Sample records for therapeutic antibody catumaxomab

  1. Catumaxomab (United States)

    Linke, Rolf; Seimetz, Diane


    Catumaxomab, a monoclonal bispecific trifunctional antibody, was approved in the european Union in April 2009 for the intraperitoneal treatment of patients with malignant ascites. The marketing authorization holder Fresenius Biotech GmbH developed catumaxomab (Removab®) together with its partner TRiOn Pharma GmbH, Germany. it is the first substance worldwide with a regulatory label for the treatment of malignant ascites due to epithelial carcinomas. Since the peritoneum is of mesothelial origin and therefore lacks epCAM expression, the intraperitoneal administration of catumaxomab is an attractive targeted immunotherapeutic approach. Catumaxomab is able to destroy epCAM positive tumor cells in the peritoneal cavity known as the main cause of malignant ascites. in addition, catumaxomab is a potential therapeutic option for several primary tumors since the epCAM molecule is expressed on the majority of epithelial carcinomas. This review focuses on the clinical development of catumaxomab and indicates future directions. PMID:20190561

  2. Antibody Engineering and Therapeutics (United States)

    Almagro, Juan Carlos; Gilliland, Gary L; Breden, Felix; Scott, Jamie K; Sok, Devin; Pauthner, Matthias; Reichert, Janice M; Helguera, Gustavo; Andrabi, Raiees; Mabry, Robert; Bléry, Mathieu; Voss, James E; Laurén, Juha; Abuqayyas, Lubna; Barghorn, Stefan; Ben-Jacob, Eshel; Crowe, James E; Huston, James S; Johnston, Stephen Albert; Krauland, Eric; Lund-Johansen, Fridtjof; Marasco, Wayne A; Parren, Paul WHI; Xu, Kai Y


    The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates. PMID:24589717

  3. Therapeutic Recombinant Monoclonal Antibodies (United States)

    Bakhtiar, Ray


    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…

  4. Palliative treatment of malignant ascites: profile of catumaxomab

    Directory of Open Access Journals (Sweden)

    Lila Ammouri


    Full Text Available Lila Ammouri, Eric E PrommerMayo Clinic Hospice and Palliative Medicine Program, Mayo Clinic College of Medicine, Mayo Clinic Hospital, Scottsdale, AZ, USAAbstract: Malignant ascites is the abnormal accumulation of fluid in the peritoneal cavity associated with several intrapelvic and intra-abdominal malignancies. The development of ascites leads to significant symptoms and poor quality of life for the cancer patient. Available therapies for palliation include treatment of the underlying disease, but when there are no treatment options, the use of diuretics, implantation of drainage catheters, and surgical shunting techniques are considered. None of these symptom palliation options affect the course of disease. The development of trifunctional antibodies, which attach to specific overexpressed surface markers on tumor cells, and trigger an immune response leading to cytoreductive effects, represents a new approach to the management of malignant ascites. The purpose of this review is to highlight current therapies for malignant ascites and review data as to the effectiveness of a new trifunctional antibody, catumaxomab.Keywords: catumaxomab, ascites, trifunctional

  5. Avian Diagnostic and Therapeutic Antibodies

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    Bradley, David Sherman [UND SMHS


    A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.

  6. Treatment of gastric peritoneal carcinomatosis by combining complete surgical resection of lesions and intraperitoneal immunotherapy using catumaxomab

    International Nuclear Information System (INIS)

    Goéré, Diane; Gras-Chaput, Nathalie; Aupérin, Anne; Flament, Caroline; Mariette, Christophe; Glehen, Olivier; Zitvogel, Laurence; Elias, Dominique


    The peritoneum is one of the most frequent sites of recurrent gastric carcinoma after curative treatment, despite the administration of pre- and/or postoperative systemic chemotherapy. Indeed, the prognosis of peritoneal carcinomatosis from gastric carcinoma continues to be poor, with a median survival of less than one year with systemic chemotherapy. Whereas the prognosis of peritoneal carcinomatosis from colorectal cancer has changed with the development of locally administered hyperthermic intraperitoneal chemotherapy (HIPEC), survival results following carcinomatosis from gastric cancer remain disappointing, yielding a 5-year survival rate of less than 20%. Innovative surgical therapies such as intraperitoneal immunotherapy therefore need to be developed for the immediate postoperative period after complete cytoreductive surgery. In a recent randomised study, a clinical effect was obtained after intraperitoneal infusion of catumaxomab in patients with malignant ascites, notably from gastric carcinoma. Catumaxomab, a nonhumanized chimeric antibody, is characterized by its unique ability to bind to three different types of cells: tumour cells expressing the epithelial cell adhesion molecule (EpCAM), T lymphocytes (CD3) and also accessory cells (Fcγ receptor). Because the peritoneum is an immunocompetent organ and up to 90% of gastric carcinomas express EpCAM, intraperitoneal infusion of catumaxomab after complete resection of all macroscopic disease (as defined in the treatment of carcinomatosis from colorectal cancer) could therefore efficiently treat microscopic residual disease. The aim of this randomized phase II study is to assess 2-year overall survival after complete resection of limited carcinomatosis synchronous with gastric carcinoma, followed by an intraperitoneal infusion of catumaxomab with different total doses administered in each of the 2 arms. Close monitoring of peri-opertive mortality, morbidity and early surgical re-intervention will be done

  7. Natural Killer (NK- and T-Cell Engaging Antibody-Derived Therapeutics

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    Christoph Stein


    Full Text Available Unmodified antibodies (abs have been successful in the treatment of hematologic malignancies, but less so for the treatment of solid tumors. They trigger anti-tumor effects through their Fc-domains, and one way to improve their efficacy is to optimize their interaction with the effectors through Fc-engineering. Another way to empower abs is the design of bispecific abs and related fusion proteins allowing a narrower choice of effector cells. Here we review frequently chosen classes of effector cells, as well as common trigger molecules. Natural Killer (NK- and T-cells are the most investigated populations in therapeutical approaches with bispecific agents until now. Catumaxomab, the first bispecific ab to receive drug approval, targets the tumor antigen Epithelial Cell Adhesion Molecule (EpCAM and recruits T-cells via a binding site for the cell surface protein CD3. The next generation of recombinant ab-derivatives replaces the broadly reactive Fc-domain by a binding domain for a single selected trigger. Blinatumomab is the first clinically successful member of this class, targeting cancer cells via CD19 and engaging T-cells by CD3. Other investigators have developed related recombinant fusion proteins to recruit effectors, such as NK-cells and macrophages. The first such agents currently in preclinical and clinical development will be discussed.

  8. Next Generation Antibody Therapeutics Using Bispecific Antibody Technology. (United States)

    Igawa, Tomoyuki


    Nearly fifty monoclonal antibodies have been approved to date, and the market for monoclonal antibodies is expected to continue to grow. Since global competition in the field of antibody therapeutics is intense, we need to establish novel antibody engineering technologies to provide true benefit for patients, with differentiated product values. Bispecific antibodies are among the next generation of antibody therapeutics that can bind to two different target antigens by the two arms of immunoglobulin G (IgG) molecule, and are thus believed to be applicable to various therapeutic needs. Until recently, large scale manufacturing of human IgG bispecific antibody was impossible. We have established a technology, named asymmetric re-engineering technology (ART)-Ig, to enable large scale manufacturing of bispecific antibodies. Three examples of next generation antibody therapeutics using ART-Ig technology are described. Recent updates on bispecific antibodies against factor IXa and factor X for the treatment of hemophilia A, bispecific antibodies against a tumor specific antigen and T cell surface marker CD3 for cancer immunotherapy, and bispecific antibodies against two different epitopes of soluble antigen with pH-dependent binding property for the elimination of soluble antigen from plasma are also described.

  9. Glycosylation profiles of therapeutic antibody pharmaceuticals. (United States)

    Wacker, Christoph; Berger, Christoph N; Girard, Philippe; Meier, Roger


    Recombinant antibodies specific for human targets are often used as therapeutics and represent a major class of drug products. Their therapeutic efficacy depends on the formation of antibody complexes resulting in the elimination of a target molecule or the modulation of specific signalling pathways. The physiological effects of antibody therapeutics are known to depend on the structural characteristics of the antibody molecule, specifically on the glycosylation which is the result of posttranslational modifications. Hence, production of therapeutic antibodies with a defined and consistent glycoform profile is needed which still remains a considerable challenge to the biopharmaceutical industry. To provide an insight into the industries capability to control their manufacturing process and to provide antibodies of highest quality, we conducted a market surveillance study and compared major oligosaccharide profiles of a number of monoclonal antibody pharmaceuticals sampled on the Swiss market. Product lot-to-lot variability was found to be generally low, suggesting that a majority of manufacturers have implemented high quality standards in their production processes. However, proportions of G0, G1 and G2 core-fucosylated chains derived from different products varied considerably and showed a bias towards the immature agalactosidated G0 form. Interestingly, differences in glycosylation caused by the production cell type seem to be of less importance compared with process related parameters such as cell growth. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. The state-of-play and future of antibody therapeutics. (United States)

    Elgundi, Zehra; Reslan, Mouhamad; Cruz, Esteban; Sifniotis, Vicki; Kayser, Veysel


    It has been over four decades since the development of monoclonal antibodies (mAbs) using a hybridoma cell line was first reported. Since then more than thirty therapeutic antibodies have been marketed, mostly as oncology, autoimmune and inflammatory therapeutics. While antibodies are very efficient, their cost-effectiveness has always been discussed owing to their high costs, accumulating to more than one billion dollars from preclinical development through to market approval. Because of this, therapeutic antibodies are inaccessible to some patients in both developed and developing countries. The growing interest in biosimilar antibodies as affordable versions of therapeutic antibodies may provide alternative treatment options as well potentially decreasing costs. As certain markets begin to capitalize on this opportunity, regulatory authorities continue to refine the requirements for demonstrating quality, efficacy and safety of biosimilar compared to originator products. In addition to biosimilars, innovations in antibody engineering are providing the opportunity to design biobetter antibodies with improved properties to maximize efficacy. Enhancing effector function, antibody drug conjugates (ADC) or targeting multiple disease pathways via multi-specific antibodies are being explored. The manufacturing process of antibodies is also moving forward with advancements relating to host cell production and purification processes. Studies into the physical and chemical degradation pathways of antibodies are contributing to the design of more stable proteins guided by computational tools. Moreover, the delivery and pharmacokinetics of antibody-based therapeutics are improving as optimized formulations are pursued through the implementation of recent innovations in the field. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Arrayed antibody library technology for therapeutic biologic discovery. (United States)

    Bentley, Cornelia A; Bazirgan, Omar A; Graziano, James J; Holmes, Evan M; Smider, Vaughn V


    Traditional immunization and display antibody discovery methods rely on competitive selection amongst a pool of antibodies to identify a lead. While this approach has led to many successful therapeutic antibodies, targets have been limited to proteins which are easily purified. In addition, selection driven discovery has produced a narrow range of antibody functionalities focused on high affinity antagonism. We review the current progress in developing arrayed protein libraries for screening-based, rather than selection-based, discovery. These single molecule per microtiter well libraries have been screened in multiplex formats against both purified antigens and directly against targets expressed on the cell surface. This facilitates the discovery of antibodies against therapeutically interesting targets (GPCRs, ion channels, and other multispanning membrane proteins) and epitopes that have been considered poorly accessible to conventional discovery methods. Copyright © 2013. Published by Elsevier Inc.

  12. Optimal Synthetic Glycosylation of a Therapeutic Antibody. (United States)

    Parsons, Thomas B; Struwe, Weston B; Gault, Joseph; Yamamoto, Keisuke; Taylor, Thomas A; Raj, Ritu; Wals, Kim; Mohammed, Shabaz; Robinson, Carol V; Benesch, Justin L P; Davis, Benjamin G


    Glycosylation patterns in antibodies critically determine biological and physical properties but their precise control is a significant challenge in biology and biotechnology. We describe herein the optimization of an endoglycosidase-catalyzed glycosylation of the best-selling biotherapeutic Herceptin, an anti-HER2 antibody. Precise MS analysis of the intact four-chain Ab heteromultimer reveals nonspecific, non-enzymatic reactions (glycation), which are not detected under standard denaturing conditions. This competing reaction, which has hitherto been underestimated as a source of side products, can now be minimized. Optimization allowed access to the purest natural form of Herceptin to date (≥90 %). Moreover, through the use of a small library of sugars containing non-natural functional groups, Ab variants containing defined numbers of selectively addressable chemical tags (reaction handles at Sia C1) in specific positions (for attachment of cargo molecules or "glycorandomization") were readily generated.

  13. Therapeutic antibodies as a treatment option for dengue fever. (United States)

    Chan, Kuan Rong; Ong, Eugenia Z; Ooi, Eng Eong


    Dengue fever is the most prevalent mosquito-borne viral disease globally with about 100 million cases of acute dengue annually. Severe dengue infection can result in a life-threatening illness. In the absence of either a licensed vaccine or antiviral drug against dengue, therapeutic antibodies that neutralize dengue virus (DENV) may serve as an effective medical countermeasure against severe dengue. However, therapeutic antibodies would need to effectively neutralize all four DENV serotypes. It must not induce antibody-dependent enhancement of DENV infection in monocytes/macrophages through Fc gamma receptor (FcγR)-mediated phagocytosis, which is hypothesized to increase the risk of severe dengue. Here, we review the strategies and technologies that can be adopted to develop antibodies for therapeutic applications. We also discuss the mechanism of antibody neutralization in the cells targeted by DENV that express Fc gamma receptor. These studies have provided significant insight toward the use of therapeutic antibodies as a potentially promising bulwark against dengue.

  14. Biodistribution mechanisms of therapeutic monoclonal antibodies in health and disease. (United States)

    Tabrizi, Mohammad; Bornstein, Gadi Gazit; Suria, Hamza


    The monoclonal antibody market continues to witness an impressive rate of growth and has become the leading source of expansion in the biologic segment within the pharmaceutical industry. Currently marketed monoclonal antibodies target a diverse array of antigens. These antigens are distributed in a variety of tissues such as tumors, lungs, synovial fluid, psoriatic plaques, and lymph nodes. As the concentration of drug at the proximity of the biological receptor determines the magnitude of the observed pharmacological responses, a significant consideration in effective therapeutic application of monoclonal antibodies is a thorough understanding of the processes that regulate antibody biodistribution. Monoclonal antibody distribution is affected by factors such as molecular weight, blood flow, tissue and tumor heterogeneity, structure and porosity, target antigen density, turnover rate, and the target antigen expression profile.

  15. Physiochemical and biochemical factors influencing the pharmacokinetics of antibody therapeutics. (United States)

    Bumbaca, Daniela; Boswell, C Andrew; Fielder, Paul J; Khawli, Leslie A


    Monoclonal antibodies are increasingly being developed to treat multiple disease areas, including those related to oncology, immunology, neurology, and ophthalmology. There are multiple factors, such as charge, size, neonatal Fc receptor (FcRn) binding affinity, target affinity and biology, immunoglobulin G (IgG) subclass, degree and type of glycosylation, injection route, and injection site, that could affect the pharmacokinetics (PK) of these large macromolecular therapeutics, which in turn could have ramifications on their efficacy and safety. This minireview examines how characteristics of the antibodies could be altered to change their PK profiles. For example, it was observed that a net charge modification of at least a 1-unit shift in isoelectric point altered antibody clearance. Antibodies with enhanced affinity for FcRn at pH 6.0 display longer serum half-lives and slower clearances than wild type. Antibody fragments have different clearance rates and tissue distribution profiles than full length antibodies. Fc glycosylation is perceived to have a minimal effect on PK while that of terminal high mannose remains unclear. More investigation is warranted to determine if injection route and/or site impacts PK. Nonetheless, a better understanding of the effects of all these variations may allow for the better design of antibody therapeutics.

  16. Single-Domain Antibodies As Therapeutics against Human Viral Diseases

    Directory of Open Access Journals (Sweden)

    Yanling Wu


    Full Text Available In full-size formats, monoclonal antibodies have been highly successful as therapeutics against cancer and immune diseases. However, their large size leads to inaccessibility of some epitopes and relatively high production costs. As an alternative, single-domain antibodies (sdAbs offer special advantages compared to full-size antibodies, including smaller size, larger number of accessible epitopes, relatively low production costs and improved robustness. Currently, sdAbs are being developed against a number of viruses, including human immunodeficiency virus-1 (HIV-1, influenza viruses, hepatitis C virus (HCV, respiratory syncytial virus (RSV, and enteric viruses. Although sdAbs are very potent inhibitors of viral infections, no sdAbs have been approved for clinical use against virial infection or any other diseases. In this review, we discuss the current state of research on sdAbs against viruses and their potential as therapeutics against human viral diseases.

  17. Preexisting Antibodies to an F(ab′)2 Antibody Therapeutic and Novel Method for Immunogenicity Assessment


    Ruppel, Jane; Brady, Ann; Elliott, Rebecca; Leddy, Cecilia; Palencia, Marco; Coleman, Daniel; Couch, Jessica A.; Wakshull, Eric


    Anti-therapeutic antibodies (ATAs) may impact drug exposure and activity and induce immune complex mediated toxicity; therefore the accurate measurement of ATA is important for the analysis of drug safety and efficacy. Preexisting ATAs to the hinge region of anti-Delta like ligand 4 (anti-DLL4) F(ab′)2, a potential antitumor therapeutic, were detected in cynomolgus monkey serum, which presented a challenge in developing assays for detecting treatment induced ATA. A total ATA assay was develop...

  18. Re-engineering therapeutic antibodies for Alzheimer's disease as blood-brain barrier penetrating bi-specific antibodies. (United States)

    Pardridge, William M


    Therapeutic antibodies are large molecule drugs that do not cross the blood-brain barrier (BBB). Therefore, drug development of therapeutic antibodies for Alzheimer's disease (AD) requires that these molecules be re-engineered to enable BBB delivery. This is possible by joining the therapeutic antibody with a transporter antibody, resulting in the engineering of a BBB-penetrating bispecific antibody (BSA). Areas covered: The manuscript covers transporter antibodies that cross the BBB via receptor-mediated transport systems on the BBB, such as the insulin receptor or transferrin receptor. Furthermore, it highlights therapeutic antibodies for AD that target the Abeta amyloid peptide, beta secretase-1, or the metabotropic glutamate receptor-1. BSAs are comprised of both the transporter antibody and the therapeutic antibody, as well as IgG constant region, which can induce immune tolerance or trigger transport via Fc receptors. Expert opinion: Multiple types of BSA molecular designs have been used to engineer BBB-penetrating BSAs, which differ in valency and spatial orientation of the transporter and therapeutic domains of the BSA. The plasma pharmacokinetics and dosing regimens of BSAs differ from that of conventional therapeutic antibodies. BBB-penetrating BSAs may be engineered in the future as new treatments of AD, as well as other neural disorders.

  19. Mass spectrometry for the biophysical characterization of therapeutic monoclonal antibodies. (United States)

    Zhang, Hao; Cui, Weidong; Gross, Michael L


    Monoclonal antibodies (mAbs) are powerful therapeutics, and their characterization has drawn considerable attention and urgency. Unlike small-molecule drugs (150-600 Da) that have rigid structures, mAbs (∼150 kDa) are engineered proteins that undergo complicated folding and can exist in a number of low-energy structures, posing a challenge for traditional methods in structural biology. Mass spectrometry (MS)-based biophysical characterization approaches can provide structural information, bringing high sensitivity, fast turnaround, and small sample consumption. This review outlines various MS-based strategies for protein biophysical characterization and then reviews how these strategies provide structural information of mAbs at the protein level (intact or top-down approaches), peptide, and residue level (bottom-up approaches), affording information on higher order structure, aggregation, and the nature of antibody complexes. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  20. Therapeutic Vaccines and Antibodies for Treatment of Orthopoxvirus Infections

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    Stuart N. Isaacs


    Full Text Available Despite the eradication of smallpox several decades ago, variola and monkeypox viruses still have the potential to become significant threats to public health. The current licensed live vaccinia virus-based smallpox vaccine is extremely effective as a prophylactic vaccine to prevent orthopoxvirus infections, but because of safety issues, it is no longer given as a routine vaccine to the general population. In the event of serious human orthopoxvirus infections, it is important to have treatments available for individual patients as well as their close contacts. The smallpox vaccine and vaccinia immune globulin (VIG were used in the past as therapeutics for patients exposed to smallpox. VIG was also used in patients who were at high risk of developing complications from smallpox vaccination. Thus post-exposure vaccination and VIG treatments may again become important therapeutic modalities. This paper summarizes some of the historic use of the smallpox vaccine and immunoglobulins in the post-exposure setting in humans and reviews in detail the newer animal studies that address the use of therapeutic vaccines and immunoglobulins in orthopoxvirus infections as well as the development of new therapeutic monoclonal antibodies.

  1. Antibody Engineering & Therapeutics 2016: The Antibody Society's annual meeting, December 11-15, 2016, San Diego, CA. (United States)

    Larrick, James W; Alfenito, Mark R; Scott, Jamie K; Parren, Paul W H I; Burton, Dennis R; Bradbury, Andrew R M; Lemere, Cynthia A; Messer, Anne; Huston, James S; Carter, Paul J; Veldman, Trudi; Chester, Kerry A; Schuurman, Janine; Adams, Gregory P; Reichert, Janice M

    Antibody Engineering & Therapeutics, the largest meeting devoted to antibody science and technology and the annual meeting of The Antibody Society, will be held in San Diego, CA on December 11-15, 2016. Each of 14 sessions will include six presentations by leading industry and academic experts. In this meeting preview, the session chairs discuss the relevance of their topics to current and future antibody therapeutics development. Session topics include bispecifics and designer polyclonal antibodies; antibodies for neurodegenerative diseases; the interface between passive and active immunotherapy; antibodies for non-cancer indications; novel antibody display, selection and screening technologies; novel checkpoint modulators / immuno-oncology; engineering antibodies for T-cell therapy; novel engineering strategies to enhance antibody functions; and the biological Impact of Fc receptor engagement. The meeting will open with keynote speakers Dennis R. Burton (The Scripps Research Institute), who will review progress toward a neutralizing antibody-based HIV vaccine; Olivera J. Finn, (University of Pittsburgh School of Medicine), who will discuss prophylactic cancer vaccines as a source of therapeutic antibodies; and Paul Richardson (Dana-Farber Cancer Institute), who will provide a clinical update on daratumumab for multiple myeloma. In a featured presentation, a representative of the World Health Organization's INN expert group will provide a perspective on antibody naming. "Antibodies to watch in 2017" and progress on The Antibody Society's 2016 initiatives will be presented during the Society's special session. In addition, two pre-conference workshops covering ways to accelerate antibody drugs to the clinic and the applications of next-generation sequencing in antibody discovery and engineering will be held on Sunday December 11, 2016.

  2. The effect of immunomodulators on the immunogenicity of TNF-blocking therapeutic monoclonal antibodies: a review

    NARCIS (Netherlands)

    Krieckaert, C.L.; Bartelds, G.M.; Lems, W.F.; Wolbink, G.J.


    Therapeutic monoclonal antibodies have revolutionized the treatment of various inflammatory diseases. Immunogenicity against these antibodies has been shown to be clinically important: it is associated with shorter response duration because of diminishing concentrations in the blood and with

  3. Projecting human pharmacokinetics of therapeutic antibodies from nonclinical data: What have we learned?


    Deng, Rong; Iyer, Suhasini; Theil, Frank-Peter; Mortensen, Deborah L; Fielder, Paul J; Prabhu, Saileta


    The pharmacokinetics (PK) of therapeutic antibodies is determined by target and non-target mediated mechanisms. These antibody-specific factors need to be considered during prediction of human PK based upon preclinical information. Principles of allometric scaling established for small molecules using data from multiple animal species cannot be directly applied to antibodies. Here, different methods for projecting human clearance (CL) from animal PK data for 13 therapeutic monoclonal antibodi...

  4. Therapeutic monoclonal antibody N-glycosylation - Structure, function and therapeutic potential. (United States)

    Cymer, Florian; Beck, Hermann; Rohde, Adelheid; Reusch, Dietmar


    Therapeutic antibodies (IgG-type) contain several post-translational modifications (PTMs) whereby introducing a large heterogeneity, both structural and functional, into this class of therapeutics. Of these modifications, glycosylation in the fragment crystallizable (Fc) region is the most heterogeneous PTM, which can affect the stability of the molecule and interactions with Fc-receptors in vivo. Hence, the glycoform distribution can affect the mode of action and have implications for bioactivity, safety and efficacy of the drug. Main topics of the manuscript include: What factors influence the (Fc) glycan pattern in therapeutic antibodies and how can these glycans be characterized? How does structure of the Fc-glycan relate to function and what methods are available to characterize those functions? Although heterogeneous in their scope, the different sections are intended to combine current knowledge on structure-function correlations of IgG glycan structures with regard to Fc (effector) functions, as well as basic aspects and methodologies for their assessment. Copyright © 2017. Published by Elsevier Ltd.

  5. Influence of FCGRT gene polymorphisms on pharmacokinetics of therapeutic antibodies. (United States)

    Passot, Christophe; Azzopardi, Nicolas; Renault, Sylvaine; Baroukh, Nadine; Arnoult, Christophe; Ohresser, Marc; Boisdron-Celle, Michèle; Gamelin, Erick; Watier, Hervé; Paintaud, Gilles; Gouilleux-Gruart, Valérie


    The neonatal Fc receptor (FcRn) encoded by FCGRT is known to be involved in the pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs). Variability in the expression of FCGRT gene and consequently in the FcRn protein level could explain differences in PK observed between patients treated with mAbs. We studied whether the previously described variable number tandem repeat (VNTR) or copy number variation (CNV) of FCGRT are associated with individual variations of PK parameters of cetuximab. VNTR and CNV were assessed on genomic DNA of 198 healthy individuals and of 94 patients treated with the therapeutic mAb. VNTR and CNV were analyzed by allele-specific PCR and duplex real-time PCR with Taqman (®) technology, respectively. The relationship between FCGRT polymorphisms (VNTR and CNV) and PK parameters of patients treated with cetuximab was studied. VNTR3 homozygote patients had a lower cetuximab distribution clearance than VNTR2/VNTR3 and VNTR3/VNTR4 patients (p = 0.021). We observed no affects of VNTR genotype on elimination clearance. One healthy person (0.5%) and 1 patient (1.1%) had 3 copies of FCGRT. The PK parameters of this patient did not differ from those of patients with 2 copies. The FCGRT promoter VNTR may influence mAbs' distribution in the body. CNV of FCGRT cannot be used as a relevant pharmacogenetic marker because of its low frequency.

  6. Avian Diagnostic and Therapeutic Antibodies to Viral Emerging Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    David Bradley


    During the current period the following key objectives were achieved: demonstration of high titer antibody production by geese following immunization with inactived H1N1 virus; completion of the epitope mapping of West Nile Virus-specific goose antibodies and initiation of epitope mapping of H1N1 flu-specific goose antibodies; advancement in scalable purification of goose antibodies.

  7. The future of antibody therapeutics: ADCs bi-specifics and RIT

    International Nuclear Information System (INIS)

    Reichert, J.


    Full text of publication follows. Antibodies are widely accepted as remarkably versatile therapeutic agents. As evidence of this, the ∼ 30 antibody products marketed worldwide had total global sales of more than 50 billion dollars in 2012, and the commercial clinical pipeline currently comprises over 350 antibody-based product candidates. In a testament to scientific ingenuity, the investigational molecules (clinical and preclinical) are notably diverse in their composition of matter and include antibodies conjugated to a variety of agents (drugs, radioisotopes), bi-specific antibodies, and fragments or domains of antibodies. The concepts that form the basis of these agents were established decades ago, but advances in technology are now allowing new opportunities for their development. In this presentation, future directions in antibody therapeutics development will be discussed, with a focus on antibody-drug conjugates, bi-specific antibodies and radioimmunotherapy. (author)

  8. The antibody response against human and chimeric anti-TNF therapeutic antibodies primarily targets the TNF binding region

    NARCIS (Netherlands)

    van Schie, K. A.; Hart, M. H.; de Groot, E. R.; Kruithof, S.; Aarden, L. A.; Wolbink, G. J.; Rispens, T.


    In a subset of patients, anti tumour necrosis factor (TNF) therapeutic antibodies are immunogenic, resulting in the formation of antidrug antibodies (ADAs). Neutralising ADAs compete with TNF for its binding site and reduces the effective serum concentration, causing clinical non-response. It is

  9. Nanoparticles for the delivery of therapeutic antibodies: Dogma or promising strategy? (United States)

    Sousa, Flávia; Castro, Pedro; Fonte, Pedro; Kennedy, Patrick J; Neves-Petersen, Maria Teresa; Sarmento, Bruno


    Over the past two decades, therapeutic antibodies have demonstrated promising results in the treatment of a wide array of diseases. However, the application of antibody-based therapy implies multiple administrations and a high cost of antibody production, resulting in costly therapy. Another disadvantage inherent to antibody-based therapy is the limited stability of antibodies and the low level of tissue penetration. The use of nanoparticles as delivery systems for antibodies allows for a reduction in antibody dosing and may represent a suitable alternative to increase antibody stability Areas covered: We discuss different nanocarriers intended for the delivery of antibodies as well as the corresponding encapsulation methods. Recent developments in antibody nanoencapsulation, particularly the possible toxicity issues that may arise from entrapment of antibodies into nanocarriers, are also assessed. In addition, this review will discuss the alterations in antibody structure and bioactivity that occur with nanoencapsulation. Expert opinion: Nanocarriers can protect antibodies from degradation, ensuring superior bioavailability. Encapsulation of therapeutic antibodies may offer some advantages, including potential targeting, reduced immunogenicity and controlled release. Furthermore, antibody nanoencapsulation may aid in the incorporation of the antibodies into the cells, if intracellular components (e.g. intracellular enzymes, oncogenic proteins, transcription factors) are to be targeted.

  10. One target, different effects: a comparison of distinct therapeutic antibodies against the same targets. (United States)

    Shim, Hyunbo


    To date, more than 30 antibodies have been approved worldwide for therapeutic use. While the monoclonal antibody market is rapidly growing, the clinical use of therapeutic antibodies is mostly limited to treatment of cancers and immunological disorders. Moreover, antibodies against only five targets (TNF-α, HER2, CD20, EGFR, and VEGF) account for more than 80 percent of the worldwide market of therapeutic antibodies. The shortage of novel, clinically proven targets has resulted in the development of many distinct therapeutic antibodies against a small number of proven targets, based on the premise that different antibody molecules against the same target antigen have distinct biological and clinical effects from one another. For example, four antibodies against TNF-α have been approved by the FDA -- infliximab, adalimumab, golimumab, and certolizumab pegol -- with many more in clinical and preclinical development. The situation is similar for HER2, CD20, EGFR, and VEGF, each having one or more approved antibodies and many more under development. This review discusses the different binding characteristics, mechanisms of action, and biological and clinical activities of multiple monoclonal antibodies against TNF-α, HER-2, CD20, and EGFR and provides insights into the development of therapeutic antibodies.

  11. Dual-color bioluminescent sensor proteins for therapeutic drug monitoring of antitumor antibodies

    NARCIS (Netherlands)

    van Rosmalen, M.; Ni, Y.; Vervoort, D.F.M.; Arts, R.; Ludwig, S.K.J.; Merkx, M.


    Monitoring the levels of therapeutic antibodies in individual patients would allow patient-specific dose optimization, with the potential for major therapeutic and financial benefits. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor)

  12. Qualitative and Quantitative Characterization of Therapeutic Antibodies by Native Mass Spectrometry

    NARCIS (Netherlands)

    Rosati, S


    This thesis describes the development of novel mass spectrometric methods for the analysis of therapeutic monoclonal antibodies. The first chapter of my thesis introduces the reader to the two main subjects discussed in this thesis: native mass spectrometry and therapeutic monoclonal antibodies.

  13. Design of therapeutic vaccines as a novel antibody therapy for cardiovascular diseases. (United States)

    Nakagami, Hironori


    Vaccines are primarily used worldwide as a preventive medicine for infectious diseases and have recently been applied to cancer. We and others have developed therapeutic vaccines designed for cardiovascular diseases that are notably different from previous vaccines. In the case of cancer vaccines, a specific protein in cancer cells is a target antigen, and the activation of cytotoxic T cells (CTL) is required to kill and remove the antigen-presenting cancer cells. Our therapeutic vaccines work against hypertension by targeting angiotensin II (Ang II) as the antigen, which is an endogenous hormone. Therapeutic vaccines must avoid CTL activation and induce the blocking antibodies for Ang II. The goal of our therapeutic vaccine for cardiovascular diseases is to induce the specific antibody response toward the target protein without inducing T-cell or antibody-mediated inflammation through the careful selection of the target antigen, carrier protein and adjuvants. The goal of our therapeutic vaccine is similar to that of antibody therapy. Recently, multiple antibody-based drugs have been developed for cancer, immune-related diseases, and dyslipidemia, which are efficient but expensive. If the effect of a therapeutic vaccine is nearly equivalent to antibody therapy as an alternative approach, the lower medical cost and improvement in drug adherence can be advantages of therapeutic vaccines. In this review, we will describe our concept of therapeutic vaccines for cardiovascular diseases and the future directions of therapeutic vaccines as novel antibody therapies. Copyright © 2017. Published by Elsevier Ltd.

  14. Immunogenicity of therapeutic antibodies : Immunological mechanisms & clinical consequences

    NARCIS (Netherlands)

    van Schie, K.A.J.


    Monoclonal antibody therapy has revolutionized the treatment of many diseases, including chronic inflammatory diseases and cancer. Antibody therapy can unfortunately also elicit an unwanted immune response, leading to anti-drug antibodies (ADA). It is well known that ADA can lower the level of free

  15. Preexisting Antibodies to an F(ab′2 Antibody Therapeutic and Novel Method for Immunogenicity Assessment

    Directory of Open Access Journals (Sweden)

    Jane Ruppel


    Full Text Available Anti-therapeutic antibodies (ATAs may impact drug exposure and activity and induce immune complex mediated toxicity; therefore the accurate measurement of ATA is important for the analysis of drug safety and efficacy. Preexisting ATAs to the hinge region of anti-Delta like ligand 4 (anti-DLL4 F(ab′2, a potential antitumor therapeutic, were detected in cynomolgus monkey serum, which presented a challenge in developing assays for detecting treatment induced ATA. A total ATA assay was developed using a bridging ELISA that detected both anti-CDR and anti-framework ATA including anti-hinge reactivity. A competition assay that could detect 500 ng/mL of anti-CDR ATA in the presence of preexisting ATA was also developed to determine ATA specific to the anti-DLL4 F(ab′2 CDR using anti-DLL4 F(ab′2 and a control F(ab′2. We used these assay methods in a cynomolgus monkey in vivo study to successfully evaluate total and anti-CDR ATA. The preexisting anti-hinge reactivity was also observed to a lesser extent in human serum, and a similar approach could be applied for specific immunogenicity assessment in clinical trials.

  16. Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential. (United States)

    Diebolder, Philipp; Keller, Armin; Haase, Stephanie; Schlegelmilch, Anne; Kiefer, Jonathan D; Karimi, Tamana; Weber, Tobias; Moldenhauer, Gerhard; Kehm, Roland; Eis-Hübinger, Anna M; Jäger, Dirk; Federspil, Philippe A; Herold-Mende, Christel; Dyckhoff, Gerhard; Kontermann, Roland E; Arndt, Michaela A E; Krauss, Jürgen


    The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro,the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.

  17. Enzymatic Inactivation of Endogenous IgG by IdeS Enhances Therapeutic Antibody Efficacy. (United States)

    Järnum, Sofia; Runström, Anna; Bockermann, Robert; Winstedt, Lena; Crispin, Max; Kjellman, Christian


    Endogenous plasma IgG sets an immunologic threshold that dictates the activity of tumor-directed therapeutic antibodies. Saturation of cellular antibody receptors by endogenous antibody limits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Here, we show how enzymatic cleavage of IgG using the bacterial enzyme IdeS can be utilized to empty both high and low affinity Fcγ-receptors and clear the entire endogenous antibody pool. Using in vitro models, tumor animal models as well as ex vivo analysis of sera collected during a previous clinical trial with IdeS, we show how clearing of competing plasma antibody levels with IdeS unblocks cellular antibody receptors. We show that therapeutic antibodies against breast cancer (trastuzumab), colon cancer (cetuximab), and lymphomas (rituximab and alemtuzumab) can be potentiated when endogenous IgG is removed. Overall, IdeS is shown to be a potent tool to reboot the human antibody repertoire and to generate a window to preferentially load therapeutic antibodies onto effector cells and thereby create an armada of dedicated tumor-seeking immune cells. Mol Cancer Ther; 16(9); 1887-97. ©2017 AACR . ©2017 American Association for Cancer Research.

  18. Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility. (United States)

    Peng, Haiyong; Nerreter, Thomas; Chang, Jing; Qi, Junpeng; Li, Xiuling; Karunadharma, Pabalu; Martinez, Gustavo J; Fallahi, Mohammad; Soden, Jo; Freeth, Jim; Beerli, Roger R; Grawunder, Ulf; Hudecek, Michael; Rader, Christoph


    Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naïve rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Recent progress of diagnostic and therapeutic approach to cancers using polyclonal or monoclonal antibodies

    International Nuclear Information System (INIS)

    Koji, Toshihiko


    Among the major topics of interest in cancer immunology, immunodiagnosis and immunotherapy with the antibodies are summarized historically and prospectively. The concept of injecting anti-tumor cell antibodies to localize tumors was first introduced in experimental systems by Pressman (1957). Since then, various trials have been achieved with human tumors using specific or nonspecific tumor-localizing antibodies diagnostically or therapeutically. In 1970's, successes in immunodiagnosis with the antibodies to oncofetal proteins also have been reported. Recently, there are numerous papers dealed with a series of external scanning or serotherapeutic trials by the use of monoclonal antibodies that bind selectively to tumor cells. Various relevant problems with them are discussed. (author)

  20. Immunological aspects of antibody formation against recombinant human therapeutics

    NARCIS (Netherlands)

    Sauerborn, M.S.


    With about 200 new products in the pipeline, recombinant human (rh) therapeutics are becoming the most dominant class of drugs. One of the reasons to create rh therapeutics was to avoid recognition by the immune system due to foreign origin. Nevertheless, rh therapeutics induced formation of

  1. Therapeutic Fc-fusion proteins and peptides as successful alternatives to antibodies. (United States)

    Beck, Alain; Reichert, Janice M


    Therapeutic antibodies have captured substantial attention due to the relatively high rate at which these products reach marketing approval, and the subsequent commercial success they frequently achieve. In the 2000s, a total of 20 antibodies (18 full-length IgG and 2 Fab) were approved by the Food and Drug Administration (FDA) or European Medicines Agency (EMA). In the 2010s to date, an additional 3 antibodies (denosumab, belimumab, ipilimumab) have been approved and one antibody-drug conjugate (brentuximab vedotin) is undergoing regulatory review and may be approved in the US by August 30, 2011. However, a less heralded group of antibody-based therapeutics comprising proteins or peptides fused with an Fc is following the success of classical antibodies.

  2. Monoclonal antibodies: A review of therapeutic applications and ...

    African Journals Online (AJOL)

    production of human anti-mouse antibodies. (HAMA) that invariably ... in the treatment of inflammatory diseases and cancer, with .... It is found to binds to a vascular integrin. (alpha-v/beta-3) ... arthritis, Crohn's disease and ulcerative colitis. [6].

  3. The therapeutic potential of plant-derived vaccines and antibodies. (United States)

    Rodgers, P B; Hamilton, W D; Adair, J R


    The production of recombinant proteins in plants is reviewed with a particular focus on plant-derived vaccines and antibodies for human healthcare. Issues relating to foreign gene expression, such as protein yield, localisation and glycosylation are also considered. Emphasis is placed on reporting progress with preclinical and clinical evaluation of plant-derived vaccines and antibodies. An assessment is made of the likely future direction of research and development in this area.

  4. Monomeric CH3: A Small, Stable Antibody Domain with Therapeutic Promise | Poster (United States)

    By Ashley DeVine, Staff Writer Antibody domains are emerging as promising biopharmaceuticals because of their relatively small size compared to full-sized antibodies, which are too large to effectively penetrate tumors and bind to sterically restricted therapeutic targets. In an article published in The Journal of Biological Chemistry, Tianlei Ying, Ph.D., Dimiter Dimitrov,

  5. Intracellular trafficking of new anticancer therapeutics: antibody-drug conjugates. (United States)

    Kalim, Muhammad; Chen, Jie; Wang, Shenghao; Lin, Caiyao; Ullah, Saif; Liang, Keying; Ding, Qian; Chen, Shuqing; Zhan, Jinbiao


    Antibody-drug conjugate (ADC) is a milestone in targeted cancer therapy that comprises of monoclonal antibodies chemically linked to cytotoxic drugs. Internalization of ADC takes place via clathrin-mediated endocytosis, caveolae-mediated endocytosis, and pinocytosis. Conjugation strategies, endocytosis and intracellular trafficking optimization, linkers, and drugs chemistry present a great challenge for researchers to eradicate tumor cells successfully. This inventiveness of endocytosis and intracellular trafficking has given considerable momentum recently to develop specific antibodies and ADCs to treat cancer cells. It is significantly advantageous to emphasize the endocytosis and intracellular trafficking pathways efficiently and to design potent engineered conjugates and biological entities to boost efficient therapies enormously for cancer treatment. Current studies illustrate endocytosis and intracellular trafficking of ADC, protein, and linker strategies in unloading and also concisely evaluate practically applicable ADCs.

  6. VGKC complex antibodies in epilepsy: diagnostic yield and therapeutic implications. (United States)

    Lilleker, James B; Jones, Matthew S; Mohanraj, Rajiv


    In a significant number of patients developing epilepsy in adult life, the aetiology of their seizures remains unclear. Antibodies directed against the voltage gated potassium channel complex (VGKC Ab) have been identified in various cohorts of patients with epilepsy, although the role of these antibodies in epilepsy pathogenesis is not fully known. We reviewed the notes of 144 patients with unexplained adult onset epilepsy who had been tested for VGKC Abs. We collected data on their clinical syndrome, investigation results and response to treatment. We identified 6 (4.2%) patients who had titres of >400 pM. One of the six patients was positive for LGI1 and another for CASPR2 subunit antibodies. All patients were given immunotherapy and experienced improvement in seizure control. No patient had the clinical syndrome of limbic encephalitis. Patients with otherwise unexplained epilepsy and positive VGKC Abs are a heterogeneous group. In our cohort there was an overall favourable response to immunotherapy but further prospective studies are needed to determine the significance of these antibodies and the optimum treatment regimen for patients. Copyright © 2013 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

  7. Antibody-based therapeutics against components of the IGF system


    Feng, Yang; Dimitrov, Dimiter S.


    The insulin-like growth factor I (IGF-I) receptor (IGF-1R) is overexpressed in most human neoplasms tested so far. Many tumors in young patients produce high levels of the IGF-1R ligands, IGF-I and IGF-II. Given the complexity of the IGF signaling pathway, its complete inhibition may require combination therapies with antibodies targeting both IGF-1R and IGF-II.

  8. Monoclonal Antibody Shows Promise as Potential Therapeutic for MERS | Poster (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.

  9. Therapeutic antibodies: market considerations, disease targets and bioprocessing. (United States)

    Elvin, John G; Couston, Ruairidh G; van der Walle, Christopher F


    Antibodies are well established in mainstream clinical practice and present an exciting area for collaborative research and development in industry and academia alike. In this review, we will provide an overview of the current market and an outlook to 2015, focussing on whole antibody molecules while acknowledging the next generation scaffolds containing variable fragments. The market will be discussed in the context of disease targets, particularly in the areas of oncology and immune disorders which generate the greatest revenue by a wide margin. Emerging targets include central nervous system disorders which will also stimulate new delivery strategies. It is becoming increasingly apparent that a better understanding of bioprocessing is required in order to optimize the steps involved in the preparation of a protein prior to formulation. The latter is outside the scope of this review and nor is it our intention to discuss protein delivery and pharmacokinetics. The challenges that lie ahead include the discovery of new disease targets and the development of robust bioprocessing operations. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

  10. Anti-SEMA3A Antibody: A Novel Therapeutic Agent to Suppress GBM Tumor Growth. (United States)

    Lee, Jaehyun; Shin, Yong Jae; Lee, Kyoungmin; Cho, Hee Jin; Sa, Jason K; Lee, Sang-Yun; Kim, Seok-Hyung; Lee, Jeongwu; Yoon, Yeup; Nam, Do-Hyun


    Glioblastoma (GBM) is classified as one of the most aggressive and lethal brain tumor. Great strides have been made in understanding the genomic and molecular underpinnings of GBM, which translated into development of new therapeutic approaches to combat such deadly disease. However, there are only few therapeutic agents that can effectively inhibit GBM invasion in a clinical framework. In an effort to address such challenges, we have generated anti-SEMA3A monoclonal antibody as a potential therapeutic antibody against GBM progression. We employed public glioma datasets, Repository of Molecular Brain Neoplasia Data and The Cancer Genome Atlas, to analyze SEMA3A mRNA expression in human GBM specimens. We also evaluated for protein expression level of SEMA3A via tissue microarray (TMA) analysis. Cell migration and proliferation kinetics were assessed in various GBM patient-derived cells (PDCs) and U87-MG cell-line for SEMA3A antibody efficacy. GBM patient-derived xenograft (PDX) models were generated to evaluate tumor inhibitory effect of anti-SEMA3A antibody in vivo. By combining bioinformatics and TMA analysis, we discovered that SEMA3A is highly expressed in human GBM specimens compared to non-neoplastic tissues. We developed three different anti-SEMA3A antibodies, in fully human IgG form, through screening phage-displayed synthetic antibody library using a classical panning method. Neutralization of SEMA3A significantly reduced migration and proliferation capabilities of PDCs and U87-MG cell-line in vitro. In PDX models, treatment with anti-SEMA3A antibody exhibited notable tumor inhibitory effect through down-regulation of cellular proliferative kinetics and tumor-associated macrophages recruitment. In present study, we demonstrated tumor inhibitory effect of SEMA3A antibody in GBM progression and present its potential relevance as a therapeutic agent in a clinical framework.

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

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


    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.

  12. IBC's 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics international conferences and the 2012 Annual Meeting of The Antibody Society: December 3-6, 2012, San Diego, CA. (United States)

    Klöhn, Peter-Christian; Wuellner, Ulrich; Zizlsperger, Nora; Zhou, Yu; Tavares, Daniel; Berger, Sven; Zettlitz, Kirstin A; Proetzel, Gabriele; Yong, May; Begent, Richard H J; Reichert, Janice M


    The 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics international conferences, and the 2012 Annual Meeting of The Antibody Society, organized by IBC Life Sciences with contributions from The Antibody Society and two Scientific Advisory Boards, were held December 3-6, 2012 in San Diego, CA. The meeting drew over 800 participants who attended sessions on a wide variety of topics relevant to antibody research and development. As a prelude to the main events, a pre-conference workshop held on December 2, 2012 focused on intellectual property issues that impact antibody engineering. The Antibody Engineering Conference was composed of six sessions held December 3-5, 2012: (1) From Receptor Biology to Therapy; (2) Antibodies in a Complex Environment; (3) Antibody Targeted CNS Therapy: Beyond the Blood Brain Barrier; (4) Deep Sequencing in B Cell Biology and Antibody Libraries; (5) Systems Medicine in the Development of Antibody Therapies/Systematic Validation of Novel Antibody Targets; and (6) Antibody Activity and Animal Models. The Antibody Therapeutics conference comprised four sessions held December 4-5, 2012: (1) Clinical and Preclinical Updates of Antibody-Drug Conjugates; (2) Multifunctional Antibodies and Antibody Combinations: Clinical Focus; (3) Development Status of Immunomodulatory Therapeutic Antibodies; and (4) Modulating the Half-Life of Antibody Therapeutics. The Antibody Society's special session on applications for recording and sharing data based on GIATE was held on December 5, 2012, and the conferences concluded with two combined sessions on December 5-6, 2012: (1) Development Status of Early Stage Therapeutic Antibodies; and (2) Immunomodulatory Antibodies for Cancer Therapy.

  13. Rheumatoid factor interference in immunogenicity assays for human monoclonal antibody therapeutics. (United States)

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


    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.

  14. Changes to International Nonproprietary Names for antibody therapeutics 2017 and beyond: of mice, men and more. (United States)

    Parren, Paul W H I; Carter, Paul J; Plückthun, Andreas

    Active pharmaceutical substances require an International Nonproprietary Name (INN) assigned by the World Health Organization (WHO) to obtain market authorization as a medicinal product. INNs are selected to represent a unique, generic name for a drug enabling unambiguous identification by stakeholders worldwide. INNs may be requested after initiating clinical development of an investigational drug. Pharmaceutical classes are indicated by a common stem or suffix. Currently, INNs for monoclonal antibody-based drugs are recognized by the suffix, -mab, preceded by a source infix such as -xi- (chimeric), -zu- (humanized) or -u- (human) designating the species from which the antibody was derived. However, many technological advances have made it increasingly difficult to accurately capture an antibody's source in its name. In 2014, the WHO and the United States Adopted Names (USAN) Council approached this challenge by implementing changes to antibody source infix definitions. Unfortunately, gaps and ambiguities in the definitions and procedures resulted in inconsistent source category assignments and widespread confusion. The Antibody Society, extensively supported by academic and industry scientists, voiced concerns leading to constructive dialog during scheduled consultations with WHO and USAN Council representatives. In June 2017, the WHO announced that use of the source infix will be discontinued for new antibody INNs effective immediately. We fully support this change as it better aligns antibody INNs with current and foreseeable future innovations in antibody therapeutics. Here we review the changes implemented. Additionally, we analyzed antibody INNs recently assigned under the previous 2014 definitions and provide recommendations for further alignment.

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

  16. A fully automated primary screening system for the discovery of therapeutic antibodies directly from B cells. (United States)

    Tickle, Simon; Howells, Louise; O'Dowd, Victoria; Starkie, Dale; Whale, Kevin; Saunders, Mark; Lee, David; Lightwood, Daniel


    For a therapeutic antibody to succeed, it must meet a range of potency, stability, and specificity criteria. Many of these characteristics are conferred by the amino acid sequence of the heavy and light chain variable regions and, for this reason, can be screened for during antibody selection. However, it is important to consider that antibodies satisfying all these criteria may be of low frequency in an immunized animal; for this reason, it is essential to have a mechanism that allows for efficient sampling of the immune repertoire. UCB's core antibody discovery platform combines high-throughput B cell culture screening and the identification and isolation of single, antigen-specific IgG-secreting B cells through a proprietary technique called the "fluorescent foci" method. Using state-of-the-art automation to facilitate primary screening, extremely efficient interrogation of the natural antibody repertoire is made possible; more than 1 billion immune B cells can now be screened to provide a useful starting point from which to identify the rare therapeutic antibody. This article will describe the design, construction, and commissioning of a bespoke automated screening platform and two examples of how it was used to screen for antibodies against two targets. © 2014 Society for Laboratory Automation and Screening.

  17. Design and Pharmacokinetic Characterization of Novel Antibody Formats for Ocular Therapeutics. (United States)

    Gadkar, Kapil; Pastuskovas, Cinthia V; Le Couter, Jennifer E; Elliott, J Michael; Zhang, Jianhuan; Lee, Chingwei V; Sanowar, Sarah; Fuh, Germaine; Kim, Hok Seon; Lombana, T Noelle; Spiess, Christoph; Nakamura, Makia; Hass, Phil; Shatz, Whitney; Meng, Y Gloria; Scheer, Justin M


    To design and select the next generation of ocular therapeutics, we performed a comprehensive ocular and systemic pharmacokinetic (PK) analysis of a variety of antibodies and antibody fragments, including a novel-designed bispecific antibody. Molecules were administrated via intravitreal (IVT) or intravenous (IV) injections in rabbits, and antibody concentrations in each tissue were determined by ELISA. A novel mathematical model was developed to quantitate the structure-PK relationship. After IVT injection, differences in vitreal half-life observed across all molecules ranged between 3.2 and 5.2 days. Modification or elimination of the fragment crystallizable (Fc) region reduced serum half-life from 9 days for the IgG to 5 days for the neonatal Fc receptor (FcRn) null mAb, to 3.1 to 3.4 days for the other formats. The F(ab')2 was the optimal format for ocular therapeutics with comparable vitreal half-life to full-length antibodies, but with minimized systemic exposure. Concomitantly, the consistency among mathematical model predictions and observed data validated the model for future PK predictions. In addition, we showed a novel design to develop bispecific antibodies, here with activity targeting multiple angiogenesis pathways. We demonstrated that protein molecular weight and Fc region do not play a critical role in ocular PK, as they do systemically. Moreover, the mathematical model supports the selection of the "ideal therapeutic" by predicting ocular and systemic PK of any antibody format for any dose regimen. These findings have important implications for the design and selection of ocular therapeutics according to treatment needs, such as maximizing ocular half-life and minimizing systemic exposure.

  18. Evolution of Therapeutic Antibodies, Influenza Virus Biology, Influenza, and Influenza Immunotherapy

    Directory of Open Access Journals (Sweden)

    Urai Chaisri


    Full Text Available This narrative review article summarizes past and current technologies for generating antibodies for passive immunization/immunotherapy. Contemporary DNA and protein technologies have facilitated the development of engineered therapeutic monoclonal antibodies in a variety of formats according to the required effector functions. Chimeric, humanized, and human monoclonal antibodies to antigenic/epitopic myriads with less immunogenicity than animal-derived antibodies in human recipients can be produced in vitro. Immunotherapy with ready-to-use antibodies has gained wide acceptance as a powerful treatment against both infectious and noninfectious diseases. Influenza, a highly contagious disease, precipitates annual epidemics and occasional pandemics, resulting in high health and economic burden worldwide. Currently available drugs are becoming less and less effective against this rapidly mutating virus. Alternative treatment strategies are needed, particularly for individuals at high risk for severe morbidity. In a setting where vaccines are not yet protective or available, human antibodies that are broadly effective against various influenza subtypes could be highly efficacious in lowering morbidity and mortality and controlling unprecedented epidemic/pandemic. Prototypes of human single-chain antibodies to several conserved proteins of influenza virus with no Fc portion (hence, no ADE effect in recipients are available. These antibodies have high potential as a novel, safe, and effective anti-influenza agent.

  19. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics

    Directory of Open Access Journals (Sweden)

    Peter Bannas


    -based human heavy chain antibodies as antitumor therapeutics.

  20. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics. (United States)

    Bannas, Peter; Hambach, Julia; Koch-Nolte, Friedrich


    chain antibodies as antitumor therapeutics.

  1. The use of anthrax and orthopox therapeutic antibodies from human origin in biodefense

    International Nuclear Information System (INIS)

    Stienstra, S.


    It is impossible to protect whole nations from the effects of bioterrorism by preventive vaccination; there are too many possible agents, costs would be exorbitantly high, and the health risks associated with complex mass vaccination programs would be unacceptable. Adequate protection, however, could be provided via a combination of rapid detection and diagnosis and the treatment of those exposed with drugs which would be beneficial in all stages of disease. Monoclonal antibodies, preferably from human origin to prevent severe complications, which neutralize or block the pathological effects of biological agents, are the optimal candidates to be deployed in case of biological warfare or a bioterrorist event. The human body is one of the better and most suitably equipped places for the generation of monoclonal antibodies which are to be used effectively in humans for treatment. Such antibodies will be of optimal physiological specificity, affinity, and pharmacological properties. In addition, the chances on severe adverse effects and cross-reactivity with human tissues will be slim. Therefore the human immune response is used by the Dutch company IQ Therapeutics, a spin-off of the Groningen University, as a basis for selecting the antibodies. People, immunised against or infected with the agent in question, donate blood cells voluntarily, which are used to generate fully human monoclonal antibodies. In this way effective therapeutics against the protective antigen (PA) and lethal factor (LF) toxin components of Bacillus anthracis are developed and currently antibodies against orthopox viruses are generated as well from donors, which have been immunized with vaccinia. Other projects are the development of therapeutic antibodies for MRSA (antibiotics resistant Staphylococcus aureus) and Enterococcus spp. Both human antibodies against the anthrax toxin components are efficacious in vitro and in pre- and post-exposure settings in mice and rabbits. The anti-LF antibody

  2. Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists. (United States)

    Liu, Tao; Zhang, Yong; Liu, Yan; Wang, Ying; Jia, Haiqun; Kang, Mingchao; Luo, Xiaozhou; Caballero, Dawna; Gonzalez, Jose; Sherwood, Lance; Nunez, Vanessa; Wang, Danling; Woods, Ashley; Schultz, Peter G; Wang, Feng


    On the basis of the 3D structure of a bovine antibody with a well-folded, ultralong complementarity-determining region (CDR), we have developed a versatile approach for generating human or humanized antibody agonists with excellent pharmacological properties. Using human growth hormone (hGH) and human leptin (hLeptin) as model proteins, we have demonstrated that functional human antibody CDR fusions can be efficiently engineered by grafting the native hormones into different CDRs of the humanized antibody Herceptin. The resulting Herceptin CDR fusion proteins were expressed in good yields in mammalian cells and retain comparable in vitro biological activity to the native hormones. Pharmacological studies in rodents indicated a 20- to 100-fold increase in plasma circulating half-life for these antibody agonists and significantly extended in vivo activities in the GH-deficient rat model and leptin-deficient obese mouse model for the hGH and hLeptin antibody fusions, respectively. These results illustrate the utility of antibody CDR fusions as a general and versatile strategy for generating long-acting protein therapeutics.

