with malignant disorders was significantly higher than that in healthy individuals (p less than 0.05). As to the ll-50 like antibodies defined by B3 (Id-B3+), the mean serum level in patients with malignant disorders was significantly higher

  12. Characterization of monoclonal antibodies directed against human thyroid stimulating hormone

    International Nuclear Information System (INIS)

    Soos, M.; Siddle, K.

    1982-01-01

    Monoclonal antibodies directed against human thyroid stimulating hormone (TSH) were obtained from hybrid myelomas, following fusion of mouse NSI myeloma cells with mouse spleen cells. Ten different antibodies were obtained from 4 separate fusions. Eight antibodies were of the IgG 1 subclass. Affinities of antibodies for TSH were in the range 2 x 10 8 -5 x 10 10 M -1 . Five of the antibodies were specific for TSH and did not react with LH, FSH or hCG. The remaining antibodies reacted with all these hormones and were assumed to recognise their common (α) subunit. The 5 specific antibodies fell into 3 subgroups recognising distinct antigenic determinants, whereas the 5 non-specific antibodies recognised a single determinant or closely related set of sites. It is concluded that these antibodies should be valuable reagents for use in sensitive and specific two-site immunoradiometric assays. (Auth.)

  13. Radiolabeled Humanized Anti-CD3 Monoclonal Antibody Visilizumab for Imaging Human T-Lymphocytes

    NARCIS (Netherlands)

    Malviya, Gaurav; D'Alessandria, Calogero; Bonanno, Elena; Vexler, Vladimir; Massari, Roberto; Trotta, Carlo; Scopinaro, Francesco; Dierckx, Rudi; Signore, Alberto

    2009-01-01

    Visilizumab is an IgG(2) humanized monoclonal antibody (mAb) characterized by non-Fc gamma R binding and specific to the CD3 antigen, expressed on more than 95% of circulating resting T-lymphocytes and on activated T-lymphocytes homing in inflamed tissues. We hypothesized that the use of a

  14. Production of human anti-HLA monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Walker, M.C.; Mercier, F.; Roger, J.; Varin, M.

    1986-03-01

    Only 40% of the several hundred anti-HLA murine monoclonal antibodies (MAbs) that have been made detect HLA-A,B,C or DR specificities previously defined by human alloantisera, the range of recognized specificities is very narrow, and few of the MAbs have proven useful as tissue typing reagents. In hopes of obtaining HLA typing reagents, the authors are developing a protocol for the production of human anti-HLA MAbs from HLA-antigen (Ag) immunized peripheral blood B cells of volunteering renal patients, immunized to one or more HLA Ags through therapeutic blood transfusions. A simple enrichment of the donor B cells has not been sufficient for anti-HLA MAb production, the authors are currently delineating the conditions necessary for increasing the number of HLA-specific donor B cells by in vitro stimulation with cells expressing the HLA Ag to which the B cell donor is immunized. For the production of MAbs, the stimulated B cells are transformed with Epstein-Barr virus and subsequently fused with KR-4 lymphoblastoid cells. Hybridomas are selected by HAT and Ouabain. Supernatants are screened for anti-HLA activity against lymphocyte targets expressing the original immunizing HLA Ag by complement mediated /sup 51/Cr release assay. Antibody specificity is determined by the complement-dependent microcytotoxicity test used for HLA typing.

  15. Monoclonal antibody 6E4 against human GAPDHS protein

    Czech Academy of Sciences Publication Activity Database

    Dorosh, Andriy

    2011-01-01

    Roč. 30, č. 3 (2011), s. 321-321 ISSN 1554-0014 Institutional research plan: CEZ:AV0Z50520701 Keywords : Monoclonal antibody * GAPDHS Subject RIV: EI - Biotechnology ; Bionics Impact factor: 0.417, year: 2011

  16. Characterization of human monoclonal antibodies that neutralize multiple poliovirus serotypes.

    Science.gov (United States)

    Puligedda, Rama Devudu; Kouiavskaia, Diana; Al-Saleem, Fetweh H; Kattala, Chandana Devi; Nabi, Usman; Yaqoob, Hamid; Bhagavathula, V Sandeep; Sharma, Rashmi; Chumakov, Konstantin; Dessain, Scott K

    2017-10-04

    Following the eradication of wild poliovirus (PV), achieving and maintaining a polio-free status will require eliminating potentially pathogenic PV strains derived from the oral attenuated vaccine. For this purpose, a combination of non-cross-resistant drugs, such as small molecules and neutralizing monoclonal antibodies (mAbs), may be ideal. We previously isolated chimpanzee and human mAbs capable of neutralizing multiple PV types (cross-neutralization). Here, we describe three additional human mAbs that neutralize types 1 and 2 PV and one mAb that neutralizes all three types. Most bind conformational epitopes and have unusually long heavy chain complementarity determining 3 domains (HC CDR3). We assessed the ability of the mAbs to neutralize A12 escape mutant PV strains, and found that the neutralizing activities of the mAbs were disrupted by different amino acid substitutions. Competitive binding studies further suggested that the specific mAb:PV interactions that enable cross-neutralization differ among mAbs and serotypes. All of the cloned mAbs bind PV in the vicinity of the "canyon", a circular depression around the 5-fold axis of symmetry through which PV recognizes its cellular receptor. We were unable to generate escape mutants to two of the mAbs, suggesting that their epitopes are important for the PV life cycle. These data indicate that PV cross-neutralization involves binding to highly conserved structures within the canyon that binds to the cellular receptor. These may be facilitated by the long HC CDR3 domains, which may adopt alternative binding configurations. We propose that the human and chimpanzee mAbs described here could have potential as anti-PV therapeutics. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Clinical application of antibody monoclonal humanized anti-EGFrnimotuzumab labeled

    International Nuclear Information System (INIS)

    Perera Pintado, Alejandro; Peña Quián, Yamilé; Batista Cuéllar, Juan F.; Prats Capote, Anaís; Torres Aroche, Leonel A.; Casacó Santana, Caridad; Sánchez Mendosa, Elvia L.; Sánchez González, Yolaine; Romero Collado, Susana; Quesada Pozo, Rodobaldo; Valladares Oviedo, Lourdes; Masquida García, Elsa M.; Leyva Montaña, René; Casacó, Angel; Ramos Suzarte, Mayra; Crombet, Tania

    2016-01-01

    Most malignant tumors are of epithelial origin. These are characterized by overexpression of the receptor of epidermal growth factor (EGFR), which the neoplastic cells escape the regulatory mechanisms are allowed, so its high concentration of membrane is generally associated with a poor prognosis . By binding an antibody specifically to this receptor, preventing binding of EGF latter and activation mechanism tyrosine kinase inhibiting cell mitosis and apoptosis causing tumor cell. For this reason, the EGFr has been considered as an attractive target for anti-tumor therapy. The humanized monoclonal antibody anti-EGFr nimotuzumab was developed by the Center of Molecular Immunology (Havana, Cuba). Numerous clinical trials have been developed in the Department of Clinical Research Center Isotopes (Cuba), in which it has been applied this antibody, both labeled with 99mTc for immuno gammagraphic detection of tumors, as labeled with 188 Re for radioimmunotherapy of gliomas high degree of malignancy. The aim of this paper is to show the experience of the Department of Clinical Research of CENTIS in various clinical trials with marking for both immuno gammagraphics detection of tumors, such as for radioimmunotherapy nimotuzumab. (author)

  18. Immunochemical identification of human trophoblast membrane antigens using monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Brown, P J; Molloy, C M; Johnson, P M [Liverpool Univ. (UK). Dept. of Immunology

    1983-11-01

    Human trophoblast membrane antigens recognised by monoclonal antibodies (H310, H315, H316 and H317) have been identified using combinations of radioimmunoprecipitation, SDS-PAGE, electroblotting, chromatographic and ELISA-type techniques. H317 is known to identify heat-stable placental-type alkaline phosphatase and accordingly was shown to react with a protein of subunit Msub(r) of 68000. H310 and H316 both recognise an antigen with a subunit Msub(r) of 34000 under reducing conditions. In non-reducing conditions, the H310/316 antigen gave oligomers of a component of Msub(r) 62000. It is unknown whether this 62000 dalton component is a dimer of the 34000 dalton protein with either itself or a second protein chain of presumed Msub(r) around 28000. H315 recognises an antigen with subunit Msub(r) of 36000; in non-reducing conditions this component readily associates to oligomeric structures. The epitope recognised by H315 may be sensitive to SDS. The two proteins recognised by H310/316 and H315 have been termed the p34 and p36 trophoblast membrane proteins, respectively.

  19. Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies

    NARCIS (Netherlands)

    Shakeel, Shabih; Westerhuis, Brenda M.; Ora, Ari; Koen, Gerrit; Bakker, Arjen Q.; Claassen, Yvonne; Wagner, Koen; Beaumont, Tim; Wolthers, Katja C.; Butcher, Sarah J.

    2015-01-01

    Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such

  20. 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

  1. [Diagnostic and therapeutic use of human anti-D (Rho) monoclonal antibodies. Evaluation and perspectives].

    Science.gov (United States)

    Rouger, P; Goossens, D; Champomier, F; Tsikas, G; Liberge, G; Leblanc, J; Richard, C; Bailleul, C; Salmon, C

    1985-12-01

    Human monoclonal antibodies will be essential in medicine. They are valuable tools for biological diagnosis and therapeutics. Our model, human monoclonal antibodies directed against the Rhesus D antigen can be used for the determination of the Rhesus D phenotype and for the suppression of Rh(D) immunisation in women. These new products require new procedures of preparation, new regulations for the quality controls, which will be discussed in this paper.

  2. Two-site sandwich radioimmunoassay of human gamma interferon with monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, E; Imai, M; Usuda, S; Tachibana, K; Okamoto, H; Ohike, Y; Nakamura, T; Miyakawa, Y; Mayumi, M [Jichi Medical School, Minamikawachi, Tochigi (Japan)

    1985-03-18

    Two monoclonal antibodies were raised against human gamma interferon (IFN-..gamma..) derived from E. coli harboring the recombinant cDNA for IFN-..gamma.., and one against a synthetic peptide representing its C-terminus amino acid sequence of 20 residues. The monoclonal antibody against the synthetic peptide reacted either with IFN-..gamma.. or the synthetic peptide. One monoclonal anti-IFN-..gamma.. did not react with the synthetic peptide, while the other showed a weak binding with the peptide. A 2-site '1-step' radioimmunoassay was developed. The assay was rapid with a sensitivity capable of detecting a few ng/ml of IFN-..gamma...

  3. Monoclonal antibodies against human trophoblast in female infertility

    Czech Academy of Sciences Publication Activity Database

    Sedláková, Alena; Elzeinová, Fatima; Bukovský, A.; Madar, J.; Ulčová-Gallová, Z.; Pěknicová, Jana

    2005-01-01

    Roč. 54, č. 3 (2005), s. 159 ISSN 0271-7352. [European Congress of Reproductive Immunology /3./. 05.09.11-05.09.15, Essex] R&D Projects: GA MZd(CZ) NR7838 Institutional research plan: CEZ:AV0Z50520514 Keywords : monoclonal antibodies * female infertility * trophoblast Subject RIV: EB - Genetics ; Molecular Biology

  4. A sensitive chain specific radioimmunoassay for human immunoglobulins using monoclonal antibodies

    International Nuclear Information System (INIS)

    Sikora, K.; Alderson, T.St.J.; Ellis, J.

    1983-01-01

    A sensitive radioimmunoassay is described for human immunoglobulins. This solid-phase assay uses commercially available monoclonal antibodies and is specific for different Ig chain types. Levels of less than 20 ng/ml Ig are detectable. The assay is suitable for the analysis of human hybridoma supernatants. (Auth.)

  5. 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

  6. 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

  7. Agonistic Human Monoclonal Antibodies against Death Receptor 4 (DR4) | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Cancer Institute is seeking parties interested in licensing human monoclonal antibodies (mAbs) that bind to death receptor 4 ("DR4"). The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its functional receptors, DR4 and DR5, have been recognized as promising targets for cancer treatment.

  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. 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

  10. Monoclonal antibodies to human factor VII: a one step immunoradiometric assay for VII:Ag.

    OpenAIRE

    Takase, T; Tuddenham, E G; Chand, S; Goodall, A H

    1988-01-01

    Three mouse monoclonal antibodies (RFF-VII/1, RFF-VII/2, and RFF-VII/3) which bind specifically to different epitopes on human factor VII antigen were raised. Two of the antibodies, RFF-VII/1 and RFF-VII/2, bound strongly to factor VII antigen (VII:Ag), but only RFF-VII/1 and RFF-VII/3 were potent inhibitors of factor VII coagulation activity (VII:C). RFF-VII/1 and RFF-VII/2 were used in a one step, double monoclonal immunoradiometric assay for VII:Ag. This was highly reproducible and detecte...

  11. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    International Nuclear Information System (INIS)

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-01-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of 3 H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes

  12. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    Energy Technology Data Exchange (ETDEWEB)

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-11-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of /sup 3/H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes.

  13. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    International Nuclear Information System (INIS)

    Holers, V.M.; Kotzin, B.L.

    1985-01-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases

  14. 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...

  15. [Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

    Science.gov (United States)

    Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

    2012-06-01

    To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

  16. Radioimmunoscintigraphy of human pancreatic carcinoma xenografts in nude mice with 131I-labeled monoclonal antibody

    International Nuclear Information System (INIS)

    Tsuda, Takatoshi; Koshiba, H.; Usui, T.; Kubota, M.; Kikuchi, Kokichi; Morita, Kazuo

    1990-01-01

    Encouraged by reports of radioimmunoimaging of colorectal carcinomas and by examining an immunohistochemical report on resected pancreas cancer tissues, we studied the diagnostic potential of radioimmunoimaging with the radioiodinelabeled monoclonal antibody (MoAb; HC-1) to a human pancreas cancer cell line (HGC25) was labeled with radioiodine and injected into athymic nude mice implanted with human pancreas cancer cells. Antibody HC-1 was cleared from the circulation and accumulated significantly in the implanted tumor sites. (author)

  17. Characterization of monoclonal antibodies against human thyrotropin and use in an immunoradiometric assay and immunohistochemistry

    International Nuclear Information System (INIS)

    Benkirane, M.; Bon, D.; Bellot, F.; Prince, P.; Delori, P.; Hassoun, J.; Carayon, P.

    1987-01-01

    Monoclonal antibodies were prepared against human thyrotropin. 13 different antibodies were characterized. Ten antibodies were of the IgG1 subclass. The affinities of the antibodies were in the range 10 9 -10 11 mol -1 .l. Four of them were specific for hTSH and did not react with hLH, hFSH, hCG or αhCG. Four reacted with these hormones and recognized the α subunit of hCG. One cross-reacted only with HFSH. The remaining four antibodies recognized the holo-hTSH only, and thus were designated as anti-conformational determinants. Monoclonal antibodies reacting with different antigenic determinants on the hTSH molecule defined seven clusters. Two of them were used to develop a simplified two-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized on tubes (anti-βTSH) and another (anti-α) labelled with 125 I. This assay was highly specific and demonstrated a sensitivity level of 0.1 μIU/ml. Two monoclonal antibodies were used in immunohistochemistry and their quality and specificity was assessed in the detection of hTSH immunoreactivity in human pituitary biological sections. 20 refs.; 6 figs.; 2 tabs

  18. 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.

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

    International Nuclear Information System (INIS)

    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

    2000-01-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG 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 μg/100 μCi of 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 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

  20. Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay.

    Science.gov (United States)

    Rubin, I; Lykkegaard, S; Olsen, A A; Selmer, J; Ballegaard, M

    1988-01-01

    Monoclonal antibodies were produced against human angiotensinogen. An enzyme linked immunosorbent assay (ELISA) was developed using a high affinity monoclonal antibody as catching antibody and a polyclonal rabbit anti human angiotensinogen antibody as detecting antibody in a "sandwich" ELISA. Linear range of the ELISA was 15-450 pmol/l of human angiotensinogen. Intra- and inter- assay variation coefficients were in the range of 2% to 8%. A correlation coefficient, r = 0.97, (n = 20), with values obtained by radioimmunoassay. This correlation coefficient, obtained by using both normal and pregnant sera, confirmed that the ELISA fulfill the requirements for clinical useful assay. Characterization of the antibodies were performed with respect to affinity constant and epitopes.

  1. A Monoclonal Antibody against Wnt-1 Induces Apoptosis in Human Cancer Cells

    Directory of Open Access Journals (Sweden)

    Biao He

    2004-01-01

    Full Text Available Aberrant activation of the Wingless-type (Wnt/β-catenin signaling pathway is associated with a variety of human cancers. Little is known regarding the role that Wnt ligands play in human carcinogenesis. To test whether a Wnt-1 signal is a survival factor in human cancer cells and thus may serve as a potential cancer therapeutic target, we investigated the effect of inhibition of Wnt-1 signaling in a variety of human cancer cell lines, including non small cell lung cancer, breast cancer, mesothelioma, and sarcoma. Both monoclonal antibody and RNA interference (RNAi were used to inhibit Wnt-1 signaling. We found that incubation of a monoclonal anti-Wnt-1 antibody induced apoptosis and caused downstream protein changes in cancer cells overexpressing Wnt-1. In contrast, apoptosis was not detected in cells lacking or having minimal Wnt-1 expression after the antibody incubation. RNAi targeting of Wnt-1 in cancer cells overexpressing Wnt-1 demonstrated similar downstream protein changes and induction of apoptosis. The antibody also suppressed tumor growth in vivo. Our results indicate that both monoclonal anti-Wnt-1 antibody and Wnt-1 siRNA inhibit Wnt-1 signaling and can induce apoptosis in human cancer cells. These findings hold promise as a novel therapeutic strategy for cancer.

  2. Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

    Science.gov (United States)

    Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

    1995-07-01

    Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

  3. A sandwich immunoassay for human prolyl 4-hydroxylase using monoclonal antibody

    International Nuclear Information System (INIS)

    Yoshida, Shinichi

    1986-01-01

    Monoclonal antibody was used in a sandwich enzyme immunoassay and in a radioimmunoassay for human serum immunoreactive prolyl 4-hydroxylase. The enzyme immunoassay utilized a monoclonal antibody as a solid phase and horseradish peroxidase-labeled rabbit antibody to human prolyl 4-hydroxylase as a conjugate. Sensitivity was 0.1 ng of enzyme per tube. With a conjugate purified by an enzyme-bound affinity column, sensitivity was increased to 0.01 ng per tube, and linearity was obtained between 0.01 to 30 ng per tube. The radioimmunoassay used a 125 I-labeled rabbit antibody (IgG) as the conjugate. Sensitivity of this technique was 0.4 ng of enzyme per tube. (Auth.)

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

    OpenAIRE

    Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Abdolalizadeh, Jalal; Kazemi, Tohid; Aghebati Maleki, Ali; Sineh sepehr, Koushan

    2013-01-01

    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 t...

  5. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    Science.gov (United States)

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

  6. Monoclonal antibodies to human mammary tumor-associated antigens and their use for radiolocalization of xenografts in athymic mice

    International Nuclear Information System (INIS)

    Colcher, D.; Schlom, J.

    1983-01-01

    The authors have utilized membrane-enriched extracts of human metastatic mammary tumor cells as immunogens to generate and characterize monoclonal antibodies reactive with determinants that would be maintained on metastatic, as well as primary, human mammary carcinoma cells. Multiple assays using tumor cells extracts, tissue sections, and live cells in culture have been employed to reveal the diversity of the monoclonal antibodies generated. Then the utility of these antibodies for radiolocalization studies was examined. (Auth.)

  7. 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.

  8. Mouse monoclonal antibodies against human c-Mpl and characterization for flow cytometry applications.

    Science.gov (United States)

    Abbott, Christina; Huang, Guo; Ellison, Aaron R; Chen, Ching; Arora, Taruna; Szilvassy, Stephen J; Wei, Ping

    2010-04-01

    Mouse monoclonal antibodies (MAbs) against human c-Mpl, the cognate receptor for thrombopoietin (TPO), were generated using hybridoma technology and characterized by various assays to demonstrate their specificity and affinity. Two such MAbs, 1.6 and 1.75, were determined to be superior for flow cytometry studies and exhibited double-digit picomolar (pM) affinities to soluble human c-Mpl protein. Both MAbs specifically bound to cells engineered to overexpress human c-Mpl protein, immortalized human hematopoietic cell lines that express endogenous c-Mpl, primary human bone marrow and peripheral blood-derived CD34(+) cells, and purified human platelets. No binding was detected on cell lines that did not express c-Mpl. Receptor competition and siRNA knock-down studies further confirmed the specificity of antibodies 1.6 and 1.75 for human c-Mpl. In contrast to these newly generated MAbs, none of eight commercially available anti-c-Mpl antibodies tested were found to bind specifically to human c-Mpl and were thus shown to be unsuitable for flow cytometry studies. Monoclonal antibodies 1.6 and 1.75 will therefore be useful flow cytometry reagents to detect cell surface c-Mpl expression.

  9. Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.

    1985-12-01

    Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae.

  10. Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

    International Nuclear Information System (INIS)

    Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.

    1985-01-01

    Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae

  11. A novel polymorphism of human complement component C3 detected by means of a monoclonal antibody

    DEFF Research Database (Denmark)

    Koch, C; Behrendt, N

    1986-01-01

    A mouse monoclonal antibody, HAV 4-1, obtained after immunization of a BALB/c mouse with purified C3F, detected a novel genetic polymorphism of human complement component C3 in a simple immunoblotting system. The frequency of HAV 4-1-positive genes was 20.1%. Reactivity of HAV 4-1 was closely...... related to C3F, but certain individuals with the C3F allele did not react with HAV 4-1. Conversely, certain C3S homozygous individuals did react with HAV 4-1. The polymorphism detected by this monoclonal antibody is therefore different from the previously described polymorphism based on charge differences....

  12. 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....

  13. 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.

  14. Clearance of 131I-labeled murine monoclonal antibody from patients' blood by intravenous human anti-murine immunoglobulin antibody

    International Nuclear Information System (INIS)

    Stewart, J.S.; Sivolapenko, G.B.; Hird, V.; Davies, K.A.; Walport, M.; Ritter, M.A.; Epenetos, A.A.

    1990-01-01

    Five patients treated with intraperitoneal 131I-labeled mouse monoclonal antibody for ovarian cancer also received i.v. exogenous polyclonal human anti-murine immunoglobulin antibody. The pharmacokinetics of 131I-labeled monoclonal antibody in these patients were compared with those of 28 other patients receiving i.p.-radiolabeled monoclonal antibody for the first time without exogenous human anti-murine immunoglobulin, and who had no preexisting endogenous human anti-murine immunoglobulin antibody. Patients receiving i.v. human anti-murine immunoglobulin antibody demonstrated a rapid clearance of 131I-labeled monoclonal antibody from their circulation. The (mean) maximum 131I blood content was 11.4% of the injected activity in patients receiving human anti-murine immunoglobulin antibody compared to 23.3% in patients not given human anti-murine immunoglobulin antibody. Intravenous human anti-murine immunoglobulin antibody decreased the radiation dose to bone marrow (from 131I-labeled monoclonal antibody in the vascular compartment) 4-fold. Following the injection of human anti-murine immunoglobulin antibody, 131I-monoclonal/human anti-murine immunoglobulin antibody immune complexes were rapidly transported to the liver. Antibody dehalogenation in the liver was rapid, with 87% of the injected 131I excreted in 5 days. Despite the efficient hepatic uptake of immune complexes, dehalogenation of monoclonal antibody was so rapid that the radiation dose to liver parenchyma from circulating 131I was decreased 4-fold rather than increased. All patients developed endogenous human anti-murine immunoglobulin antibody 2 to 3 weeks after treatment

  15. Application of monoclonal antibodies for diagnosis and treatment of human digestive cancer

    International Nuclear Information System (INIS)

    Otsuji, Eigo

    2007-01-01

    Radioimmunoscintigraphic applications of monoclonal antibodies (Mabs) for noninvasive detection and visualization of target tumors have grown immensely, and it suggests that Mabs can reach specifically to the targeted tumors in the human body. Radionuclides, cytotoxic drugs and anti-cancer drugs can be coupled to these specific MAbs to detect the extent of disease and/or to treat the tumors. Many of such immunoconjugates were studied for targeting therapy for cancer in animal experiments and some of them have applied to human. In this paper, we described the existing status of application of Mabs for diagnosis and immunotargeting therapy of digestive cancers. (author)

  16. Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

    OpenAIRE

    Fatemeh Ezzatifar; Jafar Majidi; Behzad Baradaran; Leili Aghebati Maleki; Jalal Abdolalizadeh; Mehdi Yousefi

    2015-01-01

    Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies again...

  17. Detection of anti-streptococcal, antienolase, and anti-neural antibodies in subjects with early-onset psychiatric disorders.

    Science.gov (United States)

    Nicolini, Humberto; López, Yaumara; Genis-Mendoza, Alma D; Manrique, Viana; Lopez-Canovas, Lilia; Niubo, Esperanza; Hernández, Lázaro; Bobes, María A; Riverón, Ana M; López-Casamichana, Mavil; Flores, Julio; Lanzagorta, Nuria; De la Fuente-Sandoval, Camilo; Santana, Daniel

    2015-01-01

    Infection with group A Streptococcus (StrepA) can cause post-infectious sequelae, including a spectrum of childhood-onset obsessive-compulsive (OCD) and tic disorders with autoimmune origin (PANDAS, Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections). Until now, no single immunological test has been designed that unequivocally diagnoses these disorders. In this study, we assessed the detection of serum antibodies against human brain enolase (AE), neural tissue (AN) and Streptococcus (AS) as a laboratory tool for the diagnosis of early-onset psychiatric disorders. Serum antibodies against human brain enolase, total brain proteins, and total proteins from StrepA were detected by ELISA in 37 patients with a presumptive diagnosis of PANDAS and in 12 healthy subjects from Mexico and Cuba. The antibody titers against human brain enolase (AE) and Streptococcal proteins (AS) were higher in patients than in control subjects (t-student, tAE=-2.17, P=0.035; tAS=-2.68, P=0.01, n=12 and 37/group, df=47, significance level 0.05), while the neural antibody titers did not differ between the two groups (P(t)=0.05). The number of subjects (titers> meancontrol + CI95) with simultaneous seropositivity to all three antibodies was higher in the patient group (51.4%) than in the control group (8.3%) group (X2=5.27, P=0.022, df=1, n=49). The simultaneous detection of all three of these antibodies could provide valuable information for the etiologic diagnosis of individuals with early-onset obsessive-compulsive disorders associated with streptococcal infection and, consequently, for prescribing suitable therapy.

  18. Epitope mapping of functional domains of human factor V with human and mouse monoclonal antibodies

    International Nuclear Information System (INIS)

    Annamalai, A.E.; Rao, A.K.; Chiu, H.C.; Wang, D.; Dutta-Roy, A.K.; Colman, R.W.

    1986-01-01

    The authors previously described two human monoclonal antibodies (MAbs) which inactivated factor V. The authors have now purified the predominant antibody (H2) on protein A Sepharose using a pH gradient and typed it as IgG 1 ,. Immunoprecipitation of 125 I-human factor Va with H2 demonstrated specificity for the heavy chain (D), Mr = 105,000. The authors compared using ELISA the competitive binding to factor Va, of H2, H1 and two mouse MAbs, B38 (directed to E) and B10 (to activation peptide, Cl). All four antibodies recognized distinct epitopes in factor V with steric overlap in some cases. Factor Xa showed a concentration dependent competition for binding of H1, H2 and B38 but not B10 to factor V/Va in ELISA. All MAbs bound to factor V/Va in the absence of Ca ++ . However, Ca ++ at 8 mM increased the binding of H1 and H2 to 165% and 360% and did not have any effect on the binding of either mouse MAbs. Prothrombin at a concentration of up to 400 μg/ml did not inhibit binding of any of these antibodies. Thus, both the light (E) and heavy (D) chains of factor Va but not the activation peptide (Cl) interact with factor Xa as defined by the MAbs. In addition, sites on both chains for Ca ++ are recognized by particular MAbs (H1 and H2). These studies increase their knowledge of the interactions of factor V domains in the formation of prothrombinase complex

  19. Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

    International Nuclear Information System (INIS)

    Bidart, J.M.; Troalen, F.; Salesse, R.; Bousfield, G.R.; Bohuon, C.J.; Bellet, D.H.

    1987-01-01

    We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex

  20. Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity

    International Nuclear Information System (INIS)

    Qian Weizhu; Wang Ling; Li Bohua; Wang Hao; Hou Sheng; Hong Xueyu; Zhang Dapeng; Guo Yajun

    2008-01-01

    Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application

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

    International Nuclear Information System (INIS)

    Benedictis, P. de; Minola, A.; Rota, E.; Aiello, R.; Zecchin, B.; Salomoni, A.; Foglierini, M.; Agatic, G.; Vanzetta, F.; Lavenir, R.; Lepelletier, A.; Bentley, E.; Weiss, R.; Cattoli, G.

    2016-01-01

    Full text: 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. (author)

  2. 77 FR 9678 - Prospective Grant of Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies...

    Science.gov (United States)

    2012-02-17

    .../patent applications for the technology family, to Sanomab, Ltd. The patent rights in these inventions... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Prospective Grant of Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies for the Treatment of Human...

  3. 77 FR 41792 - Prospective Grant of Co-Exclusive License: The Development of Human Anti-CD22 Monoclonal...

    Science.gov (United States)

    2012-07-16

    ... applications for the technology family, to Customized Therapeutics. The patent rights in these inventions have... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Prospective Grant of Co-Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies for the Treatment of Human...

  4. Passive vaccination with a human monoclonal antibody: generation of antibodies and studies for efficacy in Bacillus anthracis infections.

    Science.gov (United States)

    vor dem Esche, Ulrich; Huber, Maria; Zgaga-Griesz, Andrea; Grunow, Roland; Beyer, Wolfgang; Hahn, Ulrike; Bessler, Wolfgang G

    2011-07-01

    A major difficulty in creating human monoclonal antibodies is the lack of a suitable myeloma cell line to be used for fusion experiments. In order to create fully human monoclonal antibodies for passive immunization, the human mouse heteromyeloma cell line CB-F7 was evaluated. Using this cell line, we generated human monoclonal antibodies against Bacillus anthracis toxin components. Antibodies against protective antigen (PA) and against lethal factor (LF) were obtained using peripheral blood lymphocytes (PBLs) from persons vaccinated with the UK anthrax vaccine. PBL were fused with the cell line CB-F7. We obtained several clones producing PA specific Ig and one clone (hLF1-SAN) producing a monoclonal antibody (hLF1) directed against LF. The LF binding antibody was able to neutralize Anthrax toxin activity in an in vitro neutralization assay, and preliminary in vivo studies in mice also indicated a trend towards protection. We mapped the epitope of the antibody binding to LF by dot blot analysis and ELIFA using 80 synthetic LF peptides of 20 amino acid lengths with an overlapping range of 10 amino acids. Our results suggest the binding of the monoclonal antibody to the peptide regions 121-150 or 451-470 of LF. The Fab-fragment of the antibody hLF1 was cloned in Escherichia coli and could be useful as part of a fully human monoclonal antibody for the treatment of Anthrax infections. In general, our studies show the applicability of the CB-F7 line to create fully human monoclonal antibodies for vaccination. Copyright © 2010 Elsevier GmbH. All rights reserved.

  5. Characterization of human sperm protein recognized by monoclonal antibody HS-8

    Czech Academy of Sciences Publication Activity Database

    Margaryan, Hasmik; Čapková, Jana; Pěknicová, Jana

    2012-01-01

    Roč. 67, Issue Supplement s1 (2012), s. 27-28 ISSN 1046-7408. [13th International Symposium for Immunology of reproduction "From the roots to the tops of Reproductive Immunology". 22.06.2012-24.06.2012, Varna] R&D Projects: GA ČR(CZ) GA523/09/1793; GA ČR(CZ) GAP503/12/1834 Institutional research plan: CEZ:AV0Z50520701 Keywords : monoclonal antibody * GAPDHS * human sperm proteins * mass spectrometry Subject RIV: EC - Immunology

  6. 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

    , the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing......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...... whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar...

  7. Ta1, a novel 105 KD human T cell activation antigen defined by a monoclonal antibody.

    Science.gov (United States)

    Fox, D A; Hussey, R E; Fitzgerald, K A; Acuto, O; Poole, C; Palley, L; Daley, J F; Schlossman, S F; Reinherz, E L

    1984-09-01

    By using a murine monoclonal antibody produced against an IL 2-dependent human T cell line, we defined a T lineage-specific molecule, termed Ta1, that is expressed strongly on activated T lymphocytes of both the T4 and T8 subsets, as well as on T cell lines and clones, but only weakly on a fraction of resting T cells. SDS-PAGE analysis of immunoprecipitates from 125I-labeled, activated T cells demonstrates a single major band of apparent m.w. 105 KD under both reducing and nonreducing conditions. Unlike anti-IL 2 receptor antibodies, anti-Ta1 does not inhibit T cell proliferative responses to mitogen, antigen, or IL 2-containing medium. Moreover, anti-Ta1 has no effect on T cell-mediated cytotoxicity. Ta1 appears to be a novel human T cell-specific activation antigen that may serve as a useful marker of T cell activation in human disease.

  8. Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer

    International Nuclear Information System (INIS)

    Jörißen, Hannah; Klockenbring, Torsten; Bektas, Nuran; Dahl, Edgar; Hartmann, Arndt; Haaf, Anette ten; Di Fiore, Stefano; Kiefer, Hans; Thess, Andreas; Barth, Stefan

    2009-01-01

    RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression. The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3 expression in normal and cancerous human

  9. 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

  10. 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

  11. Blockade of human P2X7 receptor function with a monoclonal antibody.

    Science.gov (United States)

    Buell, G; Chessell, I P; Michel, A D; Collo, G; Salazzo, M; Herren, S; Gretener, D; Grahames, C; Kaur, R; Kosco-Vilbois, M H; Humphrey, P P

    1998-11-15

    A monoclonal antibody (MoAb) specific for the human P2X7 receptor was generated in mice. As assessed by flow cytometry, the MoAb labeled human blood-derived macrophage cells natively expressing P2X7 receptors and cells transfected with human P2X7 but not other P2X receptor types. The MoAb was used to immunoprecipitate the human P2X7 receptor protein, and in immunohistochemical studies on human lymphoid tissue, P2X7 receptor labeling was observed within discrete areas of the marginal zone of human tonsil sections. The antibody also acted as a selective antagonist of human P2X7 receptors in several functional studies. Thus, whole cell currents, elicited by the brief application of 2',3'-(4-benzoyl)-benzoyl-ATP in cells expressing human P2X7, were reduced in amplitude by the presence of the MoAb. Furthermore, preincubation of human monocytic THP-1 cells with the MoAb antagonized the ability of P2X7 agonists to induce the release of interleukin-1beta.

  12. 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.

  13. Evaluation of tumor targeting with radiolabeled F(ab2 fragment of a humanized monoclonal antibody

    Directory of Open Access Journals (Sweden)

    "Babaei MH

    2002-08-01

    Full Text Available Humanized monoclonal antibody U36 and its F(ab'2 fragment, radio labeled with 125I, were tested for tumor localization in nude mice bearing a squamous cell carcinoma xenograft line derived from a head and neck carcinoma. Monoclonal antibody IgG or F(ab'2 fragment were injected in parallel and at days 1, 2 and 3, mice were dissected for determination of isotope biodistribution. IgG as well as F(ab'2 showed highly specific localization in tumor tissue. The mean tumor uptake (n=3 is expressed as the percentage of the injected dose per gram of tumor tissue (%ID/g. %ID/g of IgG was 11.7% at day 1 and decreased to 10.9% at day 3 whereas %ID/g of F(ab'2 was 2.9% at day 1 and decreased on following days. Tumor to blood ratios (T/B at day 1 were 0.86 for IgG and 1.32 for F(ab'2 and reached a maximum at day 3 with values of 4.41 and 1.84 respectively. These findings suggest that the superior tumor to non-tumor ratios in the day of 1 render the F(ab'2 fragment more qualified for specific targeting radioisotopes to tumor xenografts in this exprimental setting.

  14. Characterisation of monoclonal antibodies for human luteinising hormone, and mapping of antigenic determinants on the hormone

    International Nuclear Information System (INIS)

    Soos, M.; Siddle, K.

    1983-01-01

    Twelve mouse monoclonal antibodies for human luteinising hormone were produced. The affinities varied from 4 X 10 7 to 1 X 10 10 l/mol. The specificity of each antibody was assessed by determining the relative reactivities with luteinising hormone, thyroid stimulating hormone, follicle stimulating hormone and chorionic gonadotrophin. Six antibodies bound to the α-subunit as shown by similar reactivity with all hormones, and the remainder to the β-subunit as shown by specificity for luteinising hormone. This latter group of antibodies cross-reacted only weakly with thyroid stimulating hormone (approximately 10%) and follicle stimulating hormone (approximately 3%). Three of these antibodies also showed low reactivity towards chorionic gonadotrophin (<10%), though the others did not (80-300%). The ability of different antibodies to bind simultaneously to luteinising hormone was examined and it was shown that several distinct antigenic determinants existed on both subunits. The characterisation of monoclonal binding sites is discussed in relation to the use of antibodies in two-site immunoradiometric assays. (Auth.)

  15. Preclinical Characterization of a Novel Monoclonal Antibody NEO-201 for the Treatment of Human Carcinomas

    Directory of Open Access Journals (Sweden)

    Massimo Fantini

    2018-01-01

    Full Text Available NEO-201 is a novel humanized IgG1 monoclonal antibody that was derived from an immunogenic preparation of tumor-associated antigens from pooled allogeneic colon tumor tissue extracts. It was found to react against a variety of cultured human carcinoma cell lines and was highly reactive against the majority of tumor tissues from many different carcinomas, including colon, pancreatic, stomach, lung, and breast cancers. NEO-201 also exhibited tumor specificity, as the majority of normal tissues were not recognized by this antibody. Functional assays revealed that treatment with NEO-201 is capable of mediating both antibody-dependent cellular cytotoxicity (ADCC and complement-dependent cytotoxicity (CDC against tumor cells. Furthermore, the growth of human pancreatic xenograft tumors in vivo was largely attenuated by treatment with NEO-201 both alone and in combination with human peripheral blood mononuclear cells as an effector cell source for ADCC. In vivo biodistribution studies in human tumor xenograft-bearing mice revealed that NEO-201 preferentially accumulates in the tumor but not organ tissue. Finally, a single-dose toxicity study in non-human primates demonstrated safety and tolerability of NEO-201, as a transient decrease in circulating neutrophils was the only related adverse effect observed. These findings indicate that NEO-201 warrants clinical testing as both a novel diagnostic and therapeutic agent for the treatment of a broad variety of carcinomas.

  16. Iodine 131 labeled GD2 monoclonal antibody in the diagnosis and therapy of human neuroblastoma

    International Nuclear Information System (INIS)

    Cheung, N.K.V.; Miraldi, F.D.

    1988-01-01

    High dose marrow ablative therapy followed by autologous bone marrow transplantation (ABMT) has prolonged survival in patients with neuroblastoma. Total body and focal irradiation play an integral role in the overall treatment of this disease. The biological basis for radiation is the radiosensitivity and the lack of sublethal repair in neuroblastoma cells. However, radiation therapy has not by itself been adequate because of the usual widespread nature of neuroblastoma and the inability to achieve selective tumor versus normal tissue delivery, especially at multiple tumor sites. Monoclonal antibodies are agents selected for their specificity for human tumors. In vivo they have the ability of targeting selectively to occult metastases. This paper discusses how the availability of radioisotopes and the development of conjugation chemistries have greatly expanded the potentials of these antibodies

  17. Expression of POTE protein in human testis detected by novel monoclonal antibodies

    International Nuclear Information System (INIS)

    Ise, Tomoko; Das, Sudipto; Nagata, Satoshi; Maeda, Hiroshi; Lee, Yoomi; Onda, Masanori; Anver, Miriam R.; Bera, Tapan K.; Pastan, Ira

    2008-01-01

    The POTE gene family is composed of 13 highly homologous paralogs preferentially expressed in prostate, ovary, testis, and placenta. We produced 10 monoclonal antibodies (MAbs) against three representative POTE paralogs: POTE-21, POTE-2γC, and POTE-22. One reacted with all three paralogs, six MAbs reacted with POTE-2γC and POTE-22, and three MAbs were specific to POTE-21. Epitopes of all 10 MAbs were located in the cysteine-rich repeats (CRRs) motifs located at the N-terminus of each POTE paralog. Testing the reactivity of each MAb with 12 different CRRs revealed slight differences among the antigenic determinants, which accounts for differences in cross-reactivity. Using MAbs HP8 and PG5 we were able to detect a POTE-actin fusion protein in human testis by immunoprecipitation followed by Western blotting. By immunohistochemistry we demonstrated that the POTE protein is expressed in primary spermatocytes, implying a role in spermatogenesis

  18. 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.

  19. Radioimmunoimaging using F(ab')2 fragment of monoclonal antibodies against human alpha-fetoprotein

    International Nuclear Information System (INIS)

    Sakahara, Harumi; Endo, Keigo; Nakashima, Tetsuo; Koizumi, Mitsuru; Ohta, Hitoya; Torizuka, Kanji; Okada, Kenichiro; Yoshida, Osamu; Nishi, Shinzo.

    1985-01-01

    Using monoclonal antibodies against human α-fetoprotein (AFP), radioiodinated F(ab') 2 fragments were compared with whole IgG as a radiotracer for radioimmunoimaging of cancer. F(ab') 2 fragments were obtained by pepsin digestion of whole IgG (IgGl). IgG and F(ab') 2 were labeled with 125 I or 131 I by the chloramine-T method with almost full retention of antibody activity. F(ab') 2 fragments were cleared more rapidly from the circulation in normal mice with a half life of 6.3 hours than whole IgG with a half life of 5.5 days. Radioactivity of F(ab') 2 in various organs also decreased faster than IgG. In nude mice transplanted with AFP-producing human testicular tumor, F(ab') 2 fragments demonstrated superior scintigrams to whole IgG at 2 days after the injection, because of the fast disappearance of background radioactivity. Although absolute accumulation of 131 I labeled F(ab') 2 in the tumor was less than that of 131 I labeled IgG, tumor to other organ ratios were much higher with F(ab') 2 than those of IgG. The tumor to blood ratio of 131 I labeled F(ab') 2 was 1.04 at day 2, whereas tumor to blood ratio of 131 I labeled IgG was 0.55 at day 2 and 0.92 at day 4, respectively. These results indicated that for the radiolabeling of monoclonal antibodies, F(ab') 2 fragments would be superior to whole IgG in the radioimmunoimaging of cancer. (author)

  20. 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

  1. 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...

  2. 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...

  3. A human/mouse chimeric monoclonal antibody against intercellular adhesion molecule-1 for tumor radioimmunoimaging

    International Nuclear Information System (INIS)

    Yamamura, Miyuki; Hinoda, Yuji; Sasaki, Shigeru; Tsujisaki, Masayuki; Imai, Kohzoh; Oriuchi, Noboru; Endo, Keigo.

    1996-01-01

    A mouse-human chimeric antibody for intercellular adhesion molecule-1 (ICAM-1) was established by using heavy chain loss mouse mutant hybridoma and human immunoglobulin expression vector. The HA58 hybridoma secreted anti-ICAM-1 monoclonal antibody (MoAb) (IgG1,κ). The gene of the mouse variable region of heavy chain was amplified and cloned by the polymerase chain reaction technique directly from the HA58 hybridoma RNA. The variable region of heavy chain was joined with an expression vector which contains human γ1 constant gene. The expression vector was transfected into heavy chain loss mutant cells HA58-7, which produced only murine immunoglobulin light chains. The resultant chimeric MoAb HA58, chHA58, retained full-binding reactivity to ICAM-1 compared with murine HA58 parental antibody. The chimeric MoAb chHA58 showed little antibody dependent cell-mediated cytotoxic activity against cultured tumor cells. Biodistribution studies with 99m Tc-labeled chHA58 in nude mice bearing human gastric carcinoma JRST cells, demonstrated that the tumor-blood ratio was 1.55 at 18 h after injection, when the tumors were clearly visible in gamma scintigraphy. These data suggest that chHA58 may be of practical use for radioimmunoimaging of a wide variety of tumors. (author)

  4. Rheumatoid factor interference in immunogenicity assays for human monoclonal antibody therapeutics.

    Science.gov (United States)

    Tatarewicz, Suzanna; Miller, Jill M; Swanson, Steven J; Moxness, Michael S

    2010-05-31

    Rheumatoid factors (RFs) are endogenous human antibodies that bind to human gamma globulins. RFs demonstrate preferential binding to aggregated gamma globulins and are involved in the clearing mechanism of immune complexes. Immunoassays designed to measure human anti-human antibodies (HAHA) after administration of monoclonal antibody therapeutics are thus vulnerable to interference from RFs. When using a sensitive electrochemiluminescent (ECL) bridging immunoassay, samples from subjects with rheumatoid arthritis demonstrated much higher baseline reactivity than healthy subjects. Interference was found to be dependent on the aggregation state of the therapeutic antibody that had been conjugated with the detection reagent (ruthenium). Size exclusion high performance liquid chromatography (SE-HPLC) demonstrated that of the total integrated peaks, as little as 0.55% high molecular weight aggregates (>600kDa) were sufficient to cause increased reactivity. Stability studies of the ruthenium and biotin conjugated therapeutic antibody indicated that storage time, temperature and buffer formulation were critical in maintaining the integrity of the reagents. Through careful SE-HPLC monitoring we were able to choose appropriate storage and buffer conditions which led to a reduction in the false reactivity rate in therapeutic-naïve serum from a rheumatoid arthritis population.

  5. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

    Science.gov (United States)

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

  6. Clinical usefulness of human-mouse chimeric Fab monoclonal antibody A7 for radioimmunoguided surgery

    International Nuclear Information System (INIS)

    Yamamoto, Kazuhito

    1999-01-01

    This study was designed to determine the clinical usefulness of radioimmunoguided surgery (RIGS) using the human-mouse chimeric Fab monoclonal antibody A7 (chA7Fab) for colorectal cancer patients. Whole murine monoclonal antibody A7 (whole A7) and chA7Fab were labelled with 125 I and 131 I, and their biodistributions were investigated experimentally and clinically. Radioactivities of the antibodies in the tissues were measured by a portable gamma detecting probe (GDP) purchased from Neoprobe Corp.. Of the four labelled antibodies used in a mouse model, 125 I-chA7Fab revealed the highest tumor/surrounding tissue ratio and all values were greater than 2.0. All tumor/surrounding tissue ratios of 131 I-chA7Fab were greater than 1.5, but the values were lower than those of 125 I-chA7Fab. Due to the limited clinical use of 125 I in Japan, 131 I was used as a radio-tracer for chA7Fab in the clinical trial. RIGS using 131 I-chA7Fab was performed on ten colorectal cancer patients. Tumor localization was intraoperatively determined in four of ten patients using the GDP. Liver metastasis and lymph node metastasis were identified in two patients and one patient, respectively. The GDP revealed tumor/surrounding tissue ratios of 1.5 or greater in eight of the ten resected tumors. Although radioimmunoguided surgery using chA7Fab is a promising tool to intraoperatively determine the tumor localization of colorectal cancer, 125 I and not 131 I should be used as a tracer for radioimmunoguided surgery to increase the accuracy of chA7Fab. (author)

  7. Human cerebrospinal fluid monoclonal N-methyl-D-aspartate receptor autoantibodies are sufficient for encephalitis pathogenesis.

