Science.gov

Sample records for bispecific monoclonal antibody

  1. Bispecific antibodies.

    PubMed

    Kontermann, Roland E; Brinkmann, Ulrich

    2015-07-01

    Bispecific antibodies (bsAbs) combine specificities of two antibodies and simultaneously address different antigens or epitopes. BsAbs with 'two-target' functionality can interfere with multiple surface receptors or ligands associated, for example with cancer, proliferation or inflammatory processes. BsAbs can also place targets into close proximity, either to support protein complex formation on one cell, or to trigger contacts between cells. Examples of 'forced-connection' functionalities are bsAbs that support protein complexation in the clotting cascade, or tumor-targeted immune cell recruiters and/or activators. Following years of research and development (R&D), the first bsAb was approved in 2009. Another bsAb entered the market in December 2014 and several more are in clinical trials. Here, we describe the potentials of bsAbs to become the next wave of antibody-based therapies, focusing on molecules in clinical development. PMID:25728220

  2. The application of mathematical modelling to the design of bispecific monoclonal antibodies.

    PubMed

    van Steeg, Tamara J; Bergmann, Kirsten Riber; Dimasi, Nazzareno; Sachsenmeier, Kris F; Agoram, Balaji

    2016-04-01

    Targeting multiple receptors with bispecific antibodies is a novel approach that may prevent the development of resistance to cancer treatments. Despite the initial promise, full clinical benefit of this technology has yet to be realized. We hypothesized that in order to optimally exploit bispecific antibody technology, thorough fundamental knowledge of their pharmacological properties compared to that of single agent combinations was needed. Therefore, we developed a mathematical model for the binding of bispecific antibodies to their targets that accounts for the spatial distribution of the binding receptors and the kinetics of binding, and is scalable for increasing valency. The model provided an adequate description of internal and literature-reported in vitro data on bispecific binding. Simulations of in vitro binding with the model indicated that bispecific antibodies are not always superior in their binding potency to combination of antibodies, and the affinity of bispecific arms must be optimized for maximum binding potency. Our results suggest that this tool can be used for the design and development of the next generation of anti-cancer bispecific compounds. PMID:26910134

  3. A Murine, Bispecific Monoclonal Antibody Simultaneously Recognizing β-Glucan and MP65 Determinants in Candida Species

    PubMed Central

    Zito, Andrea; Bromuro, Carla; Mandili, Giorgia; Chiani, Paola; Horenstein, Alberto L.; Malavasi, Fabio; Cauda, Roberto; Cassone, Antonio; Torosantucci, Antonella

    2016-01-01

    There is a real medical need of new diagnostic tools for the early recognition of invasive Candida infections. We exploited a rather simple and rapid redox methodology to construct a bispecific monoclonal antibody (bsmAb) that combines a monoclonal antibody (mAb) directed against 1,3-β-D-glucan, a well-known, pan-fungal diagnostic biomarker, with a mAb recognizing MP65, a major immunogenic mannoprotein secreted by C.albicans and other Candida species. The bsmAb (MP65/bglu mAb) was successfully produced and purified at high yields and proved to bind and reveal simultaneously, with high sensitivity, the β-glucan and MP65 antigens in both purified and native forms. The MP65/bglu mAb is the first bispecific antibody generated against a fungal microorganism and may prove useful for the concurrent detection of different and clinically significant Candida biomarkers in patient sera. PMID:26859561

  4. A Murine, Bispecific Monoclonal Antibody Simultaneously Recognizing β-Glucan and MP65 Determinants in Candida Species.

    PubMed

    Zito, Andrea; Bromuro, Carla; Mandili, Giorgia; Chiani, Paola; Horenstein, Alberto L; Malavasi, Fabio; Cauda, Roberto; Cassone, Antonio; Torosantucci, Antonella

    2016-01-01

    There is a real medical need of new diagnostic tools for the early recognition of invasive Candida infections. We exploited a rather simple and rapid redox methodology to construct a bispecific monoclonal antibody (bsmAb) that combines a monoclonal antibody (mAb) directed against 1,3-β-D-glucan, a well-known, pan-fungal diagnostic biomarker, with a mAb recognizing MP65, a major immunogenic mannoprotein secreted by C.albicans and other Candida species. The bsmAb (MP65/bglu mAb) was successfully produced and purified at high yields and proved to bind and reveal simultaneously, with high sensitivity, the β-glucan and MP65 antigens in both purified and native forms. The MP65/bglu mAb is the first bispecific antibody generated against a fungal microorganism and may prove useful for the concurrent detection of different and clinically significant Candida biomarkers in patient sera. PMID:26859561

  5. Generation of chimeric bispecific G250/anti-CD3 monoclonal antibody, a tool to combat renal cell carcinoma.

    PubMed Central

    Luiten, R. M.; Coney, L. R.; Fleuren, G. J.; Warnaar, S. O.; Litvinov, S. V.

    1996-01-01

    The monoclonal antibody (MAb) G250 binds to a tumour-associated antigen, expressed in renal cell carcinoma (RCC), which has been demonstrated to be a suitable target for antibody-mediated immunotherapy. A bispecific antibody having both G250 and anti-CD3 specificity can cross-link G250 antigen-expressing RCC target cells with T cells and can mediate lysis of such targets. Therapy studies with murine antibodies are limited by immune responses to the antibodies injected (HAMA response), which can be decreased by using chimeric antibodies. We generated a chimeric bispecific G250/anti CD3 MAb by transfecting chimeric genes of heavy and light chains for both the G250 MAb and the anti-CD3 MAb into a myeloma cell line. Cytotoxicity assays revealed that the chimeric bispecific MAb was capable of mediating lysis of RCC cell lines by cloned human CD8+T cells or by IL-2-stimulated peripheral blood lymphocytes (PBLs). Lysis mediated by the MAb was specific for target cells that expressed the G250 antigen and was effective at concentrations as low as 0.01 microgram ml-1. The chimeric bispecific G250/anti-CD3 MAb produced may be an effective adjuvant to the currently used IL-2-based therapy of advanced renal cell arcinoma. Images Figure 7 PMID:8795576

  6. Dual targeting strategies with bispecific antibodies

    PubMed Central

    2012-01-01

    Monoclonal antibodies are widely used for the treatment of cancer, inflammatory and infectious diseases and other disorders. Most of the marketed antibodies are monospecific and therefore capable of interacting and interfering with a single target. However, complex diseases are often multifactorial in nature, and involve redundant or synergistic action of disease mediators or upregulation of different receptors, including crosstalk between their signaling networks. Consequently, blockade of multiple, different pathological factors and pathways may result in improved therapeutic efficacy. This result can be achieved by combining different drugs, or use of the dual targeting strategies applying bispecific antibodies that have emerged as an alternative to combination therapy. This review discusses the various dual targeting strategies for which bispecific antibodies have been developed and provides an overview of the established bispecific antibody formats. PMID:22453100

  7. Chemiluminescence reaction kinetics-resolved multianalyte immunoassay strategy using a bispecific monoclonal antibody as the unique recognition reagent.

    PubMed

    Ouyang, Hui; Wang, Limin; Yang, Shijia; Wang, Wenwen; Wang, Lin; Liu, Fengquan; Fu, Zhifeng

    2015-03-01

    The multianalyte immunoassay (MIA) has attracted increasing attention due to its high sample throughput, short assay time, low sample consumption, and reduced overall cost. However, up to now, the reported MIA methods commonly require multiple antibodies since each antibody can recognize only one antigen. Herein, a novel bispecific monoclonal antibody (BsMcAb) that could bind methyl parathion and imidacloprid simultaneously was produced by a hybrid hybridomas strategy. A chemiluminescence (CL) reaction kinetics-resolved strategy was designed for MIA of methyl parathion and imidacloprid using the BsMcAb as the unique recognition reagent. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were adopted as the signal probes to tag the haptens of the two pesticides due to their very different CL kinetic characteristics. After competitive immunoreactions, the HRP-tagged methyl parathion hapten and the ALP-tagged imidacloprid hapten were simultaneously bound to the BsMcAb since there were two different antigen-binding sites in it. Then, two CL reactions were simultaneously triggered by adding the CL coreactants, and the signals for methyl parathion and imidacloprid detections were collected at 0.6 and 1000 s, respectively. The linear ranges for methyl parathion and imidacloprid were both 1.0-500 ng/mL, with detection limits of 0.33 ng/mL (S/N = 3). The proposed method was successfully used to detect pesticides spiked in ginseng and American ginseng with acceptable recoveries of 80-118%. This proof-of-principle work demonstrated the feasibility of MIA using only one antibody. PMID:25622025

  8. Monoclonal Antibodies.

    ERIC Educational Resources Information Center

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

    1984-01-01

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

  9. Time resolved native ion-mobility mass spectrometry to monitor dynamics of IgG4 Fab arm exchange and "bispecific" monoclonal antibody formation.