  3. Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies. (United States)

    Duong, Minh Ngoc; Matera, Eva-Laure; Mathé, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clémenceau, Béatrice; Dumontet, Charles


    Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated.

  4. Development of therapeutic antibodies to G protein-coupled receptors and ion channels: Opportunities, challenges and their therapeutic potential in respiratory diseases. (United States)

    Douthwaite, Julie A; Finch, Donna K; Mustelin, Tomas; Wilkinson, Trevor C I


    The development of recombinant antibody therapeutics continues to be a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Therapeutic drug targets such as soluble cytokines, growth factors and single transmembrane spanning receptors have been successfully targeted by recombinant monoclonal antibodies and the development of new product candidates continues. Despite this growth, however, certain classes of important disease targets have remained intractable to therapeutic antibodies due to the complexity of the target molecules. These complex target molecules include G protein-coupled receptors and ion channels which represent a large target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these important regulators of cell function. Given this opportunity, a significant effort has been applied to address the challenges of targeting these complex molecules and a number of targets are linked to the pathophysiology of respiratory diseases. In this review, we provide a summary of the importance of GPCRs and ion channels involved in respiratory disease and discuss advantages offered by antibodies as therapeutics at these targets. We highlight some recent GPCRs and ion channels linked to respiratory disease mechanisms and describe in detail recent progress made in the strategies for discovery of functional antibodies against challenging membrane protein targets such as GPCRs and ion channels. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Tofacitinib Suppresses Antibody Responses to Protein Therapeutics in Murine Hosts1 (United States)

    Onda, Masanori; Ghoreschi, Kamran; Steward-Tharp, Scott; Thomas, Craig; O’Shea, John J.; Pastan, Ira H.; FitzGerald, David J.


    Immunogenicity remains the ‘Achilles’ heel’ of protein-based therapeutics. Anti-drug antibodies produced in response to protein therapeutics can severely limit both the safety and efficacy of this expanding class of agent. Here we report that monotherapy of mice with tofacitinib (the Janus kinase inhibitor) quells antibody responses to an immunotoxin derived from the bacterial protein, Pseudomonas exotoxin A, as well as to the model antigen, keyhole limpet hemocyanin. Thousandfold reductions in IgG1 titers to both antigens were observed 21 days post-immunization. In fact, suppression was evident for all IgG isotypes and IgM. A reduction in IgG3 production was also noted with a thymus-independent type II antigen. Mechanistic investigations revealed that tofacitinib treatment led to reduced numbers of CD127+ pro-B cells. Furthermore, we observed fewer germinal center B cells and the impaired formation of germinal centers of mice treated with tofacitinib. Since normal immunoglobulin levels were still present during the tofacitinib treatment, this agent specifically reduced anti-drug antibodies, thus preserving the potential efficacy of biological therapeutics, including those that are used as cancer therapeutics. PMID:24890727

  6. Discrimination between glycosylation patterns of therapeutic antibodies using a microfluidic platform, MALDI-MS and multivariate statistics. (United States)

    Thuy, Tran Thi; Tengstrand, Erik; Aberg, Magnus; Thorsén, Gunnar


    Optimal glycosylation with respect to the efficacy, serum half-life time, and immunogenic properties is essential in the generation of therapeutic antibodies. The glycosylation pattern can be affected by several different parameters during the manufacture of antibodies and may change significantly over cultivation time. Fast and robust methods for determination of the glycosylation patterns of therapeutic antibodies are therefore needed. We have recently presented an efficient method for the determination of glycans on therapeutic antibodies using a microfluidic CD platform for sample preparation prior to matrix-assisted laser-desorption mass spectrometry analysis. In the present work, this method is applied to analyse the glycosylation patterns of three commercially available therapeutic antibodies and one intended for therapeutic use. Two of the antibodies produced in mouse myeloma cell line (SP2/0) and one produced in Chinese hamster ovary (CHO) cells exhibited similar glycosylation patterns but could still be readily differentiated from each other using multivariate statistical methods. The two antibodies with most similar glycosylation patterns were also studied in an assessment of the method's applicability for quality control of therapeutic antibodies. The method presented in this paper is highly automated and rapid. It can therefore efficiently generate data that helps to keep a production process within the desired design space or assess that an identical product is being produced after changes to the process. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Quality assurance after process changes of the production of a therapeutic antibody. (United States)

    Brass, J M; Krummen, K; Moll-Kaufmann, C


    Process development for the production of a therapeutic humanised antibody is a very complex operation. It involves recombinant genetics, verification of a strong expression system, gene amplification, characterisation of a stable host cell expression system, optimisation and design of the mammalian cell culture fermentation system and development of an efficient recovery process resulting in high yields and product quality. Rapid progress in the field and the wish of some pharmaceutical companies for outsourcing their production are the driving forces for process changes relatively late in the development phase. This literature survey is aimed at identifying the limits of acceptable process changes in up scaling of the fermentation and down stream processing of biopharmaceuticals and defining the demand in production validation to prove product equivalency and identity of the isolated, purified therapeutic antibody.

  8. A Simple and Sensitive High-Content Assay for the Characterization of Antiproliferative Therapeutic Antibodies. (United States)

    Stengl, Andreas; Hörl, David; Leonhardt, Heinrich; Helma, Jonas


    Monoclonal antibodies (mAbs) have become a central class of therapeutic agents in particular as antiproliferative compounds. Their often complex modes of action require sensitive assays during early, functional characterization. Current cell-based proliferation assays often detect metabolites that are indicative of metabolic activity but do not directly account for cell proliferation. Measuring DNA replication by incorporation of base analogues such as 5-bromo-2'-deoxyuridine (BrdU) fills this analytical gap but was previously restricted to bulk effect characterization in enzyme-linked immunosorbent assay formats. Here, we describe a cell-based assay format for the characterization of antiproliferative mAbs regarding potency and mode of action in a single experiment. The assay makes use of single cell-based high-content-analysis (HCA) for the reliable quantification of replicating cells and DNA content via 5-ethynyl-2'-deoxyuridine (EdU) and 4',6-diamidino-2-phenylindole (DAPI), respectively, as sensitive measures of antiproliferative mAb activity. We used trastuzumab, an antiproliferative therapeutic antibody interfering with HER2 cell surface receptor-mediated growth signal transduction, and HER2-overexpressing cell lines BT474 and SKBR3 to demonstrate up to 10-fold signal-to-background (S/B) ratios for treated versus untreated cells and a shift in cell cycle profiles indicating antibody-induced cell cycle arrest. The assay is simple, cost-effective, and sensitive, providing a cell-based format for preclinical characterization of therapeutic mAbs.

  9. Agonist anti-GITR antibody significantly enhances the therapeutic efficacy of Listeria monocytogenes-based immunotherapy. (United States)

    Shrimali, Rajeev; Ahmad, Shamim; Berrong, Zuzana; Okoev, Grigori; Matevosyan, Adelaida; Razavi, Ghazaleh Shoja E; Petit, Robert; Gupta, Seema; Mkrtichyan, Mikayel; Khleif, Samir N


    We previously demonstrated that in addition to generating an antigen-specific immune response, Listeria monocytogenes (Lm)-based immunotherapy significantly reduces the ratio of regulatory T cells (Tregs)/CD4 + and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. Since Lm-based immunotherapy is able to inhibit the immune suppressive environment, we hypothesized that combining this treatment with agonist antibody to a co-stimulatory receptor that would further boost the effector arm of immunity will result in significant improvement of anti-tumor efficacy of treatment. Here we tested the immune and therapeutic efficacy of Listeria-based immunotherapy combination with agonist antibody to glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) in TC-1 mouse tumor model. We evaluated the potency of combination on tumor growth and survival of treated animals and profiled tumor microenvironment for effector and suppressor cell populations. We demonstrate that combination of Listeria-based immunotherapy with agonist antibody to GITR synergizes to improve immune and therapeutic efficacy of treatment in a mouse tumor model. We show that this combinational treatment leads to significant inhibition of tumor-growth, prolongs survival and leads to complete regression of established tumors in 60% of treated animals. We determined that this therapeutic benefit of combinational treatment is due to a significant increase in tumor infiltrating effector CD4 + and CD8 + T cells along with a decrease of inhibitory cells. To our knowledge, this is the first study that exploits Lm-based immunotherapy combined with agonist anti-GITR antibody as a potent treatment strategy that simultaneously targets both the effector and suppressor arms of the immune system, leading to significantly improved anti-tumor efficacy. We believe that our findings depicted in this manuscript provide a promising and translatable strategy that can enhance the overall

  10. New class of monoclonal antibodies against severe influenza: prophylactic and therapeutic efficacy in ferrets.

    Directory of Open Access Journals (Sweden)

    Robert H E Friesen


    Full Text Available The urgent medical need for innovative approaches to control influenza is emphasized by the widespread resistance of circulating subtype H1N1 viruses to the leading antiviral drug oseltamivir, the pandemic threat posed by the occurrences of human infections with highly pathogenic avian H5N1 viruses, and indeed the evolving swine-origin H1N1 influenza pandemic. A recently discovered class of human monoclonal antibodies with the ability to neutralize a broad spectrum of influenza viruses (including H1, H2, H5, H6 and H9 subtypes has the potential to prevent and treat influenza in humans. Here we report the latest efficacy data for a representative antibody of this novel class.We evaluated the prophylactic and therapeutic efficacy of the human monoclonal antibody CR6261 against lethal challenge with the highly pathogenic avian H5N1 virus in ferrets, the optimal model of human influenza infection. Survival rates, clinically relevant disease signs such as changes in body weight and temperature, virus replication in lungs and upper respiratory tract, as well as macro- and microscopic pathology were investigated. Prophylactic administration of 30 and 10 mg/kg CR6261 prior to viral challenge completely prevented mortality, weight loss and reduced the amount of infectious virus in the lungs by more than 99.9%, abolished shedding of virus in pharyngeal secretions and largely prevented H5N1-induced lung pathology. When administered therapeutically 1 day after challenge, 30 mg/kg CR6261 prevented death in all animals and blunted disease, as evidenced by decreased weight loss and temperature rise, reduced lung viral loads and shedding, and less lung damage.These data demonstrate the prophylactic and therapeutic efficacy of this new class of human monoclonal antibodies in a highly stringent and clinically relevant animal model of influenza and justify clinical development of this approach as intervention for both seasonal and pandemic influenza.

  11. The Impact of Therapeutic Antibodies on the Management of Digestive Diseases: History, Current Practice, and Future Directions. (United States)

    Sofia, M Anthony; Rubin, David T


    The development of therapeutic antibodies represents a revolutionary change in medical therapy for digestive diseases. Beginning with the initial studies that confirmed the pathogenicity of cytokines in inflammatory bowel disease, the development and application of therapeutic antibodies brought challenges and insights into their potential and optimal use. Infliximab was the first biological drug approved for use in Crohn's disease and ulcerative colitis. The lessons learned from infliximab include the importance of immunogenicity and the influence of pharmacokinetics on disease response and outcomes. Building on this foundation, other therapeutic antibodies achieved approval for inflammatory bowel disease and many more are in development for several digestive diseases. In this review, we reflect on the history of therapeutic antibodies and discuss current practice and future directions for the field.

  12. Quantification of patient specific assay interference in different formats of enzyme linked immunoassays for therapeutic monoclonal antibodies

    NARCIS (Netherlands)

    Grebenchtchikov, N.J.; Geurts-Moespot, A.; Heijmen, L.; Laarhoven, H.W.M. van; Herpen, C.M.L. van; Thijs, A.M.J.; Span, P.N.; Sweep, F.C.


    BackgroundThe use of therapeutic monoclonal antibodies for clinical purposes has significantly increased in recent years, and so has the need to monitor antibody concentrations. This may be achieved using the well-established enzyme linked immunoassay (ELISA) methods; however, these assays are

  13. Quantification of patient-specific assay interference in different formats of enzyme-linked immunoassays for therapeutic monoclonal antibodies

    NARCIS (Netherlands)

    Grebenchtchikov, Nicolai; Geurts-Moespot, Anneke J.; Heijmen, Linda; van Laarhoven, Hanneke W. M.; van Herpen, Carla M. L.; Thijs, Annemarie M. J.; Span, Paul N.; Sweep, Fred C. G. J.


    The use of therapeutic monoclonal antibodies for clinical purposes has significantly increased in recent years, and so has the need to monitor antibody concentrations. This may be achieved using the well-established enzyme linked immunoassay (ELISA) methods; however, these assays are subject to a

  14. Molecular and Therapeutic Characterization of Anti-ectodysplasin A Receptor (EDAR) Agonist Monoclonal Antibodies* (United States)

    Kowalczyk, Christine; Dunkel, Nathalie; Willen, Laure; Casal, Margret L.; Mauldin, Elizabeth A.; Gaide, Olivier; Tardivel, Aubry; Badic, Giovanna; Etter, Anne-Lise; Favre, Manuel; Jefferson, Douglas M.; Headon, Denis J.; Demotz, Stéphane; Schneider, Pascal


    The TNF family ligand ectodysplasin A (EDA) and its receptor EDAR are required for proper development of skin appendages such as hair, teeth, and eccrine sweat glands. Loss of function mutations in the Eda gene cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition that can be ameliorated in mice and dogs by timely administration of recombinant EDA. In this study, several agonist anti-EDAR monoclonal antibodies were generated that cross-react with the extracellular domains of human, dog, rat, mouse, and chicken EDAR. Their half-life in adult mice was about 11 days. They induced tail hair and sweat gland formation when administered to newborn EDA-deficient Tabby mice, with an EC50 of 0.1 to 0.7 mg/kg. Divalency was necessary and sufficient for this therapeutic activity. Only some antibodies were also agonists in an in vitro surrogate activity assay based on the activation of the apoptotic Fas pathway. Activity in this assay correlated with small dissociation constants. When administered in utero in mice or at birth in dogs, agonist antibodies reverted several ectodermal dysplasia features, including tooth morphology. These antibodies are therefore predicted to efficiently trigger EDAR signaling in many vertebrate species and will be particularly suited for long term treatments. PMID:21730053

  15. Engineered Bovine Antibodies in the Development of Novel Therapeutics, Immunomodulators and Vaccines

    Directory of Open Access Journals (Sweden)

    Madhuri Koti


    Full Text Available Some bovine antibodies across all classes are unique, such as the CDR3 of the variable heavy-domain (VH CDR3, which is exceptionally long (up to 66 amino acids, unlike most conventional antibodies where the VH CDR3 loops range from 10 to 25 amino acids. The exceptionally long VH CDR3 is encoded by unusually long germline IGHD genes together with insertion of novel “a” nucleotide rich conserved short nucleotide sequence (CSNS specifically at the IGH V-D junction. Such an exceptionally long VH CDR3 confers unique “knob and stalk” structural architecture where the knob, formed by intra-VH CDR3 disulfide bridges, is separated by 20 Å solvent exposed stalk composed of anti-parallel beta strands. The substitution of the knob with cytokines, such as, erythropoietin and granulocyte colony stimulating factor 3 (granulocyte colony stimulating factor, results in expression of functional fusion proteins with enhanced pharmacokinetics. The beta stranded stalk can be substituted with other rigid structures, for example, repeat alpha helices to form coiled-coil that mimics the beta-stranded stalk and, thus, opens opportunities for insertion of this structure in the CDRs of antibodies across species. Given the versatility of such a structural platform in bovine antibody VH CDR3, it provides the opportunity for the development of new generation of diagnostics, therapeutics, vaccines and immunomodulating drugs.

  16. Imaging of primary and metastatic colorectal carcinoma with monoclonal antibody 791T/36 and the therapeutic potential of antibody-drug conjugates

    International Nuclear Information System (INIS)

    Pimm, M.V.; Armitage, N.C.; Ballantyne, K.; Baldwin, R.W.; Perkins, A.C.; Durrant, L.G.; Garnett, M.C.; Hardcastle, J.D.


    Monoclonal antibody 791T/36, prepared against a tumor-associated 72,000 dalton glycoprotein, reacted with cells from primary and metastatic colorectal carcinomas. I-131 or In-111-labelled antibody localized in xenografts of colorectal carcinomas established from in vitro clonogenic populations. Clinically, with I-131-labelled antibody, 8/11 colonic tumors imaged positively. Imaging was negative in four patients with benign colon disease. 5/11 rectal tumors were positively imaged, but excreted I-131 in the bladder obscured tumors in several studies. In-111-labelled antibody gave superior images and positively imaged primary and metastatic sites in 13/14 patients. Prospectively in the detection of recurrent disease, I-131 or In-111-antibody detected 29/33 separate sites in 24 patients. Seven negative patients remain disease free. There were 3 false positives; overall sensitivity was 88%, with 70% specificity. Specific localization of radiolabel was confirmed immunochemically and by counting radioactivity in resected specimens. Antibody conjugates with methotrexate, vindesine and daunomycin retained drug activity and antibody function, including xenograft localization and conjugates were therapeutically effective against xenografts. 791T/36 antibody has potential for immunodetection of primary and recurrent colorectal carcinoma and for targeting of therapeutic agents

  17. Therapeutic antibodies: A new era in the treatment of respiratory diseases? (United States)

    Sécher, T; Guilleminault, L; Reckamp, K; Amanam, I; Plantier, L; Heuzé-Vourc'h, N


    Respiratory diseases affect millions of people worldwide, and account for significant levels of disability and mortality. The treatment of lung cancer and asthma with therapeutic antibodies (Abs) is a breakthrough that opens up new paradigms for the management of respiratory diseases. Antibodies are becoming increasingly important in respiratory medicine; dozens of Abs have received marketing approval, and many more are currently in clinical development. Most of these Abs target asthma, lung cancer and respiratory infections, while very few target chronic obstructive pulmonary disease - one of the most common non-communicable causes of death - and idiopathic pulmonary fibrosis. Here, we review Abs approved for or in clinical development for the treatment of respiratory diseases. We notably highlight their molecular mechanisms, strengths, and likely future trends. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Affinity improvement of a therapeutic antibody by structure-based computational design: generation of electrostatic interactions in the transition state stabilizes the antibody-antigen complex.

    Directory of Open Access Journals (Sweden)

    Masato Kiyoshi

    Full Text Available The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1. We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen in vitro, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101 is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody. Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu. The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier benefits the durability of the antibody-antigen complex. The combination of in silico calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody.

  19. Structures of Ebola virus GP and sGP in complex with therapeutic antibodies. (United States)

    Pallesen, Jesper; Murin, Charles D; de Val, Natalia; Cottrell, Christopher A; Hastie, Kathryn M; Turner, Hannah L; Fusco, Marnie L; Flyak, Andrew I; Zeitlin, Larry; Crowe, James E; Andersen, Kristian G; Saphire, Erica Ollmann; Ward, Andrew B


    The Ebola virus (EBOV) GP gene encodes two glycoproteins. The major product is a soluble, dimeric glycoprotein (sGP) that is secreted abundantly. Despite the abundance of sGP during infection, little is known regarding its structure or functional role. A minor product, resulting from transcriptional editing, is the transmembrane-anchored, trimeric viral surface glycoprotein (GP). GP mediates attachment to and entry into host cells, and is the intended target of antibody therapeutics. Because large portions of sequence are shared between GP and sGP, it has been hypothesized that sGP may potentially subvert the immune response or may contribute to pathogenicity. In this study, we present cryo-electron microscopy structures of GP and sGP in complex with GP-specific and GP/sGP cross-reactive antibodies undergoing human clinical trials. The structure of the sGP dimer presented here, in complex with both an sGP-specific antibody and a GP/sGP cross-reactive antibody, permits us to unambiguously assign the oligomeric arrangement of sGP and compare its structure and epitope presentation to those of GP. We also provide biophysical evaluation of naturally occurring GP/sGP mutations that fall within the footprints identified by our high-resolution structures. Taken together, our data provide a detailed and more complete picture of the accessible Ebolavirus glycoprotein landscape and a structural basis to evaluate patient and vaccine antibody responses towards differently structured products of the GP gene.

  20. Utility of immunodeficient mouse models for characterizing the preclinical pharmacokinetics of immunogenic antibody therapeutics. (United States)

    Myzithras, Maria; Bigwarfe, Tammy; Li, Hua; Waltz, Erica; Ahlberg, Jennifer; Giragossian, Craig; Roberts, Simon

    Prior to clinical studies, the pharmacokinetics (PK) of antibody-based therapeutics are characterized in preclinical species; however, those species can elicit immunogenic responses that can lead to an inaccurate estimation of PK parameters. Immunodeficient (SCID) transgenic hFcRn and C57BL/6 mice were used to characterize the PK of three antibodies that were previously shown to be immunogenic in mice and cynomolgus monkeys. Four mouse strains, Tg32 hFcRn SCID, Tg32 hFcRn, SCID and C57BL/6, were administered adalimumab (Humira®), mAbX and mAbX-YTE at 1 mg/kg, and in SCID strains there was no incidence of immunogenicity. In non-SCID strains, drug-clearing ADAs appeared after 4-7 days, which affected the ability to accurately calculate PK parameters. Single species allometric scaling of PK data for Humira® in SCID and hFcRn SCID mice resulted in improved human PK predictions compared to C57BL/6 mice. Thus, the SCID mouse model was demonstrated to be a useful tool for assessing the preclinical PK of immunogenic therapeutics.

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

    Directory of Open Access Journals (Sweden)

    Cameron P Simmons


    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

  2. Impact of Shed/Soluble targets on the PK/PD of approved therapeutic monoclonal antibodies. (United States)

    Samineni, Divya; Girish, Sandhya; Li, Chunze


    Suboptimal treatment for monoclonal antibodies (mAbs) directed against endogenous circulating soluble targets and the shed extracellular domains (ECD) of the membrane-bound targets is an important clinical concern due to the potential impact of mAbs on the in vivo efficacy and safety. Consequently, there are considerable challenges in the determination of an optimal dose and/or dosing regimen. Areas covered: This review outlines the impact of shed antigen targets from membrane-bound proteins and soluble targets on the PK and/or PD of therapeutic mAbs that have been approved in the last decade. We discuss various bioanalytical techniques that have facilitated the interpretation of the PK/PD properties of therapeutic mAbs and also considered the factors that may impact such measurements. Quantitative approaches include target-mediated PK models and bi- or tri-molecular interaction PK/PD models that describe the relationships between the antibody PK and the ensuing effects on PD biomarkers, to facilitate the mAb PK/PD characterization. Expert commentary: The proper interpretation of PK/PD relationships through the integrated PK/PD modeling and bioanalytical strategy facilitates a mechanistic understanding of the disease processes and dosing regimen optimization, thereby offering insights into developing effective therapeutic regimens. This review provides an overview of the impact of soluble targets or shed ECD on mAb PK/PD properties. We provide examples of quantitative approaches that facilitate the characterization of mAb PK/PD characteristics and their corresponding bioanalytical strategies.

  3. Development of an analytical method to assess the occupational health risk of therapeutic monoclonal antibodies using LC-HRMS. (United States)

    Reinders, Lars M H; Klassen, Martin D; Jaeger, Martin; Teutenberg, Thorsten; Tuerk, Jochen


    Monoclonal antibodies are a group of commonly used therapeutics, whose occupational health risk is still discussed controversially. The long-term low-dose exposure side effects are insufficiently evaluated; hence, discussions are often based on a theoretical level or extrapolating side effects from therapeutic dosages. While some research groups recommend applying the precautionary principle for monoclonal antibodies, others consider the exposure risk too low for measures taken towards occupational health and safety. However, both groups agree that airborne monoclonal antibodies have the biggest risk potential. Therefore, we developed a peptide-based analytical method for occupational exposure monitoring of airborne monoclonal antibodies. The method will allow collecting data about the occupational exposure to monoclonal antibodies. Thus, the mean daily intake for personnel in pharmacies and the pharmaceutical industry can be determined for the first time and will help to substantiate the risk assessment by relevant data. The introduced monitoring method includes air sampling, sample preparation and detection by liquid chromatography coupled with high-resolution mass spectrometry of individual monoclonal antibodies as well as sum parameter. For method development and validation, a chimeric (rituximab), humanised (trastuzumab) and a fully humanised (daratumumab) monoclonal antibody are used. A limit of detection between 1 μg per sample for daratumumab and 25 μg per sample for the collective peptide is achieved. Graphical abstract Demonstration of the analytical workflow, from the release of monoclonal antibodies to the detection as single substances as well as sum parameter.

  4. Protocols for the analytical characterization of therapeutic monoclonal antibodies. II - Enzymatic and chemical sample preparation. (United States)

    Bobaly, Balazs; D'Atri, Valentina; Goyon, Alexandre; Colas, Olivier; Beck, Alain; Fekete, Szabolcs; Guillarme, Davy


    The analytical characterization of therapeutic monoclonal antibodies and related proteins usually incorporates various sample preparation methodologies. Indeed, quantitative and qualitative information can be enhanced by simplifying the sample, thanks to the removal of sources of heterogeneity (e.g. N-glycans) and/or by decreasing the molecular size of the tested protein by enzymatic or chemical fragmentation. These approaches make the sample more suitable for chromatographic and mass spectrometric analysis. Structural elucidation and quality control (QC) analysis of biopharmaceutics are usually performed at intact, subunit and peptide levels. In this paper, general sample preparation approaches used to attain peptide, subunit and glycan level analysis are overviewed. Protocols are described to perform tryptic proteolysis, IdeS and papain digestion, reduction as well as deglycosylation by PNGase F and EndoS2 enzymes. Both historical and modern sample preparation methods were compared and evaluated using rituximab and trastuzumab, two reference therapeutic mAb products approved by Food and Drug Administration (FDA) and European Medicines Agency (EMA). The described protocols may help analysts to develop sample preparation methods in the field of therapeutic protein analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Defining New Therapeutics Using a More Immunocompetent Mouse Model of Antibody-Enhanced Dengue Virus Infection. (United States)

    Pinto, Amelia K; Brien, James D; Lam, Chia-Ying Kao; Johnson, Syd; Chiang, Cindy; Hiscott, John; Sarathy, Vanessa V; Barrett, Alan D; Shresta, Sujan; Diamond, Michael S


    With over 3.5 billion people at risk and approximately 390 million human infections per year, dengue virus (DENV) disease strains health care resources worldwide. Previously, we and others established models for DENV pathogenesis in mice that completely lack subunits of the receptors (Ifnar and Ifngr) for type I and type II interferon (IFN) signaling; however, the utility of these models is limited by the pleotropic effect of these cytokines on innate and adaptive immune system development and function. Here, we demonstrate that the specific deletion of Ifnar expression on subsets of murine myeloid cells (LysM Cre(+) Ifnar(flox/flox) [denoted as Ifnar(f/f) herein]) resulted in enhanced DENV replication in vivo. The administration of subneutralizing amounts of cross-reactive anti-DENV monoclonal antibodies to LysM Cre(+) Ifnar(f/f) mice prior to infection with DENV serotype 2 or 3 resulted in antibody-dependent enhancement (ADE) of infection with many of the characteristics associated with severe DENV disease in humans, including plasma leakage, hypercytokinemia, liver injury, hemoconcentration, and thrombocytopenia. Notably, the pathogenesis of severe DENV-2 or DENV-3 infection in LysM Cre(+) Ifnar(f/f) mice was blocked by pre- or postexposure administration of a bispecific dual-affinity retargeting molecule (DART) or an optimized RIG-I receptor agonist that stimulates innate immune responses. Our findings establish a more immunocompetent animal model of ADE of infection with multiple DENV serotypes in which disease is inhibited by treatment with broad-spectrum antibody derivatives or innate immune stimulatory agents. Although dengue virus (DENV) infects hundreds of millions of people annually and results in morbidity and mortality on a global scale, there are no approved antiviral treatments or vaccines. Part of the difficulty in evaluating therapeutic candidates is the lack of small animal models that are permissive to DENV and recapitulate the clinical features

  6. Preclinical evaluation of VIS513, a therapeutic antibody against dengue virus, in non-human primates. (United States)

    Ong, Eugenia Z; Budigi, Yadunanda; Tan, Hwee Cheng; Robinson, Luke N; Rowley, Kirk J; Winnett, Alexander; Hobbie, Sven; Shriver, Zachary; Babcock, Gregory J; Ooi, Eng Eong


    Despite useful in vivo activity, no therapeutic against dengue virus (DENV) has demonstrated efficacy in clinical trials. Herein, we explored dosing and virological endpoints to guide the design of human trials of VIS513, a pan-serotype anti-DENV IgG1 antibody, in non-human primates (NHPs). Dosing VIS513 pre- or post-peak viremia in NHPs neutralized infectious DENV although RNAemia remained detectable post-treatment; differential interaction of human IgGs with macaque Fc-gamma receptors may delay clearance of neutralized DENV. Our findings suggest useful antiviral utility of VIS513 and highlight an important consideration when evaluating virological endpoints of trials for anti-DENV biologics. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Intracellular reprogramming of expression, glycosylation, and function of a plant-derived antiviral therapeutic monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Jeong-Hwan Lee

    Full Text Available Plant genetic engineering, which has led to the production of plant-derived monoclonal antibodies (mAb(Ps, provides a safe and economically effective alternative to conventional antibody expression methods. In this study, the expression levels and biological properties of the anti-rabies virus mAb(P SO57 with or without an endoplasmic reticulum (ER-retention peptide signal (Lys-Asp-Glu-Leu; KDEL in transgenic tobacco plants (Nicotiana tabacum were analyzed. The expression levels of mAb(P SO57 with KDEL (mAb(PK were significantly higher than those of mAb(P SO57 without KDEL (mAb(P regardless of the transcription level. The Fc domains of both purified mAb(P and mAb(PK and hybridoma-derived mAb (mAb(H had similar levels of binding activity to the FcγRI receptor (CD64. The mAb(PK had glycan profiles of both oligomannose (OM type (91.7% and Golgi type (8.3%, whereas the mAb(P had mainly Golgi type glycans (96.8% similar to those seen with mAb(H. Confocal analysis showed that the mAb(PK was co-localized to ER-tracker signal and cellular areas surrounding the nucleus indicating accumulation of the mAb(P with KDEL in the ER. Both mAb(P and mAb(PK disappeared with similar trends to mAb(H in BALB/c mice. In addition, mAb(PK was as effective as mAb(H at neutralizing the activity of the rabies virus CVS-11. These results suggest that the ER localization of the recombinant mAb(P by KDEL reprograms OM glycosylation and enhances the production of the functional antivirus therapeutic antibody in the plant.

  8. Pros and cons of different therapeutic antibody formats for recombinant antivenom development

    DEFF Research Database (Denmark)

    Laustsen, Andreas H.; Gutiérrez, José María; Knudsen, Cecilie


    Antibody technologies are being increasingly applied in the field of toxinology. Fuelled by the many advances in immunology, synthetic biology, and antibody research, different approaches and antibody formats are being investigated for the ability to neutralize animal toxins. These different...

  9. Therapeutic efficacy of antibodies lacking Fcγ receptor binding against lethal dengue virus infection is due to neutralizing potency and blocking of enhancing antibodies [corrected].

    Directory of Open Access Journals (Sweden)

    Katherine L Williams


    Full Text Available Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS are life-threatening complications following infection with one of the four serotypes of dengue virus (DENV. At present, no vaccine or antiviral therapies are available against dengue. Here, we characterized a panel of eight human or mouse-human chimeric monoclonal antibodies (MAbs and their modified variants lacking effector function and dissected the mechanism by which some protect against antibody-enhanced lethal DENV infection. We found that neutralizing modified MAbs that recognize the fusion loop or the A strand epitopes on domains II and III of the envelope protein, respectively, act therapeutically by competing with and/or displacing enhancing antibodies. By analyzing these relationships, we developed a novel in vitro suppression-of-enhancement assay that predicts the ability of modified MAbs to act therapeutically against antibody-enhanced disease in vivo. These studies provide new insight into the biology of DENV pathogenesis and the requirements for antibodies to treat lethal DENV disease.

  10. Tissue Distribution of a Therapeutic Monoclonal Antibody Determined by Large Pore Microdialysis. (United States)

    Jadhav, Satyawan B; Khaowroongrueng, Vipada; Fueth, Matthias; Otteneder, Michael B; Richter, Wolfgang; Derendorf, Hartmut


    Therapeutic monoclonal antibodies (mAbs) exhibit limited distribution to the target tissues. Determination of target tissue interstitial concentration of mAbs is an important aspect in the assessment of their pharmacokinetic/pharmacodynamics relationship especially for mAbs targeting membrane bound receptors. The pharmacokinetics of R7072, a full length mAb (IgG) targeting human insulin-like growth factor-1 receptor was evaluated following a single intravenous dose at 1, 6.25, and 25 mg/kg in healthy female SCID-beige mice. R7072 showed linear pharmacokinetics over the dose range tested and was characterized by low systemic clearance and long terminal half-life. Furthermore, interstitial distribution of R7072 was evaluated in liver, skin, kidney, and muscle tissues using large pore microdialysis (MD) after intravenous administration of 10 mg/kg dose in mice. The relative recoveries of R7072 were consistent and similar between in vitro and in vivo MD experiments. The tissue and interstitial concentrations were significantly lower compared to serum concentrations and found to be highest in liver and lowest in muscle. The interstitial concentrations of R7072 were approximately 2-fold to 4-fold lower than corresponding total tissue concentrations. Large pore MD appears to be an attractive approach for direct measurement of pharmacologically relevant concentrations of therapeutic mAbs in tissue interstitial fluid. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  11. 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.

  12. Correlation of pharmacodynamic activity, pharmacokinetics, and anti-product antibody responses to anti-IL-21R antibody therapeutics following IV administration to cynomolgus monkeys

    Directory of Open Access Journals (Sweden)

    Spaulding Vikki


    Full Text Available Abstract Background Anti-IL-21R antibodies are potential therapeutics for the treatment of autoimmune diseases. This study evaluated correlations between the pharmacodynamic (PD activity, pharmacokinetics, and anti-product antibody responses of human anti-IL-21R antibodies Ab-01 and Ab-02 following IV administration to cynomolgus monkeys. Methods The PD assay was based on the ability of recombinant human IL-21 (rhuIL-21 to induce expression of the IL-2RA gene in cynomolgus monkey whole blood samples ex vivo. Monkeys screened for responsiveness to rhuIL-21 stimulation using the PD assay, were given a single 10 mg/kg IV dosage of Ab-01, Ab-02, or a control antibody (3/group, and blood samples were evaluated for PD activity (inhibition of IL-2RA expression for up to 148 days. Anti-IL-21R antibody concentrations and anti-product antibody responses were measured in serum using immunoassays and flow cytometry. Results Following IV administration of Ab-01 and Ab-02 to cynomolgus monkeys, PD activity was observed as early as 5 minutes (first time point sampled. This PD activity had good correlation with the serum concentrations and anti-product antibody responses throughout the study. The mean terminal half-life (t1/2 was ~10.6 and 2.3 days for Ab-01 and Ab-02, respectively. PD activity was lost at ~5-13 weeks for Ab-01 and at ~2 weeks for Ab-02, when serum concentrations were relatively low. The estimated minimum concentrations needed to maintain PD activity were ~4-6 nM for Ab-01 and ~2.5 nM for Ab-02, and were consistent with the respective KD values for binding to human IL-21R. For Ab-01, there was noticeable inter-animal variability in t1/2 values (~6-14 days and the resulting PD profiles, which correlated with the onset of anti-product antibody formation. While all three Ab-01-dosed animals were positive for anti-Ab-01 antibodies, only one monkey (with the shortest t1/2 and the earliest loss of PD activity had evidence of neutralizing anti-Ab-01

  13. LC-MS/MS strategies for therapeutic antibodies and investigation into the quantitative impact of antidrug-antibodies. (United States)

    Ewles, Matthew; Mannu, Ranbir; Fox, Chris; Stanta, Johannes; Evans, Graeme; Goodwin, Lee; Duffy, James; Bell, Len; Estdale, Sian; Firth, David


    We aimed to establish novel, high-throughput LC-MS/MS strategies for quantification of monoclonal antibodies in human serum and examine the potential impact of antidrug antibodies. We present two strategies using a thermally stable immobilized trypsin. The first strategy uses whole serum digestion and the second introduces Protein G enrichment to improve the selectivity. The impact of anti-trastuzumab antibodies on the methods was tested. Whole serum digestion has been validated for trastuzumab (LLOQ 0.25 µg/ml). Protein G enrichment has been validated for trastuzumab (LLOQ 0.1 µg/ml), bevacizumab (LLOQ 0.1 µg/ml) and adalimumab (LLOQ 0.25 µg/ml). We have shown the potential for anti-drug antibodies to impact on the quantification and we have subsequently established a strategy to overcome this impact where total quantification is desired.

  14. Kinetic analysis of a monoclonal therapeutic antibody and its single-chain homolog by surface plasmon resonance. (United States)

    Patel, Rekha; Andrien, Bruce A


    Monoclonal antibodies (mAbs) and antibody fragments have become an emerging class of therapeutics since 1986. Their versatility enables them to be engineered for optimal efficiency and decreased immunogenicity, and the path to market has been set by recent regulatory approvals. One of the initial criteria for success of any protein or antibody therapeutic is to understand its binding characteristics to the target antigen. Surface plasmon resonance (SPR) has been widely used and is an important tool for ligand-antigen binding characterization. In this work, the binding kinetics of a recombinant mAb and its single-chain antibody homolog, single-chain variable fragment (scFv), was analyzed by SPR. These two proteins target the same antigen. The binding kinetics of the mAb (bivalent antibody) and scFv (monovalent scFv) for this antigen was analyzed along with an assessment of the thermodynamics of the binding interactions. Alternative binding configurations were investigated to evaluate potential experimental bias because theoretically experimental binding configuration should have no impact on binding kinetics. Self-association binding kinetics in the proteins' respective formulation solutions and antigen epitope mapping were also evaluated. Functional characterization of monoclonal and single-chain antibodies has become just as important as structural characterization in the biotechnology field.

  15. The distribution of the therapeutic monoclonal antibodies cetuximab and trastuzumab within solid tumors

    International Nuclear Information System (INIS)

    Lee, Carol M; Tannock, Ian F


    Poor distribution of some anticancer drugs in solid tumors may limit their anti-tumor activity. Here we used immunohistochemistry to quantify the distribution of the therapeutic monoclonal antibodies cetuximab and trastuzumab in relation to blood vessels and to regions of hypoxia in human tumor xenografts. The antibodies were injected into mice implanted with human epidermoid carcinoma A431 or human breast carcinoma MDA-MB-231 transfected with ERBB2 (231-H2N) that express high levels of ErbB1 and ErbB2 respectively, or wild-type MDA-MB-231, which expresses intermediate levels of ErbB1 and low levels of ErbB2. The distribution of cetuximab in A431 xenografts and trastuzumab in 231-H2N xenografts was time and dose dependent. At early intervals after injection of 1 mg cetuximab into A431 xenografts, the concentration of cetuximab decreased with increasing distance from blood vessels, but became more uniformly distributed at later times; there remained however limited distribution and binding in hypoxic regions of tumors. Injection of lower doses of cetuximab led to heterogeneous distributions. Similar results were observed with trastuzumab in 231-H2N xenografts. In MDA-MB-231 xenografts, which express lower levels of ErbB1, homogeneity of distribution of cetuximab was achieved more rapidly. Cetuximab and trastuzumab distribute slowly, but at higher doses achieve a relatively uniform distribution after about 24 hours, most likely due to their long half-lives in the circulation. There remains poor distribution within hypoxic regions of tumors

  16. Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo

    NARCIS (Netherlands)

    Labrijn, Aran F.; Buijsse, Antonio Ortiz; van den Bremer, Ewald T. J.; Verwilligen, Annemiek Y. W.; Bleeker, Wim K.; Thorpe, Susan J.; Killestein, Joep; Polman, Chris H.; Aalberse, Rob C.; Schuurman, Janine; van de Winkel, Jan G. J.; Parren, Paul W. H. I.

    Two humanized IgG4 antibodies, natalizumab and gemtuzumab, are approved for human use, and several others, like TGN1412, are or have been in clinical development. Although IgG4 antibodies can dynamically exchange half-molecules(1), Fab-arm exchange with therapeutic antibodies has not been

  17. Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo

    NARCIS (Netherlands)

    Labrijn, Aran F.; Buijsse, Antonio Ortiz; van den Bremer, Ewald T. J.; Verwilligen, Annemiek Y. W.; Bleeker, Wim K.; Thorpe, Susan J.; Killestein, Joep; Polman, Chris H.; Aalberse, Rob C.; Schuurman, Janine; van de Winkel, Jan G. J.; Parren, Paul W. H. I.


    Two humanized IgG4 antibodies, natalizumab and gemtuzumab, are approved for human use, and several others, like TGN1412, are or have been in clinical development. Although IgG4 antibodies can dynamically exchange half-molecules, Fab-arm exchange with therapeutic antibodies has not been demonstrated

  18. Pharmacokinetics and concentration-effect relationships of therapeutic monoclonal antibodies and fusion proteins. (United States)

    Ternant, David; Paintaud, Gilles


    Although monoclonal antibodies (mAbs) constitute a major advance in therapeutics, their pharmacokinetic (PK) and pharmacodynamic (PD) properties are not fully understood. Saturable mechanisms are thought to occur in distribution and elimination of mAbs, which are protected from degradation by the Brambell's receptor (FcRn). The binding of mAbs to their target antigen explains part of their nonlinear PK and PD properties. The interindividual variability in mAb PK can be explained by several factors, including immune response against the biodrug and differences in the number of antigenic sites. The concentration-effect relationships of mAbs are complex and dependent on their mechanism of action. Interindividual differences in mAb PD can be explained by factors such as genetics and clinical status. PK and concentration-effect studies are necessary to design optimal dosing regimens. Because of their above-mentioned characteristics, the interindividual variability in their dose-response relationships must be studied by PK-PD modelling.

  19. A monoclonal antibody TrkB receptor agonist as a potential therapeutic for Huntington's disease.

    Directory of Open Access Journals (Sweden)

    Daniel Todd

    Full Text Available Huntington's disease (HD is a devastating, genetic neurodegenerative disease caused by a tri-nucleotide expansion in exon 1 of the huntingtin gene. HD is clinically characterized by chorea, emotional and psychiatric disturbances and cognitive deficits with later symptoms including rigidity and dementia. Pathologically, the cortico-striatal pathway is severely dysfunctional as reflected by striatal and cortical atrophy in late-stage disease. Brain-derived neurotrophic factor (BDNF is a neuroprotective, secreted protein that binds with high affinity to the extracellular domain of the tropomyosin-receptor kinase B (TrkB receptor promoting neuronal cell survival by activating the receptor and down-stream signaling proteins. Reduced cortical BDNF production and transport to the striatum have been implicated in HD pathogenesis; the ability to enhance TrkB signaling using a BDNF mimetic might be beneficial in disease progression, so we explored this as a therapeutic strategy for HD. Using recombinant and native assay formats, we report here the evaluation of TrkB antibodies and a panel of reported small molecule TrkB agonists, and identify the best candidate, from those tested, for in vivo proof of concept studies in transgenic HD models.

  20. Monoclonal Antibody and Fusion Protein Biosimilars Across Therapeutic Areas: A Systematic Review of Published Evidence. (United States)

    Jacobs, Ira; Petersel, Danielle; Shane, Lesley G; Ng, Chee-Keng; Kirchhoff, Carol; Finch, Gregory; Lula, Sadiq


    Despite regulatory efforts to formalize guidance policies on biosimilars, there remains a need to educate healthcare stakeholders on the acknowledged definition of biosimilarity and the data that underpin it. The objectives of the study were to systematically collate published data for monoclonal antibodies and fusion protein biosimilars indicated for cancer, chronic inflammatory diseases, and other indications, and to explore differences in the type and weight (quantity and quality) of available evidence. MEDLINE, Embase, and ISI Web of Science were searched to September 2015. Conference proceedings (n = 17) were searched 2012 to July 2015. Included studies were categorized by originator, study type, and indication. To assess data strength and validity, risk of bias assessments were undertaken. Across therapeutic areas, 43 named (marketed or proposed) biosimilars were identified for adalimumab, abciximab, bevacizumab, etanercept, infliximab, omalizumab, ranibizumab, rituximab, and trastuzumab originators. Infliximab CT-P13, SB2, and etanercept SB4 biosimilars have the greatest amount of published evidence of similarity with their originators, based on results of clinical studies involving larger numbers of patients or healthy subjects (N = 1405, 743, and 734, respectively). Published data were also retrieved for marketed intended copies of etanercept and rituximab. This unbiased synthesis of the literature exposed significant differences in the extent of published evidence between molecules at preclinical, clinical, and post-marketing stages of development, providing clinicians and payers with a consolidated view of the available data and remaining gaps.

  1. Allosteric mechanism of action of the therapeutic anti-IgE antibody omalizumab. (United States)

    Davies, Anna M; Allan, Elizabeth G; Keeble, Anthony H; Delgado, Jean; Cossins, Benjamin P; Mitropoulou, Alkistis N; Pang, Marie O Y; Ceska, Tom; Beavil, Andrew J; Craggs, Graham; Westwood, Marta; Henry, Alistair J; McDonnell, James M; Sutton, Brian J


    Immunoglobulin E and its interactions with receptors FcϵRI and CD23 play a central role in allergic disease. Omalizumab, a clinically approved therapeutic antibody, inhibits the interaction between IgE and FcϵRI, preventing mast cell and basophil activation, and blocks IgE binding to CD23 on B cells and antigen-presenting cells. We solved the crystal structure of the complex between an omalizumab-derived Fab and IgE-Fc, with one Fab bound to each Cϵ3 domain. Free IgE-Fc adopts an acutely bent structure, but in the complex it is only partially bent, with large-scale conformational changes in the Cϵ3 domains that inhibit the interaction with FcϵRI. CD23 binding is inhibited sterically due to overlapping binding sites on each Cϵ3 domain. Studies of omalizumab Fab binding in solution demonstrate the allosteric basis for FcϵRI inhibition and, together with the structure, reveal how omalizumab may accelerate dissociation of receptor-bound IgE from FcϵRI, exploiting the intrinsic flexibility and allosteric potential of IgE. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Consistent manufacturing and quality control of a highly complex recombinant polyclonal antibody product for human therapeutic use. (United States)

    Frandsen, Torben P; Naested, Henrik; Rasmussen, Søren K; Hauptig, Peter; Wiberg, Finn C; Rasmussen, Lone Kjaer; Jensen, Anne Marie Valentin; Persson, Pia; Wikén, Margareta; Engström, Anders; Jiang, Yun; Thorpe, Susan J; Förberg, Cecilia; Tolstrup, Anne B


    The beneficial effect of antibody therapy in human disease has become well established mainly for the treatment of cancer and immunological disorders. The inherent monospecificity of mAbs present limitations to mAb therapy which have become apparent notably in addressing complex entities like infectious agents or heterogenic endogenous targets. For such indications mixtures of antibodies comprising a combination of specificities would convey more potent biological effect which could translate into therapeutic efficacy. Recombinant polyclonal antibodies (rpAb) consisting of a defined number of well-characterized mAbs constitute a new class of target specific antibody therapy. We have developed a cost-efficient cell banking and single-batch manufacturing concept for the production of such products and demonstrate that a complex pAb composition, rozrolimupab, comprising 25 individual antibodies can be manufactured in a highly consistent manner in a scaled-up manufacturing process. We present a strategy for the release and characterization of antibody mixtures which constitute a complete series of chemistry, manufacturing, and control (CMC) analytical methods to address identity, purity, quantity, potency, and general characteristics. Finally we document selected quality attributes of rozrolimupab based on a battery of assays at the genetic-, protein-, and functional level and demonstrate that the manufactured rozrolimupab batches are highly pure and very uniform in their composition. Copyright © 2011 Wiley Periodicals, Inc.

  3. Therapeutic potential of an anti-high mobility group box-1 monoclonal antibody in epilepsy. (United States)

    Zhao, Junli; Wang, Yi; Xu, Cenglin; Liu, Keyue; Wang, Ying; Chen, Liying; Wu, Xiaohua; Gao, Feng; Guo, Yi; Zhu, Junming; Wang, Shuang; Nishibori, Masahiro; Chen, Zhong


    Brain inflammation is a major factor in epilepsy, and the high mobility group box-1 (HMGB1) protein is known to contribute significantly to the generation of seizures. Here, we investigated the therapeutic potential of an anti-HMGB1 monoclonal antibody (mAb) in epilepsy. anti-HMGB1 mAb attenuated both acute seizure models (maximal electroshock seizure, pentylenetetrazole-induced and kindling-induced), and chronic epilepsy model (kainic acid-induced) in a dose-dependent manner. Meanwhile, the anti-HMGB1 mAb also attenuated seizure activities of human brain slices obtained from surgical resection from drug-resistant epilepsy patients. The mAb showed an anti-seizure effect with a long-term manner and appeared to be minimal side effects at even very high dose (no disrupted physical EEG rhythm and no impaired basic physical functions, such as body growth rate and thermoregulation). This anti-seizure effect of mAb results from its inhibition of translocated HMGB1 from nuclei following seizures, and the anti-seizure effect was absent in toll-like receptor 4 knockout (TLR4 -/- ) mice. Interestingly, the anti-HMGB1 mAb also showed a disease-modifying anti-epileptogenetic effect on epileptogenesis after status epileptics, which is indicated by reducing seizure frequency and improving the impaired cognitive function. These results indicate that the anti-HMGB1 mAb should be viewed as a very promising approach for the development of novel therapies to treat refractory epilepsy. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. An Integrated Solution-Based Rapid Sample Preparation Procedure for the Analysis of N-Glycans From Therapeutic Monoclonal Antibodies. (United States)

    Aich, Udayanath; Liu, Aston; Lakbub, Jude; Mozdzanowski, Jacek; Byrne, Michael; Shah, Nilesh; Galosy, Sybille; Patel, Pramthesh; Bam, Narendra


    Consistent glycosylation in therapeutic monoclonal antibodies is a major concern in the biopharmaceutical industry as it impacts the drug's safety and efficacy and manufacturing processes. Large numbers of samples are created for the analysis of glycans during various stages of recombinant proteins drug development. Profiling and quantifying protein N-glycosylation is important but extremely challenging due to its microheterogeneity and more importantly the limitations of existing time-consuming sample preparation methods. Thus, a quantitative method with fast sample preparation is crucial for understanding, controlling, and modifying the glycoform variance in therapeutic monoclonal antibody development. Presented here is a rapid and highly quantitative method for the analysis of N-glycans from monoclonal antibodies. The method comprises a simple and fast solution-based sample preparation method that uses nontoxic reducing reagents for direct labeling of N-glycans. The complete work flow for the preparation of fluorescently labeled N-glycans takes a total of 3 h with less than 30 min needed for the release of N-glycans from monoclonal antibody samples. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  5. Human antibodies to the dengue virus E-dimer epitope have therapeutic activity against Zika virus infection. (United States)

    Fernandez, Estefania; Dejnirattisai, Wanwisa; Cao, Bin; Scheaffer, Suzanne M; Supasa, Piyada; Wongwiwat, Wiyada; Esakky, Prabagaran; Drury, Andrea; Mongkolsapaya, Juthathip; Moley, Kelle H; Mysorekar, Indira U; Screaton, Gavin R; Diamond, Michael S


    The Zika virus (ZIKV) epidemic has resulted in congenital abnormalities in fetuses and neonates. Although some cross-reactive dengue virus (DENV)-specific antibodies can enhance ZIKV infection in mice, those recognizing the DENV E-dimer epitope (EDE) can neutralize ZIKV infection in cell culture. We evaluated the therapeutic activity of human monoclonal antibodies to DENV EDE for their ability to control ZIKV infection in the brains, testes, placentas, and fetuses of mice. A single dose of the EDE1-B10 antibody given 3 d after ZIKV infection protected against lethality, reduced ZIKV levels in brains and testes, and preserved sperm counts. In pregnant mice, wild-type or engineered LALA variants of EDE1-B10, which cannot engage Fcg receptors, diminished ZIKV burden in maternal and fetal tissues, and protected against fetal demise. Because neutralizing antibodies to EDE have therapeutic potential against ZIKV, in addition to their established inhibitory effects against DENV, it may be possible to develop therapies that control disease caused by both viruses.

  6. Engineering Venom’s Toxin-Neutralizing Antibody Fragments and Its Therapeutic Potential

    Directory of Open Access Journals (Sweden)

    Larissa M. Alvarenga


    Full Text Available Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be associated with adverse effects. Recombinant antibodies and smaller functional units are now emerging as credible alternatives and constitute a source of still unexploited biomolecules capable of neutralizing venoms. This review will be a walk through the technologies that have recently been applied leading to novel antibody formats with better properties in terms of homogeneity, specific activity and possible safety.

  7. 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. (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


    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.

  8. Comparison of therapeutic efficacy and biodistribution of 213Bi- and 211At-labeled monoclonal antibody MX35 in an ovarian cancer model

    DEFF Research Database (Denmark)

    Gustafsson, Anna M E; Bäck, Tom; Elgqvist, Jörgen


    The purpose of this study was to compare the therapeutic efficacy and biodistribution of the monoclonal antibody MX35 labeled with either (213)Bi or (211)At, both α-emitters, in an ovarian cancer model.......The purpose of this study was to compare the therapeutic efficacy and biodistribution of the monoclonal antibody MX35 labeled with either (213)Bi or (211)At, both α-emitters, in an ovarian cancer model....

  9. SGLT2 inhibitors provide an effective therapeutic option for diabetes complicated with insulin antibodies. (United States)

    Hayashi, Akinori; Takano, Koji; Kawai, Sayuki; Shichiri, Masayoshi


    Diabetes mellitus complicated with insulin antibodies is rare in clinical practice but usually difficult to control. A high amount of insulin antibodies, especially with low affinity and high binding capacity, leads to unstable glycemic control characterized by hyperglycemia unresponsive to large volume of insulin and unanticipated hypoglycemia. There are several treatment options, such as changing insulin preparation, immunosupression with glucocorticoids, and plasmapheresis, most of which are of limited efficacy. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a novel class of drug which decrease renal glucose reabsorption and lowers plasma glucose level independent of insulin action. We report here a case with diabetes complicated with insulin antibodies who was effectively controlled by an SGLT2 inhibitor. A 47-year-old man with type 2 diabetes treated with insulin had very poor glycemic control characterized by postprandial hyperglycemia unresponsive to insulin therapy and repetitive hypoglycemia due to insulin antibodies. Treatment with ipragliflozin, an SGLT2 inhibitor, improved HbA1c from 8.4% to 6.0% and glycated albumin from 29.4% to 17.9%. Continuous glucose monitoring revealed improvement of glycemic profile (average glucose level from 212 mg/dL to 99 mg/dL and glycemic standard deviation from 92 mg/dL to 14 mg/dL) with disappearance of hypoglycemic events. This treatment further ameliorated the characteristics of insulin antibodies and resulted in reduced insulin requirement. SGLT2 inhibitors may offer an effective treatment option for managing the poor glycemic control in diabetes complicated with insulin antibodies.

  10. Transcriptomic analysis of monocytes and macrophages derived from CLL patients which display differing abilities to respond to therapeutic antibody immune complexes

    Directory of Open Access Journals (Sweden)

    M. Burgess


    Full Text Available Chronic lymphocytic leukemia (CLL is the most common adult leukemia. While therapeutic antibodies show clinical activity in CLL patients, resistance inevitably develops resulting in treatment failure. Identifying mechanisms of antibody resistance and methods to reduce resistance would be valuable in managing CLL. Monocyte derived cells (MDCs, also known as nurse like cells (NLCs in CLL [1,2], are known to be crucial components of the CLL microenvironment network and following “maturation” in in vitro culture systems are able to provide support for the survival of the malignant B cells from CLL patients. In addition to their protective role, MDCs are key effector cells in mediating responses to therapeutic antibody therapies [3]. We have determined that macrophages from patients with early stable CLL are able to elicit superior cytotoxic response to therapeutic antibodies than macrophages derived from patients with progressive CLL. We have exploited this unique finding to gain insight into antibody resistance. Thus, we have profiled monocytes on day 0 and MDCs on day 7 from antibody sensitive and antibody resistant CLL patients (GEO accession number GEO: GSE71409. We show that there are no significant differences in transcriptomes from the monocytes or MDCs derived from sensitive or resistant patient samples. However, we show that MDCs acquire an M2-like macrophage transcriptomic signature following 7 days culture regardless of whether they were derived from sensitive or resistant patient samples. Keywords: Chronic lymphocytic leukemia, Monocyte derived cells, Antibody resistance, Microarray

  11. Characterization of crystals of an antibody-recognition fragment of the cancer differentiation antigen mesothelin in complex with the therapeutic antibody MORAb-009

    International Nuclear Information System (INIS)

    Ma, Jichun; Tang, Wai Kwan; Esser, Lothar; Pastan, Ira; Xia, Di


    The therapeutic antibody MORAb-009 disrupts the interaction of mesothelin and the ovarian cancer antigen CA-125. Crystals have been grown of the Fab fragment derived from MORAb-009 and of its complex with an N-terminal fragment of mesothelin. The mesothelin-specific monoclonal antibody MORAb-009 is capable of blocking the binding of mesothelin to CA-125 and displays promising anticancer potential. It is currently undergoing clinical trials. In order to understand the basis of the interaction between MORAb-009 and mesothelin at atomic resolution, both the Fab fragment of MORAb-009 and the complex between the Fab and an N-terminal fragment of mesothelin (residues 7–64) were crystallized. The crystals of the Fab diffracted X-rays to 1.75 Å resolution and had the symmetry of space group P4 1 2 1 2, with unit-cell parameters a = b = 140.6, c = 282.0 Å. The crystals of the mesothelin–Fab complex diffracted to 2.6 Å resolution and belonged to the hexagonal space group P6 4 , with unit-cell parameters a = b = 146.2, c = 80.9 Å. Structural analyses of these molecules are in progress

  12. 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


    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.