    Science.gov (United States)

    Kreye, Jakob; Wenke, Nina K; Chayka, Mariya; Leubner, Jonas; Murugan, Rajagopal; Maier, Nikolaus; Jurek, Betty; Ly, Lam-Thanh; Brandl, Doreen; Rost, Benjamin R; Stumpf, Alexander; Schulz, Paulina; Radbruch, Helena; Hauser, Anja E; Pache, Florence; Meisel, Andreas; Harms, Lutz; Paul, Friedemann; Dirnagl, Ulrich; Garner, Craig; Schmitz, Dietmar; Wardemann, Hedda; Prüss, Harald

    2016-10-01

    SEE ZEKERIDOU AND LENNON DOI101093/AWW213 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a recently discovered autoimmune syndrome associated with psychosis, dyskinesias, and seizures. Little is known about the cerebrospinal fluid autoantibody repertoire. Antibodies against the NR1 subunit of the NMDAR are thought to be pathogenic; however, direct proof is lacking as previous experiments could not distinguish the contribution of further anti-neuronal antibodies. Using single cell cloning of full-length immunoglobulin heavy and light chain genes, we generated a panel of recombinant monoclonal NR1 antibodies from cerebrospinal fluid memory B cells and antibody secreting cells of NMDAR encephalitis patients. Cells typically carried somatically mutated immunoglobulin genes and had undergone class-switching to immunoglobulin G, clonally expanded cells carried identical somatic hypermutation patterns. A fraction of NR1 antibodies were non-mutated, thus resembling 'naturally occurring antibodies' and indicating that tolerance induction against NMDAR was incomplete and somatic hypermutation not essential for functional antibodies. However, only a small percentage of cerebrospinal fluid-derived antibodies reacted against NR1. Instead, nearly all further antibodies bound specifically to diverse brain-expressed epitopes including neuronal surfaces, suggesting that a broad repertoire of antibody-secreting cells enrich in the central nervous system during encephalitis. Our functional data using primary hippocampal neurons indicate that human cerebrospinal fluid-derived monoclonal NR1 antibodies alone are sufficient to cause neuronal surface receptor downregulation and subsequent impairment of NMDAR-mediated currents, thus providing ultimate proof of antibody pathogenicity. The observed formation of immunological memory might be relevant for clinical relapses. © The Author (2016). Published by Oxford University Press on

  8. 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.

  9. Immunohistochemical Examination of Novel Rat Monoclonal Antibodies against Mouse and Human Podoplanin

    International Nuclear Information System (INIS)

    Kaji, Chiaki; Tsujimoto, Yuta; Kato Kaneko, Mika; Kato, Yukinari; Sawa, Yoshihiko

    2012-01-01

    This study aims to develop new monoclonal antibodies (mAbs) against mouse and human podoplanin. Rats were immunized with synthetic peptides, corresponding to amino acids 38–51 of mouse podoplanin or human podoplanin which is 100% homologous to the same site of monkey podoplanin; anti-mouse podoplanin mAb PMab-1 (IgG 2a ) and anti-human mAb NZ-1.2 (IgG 2a ) were established. In immunocytochemistry, the mouse melanoma B16-F10 and mouse podoplanin (mPDPN)-expressed CHO transfectant were stained by PMab-1; human lymphatic endothelial cells (LEC) and human podoplanin (hPDPN)-expressed squamous cell carcinoma HSC3 transfectant, were stained by NZ-1.2. Western-blot analysis detected an about 40-kDa protein in CHO-mPDPN and B16-F10 by PMab-1, and in HSC3-hPDPN and LEC by NZ-1.2. In frozen sections, PMab-1 reacted with mouse kidney, pulmonary alveoli, pulmonary pleura, and salivary gland myoepithelial cells while NZ-1.2 reacted to the human salivary gland myoepithelial cells. The immunostaining of paraffin-embedded sections also showed the reaction of PMab-1 or NZ-1.2 to the mouse or monkey kidney glomerulus, pulmonary alveoli, and lung lymphatic vessels. These results indicate that the two novel rat mAbs to the mouse and human/monkey podoplanin are useful for Western-blot and immunostaining of somatic tissues on paraffin-embedded sections as well as frozen sections

  10. Two-site sandwich immunoradiometric assay of human lymphotoxin with monoclonal antibodies and its applications

    Energy Technology Data Exchange (ETDEWEB)

    Meager, A; Parti, S; Leung, H; Woolley, J; Peil, E; Sidhu, S; Roberts, T

    1987-11-23

    Three monoclonal antibodies (MoAbs L49-15, L81-11 and L238-14) were raised against recombinant human lymphotoxin (rLT) derived from E. coli containing the cDNA sequence specifying LT. These MoAbs were ideal reagents for immunoassay of LT and a very sensitive, highly specific immunoradiometric assay (IRMA) was developed. This assay was rapid to perform and was capable of detecting as little as 10 pg/ml of LT. Application of the LT IRMA in combination with previously developed human gamma-interferon (IFN-..gamma..) and human tumour necrosis factor (TNF)-specific IRMA permitted independent estimations of these three substances to be carried out in parallel. By these means, it was found that RPMI 1788 lymphoblastoid cell line produced both LT and TNF, but not IFN-..gamma... Extensive analyses on cytokine (monokine and lymphokine) preparations derived from a variety of activated lymphocytes are also reported. Co-production of LT, TNF, and IFN-..gamma.. was a common finding, even occurring in alloantigen-specific T helper cell clones. 45 refs.; 3 figs.; 4 tabs.

  11. 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.

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

    International Nuclear Information System (INIS)

    Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki; Oshita, Masatoshi; Ideno, Shoji; Yunoki, Mikihiro; Kuhara, Motoki; Yamamoto, Naomasa; Okuno, Yoshinobu; Ikuta, Kazuyoshi

    2009-01-01

    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.

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

    Science.gov (United States)

    Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Abdolalizadeh, Jalal; Kazemi, Tohid; Aghebati Maleki, Ali; Sineh sepehr, Koushan

    2013-01-01

    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. PMID:24312838

  14. 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.

  15. First clinical evaluation of radioimmunoimaging using anti-human lung cancer monoclonal antibodies

    International Nuclear Information System (INIS)

    Zhou Qian

    1991-01-01

    Anti-human large cell lung cancer monoclonal antibodies (McAb) 2E3 and 6D1 were produced in the laboratory. Immunohistochemical studies and radiobinding assay showed these antibodies possessed high specificity against lung cancer cells. 28 patients with lung masses were investigated with 131 I-labeled McAb 6D1 and/or 2E3 scintigraphy. 19 of them were histologically proven and 13 were diagnosed primary lung carcinoma. Radioimmunoimaging visualized 10/13 of the primary lung cancers with a detection rate of 77%. Only 1 case of the non-cancer patients and a false localization, giving a true negative rate of 83%. Pathologically the squamous cell lung carcinoma had the highest localization and the small cell lung carcinoma next, but the detection rate was 100% for both. The adenocarcinoma of lung was less sensitive to these McAbs, with a detection rate of only 33% (1 of 3 cases). We conclude that radioimmunoimaging with anti-human large cell lung cancer McAbs is more specific and effective in detecting primary lung cancers and differentiating lung masses than with antibodies against other tumor associated antigens

  16. Monoclonal Antibody L1Mab-13 Detected Human PD-L1 in Lung Cancers.

    Science.gov (United States)

    Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Chang, Yao-Wen; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

    2018-04-01

    Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L 1 Mab-13 (IgG 1 , kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L 1 Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L 1 Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L 1 Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.

  17. Monoclonal antibodies reactive with human breast or ovarian carcinoma: In vivo applications

    International Nuclear Information System (INIS)

    Thor, A.D.; Edgerton, S.M.

    1989-01-01

    Monoclonal antibodies (MoAbs) are unique and useful bioprobes that allow in vivo targeting of membrane-associated or circulating antigens. Most of the clinical trials to date have used low dosages of radiolabeled MoAb given in a single dose. Newer studies have included antibody fragments, repeated injections, intraperitoneal (IP) administration, and other labels such as 90Y. Clinical MoAb trials are often arduous, expensive, and time-consuming to perform. Before human use, animal studies and extensive MoAb characterization are required. The production of pharmaceutical grade, radiolabeled MoAb is technically difficult and costly. Clinical trials require administrative and patient consent as well as extensive written protocols. These studies necessitate interdepartmental and intradepartmental cooperation and coordination. Furthermore, the use of in vivo radiolabeled probes impacts many levels of health care providers from janitorial, nursing, and technical staff to laboratories and physicians. Simple blood tests or disposal of body excretions may concern nursing or technical staff with the possibility of radiation exposure. The responsibility for study design, personnel involvement, and prospective use in patients without a definitive cancer diagnosis ultimately rests with the physician. While many issues have been addressed, additional clinical trials, consideration of safety issues, and standardization between institutions will be necessary before the use of radiolabeled MoAb for diagnosis, management, or therapy of human tumors becomes routine. Continued cooperation and funding should ensure its achievement. 136 references

  18. Monoclonal antibody DS6 detects a tumor-associated sialoglycotope expressed on human serous ovarian carcinomas.

    Science.gov (United States)

    Kearse, K P; Smith, N L; Semer, D A; Eagles, L; Finley, J L; Kazmierczak, S; Kovacs, C J; Rodriguez, A A; Kellogg-Wennerberg, A E

    2000-12-15

    A newly developed murine monoclonal antibody, DS6, immunohistochemically reacts with an antigen, CA6, that is expressed by human serous ovarian carcinomas but not by normal ovarian surface epithelium or mesothelium. CA6 has a limited distribution in normal adult tissues and is most characteristically detected in fallopian tube epithelium, inner urothelium and type 2 pneumocytes. Pre-treatment of tissue sections with either periodic acid or neuraminidase from Vibrio cholerae abolishes immunoreactivity with DS6, indicating that CA6 is a neuraminidase-sensitive and periodic acid-sensitive sialic acid glycoconjugate ("sialoglycotope"). SDS-PAGE of OVCAR5 cell lysates has revealed that the CA6 epitope is expressed on an 80 kDa non-disulfide-linked glycoprotein containing N-linked oligosaccharides. Two-dimensional non-equilibrium pH gradient gel electrophoresis indicates an isoelectric point of approximately 6.2 to 6.5. Comparison of the immunohistochemical distribution of CA6 in human serous ovarian adenocarcinomas has revealed similarities to that of CA125; however, distinct differences and some complementarity of antigen expression were revealed by double-label, 2-color immunohistochemical studies. The DS6-detected CA6 antigen appears to be distinct from other well-characterized tumor-associated antigens, including MUC1, CA125 and the histo-blood group-related antigens sLea, sLex and sTn. Copyright 2000 Wiley-Liss, Inc.

  19. Purification of human placental aromatase cytochrome P-450 with monoclonal antibody and its characterization

    International Nuclear Information System (INIS)

    Yoshida, Nobutaka; Osawa, Yoshio

    1991-01-01

    A simple and efficient method is described for the purification of microsomal aromatase cytochrome P-450 from human placenta. The enzyme was solubilized with Emulgen 913 and sodium cholate and subjected to chromatography on a column of Sepharose 4B couples with a specific monoclonal antibody, followed by hydroxyapatite column chromatography. The specific cytochrome P-450 content of purified aromatase was 13.1 (12-14.8) nmol/mg of protein. Aromatase assays were carried out with reconstituted systems of bovine liver P-450 reductase and dilauroyl-L-α-phosphatidylcholine with [1β- 3 H,4- 14 C]androstenedione as substrate. The total recovery of purified aromatase activity was 32.2%, and P-450 recovery was 17.6%. The very high K m value for 16α-hydroxytestosterone aromatization gives a reasonable indication that estriol is not the directly aromatized product in the fetoplacental unit of human pregnancy. The aromatase P-450 was subjected to SDS-polyacrylamide gel electrophoresis in increasing quantities. Silver stain detection techniques indicated a single band having a molecular mass of 55 kDa with greater than 97% purity. The stability analysis showed a half-life of over 4 years on storage at -80C

  20. LpMab-23: A Cancer-Specific Monoclonal Antibody Against Human Podoplanin.

    Science.gov (United States)

    Yamada, Shinji; Ogasawara, Satoshi; Kaneko, Mika K; Kato, Yukinari

    2017-04-01

    Human podoplanin (hPDPN), the ligand of C-type lectin-like receptor-2, is involved in cancer metastasis. Until now, many monoclonal antibodies (mAbs) have been established against hPDPN. However, it is still difficult to develop a cancer-specific mAb (CasMab) against hPDPN because the protein sequence of hPDPN expressed in cancer cells is the same as that in normal cells. Herein, we report LpMab-23 of the mouse IgG 1 subclass, a novel CasMab against hPDPN. In an immunohistochemical analysis, LpMab-23 reacted with tumor cells of human oral cancer, but did not react with normal cells such as lymphatic endothelial cells (LECs). In contrast, LpMab-17, another anti-hPDPN mAb, reacted with both tumor cells and LECs. Furthermore, flow cytometric analysis revealed that LpMab-23 reacted with hPDPN-expressing cancer cell lines (LN319, RERF-LC-AI/hPDPN, Y-MESO-14/hPDPN, and HSC3/hPDPN) but showed little reaction with normal cells (LECs and HEK-293T), although another anti-hPDPN mAb, LpMab-7, reacted with both hPDPN-expressing cancer cells and normal cells, indicating that LpMab-23 is a CasMab against hPDPN.

  1. Radioimmunodetection of human tumor xenografts by monoclonal antibody F(ab')/sub 2/ fragments

    Energy Technology Data Exchange (ETDEWEB)

    Herlyn, D.; Munz, D.L.; Herlyn, M.; Koprowski, H.; Powe, J.; Alavi, A.; Meinken, G.E.; Srivastava, S.C.

    1986-01-01

    Procedures are described for the radiolocalization of human tumors by murine monoclonal antibodies (MAb) in animal model systems. Visualization of tumor xenografts was clearer in nude mice compared to experimentally immunosuppressed mice due to the higher tumor viability. MAb localization in tumor tissue was greatly enhanced when F(ab')/sub 2/ fragments rather than intact antibody molecules were used. Although tumors could be visualized with /sup 131/I-, /sup 123/I-or /sup 111/In-labeled MAb fragments without background subtraction, tumor-to-background ratios of radioactivity were highest for /sup 131/I-labeled fragments. /sup 131/I-labeled F(ab')/sub 2/ fragments of eight MAb against human colorectal carcinoma, melanoma or lung carcinoma localized specifically only in those tumors that bound the MAb in vitro and not in unrelated tumors. Radiolabeled fragments of MAb with other specificities (anti-hepatitis virus MAb) did not localize in tumors. All MAb that inhibited tumor growth in nude mice effectively localized these tumors by ..gamma..-scintigraphy. Some MAb were effective in localizing tumors but ineffective in inhibiting their growth. The ability of the specific radiolabeled F(ab')/sub 2/ fragments to localize in tumor grafts correlated significantly with MAb binding affinity and density of antigenic sites on tumor cells together, but not with either in vitro binding parameter alone.

  2. Radioimmunodetection of human pancreatic tumor xenografts using DU-PAN II monoclonal antibody

    International Nuclear Information System (INIS)

    Nakamura, Kayoko; Kubo, Atsushi; Hashimoto, Shozo; Furuuchi, Takayuki; Abe, Osahiko; Takami, Hiroshi.

    1988-01-01

    The potential of DU-PAN II, monoclonal antibody (IgM), which was raised against the human tumor cell line, was evaluated for radioimmunodetection of human pancreatic tumors (PAN-5-JCK and EXP-58) grown in nude mice. 125 I-labeled DU-PAN II was accumulated into PAN-5-JCK producing DU-PAN II antigen with a tumor-to-blood ratio of 2.72 ± 3.00, but it did not localize in EXP-58 because of insufficient DU-PAN II. There was no significant uptake of 125 I-nonimmunized IgM in PAN-5-JCK. These facts indicated the specific tumor uptake of DU-PAN II. Excellent images of the tumor PAN-5-JCK were obtained 3 days after the injection of 125 I-DU-PAN II. Gel chromatography was also investigated with respect to the plasma taken from mice injected with antibody, or incubated with antibody in vitro. The results indicate that circulating antigen affected the tumor uptake of DU-PAN II: The more the tumor grew, the higher the amount of antigen excreted into the blood, leading to the degradation of DU-PAN II before it reached the tumor sites. Consequently, the immunoscintigram of the small tumor was remarkably clear. The catabolism and the radiolysis of the labeled IgM injected are critical points in applying immunoscintigraphy. (author)

  3. The production of high affinity monoclonal antibodies to human growth hormone

    International Nuclear Information System (INIS)

    Stuart, M.C.; Walichnowski, C.M.; Hussain, S.; Underwood, P.A.; Harman, D.F.; Rathjen, D.A.; Sturmer, S.R. von

    1983-01-01

    The primary aim of this work was to produce specific monoclonal antibodies to human growth hormone (hGH) for use in a diagnostic RIA of hGH levels in serum. Three different schedules were used for immunization of BALB/c mice and the splenocytes from each mouse were fused with myeloma cells Sp 2/0 Ag 14. Each fusion resulted in the production of hundreds of hybridomas secreting hGH-directed antibodies. Six antibodies have been fully characterized and have been grouped into pairs which recognize 3 different epitopes on the hGH molecule. One pair exhibits no cross reaction with the structurally related placental hormone, human placental lactogen (hPL), a second pair has low cross reaction with hPL (1.6-3%) and a third pair reacts equally well with hGH and hPL indicating binding to a common epitope in the 2 molecules. The highest affinity antibody, 74/6, which has an affinity constant of 4.4x10 10 l/mol and 3% cross-reactivity with hPL, has been used to establish a RIA for serum hGH measurements. Evidence is provided that hGH levels measured in this assay correlate well with those obtained in a conventional rabbit antiserum assay. (Auth.)

  4. In vitro and in vivo properties of human/mouse chimeric monoclonal antibody specific for common acute lymphocytic leukemia antigen

    International Nuclear Information System (INIS)

    Saga, T.; Endo, K.; Koizumi, M.; Kawamura, Y.; Watanabe, Y.; Konishi, J.; Ueda, R.; Nishimura, Y.; Yokoyama, M.; Watanabe, T.

    1990-01-01

    A human/mouse chimeric monoclonal antibody specific for a common acute lymphocytic leukemia antigen was efficiently obtained by ligating human heavy-chain enhancer element to the chimeric heavy- and light-chain genes. Cell binding and competitive inhibition assays of both radioiodine and indium-111- (111In) labeled chimeric antibodies demonstrated in vitro immunoreactivity identical with that of the parental murine monoclonal antibodies. The biodistribution of the radiolabeled chimeric antibody in tumor-bearing nude mice was similar to that of the parental murine antibody. Tumor accumulation of radioiodinated parental and chimeric antibodies was lower than that of 111 In-labeled antibodies, probably because of dehalogenation of the radioiodinated antibodies. Indium-111-labeled chimeric antibody clearly visualized xenografted tumor. These results suggest that a human/mouse chimeric antibody can be labeled with 111 In and radioiodine without the loss of its immunoreactivity, and that chimeric antibody localizes in vivo in the same way as the parental murine antibody

  5. Detection of human cancer in an animal model using radio-labelled tumour-associated monoclonal antibodies

    International Nuclear Information System (INIS)

    Epenetos, A.A.; Arklie, J.; Knowles, R.W.; Bodmer, W.F.

    1982-01-01

    Monoclonal antibodies to epithelial-cell antigenic determinants, labelled with 123 I and 125 I, were administered parenterally to immunodeficient mice bearing human tumours derived from a human cancer cell line. Anterior, posterior and lateral radioscans of the body were taken with a gamma scintillation camera at various times from immediately to 65 days after injection. Visual displays of the images were processed by standard computer techniques. The model used a human colon-cancer cell line, HT29, and the monoclonal antibody, AUAl, which is specific to an epithelial proliferating antigen. Tumour detection was achieved in all the mice. The smallest tumour detectable appeared to be about 1 mm in diameter. The degree of antibody uptake in a tumour depended on its size and the blood supply of its surrounding tissues. (author)

  6. Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

    Science.gov (United States)

    Ezzatifar, Fatemeh; Majidi, Jafar; Baradaran, Behzad; Aghebati Maleki, Leili; Abdolalizadeh, Jalal; Yousefi, Mehdi

    2015-01-01

    Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Results: Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. Conclusion: This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori. PMID:25789225

  7. Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

    Directory of Open Access Journals (Sweden)

    Fatemeh Ezzatifar

    2015-03-01

    Full Text Available Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Results: Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. Conclusion: This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori.

  8. Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII.

    OpenAIRE

    Broze, G J; Hickman, S; Miletich, J P

    1985-01-01

    Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII-deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on We...

  9. 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.

  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. 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.

  12. 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.

  13. Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

    Science.gov (United States)

    Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

    2016-04-22

    High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Monoclonal antibodies from rats immunized with fragment D of human fibrinogen

    International Nuclear Information System (INIS)

    Kennel, S.J.; Chen, J.P.; Lankford, P.K.; Foote, L.J.

    1981-01-01

    Fischer rats were immunized with fragment D (Fg-D) of human fibrinogen (Fg) to obtain antibody specific for neoantigens unique to this molecule. Absorption of serum with whole Fg indicated that some of the antibody produced reacted preferentially with Fg-D. Hybridoma cultures were prepared by fusion of immune rat spleen cells with mouse myeloma P3-X63-Ag8. Monoclonal antibodies obtained from these cultures fell into two classes: (a) Those reacting equally well with Fg and Fg-D. (b) Those reacting preferentially but not absolutely wth Fg-D. Antibody from hybridoma 104-14, a member of the first group had an affinity for Fg-D of 1.5 x 10 9 M -1 while antibodies from 106-59 and 106-71 (group 2) demonstrated much lower affinities of 1.0 x 10 7 and 4.7 x 10 6 M -1 , respectively. The cross reactivity of antibodies in the second group indicated that they react with protein conformations that are altered during production of Fg-D from Fg

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

    International Nuclear Information System (INIS)

    Chatal, J.F.; Douillard, J.Y.; Kremer, M.; Curtet, C.; Le Mevel, B.; Fumoleau, P.; Bourdoiseau, M.

    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 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') 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 [fr

  16. 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.).

  17. Imaging of myocardial infarction in dogs and humans using monoclonal antibodies specific for human myosin heavy chains

    International Nuclear Information System (INIS)

    Leger, J.; Chevalier, J.; Larue, C.; Gautier, P.; Planchenault, J.; Aumaitre, E.; Messner, P.; Puech, P.; Saccavini, J.C.; Pau, B.

    1991-01-01

    The use of three different monoclonal antibodies specific for human ventricular myosin heavy chains in the visualization of the location and extent of necrosis in dogs with experimental acute myocardial infarction and in humans is described. Using a classic immunohistochemical method or ex vivo analysis of heart slices in dogs with acute myocardial infarction subjected to intravenous injection of unlabeled antimyosin antibodies or antimyosin antibodies labeled with indium-111, it was observed that all antibody fragments specifically reached the targeted necrotic zone less than 2 h after antibody injection and remained bound for up to 24 h. In a limited but significant number of cases (5 of the 12 humans and 11 of 43 dogs), it was possible to image the necrotic zone in vivo as early as 2 to 4 h after antibody injection. In other cases, individual blood clearance variations retarded or even prevented in vivo necrosis detection. Higher antimyosin fixation values were obtained in the necrotic zones in dogs with a rapid blood clearance relative to that of the other dogs. It is concluded that antimyosin antibodies always reached necrotic areas within 2 h. If blood clearance was rapid, in vivo imaging of the necrotic area was possible 2 to 6 h after necrosis, even in humans. In some cases, however, uncontrolled individual variations in the timing required for sufficient blood clearance hampered this rapid in vivo detection of myocardial necrosis

  18. A Unique Report: Development of Super Anti-Human IgG Monoclone with Optical Density Over Than 3

    OpenAIRE

    Aghebati Maleki, Leili; Baradaran, Behzad; Abdolalizadeh, Jalal; Ezzatifar, Fatemeh; Majidi, Jafar

    2013-01-01

    Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity ...

  19. Immunoscintigraphy of human tumors transplanted in nude mice with radiolabeled anti-ras p21 monoclonal antibodies

    International Nuclear Information System (INIS)

    Katoh, Y.; Nakata, K.; Kohno, K.; Shima, M.; Satoh, A.; Kusumoto, Y.; Ishii, N.; Kohji, T.; Shiku, H.; Nagataki, S.

    1990-01-01

    Anti-ras p21 monoclonal antibody (RASK-3) was used for immunoscintigraphy of human cancer cell lines in nude mice. Iodine-125-labeled RASK-3 was injected into nude mice with either human colon cancers (FCC-1 or BM-314) or lung cancer (KNS-62). Clear images were obtained in all three cancers 7 days after the injection of antibody. No localization of 125 I-labeled control monoclonal antibody was observed. The ratio of tissue/blood radioactivity and % ID/g in the tumor were significantly higher than other organs by Day 8. The specific localization index examined by 131 I-RASK-3 and 125 I-control monoclonal antibody was also higher in the tumor than in other tissues. In the in vitro study, binding of RASK-3 to tumor cells increased significantly by treatment of cells with either lysolecithin or periodate-lysine-paraformaldehyde, which confirmed the intracellular localization of ras p21. The mechanism by which anti-ras p21 antibodies accumulate in tumor sites could be the necrotic changes in tumor cells or changes in membrane permeability of non-necrotic cells. These results provide a strong rationale for the utilization of ras p21 as a target antigen in the imaging of a variety of human cancers

  20. Nuclear medicine: Monoclonal antibodies

    International Nuclear Information System (INIS)

    Endo, K.; Sakahara, H.; Koizumi, M.; Kawamura, Y.; Torizuka, K.; Yokoyama, A.

    1986-01-01

    Antitumor monoclonal antibody was successfully labeled with Tc-99m by using dithiosemicarbazone (DTS) as a bifunctional chelating agent. In the first step, DTS was coupled to antibody without loss of immunoreactivity; the compound then efficiently formed a neutral 1:1 chelate with pentavalent or tetravalent Tc-99m. Imaging with Tc-99m-labeled monoclonal antibody to human osteosarcoma (OST-7) clearly displayed a small tumor in nude mice at 6 and 24 hours after intravenous administration. The tumor-to-blood ratio of the Tc-99m-labeled monoclonal antibody was higher than that of a radioiodinated antibody and similar to that of an In-111-labeled antibody. Thus, conjugation of DTS to monoclonal antibody followed by radiometalation is a simple and efficient method of preparing Tc-99m-labeled monoclonal antibody

  1. A two-site immunoradiometric assay for human pregnancy-associated plasma protein A (PAPP-A) using monoclonal antibodies

    International Nuclear Information System (INIS)

    Mowles, E.A.; Pinto-Furtado, L.G.; Bolton, A.E.

    1986-01-01

    A rapid, sensitive immunoradiometric assay has been developed for human pregnancy-associated plasma protein A (PAPP-A) using a purified mouse monoclonal antibody as the tracer and a rabbit polyclonal antibody to this protein in the solid-phase antibody preparation. The assay showed no measurable cross-reaction (< 0.1%) against a range of purified human placental proteins, and a good correlation with a previously described radioimmunoassay procedure when tested on samples taken throughout normal human pregnancies. No PAPP-A-like immunological activity could be detected in sera from non-pregnant women, confirming the absence of this protein from the circulation outside pregnancy. (Auth.)

  2. Safety testing of monoclonal antibodies in non-human primates: Case studies highlighting their impact on human risk assessment.

    Science.gov (United States)

    Brennan, Frank R; Cavagnaro, Joy; McKeever, Kathleen; Ryan, Patricia C; Schutten, Melissa M; Vahle, John; Weinbauer, Gerhard F; Marrer-Berger, Estelle; Black, Lauren E

    2018-01-01

    Monoclonal antibodies (mAbs) are improving the quality of life for patients suffering from serious diseases due to their high specificity for their target and low potential for off-target toxicity. The toxicity of mAbs is primarily driven by their pharmacological activity, and therefore safety testing of these drugs prior to clinical testing is performed in species in which the mAb binds and engages the target to a similar extent to that anticipated in humans. For highly human-specific mAbs, this testing often requires the use of non-human primates (NHPs) as relevant species. It has been argued that the value of these NHP studies is limited because most of the adverse events can be predicted from the knowledge of the target, data from transgenic rodents or target-deficient humans, and other sources. However, many of the mAbs currently in development target novel pathways and may comprise novel scaffolds with multi-functional domains; hence, the pharmacological effects and potential safety risks are less predictable. Here, we present a total of 18 case studies, including some of these novel mAbs, with the aim of interrogating the value of NHP safety studies in human risk assessment. These studies have identified mAb candidate molecules and pharmacological pathways with severe safety risks, leading to candidate or target program termination, as well as highlighting that some pathways with theoretical safety concerns are amenable to safe modulation by mAbs. NHP studies have also informed the rational design of safer drug candidates suitable for human testing and informed human clinical trial design (route, dose and regimen, patient inclusion and exclusion criteria and safety monitoring), further protecting the safety of clinical trial participants.

  3. 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

    2017-01-01

    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. PMID:28234345

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. Prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza.

    Directory of Open Access Journals (Sweden)

    Cameron P Simmons

    2007-05-01

    Full Text Available New prophylactic and therapeutic strategies to combat human infections with highly pathogenic avian influenza (HPAI H5N1 viruses are needed. We generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs and tested their efficacy for prophylaxis and therapy in a murine model of infection.Using Epstein-Barr virus we immortalized memory B cells from Vietnamese adults who had recovered from infections with HPAI H5N1 viruses. Supernatants from B cell lines were screened in a virus neutralization assay. B cell lines secreting neutralizing antibodies were cloned and the mAbs purified. The cross-reactivity of these antibodies for different strains of H5N1 was tested in vitro by neutralization assays, and their prophylactic and therapeutic efficacy in vivo was tested in mice. In vitro, mAbs FLA3.14 and FLD20.19 neutralized both Clade I and Clade II H5N1 viruses, whilst FLA5.10 and FLD21.140 neutralized Clade I viruses only. In vivo, FLA3.14 and FLA5.10 conferred protection from lethality in mice challenged with A/Vietnam/1203/04 (H5N1 in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of FLA3.14, FLA5.10, FLD20.19, and FLD21.140 provided robust protection from lethality at least up to 72 h postinfection with A/Vietnam/1203/04 (H5N1. mAbs FLA3.14, FLD21.140 and FLD20.19, but not FLA5.10, were also therapeutically active in vivo against the Clade II virus A/Indonesia/5/2005 (H5N1.These studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1

  9. 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

  10. Three-site sandwich radioimmunoassay with monoclonal antibodies for a sensitive determination of human alpha-fetoprotein

    International Nuclear Information System (INIS)

    Nomura, M.; Imai, M.; Takahashi, K.; Kumakura, T.; Tachibana, K.; Aoyagi, S.; Usuda, S.; Nakamura, T.; Miyakawa, Y.; Mayumi, M.

    1983-01-01

    Utilizing monoclonal antibodies against human alpha-fetoprotein, 3 distinct antigenic determinants were identified. These antigenic determinants, provisionally designated a, b and c, were arranged in such a manner that the binding of one determinant with the corresponding antibody did not inhibit, or only barely inhibited the binding of antibodies directed to the other 2 determinants. Monoclonal antibodies with 3 different specificities were, therefore, applied to develop a sandwich-type solid-phase radioimmunoassay of the antigen in which wells were coated with anti-a, and radiolabeled anti-b together with radiolabeled anti-c was employed to detect the bound antigen. The 3-site sandwich radioimmunoassay involving 3 different determinants gave a higher sensitivity than 2-site assays in which only anti-b or anti-c was employed as a radiolabeled reagent, because the radioactivity of the 2 labeled antibodies was added on the antigen bound to immobilized anti-a. (Auth.)

  11. Radioimaging of human mammary carcinoma xenografts in nude mice with a new monoclonal antibody

    International Nuclear Information System (INIS)

    Senekowitsch, R.; Bode, W.; Kriegel, H.; Reidel, G.; Pabst, H.W.

    1986-01-01

    A female Wistar rat aged 33 days was immunized by repeated intraperitoneal injections of a cell suspension of mammary carcinoma for eight months. Spleen cells of the immunized rat were then fused with X63-Ag8.653, a mouse myeloma line. Hybridoma supernatants were screened by ELISA using cells of mammary carcinoma (MaCa) as target cells. Initially 72 hybridomas showed positive response with MaCa cells, but no cross-reaction with normal mammary tissue was seen. Clone Ma 10-11 was chosen for its stable growth in vitro and in ascitic fluid. Monoclonal antibody obtained from ascitic fluid induced by intraperitoneal injection of 10 7 hybridoma cell into BALB/c-nu/nu mice was separated from albumin and transferrin. After separation only one band positioned at 155000 MW on SDS-PAGE slabs was detected. Radiolabeling with 131 I was achieved with the Iodogen method, the efficiency of labeling was 88%. 1.85 MBq of the intact labeled rat antibody were injected into nude mice xenografted with human mammary carcinoma and scintigrams were obtained every 48 hours p.i. up to 15 days. Scintigraphic images permitted tumor detection at 3 days p.i., but good tumor localization needed 8 days p.i.. The tumor-to-blood ratios calculated after dissection of tumor-bearing mice in groups of 3 increased from 0.97 at day 3 to 3 at day 15 p.i.. No uptake of the antibody in other organs was found. The half-life of the whole body clearance of the rat immunoglobulin was 36 h. This is significantly shorter than the half-life found for mouse immunoglobulin in nude mice. (Author)

  12. Determining the binding affinity of therapeutic monoclonal antibodies towards their native unpurified antigens in human serum.

    Directory of Open Access Journals (Sweden)

    Christine Bee

    Full Text Available Monoclonal antibodies (mAbs are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen's endogenous concentration, by combining the kinetic exclusion assay and Biacore's calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9, progranulin (PGRN, and fatty acid binding protein (FABP4. We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.

  13. Immunohistochemical Analysis of Inflammatory Rheumatoid Synovial Tissues Using Anti-Human Podoplanin Monoclonal Antibody Panel.

    Science.gov (United States)

    Suzuki, Tomoto; Takakubo, Yuya; Oki, Hiroharu; Liu, Xing; Honma, Ryusuke; Naganuma, Yasushi; Goodman, Stuart B; Kaneko, Mika K; Kato, Yukinari; Takagi, Michiaki

    2018-02-01

    Podoplanin (PDPN) is a transmembrane sialoglycoprotein, which is expressed in several normal tissues and malignant tumors. Although PDPN expression in rheumatoid arthritis (RA) has been reported, the role of PDPN in RA and other arthritic conditions has not been fully elucidated. In this study, we examined PDPN expression in inflammatory synovial tissues using an anti-human PDPN (hPDPN) monoclonal antibody (mAb) panel to select the most useful one for evaluation of synovitis. Synovial tissue samples were obtained from 11 RA patients and 9 osteoarthritis (OA) patients undergoing joint surgery. PDPN-positive cells were immunostained by a panel of PDPN mAbs (NZ-1, LpMab-3, LpMab-7, LpMab-10, LpMab-12, LpMab-13, and LpMab-17), followed by cell grading of inflammation and cell counting of PDPN-positivity by a quantitative analyzer. Immunohistochemistry showed that PDPN was markedly expressed in both macrophage-like type A and fibroblast-like type B lining cells of the hyperplastic synovial lining cell layer, and macrophages and fibroblasts in the stroma of RA. Among anti-PDPN mAbs, LpMab-12 showed the highest score. In inflammatory OA synovium, PDPN expression was also detectable. Although LpMab-12 also showed the highest score in OA, the difference was not statistically significant. The inflammatory synovitis score of RA was significantly higher than that of OA. PDPN was expressed in inflammatory lining cells and sublining stroma of RA and OA synovium. In the seven anti-hPDPN antibodies examined, LpMab-12 was the most stainable antibody for PDPN in RA synovitis. Thus, LpMab-12 for PDPN has a possible and promising specific biomarker for evaluating synovitis in RA and inflammatory OA.

  14. A human monoclonal antibody with neutralizing activity against highly divergent influenza subtypes.

    Directory of Open Access Journals (Sweden)

    Nicola Clementi

    Full Text Available The interest in broad-range anti-influenza A monoclonal antibodies (mAbs has recently been strengthened by the identification of anti-hemagglutinin (HA mAbs endowed with heterosubtypic neutralizing activity to be used in the design of "universal" prophylactic or therapeutic tools. However, the majority of the single mAbs described to date do not bind and neutralize viral isolates belonging to highly divergent subtypes clustering into the two different HA-based influenza phylogenetic groups: the group 1 including, among others, subtypes H1, H2, H5 and H9 and the group 2 including, among others, H3 subtype. Here, we describe a human mAb, named PN-SIA28, capable of binding and neutralizing all tested isolates belonging to phylogenetic group 1, including H1N1, H2N2, H5N1 and H9N2 subtypes and several isolates belonging to group 2, including H3N2 isolates from the first period of the 1968 pandemic. Therefore, PN-SIA28 is capable of neutralizing isolates belonging to subtypes responsible of all the reported pandemics, as well as other subtypes with pandemic potential. The region recognized by PN-SIA28 has been identified on the stem region of HA and includes residues highly conserved among the different influenza subtypes. A deep characterization of PN-SIA28 features may represent a useful help in the improvement of available anti-influenza therapeutic strategies and can provide new tools for the development of universal vaccinal strategies.

  15. 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

  16. Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

    Science.gov (United States)

    Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

    1992-09-01

    The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

  17. Radiolabelled monoclonal antibodies against alpha-fetoprotein for in vivo localization of human hepatocellular carcinoma by immunotomoscintigraphy

    International Nuclear Information System (INIS)

    Bergmann, J.F.; Lumbroso, J.D.; Manil, L.; Saccavini, J.C.; Rougier, P.; Assicot, M.; Mathieu, A.; Bellet, D.; Bohuon, C.

    1987-01-01

    Two high affinity monoclonal antibodies, designated AF01 and AF04, directed against distinct epitopes of human alpha-fetoprotein (AFP) and the Fab fragments of one of them, were labelled with 131 I and injected into 18 patients with AFP producing hepatocellular carcinoma (HCC) in order to carry out imaging studies by tomoscintigraphy. Twelve patients were injected with whole antibody, only three of seven patients injected with AF01 and two of five patients injected with AF04 had a positive scan. In contrast, five out of six patients injected with labelled Fab fragments of AF04 had positive imaging. These results confirm that tumour imaging of HCC using 131 I labelled monoclonal antibody against AFP is feasible. Moreover, utilization of tomoscintigraphy in place of linear scintigraphy and Fab fragments instead of whole immunoglobulin may improve the sensitivity of radioimmunolocalization. This technique provides useful information on the in vivo distribution of monoclonal antibodies directed against AFP and on the practicability of the eventual therapeutic use of anti-AFP antibodies in HCC. (orig.)

  18. Human breast tumor imaging using 111In labeled monoclonal antibody: Anthymic mouse model

    International Nuclear Information System (INIS)

    Ban An Khaw; Massachusetts General Hospital, Boston; Bailes, J.S.; Schneider, S.L.; Lancaster, J.; Lasher, J.C.; McGuire, W.L.; Powers, J.; Strauss, H.W.

    1988-01-01

    The monoclonal antibody (MoAb) 323/A3, an IgG1, was raised against the human breast tumor cell line MCF-7 and recognized a 43 Kd membrane associated glycoprotein. Histochemical studies with the antibody detected 75% of metastatic lymph nodes, 59% of primary breast tumors, and showed some staining in 20% of benign breast lesions. For radionuclide imaging, the MoAb 323/A3 was labeled with both 125 I and 111 In, via covalently coupled diethylenetriaminepentaacetic acid (DTPA) by the mixed anhydride method. The antibody activity of the DTPA modified 323/A3 was assessed by an immunoassay using viable and fixed MCF-7 target cells. Male athymic nude mice bearing BT-20 human mammary tumors were injected with dual 125 I/ 111 In labeled DTPA 323/A3 via the tail veins. The animals were imaged with a gamma camera equipped with a pinhole collimator at 1-3 h, 1, 2, 3, 4 and 5 days after the tracer administration. On day 5 or 6, the animals were killed, and the biodistribution of the radiotracers was determined for the blood, thyroid, heart, lungs, liver, spleen, kidneys, gastro-intestinal tract and tumor. Target to blood ratio at 6 days for the 111 In tracer was 24:1 in the group with a mean tumor weight of 0.492 g, and 13:1 in another group with a mean tumor weight of 0.1906 g (day 5). However, the 125 I activity showed only 3.6:1 and 5.4:1 target to blood ratios in the corresponding groups. The larger tumors localized less 111 I tracer (27.13%±7.57% injected dose/g, Mean±SD) than the smaller tumors (52.75%±22.25% ID/g). Analysis of the gamma images showed that the maximum tracer concentration occurred in the tumors at about 2 to 3 days after intravenous tracer administration. The excellent tumor resolution observed with BT-20 tumors may be due to increased 43 Kd glycoprotein antigen density in this tumor cell line. (orig.)

  19. The biodistribution of mouse monoclonal antibody ONS-M21 and the application for imaging diagnosis with its humanized antibody

    International Nuclear Information System (INIS)

    Ohkawa, Motohisa

    1997-01-01

    The mouse monoclonal antibody ONS-M21 combines with medulloblastomas and several gliomas specifically. And also we had already produced it humanized antibody. This study investigated the in vivo biodistribution of ONS-M21 and the application for imaging diagnosis using its humanized antibody. The nude mice (BALB/c nu/nu) bearing human medulloblastoma ONS-76 cells subcutaneously were injected 125 I-labeled ONS-M21 antibody via their tail vein. The radioactivities of their normal organs and the s.c. tumor were counted with γ-counter. And their autoradiograph (ARG) 6 hours after this administration was compared with gadolinium enhanced T1-weighted magnetic resonance image (Gd-T1-MRI). The brain tumor models transplanted ONS-76 cells stereotaxically was made by the nude rats (F344/N Jcl-rnu). And compared with MRI and ARG after the administration of 125 I-labeled humanized antibody into these models. The ARG indicated the accumulation of 125I -labeled ONS-M21 in the tumors which was detected by Gd-T1-MRI study. In this study, 125 I-labeled ONS-M21 remained in the tumor longer than the other normal organs. The mouse monoclonal antibody ONS-M21 have specific affinity for ONS-76 tumor in vivo. Then this humanized antibody is considerable to apply the imaging diagnosis of the malignant brain tumors. (author)

  20. 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.

  1. A neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection.

    Directory of Open Access Journals (Sweden)

    Katharine N Bossart

    2009-10-01

    Full Text Available Nipah virus is a broadly tropic and highly pathogenic zoonotic paramyxovirus in the genus Henipavirus whose natural reservoirs are several species of Pteropus fruit bats. Nipah virus has repeatedly caused outbreaks over the past decade associated with a severe and often fatal disease in humans and animals. Here, a new ferret model of Nipah virus pathogenesis is described where both respiratory and neurological disease are present in infected animals. Severe disease occurs with viral doses as low as 500 TCID(50 within 6 to 10 days following infection. The underlying pathology seen in the ferret closely resembles that seen in Nipah virus infected humans, characterized as a widespread multisystemic vasculitis, with virus replicating in highly vascular tissues including lung, spleen and brain, with recoverable virus from a variety of tissues. Using this ferret model a cross-reactive neutralizing human monoclonal antibody, m102.4, targeting the henipavirus G glycoprotein was evaluated in vivo as a potential therapeutic agent. All ferrets that received m102.4 ten hours following a high dose oral-nasal Nipah virus challenge were protected from disease while all controls died. This study is the first successful post-exposure passive antibody therapy for Nipah virus using a human monoclonal antibody.

  2. Immunotherapy of Alzheimer's disease (AD): from murine models to anti-amyloid beta (Abeta) human monoclonal antibodies.

    Science.gov (United States)

    Geylis, Valeria; Steinitz, Michael

    2006-01-01

    The deposition of amyloid beta (Abeta) protein is a key pathological feature in Alzheimer's disease (AD). In murine models of AD, both active and passive immunization against Abeta induce a marked reduction in amyloid brain burden and an improvement in cognitive functions. Preliminary results of a prematurely terminated clinical trial where AD patients were actively vaccinated with aggregated Abeta bear resemblance to those documented in murine models. Passive immunization of AD patients with anti-Abeta antibodies, in particular human antibodies, is a strategy that provides a more cautious management and control of any undesired side effects. Sera of all healthy adults contain anti-Abeta IgG autoimmune antibodies. Hence antigen-committed human B-cells are easily immortalized by Epstein-Barr virus (EBV) into anti-Abeta secreting cell lines. Two anti-Abeta human monoclonal antibodies which we recently prepared bind to the N-terminus of Abeta peptide and were shown to stain amyloid plaques in non-fixed brain sections from an AD patient. It is anticipated that specifically selected anti-Abeta human monoclonal antibodies could reduce and inhibit deposits of amyloid in brain while avoiding the cognitive decline that characterizes AD. In the future, this type of antibody may prove to be a promising immune therapy for the disease.

  3. Monoclonal antibodies and cancer

    International Nuclear Information System (INIS)

    Haisma, H.J.

    1987-01-01

    The usefulness of radiolabeled monoclonal antibodies for imaging and treatment of human (ovarian) cancer was investigated. A review of tumor imaging with monoclonal antibodies is presented. Special attention is given to factors that influence the localization of the antibodies in tumors, isotope choice and methods of radiolabeling of the monoclonal antibodies. Two monoclonal antibodies, OC125 and OV-TL3, with high specificity for human epithelial ovarian cancer are characterized. A simple radio-iodination technique was developed for clinical application of the monoclonal antibodies. The behavior of monoclonal antibodies in human tumor xenograft systems and in man are described. Imaging of tumors is complicated because of high background levels of radioactivity in other sites than the tumor, especially in the bloodpool. A technique was developed to improve imaging of human tumor xenographs in nude mice, using subtraction of a specific and a non-specific antibody, radiolabeled with 111 In, 67 Ga and 131 I. To investigate the capability of the two monoclonal antibodies, to specifically localize in human ovarian carcinomas, distribution studies in mice bearing human ovarian carcinoma xenografts were performed. One of the antibodies, OC125, was used for distribution studies in ovarian cancer patients. OC125 was used because of availability and approval to use this antibody in patients. The same antibody was used to investigate the usefulness of radioimmunoimaging in ovarian cancer patients. The interaction of injected radiolabeled antibody OC125 with circulating antigen and an assay to measure the antibody response in ovarian cancer patients after injection of the antibody is described. 265 refs.; 30 figs.; 19 tabs

  4. Experimental radioimmunoimaging of human lung small cell carcinoma xenograft H-69 by NCC-ST-433 monoclonal antibody

    International Nuclear Information System (INIS)

    Kubota, Tetsuro; Nakamura, Kayoko; Kubo, Atsushi; Hashimoto, Shozo; Watanabe, Masahiko; Ishibiki, Kyuya; Abe, Osahiko

    1989-01-01

    NCC-ST-433 monoclonal antibody raised against human gastric carcinoma xenograft (St-4) was labeled with l25 I using enzymatic and Iodogen methods. While labeling efficiency of the antibody was more excellent by enzymatic method, specific radioactivity of the antibody labeled by Iodogen method was higher than that by enzymatic method. The labeled antibody was stable in vitro and in vivo, and the labeled NCC-ST-433 was specifically accumulated in NCC-ST-433 antigen positive human tumor cell lines in vitro. The specificity of 125 I-NCC-ST-433 in vivo was found to be more excellent when this antibody was labeled by Iodogen method and acutually excellent images of H-69, a human small cell lung carcioma, were obtained 5 days after injection of 7 μg of 125 I-NCC-ST-433 per mouse. This method seemed to be promising for imaging human lung small cell carcinoma. (author)

  5. 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...... with the peptide free in solution. The reactions of the MAbs with a 5-aa motif (WCYKL) included in the sequence were examined with synthetic peptides and two of the MAbs reacted with the motif. The recognitions of recombinant full-length Nef protein were also tested. One MAb reacted with the protein in both ELISA...

  6. Monoclonal antibodies to human factor VII: production of immunodepleted plasma for VII:C assays.

    OpenAIRE

    Takase, T; Tuddenham, E G; Chand, S; Goodall, A H

    1988-01-01

    A high affinity monoclonal antibody to factor VII (RFF-VII/1), coupled to sepharose, was used to immunodeplete factor VII from normal plasma. The plasma could be used as a substrate in a one stage coagulation assay and performed as well as, or better than, commercially available factor VII deficient plasma or plasma from congenitally deficient factor VII patients. Plasma from normal donors (n = 20), patients with liver disease (n = 20), and patients receiving warfarin (n = 20), or congenitall...