    PubMed

    Debaene, François; Wagner-Rousset, Elsa; Colas, Olivier; Ayoub, Daniel; Corvaïa, Nathalie; Van Dorsselaer, Alain; Beck, Alain; Cianférani, Sarah

    2013-10-15

    Monoclonal antibodies (mAbs) and derivatives such as antibody-drug conjugates (ADC) and bispecific antibodies (bsAb), are the fastest growing class of human therapeutics. Most of the therapeutic antibodies currently on the market and in clinical trials are chimeric, humanized, and human immunoglobulin G1 (IgG1). An increasing number of IgG2s and IgG4s that have distinct structural and functional properties are also investigated to develop products that lack or have diminished antibody effector functions compared to IgG1. Importantly, wild type IgG4 has been shown to form half molecules (one heavy chain and one light chain) that lack interheavy chain disulfide bonds and form intrachain disulfide bonds. Moreover, IgG4 undergoes a process of Fab-arm exchange (FAE) in which the heavy chains of antibodies of different specificities can dissociate and recombine in bispecific antibodies both in vitro and in vivo. Here, native mass spectrometry (MS) and time-resolved traveling wave ion mobility MS (TWIM-MS) were used for the first time for online monitoring of FAE and bsAb formation using Hz6F4-2v3 and natalizumab, two humanized IgG4s which bind to human Junctional Adhesion Molecule-A (JAM-A) and alpha4 integrin, respectively. In addition, native MS analysis of bsAb/JAM-A immune complexes revealed that bsAb can bind up to two antigen molecules, confirming that the Hz6F4 family preferentially binds dimeric JAM-A. Our results illustrate how IM-MS can rapidly assess bsAb structural heterogeneity and be easily implemented into MS workflows for bsAb production follow up and bsAb/antigen complex characterization. Altogether, these results provide new MS-based methodologies for in-depth FAE and bsAb formation monitoring. Native MS and IM-MS will play an increasing role in next generation biopharmaceutical product characterization like bsAbs, antibody mixtures, and antibody-drug conjugates (ADC) as well as for biosimilar and biobetter antibodies. PMID:24007193

  10. Antigen binding properties of highly purified bispecific antibodies.

    PubMed

    Allard, W J; Moran, C A; Nagel, E; Collins, G; Largen, M T

    1992-10-01

    A panel of three bispecific monoclonal antibodies (bsMAbs) binding to follitropin (FSH) and to beta-galactosidase have been prepared by fusion of hybridoma cell lines resistant to oubain and neomycin. One of these bispecific antibodies contains heavy chains of the same IgG subclass, and two are composed of heavy chains of different IgG subclasses. We have investigated methods for the purification of bispecific antibodies from hybrid hybridoma supernatant fluids grown in serum-free medium. Following ammonium sulfate precipitation, bispecific antibodies can be purified in a single step by mixed mode ion-exchange HPLC on Bakerbond Abx columns. In one case, three species were resolved by ion-exchange HPLC and functional analysis showed that two peaks contained parental antibodies, and the third contained the bispecific. Ion-exchange HPLC purification of serum-free preparations from two other hybrid hybridomas resolved seven protein-containing peaks, only one of which was active in a bispecific ELISA. The equilibrium affinity constants for each of the parental antibodies for both FSH and beta-galactosidase were determined and found to be similar to those of the purified bsMAbs. Further, the association of FSH to one binding site on a bispecific antibody was shown to have no effect on the equilibrium binding constant for beta-galactosidase binding to the other site. Our results suggest that bsMAbs can be readily purified from hybrid hybridomas by a simple and rapid method, and the binding of antigen to one binding site on a bsMAb is independent of antigen binding to the second site. PMID:1528192

  11. Clinical pharmacology of bispecific antibody constructs.

    PubMed

    Rathi, Chetan; Meibohm, Bernd

    2015-03-01

    The confluence of rapid scientific advancements especially in protein engineering and recombinant technology, unmet medical needs, and commercial incentives have led to the development of the next generation of therapeutic proteins. Bispecific antibody constructs are one of the novel strategies that is being pursued, combining the ability to bind simultaneously to two distinct targets and the advantages of purpose-designed and optimized antibody-based scaffolds. Their pharmacokinetic and pharmacodynamic properties, including their immunogenic potential, are closely related to their structural features and ability to interact with disposition mechanisms of immunoglobulin molecules. Catumaxomab and blinatumomab are bispecific constructs that are approved for clinical use and have provided clinical pharmacology data for this novel class of therapeutics. This knowledgebase on the clinical behavior of bispecific therapeutic proteins is poised to rapidly evolve over the next few years with many development programs having entered the clinical development stage. PMID:25707960

  12. Production of bispecific antibodies: diabodies and tandem scFv.

    PubMed

    Hornig, Nora; Frber-Schwarz, Aline

    2012-01-01

    Recombinant bispecific antibodies have many different applications; especially promising is their therapeutic potential due to their ability to retarget an effector molecule or a cell to a disease-related target structure. In the last years, many formats have been developed: two commonly used are the bispecific diabody and the tandem scFv. In this chapter, the cloning, bacterial production, purification, and characterization of the two antibody formats are described in detail. PMID:22907382

  13. Nanocell targeting using engineered bispecific antibodies.

    PubMed

    Taylor, Karin; Howard, Christopher B; Jones, Martina L; Sedliarou, Ilya; MacDiarmid, Jennifer; Brahmbhatt, Himanshu; Munro, Trent P; Mahler, Stephen M

    2015-01-01

    There are many design formats for bispecific antibodies (BsAbs), and the best design choice is highly dependent on the final application. Our aim was to engineer BsAbs to target a novel nanocell (EnGeneIC Delivery Vehicle or EDV(TM)nanocell) to the epidermal growth factor receptor (EGFR). EDV(TM)nanocells are coated with lipopolysaccharide (LPS), and BsAb designs incorporated single chain Fv (scFv) fragments derived from an anti-LPS antibody (1H10) and an anti-EGFR antibody, ABX-EGF. We engineered various BsAb formats with monovalent or bivalent binding arms and linked scFv fragments via either glycine-serine (G4S) or Fc-linkers. Binding analyses utilizing ELISA, surface plasmon resonance, bio-layer interferometry, flow cytometry and fluorescence microscopy showed that binding to LPS and to either soluble recombinant EGFR or MDA-MB-468 cells expressing EGFR, was conserved for all construct designs. However, the Fc-linked BsAbs led to nanocell clumping upon binding to EDV(TM)nanocells. Clumping was eliminated when additional disulfide bonds were incorporated into the scFv components of the BsAbs, but this resulted in lower BsAb expression. The G4S-linked tandem scFv BsAb format was the optimal design with respect to EDV binding and expression yield. Doxorubicin-loaded EDV(TM)nanocells actively targeted with tandem scFv BsAb in vivo to MDA-MB-468-derived tumors in mouse xenograft models enhanced tumor regression by 40% compared to passively targeted EDV(TM)nanocells. BsAbs therefore provide a functional means to deliver EDV(TM)nanocells to target cells. PMID:25523746

  14. Nanocell targeting using engineered bispecific antibodies

    PubMed Central

    Taylor, Karin; Howard, Christopher B; Jones, Martina L; Sedliarou, Ilya; MacDiarmid, Jennifer; Brahmbhatt, Himanshu; Munro, Trent P; Mahler, Stephen M