  13. In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis. (United States)

    Salinas-Jazmín, Nohemi; González-González, Edith; Vásquez-Bochm, Luz X; Pérez-Tapia, Sonia M; Velasco-Velázquez, Marco A


    Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

  14. Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma. (United States)

    Li, Nan; Fu, Haiying; Hewitt, Stephen M; Dimitrov, Dimiter S; Ho, Mitchell


    Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma.

  15. 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


    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

  16. Therapeutic Activity of an Engineered Synthetic Killer Antiidiotypic Antibody Fragment against Experimental Mucosal and Systemic Candidiasis


    Polonelli, Luciano; Magliani, Walter; Conti, Stefania; Bracci, Luisa; Lozzi, Luisa; Neri, Paolo; Adriani, Daniela; De Bernardis, Flavia; Cassone, Antonio


    Peptides derived from the sequence of a single-chain, recombinant, antiidiotypic antibody (IdAb; KT-scFv) acting as a functional internal image of a microbicidal, wide-spectrum yeast killer toxin (KT) were synthesized and studied for their antimicrobial activity by using the KT-susceptible Candida albicans as model organism. A decapeptide containing the first three amino acids (SAS) of the light chain CDR1 was selected and optimized by alanine replacement of a single residue. This peptide exe...

  17. Immobilization of enzymes and antibodies to radiation grafted polymers for therapeutic and diagnostic applications

    Energy Technology Data Exchange (ETDEWEB)

    Hoffman, A.S.; Gombotz, W.R.; Uenoyama, S.; Dong, L.C.; Schmer, G.


    Pre-irradiation and mutual radiation grafting were employed to produce poly(methacrylic acid) (MAAc) hydrogels on polypropylene/polyethylene (PP/PE) copolymer films, and porous PP fibers of a plasma filter. A diphenyl picryl hydrazyl (DPPH) assay was developed to measure the surface peroxide concentration of the pre-irradiated PP/PE films prior to grafting. Mutually grafted porous PP fibers were used for subsequent immobilization of L-asparaginase while the mutually grafted PP/PE films were used to immobilize a schistosoma monoclonal antibody.

  18. Therapeutic effect of anti-feline TNF-alpha monoclonal antibody for feline infectious peritonitis. (United States)

    Doki, Tomoyoshi; Takano, Tomomi; Kawagoe, Kohei; Kito, Akihiko; Hohdatsu, Tsutomu


    Feline infectious peritonitis virus (FIPV) replication in macrophages/monocytes induced tumor necrosis factor (TNF)-alpha production, and that the TNF-alpha produced was involved in aggravating the pathology of FIP. We previously reported the preparation of a feline TNF-alpha (fTNF-alpha)-neutralizing mouse monoclonal antibody (anti-fTNF-alpha mAb). This anti-fTNF-alpha mAb 2-4 was confirmed to inhibit the following fTNF-alpha-induced conditions in vitro. In the present study, we investigated whether mAb 2-4 improved the FIP symptoms and survival rate of experimentally FIPV-inoculated SPF cats. Progression to FIP was prevented in 2 out of 3 cats treated with mAb 2-4, whereas all 3 cats developed FIP in the placebo control group. Plasma alpha1-glycoprotein and vascular endothelial growth factor levels were improved by the administration of mAb 2-4, and the peripheral lymphocyte count also recovered. These results strongly suggested that the anti-fTNF-alpha antibody is effective for the treatment of FIP. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. An influenza A virus (H7N9) anti-neuraminidase monoclonal antibody with prophylactic and therapeutic activity in vivo (United States)

    Wilson, Jason R.; Guo, Zhu; Reber, Adrian; Kamal, Ram P.; Music, Nedzad; Gansebom, Shane; Bai, Yaohui; Levine, Min; Carney, Paul; Tzeng, Wen-Pin; Stevens, James; York, Ian A.


    Zoonotic A(H7N9) avian influenza viruses emerged in China in 2013 and continue to be a threat to human public health, having infected over 800 individuals with a mortality rate approaching 40%. Treatment options for people infected with A(H7N9) include the use of neuraminidase (NA) inhibitors. However, like other influenza viruses, A(H7N9) can become resistant to these drugs. The use of monoclonal antibodies is a rapidly developing strategy for controlling influenza virus infection. Here we generated a murine monoclonal antibody (3c10-3) directed against the NA of A(H7N9) and show that prophylactic systemic administration of 3c10-3 fully protected mice from lethal challenge with wild-type A/Anhui/1/2013 (H7N9). Further, post-infection treatment with a single systemic dose of 3c10-3 at either 24, 48 or 72 h post A(H7N9) challenge resulted in both dose- and time-dependent protection of up to 100% of mice, demonstrating therapeutic potential for 3c10-3. Epitope mapping revealed that 3c10-3 binds near the enzyme active site of NA, and functional characterization showed that 3c10-3 inhibits the enzyme activity of NA and restricts the cell-to-cell spread of the virus in cultured cells. Affinity analysis also revealed that 3c10-3 binds equally well to recombinant NA of wild-type A/Anhui/1/2013 and to a variant NA carrying a R289K mutation known to infer NAI resistance. These results suggest that 3c10-3 has the potential to be used as a therapeutic to treat A(H7N9) infections either as an alternative to, or in combination with, current NA antiviral inhibitors. PMID:27713074

  20. Therapeutic monoclonal antibodies: scFv patents as a marker of a new class of potential biopharmaceuticals

    Directory of Open Access Journals (Sweden)

    Manuela Berto Pucca


    Full Text Available Monoclonal antibodies represent the fastest growing class of biopharmaceutical products and have a host of applications in medical research, diagnosis, therapy, and basic science. The production of recombinant monoclonal antibodies has revolutionized the generation of immunoglobulins, and their use represents a strategic breakthrough, affecting the global pharmaceutical market for therapeutic proteins. In the present work, a review of scFv, and the number of related patents, has been carried out. The results show that several countries have scFv patents, most notably the United States, China and United Kingdom. The target of these scFv antibodies was also assessed and the results demonstrate that most are directed toward cancer therapy.Anticorpos monoclonais representam a classe de maior crescimento em produtos de biofármacos e possuem várias aplicações em pesquisa médica, diagnóstico, terapias e ciência básica. A produção de anticorpos monoclonais recombinantes revolucionou a geração de imunoglobulinas e sua utilização implica em avanço estratégico, afetando o mercado farmacêutico global de proteínas terapêuticas. No presente trabalho, uma revisão sobre scFv e a relação do seu número de patentes foi analisada. Os resultados mostram que vários países apresentam patentes de scFv com destaque para os Estados Unidos, China e Reino Unido. Os alvos desses anticorpos também foram avaliados e as análises revelaram que a maioria é destinado a terapias contra o câncer.

  1. Glycoengineering of therapeutic antibodies enhances monocyte/macrophage-mediated phagocytosis and cytotoxicity. (United States)

    Herter, Sylvia; Birk, Martina C; Klein, Christian; Gerdes, Christian; Umana, Pablo; Bacac, Marina


    Therapeutic Abs possess several clinically relevant mechanisms of action including perturbation of tumor cell signaling, activation of complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cellular phagocytosis (ADCP), and induction of adaptive immunity. In view of the important role of phagocytic lineage cells in the mechanism of action of therapeutic Abs, we analyzed FcγR receptor-dependent effector functions of monocytes and macrophages triggered by glycoengineered (GE) Abs (having enhanced FcγRIIIa [CD16a] binding affinity) versus their wild-type (WT) counterparts under different experimental conditions. We first defined the precise FcγR repertoire on classical and nonclassical intermediate monocytes--M1 and M2c macrophage populations. We further show that WT and GE Abs display comparable binding and induce similar effector functions (ADCC and ADCP) in the absence of nonspecific, endogenous IgGs. However, in the presence of these IgGs (i.e., in a situation that more closely mimics physiologic conditions), GE Abs display significantly superior binding and promote stronger monocyte and macrophage activity. These data show that in addition to enhancing CD16a-dependent NK cell cytotoxicity, glycoengineering also enhances monocyte and macrophage phagocytic and cytotoxic activities through enhanced binding to CD16a under conditions that more closely resemble the physiologic setting.

  2. Medulloblastoma: evaluation of proliferative index by monoclonal antibody Mib-1, its prognostic correlation and therapeutic implications

    Directory of Open Access Journals (Sweden)

    Ferrari Antonio Fernandes


    Full Text Available In the past few years, the monoclonal antibody MIB-1 has been used by researchers in order to retrospectively study paraffin imbibed tumor fragments. The medulloblastoma is the most common malignant central nervous system tumor in childhood. The objectives were: determination of the mean Mib-1 LI value from these patients, as well as the prognostic value of the method.This retrospective study represents an analysis of the cellular proliferation index of posterior fossa medulloblastomas collected from 22 patients at A.C. Camargo Hospital, from January 1990 to December 1999. The histopathological diagnosis was confirmed by H&E and proliferative index (LI was achived with Mib-1 which detects proliferating cells during G1, G2, S and M phases.The results demostrated that the mean Mib-1 was 30,1%, and ranged from 5,2% to 62,0%.In conclusion, this method has prognostic value, has to be used as routine for patients harboring medulloblastomas and the ones who have PI greater than the mean value found in this study, should be treated aggressively.

  3. Designed Amino Acid Feed in Improvement of Production and Quality Targets of a Therapeutic Monoclonal Antibody.

    Directory of Open Access Journals (Sweden)

    Fatemeh Torkashvand

    Full Text Available Cell culture feeds optimization is a critical step in process development of pharmaceutical recombinant protein production. Amino acids are the basic supplements of mammalian cell culture feeds with known effect on their growth promotion and productivity. In this study, we reported the implementation of the Plackett-Burman (PB multifactorial design to screen the effects of amino acids on the growth promotion and productivity of a Chinese hamster ovary DG-44 (CHO-DG44 cell line producing bevacizumab. After this screening, the amino acid combinations were optimized by the response surface methodology (RSM to determine the most effective concentration in feeds. Through this strategy, the final monoclonal antibody (mAb titre was enhanced by 70%, compared to the control group. For this particular cell line, aspartic acid, glutamic acid, arginine and glycine had the highest positive effects on the final mAb titre. Simultaneously, the impact of the designed amino acid feed on some critical quality attributes of bevacizumab was examined in the group with highest productivity. The product was analysed for N-glycan profiles, charge variant distribution, and low molecular weight forms. The results showed that the target product quality has been improved using this feeding strategy. It was shown how this strategy could significantly diminish the time and number of experiments in identifying the most effective amino acids and related concentrations in target product enhancement. This model could be successfully applied to other components of culture media and feeds.

  4. Therapeutic monoclonal antibodies and the need for targeted pharmacovigilance in India. (United States)

    Kalaivani, M; Singh, Abhishank; Kalaiselvan, V


    A growing number of innovative mAb therapeutics are on the global market, and biosimilar versions have now also been approved, including in India. Although efficacy and safety is demonstrated prior to approval, targeted pharmacovigilance is essential for the identification and assessment of risk for any mAb products. We analyzed the ADR data related to mAbs reported to the NCC-PvPI through the spontaneous reporting system Vigiflow during April 2011 to February 2014 to identify mAbs with the highest number of ADR including fatal/serious ADR. Only 0.72% reports were related to mAbs. Although 15 mAbs are approved in the country, only 6 mAbs were reported through Vigiflow. Rituximab was highly reported, and no fatal/serious ADR related to any mAbs were reported during the study period. Our study shows that PvPI is effective and robust system in the detection and assessment of risks associated with the use of mAbs.

  5. Towards the implementation of quality by design to the production of therapeutic monoclonal antibodies with desired glycosylation patterns. (United States)

    del Val, Ioscani Jimenez; Kontoravdi, Cleo; Nagy, Judit M


    Quality by design (QbD) is a scheme for the development, manufacture, and approval of pharmaceutical products. The end goal of QbD is to ensure product quality by building it into the manufacturing process. The main regulatory bodies are encouraging its implementation to the manufacture of all new pharmaceuticals including biological products. Monoclonal antibodies (mAbs) are currently the leading products of the biopharmaceutical industry. It has been widely reported that glycosylation directly influences the therapeutic mechanisms by which mAbs function in vivo. In addition, glycosylation has been identified as one of the main sources of monoclonal antibody heterogeneity, and thus, a critical parameter to follow during mAb manufacture. This article reviews the research on glycosylation of mAbs over the past 2 decades under the QbD scope. The categories presented under this scope are: (a) definition of the desired clinical effects of mAbs, (b) definition of the glycosylation-associated critical quality attributes (glycCQAs) of mAbs, (c) assessment of process parameters that pose a risk for mAb glycCQAs, and (d) methods for accurately quantifying glycCQAs of mAbs. The information available in all four areas leads us to conclude that implementation of QbD to the manufacture of mAbs with specific glycosylation patterns will be a reality in the near future. We also foresee that the implementation of QbD will lead to the development of more robust and efficient manufacturing processes and to a new generation of mAbs with increased clinical efficacy. Copyright © 2010 American Institute of Chemical Engineers (AIChE).

  6. A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface

    DEFF Research Database (Denmark)

    de Jong, R. N.; Beurskens, F. J.; Verploegen, S.


    IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology......) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce...... conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD...

  7. Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins. (United States)

    Luo, Shen; Zhang, Baolin


    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.

  8. Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineered FcγReceptor IIIa-Immobilized Column. (United States)

    Kiyoshi, Masato; Caaveiro, Jose M M; Tada, Minoru; Tamura, Hiroko; Tanaka, Toru; Terao, Yosuke; Morante, Koldo; Harazono, Akira; Hashii, Noritaka; Shibata, Hiroko; Kuroda, Daisuke; Nagatoishi, Satoru; Oe, Seigo; Ide, Teruhiko; Tsumoto, Kouhei; Ishii-Watabe, Akiko


    The N-glycan moiety of IgG-Fc has a significant impact on multifaceted properties of antibodies such as in their effector function, structure, and stability. Numerous studies have been devoted to understanding its biological effect since the exact composition of the Fc N-glycan modulates the magnitude of effector functions such as the antibody-dependent cell mediated cytotoxicity (ADCC), and the complement-dependent cytotoxicity (CDC). To date, systematic analyses of the properties and influence of glycan variants have been of great interest. Understanding the principles on how N-glycosylation modulates those properties is important for the molecular design, manufacturing, process optimization, and quality control of therapeutic antibodies. In this study, we have separated a model therapeutic antibody into three fractions according to the composition of the N-glycan by using a novel FcγRIIIa chromatography column. Notably, Fc galactosylation was a major factor influencing the affinity of IgG-Fc to the FcγRIIIa immobilized on the column. Each antibody fraction was employed for structural, biological, and physicochemical analysis, illustrating the mechanism by which galactose modulates the affinity to FcγRIIIa. In addition, we discuss the benefits of the FcγRIIIa chromatography column to assess the heterogeneity of the N-glycan.

  9. Crystal clear: visualizing the intervention mechanism of the PD-1/PD-L1 interaction by two cancer therapeutic monoclonal antibodies

    Directory of Open Access Journals (Sweden)

    Shuguang Tan


    Full Text Available Abstract Antibody-based PD-1/PD-L1 blockade therapies have taken center stage in immunotherapies for cancer, with multiple clinical successes. PD-1 signaling plays pivotal roles in tumor-driven T-cell dysfunction. In contrast to prior approaches to generate or boost tumor-specific T-cell responses, antibody-based PD-1/PD-L1 blockade targets tumor-induced T-cell defects and restores pre-existing T-cell function to modulate antitumor immunity. In this review, the fundamental knowledge on the expression regulations and inhibitory functions of PD-1 and the present understanding of antibody-based PD-1/PD-L1 blockade therapies are briefly summarized. We then focus on the recent breakthrough work concerning the structural basis of the PD-1/PD-Ls interaction and how therapeutic antibodies, pembrolizumab targeting PD-1 and avelumab targeting PD-L1, compete with the binding of PD-1/PD-L1 to interrupt the PD-1/PD-L1 interaction. We believe that this structural information will benefit the design and improvement of therapeutic antibodies targeting PD-1 signaling.

  10. Contribution of enhanced engagement of antigen presentation machinery to the clinical immunogenicity of a human interleukin (IL)-21 receptor-blocking therapeutic antibody. (United States)

    Xue, L; Hickling, T; Song, R; Nowak, J; Rup, B


    Reliable risk assessment for biotherapeutics requires accurate evaluation of risk factors associated with immunogenicity. Immunogenicity risk assessment tools were developed and applied to investigate the immunogenicity of a fully human therapeutic monoclonal antibody, ATR-107 [anti-interleukin (IL)-21 receptor] that elicited anti-drug antibodies (ADA) in 76% of healthy subjects in a Phase 1 study. Because the ATR-107 target is expressed on dendritic cells (DCs), the immunogenicity risk related to engagement with DC and antigen presentation pathways was studied. Despite the presence of IL-21R on DCs, ATR-107 did not bind to the DCs more extensively than the control therapeutic antibody (PF-1) that had elicited low clinical ADA incidence. However, ATR-107, but not the control therapeutic antibody, was translocated to the DC late endosomes, co-localized with intracellular antigen-D related (HLA-DR) molecules and presented a dominant T cell epitope overlapping the complementarity determining region 2 (CDR2) of the light chain. ATR-107 induced increased DC activation exemplified by up-regulation of DC surface expression of CD86, CD274 (PD-L1) and CD40, increased expansion of activated DC populations expressing CD86(hi), CD40(hi), CD83(hi), programmed death ligand 1 (PD-L1)(hi), HLA-DR(hi) or CCR7(hi), as well as elevated secretion of tumour necrosis factor (TNF)-α by DCs. DCs exposed to ATR-107 stimulated an autologous T cell proliferative response in human donor cells, in concert with the detection of immunoglobulin (Ig)G-type anti-ATR-107 antibody response in clinical samples. Collectively, the enhanced engagement of antigen presentation machinery by ATR-107 was suggested. The approaches and findings described in this study may be relevant to identifying lower immunogenicity risk targets and therapeutic molecules. © 2015 British Society for Immunology.

  11. Antibody-mediated phagocytosis contributes to the anti-tumor activity of the therapeutic antibody daratumumab in lymphoma and multiple myeloma

    DEFF Research Database (Denmark)

    Overdijk, M. B.; Verploegen, S.; Bogels, M.


    Daratumumab (DARA) is a human CD38-specific IgG1 antibody that is in clinical development for the treatment of multiple myeloma (MM). The potential for IgG1 antibodies to induce macrophage-mediated phagocytosis, in combination with the known presence of macrophages in the tumor microenvironment...... in MM and other hematological tumors, led us to investigate the contribution of antibody-dependent, macrophage-mediated phagocytosis to DARA's mechanism of action. Live cell imaging revealed that DARA efficiently induced macrophage-mediated phagocytosis, in which individual macrophages rapidly...... and sequentially engulfed multiple tumor cells. DARA-dependent phagocytosis by mouse and human macrophages was also observed in an in vitro flow cytometry assay, using a range of MM and Burkitt's lymphoma cell lines. Phagocytosis contributed to DARA's anti-tumor activity in vivo, in both a subcutaneous...

  12. Amelioration of murine passive immune thrombocytopenia by IVIg and a therapeutic monoclonal CD44 antibody does not require the Myd88 signaling pathway.

    Directory of Open Access Journals (Sweden)

    Andrew R Crow

    Full Text Available Immune thrombocytopenia (ITP is an autoimmune bleeding disorder characterized by a low platelet count and the production of anti-platelet antibodies. The majority of ITP patients have antibodies to platelet integrin α(IIbβ₃ (GPIIbIIIa which can direct platelet phagocytosis by macrophages. One effective treatment for patients with ITP is intravenous immunoglobulin (IVIg which rapidly reverses thrombocytopenia. The exact mechanism of IVIg action in human patients is unclear, although in mouse models of passive ITP, IVIg can rapidly increase platelet counts in the absence of adaptive immunity. Another antibody therapeutic that can similarly increase platelet counts independent of adaptive immunity are CD44 antibodies. Toll-like receptors (TLRs are pattern recognition receptors which play a central role in helping direct the innate immune system. Dendritic cells, which are notable for their expression of TLRs, have been directly implicated in IVIg function as an initiator cell, while CD44 can associate with TLR2 and TLR4. We therefore questioned whether IVIg, or the therapeutic CD44 antibody KM114, mediate their ameliorative effects in a manner dependent upon normal TLR function. Here, we demonstrate that the TLR4 agonist LPS does not inhibit IVIg or KM114 amelioration of antibody-induced thrombocytopenia, and that these therapeutics do not ameliorate LPS-induced thrombocytopenia. IVIg was able to significantly ameliorate murine ITP in C3H/HeJ mice which have defective TLR4. All known murine TLRs except TLR3 utilize the Myd88 adapter protein to drive TLR signaling. Employing Myd88 deficient mice, we found that both IVIg and KM114 ameliorate murine ITP in Myd88 deficient mice to the same extent as normal mice. Thus both IVIg and anti-CD44 antibody can mediate their ameliorative effects in murine passive ITP independent of the Myd88 signaling pathway. These data help shed light on the mechanism of action of IVIg and KM114 in the amelioration of

  13. Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy

    DEFF Research Database (Denmark)

    Da Roit, F.; Engelberts, P. J.; Taylor, R. P.


    The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-delta inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated...... the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre......-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells...

  14. Antithyroglobulin antibody (United States)

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  15. Comprehensive Analysis of the Therapeutic IgG4 Antibody Pembrolizumab: Hinge Modification Blocks Half Molecule Exchange In Vitro and In Vivo. (United States)

    Yang, Xiaoyu; Wang, Fengqiang; Zhang, Ying; Wang, Larry; Antonenko, Svetlana; Zhang, Shuli; Zhang, Yi Wei; Tabrizifard, Mohammad; Ermakov, Grigori; Wiswell, Derek; Beaumont, Maribel; Liu, Liming; Richardson, Daisy; Shameem, Mohammed; Ambrogelly, Alexandre


    IgG4 antibodies are evolving as an important class of cancer immunotherapies. However, human IgG4 can undergo Fab arm (half molecule) exchange with other IgG4 molecules in vivo. The hinge modification by a point mutation (S228P) prevents half molecule exchange of IgG4. However, the experimental confirmation is still expected by regulatory agencies. Here, we report for the first time the extensive analysis of half molecule exchange for a hinge-modified therapeutic IgG4 molecule, pembrolizumab (Keytruda) targeting programmed death 1 (PD1) receptor that was approved for advanced melanoma. Studies were performed in buffer or human serum using multiple exchange partners including natalizumab (Tysabri) and human IgG4 pool. Formation of bispecific antibodies was monitored by fluorescence resonance energy transfer, exchange with Fc fragments, mixed mode chromatography, immunoassays, and liquid chromatography-mass spectrometry. The half molecule exchange was also examined in vivo in SCID (severe combined immunodeficiency) mice. Both in vitro and in vivo results indicate that the hinge modification in pembrolizumab prevented half molecule exchange, whereas the unmodified counterpart anti-PD1 wt showed active exchange activity with other IgG4 antibodies or self-exchange activity with its own molecules. Our work, as an example expected for meeting regulatory requirements, contributes to establish without ambiguity that hinge-modified IgG4 antibodies are suitable for biotherapeutic applications. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  16. Recommendations for the validation of cell-based assays used for the detection of neutralizing antibody immune responses elicited against biological therapeutics. (United States)

    Gupta, Shalini; Devanarayan, Viswanath; Finco, Deborah; Gunn, George R; Kirshner, Susan; Richards, Susan; Rup, Bonita; Song, An; Subramanyam, Meena


    The administration of biological therapeutics may result in the development of anti-drug antibodies (ADAs) in treated subjects. In some cases, ADA responses may result in the loss of therapeutic efficacy due to the formation of neutralizing ADAs (NAbs). An important characteristic of anti-drug NAbs is their direct inhibitory effect on the pharmacological activity of the therapeutic. Neutralizing antibody responses are of particular concern for biologic products with an endogenous homolog whose activity can be potentially dampened or completely inhibited by the NAbs leading to an autoimmune-type deficiency syndrome. Therefore, it is important that ADAs are detected and characterized appropriately using sensitive and reliable methods. The design, development and optimization of cell-based assays used for detection of NAbs have been published previously by Gupta et al. 2007 [1]. This paper provides recommendations on best practices for the validation of cell-based NAb assay and suggested validation parameters based on the experience of the authors. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. [Immune checkpoint inhibitors (antibodies to PD1 and PD-L1), a new therapeutic weapon against non-small cell bronchial carcinoma]. (United States)

    Berghmans, T; Grigoriu, B; Sculier, J P; Meert, A P


    Classical therapeutic strategy for advanced and metastatic non-small cell lung cancer, without activable oncogenic driver mutation, has been based mainly on cytotoxic chemotherapy with modest benefits in terms of increased survival. A better understanding of the mechanisms involved in the regulation of the immune system led to the development of antibodies directed against immune checkpoints such as PD-L1. The first encouraging clinical data from phase I studies assessing anti-PD1 and anti-PD-L1 antibodies have been confirmed in randomised phase III trials. These new drugs now constitute a standard second-line treatment for metastatic tumours and in the future, at least for pembrolizumab, in the first line. Their adjuvant role after locoregional treatment with curative intent is currently under investigation. Copyright © 2017 SPLF. Published by Elsevier Masson SAS. All rights reserved.

  18. Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy (United States)

    Roit, Fabio Da; Engelberts, Patrick J.; Taylor, Ronald P.; Breij, Esther C.W.; Gritti, Giuseppe; Rambaldi, Alessandro; Introna, Martino; Parren, Paul W.H.I.; Beurskens, Frank J.; Golay, Josée


    The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells, and antibody-dependent cellular cytotoxicity by these cells, as well as phagocytosis by macrophages or neutrophils were inhibited by ibrutinib with a half maximal effective concentration of 0.3–3 μM. Analysis of anti-CD20 mediated activation of natural killer cells isolated from patients on continued oral ibrutinib treatment suggested that repeated drug dosing inhibits these cells in vivo. Finally we show that the phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib similarly inhibited the immune cell-mediated mechanisms induced by anti-CD20 antibodies, although the effects of this drug at 10 μM were weaker than those observed with ibrutinib at the same concentration. We conclude that the design of combined treatment schedules of anti-CD20 antibodies with these kinase inhibitors should consider the multiple negative interactions between these two classes of drugs. PMID:25344523

  19. Advantages of Papio anubis for preclinical testing of immunotoxicity of candidate therapeutic antagonist antibodies targeting CD28 (United States)

    Poirier, Nicolas; Mary, Caroline; Le Bas-Bernardet, Stephanie; Daguin, Veronique; Belarif, Lyssia; Chevalier, Melanie; Hervouet, Jeremy; Minault, David; Ville, Simon; Charpy, Vianney; Blancho, Gilles; Vanhove, Bernard


    Antagonist anti-CD28 antibodies prevent T-cell costimulation and are functionally different from CTLA4Ig since they cannot block CTLA-4 and PDL-1 co-inhibitory signals. They demonstrated preclinical efficacy in suppressing effector T cells while enhancing immunoregulatory mechanisms. Because a severe cytokine release syndrome was observed during the Phase 1 study with the superagonist anti-CD28 TGN1412, development of other anti-CD28 antibodies requires careful preclinical evaluation to exclude any potential immunotoxicity side-effects. The failure to identify immunological toxicity of TGN1412 using macaques led us to investigate more relevant preclinical models. We report here that contrary to macaques, and like in man, all baboon CD4-positive T lymphocytes express CD28 in their effector memory cells compartment, a lymphocyte subtype that is the most prone to releasing cytokines after reactivation. Baboon lymphocytes are able to release pro-inflammatory cytokines in vitro in response to agonist or superagonist anti-CD28 antibodies. Furthermore, we compared the reactivity of human and baboon lymphocytes after transfer into non obese diabetic/severe combined immunodeficiency (NOD/SCID) interleukin-2rγ knockout mice and confirmed that both cell types could release inflammatory cytokines in situ after injection of agonistic anti-CD28 antibodies. In contrast, FR104, a monovalent antagonistic anti-CD28 antibody, did not elicit T cell activation in these assays, even in the presence of anti-drug antibodies. Infusion to baboons also resulted in an absence of cytokine release. In conclusion, the baboon represents a suitable species for preclinical immunotoxicity evaluation of anti-CD28 antibodies because their effector memory T cells do express CD28 and because cytokine release can be assessed in vitro and trans vivo. PMID:24598534

  20. Buffer-free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions: from protein buffer capacity prediction to bioprocess applications. (United States)

    Bahrenburg, Sven; Karow, Anne R; Garidel, Patrick


    Protein therapeutics, including monoclonal antibodies (mAbs), have significant buffering capacity, particularly at concentrations>50 mg/mL. This report addresses pH-related issues critical to adoption of self-buffered monoclonal antibody formulations. We evaluated solution conditions with protein concentrations ranging from 50 to 250 mg/mL. Samples were both buffer-free and conventionally buffered with citrate. Samples were non-isotonic or adjusted for isotonicity with NaCl or trehalose. Studies included accelerated temperature stability tests, shaking stability studies, and pH changes in infusion media as protein concentrate is added. We present averaged buffering slopes of capacity that can be applied to any mAb and present a general method for calculating buffering capacity of buffer-free, highly concentrated antibody liquid formulations. In temperature stability tests, neither buffer-free nor conventionally buffered solution conditions showed significant pH changes. Conventionally buffered solutions showed significantly higher opalescence than buffer-free ones. In general, buffer-free solution conditions showed less aggregation than conventionally buffered solutions. Shaking stability tests showed no differences between buffer-free and conventionally buffered solutions. "In-use" preparation experiments showed that pH in infusion bag medium can rapidly approximate that of self-buffered protein concentrate as concentrate is added. In summary, the buffer capacity of proteins can be predicted and buffer-free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Antibodies Against Infliximab Are Associated with De Novo Development of Antibodies to Adalimumab and Therapeutic Failure in Infliximab-to-Adalimumab Switchers with IBD

    DEFF Research Database (Denmark)

    Frederiksen, Madeline Therese; Ainsworth, Mark Andrew; Brynskov, Jørn


    BACKGROUND: A notable proportion of patients with inflammatory bowel disease (IBD) are switched from infliximab (IFX) to adalimumab (ADL). We investigated if immunogenicity of IFX influenced immunogenicity and clinical outcomes of later ADL therapy. METHODS: Single-center cohort study including all...... patients with IBD assessed for antibodies (Abs) against IFX or ADL. RESULTS: Anti-IFX Abs were evaluated in 187 patients treated with IFX as first line anti-TNF agent. Approximately, half (49%) were positive. Detected anti-IFX Abs had functional capacity as judged by a median IFX concentration below limit...... of detection (interquartile range, 0.0-0.0 μg/mL) versus 3.8 μg/mL (IQR, 1.3-7.9) in anti-IFX Ab-negative patients, P tested positive. Patients with previous anti-IFX Ab development were...

  2. Isotype Diversification of IgG Antibodies to HIV Gag Proteins as a Therapeutic Vaccination Strategy for HIV Infection. (United States)

    French, Martyn A; Abudulai, Laila N; Fernandez, Sonia


    The development of vaccines to treat and prevent human immunodeficiency virus (HIV) infection has been hampered by an incomplete understanding of "protective" immune responses against HIV. Natural control of HIV-1 infection is associated with T-cell responses against HIV-1 Gag proteins, particularly CD8⁺ T-cell responses restricted by "protective" HLA-B alleles, but other immune responses also contribute to immune control. These immune responses appear to include IgG antibodies to HIV-1 Gag proteins, interferon-a-dependant natural killer (NK) cell responses and plasmacytoid dendritic cell (pDC) responses. Here, it is proposed that isotype diversification of IgG antibodies against HIV-1 Gag proteins, to include IgG2, as well as IgG3 and IgG1 antibodies, will broaden the function of the antibody response and facilitate accessory cell responses against HIV-1 by NK cells and pDCs. We suggest that this should be investigated as a vaccination strategy for HIV-1 infection.

  3. Isotype Diversification of IgG Antibodies to HIV Gag Proteins as a Therapeutic Vaccination Strategy for HIV Infection

    Directory of Open Access Journals (Sweden)

    Sonia Fernandez


    Full Text Available The development of vaccines to treat and prevent human immunodeficiency virus (HIV infection has been hampered by an incomplete understanding of “protective” immune responses against HIV. Natural control of HIV-1 infection is associated with T-cell responses against HIV-1 Gag proteins, particularly CD8+ T-cell responses restricted by “protective” HLA-B alleles, but other immune responses also contribute to immune control. These immune responses appear to include IgG antibodies to HIV-1 Gag proteins, interferon-a-dependant natural killer (NK cell responses and plasmacytoid dendritic cell (pDC responses. Here, it is proposed that isotype diversification of IgG antibodies against HIV-1 Gag proteins, to include IgG2, as well as IgG3 and IgG1 antibodies, will broaden the function of the antibody response and facilitate accessory cell responses against HIV-1 by NK cells and pDCs. We suggest that this should be investigated as a vaccination strategy for HIV-1 infection.

  4. Characterization of product-related low molecular weight impurities in therapeutic monoclonal antibodies using hydrophilic interaction chromatography coupled with mass spectrometry. (United States)

    Wang, Shunhai; Liu, Anita P; Yan, Yuetian; Daly, Thomas J; Li, Ning


    Traditional SDS-PAGE method and its modern equivalent CE-SDS method are both widely applied to assess the purity of therapeutic monoclonal antibody (mAb) drug products. However, structural identification of low molecular weight (LMW) impurities using those methods has been challenging and largely based on empirical knowledges. In this paper, we present that hydrophilic interaction chromatography (HILIC) coupled with mass spectrometry analysis is a novel and orthogonal method to characterize such LMW impurities present within a purified mAb drug product sample. We show here that after removal of N-linked glycans, the HILIC method separates mAb-related LMW impurities with a size-based elution order. The subsequent mass measurement from a high-resolution accurate mass spectrometer provides direct and unambiguous identification of a variety of low-abundance LMW impurities within a single LC-MS analysis. Free light chain, half antibody, H2L species (antibody possessing a single light chain) and protein backbone-truncated species can all be confidently identified and elucidated in great detail, including the truncation sites and associated post-translational modifications. It is worth noting that this study provides the first example where the H2L species can be directly detected in a mAb drug product sample by intact mass analysis without prior enrichment. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Analytical characterization of ch14.18: a mouse-human chimeric disialoganglioside-specific therapeutic antibody. (United States)

    Soman, Gopalan; Kallarakal, Abraham T; Michiel, Dennis; Yang, Xiaoyi; Saptharish, Nirmala; Jiang, Hengguang; Giardina, Steve; Gilly, John; Mitra, George


    Ch14.18 is a mouse-human chimeric monoclonal antibody to the disialoganglioside (GD2) glycolipid. In the clinic, this antibody has been shown to be effective in the treatment of children with high-risk neuroblastoma, either alone or in combination therapy. Extensive product characterization is a prerequisite to addressing the potential issues of product variability associated with process changes and manufacturing scale-up. Charge heterogeneity, glycosylation profile, molecular state and aggregation, interaction (affinity) with Fcγ receptors and functional or biological activities are a few of the critical characterization assays for assessing product comparability for this antibody. In this article, we describe the in-house development and qualification of imaged capillary isoelectric focusing to assess charge heterogeneity, analytical size exclusion chromatography with online static and dynamic light scattering (DLS), batch mode DLS for aggregate detection, biosensor (surface plasmon resonance)-based Fcγ receptor antibody interaction kinetics, N-glycoprofiling with PNGase F digestion, 2-aminobenzoic acid labeling and high performance liquid chromatography and N-glycan analysis using capillary electrophoresis. In addition, we studied selected biological activity assays, such as complement-dependent cytotoxicity. The consistency and reproducibility of the assays are established by comparing the intra-day and inter-day assay results. Applications of the methodologies to address stability or changes in product characteristics are also reported. The study results reveal that the ch14.18 clinical product formulated in phosphate-buffered saline at a concentration of 5 mg/ml and stored at 2-8°C is stable for more than five years.

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

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


    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.

  7. L1 cell adhesion molecule as a potential therapeutic target in murine models of endometriosis using a monoclonal antibody approach.

    Directory of Open Access Journals (Sweden)

    Cássia G T Silveira

    Full Text Available BACKGROUND/AIMS: The neural cell adhesion molecule L1CAM is a transmembrane glycoprotein abnormally expressed in tumors and previously associated with cell proliferation, adhesion and invasion, as well as neurite outgrowth in endometriosis. Being an attractive target molecule for antibody-based therapy, the present study assessed the ability of the monoclonal anti-L1 antibody (anti-L1 mAb to impair the development of endometriotic lesions in vivo and endometriosis-associated nerve fiber growth. METHODS AND RESULTS: Endometriosis was experimentally induced in sexually mature B6C3F1 (n=34 and CD-1 nude (n=21 mice by autologous and heterologous transplantation, respectively, of endometrial fragments into the peritoneal cavity. Transplantation was confirmed four weeks post-surgery by in vivo magnetic resonance imaging and laparotomy, respectively. Mice were then intraperitoneally injected with anti-L1 mAb or an IgG isotype control antibody twice weekly, over a period of four weeks. Upon treatment completion, mice were sacrificed and endometrial implants were excised, measured and fixed. Endometriosis was histologically confirmed and L1CAM was detected by immunohistochemistry. Endometriotic lesion size was significantly reduced in anti-L1-treated B6C3F1 and CD-1 nude mice compared to mice treated with control antibody (P<0.05. Accordingly, a decreased number of PCNA positive epithelial and stromal cells was detected in autologously and heterologously induced endometriotic lesions exposed to anti-L1 mAb treatment. Anti-L1-treated mice also presented a diminished number of intraperitoneal adhesions at implantation sites compared with controls. Furthermore, a double-blind counting of anti-neurofilament L stained nerves revealed significantly reduced nerve density within peritoneal lesions in anti-L1 treated B6C3F1 mice (P=0.0039. CONCLUSIONS: Local anti-L1 mAb treatment suppressed endometriosis growth in B6C3F1 and CD-1 nude mice and exerted a potent

  8. Therapeutic Antibody-Like Immunoconjugates against Tissue Factor with the Potential to Treat Angiogenesis-Dependent as Well as Macrophage-Associated Human Diseases

    Directory of Open Access Journals (Sweden)

    Zhiwei Hu


    Full Text Available Accumulating evidence suggests that tissue factor (TF is selectively expressed in pathological angiogenesis-dependent as well as macrophage-associated human diseases. Pathological angiogenesis, the formation of neovasculature, is involved in many clinically significant human diseases, notably cancer, age-related macular degeneration (AMD, endometriosis and rheumatoid arthritis (RA. Macrophage is involved in the progression of a variety of human diseases, such as atherosclerosis and viral infections (human immunodeficiency virus, HIV and Ebola. It is well documented that TF is selectively expressed on angiogenic vascular endothelial cells (VECs in these pathological angiogenesis-dependent human diseases and on disease-associated macrophages. Under physiology condition, TF is not expressed by quiescent VECs and monocytes but is solely restricted on some cells (such as pericytes that are located outside of blood circulation and the inner layer of blood vessel walls. Here, we summarize TF expression on angiogenic VECs, macrophages and other diseased cell types in these human diseases. In cancer, for example, the cancer cells also overexpress TF in solid cancers and leukemia. Moreover, our group recently reported that TF is also expressed by cancer-initiating stem cells (CSCs and can serve as a novel oncotarget for eradication of CSCs without drug resistance. Furthermore, we review and discuss two generations of TF-targeting therapeutic antibody-like immunoconjugates (ICON and L-ICON1 and antibody-drug conjugates that are currently being tested in preclinical and clinical studies for the treatment of some of these human diseases. If efficacy and safety are proven in current and future clinical trials, TF-targeting immunoconjugates may provide novel therapeutic approaches with potential to broadly impact the treatment regimen of these significant angiogenesis-dependent, as well as macrophage-associated, human diseases.

  9. Anti-respiratory syncytial virus (RSV) G monoclonal antibodies reduce lung inflammation and viral lung titers when delivered therapeutically in a BALB/c mouse model. (United States)

    Caidi, Hayat; Miao, Congrong; Thornburg, Natalie J; Tripp, Ralph A; Anderson, Larry J; Haynes, Lia M


    RSV continues to be a high priority for vaccine and antiviral drug development. Unfortunately, no safe and effective RSV vaccine is available and treatment options are limited. Over the past decade, several studies have focused on the role of RSV G protein on viral entry, viral neutralization, and RSV-mediated pathology. Anti-G murine monoclonal antibody (mAb) 131-2G treatment has been previously shown to reduce weight loss, bronchoalveolar lavage (BAL) cell number, airway reactivity, and Th2-type cytokine production in RSV-infected mice more rapidly than a commercial humanized monoclonal antibody (mAb) against RSV F protein (Palivizumab). In this study, we have tested two human anti-RSV G mAbs, 2B11 and 3D3, by both prophylactic and therapeutic treatment for RSV in the BALB/c mouse model. Both anti-G mAbs reduced viral load, leukocyte infiltration and IFN-γ and IL-4 expression in cell-free BAL supernatants emphasizing the potential of anti-G mAbs as anti-inflammatory and antiviral strategies. Published by Elsevier B.V.

  10. Identification of the factors that govern the ability of therapeutic antibodies to provide postchallenge protection against botulinum toxin: a model for assessing postchallenge efficacy of medical countermeasures against agents of bioterrorism and biological warfare. (United States)

    Al-Saleem, Fetweh H; Nasser, Zidoon; Olson, Rebecca M; Cao, Linsen; Simpson, Lance L


    Therapeutic antibodies are one of the major classes of medical countermeasures that can provide protection against potential bioweapons such as botulinum toxin. Although a broad array of antibodies are being evaluated for their ability to neutralize the toxin, there is little information that defines the circumstances under which these antibodies can be used. In the present study, an effort was made to quantify the temporal factors that govern therapeutic antibody use in a postchallenge scenario. Experiments were done involving inhalation administration of toxin to mice, intravenous administration to mice, and direct application to murine phrenic nerve-hemidiaphragm preparations. As part of this study, several pharmacokinetic characteristics of botulinum toxin and neutralizing antibodies were measured. The core observation that emerged from the work was that the window of opportunity within which postchallenge administration of antibodies exerted a beneficial effect increased as the challenge dose of toxin decreased. The critical factor in establishing the window of opportunity was the amount of time needed for fractional redistribution of a neuroparalytic quantum of toxin from the extraneuronal space to the intraneuronal space. This redistribution event was a dose-dependent phenomenon. It is likely that the approach used to identify the factors that govern postchallenge efficacy of antibodies against botulinum toxin can be used to assess the factors that govern postchallenge efficacy of medical countermeasures against any agent of bioterrorism or biological warfare.

  11. Therapeutic Effects of Monoclonal Antibody against Dengue Virus NS1 in a STAT1 Knockout Mouse Model of Dengue Infection. (United States)

    Wan, Shu-Wen; Chen, Pei-Wei; Chen, Chin-Yu; Lai, Yen-Chung; Chu, Ya-Ting; Hung, Chia-Yi; Lee, Han; Wu, Hsuan Franziska; Chuang, Yung-Chun; Lin, Jessica; Chang, Chih-Peng; Wang, Shuying; Liu, Ching-Chuan; Ho, Tzong-Shiann; Lin, Chiou-Feng; Lee, Chien-Kuo; Wu-Hsieh, Betty A; Anderson, Robert; Yeh, Trai-Ming; Lin, Yee-Shin


    Dengue virus (DENV) is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome and is endemic to tropical and subtropical regions of the world. Our previous studies showed the existence of epitopes in the C-terminal region of DENV nonstructural protein 1 (NS1) which are cross-reactive with host Ags and trigger anti-DENV NS1 Ab-mediated endothelial cell damage and platelet dysfunction. To circumvent these potentially harmful events, we replaced the C-terminal region of DENV NS1 with the corresponding region from Japanese encephalitis virus NS1 to create chimeric DJ NS1 protein. Passive immunization of DENV-infected mice with polyclonal anti-DJ NS1 Abs reduced viral Ag expression at skin inoculation sites and shortened DENV-induced prolonged bleeding time. We also investigated the therapeutic effects of anti-NS1 mAb. One mAb designated 2E8 does not recognize the C-terminal region of DENV NS1 in which host-cross-reactive epitopes reside. Moreover, mAb 2E8 recognizes NS1 of all four DENV serotypes. We also found that mAb 2E8 caused complement-mediated lysis in DENV-infected cells. In mouse model studies, treatment with mAb 2E8 shortened DENV-induced prolonged bleeding time and reduced viral Ag expression in the skin. Importantly, mAb 2E8 provided therapeutic effects against all four serotypes of DENV. We further found that mAb administration to mice as late as 1 d prior to severe bleeding still reduced prolonged bleeding time and hemorrhage. Therefore, administration with a single dose of mAb 2E8 can protect mice against DENV infection and pathological effects, suggesting that NS1-specific mAb may be a therapeutic option against dengue disease. Copyright © 2017 by The American Association of Immunologists, Inc.

  12. Ultrasound Elasticity Imaging Predicts Therapeutic Outcomes of Patients With Crohn's Disease Treated With Anti-Tumour Necrosis Factor Antibodies. (United States)

    Orlando, Stefania; Fraquelli, Mirella; Coletta, Marina; Branchi, Federica; Magarotto, Andrea; Conti, Clara Benedetta; Mazza, Stefano; Conte, Dario; Basilisco, Guido; Caprioli, Flavio


    Ultrasound elasticity imaging is a non-invasive technique developed to evaluate fibrosis. Measuring tissue strain by ultrasound elasticity imaging can reliably detect severe ileal fibrosis in patients with Crohn's disease [CD]. We have hypothesised that a more severe range of fibrosis might influence the therapeutic response to anti-tumour necrosis factor [TNF] treatment. The aim of this study was to assess the ability of ultrasound elasticity imaging to predict the therapeutic outcome for CD patients. Consecutive patients with ileal/ileocolonic CD, starting anti-TNF treatment, were enrolled for the study. These patients underwent bowel ultrasound and ultrasound elasticity imaging at baseline and at 14 and 52 weeks after anti-TNF treatment. Bowel wall stiffness was quantified by calculating the strain ratio between the mesenteric tissue and the bowel wall. Strain ratio ≥ 2 was used to identify severe ileal fibrosis. Transmural healing at 14 and 52 weeks was defined as bowel wall thickness ≤ 3 mm. Thirty patients with CD were enrolled. Five patients underwent surgery for bowel obstruction. The frequency of surgeries was significantly greater in patients with a strain ratio ≥ 2 at baseline [p = 0.003]. A significant reduction of the bowel thickness was observed after 14 and 52 weeks of anti-TNF treatment [p < 0.005]. A significant inverse correlation was observed between the strain ratio values at baseline and the thickness variations following anti-TNF therapy [p = 0.007]; 27% of patients achieved transmural healing at 14 weeks. The baseline strain ratio was significantly lower in patients with transmural healing [p < 0.05]. This study shows that ultrasound elasticity imaging predicts therapeutic outcomes for CD patients treated with anti-TNF. Copyright © 2017 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email:

  13. The therapeutic CD38 monoclonal antibody daratumumab induces programmed cell death via fcg receptor-mediated cross-linking

    DEFF Research Database (Denmark)

    Overdijk, Marije B.; Jansen, J. H. Marco; Nederend, Maaike


    RIIb as well as activating FcgRs induce DARA cross-linking-mediated PCD. In conclusion, our in vitro and in vivo data show that FcgRmediated cross-linking of DARA induces PCD of CD38-expressing multiple myeloma tumor cells, which potentially contributes to the depth of response observed in DARA......Emerging evidence suggests that FcgR-mediated cross-linking of tumor-bound mAbs may induce signaling in tumor cells that contributes to their therapeutic activity. In this study, we show that daratumumab (DARA), a therapeutic human CD38 mAb with a broad-spectrum killing activity, is able to induce...... programmed cell death (PCD) of CD38+ multiple myeloma tumor cell lines when cross-linked in vitro by secondary Abs or via an FcgR. By comparing DARA efficacy in a syngeneic in vivo tumor model using FcRg-chain knockout or NOTAM mice carrying a signaling-inactive FcRg-chain, we found that the inhibitory Fcg...

  14. Systems approach for the selection of micro-RNAs as therapeutic biomarkers of anti-EGFR monoclonal antibody treatment in colorectal cancer (United States)

    Deyati, Avisek; Bagewadi, Shweta; Senger, Philipp; Hofmann-Apitius, Martin; Novac, Natalia


    miRNA plays an important role in tumourgenesis by regulating expression of oncogenes and tumour suppressors. Thus affects cell proliferation and differentiation, apoptosis, invasion and angiogenesis. miRNAs are potential biomarkers for diagnosis, prognosis and therapies of different forms of cancer. However, relationship between response of cancer patients towards targeted therapy and the resulting modifications of the miRNA transcriptome in the context of pathway regulation is poorly understood. With ever-increasing pathways and miRNA-mRNA interaction databases, freely available mRNA and miRNA expression data in multiple cancer therapy have produced an unprecedented opportunity to decipher the role of miRNAs in early prediction of therapeutic efficacy in diseases. Efficient translation of -omics data and accumulated knowledge to clinical decision-making are of paramount scientific and public health interest. Well-structured translational algorithms are needed to bridge the gap from databases to decisions. Herein, we present a novel SMARTmiR algorithm to prospectively predict the role of miRNA as therapeutic biomarker for an anti-EGFR monoclonal antibody i.e. cetuximab treatment in colorectal cancer.

  15. Dual Constant Domain-Fab: A novel strategy to improve half-life and potency of a Met therapeutic antibody. (United States)

    Cignetto, Simona; Modica, Chiara; Chiriaco, Cristina; Fontani, Lara; Milla, Paola; Michieli, Paolo; Comoglio, Paolo M; Vigna, Elisa


    The kinase receptor encoded by the Met oncogene is a sensible target for cancer therapy. The chimeric monovalent Fab fragment of the DN30 monoclonal antibody (MvDN30) has an odd mechanism of action, based on cell surface removal of Met via activation of specific plasma membrane proteases. However, the short half-life of the Fab, due to its low molecular weight, is a severe limitation for the deployment in therapy. This issue was addressed by increasing the Fab molecular weight above the glomerular filtration threshold through the duplication of the constant domains, in tandem (DCD-1) or reciprocally swapped (DCD-2). The two newly engineered molecules showed biochemical properties comparable to the original MvDN30 in vitro, acting as full Met antagonists, impairing Met phosphorylation and activation of downstream signaling pathways. As a consequence, Met-mediated biological responses were inhibited, including anchorage-dependent and -independent cell growth. In vivo DCD-1 and DCD-2 showed a pharmacokinetic profile significantly improved over the original MvDN30, doubling the circulating half-life and reducing the clearance. In pre-clinical models of cancer, generated by injection of tumor cells or implant of patient-derived samples, systemic administration of the engineered molecules inhibited the growth of Met-addicted tumors. Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  16. Evaluation of the therapeutic efficacy of a VEGFR2-blocking antibody using sodium-iodide symporter molecular imaging in a tumor xenograft model

    Energy Technology Data Exchange (ETDEWEB)

    Cheong, Su-Jin; Lee, Chang-Moon; Kim, Eun-Mi [Department of Nuclear Medicine, Chonbuk National University Medical School, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Research Institute of Clinical Medicine, Chonbuk National University Medical School, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Cyclotron Research Center, Chonbuk National University Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Uhm, Tai-Boong [Faculty of Biological Science, Chonbuk National University, Jeonju-si, jeonbuk 561-756 (Korea, Republic of); Jeong, Hwan-Jeong, E-mail: [Department of Nuclear Medicine, Chonbuk National University Medical School, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Research Institute of Clinical Medicine, Chonbuk National University Medical School, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Cyclotron Research Center, Chonbuk National University Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Kim, Dong Wook; Lim, Seok Tae; Sohn, Myung-Hee [Department of Nuclear Medicine, Chonbuk National University Medical School, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Research Institute of Clinical Medicine, Chonbuk National University Medical School, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Cyclotron Research Center, Chonbuk National University Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of)


    Purpose: Vascular endothelial growth factor receptor 2-blocking antibody (DC101) has inhibitory effects on tumor growth and angiogenesis in vivo. The human sodium/iodide symporter (hNIS) gene has been shown to be a useful molecular imaging reporter gene. Here, we investigated the evaluation of therapeutic efficacy by molecular imaging in reporter gene transfected tumor xenografts using a gamma imaging system. Methods: The hNIS gene was transfected into MDA-MB-231 cells using Lipofectamine. The correlation between the number of MDA-MB-231-hNIS cells and the uptake of {sup 99m}Tc-pertechnetate or {sup 125}I was investigated in vitro by gamma imaging and counting. MDA-MB-231-hNIS cells were injected subcutaneously into mice. When the tumor volume reached 180-200 mm{sup 3}, we randomly assigned five animals to each of three groups representing different tumor therapies; no DC101 (control), 100 {mu}g, or 150 {mu}g DC101/mouse. One week and 2 weeks after the first injection of DC101, gamma imaging was performed. Mice were sacrificed 2 weeks after the first injection of DC101. The tumor tissues were used for reverse transcriptase-polymerase chain reaction (RT-PCR) and CD31 staining. Results: Uptake of {sup 125}I and {sup 99m}Tc-pertechnetate into MDA-MB-231-hNIS cells in vitro showed correlation with the number of cells. In DC101 treatment groups, the mean tumor volume was smaller than that of the control mice. Furthermore, tumor uptake of {sup 125}I was lower than in the controls. The CD31 staining and RT-PCR assay results showed that vessel formation and expression of the hNIS gene were significantly reduced in the tumor tissues of treatment groups. Conclusion: This study demonstrated the power of molecular imaging using a gamma imaging system for evaluating the therapeutic efficacy of an antitumor treatment. Molecular imaging systems may be useful in evaluation and development of effective diagnostic and/or therapeutic antibodies for specific target molecules.

  17. Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli. (United States)

    Bu, Dawei; Zhou, Yuwei; Tang, Jian; Jing, Fang; Zhang, Wei


    Abnormal brain natriuretic peptide (BNP) secretion is regarded as the dominating mechanism of cerebral salt wasting syndrome (CSW), which results from a renal loss of sodium and water during intracranial disease leading to hyponatremia. Scale preparation of therapeutic single-chain variable fragment (scFv) that can neutralize elevated circulating BNP may have potential value for clinical use. In this report, we used a recently isolated humanized anti-BNP scFv fragment (3C1) as model antibody (Ab) to evaluate the potential of scale production of this therapeutic protein. The truncated gene encoding for scFv fragment cloned in pET22b (+) was mainly overexpressed as inclusion bodies in Escherichia coli (E. coli) Rosetta (DE3) pLysS cells. The insoluble fragment was solubilized and purified by Ni-NTA agarose resin under denaturation conditions, and recovered via an effective refolding buffer containing 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.5 M arginine, 2 mM GSH, 1 mM GSSG, and 5% glycerol. The refolded scFv fragment was concentrated by PEG20000, and dialyzed in PBS (containing 5% glycerol, pH 7.4). The final yield was approximately 10.2 mg active scFv fragment per liter of culture (3.4 g wet weight cells). The scFv fragment was more than 95% pure assessed by SDS-PAGE assay. Recombinant scFv fragment with His tag displayed its immunoreactivity with anti-His tag Ab by western blotting. ELISA showed the scFv fragment specifically bound to BNP, and it displayed similar activity as the traditional anti-BNP monoclonal Ab (mAb). Thus, the current strategy allows convenient small-scale production of this therapeutic protein. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. The development of immunomodulatory monoclonal antibodies as a new therapeutic modality for cancer: the Bristol-Myers Squibb experience. (United States)

    Berman, David; Korman, Alan; Peck, Ronald; Feltquate, David; Lonberg, Nils; Canetta, Renzo


    The discovery and increased understanding of the complex interactions regulating the immune system have contributed to the pharmacologic activation of antitumor immunity. The activity of effector cells, such as T and NK cells, is regulated by an array of activating and attenuating receptors and ligands. Agents that target these molecules can modulate immune responses by exerting antagonistic or agonistic effects. Several T- or NK-cell modulators have entered clinical trials, and two have been approved for use. Ipilimumab (Yervoy®, Bristol-Myers Squibb) and nivolumab (OPDIVO, Ono Pharmaceutical Co., Ltd./Bristol-Myers Squibb) were approved for the treatment of metastatic melanoma, in March 2011 in the United States, and in July 2014 in Japan, respectively. The clinical activity of these two antibodies has not been limited to tumor types considered sensitive to immunotherapy, and promising activity has been reported in other solid and hematologic tumors. Clinical development of ipilimumab and nivolumab has presented unique challenges in terms of safety and efficacy, requiring the establishment of new evaluation criteria for adverse events and antitumor effects. Guidelines intended to help oncologists properly manage treatment in view of these non-traditional features have been implemented. The introduction of this new modality of cancer treatment, which is meant to integrate with or replace the current standards of care, requires additional efforts in terms of optimization of treatment administration, identification of biomarkers and application of new clinical trial designs. The availability of immune modulators with different mechanisms of action offers the opportunity to establish immunological combinations as new standards of care. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. A comparison of targeting of neuroblastoma with mIBG and anti L1-CAM antibody mAb chCE7: therapeutic efficacy in a neuroblastoma xenograft model and imaging of neuroblastoma patients

    NARCIS (Netherlands)

    Hoefnagel, C. A.; Rutgers, M.; Buitenhuis, C. K.; Smets, L. A.; de Kraker, J.; Meli, M.; Carrel, F.; Amstutz, H.; Schubiger, P. A.; Novak-Hofer, I.