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

    Czech Academy of Sciences Publication Activity Database

    Nováková, Zora; Foss, C. A.; Copeland, B. T.; Morath, V.; Baranová, Petra; Havlínová, Barbora; Skerra, A.; Pomper, M.G.; Bařinka, Cyril

    2017-01-01

    Roč. 77, č. 7 (2017), s. 749-764 ISSN 0270-4137 R&D Projects: GA ČR GAP301/12/1513; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : monoclonal antibody * glutamate carboxypeptidase II * NAALADase Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition OBOR OECD: Endocrinology and metabolism (including diabetes, hormones) Impact factor: 3.820, year: 2016

  8. 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.

  9. Bioactivity assays and application of 125I labeled human mouse chimeric anti-CD22 monoclonal antibody SM03

    International Nuclear Information System (INIS)

    Lu Pingping; Meng Zhiyun; Dou Guifang; Wu Yingliang; Wang Minwei

    2008-01-01

    To investigate the bioactivity and application of 125 I labeled human mouse chimeric monoclonal SM03, SM03 was labeled with 125 I using Indogen method. The labeled mixture was purified by Sephacryl S-300 HR separation chromospectry. The purity and concentration of separated fractions were determined by HPLC and Protein Assay Kit, respectively. Competitive binding method and ELISA method were used for bioactivity assays. 125 I-SM03 was applied to screen cell lines which express the most abundant CD22 antigen. The purity and recovery of 125 I-SM03 were >99% and >47%, respectively. The bioactivity of 125 I- SM03 and SM03 hasn't significant difference in statistics. Ramos cell line had the strongest special radioactivity when 125 I-SM03 bound with in Raji, Daudi and Ramos cell lines. Indogen method is a good way to label Human mouse chimeric anti-CD22 monoclonal antibody SM03 and the label will not affect the activity of SM03. The 125 I-SM03 not only can be used for detect agent, but also may be put into market for NHL therapy. (authors)

  10. Radiosensitivity of a monoclonal human lung adenocarcinoma cell line with MDR phenotype induced by CDDP: an in vitro study

    International Nuclear Information System (INIS)

    Zhang Junxiang; Kong Zhaolu; Shen Zhifen; Tong Shungao; Jin Yizun

    2006-01-01

    The study was to evaluate radiosensitivity of a monoclonal human lung adenocarcinoma cell line SPC-A-1/CDDP-4 with MDR phenotype induced by cisplatin (CDDP) compared with its parental cell SPC-A-1 in vitro. The glutathione (GSH) content and the radiosensitivity of SPC-A-1/CDDP-4 and SPC-A-1 cells were investigated in aerobic and under hypoxia, respectively. The radiosensitization effect of buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, to SPC-A-1/CDDP-4 and SPC-A-1 cells was observed. The results indicated that the monoclonal human lung adenocarcinoma cell line SPC-A-1/CDDP-4 showed, to some extent, a cross-resistance to 137 Cs γ-ray, in addition to its resistance to anticancer drugs (CDDP, ADM, MTX and VCR). The GSH content of SPC-A-1/CDDP-4 cells was higher than that of SPC-A-1 cells both in aerobic and under hypoxia which might account for it. BSO had radiosensitization effect to SPC-A-1/CDDP-4 and SPC-A-1 cells both in aerobic and under hypoxia, but it was stronger under hypoxia than in aerobic and it was stronger to SPC-A-1/CDDP-4 cells than to SPC-A-1 cells. (authors)

  11. Transient human anti-mouse antibodies (HAMA) interference in CA 125 measurements during monitoring of ovarian cancer patients treated with murine monoclonal antibody.

    NARCIS (Netherlands)

    Oei, A.L.M.; Sweep, F.C.; Massuger, L.F.A.G.; Olthaar, A.J.; Thomas, C.M.G.

    2008-01-01

    OBJECTIVE: To investigate the influence of human anti-mouse antibodies (HAMA) on serial CA 125 measurements in serum of patients with epithelial ovarian cancer following single intraperitoneal (IP) therapy with Yttrium-90-labeled human milk fat globule 1 murine monoclonal antibody ((90)Y-muHMFG1) as

  12. Monoclonal antibody

    International Nuclear Information System (INIS)

    Oyamada, Hiyoshimaru

    1987-01-01

    Some aspects of monoclonal antibodies are described, centering on studies made by the author and those presented at the Second International Conference on Monoclonal Antibody Immunoconjugates for Cancer held in March this year (1987). The history of immuno-nuclear medicine and procedures for producing monoclonal antibodies are briefly outlined. Monoclonal antibodies are immunoglobulins. Here, the structure of IgG, which is used most frequently, is described. An IgG is composed of two antigen binding fragments (Fab) and one crystallizable fragment (Fc). The end portion of a Fab reacts with an antigen. One of the major applications of immuno-nuclear medicine is the diagnosis of cancer. As label nucleides, 131 I and 111 I were selected in most cases in the past while 123 I and 99m Tc are currently used more often. Advantages and disadvantages of this diagnosis method is discussed citing studies presented at the First (1986) and Second (1987) International Conference on Monoclonal Antibody Immunoconjugates for Cancer. The present status of the application of monoclonal antibodies to treatment of cancer is also described. (Nogami, K.)

  13. A natural human monoclonal antibody targeting Staphylococcus Protein A protects against Staphylococcus aureus bacteremia

    Science.gov (United States)

    Varshney, Avanish K.; Sunley, Kevin M.; Bowling, Rodney A.; Kwan, Tzu-Yu; Mays, Heather R.; Rambhadran, Anu; Zhang, Yanfeng; Martin, Rebecca L.; Cavalier, Michael C.; Simard, John

    2018-01-01

    Staphylococcus aureus can cause devastating and life-threatening infections. With the increase in multidrug resistant strains, novel therapies are needed. Limited success with active and passive immunization strategies have been attributed to S. aureus immune evasion. Here, we report on a monoclonal antibody, 514G3, that circumvents a key S. aureus evasion mechanism by targeting the cell wall moiety Protein A (SpA). SpA tightly binds most subclasses of immunoglobulins via their Fc region, neutralizing effector function. The organism can thus shield itself with a protective coat of serum antibodies and render humoral immunity ineffective. The present antibody reactivity was derived from an individual with natural anti-SpA antibody titers. The monoclonal antibody is of an IgG3 subclass, which differs critically from other immunoglobulin subclasses since its Fc is not bound by SpA. Moreover, it targets a unique epitope on SpA that allows it to bind in the presence of serum antibodies. Consequently, the antibody opsonizes S. aureus and maintains effector function to enable natural immune mediated clearance. The data presented here provide evidence that 514G3 antibody is able to successfully rescue mice from S. aureus mediated bacteremia. PMID:29364906

  14. A Unique Report: Development of Super Anti-Human IgG Monoclone with Optical Density Over Than 3

    Directory of Open Access Journals (Sweden)

    Leili Aghebati Maleki

    2013-08-01

    Full Text Available Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG. Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity with IgM & IgA by ELISA and confirmed by immunoblotting. The subclasses of the selected mAbs were determined. The best clone was injected intraperitoneally to some pristane-injected mice. Anti-IgG mAb was purified from the animals' ascitic fluid by Ion exchange chromatography and then, mAb was conjugated with HRP. Results: In the present study, over than 50 clones were obtained that 1 clone had optical density over than 3. We named this clone as supermonoclone which was selected for limiting dilution. The result of the immunoblotting, showed sharp band in IgG position and did not show any band in IgM&IgA position. Conclusion: Based on the findings of this study, the conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases.

  15. A Unique Report: Development of Super Anti-Human IgG Monoclone with Optical Density Over Than 3

    Science.gov (United States)

    Aghebati Maleki, Leili; Baradaran, Behzad; Abdolalizadeh, Jalal; Ezzatifar, Fatemeh; Majidi, Jafar

    2013-01-01

    Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity with IgM & IgA by ELISA and confirmed by immunoblotting. The subclasses of the selected mAbs were determined. The best clone was injected intraperitoneally to some pristane-injected mice. Anti-IgG mAb was purified from the animals' ascitic fluid by Ion exchange chromatography and then, mAb was conjugated with HRP. Results: In the present study, over than 50 clones were obtained that 1 clone had optical density over than 3. We named this clone as supermonoclone which was selected for limiting dilution. The result of the immunoblotting, showed sharp band in IgG position and did not show any band in IgM&IgA position. Conclusion: Based on the findings of this study, the conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases. PMID:24312857

  16. 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 r...

  17. Production and characterization of monoclonal antibodies against α-globin chain-containing human hemoglobins for detecting α-thalassemia disease.

    Science.gov (United States)

    Pakdeepak, Kanet; Pata, Supansa; Chiampanichayakul, Sawitree; Kasinrerk, Watchara; Tatu, Thanusak

    2016-01-01

    Monoclonal antibodies against α-globin containing human Hbs, named AMS-Alpha1 and AMS-Alpha 2, were produced by the hybridoma technique using spleen cells enriched by the newly developed B lymphocyte enrichment protocol. These two monoclonal antibodies were of IgM class, reacting to only intact form of human Hbs A, A2, E, and F, which contain α-globin chain. By the indirect ELISA, the AMS-Alpha1 and AMS-Alpha 2 quantified less amount of α-globin chain containing hemoglobins in HbH disease than the SEA-α thalassemia 1 carriers and normal individuals. It was thus anticipated that these monoclonal antibodies can be used for detecting Hb Bart's hydrops fetalis in which no α-globin chain is produced.

  18. 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

  19. Immunoreactivity, stability, pharmacokinetics and biodistribution of a monoclonal antibody to human leukemic B cells after three different methods of radioiodination

    International Nuclear Information System (INIS)

    Zhenping Zhu; Ghose, T.; Kralovec, Y.; Chunzheng Yang

    1994-01-01

    Dal B02, a murine monoclonal antibody against human chronic lymphocytic leukemia (CLL) was radioiodinated using chloramine T (Chl.T), Bolton-Hunter (B-H) or N-succinimidyl-p-iodobenzoate (PIB). The preparations had comparable radiochemical purity (>97%) and immunoreactive fraction (65-80%) but the Chl.T-based product was most susceptible to deiodination and loss of immunoreactivity. After i.v. injection into CLL-xenografted nude mice, the preparations had identical patterns of clearance from the blood but the PIB-based product led to more radioactivity in liver and spleen and less in the thyroid compared to the other preparations. The Chl.T-based product showed loss of immunoreactivity in circulation and less tumor-localized radioactivity 168 h after administration. The differences between the B-H-based and PIB-based products were less impressive than between PIB-based and Chl.T-based products. (author)

  20. 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...... monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly...... neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a...

  1. Secukinumab, a human anti-interleukin-17A monoclonal antibody, in patients with psoriatic arthritis (FUTURE 2): a randomised, double-blind, placebo-controlled, phase 3 trial

    NARCIS (Netherlands)

    McInnes, Iain B.; Mease, Philip J.; Kirkham, Bruce; Kavanaugh, Arthur; Ritchlin, Christopher T.; Rahman, Proton; van der Heijde, Désirée; Landewé, Robert; Conaghan, Philip G.; Gottlieb, Alice B.; Richards, Hanno; Pricop, Luminita; Ligozio, Gregory; Patekar, Manmath; Mpofu, Shephard; Bird, Paul; Hall, Stephen; Nash, Peter; Zochling, Jane; de Vlam, Kurt; Langenaken, Christine; Geusens, Piet; Beaulieu, Andre; Tremblay, Jean-Luc; McCarthy, Tim; Papp, Kim; Poulin, Yves; Cohen, Martin; Galatikova, Dagmar; Dokoupilova, Eva; Dvorak, Zdenek; Mann, Herman; Sieper, Joachim; Spieler, Wolfgang; Kurthen, Reiner; Braun, Juergen; Wollenhaupt, Juergen; Tony, Hans-Peter; Schuch, Florian; Schulze-Koops, Hendrik; Rech, Juergen; Leszczynski, Piotr; Adamski, Zygmunt; Szepietowski, Jacek; Tlustochowicz, Witold; Kaszuba, Andrzej; Szymanska, Malgorzata; Stanislav, Marina; Nesmeyanova, Olga; Vezikova, Natalia; Ershova, Olga; Izmozherova, Nadezda; Zotkin, Eugeny; Petrova, Marianna; Kastanayan, Alexander; Yakupova, Svetlana; Agafina, Alina; Asavatanabodee, Paijit; Suwannalai, Parawee; Kerrane, Jerome; Tahir, Hasan; McInnes, Iain; Edwards, Christopher; Chinoy, Hector; Marzo-Ortega, Helena; Kaul, Arvind; Sheeran, Thomas; Clunie, Gavin; Schechtman, Joy; Gaylis, Norman; Kaine, Jeffrey; Lawson, Jeffrey; El-Kadi, Hisham; Flint, Kathleen; Kivitz, Alan; Churchhill, Melvin; Sikes, David; Lowenstein, Mitchell; Halpert, Elias; Abdulky, Mary; Palmer, William; Codding, Christine; Legerton, Clarence; Singhal, Atul; Sunkureddi, Prashanth; Gough, William; Forman, Seth; Box, Jane; Khan, Mohamed; Barranco, Elizabeth

    2015-01-01

    Background Interleukin 17A is a proinflammatory cytokine that is implicated in the pathogenesis of psoriatic arthritis. We assessed the efficacy and safety of subcutaneous secukinumab, a human anti-interleukin-17A monoclonal antibody, in patients with psoriatic arthritis. Methods In this phase 3,

  2. 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 (Bertram); 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

  3. 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

    The scarcity of monoclonal human IgE antibodies with specificity for defined allergens is a bottleneck for the molecular characterisation of allergens and their epitopes. Insights into the characteristics of such antibodies may allow for analyses of the molecular basis underlying allergenicity an...

  4. 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...

  5. Safety and efficacy of ofatumumab, a fully human monoclonal anti-CD20 antibody, in patients with relapsed or refractory B-cell chronic lymphocytic leukemia

    DEFF Research Database (Denmark)

    Coiffier, Bertrand; Lepretre, Stéphane; Pedersen, Lars Møller

    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...

  6. Anti-Mycobacterium leprae monoclonal antibodies cross-react with human skin: an alternative explanation for the immune responses in leprosy

    NARCIS (Netherlands)

    Naafs, B.; Kolk, A. H.; Chin A Lien, R. A.; Faber, W. R.; van Dijk, G.; Kuijper, S.; Stolz, E.; van Joost, T.

    1990-01-01

    A panel of 17 mouse monoclonal antibodies (MoAb) raised against Mycobacterium leprae (M. leprae) antigens was used to detect antigenic determinants in normal human skin. An indirect immunoperoxidase technique was used. Eight of the MoAb detected epidermal antigens similar to patterns well known for

  7. Safety, Pharmacokinetics, Immunogenicity, and Biodistribution of (186)Re-Labeled Humanized Monoclonal Antibody BIWA 4 (Bivatuzumab( in Patients with Early-Stage Breast Cancer.

    NARCIS (Netherlands)

    Koppe, M.; Schaijk, F. van; Roos, J.C.; Leeuwen, P.; Heider, K.H.; Kuthan, H.; Bleichrodt, R.P.

    2004-01-01

    The aim of this prospective study was to evaluate the safety, pharmacokinetics, immunogenicity, and biodistribution of (186)Re-labeled humanized anti-CD44v6 monoclonal antibody (MAb( BIWA 4 (Bivatuzumab( in 9 patients with early-stage breast cancer. Radioimmunoscintigraphy (RIS( was performed within

  8. Monoclonal antibodies in oncology

    International Nuclear Information System (INIS)

    Chan, S.Y.T.; Sikora, K.

    1986-01-01

    Monoclonal antibodies (MCAs) can be used to differentiate between normal and neoplastic cells and thus exploited for diagnostic and, ultimately, therapeutic gain. The evidence for the existence of human tumour antigens is reviewed. Several areas of diagnosis are already benefiting from the application of the monoclonal technology. Immunohistology can help the pathologist with difficult diagnostic problems. New classifications of lymphoma and leukaemia can be based on specific surface molecules. Similarly, the detection of shed tumour antigens is already established as part of the routine assessment of many patients with common solid tumours. Isotopically labeled monoclonal antibodies have been used to localise primary and metastatic tumours. The use of antibodies in this way is not only a promising diagnostic tool but also the first step in studying the possibility of arming antibodies to provide therapeutic agents. Such trials are currently in progress. (Auth.)

  9. 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.

  10. 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.

  11. Experimental radioimmunotherapy of a xenografted human glioma using [sup 131]I-labeled monoclonal antibody to epidermal growth factor receptor

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Hiroshi; Nakazawa, Shozo [Nippon Medical School, Tokyo (Japan); Herlyn, D

    1993-09-01

    [sup 131]I-labeled F (ab')[sub 2] fragments of murine monoclonal antibodies (MAb) 425 specific to the epidermal growth factor receptor expressed on human gliomas were used in experimental human malignant glioma immunotherapy. Two injections of 150 [mu]Ci [sup 131]I-labeled 425 F(ab')[sub 2] achieved growth inhibition of U-87MG human malignant glioma xenografts in nude mice. This radiolabeled specific MAb F(ab')[sub 2] was significantly superior to radiolabeled fragments of an anti-hepatitis virus control MAb A5C3 in influencing tumor growth. However, similar treatment of established human malignant glioma xenografts did not inhibit progressive tumor growth significantly. No clear tumor inhibition was produced by unlabeled MAb 425F(ab')[sub 2]. These studies suggest that [sup 131]I-labeled MAbs have a significant antitumor effect where unmodified antibody is ineffective. Multiple doses of antibody may achieve an increase in labeled MAb concentration in tumors. (author).

  12. 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.

  13. 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.

  14. 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

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

    Science.gov (United States)

    Mirick, G R; Bradt, B M; Denardo, S J; Denardo, G L

    2004-12-01

    The United States Food and Drug 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 ((90)yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ((131)I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) anti-CD20 MAbs for use in radioimmunotherapy (RIT) of non-Hodgkin'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, assays were developed to determine HAGA (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.

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

    International Nuclear Information System (INIS)

    Mirick, G. R.; Bradt, B. M.; Denardo, S. J.; Denardo, G. L.

    2004-01-01

    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 ( 9 0yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ( 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

  17. 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.

  18. 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

  19. Minor human antibody response to a mouse and chimeric monoclonal antibody after a single i.v. infusion in ovarian carcinoma patients: a comparison of five assays

    NARCIS (Netherlands)

    Buist, M. R.; Kenemans, P.; van Kamp, G. J.; Haisma, H. J.

    1995-01-01

    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 mg).

  20. 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.

  1. 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.

  2. Computational study on the interactions and orientation of monoclonal human immunoglobulin G on a polystyrene surface

    Directory of Open Access Journals (Sweden)

    Javkhlantugs N

    2013-07-01

    Full Text Available Namsrai Javkhlantugs,1,2 Hexig Bayar,3 Chimed Ganzorig,1 Kazuyoshi Ueda2 1Center for Nanoscience and Nanotechnology and Department of Chemical Technology, School of Chemistry and Chemical Engineering, National University of Mongolia, Ulaanbaatar, Mongolia; 2Department of Advanced Materials Chemistry, Graduate School of Engineering, Yokohama National University, Yokohama, Japan; 3The Key Laboratory of Mammalian Reproductive Biology and Biotechnology of the Ministry of Education, Inner Mongolia University, Hohhot, Inner Mongolia Autonomous Region, People's Republic of China Abstract: Having a theoretical understanding of the orientation of immunoglobulin on an immobilized solid surface is important in biomedical pathogen-detecting systems and cellular analysis. Despite the stable adsorption of immunoglobulin on a polystyrene (PS surface that has been applied in many kinds of immunoassays, there are many uncertainties in antibody-based clinical and biological experimental methods. To understand the binding mechanism and physicochemical interactions between immunoglobulin and the PS surface at the atomic level, we investigated the binding behavior and interactions of the monoclonal immunoglobulin G (IgG on the PS surface using the computational method. In our docking simulation with the different arrangement of translational and rotational orientation of IgG onto the PS surface, three typical orientation patterns of the immunoglobulin G on the PS surface were found. We precisely analyzed these orientation patterns and clarified how the immunoglobulin G interacts with the PS surface at atomic scale in the beginning of the adsorption process. Major driving forces for the adsorption of IgG onto the PS surface come from serine (Ser, aspartic acid (Asp, and glutamic acid (Glu residues. Keywords: bionano interface, immunoassay, polystyrene, IgG, physical adsorption, simulation

  3. 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.

  4. Generation of a novel high-affinity monoclonal antibody with conformational recognition epitope on human IgM.

    Science.gov (United States)

    Sarikhani, Sina; Mirshahi, Manouchehr; Gharaati, Mohammad Reza; Mirshahi, Tooran

    2010-11-01

    As IgM is the first isotype of antibody which appears in blood after initial exposure to a foreign antigen in the pattern of primary response, detection, and quantification of this molecule in blood seems invaluable. To approach these goals, generation, and characterization of a highly specific mAb (monoclonal antibody) against human IgM were investigated. Human IgM immunoglobulins were used to immunize Balb/c mice. Spleen cells taken from the immunized animals were fused with SP2/O myeloma cells using PEG (polyethylene glycol, MW 1450) as fusogen. The hybridomas were cultured in HAT containing medium and supernatants from the growing hybrids were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with pure human IgM and the positive wells were then cloned at limiting dilutions. The best clone designated as MAN-1, was injected intraperitoneally to some Pristane-injected mice. Anti-IgM mAb was purified from the animals' ascitic fluid by protein-G sepharose followed by DEAE-cellulose ion exchange chromatography. MAN-1 interacted with human IgM with a very high specificity and affinity. The purity of the sample was tested by SDS-PAGE and the affinity constant was measured (K(a) = 3.5 x 10(9)M(-1). Immunoblotting and competitive ELISA were done and the results showed that the harvested antibody recognizes a conformational epitope on the mu chain of human IgM and there was no cross-reactivity with other subclasses of immunoglobulins. Furthermore, isotyping test was done and the results showed the subclass of the obtained mAb which was IgG(1)kappa.

  5. Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

    Science.gov (United States)

    Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Chang, Yao-Wen; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

    2017-12-01

    Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-119 (IgG 1 , kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H 2 Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H 2 Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H 2 Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H 2 Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

  6. Enavatuzumab, a Humanized Anti-TWEAK Receptor Monoclonal Antibody, Exerts Antitumor Activity through Attracting and Activating Innate Immune Effector Cells

    Directory of Open Access Journals (Sweden)

    Shiming Ye

    2017-01-01

    Full Text Available Enavatuzumab is a humanized IgG1 anti-TWEAK receptor monoclonal antibody that was evaluated in a phase I clinical study for the treatment of solid malignancies. The current study was to determine whether and how myeloid effector cells were involved in postulated mechanisms for its potent antitumor activity in xenograft models. The initial evidence for a role of effector cells was obtained in a subset of tumor xenograft mouse models whose response to enavatuzumab relied on the binding of Fc of the antibody to Fcγ receptor. The involvement of effector cells was further confirmed by immunohistochemistry, which revealed strong infiltration of CD45+ effector cells into tumor xenografts in responding models, but minimal infiltration in nonresponders. Consistent with the xenograft studies, human effector cells preferentially migrated toward in vivo-responsive tumor cells treated by enavatuzumab in vitro, with the majority of migratory cells being monocytes. Conditioned media from enavatuzumab-treated tumor cells contained elevated levels of chemokines, which might be responsible for enavatuzumab-triggered effector cell migration. These preclinical studies demonstrate that enavatuzumab can exert its potent antitumor activity by actively recruiting and activating myeloid effectors to kill tumor cells. Enavatuzumab-induced chemokines warrant further evaluation in clinical studies as potential biomarkers for such activity.

  7. Use of Re-188 labelled anti-EGFr humanized monoclonal antibody h-R3 for radioimmunotherapy of gliomas

    International Nuclear Information System (INIS)

    Perera, A.; Torres, L.A.; Lopez, G.; Casaco, A.; Batista, J.F.; Pena, Y.; Coca, M.A.; Leyva, R.; Garcia, I.

    2007-01-01

    Full text: Locally administered monoclonal antibodies labelled with radioisotopes like 90Y, 131I, 186Re, 212Bi, 211At, 177Lu and others, constitute a viable and promising alternative for management different kind of malignancies. The development of 188W/188Re generator has given the possibility of having a radionuclide showing satisfactory features for radioimmunotherapy (Eb2.12 MeV, Eg155 keV, T1/2=16.9 h and easy to make labelling approaches, similar to used for 99mTc). Neuroepithelial-derived tumours show an increased expression of the EGF receptor with regard to adjacent normal tissue. This overexpression could be related with the autocrine stimulation of the neoplasm by EGF and TGFb. Humanized monoclonal antibody h-R3 has shown a high affinity for this EGF receptor, blocking the binding of EGF to it receptor and inducing apoptosis. Thus, it could be a good candidate for radioimmunotherapy of neuroepithelial malignancies. The aim of the present work was to label monoclonal antibody h-R3 with 188Re, to assess it in an animal model and evaluate its internal dosimetry and toxicity in patients with grade III-IV gliomas through a phase I clinical trial. Schwarz's direct labelling method was employed. Briefly, a 2000-fold molar excess of 2-mercaptoethanol was used to reduce bisulfide bonds of the antibody. The amount of sodium glucoheptonate, ascorbic acid and stannous fluoride were varied to achieve optimum labelling yield. 188Re-labeling yield was proportional to the volume of stannous glucoheptonate solution added to the formulation. Radiochemical purity of 188Re-h-R3 was 98.0±0.4%. Challenge against 300-fold molar excess of L-cysteine was made to assess the stability of the tracer. There was no significant difference between stability of 188Re-h-R3 and 99mTc-h-R3 against cysteine challenge up to 24 h. Animal biodistribution study was performed at 3 and 24 h after intravenous administration of 188Re-h-R3 through tale vein of Male Wistar rats. The results were

  8. A variety of human monoclonal antibodies against epidermal growth factor receptor isolated from a phage antibody library.

    Science.gov (United States)

    Kurosawa, Gene; Kondo, Mariko; Kurosawa, Yoshikazu

    2016-11-04

    When the technology for constructing human antibody (Ab) libraries using a phage-display system was developed, many researchers in Ab-related fields anticipated that it would be widely applied to the development of pharmaceutical drugs against various diseases, including cancers. However, successful examples of such applications are very limited. Moreover, researchers who utilize phage-display technology now show divergent ways of thinking about phage Ab libraries. For example, there is debate about what should be the source of V H and V L genes for the construction of libraries to cover the whole repertoire of Abs present in the human body. In the immune system, the introduction of mutations into V genes followed by selection based on binding activity, termed Ab maturation, is required for the production of Abs exhibiting high affinity to the antigen (Ag). Therefore, introduction of mutations and selection are required for isolation of Abs with high affinity after isolation of clones from phage Ab libraries. We constructed a large human Ab library termed AIMS, developed a screening method termed ICOS, and succeeded in isolating many human monoclonal Abs (mAbs) that specifically and strongly bind to various tumor-associated Ags. Eight anti-EGFR mAbs were included, which we characterized. These mAbs showed various different activities against EGFR-expressing cancer cells. In this paper, we describe these data and discuss the possibility and necessity that the mAbs isolated from the AIMS library might be developed as therapeutic drugs against cancers without introduction of mutations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Monoclonal antibodies directed to human insulin-like growth factor I (IGF I)

    International Nuclear Information System (INIS)

    Laubli, U.K.; Baier, W.; Celio, M.R.; Binz, H.; Humbel, R.E.

    1982-01-01

    Mouse hybridomas secreting antibodies to human insulin-like growth factor I (IGF I) were produced by fusion of spleen cells of hyperimmunised mice with FO mouse-myeloma cells. Eight clones producing antibodies against human IGF I have been isolated, two of which have been characterised. One was used in a radioimmunoassay, the other for immunopurification of IGF. (Auth.)

  10. Novel anti-c-Mpl monoclonal antibodies identified multiple differentially glycosylated human c-Mpl proteins in megakaryocytic cells but not in human solid tumors.

    Science.gov (United States)

    Zhan, Jinghui; Felder, Barbara; Ellison, Aaron R; Winters, Aaron; Salimi-Moosavi, Hossein; Scully, Sheila; Turk, James R; Wei, Ping

    2013-06-01

    Thrombopoietin and its cognate receptor, c-Mpl, are the primary molecular regulators of megakaryocytopoiesis and platelet production. To date the pattern of c-Mpl expression in human solid tumors and the distribution and biochemical properties of c-Mpl proteins in hematopoietic tissues are largely unknown. We have recently developed highly specific mouse monoclonal antibodies (MAb) against human c-Mpl. In this study we used these antibodies to demonstrate the presence of full-length and truncated human c-Mpl proteins in various megakaryocytic cell types, and their absence in over 100 solid tumor cell lines and in the 12 most common primary human tumor types. Quantitative assays showed a cell context-dependent distribution of full-length and truncated c-Mpl proteins. All forms of human c-Mpl protein were found to be modified with extensive N-linked glycosylation but different degrees of sialylation and O-linked glycosylation. Of note, different variants of full-length c-Mpl protein exhibiting differential glycosylation were expressed in erythromegakaryocytic leukemic cell lines and in platelets from healthy human donors. This work provides a comprehensive analysis of human c-Mpl mRNA and protein expression on normal and malignant hematopoietic and non-hematopoietic cells and demonstrates the multiple applications of several novel anti-c-Mpl antibodies.

  11. 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.

  12. Serological analysis of human anti-human antibody responses in colon cancer patients treated with repeated doses of humanized monoclonal antibody A33.

    Science.gov (United States)

    Ritter, G; Cohen, L S; Williams, C; Richards, E C; Old, L J; Welt, S

    2001-09-15

    Mouse monoclonal antibody A33 (mAb A33) recognizes a M(r) 43,000 cell surface glycoprotein (designated A33) expressed in human colonic epithelium and colon cancer but absent from most other normal tissues. In patients, mAb A33 localizes with high specificity to colon cancer and is retained for up to 6 weeks in the cancer but cleared rapidly from normal colon (5-6 days). As a carrier of (125)I or (131)I, mAb A33 has shown antitumor activity. Induction of strong human anti-mouse antibody (immunoglobulin; HAMA) responses in patients, however, limits the use of the murine mAb A33 to very few injections. A humanized version of this antibody (huAb A33) has been prepared for Phase I and II clinical studies in patients with colon cancer. In those studies, immunogenicity of huAb A33 has been monitored using a novel, highly sensitive BIACORE method, which allows measurement of human anti-human antibodies (HAHAs) without the use of secondary reagents. We found that 63% (26 of 41) of the patients treated with repeated doses of huAb A33 developed HAHAs against a conformational antigenic determinant located in the V(L) and V(H) regions of huAb A33. Detailed serological analysis showed two distinct types of HAHAs. HAHA of type I (49% of patients) was characterized by an early onset with peak HAHA levels after 2 weeks of treatment, which declined with ongoing huAb A33 treatment. HAHA of type II (17% of patients) was characterized by a typically later onset of HAHA than in type I and by progressively increasing HAHA levels with each subsequent huAb A33 administration. Colon cancer patients with type I HAHAs did not develop infusion-related adverse events. In contrast, HAHA of type II was indicative of infusion-related adverse events. By using this new method, we were able to distinguish these two types of HAHAs in patients while on antibody treatment, allowing patients to be removed from study prior to the onset of severe infusion-related adverse events.

  13. Recombinant human monoclonal autoantibodies specific for citrulline-containing peptides from phage display libraries derived from patients with rheumatoid arthritis.

    NARCIS (Netherlands)

    Raats, J.M.H.; Wijnen, E.M.; Pruijn, G.J.M.; Hoogen, F.H.J. van den; Venrooij, W.J.W. van

    2003-01-01

    OBJECTIVE: To isolate and characterize monoclonal autoantibodies (Mab) directed to citrullinated antigens from patients with rheumatoid arthritis (RA). METHODS: Using lymphocytes from bone marrow or peripheral blood from RA patients, we constructed antibody fragment libraries representing the

  14. 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.

  15. Immunoscintigraphy of human pancreatic carcinoma in nude mice with I-131-F(ab')/sub 2/-fragments of monoclonal antibodies

    International Nuclear Information System (INIS)

    Senekowitsch, R.; Maul, F.D.; Wenisch, H.J.C.; Kriegel, H.; Hor, G.

    1985-01-01

    In the present study radioiodinated F(ab')/sub 2/-fragments of CA19-9 and antibody that reacts specifically with human gastrointestinal cancer were examined for their ability to detect human pancreatic carcinoma hosted in nude mice. Tumor-bearing mice received 80μCi of I-131-F(ab')/sub 2/ with a specific activity of 1.8μCi/μg. All mice were imaged after the injection and every 24hr up to 6 days. The retained radioactivity was also registered with a whole-body counter immediately after imaging. As a control F(ab's)/sub 2/ of a nonspecific antibody were administered in parallel to another group of animals bearing the same tumor. Three animals of each group were killed at 1,2,4 and 8 days for determination of the distribution of both labeled antibody-fragments. On scintigraphic images obtained with the CA19-9-F(ab')/sub 2/ the tumors could be visualized 24hr after injection, the best dilineation however was achieved 96hr p.i.. The biodistribution data exhibited a more rapid blood clearance for the specific fragments compared to that for the unspecific ones. Tumors showed an increase in uptake up to 48hr reaching 1.7% of the injected dose per gram, declining to values of 0.08%/g at day 6 p.i.. The highest tumor-to-blood ratios were found after 96h. They were 7 for the CA19-9-fragments compared to 1.5 for the unspecific fragments. The whole body counting revealed a more rapid excretion for the fragments of the specific monoclonal antibodies than for the unspecific ones. In summary the authors were able to show that CA19-9-F(ab')/sub 2/-fragments can be used for immunodetection of human pancreatic carcinoma hosted in nude mice

  16. 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.

  17. 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

    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...... 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...... class II-positive monocytic (I937) and T leukemic (HUT78) tumor cell lines and Epstein-Barr virus (EBV) transformed B-lymphoid cell lines (EBV-LCL). Class II-negative cell lines (U-937 and the EBV-LCL mutant line 616) were not induced to aggregate. An HLA-G-transfected EBV-LCL, 221-AGN...

  18. Radioimmunoimaging of human colon and gastric cancers xenografts by NCC-ST-439 and NCC-ST-433 monoclonal antibodies

    International Nuclear Information System (INIS)

    Nakamura, Kayoko; Tsukatani, Yasushi; Nishiguchi, Iku

    1987-01-01

    Both NCC-ST-439 and NCC-ST-433 are monoclonal antibodies raised against human gastric cancer (St-4) xenografts in nude mice. Imaging and localization experiments were performed by injecting I-125 labeled antibodies into nude mice bearing CO-4 (colon carcinoma) and H-111 (gastric carcinoma). There was uptake of NCC-ST-439 (polymer) into the CO-4, though it was not clearly visualized until 5 days post injection. By injecting NCC-ST-439 (monomer), CO-4 was better seen at day 3, while average accumulation into the tumors decreased compared with NCC-ST-439 (polymer). High radioactivities were observed in the liver and spleen, which was probably due to the immunocomplex with the antigen in the blood. NCC-ST-433 was selectively accumulated into the H-111 with tumor to blood ratio 7.8 at day 7, without significant uptake into the liver and spleen. Significant correlation was also found between the tumor uptake level of NCC-ST-433 and size of tumors. Excellent images of H-111 were obtained 3 days after the injection. NCC-ST-433 holds promise for the radioimmunodetection of gastric cancers. (author)

  19. Radioimmunoimaging of experimental thrombi in dogs using technetium-99m-labeled monoclonal antibody fragments reactive with human platelets

    International Nuclear Information System (INIS)

    Som, P.; Oster, Z.H.; Zamora, P.O.; Yamamoto, K.; Sacker, D.F.; Brill, A.B.; Newell, K.D.; Rhodes, B.A.

    1986-01-01

    Monoclonal antibody 50H.19, which reacts with human platelets, was converted to fragments, pretinned, and made into kits for subsequent radiolabeling with /sup 99m/Tc. The antibody, which cross-reacts with dog platelets, was used to evaluate in vitro binding to blood clots and in vivo in experimental thrombi in dogs. After radiolabeling, 97.4 +/- 6.4% of the /sup 99m/Tc was antibody-associated. The preparations retained immunoreactivity, as determined by: binding studies using whole blood and determining the ratio of cell-to-plasma radioactivity (ratios of 57.6-61.2) and binding of the antibody to clots (clot/serum ratios were 57.2-74.6%). Approximately 50% of the radioactivity was cleared from the blood in 3-6 min and 18-24% was excreted in urine within 3 hr. Experimental thrombi in dogs could be visualized consistently within 2-3 hr postinjection in peripheral veins and arteries, pulmonary arteries, and the right ventricle. In addition, damage to blood vessel intima without visible thrombi could also be detected. This method has the following advantages: short and simple pre-imaging preparation, and rapid visualization of thrombi with no need for blood-pool subtraction or delayed imaging

  20. 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

  1. Radioimmunoimaging of human breast carcinoma xenografts in nude mouse model with 111In-labeled new monoclonal antibody EBA-1 and F(ab')2 fragments

    International Nuclear Information System (INIS)

    Yemul, Shrishailam; Leon, J.A.; Pozniakoff, Ted; Esser, P.D.; Estabrook, Alison; Met-Path Inc., Teterboro, NJ

    1993-01-01

    Radioimmunoimaging characteristics of a new monoclonal antibody EBA-1 and its F(ab') 2 fragments utilizing nu/nu mice bearing human breast carcinoma xenografts are described. 111 In-DPTA conjugates of EBA-1 localized with tumor/blood ratios of 0.99 ± 0.10 (P 2 radioconjugates at 48 h. These results suggest that EBA-1 and its F(ab') 2 might be useful reagents in radioimmunoimaging and radioimmunotherapy. (author)

  2. Radioimmunoscintigraphy of colorectal carcinoma using technetium-99m-labeled, totally human monoclonal antibody 88BV59H21-2.

    Science.gov (United States)

    Gulec, S A; Serafini, A N; Moffat, F L; Vargas-Cuba, R D; Sfakianakis, G N; Franceschi, D; Crichton, V Z; Subramanian, R; Klein, J L; De Jager, R L

    1995-12-01

    Radioimmunoscintigraphy (RIS) using human monoclonal antibodies offers the important clinical advantage of repeated imaging over murine monoclonal antibodies by eliminating the cross-species antibody response. This article reports a Phase I-II clinical trial with Tc-99m-labeled, totally human monoclonal antibody 88BV59H21-2 in patients with colorectal carcinoma. The study population consisted of 34 patients with colorectal cancer (20 men and 14 women; age range, 44-81 years). Patients were administered 5-10 mg antibody labeled with 21-41 mCi Tc-99m by the i.v. route and imaged at 3-10 and 16-24 h after infusion using planar and single-photon emission computed tomographic (CT) techniques. Pathological confirmation was obtained in 25 patients who underwent surgery. Human antihuman antibody (HAHA) titers were checked prior to and 1 and 3 months after the infusion. RIS with Tc-99m-labeled 88BV59H21-2 revealed a better detection rate in the abdomen-pelvis region compared with axial CT. The combined use of both modalities increased the sensitivity in both the liver and abdomen-pelvis regions. Ten patients developed mild adverse reactions (chills and fever). No HAHA response was detected in this series. Tc-99m-labeled human monoclonal antibody 88BV59H21-2 RIS shows promise as a useful diagnostic modality in patients with colorectal cancer. RIS alone or in combination with CT is more sensitive than CT in detecting tumor within the abdomen and pelvis. Repeated RIS studies may be possible, due to the lack of a HAHA response.

  3. Monoclonal antibody to an external epitope of the human mdr1 P-glycoprotein

    NARCIS (Netherlands)

    Arceci, R. J.; Stieglitz, K.; Bras, J.; Schinkel, A.; Baas, F.; Croop, J.

    1993-01-01

    A membrane glycoprotein, termed P-glycoprotein, has been shown to be responsible for cross-resistance to a broad range of structurally and functionally distinct cytotoxic agents. P-glycoprotein, encoded in humans by the mdr1 gene, functions as an energy-dependent efflux pump to exclude these

  4. 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

  5. Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies

    Science.gov (United States)

    Avery, Lindsay B.; Wang, Mengmeng; Kavosi, Mania S.; Joyce, Alison; Kurz, Jeffrey C.; Fan, Yao-Yun; Dowty, Martin E.; Zhang, Minlei; Zhang, Yiqun; Cheng, Aili; Hua, Fei; Jones, Hannah M.; Neubert, Hendrik; Polzer, Robert J.; O'Hara, Denise M.

    2016-01-01

    ABSTRACT Therapeutic antibodies continue to develop as an emerging drug class, with a need for preclinical tools to better predict in vivo characteristics. Transgenic mice expressing human neonatal Fc receptor (hFcRn) have potential as a preclinical pharmacokinetic (PK) model to project human PK of monoclonal antibodies (mAbs). Using a panel of 27 mAbs with a broad PK range, we sought to characterize and establish utility of this preclinical animal model and provide guidance for its application in drug development of mAbs. This set of mAbs was administered to both hemizygous and homozygous hFcRn transgenic mice (Tg32) at a single intravenous dose, and PK parameters were derived. Higher hFcRn protein tissue expression was confirmed by liquid chromatography-high resolution tandem mass spectrometry in Tg32 homozygous versus hemizygous mice. Clearance (CL) was calculated using non-compartmental analysis and correlations were assessed to historical data in wild-type mouse, non-human primate (NHP), and human. Results show that mAb CL in hFcRn Tg32 homozygous mouse correlate with human (r2 = 0.83, r = 0.91, p PK studies, enhancement of the early selection of lead molecules, and ultimately a decrease in the time for a drug candidate to reach the clinic. PMID:27232760

  6. 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

    -reactivities predicted by primary structure analyses of different isoforms and PR10 proteins were verified by allergen chip-based analyses. CONCLUSIONS: The obtained results demonstrate that hybrid IgE repertoires represent a source for human antibodies with genuine paratopes. The IgE-derived information about the Ig...... generation and epitope delineation of a human monoclonal IgE against the prototypic allergen Bet v 1. METHODS: Phage-display scFv hybrid libraries of allergic donor-derived VH epsilon and synthetic VL were established from 107 mononuclear cells. An obtained scFv was converted into human immunoglobulin...

  7. Glutamine synthetase gene knockout-human embryonic kidney 293E cells for stable production of monoclonal antibodies.

    Science.gov (United States)

    Yu, Da Young; Lee, Sang Yoon; Lee, Gyun Min

    2018-05-01

    Previously, it was inferred that a high glutamine synthetase (GS) activity in human embryonic kidney (HEK) 293E cells results in elevated resistance to methionine sulfoximine (MSX) and consequently hampers GS-mediated gene amplification and selection by MSX. To overcome this MSX resistance in HEK293E cells, a GS-knockout HEK293E cell line was generated using the CRISPR/Cas9 system to target the endogenous human GS gene. The GS-knockout in the HEK293E cell line (RK8) was confirmed by Western blot analysis of GS and by observation of glutamine-dependent growth. Unlike the wild type HEK293E cells, the RK8 cells were successfully used as host cells to generate a recombinant HEK293E cell line (rHEK293E) producing a monoclonal antibody (mAb). When the RK8 cells were transfected with the GS expression vector containing the mAb gene, rHEK293E cells producing the mAb could be selected in the absence as well as in the presence of MSX. The gene copies and mRNA expression levels of the mAb in rHEK293E cells were also quantified using qRT-PCR. Taken together, the GS-knockout HEK293E cell line can be used as host cells to generate stable rHEK293E cells producing a mAb through GS-mediated gene selection in the absence as well as in the presence of MSX. © 2018 Wiley Periodicals, Inc.

  8. Chimpanzee-Human Monoclonal Antibodies for Treatment of Chronic Poliovirus Excretors and Emergency Postexposure Prophylaxis▿‡

    Science.gov (United States)

    Chen, Zhaochun; Chumakov, Konstantin; Dragunsky, Eugenia; Kouiavskaia, Diana; Makiya, Michelle; Neverov, Alexander; Rezapkin, Gennady; Sebrell, Andrew; Purcell, Robert

    2011-01-01

    Six poliovirus-neutralizing Fabs were recovered from a combinatorial Fab phage display library constructed from bone marrow-derived lymphocytes of immunized chimpanzees. The chimeric chimpanzee-human full-length IgGs (hereinafter called monoclonal antibodies [MAbs]) were generated by combining a chimpanzee IgG light chain and a variable domain of heavy chain with a human constant Fc region. The six MAbs neutralized vaccine strains and virulent strains of poliovirus. Five MAbs were serotype specific, while one MAb cross-neutralized serotypes 1 and 2. Epitope mapping performed by selecting and sequencing antibody-resistant viral variants indicated that the cross-neutralizing MAb bound between antigenic sites 1 and 2, thereby covering the canyon region containing the receptor-binding site. Another serotype 1-specific MAb recognized a region located between antigenic sites 2 and 3 that included parts of capsid proteins VP1 and VP3. Both serotype 2-specific antibodies recognized antigenic site 1. No escape mutants to serotype 3-specific MAbs could be generated. The administration of a serotype 1-specific MAb to transgenic mice susceptible to poliovirus at a dose of 5 μg/mouse completely protected them from paralysis after challenge with a lethal dose of wild-type poliovirus. Moreover, MAb injection 6 or 12 h after virus infection provided significant protection. The MAbs described here could be tested in clinical trials to determine whether they might be useful for treatment of immunocompromised chronic virus excretors and for emergency protection of contacts of a paralytic poliomyelitis case. PMID:21345966

  9. 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.

  10. 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.

  11. A first‐in‐human pharmacodynamic and pharmacokinetic study of a fully human anti‐glucagon receptor monoclonal antibody in normal healthy volunteers

    Science.gov (United States)

    Kostic, Ana; King, Thomas Alexander; Yang, Feng; Chan, Kuo‐Chen; Yancopoulos, George D.; Gromada, Jesper

    2017-01-01

    Aims Glucagon receptor (GCGR) blockers are being investigated as potential therapeutics for type 1 and type 2 diabetes. Here we report the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of REGN1193, a fully human glucagon receptor blocking monoclonal antibody from a first‐in‐human healthy volunteer randomized double‐blinded trial. Methods Healthy men and women received single ascending doses of REGN1193 ranging from 0.05 to 0.6 mg/kg (n = 42) or placebo (n = 14) intravenously. Safety, tolerability and PK were assessed over 106 days. The glucose‐lowering effect of REGN1193 was assessed after induction of hyperglycaemia by serial glucagon challenges. Results REGN1193 was generally well tolerated. There were small (50 mg/dL, and did not require treatment or medical assistance. Concentration‐time profiles suggest a 2‐compartment disposition and marked nonlinearity, consistent with target‐mediated clearance. REGN1193 inhibited the glucagon‐stimulated glucose increase in a dose‐dependent manner. The 0.6 mg/kg dose inhibited the glucagon‐induced glucose area under the curve for 0 to 90 minutes (AUC0‐90 minutes) by 80% to 90% on days 3 and 15, while blunting the increase in C‐peptide. REGN1193 dose‐dependently increased total GLP‐1, GLP‐2 and glucagon, with plasma levels returning to baseline by day 29 in all dose groups. Conclusion REGN1193, a GCGR‐blocking monoclonal antibody, produced a safety, tolerability and PK/PD profile suitable for further clinical development. The occurrence of transient elevations in serum hepatic aminotransferases observed here and reported with several small molecule glucagon receptor antagonists suggests an on‐target effect of glucagon receptor blockade. The underlying mechanism is unknown. PMID:28755409

  12. Production and properties of monoclonal antibodies to human blood coagulation factor VII and factor VIIa

    International Nuclear Information System (INIS)

    Mann, P.; Nesbitt, J.A.; Ge, M.; Kisiel, W.