    2015-01-01

    There are many design formats for bispecific antibodies (BsAbs), and the best design choice is highly dependent on the final application. Our aim was to engineer BsAbs to target a novel nanocell (EnGeneIC Delivery Vehicle or EDVTMnanocell) to the epidermal growth factor receptor (EGFR). EDVTMnanocells are coated with lipopolysaccharide (LPS), and BsAb designs incorporated single chain Fv (scFv) fragments derived from an anti-LPS antibody (1H10) and an anti-EGFR antibody, ABX-EGF. We engineered various BsAb formats with monovalent or bivalent binding arms and linked scFv fragments via either glycine-serine (G4S) or Fc-linkers. Binding analyses utilizing ELISA, surface plasmon resonance, bio-layer interferometry, flow cytometry and fluorescence microscopy showed that binding to LPS and to either soluble recombinant EGFR or MDA-MB-468 cells expressing EGFR, was conserved for all construct designs. However, the Fc-linked BsAbs led to nanocell clumping upon binding to EDVTMnanocells. Clumping was eliminated when additional disulfide bonds were incorporated into the scFv components of the BsAbs, but this resulted in lower BsAb expression. The G4S-linked tandem scFv BsAb format was the optimal design with respect to EDV binding and expression yield. Doxorubicin-loaded EDVTMnanocells actively targeted with tandem scFv BsAb in vivo to MDA-MB-468-derived tumors in mouse xenograft models enhanced tumor regression by 40% compared to passively targeted EDVTMnanocells. BsAbs therefore provide a functional means to deliver EDVTMnanocells to target cells. PMID:25523746

  15. Redirecting adenoviruses to tumour cells using therapeutic antibodies: Generation of a versatile human bispecific adaptor.

    PubMed

    Vasiljevic, Snezana; Beale, Emma V; Bonomelli, Camille; Easthope, Iona S; Pritchard, Laura K; Seabright, Gemma E; Caputo, Alessandro T; Scanlan, Christopher N; Dalziel, Martin; Crispin, Max

    2015-12-01

    Effective use of adenovirus-5 (Ad5) in cancer therapy is heavily dependent on the degree to which the virus's natural tropism can be subverted to one that favours tumour cells. This is normally achieved through either engineering of the viral fiber knob or the use of bispecific adaptors that display both adenovirus and tumour antigen receptors. One of the main limitations of these strategies is the need to tailor each engineering event to any given tumour antigen. Here, we explore bispecific adaptors that can utilise established anti-cancer therapeutic antibodies. Conjugates containing bacterially derived antibody binding motifs are efficient at retargeting virus to antibody targets. Here, we develop a humanized strategy whereby we synthesise a re-targeting adaptor based on a chimeric Ad5 ligand/antibody receptor construct. This adaptor acts as a molecular bridge analogous to therapeutic antibody mediated cross-linking of cytotoxic effector and tumour cells during immunotherapy. As a proof or principle, we demonstrate how this adaptor allows efficient viral recognition and entry into carcinoma cells through the therapeutic monoclonal antibodies Herceptin/trastuzumab and bavituximab. We show that targeting can be augmented by use of contemporary antibody enhancement strategies such as the selective elimination of competing serum IgG using "receptor refocusing" enzymes and we envisage that further improvements are achievable by enhancing the affinities between the adaptor and its ligands. Humanized bispecific adaptors offer the promise of a versatile retargeting technology that can exploit both clinically approved adenovirus and therapeutic antibodies. PMID:26391350

  16. Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity.

    PubMed

    Wagner, Koen; Kwakkenbos, Mark J; Claassen, Yvonne B; Maijoor, Kelly; Bhne, Martino; van der Sluijs, Koenraad F; Witte, Martin D; van Zoelen, Diana J; Cornelissen, Lisette A; Beaumont, Tim; Bakker, Arjen Q; Ploegh, Hidde L; Spits, Hergen

    2014-11-25

    Bispecific antibodies have therapeutic potential by expanding the functions of conventional antibodies. Many different formats of bispecific antibodies have meanwhile been developed. Most are genetic modifications of the antibody backbone to facilitate incorporation of two different variable domains into a single molecule. Here, we present a bispecific format where we have fused two full-sized IgG antibodies via their C termini using sortase transpeptidation and click chemistry to create a covalently linked IgG antibody heterodimer. By linking two potent anti-influenza A antibodies together, we have generated a full antibody dimer with bispecific activity that retains the activity and stability of the two fusion partners. PMID:25385586

  17. Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity

    PubMed Central

    Wagner, Koen; Kwakkenbos, Mark J.; Claassen, Yvonne B.; Maijoor, Kelly; Bhne, Martino; van der Sluijs, Koenraad F.; Witte, Martin D.; van Zoelen, Diana J.; Cornelissen, Lisette A.; Beaumont, Tim; Bakker, Arjen Q.; Ploegh, Hidde L.; Spits, Hergen

    2014-01-01

    Bispecific antibodies have therapeutic potential by expanding the functions of conventional antibodies. Many different formats of bispecific antibodies have meanwhile been developed. Most are genetic modifications of the antibody backbone to facilitate incorporation of two different variable domains into a single molecule. Here, we present a bispecific format where we have fused two full-sized IgG antibodies via their C termini using sortase transpeptidation and click chemistry to create a covalently linked IgG antibody heterodimer. By linking two potent anti-influenza A antibodies together, we have generated a full antibody dimer with bispecific activity that retains the activity and stability of the two fusion partners. PMID:25385586

  18. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

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

  19. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

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

  20. An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants.

    PubMed

    Paul, Suparna; Connor, Judy; Nesspor, Tom; Haytko, Peter; Boakye, Ken; Chiu, Mark L; Jiang, Haiyan

    2016-05-01

    Bispecific antibody generation is actively pursued for therapeutic and research antibody development. Although there are multiple strategies for generating bispecific antibodies (bsAbs); the common challenge is to develop a scalable method to prepare bsAbs with high purity and yield. The controlled Fab-arm exchange (cFAE) method combines two parental monoclonal antibodies (mAbs), each with a matched point mutation, F405L and K409R in the respective CH3 domains. The conventional process employs two steps: the purification of two parental mAbs from culture supernatants followed by cFAE. Following a reduction/oxidation reaction, the bispecific mAb is formed with greater than 95% heterodimerization efficiency. In this study, cFAE was initiated in culture supernatants expressing the two parental mAbs, thereby eliminating the need to first purify the parental mAbs. The bsAbs formed in culture supernatant was then purified using a Protein A affinity chromatography. The BsAbs generated in this manner had efficiency comparable to the conventional method using purified parental mAbs. BsAbs prepared by two different routes showed indistinguishable characteristics by SDS capillary electrophoresis, analytical size exclusion, and cation exchange chromatography. This alternative method significantly shortened timelines and reduced resources required for bsAb generation, providing an improved process with potential benefits in large-scale bsAb preparation, as well as for HTP small-scale bsAb matrix selection. PMID:26826313

  1. Bispecific antibody: a tool for diagnosis and treatment of disease.

    PubMed Central

    Songsivilai, S; Lachmann, P J

    1990-01-01

    Antibodies with two distinct binding specificities have great potential for a wide range of clinical applications as targeting agents for in vitro and in vivo immunodiagnosis and therapy, and for improving immunoassays. They have shown great promise for targeting cytotoxic effector cells, delivering radionuclides, toxins or cytotoxic drugs to specific targets, particularly tumour cells. We discuss potential applications of bispecific antibodies, the theoretical basis and problems associated with their production and purification, cell fusion and chemical conjugation techniques, and propose a new manufacturing strategy by genetic engineering. This approach has enormous potential applications for producing tailor-made bispecific antibodies, and will enable widespread clinical uses of these antibodies both for diagnostic purposes and therapy. PMID:2180597

  2. Anti-CD20/CD3 T cell-dependent bispecific antibody for the treatment of B cell malignancies.

    PubMed

    Sun, Liping L; Ellerman, Diego; Mathieu, Mary; Hristopoulos, Maria; Chen, Xiaocheng; Li, Yijin; Yan, Xiaojie; Clark, Robyn; Reyes, Arthur; Stefanich, Eric; Mai, Elaine; Young, Judy; Johnson, Clarissa; Huseni, Mahrukh; Wang, Xinhua; Chen, Yvonne; Wang, Peiyin; Wang, Hong; Dybdal, Noel; Chu, Yu-Waye; Chiorazzi, Nicholas; Scheer, Justin M; Junttila, Teemu; Totpal, Klara; Dennis, Mark S; Ebens, Allen J

    2015-05-13

    Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell-recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell-targeting anti-CD20/CD3 T cell-dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using "knobs-into-holes" technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies. PMID:25972002

  3. [Progress in monoclonal antibody-based immunotherapy for cancer treatment].