    Iodine-131 labelled anti L1-CAM antibody mAb chCE7 was compared with the effective neuroblastoma-seeking agent 131I-labelled metaiodobenzylguanidine (MIBG) with regard to (a) its therapeutic efficacy in treating nude mice with neuroblastoma xenografts and (b) its tumour targeting ability in

  20. Heavy chain only antibodies

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud


    Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen...

  1. Incorporation of FcRn-mediated disposition model to describe the population pharmacokinetics of therapeutic monoclonal IgG antibody in clinical patients. (United States)

    Ng, Chee M


    The two-compartment linear model used to describe the population pharmacokinetics (PK) of many therapeutic monoclonal antibodies (TMAbs) offered little biological insight to antibody disposition in humans. The purpose of this study is to develop a semi-mechanistic FcRn-mediated IgG disposition model to describe the population PK of TMAbs in clinical patients. A standard two-compartment linear PK model from a previously published population PK model of pertuzumab was used to simulate intensive PK data of 100 subjects for model development. Two different semi-mechanistic FcRn-mediated IgG disposition models were developed and First Order Conditional Estimation (FOCE) with the interaction method in NONMEM was used to obtain the final model estimates. The performances of these models were then compared with the two-compartment linear PK model used to simulate the data for model development. A semi-mechanistic FcRn-mediated IgG disposition model consisting of a peripheral tissue compartment and FcRn-containing endosomes in the central compartment best describes the simulated pertuzumab population PK data. This developed semi-mechanistic population PK model had the same number of model parameters, produced very similar concentration-time profiles but provided additional biological insight to the FcRn-mediated IgG disposition in human subjects compared with the standard linear two-compartment linear PK model. This first reported semi-mechanistic model may serve as an important model framework for developing future population PK models of TMAbs in clinical patients. Copyright © 2015 John Wiley & Sons, Ltd.

  2. Therapeutic effects of anti-CD115 monoclonal antibody in mouse cancer models through dual inhibition of tumor-associated macrophages and osteoclasts.

    Directory of Open Access Journals (Sweden)

    Laetitia Fend

    Full Text Available Tumor progression is promoted by Tumor-Associated Macrophages (TAMs and metastasis-induced bone destruction by osteoclasts. Both myeloid cell types depend on the CD115-CSF-1 pathway for their differentiation and function. We used 3 different mouse cancer models to study the effects of targeting cancer host myeloid cells with a monoclonal antibody (mAb capable of blocking CSF-1 binding to murine CD115. In mice bearing sub-cutaneous EL4 tumors, which are CD115-negative, the anti-CD115 mAb depleted F4/80(+ CD163(+ M2-type TAMs and reduced tumor growth, resulting in prolonged survival. In the MMTV-PyMT mouse model, the spontaneous appearance of palpable mammary tumors was delayed when the anti-CD115 mAb was administered before malignant transition and tumors became palpable only after termination of the immunotherapy. When administered to mice already bearing established PyMT tumors, anti-CD115 treatment prolonged their survival and potentiated the effect of chemotherapy with Paclitaxel. As shown by immunohistochemistry, this therapeutic effect correlated with the depletion of F4/80(+CD163(+ M2-polarized TAMs. In a breast cancer model of bone metastasis, the anti-CD115 mAb potently blocked the differentiation of osteoclasts and their bone destruction activity. This resulted in the inhibition of cancer-induced weight loss. CD115 thus represents a promising target for cancer immunotherapy, since a specific blocking antibody may not only inhibit the growth of a primary tumor through TAM depletion, but also metastasis-induced bone destruction through osteoclast inhibition.

  3. Anti-S1P Antibody as a Novel Therapeutic Strategy for VEGFR TKI-Resistant Renal Cancer. (United States)

    Zhang, Liang; Wang, Xiaoen; Bullock, Andrea J; Callea, Marcella; Shah, Harleen; Song, Jiaxi; Moreno, Kelli; Visentin, Barbara; Deutschman, Douglas; Alsop, David C; Atkins, Michael B; Mier, James W; Signoretti, Sabina; Bhasin, Manoj; Sabbadini, Roger A; Bhatt, Rupal S


    VEGFR2 tyrosine kinase inhibition (TKI) is a valuable treatment approach for patients with metastatic renal cell carcinoma (RCC). However, resistance to treatment is inevitable. Identification of novel targets could lead to better treatment for patients with TKI-naïve or -resistant RCC. In this study, we performed transcriptome analysis of VEGFR TKI-resistant tumors in a murine model and discovered that the SPHK-S1P pathway is upregulated at the time of resistance. We tested sphingosine-1-phosphate (S1P) pathway inhibition using an anti-S1P mAb (sphingomab), in two mouse xenograft models of RCC, and assessed tumor SPHK expression and S1P plasma levels in patients with metastatic RCC. Resistant tumors expressed several hypoxia-regulated genes. The SPHK1 pathway was among the most highly upregulated pathways that accompanied resistance to VEGFR TKI therapy. SPHK1 was expressed in human RCC, and the product of SPHK1 activity, S1P, was elevated in patients with metastatic RCC, suggesting that human RCC behavior could, in part, be due to overproduction of S1P. Sphingomab neutralization of extracellular S1P slowed tumor growth in both mouse models. Mice bearing tumors that had developed resistance to sunitinib treatment also exhibited tumor growth suppression with sphingomab. Sphingomab treatment led to a reduction in tumor blood flow as measured by MRI. Our findings suggest that S1P inhibition may be a novel therapeutic strategy in patients with treatment-naïve RCC and also in the setting of resistance to VEGFR TKI therapy. ©2015 American Association for Cancer Research.

  4. Comparison of originator and biosimilar therapeutic monoclonal antibodies using comprehensive two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry. (United States)

    Sorensen, Matthew; Harmes, David C; Stoll, Dwight R; Staples, Gregory O; Fekete, Szabolcs; Guillarme, Davy; Beck, Alain


    As research, development, and manufacturing of biosimilar protein therapeutics proliferates, there is great interest in the continued development of a portfolio of complementary analytical methods that can be used to efficiently and effectively characterize biosimilar candidate materials relative to the respective reference (i.e., originator) molecule. Liquid phase separation techniques such as liquid chromatography and capillary electrophoresis are powerful tools that can provide both qualitative and quantitative information about similarities and differences between reference and biosimilar materials, especially when coupled with mass spectrometry. However, the inherent complexity of these protein materials challenges even the most modern one-dimensional (1D) separation methods. Two-dimensional (2D) separations present a number of potential advantages over 1D methods, including increased peak capacity, 2D peak patterns that can facilitate unknown identification, and improvement in the compatibility of some separation methods with mass spectrometry. In this study, we demonstrate the use of comprehensive 2D-LC separations involving cation-exchange (CEX) and reversed-phase (RP) separations in the first and second dimensions to compare 3 reference/biosimilar pairs of monoclonal antibodies (cetuximab, trastuzumab and infliximab) that cover a range of similarity/disimilarity in a middle-up approach. The second dimension RP separations are coupled to time-of-flight mass spectrometry, which enables direct identification of features in the chromatograms obtained from mAbs digested with the IdeS enzyme, or digestion with IdeS followed by reduction with dithiothreitol. As many as 23 chemically unique mAb fragments were detected in a single sample. Our results demonstrate that these rich datasets enable facile assesment of the degree of similarity between reference and biosimilar materials.

  5. Analysis of the therapeutic gain in the treatment of human osteosarcoma microcolonies in vitro with 211At-labelled monoclonal antibody. (United States)

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


    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.

  6. A new therapeutic approach for type 1 diabetes: rationale for GNbAC1 an anti-HERV-W-Env monoclonal antibody. (United States)

    Curtin, Francois; Bernard, Corinne; Levet, Sandrine; Perron, Hervé; Porchet, Hervé; Médina, Julie; Malpass, Sam; Lloyd, David; Simpson, Richard


    We describe a newly identified therapeutic target for type 1 diabetes: an envelope protein of endogenous retroviral origin called Human Endogenous Retrovirus W Envelope (HERV-W-Env). HERV-W-Env was found to be detected in the blood of around 60% of type 1 diabetes (T1D) patients and is expressed in acinar pancreatic cells of 75% of T1D patients at post-mortem examination. Preclinical experiments showed that this protein displays direct cytotoxicity on human β-islet cells. In vivo HERV-W-Env impairs the insulin and glucose metabolism in transgenic mice expressing HERV-W-Env. GNbAC1, an IgG4 monoclonal antibody has been developed to specifically target HERV-W-Env and to neutralize the effect of HERV-W-Env in vitro and in vivo. GNbAC1 is currently in clinical development for multiple sclerosis and more than 300 subjects have been administered with GNbAC1 so far. GNbAC1 is now tested in T1D in the RAINBOW-T1D study: a randomized placebo controlled study with the objective of showing the safety and pharmacodynamics response of GNbAC1 in patients suffering from T1D with a maximum duration of 4 years. GNbAC1 is tested versus placebo at the dose of 6 mg/kg in 60 patients during 6 repeated administrations during 6 months, a 6-month open-label extension will follow. The primary endpoint will assess safety and secondary endpoints the pharmacodynamic responses to GNbAC1. GNbAC1 targeting HERV-W-Env is currently in clinical development in T1D with the first safety and pharmacodynamic study. If the study results are positive, this may open the door to the development of an innovative non-immunomodulatory disease modifying treatment for T1D. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  7. Therapeutic preparations of IVIg contain naturally occurring anti-HLA-E antibodies that react with HLA-Ia (HLA-A/-B/-Cw) alleles. (United States)

    Ravindranath, Mepur H; Terasaki, Paul I; Pham, Tho; Jucaud, Vadim; Kawakita, Satoru


    The US Food and Drug Administration approved intravenous immunoglobulin (IVIg), extracted from the plasma of thousands of blood donors, for removing HLA antibodies (Abs) in highly sensitized patients awaiting organ transplants. Since the blood of healthy individuals has HLA Abs, we tested different IVIg preparations for reactivity to HLA single antigen Luminex beads. All preparations showed high levels of HLA-Ia and -Ib reactivity. Since normal nonalloimmunized males have natural antibodies to the heavy chains (HCs) of HLA antigens, the preparations were then tested against iBeads coated only with intact HLA antigens. All IVIg preparations varied in level of antibody reactivity to intact HLA antigens. We raised monoclonal Abs against HLA-E that mimicked IVIg's HLA-Ia and HLA-Ib reactivity but reacted only to HLA-I HCs. Inhibition experiments with synthetic peptides showed that HLA-E shares epitopes with HLA-Ia alleles. Importantly, depleting anti-HLA-E Abs from IVIg totally eliminated the HLA-Ia reactivity of IVIg. Since anti-HLA-E mAbs react with HLA-Ia, they might be useful in suppressing HLA antibody production, similar to the way anti-RhD Abs suppress production. At the same time, anti-HLA-E mAb, which reacts only to HLA-I HCs, is unlikely to produce transfusion-related acute lung injury, in contrast to antibodies reacting to intact-HLA.

  8. Relationship between potential aggregation-prone regions and HLA-DR-binding T-cell immune epitopes: implications for rational design of novel and follow-on therapeutic antibodies. (United States)

    Kumar, Sandeep; Mitchell, Mark A; Rup, Bonita; Singh, Satish K


    Aggregation and unwanted immunogenicity are hurdles to avoid in successful commercial development of antibody-based therapeutics. In this article, the relationship between aggregation-prone regions (APRs), capable of forming cross-β motifs/amyloid fibrils, and major histocompatibility complex class II-restricted human leukocyte antigen (HLA)-DR-binding T-cell immune epitopes (TcIEs) is analyzed using amino acid sequences of 25 therapeutic antibodies, 55 TcIEs recognized by T-regulatory cells (tregitopes), 1000 randomly generated 15-residue-long peptides, 2257 human self-TcIEs (autoantigens), and 11 peptides in HLA-peptide cocrystal structures. Sequence analyses from these diverse sources consistently show a high level of correlation between APRs and TcIEs: approximately one-third of TcIEs contain APRs, but the majority of APRs occur within TcIE regions (TcIERs). Tregitopes also contain APRs. Most APR-containing TcIERs can bind multiple HLA-DR alleles, suggesting that aggregation-driven adverse immune responses could impact a broad segment of patient population. This article has identified common molecular sequence-structure loci that potentially contribute toward both manufacturability and safety profiles of the therapeutic antibodies, thereby laying a foundation for simultaneous optimization of these attributes in novel and follow-on candidates. Incidence of APRs within TcIERs is not special to biotherapeutics, self-TcIEs from human proteins, involved in various diseases, also contain predicted APRs and experimentally proven amyloid-fibril-forming peptide sequence portions. Copyright © 2012 Wiley Periodicals, Inc.

  9. Expression of recombinant Antibodies

    Directory of Open Access Journals (Sweden)

    André eFrenzel


    Full Text Available Recombinant antibodies are highly specific detection probes in research, diagnostics and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines and transgenic plants are promising to obtain antibodies with human-like post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  10. Antibodies and Selection of Monoclonal Antibodies. (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.

  11. Human monoclonal antibodies: the residual challenge of antibody immunogenicity. (United States)

    Waldmann, Herman


    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.

  12. Postvaccination C-Reactive Protein and C5/gp41732-744 Antibody Level Fold-Changes Over Baseline Are Independent Predictors of Therapeutic HIV Vaccine Effect in a Phase 2 Clinical Study of Vacc-4x. (United States)

    Huang, Yunda; Zhang, Lily; Jolliffe, Darren; Sanchez, Brittany; Stjernholm, Grete; Jelmert, Øyvind; Ökvist, Mats; Sommerfelt, Maja A


    Therapeutic vaccination has the potential to contribute to functional HIV cure strategies. However, to show functional HIV cure, study participants must be taken off combination antiretroviral therapy (cART). The availability of suitable biomarkers that can predict viral load (VL) or CD4 count outcomes following therapeutic HIV vaccination would reduce the risks associated with cART interruption in such studies. This report sought to determine baseline and postvaccination biomarker predictors of vaccine effect (VE) on VL and CD4 counts following cART interruption in a double-blind, randomized phase 2 study of the peptide-based therapeutic HIV vaccine, Vacc-4x (n = 93), versus placebo (n = 43). Antibody responses to a novel envelope glycoprotein antigen, C5/gp41 732-744 , and three safety marker measurements [C-reactive protein (CRP), white blood cell, and lactate dehydrogenase] were considered. Interaction tests in univariate and multivariate linear regression models were used to estimate the effect of biomarkers on VE, defined as the VL or CD4 count difference in Vacc-4x versus placebo groups. The reported q-values (considered significant for hypothesis-generating purposes if ≤0.2) accounted for multiple comparisons using the false discovery rate method. Data were analyzed from all available 58 Vacc-4x and 25 placebo recipients before cART resumption. Lower postvaccination fold-change over baseline of CRP concentration (interaction p- (q-) value = 0.005 (0.11) for VL) and higher fold-change of anti-C5/gp41 732-744 antibody levels (0.005 (0.11) for VL and 0.009 (0.20) for CD4) were associated with Vacc-4x benefit. These findings suggest potential roles for inflammation and immune activation markers in predicting therapeutic HIV VE.

  13. Alternative affinity tools: more attractive than antibodies?

    NARCIS (Netherlands)

    Ruigrok, V.J.B.; Levisson, M.; Eppink, M.H.M.; Smidt, H.; Oost, van der J.


    Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids

  14. Monoclonal antibodies in oncology

    International Nuclear Information System (INIS)

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


    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.)

  15. Rationale for combination of therapeutic antibodies targeting tumor cells and immune checkpoint receptors: Harnessing innate and adaptive immunity through IgG1 isotype immune effector stimulation. (United States)

    Ferris, Robert L; Lenz, Heinz-Josef; Trotta, Anna Maria; García-Foncillas, Jesús; Schulten, Jeltje; Audhuy, François; Merlano, Marco; Milano, Gerard


    Immunoglobulin (Ig) G1 antibodies stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). Cetuximab, an IgG1 isotype monoclonal antibody, is a standard-of-care treatment for locally advanced and recurrent and/or metastatic squamous cell carcinoma of the head and neck (SCCHN) and metastatic colorectal cancer (CRC). Here we review evidence regarding the clinical relevance of cetuximab-mediated ADCC and other immune functions and provide a biological rationale concerning why this property positions cetuximab as an ideal partner for immune checkpoint inhibitors (ICIs) and other emerging immunotherapies. We performed a nonsystematic review of available preclinical and clinical data involving cetuximab-mediated immune activity and combination approaches of cetuximab with other immunotherapies, including ICIs, in SCCHN and CRC. Indeed, cetuximab mediates ADCC activity in the intratumoral space and primes adaptive and innate cellular immunity. However, counterregulatory mechanisms may lead to immunosuppressive feedback loops. Accordingly, there is a strong rationale for combining ICIs with cetuximab for the treatment of advanced tumors, as targeting CTLA-4, PD-1, and PD-L1 can ostensibly overcome these immunosuppressive counter-mechanisms in the tumor microenvironment. Moreover, combining ICIs (or other immunotherapies) with cetuximab is a promising strategy for boosting immune response and enhancing response rates and durability of response. Cetuximab immune activity-including, but not limited to, ADCC-provides a strong rationale for its combination with ICIs or other immunotherapies to synergistically and fully mobilize the adaptive and innate immunity against tumor cells. Ongoing prospective studies will evaluate the clinical effect of these combination regimens and their immune effect in CRC and SCCHN and in other indications. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Comparison of cell-based and non-cell-based assay platforms for the detection of clinically relevant anti-drug neutralizing antibodies for immunogenicity assessment of therapeutic proteins. (United States)

    Hu, Jenny; Wala, Iwona; Han, Hong; Nagatani, Janice; Barger, Troy; Civoli, Francesca; Kaliyaperumal, Arunan; Zhuang, Yao; Gupta, Shalini


    Anti-drug neutralizing antibodies (NAbs) formed due to unwanted immunogenicity of a therapeutic protein point towards a mature immune response. NAb detection is important in interpreting the therapeutic's efficacy and safety in vivo. In vitro cell-based NAb assays provide a physiological system for NAb detection, however are complex assays. Non-cell-based competitive ligand binding (CLB) approaches are also employed for NAb detection. Instead of cells, CLB assays use soluble receptor and conjugated reagents and are easier to perform, however have reduced physiological relevance. The aim of this study was to compare the performance of CLB assays to established cell-based assays to determine the former's ability to detect clinically relevant NAbs towards therapeutics that (i) acted as an agonist or (ii) acted as antagonists by binding to a target receptor. We performed a head-to-head comparison of the performance of cell-based and CLB NAb assays for erythropoietin (EPO) and two anti-receptor monoclonal antibodies (AMG-X and AMG 317). Clinically relevant NAb-positive samples identified previously by a cell-based assay were assessed in the corresponding CLB format(s). A panel of 12 engineered fully human anti-EPO monoclonal antibodies (MAbs) was tested in both EPO NAb assay formats. Our results showed that the CLB format was (i) capable of detecting human anti-EPO MAbs of differing neutralizing capabilities and affinities and (ii) provided similar results as the cell-based assay for detecting NAbs in patient samples. The cell-based and CLB assays also behaved comparably in detecting NAbs in clinical samples for AMG-X. In the case of anti-AMG 317 NAbs, the CLB format failed to detect NAbs in more than 50% of the tested samples. We conclude that assay sensitivity, drug tolerance and the selected assay matrix played an important role in the inability of AMG 317 CLB assays to detect clinically relevant NAbs. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Avidin-conjugated polymers with monobiotinylated antibody fragments: a new strategy for the noncovalent attachment of recombinant proteins for polymer therapeutics

    Czech Academy of Sciences Publication Activity Database

    Laga, Richard; Pola, Robert; Ulbrich, Karel; Hořejší, Magdalena; Sieglová, Irena; Král, Vlastimil; Fábry, Milan; Pechar, Michal


    Roč. 28, č. 3 (2013), s. 289-299 ISSN 0883-9115 R&D Projects: GA ČR GCP207/12/J030; GA AV ČR IAAX00500803 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:61389013 ; RVO:68378050 Keywords : avidin-biotin complex * polymer therapeutics * drug targeting Subject RIV: CD - Macromolecular Chemistry Impact factor: 2.500, year: 2013

  18. Antimitochondrial antibody (United States)

    ... page: // Antimitochondrial antibody To use the sharing features on this page, please enable JavaScript. Antimitochondrial antibodies (AMA) are substances ( antibodies ) that form against mitochondria. ...

  19. Dissecting Immunogenicity of Monoclonal Antibodies

    National Research Council Canada - National Science Library

    Snyder, Christopher


    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...

  20. A truncated receptor-binding domain of MERS-CoV spike protein potently inhibits MERS-CoV infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines.

    Directory of Open Access Journals (Sweden)

    Lanying Du

    Full Text Available An emerging respiratory infectious disease with high mortality, Middle East respiratory syndrome (MERS, is caused by a novel coronavirus (MERS-CoV. It was first reported in 2012 in Saudi Arabia and has now spread to eight countries. Development of effective therapeutics and vaccines is crucial to save lives and halt the spread of MERS-CoV. Here, we show that a recombinant protein containing a 212-amino acid fragment (residues 377-588 in the truncated receptor-binding domain (RBD: residues 367-606 of MERS-CoV spike (S protein fused with human IgG Fc fragment (S377-588-Fc is highly expressed in the culture supernatant of transfected 293T cells. The purified S377-588-Fc protein efficiently binds to dipeptidyl peptidase 4 (DPP4, the receptor of MERS-CoV, and potently inhibited MERS-CoV infection, suggesting its potential to be further developed as a therapeutic modality for treating MERS-CoV infection and saving the patients' lives. The recombinant S377-588-Fc is able to induce in the vaccinated mice strong MERS-CoV S-specific antibodies, which blocks the binding of RBD to DPP4 receptor and effectively neutralizes MERS-CoV infection. These findings indicate that this truncated RBD protein shows promise for further development as an effective and safe vaccine for the prevention of MERS-CoV infection.

  1. In vivo time-related evaluation of a therapeutic neutralization monoclonal antibody against lethal enterovirus 71 infection in a mouse model.

    Directory of Open Access Journals (Sweden)

    Zhiqun Li

    Full Text Available Enterovirus 71 (EV71 is a neurotropic virus capable of inducing severe neurological symptoms and death. No direct targeting antivirals are useful in the treatment of severe EV71 infection. Because of low toxicity and good specificity, monoclonal antibodies (MAb are a potential candidate for the treatment of viral infections. Therefore, we developed an EV71-specific conformational MAb with high in vitro cross-neutralization activity to heterologous EV71 subgenotypes. The in vivo treatment experiment at different days post-infection indicated that a single treatment of MAb CT11F9 within day 3 post-infection fully protected mice from morbidity and mortality (0% PBS vs. 100% at 10 µg/g per body weight ***P<0.0001. Immunohistochemical and histological analysis confirmed that CT11F9 significantly prohibited EV71 VP1 expression in various tissues and prevented EV71-induced myonecrosis. Moreover, thrice-treatment at day 4, 5, 6 post-infection was associated with an increased survival rate (18.2% single vs. 50% thrice at 20 µg/g per body weight, and the mice recovered from limb paralysis. Competitive ELISA also confirmed that CT11F9-recognized epitopes were immunodominant in humans. In conclusion, MAb CT11F9 is an ideal candidate to be humanized and used in severe EV71 infection.

  2. Antibody mimetics: promising complementary agents to animal-sourced antibodies. (United States)

    Baloch, Abdul Rasheed; Baloch, Abdul Wahid; Sutton, Brian J; Zhang, Xiaoying


    Despite their wide use as therapeutic, diagnostic and detection agents, the limitations of polyclonal and monoclonal antibodies have inspired scientists to design the next generation biomedical agents, so-called antibody mimetics that offer many advantages over conventional antibodies. Antibody mimetics can be constructed by protein-directed evolution or fusion of complementarity-determining regions through intervening framework regions. Substantial progress in exploiting human, butterfly (Pieris brassicae) and bacterial systems to design and select mimetics using display technologies has been made in the past 10 years, and one of these mimetics [Kalbitor® (Dyax)] has made its way to market. Many challenges lie ahead to develop mimetics for various biomedical applications, especially those for which conventional antibodies are ineffective, and this review describes the current characteristics, construction and applications of antibody mimetics compared to animal-sourced antibodies. The possible limitations of mimetics and future perspectives are also discussed.

  3. 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


    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)

  4. Prophylactic and therapeutic efficacy of avian antibodies against influenza virus H5N1 and H1N1 in mice.

    Directory of Open Access Journals (Sweden)

    Huan H Nguyen

    Full Text Available BACKGROUND: Pandemic influenza poses a serious threat to global health and the world economy. While vaccines are currently under development, passive immunization could offer an alternative strategy to prevent and treat influenza virus infection. Attempts to develop monoclonal antibodies (mAbs have been made. However, passive immunization based on mAbs may require a cocktail of mAbs with broader specificity in order to provide full protection since mAbs are generally specific for single epitopes. Chicken immunoglobulins (IgY found in egg yolk have been used mainly for treatment of infectious diseases of the gastrointestinal tract. Because the recent epidemic of highly pathogenic avian influenza virus (HPAIV strain H5N1 has resulted in serious economic losses to the poultry industry, many countries including Vietnam have introduced mass vaccination of poultry with H5N1 virus vaccines. We reasoned that IgY from consumable eggs available in supermarkets in Vietnam could provide protection against infections with HPAIV H5N1. METHODS AND FINDINGS: We found that H5N1-specific IgY that are prepared from eggs available in supermarkets in Vietnam by a rapid and simple water dilution method cross-protect against infections with HPAIV H5N1 and related H5N2 strains in mice. When administered intranasally before or after lethal infection, the IgY prevent the infection or significantly reduce viral replication resulting in complete recovery from the disease, respectively. We further generated H1N1 virus-specific IgY by immunization of hens with inactivated H1N1 A/PR/8/34 as a model virus for the current pandemic H1N1/09 and found that such H1N1-specific IgY protect mice from lethal influenza virus infection. CONCLUSIONS: The findings suggest that readily available H5N1-specific IgY offer an enormous source of valuable biological material to combat a potential H5N1 pandemic. In addition, our study provides a proof-of-concept for the approach using virus

  5. Identification of a new epitope in uPAR as a target for the cancer therapeutic monoclonal antibody ATN-658, a structural homolog of the uPAR binding integrin CD11b (αM.

    Directory of Open Access Journals (Sweden)

    Xiang Xu

    Full Text Available The urokinase plasminogen activator receptor (uPAR plays a role in tumor progression and has been proposed as a target for the treatment of cancer. We recently described the development of a novel humanized monoclonal antibody that targets uPAR and has anti-tumor activity in multiple xenograft animal tumor models. This antibody, ATN-658, does not inhibit ligand binding (i.e. uPA and vitronectin to uPAR and its mechanism of action remains unclear. As a first step in understanding the anti-tumor activity of ATN-658, we set out to identify the epitope on uPAR to which ATN-658 binds. Guided by comparisons between primate and human uPAR, epitope mapping studies were performed using several orthogonal techniques. Systematic site directed and alanine scanning mutagenesis identified the region of aa 268-275 of uPAR as the epitope for ATN-658. No known function has previously been attributed to this epitope Structural insights into epitope recognition were obtained from structural studies of the Fab fragment of ATN-658 bound to uPAR. The structure shows that the ATN-658 binds to the DIII domain of uPAR, close to the C-terminus of the receptor, corroborating the epitope mapping results. Intriguingly, when bound to uPAR, the complementarity determining region (CDR regions of ATN-658 closely mimic the binding regions of the integrin CD11b (αM, a previously identified uPAR ligand thought to be involved in leukocyte rolling, migration and complement fixation with no known role in tumor progression of solid tumors. These studies reveal a new functional epitope on uPAR involved in tumor progression and demonstrate a previously unrecognized strategy for the therapeutic targeting of uPAR.

  6. A comparison of targetting of neuroblastoma with MIBG and anti L1-CAM antibody mAb chCE7: therapeutic efficacy in a neuroblastoma xenograft model and imaging of neuroblastoma patients

    International Nuclear Information System (INIS)

    Hoefnagel, C.A.; Rutgers, M.; Buitenhuis, C.K.M.; Smets, L.A.; Kraker, J. de; Meli, M.; Carrel, F.; Schubiger, P.A.; Novak-Hofer, I.; Amstutz, H.


    Modine-131 labelled anti L1-CAM antibody mAb chCE7 was compared with the effective neuroblastoma-seeking agent 131 I-labelled metaiodobenzylguanidine (MIBG) with regard to (a) its therapeutic efficacy in treating nude mice with neuroblastoma xenografts and (b) its tumour targetting ability in neuroblastoma patients. The SK-N-SH tumour cells used in the mouse experiments show good MIBG uptake and provide a relatively low number of 6,300 binding sites/cell for mAb chCE7. Tumours were treated with single injections of 131 I-MIBG (110 MBq) and with 131 I-labelled mAb chCE7 (17 MBq) and both agents showed antitumour activity. After therapy with 131 I-chCE7, the subcutaneous tumours nearly disappeared; treatment with 131 I-MIBG was somewhat less effective, resulting in a 70% reduction in tumour volume. A calculated tumour regrowth delay of 9 days occurred with a radioactivity dose of 17 MBq of an irrelevant control antibody mAb 35, which does not bind to SK-N-SH cells, compared with a regrowth delay of 34 days with 131 I-mAb chCE7 and of 24 days with 131 I-MIBG. General toxicity appeared to be mild, as assessed by a transient, approximate 10% maximum decrease in body weight during the treatments. The superior growth inhibition achieved by 131 I-chCE7 compared with 131 I-MIBG can be explained by its prolonged retention in the tumours, due to slower normal tissue and plasma clearance. Cross-reaction of mAb chCE7 with L1-CAM present in normal human tissues was investigated by direct binding of radioiodinated mAb to frozen tissue sections. Results showed a strong reaction with normal human brain tissue and weak but detectable binding to normal adult kidney sections. Seven patients with recurrent neuroblastoma were sequentially imaged with 131 I-MIBG and 131 I-chCE7. The results underlined the heterogeneity of neuroblastoma and showed the two imaging modalities to be complementary. 131 I-chCE7 scintigraphy may have clinical utility in detecting metastases which do not

  7. Antibody biotechnology

    African Journals Online (AJOL)



    Jul 6, 2009 ... Another milestone in the history of antibodies was the work of Porter and Edelman ... transgenic animals (Lonberg et al., 1994; Green et al.,. 1994) or .... create and to screen human recombinant antibodies libraries, that is ...

  8. Antithyroid microsomal antibody (United States)

    Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also been linked with an increased risk ...

  9. Antibodies to watch in 2018 (United States)

    Kaplon, Hélène; Reichert, Janice M.


    ABSTRACT The pace of antibody therapeutics development accelerated in 2017, and this faster pace is projected to continue through 2018. Notably, the annual number of antibody therapeutics granted a first approval in either the European Union (EU) or United States (US) reached double-digits (total of 10) for the first time in 2017. The 10 antibodies granted approvals are: brodalumab, dupilumab, sarilumab, guselkumab, benralizumab, ocrelizumab, inotuzumab ozogamicin, avelumab, duvalumab, and emicizumab. Brodalumab, however, had already been approved in Japan in 2016. As of December 1, 2017, nine antibody therapeutics (ibalizumab, burosumab, tildrakizumab, caplacizumab, erenumab, fremanezumab, galcanezumab, romosozumab, mogamulizumab) were in regulatory review in the EU or US, and regulatory actions on their marketing applications are expected by the end of 2018. Based on company announcements and estimated clinical study primary completion dates, and assuming the study results are positive, marketing applications for at least 12 antibody therapeutics that are now being evaluated in late-stage clinical studies may be submitted by the end of 2018. Of the 12 candidates, 8 are for non-cancer indications (lanadelumab, crizanlizumab, ravulizumab, eptinezumab, risankizumab, satralizumab, brolucizumab, PRO140) and 4 are for cancer (sacituzumab govitecan, moxetumomab pasudotox, cemiplimab, ublituximab). Additional antibody therapeutics to watch in 2018 include 19 mAbs undergoing evaluation in late-stage studies with primary completion dates in late 2017 or during 2018. Of these mAbs, 9 are for non-cancer indications (lampalizumab, roledumab, emapalumab, fasinumab, tanezumab, etrolizumab, NEOD001, gantenerumab, anifrolumab) and 10 are for cancer indications (tremelimumab, isatuximab, BCD-100, carotuximab, camrelizumab, IBI308, glembatumumab vedotin, mirvetuximab soravtansine, oportuzumab monatox, L19IL2/L19TNF). Positive clinical study results may enable marketing application

  10. Thyroid Antibodies (United States)

    ... PF4 Antibody Hepatitis A Testing Hepatitis B Testing Hepatitis C Testing HER2/neu Herpes Testing High-sensitivity C-reactive Protein (hs-CRP) Histamine Histone Antibody HIV Antibody and HIV Antigen (p24) HIV Antiretroviral Drug Resistance Testing, Genotypic HIV Viral Load HLA Testing HLA- ...

  11. Antibody or Antibody Fragments : Implications for Molecular Imaging and Targeted Therapy of Solid Tumors

    NARCIS (Netherlands)

    Xenaki, Katerina T; Oliveira, Sabrina; van Bergen En Henegouwen, Paul M P


    The use of antibody-based therapeutics has proven very promising for clinical applications in cancer patients, with multiple examples of antibodies and antibody-drug conjugates successfully applied for the treatment of solid tumors and lymphomas. Given reported recurrence rates, improvements are

  12. A systematic study of the effect of low pH acid treatment on anti-drug antibodies specific for a domain antibody therapeutic: Impact on drug tolerance, assay sensitivity and post-validation method assessment of ADA in clinical serum samples. (United States)

    Kavita, Uma; Duo, Jia; Crawford, Sean M; Liu, Rong; Valcin, Joan; Gleason, Carol; Dong, Huijin; Gadkari, Snaehal; Dodge, Robert W; Pillutla, Renuka C; DeSilva, Binodh S


    We developed a homogeneous bridging anti-drug antibody (ADA) assay on an electro chemiluminescent immunoassay (ECLIA) platform to support the immunogenicity evaluation of a dimeric domain antibody (dAb) therapeutic in clinical studies. During method development we evaluated the impact of different types of acid at various pH levels on polyclonal and monoclonal ADA controls of differing affinities and on/off rates. The data shows for the first time that acids of different pH can have a differential effect on ADA of various affinities and this in turn impacts assay sensitivity and drug tolerance as defined by these surrogate controls. Acid treatment led to a reduction in signal of intermediate and low affinity ADA, but not high affinity or polyclonal ADA. We also found that acid pretreatment is a requisite for dissociation of drug bound high affinity ADA, but not for low affinity ADA-drug complexes. Although we were unable to identify an acid that would allow a 100% retrieval of ADA signal post-treatment, use of glycine pH3.0 enabled the detection of low, intermediate and high affinity antibodies (Abs) to various extents. Following optimization, the ADA assay method was validated for clinical sample analysis. Consistencies within various parameters of the clinical data such as dose dependent increases in ADA rates and titers were observed, indicating a reliable ADA method. Pre- and post-treatment ADA negative or positive clinical samples without detectable drug were reanalyzed in the absence of acid treatment or presence of added exogenous drug respectively to further assess the effectiveness of the final acid treatment procedure. The overall ADA results indicate that assay conditions developed and validated based on surrogate controls sufficed to provide a reliable clinical data set. The effect of low pH acid treatment on possible pre-existing ADA or soluble multimeric target in normal human serum was also evaluated, and preliminary data indicate that acid type and

  13. Micellar and antibody-targeted polymer therapeutics

    Czech Academy of Sciences Publication Activity Database

    Etrych, Tomáš; Chytil, Petr; Kovář, Lubomír; Říhová, Blanka; Ulbrich, Karel


    Roč. 295, - (2010), s. 1-12 ISSN 1022-1360. [Prague Meetings on Macromolecules /73./ New Frontiers in Macromolecular Science: From Macromolecular Concepts of Living Matter to Polymers for Better Quality of Life. Prague, 05.07.2009-09.07.2009] R&D Projects: GA AV ČR IAA400500806; GA AV ČR IAAX00500803; GA MŠk 1M0505 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50200510 Keywords : doxorubicin * drug delivery systems * HPMA copolymers Subject RIV: CD - Macromolecular Chemistry

  14. Antiprothrombin Antibodies

    Directory of Open Access Journals (Sweden)

    Polona Žigon


    Full Text Available In patients with the antiphospholipid syndrome (APS, the presence of a group of pathogenic autoantibodies called antiphospholipid antibodies causes thrombosis and pregnancy complications. The most frequent antigenic target of antiphospholipid antibodies are phospholipid bound β2-glycoprotein 1 (β2GPI and prothrombin. The international classification criteria for APS connect the occurrence of thrombosis and/or obstetric complications together with the persistence of lupus anticoagulant, anti-cardiolipin antibodies (aCL and antibodies against β2GPI (anti-β2GPI into APS. Current trends for the diagnostic evaluation of APS patients propose determination of multiple antiphospholipid antibodies, among them also anti-prothrombin antibodies, to gain a common score which estimates the risk for thrombosis in APS patients. Antiprothrombin antibodies are common in APS patients and are sometimes the only antiphospholipid antibodies being elevated. Methods for their determination differ and have not yet been standardized. Many novel studies confirmed method using phosphatidylserine/prothrombin (aPS/PT ELISA as an antigen on solid phase encompass higher diagnostic accuracy compared to method using prothrombin alone (aPT ELISA. Our research group developed an in-house aPS/PT ELISA with increased analytical sensitivity which enables the determination of all clinically relevant antiprothrombin antibodies. aPS/PT exhibited the highest percentage of lupus anticoagulant activity compared to aCL and anti-β2GPI. aPS/PT antibodies measured with the in-house method associated with venous thrombosis and presented the strongest independent risk factor for the presence of obstetric complications among all tested antiphospholipid antibodies

  15. Human antibody technology and the development of antibodies against cytomegalovirus. (United States)

    Ohlin, Mats; Söderberg-Nauclér, Cecilia


    Cytomegalovirus (CMV) is a virus that causes chronic infections in a large set of the population. It may cause severe disease in immunocompromised individuals, is linked to immunosenescence and implied to play an important role in the pathogenesis of cardiovascular diseases and cancer. Modulation of the immune system's abilities to manage the virus represent a highly viable therapeutic option and passive immunotherapy with polyclonal antibody preparations is already in clinical use. Defined monoclonal antibodies offer many advantages over polyclonal antibodies purified from serum. Human CMV-specific monoclonal antibodies have consequently been thoroughly investigated with respect to their potential in the treatment of diseases caused by CMV. Recent advances in human antibody technology have substantially expanded the breadth of antibodies for such applications. This review summarizes the fundamental basis for treating CMV disease by use of antibodies, the basic technologies to be used to develop such antibodies, and relevant human antibody specificities available to target this virus. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Antibodies to watch in 2014. (United States)

    Reichert, Janice M


    Since 2010, mAbs has documented the biopharmaceutical industry's progress in transitioning antibody therapeutics to first Phase 3 clinical studies and regulatory review, and its success at gaining first marketing approvals for antibody-based products. This installment of the "Antibodies to watch" series outlines events anticipated to occur between December 2013 and the end of 2014, including first regulatory actions on marketing applications for vedolizumab, siltuximab, and ramucirumab, as well as the Fc fusion proteins Factor IX-Fc and Factor VIII-Fc; and the submission of first marketing applications for up to five therapeutics (secukinumab, ch14.18, onartuzumab, necitumumab, gevokizumab). Antibody therapeutics in Phase 3 studies are described, with an emphasis on those with study completion dates in 2014, including antibodies targeting interleukin-17a or the interleukin-17a receptor (secukinumab, ixekizumab, brodalumab), proprotein convertase subtilisin/kexin type 9 (alirocumab, evolocumab, bococizumab), and programmed death 1 receptor (lambrolizumab, nivolumab). Five antibodies with US Food and Drug Administration's Breakthrough Therapy designation (obinutuzumab, ofatumumab, lambrolizumab, bimagrumab, daratumumab) are also discussed.

  17. Stratification of Antibody-Positive Subjects by Antibody Level Reveals an Impact of Immunogenicity on Pharmacokinetics


    Zhou, Lei; Hoofring, Sarah A.; Wu, Yu; Vu, Thuy; Ma, Peiming; Swanson, Steven J.; Chirmule, Narendra; Starcevic, Marta


    The availability of highly sensitive immunoassays enables the detection of antidrug antibody (ADA) responses of various concentrations and affinities. The analysis of the impact of antibody status on drug pharmacokinetics (PK) is confounded by the presence of low-affinity or low-concentration antibody responses within the dataset. In a phase 2 clinical trial, a large proportion of subjects (45%) developed ADA following weekly dosing with AMG 317, a fully human monoclonal antibody therapeutic....

  18. Monoclonal antibodies for treating cancer

    International Nuclear Information System (INIS)

    Dillman, R.O.


    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

  19. Monoclonal antibody therapy of inflammatory bowel disease

    NARCIS (Netherlands)

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


    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

  20. Conference scene: progress with promising human antibodies. (United States)

    Larrick, James W


    Antibodies and antibody-based therapeutics have become big business, with annual sales over US$50 billion, accounting for >6% of worldwide pharmaceutical revenues. Ten molecules have blockbuster status (>US$1 billion), with six generating more than US$6 billion in sales. In excess of 300 products based on this rapidly maturing technology are in clinical trials. The generation and manufacture of human antibodies is now routine, although the cost of goods remains an issue. Optimizing combinations of antibodies with other therapeutics (e.g., chemotherapy) is a major short-term goal, while target validation and product differentiation remain significant hurdles if growth is to continue. Some of the notable highlights of the recent 16th International Conference on Human Antibodies and Hybridomas meeting in Cannes, France are described below. The conference was sponsored by the international journal Human Antibodies, in association with the Integrative Medical Sciences Association (IMSA). The Program Chairman was Professor Mark Glassy, IMSA, San Diego, CA, USA.

  1. Monoclonal antibody

    International Nuclear Information System (INIS)

    Oyamada, Hiyoshimaru


    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.)

  2. Polyclonal and monoclonal antibodies in clinic. (United States)

    Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses


    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.

  3. Antibody therapies for lymphoma in children

    NARCIS (Netherlands)

    de Zwart, Verena; Gouw, Samantha C.; Meyer-Wentrup, Friederike A. G.


    Lymphomas are the third most common malignancy in childhood. Cure rates are high but have reached a plateau. Therefore new treatment modalities should be developed. Antibody therapy is a successful new treatment option in adult lymphoma. However, none of the therapeutic antibodies available for

  4. Anti-influenza M2e antibody (United States)

    Bradbury, Andrew M [Santa Fe, NM


    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  5. Clinical use of antibodies

    International Nuclear Information System (INIS)

    Baum, R.P.; Hoer, Gustav; Cox, P.H.; Buraggi, G.L.


    Use of monoclonal antibodies as tumour specific carrier molecules for therapeutic agents or as in vivo diagnostic reagents when labelled with radionuclides or NMR signal enhancers is attracting more and more attention. The potential is enormous but the technical problems are also considerable requiring the concerted action of many different scientific disciplines. This volume is based upon a symposium organised in Frankfurt in 1990 under the auspices of the European Association of Nuclear Medicines' Specialist Task Groups on Cardiology and the Utility of Labelled Antibodies. It gives a multidisciplinary review of the state of the art and of problems to be solved as well as recording the not inconsiderable successes which have been booked to date. The book will be of value as a reference to both clinicians and research scientists. refs.; figs.; tabs

  6. Designing two-in-one antibodies. (United States)

    Valladares, Ignacio Garcia; Espinoza, Luis R


    Evaluation of: Bostrom J, Shang-Fan Y, Kan D et al.: Variants of the antibody Herceptin that interact with HER2 and VEGF at the antigen binding site. Science 323, 1610-1614 (2009). The longstanding held notion that one antibody equals one antigen and, hence, one function has been challenged in recent years. Improved technology in antibody production, especially the accumulation of sequence data of immunoglobulin genes and the advent of PCR have made it possible to clone antibody gene repertoires. The current paper provides further challenge to the notion of one antibody = one antigen by developing 'two-in-one' antibodies with an antigen-binding site that binds two distinct proteins with high affinity. A therapeutic variant antibody of Herceptin (Genentech, CA, USA) was isolated that binds the human EGF receptor (HER)2 and also to VEGF. This development may represent a breakthrough discovery and may have significant implications in the therapy of malignant, infectious, allergic and autoimmune disorders.

  7. Clinical efficacy and management of monoclonal antibodies targeting CD38 and SLAMF7 in multiple myeloma

    DEFF Research Database (Denmark)

    van de Donk, Niels W C J; Moreau, Philippe; Plesner, Torben


    Immunotherapeutic strategies are emerging as promising therapeutic approaches in multiple myeloma (MM), with several monoclonal antibodies in advanced stages of clinical development. Of these agents, CD38-targeting antibodies have marked single agent activity in extensively pretreated MM...... of therapeutic antibodies with immunofixation and serum protein electrophoresis assays may lead to underestimation of complete response. Strategies to mitigate interference, based on shifting the therapeutic antibody band, are in development. Furthermore, daratumumab, and probably also other CD38-targeting...

  8. Catalytic Antibodies

    Indian Academy of Sciences (India)

    biological processes and is intended to catalyze a reaction for which no real enzyme is ... the reaction. In order to enhance the rates of chemical reactions, enzymes, ..... of such antibodies has already been exploited in the production of a biosensor. ..... tant to the pharmaceutical and fine chemical industries for the synthesis ...

  9. High throughput screening for antibody induced complement-dependent cytotoxicity in early antibody discovery using homogeneous macroconfocal fluorescence imaging

    NARCIS (Netherlands)

    Gerritsen, Arnout F.; Bosch, Martijn; de Weers, Michel; van de Winkel, Jan G. J.; Parren, Paul W. H. I.


    Complement-dependent cytotoxicity (CDC) represents an important Fc-mediated effector function of antibodies and is a quality often sought in candidates for therapeutic antibody development in cancer. Antibodies inducing potent CDC are relatively rare as the ability to induce CDC is strongly

  10. Current status of radioligand antibodies in the treatment of malignancy

    International Nuclear Information System (INIS)

    Maners, A.W.; Sanders, M.M.; Pappas, A.A.


    Monoclonal anti-tumor antibodies labeled with a radioactive moiety present an exciting new approach to cancer therapy. With the advent of hybridoma technology, monoclonal antibodies can now be produced in quantity. Indeed, antibodies against tumor-related and tumor-specific antigens have been produced, labeled with a radioactive substance, and used therapeutically. The rationale for this therapeutic approach and the results of human clinical trials will be reported herein.27 references

  11. Cooverexpression of EpCAM and c-myc genes in malignant breast ...

    Indian Academy of Sciences (India)


    catumaxomab, an efficient monoclonal antibody approved in. European Market in 2009, to reduce the rate of metastasis in patients with metastatic breast cancer. ..... + tumours. However, a broader study is required to assess this hypothesis.

  12. Immunogenicity of anti-tumor necrosis factor antibodies - toward improved methods of anti-antibody measurement

    NARCIS (Netherlands)

    Aarden, Lucien; Ruuls, Sigrid R.; Wolbink, Gertjan


    To date, millions of people have been treated with therapeutic monoclonal antibodies (TmAbs) for various indications. It is becoming increasingly clear that TmAbs can be immunogenic, which may reduce efficacy or induce adverse effects. Over the years, the importance of antibody formation has been

  13. Antiparietal cell antibody test (United States)

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; ...

  14. VHH Antibodies: Reagents for Mycotoxin Detection in Food Products

    Directory of Open Access Journals (Sweden)

    Jia Wang


    Full Text Available Mycotoxins are the toxic secondary metabolites produced by fungi and they are a worldwide public health concern. A VHH antibody (or nanobody is the smallest antigen binding entity and is produced by heavy chain only antibodies. Compared with conventional antibodies, VHH antibodies overcome many pitfalls typically encountered in clinical therapeutics and immunodiagnostics. Likewise, VHH antibodies are particularly useful for monitoring mycotoxins in food and feedstuffs, as they are easily genetic engineered and have superior stability. In this review, we summarize the efforts to produce anti-mycotoxins VHH antibodies and associated assays, presenting VHH as a potential tool in mycotoxin analysis.

  15. Binding assays for the quantitative detection of P. brevis polyether neurotoxins in biological samples and antibodies as therapeutic aids for polyether marine intoxication. Annual report, 1 December 1987-30 November 1988

    Energy Technology Data Exchange (ETDEWEB)

    Baden, D.G.


    The polyether lipid-soluble toxins isolated from the marine dinoflagellate Ptychodiscus brevis (formerly Gymnodinium breve) can be detected using two separate types of specific binding reaction. Using tritiated PbTx-3 as a specific probe for binding to voltage-dependent sodium channels in rat brain synaptosomes or to specific polyclonal antibodies, binding equilibria and displacement by unlabeled brevetoxins were compared. Labeled toxin can be displaced in a competitive manner by any of the other 5 naturally-occurring toxins; the quantitative displacement ability of each appears to reflect individual potency in fish bioassay. A comparison of ED50 in Radioimmunoassay and ED50 in synaptosome binding assay indicates that the former assay is useful for detection of toxins which possess the structural backbone of PbTx-3, the immunizing hapten. Thus, the two assays have quantitative applicability; the sodium channel with respect to potency and the antibodies with respect to structure. Microtiter plate assays utilizing each specific brevetoxin binding component and enzyme-linked toxin hapten have been successful and indicate a general applicability of colorimetric prototypes. There, is however, considerable manipulation required to decrease non-specific binding of the hydrophobic toxin-enzyme complex to the plates. Preliminary studies aimed at producing monoclonal antibodies have been explored using brevetoxins linked to keyhole limpet hemocyanin.

  16. Targeting Malignant Brain Tumors with Antibodies

    Directory of Open Access Journals (Sweden)

    Rok Razpotnik


    Full Text Available Antibodies have been shown to be a potent therapeutic tool. However, their use for targeting brain diseases, including neurodegenerative diseases and brain cancers, has been limited, particularly because the blood–brain barrier (BBB makes brain tissue hard to access by conventional antibody-targeting strategies. In this review, we summarize new antibody therapeutic approaches to target brain tumors, especially malignant gliomas, as well as their potential drawbacks. Many different brain delivery platforms for antibodies have been studied such as liposomes, nanoparticle-based systems, cell-penetrating peptides (CPPs, and cell-based approaches. We have already shown the successful delivery of single-chain fragment variable (scFv with CPP as a linker between two variable domains in the brain. Antibodies normally face poor penetration through the BBB, with some variants sufficiently passing the barrier on their own. A “Trojan horse” method allows passage of biomolecules, such as antibodies, through the BBB by receptor-mediated transcytosis (RMT. Such examples of therapeutic antibodies are the bispecific antibodies where one binding specificity recognizes and binds a BBB receptor, enabling RMT and where a second binding specificity recognizes an antigen as a therapeutic target. On the other hand, cell-based systems such as stem cells (SCs are a promising delivery system because of their tumor tropism and ability to cross the BBB. Genetically engineered SCs can be used in gene therapy, where they express anti-tumor drugs, including antibodies. Different types and sources of SCs have been studied for the delivery of therapeutics to the brain; both mesenchymal stem cells (MSCs and neural stem cells (NSCs show great potential. Following the success in treatment of leukemias and lymphomas, the adoptive T-cell therapies, especially the chimeric antigen receptor-T cells (CAR-Ts, are making their way into glioma treatment as another type of cell

  17. Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody-antigen interaction.

    Directory of Open Access Journals (Sweden)

    Takayoshi Matsuda

    Full Text Available Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody-antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab of the anti-EGFR antibody 059-152 were rapidly determined in a day by using a 30-μl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355EGFR, Gln384EGFR, H409EGFR, and Lys465EGFR, so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development.

  18. Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells

    NARCIS (Netherlands)

    D.D. Drabek (Dubravka); R. Janssens (Rick); Boer, E. (Ernie de); Rademaker, R. (Rik); Kloess, J. (Johannes); J.J. Skehel (John ); Grosveld, F. (Frank)


    textabstractSeveral technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and

  19. Monoclonal antibodies in oncology. Review article

    Energy Technology Data Exchange (ETDEWEB)

    Chan, S Y.T.; Sikora, K


    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. Macromolecular therapeutics. (United States)

    Yang, Jiyuan; Kopeček, Jindřich


    This review covers water-soluble polymer-drug conjugates and macromolecules that possess biological activity without attached low molecular weight drugs. The main design principles of traditional and backbone degradable polymer-drug conjugates as well as the development of a new paradigm in nanomedicines - (low molecular weight) drug-free macromolecular therapeutics are discussed. To address the biological features of cancer, macromolecular therapeutics directed to stem/progenitor cells and the tumor microenvironment are deliberated. Finally, the future perspectives of the field are briefly debated. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. The Effect of Induced Antibodies with Respect to Neutralization, Clearance Rate and Functional Activity in a Rabbit/Infliximab Model

    DEFF Research Database (Denmark)

    Henriksen, Maiken Lumby; Teisner, Ane; Kjeldsen, Jens


    Background: Therapeutic antibodies are a developing field for treatment of an expanding number of inflammatory diseases, including Crohn's disease. Treatment with monoclonal antibodies is frequently hampered by development of anti-drug antibodies (ADAs) that may compromise the treatment. Materials...... in patients. The applied assay technology is easily adapted to human plasma samples and/or other therapeutic antibodies, including fully humanized antibodies, for which immunogenicity also is observed....

  2. Therapeutic Plasmapheresis in Kidney Transplantation

    Directory of Open Access Journals (Sweden)

    Zeynep Kendi Celebi


    Full Text Available In 1960's, with succesfully renal transplantations, acute rejection became to be a serious problem for graft survival. From 1965 to 2010, with the introduction of new immunosuppressant agents such as cyclosporine, mycophenolate mofetile and tacrolimus, the acute rejection rates declined from 80% to 10% . There is an ongoing gradual improvement in allograft survival. Use of Therapeutic plasma exchange (TPE is not evidence based treatment, but TPE is necessary for pre- and also post transplantation in patients with DSA. TPE is also a main treatment for antibody mediated rejection (AMR , but in clinical practice the duration and frequency of TPE and individual difference of antibody production is unclear. There is a requirement for more specific antibody elimination. Further randomised controlled studies are needed to elucidate TPE use before and after kidney transplantation. [Dis Mol Med 2013; 1(1.000: 8-10

  3. Therapeutic Nanodevices (United States)

    Lee, Stephen; Ruegsegger, Mark; Barnes, Philip; Smith, Bryan; Ferrari, Mauro

    Therapeutic nanotechnology offers minimally invasive therapies with high densities of function concentrated in small volumes, features that may reduce patient morbidity and mortality. Unlike other areas of nanotechnology, novel physical properties associated with nanoscale dimensionality are not the raison d'être of therapeutic nanotechnology, whereas the aggregation of multiple biochemical (or comparably precise) functions into controlled nanoarchitectures is. Multifunctionality is a hallmark of emerging nanotherapeutic devices, and multifunctionality can allow nanotherapeutic devices to perform multistep work processes, with each functional component contributing to one or more nanodevice subroutine such that, in aggregate, subroutines sum to a cogent work process. Cannonical nanotherapeutic subroutines include tethering (targeting) to sites of disease, dispensing measured doses of drug (or bioactive compound), detection of residual disease after therapy and communication with an external clinician/operator. Emerging nanotherapeutics thus blur the boundaries between medical devices and traditional pharmaceuticals. Assembly of therapeutic nanodevices generally exploits either (bio)material self-assembly properties or chemoselective bioconjugation techniques, or both. Given the complexity, composition, and the necessity for their tight chemical and structural definition inherent in the nature of nanotherapeutics, their cost of goods (COGs) might exceed that of (already expensive) biologics. Early therapeutic nanodevices will likely be applied to disease states which exhibit significant unmet patient need (cancer and cardiovascular disease), while application to other disease states well-served by conventional therapy may await perfection of nanotherapeutic design and assembly protocols.