    1986-01-01

    Human factor VII is a trace vitamin K-dependent protein that circulates in blood as a single-chain precursor to a serine protease. Upon activation, two-chain factor VIIa activates factor x in the presence of tissue factor and calcium. Purified preparations of single-chain (SC) human factor VII and two-chain (TC) factor VIIa were utilized to immunize Balb/c mice. Spleen cells from these immunized mice were fused to a non-secreting NS-1 derivative of X63-Ag8 myeloma cells and grown in selective medium. Analysis of culture supernatants by EIA revealed several hybridomas that were secreting IgG specific for Sc-factor VII and TC-factor VIIa. In addition, several hybridomas secreted IgG that reacted equally well with factor VII and factor VIIa. One of the latter McAb (A-29) reacted with the heavy chain of factor VIIa and the intact factor VII molecule equally as judged by Western blotting. A-29 was produced in ascites fluid, purified and coupled to activated CH-Sepharose. Application of one liter of normal human plasma to 10 ml of this immunoadsorbent column, elution of factor VII and subsequent Western blot using 125 I-rabbit anti-human factor VII indicated a single species of factor VII(M/sub r/ = 50 KDa) in normal plasma. These specific factor VII/VIIa McAbs may prove useful in the analysis of these factors, and in the separation of SC-factor VII from TC-factor VIIa

  13. 92R Monoclonal Antibody Inhibits Human CCR9+ Leukemia Cells Growth in NSG Mice Xenografts.

    Science.gov (United States)

    Somovilla-Crespo, Beatriz; Martín Monzón, Maria Teresa; Vela, Maria; Corraliza-Gorjón, Isabel; Santamaria, Silvia; Garcia-Sanz, Jose A; Kremer, Leonor

    2018-01-01

    CCR9 is as an interesting target for the treatment of human CCR9 + -T cell acute lymphoblastic leukemia, since its expression is limited to immature cells in the thymus, infiltrating leukocytes in the small intestine and a small fraction of mature circulating T lymphocytes. 92R, a new mouse mAb (IgG2a isotype), was raised using the A-isoform of hCCR9 as immunogen. Its initial characterization demonstrates that binds with high affinity to the CCR9 N-terminal domain, competing with the previously described 91R mAb for receptor binding. 92R inhibits human CCR9 + tumor growth in T and B-cell deficient Rag2 -/- mice. In vitro assays suggested complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity as possible in vivo mechanisms of action. Unexpectedly, 92R strongly inhibited tumor growth also in a model with compromised NK and complement activities, suggesting that other mechanisms, including phagocytosis or apoptosis, might also be playing a role on 92R-mediated tumor elimination. Taken together, these data contribute to strengthen the hypothesis of the immune system's opportunistic nature.

  14. MOR103, a human monoclonal antibody to granulocyte–macrophage colony-stimulating factor, in the treatment of patients with moderate rheumatoid arthritis

    DEFF Research Database (Denmark)

    Behrens, Frank; Tak, Paul P; Ostergaard, Mikkel

    2015-01-01

    OBJECTIVES: To determine the safety, tolerability and signs of efficacy of MOR103, a human monoclonal antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF), in patients with rheumatoid arthritis (RA). METHODS: Patients with active, moderate RA were enrolled in a randomised...... placebo and MOR103 0.3, 1.0 and 1.5 mg/kg, respectively). Treatment emergent adverse events (AEs) in the MOR103 groups were mild or moderate in intensity and generally reported at frequencies similar to those in the placebo group. The most common AE was nasopharyngitis. In two cases, AEs were classified...... with active RA. The data support further investigation of this monoclonal antibody to GM-CSF in RA patients and potentially in those with other immune-mediated inflammatory diseases. TRIAL REGISTRATION NUMBER: NCT01023256....

  15. Characterization of sporozoite surface antigens of Plasmodium falciparum, using monoclonal antibodies. Part of a coordinated programme on the preparation of irradiated vaccines against some human diseases

    International Nuclear Information System (INIS)

    Groot, M.

    1982-10-01

    Sporozoites are considered as a source of potential vaccine. Characterization of their antigens is therefore important and can be achieved by monoclonal antibodies. The purpose of this project is to study the production of monoclonal antibodies against sporozoites of P. falciparum. Various infections of mosquitoes were carried out during the period 1981-1982 to obtain antigens for the production of hybridomas. Hybridomas were produced from mice immunized through the bites of infected mosquitoes and by intravenous inoculation. The anti-sporozoite activity of the hybridomas was tested by an immunofluorescent antibody test using P. falciparum sporozoites as antigens. Positive immunofluorescence was seen in hybridoma cell lines tested with P. falciparum, whereas negative results were obtained when the cell lines were cross-reacted with other human species (P. vivax) and with a rodent malaria parasite (P. berghei)

  16. Study of rat kidney transamidinase structure and regulation with monoclonal antibodies and the purification and characterization of human kidney transamidinase

    International Nuclear Information System (INIS)

    Gross, M.D.

    1985-01-01

    The isolation of monoclonal antibodies to transamidinase made possible the development of an immunosorbent inhibition assay for transamidinase protein using a 125 I-labeled monoclonal antibody. This assay is a more direct measurement of transamidinase protein than the determination of the amount of polyclonal antibody required to precipitate the transamidinase activities. Rats were fed diets supplemented with creatine and/or glycine, and the amounts of transamidinase protein were determined with the assay using the monoclonal antibody. The transamidinase activities of kidneys from the rats fed the various supplemented diets ranged from 10 to 40% of the control values, whereas, the amounts of transamidinase protein were, in all instances no lower than 66% of the control values. Purified homogeneous rat kidney transamidinase and rat kidney supernatants were subjected to isoelectric focussing and four to five fractions of the enzyme were obtained. Polyclonal antibodies, but not the monoclonal antibodies were found by Western blotting experiments to recognize all the forms of the enzyme obtained by the isoelectric focussing. The author concluded that the monoclonal antibodies recognized forms of the enzyme that changed very little in amount, relative to the alterations in enzyme activities, when rats were fed a diet containing creatine

  17. Development of novel monoclonal antibodies that define differentiation stages of human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Andersen, Ditte Caroline; Kortesidis, Angela; Zannettino, Andrew C W

    2011-01-01

    Human mesenchymal stem cells (hMSC) are currently being introduced for cell therapy, yet, antibodies specific for native and differentiated MSCs are required for their identification prior to clinical use. Herein, high quality antibodies against MSC surface proteins were developed by immunizing...... fewer differentiated alkaline phosphatase(+) cells compared to STRO-1(+/-)/Collagen VI(+) hMSC, suggesting that Collagen VI on the cell membrane exclusively defines differentiated MSCs. In conclusion, we have generated a panel of high quality antibodies to be used for characterization of MSCs...... mice with hMSC, and by using a panel of subsequent screening methods. Flow cytometry analysis revealed that 83.5, 1.1, and 8.5% of primary cultures of hMSC were double positive for STRO-1 and either of DJ 3, 9, and 18, respectively. However, none of the three DJ antibodies allowed enrichment...

  18. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms.

    Science.gov (United States)

    Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M

    2016-01-01

    Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies' potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease.

  19. 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

  20. 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.

  1. A model-based meta-analysis of monoclonal antibody pharmacokinetics to guide optimal first-in-human study design

    Science.gov (United States)

    Davda, Jasmine P; Dodds, Michael G; Gibbs, Megan A; Wisdom, Wendy; Gibbs, John P

    2014-01-01

    The objectives of this retrospective analysis were (1) to characterize the population pharmacokinetics (popPK) of four different monoclonal antibodies (mAbs) in a combined analysis of individual data collected during first-in-human (FIH) studies and (2) to provide a scientific rationale for prospective design of FIH studies with mAbs. The data set was composed of 171 subjects contributing a total of 2716 mAb serum concentrations, following intravenous (IV) and subcutaneous (SC) doses. mAb PK was described by an open 2-compartment model with first-order elimination from the central compartment and a depot compartment with first-order absorption. Parameter values obtained from the popPK model were further used to generate optimal sampling times for a single dose study. A robust fit to the combined data from four mAbs was obtained using the 2-compartment model. Population parameter estimates for systemic clearance and central volume of distribution were 0.20 L/day and 3.6 L with intersubject variability of 31% and 34%, respectively. The random residual error was 14%. Differences (> 2-fold) in PK parameters were not apparent across mAbs. Rich designs (22 samples/subject), minimal designs for popPK (5 samples/subject), and optimal designs for non-compartmental analysis (NCA) and popPK (10 samples/subject) were examined by stochastic simulation and estimation. Single-dose PK studies for linear mAbs executed using the optimal designs are expected to yield high-quality model estimates, and accurate capture of NCA estimations. This model-based meta-analysis has determined typical popPK values for four mAbs with linear elimination and enabled prospective optimization of FIH study designs, potentially improving the efficiency of FIH studies for this class of therapeutics. PMID:24837591

  2. An Enhanced Pre- and Postnatal Development Study in Cynomolgus Monkeys with Tabalumab: A Human IgG4 Monoclonal Antibody.

    Science.gov (United States)

    Breslin, William J; Hilbish, Kim G; Martin, Jennifer A; Halstead, Carolyn A; Newcomb, Deanna L; Chellman, Gary J

    2015-06-01

    Tabalumab, a human IgG4 monoclonal antibody (mAb) with neutralizing activity against both soluble and membrane B-cell activating factor (BAFF), has been under development for the treatment of autoimmune diseases. The purpose of this study was to determine the potential adverse effects of maternal tabalumab exposure on pregnancy, parturition, and lactation of the mothers and on the growth, viability, and development of the offspring through postnatal day (PND) 204. Tabalumab was administered by subcutaneous injection to presumed pregnant cynomolgus monkeys (16-19 per group) every 2 weeks from gestation day (GD) 20 to 22 until parturition at doses of 0, 0.3, or 30 mg/kg. Evaluations in mothers and infants included clinical signs, body weight, toxicokinetics, blood lymphocyte phenotyping, T-cell-dependent antibody response (infants only), antitherapeutic antibody (ATA), organ weights (infants only), and gross and microscopic histopathology. Infants were also examined for external and visceral morphologic and neurobehavioral development. There were no adverse tabalumab-related effects on maternal or infant endpoints. An expected pharmacological decrease in peripheral blood B-lymphocytes occurred in adults and infants; however, B-cell recovery was evident by PND154 in adults and infants at 0.3 mg/kg and by PND204 in infants at 30 mg/kg. At 30 mg/kg, a reduced IgM antibody response to T-cell-dependent antigen keyhole limpet hemocyanin (KLH) was observed following primary immunization. Following secondary KLH immunization, all infants in both dose groups mounted anti-KLH IgM and IgG antibody responses similar to control. Placental and mammary transfer of tabalumab was demonstrated. In conclusion, the no-observed-adverse-effect level for maternal and developmental toxicity was 30 mg/kg, the highest dose tested. Exposures at 30 mg/kg provide a margin of safety of 16× the anticipated clinical exposure. © 2015 Wiley Periodicals, Inc.

  3. Population PK and IgE pharmacodynamic analysis of a fully human monoclonal antibody against IL4 receptor.

    Science.gov (United States)

    Kakkar, Tarundeep; Sung, Cynthia; Gibiansky, Leonid; Vu, Thuy; Narayanan, Adimoolam; Lin, Shao-Lee; Vincent, Michael; Banfield, Christopher; Colbert, Alex; Hoofring, Sarah; Starcevic, Marta; Ma, Peiming

    2011-10-01

    For AMG 317, a fully human monoclonal antibody to interleukin receptor IL-4Rα, we developed a population pharmacokinetic (PK) model by fitting data from four early phase clinical trials of intravenous and subcutaneous (SC) routes simultaneously, investigated important PK covariates, and explored the relationship between exposure and IgE response. Data for 294 subjects and 2183 AMG 317 plasma concentrations from three Phase 1 and 1 Phase 2 studies were analyzed by nonlinear mixed effects modeling using first-order conditional estimation with interaction. The relationship of IgE response with post hoc estimates of exposure generated from the final PK model was explored based on data from asthmatic patients. The best structural model was a two-compartment quasi-steady-state target-mediated drug disposition model with linear and non-linear clearances. For a typical 80-kg, 40-year subject, linear clearance was 35.0 mL/hr, central and peripheral volumes of distribution were 1.78 and 5.03 L, respectively, and SC bioavailability was 24.3%. Body weight was an important covariate on linear clearance and central volume; age influenced absorption rate. A significant treatment effect was observable between the cumulative AUC and IgE response measured. The population PK model adequately described AMG 317 PK from IV and SC routes over a 60-fold range of doses with two dosing strengths across multiple studies covering healthy volunteers and patients with mild to severe asthma. IgE response across a range of doses and over the sampling time points was found to be related to cumulative AMG 317 exposure.

  4. Novel Monoclonal Antibody LpMab-17 Developed by CasMab Technology Distinguishes Human Podoplanin from Monkey Podoplanin.

    Science.gov (United States)

    Kato, Yukinari; Ogasawara, Satoshi; Oki, Hiroharu; Honma, Ryusuke; Takagi, Michiaki; Fujii, Yuki; Nakamura, Takuro; Saidoh, Noriko; Kanno, Hazuki; Umetsu, Mitsuo; Kamata, Satoshi; Kubo, Hiroshi; Yamada, Mitsuhiro; Sawa, Yoshihiko; Morita, Kei-Ichi; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika Kato

    2016-04-01

    Podoplanin (PDPN) is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregation-stimulating (PLAG) domain in its N-terminus. Among the three PLAG domains, O-glycan on Thr52 of PLAG3 is critical for the binding with C-type lectin-like receptor-2 (CLEC-2) and is essential for platelet-aggregating activity of PDPN. Although many anti-PDPN monoclonal antibodies (mAbs) have been established, almost all mAbs bind to PLAG domains. We recently established CasMab technology to produce mAbs against membranous proteins. Using CasMab technology, we produced a novel anti-PDPN mAb, LpMab-17, which binds to non-PLAG domains. LpMab-17 clearly detected endogenous PDPN of cancer cells and normal cells in Western-blot, flow cytometry, and immunohistochemistry. LpMab-17 recognized glycan-deficient PDPN in flow cytometry, indicating that the interaction between LpMab-17 and PDPN is independent of its glycosylation. The minimum epitope of LpMab-17 was identified as Gly77-Asp82 of PDPN using enzyme-linked immunosorbent assay. Of interest, LpMab-17 did not bind to monkey PDPN, whereas the homology is 94% between human PDPN and monkey PDPN, indicating that the epitope of LpMab-17 is unique compared with the other anti-PDPN mAbs. The combination of different epitope-possessing mAbs could be advantageous for the PDPN-targeting diagnosis or therapy.

  5. 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.

  6. 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

  7. HCMV Infection of Human Trophoblast Progenitor Cells of the Placenta Is Neutralized by a Human Monoclonal Antibody to Glycoprotein B and Not by Antibodies to the Pentamer Complex

    Directory of Open Access Journals (Sweden)

    Martin Zydek

    2014-03-01

    Full Text Available Human cytomegalovirus (HCMV is the major viral cause of congenital infection and birth defects. Primary maternal infection often results in virus transmission, and symptomatic babies can have permanent neurological deficiencies and deafness. Congenital infection can also lead to intrauterine growth restriction, a defect in placental transport. HCMV replicates in primary cytotrophoblasts (CTBs, the specialized cells of the placenta, and inhibits differentiation/invasion. Human trophoblast progenitor cells (TBPCs give rise to the mature cell types of the chorionic villi, CTBs and multi-nucleated syncytiotrophoblasts (STBs. Here we report that TBPCs are fully permissive for pathogenic and attenuated HCMV strains. Studies with a mutant virus lacking a functional pentamer complex (gH/gL/pUL128-131A showed that virion entry into TBPCs is independent of the pentamer. In addition, infection is blocked by a potent human neutralizing monoclonal antibody (mAb, TRL345, reactive with glycoprotein B (gB, but not mAbs to the pentamer proteins pUL130/pUL131A. Functional studies revealed that neutralization of infection preserved the capacity of TBPCs to differentiate and assemble into trophospheres composed of CTBs and STBs in vitro. Our results indicate that mAbs to gB protect trophoblast progenitors of the placenta and could be included in antibody treatments developed to suppress congenital infection and prevent disease.

  8. Detection of Human Epididymis Protein 4 (HE4) in Human Serum Samples Using a Specific Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assay (ELISA).

    Science.gov (United States)

    Zhou, Lijun; Lv, Zhiqiang; Shao, Jing; Xu, Ying; Luo, Xiaohong; Zhang, Yuming; Hu, Yang; Zhang, Wenji; Luo, Shuhong; Fang, Jianmin; Wang, Ying; Duan, Chaohui; Huang, Ruopan

    2016-09-01

    The human epididymis protein 4 (HE4) may have high specificity in the detection of malignant diseases, making the development of an immunoassay for HE4 essential. In our study, a fusion gene was constructed encoded with the HE4 protein. This protein was then produced in the bacterial cells (Escherichia coli) and used to immunize mice in order to eventually generate hybridomas specific to HE4. The hybridoma supernatants were then screened, and four positive anti-HE4 cell lines were selected. These cell lines produce monoclonal antibodies against HE4 epitopes, as demonstrated in the Western blot as well as by direct enzyme-linked immunosorbent assay (ELISA). Using the developed antibodies, we successfully identified several good antibody pairs from the hybridomas, which allowed for the development of a sandwich ELISA to measure HE4 levels. By using the HE4 ELISA, we measured HE4 levels of 60 clinical human serum samples. Compared with the Food and Drug Administration (FDA) approved kit (Roche), our results showed a strong positive correlation to those of the FDA-approved kit. In summary, highly sensitive antibody pairs were screened against HE4, and a sandwich ELISA was developed as an accurate analytical tool for the detection of HE4 in human serum, which could be especially valuable for diagnosing ovarian carcinomas. © 2015 Wiley Periodicals, Inc.

  9. Development of Novel Monoclonal Antibodies that Define Differentiation Stages of Human Stromal (Mesenchymal) Stem Cells

    Science.gov (United States)

    Andersen, Ditte C.; Kortesidis, Angela; Zannettino, Andrew C.W.; Kratchmarova, Irina; Chen, Li; Jensen, Ole N.; Teisner, Børge; Gronthos, Stan; Jensen, Charlotte H.; Kassem, Moustapha

    2011-01-01

    Human mesenchymal stem cells (hMSC) are currently being introduced for cell therapy, yet, antibodies specific for native and differentiated MSCs are required for their identification prior to clinical use. Herein, high quality antibodies against MSC surface proteins were developed by immunizing mice with hMSC, and by using a panel of subsequent screening methods. Flow cytometry analysis revealed that 83.5, 1.1, and 8.5% of primary cultures of hMSC were double positive for STRO-1 and either of DJ 3, 9, and 18, respectively. However, none of the three DJ antibodies allowed enrichment of clonogenic hMSC from BMMNCs as single reagents. Using mass-spectrometric analysis, we identified the antigen recognised by DJ3 as CD44, whereas DJ9 and DJ18 recognized HLA-DRB1 and Collagen VI, respectively. The identified proteins were highly expressed throughout in vitro osteogenic- and adipogenic differentiation. Interestingly, undifferentiated cells revealed a sole cytoplasmic distribution pattern of Collagen VI, which however changed to an extracellular matrix appearance upon osteogenic- and adipogenic differentiation. In relation to this, we found that STRO-1+/-/Collagen VI- sorted hMSC contained fewer differentiated alkaline phosphatase + cells compared to STRO-1+/-/Collagen VI+ hMSC, suggesting that Collagen VI on the cell membrane exclusively defines differentiated MSCs. In conclusion, we have generated a panel of high quality antibodies to be used for characterization of MSCs, and in addition our results may suggest that the DJ18 generated antibody against Collagen VI can be used for negative selection of cultured undifferentiated MSCs. PMID:21614487

  10. Monoclonal antibody 1.6.1 against human MPL receptor allows HSC enrichment of CB and BM CD34(+)CD38(-) populations.

    Science.gov (United States)

    Petit Cocault, Laurence; Fleury, Maud; Clay, Denis; Larghero, Jérôme; Vanneaux, Valérie; Souyri, Michèle

    2016-04-01

    Thrombopoietin (TPO) and its receptor Mpl (CD110) play a crucial role in the regulation of hematopoietic stem cells (HSCs). Functional study of Mpl-expressing HSCs has, however, been hampered by the lack of efficient monoclonal antibodies, explaining the very few data available on Mpl(+) HSCs during human embryonic development and after birth. Investigating the main monoclonal antibodies used so far to sort CD110(+) cells from cord blood (CB) and adult bone marrow (BM), we found that only the recent monoclonal antibody 1.6.1 engineered by Immunex Corporation was specific. Using in vitro functional assays, we found that this antibody can be used to sort a CD34(+)CD38(-)CD110(+) population enriched in hematopoietic progenitor stem cells, both in CB and in adult BM. In vivo injection into NSG mice further indicated that the CB CD34(+)CD38(-)CD110(+) population is highly enriched in HSCs compared with both CD34(+)CD38(-)CD110(-) and CD34(+)CD38(-) populations. Together our results validate MAb1.6.1 as an important tool, which has so far been lacking, in the HSC field. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  11. Characterization of recombinant yellow fever-dengue vaccine viruses with human monoclonal antibodies targeting key conformational epitopes.

    Science.gov (United States)

    Lecouturier, Valerie; Berry, Catherine; Saulnier, Aure; Naville, Sophie; Manin, Catherine; Girerd-Chambaz, Yves; Crowe, James E; Jackson, Nicholas; Guy, Bruno

    2018-04-26

    The recombinant yellow fever-17D-dengue virus, live, attenuated, tetravalent dengue vaccine (CYD-TDV) is licensed in several dengue-endemic countries. Although the vaccine provides protection against dengue, the level of protection differs by serotype and warrants further investigation. We characterized the antigenic properties of each vaccine virus serotype using highly neutralizing human monoclonal antibodies (hmAbs) that bind quaternary structure-dependent epitopes. Specifically, we monitored the binding of dengue virus-1 (DENV-1; 1F4), DENV-2 (2D22) or DENV-3 (5J7) serotype-specific or DENV-1-4 cross-reactive (1C19) hmAbs to the four chimeric yellow fever-dengue vaccine viruses (CYD-1-4) included in phase III vaccine formulations using a range of biochemical and functional assays (dot blot, ELISA, surface plasmon resonance and plaque reduction neutralization assays). In addition, we used the "classic" live, attenuated DENV-2 vaccine serotype, immature CYD-2 viruses and DENV-2 virus-like particles as control antigens for anti-serotype-2 reactivity. The CYD vaccine serotypes were recognized by each hmAbs with the expected specificity, moreover, surface plasmon resonance indicated a high functional affinity interaction with the CYD serotypes. In addition, the hmAbs provided similar protection against CYD and wild-type dengue viruses in the in vitro neutralization assay. Overall, these findings demonstrate that the four CYD viruses used in clinical trials display key conformational and functional epitopes targeted by serotype-specific and/or cross-reactive neutralizing human antibodies. More specifically, we showed that CYD-2 displays serotype- specific epitopes present only on the mature virus. This indicates that the CYD-TDV has the ability to elicit antibody specificities which are similar to those induced by the wild type DENV. Future investigations will be needed to address the nature of CYD-TDV-induced responses after vaccine administration, and how these

  12. Predominant antitumor effects by fully human anti-TRAIL-receptor2 (DR5) monoclonal antibodies in human glioma cells in vitro and in vivo

    Science.gov (United States)

    Nagane, Motoo; Shimizu, Saki; Mori, Eiji; Kataoka, Shiro; Shiokawa, Yoshiaki

    2010-01-01

    Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2 L) preferentially induces apoptosis in human tumor cells through its cognate death receptors DR4 or DR5, thereby being investigated as a potential agent for cancer therapy. Here, we applied fully human anti-human TRAIL receptor monoclonal antibodies (mAbs) to specifically target one of death receptors for TRAIL in human glioma cells, which could also reduce potential TRAIL-induced toxicity in humans. Twelve human glioma cell lines treated with several fully human anti-human TRAIL receptor mAbs were sensitive to only anti-DR5 mAbs, whereas they were totally insensitive to anti-DR4 mAb. Treatment with anti-DR5 mAbs exerted rapid cytotoxicity and lead to apoptosis induction. The cellular sensitivity was closely associated with cell-surface expression of DR5. Expression of c-FLIPL, Akt, and Cyclin D1 significantly correlated with sensitivity to anti-DR5 mAbs. Primary cultures of glioma cells were also relatively resistant to anti-DR5 mAbs, exhibiting both lower DR5 and higher c-FLIPL expression. Downregulation of c-FLIPL expression resulted in the sensitization of human glioma cells to anti-DR5 mAbs, whereas overexpression of c-FLIPL conferred resistance to anti-DR5 mAb. Treatment of tumor-burden nude mice with the direct agonist anti-DR5 mAb KMTR2 significantly suppressed growth of subcutaneous glioma xenografts leading to complete regression. Similarly, treatment of nude mice bearing intracerebral glioma xenografts with KMTR2 significantly elongated lifespan without tumor recurrence. These results suggest that DR5 is the predominant TRAIL receptor mediating apoptotic signals in human glioma cells, and sensitivity to anti-DR5 mAbs was determined at least in part by the expression level of c-FLIPL and Akt. Specific targeting of death receptor pathway through DR5 using fully human mAbs might provide a novel therapeutic strategy for intractable malignant gliomas. PMID:20511188

  13. 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

    monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly...

  14. 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.

  15. Pharmacokinetics, biodistribution and dosimetry of 99mTc-labeled anti-human epidermal growth factor receptor humanized monoclonal antibody R3 in rats

    International Nuclear Information System (INIS)

    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 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 (1(2α)) ) of 0.250 h and a mean elimination (t (1(2β)) ) 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 99m Tc-labeled humanized MAb R3 conjugate in patients should be supported

  16. Development of a monoclonal antibody that specifically detects tissue inhibitor of metalloproteinase-4 (TIMP-4) in formalin-fixed, paraffin-embedded human tissues.

    Science.gov (United States)

    Donover, P Scott; Wojciechowski, Brian S; Thirumaran, Rajesh; Zemba-Palko, Vlasta; Prendergast, George C; Wallon, U Margaretha

    2010-08-01

    Overexpression of the extracellular metalloproteinase inhibitor TIMP-4 in estrogen receptor-negative breast cancers was found recently to be associated with a poor prognosis for survival. To pursue exploration of the theranostic applications of TIMP-4, specific antibodies with favorable properties for immunohistochemical use and other clinical assays are needed. Here we report the characterization of a monoclonal antibody (clone 9:4-7) specific for full-length human TIMP-4 with suitable qualities. The antibody was determined to be an IgG(2b) immunoglobulin. In enzyme-linked immunosorbent assay (ELISA) and immunoblotting assays, it did not exhibit any detectable crossreactivity with recombinant forms of the other human TIMPs 1, 2, and 3. In contrast, the antibody displayed high specificity and sensitivity for TIMP-4 including in formalin-fixed and paraffin-embedded specimens of human breast specimens. An analysis of tissue microarrays of human cancer and corresponding normal tissues revealed specific staining patterns with excellent signal-to-noise ratios. This study documents TIMP-4 monoclonal antibody clone 9:4-7 as an effective tool for preclinical and clinical investigations. Published 2010 Wiley-Liss, Inc.

  17. 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

  18. Radiolabeled monoclonal antibody 15 and its fragments for localization and imaging of xenografts of human lung cancer

    International Nuclear Information System (INIS)

    Endo, K.; Kamma, H.; Ogata, T.

    1988-01-01

    Monoclonal antibody (MAb) 15 and its F(ab')2 and Fab fragments were radioiodinated, and their biodistribution and imaging were compared in BALB/c nude mice bearing a xenograft of a human lung cancer (TKB-2). Association constants for 125I-labeled MAb 15 IgG, F(ab')2, and Fab were 1.9 X 10(9), 1.8 X 10(9), and 3.7 X 10(8) M-1, respectively. Immunoreactive fractions ranged from 0.59 to 0.50. Cultured TKB-2 cells expressed 1.1 X 10(4) binding sites/cell for MAb 15 IgG in vitro. The binding of a control antibody and the binding of its fragments to TKB-2 cells were less than 3% of the input doses. The mice with the TKB-2 tumors were given simultaneous injections of 10 microCi of 131I-labeled MAb 15 or its fragments and 10 microCi of 125I-labeled control IgG or its fragments. With MAb 15 IgG, the percentage of the injected dose bound per gram of tissue (ID/g) of the tumor was 3.68% at day 7, when the localization index (LI) was 4.38. At day 2 after MAb 15 F(ab')2 injection, 1.12% of the ID/g was localized in the tumor and the LI was 3.04. After MAb 15 Fab injection, the percentage of the ID/g of the tumor was 0.31% and the LI was 2.58 at day 1. MAb 15 IgG, F(ab')2, and Fab cleared from the blood early, with a half-life of 33, 16, and 9 hours, respectively. The distributions of MAb 15 and its fragments in the normal organs did not differ from those of the control. Radioimaging with 100 microCi of 131I-labeled MAb 15 and its fragments showed that 42%, 44%, and 32% of the total-body count were localized in the tumor with IgG at day 7, F(ab')2 at day 2, or Fab at day 1, respectively. Because the radioactivity remaining in the tumor with Fab was low, the image was insufficient. Throughout the period, less than 10% of the control IgG and its fragments remained in the tumor. Microautoradiography confirmed the binding of MAb 15 and its fragments to the tumor cells

  19. 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.

  20. Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

    Directory of Open Access Journals (Sweden)

    Andrabi Raiees

    2012-09-01

    Full Text Available Abstract Background Analysis of human monoclonal antibodies (mAbs developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3 is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5 binding and presence of epitopes recognized by broadly neutralizing antibodies. Methods Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females within the age range of 20–57 years (median = 33 years were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. Results We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL, suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. Conclusions Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope

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

    International Nuclear Information System (INIS)

    Ryan, Patricia C.; Sleeman, Matthew A.; Rebelatto, Marlon; Wang, Bing; Lu, Hong; Chen, Xiaomin; Wu, Chi-Yuan; Hinrichs, Mary Jane; Roskos, Lorin; Towers, Heidi; McKeever, Kathleen; Dixit, Rakesh

    2014-01-01

    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.

  2. 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...

  3. Safety and pharmacokinetics of the Fc-modified HIV-1 human monoclonal antibody VRC01LS: A Phase 1 open-label clinical trial in healthy adults

    OpenAIRE

    Gaudinski, Martin R.; Coates, Emily E.; Houser, Katherine V.; Chen, Grace L.; Yamshchikov, Galina; Saunders, Jamie G.; Holman, LaSonji A.; Gordon, Ingelise; Plummer, Sarah; Hendel, Cynthia S.; Conan-Cibotti, Michelle; Lorenzo, Margarita Gomez; Sitar, Sandra; Carlton, Kevin; Laurencot, Carolyn

    2018-01-01

    Background VRC01 is a human broadly neutralizing monoclonal antibody (bnMAb) against the CD4-binding site of the HIV-1 envelope glycoprotein (Env) that is currently being evaluated in a Phase IIb adult HIV-1 prevention efficacy trial. VRC01LS is a modified version of VRC01, designed for extended serum half-life by increased binding affinity to the neonatal Fc receptor. Methods and findings This Phase I dose-escalation study of VRC01LS in HIV-negative healthy adults was conducted by the Vaccin...

  4. 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...

  5. A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display

    OpenAIRE

    Baskar, Sivasubramanian; Suschak, Jessica M.; Samija, Ivan; Srinivasan, Ramaprasad; Childs, Richard W.; Pavletic, Steven Z.; Bishop, Michael R.; Rader, Christoph

    2009-01-01

    Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only potentially curative treatment available for patients with B-cell chronic lymphocytic leukemia (B-CLL). Here, we show that post-alloHSCT antibody repertoires can be mined for the discovery of fully human monoclonal antibodies to B-CLL cell-surface antigens. Sera collected from B-CLL patients at defined times after alloHSCT showed selective binding to primary B-CLL cells. Pre-alloHSCT sera, donor sera, and control sera w...

  6. Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody

    OpenAIRE

    Donahue, Renee N.; Lepone, Lauren M.; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A.; Heery, Christopher R.; Gulley, James L.; Schlom, Jeffrey

    2017-01-01

    Background Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range...

  7. Experimental study on 211At labelled monoclonal antibody 3H11 and its Fab fragment radioimmunotherapy for human gastric cancer xenografts in nude mice

    International Nuclear Information System (INIS)

    Jin Jiannan; Liu Ning; Zhang Shuyuan; Zhang Shiyuan; Luo Deyuan; Zhou Maolun

    1996-01-01

    Experimental radioimmunotherapy investigation of α-emitting radionuclide 211 At labelled anti-gastric cancer monoclonal antibody 3H11 and its Fab fragment for nude mice carrying human gastric cancer xenografts was conducted. Three i.p. injections of 14.8 or 22.2 kBq/g mouse were given, once every 5 days. The results showed that the growth of tumor xenografts was inhibited efficiently. The most evident therapy effect was observed at 15 days after treatment, and the tumor inhibition rates were 65% and 72%, respectively. No radiation injury of important organs was found

  8. Preliminary study on guiding therapy of subcutaneous human hetero graft in nude mice by 211-At labelled monoclonal antibody against gastric cancer

    International Nuclear Information System (INIS)

    Luo Lin; Wang Fangyuan

    1993-01-01

    In short time, 211 At labelled monoclonal antibody 3H 11 inhibited the growth of human gastric cancer grafted in nude mice effectively. The most evident inhibition was observed at the 9th day after treatment, and the tumor inhibition rates were 80%, 93%, 48% in the groups of intravenous injection, internal tumor injection (both 211 At-3H 11 5 μCi per animal), Na 211 At (5 μCi per animal) treatment respectively. On the 20th day, when animals were killed, the tumor inhibition rates were 66%, 81%, 6%, respectively. Inhibition treated with Na 211 At showed obviously lower than those at the 9th day

  9. Tumour localization and pharmacokinetics of iodine-125 human monoclonal IgM antibody (COU-1) and its monomeric and half-monomeric fragments analysed in nude mice grafted with human tumour

    International Nuclear Information System (INIS)

    Ditzel, H.; Erb, K.; Rasmussen, J.W.; Jensenius, J.C.

    1992-01-01

    Human monoclonal IgM antibodies reactive with cancer-associated antigens may not have the optimal imaging capability due to their large size. Fragmentation of human IgM is less than straight-forward due to the loss of immunoreactivity. From the human monoclonal IgM antibody COU-1 we have prepared monomeric and half-monomeric fragments, which retain the ability to bind to colon cancer cells in vitro. The pharmacokinetics and tumour localization were evaluated in nude mice bearing human colon adenocarcinoma and human melanoma grafts. Faster clearance from the circulation was seen for the smaller half-monomeric fragment with a half-life (rapid phase/slow phase) of 2 h/16 h compared with the intact antibody, 4 h/25 h, and the monomeric fragment, 3 h/27 h. Intact COU-1 as well as the fragments accumulated in the colon tumour graft. Higher amounts of radioactivity were found in the colon tumour as compared to normal organs for intact COU-1 at days 4 and 6, for the monomeric fragment at day 4, and for the half-monomeric fragment at day 2 after injection. This investigation demonstrates the favourable biodistribution of the half monomeric COU-1 fragment. The fast clearance of this fragment resulted in a tumour-to-muscle ratio as high as 22 on day 2 after injection. Also, only this fragment gave a positive tumour-to-blood ratio. Normal IgM and its fragments were used as controls. Radioimmunoscintigraphy demonstrated the colon tumour discriminatory properties of each of the three iodine-labelled antibody preparations. The results compare favourably with previously reported investigations of the localization of human monoclonal antibodies and suggest that fragments of human monoclonal IgM antibodies may be useful tools for the immunodetection of cancer in patients. (orig.)

  10. 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...... peripheral blood lymphocytes caused by the low abundance of antigen-specific B cells in the circulation. The preselection of B cells is based on the specificity of the surface Ig receptor and is accomplished using the antigen of interest conjugated to magnetic beads. This method should significantly increase...... the frequency of antibody phage particles of interest in the library and allow for efficient isolation monoclonal antibodies with the predefined specificity....

  11. Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

    Science.gov (United States)

    Bidlingmaier, Scott; Su, Yang; Liu, Bin

    2015-01-01

    Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

  12. Human monoclonal antibody 99mTc-88BV59: detection of colorectal cancer, recurrent or metastatic disease and immunogenicity assessment

    International Nuclear Information System (INIS)

    Krause, B.J.; Baum, R.P.; Staib-Sebler, E.; Lorenz, M.; Niesen, A.; Hoer, G.

    1997-01-01

    This study presents immunoscintigraphic results in 24 patients suffering from primary colorectal cancer, recurrent or metastatic disease after the injection of 1197-1351 MBq technetium-99m labelled totally human monoclonal antibody 88BV59. Labelling efficacy of 99m Tc-88BV59 ranged from 97% to 99%. Immunoscintigraphy was performed 18-20 h after injection. Scintigraphic findings were compared with those of computed tomography (CT). Patients underwent surgery in order to evaluate immunoscintigraphic findings histologically. Sera of the patients (before injection and 1 and 3 months post infusion) were analysed for the presence of human anti-human antibodies (HAHA). None of the patients showed a HAHA response as assessed by a solid-phase ELISA assay. The antibody scan detected about 25% more lesions than CT. In the detection of extrahepatic disease, the sensitivity of the antibody scan proved to be 68%, whereas the sensitivity of CT was 41%. (orig.). With 3 figs., 1 tab

  13. Human monoclonal antibody 99mTc-88BV59: detection of colorectal cancer, recurrent or metastatic disease and immunogenicity assessment.

    Science.gov (United States)

    Krause, B J; Baum, R P; Staib-Sebler, E; Lorenz, M; Niesen, A; Hör, G

    1997-01-01

    This study presents immunoscintigraphic results in 24 patients suffering from primary colorectal cancer, recurrent or metastatic disease after the injection of 1197-1351 MBq technetium-99m labelled totally human monoclonal antibody 88BV59. Labelling efficacy of 99mTc-88BV59 ranged from 97% to 99%. Immunoscintigraphy was performed 18-20 h after injection. Scintigraphic findings were compared with those of computed tomography (CT). Patients underwent surgery in order to evaluate immunoscintigraphic findings histologically. Sera of the patients (before injection and 1 and 3 months post infusion) were analysed for the presence of human anti-human antibodies (HAHA). None of the patients showed a HAHA response as assessed by a solid-phase ELISA assay. The antibody scan detected about 25% more lesions than CT. In the detection of extrahepatic disease, the sensitivity of the antibody scan proved to be 68%, whereas the sensitivity of CT was 41%.

  14. Human monoclonal antibody {sup 99m}Tc-88BV59: detection of colorectal cancer, recurrent or metastatic disease and immunogenicity assessment

    Energy Technology Data Exchange (ETDEWEB)

    Krause, B.J. [Department of Nuclear Medicine, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany); Baum, R.P. [Department of Nuclear Medicine, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany); Staib-Sebler, E. [Department of General and Abdominal Surgery, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany); Lorenz, M. [Department of General and Abdominal Surgery, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany); Niesen, A. [Department of Nuclear Medicine, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany); Hoer, G. [Department of Nuclear Medicine, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany)

    1997-01-01

    This study presents immunoscintigraphic results in 24 patients suffering from primary colorectal cancer, recurrent or metastatic disease after the injection of 1197-1351 MBq technetium-99m labelled totally human monoclonal antibody 88BV59. Labelling efficacy of {sup 99m}Tc-88BV59 ranged from 97% to 99%. Immunoscintigraphy was performed 18-20 h after injection. Scintigraphic findings were compared with those of computed tomography (CT). Patients underwent surgery in order to evaluate immunoscintigraphic findings histologically. Sera of the patients (before injection and 1 and 3 months post infusion) were analysed for the presence of human anti-human antibodies (HAHA). None of the patients showed a HAHA response as assessed by a solid-phase ELISA assay. The antibody scan detected about 25% more lesions than CT. In the detection of extrahepatic disease, the sensitivity of the antibody scan proved to be 68%, whereas the sensitivity of CT was 41%. (orig.). With 3 figs., 1 tab.

  15. 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.

  16. 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.

  17. Purification of HBsAg produced by the human hepatoma cell line PLC/PRE/5 by affinity chromatography using monoclonal antibodies and application for ELISA diagnostic.

    Science.gov (United States)

    Merten, O W; Reiter, S; Scheirer, W; Katinger, H

    1983-01-01

    The human cell line PLC/PRF/5 (5) was used for the production of hepatitis B surface antigen subtype ad (HBsAg ad) and purified by affinity chromatography (AC) with monoclonal antibodies (mAb). mAb to HBsAg from mouse ascites have been purified by Protein A - AC prior coupling to AH-Sepharose 4B (Pharmacia). The combined procedure of ammonium-sulphate-precipitation of HBsAg from culture supernatants and immunosorbent-AC leads to approx. 700-fold purification. ELISA results using the mAb and the HBsAg for diagnostics of human serum, positive for anti-HBsAg-antibodies correlate with the RIA (AUSAB, Abbott).

  18. Expression of blood group I and i active carbohydrate sequences on cultured human and animal cell lines assessed by radioimmunoassays with monoclonal cold agglutinins

    International Nuclear Information System (INIS)

    Childs, R.A.; Kapadia, A.; Feizi, T.

    1980-01-01

    Human monoclonal anti-I and anti-i, reactive with known carbohydrate sequences, have been used as reagents to quantitate (by radioimmunoassay) and visualize (by immunofluorescence) the expression of the various blood group I and i antigenic determinants in a variety of cultured cell lines commonly used in laboratory investigations. It has been shown that the antigens they recognize are widely distributed on the surface of human and animal cell lines, expressed in varying amounts in different cell lines and on individual cells within a given cell line. In two cell lines, a transformation-associated increase in the expression of I antigen was observed. Because of their precise specificity for defined carbohydrate chain domains, these autoantibodies have become valuable reagents in biological chemistry. (orig.) [de

  19. Influenza human monoclonal antibody 1F1 interacts with three major antigenic sites and residues mediating human receptor specificity in H1N1 viruses.

    Directory of Open Access Journals (Sweden)

    Tshidi Tsibane

    Full Text Available Most monoclonal antibodies (mAbs to the influenza A virus hemagglutinin (HA head domain exhibit very limited breadth of inhibitory activity due to antigenic drift in field strains. However, mAb 1F1, isolated from a 1918 influenza pandemic survivor, inhibits select human H1 viruses (1918, 1943, 1947, and 1977 isolates. The crystal structure of 1F1 in complex with the 1918 HA shows that 1F1 contacts residues that are classically defined as belonging to three distinct antigenic sites, Sa, Sb and Ca(2. The 1F1 heavy chain also reaches into the receptor binding site (RBS and interacts with residues that contact sialoglycan receptors and determine HA receptor specificity. The 1F1 epitope is remarkably similar to the previously described murine HC63 H3 epitope, despite significant sequence differences between H1 and H3 HAs. Both antibodies potently inhibit receptor binding, but only HC63 can block the pH-induced conformational changes in HA that drive membrane fusion. Contacts within the RBS suggested that 1F1 may be sensitive to changes that alter HA receptor binding activity. Affinity assays confirmed that sequence changes that switch the HA to avian receptor specificity affect binding of 1F1 and a mAb possessing a closely related heavy chain, 1I20. To characterize 1F1 cross-reactivity, additional escape mutant selection and site-directed mutagenesis were performed. Residues 190 and 227 in the 1F1 epitope were found to be critical for 1F1 reactivity towards 1918, 1943 and 1977 HAs, as well as for 1I20 reactivity towards the 1918 HA. Therefore, 1F1 heavy-chain interactions with conserved RBS residues likely contribute to its ability to inhibit divergent HAs.

  20. 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

    International Nuclear Information System (INIS)

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga; Luque, Daniel; Terrón, María C.; Calder, Lesley J.; Melero, José A.

    2014-01-01

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV 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 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 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

  1. 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.

  2. 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.

  3. 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 

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

    International Nuclear Information System (INIS)

    Hu, Weibin; Chen, Aizhong; Miao, Yi; Xia, Shengli; Ling, Zhiyang; Xu, Ke; Wang, Tongyan; Xu, Ying; Cui, Jun; Wu, Hongqiang; Hu, Guiyu; Tian, Lin; Wang, Lingling; Shu, Yuelong; Ma, Xiaowei; Xu, Bianli; Zhang, Jin; Lin, Xiaojun; Bian, Chao; Sun, Bing

    2013-01-01

    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.

  5. 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.

  6. Radiolabeled monoclonal antibodies: a review

    International Nuclear Information System (INIS)

    Toledo e Souza, I.T. de; Okada, H.

    1990-05-01

    Since the description by Kohler and Milstein 1975 of their technique for producing monoclonal antibodies of predefined specificity, it has become a mainstay in most laboratories that utilize immunochemical techniques to study problems in basic, applied or clinical research. Paradoxically, the very success of monoclonal antibodies has generated a literature which is now so vast and scattered that it has become difficult to obtain a perspective. This brief review represents the distillation of many publications relating to the production and use of monoclonaal antibodies as radiopharmaceuticals. Significant advances were made possible in the last few years by combined developments in the fields of tumor-associated antigens and of monoclonal antibodies. In fact monoclonal antibodies against some well defined tumor-associated antigens, has led to significantly greater practical possibilities for producing highly specific radiolabeled antibodies as radiopharmaceuticals for diagnosis and therapy of human tumors. One of the main requirements of this methodology is the availability of stable radiopharmaceutical reagents which after labeling in vivo injection retain the capacity of specific interaction with the defined antigen and their molecular integrity. Since injection into human is the objetive of this kind of study all the specifications of radiopharmaceutical have to be fulfilled e.g. sterility, apirogenicity and absence of toxicity. (author) [pt

  7. Fiscal 2000 achievement report on the venture business assisting type regional consortium - Minor business creation base type. Commercialization of human monoclonal antibody as investigational reagent; 2000 nendo chiiki consortium kenkyu kaihatsu jigyo seika hokokusho. Hitogata monoclonal kotai no kenkyuyo shiyaku to shite no seihinka

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    Technical seeds of in vitro immunization technologies for human peripheral lymphocytes and technologies for human cell fusion, both developed by Kita-Kyushu National College of Technology, are used, and human monoclonal antibodies are prepared easy to connect to tumor markers, allergens, and microbes. The project aims further to develop a technology for small-scale high-density production of such antibodies and to supply them as human monoclonal antibody reagents meeting the needs of bio-researchers in the fields of medicine, pharmacy, biology, agriculture, or the like. A human fusion partner cell strain SK-729-1 clone is obtained, which is high in ability to produce antibodies. Since the obtained cell is culturable in a serumless medium, a fusion cell strain prepared using the said cell strain is also culturable in a serumless medium. Hence: advanced refining now available for human monoclonal antibodies. Nine types of antibodies are acquired. Furthermore, it is made possible to predict the antibody reaction and the antibody amount in a simplified way. Containers for antibody preservation are screened, and a container is selected in a freezing/thawing test, capable of preserving 90% or more of antibodies. A high-density culture that uses the hollow fiber cartridge is now feasible. (NEDO)

  8. 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.

  9. A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display.

    Science.gov (United States)

    Baskar, Sivasubramanian; Suschak, Jessica M; Samija, Ivan; Srinivasan, Ramaprasad; Childs, Richard W; Pavletic, Steven Z; Bishop, Michael R; Rader, Christoph

    2009-11-12

    Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only potentially curative treatment available for patients with B-cell chronic lymphocytic leukemia (B-CLL). Here, we show that post-alloHSCT antibody repertoires can be mined for the discovery of fully human monoclonal antibodies to B-CLL cell-surface antigens. Sera collected from B-CLL patients at defined times after alloHSCT showed selective binding to primary B-CLL cells. Pre-alloHSCT sera, donor sera, and control sera were negative. To identify post-alloHSCT serum antibodies and subsequently B-CLL cell-surface antigens they recognize, we generated a human antibody-binding fragment (Fab) library from post-alloHSCT peripheral blood mononuclear cells and selected it on primary B-CLL cells by phage display. A panel of Fab with B-CLL cell-surface reactivity was strongly enriched. Selection was dominated by highly homologous Fab predicted to bind the same antigen. One Fab was converted to immunoglobulin G1 and analyzed for reactivity with peripheral blood mononuclear cells from B-CLL patients and healthy volunteers. Cell-surface antigen expression was restricted to primary B cells and up-regulated in primary B-CLL cells. Mining post-alloHSCT antibody repertoires offers a novel route to discover fully human monoclonal antibodies and identify antigens of potential therapeutic relevance to B-CLL and possibly other cancers. Trials described herein were registered at www.clinicaltrials.gov as nos. NCT00055744 and NCT00003838.