    PubMed

    Guo, Yajun

    2015-06-01

    More than 100 years ago, Paul Ehrlich first proposed the "magic bullets" concept in which antibody targeting disease related antigen can fight against human disease. Since then, with the development of hybridoma technology for monoclonal antibody production and cancer serum therapy, immunotherapy based monoclonal antibody bas been used in chinical practice to treat hematological and solid tumor. Up to now, more than 20 recombinant antibody drugs were approved for cancer treatment worldwide. In recent years, the next-generation antibody drug, including immune checkpoint antagonists, bi-specific antibody, and antibody drug conjugates have successfully cured various malignant tumor. This review recalled the history of monoclonal antibody as potent immunotherapy of cancer firstly, and focused on the next-generation antibody drug's mechanism of action, construction strategies, and the side effects in clinic. Lastly, the future trend of anti-tumor antibody drug was also discussed. PMID:26672362

  4. Monoclonal antibodies and cancer therapy

    SciTech Connect

    Reisfeld, R.A.; Sell, S.

    1985-01-01

    These proceedings collect papers on the subject of monoclonal antibodies. Topics include: Monoclonal antibody, biochemical effects and cancer therapeutic potential of tunicamycin, use of monoclonal antibodies for detection of lymph node metastases, active specific immunotherapy, and applications of monoclonal antibodies to investigations of growth factors.

  5. Quantification of cell surface proteins with bispecific antibodies.

    PubMed

    Panke, C; Weininger, D; Haas, A; Schelter, F; Schlothauer, T; Bader, S; Sircar, R; Josel, H P; Baer, U; Burtscher, H; Mundigl, O; Grote, M; Brinkmann, U; Sustmann, C

    2013-10-01

    Flow cytometry is an established method for fast and accurate quantitation of cellular protein levels and requires fluorescently labeled antibodies as well as calibration standards. A critical step for quantitation remains the production of suitable detection antibodies with a precisely defined ratio of antigen-binding sites to fluorophores. Problems often arise as a consequence of inefficient and unspecific labeling which can influence antibody properties. In addition, the number of incorporated fluorophores necessitates a special normalization step for quantitation. To address these problems, we constructed different mono- and bivalent bispecific antibodies with binding site(s) for the cell surface antigens, cMET, EGFR1/HER1, ErbB2/HER2 or ErbB3/HER3 and with an additional digoxigenin-binding single-chain Fv fusion. The fluorophore Cy5 was covalently coupled to digoxigenin and quantitatively bound by the bispecific antibody. A panel of tumor cell lines was assessed under different culture conditions for absolute receptor expression levels of the indicated antigens and the data were set in relation to mRNA, gene count and immunoblot data. We could reproducibly quantify these receptors, omit the otherwise required normalization step and demonstrate the superiority of a 1 + 1 bispecific antibody. The same antibodies were also used to quantify the number of proteins in intracellular vesicles in confocal microscopy. The antibodies can be stored like regular antibodies and can be coupled with different digoxigenin-labeled fluorophores which makes them excellent tools for FACS and imaging-based experiments. PMID:23960142

  6. Bispecific Antibody Pretargeting for Improving Cancer Imaging and Therapy

    SciTech Connect

    Sharkey, Robert M.

    2005-02-04

    The main objective of this project was to evaluate pretargeting systems that use a bispecific antibody (bsMAb) to improve the detection and treatment of cancer. A bsMAb has specificity to a tumor antigen, which is used to bind the tumor, while the other specificity is to a peptide that can be radiolabeled. Pretargeting is the process by which the unlabeled bsMAb is given first, and after a sufficient time (1-2 days) is given for it to localize in the tumor and clear from the blood, a small molecular weight radiolabeled peptide is given. According to a dynamic imaging study using a 99mTc-labeled peptide, the radiolabeled peptide localizes in the tumor in less than 1 hour, with > 80% of it clearing from the blood and body within this same time. Tumor/nontumor targeting ratios that are nearly 50 times better than that with a directly radiolabeled Fab fragment have been observed (Sharkey et al., ''Signal amplification in molecular imaging by a multivalent bispecific nanobody'' submitted). The bsMAbs used in this project have been composed of 3 antibodies that will target antigens found in colorectal and pancreatic cancers (CEA, CSAp, and MUC1). For the ''peptide binding moiety'' of the bsMAb, we initially examined an antibody directed to DOTA, but subsequently focused on another antibody directed against a novel compound, HSG (histamine-succinyl-glycine).

  7. The therapeutic monoclonal antibody market

    PubMed Central

    Ecker, Dawn M; Jones, Susan Dana; Levine, Howard L

    2015-01-01

    Since the commercialization of the first therapeutic monoclonal antibody product in 1986, this class of biopharmaceutical products has grown significantly so that, as of November 10, 2014, forty-seven monoclonal antibody products have been approved in the US or Europe for the treatment of a variety of diseases, and many of these products have also been approved for other global markets. At the current approval rate of ∼ four new products per year, ∼70 monoclonal antibody products will be on the market by 2020, and combined world-wide sales will be nearly $125 billion. PMID:25529996

  8. The therapeutic monoclonal antibody market.

    PubMed

    Ecker, Dawn M; Jones, Susan Dana; Levine, Howard L

    2015-01-01

    Since the commercialization of the first therapeutic monoclonal antibody product in 1986, this class of biopharmaceutical products has grown significantly so that, as of November 10, 2014, forty-seven monoclonal antibody products have been approved in the US or Europe for the treatment of a variety of diseases, and many of these products have also been approved for other global markets. At the current approval rate of ∼ four new products per year, ∼ 70 monoclonal antibody products will be on the market by 2020, and combined world-wide sales will be nearly $125 billion. PMID:25529996

  9. Combination of a bispecific antibody and costimulatory antibody-ligand fusion proteins for targeted cancer immunotherapy.

    PubMed

    Hornig, Nora; Kermer, Vanessa; Frey, Katharina; Diebolder, Philipp; Kontermann, Roland E; Mller, Dafne

    2012-06-01

    Initiation of a tumor-directed immune response and appropriate modulation of its progress are key issues in cancer immunotherapy. Combinatorial strategies addressing both aspects might therefore be especially suitable. Here, we report a targeted approach combining a bispecific antibody with 2 costimulatory antibody-ligand fusion proteins. According to the concept, the bispecific antibody (scDbFAPCD3) retargets T cells in a MHC-independent manner to tumor cells, providing an artificial first signal that allows the costimulatory antibody-ligand fusion proteins (B7.2-Db and scFv-4-1BBL) likewise targeted to the tumor cells to modulate the T-cell response. In our model system, the target cells coexpress the fibroblast activation protein (FAP) and endoglin as antigens. ScDbFAPCD3 and B7.2-Db are targeted to FAP although by different antibody moieties, whereas scFv-4-1BBL is directed against endoglin. ScDbFAPCD3-induced T-cell stimulation could be enhanced by the addition of either B7.2-Db or scFv-4-1BBL and even further by the combination of both as shown in terms of cytokine release (interleukin-2/interferon ?), proliferation and activation marker expression (CD25). By combined costimulation, overall T-cell population strongly increased in activation-experienced memory phenotype accompanied by a decrease in naive phenotype. ScFv-4-1BBL-mediated costimulation of naive CD8+ T cells promoted the expansion and development of cytotoxic T cells with strong effector potential. Thus, combining a bispecific antibody with antibody-ligand fusion protein-mediated CD28 and 4-1BB costimulation in a targeted approach shows great potential to generate and shape an immune response at the tumor site. Therefore, the adaptation of this approach to other immune modulatory ligands and tumor-relevant targets seems to be promising. PMID:22576347