  4. Boosting antibody developability through rational sequence optimization. (United States)

    Seeliger, Daniel; Schulz, Patrick; Litzenburger, Tobias; Spitz, Julia; Hoerer, Stefan; Blech, Michaela; Enenkel, Barbara; Studts, Joey M; Garidel, Patrick; Karow, Anne R


    The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and long-term stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability.

  5. Reshaping Human Antibodies: Grafting an Antilysozyme Activity (United States)

    Verhoeyen, Martine; Milstein, Cesar; Winter, Greg


    The production of therapeutic human monoclonal antibodies by hybridoma technology has proved difficult, and this has prompted the ``humanizing'' of mouse monoclonal antibodies by recombinant DNA techniques. It was shown previously that the binding site for a small hapten could be grafted from the heavy-chain variable domain of a mouse antibody to that of a human myeloma protein by transplanting the hypervariable loops. It is now shown that a large binding site for a protein antigen (lysozyme) can also be transplanted from mouse to human heavy chain. The success of such constructions may be facilitated by an induced-fit mechanism.

  6. HIV antibodies for treatment of HIV infection. (United States)

    Margolis, David M; Koup, Richard A; Ferrari, Guido


    The bar is high to improve on current combination antiretroviral therapy (ART), now highly effective, safe, and simple. However, antibodies that bind the HIV envelope are able to uniquely target the virus as it seeks to enter new target cells, or as it is expressed from previously infected cells. Furthermore, the use of antibodies against HIV as a therapeutic may offer advantages. Antibodies can have long half-lives, and are being considered as partners for long-acting antiretrovirals for use in therapy or prevention of HIV infection. Early studies in animal models and in clinical trials suggest that such antibodies can have antiviral activity but, as with small-molecule antiretrovirals, the issues of viral escape and resistance will have to be addressed. Most promising, however, are the unique properties of anti-HIV antibodies: the potential ability to opsonize viral particles, to direct antibody-dependent cellular cytotoxicity (ADCC) against actively infected cells, and ultimately the ability to direct the clearance of HIV-infected cells by effector cells of the immune system. These distinctive activities suggest that HIV antibodies and their derivatives may play an important role in the next frontier of HIV therapeutics, the effort to develop treatments that could lead to an HIV cure. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  7. Clinical significance of BRAF non-V600E mutations on the therapeutic effects of anti-EGFR monoclonal antibody treatment in patients with pretreated metastatic colorectal cancer: the Biomarker Research for anti-EGFR monoclonal Antibodies by Comprehensive Cancer genomics (BREAC) study. (United States)

    Shinozaki, Eiji; Yoshino, Takayuki; Yamazaki, Kentaro; Muro, Kei; Yamaguchi, Kensei; Nishina, Tomohiro; Yuki, Satoshi; Shitara, Kohei; Bando, Hideaki; Mimaki, Sachiyo; Nakai, Chikako; Matsushima, Koutatsu; Suzuki, Yutaka; Akagi, Kiwamu; Yamanaka, Takeharu; Nomura, Shogo; Fujii, Satoshi; Esumi, Hiroyasu; Sugiyama, Masaya; Nishida, Nao; Mizokami, Masashi; Koh, Yasuhiro; Abe, Yukiko; Ohtsu, Atsushi; Tsuchihara, Katsuya


    Patients with BRAF V600E -mutated metastatic colorectal cancer (mCRC) have a poorer prognosis as well as resistance to anti-EGFR antibodies. However, it is unclear whether BRAF mutations other than BRAF V600E (BRAF non-V600E mutations) contribute to anti-EGFR antibody resistance. This study was composed of exploratory and inference cohorts. Candidate biomarkers identified by whole exome sequencing from super-responders and nonresponders in the exploratory cohort were validated by targeted resequencing for patients who received anti-EGFR antibody in the inference cohort. In the exploratory cohort, 31 candidate biomarkers, including KRAS/NRAS/BRAF mutations, were identified. Targeted resequencing of 150 patients in the inference cohort revealed 40 patients with RAS (26.7%), 9 patients with BRAF V600E (6.0%), and 7 patients with BRAF non-V600E mutations (4.7%), respectively. The response rates in RAS, BRAF V600E , and BRAF non-V600E were lower than those in RAS/BRAF wild-type (2.5%, 0%, and 0% vs 31.9%). The median PFS in BRAF non-V600E mutations was 2.4 months, similar to that in RAS or BRAF V600E mutations (2.1 and 1.6 months) but significantly worse than that in wild-type RAS/BRAF (5.9 months). Although BRAF non-V600E mutations identified were a rare and unestablished molecular subtype, certain BRAF non-V600E mutations might contribute to a lesser benefit of anti-EGFR monoclonal antibody treatment.

  8. Stratification of antibody-positive subjects by antibody level reveals an impact of immunogenicity on pharmacokinetics. (United States)

    Zhou, Lei; Hoofring, Sarah A; Wu, Yu; Vu, Thuy; Ma, Peiming; Swanson, Steven J; Chirmule, Narendra; Starcevic, Marta


    The availability of highly sensitive immunoassays enables the detection of antidrug antibody (ADA) responses of various concentrations and affinities. The analysis of the impact of antibody status on drug pharmacokinetics (PK) is confounded by the presence of low-affinity or low-concentration antibody responses within the dataset. In a phase 2 clinical trial, a large proportion of subjects (45%) developed ADA following weekly dosing with AMG 317, a fully human monoclonal antibody therapeutic. The antibody responses displayed a wide range of relative concentrations (30 ng/mL to >13 μg/mL) and peaked at various times during the study. To evaluate the impact of immunogenicity on PK, AMG 317 concentration data were analyzed following stratification by dose group, time point, antibody status (positive or negative), and antibody level (relative concentration). With dose group as a stratifying variable, a moderate reduction in AMG 317 levels (AMG 317 levels was revealed when antibody data was stratified by both time point and antibody level. In general, high ADA concentrations (>500 ng/mL) and later time points (week 12) were associated with significantly (up to 97%) lower trough AMG 317 concentrations. The use of quasi-quantitative antibody data and appropriate statistical methods was critical for the most comprehensive evaluation of the impact of immunogenicity on PK.

  9. Antibody-Based Immunotoxins for the Treatment of Cancer


    Nurit Becker; Itai Benhar


    Antibody-based immunotoxins comprise an important group in targeted cancer therapeutics. These chimeric proteins are a form of biological guided missiles that combine a targeting moiety with a potent effector molecule. The targeting moiety is mostly a monoclonal antibody (MAb) or a recombinant antibody-based fragment that confers target specificity to the immunotoxin. The effector domain is a potent protein toxin of bacterial or plant origin, which, following binding to the target cells, unde...

  10. Anti-B cell antibody therapies for inflammatory rheumatic diseases

    DEFF Research Database (Denmark)

    Faurschou, Mikkel; Jayne, David R W


    Several monoclonal antibodies targeting B cells have been tested as therapeutics for inflammatory rheumatic diseases. We review important observations from randomized clinical trials regarding the efficacy and safety of anti-B cell antibody-based therapies for rheumatoid arthritis, systemic lupus...... and functions in rheumatic disorders. Future studies should also evaluate how to maintain disease control by means of conventional and/or biologic immunosuppressants after remission-induction with anti-B cell antibodies....

  11. Postbooster Antibodies from Humans as Source of Diphtheria Antitoxin. (United States)

    Bermejo-Martin, Jesús F; Avila-Alonso, Ana; González-Rivera, Milagros; Tamayo, Eduardo; Eiros, Jose María; Almansa, Raquel


    Diphtheria antitoxin for therapeutic use is in limited supply. A potential source might be affinity-purified antibodies originally derived from plasma of adults who received a booster dose of a vaccine containing diphtheria toxoid. These antibodies might be useful for treating even severe cases of diphtheria.

  12. Immunoprophylaxis in fish by injection of mouse antibody genes

    DEFF Research Database (Denmark)

    Lorenzen, Niels; Cupit, P.M.; Einer-Jensen, Katja


    Antibodies are a crucial part of the body's specific defense against infectious diseases and have considerable potential as therapeutic and prophylactic agents in humans and animals, The development of recombinant single-chain antibodies allows a genetic application strategy for prevention of inf...

  13. Therapeutic ultrasound

    International Nuclear Information System (INIS)

    Crum, Lawrence A


    The use of ultrasound in medicine is now quite commonplace, especially with the recent introduction of small, portable and relatively inexpensive, hand-held diagnostic imaging devices. Moreover, ultrasound has expanded beyond the imaging realm, with methods and applications extending to novel therapeutic and surgical uses. These applications broadly include: tissue ablation, acoustocautery, lipoplasty, site-specific and ultrasound mediated drug activity, extracorporeal lithotripsy, and the enhancement of natural physiological functions such as wound healing and tissue regeneration. A particularly attractive aspect of this technology is that diagnostic and therapeutic systems can be combined to produce totally non-invasive, imageguided therapy. This general lecture will review a number of these exciting new applications of ultrasound and address some of the basic scientific questions and future challenges in developing these methods and technologies for general use in our society. We shall particularly emphasize the use of High Intensity Focused Ultrasound (HIFU) in the treatment of benign and malignant tumors as well as the introduction of acoustic hemostasis, especially in organs which are difficult to treat using conventional medical and surgical techniques. (amum lecture)

  14. Monoclonal antibody therapy of inflammatory bowel disease

    NARCIS (Netherlands)

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


    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

  15. Antibody or Antibody Fragments: Implications for Molecular Imaging and Targeted Therapy of Solid Tumors

    Directory of Open Access Journals (Sweden)

    Katerina T. Xenaki


    Full Text Available The use of antibody-based therapeutics has proven very promising for clinical applications in cancer patients, with multiple examples of antibodies and antibody–drug conjugates successfully applied for the treatment of solid tumors and lymphomas. Given reported recurrence rates, improvements are clearly still necessary. A major factor limiting the efficacy of antibody-targeted cancer therapies may be the incomplete penetration of the antibody or antibody–drug conjugate into the tumor. Incomplete tumor penetration also affects the outcome of molecular imaging, when using such targeting agents. From the injection site until they arrive inside the tumor, targeting molecules are faced with several barriers that impact intratumoral distribution. The primary means of antibody transport inside tumors is based on diffusion. The diffusive penetration inside the tumor is influenced by both antibody properties, such as size and binding affinity, as well as tumor properties, such as microenvironment, vascularization, and targeted antigen availability. Engineering smaller antibody fragments has shown to improve the rate of tumor uptake and intratumoral distribution. However, it is often accompanied by more rapid clearance from the body and in several cases also by inherent destabilization and reduction of the binding affinity of the antibody. In this perspective, we discuss different cancer targeting approaches based on antibodies or their fragments. We carefully consider how their size and binding properties influence their intratumoral uptake and distribution, and how this may affect cancer imaging and therapy of solid tumors.

  16. Antibody conjugate radioimmunotherapy of superficial bladder cancer

    International Nuclear Information System (INIS)

    Perkins, Alan; Hopper, Melanie; Murray, Andrea; Frier, Malcolm; Bishop, Mike


    The administration of antibody conjugates for cancer therapy is now proving to be of clinical value. We are currently undertaking a programme of clinical studies using the monoclonal antibody C 595 (gG3) which reacts with the MUC1 glycoprotein antigen that is aberrantly expressed in a high proportion of bladder tumours. Radio immuno conjugates of the C 595 antibody have been produced with high radiolabelling efficiency and immuno reactivity using Tc-99 m and In-111 for diagnostic imaging, and disease staging and the cytotoxic radionuclides Cu-67 and Re-188 for therapy of superficial bladder cancer. A Phase I/II therapeutic trail involving the intravesical administration of antibody directly into the bladder has now begun. (author)

  17. New Strategies Using Antibody Combinations to Increase Cancer Treatment Effectiveness

    Directory of Open Access Journals (Sweden)

    Isabel Corraliza-Gorjón


    Full Text Available Antibodies have proven their high value in antitumor therapy over the last two decades. They are currently being used as the first-choice to treat some of the most frequent metastatic cancers, like HER2+ breast cancers or colorectal cancers, currently treated with trastuzumab (Herceptin and bevacizumab (Avastin, respectively. The impressive therapeutic success of antibodies inhibiting immune checkpoints has extended the use of therapeutic antibodies to previously unanticipated tumor types. These anti-immune checkpoint antibodies allowed the cure of patients devoid of other therapeutic options, through the recovery of the patient’s own immune response against the tumor. In this review, we describe how the antibody-based therapies will evolve, including the use of antibodies in combinations, their main characteristics, advantages, and how they could contribute to significantly increase the chances of success in cancer therapy. Indeed, novel combinations will consist of mixtures of antibodies against either different epitopes of the same molecule or different targets on the same tumor cell; bispecific or multispecific antibodies able of simultaneously binding tumor cells, immune cells or extracellular molecules; immunomodulatory antibodies; antibody-based molecules, including fusion proteins between a ligand or a receptor domain and the IgG Fab or Fc fragments; autologous or heterologous cells; and different formats of vaccines. Through complementary mechanisms of action, these combinations could contribute to elude the current limitations of a single antibody which recognizes only one particular epitope. These combinations may allow the simultaneous attack of the cancer cells by using the help of the own immune cells and exerting wider therapeutic effects, based on a more specific, fast, and robust response, trying to mimic the action of the immune system.

  18.  Variable fragments of heavy chain antibodies (VHHs: a new magic bullet molecule of medicine?

    Directory of Open Access Journals (Sweden)

    Dorota Smolarek


    Full Text Available  Serum of animals belonging to the Camelidae family (camels and llamas contains fully active antibodies that are naturally devoid of light chains. Variable domains derived from heavy chain antibodies (hcAb called VHHs or nanobodies™ can bind antigens as effectively as full-length antibodies and are easy to clone and express. Because of their potential, VHHs are being intensively studied as potential therapeutic, diagnostic and imaging tools. The paper reviews the molecular background of heavy chain antibodies and describes methods of obtaining recombinant fragments of heavy chain antibodies as well as their therapeutic, diagnostic and other applications.

  19. 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.

  20. Lyme disease antibody (United States)

    ... JavaScript. The Lyme disease blood test looks for antibodies in the blood to the bacteria that causes ... needed. A laboratory specialist looks for Lyme disease antibodies in the blood sample using the ELISA test . ...

  1. Antinuclear antibody panel (United States)

    ... page: // Antinuclear antibody panel To use the sharing features on this page, please enable JavaScript. The antinuclear antibody panel is a blood test that looks at ...

  2. Acetylcholine receptor antibody (United States)

    ... Acetylcholine receptor antibody To use the sharing features on this page, please enable JavaScript. Acetylcholine receptor antibody is a protein found in the blood of ...

  3. Nuclear medicine: Monoclonal antibodies

    International Nuclear Information System (INIS)

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


    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

  4. Platelet antibodies blood test (United States)

    This blood test shows if you have antibodies against platelets in your blood. Platelets are a part of the blood ... Chernecky CC, Berger BJ. Platelet antibody - blood. In: Chernecky ... caused by platelet destruction, hypersplenism, or hemodilution. ...

  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


    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. Aggregates in monoclonal antibody manufacturing processes. (United States)

    Vázquez-Rey, María; Lang, Dietmar A


    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.

  7. Hepatitis A virus antibody

    International Nuclear Information System (INIS)

    Novak, J.; Kselikova, M.; Urbankova, J.


    A description is presented of a radioimmunoassay designed to prove the presence of the antibody against the hepatitis A virus (HA Ab, anti-Ha) using an Abbott HAVAB set. This proof as well as the proof of the antibody against the nucleus of the hepatitis B virus is based on competition between a normal antibody against hepatitis A virus and a 125 I-labelled antibody for the binding sites of a specific antigen spread all over the surface of a tiny ball; this is then indirect proof of the antibody under investigation. The method is described of reading the results from the number of impulses per 60 seconds: the higher the titre of the antibody against the hepatitis A virus in the serum examined, the lower the activity of the specimen concerned. The rate is reported of incidence of the antibody against the hepatitis A virus in a total of 68 convalescents after hepatitis A; the antibody was found in 94.1%. The immunoglobulin made from the convalescents' plasma showed the presence of antibodies in dilutions as high as 1:250 000 while the comparable ratio for normal immunoglobulin Norga was only 1:2500. Differences are discussed in the time incidence of the antibodies against the hepatitis A virus, the antibodies against the surface antigen of hepatitis B, and the antibody against the nucleus of the hepatitis V virus. (author)

  8. Microbial platform technology for recombinant antibody fragment production: A review. (United States)

    Gupta, Sanjeev Kumar; Shukla, Pratyoosh


    Recombinant antibody fragments are being used for the last few years as an important therapeutic protein to cure various critical and life threatening human diseases. Several expression platforms now days employed for the production of these recombinant fragments, out of which bacterial system has emerged a promising host for higher expression. Since, a small antibody fragment unlike full antibody does not require human-like post-translational modification therefore it is potentially expressed in prokaryotic production system. Recently, small antibody fragments such as scFvs (single-chain variable fragments) and Fabs (antibody fragments) which does not require glycosylation are successfully produced in bacteria and have commercially launched for therapeutic use as these fragments shows better tissue penetration and less immunogenic to human body compared to full-size antibody. Recently developed Wacker's ESETEC secretion technology is an efficient technology for the expression and secretion of the antibody fragment (Fab) exceeded up to 4.0 g/L while scFv up to 3.5 g/L into the fermentation broth. The Pfenex system and pOP prokaryotic expression vector are another platform used for the considerably good amount of antibody fragment production successfully. In this review, we summarize the recent progress on various expression platforms and cloning approaches for the production of different forms of antibody fragments in E. coli.

  9. Macrophages are critical effectors of antibody therapies for cancer. (United States)

    Weiskopf, Kipp; Weissman, Irving L


    Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fcγ receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein α (SIRPα) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients.

  10. Anti-insulin antibody test (United States)

    Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... Normally, there are no antibodies against insulin in your blood. ... different laboratories. Some labs use different measurements or ...

  11. Monoclonal antibodies and cancer

    International Nuclear Information System (INIS)

    Haisma, H.J.


    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

  12. Antibody-Based Therapies in Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Yu-Tzu Tai


    Full Text Available The unmet need for improved multiple myeloma (MM therapy has stimulated clinical development of monoclonal antibodies (mAbs targeting either MM cells or cells of the bone marrow (BM microenvironment. In contrast to small-molecule inhibitors, therapeutic mAbs present the potential to specifically target tumor cells and directly induce an immune response to lyse tumor cells. Unique immune-effector mechanisms are only triggered by therapeutic mAbs but not by small molecule targeting agents. Although therapeutic murine mAbs or chimeric mAbs can cause immunogenicity, the advancement of genetic recombination for humanizing rodent mAbs has allowed large-scale production and designation of mAbs with better affinities, efficient selection, decreasing immunogenicity, and improved effector functions. These advancements of antibody engineering technologies have largely overcome the critical obstacle of antibody immunogenicity and enabled the development and subsequent Food and Drug Administration (FDA approval of therapeutic Abs for cancer and other diseases.

  13. [VGKC-complex antibodies]. (United States)

    Watanabe, Osamu


    Various antibodies are associated with voltage-gated potassium channels (VGKCs). Representative antibodies to VGKCs were first identified by radioimmunoassays using radioisotope-labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were detected only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in patients with Morvan's syndrome and in those with a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins (for example LGI-1 and CASPR-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now commonly known as VGKC-complex antibodies. In general, LGI-1 antibodies are most commonly detected in patients with limbic encephalitis with syndrome of inappropriate secretion of antidiuretic hormone. CASPR-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability. Furthermore, VGKC-complex antibodies are tightly associated with chronic idiopathic pain. Hyperexcitability of nociceptive pathways has also been implicated. These antibodies may be detected in sera of some patients with neurodegenerative diseases (for example, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease).

  14. Radiolabeled antibody imaging

    International Nuclear Information System (INIS)

    Wahl, R.L.


    Radiolabeled antibodies, in particular monoclonal antibodies, offer the potential for the specific nuclear imaging of malignant and benign diseases in man. If this imaging potential is realized, they may also have a large role in cancer treatment. This paper reviews: (1) what monoclonal antibodies are and how they differ from polyclonal antibodies, (2) how they are produced and radiolabeled, (3) the results of preclinical and clinical trials in cancer imaging, including the utility of SPECT and antibody fragments, (4) the role of antibodies in the diagnosis of benign diseases, (5) alternate routes of antibody delivery, (6) the role of these agents in therapy, and (7) whether this technology ''revolutionizes'' the practice of nuclear radiology, or has a more limited complementary role in the imaging department

  15. Exploration of novel strategies to enhance monoclonal antibodies targeting

    International Nuclear Information System (INIS)

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


    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

  16. Immunogenicity of anti-tumor necrosis factor antibodies-toward improved methods of anti-antibody measurement. (United States)

    Aarden, Lucien; Ruuls, Sigrid R; Wolbink, Gertjan


    To date, millions of people have been treated with therapeutic monoclonal antibodies (TmAbs) for various indications. It is becoming increasingly clear that TmAbs can be immunogenic, which may reduce efficacy or induce adverse effects. Over the years, the importance of antibody formation has been questioned and sometimes minimized, as few antibody responses to TmAbs (HACA or HAHA) were reported. However, the methods to detect and quantify such antibodies used in the past have been problematic. Only recently, methods have been developed that have adequate sensitivity and are not seriously disturbed by false-positive reactions caused by rheumatoid factors, natural antibodies to Fab or F(ab')2 fragments, or Fc interactions of IgG4. The large number of treated patients, in combination with these new assays, presents a unique opportunity to study the anti-antibody immune response in man, possibly allowing us to manipulate immunogenicity in the future.

  17. A mechanistic compartmental model for total antibody uptake in tumors. (United States)

    Thurber, Greg M; Dane Wittrup, K


    Antibodies are under development to treat a variety of cancers, such as lymphomas, colon, and breast cancer. A major limitation to greater efficacy for this class of drugs is poor distribution in vivo. Localization of antibodies occurs slowly, often in insufficient therapeutic amounts, and distributes heterogeneously throughout the tumor. While the microdistribution around individual vessels is important for many therapies, the total amount of antibody localized in the tumor is paramount for many applications such as imaging, determining the therapeutic index with antibody drug conjugates, and dosing in radioimmunotherapy. With imaging and pretargeted therapeutic strategies, the time course of uptake is critical in determining when to take an image or deliver a secondary reagent. We present here a simple mechanistic model of antibody uptake and retention that captures the major rates that determine the time course of antibody concentration within a tumor including dose, affinity, plasma clearance, target expression, internalization, permeability, and vascularization. Since many of the parameters are known or can be estimated in vitro, this model can approximate the time course of antibody concentration in tumors to aid in experimental design, data interpretation, and strategies to improve localization. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Progress and Challenges in the Design and Clinical Development of Antibodies for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Juan C. Almagro


    Full Text Available The remarkable progress in engineering and clinical development of therapeutic antibodies in the last 40 years, after the seminal work by Köhler and Milstein, has led to the approval by the United States Food and Drug Administration (FDA of 21 antibodies for cancer immunotherapy. We review here these approved antibodies, with emphasis on the methods used for their discovery, engineering, and optimization for therapeutic settings. These methods include antibody engineering via chimerization and humanization of non-human antibodies, as well as selection and further optimization of fully human antibodies isolated from human antibody phage-displayed libraries and immunization of transgenic mice capable of generating human antibodies. These technology platforms have progressively led to the development of therapeutic antibodies with higher human content and, thus, less immunogenicity. We also discuss the genetic engineering approaches that have allowed isotype switching and Fc modifications to modulate effector functions and bioavailability (half-life, which together with the technologies for engineering the Fv fragment, have been pivotal in generating more efficacious and better tolerated therapeutic antibodies to treat cancer.

  19. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses. (United States)

    Tay, Matthew Zirui; Liu, Pinghuang; Williams, LaTonya D; McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T; Dennison, S Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S Munir; Moody, M Anthony; Hope, Thomas J; Haynes, Barton F; Tomaras, Georgia D


    -induced antibodies and therapeutic antibodies will enable a better understanding of their capacity to prevent and/or control HIV-1 infection in vivo.

  20. Innovations in monoclonal antibody tumor targeting; Diagnostic and therapeutic implications

    International Nuclear Information System (INIS)

    Schlom, J.


    The detection of carcinoma lesions not identified by conventional radiological and physical examinations remains a major problem in the treatment of many carcinomas. This article reviews potential new modalities and areas of research for carcinoma diagnosis and treatment. A case history is presented

  1. IL-8 as antibody therapeutic target in inflammatory diseases

    DEFF Research Database (Denmark)

    Skov, Lone; Beurskens, Frank J; Zachariae, Claus O C


    IL-8 is a chemokine that has been implicated in a number of inflammatory diseases involving neutrophil activation. HuMab 10F8 is a novel fully human mAb against IL-8, which binds a discontinuous epitope on IL-8 overlapping the receptor binding site, and which effectively neutralizes IL-8-dependen...

  2. Therapeutic radionuclides: Making the right choice

    International Nuclear Information System (INIS)

    Srivastava, S.C.


    Recently, there has been a resurgence of interest in nuclear medicine therapeutic procedures. Using unsealed sources for therapy is not a new concept; it has been around since the beginnings of nuclear medicine. Treatment of thyroid disorders with radioiodine is a classic example. The availability of radionuclides with suitable therapeutic properties for specific applications, as well as methods for their selective targeting to diseased tissue have, however, remained the main obstacles for therapy to assume a more widespread role in nuclear medicine. Nonetheless, a number of new techniques that have recently emerged, (e.g., tumor therapy with radiolabeled monoclonal antibodies, treatment of metastatic bone pain, etc.) appear to have provided a substantial impetus to research on production of new therapeutic radionuclides. Although there are a number of new therapeutic approaches requiring specific radionuclides, only selected broad areas will be used as examples in this article

  3. Recent advances in the construction of antibody-drug conjugates (United States)

    Chudasama, Vijay; Maruani, Antoine; Caddick, Stephen


    Antibody-drug conjugates (ADCs) comprise antibodies covalently attached to highly potent drugs using a variety of conjugation technologies. As therapeutics, they combine the exquisite specificity of antibodies, enabling discrimination between healthy and diseased tissue, with the cell-killing ability of cytotoxic drugs. This powerful and exciting class of targeted therapy has shown considerable promise in the treatment of various cancers with two US Food and Drug Administration approved ADCs currently on the market (Adcetris and Kadcyla) and approximately 40 currently undergoing clinical evaluation. However, most of these ADCs exist as heterogeneous mixtures, which can result in a narrow therapeutic window and have major pharmacokinetic implications. In order for ADCs to deliver their full potential, sophisticated site-specific conjugation technologies to connect the drug to the antibody are vital. This Perspective discusses the strategies currently used for the site-specific construction of ADCs and appraises their merits and disadvantages.

  4. Pharmacokinetic and pharmacodynamic considerations for the next generation protein therapeutics. (United States)

    Shah, Dhaval K


    Increasingly sophisticated protein engineering efforts have been undertaken lately to generate protein therapeutics with desired properties. This has resulted in the discovery of the next generation of protein therapeutics, which include: engineered antibodies, immunoconjugates, bi/multi-specific proteins, antibody mimetic novel scaffolds, and engineered ligands/receptors. These novel protein therapeutics possess unique physicochemical properties and act via a unique mechanism-of-action, which collectively makes their pharmacokinetics (PK) and pharmacodynamics (PD) different than other established biological molecules. Consequently, in order to support the discovery and development of these next generation molecules, it becomes important to understand the determinants controlling their PK/PD. This review discusses the determinants that a PK/PD scientist should consider during the design and development of next generation protein therapeutics. In addition, the role of systems PK/PD models in enabling rational development of the next generation protein therapeutics is emphasized.

  5. Next-Generation Sequencing of Antibody Display Repertoires

    Directory of Open Access Journals (Sweden)

    Romain Rouet


    Full Text Available In vitro selection technology has transformed the development of therapeutic monoclonal antibodies. Using methods such as phage, ribosome, and yeast display, high affinity binders can be selected from diverse repertoires. Here, we review strategies for the next-generation sequencing (NGS of phage- and other antibody-display libraries, as well as NGS platforms and analysis tools. Moreover, we discuss recent examples relating to the use of NGS to assess library diversity, clonal enrichment, and affinity maturation.

  6. Discovery Of Human Antibodies Against Spitting Cobra Toxins

    DEFF Research Database (Denmark)

    Bojsen-Møller, Laura; Lohse, Brian; Harrison, Robert

    Current snakebite envenoming treatment options consist of animal-derived antisera and are associated with severe adverse reactions due to the heterologous nature of the animal-derived antibodies present in these antisera, and the presence of therapeutically irrelevant antibodies. The African...... spitting cobras are among the most medically important snakes in sub-Saharan regions due to the severity of the clinical outcomes caused by their cytotoxic venom, which is derived from cytotoxins of the 3FTx toxin family and PLA2. Here we report the results of our progress in identifying human antibodies...... targeting relevant toxins from the venom of the black necked spitting cobra (Naja nigricolis)....

  7. Antibody engineering: methods and protocols

    National Research Council Canada - National Science Library

    Chames, Patrick


    "Antibody Engineering: Methods and Protocols, Second Edition was compiled to give complete and easy access to a variety of antibody engineering techniques, starting from the creation of antibody repertoires and efficient...

  8. What Is Antiphospholipid Antibody Syndrome? (United States)

    ... Back To Health Topics / Antiphospholipid Antibody Syndrome Antiphospholipid Antibody Syndrome Also known as What Is Antiphospholipid (AN-te-fos-fo-LIP-id) antibody syndrome (APS) is an autoimmune disorder. Autoimmune disorders ...

  9. Recent Advances in Monoclonal Antibody Therapies for Multiple Sclerosis (United States)

    Stavropoulos, Nikolaos; Wittenberg, Nathan J.; Dasari, Harika; Abdelrahim, Murtada A.; Henley, John R.; Oh, Sang-Hyun; Warrington, Arthur E.; Rodriguez, Moses


    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

  10. Antiviral Therapy by HIV-1 Broadly Neutralizing and Inhibitory Antibodies

    Directory of Open Access Journals (Sweden)

    Zhiqing Zhang


    Full Text Available Human immunodeficiency virus type 1 (HIV-1 infection causes acquired immune deficiency syndrome (AIDS, a global epidemic for more than three decades. HIV-1 replication is primarily controlled through antiretroviral therapy (ART but this treatment does not cure HIV-1 infection. Furthermore, there is increasing viral resistance to ART, and side effects associated with long-term therapy. Consequently, there is a need of alternative candidates for HIV-1 prevention and therapy. Recent advances have discovered multiple broadly neutralizing antibodies against HIV-1. In this review, we describe the key epitopes on the HIV-1 Env protein and the reciprocal broadly neutralizing antibodies, and discuss the ongoing clinical trials of broadly neutralizing and inhibitory antibody therapy as well as antibody combinations, bispecific antibodies, and methods that improve therapeutic efficacy by combining broadly neutralizing antibodies (bNAbs with latency reversing agents. Compared with ART, HIV-1 therapeutics that incorporate these broadly neutralizing and inhibitory antibodies offer the advantage of decreasing virus load and clearing infected cells, which is a promising prospect in HIV-1 prevention and treatment.

  11. Dual antibody therapy to harness the innate anti-tumor immune response to enhance antibody targeting of tumors. (United States)

    Chester, Cariad; Marabelle, Aurelien; Houot, Roch; Kohrt, Holbrook E


    Cancer immunotherapy is a rapidly evolving field that offers a novel paradigm for cancer treatment: therapies focus on enhancing the immune system's innate and adaptive anti-tumor response. Early immunotherapeutics have achieved impressive clinical outcomes and monoclonal antibodies are now integral to therapeutic strategies in a variety of cancers. However, only recently have antibodies targeting innate immune cells entered clinical development. Innate immune effector cells play important roles in generating and maintaining antitumor immunity. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are important innate immune mechanisms for tumor eradication. These cytolytic processes are initiated by the detection of a tumor-targeting antibody and can be augmented by activating co-stimulatory pathways or blocking inhibitory signals on innate immune cells. The combination of FDA-approved monoclonal antibodies with innate effector-targeting antibodies has demonstrated potent preclinical therapeutic synergy and early-phase combinatorial clinical trials are ongoing. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Therapeutic target for protozoal diseases (United States)

    Rathore, Dharmendar [Blacksburg, VA; Jani, Dewal [Blacksburg, VA; Nagarkatti, Rana [Blacksburg, VA


    A novel Fasciclin Related Adhesive Protein (FRAP) from Plasmodium and related parasites is provided as a target for therapeutic intervention in diseases caused by the parasites. FRAP has been shown to play a critical role in adhesion to, or invasion into, host cells by the parasite. Furthermore, FRAP catalyzes the neutralization of heme by the parasite, by promoting its polymerization into hemozoin. This invention provides methods and compositions for therapies based on the administration of protein, DNA or cell-based vaccines and/or antibodies based on FRAP, or antigenic epitopes of FRAP, either alone or in combination with other parasite antigens. Methods for the development of compounds that inhibit the catalytic activity of FRAP, and diagnostic and laboratory methods utilizing FRAP are also provided.

  13. Radiolabelled antibodies in imaging

    International Nuclear Information System (INIS)

    Khaw, B.A.; Haber, E.


    Recent technological advances make it possible to produce pure (monoclonal) antibodies in unlimited quantities without the need for continuous immunization of animals and to label these antibodies with a variety of radionuclides which can be traced by single-photon computed tomography. An outline review of the state of the art is presented, with particular reference to the imaging of myocardial infarcts and to tumour imaging studies using labelled monoclonal antibodies (sup(99m)Tc and 125 I). Lengthy bibliography. (U.K.)

  14. A novel antibody engineering strategy for making monovalent bispecific heterodimeric IgG antibodies by electrostatic steering mechanism. (United States)

    Liu, Zhi; Leng, Esther C; Gunasekaran, Kannan; Pentony, Martin; Shen, Min; Howard, Monique; Stoops, Janelle; Manchulenko, Kathy; Razinkov, Vladimir; Liu, Hua; Fanslow, William; Hu, Zhonghua; Sun, Nancy; Hasegawa, Haruki; Clark, Rutilio; Foltz, Ian N; Yan, Wei


    Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Physiologically Based Pharmacokinetic Modeling of Therapeutic Proteins. (United States)

    Wong, Harvey; Chow, Timothy W


    Biologics or therapeutic proteins are becoming increasingly important as treatments for disease. The most common class of biologics are monoclonal antibodies (mAbs). Recently, there has been an increase in the use of physiologically based pharmacokinetic (PBPK) modeling in the pharmaceutical industry in drug development. We review PBPK models for therapeutic proteins with an emphasis on mAbs. Due to their size and similarity to endogenous antibodies, there are distinct differences between PBPK models for small molecules and mAbs. The high-level organization of a typical mAb PBPK model consists of a whole-body PBPK model with organ compartments interconnected by both blood and lymph flows. The whole-body PBPK model is coupled with tissue-level submodels used to describe key mechanisms governing mAb disposition including tissue efflux via the lymphatic system, elimination by catabolism, protection from catabolism binding to the neonatal Fc (FcRn) receptor, and nonlinear binding to specific pharmacological targets of interest. The use of PBPK modeling in the development of therapeutic proteins is still in its infancy. Further application of PBPK modeling for therapeutic proteins will help to define its developing role in drug discovery and development. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  16. Metal chelate conjugated monoclonal antibodies, wherein the metal is an α emitter

    International Nuclear Information System (INIS)

    Gansow, O.A.; Strand, M.


    Methods of manufacturing and purifying metal chelate conjugated monoclonal antibodies are described, wherein the chelated metal emits alpha radiation. The conjugates are suited for therapeutic uses being substantially free of nonchelated radiometal. (author)

  17. Future of antibody purification. (United States)

    Low, Duncan; O'Leary, Rhona; Pujar, Narahari S


    Antibody purification seems to be safely ensconced in a platform, now well-established by way of multiple commercialized antibody processes. However, natural evolution compels us to peer into the future. This is driven not only by a large, projected increase in the number of antibody therapies, but also by dramatic improvements in upstream productivity, and process economics. Although disruptive technologies have yet escaped downstream processes, evolution of the so-called platform is already evident in antibody processes in late-stage development. Here we perform a wide survey of technologies that are competing to be part of that platform, and provide our [inherently dangerous] assessment of those that have the most promise.

  18. Serum herpes simplex antibodies (United States)

    ... causes cold sores (oral herpes). HSV-2 causes genital herpes. How the Test is Performed A blood sample ... person has ever been infected with oral or genital herpes . It looks for antibodies to herpes simplex virus ...

  19. Antibody tumor penetration (United States)

    Thurber, Greg M.; Schmidt, Michael M.; Wittrup, K. Dane


    Antibodies have proven to be effective agents in cancer imaging and therapy. One of the major challenges still facing the field is the heterogeneous distribution of these agents in tumors when administered systemically. Large regions of untargeted cells can therefore escape therapy and potentially select for more resistant cells. We present here a summary of theoretical and experimental approaches to analyze and improve antibody penetration in tumor tissue. PMID:18541331

  20. Monoclonal antibodies based on hybridoma technology. (United States)

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


    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.

  1. Antibodies: From novel repertoires to defining and refining the structure of biologically important targets. (United States)

    Conroy, Paul J; Law, Ruby H P; Caradoc-Davies, Tom T; Whisstock, James C


    Antibodies represent a highly successful class of molecules that bind a wide-range of targets in therapeutic-, diagnostic- and research-based applications. The antibody repertoire is composed of the building blocks required to develop an effective adaptive immune response against foreign insults. A number of species have developed novel genetic and structural mechanisms from which they derive these antibody repertoires, however, traditionally antibodies are isolated from human, and rodent sources. Due to their high-value therapeutic, diagnostic, biotechnological and research applications, much innovation has resulted in techniques and approaches to isolate novel antibodies. These approaches are bolstered by advances in our understanding of species immune repertoires, next generation sequencing capacity, combinatorial antibody discovery and high-throughput screening. Structural determination of antibodies and antibody-antigen complexes has proven to be pivotal to our current understanding of the immune repertoire for a range of species leading to advances in man-made libraries and fine tuning approaches to develop antibodies from immune-repertoires. Furthermore, the isolation of antibodies directed against antigens of importance in health, disease and developmental processes, has yielded a plethora of structural and functional insights. This review highlights the significant contribution of antibody-based crystallography to our understanding of adaptive immunity and its application to providing critical information on a range of human-health related indications. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. A novel anti-GPC3 monoclonal antibody (YP7) | Center for Cancer Research (United States)

    Glypican-3 (GPC3) is an emerging therapeutic target in hepatoma. A novel anti-GPC3 monoclonal antibody (YP7) has been generated through a combination of peptide immunization and high-throughput flow cytometry screening. YP7 binds cell-surface-associated GPC3 with high affinity and exhibits significant hepatoma xenograft growth inhibition in nude mice. The new antibody may have

  3. Radiolabeled antibodies and RGD-peptides for the treatment of ovarian cancer.

    NARCIS (Netherlands)

    Janssen, M.L.H.


    In this thesis, preclinical studies on new treatment modalities for ovarian cancer are descibed, applying radiolabeled antibodies and radiolabeled RGD-peptides. In chapter 2 a study is described comparing the therapeutic efficacy of the antibody HMFG1 radiolabeled with several beta-emitting

  4. Animal models of antineutrophil cytoplasm antibody-associated vasculitis.

    LENUS (Irish Health Repository)

    Salama, Alan D


    To provide an update on the experimental models that have been developed recapitulating clinical antineutrophil cytoplasm antibody (ANCA) associated vasculitis. The application of the models in the study of pathogenesis, and the therapeutic implications of this, are covered in the article by van Timmeren and Heeringa in this issue.

  5. Recent developments in monoclonal antibody radiolabeling techniques

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.; Mease, R.C.


    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.

  6. Recent developments in monoclonal antibody radiolabeling techniques

    International Nuclear Information System (INIS)

    Srivastava, S.C.; Mease, R.C.


    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

  7. Myasthenia gravis: subgroup classification and therapeutic strategies. (United States)

    Gilhus, Nils Erik; Verschuuren, Jan J


    Myasthenia gravis is an autoimmune disease that is characterised by muscle weakness and fatigue, is B-cell mediated, and is associated with antibodies directed against the acetylcholine receptor, muscle-specific kinase (MUSK), lipoprotein-related protein 4 (LRP4), or agrin in the postsynaptic membrane at the neuromuscular junction. Patients with myasthenia gravis should be classified into subgroups to help with therapeutic decisions and prognosis. Subgroups based on serum antibodies and clinical features include early-onset, late-onset, thymoma, MUSK, LRP4, antibody-negative, and ocular forms of myasthenia gravis. Agrin-associated myasthenia gravis might emerge as a new entity. The prognosis is good with optimum symptomatic, immunosuppressive, and supportive treatment. Pyridostigmine is the preferred symptomatic treatment, and for patients who do not adequately respond to symptomatic therapy, corticosteroids, azathioprine, and thymectomy are first-line immunosuppressive treatments. Additional immunomodulatory drugs are emerging, but therapeutic decisions are hampered by the scarcity of controlled studies. Long-term drug treatment is essential for most patients and must be tailored to the particular form of myasthenia gravis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Antibody-mediated immunotherapy against chronic hepatitis B virus infection. (United States)

    Gao, Ying; Zhang, Tian-Ying; Yuan, Quan; Xia, Ning-Shao


    The currently available drugs to treat hepatitis B virus (HBV) infection include interferons and nucleos(t)ide analogs, which can only induce disease remission and are inefficient for the functional cure of patients with chronic HBV infection (CHB). Since high titers of circulating hepatitis B surface antigen (HBsAg) may be essential to exhaust the host anti-HBV immune response and they cannot be significantly reduced by current drugs, new antiviral strategies aiming to suppress serum hepatitis B surface antigen (HBsAg) could help restore virus-specific immune responses and promote the eradication of the virus. As an alternative strategy, immunotherapy with HBsAg-specific antibodies has shown some direct HBsAg suppression effects in several preclinical and clinical trial studies. However, most described previously HBsAg-specific antibodies only had very short-term HBsAg suppression effects in CHB patients and animal models mimicking persistent HBV infection. More-potent antibodies with long-lasting HBsAg clearance effects are required for the development of the clinical application of antibody-mediated immunotherapy for CHB treatment. Our recent study described a novel mAb E6F6 that targets a unique epitope on HBsAg. It could durably suppress the levels of HBsAg and HBV DNA via Fcγ receptor-dependent phagocytosis in vivo. In this commentary, we summarize the current research progress, including the therapeutic roles and mechanisms of antibody-mediated HBV clearance as well as the epitope-determined therapeutic potency of the antibody. These insights may provide some clues and guidance to facilitate the development of therapeutic antibodies against persistent viral infection.

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

    International Nuclear Information System (INIS)

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


    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.''

  10. Radiolabelled antibody imaging

    International Nuclear Information System (INIS)

    Perkins, A.C.


    A steadily growing number of tumor-associated antigens are used to raise antibodies used for the detection of human tumors by external imaging, a technique termed immunoscintigraphy. The majority of these clinical antibody studies are performed using Iodine-131, which is cheap, readily available and easily attached to protein. It has the disadvantage of having a high energy gamma emission (365 keV) which is poorly detected by modern cameras, so that increasing use is now being made of more appropriate labels with lower energies for imaging, such as Iodine-123, Indium-111 and Technetium-99m. A number of research centres in the United Kingdom are currently involved in the production of tumor-associated monoclonal antibodies, only a small number of which are finally selected for diagnostic use. These developments represent a major area of advancement in Nuclear Medicine and when used for imaging are capable of providing diagnostic information complimentary to other diagnostic techniques

  11. Severe antiphospholipid antibody syndrome - response to plasmapheresis and rituximab. (United States)

    Gkogkolou, Paraskevi; Ehrchen, Jan; Goerge, Tobias


    Antiphospholipid antibody syndrome (APS) is a systemic autoimmune disease characterized by arterial and/or venous thrombosis, recurrent abortions and detection of antiphospholipid antibodies. In fulminant cases, involvement of multiple organs can lead to significant morbidity and even fatal outcomes, so that a rapid, interdisciplinary treatment is needed. Here, we describe the case of a 39-year-old woman with a severe hard-to-treat APS with arterial occlusion and progressive skin necrosis, who was successfully treated with a combination therapy with plasmapheresis and rituximab. The treatment led to complete remission of the skin lesions for over a year. Clinical response correlated with a long-lasting reduction of antiphospholipid antibodies and B-cell depletion. This case demonstrates the use of antiphospholipid antibodies for monitoring APS-activity and shows that this severe vascular disease requires rigorous therapeutic approaches.

  12. Radioimmunoimaging of experimental gliomas using radiolabelled monoclonal antibodies

    International Nuclear Information System (INIS)

    Glaessner, H.


    The biodistribution and tumour uptake of radiolabelled (131 I) glioma-seeking monoclonal antibodies (14 AC1) and their F(ab') 2 fragments were investigated in nude mice having received glioma transplants. Radioimmunoimaging by external scintigraphy at 48 and 96 hours pointed to a superior tumour localisation by the fragments that was clearly related to the dose. Wholebody determinations of the biokinetic behaviour led to the following results: Faster clearance anc more ready elimination from the blood pool for the fragments, preferential uptake in the tumour; intact antibodies; binding in the liver, spleen and lungs. The study confirmed the value of fragments of monoclonal antibodies in the diagnosis of tumours and pointed to the possibility of using intact monoclonal antibodies as carriers of radioisotopes and cytotoxic drugs within the scope of therapeutic programmes. (TRV) [de

  13. Antibody informatics for drug discovery

    DEFF Research Database (Denmark)

    Shirai, Hiroki; Prades, Catherine; Vita, Randi


    to the antibody science in every project in antibody drug discovery. Recent experimental technologies allow for the rapid generation of large-scale data on antibody sequences, affinity, potency, structures, and biological functions; this should accelerate drug discovery research. Therefore, a robust bioinformatic...... infrastructure for these large data sets has become necessary. In this article, we first identify and discuss the typical obstacles faced during the antibody drug discovery process. We then summarize the current status of three sub-fields of antibody informatics as follows: (i) recent progress in technologies...... for antibody rational design using computational approaches to affinity and stability improvement, as well as ab-initio and homology-based antibody modeling; (ii) resources for antibody sequences, structures, and immune epitopes and open drug discovery resources for development of antibody drugs; and (iii...

  14. Antithyroglobulin Antibodies and Antimicrosomal Antibodies in Various Thyroid Diseases

    International Nuclear Information System (INIS)

    Lee, Gwon Jun; Hong, Key Sak; Choi, Kang Won; Lee, Kyu; Koh, Chang Soon; Lee, Mun Ho; Park, Sung Hoe; Chi, Je Geun; Lee, Sang Kook


    The authors investigated the incidence of antithyroglobulin antibodies and antibodies and antimicrosomal antibodies measured by tanned red cell hemagglutination method in subjects suffering from various thyroid disorders. 1) In 15 normal patients, neither suffering from any thyroid diseases nor from any other autoimmune disorders, the antithyroglobulin antibodies were all negative, but the antimicrosomal antibody was positive only in one patient (6.7%). 2) The antithyroglobulin antibodies were positive in 31.5% (34 patients) of 108 patients with various thyroid diseases, and the antimicrosomal antibodies were positive in 37.0% (40 patients). 3) of the 25 patients with Graves' diseases, 7 patients (28.0%) showed positive for the antithyroglobulin antibodies, and 9 (36.0%) for the antimicrosomal antibodies. There was no definite differences in clinical and thyroid functions between the groups with positive and negative results. 4) Both antibodies were positive in 16 (88.9%) and 17 (94.4%) patients respectively among 18 patients with Hashimoto's thyroiditis, all of them were diagnosed histologically. 5) Three out of 33 patients with thyroid adenoma showed positive antibodies, and 3 of 16 patients with thyroid carcinoma revealed positive antibodies. 6) TRCH antibodies demonstrated negative results in 2 patients with subacute thyroiditis, but positive in one patient with idiopathic primary myxedema. 7) The number of patients with high titers(>l:802) was 16 for antithyroglobulin antibody, and 62.5% (10 patients) of which was Hashimoto's thyroiditis. Thirteen (65.0) of 20 patients with high titers (>l:802) for antimicrosomal antibody was Hashimoto's thyroiditis. TRCH test is a simple, sensitive method, and has high reliability and reproducibility. The incidences and titers of antithyroglobulin antibody and antimicrosomal antibody are especially high in Hashimoto's thyroiditis.

  15. Antithyroglobulin Antibodies and Antimicrosomal Antibodies in Various Thyroid Diseases

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Gwon Jun; Hong, Key Sak; Choi, Kang Won; Lee, Kyu; Koh, Chang Soon; Lee, Mun Ho; Park, Sung Hoe; Chi, Je Geun; Lee, Sang Kook [Seoul National University College of Medicine, Seoul (Korea, Republic of)


    The authors investigated the incidence of antithyroglobulin antibodies and antibodies and antimicrosomal antibodies measured by tanned red cell hemagglutination method in subjects suffering from various thyroid disorders. 1) In 15 normal patients, neither suffering from any thyroid diseases nor from any other autoimmune disorders, the antithyroglobulin antibodies were all negative, but the antimicrosomal antibody was positive only in one patient (6.7%). 2) The antithyroglobulin antibodies were positive in 31.5% (34 patients) of 108 patients with various thyroid diseases, and the antimicrosomal antibodies were positive in 37.0% (40 patients). 3) of the 25 patients with Graves' diseases, 7 patients (28.0%) showed positive for the antithyroglobulin antibodies, and 9 (36.0%) for the antimicrosomal antibodies. There was no definite differences in clinical and thyroid functions between the groups with positive and negative results. 4) Both antibodies were positive in 16 (88.9%) and 17 (94.4%) patients respectively among 18 patients with Hashimoto's thyroiditis, all of them were diagnosed histologically. 5) Three out of 33 patients with thyroid adenoma showed positive antibodies, and 3 of 16 patients with thyroid carcinoma revealed positive antibodies. 6) TRCH antibodies demonstrated negative results in 2 patients with subacute thyroiditis, but positive in one patient with idiopathic primary myxedema. 7) The number of patients with high titers(>l:802) was 16 for antithyroglobulin antibody, and 62.5% (10 patients) of which was Hashimoto's thyroiditis. Thirteen (65.0) of 20 patients with high titers (>l:802) for antimicrosomal antibody was Hashimoto's thyroiditis. TRCH test is a simple, sensitive method, and has high reliability and reproducibility. The incidences and titers of antithyroglobulin antibody and antimicrosomal antibody are especially high in Hashimoto's thyroiditis.

  16. Prediction of Antibody Epitopes

    DEFF Research Database (Denmark)

    Nielsen, Morten; Marcatili, Paolo


    Antibodies recognize their cognate antigens in a precise and effective way. In order to do so, they target regions of the antigenic molecules that have specific features such as large exposed areas, presence of charged or polar atoms, specific secondary structure elements, and lack of similarity...... to self-proteins. Given the sequence or the structure of a protein of interest, several methods exploit such features to predict the residues that are more likely to be recognized by an immunoglobulin.Here, we present two methods (BepiPred and DiscoTope) to predict linear and discontinuous antibody...

  17. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    Yeast surface display is an effective tool for antibody affinity maturation because yeast can be used as an all-in-one workhorse to assemble, display and screen diversified antibody libraries. By employing the natural ability of yeast Saccharomyces cerevisiae to efficiently recombine multiple DNA...... laboratory conditions. A particular emphasis was put on using molecular techniques in conjunction with microenvironmental measurements (O2, pH, irradiance), a combination that is rarely found but provides a much more detailed understanding of “cause and effect” in complex natural systems...

  18. Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies

    Directory of Open Access Journals (Sweden)

    Fa Yang


    Full Text Available With the development of molecular cloning technology and the deep understanding of antibody engineering, there are diverse bispecific antibody formats from which to choose to pursue the optimal biological activity and clinical purpose. The single-chain-based bispecific antibodies usually bridge tumor cells with immune cells and form an immunological synapse because of their relatively small size. Bispecific antibodies in the IgG format include asymmetric bispecific antibodies and homodimerized bispecific antibodies, all of which have an extended blood half-life and their own crystalline fragment (Fc-mediated functions. Besides retargeting effector cells to the site of cancer, new applications were established for bispecific antibodies. Bispecific antibodies that can simultaneously bind to cell surface antigens and payloads are a very ideal delivery system for therapeutic use. Bispecific antibodies that can inhibit two correlated signaling molecules at the same time can be developed to overcome inherent or acquired resistance and to be more efficient angiogenesis inhibitors. Bispecific antibodies can also be used to treat hemophilia A by mimicking the function of factor VIII. Bispecific antibodies also have broad application prospects in bone disorders and infections and diseases of the central nervous system. The latest developments of the formats and application of bispecific antibodies will be reviewed. Furthermore, the challenges and perspectives are summarized in this review.

  19. Fetal neonatal hyperthyroidism: diagnostic and therapeutic approachment (United States)

    Kurtoğlu, Selim; Özdemir, Ahmet


    Fetal and neonatal hyperthyroidism may occur in mothers with Graves’ disease. Fetal thyrotoxicosis manifestation is observed with the transition of TSH receptor stimulating antibodies to the fetus from the 17th–20th weeks of pregnancy and with the fetal TSH receptors becoming responsive after 20 weeks. The diagnosis is confirmed by fetal tachycardia, goiter and bone age advancement in pregnancy and maternal treatment is conducted in accordance. The probability of neonatal hyperthyroidism is high in the babies of mothers that have ongoing antithyroid requirement and higher antibody levels in the last months of pregnancy. Clinical manifestation may be delayed by 7–17 days because of the antithyroid drugs taken by the mother. Neonatal hyperthyroidism symptoms can be confused with sepsis and congenital viral infections. Herein, the diagnosis and therapeutic approach are reviewed in cases of fetal neonatal hyperthyroidism. PMID:28439194

  20. Tracking of multimodal therapeutic nanocomplexes targeting breast cancer in vivo. (United States)

    Bardhan, Rizia; Chen, Wenxue; Bartels, Marc; Perez-Torres, Carlos; Botero, Maria F; McAninch, Robin Ward; Contreras, Alejandro; Schiff, Rachel; Pautler, Robia G; Halas, Naomi J; Joshi, Amit


    Nanoparticle-based therapeutics with local delivery and external electromagnetic field modulation holds extraordinary promise for soft-tissue cancers such as breast cancer; however, knowledge of the distribution and fate of nanoparticles in vivo is crucial for clinical translation. Here we demonstrate that multiple diagnostic capabilities can be introduced in photothermal therapeutic nanocomplexes by simultaneously enhancing both near-infrared fluorescence and magnetic resonance imaging (MRI). We track nanocomplexes in vivo, examining the influence of HER2 antibody targeting on nanocomplex distribution over 72 h. This approach provides valuable, detailed information regarding the distribution and fate of complex nanoparticles designed for specific diagnostic and therapeutic functions.

  1. Addressing the Immunogenicity of the Cargo and of the Targeting Antibodies with a Focus on Deimmunized Bacterial Toxins and on Antibody-Targeted Human Effector Proteins (United States)

    Grinberg, Yehudit; Benhar, Itai


    Third-generation immunotoxins are composed of a human, or humanized, targeting moiety, usually a monoclonal antibody or an antibody fragment, and a non-human effector molecule. Due to the non-human origin of the cytotoxic domain, these molecules stimulate potent anti-drug immune responses, which limit treatment options. Efforts are made to deimmunize such immunotoxins or to combine treatment with immunosuppression. An alternative approach is using the so-called “human cytotoxic fusion proteins”, in which antibodies are used to target human effector proteins. Here, we present three relevant approaches for reducing the immunogenicity of antibody-targeted protein therapeutics: (1) reducing the immunogenicity of the bacterial toxin, (2) fusing human cytokines to antibodies to generate immunocytokines and (3) addressing the immunogenicity of the targeting antibodies. PMID:28574434

  2. Generation of anti-idiotype scFv for pharmacokinetic measurement in lymphoma patients treated with chimera anti-CD22 antibody SM03.

    Directory of Open Access Journals (Sweden)

    Qi Zhao

    Full Text Available Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.

  3. Aligning physics and physiology: Engineering antibodies for radionuclide delivery. (United States)

    Tsai, Wen-Ting K; Wu, Anna M


    The exquisite specificity of antibodies and antibody fragments renders them excellent agents for targeted delivery of radionuclides. Radiolabeled antibodies and fragments have been successfully used for molecular imaging and radioimmunotherapy (RIT) of cell surface targets in oncology and immunology. Protein engineering has been used for antibody humanization essential for clinical applications, as well as optimization of important characteristics including pharmacokinetics, biodistribution, and clearance. Although intact antibodies have high potential as imaging and therapeutic agents, challenges include long circulation time in blood, which leads to later imaging time points post-injection and higher blood absorbed dose that may be disadvantageous for RIT. Using engineered fragments may address these challenges, as size reduction and removal of Fc function decreases serum half-life. Radiolabeled fragments and pretargeting strategies can result in high contrast images within hours to days, and a reduction of RIT toxicity in normal tissues. Additionally, fragments can be engineered to direct hepatic or renal clearance, which may be chosen based on the application and disease setting. This review discusses aligning the physical properties of radionuclides (positron, gamma, beta, alpha, and Auger emitters) with antibodies and fragments and highlights recent advances of engineered antibodies and fragments in preclinical and clinical development for imaging and therapy. Copyright © 2018 John Wiley & Sons, Ltd.