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

    International Nuclear Information System (INIS)

    Pan, Yang; Sasaki, Tadahiro; Kubota-Koketsu, Ritsuko; Inoue, Yuji; Yasugi, Mayo; Yamashita, Akifumi; Ramadhany, Ririn; Arai, Yasuha; Du, Anariwa; Boonsathorn, Naphatsawan; Ibrahim, Madiha S.

    2014-01-01

    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

  11. 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.

  12. Penetration and binding of monoclonal antibody in human osteosarcoma multicell spheroids. Comparison of confocal laser scanning microscopy and autoadiography

    International Nuclear Information System (INIS)

    Hjelstuen, M.H.; Rasch-Halvorsen, K.; Brekken, C.; Bruland, Oe.; Davies, C. de L.

    1996-01-01

    Penetration and binding of monoclonal antibody (MAb) in multicell osteosarcoma spheroids have been studied by autoradiography and confocal laser scanning microscopy (CLSM). Optical sectioning of the 3-dimensional spheroids was performed by CLSM. Owing to attenuation of fluorescence intensity, FITC-labelled MAb could not be detected at depths greater than 60 μm within the spheroids. The antibody uptake seen in autoradiographs and CLSM images 60 μm within the spheroids were essentially identical. MAb had reached all parts of the spheroids within 6 h. Quantitative measurements of the fluorescence intensity of FITC-labelled MAb seen in confocal images and measurements of MAb bound per cell using flow cytometry, showed that maximum uptake was reached after 6 h. The possibility to perform both quantatitive and qualitative measurements makes CLSM a promising method for studying antibody uptake in thick tissue samples. (orig.)

  13. Radioimmunoimaging of human colon carcinoma grafted into nudemice using 131I-labeled monoclonal anticea antibody and its F(ab')2 fragments

    International Nuclear Information System (INIS)

    Liu Guangda

    1988-01-01

    131 I-labeled monoclonal anti-CEA antibody and its F(ab') 2 fragments were injected into nude mice bearing human colon carcinoma xenografts for tumor localization and radioimmunoimaging studies. Transplanted tumors were visualized in 12 hours after injection of the labeled anti-CEA or its F(ab') 2 by gamma camera. Biodistribution data indicated that F(ab') 2 fragments were cleared more rapidly from blood (T 1/2 = 13.3 h for F(ab') 2 , T 1/2 = 21.1 h for intact antibody) over 6-24 h and had higher tumor blood ratios. The intact antibody was concentrated in the tumor better than F(ab') 2 . In double-label experiments, a nonspecific localization of the control ( 125 I-labeled anti-HCG) in the tumor was also observed

  14. 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

    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....... There was no effect of treatment on plasma lipid profile, vascular FDG-PET signal or the amount of atherosclerosis in any of the examined arteries. However, immunostaining of coronary lesions revealed reduced cathepsin S positivity in the treated group compared with placebo (4.8% versus 8.2% of intima area, p = 0.......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....

  15. 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

    International Nuclear Information System (INIS)

    Boonsathorn, Naphatsawan; Panthong, Sumolrat; Koksunan, Sarawut; Chittaganpitch, Malinee; Phuygun, Siripaporn; Waicharoen, Sunthareeya; Prachasupap, Apichai; Sasaki, Tadahiro; Kubota-Koketsu, Ritsuko; Yasugi, Mayo; Ono, Ken-ichiro; Arai, Yasuha

    2014-01-01

    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

  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. Recombinant Human Respiratory Syncytial Virus (RSV) Monoclonal Antibody Fab is Effective Therapeutically when Introduced Directly into the Lungs of RSV-Infected Mice

    Science.gov (United States)

    Crowe, James E., Jr.; Murphy, Brian R.; Chanock, Robert M.; Williamson, R. Anthony; Barbas, Carlos F., III; Burton, Dennis R.

    1994-02-01

    Previously, recombinant human respiratory syncytial virus (RSV) monoclonal antibody Fabs were generated by antigen selection from random combinatorial libraries displayed at the tip of filamentous phage. Two such Fabs, which exhibited high binding affinity for RSV F glycoprotein (a major protective antigen), were evaluated for therapeutic efficacy in infected mice just before or at the time of peak virus replication in the lungs. Fab 19, which neutralized RSV infectivity with high efficiency in tissue culture, was effective therapeutically when delivered directly into the lungs by intranasal instillation under anesthesia. In contrast, RSV Fab 126, which failed to neutralize virus in cell culture, did not exhibit a therapeutic effect under these conditions. The amount of Fab 19 required to effect a 5000- to 12,000-fold reduction in titer of RSV in the lungs within 24 hr was rather small. In four separate experiments, a single instillation of 12.9-50 μg of RSV Fab 19 was sufficient to achieve such a reduction in pulmonary virus in a 25g mouse. The use of Fabs instead of the whole immunoglobulin molecules from which they are derived reduced the protein content of a therapeutic dose. This is important because the protein load that can be delivered effectively into the lungs is limited. The therapeutic effect of a single treatment with Fab 19 was not sustained, so that a rebound in pulmonary virus titer occurred on the 2nd day after treatment. This rebound in pulmonary RSV titer could be prevented by treating infected mice with a single dose of Fab 19 daily for 3 days. These observations suggest that human monoclonal Fabs grown in Escherichia coli may prove useful in the treatment of serious RSV disease as well as diseases caused by other viruses where replication in vivo is limited primarily to the lumenal lining of the respiratory tract.

  18. Early fetal acquisition of the chromaffin and neuronal immunophenotype by human adrenal medullary cells. An immunohistological study using monoclonal antibodies to chromogranin A, synaptophysin, tyrosine hydroxylase, and neuronal cytoskeletal proteins.

    NARCIS (Netherlands)

    Molenaar, W M; Lee, V M; Trojanowski, J Q

    1990-01-01

    The development of chromaffin and neuronal features in the adrenal medulla was studied in normal human fetuses with gestational ages (GAs) of 6-34 weeks. Monoclonal antibodies specific for chromogranin A, synaptophysin, and tyrosine hydroxylase; for different subunits and phosphoisoforms of

  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. Antiglycopeptide Mouse Monoclonal Antibody LpMab-21 Exerts Antitumor Activity Against Human Podoplanin Through Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity.

    Science.gov (United States)

    Kato, Yukinari; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Uchida, Hiroaki; Tahara, Hideaki; Yamada, Shinji; Yanaka, Miyuki; Nakamura, Takuro; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Honma, Ryusuke; Takagi, Michiaki; Ogasawara, Satoshi; Murata, Takeshi; Kaneko, Mika K

    2017-02-01

    The interaction between podoplanin (PDPN) and C-type lectin-like receptor 2 (CLEC-2) is involved in tumor malignancy. We have established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-21, one of the mouse antipodoplanin mAbs, is of the IgG 2a subclass, and its minimum epitope was determined to be Thr76-Arg79 of the human podoplanin. Importantly, sialic acid is linked to Thr76; therefore, LpMab-21 is an antiglycopeptide mAb (GpMab). In this study, we investigated whether LpMab-21 shows antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cell lines in vitro and also studied its antitumor activities using a xenograft model. LpMab-21 showed high ADCC and CDC activities against not only podoplanin-expressing Chinese hamster ovary cells but also LN319 glioblastoma cells and PC-10 lung cancer cells, both of which endogenously express podoplanin. Furthermore, LpMab-21 decreased tumor growth in vivo, indicating that LpMab-21 could be useful for antibody therapy against human podoplanin-expressing cancers.

  1. Preparation and radiolabeling of humanized anti-HER1 monoclonal antibody nimotuzumab Fab' fragment with 68Ga and 90Y

    International Nuclear Information System (INIS)

    Alonso Martinez, L. M.; Xiques Castillo, A.; Leyva Montanna, R.; Perez-Malo Cruz, M.; Zamora Barrabi, M.; Manresa Sanchez, Y.

    2013-01-01

    Antibody-based targeted delivery of radioisotopes to malignant tissues is a promising approach in cancer diagnostics and therapy. However, intact antibody molecules are large glycoproteins (∼150 kDa) that have limited application in molecular imaging and therapy due to their relatively slow clearance from the circulation leading to a high background signal rather both cases the sensitivity can be increased with the use of enzymatically produced Fab' fragments. In this work, the ability to get labeled with 62 Ga and 90 Y of a monoclonal antibody (mAb) Fab' fragment against the transmembrane receptor tyrosine kinase HER-1 was studied for future applications in PET imaging and radioimmunotherapy of tumors. In order to obtain the Fab' fragment the mAb was cleaved with pepsin in molar excess. After separating the reaction mixture in two steps using affinity and ion-exchange chromatography, the Fab' fragment was finally obtained by reduction of the F(ab') 2 with a molar excess of 2-mercaptoethanol followed by a size exclusion purification step. The Fab' fragment was derivatized with 1,4,7,10-tetraaza cyclododecane-1,4,7,10-tetraacetic acid mono N-hydroxysuccinimide commercial ester (DOTA-NHS-ester) applying a simple procedure and the number of DOTA groups linked to Fab' were determinate. The labeling of the conjugate with 68 Ga and 90 Y from 'in-house generators yielded radiochemically pure probes that can become a suitable radioimmunoconjugated in a near future. (Author)

  2. Increased specificity in human cardiac-myosin radioimmunoassay utilizing two monoclonal antibodies in a double sandwich assay

    International Nuclear Information System (INIS)

    Katus, H.A.; Hurrell, J.G.; Matsueda, G.R.; Ehrlich, P.; Zurawski, V.R. Jr.; Khaw, B.-A.; Haber, E.

    1982-01-01

    An immunoradiometric assay that simultaneously measured two different epitopes on the same molecule was devised to differential between cardiac- and skeletal-myosin light chains. Three monoclonal antibodies were examined that were 100% (lC5), 25% (2B9) and 17% (4F10) cross reactive, respectively, between the two antigens. One antibody of the pair to be studied was immobilized to cyanogen bromide-activated Sepharose 4B while the other was iodinated with 125 I using the lactoperoxidase method. The antigen was mixed with the immobilized antibody, the labeled antibody was added and the precipitate then washed and counted in a gamma counter. When both antibodies of the pair to be studied (immobilized and labeled) were the same (2B9), no radioactivity above background was bound to the precipitate, indicating that the second antibody could not bind to an already occupied epitope. When two different antibodies were employed, the specificity of the assay increased over that of a single antibody. The cross reactivity of a pair approximated the product of the cross reactivities of the individual antibodies. Thus, lC5 and 2B9 were 25% cross reactive together, lC5 and 4F10 17% cross reactive, and 2B9 and 4F10 4.3% cross reactive. (author)

  3. 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...... demonstrated co-expression of the antigen defined by F101.01 and the pan-T cell antigens defined by CD2, CD3, CD5, and CD7 antibodies. Cells stained with CD4 and CD8 antibodies were both included in the F101.01-positive population, whereas CD16-positive natural killer cells (NK), B cells (CD19 and CD20......), and myeloid cells (CD13 and CD33) were excluded. The target antigen of F101.01 co-modulated with the CD3-defined antigen (T3) and the TCR recognized by the MoAb WT-31. CD3 antibody and WT-31 both blocked binding of F101.01. F101.01 precipitated the TCR-T3 complex from lysates of 125I-labelled peripheral blood...

  4. 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.

  5. 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.

  6. 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 

  7. 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.

  8. Prognosis in monoclonal proteinaemia

    NARCIS (Netherlands)

    Schaar, Cornelis Gerardus

    2006-01-01

    Monoclonal proteinaemia (M-proteinemia) is associated with multiple myeloma (MM) or other hematological malignancies. In the absence of these diseases the term MGUS (Monoclonal Gammopathy of Undetermined Significance) is used. During 1991-1993 1464 patients with newly diagnosed M-proteinemia in the

  9. 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…

  10. Biodistribution of 99mTc-labeled anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody h-R3 in a xenograft model of human lung adenocarcinoma

    International Nuclear Information System (INIS)

    Morales-Morales, Alejo; Duconge, Jorge; Caballero-Torres, Idania; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Iznaga-Escobar, Normando

    1999-01-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody (MAb) h-R3 is an (IgG 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 μg/100 μCi of 99m Tc-labeled MAbs. Immunoreactivity of 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 99m Tc-labeled humanized antibody h-R3, a phase I diagnostic clinical trial of tumor with epithelial origin should be pursued

  11. 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.

  12. Pharmacokinetics and immunogenicity investigation of a human anti-interleukin-17 monoclonal antibody in non-naïve cynomolgus monkeys.

    Science.gov (United States)

    Han, Chao; Gunn, George R; Marini, Joseph C; Shankar, Gopi; Han Hsu, Helen; Davis, Hugh M

    2015-05-01

    The pharmacokinetics (PK) of biologic therapeutics, especially monoclonal antibodies (mAbs), in monkeys generally presents the most relevant predictive PK information for humans. However, human mAbs, xenogeneic proteins to monkeys, are likely to be immunogenic. Monkeys previously treated with a human mAb (non-naïve) may have developed antidrug antibodies (ADAs) that cross-react with another test mAb in subsequent studies. Unlike PK studies for small-molecule therapeutics, in which animals may be reused, naïve monkeys have been used almost exclusively for preclinical PK studies of biologic therapeutics to avoid potential pre-existing immunologic cross-reactivity issues. The propensity and extent of pre-existing ADAs have not been systematically investigated to date. In this study, the PK and immunogenicity of mAb A, a human anti-human interkeukin-17 mAb, were investigated in a colony of 31 cynomolgus monkeys previously exposed to other human mAbs against different targets. We screened the monkeys for pre-existing antibodies to mAb A prior to the PK study and showed that 44% of the monkeys had pre-existing cross-reactive antibodies to mAb A, which could affect the PK characterization of the antibody. In the subcolony of monkeys without measurable pre-existing ADAs, PK and immunogenicity of mAb A were successfully characterized. The impact of ADAs on mAb A PK was also demonstrated in the monkeys with pre-existing ADAs. Here we report the results and propose a pragmatic approach for the use of non-naïve monkeys when conducting PK studies of biologic therapeutics. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  13. Kinetic data of in-vivo labeled granulocytes in humans with a murine Tc-99m-labelled monoclonal antibody

    International Nuclear Information System (INIS)

    Becker, W.; Boerner, W.; Borst, U.; Schaefer, R.; Fischbach, W.; Pasurka, B.

    1989-01-01

    Twenty-five patients were examined in vivo with 99m Tc labelled monoclonal antibodies; 15 with suspected infections with an antigranulocyte antibody (BW 250/183), 10 with suspected recurrence of a colorectal carcinoma with an anti CEA antibody (BW 431/26). Both antibodies were IgG1 isotypes. In the patients with suspected infections no change of the peripheral leukocyte count could be observed after the antibody injection (1 mg, n=9; 0.05 mg, n=1; 0.25 mg, n=6). In 2 patients examined with the anti CEA antibody (2 mg), a significant decrease of the peripheral leukocyte count could be observed. The recovery rate of the 99m Tc antibody labelled granulocytes was calculated to be about 10%. The increase of the antibody-antigen binding was calculated to be 0.2%/min. In vivo the organ distribution curves demonstrated an increase of 99m Tc activity over spleen and bone marrow of 1.1%/min, which was interpreted as antigen-antibody reactivity. The organ distribution curves of the anti granulocyte antibody over spleen and bone marrow showed typical binding characteristics to the local granulocyte epitopes. The curves over other organs showed a simple perfusion pattern. The curves of the anti CEA antibody showed a perfusion pattern over all the examined organs. A sham dialysis model in one patient with renal insufficiency undergoing regular dialysis treatment demonstrated the viability of 99m Tc antibody labelled granulocytes in vivo. The kinetic patterns of the 99m Tc antibody in patients with Crohn's disease were interpreted as CEA binding of the antibody in the bowel wall. (orig.)

  14. Broadly-reactive human monoclonal antibodies elicited following pandemic H1N1 influenza virus exposure protect mice from highly pathogenic H5N1 challenge.

    Science.gov (United States)

    Nachbagauer, Raffael; Shore, David; Yang, Hua; Johnson, Scott K; Gabbard, Jon D; Tompkins, S Mark; Wrammert, Jens; Wilson, Patrick C; Stevens, James; Ahmed, Rafi; Krammer, Florian; Ellebedy, Ali H

    2018-06-13

    Broadly cross-reactive antibodies that recognize conserved epitopes within the influenza virus hemagglutinin (HA) stalk domain are of particular interest for their potential use as therapeutic and prophylactic agents against multiple influenza virus subtypes including zoonotic virus strains. Here, we characterized four human HA stalk-reactive monoclonal antibodies (mAbs) for their binding breadth and affinity, in vitro neutralization capacity, and in vivo protective potential against an highly pathogenic avian influenza virus. The monoclonal antibodies were isolated from individuals shortly following infection with (70-1F02 and 1009-3B05) or vaccination against (05-2G02 and 09-3A01) A(H1N1)pdm09. Three of the mAbs bound HAs from multiple strains of group 1 viruses, and one mAb, 05-2G02, bound to both group 1 and group 2 influenza A HAs. All four antibodies prophylactically protected mice against a lethal challenge with the highly pathogenic A/Vietnam/1203/04 (H5N1) strain. Two mAbs, 70-1F02 and 09-3A01, were further tested for their therapeutic efficacy against the same strain and showed good efficacy in this setting as well. One mAb, 70-1F02, was co-crystallized with H5 HA and showed similar heavy chain only interactions as a the previously described anti-stalk antibody CR6261. Finally, we showed that antibodies that compete with these mAbs are prevalent in serum from an individual recently infected with A(H1N1)pdm09 virus. The antibodies described here can be developed into broad-spectrum antiviral therapeutics that could be used to combat infections with zoonotic or emerging pandemic influenza viruses. IMPORTANCE The rise in zoonotic infections of humans with emerging influenza viruses is a worldwide public health concern. The majority of recent zoonotic human influenza cases were caused by H7N9 and H5Nx viruses and were associated with high morbidity and mortality. In addition, seasonal influenza viruses are estimated to cause up to 650,000 deaths annually

  15. Evaluation of dry blood spot technique for quantification of an Anti-CD20 monoclonal antibody drug in human blood samples.

    Science.gov (United States)

    Lin, Yong-Qing; Zhang, Yilu; Li, Connie; Li, Louis; Zhang, Kelley; Li, Shawn

    2012-01-01

    To evaluate the dried blood spot (DBS) technique in ELISA quantification of larger biomolecular drugs, an anti-CD20 monoclonal antibody drug was used as an example. A method for the quantification of the anti-CD20 drug in human DBS was developed and validated. The drug standard and quality control samples prepared in fresh human blood were spotted on DBS cards and then extracted. A luminescent ELISA was used for quantification of the drug from DBS samples. The assay range of the anti-CD20 drug standards in DBS was 100-2500ng/mL. The intra-assay precision (%CV) ranged from 0.4% to 10.1%, and the accuracy (%Recovery) ranged from 77.9% to 113.9%. The inter assay precision (%CV) ranged from 5.9% to 17.4%, and the accuracy ranged from 81.5% to 110.5%. The DBS samples diluted 500 and 50-fold yielded recovery of 88.7% and 90.7%, respectively. The preparation of DBS in higher and lower hematocrit (53% and 35%) conditions did not affect the recovery of the drug. Furthermore, the storage stability of the anti-CD20 drug on DBS cards was tested at various conditions. It was found that the anti-CD20 drug was stable for one week in DBS stored at room temperature. However, it was determined that the stability was compro]mised in DBS stored at high humidity, high temperature (55°C), and exposed to direct daylight for a week, as well as for samples stored at room temperature and high humidity conditions for a month. Stability did not change significantly in samples that underwent 3 freeze/thaw cycles. Our results demonstrated a successful use of DBS technique in ELISA quantification of an anti-CD20 monoclonal antibody drug in human blood. The stability data provides information regarding sample storage and shipping for future clinical studies. It is, therefore, concluded that the DBS technique is applicable in the quantification of other large biomolecule drugs or biomarkers. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. 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

  17. 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.

  18. Fully human monoclonal antibodies from antibody secreting cells after vaccination with Pneumovax®23 are serotype specific and facilitate opsonophagocytosis.

    Science.gov (United States)

    Smith, Kenneth; Muther, Jennifer J; Duke, Angie L; McKee, Emily; Zheng, Nai-Ying; Wilson, Patrick C; James, Judith A

    2013-05-01

    B lymphocyte memory generates antibody-secreting cells (ASCs) that represent a source of protective antibodies that may be exploited for therapeutics. Here we vaccinated four donors with Pneumovax®23 and produced human monoclonal antibodies (hmAbs) from ASCs. We have cloned 137 hmAbs and the specificities of these antibodies encompass 19 of the 23 serotypes in the vaccine, as well as cell wall polysaccharide (CWPS). Although the majority of the antibodies are serotype specific, 12% cross-react with two serotypes. The Pneumovax®23 ASC antibody sequences are highly mutated and clonal, indicating an anamnestic response, even though this was a primary vaccination. Hmabs from 64% of the clonal families facilitate opsonophagocytosis. Although 9% of the total antibodies bind to CWPS impurity in the vaccine, none of these clonal families showed opsonophagocytic activity. Overall, these studies have allowed us to address unanswered questions in the field of human immune responses to polysaccharide vaccines, including the cross-reactivity of individual antibodies between serotypes and the percentage of antibodies that are protective after vaccination with Pneumovax®23. Copyright © 2012 Elsevier GmbH. All rights reserved.

  19. Detection of cyclobutane thymine dimers in DNA of human cells with monoclonal antibodies raised against a thymine dimer-containing tetranucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Roza, L; Wulp, K.J.M. van der; MacFarlane, S J; Lohman, P H.M.; Baan, R A

    1988-11-01

    A hybrid cell line (hybridoma) has been isolated after fusion between mouse-plasmacytoma cells and spleen cells from mice immunized with a thymine dimer-containing tetranucleotide coupled to a carrier protein. Monoclonal antibodies produced by this hybridoma were characterized by testing the effect of various inhibitors in a competitive enzyme-linked immunosorbent assay (ELISA). The antibodies have a high specificity for thymine dimers in single-stranded DNA or poly(dT), but do not bind UV-irradiated d(TpC)/sub 5/. Less binding is observed with short thymine dimer-containing sequences. In vitro treatment of UV-irradiated DNA with photoreactivating enzyme in the presence of light, or with Micrococcus luteus UV-endonuclease results in disappearance of antigenicity. Antibody-binding to DNA isolated from UV-irradiated human fibroblasts (at 254 nm) is linear with dose. Removal of thymine dimers in these cells during a post-irradiation incubation, as detected with the antibodies, is fast initially but the rate rapidly decreases (about 50% residual dimers at 20 h after 10 J/m/sup 2/). The induction of thymine dimers in human skin irradiated with low doses of UV-B, too, was demonstrated immunochemically, by ELISA as well as by quantitative immunofluorescence microscopy.

  20. Application of four anti-human interferon-alpha monoclonal antibodies for immunoassay and comparative analysis of natural interferon-alpha mixtures

    International Nuclear Information System (INIS)

    Andersson, G.; Lundgren, E.; Ekre, H.P.

    1991-01-01

    Four different mouse monoclonal antibodies to human interferon-alpha (IFN-alpha) were evaluated for application in quantitative and comparative analysis of natural IFN-alpha mixtures. Binding to IFN-alpha subtypes in solution revealed individual reactivity patterns. These patterns changed if the IFN-alpha molecules were immobilized either passively to a surface or bound by another antibody. Also, substitution of a single amino acid in IFN-alpha 2 affected the binding, apparently by altering the conformation. Isoelectric focusing of three natural IFN-alpha preparations from different sources, followed by immunoblotting, resulted in individual patterns with each of the four mAbs and also demonstrated variation in the composition of the IFN-alpha preparations. None of the mAbs was subtype specific, but by combining the different mAbs, and also applying polyclonal anti-human IFN-alpha antibodies, it was possible to design sensitive sandwich ELISAs with broad or more limited IFN-alpha subtype specificity

  1. 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; Qiu, Qi; Pow Sang, Luis; Shashikumar, Soumya; Casares, Sofia; Brumeanu, Teodor-D

    2018-02-01

    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-2Rgc 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 ( 180 WGIHHPPNSKEQ QNLY 195 ) of hemagglutinin (HA) envelope protein of PR8/A/34 (H1N1) virus with high homology among seven influenza type A viruses. A single administration of HA 180-195 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.

  2. Monoclonal antibodies: an overview of their advantages and limitations in nuclear medicine

    International Nuclear Information System (INIS)

    Revillard, J.P.; Cohen, J.

    1982-01-01

    The following topics were reviewed: antigen recognition by the immune system; development of immunoassays for antigenic components of biological fluids; monoclonal antibodies against infectious agents; monochonal antibodies against tumor and differentiation antigens; human monoclonal antibodies

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

    Directory of Open Access Journals (Sweden)

    Genna E Davies

    Full Text Available 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.

  4. Open-label, dose escalation phase I study in healthy volunteers to evaluate the safety and pharmacokinetics of a human monoclonal antibody to Clostridium difficile toxin A.

    Science.gov (United States)

    Taylor, Claribel P; Tummala, Sanjeev; Molrine, Deborah; Davidson, Lisa; Farrell, Richard J; Lembo, Anthony; Hibberd, Patricia L; Lowy, Israel; Kelly, Ciaran P

    2008-06-25

    Recent data suggest that Clostridium difficile-associated diarrhea is becoming more severe and difficult to treat. Antibody responses to C. difficile toxin A are protective against symptomatic disease and recurrence. We examined the safety and pharmacokinetics (pk) of a novel neutralizing human monoclonal antibody against C. difficile toxin A (CDA1) in healthy adults. Five cohorts with 6 subjects each received a single intravenous infusion of CDA1 at escalating doses of 0.3, 1, 5, 10, and 20 mg/kg. Safety evaluations took place on days 1, 2, 3, 7, 14, 28, and 56 post-infusion. Samples for pk analysis were obtained before and after infusion, and at each safety evaluation. Serum CDA1 antibody concentrations and human anti-human antibody (HAHA) titers were measured with enzyme-linked immunosorbent assays. A noncompartmental model was used for pk analysis. Thirty subjects were enrolled. The median age was 27.5 yrs. There were no serious adverse events (AE) related to CDA1. Twenty-one of the 48 reported non-serious adverse events were possibly related to CDA1, and included transient blood pressure changes requiring no treatment, nasal congestion, headache, abdominal cramps, nausea, and self-limited diarrhea. Serum CDA1 concentrations increased with escalating doses: mean C(max) ranged from 6.82 microg/ml for the 0.3 mg/kg cohort to 511 microg/ml for the 20 mg/kg cohort. The geometric mean values of the half-life of CDA1 ranged between 25.3 and 31.8 days, and the volume of distribution approximated serum. No subject formed detectable HAHA titers. Administration of CDA1 as a single intravenous infusion was safe and well tolerated. C(max) increased proportionally with increasing doses. A randomized study of CDA1 in patients with C. difficile associated diarrhea is underway.

  5. Safety, pharmacokinetics, immunogenicity, and biodistribution of (186)Re-labeled humanized monoclonal antibody BIWA 4 (Bivatuzumab) in patients with early-stage breast cancer.

    Science.gov (United States)

    Koppe, Manuel; Schaijk, Frank van; Roos, Jan; Leeuwen, Paul van; Heider, Karl-Heinz; Kuthan, Hartmut; Bleichrodt, Robert

    2004-12-01

    The aim of this prospective study was to evaluate the safety, pharmacokinetics, immunogenicity, and biodistribution of (186)Re-labeled humanized anti-CD44v6 monoclonal antibody (MAb( BIWA 4 (Bivatuzumab( in 9 patients with early-stage breast cancer. Radioimmunoscintigraphy (RIS( was performed within 1, 24, and 72 hours after administration. BIWA 4 concentration in plasma (ELISA and radioactivity measurements( and the development of human antihuman antibody (HAHA( responses was determined. The biodistribution of (186)Re-BIWA 4 was determined by radioactivity measurements in tumor and normal tissue biopsies obtained during surgery 1 week after administration. Administration of (186)Re-BIWA 4 was well tolerated by all patients and no HAHA responses were observed. The mean t(1/2) in plasma of BIWA 4 (ELISA( was 81 hours (range, 67-97(, whereas the mean radioactivity t(1/2) tended to be longer, at 105 hours (range, 90-114(. RIS unmistakably showed the tumor in 3 patients. Less clear identifications were established in 3 additional patients. In 2 patients, the tumor was wrongly identified in the contralateral breast. Median tumor CD44v6 expression, as determined by immunohistochemistry, was 70% (range, 10-90%). Mean tumor uptake was 2.96% ID/kg (range, 0.92-6.27(, with no apparent correlation with either tumor CD44v6 expression, tumor-cell cellularity, or tumor diameter. Tumor-to-nontumor ratios were unfavorable for blood, bone marrow, mammary gland tissue, and skin. The (186)Re-labeled humanized MAb BIWA 4 can safely be administered to patients with early-stage breast cancer. Tumorto- nontumor ratios were unfavorable, with no apparent correlation with CD44v6 expression, tumor-cell cellularity, or tumor diameter. BIWA 4, therefore, appears to have limitations as a vehicle for radioimmunotherapy in patients with breast cancer.

  6. 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.

  7. Inactivation of human osteosarcoma cells in vitro by 211At-TP-3 monoclonal antibody: Comparison with astatine-211 and external-beam X rays

    International Nuclear Information System (INIS)

    Larsen, R.H.; Bruland, O.S.; Hoff, P.; Alstad, J.; Lindmo, T.; Rofstad, E.K.

    1994-01-01

    The potential usefulness of α-particle radioimmunotherapy in the treatment of osteosarcoma was studied in vitro by using the monoclonal antibody TP-3 and cells of three human osteosarcoma cell lines (OHS, SAOS and KPDX) differing in antigen expression. Cell survival curves were established after treatment with (a) 211 At-TP-3 of different specific activities, (b) 211 At-labeled bovine serum albumin (BSA), (c) free 211 At and (d) external-beam X rays. The three osteosarcoma cell lines showed similar survival curves, whether treated with external-beam X rays, 211 At-BSA or free 211 At. The D o 's were lower for free 211 At than for 211 At-BSA. The survival curves for 211 At-TP-3 treatment, on the other hand, differed significantly among the cell lines, suggesting that sensitivity to 211 At-TP-3 treatment was governed by cellular properties other than sensitivity to external-beam X rays. The cellular property most important for sensitivity to 211 At-TP-3 treatment was the antigen expression. Cell inactivation after 211 At-TP-3 treatment increased substantially with increasing specific activity of the 211 At-TP-3. At high specific activities, the cytotoxic effect of 211 At-TP-3 was significantly higher than that of 211 At-BSA. In conclusion, 211 At-TP-3 has the potential to give clinically favorable therapeutic ratios in the treatment of osteosarcoma. 39 refs., 5 figs., 2 tabs

  8. Monoclonal antibody-based dipstick assay: a reliable field applicable technique for diagnosis of Schistosoma mansoni infection using human serum and urine samples.

    Science.gov (United States)

    Demerdash, Zeinab; Mohamed, Salwa; Hendawy, Mohamed; Rabia, Ibrahim; Attia, Mohy; Shaker, Zeinab; Diab, Tarek M

    2013-02-01

    A field applicable diagnostic technique, the dipstick assay, was evaluated for its sensitivity and specificity in diagnosing human Schistosoma mansoni infection. A monoclonal antibody (mAb) against S. mansoni adult worm tegumental antigen (AWTA) was employed in dipstick and sandwich ELISA for detection of circulating schistosome antigen (CSA) in both serum and urine samples. Based on clinical and parasitological examinations, 60 S. mansoni-infected patients, 30 patients infected with parasites other than schistosomiasis, and 30 uninfected healthy individuals were selected. The sensitivity and specificity of dipstick assay in urine samples were 86.7% and 90.0%, respectively, compared to 90.0% sensitivity and 91.7% specificity of sandwich ELISA. In serum samples, the sensitivity and specificity were 88.3% and 91.7% for dipstick assay vs. 91.7% and 95.0% for sandwich ELISA, respectively. The diagnostic efficacy of dipstick assay in urine and serum samples was 88.3% and 90.0%, while it was 90.8% and 93.3% for sandwich ELISA, respectively. The diagnostic indices of dipstick assay and ELISA either in serum or in urine were statistically comparable (P>0.05). In conclusion, the dipstick assay offers an alternative simple, rapid, non-invasive technique in detecting CSA or complement to stool examinations especially in field studies.

  9. Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

    Science.gov (United States)

    Sabin, Charles; Corti, Davide; Buzon, Victor; Seaman, Mike S; Lutje Hulsik, David; Hinz, Andreas; Vanzetta, Fabrizia; Agatic, Gloria; Silacci, Chiara; Mainetti, Lara; Scarlatti, Gabriella; Sallusto, Federica; Weiss, Robin; Lanzavecchia, Antonio; Weissenhorn, Winfried

    2010-11-18

    The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

  10. Radioimmunoscintigraphy of recurrent, metastatic, or occult colorectal cancer with technetium 99m-labeled totally human monoclonal antibody 88BV59: results of pivotal, phase III multicenter studies.

    Science.gov (United States)

    Serafini, A N; Klein, J L; Wolff, B G; Baum, R; Chetanneau, A; Pecking, A; Fischman, A J; Hoover, H C; Wynant, G E; Subramanian, R; Goroff, D K; Hanna, M G

    1998-05-01

    To assess the performance and potential clinical impact of a totally human monoclonal antibody, 88BV59 (HumaSPECT) (INTRACEL, Corp, Rockville, MD), in 202 assessable presurgical patients with recurrent, metastatic, or occult colorectal cancer. 88BV59, labeled with technetium Tc 99m (99mTc) (HumaSPECT-Tc), was injected intravenously, and planar and single photon emission tomography (SPECT) images were obtained 14 to 20 hours postinjection. Surgical and pathologic verification of tumor were used as the standard against which the performance of HumaSPECT-Tc imaging and computed tomography (CT) analysis were evaluated. All patients entered onto the recurrent disease study had at least one tumor site defined on CT. The sensitivity of HumaSPECT-Tc in those CT-positive patients was 87%. The specificity of HumaSPECT-Tc was 57% compared with 17% for CT and the difference was statistically significant (P HAHA) response (90 ng/mL) at 9 weeks postinfusion was observed. HumaSPECT-Tc can provide important and accurate information about the presence and location of disease in patients with a high clinical suspicion of metastatic or recurrent colorectal cancer and either positive (known disease) or negative (occult disease) CT scans.

  11. Safety, pharmacokinetic, and functional effects of the nogo-a monoclonal antibody in amyotrophic lateral sclerosis: a randomized, first-in-human clinical trial.

    Directory of Open Access Journals (Sweden)

    Vincent Meininger

    Full Text Available The neurite outgrowth inhibitor, Nogo-A, has been shown to be overexpressed in skeletal muscle in amyotrophic lateral sclerosis (ALS; it is both a potential biomarker and therapeutic target. We performed a double-blind, two-part, dose-escalation study, in subjects with ALS, assessing safety, pharmacokinetics (PK and functional effects of ozanezumab, a humanized monoclonal antibody against Nogo-A. In Part 1, 40 subjects were randomized (3∶1 to receive single dose intravenous ozanezumab (0.01, 0.1, 1, 5, or 15 mg/kg or placebo. In Part 2, 36 subjects were randomized (3∶1 to receive two repeat doses of intravenous ozanezumab (0.5, 2.5, or 15 mg/kg or placebo, approximately 4 weeks apart. The primary endpoints were safety and tolerability (adverse events [AEs], vital signs, electrocardiogram (ECG, and clinical laboratory tests. Secondary endpoints included PK, immunogenicity, functional endpoints (clinical and electrophysiological, and biomarker parameters. Overall, ozanezumab treatment (0.01-15 mg/kg was well tolerated. The overall incidence of AEs in the repeat dose 2.5 mg/kg and 15 mg/kg ozanezumab groups was higher than in the repeat dose placebo group and repeat dose 0.5 mg/kg ozanezumab group. The majority were considered not related to study drug by the investigators. Six serious AEs were reported in three subjects receiving ozanezumab; none were considered related to study drug. No study drug-related patterns were identified for ECG, laboratory, or vital signs parameters. One subject (repeat dose 15 mg/kg ozanezumab showed a weak, positive anti-ozanezumab-antibody result. PK results were generally consistent with monoclonal antibody treatments. No apparent treatment effects were observed for functional endpoints or muscle biomarkers. Immunohistochemical staining showed dose-dependent co-localization of ozanezumab with Nogo-A in skeletal muscle. In conclusion, single and repeat dose ozanezumab treatment was well tolerated and demonstrated

  12. 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....

  13. Preclinical pharmacokinetics, interspecies scaling, and pharmacokinetics of a Phase I clinical trial of TTAC-0001, a fully human monoclonal antibody against vascular endothelial growth factor 2

    Directory of Open Access Journals (Sweden)

    Lee WS

    2018-03-01

    Full Text Available Weon Sup Lee,1 Sang Ryeol Shim,1 Seon Young Lee,1 Jin San Yoo,1 Sung Kweon Cho2 1PharmAbcine, Inc., Daejeon, Republic of Korea; 2Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, Republic of Korea Background: VEGF is a highly selective mitogen that serves as the central regulator of tumor angiogenesis by mediating endothelial proliferation, permeability, and survival. Tanibirumab (TTAC-0001 is a fully human IgG1 monoclonal antibody derived from a fully human naïve single-chain variable fragment (ScFv phage library that was developed to inhibit the effects of VEGF in the treatment of solid tumors, especially those of the brain. Methods: In the present study, we conducted intravenous pharmacokinetic studies of TTAC-0001 in mice, rats, and cynomolgus monkeys. At the doses studied (3 mg/kg, 10 mg/kg, 30 mg/kg, TTAC-0001 exhibited dose proportionality in mice and monkeys. At a dose of ~10 mg/kg, the clearance of TTAC-0001 from serum was 0.017 mL/h in mice, 0.35 mL/h in rats, and 2.19 mL/h in cynomolgus monkeys, and the terminal half-life ranged from 20–30 h among the three species. Pharmacokinetic data in mice, rats, and cynomolgus monkeys were used to predict the pharmacokinetics of TTAC-0001 in humans using allometric scaling. The predicted serum clearance of TTAC-0001 in humans was 102.45 mL/h and the terminal half-life was 27.52 h. Results: The maximum life span-corrected clearance value was 72.92 mL/h. The observed clearance in humans was more similar to the predicted scaled clearance. Conclusion: We investigated the pharmacokinetics of TTAC-0001 in mice, rats, and cynomolgus monkeys after intravenous administration. At the doses studied, TTAC-0001 exhibited dose proportionality in mice and monkeys. The scaled pharmacokinetics of TTAC-0001 reported here was useful for designing first-in-human studies. Allometric scaling in the therapeutic antibody is feasible. Keywords: VEGF2, tanibirumab, pharmacokinetics

  14. Immunoglobulin variable region sequences of two human monoclonal antibodies directed to an onco-developmental carbohydrate antigen, lactotetraosylceramide (LcOse4Cer).

    Science.gov (United States)

    Yago, K; Zenita, K; Ohwaki, I; Harada, R; Nozawa, S; Tsukazaki, K; Iwamori, M; Endo, N; Yasuda, N; Okuma, M

    1993-11-01

    A human monoclonal antibody, 11-50, was generated and was shown to recognize an onco-developmental carbohydrate antigen, LcOse4Cer. The isotype of this antibody was IgM, lambda, similar to the previously known human anti-LcOse4 antibodies, such as IgMWOO and HMST-1. We raised a murine anti-idiotypic antibody G3 (IgG1, kappa) against 11-50, and tested its reactivity towards the affinity purified human polyclonal anti-LcOse4 antibodies prepared from pooled human sera using a Gal beta 1-->3GlcNAc beta-immobilized column. The results indicated that at least a part of the human polyclonal anti-LcOse4 antibodies shared the G3 idiotype with 11-50. We further analyzed the sequence of variable regions of the two anti-LcOse4 antibodies, 11-50 and HMST-1. Sequence analysis of the heavy chain variable regions indicated that the VH regions of these two antibodies were highly homologous to each other (93.5% at the nucleic acid level), and these antibodies utilized the germline genes VH1.9III and hv3005f3 as the VH segments, which are closely related germline genes of the VHIII family. It was noted that these germline VH genes are frequently utilized in fetal B cells. The JH region of both antibodies was encoded by the JH4 gene. For the light chain, the V lambda segments of the two antibodies were 96.3% homologous to each other at the nucleic acid level. The V lambda segments of both antibodies showed the highest homology to the rearranged V lambda gene called V lambda II.DS among reported V lambda genes, while the exact germline V lambda genes encoding the two antibodies were not yet registered in available sequence databanks. The amino acid sequences of the J lambda segments of both antibodies were identical. These results indicate that the two human antibodies recognizing the onco-developmental carbohydrate antigen Lc4 are encoded by the same or very homologous germline genes.

  15. 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

  16. The biological activity of human CD20 monoclonal antibodies is linked to unique epitopes on CD20

    NARCIS (Netherlands)

    Teeling, J.L.; Mackus, W.J.M.; Wiegman, L.; Brakel, van den J.H.N.; Beers, S.A.; French, R.R.; Meerten, van T.; Ebeling, S.; Vink, T.; Slootstra, J.W.; Parren, P.; Glennie, M.J.; Winkel, van de J.G.J.

    2006-01-01

    We have previously defined a panel of fully human CD20 mAb. Most of these were unexpectedly efficient in their ability to recruit C1q to the surface of CD20-positive cells and mediate tumor lysis via activation of the classical pathway of complement. This complement-dependent cytotoxicity (CDC)

  17. Analysis of the therapeutic gain in the treatment of human osteosarcoma microcolonies in vitro with 211At-labelled monoclonal antibody.

    Science.gov (United States)

    Larsen, R H; Bruland, O S; Hoff, P; Alstad, J; Rofstad, E K

    1994-06-01

    Microcolonies were obtained by culturing cells of two human osteosarcoma lines (OHS and KPDX) and one human melanoma line (WIX-c) for either 24 or 72 h. The microcolonies were treated with either alpha-particle radiation emitted by the 211At-labelled monoclonal antibody (MAb) TP-3 or external beam X-rays. Survival of microcolonies was assayed by colony formation. Therapeutic gain factor (TGF) values were calculated for two survival levels, 50% and 20% microcolony regeneration (i.e. at least one cell in 50% or 20% of the colonies survived the treatments). The TGF values were affected by the specific activity of the 211At-MAb conjugate, the antigen expression of the cells and the size and growth pattern of the microcolonies. Treatment with 211At-TP-3 gave TGF values that varied from 1.3 +/- 0.4 to 4.5 +/- 0.7 (mean +/- s.e.). The antigen-rich OHS cell line had on average 1.6 times higher TGF than the antigen-poor KPDX cell line. The TGF increased significantly with colony size for the densely packed colonies of the KPDX cell line but not for the OHS cell line, which had colonies with cells growing in a more scattered pattern. Control experiments with the two non-specific 211At forms, free 211At and 211At-labelled bovine serum albumin, gave TGF values from 0.6 +/- 0.1 to 1.0 +/- 0.3. This study suggests that in vivo evaluation of 211At-MAbs using relevant tumour models is desirable.

  18. Development and characterization of anti-glycopeptide monoclonal antibodies against human podoplanin, using glycan-deficient cell lines generated by CRISPR/Cas9 and TALEN.

    Science.gov (United States)

    Kaneko, Mika K; Nakamura, Takuro; Honma, Ryusuke; Ogasawara, Satoshi; Fujii, Yuki; Abe, Shinji; Takagi, Michiaki; Harada, Hiroyuki; Suzuki, Hiroyoshi; Nishioka, Yasuhiko; Kato, Yukinari

    2017-02-01

    Human podoplanin (hPDPN), which binds to C-type lectin-like receptor-2 (CLEC-2), is involved in platelet aggregation and cancer metastasis. The expression of hPDPN in cancer cells or cancer-associated fibroblasts indicates poor prognosis. Human lymphatic endothelial cells, lung-type I alveolar cells, and renal glomerular epithelial cells express hPDPN. Although numerous monoclonal antibodies (mAbs) against hPDPN are available, they recognize peptide epitopes of hPDPN. Here, we generated a novel anti-hPDPN mAb, LpMab-21. To characterize the hPDPN epitope recognized by the LpMab-21, we established glycan-deficient CHO-S and HEK-293T cell lines, using the CRISPR/Cas9 or TALEN. Flow cytometric analysis revealed that the minimum hPDPN epitope, in which sialic acid is linked to Thr76, recognized by LpMab-21 is Thr76-Arg79. LpMab-21 detected hPDPN expression in glioblastoma, oral squamous carcinoma, and seminoma cells as well as in normal lymphatic endothelial cells. However, LpMab-21 did not react with renal glomerular epithelial cells or lung type I alveolar cells, indicating that sialylation of hPDPN Thr76 is cell-type-specific. LpMab-21 combined with other anti-hPDPN antibodies that recognize different epitopes may therefore be useful for determining the physiological function of sialylated hPDPN. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  19. 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.

  20. Targeted radiotherapy potentiates the cytotoxicity of a novel anti-human DR5 monoclonal antibody and the adenovirus encoding soluble TRAIL in prostate cancer

    International Nuclear Information System (INIS)

    Arafat, W.; Arafat, W.; Zhou, T.; Naoum, G.E.; Buchsbaum, D.J.

    2015-01-01

    TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces a death signal following binding to death receptors (DR4, DR5). We have developed a novel anti-human DR-5 monoclonal antibody (TRA-8) and adenoviral encoding TRAIL (Ad/TRAIL). Herein, we are testing the combined effect of radiotherapy and TRA-8 or Ad TRAIL in prostate cancer cells. Human prostate cancer cell lines LnCap, PC-3 and DU145 were used in this study. Cells were treated either with TRA-8 alone or Ad/TRAIL, radiation alone, or a combination of each at different doses and intervals. Cell survival using the MTS assay and colony forming assay were used to determine radiosensitization. Immunohistochemistry was used to detect bax and bcl-2. Real-time PCR was performed on mRNA of treated prostate cancer cell lines. Finally, a murine model of subcutaneous prostate cancer was used to evaluate the in vivo effect. Cell survival assays detected by MTS assay showed that prostate cell lines treated with a combination of radiation and TRA-8 showed significantly lower survival than cells treated with either radiation or TRA-8 alone. Colony forming assay and cell proliferation assays showed increased killing after combination treatment with TRA-8 or Ad/TRAIL and radiation, than either single agent alone. Mechanistic studies showed that the killing effect was due to induction of apoptosis mostly by increased expression of bax in TRA-8 or Ad/TRAIL treated cells. Additionally, RT-PCR showed an increased copy number of bax in most cells treated with TRA-8 and radiation. It is concluded that radiation and TRA-8 or Ad/ TRAIL produced a synergistic effect in refractory prostrate cancer.

  1. Monoclonal antibodies for treating cancer

    International Nuclear Information System (INIS)

    Dillman, R.O.