  10. Bispecific Antibodies that Mediate Killing of Cells Infected with Human Immunodeficiency Virus of Any Strain

    NASA Astrophysics Data System (ADS)

    Berg, Jorg; Lotscher, Erika; Steimer, Kathelyn S.; Capon, Daniel J.; Baenziger, Jurg; Jack, Hans-Martin; Wabl, Matthias

    1991-06-01

    Although AIDS patients lose human immunodeficiency virus (HIV)-specific cytotoxic T cells, their remaining CD8-positive T lymphocytes maintain cytotoxic function. To exploit this fact we have constructed bispecific antibodies that direct cytotoxic T lymphocytes of any specificity to cells that express gp120 of HIV. These bispecific antibodies comprise one heavy/light chain pair from an antibody to CD3, linked to a heavy chain whose variable region has been replaced with sequences from CD4 plus a second light chain. CD3 is part of the antigen receptor on T cells and is responsible for signal transduction. In the presence of these bispecific antibodies, T cells of irrelevant specificity effectively lyse HIV-infected cells in vitro.

  11. Rational design and generation of recombinant control reagents for bispecific antibodies through CDR mutagenesis

    PubMed Central

    Choi, Bryan D.; Gedeon, Patrick C.; Kuan, Chien-Tsun; Sanchez-Perez, Luis; Archer, Gary E.; Bigner, Darell D.; Sampson, John H.

    2013-01-01

    Developments in the field of bispecific antibodies have progressed rapidly in recent years, particularly in their potential role for the treatment of malignant disease. However, manufacturing stable molecules has proven to be costly and time-consuming, which in turn has hampered certain aspects of preclinical evaluation including the unavailability of appropriate negative controls. Bispecific molecules (e.g., bispecific tandem scFv) exhibit two specificities, often against a tumor antigen as well as an immune-activation ligand such as CD3. While for IgG antibodies, isotype-matched controls are well accepted, when considering smaller antibody fragments it is not possible to adequately control for their biological activity through the use of archetypal isotypes, which differ dramatically in affinity, size, structure, and design. Here, we demonstrate a method for the rapid production of negative control tandem scFvs through complementarity determining region (CDR) mutagenesis, using a recently described bispecific T-cell engager (BiTE) targeting a tumor-specific mutation of the epidermal growth factor receptor (EGFRvIII) as an example. Four independent control constructs were developed by this method through alteration of residues spanning individual CDR domains. Importantly, while target antigen affinity was completely impaired, CD3 binding affinity was conserved in each molecule. These results have a potential to enhance the sophistication by which bispecific antibodies can be evaluated in the preclinical setting and may have broader applications for an array of alternative antibody-derived therapeutic platforms. PMID:23806556

  12. Heavy chain-only antibodies and tetravalent bispecific antibody neutralizing Staphylococcus aureus leukotoxins

    PubMed Central

    Laventie, Benot-Joseph; Rademaker, Hendrik Jan; Saleh, Maher; de Boer, Ernie; Janssens, Rick; Bourcier, Tristan; Subilia, Audrey; Marcellin, Luc; van Haperen, Rien; Lebbink, Joyce H. G.; Chen, Tao; Prvost, Gilles; Grosveld, Frank; Drabek, Dubravka

    2011-01-01

    PantonValentine leukocidin (PVL) is a pore-forming toxin associated with current outbreaks of community-associated methicillin-resistant strains and implicated directly in the pathophysiology of Staphylococcus aureus-related diseases. Humanized heavy chain-only antibodies (HCAb) were generated against S. aureus PVL from immunized transgenic mice to neutralize toxin activity. The active form of PVL consists of the two components, LukS-PV and LukF-PV, which induce osmotic lysis following pore formation in host defense cells. One antiLukS-PV HCAb, three antiLukF-PV HCAbs with affinities in the nanomolar range, and one engineered tetravalent bispecific HCAb were tested in vitro and in vivo, and all prevented toxin binding and pore formation. AntiLukS-PV HCAb also binds to ?-hemolysin C (HlgC) and inhibits HlgC/HlgB pore formation. Experiments in vivo in a toxin-induced rabbit endophthalmitis model showed that these HCAbs inhibit inflammatory reactions and tissue destruction, with the tetravalent bispecific HCAb performing best. Our findings show the therapeutic potential of HCAbs, and in particular, bispecific antibodies. PMID:21930905

  13. A Novel Antibody Engineering Strategy for Making Monovalent Bispecific Heterodimeric IgG Antibodies by Electrostatic Steering Mechanism*

    PubMed Central

    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

    2015-01-01

    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. PMID:25583986

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

    PubMed

    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

    2015-03-20

    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. PMID:25583986

  15. In vitro cytotoxic targeting by human mononuclear cells and bispecific antibody 2B1, recognizing c-erbB-2 protooncogene product and Fc gamma receptor III.

    PubMed

    Hsieh-Ma, S T; Eaton, A M; Shi, T; Ring, D B

    1992-12-15

    Bispecific murine monoclonal antibody 2B1, possessing dual specificity for the human c-erbB-2 protooncogene product and human Fc gamma receptor III (CD16) was evaluated for the ability to promote specific lysis of c-erbB-2-positive tumor cells in vitro. In short-term 51Cr release assays with human mononuclear cells as effectors and SK-Br-3 human breast cancer cells as targets, neither parental antibody of 2B1 mediated significant specific lysis, but bispecific antibody was as active as a chemical heteroconjugate, with 5 ng/ml of 2B1 causing half-maximal lysis at an effector/target ratio of 20:1 and 2 ng/ml 2B1 causing half-maximal lysis at an E/T ratio of 40:1. The cytotoxic targeting activity of 2B1 F(ab')2 fragment was the same as that of whole bispecific antibody, and the activity of whole 2B1 was not reduced when assays were performed in 100% autologous human serum, indicating that 2B1 binds effector cells through the CD16-binding site derived from parental antibody 3G8 rather than through its Fc portion. Variable inhibition of 2B1-mediated lysis was observed when autologous polymorphonuclear leukocytes from different donors were added to mononuclear effector cells at a 2:1 ratio; this inhibition was overcome at higher antibody concentration. 2B1 bispecific monoclonal antibody was also able to mediate targeted cytolysis using whole human blood as a source of effector cells or using effector or target cells derived from ovarian cancer patients. PMID:1360872

  16. Unexpected recombinations in single chain bispecific anti-CD3-anti-CD33 antibodies can be avoided by a novel linker module.