  4. Compositions, antibodies, asthma diagnosis methods, and methods for preparing antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hongjun; Zangar, Richard C.


    Methods for preparing an antibody are provided with the method including incorporating 3-bromo-4-hydroxy-benzoic acid into a protein to form an antigen, immunizing a mammalian host with the antigen, and recovering an antibody having an affinity for the antigen from the host. Antibodies having a binding affinity for a monohalotyrosine are provided as well as composition comprising an antibody bound with monohalotyrosine. Compositions comprising a protein having a 3-bromo-4-hydroxy-benzoic acid moiety are also provided. Methods for evaluating the severity of asthma are provide with the methods including analyzing sputum of a patient using an antibody having a binding affinity for monohalotyrosine, and measuring the amount of antibody bound to protein. Methods for determining eosinophil activity in bodily fluid are also provided with the methods including exposing bodily fluid to an antibody having a binding affinity for monohalotyrosine, and measuring the amount of bound antibody to determine the eosinophil activity.

  5. Prediction of antibody persistency from antibody titres to natalizumab

    DEFF Research Database (Denmark)

    Jensen, Poul Erik H; Koch-Henriksen, Nils; Sellebjerg, Finn


    In a subgroup of patients with multiple sclerosis natalizumab therapy causes generation of anti-natalizumab antibodies that may be transient or persistent. It is recommended to discontinue natalizumab therapy in persistently antibody-positive patients.......In a subgroup of patients with multiple sclerosis natalizumab therapy causes generation of anti-natalizumab antibodies that may be transient or persistent. It is recommended to discontinue natalizumab therapy in persistently antibody-positive patients....

  6. ANA (Antinuclear Antibody Test) (United States)

    ... as ratios. For example, the result 1:320 means that one part blood sample was mixed with 320 parts of a diluting ... name "antinuclear". My doctor told me my ANA test is ... normal concentration of these antibodies. This is one of the tools in diagnosing lupus as well ...

  7. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

    Sondergeld, P.; van de Donk, N. W. C. J.; Richardson, P. G.


    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...

  8. Antibodies Targeting EMT (United States)


    these unusual antibodies can effectively be displayed on the cell surface. 5 Additionally, we successfully prepared cDNA from lymphocytes derived...from cow peripheral blood, spleen, and lymph nodes, amplified this cDNA by PCR with VH gene specific primers, and this “library” has been cloned into

  9. Antibody Blood Tests (United States)

    ... out for sure? If antibody tests and/or symptoms suggest celiac disease, the physician needs to establish the diagnosis by ... who is still experiencing symptoms, to establish the diagnosis or to rule out celiac disease as a part of establishing another diagnosis. Find ...

  10. Antinuclear Antibodies (ANA) (United States)

    ... MACRA MACRAlerts MACRA FAQs MACRA Glossary MACRA Resources Position Statements Insurance Advocacy Current Issues Tools & Resources Practice Resources ... a medical or health condition. Resources Antinuclear Antibodies (ANA) in Spanish (Español) Download Print-Friendly PDF ... Join Donate © 2018 American College ...


    Directory of Open Access Journals (Sweden)

    Oliinyk O. S.


    Full Text Available Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, and 6 unique restriction patterns were found. Single-chain antibodies were expressed in Escherichia coli XL1-blue. The recombinant proteins were characterized by immunoblotting of bacterial extracts and detection with an anti-E-tag antibody. The toxin B-subunit-binding function of the single-chain antibody was shown by ELISA. The affinity constants for different clones were found to be from 106 to 108 М–1. Due to the fact, that these antibody fragments recognized epitopes in the receptor-binding Bsubunit of diphtheria toxin, further studies are interesting to evaluate their toxin neutralization properties and potential for therapeutic applications. Obtained scFv-antibodies can also be used for detection and investigation of biological properties of diphtheria toxin.

  12. Antibody-Based Agents in the Management of Antibiotic-Resistant Staphylococcus aureus Diseases (United States)

    Speziale, Pietro; Rindi, Simonetta


    Staphylococcus aureus is a human pathogen that can cause a wide spectrum of diseases, including sepsis, pneumonia, arthritis, and endocarditis. Ineffective treatment of a number of staphylococcal infections with antibiotics is due to the development and spread of antibiotic-resistant strains following decades of antibiotic usage. This has generated renewed interest within the scientific community in alternative therapeutic agents, such as anti-S. aureus antibodies. Although the role of antibodies in the management of S. aureus diseases is controversial, the success of this pathogen in neutralizing humoral immunity clearly indicates that antibodies offer the host extensive protection. In this review, we report an update on efforts to develop antibody-based agents, particularly monoclonal antibodies, and their therapeutic potential in the passive immunization approach to the treatment and prevention of S. aureus infections. PMID:29533985

  13. Manufacturing of recombinant therapeutic proteins in microbial systems. (United States)

    Graumann, Klaus; Premstaller, Andreas


    Recombinant therapeutic proteins have gained enormous importance for clinical applications. The first recombinant products have been produced in E. coli more than 20 years ago. Although with the advent of antibody-based therapeutics mammalian expression systems have experienced a major boost, microbial expression systems continue to be widely used in industry. Their intrinsic advantages, such as rapid growth, high yields and ease of manipulation, make them the premier choice for expression of non-glycosylated peptides and proteins. Innovative product classes such as antibody fragments or alternative binding molecules will further expand the use of microbial systems. Even more, novel, engineered production hosts and integrated technology platforms hold enormous potential for future applications. This review summarizes current applications and trends for development, production and analytical characterization of recombinant therapeutic proteins in microbial systems.

  14. Anti-smooth muscle antibody (United States)

    ... gov/ency/article/003531.htm Anti-smooth muscle antibody To use the sharing features on this page, please enable JavaScript. Anti-smooth muscle antibody is a blood test that detects the presence ...

  15. Tabhu: tools for antibody humanization.

    KAUST Repository

    Olimpieri, Pier Paolo; Marcatili, Paolo; Tramontano, Anna


    for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps of the humanization experiment protocol. AVAILABILITY: http

  16. Antibodies from plants for bionanomaterials


    Edgue, G.; Twyman, R.M.; Beiss, V.; Fischer, R.; Sack, M.


    Antibodies are produced as part of the vertebrate adaptive immune response and are not naturally made by plants. However, antibody DNA sequences can be introduced into plants, and together with laboratory technologies that allow the design of antibodies recognizing any conceivable molecular structure, plants can be used as green factories' to produce any antibody at all. The advent of plant-based transient expression systems in particular allows the rapid, convenient, and safe production of a...

  17. Use of polyclonal/monoclonal antibody therapies in transplantation. (United States)

    Yeung, Melissa Y; Gabardi, Steven; Sayegh, Mohamed H


    For over thirty years, antibody (mAb)-based therapies have been a standard component of transplant immunosuppression, and yet much remains to be learned in order for us to truly harness their therapeutic capabilities. Current mAbs used in transplant directly target and destroy graft-destructive immune cells, interrupt cytokine and costimulation-dependent T and B cell activation, and prevent down-stream complement activation. Areas covered: This review summarizes our current approaches to using antibody-based therapies to prevent and treat allograft rejection. It also provides examples of promising novel mAb therapies, and discusses the potential for future mAb development in transplantation. Expert opinion: The broad capability of antibodies, in parallel with our growing ability to synthetically modulate them, offers exciting opportunities to develop better biologic therapeutics. In order to do so, we must further our understanding about the basic biology underlying allograft rejection, and gain better appreciation of how characteristics of therapeutic antibodies affect their efficacy.

  18. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    Zegers, N.D.


    Synthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps that lead to the

  19. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    N.D. Zegers (Netty)


    textabstractSynthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps

  20. Monoclonal antibodies to Pneumocystis carinii

    DEFF Research Database (Denmark)

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


    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...

  1. Introduction to current and future protein therapeutics: a protein engineering perspective. (United States)

    Carter, Paul J


    Protein therapeutics and its enabling sister discipline, protein engineering, have emerged since the early 1980s. The first protein therapeutics were recombinant versions of natural proteins. Proteins purposefully modified to increase their clinical potential soon followed with enhancements derived from protein or glycoengineering, Fc fusion or conjugation to polyethylene glycol. Antibody-based drugs subsequently arose as the largest and fastest growing class of protein therapeutics. The rationale for developing better protein therapeutics with enhanced efficacy, greater safety, reduced immunogenicity or improved delivery comes from the convergence of clinical, scientific, technological and commercial drivers that have identified unmet needs and provided strategies to address them. Future protein drugs seem likely to be more extensively engineered to improve their performance, e.g., antibodies and Fc fusion proteins with enhanced effector functions or extended half-life. Two old concepts for improving antibodies, namely antibody-drug conjugates and bispecific antibodies, have advanced to the cusp of clinical success. As for newer protein therapeutic platform technologies, several engineered protein scaffolds are in early clinical development and offer differences and some potential advantages over antibodies. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Characterization of Tumor-Avid Antibody Fragments Genetically Engineered for Mono-Specific Radionuclide Chelation

    International Nuclear Information System (INIS)

    Quinn, T.P.


    The successful clinical application of targeted-radiopharmaceuticals depends on the development of molecules that optimize tumor specific radionuclide deposition and minimize non-specific organ irradiation. To this end, this proposal outlines a research effort to identify and evaluate novel antibodies and antibody fragments that bind breast tumors. The tumor-avid antibodies will be investigated for as imaging and therapeutic agents and to gain a better understanding of the pharmacokinetics and metabolism of radiolabeled tumor-avid antibody fragments through the use of site-specifically labeled molecules. Antibodies or antibody fragments, that bind breast carcinoma carbohydrate antigens, will be obtained from hybridoma or bacteriophage library screening. More specifically, antibody fragments that bind the carcinoma-associated Thomsen-Friedenreich (T) antigen will be radiolabeled with 99m Tc and 188 Re at a natural amino acid chelation site and will be investigated in vivo for their abilities to target human breast tumors. In addition, site-specific radiolabeled antibody fragments will be biosynthesized using misacylated suppressor tRNAs. Homogeneously radiolabeled populations of antibody fragments will be used to investigate the effects of radionuclide location and chelation chemistries on their biodistribution and metabolism. It is hypothesized that site-specifically radiolabeled antibody fragments will possess enhanced tumor imaging and therapeutic properties due to optimal label location and conjugation chemistries. New insights into the factors that govern antibody metabolism in vivo are also expected from this work. Results from these studies should enhance our ability to design and synthesize radiolabeled antibody fragments that have improved pharmacokinetic properties. The studies in this proposal involve basic research into the development of antibody-based radiopharmaceuticals, with the ultimate goal of application in humans. This type of basic nuclear

  3. Effects of altered FcγR binding on antibody pharmacokinetics in cynomolgus monkeys (United States)

    Leabman, Maya K; Meng, Y Gloria; Kelley, Robert F; DeForge, Laura E; Cowan, Kyra J; Iyer, Suhasini


    Antibody interactions with Fcγ receptors (FcγRs), like FcγRIIIA, play a critical role in mediating antibody effector functions and thereby contribute significantly to the biologic and therapeutic activity of antibodies. Over the past decade, considerable work has been directed towards production of antibodies with altered binding affinity to FcγRs and evaluation of how the alterations modulate their therapeutic activity. This has been achieved by altering glycosylation status at N297 or by engineering modifications in the crystallizable fragment (Fc) region. While the effects of these modifications on biologic activity and efficacy have been examined, few studies have been conducted to understand their effect on antibody pharmacokinetics (PK). We present here a retrospective analysis in which we characterize the PK of three antibody variants with decreased FcγR binding affinity caused by amino acid substitutions in the Fc region (N297A, N297G, and L234A/L235A) and three antibody variants with increased FcγRIIIA binding affinity caused by afucosylation at N297, and compare their PK to corresponding wild type antibody PK in cynomolgus monkeys. For all antibodies, PK was examined at a dose that was known to be in the linear range. Since production of the N297A and N297G variants in Chinese hamster ovary cells results in aglycosylated antibodies that do not bind to FcγRs, we also examined the effect of expression of an aglycosylated antibody, without sequence change(s), in E. coli. All the variants demonstrated similar PK compared with that of the wild type antibodies, suggesting that, for the six antibodies presented here, altered FcγR binding affinity does not affect PK. PMID:24492343

  4. Antibody Selection for Cancer Target Validation of FSH-Receptor in Immunohistochemical Settings

    Directory of Open Access Journals (Sweden)

    Nina Moeker


    Full Text Available Background: The follicle-stimulating hormone (FSH-receptor (FSHR has been reported to be an attractive target for antibody therapy in human cancer. However, divergent immunohistochemical (IHC findings have been reported for FSHR expression in tumor tissues, which could be due to the specificity of the antibodies used. Methods: Three frequently used antibodies (sc-7798, sc-13935, and FSHR323 were validated for their suitability in an immunohistochemical study for FSHR expression in different tissues. As quality control, two potential therapeutic anti-hFSHR Ylanthia® antibodies (Y010913, Y010916 were used. The specificity criteria for selection of antibodies were binding to native hFSHR of different sources, and no binding to non-related proteins. The ability of antibodies to stain the paraffin-embedded Flp-In Chinese hamster ovary (CHO/FSHR cells was tested after application of different epitope retrieval methods. Results: From the five tested anti-hFSHR antibodies, only Y010913, Y010916, and FSHR323 showed specific binding to native, cell-presented hFSHR. Since Ylanthia® antibodies were selected to specifically recognize native FSHR, as required for a potential therapeutic antibody candidate, FSHR323 was the only antibody to detect the receptor in IHC/histochemical settings on transfected cells, and at markedly lower, physiological concentrations (ex., in Sertoli cells of human testes. The pattern of FSH323 staining noticed for ovarian, prostatic, and renal adenocarcinomas indicated that FSHR was expressed mainly in the peripheral tumor blood vessels. Conclusion: Of all published IHC antibodies tested, only antibody FSHR323 proved suitable for target validation of hFSHR in an IHC setting for cancer. Our studies could not confirm the previously reported FSHR overexpression in ovarian and prostate cancer cells. Instead, specific overexpression in peripheral tumor blood vessels could be confirmed after thorough validation of the antibodies used.

  5. Pathogenesis of Graves' disease and therapeutic implications

    International Nuclear Information System (INIS)

    Seif, F.J.


    Graves' disease presents itself clinically mainly as hyperthyroidism and infiltrative ophthalmopathy and to a minimal extent also as dermopathy and acropachy. Autoimmune processes are the basic pathogenesis. Stimulating antibodies against the TSH receptor cause hyperthyroidism. Autoantibodies and autoreactive T lymphocytes against primarily thyroidal antigens cross-react with similar antigens of the eye muscles and orbital connective tissue, thus spreading the disease from the thyroid to the eyes. The therapeutic goal comprises not only the treatment of hyperthyroidism, but also the induction of a steady immuntolerance in order to minimize the irreversible damage to the eye. The therapeutic armamentarium is formed by antithyroid drugs, glucocorticoids, retrobulbar radition and thyroid ablation, either by nearly total thyroidectomy or by radioiodine. The different indications for both ablative procedures are discussed. (orig.) [de

  6. Variable Domain N-Linked Glycans Acquired During Antigen-Specific Immune Responses Can Contribute to Immunoglobulin G Antibody Stability

    Directory of Open Access Journals (Sweden)

    Fleur S. van de Bovenkamp


    Full Text Available Immunoglobulin G (IgG can contain N-linked glycans in the variable domains, the so-called Fab glycans, in addition to the Fc glycans in the CH2 domains. These Fab glycans are acquired following introduction of N-glycosylation sites during somatic hypermutation and contribute to antibody diversification. We investigated whether Fab glycans may—in addition to affecting antigen binding—contribute to antibody stability. By analyzing thermal unfolding profiles of antibodies with or without Fab glycans, we demonstrate that introduction of Fab glycans can improve antibody stability. Strikingly, removal of Fab glycans naturally acquired during antigen-specific immune responses can deteriorate antibody stability, suggesting in vivo selection of stable, glycosylated antibodies. Collectively, our data show that variable domain N-linked glycans acquired during somatic hypermutation can contribute to IgG antibody stability. These findings indicate that introducing Fab glycans may represent a mechanism to improve therapeutic/diagnostic antibody stability.

  7. 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


    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)

  8. 9th annual European Antibody Congress, November 11-13, 2013, Geneva, Switzerland. (United States)

    Reichert, Janice M; Beck, Alain; Lugovskoy, Alexey A; Wurch, Thierry; Coats, Steven; Brezski, Randall J


    The annual European Antibody Congress (EAC) has traditionally been the key event for updates on critical scientific advances in the antibody field, and 2013 was no exception. Organized by Terrapinn, the well-attended meeting featured presentations on considerations for developing antibodies and antibody-like therapeutics, with separate tracks for antibody-drug conjugates, naked antibodies, and multispecific antibodies or protein scaffolds. The overall focus of the EAC was current approaches to enhance the functionality of therapeutic antibodies or other targeted proteins, with the ultimate goal being improvement of the safety and efficacy of the molecules as treatments for cancer, immune-mediated disorders and other diseases. Roundtable discussion sessions gave participants opportunities to engage in group discussions with industry leaders from companies such as Genmab, Glenmark Pharmaceuticals, MedImmune, Merrimack Pharmaceuticals, and Pierre Fabre. As the 2013 EAC was co-located with the World Biosimilar Congress, participants also received an update on European Medicines Agency guidelines and thoughts on the future direction and development of biosimilar antibodies in the European Union.

  9. Delta antibody radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Kselikova, M; Urbankova, J


    The principle and procedure are described of the radioimmunoassay of delta antibody (delta-Ab) using the ABBOTT ANTI-DELTA kit by Abbott Co. A description is given of the kit, the working procedure and the method of evaluation. The results are reported of the incidence of delta-Ab in sera of patients with viral hepatitis B, in haemophiliacs, carriers of the hepatitis B virus surface antigen (HBsAg) and blood donors. The presence was detected of delta-Ab in one HBsAg carrier. The necessity is emphasized of delta-Ab determinations in the blood of donors in view of the antibody transfer with blood and blood preparations.

  10. [Antibody therapy for Alzheimer's disease]. (United States)

    Tabira, Takeshi; Matsumoto, Shin-Ei; Jin, Haifeng


    In order to avoid Abeta-induced autoimmune encephalitis, several monoclonal and polyclonal antibodies are in clinical trials. These are bapineuzumab, solanezumab, ponezumab, gantenerumab, BAN2401, gammaguard and octagam. Since each antibody has a different antigen epitope of Abeta, anti-amyloid activities are different. It is unknown which antibody is effective for Alzheimer disease, and we must wait for the result of clinical trials. Some patients who developed tissue amyloid plaque immuno-reactive (TAPIR) antibody showed slower decline after AN-1792 vaccination. We developed TAPIR-like monoclonal antibody, which was found to react with Abeta oligomers preferentially.

  11. Monoclonal antibodies in the treatment of non-Hodgkin's lymphoma. (United States)

    Fanale, Michelle A; Younes, Anas


    Antibody-based therapeutic approaches have had a significant impact in the treatment of non-Hodgkin's lymphoma (NHL). Rituximab's development as an anti-CD20 antibody heralded a new era in treatment approaches for NHL. While rituximab was first shown to be effective in the treatment of relapsed follicular lymphoma, it is now standard monotherapy for front-line treatment of follicular lymphoma, and is also used in conjunction with chemotherapy for other indolent, intermediate and aggressive B-cell lymphomas. The development of rituximab has led to intense interest in this type of therapeutic approach and to development and approval of the radioimmunoconjugates of rituximab, (90)Y-ibritumomab tiuxetan and (131)I-tositumomab, which have added to the repertoire of treatments for relapsed follicular lymphoma and increased interest in developing other conjugated antibodies. Since rituximab is a chimeric antibody, there is a need to develop fully humanised antibodies, such as IMMU-106 (hA20), in order to minimise infusion reactions and eliminate the development of human antibodies against the drug. Further clinical evaluation of antibodies has been based largely on our knowledge of antigen expression on the surface of lymphoma cells and has led to the development of antibodies against CD22 (unconjugated epratuzumab and calicheamicin conjugated CMC-544 [inotuzumab ozogamicin]), CD80 (galiximab), CD52 (alemtuzumab), CD2 (MEDI-507 [siplizumab]), CD30 (SGN-30 and MDX-060 [iratumumab]), and CD40 (SGN-40). Furthermore, the VEGF (vascular endothelial growth factor) inhibitor bevacizumab, which was first approved for the treatment of colon cancer is currently under investigation in NHL, and agonists rather than antibodies to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) [rApo2L/TRAIL, HGS-ETR1{mapatumumab}, HGS-ETR2] are currently being investigated as treatments for both advanced solid tumours and NHL. Knowledge of the ability of cancer cells to become

  12. Exogenous insulin antibody syndrome (EIAS): a clinical syndrome associated with insulin antibodies induced by exogenous insulin in diabetic patients. (United States)

    Hu, Xiaolei; Chen, Fengling


    Insulin has been used for diabetes therapy and has achieved significant therapeutic effect. In recent years, the use of purified and recombinant human insulin preparations has markedly reduced, but not completely suppressed, the incidence of insulin antibodies (IAs). IAs induced by exogenous insulin in diabetic patients is associated with clinical events, which is named exogenous insulin antibody syndrome (EIAS). The present review is based on our research and summarizes the characterization of IAs, the factors affecting IA development, the clinical significance of IAs and the treatments for EIAS. © 2018 The authors.

  13. Prediction of site-specific interactions in antibody-antigen complexes: the proABC method and server.

    KAUST Repository

    Olimpieri, Pier Paolo; Chailyan, Anna; Tramontano, Anna; Marcatili, Paolo


    MOTIVATION: Antibodies or immunoglobulins are proteins of paramount importance in the immune system. They are extremely relevant as diagnostic, biotechnological and therapeutic tools. Their modular structure makes it easy to re-engineer them

  14. Chemoradiotherapy of esophagus cancers: prognostic value of anti-P53 and anti-ras circulating antibodies

    International Nuclear Information System (INIS)

    Blanchard, P.; Quero, L.; Pacaud, V.; Baruch-Hennequin, V.; Maylin, C.; Hennequin, C.; Schlageter, M.H.


    The presence of anti-p53 antibodies makes suspect a resistance to the chemoradiotherapy and has to make envisage other approaches that the chemo - radiotherapy by 5-fluoro-uracil-cisplatin (surgery, intensification, targeted therapeutic). (N.C.)

  15. A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library. (United States)

    Adler, Adam S; Bedinger, Daniel; Adams, Matthew S; Asensio, Michael A; Edgar, Robert C; Leong, Renee; Leong, Jackson; Mizrahi, Rena A; Spindler, Matthew J; Bandi, Srinivasa Rao; Huang, Haichun; Tawde, Pallavi; Brams, Peter; Johnson, David S


    Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of "randomly paired" scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.

  16. FcγRII-binding Centyrins mediate agonism and antibody-dependent cellular phagocytosis when fused to an anti-OX40 antibody. (United States)

    Zhang, Di; Whitaker, Brian; Derebe, Mehabaw G; Chiu, Mark L


    Immunostimulatory antibodies against the tumor necrosis factor receptors (TNFR) are emerging as promising cancer immunotherapies. The agonism activity of such antibodies depends on crosslinking to Fc gamma RIIB receptor (FcγRIIB) to enable the antibody multimerization that drives TNFR activation. Previously, Fc engineering was used to enhance the binding of such antibodies to Fcγ receptors. Here, we report the identification of Centyrins as alternative scaffold proteins with binding affinities to homologous FcγRIIB and FcγRIIA, but not to other types of Fcγ receptors. One Centyrin, S29, was engineered at distinct positions of an anti-OX40 SF2 antibody to generate bispecific and tetravalent molecules named as mAbtyrins. Regardless of the position of S29 on the SF2 antibody, SF2-S29 mAbtyrins could bind FcγRIIB and FcγRIIA specifically while maintaining binding to OX40 receptors. In a NFκB reporter assay, attachment of S29 Centyrin molecules at the C-termini, but not the N-termini, resulted in SF2 antibodies with increased agonism owing to FcγRIIB crosslinking. The mAbtyrins also showed agonism in T-cell activation assays with immobilized FcγRIIB and FcγRIIA, but this activity was confined to mAbtyrins with S29 specifically at the C-termini of antibody heavy chains. Furthermore, regardless of the position of the molecule, S29 Centyrin could equip an otherwise Fc-silent antibody with antibody-dependent cellular phagocytosis activity without affecting the antibody's intrinsic antibody-dependent cell-meditated cytotoxicity and complement-dependent cytotoxicity. In summary, the appropriate adoption FcγRII-binding Centyrins as functional modules represents a novel strategy to engineer therapeutic antibodies with improved functionalities.

  17. Quantitative relationship between antibody affinity and antibody avidity

    International Nuclear Information System (INIS)

    Griswold, W.R.


    The relationship between antibody avidity, measured by the dissociation of the antigen-antibody bond in antigen excess, and antibody affinity was studied. Complexes of radiolabelled antigen and antibody of known affinity were prepared in vitro and allowed to stand for seven days to reach equilibrium. Then nonlabelled antigen in one hundred fold excess was added to dissociate the complexes. After an appropriate incubation the fraction of antigen bound to antibody was measured by the ammonium sulfate precipitation method. The dissociation index was the fraction bound in the experimental sample divided by the fraction bound in the control. The correlation coefficient between the dissociation index and the antibody binding constant was 0.92 for early dissociation and 0.98 for late dissociation. The regression equation relating the binding constant to the dissociation index was K = 6.4(DI) + 6.25, where DI is the late dissociation index and K is the logarithm to the base 10 of the binding constant. There is a high correlation between avidity and affinity of antibody. Antibody affinity can be estimated from avidity data. The stability of antigen-antibody complexes can be predicted from antibody affinity

  18. Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein.

    Directory of Open Access Journals (Sweden)

    Luis Mario Rodríguez-Martínez

    Full Text Available Current Ebola virus (EBOV detection methods are costly and impractical for epidemic scenarios. Different immune-based assays have been reported for the detection and quantification of Ebola virus (EBOV proteins. In particular, several monoclonal antibodies (mAbs have been described that bind the capsid glycoprotein (GP of EBOV GP. However, the currently available platforms for the design and production of full-length mAbs are cumbersome and costly. The use of antibody fragments, rather than full-length antibodies, might represent a cost-effective alternative for the development of diagnostic and possibly even therapeutic alternatives for EBOV.We report the design and expression of three recombinant anti-GP mAb fragments in Escherichia coli cultures. These fragments contained the heavy and light variable portions of the three well-studied anti-GP full-length mAbs 13C6, 13F6, and KZ52, and are consequently named scFv-13C6, scFv-13F6, and Fab-KZ52, respectively. All three fragments exhibited specific anti-GP binding activity in ELISA experiments comparable to that of full-length anti-GP antibodies (i.e., the same order of magnitude and they are easily and economically produced in bacterial cultures.Antibody fragments might represent a useful, effective, and low cost alternative to full-length antibodies in Ebola related capture and diagnostics applications.

  19. Affinity chromatography: A versatile technique for antibody purification. (United States)

    Arora, Sushrut; Saxena, Vikas; Ayyar, B Vijayalakshmi


    Antibodies continue to be extremely utilized entities in myriad applications including basic research, imaging, targeted delivery, chromatography, diagnostics, and therapeutics. At production stage, antibodies are generally present in complex matrices and most of their intended applications necessitate purification. Antibody purification has always been a major bottleneck in downstream processing of antibodies, due to the need of high quality products and associated high costs. Over the years, extensive research has focused on finding better purification methodologies to overcome this holdup. Among a plethora of different techniques, affinity chromatography is one of the most selective, rapid and easy method for antibody purification. This review aims to provide a detailed overview on affinity chromatography and the components involved in purification. An array of support matrices along with various classes of affinity ligands detailing their underlying working principles, together with the advantages and limitations of each system in purifying different types of antibodies, accompanying recent developments and important practical methodological considerations to optimize purification procedure are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Effects of genetic engineering on the pharmacokinetics of antibodies

    International Nuclear Information System (INIS)

    Colcher, D.; Goel, A.; Pavlinkova, G.; Beresford, G.; Booth, B.; Batra, S.K.


    Monoclonal antibodies (MAbs) may be considered 'magic bullets' due to their ability to recognize and eradicate malignant cells. MAbs, however, have practical limitations for their rapid application in the clinics. The structure of the antibody molecules can be engineered to modify functional domains such as antigen-binding sites and/or effectors functions. Advanced in genetic engineering have provided rapid progress the development of new immunoglobulin constructs of MAbs with defined research and therapeutic application. Recombinant antibody constructs are being engineered, such as human mouse chimeric, domain-dispositioned, domain-deleted, humanized and single-chain Fv fragments. Genetically-engineered antibodies differ in size and rate of catabolism. Pharmacokinetics studies show that the intact IgG (150 kD), enzymatically derived fragments Fab' (50 kD) and single chain Fv (28 kD) have different clearance rates. These antibody forms clear 50% from the blood pool in 2.1 days, 30 minutes and 10 minutes, respectively. Genetically-engineered antibodies make a new class of immunotherapeutic tracers for cancer treatment

  1. Pulmonary biology of anti-interleukin 5 antibodies

    Directory of Open Access Journals (Sweden)

    RW Egan


    Full Text Available Interleukin 5 (IL-5 is a critical cytokine for the maturation of eosinophil precursors to eosinophils in the bone marrow and those eosinophils then accumulate in the lungs during asthma. We have studied anti IL-5 antibodies on allergic responses in mice, guinea pigs and monkeys and are extending this experiment into humans with a humanized antibody. In a monkey model of pulmonary inflammation and airway hyperreactivity, we found that the TRFK-5 antibody blocked both responses for three months following a single dose of 0.3 mg/kg, i.v. This antibody also blocked lung eosinophilia in mice by inhibiting release from the bone marrow. To facilitate multiple dosing and to reduce immunogenicity in humans, we prepared Sch 55700, a humanized antibody against IL-5. Sch 55700 was also active against lung eosinophilia in allergic monkeys and mice and against pulmonary eosinophilia and airway hyperresponsiveness in guinea pigs. Furthermore, as opposed to steroids, Sch 55700 did not cause immunosuppression in guinea pigs. Studies with this antibody in humans will be critical to establishing the therapeutic potential of IL-5 inhibition.

  2. A modular platform for targeted RNAi therapeutics. (United States)

    Kedmi, Ranit; Veiga, Nuphar; Ramishetti, Srinivas; Goldsmith, Meir; Rosenblum, Daniel; Dammes, Niels; Hazan-Halevy, Inbal; Nahary, Limor; Leviatan-Ben-Arye, Shani; Harlev, Michael; Behlke, Mark; Benhar, Itai; Lieberman, Judy; Peer, Dan


    Previous studies have identified relevant genes and signalling pathways that are hampered in human disorders as potential candidates for therapeutics. Developing nucleic acid-based tools to manipulate gene expression, such as short interfering RNAs 1-3 (siRNAs), opens up opportunities for personalized medicine. Yet, although major progress has been made in developing siRNA targeted delivery carriers, mainly by utilizing monoclonal antibodies (mAbs) for targeting 4-8 , their clinical translation has not occurred. This is in part because of the massive development and production requirements and the high batch-to-batch variability of current technologies, which rely on chemical conjugation. Here we present a self-assembled modular platform that enables the construction of a theoretically unlimited repertoire of siRNA targeted carriers. The self-assembly of the platform is based on a membrane-anchored lipoprotein that is incorporated into siRNA-loaded lipid nanoparticles that interact with the antibody crystallizable fragment (Fc) domain. We show that a simple switch of eight different mAbs redirects the specific uptake of siRNAs by diverse leukocyte subsets in vivo. The therapeutic potential of the platform is demonstrated in an inflammatory bowel disease model by targeting colon macrophages to reduce inflammatory symptoms, and in a Mantle Cell Lymphoma xenograft model by targeting cancer cells to induce cell death and improve survival. This modular delivery platform represents a milestone in the development of precision medicine.

  3. A modular platform for targeted RNAi therapeutics (United States)

    Kedmi, Ranit; Veiga, Nuphar; Ramishetti, Srinivas; Goldsmith, Meir; Rosenblum, Daniel; Dammes, Niels; Hazan-Halevy, Inbal; Nahary, Limor; Leviatan-Ben-Arye, Shani; Harlev, Michael; Behlke, Mark; Benhar, Itai; Lieberman, Judy; Peer, Dan


    Previous studies have identified relevant genes and signalling pathways that are hampered in human disorders as potential candidates for therapeutics. Developing nucleic acid-based tools to manipulate gene expression, such as short interfering RNAs1-3 (siRNAs), opens up opportunities for personalized medicine. Yet, although major progress has been made in developing siRNA targeted delivery carriers, mainly by utilizing monoclonal antibodies (mAbs) for targeting4-8, their clinical translation has not occurred. This is in part because of the massive development and production requirements and the high batch-to-batch variability of current technologies, which rely on chemical conjugation. Here we present a self-assembled modular platform that enables the construction of a theoretically unlimited repertoire of siRNA targeted carriers. The self-assembly of the platform is based on a membrane-anchored lipoprotein that is incorporated into siRNA-loaded lipid nanoparticles that interact with the antibody crystallizable fragment (Fc) domain. We show that a simple switch of eight different mAbs redirects the specific uptake of siRNAs by diverse leukocyte subsets in vivo. The therapeutic potential of the platform is demonstrated in an inflammatory bowel disease model by targeting colon macrophages to reduce inflammatory symptoms, and in a Mantle Cell Lymphoma xenograft model by targeting cancer cells to induce cell death and improve survival. This modular delivery platform represents a milestone in the development of precision medicine.

  4. TRAIL death receptors and cancer therapeutics

    International Nuclear Information System (INIS)

    Huang Ying; Sheikh, M. Saeed


    Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) also known as Apo2L is an apoptotic molecule that belongs to the tumor necrosis factor superfamily of cytokines. It mediates its apoptotic effects via its cognate death receptors including DR4 and DR5. Agonistic monoclonal antibodies have also been developed that selectively activate TRAIL death receptors to mediate apoptosis. Multiple clinically relevant agents also upregulate the expression of TRAIL death receptors, and cooperate with TRAIL as well as DR4 and DR5-specific agonistic antibodies to exhibit tumor cell killing. TRAIL is currently in phase I clinical trials, whereas DR4 and DR5-specific agonistic antibodies have been tested in phase I and II studies. Thus, TRAIL has clearly distinguished itself from the other family members including TNF-alpha and FasL both of which could not make it to the clinic due to their toxic nature. It is therefore, evident that the future of TRAIL-based therapeutic approaches looks brighter

  5. In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody (United States)

    Richard, Gabrielle; Meyers, Ashley J.; McLean, Michael D.; Arbabi-Ghahroudi, Mehdi; MacKenzie, Roger; Hall, J. Christopher


    Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. PMID:23894495

  6. [Study of anti-idiotype antibodies to human monoclonal antibody]. (United States)

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


    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

  7. CD44 antibodies and immune thrombocytopenia in the amelioration of murine inflammatory arthritis.

    Directory of Open Access Journals (Sweden)

    Patrick J Mott

    Full Text Available Antibodies to CD44 have been used to successfully ameliorate murine models of autoimmune disease. The most often studied disease model has been murine inflammatory arthritis, where a clear mechanism for the efficacy of CD44 antibodies has not been established. We have recently shown in a murine passive-model of the autoimmune disease immune thrombocytopenia (ITP that some CD44 antibodies themselves can induce thrombocytopenia in mice, and the CD44 antibody causing the most severe thrombocytopenia (IM7, also is known to be highly effective in ameliorating murine models of arthritis. Recent work in the K/BxN serum-induced model of arthritis demonstrated that antibody-induced thrombocytopenia reduced arthritis, causing us to question whether CD44 antibodies might primarily ameliorate arthritis through their thrombocytopenic effect. We evaluated IM7, IRAWB14.4, 5035-41.1D, KM201, KM114, and KM81, and found that while all could induce thrombocytopenia, the degree of protection against serum-induced arthritis was not closely related to the length or severity of the thrombocytopenia. CD44 antibody treatment was also able to reverse established inflammation, while thrombocytopenia induced by an anti-platelet antibody targeting the GPIIbIIIa platelet antigen, could not mediate this effect. While CD44 antibody-induced thrombocytopenia may contribute to some of its therapeutic effect against the initiation of arthritis, for established disease there are likely other mechanisms contributing to its efficacy. Humans are not known to express CD44 on platelets, and are therefore unlikely to develop thrombocytopenia after CD44 antibody treatment. An understanding of the relationship between arthritis, thrombocytopenia, and CD44 antibody treatment remains critical for continued development of CD44 antibody therapeutics.

  8. Interplay between Natural Killer Cells and Anti-HER2 Antibodies: Perspectives for Breast Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Aura Muntasell


    Full Text Available Overexpression of the human epidermal growth factor receptor 2 (HER2 defines a subgroup of breast tumors with aggressive behavior. The addition of HER2-targeted antibodies (i.e., trastuzumab, pertuzumab to chemotherapy significantly improves relapse-free and overall survival in patients with early-stage and advanced disease. Nonetheless, considerable proportions of patients develop resistance to treatment, highlighting the need for additional and co-adjuvant therapeutic strategies. HER2-specific antibodies can trigger natural killer (NK cell-mediated antibody-dependent cellular cytotoxicity and indirectly enhance the development of tumor-specific T cell immunity; both mechanisms contributing to their antitumor efficacy in preclinical models. Antibody-dependent NK cell activation results in the release of cytotoxic granules as well as the secretion of pro-inflammatory cytokines (i.e., IFNγ and TNFα and chemokines. Hence, NK cell tumor suppressive functions include direct cytolytic killing of tumor cells as well as the regulation of subsequent antitumor adaptive immunity. Albeit tumors with gene expression signatures associated to the presence of cytotoxic lymphocyte infiltrates benefit from trastuzumab-based treatment, NK cell-related biomarkers of response/resistance to HER2-specific therapeutic antibodies in breast cancer patients remain elusive. Several variables, including (i the configuration of the patient NK cell repertoire; (ii tumor molecular features (i.e., estrogen receptor expression; (iii concomitant therapeutic regimens (i.e., chemotherapeutic agents, tyrosine kinase inhibitors; and (iv evasion mechanisms developed by progressive breast tumors, have been shown to quantitatively and qualitatively influence antibody-triggered NK cell responses. In this review, we discuss possible interventions for restoring/enhancing the therapeutic activity of HER2 therapeutic antibodies by harnessing NK cell antitumor potential through

  9. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels. (United States)

    Wilkinson, Trevor C I


    The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  10. Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS.

    Directory of Open Access Journals (Sweden)

    Sung Sun Yim

    Full Text Available Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS. First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6. These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.

  11. Clinical relevance of anti-exenatide antibodies: safety, efficacy and cross-reactivity with long-term treatment

    NARCIS (Netherlands)

    Fineman, M.S.; Mace, K.F.; Diamant, M.; Darsow, T.; Cirincione, B.B.; Porter, T.K.B.; Kinninger, L.A.; Trautmann, M.E.


    Aims: Antibody formation to therapeutic peptides is common. This analysis characterizes the time-course and cross-reactivity of anti-exenatide antibodies and potential effects on efficacy and safety. Methods: Data from intent-to-treat patients in 12 controlled (n = 2225,12-52weeks) and 5

  12. Microbials for the production of monoclonal antibodies and antibody fragments. (United States)

    Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph


    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.

  13. Fully human monoclonal antibodies from antibody secreting cells after vaccination with Pneumovax®23 are serotype specific and facilitate opsonophagocytosis. (United States)

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


    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.

  14. Radioimmunoassay with heterologous antibody (hetero-antibody RIA)

    International Nuclear Information System (INIS)

    Iwasawa, Atsushi; Hayashi, Hiroaki; Itoh, Zen; Wakabayashi, Katsumi


    To develop a homologous radioimmunoassay (RIA) for a hormone of a small or rare animal often meets difficulty in collecting a large amount of purified antigen required for antibody production. On the other hand, to employ a heterologous RIA to estimate the hormone often gives poor sensitivity. To overcome this difficulty, a 'hetero-antibody' RIA was studied. In a hetero-antibody RIA system, a purified preparation of a hormone is used for radioiodination and standardization and a heterologous antibody to the hormone is used for the first antibody. Canine motilin and rat LH were selected as examples, and anti-porcine motilin and anti-hCG, anti-hCGβ or anti-ovine LHβ was used as the heterologous antibody. The sensitivities of the hetero-antibody RIAs were much higher than those of heterologous RIAs in any case, showing that these hetero-antibody RIA systems were suitable for practical use. To clarify the principle of hetero-antibody RIA, antiserum to porcine motilin was fractionated on an affinity column where canine motilin was immobilized. The fraction bound had greater constants of affinity with both porcine and canine motilins than the rest of the antibody fractions. This fraction also reacted with a synthetic peptide corresponding to the C-terminal sequence common to porcine and canine motilins in a competitive binding test with labeled canine motilin. These results suggest that an antibody population having high affinity and cross-reactivity is present in polyclonal antiserum and indicate that the population can be used in hetero-antibody RIA at an appropriate concentration. (author)

  15. Monoclonal Antibodies Radiolabeling with Rhenium-188 for Radioimmunotherapy (United States)

    Martini, Petra; Pasquali, Micol


    Rhenium-188, obtained from an alumina-based tungsten-188/rhenium-188 generator, is actually considered a useful candidate for labeling biomolecules such as antibodies, antibody fragments, peptides, and DNAs for radiotherapy. There is a widespread interest in the availability of labeling procedures that allow obtaining 188Re-labeled radiopharmaceuticals for various therapeutic applications, in particular for the rhenium attachment to tumor-specific monoclonal antibodies (Mo)Abs for immunotherapy. Different approaches have been developed in order to obtain 188Re-radioimmunoconjugates in high radiochemical purity starting from the generator eluted [188Re]ReO4−. The aim of this paper is to provide a short overview on 188Re-labeled (Mo)Abs, focusing in particular on the radiolabeling methods, quality control of radioimmunoconjugates, and their in vitro stability for radioimmunotherapy (RIT), with particular reference to the most important contributions published in literature in this topic. PMID:28951872

  16. Rationalization and Design of the Complementarity Determining Region Sequences in an Antibody-Antigen Recognition Interface (United States)

    Chen, Ing-Chien; Lee, Yu-Ching; Chen, Jun-Bo; Tsai, Keng-Chang; Chen, Ching-Tai; Chang, Jeng-Yih; Yang, Ei-Wen; Hsu, Po-Chiang; Jian, Jhih-Wei; Hsu, Hung-Ju; Chang, Hung-Ju; Hsu, Wen-Lian; Huang, Kai-Fa; Ma, Alex Che; Yang, An-Suei


    Protein-protein interactions are critical determinants in biological systems. Engineered proteins binding to specific areas on protein surfaces could lead to therapeutics or diagnostics for treating diseases in humans. But designing epitope-specific protein-protein interactions with computational atomistic interaction free energy remains a difficult challenge. Here we show that, with the antibody-VEGF (vascular endothelial growth factor) interaction as a model system, the experimentally observed amino acid preferences in the antibody-antigen interface can be rationalized with 3-dimensional distributions of interacting atoms derived from the database of protein structures. Machine learning models established on the rationalization can be generalized to design amino acid preferences in antibody-antigen interfaces, for which the experimental validations are tractable with current high throughput synthetic antibody display technologies. Leave-one-out cross validation on the benchmark system yielded the accuracy, precision, recall (sensitivity) and specificity of the overall binary predictions to be 0.69, 0.45, 0.63, and 0.71 respectively, and the overall Matthews correlation coefficient of the 20 amino acid types in the 24 interface CDR positions was 0.312. The structure-based computational antibody design methodology was further tested with other antibodies binding to VEGF. The results indicate that the methodology could provide alternatives to the current antibody technologies based on animal immune systems in engineering therapeutic and diagnostic antibodies against predetermined antigen epitopes. PMID:22457753

  17. Tabhu: tools for antibody humanization

    DEFF Research Database (Denmark)

    Olimpieri, Pier Paolo; Marcatili, Paolo; Tramontano, Anna


    Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can...... elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity...... and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps...

  18. Cancer imaging with radiolabeled antibodies

    International Nuclear Information System (INIS)

    Goldenberg, D.M.


    This book presents a perspective of the use of antibodies to target diagnostic isotopes to tumors. Antibodies with reasonable specificity can be developed against almost any substance. If selective targeting to cancer cells can be achieved, the prospects for a selective therapy are equally intriguing. But the development of cancer detection, or imaging, with radiolabeled antibodies has depended upon advances in a number of different areas, including cancer immunology and immunochemistry for identifying suitable antigen targets and antibodies to these targets, tumor biology for model systems, radiochemistry for he attachment of radionuclides to antibodies, molecular biology for reengineering the antibodies for safer and more effective use in humans, and nuclear medicine for providing the best imaging protocols and instrumentation to detect minute amounts of elevated radioactivity against a background of considerable noise. Accordingly, this book has been organized to address the advances that are being made in many of these areas

  19. Catalytic immunoglobulin gene delivery in a mouse model of Alzheimer's disease: prophylactic and therapeutic applications. (United States)

    Kou, Jinghong; Yang, Junling; Lim, Jeong-Eun; Pattanayak, Abhinandan; Song, Min; Planque, Stephanie; Paul, Sudhir; Fukuchi, Ken-Ichiro


    Accumulation of amyloid beta-peptide (Aβ) in the brain is hypothesized to be a causal event leading to dementia in Alzheimer's disease (AD). Aβ vaccination removes Aβ deposits from the brain. Aβ immunotherapy, however, may cause T cell- and/or Fc-receptor-mediated brain inflammation and relocate parenchymal Aβ deposits to blood vessels leading to cerebral hemorrhages. Because catalytic antibodies do not form stable immune complexes and Aβ fragments produced by catalytic antibodies are less likely to form aggregates, Aβ-specific catalytic antibodies may have safer therapeutic profiles than reversibly-binding anti-Aβ antibodies. Additionally, catalytic antibodies may remove Aβ more efficiently than binding antibodies because a single catalytic antibody can hydrolyze thousands of Aβ molecules. We previously isolated Aβ-specific catalytic antibody, IgVL5D3, with strong Aβ-hydrolyzing activity. Here, we evaluated the prophylactic and therapeutic efficacy of brain-targeted IgVL5D3 gene delivery via recombinant adeno-associated virus serotype 9 (rAAV9) in an AD mouse model. One single injection of rAAV9-IgVL5D3 into the right ventricle of AD model mice yielded widespread, high expression of IgVL5D3 in the unilateral hemisphere. IgVL5D3 expression was readily detectable in the contralateral hemisphere but to a much lesser extent. IgVL5D3 expression was also confirmed in the cerebrospinal fluid. Prophylactic and therapeutic injection of rAAV9-IgVL5D3 reduced Aβ load in the ipsilateral hippocampus of AD model mice. No evidence of hemorrhages, increased vascular amyloid deposits, increased proinflammatory cytokines, or infiltrating T-cells in the brains was found in the experimental animals. AAV9-mediated anti-Aβ catalytic antibody brain delivery can be prophylactic and therapeutic options for AD.

  20. Clinical Manifestations of an Anti-Drug Antibody Response: Autoimmune Reactions. (United States)

    Swanson, Steven J


    Antibodies can be generated against a therapeutic protein upon administration to human subjects. When the therapeutic protein closely mimics one of the subject's endogenous proteins, those antibodies might bind to the endogenous protein in addition to the therapeutic protein. This scenario results when tolerance to the endogenous protein is broken. The consequences of breaking tolerance include an autoimmune response where antibodies are generated against the endogenous protein. These autoantibodies could have significant clinical relevance depending on several factors, including the redundancy of action of the endogenous protein as well as the concentration, binding affinity, and neutralizing potential of the antibodies. The consequences of a therapeutic-protein-induced autoimmune reaction can be challenging to manage as the stimulus for further perpetuation of the immune response can shift from the therapeutic protein to the endogenous protein. The potential for inducing an autoimmune response is one of the reasons that the immune response to a therapeutic protein should be monitored if it persists through the end of the study.

  1. Antibody drug conjugates - Trojan horses in the war on cancer. (United States)

    Iyer, U; Kadambi, V J


    Antibody drug conjugates (ADCs) consist of an antibody attached to a cytotoxic drug by means of a linker. ADCs provide a way to couple the specificity of a monoclonal antibody (mAb) to the cytotoxicity of a small-molecule drug and, therefore, are promising new therapies for cancer. ADCs are prodrugs that are inactive in circulation but exert their cytotoxicity upon binding to the target cancer cell. Earlier unsuccessful attempts to generate ADCs with therapeutic value have emphasized the important role each component plays in determining the efficacy and safety of the final ADC. Scientific advances in engineering antibodies for maximum efficacy as anticancer agents, identification of highly cytotoxic molecules, and generation of linkers with increased stability in circulation have all contributed to the development of the many ADCs that are currently in clinical trials. This review discusses parameters that guide the selection of the components of an ADC to increase its therapeutic window, provides a brief look at ADCs currently in clinical trials, and discusses future challenges in this field. Copyright © 2011. Published by Elsevier Inc.

  2. Tumor imaging with monoclonal antibodies

    International Nuclear Information System (INIS)

    Haisma, H.; Hilgers, J.


    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

  3. Human anti-luteinizing hormone-releasing hormone antibodies in patients treated with synthetic luteinizing hormone-releasing hormone

    International Nuclear Information System (INIS)

    Meakin, J.L.; Keogh, E.J.; Martin, C.E.


    One hundred sixty-three patients who were given synthetic LH-RH therapeutically underwent monitoring of serum IgG anti-LH-RH antibodies. Five of the patients showed specific binding to antibodies. Development of anti-LH-RH antibodies was not limited to those patients with a congenital deficiency of LH-RH. Urticarial responses occurred in four patients, only one of whom had IgG antibodies. Patients who had IgG antibodies or an urticarial response underwent monitoring of their serum IgE anti-LH-RH antibodies, but none had a positive binding response. The refractory state which has been reported in patients in whom similar antibodies to LH-RH develop was not invariably observed among these patients

  4. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries. (United States)

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard


    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

  5. Therapeutic HIV Peptide Vaccine

    DEFF Research Database (Denmark)

    Fomsgaard, Anders


    Therapeutic vaccines aim to control chronic HIV infection and eliminate the need for lifelong antiretroviral therapy (ART). Therapeutic HIV vaccine is being pursued as part of a functional cure for HIV/AIDS. We have outlined a basic protocol for inducing new T cell immunity during chronic HIV-1...... infection directed to subdominant conserved HIV-1 epitopes restricted to frequent HLA supertypes. The rationale for selecting HIV peptides and adjuvants are provided. Peptide subunit vaccines are regarded as safe due to the simplicity, quality, purity, and low toxicity. The caveat is reduced immunogenicity...

  6. Complement inhibition enables tumor delivery of LCMV glycoprotein pseudotyped viruses in the presence of antiviral antibodies

    Directory of Open Access Journals (Sweden)

    Laura Evgin


    Full Text Available The systemic delivery of therapeutic viruses, such as oncolytic viruses or vaccines, is limited by the generation of neutralizing antibodies. While pseudotyping of rhabdoviruses with the lymphocytic choriomeningitis virus glycoprotein has previously allowed for multiple rounds of delivery in mice, this strategy has not translated to other animal models. For the first time, we provide experimental evidence that antibodies generated against the lymphocytic choriomeningitis virus glycoprotein mediate robust complement-dependent viral neutralization via activation of the classical pathway. We show that this phenotype can be capitalized upon to deliver maraba virus pseudotyped with the lymphocytic choriomeningitis virus glycoprotein in a Fischer rat model in the face of neutralizing antibody through the use of complement modulators. This finding changes the understanding of the humoral immune response to arenaviruses, and also describes methodology to deliver viral vectors to their therapeutic sites of action without the interference of neutralizing antibody.

  7. Tubulin Inhibitor-Based Antibody-Drug Conjugates for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Hao Chen


    Full Text Available Antibody-drug conjugates (ADCs are a class of highly potent biopharmaceutical drugs generated by conjugating cytotoxic drugs with specific monoclonal antibodies through appropriate linkers. Specific antibodies used to guide potent warheads to tumor tissues can effectively reduce undesired side effects of the cytotoxic drugs. An in-depth understanding of antibodies, linkers, conjugation strategies, cytotoxic drugs, and their molecular targets has led to the successful development of several approved ADCs. These ADCs are powerful therapeutics for cancer treatment, enabling wider therapeutic windows, improved pharmacokinetic/pharmacodynamic properties, and enhanced efficacy. Since tubulin inhibitors are one of the most successful cytotoxic drugs in the ADC armamentarium, this review focuses on the progress in tubulin inhibitor-based ADCs, as well as lessons learned from the unsuccessful ADCs containing tubulin inhibitors. This review should be helpful to facilitate future development of new generations of tubulin inhibitor-based ADCs for cancer therapy.

  8. Targeted Delivery of siRNA Therapeutics to Malignant Tumors

    Directory of Open Access Journals (Sweden)

    Qixin Leng


    Full Text Available Over the past 20 years, a diverse group of ligands targeting surface biomarkers or receptors has been identified with several investigated to target siRNA to tumors. Many approaches to developing tumor-homing peptides, RNA and DNA aptamers, and single-chain variable fragment antibodies by using phage display, in vitro evolution, and recombinant antibody methods could not have been imagined by researchers in the 1980s. Despite these many scientific advances, there is no reason to expect that the ligand field will not continue to evolve. From development of ligands based on novel or existing biomarkers to linking ligands to drugs and gene and antisense delivery systems, several fields have coalesced to facilitate ligand-directed siRNA therapeutics. In this review, we discuss the major categories of ligand-targeted siRNA therapeutics for tumors, as well as the different strategies to identify new ligands.

  9. Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity. (United States)

    Prendergast, Jillian M; Galvao da Silva, Ana Paula; Eavarone, David A; Ghaderi, Darius; Zhang, Mai; Brady, Dane; Wicks, Joan; DeSander, Julie; Behrens, Jeff; Rueda, Bo R

    Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity.

  10. A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies. (United States)

    Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei


    Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Incomplete Antibodies May Reduce ABO Cross-Match Incompatibility: A Pilot Study

    Directory of Open Access Journals (Sweden)

    Mehmet Özen


    Full Text Available Objective: Any erythrocyte transfusion among humans having type A or B blood groups is impossible due to antibodies causing fatal transfusion complications. A cross-match test is performed to prevent immune transfusion complications before transfusion. Our hypothesis is that the fragment antibody (Fab part of the antibody (incomplete antibody may be used to prevent an immune stimulus related to the complete antibody. Therefore, we designed a pilot study to evaluate the effectiveness of these incomplete antibodies using cross-match tests. Materials and Methods: Pepsin enzyme and staphylococcal protein A columns were used to cut anti-A and anti-B monoclonal antibodies and purify their Fab (2 fragments, respectively. An Rh-positive erythrocyte suspension with purified anti-A Fab (2 solution and B Rh-positive erythrocyte suspension with purified anti-B Fab (2 solution were combined correspondingly. Cross-match tests were performed by tube and gel centrifugation methods. The agglutination levels due to the anti-A and anti-B Fab (2 antibodies and their effects on the agglutination normally observed with complete antibodies were then measured. Results: No agglutination for the purified incomplete anti-A Fab (2 with A Rh+ erythrocyte and anti-B Fab (2 with B Rh+ erythrocyte combinations was observed in the tube cross-match tests. These agglutination levels were 1+ in two wells in the gel centrifugation cross-match tests. Fab (2-treated erythrocytes were also resistant to the agglutination that normally occurs with complete antibodies. Conclusion: We determined that the Fab (2 fragments of antibodies may not only be used to obtain a mild or negative reaction when compared to complete antibodies, but they might also be used for decreasing ABO incompatibility. Incomplete antibodies might be a therapeutic option in autoimmune hemolytic anemia and they may also be used in solid organ or hematopoietic stem cell transplantation. Therefore, we have planned an

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

    Directory of Open Access Journals (Sweden)

    Felix Hart


    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.

  13. Immunity War: A Novel Therapy for Lymphoma Using T-cell Bispecific Antibodies. (United States)

    Prakash, Ajay; Diefenbach, Catherine S


    The activity of T cell mediated immunotherapies in B cell lymphoma has been limited to date. The novel bi-specific antibody CD20-TCB, has a 2:1 antibody design to maximize T cell engagement, and demonstrates activity in preclinical models. This may represent a novel therapeutic approach for patients with relapsed/refractory NHL. Copyright ©2018, American Association for Cancer Research.

  14. Antibody-mediated delivery of interleukin 4 to the neo-vasculature reduces chronic skin inflammation


    Hemmerle Teresa; Zgraggen Silvana; Matasci Mattia; Halin Cornelia; Detmar Michael; Neri Dario


    BACKGROUND: The antibody mediated delivery of cytokines (quot;immunocytokinesquot;) to sites of pathological angiogenesis represents an attractive strategy for the development of innovative biopharmaceuticals capable of modulating the activity of the immune system in cancer and in chronic inflammatory conditions. OBJECTIVE: Recombinant IL4 has previously been shown to be therapeutically active in patients with psoriasis. The antibody mediated delivery of this cytokine to sites of chronic skin...