    1989-01-01

    The purpose of this study is to assess the current status of in-vivo use of monoclonal antibodies for treating cancer. Publications appearing between 1980 and 1988 were identified by computer searches using MEDLINE and CANCERLIT, by reviewing the table of contents of recently published journals, and by searching bibliographies of identified books and articles. More than 700 articles, including peer-reviewed articles and book chapters, were identified and selected for analysis. The literature was reviewed and 235 articles were selected as relevant and representative of the current issues and future applications for in-vivo monoclonal antibodies for cancer therapy and of the toxicity and efficacy which has been associated with clinical trials. Approaches include using antibody alone (interacting with complement or effector cells or binding directly with certain cell receptors) and immunoconjugates (antibody coupled to radioisotopes, drugs, toxins, or other biologicals). Most experience has been with murine antibodies. Trials of antibody alone and radiolabeled antibodies have confirmed the feasibility of this approach and the in-vivo trafficking of antibodies to tumor cells. However, tumor cell heterogeneity, lack of cytotoxicity, and the development of human antimouse antibodies have limited clinical efficacy. Although the immunoconjugates are very promising, heterogeneity and the antimouse immune response have hampered this approach as has the additional challenge of chemically or genetically coupling antibody to cytotoxic agents. As a therapeutic modality, monoclonal antibodies are still promising but their general use will be delayed for several years. New approaches using human antibodies and reducing the human antiglobulin response should facilitate treatment. 235 references

  2. Monoclonal antibody PAL-E specific for endothelium

    NARCIS (Netherlands)

    Schlingemann, R. O.; Dingjan, G. M.; Emeis, J. J.; Blok, J.; Warnaar, S. O.; Ruiter, D. J.

    1985-01-01

    A monoclonal antibody, PAL-E, is described that is specific for endothelial cells. The monoclonal antibody, an IgG2a, markedly stains endothelium of capillaries, medium-sized and small veins, and venules in frozen sections of human and some animal tissues tested. It reacts not at all or only weakly

  3. Safety, biodistribution, pharmacokinetics, and immunogenicity of 99mTc-labeled humanized monoclonal antibody BIWA 4 (bivatuzumab) in patients with squamous cell carcinoma of the head and neck.

    Science.gov (United States)

    Colnot, David R; Roos, Jan C; de Bree, Remco; Wilhelm, Abraham J; Kummer, J Alain; Hanft, Gertraud; Heider, Karl-Heinz; Stehle, Gerd; Snow, Gordon B; van Dongen, Guus A M S

    2003-09-01

    Previous studies have shown the potential of murine and chimeric anti-CD44v6 monoclonal antibodies (MAbs) for radioimmunotherapy (RIT) of head and neck squamous cell carcinoma (HNSCC). A limitation of these MAbs, however, appeared to be their immunogenicity. Therefore, humanized monoclonal antibody BIWA 4 (bivatuzumab), with an intermediate affinity for CD44v6, was recently selected. As a prelude to RIT, we evaluated the safety, tumor-targeting potential, pharmacokinetics, and immunogenicity of technetium-99m-labeled BIWA 4 in patients undergoing operations for primary HNSCC in this study. Ten patients were treated at BIWA 4 dose levels of 25 mg (n=3), 50 mg (n=4), and 100 mg (n=3). Patients received 2 mg of 750 MBq 99mTc-BIWA 4, together with 23-, 48-, and 98-mg unlabeled BIWA 4, respectively. Radioimmunoscintigraphy (RIS) was performed within 1 h and after 21 h, and patients underwent surgery at 48 h after injection. Biodistribution of 99mTc-BIWA 4 was evaluated by radioactivity measurements in blood, bone marrow, and in biopsies of a surgical specimen obtained 48 h after injection. BIWA 4 concentration in blood was assessed by ELISA and high performance liquid chromatography and related to soluble CD44v6 levels in serum samples. The development of human anti-human antibody (HAHA) responses was determined. Administration of 99mTc-BIWA 4 was well tolerated by all patients and no HAHA responses were observed. A mean t1/2 in plasma of 54.8 +/- 11.5 h, 76.1 +/- 21.8 h, and 68.5 +/- 21.2 h was found for the 25-, 50-, and 100-mg dose group, respectively. No complex formation of BIWA 4 with soluble CD44v6 in blood was observed. RIS showed targeting of primary tumors and lymph node metastases in 8 of 10 and 1 of 5 patients, respectively. The highest tumor uptake and tumor to nontumor ratios were observed for the 50-mg dose group. Tumor uptake was 12.9 +/- 5.9, 26.2 +/- 3.1, and 15.4 +/- 1.9% of the injected dose (ID)/kg for the 25-, 50-, and 100-mg dose group

  4. Heterologous radioimmunoassay for llama and alpaca luteinizing hormone with a monoclonal antibody, and equine standard and a human tracer

    International Nuclear Information System (INIS)

    Aba, M.A.; Forsberg, M.

    1995-01-01

    A radioimmunoassay for llama and alpaca LH was developed using a human I 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 μ3g L -1 . The intra-assay coefficient of variation was 37% at 1 μg L -1 , declined to 15% at 4 μg L -1 and was below 6% for concentrations up to 32 μg L -1 . The inter-assay coefficients of variation for 3 control samples were 20% (2.8 μg L -1 ), 16% (7.1 μg L -1 ) and 9.8% (19 μg L -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 μL to 100 μL (in the standard curve the increased volume was compensated for by human LH free serum). With this protocol, the assay sensitivity was 0,5 μg L -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

  5. Experimental immunotargeting therapy for esophageal squamous cell carcinoma using anti-human esophageal monoclonal antibody KIS1

    International Nuclear Information System (INIS)

    Fujii, Teruhiko; Yamana, Hideaki; Higaki, Kensaku; Fujita, Hiromasa; Shirouzu, Kazuo; Morimatsu, Minoru

    1997-01-01

    In recent years, several MoAbs with high specificity to tumor associated antigens, have been produced and investigated for diagnosis and immunotherapy of tumors. We produced murine MoAb KIS1 against human squamous cell carcinoma of the esophagus, and we evaluated it and its F (ab') 2 fragment for experimental radioimmunotherapy (RIT), RIT combined with hyperthermia (HT) and KIS1-vindesine (VDS) conjugate using tumor bearing nude mice. KIS1 has been shown to react specifically with an antigen of human squamous cell carcinoma. Scintigraphy produced high quality tumor images on 3 days following the injection of 131 I-KIS1F (ab') 2 . By 14 days following injection, tumor bearing mice treated with RIT+HT group showed significant tumor growth inhibition about 1.5, 2.1 and 1.7 times greater than that of the KIS1-VDS group, 131 I-intact KIS1 group and 131 I-KIS1F (ab') 2 group. These results suggest that RIT combined with hyperthermia may be clinically useful for tumor targeting therapy for squamous cell carcinoma of the esophagus. (author)

  6. Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody.

    Science.gov (United States)

    Donahue, Renee N; Lepone, Lauren M; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A; Heery, Christopher R; Gulley, James L; Schlom, Jeffrey

    2017-01-01

    Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range of human tumor cells, and clinical studies have demonstrated anti-tumor activity versus a range of human cancers. This study was designed to investigate the effect on immune cell subsets in the peripheral blood of cancer patients prior to and following multiple administrations of avelumab. One hundred twenty-three distinct immune cell subsets in the peripheral blood of cancer patients ( n  = 28) in a phase I trial were analyzed by flow cytometry prior to and following one, three, and nine cycles of avelumab. Changes in soluble (s) CD27 and sCD40L in plasma were also evaluated. In vitro studies were also performed to determine if avelumab would mediate ADCC of PBMC. No statistically significant changes in any of the 123 immune cell subsets analyzed were observed at any dose level, or number of doses, of avelumab. Increases in the ratio of sCD27:sCD40L were observed, suggesting potential immune activation. Controlled in vitro studies also showed lysis of tumor cells by avelumab versus no lysis of PBMC from five donors. These studies demonstrate the lack of any significant effect on multiple immune cell subsets, even those expressing PD-L1, following multiple cycles of avelumab. These results complement prior studies showing anti-tumor effects of avelumab and comparable levels of adverse events with avelumab versus other anti-PD-1/PD-L1 MAbs. These studies provide the rationale to further exploit the potential ADCC mechanism of action of avelumab as well as other human IgG1 checkpoint

  7. Safety and pharmacokinetics of the Fc-modified HIV-1 human monoclonal antibody VRC01LS: A Phase 1 open-label clinical trial in healthy adults.

    Directory of Open Access Journals (Sweden)

    Martin R Gaudinski

    2018-01-01

    Full Text Available VRC01 is a human broadly neutralizing monoclonal antibody (bnMAb against the CD4-binding site of the HIV-1 envelope glycoprotein (Env that is currently being evaluated in a Phase IIb adult HIV-1 prevention efficacy trial. VRC01LS is a modified version of VRC01, designed for extended serum half-life by increased binding affinity to the neonatal Fc receptor.This Phase I dose-escalation study of VRC01LS in HIV-negative healthy adults was conducted by the Vaccine Research Center (VRC at the National Institutes of Health (NIH Clinical Center (Bethesda, MD. The age range of the study volunteers was 21-50 years; 51% of study volunteers were male and 49% were female. Primary objectives were safety and tolerability of VRC01LS intravenous (IV infusions at 5, 20, and 40 mg/kg infused once, 20 mg/kg given three times at 12-week intervals, and subcutaneous (SC delivery at 5 mg/kg delivered once, or three times at 12-week intervals. Secondary objectives were pharmacokinetics (PK, serum neutralization activity, and development of antidrug antibodies. Enrollment began on November 16, 2015, and concluded on August 23, 2017. This report describes the safety data for the first 37 volunteers who received administrations of VRC01LS. There were no serious adverse events (SAEs or dose-limiting toxicities. Mild malaise and myalgia were the most common adverse events (AEs. There were six AEs assessed as possibly related to VRC01LS administration, and all were mild in severity and resolved during the study. PK data were modeled based on the first dose of VRC01LS in the first 25 volunteers to complete their schedule of evaluations. The mean (±SD serum concentration 12 weeks after one IV administration of 20 mg/kg or 40 mg/kg were 180 ± 43 μg/mL (n = 7 and 326 ± 35 μg/mL (n = 5, respectively. The mean (±SD serum concentration 12 weeks after one IV and SC administration of 5 mg/kg were 40 ± 3 μg/mL (n = 2 and 25 ± 5 μg/mL (n = 9, respectively. Over the 5-40 mg

  8. Cloning of the first human anti-JCPyV/VP1 neutralizing monoclonal antibody: epitope definition and implications in risk stratification of patients under natalizumab therapy.

    Science.gov (United States)

    Diotti, Roberta Antonia; Mancini, Nicasio; Clementi, Nicola; Sautto, Giuseppe; Moreno, Guisella Janett; Criscuolo, Elena; Cappelletti, Francesca; Man, Petr; Forest, Eric; Remy, Louise; Giannecchini, Simone; Clementi, Massimo; Burioni, Roberto

    2014-08-01

    JC virus (JCPyV) has gained novel clinical importance as cause of progressive multifocal leukoencephalopathy (PML), a rare demyelinating disease recently associated to immunomodulatory drugs, such as natalizumab used in multiple sclerosis (MS) cases. Little is known about the mechanisms leading to PML, and this makes the need of PML risk stratification among natalizumab-treated patients very compelling. Clinical and laboratory-based risk-stratification markers have been proposed, one of these is represented by the JCPyV-seropositive status, which includes about 54% of MS patients. We recently proposed to investigate the possible protective role of neutralizing humoral immune response in preventing JCPyV reactivation. In this proof-of-concept study, by cloning the first human monoclonal antibody (GRE1) directed against a neutralizing epitope on JCPyV/VP1, we optimized a robust anti-JCPyV neutralization assay. This allowed us to evaluate the neutralizing activity in JCPyV-positive sera from MS patients, demonstrating the lack of correlation between the level of anti-JCPyV antibody and anti-JCPyV neutralizing activity. Relevant consequences may derive from future clinical studies induced by these findings; indeed the study of the serum anti-JCPyV neutralizing activity could allow not only a better risk stratification of the patients during natalizumab treatment, but also a better understanding of the pathophysiological mechanisms leading to PML, highlighting the contribution of peripheral versus central nervous system JCPyV reactivation. Noteworthy, the availability of GRE1 could allow the design of novel immunoprophylactic strategies during the immunomodulatory treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. 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 [ 3 H] 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.

  10. Radiotoxicity of systemically administered 211At-labeled human/mouse chimeric monoclonal antibody: a long-term survival study with histologic analysis

    International Nuclear Information System (INIS)

    McLendon, Roger E.; Archer, Gary E.; Larsen, Roy H.; Akabani, Gamal; Bigner, Darell D.; Zalutsky, Michael R.

    1999-01-01

    Purpose: The antitenascin human/mouse chimeric monoclonal antibody labeled with the α-particle-emitting radionuclide 211 At is of interest as an endo radiotherapeutic agent for the treatment of brain tumors. To facilitate the investigation of 211 At-labeled chimeric 81C6 in patients, the long-term radiotoxicity of this radiopharmaceutical has been evaluated. Methods and Materials: Antibody labeling was performed using N-succinimidyl 3-[ 211 At]astato-benzoate. After an initial dose-finding experiment, a second toxicity study was carried out at 4 dose levels in groups of 30 non thyroid blocked B6C3F 1 mice per group (15 males, 15 females). Male mice received either saline or 15-81 kBq/g and females received either saline or 16-83 kBq/g of 211 At-labeled antibody. Ten animals (5 males, 5 females) were followed for 6 months and the remainder for 1 year. Results: The lethal dose in 10% of animals (LD 10 ) for 211 At-labeled chimeric 81C6 was 46 kBq/g in females and 102 kBq/g in males. Toxic effects--perivascular fibrosis of the intraventricular septum of the heart, bone marrow suppression, splenic white pulp atrophy, and spermatic maturational delay--generally were confined to a few animals receiving the highest doses of labeled antibody. Conclusions: The LD 10 of 211 At-labeled chimeric 81C6 in this mouse strain was about half that of [ 211 At]astatide. These results establish the preclinical maximum tolerated dose of 211 At-labeled chimeric 81C6 and define in the mouse the target organs for toxicity. These studies will be useful for determining starting doses for clinical studies with 211 At-labeled chimeric 81C6

  11. Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

    Directory of Open Access Journals (Sweden)

    Charles Sabin

    Full Text Available The human monoclonal antibody (mAb HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1 of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

  12. Human pharmacokinetics, biodistribution and dosimetry of 99m Tc labelled monoclonal antibody ior egf/r3 in patients with tumors of epithelial origin: preliminary results

    International Nuclear Information System (INIS)

    Iznaga-Escobar, Normando E.; Morales, Alejo; Ramos, Mayra; Perez, Niuvis; Torres, Leonel A.; Alavarez, Ivette; Rodriguez, Nelson; Fraxedas, Roberto; Rodriguez, Oscar; Stabin, Michael G.

    1997-01-01

    Human pharmacokinetics, biodistribution and internal radiation dosimetry to normal organs and total body of 99m Tc-labeled monoclonal antibody ior egf/r 3 was investigated following intravenous injection in 5 patients. Following administration, blood and urine samples were collected from 4 patients up to 24 hr after injection Pharmacokinetics obtained from whole blood radioactivity showed blood disappearance described most properly by a biexponential model with a mean distribution half-life value of 0.14±0.02 hr and elimination half-life value of 31.0±13.6 hr. Whole body anterior and posterior scans were obtained at 10 min, 1,3,5 and 24 hr after injection. ROIs were drawn over the heart, liver spleen and bladder to measure the activity in the source organs. Time-activity curves for each source organ were fitted to mono- or biexponential functions by non-linear least squares regression using the flexible polyhedral method and integrated to determine organ residence times. The mean absorbed dose to the whole body and various normal organs were then estimated from residence times and from blood and urine samples using the MIRD method. The effective dose equivalent (EDE) and effective dose (ED) were calculated. Estimates of radiation absorbed dose to normal organs in rads/mCi administered (mean ± SD, n=4) were: whole body, 0.0185± 0.0023, gallbladder wall, 0.0755± 0.00761, spleen, 0.0637± 0.0167 and liver, 0.276± 0.029. The effective dose equivalent and effective dose estimates for adults were 0.039± 0.008 and 0.028± 0.004 rem/m Ci administered. (author). 15 refs., 3 figs., 3 tabs

  13. Nuclear Factor κB is required for tumor growth inhibition mediated by enavatuzumab (PDL192, a humanized monoclonal antibody to TweakR

    Directory of Open Access Journals (Sweden)

    James W. Purcell

    2014-01-01

    Full Text Available TweakR is a TNF receptor family member, whose natural ligand is the multifunctional cytokine TWEAK. The growth inhibitory activity observed following TweakR stimulation in certain cancer cell lines and the overexpression of TweakR in many solid tumor types led to the development of enavatuzumab (PDL192, a humanized IgG1 monoclonal antibody to TweakR. The purpose of this study was to determine the mechanism of action of enavatuzumab’s tumor growth inhibition and to provide insight into the biology behind TweakR as a cancer therapeutic target. A panel of 105 cancer lines was treated with enavatuzumab in vitro; and 29 cell lines of varying solid tumor backgrounds had >25% growth inhibition in response to the antibody. Treatment of sensitive cell lines with enavatuzumab resulted in the in vitro and in vivo (xenograft activation of both classical (p50, p65 and non-classical (p52, RelB NFκB pathways. Using NFκB DNA binding functional ELISAs and microarray analysis, we observed increased activation of NFκB subunits and NFκB regulated genes in sensitive cells over that observed in resistant cell lines. Inhibiting NFκB subunits (p50, p65, RelB, p52 and upstream kinases (IKK1, IKK2 with siRNA and chemical inhibitors consistently blocked enavatuzumab’s activity. Furthermore, enavatuzumab treatment resulted in NFκB-dependent reduction in cell-division as seen by the activation of the cell cycle inhibitor p21 both in vitro and in vivo. The finding that NFκB drives the growth inhibitory activity of enavatuzumab suggests that targeting TweakR with enavatuzumab may represent a novel cancer treatment strategy.

  14. Human pharmacokinetics, biodistribution and dosimetry of {sup 99m} Tc labelled monoclonal antibody ior egf/r3 in patients with tumors of epithelial origin: preliminary results

    Energy Technology Data Exchange (ETDEWEB)

    Iznaga-Escobar, Normando E.; Morales, Alejo; Ramos, Mayra; Perez, Niuvis [Center of Molecular Immunology (CIM), Havana (Cuba); Torres, Leonel A.; Alavarez, Ivette [Center of Clinical Researches (CIC), Havana (Cuba); Rodriguez, Nelson [Center of Medical-Surgical Researches (CIMEQ), Havana (Cuba); Fraxedas, Roberto [Institute of Nephrology (INEF), Havana (Cuba); Rodriguez, Oscar [Orthopedic Hospital Frank Pais, Havana (Cuba); Stabin, Michael G. [Radiation Internal Dose Information Center (RIDIC), Oak Ridge, TN (United States)

    1997-12-01

    Human pharmacokinetics, biodistribution and internal radiation dosimetry to normal organs and total body of {sup 99m} Tc-labeled monoclonal antibody ior egf/r{sup 3} was investigated following intravenous injection in 5 patients. Following administration, blood and urine samples were collected from 4 patients up to 24 hr after injection Pharmacokinetics obtained from whole blood radioactivity showed blood disappearance described most properly by a biexponential model with a mean distribution half-life value of 0.14{+-}0.02 hr and elimination half-life value of 31.0{+-}13.6 hr. Whole body anterior and posterior scans were obtained at 10 min, 1,3,5 and 24 hr after injection. ROIs were drawn over the heart, liver spleen and bladder to measure the activity in the source organs. Time-activity curves for each source organ were fitted to mono- or biexponential functions by non-linear least squares regression using the flexible polyhedral method and integrated to determine organ residence times. The mean absorbed dose to the whole body and various normal organs were then estimated from residence times and from blood and urine samples using the MIRD method. The effective dose equivalent (EDE) and effective dose (ED) were calculated. Estimates of radiation absorbed dose to normal organs in rads/mCi administered (mean {+-} SD, n=4) were: whole body, 0.0185{+-} 0.0023, gallbladder wall, 0.0755{+-} 0.00761, spleen, 0.0637{+-} 0.0167 and liver, 0.276{+-} 0.029. The effective dose equivalent and effective dose estimates for adults were 0.039{+-} 0.008 and 0.028{+-} 0.004 rem/m Ci administered. (author). 15 refs., 3 figs., 3 tabs.

  15. Selective modification of NMR relaxation time in human colorectal carcinoma by using gadolinium-diethylenetriaminepentaacetic acid conjugated with monoclonal antibody 19-9.

    Science.gov (United States)

    Curtet, C; Tellier, C; Bohy, J; Conti, M L; Saccavini, J C; Thedrez, P; Douillard, J Y; Chatal, J F; Koprowski, H

    1986-01-01

    Monoclonal antibody 19-9 (mAb 19-9) against human colon adenocarcinoma was conjugated with gadolinium X diethylenetriaminepentaacetic acid (Gd X DTPA) and used as a contrast agent in nuclear magnetic resonance (NMR) in an effort to improve tumor target selectivity in nude mice. The data indicate that Gd X DTPA-mAb 19-9 in solution decreased the T1 relaxation of water protons at 90 MHz in direct proportion to the gadolinium concentration, and this effect was greater than in Gd X DTPA solutions. T1 relaxation time at 90 MHz, measured in tumors removed from nude mice 24 hr after injection of Gd X DTPA-mAb 19-9 (Gd, 20 mumol/kg; 16 DTPA molecules per mAb molecule), was significantly decreased (by 15%) as compared with the control group. Similar results were obtained in tumors from mice injected with Gd X DTPA-mAb 19-9 solutions in which Gd was used at 2, 6, or 10 mumol/kg (16 DTPA molecules per mAb molecule). These doses are lower than those commonly used for Gd X DTPA (10-100 mumol/kg) as contrast agent. Tumor localization by the Gd X DTPA-mAb 19-9 complex containing radioactive Gd (0.3 microCi/microgram of 153Gd) to confirm scintigraphy revealed significant concentrations of the complex (5% of the injected dose per gram of tissue) in the tumor. Scan images recorded in planar scintigraphy at day 5 showed good visualization of tumors. Images PMID:3459174

  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. Epitope mapping and characterization of a novel CD4-induced human monoclonal antibody capable of neutralizing primary HIV-1 strains

    International Nuclear Information System (INIS)

    Xiang Shihua; Wang Liping; Abreu, Mariam; Huang, C.-C.; Kwong, Peter D.; Rosenberg, Eric; Robinson, James E.; Sodroski, Joseph

    2003-01-01

    Human immunodeficiency virus (HIV-1) enters target cells by binding its gp120 exterior envelope glycoprotein to CD4 and one of the chemokine receptors, CCR5 or CXCR4. CD4-induced (CD4i) antibodies bind gp120 more efficiently after CD4 binding and block the interaction with the chemokine receptor. Examples of CD4i antibodies are limited, and the prototypes of the CD4i antibodies exhibit only weak neutralizing activity against primary, clinical HIV-1 isolates. Here we report the identification of a novel antibody, E51, that exhibits CD4-induced binding to gp120 and neutralizes primary HIV-1 more efficiently than the prototypic CD4i antibodies. The E51 antibody blocks the interaction of gp120-CD4 complexes with CCR5 and binds to a highly conserved, basic gp120 element composed of the β19-strand and surrounding structures. Thus, on primary HIV-1 isolates, this gp120 region, which has been previously implicated in chemokine receptor binding, is accessible to a subset of CD4i antibodies

  18. Nucleotide sequences of the cDNAs encoding the V-regions of H- and L-chains of a human monoclonal antibody with broad reactivity to malignant tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Kishimoto, Toshimitsu; Okajima, Hideki; Okumoto, Takeki [Yoshitomi Pharmaceutical Industries, Ltd., Saitama (Japan); Taniguchi, Masaru [Chiba Univ. (Japan)

    1989-06-12

    The human monoclonal antibody secreted from 4G12 hybridoma cells has broad reactivity to malignant tumor cells, especially for lung squamous cell carcinomas, and recognizes a new tumor-associated and differentiation antigen. The antigen detected by 4G12 is a glycoprotein with MW 195,000 and MW 65,000 under nonreducing and reducing conditions, respectively. Screening of a 4G12 {lambda}gt10 cDNA library with constant region probes for human immunoglobulin yielded full length clones for H- and L-chains. Nucleotide sequences revealed that subtypes of the variable regions were V{sub HIII} and {lambda}{sub 1}, respectively.

  19. Monoclonal antibodies in oncology. Review article

    Energy Technology Data Exchange (ETDEWEB)

    Chan, S Y.T.; Sikora, K

    1986-05-01

    Monoclonal antibodies (MCAs) can be used to differentiate between normal and neoplastic cells and thus exploited for diagnostic and, ultimately, therapeutic gain. The evidence for the existence of human tumour antigens is reviewed. Several areas of diagnosis are already benefiting from the application of the monoclonal technology. Immunohistology can help the pathologist with difficult diagnostic problems. New classifications of lymphoma and leukaemia can be based on specific surface molecules. Similarly, the detection of shed tumour antigens is already established as part of the routine assessment of many patients with common solid tumours. Isotopically labeled monoclonal antibodies have been used to localise primary and metastatic tumours. The use of antibodies in this way is not only a promising diagnostic tool but also the first step in studying the possibility of arming antibodies to provide therapeutic agents. Such trials are currently in progress. 69 refs.; 7 figs.; 3 tabs.

  20. 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...

  1. 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...

  2. 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,

  3. 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...

  4. 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 molecules...

  5. Monoclonal antibody hapten radiopharmaceutical delivery

    International Nuclear Information System (INIS)

    Goodwin, D.A.; McTigue, M.

    1986-01-01

    One hundred μg of monoclonal antibody (MoAb) CHA255 with a binding constant Kb of 4 x 10 9 was complexed with indium-111 labelled BLEDTA II, BLEDTA IV, benzyl EDTA, and an EDTA conjugate of Fab. The 24-h tumour and organ distribution of BALB/c mice bearing KHJJ tumours was studied for each compound alone, the antibody complex, and 3 h following a chelate chase of the antibody complex. Whole body biological half-life was measured for 7 days with and without a chelate chase for each antibody complex. The 24-h whole body counts dropped 20 to 60% and blood concentration fell over 89% within 3 h of administering the chelate chase. Theoretical equivalent human organ doses were calculated from the 24-h organ concentrations, effective half-life, and MIRD 11 S values (absorbed dose per cumulated activity). Liver and spleen were the target organs, with the dose ranging from 0.50 to 3.91 rads mCi -1 . The reduction in organ radiation dose varied up to 95% following the chelate chase. Rapid selective renal clearance of chelate labelled radiopharmaceuticals by competitive inhibition (chelate chase) of their reversible binding to monoclonal antibodies enhances tumour imaging and improves the radiation dosimetry. (author)

  6. Effect of four monthly doses of a human monoclonal anti-FGF23 antibody (KRN23 on quality of life in X-linked hypophosphatemia

    Directory of Open Access Journals (Sweden)

    Mary D. Ruppe

    2016-12-01

    Full Text Available X-linked hypophosphatemia (XLH is characterized by lower extremity deformities that lead to bone and/or joint pain that result from decreased renal tubular reabsorption leading to hypophosphatemia caused by elevated levels of fibroblast growth factor 23 (FGF23. Objective: Validate the use of SF-36v2 Health Survey (SF-36v2 and the Western Ontario and McMaster Osteoarthritis Index (WOMAC to measure previously unstudied health-related quality of life (HRQoL in XLH patients and determine the change in HRQoL before and after treatment with KRN23, a human monoclonal anti-FGF23 antibody. Methods: Twenty-eight adult outpatients with XLH received up to four doses of KRN23 administered subcutaneously every 28 days. General HRQoL was measured with the SF-36v2 and condition-related HRQoL with the WOMAC at baseline and study endpoint as a secondary outcome of a Phase 1/2, open-label, multicenter, dose-escalation trial. Results: Testing for scale discriminant validity and convergent-divergent validity supported the use of these scales in the assessment of HRQoL in XLH. Both instruments indicated impairment of physical function at baseline with all mean scores showing a trend to improved health at study endpoint compared to baseline. When corrected for multiple comparisons, the score for Role Limitations due to physical health on the SF-36v2 which measures the patient's perception of their own chronic functional impairments due to poor physical health remained significantly improved (P < 0.05, increasing to the mean score of US adults. For the WOMAC, Physical Functioning and Stiffness scores were significantly improved (P < 0.05. Conclusion: KRN23 administration was associated with significantly improved patient perception of their Physical Functioning and Stiffness due to their disease. This study demonstrates that the SF-36v2 and WOMAC are valid tools for assessing HRQoL in XLH. Keywords: X-linked hypophosphatemia, KRN23, Health-related quality of life

  7. 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

  8. Imaging of non-small cell lung cancers with a monoclonal antibody, KC-4G3, which recognizes a human milk fat globule antigen

    International Nuclear Information System (INIS)

    Dienhart, D.G.; Schmelter, R.F.; Lear, J.L.; Miller, G.J.; Glenn, S.D.; Bloedow, D.C.; Kasliwal, R.; Moran, P.; Seligman, P.; Murphy, J.R.

    1990-01-01

    To determine the role of lung cancer tumor imaging with monoclonal antibodies directed against high molecular weight human milk fat globule antigens, we administered i.v. 111In-KC-4G3 to 24 patients with advanced non-small cell lung cancer. One mg of 111In-KC-4G3 was mixed with 0, 9, 49, 99, or 499 mg of unlabeled KC-4G3 and infused i.v. over 1 to 5 h. The mean 111In-KC-4G3 radiochemical purity was greater than 97% and the resultant immunoreactivity averaged 62%. Successful imaging of cancer sites was accomplished in 92% of 24 patients, and 57% of 91 total lesions were visualized. Successful localization of tumor sites related to size (P less than 0.001), with 81% of lesions greater than 3.0 cm in diameter, 50% of lesions 1.5 to 3 cm, and 6% of lesions less than 1.5 cm successfully imaging, and to location (P less than 0.05), with 69% of pulmonary lesions, 80% of soft tissue lesions, and only 32% of bone metastases being visualized. Nonspecific reticulo-endothelial uptake of radioactivity was a major problem. Approximately 35% of 111In was chelated to serum transferrin by 24 and 48 h after infusion. The mean t 1/2 beta for plasma radioisotope and immunoreactive KC-4G3 was 29 and 27 h, respectively. There was no correlation between total infused antibody dose and imaging success or between total dose and effect on 111In and KC-4G3 kinetics. Circulating free KC-4 antigen was measurable in all but one patient before study. Tumor biopsy following infusion could demonstrate antibody presence but not saturable antigen binding. We conclude that (a) 111In-KC-4G3 demonstrates successful tumor localization in non-small cell lung cancers bearing generally high expression of its antigen and (b) further investigations to diminish nonspecific radioactivity for imaging and utilization of high dose radiolabeled antibody for therapeutic intent are warranted

  9. Evaluation of (89Zr-labeled human anti-CD147 monoclonal antibody as a positron emission tomography probe in a mouse model of pancreatic cancer.

    Directory of Open Access Journals (Sweden)

    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

  10. 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

  11. In vitro evaluation of the monoclonal antibody Cu-64-IgG M75 against human carbonic anhydrase IX and its in vivo imaging

    Czech Academy of Sciences Publication Activity Database

    Čepa, Adam; Ráliš, Jan; Král, Vlastimil; Paurová, M.; Kučka, Jan; Humajová, J.; Lázníček, M.; Lebeda, Ondřej

    2018-01-01

    Roč. 133, č. March (2018), s. 9-13 ISSN 0969-8043 Institutional support: RVO:61389005 ; RVO:68378050 ; RVO:61389013 Keywords : IgG M75 * immunoaffinity * imaging * monoclonal atibody * Cu-64 Subject RIV: FP - Other Medical Disciplines; CD - Macromolecular Chemistry (UMCH-V); EB - Genetics ; Molecular Biology (UMG-J) OBOR OECD: Radiology, nuclear medicine and medical imaging; Polymer science (UMCH-V); Biochemical research methods (UMG-J) Impact factor: 1.128, year: 2016

  12. 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

  13. 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.

  14. [International classification of various types of monoclonal antibodies].

    Science.gov (United States)

    Scheen, A J

    2009-01-01

    Significant advances in the development of monoclonal antibodies ("mabs") have been acknowledged during the last two decades. Successive developments led to the marketing of murine antibodies ("o-mab" first, followed by chimeric antibodies ("xi-mab"), humanised antibodies ("zu-mab") and, finally, human monoclonal antibodies ("u-mab"). In order to facilitate the distinction between the various monoclonal antibodies used in clinical practice, an international nomenclature has been proposed with the use of a specific suffix corresponding to the origine/source of "mabs" preceded by an infix referring to the medicine's target. The efforts in developing new types of monoclonal antibodies aimed at improving their pharmacokinetics (longer half-life), pharmacodynamics (better efficacy because of stronger affinity to human receptor), and safety profile (less antigenic and immunogenic reactions). These progresses could be obtained thanks to the remarkable development of molecular biotechnology.

  15. Rat Monoclonal Antibodies Specific for LST1 Proteins

    OpenAIRE

    Schiller, Christian; Nitschké, Maximilian J. E.; Seidl, Alexander; Kremmer, Elisabeth; Weiss, Elisabeth H.

    2009-01-01

    The LST1 gene is located in the human MHC class III region and encodes transmembrane and soluble isoforms that have been suggested to play a role in the regulation of the immune response and are associated with inflammatory diseases such as rheumatoid arthritis. Here we describe the generation and characterization of the first monoclonal antibodies against LST1. Two hybridoma lines secreting monoclonal antibodies designated 7E2 and 8D12 were established. The 7E2 antibody detects recombinant a...

  16. 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

    D3 monoclonal antibody shows a function-dependent reactivity to an extracellular epitope of the ABCG2 transporter. In the current experiments we have further characterized the 5D3-ABCG2 interaction. The effect of chemical cross-linking and the modulation of extracellular S-S bridges...... on the transporter function and 5D3 reactivity of ABCG2 were investigated in depth. We found that several protein cross-linkers greatly increased 5D3 labeling in ABCG2 expressing HEK cells; however, there was no correlation between covalent dimer formation, the inhibition of transport activity, and the increase in 5...

  17. 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...... with no indication of any alternative pathway of infection, as evidenced by abrogation of enhancement by anti-CD4 MAb or soluble recombinant CD4, and also the inability of anti-Le(y) MAb to mediate HIV infection of HSB-2 cells in which HTLV-1/HIV pseudovirus infection was enhanced. While F(ab)2 fragments of ABL 364...

  18. Effect of human ZP3 monoclonal antibody on expression of GDF-9 and number of theca cells in ovary of mice (Mus musculus

    Directory of Open Access Journals (Sweden)

    Lilik Indahwati, M.Sc.

    2018-06-01

    Full Text Available الملخص: أهداف البحث: تهدف هذه الدراسة إلى التأكد من تأثير الأجسام المضادة أحادية المنشأ للمنطقة البشرية الشفافة ٣ على التعبير عن ج د ف-٩ وكمية خلايا ثيكا في المبيض لفئران موس العضلية. طرق البحث: استخدمنا تجربة حقيقية لما بعد الاختبار- تصميم مجموعة التحكم فقط، التي شملت ٤٨ فأرا تم تقسيمهم إلى مجموعة التحكم والمجموعة العلاجية للأجسام المضادة أحادية المنشأ للمنطقة البشرية الشفافة ٣ (٢٠ ميكروغرام،٤٠و ميكروغرام، و٦٠ ميكروغرام. قتلت كل مجموعة من الفئران في اليوم ١٠ و١٥ و٢٠. كما تم إجراء قياس تعبير ج د ف-٩ باستخدام الكيمياء النسيجية المناعية وتم قياس كمية خلايا ثيكا. النتائج: تحليل تفاعل الأجسام المضادة أحادية المنشأ للمنطقة البشرية الشفافة ٣ عند جرعة ٢٠ ميكروغرام -٦٠ ميكروغرام على التعبير عن ج د ف -٩ وكمية خلايا ثيكا لم يظهر اختلافات كبيرة. ولوحظ اكتشاف مماثل أيضا في الفترة من ١٠-٢٠- يوما. ولم نجد للأجسام المضادة أحادية المنشأ للمنطقة البشرية الشفافة ٣ أي تعبير عن ج د ف -٩ وخلايا ثيكا. الاستنتاجات: أظهرت هذه الدراسة أن الأجسام المضادة أحادية المنشأ للمنطقة البشرية الشفافة ٣ يمكن اعتبارها طريقة مناعية لمنع الحمل فاعلة وآمنة. Abstract: Objectives: This study investigated the effects of a human ZP3 monoclonal antibody (mAb hZP3 on

  19. 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.

  20. Monoclonal TCR-redirected tumor cell killing.

    Science.gov (United States)

    Liddy, Nathaniel; Bossi, Giovanna; Adams, Katherine J; Lissina, Anna; Mahon, Tara M; Hassan, Namir J; Gavarret, Jessie; Bianchi, Frayne C; Pumphrey, Nicholas J; Ladell, Kristin; Gostick, Emma; Sewell, Andrew K; Lissin, Nikolai M; Harwood, Naomi E; Molloy, Peter E; Li, Yi; Cameron, Brian J; Sami, Malkit; Baston, Emma E; Todorov, Penio T; Paston, Samantha J; Dennis, Rebecca E; Harper, Jane V; Dunn, Steve M; Ashfield, Rebecca; Johnson, Andy; McGrath, Yvonne; Plesa, Gabriela; June, Carl H; Kalos, Michael; Price, David A; Vuidepot, Annelise; Williams, Daniel D; Sutton, Deborah H; Jakobsen, Bent K

    2012-06-01

    T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

  1. 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).

  2. Nuclear oncology with monoclonal antibodies and peptides

    International Nuclear Information System (INIS)

    Hosono, Makoto

    1998-01-01

    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

  3. 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.

  4. Dissecting Immunogenicity of Monoclonal Antibodies

    National Research Council Canada - National Science Library

    Snyder, Christopher

    2003-01-01

    The potential of monoclonal antibodies, (mAbs), for use in therapeutic and diagnostic applications has not been fully realized in part due to counter-immune responses that often arise in patient recipients of mAb...

  5. Systemic radiotherapy with monoclonal antibodies

    International Nuclear Information System (INIS)

    Sautter-Bihl, M.L.; Matzku, S.; Bihl, H.

    1993-01-01

    In this experimental study, feasibility and efficiency of systematic radiotherapy with the I-131 labelled monoclonal antibody BW575/9 (radioimmunotherapy) are investigated using human SK-N-SH neuroblastoma transplated into nude mice. Series of six nude mice were treated with intravenous application of 400 μCi (group 1), 700 μCi (group 2) of the I-131 labelled and of the unlabelled MAb (group 3). An untreated group (group 4) served as control. Tumors of group (3) and (4) showed an identical growth. In group (1), tumor growth was arrested for seven days. In group (2), the tumor showed complete regression after eight days which lasted for 55 days. Thereafter, the tumor started to regrow. This growth characteristics are correlated with the doses achieved in the tumor using a medical radiation dose (MIRD) formulation. The biodistribution data necessary for MIRD calculation were obtained by previously performed experiments with the I-125 labelled MAb. The doses assessed in the tumor turned out to be five to ten times greater than those in normal tissues (liver, bone, etc.) These results confirm feasibility, selectivity and efficiency of radioimmunotherapy in the above described model. Moreover, this in vivo model seems suitable for further investigations concerning fundamental issues of radioimunotherapy. (orig.) [de

  6. Linear pharmacokinetic parameters for monoclonal antibodies are similar within a species and across different pharmacological targets: A comparison between human, cynomolgus monkey and hFcRn Tg32 transgenic mouse using a population-modeling approach.

    Science.gov (United States)

    Betts, Alison; Keunecke, Anne; van Steeg, Tamara J; van der Graaf, Piet H; Avery, Lindsay B; Jones, Hannah; Berkhout, Jan

    2018-04-10

    The linear pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs) can be considered a class property with values that are similar to endogenous IgG. Knowledge of these parameters across species could be used to avoid unnecessary in vivo PK studies and to enable early PK predictions and pharmacokinetic/pharmacodynamic (PK/PD) simulations. In this work, population-pharmacokinetic (popPK) modeling was used to determine a single set of 'typical' popPK parameters describing the linear PK of mAbs in human, cynomolgus monkey and transgenic mice expressing the human neonatal Fc receptor (hFcRn Tg32), using a rich dataset of 27 mAbs. Non-linear PK was excluded from the datasets and a 2-compartment model was applied to describe mAb disposition. Typical human popPK estimates compared well with data from comparator mAbs with linear PK in the clinic. Outliers with higher than typical clearance were found to have non-specific interactions in an affinity-capture self-interaction nanoparticle spectroscopy assay, offering a potential tool to screen out these mAbs at an early stage. Translational strategies were investigated for prediction of human linear PK of mAbs, including use of typical human popPK parameters and allometric exponents from cynomolgus monkey and Tg32 mouse. Each method gave good prediction of human PK with parameters predicted within 2-fold. These strategies offer alternative options to the use of cynomolgus monkeys for human PK predictions of linear mAbs, based on in silico methods (typical human popPK parameters) or using a rodent species (Tg32 mouse), and call into question the value of completing extensive in vivo preclinical PK to inform linear mAb PK.

  7. An anti-CCR5 monoclonal antibody and small molecule CCR5 antagonists synergize by inhibiting different stages of human immunodeficiency virus type 1 entry

    International Nuclear Information System (INIS)

    Safarian, Diana; Carnec, Xavier; Tsamis, Fotini; Kajumo, Francis; Dragic, Tatjana

    2006-01-01

    HIV-1 coreceptors are attractive targets for novel antivirals. Here, inhibition of entry by two classes of CCR5 antagonists was investigated. We confirmed previous findings that HIV-1 isolates vary greatly in their sensitivity to small molecule inhibitors of CCR5-mediated entry, SCH-C and TAK-779. In contrast, an anti-CCR5 monoclonal antibody (PA14) similarly inhibited entry of diverse viral isolates. Sensitivity to small molecules was V3 loop-dependent and inversely proportional to the level of gp120 binding to CCR5. Moreover, combinations of the MAb and small molecules were highly synergistic in blocking HIV-1 entry, suggesting different mechanisms of action. This was confirmed by time course of inhibition experiments wherein the PA14 MAb and small molecules were shown to inhibit temporally distinct stages of CCR5 usage. We propose that small molecules inhibit V3 binding to the second extracellular loop of CCR5, whereas PA14 preferentially inhibits subsequent events such as CCR5 recruitment into the fusion complex or conformational changes in the gp120-CCR5 complex that trigger fusion. Importantly, our findings suggest that combinations of CCR5 inhibitors with different mechanisms of action will be central to controlling HIV-1 infection and slowing the emergence of resistant strains

  8. Antitumor efficacy of triple monoclonal antibody inhibition of epidermal growth factor receptor (EGFR) with MM151 in EGFR-dependent and in cetuximab-resistant human colorectal cancer cells

    Science.gov (United States)

    Napolitano, Stefania; Martini, Giulia; Martinelli, Erika; Della Corte, Carminia Maria; Morgillo, Floriana; Belli, Valentina; Cardone, Claudia; Matrone, Nunzia; Ciardiello, Fortunato; Troiani, Teresa

    2017-01-01

    Purpose We investigated the effect of triple monoclonal antibody inhibition of EGFR to overcome acquired resistance to first generation of anti-EGFR inhibitors. Experimental design MM151 is a mixture of three different monoclonal IgG1 antibodies directed toward three different, non-overlapping, epitopes of the EGFR. We performed an in vivo study by using human CRC cell lines (SW48, LIM 1215 and CACO2) which are sensitive to EGFR inhibitors, in order to evaluate the activity of MM151 as compared to standard anti-EGFR mAbs, such as cetuximab, as single agent or in a sequential strategy of combination MM151 with irinotecan (induction therapy) followed by MM151 with a selective MEK1/2 inhibitor (MEKi) (maintenance therapy). Furthermore, the ability of MM151 to overcome acquired resistance to cetuximab has been also evaluated in cetuximab-refractory CRC models. Results MM151 shown stronger antitumor activity as compared to cetuximab. The maintenance treatment with MM151 plus MEKi resulted the most effective therapeutic modality. In fact, this combination caused an almost complete suppression of tumor growth in SW48, LIM 1215 and CACO2 xenografts model at 30 week. Moreover, in this treatment group, mice with no evidence of tumor were more than double as compared to single agent treated mice. Its superior activity has also been demonstrated, in cetuximab-refractory CRC models. Conclusions These results provide experimental evidence that more efficient and complete EGFR blockade may determine better antitumor activity and could contribute to prevent and/or overcome acquired resistance to EGFR inhibitors. PMID:29137301

  9. Identification and determination of the viability of Giardia lamblia cysts and Cryptosporidium parvum and Cryptosporidium hominis oocysts in human fecal and water supply samples by fluorescent in situ hybridization (FISH) and monoclonal antibodies.

    Science.gov (United States)

    Lemos, Vanessa; Graczyk, Thaddeus K; Alves, Margarida; Lobo, Maria Luísa; Sousa, Maria C; Antunes, Francisco; Matos, Olga

    2005-12-01

    In the present study, fluorescent in situ hybridization (FISH) and monoclonal antibodies (MAbs) were evaluated for species-specific detection and viability determination of Giardia lamblia, Cryptosporidium parvum, and Cryptosporidium hominis in human fecal and water supply samples. A total of 50 fecal human samples positive for G. lamblia cysts, 38 positive for C. parvum, and 23 positive for C. hominis were studied. Also, 18 water supply samples positive for Giardia spp. and Cryptosporidium spp. by the United States Environmental Protection Agency (USEPA) Method 1623 were studied by FISH and fluorescein isothiocyanate (FITC)-conjugated MAbs. Eighteen percent of the fecal samples parasitologically positive for G. lamblia presented viable and nonviable cysts, and 5% of those positive for Cryptosporidium spp. presented viable and nonviable oocysts. Of the 18 water supply samples analyzed, 6 (33%) presented Giardia spp. viable and nonviable cysts and 2 (11%) presented viable and nonviable Cryptosporidium spp. oocysts. G. lamblia identification was confirmed by polymerase chain reaction (PCR) and sequencing of the beta-giardin gene in the fecal and water samples found positive by FISH and FITC-conjugated MAbs. C. parvum and Cryptosporidium muris were identified, by PCR and sequencing of the small subunit of ribosomal RNA gene, in seven and one water samples, respectively. Our results confirm that this technique enables simultaneous visualization, species-specific identification, and viability determination of the organisms present in human fecal and water supply samples.

  10. 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

  11. 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

  12. Exploration of novel strategies to enhance monoclonal antibodies targeting

    International Nuclear Information System (INIS)

    Khawli, L.A.; Epstein, A.L.

    1997-01-01

    This paper highlights the major obstacles and prospects of antibody targeting for the radio imaging and therapy of human malignant lymphomas and more challenging solid tumors. To improve the therapeutic potential of monoclonal antibodies, the authors have focused their attention on the development of new and successful methods to augment antibody uptake in the tumor. These approaches include the use of radiolabeled streptavidin to target biotinylated monoclonal antibodies already bound to tumor, pretreatment with vasoactive immunoconjugates, and the use of chemically modified antibodies. Because of the promising preclinical data obtained with these three newer approaches, plans are underway to test them in the clinic. More generally, these approaches are applicable to the use of other monoclonal antibody/tumor systems for the diagnosis and therapy of human cancers and related diseases

  13. Generation of neutralizing monoclonal antibodies against a conformational epitope of human adenovirus type 7 (HAdv-7 incorporated in capsid encoded in a HAdv-3-based vector.