    PubMed

    Stamova, Slava; Cartellieri, Marc; Feldmann, Anja; Arndt, Claudia; Koristka, Stefanie; Bartsch, Holger; Bippes, Claudia C; Wehner, Rebekka; Schmitz, Marc; von Bonin, Malte; Bornhuser, Martin; Ehninger, Gerhard; Bachmann, Michael

    2011-12-01

    CD33 is an attractive immunotarget on the surface of tumor cells from patients with acute myeloid leukemia (AML). In a first attempt for immunotargeting of AML blasts we constructed two bispecific antibodies in the single chain bispecific diabody (scBsDb) format by fusing the variable domains of monoclonal antibodies directed against CD3 and CD33. Unfortunately, protein expression of both scBsDbs resulted in varying mixtures of fragmented and full length proteins. As the non-functional fragments competed with the functional full length antibodies we tried to understand the reason for the fragmentation. We found that the anti-CD3 and anti-CD33 antibody genes show striking sequence homologies: during B cell development the same V(h) J558 heavy and V(l) kk4 light chain genes were selected. Moreover, the closely related D genes DSP2 (9 and 11) were combined with the same JH4 gene. And finally, during VJ recombination of the light chain the same JK5 element was selected. These homologies between the two monoclonal antibodies were the reason for recombinations in the cell lines generated for expression of the scBsDbs. Finally, we solved this problem by (i) rearranging the order of the heavy and light chains of the anti-CD3 and anti-CD33 domains, and (ii) a replacement of one of the commonly used glycine serine linkers with a novel linker domain. The resulting bispecific antibody in a single chain bispecific tandem format (scBsTaFv) was stable and capable of redirecting T cells to CD33-positive tumor cells including AML blasts of patients. PMID:22014687

  17. Fragmentation of monoclonal antibodies

    PubMed Central

    Vlasak, Josef

    2011-01-01

    Fragmentation is a degradation pathway ubiquitously observed in proteins despite the remarkable stability of peptide bond; proteins differ only by how much and where cleavage occurs. The goal of this review is to summarize reports regarding the non-enzymatic fragmentation of the peptide backbone of monoclonal antibodies (mAbs). The sites in the polypeptide chain susceptible to fragmentation are determined by a multitude of factors. Insights are provided on the intimate chemical mechanisms that can make some bonds prone to cleavage due to the presence of specific side-chains. In addition to primary structure, the secondary, tertiary and quaternary structures have a significant impact in modulating the distribution of cleavage sites by altering local flexibility, accessibility to solvent or bringing in close proximity side chains that are remote in sequence. This review focuses on cleavage sites observed in the constant regions of mAbs, with special emphasis on hinge fragmentation. The mechanisms responsible for backbone cleavage are strongly dependent on pH and can be catalyzed by metals or radicals. The distribution of cleavage sites are different under acidic compared to basic conditions, with fragmentation rates exhibiting a minimum in the pH range 56; therefore, the overall fragmentation pattern observed for a mAb is a complex result of structural and solvent conditions. A critical review of the techniques used to monitor fragmentation is also presented; usually a compromise has to be made between a highly sensitive method with good fragment separation and the capability to identify the cleavage site. The effect of fragmentation on the function of a mAb must be evaluated on a case-by-case basis depending on whether cleavage sites are observed in the variable or constant regions, and on the mechanism of action of the molecule. PMID:21487244

  18. Bispecific antibodies and trispecific immunocytokines for targeting the immune system against cancer: preparing for the future.

    PubMed

    Fournier, Philippe; Schirrmacher, Volker

    2013-02-01

    Monoclonal anti-tumor antibodies (mAbs) that are clinically effective usually recruit, via their constant fragment (Fc) domain, Fc receptor (FcR)-positive accessory cells of the immune system and engage these additionally against the tumor. Since T cells are FcR negative, these important cells are not getting involved. In contrast to mAbs, bispecific antibodies (bsAbs) can be designed in such a way that they involve T cells. bsAbs are artificially designed molecules that bind simultaneously to two different antigens, one on the tumor cell, the other one on an immune effector cell such as CD3 on T cells. Such dual antibody constructs can cross-link tumor cells and T cells. Many such bsAb molecules at the surface of tumor cells can thus build a bridge to T cells and aggregate their CD3 molecules, thereby activating them for cytotoxic activity. BsAbs can also contain a third binding site, for instance a Fc domain or a cytokine that would bind to its respective cytokine receptor. The present review discusses the pros and cons for the use of the Fc fragment during the development of bsAbs using either cell-fusion or recombinant DNA technologies. The recombinant antibody technology allows the generation of very efficient bsAbs containing no Fc domain such as the bi-specific T-cell engager (BiTE). The strong antitumor activity of these molecules makes them very interesting new cancer therapeutics. Over the last decade, we have developed another concept, namely to combine bsAbs and multivalent immunocytokines with a tumor cell vaccine. The latter are patient-derived tumor cells modified by infection with a virus. The virus-Newcastle Disease Virus (NDV)-introduces, at the surface of the tumor cells, viral molecules that can serve as general anchors for the bsAbs. Our strategy aims at redirecting, in an Fc-independent fashion, activities of T cells and accessory cells against autologous tumor antigens. It creates very promising perspectives for a new generation of efficient and safe cancer therapeutics that should confer long-lasting anti-tumor immunity. PMID:23329400

  19. Cooperative mixtures of bispecific F(ab')2 antibodies for delivering saporin to lymphoma in vitro and in vivo

    SciTech Connect

    French, R.R.; Courtenay, A.E.; Ingamells, S.; Stevenson, G.T.; Glennie, M.J. )

    1991-05-01

    We report that selected combinations of two or more monoclonal bispecific F(ab')2 antibodies (BsAbs) far outperform single derivatives in the delivery of the ribosome-inactivating protein, saporin, to guinea pig L2C leukemic cells. Throughout the work, BsAbs were constructed by thioether-linking the hinges of two Fab'gamma, one from monoclonal anti-L2C-idiotype antibody and the other from anti-saporin antibody. The latter was either affinity-purified rabbit polyclonal or one of a panel of five mouse monoclonal antibodies. In vitro cytotoxicity studies showed that, though all derivatives were effective, the BsAb made with the polyclonal antibody was always 10 to 20 times more potent than those made with a monoclonal antibody in yielding 50% inhibition of (3H)leucine uptake. This superior activity could be matched by selective mixtures of two or more of the monoclonal derivatives. Furthermore, in immunotherapeutic delivery of saporin to tumor, a pair of BsAbs performed significantly better than did either individually. Binding and uptake studies with radiolabeled saporin demonstrated a 20-fold increase in functional affinity when saporin was held at the cell surface by an appropriate BsAb mixture rather than by a single BsAb. In contrast, only small differences were recorded in the rate at which saporin was internalized as a result of the same maneuver. We conclude that the improved performance of combinations of BsAbs arises from their ability to provide multiple linkages between saporin molecules and cell surfaces, increasing the functional affinity with which saporin is tethered to the cell, but, in this system at least, having only a minor effect on the rate at which it is internalized. Cocktails of two or more BsAbs, selected to bind to multiple epitopes on ribosome-inactivating proteins could provide an important new strategy in immunotherapy.

  20. Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates.

    PubMed

    Yu, Y Joy; Atwal, Jasvinder K; Zhang, Yin; Tong, Raymond K; Wildsmith, Kristin R; Tan, Christine; Bien-Ly, Nga; Hersom, Maria; Maloney, Janice A; Meilandt, William J; Bumbaca, Daniela; Gadkar, Kapil; Hoyte, Kwame; Luk, Wilman; Lu, Yanmei; Ernst, James A; Scearce-Levie, Kimberly; Couch, Jessica A; Dennis, Mark S; Watts, Ryan J

    2014-11-01

    Using therapeutic antibodies that need to cross the blood-brain barrier (BBB) to treat neurological disease is a difficult challenge. We have shown that bispecific antibodies with optimized binding to the transferrin receptor (TfR) that target ?-secretase (BACE1) can cross the BBB and reduce brain amyloid-? (A?) in mice. Can TfR enhance antibody uptake in the primate brain? We describe two humanized TfR/BACE1 bispecific antibody variants. Using a human TfR knock-in mouse, we observed that anti-TfR/BACE1 antibodies could cross the BBB and reduce brain A? in a TfR affinity-dependent fashion. Intravenous dosing of monkeys with anti-TfR/BACE1 antibodies also reduced A? both in cerebral spinal fluid and in brain tissue, and the degree of reduction correlated with the brain concentration of anti-TfR/BACE1 antibody. These results demonstrate that the TfR bispecific antibody platform can robustly and safely deliver therapeutic antibody across the BBB in the primate brain. PMID:25378646

  1. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2010-06-22

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  2. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2013-04-09

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  3. Monoclonal antibodies for treating cancer

    SciTech Connect

    Dillman, R.O. )

    1989-10-01

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

  4. Efficient tumor cell lysis by autologous, tumor-resident T lymphocytes in primary ovarian cancer samples by an EP-CAM-/CD3-bispecific antibody.