  15. Understanding and modulating opalescence and viscosity in a monoclonal antibody formulation


    Salinas, Branden A; Sathish, Hasige A; Bishop, Steven M; Harn, Nick; Carpenter, John F; Randolph, Theodore W


    Opalescence and high viscosities can pose challenges for high concentration formulation of antibodies. Both phenomena result from protein-protein intermolecular interactions that can be modulated with solution ionic strength. We studied a therapeutic monoclonal antibody that exhibits high viscosity in solutions at low ionic strength (~20 centipoise (cP) at 90 mg/mL and 23°C) and significant opalescence at isotonic ionic strength (approximately 100 nephelometric turbidity units at 90 mg/mL and...

  16. Marketing therapeutic recreation services. (United States)

    Thorn, B E


    The use of marketing strategies can enhance the delivery of therapeutic recreation services. This article discusses how agencies can adapt marketing techniques and use them to identify potential markets, improve image, evaluate external pressures, and maximize internal strengths. Four variables that can be controlled and manipulated in a proposed marketing plan are product, price, place and promotion.

  17. Therapeutic applications of radiopharmaceuticals

    International Nuclear Information System (INIS)

    Baker, W.J.; Datz, F.L.; Beightol, R.W.


    Whether a radiopharmaceutical has diagnostic or therapeutic application depends on both the isotope and pharmaceutical used. For diagnostic applications, the isotope should undergo only γ-decay, since usually only γ-radiation is detected by nuclear medicine cameras. The half-life should be just long enough to allow the procedure to be performed. In contrast, the isotope needed for therapeutic purposes should have particulate radiation, such as a β-particle (electron), since these are locally absorbed an increase the local radiation dose. γ-Radiation, which penetrates the tissues, produces less radiation dose than do Β-particles. Several references dealing with radioactive decay, particulate interactions, and diagnostic and therapeutic applications of radiopharmaceuticals are available. Radiopharmaceuticals can legally be used only by physicians who are qualified by specific training in the safe handling of radionuclides. The experience and training of these physicians must be approved by the Nuclear Regulatory Commission or Agreement State Agency authorized to license the use of radiopharmaceuticals. A list of all byproduct material and procedures is available in the Code of Federal Regulations. Of the many radiopharmaceuticals available for diagnostic and therapeutic use, only those commonly used are discussed in this chapter

  18. Potential immunological markers for diagnosis and therapeutic assessment of toxocariasis

    Directory of Open Access Journals (Sweden)

    Guita Rubinsky-Elefant


    Full Text Available In human toxocariasis, there are few approaches using immunological markers for diagnosis and therapeutic assessment. An immunoblot (IB assay using excretory-secretory Toxocara canis antigen was standardized for monitoring IgG, IgE and IgA antibodies in 27 children with toxocariasis (23 visceral, three mixed visceral and ocular, and one ocular form for 22-116 months after chemotherapy. IB sensitivity was 100% for IgG antibodies to bands of molecular weight 29-38, 48-54, 95-116, 121-162, >205 kDa, 80.8% for IgE to 29-38, 48-54, 95-121, > 205 kDa, and 65.4% for IgA to 29-38, 48-54, 81-93 kDa. Candidates for diagnostic markers should be IgG antibodies to bands of low molecular weight (29-38 and 48-54 kDa. One group of patients presented the same antibody reactivity to all bands throughout the follow-up study; in the other group, antibodies decayed partially or completely to some or all bands, but these changes were not correlated with time after chemotherapy. Candidates for monitoring patients after chemotherapy may be IgG antibodies to > 205 kDa fractions, IgA to 29-38, 48-54, 81-93 kDa and IgE to 95-121 kDa. Further identification of antigen epitopes related to these markers will allow the development of sensitive and specific immunoassays for the diagnosis and therapeutic assessment of toxocariasis.

  19. Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye. (United States)

    Nath, Nidhi; Godat, Becky; Zimprich, Chad; Dwight, Stephen J; Corona, Cesear; McDougall, Mark; Urh, Marjeta


    Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be

  20. Red Blood Cell Antibody Identification (United States)

    ... antibodies may or may not be associated with adverse reactions, and identification of the specific type of RBC ... the only things that can cause a transfusion reaction. The recipient's immune ... or to drugs that the donor may have taken. Rarely, antibodies in the plasma ...

  1. Targeting of human glioma xenografts in vivo utilizing radiolabeled antibodies

    International Nuclear Information System (INIS)

    Williams, J.A.; Wessels, B.W.; Wharam, M.D.; Order, S.E.; Wanek, P.M.; Poggenburg, J.K.; Klein, J.L.


    Radiolabeled antibodies provide a potential basis for selective radiotherapy of human gliomas. We have measured tumor targeting by radiolabeled monoclonal and polyclonal antibodies directed against neuroectodermal and tumor-associated antigens in nude mice bearing human glioma xenografts. Monoclonal P96.5, a mouse IgG2a immunoglobulin, defines an epitope of a human melanoma cell surface protein, and specifically binds the U-251 human glioma as measured by immunoperoxidase histochemistry. 111In-radiolabeled P96.5 specifically targets the U-251 human glioma xenograft and yields 87.0 microCuries (microCi) of tumor activity per gram per 100 microCi injected activity compared to 4.5 microCi following administration of radiolabeled irrelevant monoclonal antibody. Calculations of targeting ratios demonstrate deposited dose to be 11.6 times greater with radiolabeled P96.5 administration compared to irrelevant monoclonal antibody. The proportion of tumor dose found in normal organs is less than 10%, further supporting specific targeting of the human glioma xenograft by this antibody. Monoclonal antibody ZME018, which defines a second melanoma-associated antigen, and polyclonal rabbit antiferritin, which defines a tumor-associated antigen, demonstrate positive immunoperoxidase staining of the tumor, but comparatively decreased targeting. When compared to the 111In-radiolabeled antibody, 90Y-radiolabeled P96.5 demonstrates comparable tumor targeting and percentages of tumor dose found in normal organs. To test the therapeutic potential of 90Y-radiolabeled P96.5, tumors and normal sites were implanted with miniature thermoluminescent dosimeters (TLD). Seven days following administration of 100 microCi 90Y-radiolabeled P96.5, average absorbed doses of 3770, 980, 353, and 274 cGy were observed in tumor, liver, contralateral control site, and total body, respectively

  2. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo


    The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides...... can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery....... To better understand the underlying mechanisms of antibody-antigen interaction here we present a pipeline developed by us to structurally classify immunoglobulin antigen binding sites and to infer key sequence residues and other variables that have a prominent role in each structural class....

  3. The European AntibotABE Framework Program and Its Update: Development of Innovative Botulinum Antibodies

    Directory of Open Access Journals (Sweden)

    Christine Rasetti-Escargueil


    Full Text Available The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and LC of the three BoNT serotypes were targeted to achieve a synergistic effect (oligoclonal antibodies. For antibody isolation, macaques were immunized with the recombinant and non-toxic BoNT/A, B or E, HC or LC, followed by the generation of immune phage-display libraries. Antibodies were selected from these libraries against the holotoxin and further analyzed in in vitro and ex vivo assays. For each library, the best ex vivo neutralizing antibody fragments were germline-humanized and expressed as immunoglobulin G (IgGs. The IgGs were tested in vivo, in a standardized model of protection, and challenged with toxins obtained from collections of Clostridium strains. Protective antibody combinations against BoNT/A and BoNT/B were evidenced and for BoNT/E, the anti-LC antibody alone was found highly protective. The combination of these five antibodies as an oligoclonal antibody cocktail can be clinically and regulatorily developed while their high “humanness” predicts a high tolerance in humans.

  4. Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences. (United States)

    Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F


    The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human Fab (fragment antigen binding) antibody libraries. In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final Fab products that are used for cloning.

  5. Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences. (United States)

    Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F


    The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human scFv (single chain antibody fragment) libraries using a short linker (GGSSRSS) or a long linker (GGSSRSSSSGGGGSGGGG). In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products that are used for cloning.

  6. Generation and analyses of human synthetic antibody libraries and their application for protein microarrays

    DEFF Research Database (Denmark)

    Säll, Anna; Walle, Maria; Wingren, Christer


    in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated. The specificities......Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments...... for diagnostics and therapeutics. In addition, this work provides a great example on how a synthetic approach can be used to optimize library designs. By having precise control of the diversity introduced into the antigen-binding sites, synthetic libraries offer increased understanding of how different diversity...

  7. Radiolabeled antibodies in cancer. Oncology Overview

    International Nuclear Information System (INIS)


    Oncology Overviews are a service of the International Cancer Research Data Bank (ICRDB) Program of the National Cancer Institute, intended to facilitate and promote the exchange of information between cancer scientists by keeping them aware of literature related to their research being published by other laboratories through the world. Each Oncology Overview represents a survey of the literature associated with a selected area of cancer research. It contains abstracts of articles which have been selected and organized by researchers associated with the field. Contents: Radiolabeled antibodies--labeling and imaging techniques; Radiolabeled antibodies--carcinoembryonic antigen; Radiolabeled antibodies--alpha-fetoprotein; Radiolabeled antibodies--human chorionic gonadotropin; Radiolabeled antibodies--ferritin; Radiolabeled antibodies--imaging of colorectal tumors; Radiolabeled antibodies--imaging of malignant melanoma; Radiolabeled antibodies--imaging of urogenital tumors; Radiolabeled antibodies--imaging of thyroid tumors; Radiolabeled antibodies--other clinical studies; Radiolabeled antibodies--selected preclinical studies; Radiolabeled antibodies--reviews

  8. New perspectives on recombinant human antibodies

    NARCIS (Netherlands)

    J. de Kruif (John); A.-R. van der Vuurst de Vries (Anne); L. Cilenti (L.); E. Boel (E.); W. van Ewijk (Willem); T. Logtenberg (Ton)


    textabstractThe limited potential of murine monoclonal antibodies for human immunotherapy has driven recent progress in recombinant antibody technology. Here, de Kruif and colleagues report on advances in the development and use of phage-antibody-display libraries.

  9. Measurement of antibodies to tubulin by radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Mead, G M; Cowin, P; Whitehouse, J M.A. [CRC Medical Oncology Unit, Southampton General Hospital, Southampton, U.K.


    A solid-phase double antibody radioimmunoassay capable of measuring antibody to tubulin, the principal component of microtubules, is described. This assay is simple, combining sensitivity with specificity and also allowing determination of antibody subclasses.

  10. Investigating interactions between phospholipase B-Like 2 and antibodies during Protein A chromatography. (United States)

    Tran, Benjamin; Grosskopf, Vanessa; Wang, Xiangdan; Yang, Jihong; Walker, Don; Yu, Christopher; McDonald, Paul


    Purification processes for therapeutic antibodies typically exploit multiple and orthogonal chromatography steps in order to remove impurities, such as host-cell proteins. While the majority of host-cell proteins are cleared through purification processes, individual host-cell proteins such as Phospholipase B-like 2 (PLBL2) are more challenging to remove and can persist into the final purification pool even after multiple chromatography steps. With packed-bed chromatography runs using host-cell protein ELISAs and mass spectrometry analysis, we demonstrated that different therapeutic antibodies interact to varying degrees with host-cell proteins in general, and PLBL2 specifically. We then used a high-throughput Protein A chromatography method to further examine the interaction between our antibodies and PLBL2. Our results showed that the co-elution of PLBL2 during Protein A chromatography is highly dependent on the individual antibody and PLBL2 concentration in the chromatographic load. Process parameters such as antibody resin load density and pre-elution wash conditions also influence the levels of PLBL2 in the Protein A eluate. Furthermore, using surface plasmon resonance, we demonstrated that there is a preference for PLBL2 to interact with IgG4 subclass antibodies compared to IgG1 antibodies. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Red blood cell antibodies in pregnancy and their clinical consequences: synergistic effects of multiple specificities. (United States)

    Nordvall, Maria; Dziegiel, Morten; Hegaard, Hanne Kristine; Bidstrup, Mogens; Jonsbo, Finn; Christensen, Birgit; Hedegaard, Morten


    The objective was to determine clinical consequences of various specificities for the infant/fetus. The population was patients referred between 1998 and 2005 to the tertiary center because of detected red blood cell (RBC) alloimmunization. Altogether 455 infants were delivered by 390 alloimmunized women. This was a retrospective cohort study. Data were obtained from the blood bank register and the obstetric and neonatal database. As indicators of hemolytic activity of the antibodies, the frequency of the therapeutic interventions intrauterine transfusion, exchange transfusion, and simple transfusion was used. Anti-D was the most common antibody (46.6%), followed by anti-K (15.4%). A combination of antibodies was detected in 27%. All three types of therapeutic intervention were significantly more frequent in women with anti-D plus an additional antibody than in women with anti-D as the sole antibody. The anti-D titer closely paralleled the clinical importance of the antibody. One case of anti-s with a titer of 512 required all three types of transfusion. Anti-D was the single most frequent and harmful specificity closely followed by anti-K. Combinations of antibody specificities were more harmful than single specificities, and a potentially synergistic effect should be considered.


    Directory of Open Access Journals (Sweden)

    C. M. C. Falcão


    Full Text Available Pythiosis, a disease caused by the oomycete Pythium insidiosum, often presents inefficient response to chemotherapy. It is a consensus that, in spite the several therapeutic protocols, a combination of surgery, chemotherapy and immunotherapy should be used. Surgical excision requires the removal of the entire affected area, with a wide margin of safety. The use of antifungal drugs has resulted in variable results, both in vitro and in vivo, and presents low therapeutic efficiency due to differences in the agent characteristics, which differ from true fungi. Immunotherapy is a non-invasive alternative for the treatment of pythiosis, which aims at modifying the immune response of the host, thereby producing an effective response to the agent. Photodynamic therapy has emerged as a promising technique, with good activity against P. insidiosum in vitro and in vivo. However, more studies are necessary to increase the efficiency of the current treatment protocols and consequently improve the cure rates. This paper aims to conduct a review covering the conventional and recent therapeutic methods against P. insidiosum infections

  13. OptMAVEn--a new framework for the de novo design of antibody variable region models targeting specific antigen epitopes.

    Directory of Open Access Journals (Sweden)

    Tong Li

    Full Text Available Antibody-based therapeutics provides novel and efficacious treatments for a number of diseases. Traditional experimental approaches for designing therapeutic antibodies rely on raising antibodies against a target antigen in an immunized animal or directed evolution of antibodies with low affinity for the desired antigen. However, these methods remain time consuming, cannot target a specific epitope and do not lead to broad design principles informing other studies. Computational design methods can overcome some of these limitations by using biophysics models to rationally select antibody parts that maximize affinity for a target antigen epitope. This has been addressed to some extend by OptCDR for the design of complementary determining regions. Here, we extend this earlier contribution by addressing the de novo design of a model of the entire antibody variable region against a given antigen epitope while safeguarding for immunogenicity (Optimal Method for Antibody Variable region Engineering, OptMAVEn. OptMAVEn simulates in silico the in vivo steps of antibody generation and evolution, and is capable of capturing the critical structural features responsible for affinity maturation of antibodies. In addition, a humanization procedure was developed and incorporated into OptMAVEn to minimize the potential immunogenicity of the designed antibody models. As case studies, OptMAVEn was applied to design models of neutralizing antibodies targeting influenza hemagglutinin and HIV gp120. For both HA and gp120, novel computational antibody models with numerous interactions with their target epitopes were generated. The observed rates of mutations and types of amino acid changes during in silico affinity maturation are consistent with what has been observed during in vivo affinity maturation. The results demonstrate that OptMAVEn can efficiently generate diverse computational antibody models with both optimized binding affinity to antigens and reduced

  14. Approaches to Preventative and Therapeutic HIV vaccines (United States)

    Gray, Glenda E.; Laher, Fatima; Lazarus, Erica; Ensoli, Barbara; Corey, Lawrence


    Novel strategies are being researched to discover vaccines to prevent and treat HIV-1. Nonefficacious preventative vaccine approaches include bivalent recombinant gp120 alone, HIV gene insertion into an Adenovirus 5 (Ad5) virus vector and the DNA prime/Ad5 boost vaccine regimen. However, the ALVAC-HIV prime/AIDSVAX® B/E gp120 boost regimen showed 31.2% efficacy at 3.5 years, and is being investigated as clade C constructs with an additional boost. Likewise, although multiple therapeutic vaccines have failed in the past, in a non-placebo controlled trial, a Tat vaccine demonstrated immune cell restoration, reduction of immune activation, and reduced HIV-1 DNA viral load. Monoclonal antibodies for passive immunization or treatment show promise, with VRC01 entering advanced clinical trials. PMID:26985884

  15. Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR. (United States)

    Suzuki, Takuo; Ishii-Watabe, Akiko; Tada, Minoru; Kobayashi, Tetsu; Kanayasu-Toyoda, Toshie; Kawanishi, Toru; Yamaguchi, Teruhide


    The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcgammaRI binding region of the Fc domain.

  16. Antibody Production in Plants and Green Algae. (United States)

    Yusibov, Vidadi; Kushnir, Natasha; Streatfield, Stephen J


    Monoclonal antibodies (mAbs) have a wide range of modern applications, including research, diagnostic, therapeutic, and industrial uses. Market demand for mAbs is high and continues to grow. Although mammalian systems, which currently dominate the biomanufacturing industry, produce effective and safe recombinant mAbs, they have a limited manufacturing capacity and high costs. Bacteria, yeast, and insect cell systems are highly scalable and cost effective but vary in their ability to produce appropriate posttranslationally modified mAbs. Plants and green algae are emerging as promising production platforms because of their time and cost efficiencies, scalability, lack of mammalian pathogens, and eukaryotic posttranslational protein modification machinery. So far, plant- and algae-derived mAbs have been produced predominantly as candidate therapeutics for infectious diseases and cancer. These candidates have been extensively evaluated in animal models, and some have shown efficacy in clinical trials. Here, we review ongoing efforts to advance the production of mAbs in plants and algae.

  17. Rhenium radioisotopes for therapeutic radiopharmaceutical development

    International Nuclear Information System (INIS)

    Knapp, F.F. Jr.; Beets, A.L.; Pinkert, J.; Kropp, J.; Lin, W.Y.; Wang, S.Y.


    Rhenium-186 and rhenium-188 represent two important radioisotopes which are of interest for a variety of therapeutic applications in oncology, nuclear medicine and interventional cardiology. Rhenium-186 is directly produced in a nuclear reactor and the 90 hour half-life allows distribution to distant sites. The relatively low specific activity of rhenium-186 produced in most reactors, however, permits use of phosphonates, but limits use for labelled peptides and antibodies. Rhenium-188 has a much shorter 16.9 hour half-life which makes distribution from direct reactor production difficult. However, rhenium-188 can be obtained carrier-free from a tungsten-188/rhenium-188 generator, which has a long useful shelf-life of several months which is cost-effective, especially for developing regions. In this paper we discuss the issues associated with the production of rhenium-186- and rhenium-188 and the development and use of various radiopharmaceuticals and devices labelled with these radioisotopes for bone pain palliation, endoradiotherapy of tumours by selective catheterization and tumour therapy using radiolabelled peptides and antibodies, radionuclide synovectomy and the new field of vascular radiation therapy. (author)

  18. Development of antibody-based c-Met inhibitors for targeted cancer therapy

    Directory of Open Access Journals (Sweden)

    Lee D


    Full Text Available Dongheon Lee, Eun-Sil Sung, Jin-Hyung Ahn, Sungwon An, Jiwon Huh, Weon-Kyoo You Hanwha Chemical R&D Center, Biologics Business Unit, Daejeon, Republic of Korea Abstract: Signaling pathways mediated by receptor tyrosine kinases (RTKs and their ligands play important roles in the development and progression of human cancers, which makes RTK-mediated signaling pathways promising therapeutic targets in the treatment of cancer. Compared with small-molecule compounds, antibody-based therapeutics can more specifically recognize and bind to ligands and RTKs. Several antibody inhibitors of RTK-mediated signaling pathways, such as human epidermal growth factor receptor 2, vascular endothelial growth factor, epidermal growth factor receptor or vascular endothelial growth factor receptor 2, have been developed and are widely used to treat cancer patients. However, since the therapeutic options are still limited in terms of therapeutic efficacy and types of cancers that can be treated, efforts are being made to identify and evaluate novel RTK-mediated signaling pathways as targets for more efficacious cancer treatment. The hepatocyte growth factor/c-Met signaling pathway has come into the spotlight as a promising target for development of potent cancer therapeutic agents. Multiple antibody-based therapeutics targeting hepatocyte growth factor or c-Met are currently in preclinical or clinical development. This review focuses on the development of inhibitors of the hepatocyte growth factor/c-Met signaling pathway for cancer treatment, including critical issues in clinical development and future perspectives for antibody-based therapeutics. Keywords: hepatocyte growth factor, ligands, receptor tyrosine kinase, signaling pathway, therapeutic agent

  19. Antibody guided irradiation of brain glioma by arterial infusion of radioactive monoclonal antibody against epidermal growth factor receptor and blood group A antigen

    Energy Technology Data Exchange (ETDEWEB)

    Epenetos, A.A.; Courtenay-Luck, N.; Pickering, D.; Hooker, G.; Lavender, J.P.; McKenzie, C.G. (Hammersmith Hospital, London (UK)); Durbin, H. (Imperial Cancer Research Fund, London (UK). Labs.)


    In a patient with recurrent grade IV glioma of the brain resistant to conventional treatment an antibody guided isotopic scan showed uptake by the tumour of a monoclonal antibody (9A) that was developed against epidermal growth factor receptor but cross reacted with blood group A antigen. As a therapeutic attempt antibody labelled with 1665 MBq (45.0 mCi) iodine-131 was delivered to the tumour area by infusion into the internal carotid artery. Computed tomography showed regression of the tumour after treatment, and an appreciable and sustained clinical improvement was noted without any toxicity. Delivery of irradiation guided by monoclonal antibody delivered by arterial infusion of the tumour area may be of clinical value in the treatment of brain gliomas resistant to conventional forms of treatment.

  20. Stroke and Therapeutic Hypothermia

    Directory of Open Access Journals (Sweden)

    Ozlem Ozkan Kuscu


    Full Text Available Stroke is significant cause of morbidity and mortality caused by disruption of blood flow. Neural injury occurs with two stage; while primary neural injury occurs with disruption of blood flow, after days and hours with metabolic processes secondary injury develops in tissues which is non injured in the first stage. Therefore it is important to prevent and treat the secondary injury as much as preventing and treating the primary neural injury. In this article developing pathophysiological changes after stroke, mechanisms of therapeutic hypothermia, application methods, the factors that determine the effectiveness, side effects and complications were reviewed. [Archives Medical Review Journal 2016; 25(3.000: 351-368

  1. The Effect of Induced Antibodies with Respect to Neutralization, Clearance Rate and Functional Activity in a Rabbit/Infliximab Model

    DEFF Research Database (Denmark)

    Henriksen, Maiken Lumby; Søgaard Teisner, Ane; Kjeldsen, Jens


    BACKGROUND: Therapeutic antibodies are a developing field for treatment of an expanding number of inflammatory diseases, including Crohn's disease. Treatment with monoclonal antibodies is frequently hampered by development of anti-drug antibodies (ADAs) that may compromise the treatment. MATERIALS...... AND METHODS: We addressed this issue in a rabbit model of treatment with the anti-tumor-necrosis factor alpha (TNFα) antibody, infliximab (IFX). We developed an inhibition ELISA to selectively measure absolute concentrations of neutralizing antibodies and another ELISA for measuring the concentration...... of functional IFX in the circulation. RESULTS: We found that the concentration of functional IFX was inversely proportional to the concentration of neutralizing antibodies. CONCLUSION: Administration of IFX to rabbits showed diversity in immune responses/tolerance toward IFX, corresponding to responses observed...

  2. Development of an EGFRvIII specific recombinant antibody

    Directory of Open Access Journals (Sweden)

    Li Gordon


    determined by enzyme-linked immunosorbent assay (ELISA. Conclusion This recombinant antibody thus holds great potential to be used as a research reagent and diagnostic tool in research laboratories and clinics because of its high quality, easy viability and unique versatility. This antibody is also a strong candidate to be investigated for further in vivo therapeutic studies.

  3. Monoclonal antibodies technology. Protocols

    International Nuclear Information System (INIS)

    Acevado Castro, B.E.


    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

  4. A novel Antibody based approach to Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Yoshikazu Kurosawa


    Full Text Available Cancer is one of the leading causes of death among the human race. No valid modalities of treatment other than surgical treatment have been established for this disease. We aimed to identify and to characterize cancer using large number of human monoclonal antibodies (mAbs which are specific against their surface for new molecular targeted immunotherapy. In order to find proper targets for therapeutic antibodies against cancers we developed a screening strategy. We used a huge phage library of human antibodies. At the first step we comprehensively isolated many monoclonal antibodies (mAbs that specifically bound to surface of cancer cells. Development of ICOS (Isolation of antigen/antibody complexes through organic solvent method allowed us to succeed in isolation of a huge number of mAbs with various characteristics (Y Akahori et al. 2009. At the next step we selected clones that showed tumor-specific staining patterns in immunohistochemical (IHC analysis by using many fresh cancer tissues reseted. Many surgeons took part in this project. Finally the antigens recognized by these clones were identified by immunoprecipitation (IP followed by analysis with mass (MS spectrometry (G Kurosawa et al. 2009. We have succeeded in identification of 29 tumor-associated antigens (TAAs and in isolation of 441 human mAbs that specifically bound to one of the 29 TAAs (G Kurosawa et al. 2008. In these screenings of the library, rounds of the selection process, mixing of cells and phage particles centrifugation growth of phages, were repeated three to four times in each screening. Therefore, numbers of phages of the clones whose antigens were abundantly present on the cell surface increased during the screenings. Recently we developed a new method for isolation of clones whose antigens were less abundantly present on the cell surface. Hence, we would like to talk on these methodology and discuss regarding this “A novel antibody based approach to Cancer

  5. Efficacy of Wnt-1 monoclonal antibody in sarcoma cells

    International Nuclear Information System (INIS)

    Mikami, Iwao; Koizumi, Kiyoshi; Jablons, David M; You, Liang; He, Biao; Xu, Zhidong; Batra, Sonny; Lee, Amie Y; Mazieres, Julien; Reguart, Noemi; Uematsu, Kazutsugu


    Sarcomas are one of the most refractory diseases among malignant tumors. More effective therapies based on an increased understanding of the molecular biology of sarcomas are needed as current forms of therapy remain inadequate. Recently, it has been reported that Wnt-1/β-catenin signaling inhibits apoptosis in several cancers. In this study, we investigated the efficacy of a monoclonal anti-Wnt-1 antibody in sarcoma cells. We treated cell lines A-204, SJSA-1, and fresh primary cultures of lung metastasis of sarcoma with a monoclonal anti-Wnt-1 antibody. Wnt-1 siRNA treatment was carried out in A-204. We assessed cell death using Crystal Violet staining. Apoptosis induction was estimated by flow cytometry analysis (Annexin V and PI staining). Cell signaling changes were determined by western blotting analysis. We detected Wnt-1 expression in all tissue samples and cell lines. Significant apoptosis induction was found in monoclonal anti-Wnt-1 antibody treated cells compared to control monoclonal antibody treated cells (p < 0.02). Similarly, we observed increased apoptosis in Wnt-1 siRNA treated cells. Blockade of Wnt-1 signaling in both experiments was confirmed by analyzing intracellular levels of Dishevelled-3 and of cytosolic β-catenin. Furthermore, the monoclonal anti-Wnt-1 antibody also induced cell death in fresh primary cultures of metastatic sarcoma in which Wnt-1 signaling was active. Our results indicate that Wnt-1 blockade by either monoclonal antibody or siRNA induces cell death in sarcoma cells. These data suggest that Wnt-1 may be a novel therapeutic target for the treatment of a subset of sarcoma cells in which Wnt-1/β-catenin signaling is active

  6. Female Infertility and Serum Auto-antibodies: a Systematic Review. (United States)

    Deroux, Alban; Dumestre-Perard, Chantal; Dunand-Faure, Camille; Bouillet, Laurence; Hoffmann, Pascale


    On average, 10 % of infertile couples have unexplained infertility. Auto-immune disease (systemic lupus erythematosus, anti-phospholipid syndrome) accounts for a part of these cases. In the last 20 years, aspecific auto-immunity, defined as positivity of auto-antibodies in blood sample without clinical or biological criteria for defined diseases, has been evoked in a subpopulation of infertile women. A systematic review was performed (PUBMED) using the MESH search terms "infertility" and "auto-immunity" or "reproductive technique" or "assisted reproduction" or "in vitro fertilization" and "auto-immunity." We retained clinical and physiopathological studies that were applicable to the clinician in assuming joint management of both infertility associated with serum auto-antibodies in women. Thyroid auto-immunity which affects thyroid function could be a cause of infertility; even in euthyroidia, the presence of anti-thyroperoxydase antibodies and/or thyroglobulin are related to infertility. The presence of anti-phospholipid (APL) and/or anti-nuclear (ANA) antibodies seems to be more frequent in the population of infertile women; serum auto-antibodies are associated with early ovarian failure, itself responsible for fertility disorders. However, there exist few publications on this topic. The methods of dosage, as well as the clinical criteria of unexplained infertility deserve to be standardized to allow a precise response to the question of the role of serum auto-antibodies in these women. The direct pathogenesis of this auto-immunity is unknown, but therapeutic immunomodulators, prescribed on a case-by-case basis, could favor pregnancy even in cases of unexplained primary or secondary infertility.

  7. BRCAA1 antibody- and Her2 antibody-conjugated amphiphilic polymer engineered CdSe/ZnS quantum dots for targeted imaging of gastric cancer (United States)

    Li, Chao; Ji, Yang; Wang, Can; Liang, Shujing; Pan, Fei; Zhang, Chunlei; Chen, Feng; Fu, Hualin; Wang, Kan; Cui, Daxiang


    Successful development of safe and highly effective nanoprobes for targeted imaging of in vivo early gastric cancer is a great challenge. Herein, we choose the CdSe/ZnS (core-shell) quantum dots (QDs) as prototypical materials, synthesized one kind of a new amphiphilic polymer including dentate-like alkyl chains and multiple carboxyl groups, and then used the prepared amphiphilic polymer to modify QDs. The resultant amphiphilic polymer engineered QDs (PQDs) were conjugated with BRCAA1 and Her2 monoclonal antibody, and prepared BRCAA1 antibody- and Her2 antibody-conjugated QDs were used for in vitro MGC803 cell labeling and in vivo targeted imaging of gastric cancer cells. Results showed that the PQDs exhibited good water solubility, strong photoluminescence (PL) intensity, and good biocompatibility. BRCAA1 antibody- and Her2 antibody-conjugated QD nanoprobes successfully realized targeted imaging of in vivo gastric cancer MGC803 cells. In conclusion, BRCAA1 antibody- and Her2 antibody-conjugated PQDs have great potential in applications such as single cell labeling and in vivo tracking, and targeted imaging and therapeutic effects' evaluation of in vivo early gastric cancer cells in the near future.

  8. Tabhu: tools for antibody humanization.

    KAUST Repository

    Olimpieri, Pier Paolo


    SUMMARY: Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity into a suitable human template. Unfortunately, this procedure may results in a partial or complete loss of affinity of the grafted molecule that can be restored by back-mutating some of the residues of human origin to the corresponding murine ones. This trial-and-error procedure is hard and involves expensive and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps of the humanization experiment protocol. AVAILABILITY: CONTACT:, SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

  9. Radiolabeled monoclonal antibodies: a review

    International Nuclear Information System (INIS)

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


    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

  10. Method of stably radiolabeling antibodies with technetium and rhenium

    International Nuclear Information System (INIS)

    Paik, C.H.; Reba, R.C.; Eckelman, W.C.


    A method is described for labeling antibodies or antibody fragments with radionuclides of technetium or rhenium to obtain stable labeling, comprising: reacting a reduced radioisotope of technetium or rhenium with an antibody or antibody fragment, or a diethylenetriaminepentaacetic acid conjugated antibody or antibody fragment, in the presence of free or carrier-bound diethylenetriaminepentaacetic acid (DTPA). The amount of DTPA is sufficient to substantially completely inhibit binding of the reduced technetium or rhenium to nonstable binding sites of the antibody or antibody fragment, or the DTPA-conjugated antibody or antibody fragment. The resultant stably labeled antibody or antibody fragment, or DTPA[conjugated antibody or antibody fragment is recovered

  11. Pharmacogenetics approach to therapeutics. (United States)

    Koo, Seok Hwee; Lee, Edmund Jon Deoon


    1. Pharmacogenetics refers to the study of genetically controlled variations in drug response. Functional variants caused by single nucleotide polymorphisms (SNPs) in genes encoding drug-metabolising enzymes, transporters, ion channels and drug receptors have been known to be associated with interindividual and interethnic variation in drug response. Genetic variations in these genes play a role in influencing the efficacy and toxicity of medications. 2. Rapid, precise and cost-effective high-throughput technological platforms are essential for performing large-scale mutational analysis of genetic markers involved in the aetiology of variable responses to drug therapy. 3. The application of a pharmacogenetics approach to therapeutics in general clinical practice is still far from being achieved today owing to various constraints, such as limited accessibility of technology, inadequate knowledge, ambiguity of the role of variants and ethical concerns. 4. Drug actions are determined by the interplay of several genes encoding different proteins involved in various biochemical pathways. With rapidly emerging SNP discovery technological platforms and widespread knowledge on the role of SNPs in disease susceptibility and variability in drug response, the pharmacogenetics approach to therapeutics is anticipated to take off in the not-too-distant future. This will present profound clinical, economic and social implications for health care.

  12. Therapeutic nuclear medicine

    International Nuclear Information System (INIS)

    Baum, Richard P.


    Discusses all aspects of radionuclide therapy, including basic principles, newly available treatments, regulatory requirements, and future trends. Provides the knowledge required to administer radionuclide therapy safely and effectively in the individual patient. Explains the role of the therapeutic nuclear physician in effectively coordinating a diverse multidisciplinary team. Written by leading experts. The recent revolution in molecular biology offers exciting new opportunities for targeted radionuclide therapy. The selective irradiation of tumor cells through molecular biological mechanisms is now permitting the radiopharmaceutical control of tumors that are unresectable and unresponsive to either chemotherapy or conventional radiotherapy. In this up-to-date, comprehensive book, world-renowned experts discuss the basic principles of radionuclide therapy, explore in detail the available treatments, explain the regulatory requirements, and examine likely future developments. The full range of clinical applications is considered, including thyroid cancer, hematological malignancies, brain tumors, liver cancer, bone and joint disease, and neuroendocrine tumors. The combination of theoretical background and practical information will provide the reader with all the knowledge required to administer radionuclide therapy safely and effectively in the individual patient. Careful attention is also paid to the important role of the therapeutic nuclear physician in delivering the effective coordination of a diverse multidisciplinary team that is essential to the safe provision of treatment.

  13. Mechanisms of Plasma Therapeutics (United States)

    Graves, David


    In this talk, I address research directed towards biomedical applications of atmospheric pressure plasma such as sterilization, surgery, wound healing and anti-cancer therapy. The field has seen remarkable growth in the last 3-5 years, but the mechanisms responsible for the biomedical effects have remained mysterious. It is known that plasmas readily create reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and RNS (or RONS), in addition to a suite of other radical and non-radical reactive species, are essential actors in an important sub-field of aerobic biology termed ``redox'' (or oxidation-reduction) biology. It is postulated that cold atmospheric plasma (CAP) can trigger a therapeutic shielding response in tissue in part by creating a time- and space-localized, burst-like form of oxy-nitrosative stress on near-surface exposed cells through the flux of plasma-generated RONS. RONS-exposed surface layers of cells communicate to the deeper levels of tissue via a form of the ``bystander effect,'' similar to responses to other forms of cell stress. In this proposed model of CAP therapeutics, the plasma stimulates a cellular survival mechanism through which aerobic organisms shield themselves from infection and other challenges.

  14. Therapeutic nuclear medicine

    Energy Technology Data Exchange (ETDEWEB)

    Baum, Richard P. (ed.) [ENETS Center of Excellence, Bad Berka (Germany). THERANOSTICS Center for Molecular Radiotherapy and Molecular Imaging


    Discusses all aspects of radionuclide therapy, including basic principles, newly available treatments, regulatory requirements, and future trends. Provides the knowledge required to administer radionuclide therapy safely and effectively in the individual patient. Explains the role of the therapeutic nuclear physician in effectively coordinating a diverse multidisciplinary team. Written by leading experts. The recent revolution in molecular biology offers exciting new opportunities for targeted radionuclide therapy. The selective irradiation of tumor cells through molecular biological mechanisms is now permitting the radiopharmaceutical control of tumors that are unresectable and unresponsive to either chemotherapy or conventional radiotherapy. In this up-to-date, comprehensive book, world-renowned experts discuss the basic principles of radionuclide therapy, explore in detail the available treatments, explain the regulatory requirements, and examine likely future developments. The full range of clinical applications is considered, including thyroid cancer, hematological malignancies, brain tumors, liver cancer, bone and joint disease, and neuroendocrine tumors. The combination of theoretical background and practical information will provide the reader with all the knowledge required to administer radionuclide therapy safely and effectively in the individual patient. Careful attention is also paid to the important role of the therapeutic nuclear physician in delivering the effective coordination of a diverse multidisciplinary team that is essential to the safe provision of treatment.

  15. Molecular imaging of rheumatoid arthritis by radiolabelled monoclonal antibodies: new imaging strategies to guide molecular therapies

    Energy Technology Data Exchange (ETDEWEB)

    Malviya, G.; Dierckx, R.A. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Conti, F. [Rheumatology Unit, I Faculty of Medicine and Surgery, Sapienza University of Rome (Italy); Chianelli, M. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Unit of Nuclear Medicine, Regina apostolorum Hospital, Albano, Rome (Italy); Scopinaro, F. [Nuclear Medicine Department, Sapienza University of Rome, St. Andrea Hospital, Rome (Italy); Signore, A. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Nuclear Medicine Department, Sapienza University of Rome, St. Andrea Hospital, Rome (Italy)


    The closing of the last century opened a wide variety of approaches for inflammation imaging and treatment of patients with rheumatoid arthritis (RA). The introduction of biological therapies for the management of RA started a revolution in the therapeutic armamentarium with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion molecules, activation markers, antigens or receptors, to interfere with specific inflammation pathways at the molecular level, leading to immune-modulation of the underlying pathogenic process. These new generation of mAbs can also be radiolabelled by using direct or indirect method, with a variety of nuclides, depending upon the specific diagnostic application. For studying rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-{alpha}, anti-CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody, have been radiolabelled mainly with {sup 99m}Tc or {sup 111}In. Scintigraphy with these radiolabelled antibodies may offer an exciting possibility for the study of RA patients and holds two types of information: (1) it allows better staging of the disease and diagnosis of the state of activity by early detection of inflamed joints that might be difficult to assess; (2) it might provide a possibility to perform 'evidence-based biological therapy' of arthritis with a view to assessing whether an antibody will localise in an inflamed joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection of patients to be treated since biological therapies can be associated with severe side-effects and are considerably expensive. This article reviews the use of radiolabelled mAbs in the study of RA with particular emphasis on the use of different radiolabelled monoclonal antibodies for

  16. Molecular imaging of rheumatoid arthritis by radiolabelled monoclonal antibodies: new imaging strategies to guide molecular therapies

    International Nuclear Information System (INIS)

    Malviya, G.; Dierckx, R.A.; Conti, F.; Chianelli, M.; Scopinaro, F.; Signore, A.


    The closing of the last century opened a wide variety of approaches for inflammation imaging and treatment of patients with rheumatoid arthritis (RA). The introduction of biological therapies for the management of RA started a revolution in the therapeutic armamentarium with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion molecules, activation markers, antigens or receptors, to interfere with specific inflammation pathways at the molecular level, leading to immune-modulation of the underlying pathogenic process. These new generation of mAbs can also be radiolabelled by using direct or indirect method, with a variety of nuclides, depending upon the specific diagnostic application. For studying rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-α, anti-CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody, have been radiolabelled mainly with 99m Tc or 111 In. Scintigraphy with these radiolabelled antibodies may offer an exciting possibility for the study of RA patients and holds two types of information: (1) it allows better staging of the disease and diagnosis of the state of activity by early detection of inflamed joints that might be difficult to assess; (2) it might provide a possibility to perform 'evidence-based biological therapy' of arthritis with a view to assessing whether an antibody will localise in an inflamed joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection of patients to be treated since biological therapies can be associated with severe side-effects and are considerably expensive. This article reviews the use of radiolabelled mAbs in the study of RA with particular emphasis on the use of different radiolabelled monoclonal antibodies for therapy decision-making and

  17. Long-term measurement of anti-adalimumab using pH-shift-anti-idiotype antigen binding test shows predictive value and transient antibody formation

    NARCIS (Netherlands)

    van Schouwenburg, Pauline A.; Krieckaert, Charlotte L.; Rispens, Theo; Aarden, Lucien; Wolbink, Gerrit Jan; Wouters, Diana


    Therapeutic monoclonal antibodies are effective drugs for many different diseases. However, the formation of anti-drug antibodies (ADA) against a biological can result in reduced clinical response in some patients. Measurement of ADA in the presence of (high) drug levels is difficult due to drug

  18. Ribosome display: next-generation display technologies for production of antibodies in vitro. (United States)

    He, Mingyue; Khan, Farid


    Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.

  19. Chemokine Receptor-Specific Antibodies in Cancer Immunotherapy: Achievements and Challenges (United States)

    Vela, Maria; Aris, Mariana; Llorente, Mercedes; Garcia-Sanz, Jose A.; Kremer, Leonor


    The 1990s brought a burst of information regarding the structure, expression pattern, and role in leukocyte migration and adhesion of chemokines and their receptors. At that time, the FDA approved the first therapeutic antibodies for cancer treatment. A few years later, it was reported that the chemokine receptors CXCR4 and CCR7 were involved on directing metastases to liver, lung, bone marrow, or lymph nodes, and the over-expression of CCR4, CCR6, and CCR9 by certain tumors. The possibility of inhibiting the interaction of chemokine receptors present on the surface of tumor cells with their ligands emerged as a new therapeutic approach. Therefore, many research groups and companies began to develop small molecule antagonists and specific antibodies, aiming to neutralize signaling from these receptors. Despite great expectations, so far, only one anti-chemokine receptor antibody has been approved for its clinical use, mogamulizumab, an anti-CCR4 antibody, granted in Japan to treat refractory adult T-cell leukemia and lymphoma. Here, we review the main achievements obtained with anti-chemokine receptor antibodies for cancer immunotherapy, including discovery and clinical studies, proposed mechanisms of action, and therapeutic applications. PMID:25688243

  20. IgY antibodies in human nutrition for disease prevention. (United States)

    Müller, Sandra; Schubert, Andreas; Zajac, Julia; Dyck, Terry; Oelkrug, Christopher


    Oral administration of preformed specific antibodies is an attractive approach against infections of the digestive system in humans and animals in times of increasing antibiotic resistances. Previous studies showed a positive effect of egg yolk IgY antibodies on bacterial intoxications in animals and humans. Immunization of chickens with specific antigens offers the possibility to create various forms of antibodies. Research shows that orally applied IgY's isolated from egg yolks can passively cure or prevent diseases of the digestive system. The use of these alternative therapeutic drugs provides further advantages: (1) The production of IgY's is a non-invasive alternative to current methods; (2) The keeping of chickens is inexpensive; (3) The animals are easy to handle; (4) It avoids repetitive bleeding of laboratory animals; (5) It is also very cost effective regarding the high IgY concentration within the egg yolk. Novel targets of these antigen specific antibodies are Helicobacter pylori and also molecules involved in signaling pathways in gastric cancer. Furthermore, also dental caries causing bacteria like Streptococcus mutans or opportunistic Pseudomonas aeruginosa in cystic fibrosis patients are possible targets. Therefore, IgY's included in food for human consumption may be able to prevent or cure human diseases.

  1. PCSK9 Antibodies for the Treatment of Hypercholesterolemia

    Directory of Open Access Journals (Sweden)

    Ioanna Gouni-Berthold


    Full Text Available The serine protease proprotein convertase subtilisin/kexin type 9 (PCSK9 binds to the low-density lipoprotein (LDL receptor (LDLR and directs it to lysosomes for intracellular degradation. This results in decreased numbers of LDLR available on the hepatic cell surface to bind LDL particles and remove them from the circulation and therefore to a subsequent increase in circulating LDL-cholesterol (LDL-C plasma levels. Since 2003, when the role of PCSK9 in LDL-C metabolism was discovered, there have been major efforts to develop efficient and safe methods to inhibit it. Amongst those, monoclonal antibodies against PCSK9 are the furthest in development, with multiple phase 3 trials already published and with cardiovascular endpoint trials currently underway. Two fully human monoclonal antibodies, evolocumab (AMG 145 and alirocumab (REGN727/SAR236553, have been extensively studied in a wide range of subjects, such as those with statin intolerance, as an add-on to statin therapy, as a monotherapy and in patients with familial hypercholesterolemia. PCSK9 antibodies result in a consistent and robust decrease in LDL-C plasma levels ranging from 40% to 70%, either on top of statins or as a monotherapy. If the safety data from the on-going phase 3 trials remain as reassuring as the data available till now, PCSK9 antibodies will offer a novel, powerful therapeutic option to decrease LDL-C plasma levels and, hopefully, cardiovascular risk.

  2. Antibody-Based Immunotoxins for the Treatment of Cancer

    Directory of Open Access Journals (Sweden)

    Nurit Becker


    Full Text Available Antibody-based immunotoxins comprise an important group in targeted cancer therapeutics. These chimeric proteins are a form of biological guided missiles that combine a targeting moiety with a potent effector molecule. The targeting moiety is mostly a monoclonal antibody (MAb or a recombinant antibody-based fragment that confers target specificity to the immunotoxin. The effector domain is a potent protein toxin of bacterial or plant origin, which, following binding to the target cells, undergoes internalization and causes cell death. Over time and following research progression, immunotoxins become better fitted to their purpose, losing immunogenic fragments and non-specific targeting moieties. Many immunotoxins have gone through clinical evaluation. Some of these have been shown to be active and work is progressing with them in the form of further clinical trials. Others, mostly developed in the previous century, failed to generate a response in patients, or even caused undesired side effects. This article reviews the antibody and protein-toxin based immunotoxins that were clinically evaluated up to the present day.

  3. Radioiodination of antibodies for tumor imaging

    International Nuclear Information System (INIS)

    Saha, G.B.


    In view of the great potential of radioiodinated antibody for the detection and treatment of cancer, the present article deals with the various techniques of radioiodination of antibody and their uses. Topics include methods of iodination of antibody, advantages and disadvantages of different methods, and effects of radioiodination on the antibody molecules with respect to their physiochemical and immunologic reactivity. In addition, the clinical usefulness of radioiodinated antibodies is discussed. (Auth.)

  4. Intravenous cidofovir for resistant cutaneous warts in a patient with psoriasis treated with monoclonal antibodies.

    LENUS (Irish Health Repository)

    McAleer, M A


    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.

  5. How does the recombinant human interferon beta induce antibodies in immune tolerant mice?

    NARCIS (Netherlands)

    Kijanka, G.M.


    Therapeutic proteins revolutionized the treatment of severe diseases like multiple sclerosis, diabetes, haemophilia and many more. Unfortunately, their usage is often limited due to the formation of anti drug antibodies (ADAs), which may block the activity of these protein drugs and may lead to

  6. A partner monoclonal antibody to Moab 730 kills 100% of DU145 ...

    Indian Academy of Sciences (India)

    A number of therapeutic options are available for patients with prostate carcinoma till the time that the tumour is hormone dependent. However, no fully effective therapy is available for the treatment of androgen-independent prostate carcinomas. Antibodies directed at epitopes unique to or overexpressed on the cancer cells ...

  7. Monoclonal antibodies in the treatment of metastatic colorectal cancer: a review

    NARCIS (Netherlands)

    Tol, Jolien; Punt, Cornelis J. A.


    Two groups of agents targeting either the vascular endothelial growth factor (VEGF) receptor or the epidermal growth factor receptor (EGFR) have been added to the therapeutic arsenal against metastatic colorectal cancer (mCRC). Currently available agents in these groups are the anti-VEGF antibody

  8. Antibodies from plants for bionanomaterials. (United States)

    Edgue, Gueven; Twyman, Richard M; Beiss, Veronique; Fischer, Rainer; Sack, Markus


    Antibodies are produced as part of the vertebrate adaptive immune response and are not naturally made by plants. However, antibody DNA sequences can be introduced into plants, and together with laboratory technologies that allow the design of antibodies recognizing any conceivable molecular structure, plants can be used as 'green factories' to produce any antibody at all. The advent of plant-based transient expression systems in particular allows the rapid, convenient, and safe production of antibodies, ranging from laboratory-scale expression to industrial-scale manufacturing. The key features of plant-based production include safety, speed, low cost, and convenience, allowing newcomers to rapidly master the technology and use it to its full advantage. Manufacturing in plants has recently achieved significant milestones and offers more than just an alternative to established microbial and mammalian cell platforms. The use of plants for product development in particular offers the power and flexibility to easily coexpress many different genes, allowing the plug-and-play construction of novel bionanomaterials, perfectly complementing existing approaches based on plant virus-like particles. As well as producing single antibodies for applications in medicine, agriculture, and industry, plants can be used to produce antibody-based supramolecular structures and scaffolds as a new generation of green bionanomaterials that promise a bright future based on clean and renewable nanotechnology applications. WIREs Nanomed Nanobiotechnol 2017, 9:e1462. doi: 10.1002/wnan.1462 For further resources related to this article, please visit the WIREs website. © 2017 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals, Inc.

  9. Monoclonal Antibodies for Non-Hodgkin's Lymphoma: State of the Art and Perspectives

    Directory of Open Access Journals (Sweden)

    Giulia Motta


    Full Text Available Monoclonal antibodies have been the most successful therapeutics ever brought to cancer treatment by immune technologies. The use of monoclonal antibodies in B-cell Non-Hodgkin's lymphomas (NHL represents the greatest example of these advances, as the introduction of the anti-CD20 antibody rituximab has had a dramatic impact on how we treat this group of diseases today. Despite this success, several questions about how to optimize the use of monoclonal antibodies in NHL remain open. The best administration schedules, as well as the optimal duration of rituximab treatment, have yet to be determined. A deeper knowledge of the mechanisms underlying resistance to rituximab is also necessary in order to improve the activity of this and of similar therapeutics. Finally, new antibodies and biological agents are entering the scene and their advantages over rituximab will have to be assessed. We will discuss these issues and present an overview of the most significant clinical studies with monoclonal antibodies for NHL treatment carried out to date.

  10. Antibodies to the extracellular pore loop of TRPM8 act as antagonists of channel activation.

    Directory of Open Access Journals (Sweden)

    Silke Miller

    Full Text Available The mammalian transient receptor potential melastatin channel 8 (TRPM8 is highly expressed in trigeminal and dorsal root ganglia. TRPM8 is activated by cold temperature or compounds that cause a cooling sensation, such as menthol or icilin. TRPM8 may play a role in cold hypersensitivity and hyperalgesia in various pain syndromes. Therefore, TRPM8 antagonists are pursued as therapeutics. In this study we explored the feasibility of blocking TRPM8 activation with antibodies. We report the functional characterization of a rabbit polyclonal antibody, ACC-049, directed against the third extracellular loop near the pore region of the human TRPM8 channel. ACC-049 acted as a full antagonist at recombinantly expressed human and rodent TRPM8 channels in cell based agonist-induced 45Ca2+ uptake assays. Further, several poly-and monoclonal antibodies that recognize the same region also blocked icilin activation of not only recombinantly expressed TRPM8, but also endogenous TRPM8 expressed in rat dorsal root ganglion neurons revealing the feasibility of generating monoclonal antibody antagonists. We conclude that antagonist antibodies are valuable tools to investigate TRPM8 function and may ultimately pave the way for development of therapeutic antibodies.

  11. Genetic engineering of cell lines using lentiviral vectors to achieve antibody secretion following encapsulated implantation. (United States)

    Lathuilière, Aurélien; Bohrmann, Bernd; Kopetzki, Erhard; Schweitzer, Christoph; Jacobsen, Helmut; Moniatte, Marc; Aebischer, Patrick; Schneider, Bernard L


    The controlled delivery of antibodies by immunoisolated bioimplants containing genetically engineered cells is an attractive and safe approach for chronic treatments. To reach therapeutic antibody levels there is a need to generate renewable cell lines, which can long-term survive in macroencapsulation devices while maintaining high antibody specific productivity. Here we have developed a dual lentiviral vector strategy for the genetic engineering of cell lines compatible with macroencapsulation, using separate vectors encoding IgG light and heavy chains. We show that IgG expression level can be maximized as a function of vector dose and transgene ratio. This approach allows for the generation of stable populations of IgG-expressing C2C12 mouse myoblasts, and for the subsequent isolation of clones stably secreting high IgG levels. Moreover, we demonstrate that cell transduction using this lentiviral system leads to the production of a functional glycosylated antibody by myogenic cells. Subsequent implantation of antibody-secreting cells in a high-capacity macroencapsulation device enables continuous delivery of recombinant antibodies in the mouse subcutaneous tissue, leading to substantial levels of therapeutic IgG detectable in the plasma.

  12. Therapeutic and diagnostic nanomaterials

    CERN Document Server

    Devasena T


    This brief highlights nanoparticles used in the diagnosis and treatment of prominent diseases and toxic conditions. Ecofriendly methods which are ideal for the synthesis of medicinally valued nanoparticles are explained and the characteristic features of these particles projected. The role of these particles in the therapeutic field, and the induced biological changes in some diseases are discussed. The main focus is on inflammation, oxidative stress and cellular membrane integrity alterations. The effect of nanoparticles on these changes produced by various agents are highlighted using in vitro and in vivo models. The mechanism of nanoparticles in ameliorating the biological changes is supported by relevant images and data. Finally, the brief demonstrates recent developments on the use of nanoparticles in diagnosis or sensing of some biological materials and biologically hazardous environmental materials.

  13. [Therapeutic education didactic techniques]. (United States)

    Valverde, Maite; Vidal, Mercè; Jansa, Margarida


    This article includes an introduction to the role of Therapeutic Education for Diabetes treatment according to the recommendations of the American Diabetes Association (ADA), the Diabetes Education Study Group (DESG) of the "European Association for Study of Diabetes (EASD) and the clinical Practice Guidelines (CPG) of the Spanish Ministry of Health. We analyze theoretical models and the differences between teaching vs. learning as well as current trends (including Internet), that can facilitate meaningful learning of people with diabetes and their families and relatives. We analyze the differences, similarities, advantages and disadvantages of individual and group education. Finally, we describe different educational techniques (metaplan, case method, brainstorming, role playing, games, seminars, autobiography, forums, chats,..) applicable to individual, group or virtual education and its application depending on the learning objective.

  14. Dashboard systems: Pharmacokinetic/pharmacodynamic mediated dose optimization for monoclonal antibodies. (United States)

    Mould, Diane R; Dubinsky, Marla C


    Many marketed drugs exhibit high variability in exposure and response. While these drugs are efficacious in their approved indications, finding appropriate dose regimens for individual patients is not straightforward. Similar dose adjustment problems are also seen with drugs that have a complex relationship between exposure and response and/or a narrow therapeutic window. This is particularly true for monoclonal antibodies, where prolonged dosing at a sub-therapeutic dose can also elicit anti-drug antibodies which will further compromise safety and efficacy. Thus, finding appropriate doses quickly would represent a substantial improvement in healthcare. Dashboard systems, which are decision-support tools, offer an improved, convenient means of tailoring treatment for individual patients. This article reviews the clinical need for this approach, particularly with monoclonal antibodies, the design, development, and testing of such systems, and the likely benefits of dashboard systems in clinical practice. We focus on infliximab for reference. © 2015, The American College of Clinical Pharmacology.

  15. Human anti-rhesus D IgG1 antibody produced in transgenic plants

    DEFF Research Database (Denmark)

    Bouquin, Thomas; Thomsen, Mads; Nielsen, Leif Kofoed


    antigen, which is responsible for alloimmunization of RhD- mothers carrying an RhD+ fetus. Anti-RhD extracted from plants specifically reacted with RhD+ cells in antiglobulin technique, and elicited a respiratory burst in human peripheral blood mononuclear cells. Plant-derived antibody had equivalent......Transgenic plants represent an alternative to cell culture systems for producing cheap and safe antibodies for diagnostic and therapeutic use. To evaluate the functional properties of a 'plantibody', we generated transgenic Arabidopsis plants expressing full-length human IgG1 against the Rhesus D...... properties to CHO cell-produced anti-RhD antibody, indicating its potential usefulness in diagnostic and therapeutic programs....