    Directory of Open Access Journals (Sweden)

    Minglong Liu

    Full Text Available The generation of monoclonal antibodies (MAbs by epitope-based immunization is difficult because the immunogenicity of simple peptides is poor and T cells must be potently stimulated and immunological memory elicited. A strategy in which antigen is incorporated into the adenoviral capsid protein has been used previously to develop antibody responses against several vaccine targets and may offer a solution to this problem. In this study, we used a similar strategy to develop HAdv-7-neutralizing MAbs using rAdMHE3 virions into which hexon hypervariable region 5 (HVR5 of adenovirus type 7 (HAdv-7 was incorporated. The epitope mutant rAdMHE3 was generated by replacing HVR5 of Ad3EGFP, a recombinant HAdv-3-based vector expressing enhanced green fluorescence protein, with HVR5 of HAdv-7. We immunized BALB/c mice with rAdMHE3 virions and produced 22 different MAbs against them, four of which showed neutralizing activity against HAdv-7 in vitro. Using an indirect enzyme-linked immunosorbent assay (ELISA analysis and an antibody-binding-competition ELISA with Ad3EGFP, HAdv-7, and a series of chimeric adenoviral particles containing epitope mutants, we demonstrated that the four MAbs recognize the neutralization site within HVR5 of the HAdv-7 virion. Using an immunoblotting analysis and ELISA with HAdv-7, recombinant peptides, and a synthetic peptide, we also showed that the neutralizing epitope within HVR5 of the HAdv-7 virion is a conformational epitope. These findings suggest that it is feasible to use a strategy in which antigen is incorporated into the adenoviral capsid protein to generate neutralizing MAbs. This strategy may also be useful for developing therapeutic neutralizing MAbs and designing recombinant vector vaccines against HAdv-7, and in structural analysis of adenoviruses.

  14. Identification of a second T-cell antigen receptor in human and mouse by an anti-peptide γ-chain-specific monoclonal antibody

    International Nuclear Information System (INIS)

    Ioannides, C.G.; Itoh, K.; Fox, F.E.; Pahwa, R.; Good, R.A.; Platsoucas, C.D.

    1987-01-01

    The authors developed a monoclonal antibody (mAb) (9D7) against a synthetic peptide (P13K) selected from the deduced amino acid sequence of the constant region of the λ chain of the murine T-cell antigen receptor (TCR) (amino acids 118-130). Using this mAb, they identified a putative second TCR expressed on peripheral blood lymphocytes from a patient with severe combined immunodeficiency (SCID) that were propagated in culture with recombinant interleukin 2 (rIL-2) and Con A. This mAb immunoprecipitated two polypeptide chains of 40 and 58 kDa under nonreducing conditions and of 40 and 56 kDa under reducing conditions from 125 I-labeled denatured lysates of T3 + WT31 - lymphocytes expanded in culture from a SCID patient. Chemical crosslinking of 125 I-labeled cells followed by immunoprecipitation with anti-Leu-4 mAb under nonreducing or reducing conditions revealed that the 40- and 56-kDa polypeptide chains were associated with the T3 differentiation antigen. These experiments were done with polyclonal cell populations. Cloned T3 + WT31 - cell populations are required to determine whether the TCR contains two λ polypeptide chains. Using the same 9D7 anti-P18K mAb and immunoblotting analysis, they identified a 35 kDa γ-chain polypeptide under reducing conditions expressed on purified L3T4 - Lyt2 - BALB/c mouse thymocytes. This γ-chain TCR is disulfide linked and has a molecular mass of 80 kDa under nonreducing conditions

  15. First-in-human phase 1 of YS110, a monoclonal antibody directed against CD26 in advanced CD26-expressing cancers.

    Science.gov (United States)

    Angevin, Eric; Isambert, Nicolas; Trillet-Lenoir, Véronique; You, Benoit; Alexandre, Jérôme; Zalcman, Gérard; Vielh, Philippe; Farace, Françoise; Valleix, Fanny; Podoll, Thomas; Kuramochi, Yu; Miyashita, Itaru; Hosono, Osamu; Dang, Nam H; Ohnuma, Kei; Yamada, Taketo; Kaneko, Yutaro; Morimoto, Chikao

    2017-04-25

    YS110 is a humanised IgG1 monoclonal antibody with high affinity to the CD26 antigen. YS110 demonstrated preclinical anti-tumour effects without significant side effects. This FIH study was designed to determine the maximal tolerated dose (MTD) and recommended phase 2 dose (RP2D) to assess the tolerance, pharmacokinetics (PK) and pharmacodynamics profiles of YS110 and preliminary efficacy. YS110 were initially administered intravenously once every 2 weeks (Q2W) for three doses and then, based on PK data, once every week (Q1W) for five doses in patients with CD26-expressing solid tumours. Thirty-three patients (22 mesothelioma) received a median of 3 (range 1-30) YS110 infusions across six dose levels (0.1-6 mg kg -1 ). MTD was not reached and two dose-limiting toxicities (infusion hypersensitivity reactions) led to the institution of a systemic premedication. Low-grade asthenia (30.3%), hypersensitivity (27.3%), nausea (15.2%), flushing (15.2%), chills (12.1%) and pyrexia (12.1%) were reported as ADRs. Pharmacokinetic parameters (AUC and C max ) increased in proportion with the dose. sCD26/DPPIV assays indicated CD26 modulation. Prolonged stable diseases were observed in 13 out of 26 evaluable patients. YS110 is well tolerated up to 6 mg kg -1 Q1W, which has been defined as the RP2D, with encouraging prolonged disease stabilisations observed in a number of patients with advanced/refractory mesothelioma.

  16. 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.

  17. Production and characterization of monoclonal antibodies (mAbs) against human serum albumin (HSA) for the development of an immunoaffinity system with oriented anti-HSA mAbs as immobilized ligand.

    Science.gov (United States)

    Rajak, Poonam; Vijayalakshmi, M A; Jayaprakash, N S

    2013-05-05

    Proteins present in human serum are of immense importance in the field of biomarker discovery. But, the presence of high-abundant proteins like albumin makes the analysis more challenging because of masking effect on low-abundant proteins. Therefore, removal of albumin using highly specific monoclonal antibodies (mAbs) can potentiate the discovery of low-abundant proteins. In the present study, mAbs against human serum albumin (HSA) were developed and integrated in to an immunoaffinity based system for specific removal of albumin from the serum. Hybridomas were obtained by fusion of Sp2/0 mouse myeloma cells with spleen cells from the mouse immunized with HSA. Five clones (AHSA1-5) producing mAbs specific to HSA were established and characterized by enzyme linked immunosorbent assay (ELISA) and immunoblotting for specificity, sensitivity and affinity in terms of antigen binding. The mAbs were able to bind to both native albumin as well as its glycated isoform. Reactivity of mAbs with different mammalian sera was tested. The affinity constant of the mAbs ranged from 10(8) to 10(9)M(-1). An approach based on oriented immobilization was followed to immobilize purified anti-HSA mAbs on hydrazine activated agarose gel and the dynamic binding capacity of the column was determined. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Production and characterization of a murine monoclonal IgM antibody to human C1q receptor (C1qR)

    International Nuclear Information System (INIS)

    Ghebrehiwet, B.

    1986-01-01

    A hybridoma cell line that produces a monoclonal antibody (MAb) to cell surface C1q receptor (C1qr) has been produced by fusion of the P3 x 63-Ag8.653 mouse myeloma cell line with the spleen cells of a CD-1 mouse that had been hyperimmunized with viable Raji cell suspensions (5 x 10 7 cells/inoculum). This MAb, designated II1/D1, is an IgM antibody with lambda-light chain specificity. Radiolabeled or unlabeled, highly purified II1/D1 was used to determine that: a) this antibody competes for C1q binding sites on C1qR-bearing cells; b) the molecule recognized by this MAb is the C1qR; and c) cells that are known to bind C1q also bind II1/D1 in a specific manner. Western blot analysis of solubilized Raji, or U937 cell membranes, showed that the 125 I-MAb detected a major protein band of approximately 85000 m.w. in its unreduced state, indicating that the C1qR is similar, if not identical, in both types of cells. Analyses of 125 I-II/D1 binding experiments revealed that the antibody bound to Raji cells or u937 cells in a specific manner. Uptake of the antibody was saturable, with equilibrium virtually attained within 35 min. Scatchard analysis of the binding data using the intact MAb suggests that the affinity constant K/sub D/ is 2.9 x 10 -10 M, and at apparent saturation, 24.6 ng of the antibody were bound per 2 x 10 6 cells, giving an estimated 7.8 x 10 3 antibody molecules bound per cell. That the II1/D1 antibody is specifically directed to the C1q was further evidenced by an ELISA in which the ability of C1qR-bearing cells to bind the MAb was abrogated by c-C1q in a specific dose-dependent manner

  19. 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 ...

  20. Monoclonal anti-melanoma antibodies and their possible clinical use

    International Nuclear Information System (INIS)

    Hellstroem, K.E.; Hellstroem, Ingegerd; Washington Univ., Seattle; Washington Univ., Seattle

    1985-01-01

    Cell surface antigens of human melanoma, as defined by monoclonal antibodies, are discussed and in particular the three antigens p97, a GD3 ganglioside and a proteoglycan. The potential diagnostic uses of antibodies to melanoma antigens are reviewed including in vitro diagnosis by immuno-histology, in vitro diagnosis by serum assays and in vivo diagnosis by tumour imaging using radioactively labelled antibodies. The potential therapeutic uses of monoclonal antibodies to melanoma antigens are also reviewed including targets for antibody therapy, the use of antibodies alone, radiolabelled antibodies, antibody-toxin conjugates, antibody-drug conjugates, anti-idiotypic antibodies and vaccines. (UK)

  1. 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.

  2. Making Recombinant Monoclonal Antibody And Radiolabelling For Medical Purpose

    International Nuclear Information System (INIS)

    Nguyen Thi Thu; Duong Van Dong; Vo Thi Cam Hoa; Bui Van Cuong; Chu Van Khoa; Vu Bich Huong; Le Quang Huan

    2008-01-01

    Recombinant monoclonal antibody labeling with 131 I specific to tumor cell has been studied and prepared for treatment of Hodgkin lymphoma. In this study, a recombinant monoclonal antibody with two specific properties is a hybrid molecule created by coupling an antibody variable fragments with peptide melittin. The gene coding the antibody fragment has been obtained from human synthetic Fv libraries using for panning and screening on populations of lymphocytes fragmented from human blood cells with Hodgkin diseases. The gene encoding peptit melittin has been cloned from honeybee Apis cerana DNA. The gene coding recombinant monoclonal antibody has been expressed in E.coli BL21 (DE3) at 37 o C and was induced with 0.6 mM IPTG. The recombinant compound has been purified by affinity chromatography with HiTrap affinity column. The obtained recombinant monoclonal antibody has showed cytolytic activities when added to cell culture medium for LU cancer cell line with the amount of 100 - 200 mg/ml. This monoclonal antibody is labeled with 131 I using chloramine T procedure. ChT mass for the oxidation of 50 μg monoclonal antibody in 76 MBq was 10 μg. Sodium metabisulfite was used as a reducing agent. Reaction time was above 3 mins. The radiochemical purity was determined using electrophoresis and TLC methods. Radiochemical yield was > 97%. Radiochemical purity after purification was > 99%. Nuclear purity was > 99%. Stability of the label antibody was 12 days. This is the product promise potential used in the diagnostic and therapeutic of Hodgkin lymphoma. (author)

  3. Tumor imaging with monoclonal antibodies

    International Nuclear Information System (INIS)

    Haisma, H.; Hilgers, J.

    1987-01-01

    Many monoclonal antibodies directed against tumor-associated antigens have been identified, but so far none of these are tumor specific. Polyclonal and monoclonal antibodies have been used for imaging of a wide variety of tumors with success. Radiolabeling of antibody is usually done with iodine isotopes of which 123 I is the best candidate for radioimmunodetection purposes. The labeling of antibodies through chelates makes it possible to use metal radioisotopes like 111 In, which is the best radioisotope for imaging with monoclonal antibodies due to its favorable half-life of 2.5 days. Usually imaging cannot be performed within 24 h after injection, but clearance of antibody can be increased by using F(ab) 2 of Fab. Another approach is to clear non-bound antibody by a second antibody, directed against the first. The detection limit of immunoimaging is about 2 cm, but will be improved by tomography or SPECT. There is still a high false positive and false negative rate, which makes it impossible to use radioimmunodetection as the only technique for diagnosis of tumors. In combination with other detection techniques, tumor imaging with monoclonal antibodies can improve diagnosis. 44 refs.; 3 tabs

  4. 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

    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......) were positively correlated to the false positive signals in the PP14 ELISA (r = 0.923; p detected in the donor sera, but levels and frequencies were lower compared to that of HABIA. Furthermore, HABIA were...

  5. 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

    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......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...

  6. 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.

  7. Localization of MHC class II/human cartilage glycoprotein-39 complexes in synovia of rheumatoid arthritis patients using complex-specific monoclonal antibodies

    NARCIS (Netherlands)

    Steenbakkers, Peter G. A.; Baeten, Dominique; Rovers, Eric; Veys, Eric M.; Rijnders, Antonius W. M.; Meijerink, Jan; de Keyser, Filip; Boots, Annemieke M. H.

    2003-01-01

    Recently human cartilage gp-39 (HC gp-39) was identified as a candidate autoantigen in rheumatoid arthritis (RA). To further investigate the relevance of this Ag in RA, we have generated a set of five mAbs to a combination epitope of complexes of HC gp-39(263-275) and the RA-associated DR alpha beta

  8. 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

    with P. aeruginosa at doses as low as 5 microg/animal. Furthermore, a high efficacy of KBPA101 in protection from local respiratory infections in an acute lung infection model in mice was demonstrated. Preclinical toxicology evaluation on human tissue, in rabbits, and in mice did not indicate any...

  9. A protein-conjugate approach to develop a monoclonal antibody-based antigen detection test for the diagnosis of human brucellosis

    NARCIS (Netherlands)

    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

  10. 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.

  11. 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.

  12. Phase 2, multicenter, open-label study of tigatuzumab (CS-1008), a humanized monoclonal antibody targeting death receptor 5, in combination with gemcitabine in chemotherapy-naive patients with unresectable or metastatic pancreatic cancer

    International Nuclear Information System (INIS)

    Forero-Torres, Andres; Infante, Jeffrey R; Waterhouse, David; Wong, Lucas; Vickers, Selwyn; Arrowsmith, Edward; He, Aiwu Ruth; Hart, Lowell; Trent, David; Wade, James; Jin, Xiaoping; Wang, Qiang; Austin, TaShara; Rosen, Michael; Beckman, Robert; Roemeling, Reinhard von; Greenberg, Jonathan; Saleh, Mansoor

    2013-01-01

    Tigatuzumab is the humanized version of the agonistic murine monoclonal antibody TRA-8 that binds to the death receptor 5 and induces apoptosis of human cancer cell lines via the caspase cascade. The combination of tigatuzumab and gemcitabine inhibits tumor growth in murine pancreatic xenografts. This phase 2 trial evaluated the efficacy of tigatuzumab combined with gemcitabine in 62 chemotherapy-naive patients with histologically or cytologically confirmed unresectable or metastatic pancreatic cancer. Patients received intravenous tigatuzumab (8 mg/kg loading dose followed by 3 mg/kg weekly) and gemcitabine (1000 mg/m 2 once weekly for 3 weeks followed by 1 week of rest) until progressive disease (PD) or unacceptable toxicity occurred. The primary end point was progression-free survival (PFS) at 16 weeks. Secondary end points included objective response rate (ORR) (complete responses plus partial responses), duration of response, and overall survival (OS). Safety of the combination was also evaluated. Mean duration of treatment was 18.48 weeks for tigatuzumab and 17.73 weeks for gemcitabine. The PFS rate at 16 weeks was 52.5% (95% confidence interval [CI], 39.3–64.1%). The ORR was 13.1%; 28 (45.9%) patients had stable disease and 14 (23%) patients had PD. Median PFS was 3.9 months (95% CI, 2.2–5.4 months). Median OS was 8.2 months (95% CI, 5.1–9.6 months). The most common adverse events related to tigatuzumab were nausea (35.5%), fatigue (32.3%), and peripheral edema (19.4%). Tigatuzumab combined with gemcitabine was well tolerated and may be clinically active for the treatment of chemotherapy-naive patients with unresectable or metastatic pancreatic cancer

  13. Monoclonal antibodies technology. Protocols

    International Nuclear Information System (INIS)

    Acevado Castro, B.E.

    1997-01-01

    Full text: Immunization. The first step in preparing useful monoclonal antibodies (MAbs) is to immunize an animal (Balb/c for example) with an appropriate antigen. Methods (only for soluble antigen): Solubilize selected antigen in Phosphate buffer solution (PBS) at pH 7.2-7.4, ideally at a final concentration per animal between 10 to 50 μg/ml. It is recommended that the antigen under consideration be incorporated into the emulsion adjuvants in 1:1 volumetric relation. We commonly use Frend's adjuvant (FA) to prepared immunized solution. The first immunization should be prepared with complete FA, and the another could be prepared with incomplete FA. It is recommended to inject mice with 0.2 ml intraperitoneal (ip) or subcutaneous (sc). Our experience suggests the sc route is the preferred route. A minimum protocol for immunizing mice to generate cells for preparing hybridomas is s follows: immunize sc on day 0, boost sc on day 21, take a trial bleeding on day 26; if antibody titters are satisfactory, boost ip on day 35 with antigen only, and remove the spleen to obtain cells for fusion on day 38. Fusion protocol. The myeloma cell line we are using is X63 Ag8.653. At the moment of fusion myeloma cells need a good viability (at least a 95%). 1. Remove the spleen cells from immunized mice using sterile conditions. An immune spleen should yield between 7 a 10x10 7 nucleated cells. 2. Place the spleen in 20 ml of serum-free RPMI 1640 in a Petri dish. Using a needle and syringe, inject the spleen with medium to distend and disrupt the spleen stroma and free the nucleated cells. 3. Flush the cell suspension with a Pasteur pipet to disperse clumps of cells. 4. Centrifuge the spleen cell suspension at 250g for 10 min. Resuspend the pellet in serum-free RPMI 1640. Determine cell concentration using Neuhabuer chamber. 5. Mix the myeloma cells and spleen cells in a conical 50-ml tube in serum-free RPMI 1640, 1 x10 7 spleen cells to 1x10 6 myeloma cells (ratio 10:1). Centrifuge

  14. Application of murine monoclonal antibodies to the serodiagnosis of tuberculosis

    International Nuclear Information System (INIS)

    Ivanyl, J.; Coates, A.R.M.; Krambovitis, E.

    1982-01-01

    The immune response during infectious diseases leads to a rise in antibody titre to the various different antigenic determinants of the causative organism. The response is further complicated by the fact that it is relatively unusual for one individual to respond to all antigenic components of an organism. Demonstration of the specific immune response of an infected host by serological tests is often hampered by the broad cross-reactivity between several bacterial antigens. The authors report on a serodiagnostic application of murine monoclonal antibodies (MAB), specific for a human pathogen, M. tuberculosis by a technique which is applicable in principle to the serodiagnosis of many other infectious diseases. The serum diagnostic test is based on the competitive inhibition by human sera of the binding of 125 I-labelled murine monoclonal antibodies to M. tuberculosis-coated polyvinyl plates. Five monoclonal antibodies binding to distinct antigenic determinants of the organism were used as structural probes which conferred their stringent combining site specificities to the polyclonal mixture of antibodies from patients' sera. When compared with healthy controls, increased titres of inhibitory antibodies were found in about 70% of patients with active tuberculosis. The diagnostic value of the individual monoclonal antibodies as well as the benefit from the use of multiple specificity probes has been qualified

  15. 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

    Science.gov (United States)

    Goodman, Simon L.; Grote, Hans Juergen; Wilm, Claudia

    2012-01-01

    Summary 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. PMID:23213423

  16. 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.

  17. The human CD38 monoclonal antibody daratumumab shows anti-tumor 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

    2016-01-01

    Purpose To establish a proof-of-concept for the efficacy of the anti-CD38 antibody daratumumab in the poor prognosis CD38+ 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, while 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 down-regulation 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. PMID:27637890

  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 1 study to evaluate the safety and LDL cholesterol-lowering effects of RG7652, a fully human monoclonal antibody against proprotein convertase subtilisin/kexin type 9.

    Science.gov (United States)

    Baruch, Amos; Luca, Diana; Kahn, Robert S; Cowan, Kyra J; Leabman, Maya; Budha, Nageshwar R; Chiu, Cecilia P C; Wu, Yan; Kirchhofer, Daniel; Peterson, Andrew; Davis, John C; Tingley, Whittemore G

    2017-07-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) downregulates low-density lipoprotein (LDL) receptors, thereby leading to a rise in circulating LDL cholesterol (LDL-C). RG7652 is a fully human monoclonal antibody against PCSK9. This placebo-controlled, phase 1 ascending-dose study in healthy subjects evaluated the safety of RG7652 and its efficacy as a potential LDL-C-lowering drug. Anti-PCSK9 antibody therapy safely and effectively reduces LDL-C. Subjects (N = 80) were randomized into 10 cohorts. Six sequential single-dose cohorts received 10, 40, 150, 300, 600, or 800 mg of RG7652 via subcutaneous injection. Four multiple-dose cohorts received 40 or 150 mg of RG7652 once weekly for 4 weeks, either with or without statin therapy (atorvastatin). Adverse events (AEs) were generally mild; the most common AEs were temporary injection-site reactions. No serious AEs, severe AEs, AEs leading to study-drug discontinuation, or dose-limiting toxicities were reported. RG7652 monotherapy reduced mean LDL-C levels by up to 64% and as much as 100 mg/dL at week 2; the effect magnitude and duration increased with dose (≥57 days following a single RG7652 dose ≥300 mg). Exploratory analyses showed reduced oxidized LDL, lipoprotein(a), and lipoprotein-associated phospholipase A2 with RG7652. Antidrug antibody against RG7652 tested positive in 2 of 60 (3.3%) RG7652-treated and in 4 of 20 (20.0%) placebo-treated subjects. Simultaneous atorvastatin administration did not appear to impact the pharmacokinetic profile or lipid-lowering effects of RG7652. Overall, RG7652 elicited substantial and sustained dose-related LDL-C reductions with an acceptable safety profile and minimal immunogenicity. © 2017 Wiley Periodicals, Inc.

  20. The value of gamma camera and computed tomography data set coregistration to assess Lewis Y antigen targeting in small cell lung cancer by 111Indium-labeled humanized monoclonal antibody 3S193

    International Nuclear Information System (INIS)

    Quaia, Emilio; Krug, Lee M.; Pandit-Taskar, Neeta; Nagel, Andrew; Reuter, Victor E.; Humm, John; Divgi, Chaitanya

    2008-01-01

    Aim: To assess the value of data set coregistration of gamma camera and computed tomography (CT) in the assessment of targeting of humanized monoclonal antibody 3S193 labeled with indium-111 ( 111 In-hu3S193) to small cell lung cancer (SCLC). Methods and materials: Ten patients (6 male and 4 female; mean age ± S.D., 60 ± 4 years), from an overall population of 20 patients with SCLCs expressing Lewis Y antigen at immunohistochemical analysis, completed a four weekly injections of 111 In-hu3S193 and underwent gamma camera imaging. All had had, as part of their baseline evaluation, Fluorine18 fluoro-2-deoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT). Two readers in consensus retrospectively coregistered the gamma camera images with the CT component of the FDG PET/CT by automatic or manual alignment. The resulting image sets were visually examined and SCLC lesions targeting at coregistered gamma camera and CT was correlated side-by-side with the 18 F-FDG uptake. Results: A total number of 31 lesions from SCLC with a thoracic (n = 13) or extrathoracic location (n = 18) were all positive on FDG PET/CT. Coregistration of the gamma camera to the CT demonstrated targeting of antibody to all lesions >2 cm (n = 20) and in a few lesions ≤2 cm (n = 2), with no visualization of most lesions ≤2 cm (n = 9). No 111 In-hu3S193 uptake in normal tissues was observed. Conclusion: Coregistration of antibody gamma camera imaging to FDG PET/CT is feasible and allows valuable assessment of 111 In-hu3S193 antibody targeting to SCLC lesions >2 cm, while lesions ≤2 cm reveal a limited targeting

  1. Targeting of indium 111-labeled bivalent hapten to human melanoma mediated by bispecific monoclonal antibody conjugates: Imaging of tumors hosted in nude mice

    International Nuclear Information System (INIS)

    Le Doussal, J.M.; Gruaz-Guyon, A.; Martin, M.; Gautherot, E.; Delaage, M.; Barbet, J.

    1990-01-01

    Antibody conjugates were prepared by coupling F(ab')2 or Fab' fragments of an antibody specific for the human high molecular weight-melanoma associated antigen to Fab' fragments of an antibody specific for indium-diethylenetriaminepentaacetate complexes. Monovalent and bivalent haptens were synthesized by reacting the dipeptide tyrosyl-lysine with diethylenetriaminepentaacetic cyclic anhydride. In vitro, the antibody conjugate mediated binding of the 111In-labeled haptens to melanoma cells. In vivo, it allowed specific localization of the haptens in A375 tumors. The bivalent hapten exhibited much higher efficiency at targeting 111In onto cells, both in vitro and in vivo. Antibody conjugate and hapten doses (2 micrograms and 1 pmol, respectively) and the delay between antibody conjugate and tracer injections (24 h) were adjusted to maximize tumor uptake (4% injected dose/g) and tumor to normal tissue contrast (greater than 3) obtained 3 h after injection of the 111In-labeled bivalent hapten. This two-step technique, when compared to direct targeting of 111In-labeled F(ab')2 fragments, provided lower localization of injected activity into the tumor (x 0.25), but higher tumor/tissue ratios, especially with respect to liver (x 7), spleen (x 8), and kidneys (x 10). In addition, high contrast images were obtained within 3 hours, instead of days. Thus, antibody conjugate-mediated targeting of small bivalent haptens, labeled with short half-life isotopes, is proposed as a general method for improving tumor radioimmunolocalization

  2. 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.

  3. Effect of radiation on the expression of tumor-associated antigens of human lung adenocarcinoma cells. Immunological study using monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Hareyama, Masato

    1988-12-01

    We studied the effects of irradiation on the expression of a tumor-associated antigen (YH206 antigen) of cultured human lung adenocarcinoma A549 cells by using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. YH206 antigen is preferentially expressed on adenocarcinoma cells. Irradiation of A549 cells remarkably increased the expression of YH206 antigen on the cell surface and the level of the antigen in the culture supernatant as well as in the cell lysate, whereas it significantly affected the expression of HLA (MHC-class I) antigen on the same cells. The expression of HLA antigen on the cell was also increased after treatment of the cells with interferon-..gamma... In an additional experiment, cells were stained simultaneously for surface antigens (fluorescein coupled antibodies) and for DNA content (propidium iodide), and then dual parameter measurements were performed by flow cytometry to analyse the relationship between antigen levels and the cell cycle. YH206 antigen and HLA antigen increased more in the S and G/sub 2//M phases of the cell cycle than in G/sub 0//G/sub 1/. The expression of YH206 antigen was enhanced in the S and G/sub 2//M phases by irradiation, whereas the expression of HLA antigen was enhanced in each phase of the cell cycle with irradiation or IFN. These results suggest that irradiation plays a key role in the change of the expression of certain tumor-associated antigens.

  4. Ablation of human colon carcinoma in nude mice by 131I-labeled monoclonal anti-carcinoembryonic antigen antibody F(ab')2 fragments

    International Nuclear Information System (INIS)

    Buchegger, F.; Pfister, C.; Fournier, K.; Prevel, F.; Schreyer, M.; Carrel, S.; Mach, J.P.

    1989-01-01

    Pooled F(ab')2 fragments of three MAbs against distinct epitopes of carcinoembryonic antigen (CEA) were used for radioimmunotherapy of nude mice bearing a subcutaneous human colon carcinoma xenograft. 9-10 d after transplantation when tumor nodules were in exponential growth, 36 mice were treated by intravenous injection of different amounts of 131 I-labeled MAb F(ab')2. All 14 mice injected with a single dose of 2,200 (n = 10) or 2,800 microCi (n = 4) showed complete tumor remission. 8 of the 10 mice treated with 2,200 microCi survived in good health for 1 yr when they were killed and shown to be tumor free. Four of nine other mice treated with four fractionated doses of 400 microCi showed no tumor relapse for more than 9 mo. In contrast, all 15 mice injected with 1,600-3,000 microCi 131 I-control IgG F(ab')2 showed tumor growth retardation of only 1-4 wk, and 15 of 16 mice injected with unlabeled anti-CEA MAb F(ab')2 showed unmodified tumor progression as compared with untreated mice. From tissue radioactivity distributions it was calculated that by an injection of 2,200 microCi 131 I-MAb F(ab')2 a mean dose of 8,335 rad was selectively delivered to the tumor, while the tissue-absorbed radiation doses for the normal organs were: peripheral blood, 2,093; stomach, 1,668; kidney, 1,289; lung, 1,185; liver, 617; spleen, 501; small intestine, 427; large intestine, 367; bone, 337; and muscle, 198. These treatments were well tolerated since out of 19 mice with complete tumor remission only 4 required bone marrow transplantation and 17 were in good health for 6-12 mo of observation

  5. 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)

  6. Human pharmacokinetics, biodistribution and dosimetry of the kit of monoclonal antibody IOR EGF/R3 labelled with 99m Tc

    International Nuclear Information System (INIS)

    Torres, L.A.; Ramos, M.; Perera, A.; Hernandez, A.; Iznaga, M.E. N.

    1998-01-01

    The aim of this work was to assess the human pharmacokinetics, biodistribution and dosimetry of the 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)

  7. Production of Monoclonal Antibodies specific for Progesterone

    OpenAIRE

    YÜCEL, Fatıma

    2014-01-01

    Progesterone levels in milk and serum are indicators of pregnancy in cattle. The progesterone level reaches a peak on the 21 st and 22 nd days of pregnancy. Monoclonal antibodies specific to progesterone could be used for the immunodetection of milk and serum progesterone levels. We report here the development of hybrid cells prdoducing monoclonal antibodies specific for progesterone using hybridoma technology. Hybridoma cells secreting monoclonal antibodies against progesterone (MAM 2H1...

  8. Mapping of monoclonal antibody- and receptor-binding domains on human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using a surface plasmon resonance-based biosensor.

    Science.gov (United States)

    Laricchia-Robbio, L; Liedberg, B; Platou-Vikinge, T; Rovero, P; Beffy, P; Revoltella, R P

    1996-10-01

    An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct

  9. Monoclonal antibody-based immunoassays.

    Science.gov (United States)

    Appleby, P; Reischl, U

    1998-01-01

    An immunoassay may be defined as an assay that employs an immunological reagent, usually an antibody, to confer specificity for the ligand being measured. As a corollary to this, the discovery, and subsequent development, of monoclonal antibodies (MAbs) has greatly expanded the application and use of immunoassays. Polyclonal reagents, with their associated problems of specificity and quality control, have now been largely replaced by readily available MAbs of potential immortality and well-defined specificity and affinity. This has resulted, in the last two decades, in a great expansion in the range of immunoassays available and also a significant improvement in their reproducibility and reliability.

  10. Boronated monoclonal antibody conjugates for neutron capture therapy

    International Nuclear Information System (INIS)

    Borg, D.C.; Elmore, J.J. Jr.; Ferrone, S.

    1986-01-01

    This paper describes the effectiveness of 10 B-labeled monoclonal antibodies against Colo-38 human melanoma in vitro. The authors obtained high boron to antibody ratios while maintaining antibody activity by using dextran intermediate carriers to link 10 B to the antibody. They developed a double cell quasi-competitive binding bioassay to minimize the effects of nonspecific binding of boronated complexes to cells. 1 fig., 2 tabs

  11. 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.

  12. Use of AN Eosinophil Specific Monoclonal Antibody in Assessing Eosinophil Function.

    Science.gov (United States)

    Minkoff, Marjorie Sue

    A monoclonal antibody to an eosinophil specific determinant is very important in assessing eosinophil function during helminthic infection. Eosinophils induced by Schistosoma mansoni infection in BALB/c mice were used to induce C57B1/6 immunocytes for production of hybridomas secreting eosinophil monoclonal antibodies. These antibodies were shown to react with an eosinophil surface epitope but not with neutrophils or macrophages as determined by ELISA, immunodiffusion, immunofluorescence, and immunoblot assay. Affinity chromatography with eosinophil chemotactic factor-sepharose consistently selected out a { rm M_ R} 67,000 protein from solubilized eosinophil membrane antigens but not from neutrophil and macrophage antigens. In vitro studies showed that the eosinophil-specific monoclonal antibodies abrogated antibody-dependent eosinophil -mediated killing of S. mansoni schistosomula using mouse, rat or human eosinophils. Neutrophil and macrophage killing activities were unaffected. The monoclonal antibodies effected complement-dependent lysis of mouse and rat eosinophils but not of human eosinophils. ECF-treated eosinophils showed enhanced killing of schistosomula which was blocked by the monoclonal antibody. Murine and human eosinophils preincubated with monoclonal antibody exhibited decreased chemotaxis to ECF at optimal chemotactic concentrations. The monoclonal antibody also blocked eosinophil binding to ECF- sepharose beads. In vivo induction of peripheral blood eosinophilia by injection of S. mansoni eggs was suppressed by injections of monoclonal antibodies 2CD13 and 2QD45 in mouse and rat experimental models. Eosinophilia induced by keyhole limpet hemocyanin- cyclophosphamide treatment was also suppressed by monoclonal antibody in both murine and rat systems. Pulmonary granulomas in mice given egg injection and monoclonal antibody were smaller and contained fewer eosinophils than those granulomas from mice given eggs only. In immuno-biochemical studies, the

  13. Uses of monoclonal antibody 8H9

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, Nai-Kong V.

    2018-04-10

    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 a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

  14. A novel antibody-dependent cellular cytotoxicity epitope in gp120 is identified by two monoclonal antibodies isolated from a long-term survivor of human immunodeficiency virus type 1 infection.

    Science.gov (United States)

    Alsmadi, O; Herz, R; Murphy, E; Pinter, A; Tilley, S A

    1997-01-01

    Two monoclonal antibodies (MAbs), 42F and 43F, were isolated some 14 months apart from a single long-term survivor of human immunodeficiency virus type 1 (HIV-1) infection. These MAbs were found to be indistinguishable in terms of their isotypes, specificities, affinities, and biological activities. Both 42F and 43F directed substantial antibody-dependent cellular cytotoxicity (ADCC) against cells infected with four divergent lab-adapted strains of HIV-1, but no neutralizing activity against these strains was detectable. The ability of MAbs 42F and 43F, as well as that of MAbs against two other gp120 epitopes, to direct ADCC against uninfected CD4+ cells to which recombinant gp120SF2 had been adsorbed (i.e., "innocent bystanders") was demonstrated to be less efficient by at least an order of magnitude than their ability to direct ADCC against HIV-1-infected cells. Flow cytometry analyses showed that 42F and 43F also bind to native primary isolate Envs from clades B and E expressed on cell surfaces. By direct binding and competition assays, it was demonstrated that the 42F/43F epitope lies in a domain of gp120 outside the previously described CD4-binding site and V3 loop ADCC epitope clusters. Immunoblot analysis revealed that the 42F/43F epitope is not dependent on disulfide bonds or N-linked glycans in gp120. Epitope mapping of 42F and 43F by binding to linear peptides demonstrated specificity of these MAbs for a sequence of 10 amino acids in the C5 domain comprising residues 491 to 500 (Los Alamos National Laboratory numbering for the HXB2 strain). Thus, 42F and 43F define a new ADCC epitope in gp120. Because of the relative conservation of this epitope and the fact that it appears to have been significantly immunogenic in the individual from which these MAbs were derived, it may prove to be a useful component of HIV vaccines. Furthermore, these MAbs may be used as tools to probe the potential importance of ADCC as an antiviral activity in HIV-1 infection. PMID

  15. Monoclonal antibodies for radioimmunodetection of tumours and for targeting

    International Nuclear Information System (INIS)

    Baldwin, R.W.; Embleton, M.J.; Pimm, M.V.

    1983-01-01

    A monoclonal antibody 791T/36 prepared against human osteogenic sarcoma has been used to detect primary and metastatic colorectal carcinomas by external imaging of patients following injection of 131 I-labelled antibody. In 10 of 11 patients radiolabelled 791T/36 antibody localized in tumours, the tumour/non tumour ratio of radioactivity ranging from 1.5:1 to 8.1. 791T/36 antibody was also evaluated for its potential for targeting anti-tumour agents including cytotoxic drugs (Vindesine) and immunomodulating agents (interferon). Vindesine-791T/36 conjugates were preferentially cytotoxic in vitro for target cells expressing the 791T/36 anti-body defined antigen. Also interferon conjugated to 791T/36 antibody, like free interferon activated peripheral blood natural killer cell activity. These in vitro tests together with related studies on antibody localization in vivo indicate the potential of monoclonal antibody targeting of anti-tumour agents

  16. Localisation of metastatic carcinoma by a radiolabelled monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Smedley, H M; Ritson, A; Wraight, P; Sikora, K [Addenbrooke' s Hospital, Cambridge (UK); Hinchingbrooke Hospital, Huntingdon (UK)); Finan, P [St. James Hospital, Leeds (UK); Lennox, E S; Takei, F [Medical Research Council, Cambridge (UK)

    1983-02-01

    Rat monoclonal antibodies were prepared by immunising rats with human colorectal carcinoma cell membranes and fusing splenic lymphocytes with a rat myeloma. Hybridoma supernatants were screened by binding assays on membranes prepared from colorectal carcinoma tissue. One hybridoma supernatant, containing a monoclonal antibody with high binding activity on malignant compared to normal colon sections, was grown in large quantities in serum-free medium. After ammonium sulphate precipitation the antibody was purified by ion-exchange chromatography and labelled with /sup 131/I. Radiolabelled antibody was administered i.v. to 27 patients with colonic and other tumours. Scintigrams were obtained at 48 h. Computerised subtraction of the blood pool image revealed localised areas of uptake corresponding with areas of known disease in 13/16 patients with colorectal carcinoma and 3/4 patients with breast cancer.

  17. Monoclonal antibodies based on hybridoma technology.

    Science.gov (United States)

    Yagami, Hisanori; Kato, Hiroshi; Tsumoto, Kanta; Tomita, Masahiro

    2013-03-01

    Based on the size and scope of the present global market for medicine, monoclonal antibodies (mAbs) have a very promising future, with applications for cancers through autoimmune ailments to infectious disease. Since mAbs recognize only their target antigens and not other unrelated proteins, pinpoint medical treatment is possible. Global demand is dramatically expanding. Hybridoma technology, which allows production of mAbs directed against antigens of interest is therefore privileged. However, there are some pivotal points for further development to generate therapeutic antibodies. One is selective generation of human mAbs. Employment of transgenic mice producing human antibodies would overcome this problem. Another focus is recognition sites and conformational epitopes in antigens may be just as important as linear epitopes, especially when membrane proteins such as receptors are targeted. Recognition of intact structures is of critical importance for medical purposes. In this review, we describe patent related information for therapeutic mAbs based on hybridoma technology and also discuss new advances in hybridoma technology that facilitate selective production of stereospecific mAbs.

  18. 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

  19. Monoclonal gammopathy in rheumatic diseases.

    Science.gov (United States)

    Yang, Yue; Chen, Long; Jia, Yuan; Liu, Yang; Wen, Lei; Liang, Yaoxian; An, Yuan; Chen, Shi; Su, Yin; Li, Zhanguo

    2018-07-01

    To analyze the clinical spectrum, laboratory characteristics, and outcomes of monoclonal gammopathy (MG) in patients with rheumatic diseases. Screening for the presence of MG was performed in 872 inpatients with rheumatic diseases from January 2010 to July 2017. A total of 41 patients were enrolled. Their clinical and biological features in addition to outcomes were described. For each patient with primary Sjögren syndrome (pSS), 2 age- and sex-matched pSS patients without MG were selected as controls. Risk factors for the presence of MG and malignant hematological neoplasias were assessed. MG was observed in patients with SS, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, primary biliary cirrhosis, polymyositis, hypomyopathic dermatomyositis, psoriatic arthritis, ANCA-associated vasculitis, polyarteritis nodosa, and polymyalgia rheumatic, with SS the most frequent type. Serum M protein was detected in 37 patients. The monoclonal bands identified in serum were 16 IgG (5 κ, 11 λ), 11 IgA (6 κ, 5 λ), 6 IgM (5 κ, 1 λ), and 4 free λ chains. M components were observed in urine in the other 4 patients. High ESR, albumin/globulin inversion, rheumatoid factor positivity, hypergammaglobulinemia, and hypocomplementemia were common features, presented in more than half of the 41 patients. Patients with pSS, when complicated with MG, showed a higher rate of abnormal urine NAG (71.4 vs 15.8%, P = 0.025), higher levels of ESR [55.0 (53.5) mm/h vs 21.0 (31.8) mm/h, P = 0.001], ESSDAI [26.0 (25.0) vs 12.0 (9.0), P = 0.006], and ClinESSDAI scores [24.0 (25.0) vs 10.5 (10.0), P = 0.011]. Multivariate analysis revealed that the disease activity, assessed by either ESSDAI [adjusted OR 1.127 (95%CI 1.015-1.251), P = 0.025] or ClinESSDAI [adjusted OR 1.121 (95%CI 1.011-1.242), P = 0.030], was the only independent risk factor for the presence of MG. During the follow-up, 2 patients had transient serum M protein, 2 had isotype

  20. Clinical Translation and Validation of a Predictive Biomarker for Patritumab, an Anti-human Epidermal Growth Factor Receptor 3 (HER3) Monoclonal Antibody, in Patients With Advanced Non-small Cell Lung Cancer.

    Science.gov (United States)

    Mendell, Jeanne; Freeman, Daniel J; Feng, Wenqin; Hettmann, Thore; Schneider, Matthias; Blum, Sabine; Ruhe, Jens; Bange, Johannes; Nakamaru, Kenji; Chen, Shuquan; Tsuchihashi, Zenta; von Pawel, Joachim; Copigneaux, Catherine; Beckman, Robert A

    2015-03-01

    During early clinical development, prospective identification of a predictive biomarker and validation of an assay method may not always be feasible. Dichotomizing a continuous biomarker measure to classify responders also leads to challenges. We present a case study of a prospective-retrospective approach for a continuous biomarker identified after patient enrollment but defined prospectively before the unblinding of data. An analysis of the strengths and weaknesses of this approach and the challenges encountered in its practical application are also provided. HERALD (NCT02134015) was a double-blind, phase 2 study in patients with non-small cell lung cancer (NSCLC) randomized to erlotinib with placebo or with high or low doses of patritumab, a monoclonal antibody targeted against human epidermal growth factor receptor 3 (HER3). While the primary objective was to assess safety and progression-free survival (PFS), a secondary objective was to determine a single predictive biomarker hypothesis to identify subjects most likely to benefit from the addition of patritumab. Although not identified as the primary biomarker in the study protocol, on the basis of preclinical results from 2 independent laboratories, expression levels of the HER3 ligand heregulin (HRG) were prospectively declared the predictive biomarker before data unblinding but after subject enrollment. An assay to measure HRG mRNA was developed and validated. Other biomarkers, such as epidermal growth factor receptor (EGFR) mutation status, were also evaluated in an exploratory fashion. The cutoff value for high vs. low HRG mRNA levels was set at the median delta threshold cycle. A maximum likelihood analysis was performed to evaluate the provisional cutoff. The relationship of HRG values to PFS hazard ratios (HRs) was assessed as a measure of internal validation. Additional NSCLC samples were analyzed to characterize HRG mRNA distribution. The subgroup of patients with high HRG mRNA levels ("HRG

  1. Relationship between hyperthyroidism and monoclonal gammapathy

    International Nuclear Information System (INIS)

    Canas, Carlos Alberto

    2007-01-01

    A 66-year-old man with primary hyperparathyroidism (PHPT) and monoclonal gammapathy associated to it of uncertain significance (MGUS). A possible pathogenic relationship between HPTP and MGUS is analyzed. Interleukin 6 could play a pivotal role.

  2. Monoclonal antibody therapy of inflammatory bowel disease

    NARCIS (Netherlands)

    van Deventer, S. J.; Camoglio, L.

    1997-01-01

    Animal models of inflammatory bowel disease have provided insight in the regulation of mucosal inflammation. This has resulted in novel therapeutic approaches that specifically target a single inflammatory mediator. Monoclonal antibody therapy has been used in steroid refractory Crohn's disease

  3. Cuban Monoclonal Antibodies for Radioimmunodiagnosis and Radioimmunotherapy of Cancer Diseases

    International Nuclear Information System (INIS)

    Casaco, A.

    2009-01-01

    The Centre of Molecular Immunology produces monoclonal antibodies for treating cancer diseases. We are mainly focus on two target systems; one is the epidermal growth factor receptor (EGF-R) because there is a tremendous relationship between the EGF/EGF-R system and several human tumours such as lung, head and neck, ovarian breast and brain cancers; the second one is the ganglioside system, the relevance of certain gangliosides in tumour growth and metastatic dissemination has been well documented, GM3(NeuGc) ganglioside is particularly interesting due to its restrictive expression in normal human tissues. Nimotuzumab (h-R3) is a humanized monoclonal antibody (mAb) that was obtained by complementarity-determining regions grafting of a murine mAb (ior egf/r3) to a human framework having remarkable antiproliferative, pro-apoptotic, and antiangiogenic effects. A Phase I clinical trial was performed to evaluate the toxicity and clinical effect of an intracavitary (intracerebral) administration of a single dose of nimotuzumab (h-R3) labelled with increasing doses of 188Re. All patients bearing astrocytomas grade III/IV should be treated previously with conventional therapies and have an EGF-R overexpression in the tumour, demonstrated by immunohistochemical study. Maximal tolerated dose was 3 mg of the h-R3 labelled with 10 mCi of 188 Re. The radioimmunoconjugate showed a high retention in the surgical created resection cavity and the brain adjacent tissues with a mean value of 85.5% of the injected dose one hour post-administration. This radioimmunoconjugate may be relatively safe and a promising therapeutic approach for treating high grade gliomas. GM3(NeuGc) ganglioside is particularly interesting due to its restrictive expression in normal human tissues according to immunohistochemical studies, using either polyclonal or monoclonal antibodies. But both immunohistochemical and biochemical methods have strongly suggested its over-expression in human breast and colon

  4. Breast cancer imaging with mouse monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Major, P.; Wang Taqui; Unger, M.; Rosenthall, L.

    1989-10-01

    The localization of /sup 111/In-labelled MA5 monoclonal antibody, reactive with a breast tumor associated antigen, was studied in 17 patients. MA5 was selected because (1) it reacts with >95% of primary and metastatic lesions, (2) the recognized antigen is present on the cell surface in vivo and (3) MA5 gives excellent localization in human breast tumor xenografts. Each patient received 2 mg antibody labeled with 5 mCi /sup 111/In and in some cases, 3 mg or 18 mg unlabeled carrier antibody. No serious allergic reactions were noted. There was a large uptake in the liver, less significant uptake in the spleen and bone and minimal accumulation in the bowel. Bone lesions, primary tumors, soft tissue recurrences and lung metastases larger than 3 cm diameter were imaged, while only 1 lesion smaller than 3 cm was detected. Non specific accumulation of tracer was noted at the site of a port-a-cath, in a hematoma, in fibrocystic lesions, and at sites of previous radiation treatment. Extensive fibrosis and poor vascularization characteristic of breast tumors may explain in part the limited sensitivity of the imaging. (orig.).

  5. Monoclonal for cancer detection and therapy

    International Nuclear Information System (INIS)

    Baldwin, R.W.; Byers, V.S.

    1985-01-01

    This book contains 18 chapters. Some of the chapter titles are: Monoclonal Antibodies to Breast Cancer and Their Application; Clinical Applications of Radioimmunolocalisation; Localisation of Cancer of the Ovary and Metastases Using 123 I-labelled Monoclonal Antibody HMFG-2 Compared to Surgical Findings; Interest of Globotriaosylceramide Membrane Antigen as Target for Immunotoxins; and Analysis, Results and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy

  6. 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.