    PubMed

    Wimberger, Pauline; Xiang, Wei; Mayr, Doris; Diebold, Joachim; Dreier, Torsten; Baeuerle, Patrick A; Kimmig, Rainer

    2003-06-10

    The epithelial cell adhesion molecule (Ep-CAM) is expressed on the surface of most human carcinomas, including ovarian, breast, lung, prostate and colorectal carcinoma. Ep-CAM was shown to be a valid target for monoclonal antibody-based therapies. We have investigated whether an Ep-CAM-/CD3-bispecific single-chain antibody called bscEp-CAM x CD3 is effective in tumor cell elimination within the cellular microenvironment of primary ovarian cancer tissue. The ex vivo elimination of ovarian cancer cells in tumor preparations from 21 patients was monitored by flow cytometry using Ep-CAM/CA-125 double-labeling or Ep-CAM single-labeling combined with propidium iodide uptake of cells. Methodology was established by the ovarian cancer cell line OvCAR. A total of 17 (81%) patient samples showed a dose-dependent tumor cell elimination by bscEp-CAM x CD3. High and specific tumor cell lysis was seen at bscEp-CAM x CD3 concentrations as low as 1 ng/ml, at very low effector:target ratios and in the absence of T cell costimulation. The high efficacy of the bispecific antibody may be due to the non-restricted activation of tumor-resident cytotoxic T lymphocytes. In clinical trials, the ex vivo data with the T cell-recruiting bispecific antibody bscEp-CAM x CD3 may translate into a high response rate and efficacy of tumor cell elimination. PMID:12673686

  5. Detection of Campylobacter species using monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Young, Colin R.; Lee, Alice; Stanker, Larry H.

    1999-01-01

    A panel of species specific monoclonal antibodies were raised to Campylobacter coli, Campylobacter jejuni and Campylobacter lari. The isotypes, and cross-reactivity profiles of each monoclonal antibody against an extensive panel of micro- organisms, were determined.

  6. Monoclonal antibodies in haematopathology

    SciTech Connect

    Grignani, F.; Martelli, M.F.; Mason, D.Y.

    1985-01-01

    This book contains over 40 selections. Some of the titles are: Oncogene (c-myc, c-myb) amplification in acute myelogenous leukaemia; Ultrastructural characterization of leukaemic cells with monoloclonal antibodies; Origin of B-cell malignancies; Immunohistology of gut lymphomas; and Spurious evidence of lineage infidelity in monocytic leukaemia.

  7. Production of bispecific antibodies in knobs-into-holes using a cell-free expression system

    PubMed Central

    Xu, Yiren; Lee, John; Tran, Cuong; Heibeck, Tyler H; Wang, Willie D; Yang, Junhao; Stafford, Ryan L; Steiner, Alexander R; Sato, Aaron K; Hallam, Trevor J; Yin, Gang

    2015-01-01

    Bispecific antibodies have emerged in recent years as a promising field of research for therapies in oncology, inflammable diseases, and infectious diseases. Their capability of dual target recognition allows for novel therapeutic hypothesis to be tested, where traditional mono-specific antibodies would lack the needed mode of target engagement. Among extremely diverse architectures of bispecific antibodies, knobs-into-holes (KIHs) technology, which involves engineering CH3 domains to create either a knob or a hole in each heavy chain to promote heterodimerization, has been widely applied. Here, we describe the use of a cell-free expression system (Xpress CF) to produce KIH bispecific antibodies in multiple scaffolds, including 2-armed heterodimeric scFv-KIH and one-armed asymmetric BiTE-KIH with tandem scFv. Efficient KIH production can be achieved by manipulating the plasmid ratio between knob and hole, and further improved by addition of prefabricated knob or hole. These studies demonstrate the versatility of Xpress CF in KIH production and provide valuable insights into KIH construct design for better assembly and expression titer. PMID:25427258

  8. Lysis of prostate carcinoma cells by trifunctional bispecific antibodies (alpha EpCAM x alpha CD3).

    PubMed

    Riesenberg, R; Buchner, A; Pohla, H; Lindhofer, H

    2001-07-01

    Bispecific monoclonal antibodies (bsAbs) are a promising immunotherapeutic option for treatment of cancer, especially in situations of minimal residual disease. The combination of an anti-CD3 and anti-tumor-associated antigen antibody redirects cytotoxic T-lymphocytes towards malignant cells. Using a trifunctional bispecific antibody against EpCAM x CD3, that additionally activates Fc gamma R(+) accessory cells via its Fc region, we investigated the interaction between three EpCAM(+) prostate carcinoma cell lines and peripheral blood mononuclear cells (PBMCs) of healthy donors and patients with prostate carcinoma (PC). Visualization was performed by double immunocytochemical methods and computerized sequential video microscopy. Tumor cells and PBMCs supplemented with alpha EpCAM x alpha CD3 in 16-well chamber slides resulted in lysis of tumor cells within 1--3 days without any differences between patient and healthy donor PBMCs. The characteristic necrotic way of tumor cell killing (rounding, swelling, disrupting) could be observed in computerized sequences of video frames. Simultaneously, we could not reveal any form of apoptotic signal using three different apoptotic markers (TUNEL, M30 cyto death, anti-active caspase 3). Within the first 48 hr we observed typical PBMC cluster formation with increasing cell proliferation. PBMCs surrounding the tumor cells were not dominated by CD4(+), CD8(+), or CD14(+) cells. Lymphocytes with pore-forming perforin proteins concentrated towards the tumor target cells. Our combination of double immunocytochemical and computerized video microscopic techniques may serve as an important improvement of validity of cell-cell interaction experiments using in vitro models. (J Histochem Cytochem 49:911-917, 2001) PMID:11410615

  9. Development of a bispecific antibody tetramerized through hetero-associating peptides.

    PubMed

    Osaki, Tomohiro; Fujisawa, Shingo; Kitaguchi, Masahiro; Kitamura, Masaya; Nakanishi, Takeshi

    2015-11-01

    The specific assembly of self-associating peptides can be useful in building a functional antibody complex from small antibody fragments. We have focused on the exceedingly specific heterotetrameric assembly of Lin-2 and Lin-7 (L27) domains, which work as protein-protein interaction modules in many scaffold proteins. Here, we describe a novel method for constructing a highly functional antibody based on the hetero-association of L27 domains. In this study, we used a bacterial expression system to produce a bispecific antibody that was heterotetramerized through L27 domains and that targeted both epidermal growth factor receptor (EGFR) and Fc? receptor III (Fc?RIII or CD16). Gel electrophoresis, mass spectrometry and gel filtration analyses revealed that the constructed recombinant antibody was a disulfide-linked heterotetramer. The tetramerized antibody bound to EGFR and CD16 simultaneously, according to results from flow cytometry and surface plasmon resonance spectroscopy, respectively. Furthermore, we demonstrated that the bispecific antibody showed cytotoxic activity against EGFR-expressing tumor cells by using CD16-positive lymphocytes as effectors, and its cytotoxicity was comparable to that of a commercial therapeutic antibody. Taken together, the results show that our method has high potential for the cost-efficient production of highly active therapeutic antibodies. PMID:26337767

  10. Monoclonal antibodies to mitotic cells.

    PubMed Central

    Davis, F M; Tsao, T Y; Fowler, S K; Rao, P N

    1983-01-01

    Certain proteins or activities are present in mitotic cells but not in interphase cells. These proteins may be synthesized or activated, or both, just prior to mitosis and are responsible for the breakdown of the nuclear envelope and the condensation of chromosomes. To learn more about the nature of these proteins, we raised monoclonal antibodies to mitotic cells. Spleen cells from mice immunized with a 0.15 M NaCl extract of synchronized mitotic HeLa cells were fused with SP2/0-Ag14 mouse myeloma cells, and hybrids were selected in medium containing hypoxanthine, methotrexate, thymidine, and glycine. Two different hybridoma clones secreting antibodies reactive with mitotic and meiotic cells from every species tested were isolated. Chromosomes as well as cytoplasm in mitotic cells reacted with the antibodies, as detected by indirect immunofluorescence. The proteins from mitotic cells were separated by electrophoresis in NaDodSO4/polyacrylamide slab gels, transferred to nitrocellulose sheets, and stained immunochemically. The two antibodies, designated MPM-1 and MPM-2, recognize a family of polypeptides with apparent molecular masses of 0.40 to greater than 200 kilodaltons (kDa). Both antibodies reacted strongly with three polypeptide bands of 182 kDa, 118 kDa, and 70 kDa. Only mitotic cells exhibited the protein bands that were recognized by the antibodies. All these bands were found to be phosphoproteins as shown by 32P labeling and autoradiography and their removal by alkaline phosphatase treatment. Images PMID:6574461