  16. Identification and evaluation of nextgeneration PTM-specific antibodies

    DEFF Research Database (Denmark)

    Persson, Nina Emilia

    -chain fragment variable (scFv)clones. Two different analyses are performed on the same microarray. There is no need for anypurification or enrichment before screening. In the first analysis, the ability of the individualscFv clone to bind to the soluble form of the antigens is evaluated. Favouring selection....... Including antibodies target several different categories of antigens suchas proteins, glycoproteins and glycolipids. Glycoproteins have become highlighted in cancerresearch since they are frequently involved in the initiation and spreading of cancer. One form ofglycosylation of proteins is the O...... for the patients. Severalmonoclonal antibodies have been generated against the Tn- and STn-antigens, but none has yetreached approval for therapeutic or diagnostic use. Indicating the need for a new generation ofantibodies against this type aberrant glycosylation.The two major techniques used for the production...

  17. Biomimetic Nanosponges for Treating Antibody-Mediated Autoimmune Diseases. (United States)

    Jiang, Yao; Fang, Ronnie H; Zhang, Liangfang


    Autoimmune diseases are characterized by overactive immunity, where the body's defense system launches an attack against itself. If left unchecked, this can result in the destruction of healthy tissue and significantly affect patient well-being. In the case of type II autoimmune hypersensitivities, autoreactive antibodies attack the host's own cells or extracellular matrix. Current clinical treatment modalities for managing this class of disease are generally nonspecific and face considerable limitations. In this Topical Review, we cover emerging therapeutic strategies, with an emphasis on novel nanomedicine platforms. Specifically, the use of biomimetic cell membrane-coated nanosponges that are capable of specifically binding and neutralizing pathological antibodies will be explored. There is significant untapped potential in the application of nanotechnology for the treatment of autoimmune diseases, and continued development along this line may help to eventually change the clinical landscape.

  18. Influence of antidrug antibodies on plectasin efficacy and pharmacokinetics

    DEFF Research Database (Denmark)

    Brinch, Karoline Sidelmann; Frimodt-Møller, Niels; Hoiby, Niels


    Plectasin is a 4.4-kDa antimicrobial peptide with the potential to be a treatment of infections caused by gram-positive bacteria. Since plectasin is a large molecule compared to conventional antibiotics, the development of antidrug antibodies (ADAs) could be anticipated. The immunogenic properties....... Additionally, mice were immunized with plectasin in Freund's incomplete adjuvant (FIA). Ninety-two percent of these mice developed ADAs after repeated immunizations, with two-thirds having high levels of antibodies. An agar diffusion bioassay showed that sera from animals immunized with plectasin did...... for the treatment of Streptococcus pneumoniae infections both in naïve animals and in animals with ADAs. No difference in bacterial counts was seen when the animals were treated with plectasin at 2.5 mg/kg of body weight, a dose below the expected therapeutic level. When animals were treated with plectasin at 0...

  19. Designing proteins for therapeutic applications. (United States)

    Lazar, Greg A; Marshall, Shannon A; Plecs, Joseph J; Mayo, Stephen L; Desjarlais, John R


    Protein design is becoming an increasingly useful tool for optimizing protein drugs and creating novel biotherapeutics. Recent progress includes the engineering of monoclonal antibodies, cytokines, enzymes and viral fusion inhibitors.

  20. Intraperitoneal delivery of monoclonal antibodies: enhanced regional delivery advantage using intravenous unlabeled anti-mouse antibody

    International Nuclear Information System (INIS)

    Wahl, R.L.; Fisher, S.


    Radiolabeled monoclonal antibodies (MAb) delivered intraperitoneally expose cells in contact with peritoneal fluid to considerably higher levels of MAb than if the MAb dose were given intravenously. This regional delivery advantage for intact MAb is present mainly due to the relatively slow exit of MAb from the peritoneal fluid to the blood. Eventually, following i.p. injection, blood levels of MAb rise resulting in exposure of the animal to high systemic MAb levels and potential toxicity. In this series of experiments, systemic exposure was minimized by the administration of unlabeled goat polyclonal anti-mouse antibody intravenously from 1 1/2 to 6 h following i.p. MAb injection. This maneuver results in the formation of immune complexes with their subsequent clearance and dehalogenation by the reticuloendothelial system, thus minimizing systemic MAb exposure. This approach, of increasing systemic clearance of MAb, did not alter intraperitoneal MAb levels and thus significantly increased the regional delivery advantage to the peritoneal cavity by 70-100%. This approach provides an immunologic rationale for the further enhancement of MAb delivery to i.p. foci of malignant disease and may have diagnostic and therapeutic utility. (author)

  1. Enhanced antibody-dependent cellular phagocytosis by chimeric monoclonal antibodies with tandemly repeated Fc domains. (United States)

    Nagashima, Hiroaki; Ootsubo, Michiko; Fukazawa, Mizuki; Motoi, Sotaro; Konakahara, Shu; Masuho, Yasuhiko


    We previously reported that chimeric monoclonal antibodies (mAbs) with tandemly repeated Fc domains, which were developed by introducing tandem repeats of Fc domains downstream of 2 Fab domains, augmented binding avidities for all Fcγ receptors, resulting in enhanced antibody (Ab)-dependent cellular cytotoxicity. Here we investigated regarding Ab-dependent cellular phagocytosis (ADCP) mediated by these chimeric mAbs, which is considered one of the most important mechanisms that kills tumor cells, using two-color flow cytometric methods. ADCP mediated by T3-Ab, a chimeric mAb with 3 tandemly repeated Fc domains, was 5 times more potent than that by native anti-CD20 M-Ab (M-Ab hereafter). Furthermore, T3-Ab-mediated ADCP was resistant to competitive inhibition by intravenous Ig (IVIG), although M-Ab-mediated ADCP decreased in the presence of IVIG. An Fcγ receptor-blocking study demonstrated that T3-Ab mediated ADCP via both FcγRIA and FcγRIIA, whereas M-Ab mediated ADCP exclusively via FcγRIA. These results suggest that chimeric mAbs with tandemly repeated Fc domains enhance ADCP as well as ADCC, and that Fc multimerization may significantly enhance the efficacy of therapeutic Abs. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. Monoclonal antibody hapten radiopharmaceutical delivery

    International Nuclear Information System (INIS)

    Goodwin, D.A.; McTigue, M.


    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)

  3. Coagulation factor VII variants resistant to inhibitory antibodies. (United States)

    Branchini, Alessio; Baroni, Marcello; Pfeiffer, Caroline; Batorova, Angelika; Giansily-Blaizot, Muriel; Schved, Jean F; Mariani, Guglielmo; Bernardi, Francesco; Pinotti, Mirko


    Replacement therapy is currently used to prevent and treat bleeding episodes in coagulation factor deficiencies. However, structural differences between the endogenous and therapeutic proteins might increase the risk for immune complications. This study was aimed at identifying factor (F)VII variants resistant to inhibitory antibodies developed after treatment with recombinant activated factor VII (rFVIIa) in a FVII-deficient patient homozygous for the p.A354V-p.P464Hfs mutation, which predicts trace levels of an elongated FVII variant in plasma. We performed fluorescent bead-based binding, ELISA-based competition as well as fluorogenic functional (activated FX and thrombin generation) assays in plasma and with recombinant proteins. We found that antibodies displayed higher affinity for the active than for the zymogen FVII (half-maximal binding at 0.54 ± 0.04 and 0.78 ± 0.07 BU/ml, respectively), and inhibited the coagulation initiation phase with a second-order kinetics. Isotypic analysis showed a polyclonal response with a large predominance of IgG1. We hypothesised that structural differences in the carboxyl-terminus between the inherited FVII and the therapeutic molecules contributed to the immune response. Intriguingly, a naturally-occurring, poorly secreted and 5-residue truncated FVII (FVII-462X) escaped inhibition. Among a series of truncated rFVII molecules, we identified a well-secreted and catalytically competent variant (rFVII-464X) with reduced binding to antibodies (half-maximal binding at 0.198 ± 0.003 BU/ml) as compared to the rFVII-wt (0.032 ± 0.002 BU/ml), which led to a 40-time reduced inhibition in activated FX generation assays. Taken together our results provide a paradigmatic example of mutation-related inhibitory antibodies, strongly support the FVII carboxyl-terminus as their main target and identify inhibitor-resistant FVII variants.

  4. Quality control of radiolabeled antibodies through simultaneous determination of antibody concentration and specific activity using time-resolved interaction analysis and reverse kinetic fit

    International Nuclear Information System (INIS)

    Andersson, K.; Mihaylova, D.; Wang, E.; Abrahamsen, L.; Buijs, J.; Bjoerkelund, H.


    Full text of publication follows. With the advent of efficient methods for producing proteins that bind to a defined target, the number of radiolabeled proteins, and in particular antibodies, used for medical imaging and cancer therapy is increasing rapidly. In line with this increase, focus should be put on methods for the quality control (QC). Proper antibody quality is of fundamental importance to guarantee safety and consistent efficacy for the patient. Adequate QC procedures exist for small radiolabeled synthetic compounds like FDG, but antibody based radiopharmaceuticals are different. Proteins are much more complex and fragile than the synthetic compounds, and hence require new methods for adequate characterization and QC. Yet another complication is the labeling where there is a risk that a subpopulation of the protein is damaged to the level that it no longer binds the target. Therefore, a new toolbox is required to fulfill the quality characterization of radiolabeled antibodies. We have developed a QC assay for the simultaneous determination of antibody function, concentration and specific activity. The assay is based on time-resolved detection of the antibody interaction with antigen-coated magnetic beads in LigandTracer instruments. The resulting binding curve is evaluated using reverse kinetic fits, where the known interaction parameters of the antibody-antigen interaction are set constant while as the concentration and signal level are fitted. The assay takes approximately 2 hours and the majority of the time constitutes automated data collection in the instrument. The QC assay has been tested on multiple antibody-antigen interactions and consistently provides repeatable results for concentration and specific activity, both with coefficient of variation (CV) less than 15%. We believe that this QC assay can improve the quality of radiolabeled therapeutic antibodies. (authors)

  5. Bioanalysis of antibody-drug conjugates: American Association of Pharmaceutical Scientists Antibody-Drug Conjugate Working Group position paper. (United States)

    Gorovits, Boris; Alley, Stephen C; Bilic, Sanela; Booth, Brian; Kaur, Surinder; Oldfield, Phillip; Purushothama, Shobha; Rao, Chetana; Shord, Stacy; Siguenza, Patricia


    Antibody-drug conjugates (ADCs) typically consist of a cytotoxic drug covalently bound to an antibody by a linker. These conjugates have the potential to substantially improve efficacy and reduce toxicity compared with cytotoxic small-molecule drugs. Since ADCs are generally complex heterogeneous mixtures of multiple species, these novel therapeutic products present unique bioanalytical challenges. The growing number of ADCs being developed across the industry suggests the need for alignment of the bioanalytical methods or approaches used to assess the multiple species and facilitate consistent interpretation of the bioanalytical data. With limited clinical data, the current strategies that can be used to provide insight into the relationship between the multiple species and the observed clinical safety and efficacy are still evolving. Considerations of the bioanalytical strategies for ADCs based on the current industry practices that take into account the complexity and heterogeneity of ADCs are discussed.

  6. Immunological mechanism underlying the immune response to tecombinant human protein therapeutics

    NARCIS (Netherlands)

    Sauerborn, M.S.; Brinks, V.; Jiskoot, W.; Schellekens, H.


    Recombinant human (rhu) protein therapeutics are powerful tools to treat several severe diseases such as multiple sclerosis and diabetes mellitus, among others. A major drawback of these proteins is the production of anti-drug antibodies (ADAs). In some cases, these ADAs have neutralizing capacity

  7. Exogenous insulin antibody syndrome (EIAS: a clinical syndrome associated with insulin antibodies induced by exogenous insulin in diabetic patients

    Directory of Open Access Journals (Sweden)

    Xiaolei Hu


    Full Text Available Insulin has been used for diabetes therapy and has achieved significant therapeutic effect. In recent years, the use of purified and recombinant human insulin preparations has markedly reduced, but not completely suppressed, the incidence of insulin antibodies (IAs. IAs induced by exogenous insulin in diabetic patients is associated with clinical events, which is named exogenous insulin antibody syndrome (EIAS. The present review is based on our research and summarizes the characterization of IAs, the factors affecting IA development, the clinical significance of IAs and the treatments for EIAS.

  8. Emerging Technologies and Generic Assays for the Detection of Anti-Drug Antibodies

    Directory of Open Access Journals (Sweden)

    Michael A. Partridge


    Full Text Available Anti-drug antibodies induced by biologic therapeutics often impact drug pharmacokinetics, pharmacodynamics response, clinical efficacy, and patient safety. It is critical to assess the immunogenicity risk of potential biotherapeutics in producing neutralizing and nonneutralizing anti-drug antibodies, especially in clinical phases of drug development. Different assay methodologies have been used to detect all anti-drug antibodies, including ELISA, radioimmunoassay, surface plasmon resonance, and electrochemiluminescence-based technologies. The most commonly used method is a bridging assay, performed in an ELISA or on the Meso Scale Discovery platform. In this report, we aim to review the emerging new assay technologies that can complement or address challenges associated with the bridging assay format in screening and confirmation of ADAs. We also summarize generic anti-drug antibody assays that do not require drug-specific reagents for nonclinical studies. These generic assays significantly reduce assay development efforts and, therefore, shorten the assay readiness timeline.


    Directory of Open Access Journals (Sweden)

    E. N. Gorshkova


    Full Text Available Almost 20 years ago, a unique class of antibodies devoid of L chains was discovered in Camelidae blood serum. Only one variable domain is responsible for antigen recognition in these unusual antibodies. A recombinant protein, which is analogue to such antigen-recognizing variable domain was called the single domain antibody (sdAb, “nanobody” or “nanoantibody”. The single-domain antibodies and their derivatives have been widely used in the field of biology, toxicology and medicine offering new opportunities for diagnosis and treatment of cancer, autoimmune diseases, infectious diseases, and for toxin neutralization. This review focuses on latest researches in the field and concerns some prospectives for creation of nanoantibody-based diagnostic and therapeutic drugs.

  10. Criteria for the use of monoclonal antibodies, legislation and ethical considerations

    International Nuclear Information System (INIS)

    Cox, P.H.


    The criteria governing the in vivo use of monoclonal antibodies in humans are based upon a number of legal requirements with respect to radiation hygiene, pharmaceutical legislation, radiopharmaceutical legislation and regulations with respect to products arising from biotechnology. This in itself has led to a complicated situation which has undoubtedly restricted the development of valuable diagnostic and potential therapeutic agents. From the ethical point of view there are also important considerations, firstly with respect to the methods of producing antibodies, which has resulted in the discontinuation of the raising of antibodies in murine ascites, and secondly in consideration of the ethics of administering labelled antibodies to healthy volunteers and to patients who may not necessarily benefit personally from the procedure. These factors must be evaluated in the light of the EEC document 'Good clinical practice for trials in medicinal products in the European Community from the CPMP working party on Efficacy of Medicinal Products'. (author)

  11. DNA-templated antibody conjugation for targeted drug delivery to cancer cells

    DEFF Research Database (Denmark)

    Liu, Tianqiang


    -templated organic synthesis due to the wide existence of the 3-histidine cluster in most wild-type proteins. In this thesis, three projects that relate to targeted drug delivery to cancer cells based on the DTPC method is described. The first project was a delivery system which uses transferrin as the targeting....... The study shows that DNA is a highly useful tool for the assembly of proteins with potential therapeutic applications. The DNA-templated protein conjugation shows a promising application in constructing antibody-toxin conjugates or antibody-drug conjugates. In addition, DNA strands used for antibody...... either antibody engineering or special expression systems and are both time and labor consuming. To avoid the drawbacks caused by conventional chemical modification and recombinant methodologies, an ideal site specific conjugation technique would use natural amino acid residues to the protein by a new...

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

    Directory of Open Access Journals (Sweden)

    Michał Skowicki


    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.

  13. Uses of monoclonal antibody 8H9 (United States)

    Cheung, Nai-Kong V.


    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.

  14. [Nuclear transfer and therapeutic cloning]. (United States)

    Xu, Xiao-Ming; Lei, An-Min; Hua, Jin-Lian; Dou, Zhong-Ying


    Nuclear transfer and therapeutic cloning have widespread and attractive prospects in animal agriculture and biomedical applications. We reviewed that the quality of oocytes and nuclear reprogramming of somatic donor cells were the main reasons of the common abnormalities in cloned animals and the low efficiency of cloning and showed the problems and outlets in therapeutic cloning, such as some basic problems in nuclear transfer affected clinical applications of therapeutic cloning. Study on isolation and culture of nuclear transfer embryonic stem (ntES) cells and specific differentiation of ntES cells into important functional cells should be emphasized and could enhance the efficiency. Adult stem cells could help to cure some great diseases, but could not replace therapeutic cloning. Ethics also impeded the development of therapeutic cloning. It is necessary to improve many techniques and reinforce the research of some basic theories, then somatic nuclear transfer and therapeutic cloning may apply to agriculture reproduction and benefit to human life better.

  15. Refolding Technologies for Antibody Fragments

    Directory of Open Access Journals (Sweden)

    Tsutomu Arakawa


    Full Text Available Refolding is one of the production technologies for pharmaceutical grade antibody fragments. Detergents and denaturants are primarily used to solubilize the insoluble proteins. The solubilized and denatured proteins are refolded by reducing the concentration of the denaturants or detergents. Several refolding technologies have been used for antibody fragments, comprising dilution, dialysis, solid phase solvent exchange and size exclusion chromatography, as reviewed here. Aggregation suppressor or folding-assisting agents, including arginine hydrochloride, ionic liquids and detergents or denaturants at low concentrations, are included in the refolding solvent to enhance refolding yield.

  16. Serum Antibody Biomarkers for ASD (United States)


    typically developing control. US, unaffected sibling control. 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a...typically developing (TD) children (e.g., Warren et al., 1990; Singh, 2009). The goal of this study is to identify a serum antibody biomarker for ASD using...50% less IgG1 antibody in ASD boys vs . TD boys (p=0.0096). The level of ASD1 binding to the AM group was the same as to the ASD boys. These data

  17. Monoclonal antibody-based immunoassays. (United States)

    Appleby, P; Reischl, U


    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.

  18. Antibody profiling sensitivity through increased reporter antibody layering (United States)

    Apel, William A; Thompson, Vicki S


    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  19. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.


    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  20. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.


    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  1. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S


    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  2. Molecularly targeted therapeutic radiopharmaceuticals

    International Nuclear Information System (INIS)

    Saw, M.M.


    Full text: It is generally agreed that current focus of nuclear medicine development should be on molecular imaging and therapy. Though, the widespread use of the terminology 'molecular imaging' is quite recent, nuclear medicine has used molecular imaging techniques for more than 20 years ago. A variety of radiopharmaceuticals have been introduced for the internal therapy of malignant and inflammatory lesions in nuclear medicine. In the field of bio/medical imaging, nuclear medicine is one of the disciplines which has the privilege of organized and well developed chemistry/ pharmacy section; radio-chemistry/radiopharmacy. Fundamental principles have been developed more than 40 years ago and advanced research is going well into postgenomic era. The genomic revolution and dramatically increased insight in the molecular mechanisms underlying pathology have led to paradigm shift in drug development. Likewise does in the nuclear medicine. Here, the author will present current clinical and pre-clinical therapeutic radiopharmaceuticals based on molecular targets such as membrane-bound receptors, enzymes, nucleic acids, sodium iodide symporter, etc, in correlation with fundamentals of radiopharmacy. (author)

  3. Rethinking Therapeutic Misconception in Biobanking

    DEFF Research Database (Denmark)

    Tupasela, Aaro; Snell, Karoliina; Cañada, Jose


    Some authors have noted that in biobank research participants may be guided by what is called therapeutic misconception, whereby participants attribute therapeutic intent to research procedures.This article argues that the notion of therapeutic misconception is increasingly less justified when...... underpinnings for the need to separate research and treatment, and thus the notion of therapeutic misconception in the fi rst place. We call this tension between research and treatment ambivalent research advancement to highlight the difficulties that various actors have in managing such shifts within...

  4. Therapeutic cloning: The ethical limits

    International Nuclear Information System (INIS)

    Whittaker, Peter A.


    A brief outline of stem cells, stem cell therapy and therapeutic cloning is given. The position of therapeutic cloning with regard to other embryonic manipulations - IVF-based reproduction, embryonic stem formation from IVF embryos and reproductive cloning - is indicated. The main ethically challenging stages in therapeutic cloning are considered to be the nuclear transfer process including the source of eggs for this and the destruction of an embryo to provide stem cells for therapeutic use. The extremely polarised nature of the debate regarding the status of an early human embryo is noted, and some potential alternative strategies for preparing immunocompatible pluripotent stem cells are indicated

  5. Therapeutic cloning in the mouse (United States)

    Mombaerts, Peter


    Nuclear transfer technology can be applied to produce autologous differentiated cells for therapeutic purposes, a concept termed therapeutic cloning. Countless articles have been published on the ethics and politics of human therapeutic cloning, reflecting the high expectations from this new opportunity for rejuvenation of the aging or diseased body. Yet the research literature on therapeutic cloning, strictly speaking, is comprised of only four articles, all in the mouse. The efficiency of derivation of embryonic stem cell lines via nuclear transfer is remarkably consistent among these reports. However, the efficiency is so low that, in its present form, the concept is unlikely to become widespread in clinical practice. PMID:12949262

  6. Origin, diversity and maturation of human antiviral antibodies analyzed by high-throughput sequencing

    Directory of Open Access Journals (Sweden)

    Ponraj ePrabakaran


    Full Text Available Our understanding of how antibodies are generated and function could help develop effective vaccines and antibody-based therapeutics against viruses such as HIV-1, SARS Coronavirus (CoV, and Hendra and Nipah viruses (henipaviruses. Although broadly neutralizing antibodies (bnAbs against the HIV-1 were observed in patients, elicitation of such bnAbs remains a major challenge when compared to other viral targets. We previously hypothesized that HIV-1 could have evolved a strategy to evade the immune system due to absent or very weak binding of germline antibodies to the conserved epitopes that may not be sufficient to initiate and/or maintain an effective immune response. To further explore our hypothesis, we used the 454 sequence analysis of a large naïve library of human IgM antibodies which had been used for selecting antibodies against SARS Coronavirus (CoV receptor-binding domain (RBD, and soluble G proteins (sG of Hendra and Nipah viruses (henipaviruses. We found that the human IgM repertoires from the 454 sequencing have diverse germline usages, recombination patterns, junction diversity and a lower extent of somatic mutation. In this study, we identified germline intermediates of antibodies specific to HIV-1 and other viruses as observed in normal individuals, and compared their genetic diversity and somatic mutation level along with available structural and functional data. Further computational analysis will provide framework for understanding the underlying genetic and molecular determinants related to maturation pathways of antiviral bnAbs that could be useful for applying novel approaches to the design of effective vaccine immunogens and antibody-based therapeutics.

  7. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms. (United States)

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


    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.

  8. A high-yielding, generic fed-batch process for recombinant antibody production of GS-engineered cell lines

    DEFF Research Database (Denmark)

    Fan, Li; Zhao, Liang; Sun, Yating


    An animal component-free and chemically defined fed-batch process for GS-engineered cell lines producing recombinant antibodies has been developed. The fed-batch process relied on supplying sufficient nutrients to match their consumption, simultaneously minimizing the accumulation of byproducts....... This generic and high-yielding fed-batch process would shorten development time, and ensure process stability, thereby facilitating the manufacture of therapeutic antibodies by GS-engineered cell lines....

  9. A robust robotic high-throughput antibody purification platform. (United States)

    Schmidt, Peter M; Abdo, Michael; Butcher, Rebecca E; Yap, Min-Yin; Scotney, Pierre D; Ramunno, Melanie L; Martin-Roussety, Genevieve; Owczarek, Catherine; Hardy, Matthew P; Chen, Chao-Guang; Fabri, Louis J


    Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Tumor detection using radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

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


    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

  11. Bispecific antibodies targeting human CD73

    DEFF Research Database (Denmark)


    The present invention relates to a bispecific antibody targeting CD73. In particular, the present invention relates to a bispecific antibody targeting different epitopes on CD73 or a bispecific antibody targeting an epitope on CD73 and an epitope on a different antigen.......The present invention relates to a bispecific antibody targeting CD73. In particular, the present invention relates to a bispecific antibody targeting different epitopes on CD73 or a bispecific antibody targeting an epitope on CD73 and an epitope on a different antigen....

  12. NMR Detection of Semi-Specific Antibody Interactions in Serum Environments

    Directory of Open Access Journals (Sweden)

    Saeko Yanaka


    Full Text Available Although antibody functions are executed in heterogeneous blood streams characterized by molecular crowding and promiscuous intermolecular interaction, detailed structural characterizations of antibody interactions have thus far been performed under homogeneous in vitro conditions. NMR spectroscopy potentially has the ability to study protein structures in heterogeneous environments, assuming that the target protein can be labeled with NMR-active isotopes. Based on our successful development of isotope labeling of antibody glycoproteins, here we apply NMR spectroscopy to characterize antibody interactions in heterogeneous extracellular environments using mouse IgG-Fc as a test molecule. In human serum, many of the HSQC peaks originating from the Fc backbone exhibited attenuation in intensity of various magnitudes. Similar spectral changes were induced by the Fab fragment of polyclonal IgG isolated from the serum, but not by serum albumin, indicating that a subset of antibodies reactive with mouse IgG-Fc exists in human serum without preimmunization. The metaepitopes recognized by serum polyclonal IgG cover the entire molecular surface of Fc, including the binding sites to Fc receptors and C1q. In-serum NMR observation will offer useful tools for the detailed characterization of biopharamaceuticals, including therapeutic antibodies in physiologically relevant heterogeneous environments, also giving deeper insight into molecular recognition by polyclonal antibodies in the immune system.

  13. Antibodies and antiestrogens combined with boron for use in the neutron capture therapy

    International Nuclear Information System (INIS)

    Abraham, R.


    The ZR-75-1 cell line developed from a mammary carcinoma was chosen to characterise the binding of antiestrogen U23.469-M to the cell, which was subsequently compared to that of a derivative combined with boron. It was found that the original U23.469-M showed antiestrogenic activity, while this effect was largely abolished after the substance had been modified using b-decachloro-o-carborane. In this study, boron-conjugated antibodies were produced in order to find out whether those modified immunoglobulins would be suitable to bind sufficient quantities of boron to the tumour cells. It was calculated by experts on radiation biology that a minimum of 1000 boron atoms is required for a tumour-specific antibody to be therapeutically effective. When oxidated dextran of a molecular weight of 33 kD was used as a linking molecule, a reproducible method could be developed that permitted more than 1000 boron atoms to be bound per antibody. In one of the monoclonal antibodies tested here a combination with boron could, however, only be achieved at the expense of complete inactivation. A model was developed allowing to significantly increase the number modified antibodies attached to any one tumour cell. The cell binding experiments and radioimmunoassays then carried out were able to show that the number of antibodies bound to tumour cells can be increased to different degrees, depending on the monoclonal antibody used in each case. (orig./MG) [de

  14. Antibody Fragments as Potential Biopharmaceuticals for Cancer Therapy: Success and Limitations. (United States)

    Kholodenko, Roman V; Kalinovsky, Daniel V; Doronin, Igor I; Ponomarev, Eugene D; Kholodenko, Irina V


    Monoclonal antibodies (mAbs) are an important class of therapeutic agents approved for the therapy of many types of malignancies. However, in certain cases applications of conventional mAbs have several limitations in anticancer immunotherapy. These limitations include insufficient efficacy and adverse effects. The antigen-binding fragments of antibodies have a considerable potential to overcome the disadvantages of conventional mAbs, such as poor penetration into solid tumors and Fc-mediated bystander activation of the immune system. Fragments of antibodies retain antigen specificity and part of functional properties of conventional mAbs and at the same time have much better penetration into the tumors and a greatly reduced level of adverse effects. Recent advantages in antibody engineering allowed to produce different types of antibody fragments with improved structure and properties for efficient elimination of tumor cells. These molecules opened up new perspectives for anticancer therapy. Here we will overview the structural features of the various types of antibody fragments and their applications for anticancer therapy as separate molecules and as part of complex conjugates or structures. Mechanisms of antitumor action of antibody fragments as well as their advantages and disadvantages for clinical application will be discussed in this review. Copyright© Bentham Science Publishers; For any queries, please email at

  15. Non-HLA antibodies post-transplantation: clinical relevance and treatment in solid organ transplantation. (United States)

    Dragun, Duska; Hegner, Bjorn


    Antibodies and B cells are increasingly recognized as major modulators of allograft function and survival. Improved immunohistochemical and serologic diagnostic procedures have been developed to monitor antibody responses against HLA antigens during the last decade. Acute and chronic allograft rejection can occur in HLA-identical sibling transplants implicating the importance of immune response against non-HLA targets. Non-HLA anti-bodies may occur as alloantiboides, yet they seem to be predominantly autoantibodies. Antigenic targets of non-HLA antibodies described thus far include various minor histocompatibility antigens, vascular receptors, adhesion molecules, and intermediate filaments. Non-HLA antibodies may function as complement- and non-complement-fixing antibodies and they may induce a wide variety of allograft injuries, reflecting the complexity of their acute and chronic actions. Refined approaches considering the subtle mechanistic differences in the individual antibody responses directed against non-HLA antigens may help to define patients at particular risk for irreversible acute or chronic allograft injuries and improve over-all outcomes. We attempted to summarize the current state of research, development in diagnostic and therapeutic strategies, and to address some emerging problems in the area of humoral response against non-HLA antigens beyond ABO blood group and MHC class I chain-related gene A and B (MICA and MICB) antigens in solid organ transplantation. Copyright (c) 2009 S. Karger AG, Basel.

  16. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    Directory of Open Access Journals (Sweden)

    Tal Noy-Porat


    Full Text Available Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1 that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  17. Generation and analyses of human synthetic antibody libraries and their application for protein microarrays. (United States)

    Säll, Anna; Walle, Maria; Wingren, Christer; Müller, Susanne; Nyman, Tomas; Vala, Andrea; Ohlin, Mats; Borrebaeck, Carl A K; Persson, Helena


    Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated. The specificities of these antibodies were analyzed in a variety of immunochemical assays and a subset was further evaluated for functionality in protein microarray applications. This high-throughput approach demonstrates the ability to rapidly generate a wealth of reagents not only for proteome research, but potentially also for diagnostics and therapeutics. In addition, this work provides a great example on how a synthetic approach can be used to optimize library designs. By having precise control of the diversity introduced into the antigen-binding sites, synthetic libraries offer increased understanding of how different diversity contributes to antibody binding reactivity and stability, thereby providing the key to future library optimization. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail:

  18. Antibody response to the extracellular adherence protein (Eap) of Staphylococcus aureus in healthy and infected individuals. (United States)

    Joost, Insa; Jacob, Susanne; Utermöhlen, Olaf; Schubert, Uwe; Patti, Joseph M; Ong, Mei-Fang; Gross, Jürgen; Justinger, Christoph; Renno, Jörg H; Preissner, Klaus T; Bischoff, Markus; Herrmann, Mathias


    The extracellular adherence protein (Eap) from Staphylococcus aureus has been suggested as a vaccine candidate and for therapeutic use due to its immunomodulating and antiangiogenic properties; however, little is known about anti-Eap antibodies in humans. We determined anti-Eap antibody titers by enzyme-linked immunosorbent assay and Western blot and measured serum samples from 92 patients with proven S. aureus infections and 93 healthy controls. The functionality of antibodies was assessed by a phagocytosis assay using Eap-coated fluorescent microspheres. Antibodies were detected in all human samples, but not in mice. Patients showed significantly higher titers than controls [immunoglobulin M (IgM), P=0.007; IgG, PEap alone was sufficient to promote phagocytosis by peripheral blood mononuclear cell and granulocytes that was moderately enhanced in the presence of human serum, but no correlation was found with the levels of anti-Eap antibodies. Anti-Eap antibodies are prevalent in all tested humans and correlate with the severity of S. aureus infection; however, they do not seem to provide protection against invasive infections. Before considering Eap for therapy or as a vaccine candidate, further studies are warranted to assess the impact of the interference between Eap and its specific antibodies. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  19. Purinergic Signalling: Therapeutic Developments

    Directory of Open Access Journals (Sweden)

    Geoffrey Burnstock


    Full Text Available Purinergic signalling, i.e., the role of nucleotides as extracellular signalling molecules, was proposed in 1972. However, this concept was not well accepted until the early 1990’s when receptor subtypes for purines and pyrimidines were cloned and characterised, which includes four subtypes of the P1 (adenosine receptor, seven subtypes of P2X ion channel receptors and 8 subtypes of the P2Y G protein-coupled receptor. Early studies were largely concerned with the physiology, pharmacology and biochemistry of purinergic signalling. More recently, the focus has been on the pathophysiology and therapeutic potential. There was early recognition of the use of P1 receptor agonists for the treatment of supraventricular tachycardia and A2A receptor antagonists are promising for the treatment of Parkinson’s disease. Clopidogrel, a P2Y12 antagonist, is widely used for the treatment of thrombosis and stroke, blocking P2Y12 receptor-mediated platelet aggregation. Diquafosol, a long acting P2Y2 receptor agonist, is being used for the treatment of dry eye. P2X3 receptor antagonists have been developed that are orally bioavailable and stable in vivo and are currently in clinical trials for the treatment of chronic cough, bladder incontinence, visceral pain and hypertension. Antagonists to P2X7 receptors are being investigated for the treatment of inflammatory disorders, including neurodegenerative diseases. Other investigations are in progress for the use of purinergic agents for the treatment of osteoporosis, myocardial infarction, irritable bowel syndrome, epilepsy, atherosclerosis, depression, autism, diabetes, and cancer.

  20. Dental therapeutic systems. (United States)

    Iqbal, Zeenat; Jain, Nilu; Jain, Gaurav K; Talegaonkar, Sushama; Ahuja, Alka; Khar, Roop K; Ahmad, Farhan J


    The recognition of periodontal diseases as amenable to local antibiotherapy has resulted in a paradigmatic shift in treatment modalities of dental afflictions. Moreover the presence of antimicrobial resistance, surfacing of untoward reactions owing to systemic consumption of antibiotics has further advocated the use of local delivery of physiologically active substances into the periodontal pocket. While antimicrobials polymerized into acrylic strips, incorporated into biodegradable collagen and hollow permeable cellulose acetate fibers, multiparticulate systems, bio-absorbable dental materials, biodegradable gels/ointments, injectables, mucoadhesive microcapsules and nanospheres will be more amenable for direct placement into the periodontal pockets the lozenges, buccoadhesive tablets, discs or gels could be effectively used to mitigate the overall gingival inflammation. Whilst effecting controlled local delivery of a few milligram of an antibacterial agent within the gingival crevicular fluid for a longer period of time, maintaining therapeutic concentrations such delivery devices will circumvent all adverse effects to non- oral sites. Since the pioneering efforts of Goodson and Lindhe in 1989, delivery at gingival and subgingival sites has witnessed a considerable progress. The interest in locally active systems is evident from the patents being filed and granted. The present article shall dwell in reviewing the recent approaches being proffered in the field. Patents as by Shefer, et al. US patent, 6589562 dealing with multicomponent biodegradable bioadhesive controlled release system for oral care products, Lee, et al. 2001, US patent 6193994, encompassing a locally administrable, biodegradable and sustained-release pharmaceutical composition for periodontitis and process for preparation thereof and method of treating periodontal disease as suggested by Basara in 2004via US patent 6830757, shall be the types of intellectual property reviewed and presented in

  1. Purification of immunoreactive radiolabeled moniclonal antibodies with anti-iodiotypic moniclonal antibodies

    International Nuclear Information System (INIS)

    Temponi, M.; Pupa, S.; Ferrone, S.


    A method is described to purify immunoreactive moniclonal antibodies from radiolabeled monoclonal antibody preparations. The method is based on incubation of radiolabeled monoclonal antibodies with insolubilized anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of monoclonal antibodies to be purified an elution of bound monoclonal antibodies with a low pH buffer. The immunoreactive fraction of the purified monoclonal antibodies was at least 82%; the yeald was at least 73%. The purification procedure did not cause any detectable change in the affinity constant of the eluted monoclonal antibodies. The method is simple and rapid; the requirement for anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of the antibodies to be purified is not likely to represent a major limitation in the broad application of the present method, since the hybridoma technology has greatly facilitated the development of anti-idiotypic monoclonal antibodies. (author). 12 refs.; 4 figs.; 1 tab

  2. Therapeutical aspect of trichomoniasis

    Directory of Open Access Journals (Sweden)

    Vukićević Jelica


    Full Text Available Trichomoniasis is frequent, parasitic and sexually transmitted infection of genitourinary tract. It is treated by metronidazole (5-nitroimidazole according to protocol recommended by Center for Disease Control (CDC formerly called: Communicable Disease Center [19]. The resistance of Trichomonas vaginalis (TV strains to metronidazole (MND was described in USA in 1960, and later on in many European countries [8, 9, 10, 11, 12, 13]. In these cases, due to persistent trichomonas infection, it is necessary to repeat MND treatment with moderate modification of dose and/or length of its application. Nevertheless, oncogenic and toxic effects of MND have to be taken into consideration. OBJECT The aim of this study was to investigate and analyze the incidence of TV in STD and lower susceptibility of certain TV strains to MND were analyzed. MATERIAL AND METHODS In three-year period (1999-2001 612 patients (244 females and 368 males suspected of STD were examined clinically and microbiologically at the Institute of Dermatovenereology in Belgrade. The patients detected for TV were treated according to CDC protocol. The affected were considered cured if there was no manifest clinical infection, and no TV verified by microbiological test. Results TV was isolated in 216 patients (35.29 % of all subjects. Trichomonas infection was found in 90 (36.88 % out of 244 tested females and in 126 (32.34 % of 368 males. Clinically manifested infection, with extensive urethral and vaginal secretion, was recorded in 161 patients, while the asymptomatic form was found in 55 subjects. This result indicates the predominance of manifested trichomonas infections (75.54 % of cases. The difference of distribution of clinical forms of trichomoniasis, in relation to sex, was not statistically significant (c2=0.854; p>0.05. The patients with verified trichomonas infection were treated by metronidazole according to CDC protocol. The recommended therapeutical scheme consisted of three

  3. Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor-α

    Directory of Open Access Journals (Sweden)

    Ceran Ceyhan


    , but antagonistically on BT-474 cells. A representative anti-HER2 antibody inhibited Akt and ERK1/2 phosphorylation leading to cyclin D1 accumulation and growth arrest in SK-BR-3 cells, independently from TNF-α. Conclusions Novel antibodies against extracellular domain of HER2 may serve as potent anti-cancer bioactive molecules. Cell-dependent synergy and antagonism between anti-HER2 antibodies and TNF-α provide evidence for a complex interplay between HER2 and TNF-α signaling pathways. Such complexity may drastically affect the outcome of HER2-directed therapeutic interventions.

  4. Dengue antibodies in blood donors. (United States)

    Ribas-Silva, Rejane Cristina; Eid, Andressa Ahmad


    Dengue is an urban arbovirus whose etiologic agent is a virus of the genus Flavorius with four distinct antigen serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) that is transmitted to humans through the bite of the mosquito Aedes aegypti. The Campo Mourão region in Brazil is endemic for dengue fever. OBTECTIVE: The aim of this study was to evaluate the presence of IgG and IgM antibodies specific to the four serotypes of dengue in donors of the blood donor service in the city of Campo Mourão. Epidemiological records were evaluated and 4 mL of peripheral blood from 213 blood donors were collected in tubes without anticoagulant. Serum was then obtained and immunochromatographic tests were undertaken (Imuno-Rápido Dengue IgM/IgG(TM)). Individuals involved in the study answered a social and epidemiological questionnaire on data which included age, gender and diagnosis of dengue. Only three (1.4%) of the 213 blood tests were positive for IgG anti-dengue antibodies. No donors with IgM antibody, which identifies acute infection, were identified. The results of the current analysis show that the introduction of quantitative or molecular serological methods to determine the presence of anti-dengue antibodies or the detection of the dengue virus in blood donors in endemic regions should be established so that the quality of blood transfusions is guaranteed.

  5. Immunogenicity of biologically-derived therapeutics: assessment and interpretation of nonclinical safety studies. (United States)

    Ponce, Rafael; Abad, Leslie; Amaravadi, Lakshmi; Gelzleichter, Thomas; Gore, Elizabeth; Green, James; Gupta, Shalini; Herzyk, Danuta; Hurst, Christopher; Ivens, Inge A; Kawabata, Thomas; Maier, Curtis; Mounho, Barbara; Rup, Bonita; Shankar, Gopi; Smith, Holly; Thomas, Peter; Wierda, Dan


    An evaluation of potential antibody formation to biologic therapeutics during the course of nonclinical safety studies and its impact on the toxicity profile is expected under current regulatory guidance and is accepted standard practice. However, approaches for incorporating this information in the interpretation of nonclinical safety studies are not clearly established. Described here are the immunological basis of anti-drug antibody formation to biopharmaceuticals (immunogenicity) in laboratory animals, and approaches for generating and interpreting immunogenicity data from nonclinical safety studies of biotechnology-derived therapeutics to support their progression to clinical evaluation. We subscribe that immunogenicity testing strategies should be adapted to the specific needs of each therapeutic development program, and data generated from such analyses should be integrated with available clinical and anatomic pathology, pharmacokinetic, and pharmacodynamic data to properly interpret nonclinical studies.

  6. Podoplanin - an emerging cancer biomarker and therapeutic target. (United States)

    Krishnan, Harini; Rayes, Julie; Miyashita, Tomoyuki; Ishii, Genichiro; Retzbach, Edward P; Sheehan, Stephanie A; Takemoto, Ai; Chang, Yao-Wen; Yoneda, Kazue; Asai, Jun; Jensen, Lasse; Chalise, Lushun; Natsume, Atsushi; Goldberg, Gary S


    Podoplanin (PDPN) is a transmembrane receptor glycoprotein that is upregulated on transformed cells, cancer associated fibroblasts (CAFs), and inflammatory macrophages that contribute to cancer progression. In particular, PDPN increases tumor cell clonal capacity, epithelial mesenchymal transition (EMT), migration, invasion, metastasis, and inflammation. Antibodies, CAR-T cells, biologics, and synthetic compounds that target PDPN can inhibit cancer progression and septic inflammation in preclinical models. This review describes recent advances in how PDPN may be used as a biomarker and therapeutic target for many types of cancer including glioma, squamous cell carcinoma, mesothelioma, and melanoma. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  7. Genetically engineered multivalent single chain antibody constructs for cancer therapy

    International Nuclear Information System (INIS)

    Surinder Batra


    Current therapeutic approaches against the advanced stages of human solid tumors are palliative rather than curative. Many modalities, including, surgery, radiation, and chemotherapy, either alone or in combination have met with only modest success for advanced metastatic cancers. Radioimmunotherapy (RIT) combines the specificity of monoclonal antibodies with cytotoxic effects of radioisotopes. It is the ''smart'' way of delivering radiation to the known and occult metastatic cancer cells and is independent of drug toxicity and/or hormone resistance. The tumor associated glycoprotein-72 (TAG-72) containing the unique disaccharide sialyl-Tn, is highly expressed in majority of adenocarcinomas, including carcinomas of the prostate, breast, ovaries, pancreas and colon (80-90%) compared to undetectable expression in normal tissues. Monoclonal antibody CC49, reactive with TAG-72, after conjugation to potent gamma- and beta-emitting radionuclides, has been useful in selective systemic radiolocalization of disease and therapy of primary and metastatic tumor sites. However, limited therapeutic responses were observed in patients. Limited success of antibody based delivery of radioisotopes can be attributed to several factors including undesirable pharmacokinetics, poor tumor uptake and high immunogenicity of intact antibodies (IgGs). The primary factors contributing towards the failure of RIT include: (1) longer serum half-lives of the intact IgG molecules resulting in the radiotoxicity, (2) generation of human antibodies against murine antibodies (HAMA) that limits the frequency of dose administration, (3) poor diffusion rates of intact IgG due to the large size and (4) high interstitial fluid pressures (IFP) encountered in solid tumors. The major goal of our multidisciplinary project was to develop specific novel radiopharmaceuticals, with desired pharmacokinetics, for the diagnosis and therapy of solid tumors. To overcome the low uptake of radioactivity by tumors and to

  8. Antibodies against glycolipids enhance antifungal activity of macrophages and reduce fungal burden after infection with Paracoccidioides brasiliensis

    Directory of Open Access Journals (Sweden)

    Renata Amelia eBueno


    Full Text Available Paracoccidioidomycosis is a fungal disease endemic in Latin America. Polyclonal antibodies to acidic glycosphingolipids (GSLs from Paracoccidioides brasiliensis opsonized yeast forms in vitro increasing phagocytosis and reduced the fungal burden of infected animals. Antibodies to GSL were active in both prophylactic and therapeutic protocols using a murine intratracheal infection model. Pathological examination of the lungs of animals treated with antibodies to GSL showed well-organized granulomas and minimally damaged parenchyma compared to the untreated control. Murine peritoneal macrophages activated by IFN-γ and incubated with antibodies against acidic GSLs more effectively phagocytosed and killed P. brasiliensis yeast cells as well as produced more nitric oxide compared to controls. The present work discloses a novel target of protective antibodies against P. brasiliensis adding to other well-studied mediators of the immune response to this fungus.

  9. Progranulin antibodies in autoimmune diseases. (United States)

    Thurner, Lorenz; Preuss, Klaus-Dieter; Fadle, Natalie; Regitz, Evi; Klemm, Philipp; Zaks, Marina; Kemele, Maria; Hasenfus, Andrea; Csernok, Elena; Gross, Wolfgang L; Pasquali, Jean-Louis; Martin, Thierry; Bohle, Rainer Maria; Pfreundschuh, Michael


    Systemic vasculitides constitute a heterogeneous group of diseases. Autoimmunity mediated by B lymphocytes and their humoral effector mechanisms play a major role in ANCA-associated vasculitis (AAV) as well as in non-ANCA associated primary systemic vasculitides and in the different types of autoimmune connective tissue disorders and rheumatoid arthritis. In order to detect autoantibodies in systemic vasculitides, we screened protein macroarrays of human cDNA expression libraries with sera from patients with ANCA-associated and ANCA-negative primary systemic vasculitides. This approach led to the identification of antibodies against progranulin, a 88 kDA secreted glycoprotein with strong anti-inflammatory activity in the course of disease of giant-cell arteritis/polymyalgia rheumatica (14/65), Takayasu's arteritis (4/13), classical panarteritis nodosa (4/10), Behcet's disease (2/6) and in the course of disease in granulomatosis with polyangiitis (31/75), Churg-Strauss syndrome (7/23) and in microscopic polyangiitis (7/19). In extended screenings the progranulin antibodies were also detected in other autoimmune diseases such as systemic lupus erythematosus (39/91) and rheumatoid arthritis (16/44). Progranulin antibodies were detected only in 1 of 97 healthy controls. Anti-progranulin positive patients with systemic vasculitides, systemic lupus erythematosus or rheumatoid arthritis had significant lower progranulin plasma levels, indicating a neutralizing effect. In light of the anti-inflammatory effects of progranulin, progranulin antibodies might exert pro-inflammatory effects thus contributing to the pathogenesis of the respective autoimmune diseases and might serve as a marker for disease activity. This hypothesis is supported by the fact that a positive progranulin antibody status was associated with active disease in granulomatosis with polyangiitis. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Anti-VEGF antibody conjugated CdHgTe quantum dots as a fluorescent probe for imaging in living mouse

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Lili; Cui, Hongjing [Department of Analytical Chemistry, China Pharmaceutical University, Nanjing (China); Liu, Yu [Department of Biochemistry, School of Life Science and Technology, China Pharmaceutical University, Nanjing (China); Zhong, Wenying, E-mail: [Department of Analytical Chemistry, China Pharmaceutical University, Nanjing (China); Key laboratory of Biomedical Functional Materials, China Pharmaceutical University, Nanjing (China)


    The dual-function anti-VEGF antibody conjugated CdHgTe quantum dots with good targeting property was successfully prepared. In this system, anti-VEGF antibody is not only a target agent but also a therapeutic drug. The anti-VEGF antibody conjugated near-infrared quantum dots can achieve the purposes of detection and treatment at the same time. As-prepared dual-function fluorescent probe in this work has been successfully applied for in vivo and in vitro imaging, which is promising in rapid tumor detection.

  11. Multiplex serology of paraneoplastic antineuronal antibodies

    NARCIS (Netherlands)

    P. Maat (Peter); E. Brouwer (Eric); E. Hulsenboom (Esther); M.M. van Duijn (Martijn); M.W.J. Schreurs (Marco); H. Hooijkaas (Herbert); P.A. Smitt (Peter)


    textabstractParaneoplastic neurological syndromes (PNS) are devastating neurological disorders secondary to cancer, associated with onconeural autoantibodies. Such antibodies are directed against neuronal antigens aberrantly expressed by the tumor. The detection of onconeural antibodies in a patient

  12. [Neuroimmunological diseases associated with VGKC complex antibodies]. (United States)

    Watanabe, Osamu


    Antibodies to voltage-gated potassium channels(VGKC) were first identified by radioimmunoassay of radioisotope labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were found only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in Morvan's syndrome and in a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins(for example LGI-1, Caspr-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now usually known as VGKC-complex antibodies. In general, LGI-1 antibodies are most common in limbic encephalitis with SIADH. Caspr-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability.

  13. Therapeutic Inertia and Treatment Intensification. (United States)

    Josiah Willock, Robina; Miller, Joseph B; Mohyi, Michelle; Abuzaanona, Ahmed; Muminovic, Meri; Levy, Phillip D


    This review aims to emphasize how therapeutic inertia, the failure of clinicians to intensify treatment when blood pressure rises or remains above therapeutic goals, contributes to suboptimal blood pressure control in hypertensive populations. Studies reveal that the therapeutic inertia is quite common and contributes to suboptimal blood pressure control. Quality improvement programs and standardized approaches to support antihypertensive treatment intensification are ways to combat therapeutic inertia. Furthermore, programs that utilize non-physician medical professionals such as pharmacists and nurses demonstrate promise in mitigating the effects of this important problem. Therapeutic inertia impedes antihypertensive management and requires a broad effort to reduce its effects. There is an ongoing need for renewed focus and research in this area to improve hypertension control.

  14. Exubera. Inhale therapeutic systems. (United States)

    Bindra, Sanjit; Cefalu, William T


    Inhale, in colaboration with Pfizer and Aventis Pharma (formerly Hoechst Marion Roussel; HMR), is developing an insulin formulation utilizing its pulmonary delivery technology for macromolecules for the potential treatment of type I and II diabetes. By July 2001, the phase III program had been completed and the companies had begun to assemble data for MAA and NDA filings; however, it was already clear at this time that additional data might be required for filing. By December 2001, it had been decided that the NDA should include an increased level of controlled, long-term pulmonary safety data in diabetic patients and a major study was planned to be completed in 2002, with the NDA filed thereafter (during 2002). US-05997848 was issued to Inhale Therapeutic Systems in December 1999, and corresponds to WO-09524183, filed in February 1995. Equivalent applications have appeared to date in Australia, Brazil, Canada, China, Czech Republic, Europe, Finland, Hungary, Japan, Norway, New Zealand, Poland and South Africa. This family of applications is specific to pulmonary delivery of insulin. In February 1999, Lehman Brothers gave this inhaled insulin a 60% probability of reaching market, with a possible launch date of 2001. The analysts estimated peak sales at $3 billion in 2011. In May 2000, Aventis predicted that estimated peak sales would be in excess of $1 billion. In February 2000, Merrill Lynch expected product launch in 2002 and predicted that it would be a multibillion-dollar product. Analysts Merril Lynch predicted, in September and November 2000, that the product would be launched by 2002, with sales in that year of e75 million, rising to euro 500 million in 2004. In April 2001, Merrill Lynch predicted that filing for this drug would occur in 2001. Following the report of the potential delay in regulatory filing, issued in July 2001, Deutsche Banc Alex Brown predicted a filing would take place in the fourth quarter of 2002 and launch would take place in the first

  15. Frizzled Receptors as Potential Therapeutic Targets in Human Cancers

    Directory of Open Access Journals (Sweden)

    Chui-Mian Zeng


    Full Text Available Frizzled receptors (FZDs are a family of seven-span transmembrane receptors with hallmarks of G protein-coupled receptors (GPCRs that serve as receptors for secreted Wingless-type (WNT ligands in the WNT signaling pathway. Functionally, FZDs play crucial roles in regulating cell polarity, embryonic development, cell proliferation, formation of neural synapses, and many other processes in developing and adult organisms. In this review, we will introduce the basic structural features and review the biological function and mechanism of FZDs in the progression of human cancers, followed by an analysis of clinical relevance and therapeutic potential of FZDs. We will focus on the development of antibody-based and small molecule inhibitor-based therapeutic strategies by targeting FZDs for human cancers.

  16. Therapeutic Targets of Triglyceride Metabolism as Informed by Human Genetics. (United States)

    Bauer, Robert C; Khetarpal, Sumeet A; Hand, Nicholas J; Rader, Daniel J


    Human genetics has contributed to the development of multiple drugs to treat hyperlipidemia and coronary artery disease (CAD), most recently including antibodies targeting PCSK9 to reduce LDL cholesterol. Despite these successes, a large burden of CAD remains. Genetic and epidemiological studies have suggested that circulating triglyceride (TG)-rich lipoproteins (TRLs) are a causal risk factor for CAD, presenting an opportunity for novel therapeutic strategies. We discuss recent unbiased human genetics testing, including genome-wide association studies (GWAS) and whole-genome or -exome sequencing, that have identified the lipoprotein lipase (LPL) and hepatic lipogenesis pathways as important mechanisms in the regulation of circulating TRLs. Further strengthening the causal relationship between TRLs and CAD, findings such as these may provide novel targets for much-needed potential therapeutic interventions. Copyright © 2016. Published by Elsevier Ltd.

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

    Directory of Open Access Journals (Sweden)

    Biao He


    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.

  18. Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier. (United States)

    Couch, Jessica A; Yu, Y Joy; Zhang, Yin; Tarrant, Jacqueline M; Fuji, Reina N; Meilandt, William J; Solanoy, Hilda; Tong, Raymond K; Hoyte, Kwame; Luk, Wilman; Lu, Yanmei; Gadkar, Kapil; Prabhu, Saileta; Ordonia, Benjamin A; Nguyen, Quyen; Lin, Yuwen; Lin, Zhonghua; Balazs, Mercedesz; Scearce-Levie, Kimberly; Ernst, James A; Dennis, Mark S; Watts, Ryan J


    Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting antibody uptake in brain. Nevertheless, there are limited data on the therapeutic properties including safety liabilities that will enable successful development of TfR-based therapeutics. We evaluate TfR/BACE1 bispecific antibody variants in mouse and show that reducing TfR binding affinity improves not only brain uptake but also peripheral exposure and the safety profile of these antibodies. We identify and seek to address liabilities of targeting TfR with antibodies, namely, acute clinical signs and decreased circulating reticulocytes observed after dosing. By eliminating Fc effector function, we ameliorated the acute clinical signs and partially rescued a reduction in reticulocytes. Furthermore, we show that complement mediates a residual decrease in reticulocytes observed after Fc effector function is eliminated. These data raise important safety concerns and potential mitigation strategies for the development of TfR-based therapies that are designed to cross the blood-brain barrier.

  19. Delivery of Flightless I Neutralizing Antibody from Porous Silicon Nanoparticles Improves Wound Healing in Diabetic Mice. (United States)

    Turner, Christopher T; McInnes, Steven J P; Melville, Elizabeth; Cowin, Allison J; Voelcker, Nicolas H


    Flightless I (Flii) is elevated in human chronic wounds and is a negative regulator of wound repair. Decreasing its activity improves healing responses. Flii neutralizing antibodies (FnAbs) decrease Flii activity in vivo and hold significant promise as healing agents. However, to avoid the need for repeated application in a clinical setting and to protect the therapeutic antibody from the hostile environment of the wound, suitable delivery vehicles are required. In this study, the use of porous silicon nanoparticles (pSi NPs) is demonstrated for the controlled release of FnAb to diabetic wounds. We achieve FnAb loading regimens exceeding 250 µg antibody per mg of vehicle. FnAb-loaded pSi NPs increase keratinocyte proliferation and enhance migration in scratch wound assays. Release studies confirm the functionality of the FnAb in terms of Flii binding. Using a streptozotocin-induced model of diabetic wound healing, a significant improvement in healing is observed for mice treated with FnAb-loaded pSi NPs compared to controls, including FnAb alone. FnAb-loaded pSi NPs treated with proteases show intact and functional antibody for up to 7 d post-treatment, suggesting protection of the antibodies from proteolytic degradation in wound fluid. pSi NPs may therefore enable new therapeutic approaches for the treatment of diabetic ulcers. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Radioimmunoassay method for detection of gonorrhea antibodies

    International Nuclear Information System (INIS)


    A novel radioimmunoassay for the detection of gonorrhea antibodies in serum is described. A radionuclide is bound to gonorrhea antigens produced by a growth culture. In the presence of gonorrhea antibodies in the serum, an antigen-antibody conjugate is formed, the concentration of which can be measured with conventional radiometric methods. The radioimmunoassay is highly specific