  7. 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

  8. Application of 99mTc-labeled chimeric Fab fragments of monoclonal antibody A7 for radioimmunoscintigraphy of pancreatic cancer

    International Nuclear Information System (INIS)

    Matsumura, Hiroomi

    1999-01-01

    Pancreatic cancer is one of the most lethal diseases and its prognosis is still poor. To improve the survival rate, it is essential to develop new technologies for early and definitive diagnosis. In this study, chimeric Fab fragments of monoclonal antibody A7 were successfully radio-labeled with 99m Tc, preventing depression of the antigen-binding activity. 99m Tc-labeled monoclonal antibody A7, 99m Tc-labeled chimeric Fab fragments of monoclonal antibody A7, 99m Tc-labeled normal mouse IgG and 99m Tc-labeled Fab fragments of normal mouse IgG were injected intravenously into nude mice bearing human pancreatic cancer xenografts and the radioactivity was subsequently measured. The tumor accumulation was significantly higher with labeled monoclonal antibody A7 than with normal mouse IgG, and higher with chimeric Fab fragments of monoclonal antibody A7 than with Fab fragments of normal mouse IgG. The tumor/blood ratio of radioactivity increased rapidly over time with chimeric Fab fragments of monoclonal antibody A7. These results suggest that chimeric Fab fragments of monoclonal antibody A7 may be useful for diagnosing pancreatic cancer by means of radioimmunoscintigraphy. (author)

  9. Behavioral and neural effects of intra-striatal infusion of anti-streptococcal antibodies in rats

    Science.gov (United States)

    Lotan, Dafna; Benhar, Itai; Alvarez, Kathy; Mascaro-Blanco, Adita; Brimberg, Lior; Frenkel, Dan; Cunningham, Madeleine W.; Joel, Daphna

    2014-01-01

    Group A β-hemolytic streptococcal (GAS) infection is associated with a spectrum of neuropsychiatric disorders. The leading hypothesis regarding this association proposes that a GAS infection induces the production of auto-antibodies, which cross-react with neuronal determinants in the brain through the process of molecular mimicry. We have recently shown that exposure of rats to GAS antigen leads to the production of anti-neuronal antibodies concomitant with the development of behavioral alterations. The present study tested the causal role of the antibodies by assessing the behavior of naïve rats following passive transfer of purified antibodies from GAS-exposed rats. Immunoglobulin G (IgG) purified from the sera of GAS-exposed rats was infused directly into the striatum of naïve rats over a 21-day period. Their behavior in the induced-grooming, marble burying, food manipulation and beam walking assays was compared to that of naïve rats infused with IgG purified from adjuvant-exposed rats as well as of naïve rats. The pattern of in vivo antibody deposition in rat brain was evaluated using immunofluorescence and colocalization. Infusion of IgG from GAS-exposed rats to naïve rats led to behavioral and motor alterations partially mimicking those seen in GAS-exposed rats. IgG from GAS-exposed rats reacted with D1 and D2 dopamine receptors and 5HT-2A and 5HT-2C serotonin receptors in vitro. In vivo, IgG deposits in the striatum of infused rats colocalized with specific brain proteins such as dopamine receptors, the serotonin transporter and other neuronal proteins. Our results demonstrate the potential pathogenic role of autoantibodies produced following exposure to GAS in the induction of behavioral and motor alterations, and support a causal role for autoantibodies in GAS-related neuropsychiatric disorders. PMID:24561489

  10. Production of anti-streptococcal liamocins from agricultural biomass by Aureobasidium pullulans.

    Science.gov (United States)

    Leathers, Timothy D; Price, Neil P J; Manitchotpisit, Pennapa; Bischoff, Kenneth M

    2016-12-01

    Liamocins are unique heavier-than-water "oils" produced by certain strains of the fungus Aureobasidium pullulans. Liamocins have antibacterial activity with specificity for Streptococcus sp. Previous studies reported that liamocin yields were highest from strains of A. pullulans belonging to phylogenetic clades 8, 9, and 11, cultured on medium containing sucrose. In this study, 27 strains from these clades were examined for the first time for production of liamocins from agricultural biomass substrates. Liamocin yields were highest from strains in phylogenetic clade 11, and yields were higher from cultures grown on sucrose than from those grown on pretreated wheat straw. However, when supplementary enzymes (cellulase, β-glucosidase, and xylanase) were added, liamocin production on pretreated wheat straw was equivalent to that on sucrose. Liamocins produced from wheat straw were free of the melanin contamination common in sucrose-grown cultures. Furthermore, MALDI-TOF MS analysis showed that liamocins produced from wheat straw were under-acetylated, resulting in higher proportions of the mannitol A1 and B1 species of liamocin, the latter of which has the highest biological activity against Streptococcus sp.

  11. Isolation and characterization of a monoclonal anti-protein kinase CK2 beta-subunit antibody of the IgG class for the direct detection of CK2 beta-subunit in tissue cultures of various mammalian species and human tumors

    DEFF Research Database (Denmark)

    Nastainczyk, W; Schmidt-Spaniol, I; Boldyreff, B

    1995-01-01

    A murine monoclonal anti-protein kinase CK2 beta antibody was isolated and characterized. The antibody detects 1 pmol of purified recombinant CK2 beta-subunit after analysis on SDS-PAGE. Alternatively undenatured CK2 beta-subunit was detected by an ELISA assay either as recombinant CK2 beta......-subunit or in the CK2 holoenzyme (alpha 2 beta 2). Here, concentrations of the first antibody of 1 ng/ml still allowed the detection of the subunit. Immunoblotting of crude cellular extracts from various tissue cultures (man, mouse, and hamster), from human tumors, and the nonneoplastic tissue allowed the detection...... of the CK2 beta-subunit. The detected epitope of this antibody was, as determined by the epitope analysis technique, 123GLSDI127....

  12. Tumor detection using radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

    Moldofsky, P.J.; Powe, J.; Hammond, N.D.

    1987-01-01

    Radioisotope conjugated to monoclonal antibody products has been used for imaging tumors targeted by the antibody. As imaging progresses, new sets of procedural and technical questions arise. In this chapter, we discuss several current problems in imaging tumor with radiolabeled monoclonal antibody. These include (1) methods for selection of specific antibody and, once the particular antibody is selected, which fragment form is to be used; (2) imaging procedures: what are the optimum imaging parameters, such as optimum time for imaging after administration of tracer and considerations regarding background subtraction; and (3) noninvasive quantitative techniques: quantitation of localization of antibody indirectly from quantitative information in the images.100 references

  13. 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.

  14. 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...... antibody, for psoriasis treatment....

  15. Porcine humoral immune responses to multiple injections of murine monoclonal antibodies

    DEFF Research Database (Denmark)

    Lohse, Louise; Nielsen, Jens; Kamstrup, Søren

    2005-01-01

    In humans and cattle, multiple injections of murine monoclonal antibodies (m-mAbs) induce anti-mouse antibody responses. The objectives of the present. study were to investigate whether a similar response could be seen when pigs were subjected to m-mAb therapy, and to study the kinetics of such a...

  16. Monoclonal Antibody Fragments for Targeting Therapeutics to Growth Plate Cartilage | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    Researchers at The Eunice Kennedy Shriver National Institute on Child Health and Human Development (NICHD) have discovered monoclonal antibodies that bind to matrilin-3, a protein specifically expressed in cartilage tissue, that could be used for treating or inhibiting growth plate disorders, such as a skeletal dysplasia or short stature. The monoclonal antibodies can also be used to target therapeutic agents, such as anti-arthritis agents, to cartilage tissue. NICHD seeks statements of capability or interest from parties interested in collaborative research to co-develop, evaluate, or commercialize treatment of skeletal disorders using targeting antibodies.

  17. 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.

  18. 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.

  19. Improved radioimaging and tumor localization with monoclonal F(ab')2

    International Nuclear Information System (INIS)

    Wahl, R.L.; Parker, C.W.; Philpott, G.W.

    1983-01-01

    Monoclonal anti-tumor antibodies have great promise for radioimmunodetection and localization of tumors. Fab and F(ab')2 fragments, which lack the Fc fragment of antibody (Ab), are cleared more rapidly from the circulation and may have less nonspecific tissue binding than intact Ab. In radioimaging studies using a murine monoclonal antibody to carcinoembryonic antigen in a human colon carcinoma xenografted into hamsters, F(ab')2 fragments were shown superior to Fab fragments and intact antibody for scintiscanning. In double-label experiments with anti-CEA antibody and control monoclonal IgG, F(ab')2 fragments were found to give better and more rapid specific tumor localization than intact antibody or Fab fragments. F(ab')2 fragments offer significant promise for tumor imaging and possibly therapy

  20. Radiolabeled monoclonal antibodies for imaging and therapy: Potential, problems, and prospects: Scientific highlights

    International Nuclear Information System (INIS)

    Srivastava, S.C.; Buraggi, G.L.

    1986-01-01

    This meeting focused on areas of research on radiolabeled monoclonal antibodies. Topics covered included the production, purification, and fragmentation of monoclonal antibodies and immunochemistry of hybridomas; the production and the chemistry of radionuclides; the radiohalogenation and radiometal labeling techniques; the in-vivo pharmacokinetics of radiolabeled antibodies; the considerations of immunoreactivity of radiolabeled preparations; the instrumentation and imaging techniques as applied to radioimmunodetection; the radiation dosimetry in diagnostic and therapeutic use of labeled antibodies; the radioimmunoscintigraphy and radioimmunotherapy studies; and perspectives and directions for future research. Tutorial as well as scientific lectures describing the latest research data on the above topics were presented. Three workshop panels were convened on ''Methods for Determining Immunoreactivity of Radiolabeled Monoclonal Antibodies - Problems and Pitfalls,'' Radiobiological and Dosimetric Considerations for Immunotherapy with Labeled Antibodies,'' and ''The Human Anti-Mouse Antibody Response in Patients.''

  1. Comparative tumour localization properties of radiolabelled monoclonal antibody preparations of defined immunoreactivities

    International Nuclear Information System (INIS)

    Pimm, M.V.; Baldwin, R.W.

    1987-01-01

    The immunoreactive fraction of an anti-CEA monoclonal antibody preparation has been progressively decreased by the addition of increasing proportions of impurity in the form of immunologically inert mouse immunoglobulin. Following radioiodination, the immunoreactive fractions of the preparations were determined and their localization in a human tumour xenograft in nude mice was assessed. There was a progressive decline in tumour localization, from tumour to blood ratios of 2:1 with unadulterated antibody to 0.6:1 with preparations only 15% with respect to the initial antibody. These findings demonstrate that the immunoreactive fraction of monoclonal antibody preparations is a major limiting factor in tumour localization and this has implications for experimental and clinical applications of monoclonal antibodies. (orig.)

  2. The development of methods for obtaining monoclonal antibody-producing cells

    Directory of Open Access Journals (Sweden)

    Michał Skowicki

    2016-04-01

    Full Text Available Monoclonal antibodies (mAbs are biomolecules of great scientific and practical significance. In contrast to polyclonal antibodies from immune sera, they are homogeneous and monospecific, since they are produced by hybridoma cells representing a clone arising from a single cell. The successful technology was described for the first time in 1975; the inventors were later awarded the Nobel Prize. Currently, mAbs are broadly used as a research tool, in diagnostics and medicine in particular for the treatment of cancer or in transplantology. About 47 therapeutics based on monoclonal antibodies are now available in the US and Europe, and the number is still growing. Production of monoclonal antibodies is a multistage, time-consuming and costly process. Growing demand for these molecules creates space for research focused on improvements in hybridoma technology. Lower costs, human labor, and time are important goals of these attempts. In this article, a brief review of current methods and their advances is given.

  3. Generation of a Monoclonal Antibody against Mycoplasma spp. following Accidental Contamination during Production of a Monoclonal Antibody against Lawsonia intracellularis

    OpenAIRE

    Hwang, Jeong-Min; Lee, Ji-Hye; Yeh, Jung-Yong

    2012-01-01

    This report describes Mycoplasma contamination of Lawsonia intracellularis cultures that led to the unintended acquisition of a monoclonal antibody against Mycoplasma spp. during the attempted generation of a monoclonal antibody against L. intracellularis.

  4. Preparation of Ga-67 labeled monoclonal antibodies using deferoxamine as a bifunctional chelating agent

    International Nuclear Information System (INIS)

    Endo, K.; Furukawa, T.; Ohmomo, Y.

    1984-01-01

    Ga-67 labeled monoclonal IgG or F(ab')/sub 2/ fragments against α-fetoprotein and β-subunit of human choriogonadotropin (HCG), were prepared using Deferoxamine (DFO) as a bifunctional chelating agent. DFO, a well-known iron chelating agent, was conjugated with monoclonal antibodies (Ab) by a glutaraldehyde two step method and the effect of conjugation on the Ab activities was examined by RIA and Scatchard plot analysis. In both monoclonal Ab preparations, the conjugation reaction was favored as the pH increased. However, Ab-binding activities decreased as the molecular ratios of DFO to Ab increased. Preserved Ab activities were observed when Ab contained DFO per Ab molecule less than 2.1. At a ratio of over 3.3 DFO molecules per Ab, the maximal binding capacity rather than the affinity constant decreased. The inter-molecular cross linkage seemed to be responsible for the deactivation of binding activities. The obtained DFO-Ab conjugates, were then easily labeled with high efficiency and reproducibility and Ga-67 DFO-Ab complexes were highly stable both in vitro and in vivo. Thus, biodistribution of Ga-67 labeled F(ab')/sub 2/ fragments of monoclonal Ab to HCG β-subunit was attempted in nude mice transplanted with HCG-producing human teratocarcinoma. Tumor could be visualized, in spite of relatively high background imaging of liver, kidney and spleen. The use of DFO as a bifunctional chelating agent provided good evidence for its applicability to labeling monoclonal Ab with almost full retention of Ab activities. Further, availability of Ga-68 will make Ga-68 DFO-monoclonal Ab a very useful tool for positron tomography imaging of various tumors

  5. 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.

  6. Mass-Production and Characterization of Anti-CD20 Monoclonal Antibody in Peritoneum of Balb/c Mice

    Science.gov (United States)

    Sineh sepehr, Koushan; Baradaran, Behzad; Majidi, Jafar; Abdolalizadeh, Jalal; Aghebati, leili; Zare Shahneh, Fatemeh

    2013-01-01

    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. PMID:24312821

  7. 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

  8. 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.

  9. Monoclonal antibody technologies and rapid detection assays

    Science.gov (United States)

    Novel methodologies and screening strategies will be outlined on the use of hybridoma technology for the selection of antigen specific monoclonal antibodies. The development of immunoassays used for diagnostic detection of prions and bacterial toxins will be discussed and examples provided demonstr...

  10. Monoclonal antibody therapy of inflammatory bowel disease

    NARCIS (Netherlands)

    van Deventer, S. J.; Camoglio, L.

    1996-01-01

    Several anti-inflammatory drugs have therapeutic efficacy in inflammatory bowel disease, but their targets remain incompletely characterized. The development of monoclonal antibodies that either recognize epitopes on immune-competent cells, or neutralize pro-inflammatory cytokines, has helped to

  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. Enhancement by gamma-interferon of in vivo tumor radiolocalization by a monoclonal antibody against HLA-DR antigen

    International Nuclear Information System (INIS)

    Rowlinson, G.; Balkwill, F.; Snook, D.; Hooker, G.; Epenetos, A.A.

    1986-01-01

    Athymic nu/nu (nude) mice bearing s.c. human breast tumors were treated systemically with recombinant human gamma-interferon. These tumors were phenotypically negative for HLA-DR prior to therapy, but after 4 days of treatment, 80% of the cells expressed this antigen in vivo as assessed by immunoperoxidase (F. R. Balkwill et al., Eur. J. Cancer Clin. Oncol., in press, 1986). A radioiodine-labeled murine monoclonal antibody (TAL-1B5) against HLA-DR specifically localized to the tumors in recombinant human gamma-interferon-treated but not in control mice. An isotype-identical murine monoclonal antibody that did not react with control or recombinant human gamma-interferon-treated tumors did not show any specific localization. These results demonstrate that specific localization to tumors of radio-labeled monoclonal antibodies to HLA-DR can be facilitated by systemic therapy with gamma-interferon

  13. MOR103, a human monoclonal antibody to granulocyte–macrophage colony-stimulating factor, in the treatment of patients with moderate rheumatoid arthritis: results of a phase Ib/IIa randomised, double-blind, placebo-controlled, dose-escalation trial

    Science.gov (United States)

    Behrens, Frank; Tak, Paul P; Østergaard, Mikkel; Stoilov, Rumen; Wiland, Piotr; Huizinga, Thomas W; Berenfus, Vadym Y; Vladeva, Stoyanka; Rech, Juergen; Rubbert-Roth, Andrea; Korkosz, Mariusz; Rekalov, Dmitriy; Zupanets, Igor A; Ejbjerg, Bo J; Geiseler, Jens; Fresenius, Julia; Korolkiewicz, Roman P; Schottelius, Arndt J; Burkhardt, Harald

    2015-01-01

    Objectives To determine the safety, tolerability and signs of efficacy of MOR103, a human monoclonal antibody to granulocyte–macrophage colony-stimulating factor (GM-CSF), in patients with rheumatoid arthritis (RA). Methods Patients with active, moderate RA were enrolled in a randomised, multicentre, double-blind, placebo-controlled, dose-escalation trial of intravenous MOR103 (0.3, 1.0 or 1.5 mg/kg) once a week for 4 weeks, with follow-up to 16 weeks. The primary outcome was safety. Results Of the 96 randomised and treated subjects, 85 completed the trial (n=27, 24, 22 and 23 for pooled placebo and MOR103 0.3, 1.0 and 1.5 mg/kg, respectively). Treatment emergent adverse events (AEs) in the MOR103 groups were mild or moderate in intensity and generally reported at frequencies similar to those in the placebo group. The most common AE was nasopharyngitis. In two cases, AEs were classified as serious because of hospitalisation: paronychia in a placebo subject and pleurisy in a MOR103 0.3 mg/kg subject. Both patients recovered fully. In exploratory efficacy analyses, subjects in the MOR103 1.0 and 1.5 mg/kg groups showed significant improvements in Disease Activity Score-28 scores and joint counts and significantly higher European League Against Rheumatism response rates than subjects receiving placebo. MOR103 1.0 mg/kg was associated with the largest reductions in disease activity parameters. Conclusions MOR103 was well tolerated and showed preliminary evidence of efficacy in patients with active RA. The data support further investigation of this monoclonal antibody to GM-CSF in RA patients and potentially in those with other immune-mediated inflammatory diseases. Trial registration number NCT01023256 PMID:24534756

  14. Imaging of melanoma with 131I-labeled monoclonal antibodies

    International Nuclear Information System (INIS)

    Larson, S.M.; Brown, J.P.; Wright, P.W.; Carrasquillo, J.A.; Hellstroem, I.; Hellstroem, K.E.

    1983-01-01

    Mouse monoclonal antibodies and Fab fragments specific for p97, a melanoma-associated antigen, were used to image metastatic human melanoma. Preclinical studies in athymic mice showed antigen-specific uptake in melanoma xenografts, and toxicity tests in rabbits gave no evidence for tissue damage after injection of up to 100 times the amount of antibody used in humans. Six patients received 1 mg labeled antibody, and one patient received 1 mg of labeled Fab. No. toxic side effects were observed. All of the six patients had positive scans, visualizing 22 of 25 (88%) of lesions larger than 1.5 cm. In tumors from two patients, greater uptake of p97-specific, versus control IgG and Fab, respectively, was documented by biopsy. Antibodies to mouse immunoglobulin appeared in three patients receiving 1 mg or more of radiolabeled mouse antibody

  15. Primary hepatocellular carcinoma localised by a radiolabelled monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Markham, N; Ritson, A; James, O; Curtin, N; Bassendine, M; Sikora, K

    1986-01-01

    A rat monoclonal antibody, YPC2/38.8, was selected from a panel of antibodies derived by immunising rats with fresh human colorectal carcinoma. It was found to bind to a 30,000 dalton protein present on the cell surface of normal colon and liver. This protein was increased 10-fold on primary hepatocellular carcinoma (PHC) cells. After labelling with /sup 131/I, YPC2/38.8 was shown to localise human PHCs grown as xenografts in immunosuppressed mice. The authors conclude that YPC2/38.8 may have potential for diagnostic localisation and possibly thence for the selective targeting of drugs or toxins in patients with PHC arising in a liver unaffected by significant parenchymal disease. 16 refs.; 4 figs.; 1 table.

  16. 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.

  17. Monkey-derived monoclonal antibodies against Plasmodium falciparum

    International Nuclear Information System (INIS)

    Stanley, H.A.; Reese, R.T.

    1985-01-01

    A system has been developed that allows efficient production of monkey monoclonal antibodies from owl monkeys. Splenocytes or peripheral blood lymphocytes from monkeys immune to the human malarial parasite, Plasmodium falciparum, were fused with P3X63 Ag8.653 mouse myelomas. The resulting hybridomas were screened by an indirect fluorescent antibody test for the production of monkey monoclonal antibodies (mAb) reactive with P. falciparum. Most of the mAb reacted with the P. falciparum merozoites and immunoprecipitated a parasite-derived glycoprotein having a relative molecular weight of 185,000. These mAb gave a minimum of five different immunoprecipitation patterns, thus demonstrating that a large number of polypeptides obtained when parasitized erythrocytes are solubilized share epitopes with this large glycoprotein. In addition, mAb were obtained that reacted with antigens associated with the infected erythrocyte membrane. One of these mAb bound a M/sub r/ 95,000 antigen. Radioimmunoprecipitation assays using 125 T-antibodies were done

  18. Perfusion of tumor-bearing kidneys as a model for scintigraphic screening of monoclonal antibodies

    International Nuclear Information System (INIS)

    van Dijk, J.; Oosterwijk, E.; van Kroonenburgh, M.J.; Jonas, U.; Fleuren, G.J.; Pauwels, E.K.; Warnaar, S.O.

    1988-01-01

    Tumor-bearing human kidneys were used in an ex vivo perfusion model to screen monoclonal antibodies, recognizing renal cell carcinoma-associated antigens for diagnostic potential in vivo. Perfusion of tumor-bearing kidneys with /sup 99m/Tc-labeled G250 and RC38 antibody resulted in visualization of the tumor, whereas perfusion with two other monoclonal antibodies, RC2 and RC4, did not lead to tumor visualization. Uptake of radiolabel in normal kidney tissue was low for G250 and RC38 antibody. Tumor-to-kidney tissue ratios after perfusion with G250 and RC38 antibody were 2.7 and 2.2, respectively. After rinsing for 3 hr with unlabeled perfusion fluid the tumor-to-kidney tissue ratios increased to 8.6 for G250 antibody and to 2.7 for RC38 antibody. We conclude that perfusion of tumor-bearing human kidneys with radiolabeled monoclonal antibodies is a relatively simple way to evaluate renal cell carcinoma associated monoclonal antibodies as diagnostic agents in vivo

  19. Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase.

    Science.gov (United States)

    Jastreboff, M M; Todd, M B; Malech, H L; Bertino, J R

    1985-01-29

    Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.

  20. Ki-67 immunoreactivity in meningiomas--determination of the proliferative potential of meningiomas using the monoclonal antibody Ki-67

    DEFF Research Database (Denmark)

    Madsen, C; Schrøder, H D

    1997-01-01

    The proliferative potential of 66 human intracranial meningiomas (15 benign, 15 atypical, 15 recurrent, 13 bone-invasive, and 8 brain-invasive) was investigated by means of immunohisto-chemistry using the monoclonal antibody Ki-67. This antibody recognizes a nuclear antigen present in human cells...

  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. Quantitative imaging with radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

    Moldofsky, P.J.; Hammond, N.D.

    1988-01-01

    The ability to image tumor by using radiolabeled monoclonal antibody products has been widely demonstrated. The questions of safety and efficacy remain open and require further experience, but at least in some clinical situations radioimmunoimaging has provided clinically useful information. Imaging tumor with radiolabeled monoclonal and polyclonal antibodies has been widely reported, and several summaries have recently appeared. For extensive review of recent clinical imaging the reader is referred to these excellent sources. Having demonstrated the possibility of imaging tumor with radiolabeled antibody, the question now apparent is: will the imaging modality provide information new and different from the already available with established techniques in computed tomography, magnetic resonance imaging, and standard nuclear medicine?

  3. [Monoclonal antibodies in diagnosis of acute leukemias].

    Science.gov (United States)

    Krawczyńska, A; Robak, T

    1996-01-01

    Immunophenotyping has become an essential component for the study of acute myeloblastic (AML) and lymphoblastic (ALL) leukaemias. The recent development of highly specific monoclonal antibodies (Mc Ab) to differentiation antigens (CD) of haematopoetic cells have made it readily available to clinical laboratories in most major hospitals. Immunophenotyping complements standard morphology by providing information on lineage, stage of differentiation and clonality. In addition some of the flow cytometry findings have independent prognostic significance. Monoclonal antibodies useful in defining lineage (B-cell versus T-cell) and stages of differentiation of ALL. It can be also used in identifying characteristic feature of AML and aiding in lineage determination in acute leukaemias that are morphologically undifferentiated. Surface immunophenotyping is especially helpful for recognizing mixed lineage acute leukaemia and diagnosing certain rare entities such as erythroleukaemia (M6), acute megakaryocytic leukaemia (M7) and minimally differentiation acute myeloid leukaemia.

  4. Recent developments in monoclonal antibody radiolabeling techniques

    International Nuclear Information System (INIS)

    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

  5. Taking aim at cancer with monoclonal antibodies

    International Nuclear Information System (INIS)

    Klausner, A.

    1986-01-01

    Conjugating radioisotopes to monoclonal antibodies could have certain advantages in cancer therapy. Radioactive compounds have the double-edged ability to kill cells that are up to centimeter or more away. This is a plausible way to overcome tumor heterogeneity, but it also means that normal cells near the tumor could be affected. Hybritech (San Diego, CA) has been supplying antibody linked to the radioisotope yttrium-90 for a number of clinical trials. Work at Johns Hopkins University (Baltimore, MD) has focused on polyclonal antibodies to hepatoma. Monoclonal antibodies will be used there soon, and trials could be expanded eventually to include breast, lung, and prostate cancer as well. Hybritech also expects that the yttrium-antibody conjugates developed with NCI will enter the clinic later this year for treating leukemia and lymphoma systems; treatments for melanomas should follow

  6. Safety and immunotoxicity assessment of immunomodulatory monoclonal antibodies

    Science.gov (United States)

    Morton, Laura Dill; Spindeldreher, Sebastian; Kiessling, Andrea; Allenspach, Roy; Hey, Adam; Muller, Patrick Y; Frings, Werner; Sims, Jennifer

    2010-01-01

    Most therapeutic monoclonal antibodies (mAbs) licensed for human use or in clinical development are indicated for treatment of patients with cancer and inflammatory/autoimmune disease and as such, are designed to directly interact with the immune system. A major hurdle for the development and early clinical investigation of many of these immunomodulatory mAbs is their inherent risk for adverse immune-mediated drug reactions in humans such as infusion reactions, cytokine storms, immunosuppression and autoimmunity. A thorough understanding of the immunopharmacology of a mAb in humans and animals is required to both anticipate the clinical risk of adverse immunotoxicological events and to select a safe starting dose for first-in-human (FIH) clinical studies. This review summarizes the most common adverse immunotoxicological events occurring in humans with immunomodulatory mAbs and outlines non-clinical strategies to define their immunopharmacology and assess their immunotoxic potential, as well as reduce the risk of immunotoxicity through rational mAb design. Tests to assess the relative risk of mAb candidates for cytokine release syndrome, innate immune system (dendritic cell) activation and immunogenicity in humans are also described. The importance of selecting a relevant and sensitive toxicity species for human safety assessment in which the immunopharmacology of the mAb is similar to that expected in humans is highlighted, as is the importance of understanding the limitations of the species selected for human safety assessment and supplementation of in vivo safety assessment with appropriate in vitro human assays. A tiered approach to assess effects on immune status, immune function and risk of infection and cancer, governed by the mechanism of action and structural features of the mAb, is described. Finally, the use of immunopharmacology and immunotoxicity data in determining a minimum anticipated biologic effect Level (MABEL) and in the selection of safe human

  7. Application of a monoclonal antibody to a comparative study of alpha-lactalbumins from various species

    International Nuclear Information System (INIS)

    Kaminogawa, S.; Shimoda, M.; Kurisaki, J.; Yamauchi, K.

    1989-01-01

    A monoclonal antibody to bovine alpha-lactalbumin was prepared and purified. The binding ability of alpha-lactalbumin from different species (cow, goat, giraffe, horse, pig, human, monkey, and guinea pig) was examined by a competitive radioimmunoassay. The order in strength of the binding affinity was cow goat, giraffe, horse, cynomolgus monkey and human, pig, and guinea pig. The order of evolutional divergence calculated from the amino acid composition was cow, goat, giraffe, horse, pig, guinea pig and human, and monkey. The orders in both cases were similar. Hence, it is suggested that immunological divergence as deduced by a monoclonal antibody is likely to be close to the evolutional divergence of alpha-lactalbumin

  8. Lymphocyte targeting with /sup 111/In-labelled monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Loutfi, I.; Batchelor, J.R.; Lavender, J.P.; Epenetos, A.A.

    1988-01-01

    In vitro tests were conducted using human T and B cell lines, as well as whole blood, to establish the usefulness of 2 murine monoclonal antibodies (MAbs), an anti-CD5 (Pan T) and a Pan B, for potential radioimmunolocalization and therapy. Both MAbs showed specificity for the cell line in question as tested by indirect immunofluorescence and radioimmunoassay. Assays carried out on whole blood showed 40-70% of the added activity of /sup 111/In-labelled Pan B antibody binding to B cells and 20-24% of /sup 111/In-Pan T antibody binding to T cells. The amount of internalised /sup 111/In-labelled Pan B was 6% of total amount at 24 hr indicating a slow internalization process. These results should allow for in vivo targeting of normal and neoplastic B and T cells.

  9. Microbials for the production of monoclonal antibodies and antibody fragments.

    Science.gov (United States)

    Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph

    2014-01-01

    Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Iodination of monoclonal antibodies, proteins and peptide using iodogen

    Energy Technology Data Exchange (ETDEWEB)

    Zhanpo, Niu [Chinese Academy of Medical Sciences, Beijing, BJ (China). PUMC Hospital; and others

    1988-05-01

    The use of the iodinating reagent 1,3,4,6-tetrachloro-3{alpha}, 6{alpha}-diphenylglycholuril (Iodogen) to label monoclonal antibodies (McAbs). Proteins and peptides was invesrigated with McAbs identified as mouse IgG and IgM, arginine-vasopressin (AVP), glucagon (Glu), human insulin(hI) and albumin(Alb). The labeled products were purified by gel chromatography and their immunoreactivity were detected by RIA or IRMA> Comparison of the Iodogen method with the lactoperoxides and chloramine-T methods showed that the Iodogen method had a number of advantages: (1) technically simpler ; (2) a high labeling efficiency could be obtained; (3) the immunoreactivity of the products was minimally affected; (4) the products were stable for up to 4 months.

  11. Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody.

    Science.gov (United States)

    Hu, Francis Jingxin; Uhlen, Mathias; Rockberg, Johan

    2014-01-25

    One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

    DEFF Research Database (Denmark)

    Skjødt, K; Schou, C; Koch, C

    1984-01-01

    A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody. Using this technique we have been ...... I molecules. In other experiments using the same technique we demonstrated the reaction of a monoclonal antibody specific for chicken Ig light chains. Udgivelsesdato: 1984-Aug-3...

  13. Development and Characterization of Canine Distemper Virus Monoclonal Antibodies.

    Science.gov (United States)

    Liu, Yuxiu; Hao, Liying; Li, Xiangdong; Wang, Linxiao; Zhang, Jianpo; Deng, Junhua; Tian, Kegong

    2017-06-01

    Five canine distemper virus monoclonal antibodies were developed by immunizing BALB/c mice with a traditional vaccine strain Snyder Hill. Among these monoclonal antibodies, four antibodies recognized both field and vaccine strains of canine distemper virus without neutralizing ability. One monoclonal antibody, 1A4, against hemagglutinin protein of canine distemper virus was found to react only with vaccine strain virus but not field isolates, and showed neutralizing activity to vaccine strain virus. These monoclonal antibodies could be very useful tools in the study of the pathogenesis of canine distemper virus and the development of diagnostic reagents.

  14. MOR103, a human monoclonal antibody to granulocyte-macrophage colony-stimulating factor, in the treatment of patients with moderate rheumatoid arthritis: results of a phase Ib/IIa randomised, double-blind, placebo-controlled, dose-escalation trial.

    Science.gov (United States)

    Behrens, Frank; Tak, Paul P; Østergaard, Mikkel; Stoilov, Rumen; Wiland, Piotr; Huizinga, Thomas W; Berenfus, Vadym Y; Vladeva, Stoyanka; Rech, Juergen; Rubbert-Roth, Andrea; Korkosz, Mariusz; Rekalov, Dmitriy; Zupanets, Igor A; Ejbjerg, Bo J; Geiseler, Jens; Fresenius, Julia; Korolkiewicz, Roman P; Schottelius, Arndt J; Burkhardt, Harald

    2015-06-01

    To determine the safety, tolerability and signs of efficacy of MOR103, a human monoclonal antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF), in patients with rheumatoid arthritis (RA). Patients with active, moderate RA were enrolled in a randomised, multicentre, double-blind, placebo-controlled, dose-escalation trial of intravenous MOR103 (0.3, 1.0 or 1.5 mg/kg) once a week for 4 weeks, with follow-up to 16 weeks. The primary outcome was safety. Of the 96 randomised and treated subjects, 85 completed the trial (n=27, 24, 22 and 23 for pooled placebo and MOR103 0.3, 1.0 and 1.5 mg/kg, respectively). Treatment emergent adverse events (AEs) in the MOR103 groups were mild or moderate in intensity and generally reported at frequencies similar to those in the placebo group. The most common AE was nasopharyngitis. In two cases, AEs were classified as serious because of hospitalisation: paronychia in a placebo subject and pleurisy in a MOR103 0.3 mg/kg subject. Both patients recovered fully. In exploratory efficacy analyses, subjects in the MOR103 1.0 and 1.5 mg/kg groups showed significant improvements in Disease Activity Score-28 scores and joint counts and significantly higher European League Against Rheumatism response rates than subjects receiving placebo. MOR103 1.0 mg/kg was associated with the largest reductions in disease activity parameters. MOR103 was well tolerated and showed preliminary evidence of efficacy in patients with active RA. The data support further investigation of this monoclonal antibody to GM-CSF in RA patients and potentially in those with other immune-mediated inflammatory diseases. NCT01023256. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  15. Effect of producer cell line on functional activity of anti-D monoclonal antibodies destined for prevention of rhesus sensitization.

    Science.gov (United States)

    Olovnikova, N I; Ershler, M A; Belkina, E V; Nikolaeva, T L; Miterev, G Yu

    2009-04-01

    The ability of anti-D antibodies to cause antigen-specific immunosuppression depends on their interaction with low-affinity Fcgamma-receptors. Human monoclonal antibodies to D antigen of the rhesus system were investigated by antibody-dependent cytotoxicity assay in order to estimate their ability to induce hemolysis mediated by low-affinity Fcgamma receptors. We demonstrate that affinity of monoclonal antibodies to receptors of this type does not depend on primary structure of Fc-fragment, but depends on the producer cell line which expresses the antibodies. Monoclonal IgG1 antibodies interacting with FcgammaRIIa and FcgammaRIII lost this property, if they were secreted by human-mouse heterohybridoma, but not by human B-cell line. On the opposite, monoclonal antibodies that could not activate low-affinity Fcgamma receptors were highly active after human cells fusion with rat myeloma YB2/0. Hemolytic activity of IgG3 remained unchanged after fusion of human cells with rodent cells.

  16. Monoclonal Idiotope Vaccine against Streptococcus pneumoniae Infection

    Science.gov (United States)

    McNamara, Mary K.; Ward, Ronald E.; Kohler, Heinz

    1984-12-01

    A monoclonal anti-idiotope antibody coupled to a carrier protein was used to immunize BALB/c mice against a lethal Streptococcus pneumoniae infection. Vaccinated mice developed a high titer of antibody to phosphorylcholine, which is known to protect against infection with Streptococcus pneumoniae. Measurement of the median lethal dose of the bacteria indicated that anti-idiotope immunization significantly increased the resistance of BALB/c mice to the bacterial challenge. Antibody to an idiotope can thus be used as an antigen substitute for the induction of protective immunity.

  17. Target antigens for Hs-14 monoclonal antibody and their various expression in normozoospermic and asthenozoospermic men

    Czech Academy of Sciences Publication Activity Database

    Čapková, Jana; Margaryan, Hasmik; Kubátová, Alena; Novák, Petr; Pěknicová, Jana

    2015-01-01

    Roč. 25, č. 11 (2015) ISSN 2051-4190 R&D Projects: GA ČR(CZ) GAP503/12/1834; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 ; RVO:61388971 Keywords : acrosome * human spermatozoa * monoclonal antibody * asthenozoospermia * transitional endoplasmic reticulum ATPase Subject RIV: CE - Biochemistry http://link.springer.com/article/10.1186/s12610-015-0025-0

  18. Rapid Identification of Dengue Virus Serotypes Using Monoclonal Antibodies in an Indirect Immunofluorescence Test.

    Science.gov (United States)

    1982-06-18

    encephalitis(TBH-28), West Nile(E-101), Yellow fever(French neurotropic and 17D strains), and Zika . Two Sandfly Fever viruses (213452 and Candiru) were...were provided as first passage isolates ( Aedes pseudoscutellaris cells, AP-61) or human serum from recent dengue virus patients. African isolates... viruses of the Phlebovirus genus (Table 1). Several monoclonal antibody preparations reacted solely with dengue virus serotypes. Two preparations (13E7 and

  19. 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). PMID:24611141

  20. Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts

    International Nuclear Information System (INIS)

    Flier, J.S.; Usher, P.; Moses, A.C.

    1986-01-01

    Insulin and insulin-like growth factor type I (IGF-I) stimulate an overlapping spectrum of biological responses in human skin fibroblasts. Although insulin and IGF-I are known to stimulate the incorporation of [ 3 H]thymidine into DNA in these cells, the identify of the receptor(s) that mediates this effect has not been fully clarified. The mouse anti-human IGF-I receptor antibody αIR-3 binds with specificity to IGF-I but not to insulin receptors in human placental membranes; it also specifically inhibits the binding of 125 I-labeled IGF-I but not 125 I-labeled insulin to suspensions of human skin fibroblasts in a dose-dependent manner. αIR-3 competitively inhibits IGF-I-mediated stimulation of [ 3 H]thymidine incorporation into DNA. This inhibition is dependent on the concentration of αIR-3 and in the presence of a fixed antibody concentration can be partially overcome by high concentrations of IGF-I. In contrast, at concentrations of 3 H]thymidine incorporation is not inhibited by αIR-3. However, the incremental effects of higher concentrations (> 1 μg/ml) of insulin on [ 3 H]thymidine incorporation are inhibited by αIR-3. αIR-3 is a highly specific antagonist of IGF-I receptor-mediated mitogenesis in human skin fibroblasts. By using this antibody, it is shown directly that insulin can act through the IGF-I receptor to stimulate DNA synthesis but can also activate this effect through the insulin receptor itself

  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. Kinetics of intralymphatically delivered monoclonal antibodies

    International Nuclear Information System (INIS)

    Wahl, R.L.; Geatti, O.; Liebert, M.; Beers, B.; Jackson, G.; Laino, L.; Kronberg, S.; Wilson, B.S.; Beierwaltes, W.H.

    1985-01-01

    Radiolabeled monoclonal antibody (MoAb) administration subcutaneously (sq), so that preferential uptake is to the lymphatics, holds significant promise for the detection of lymph node metastases. Only limited information is available about clearance rates of intralymphatically administered MoAbs. I-131 labeled intact IgG (225.28S), F(ab's)2 (225.28S) or IgM (FT162) were administered sq to anesthetized Balb/C mice. Eight mice were studied with each MoAb, 4 with a foot-pad injection, 4 with an anterior abdominal injection. Gamma camera images were collected into a computer, over the first 6 hrs after injection with the animals anesthetized and immobile. Animals were then allowed to move about freely. Additional images were then acquired out to 48 hrs. Regions of interest wre selected over the injection site and the kinetics of antibody egress determined. Clearance rates from local sq injection sites are influenced by motion and somewhat by location. The class and fragment status of the MoAb appear relatively less important in determining clearance rates from sq injections than they are in determining whole-body clearance after iv injections. Additional studies using Fab fragments and additional monoclonals will be useful in extending these observations

  3. Bone marrow dosimetry for monoclonal antibody therapy

    International Nuclear Information System (INIS)

    Bigler, R.E.; Zanzonico, P.B.; Leonard, R.

    1986-01-01

    Immunoglobulins must permeate through the basement membrane of capillaries in order to enter the extracellular space (ECS) of tissue. Since the process is quite slow, the blood plasma activity in various organs contributes considerably to the radiation dose of the dose-limiting tissues. In bone marrow the basement membrane is absent and the blood circulation is functionally open. Therefore, blood plasma and marrow ECS maintain equal concentrations of labeled immunoglobulins. A combination of factors including intravenous administration, slow absorption into most tissues, slow breakdown and elimination of labeled immunoglobulin, and rapid entry into bone marrow ECS as well as known radiosensitivity of marrow led the authors to expect this tissue would prove to be the primary tissue at risk for systemic monoclonal antibody therapy. They have developed and applied in a Phase I clinical study of 131 I labeled CEA antibody a procedure for estimation of radiation dose to red bone marrow. Serieal measurements of blood plasma and total body retention are carried out. Binding of labeled antibody to the cellular components of blood is verified to be very low. They have observed bone marrow depression at doses greater than 400 rad. If no special procedures are used to reconstitute marrow after radiation treatment, this level represents a much greater than generally recognized limitation to radiolabeled monoclonal antibody therapy. 25 references, 4 tables

  4. Growth inhibition of tumor cells in vitro by using monoclonal antibodies against gonadotropin-releasing hormone receptor.

    Science.gov (United States)

    Lee, Gregory; Ge, Bixia

    2010-07-01

    As the continuation of a previous study, synthetic peptides corresponding to the extracellular domains of human gonadotropin-releasing hormone (GnRH) receptor were used to generate additional monoclonal antibodies which were further characterized biochemically and immunologically. Among those identified to recognize GnRH receptor, monoclonal antibodies designated as GHR-103, GHR-106 and GHR-114 were found to exhibit high affinity (Kd L37), when cancer cells were incubated with GnRH or GHR-106. The widespread expressions of GnRH receptor in almost all of the studied human cancer cell lines were also demonstrated by RT-PCR and Western blot assay, as well as indirect immunofluorescence assay with either of these monoclonal antibodies as the primary antibody. In view of the longer half life of antibodies as compared to that of GnRH or its analogs, anti-GnRH receptor monoclonal antibodies in humanized forms could function as GnRH analogs and serve as an ideal candidate of anti-cancer drugs for therapeutic treatments of various cancers in humans as well as for fertility regulations.

  5. Monoclonal antibody-assisted structure-function analysis of the carbohydrate recognition domain of surfactant protein D

    DEFF Research Database (Denmark)

    Hartshorn, Kevan L; White, Mitchell R; Rynkiewicz, Michael

    2010-01-01

    Surfactant protein D (SP-D) plays important roles in host defense against a variety of pathogens including influenza A virus (IAV). Ligand binding by SP-D is mediated by the trimeric neck and carbohydrate recognition domain (NCRD). We used monoclonal antibodies (mAbs) against human SP-D and a panel...

  6. Monoclonal carcinoembryonic antigen radioimmunoassay in prostatic cancer: Validation of the method and comparison to some other tumor-associated markers

    International Nuclear Information System (INIS)

    Stefanovic, V.; Ignjatovic, M.; Milosavljevic, B.; Dinic, A.; Nis Univ.

    1987-01-01

    The use of a monoclonal CEA RIA and of some other biological markers for a diagnosis of prostatic cancer was investigated. The increased level of serum CEA was found in both prostatic cancer and in non-malignant disease. The low sensitivity of the CEA monoclonal assay precludes its use for a clinical diagnosis of prostatic cancer. The simultaneous use of some other biological markers (PAP, TPA, β2-microglobulin and ferritin) did increase sensitivity. However, further studies should be directed to a much more specific and sensitive marker of the human prostatic adenocarcinoma. (orig.) [de

  7. Monoclonal carcinoembryonic antigen radioimmunoassay in prostatic cancer: Validation of the method and comparison to some other tumor-associated markers

    Energy Technology Data Exchange (ETDEWEB)

    Stefanovic, V; Ignjatovic, M; Milosavljevic, B; Dinic, A

    1987-04-01

    The use of a monoclonal CEA RIA and of some other biological markers for a diagnosis of prostatic cancer was investigated. The increased level of serum CEA was found in both prostatic cancer and in non-malignant disease. The low sensitivity of the CEA monoclonal assay precludes its use for a clinical diagnosis of prostatic cancer. The simultaneous use of some other biological markers (PAP, TPA, ..beta..2-microglobulin and ferritin) did increase sensitivity. However, further studies should be directed to a much more specific and sensitive marker of the human prostatic adenocarcinoma.

  8. Biotechnology and genetic engineering in the new drug development. Part II. Monoclonal antibodies, modern vaccines and gene therapy.

    Science.gov (United States)

    Stryjewska, Agnieszka; Kiepura, Katarzyna; Librowski, Tadeusz; Lochyński, Stanisław

    2013-01-01

    Monoclonal antibodies, modern vaccines and gene therapy have become a major field in modern biotechnology, especially in the area of human health and fascinating developments achieved in the past decades are impressive examples of an interdisciplinary interplay between medicine, biology and engineering. Among the classical products from cells one can find viral vaccines, monoclonal antibodies, and interferons, as well as recombinant therapeutic proteins. Gene therapy opens up challenging new areas. In this review, a definitions of these processes are given and fields of application and products, as well as the future prospects, are discussed.

  9. A monoclonal antibody that specifically recognizes m6A nucleoside

    OpenAIRE

    Espuny, Ruth; Castro, Ana; Codony, Carles; Eritja Casadellà, Ramón; Bach-Elias, Montse

    1998-01-01

    A hybridoma against the nucleoside m6A has been obtained from mouse spleen. This hybridoma was named H65 and it secretes monoclonal antibodies anti-m6A. The competition assays showed that the monoclonal antibody was highly specific for m6A nucleoside.

  10. A single dose of a neuron-binding human monoclonal antibody improves brainstem NAA concentrations, a biomarker for density of spinal cord axons, in a model of progressive multiple sclerosis.

    Science.gov (United States)

    Wootla, Bharath; Denic, Aleksandar; Watzlawik, Jens O; Warrington, Arthur E; Rodriguez, Moses

    2015-04-29

    Intracerebral infection of susceptible mouse strains with Theiler's murine encephalomyelitis virus (TMEV) results in chronic demyelinating disease with progressive axonal loss and neurologic dysfunction similar to progressive forms of multiple sclerosis (MS). We previously showed that as the disease progresses, a marked decrease in brainstem N-acetyl aspartate (NAA; metabolite associated with neuronal integrity) concentrations, reflecting axon health, is measured. We also demonstrated stimulation of neurite outgrowth by a neuron-binding natural human antibody, IgM12. Treatment with either the serum-derived or recombinant human immunoglobulin M 12 (HIgM12) preserved functional motor activity in the TMEV model. In this study, we examined IgM-mediated changes in brainstem NAA concentrations and central nervous system (CNS) pathology. (1)H-magnetic resonance spectroscopy (MRS) showed that treatment with HIgM12 significantly increased brainstem NAA concentrations compared to controls in TMEV-infected mice. Pathologic analysis demonstrated a significant preservation of axons in the spinal cord of animals treated with HIgM12. This study links drug efficacy of slowing deficits with axon preservation and NAA concentrations in the brainstem in a model of progressive MS. HIgM12-mediated changes of NAA concentrations in the brainstem are a surrogate marker of axon injury/preservation throughout the spinal cord. This study provides proof-of-concept that a neuron-reactive human IgM can be therapeutic and provides a biomarker for clinical trials.

  11. Treatment with anti-interferon-δ monoclonal antibodies modifies experimental autoimmune encephalomyelitis in interferon-δ receptor knockout mice

    DEFF Research Database (Denmark)

    Espejo, C.; Penkowa, Milena; Saez-Torres, I.

    2001-01-01

    Neuroinflammation, neuronal degeneration, regeneration, monoclonal