  11. Bispecific antibodies, nanoparticles and cells: bringing the right cells to get the job done

    PubMed Central

    Tang, Junnan; Shen, Deliang; Zhang, Jinying; Ligler, Frances S; Cheng, Ke

    2016-01-01

    Pre-arming therapeutic cells with bispecific antibodies (BiAbs) before infusion can home the cells to specific tissue antigens in the body. With the development of nanotechnology, we developed a novel strategy, namely magnetic bispecific cell engager (MagBICE), that combines BiAbs with biodegradable iron nanoparticles. Compared to conventional BiAbs, the latter enables magnetic targeting and imaging. This editorial discusses current knowledge of BiAbs and their applications in targeting activated T cells to cancerous tissues or targeting bone marrow-derived stem cells to myocardial infarction. We will also discuss the fabrication of MagBICE and its application in treating rodents with myocardial infarction. PMID:26004388

  12. Monoclonal antibody therapeutics with up to five specificities

    PubMed Central

    LaFleur, David W.; Abramyan, Donara; Kanakaraj, Palanisamy; Smith, Rodger G.; Shah, Rutul R.; Wang, Geping; Yao, Xiao-Tao; Kankanala, Spandana; Boyd, Ernie; Zaritskaya, Liubov; Nam, Viktoriya; Puffer, Bridget A.; Buasen, Pete; Kaithamana, Shashi; Burnette, Andrew F.; Krishnamurthy, Rajesh; Patel, Dimki; Roschke, Viktor V.; Kiener, Peter A.; Hilbert, David M.; Barbas III, Carlos F.

    2013-01-01

    The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvβ3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model. PMID:23575268

  13. Anti-CD22/CD20 Bispecific Antibody with Enhanced Trogocytosis for Treatment of Lupus

    PubMed Central

    Rossi, Edmund A.; Chang, Chien-Hsing; Goldenberg, David M.

    2014-01-01

    The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma, leukemia and autoimmune diseases, treating currently over 1500 cases of non-Hodgkin lymphoma, acute lymphoblastic leukemias, Waldenström’s macroglobulinemia, Sjögren’s syndrome, and systemic lupus erythematosus. Because epratuzumab reduces on average only 35% of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. We reported recently that epratuzumab mediates Fc/FcR-dependent membrane transfer from B cells to effector cells via trogocytosis, resulting in a substantial reduction of multiple BCR modulators, including CD22, CD19, CD21, and CD79b, as well as key cell adhesion molecules, including CD44, CD62L, and β7 integrin, on the surface of B cells in peripheral blood mononuclear cells obtained from normal donors or SLE patients. Rituximab has clinical activity in lupus, but failed to achieve primary endpoints in a Phase III trial. This is the first study of trogocytosis mediated by bispecific antibodies targeting neighboring cell-surface proteins, CD22, CD20, and CD19, as demonstrated by flow cytometry and immunofluorescence microscopy. We show that, compared to epratuzumab, a bispecific hexavalent antibody comprising epratuzumab and veltuzumab (humanized anti-CD20 mAb) exhibits enhanced trogocytosis resulting in major reductions in B-cell surface levels of CD19, CD20, CD21, CD22, CD79b, CD44, CD62L and β7-integrin, and with considerably less immunocompromising B-cell depletion that would result with anti-CD20 mAbs such as veltuzumab or rituximab, given either alone or in combination with epratuzumab. A CD22/CD19 bispecific hexavalent antibody, which exhibited enhanced trogocytosis of some antigens and minimal B-cell depletion, may also be therapeutically useful. The bispecific antibody is a candidate for improved treatment of lupus and other autoimmune diseases, offering advantages over administration of the two parental antibodies in combination. PMID:24841238

  14. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V

    2013-08-06

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

  15. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2010-06-15

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

  16. The future of monoclonal antibody technology

    PubMed Central

    Zider, Alexander

    2010-01-01

    With the rapid growth of monoclonal antibody-based products, new technologies have emerged for creating modified forms of antibodies, including fragments, conjugates and multi-specific antibodies. We created a database of 450 therapeutic antibodies in development to determine which technologies and indications will constitute the next generation of antibody products. We conclude that the antibodies of the future will closely resemble the antibodies that have already been approved for commercial sale. PMID:20676053

  17. Application of 300 enhanced fluorescence on a plasmonic chip modified with a bispecific antibody to a sensitive immunosensor.

    PubMed

    Tawa, Keiko; Umetsu, Mitsuo; Nakazawa, Hikaru; Hattori, Takamitsu; Kumagai, Izumi

    2013-09-11

    The grating substrate covered with a metal layer, a plasmonic chip, and a bispecific antibody can play a key role in the sensitive detection of a marker protein with an immunosensor, because of the provision of an enhanced fluorescence signal and the preparation of a sensor surface densely modified with capture antibody, respectively. In this study, one of the tumor markers, a soluble epidermal growth factor receptor (sEGFR), was selected as the target to be detected. The ZnO- and silver-coated plasmonic chip with precise regularity and the appropriate duty ratio in the periodic structure further enhanced the fluorescence intensity. As for sensor surface modification with capture antibody, a bispecific antibody (anti-sEGFR and anti-ZnO antibody), the concentrated bispecific antibody solution was found to nonlinearly form a surface densely immobilized with antibody, because the binding process of a bispecific antibody to the ZnO surface can be a competitive process with adsorption of phosphate. As a result, the interface on the plasmonic chip provided a 300 enhanced fluorescence signal compared with that on a ZnO-coated glass slide, and therefore sEGFR was found to be quantitatively detected in a wide concentration range from 10 nM to 700 fM on our plasmonic surface. PMID:23945148

  18. Cloned transgenic farm animals produce a bispecific antibody for T cell-mediated tumor cell killing

    PubMed Central

    Grosse-Hovest, Ludger; Müller, Sigrid; Minoia, Rosa; Wolf, Eckhard; Zakhartchenko, Valeri; Wenigerkind, Hendrik; Lassnig, Caroline; Besenfelder, Urban; Müller, Mathias; Lytton, Simon D.; Jung, Gundram; Brem, Gottfried

    2004-01-01

    Complex recombinant antibody fragments for modulation of immune function such as tumor cell destruction have emerged at a rapid pace and diverse anticancer strategies are being developed to benefit patients. Despite improvements in molecule design and expression systems, the quantity and stability, e.g., of single-chain antibodies produced in cell culture, is often insufficient for treatment of human disease, and the costs of scale-up, labor, and fermentation facilities are prohibitive. The ability to yield mg/ml levels of recombinant antibodies and the scale-up flexibility make transgenic production in plants and livestock an attractive alternative to mammalian cell culture as a source of large quantities of biotherapeutics. Here, we report on the efficient production of a bispecific single-chain antibody in the serum of transgenic rabbits and a herd of nine cloned, transgenic cattle. The bispecific protein, designated r28M, is directed to a melanoma-associated proteoglycan and the human CD28 molecule on T cells. Purified from the serum of transgenic animals, the protein is stable and fully active in mediating target cell-restricted T cell stimulation and tumor cell killing. PMID:15105446

  19. Improved monoclonal antibodies to halodeoxyuridine

    DOEpatents

    Vanderlaan, M.; Dolbeare, F.A.; Gray, J.W.; Thomas, C.B.

    1983-10-18

    The development, method of production, characterization and methods of use of two hybridomas, CIdU-1 (ATCC Accession No. HB-8321) and CIdU-2 (ATCC Accession No. HB-8320), are described. These secrete IgG/sub 1/(K) immunoglobulins that react with halodeoxyuridine (HdU or halodU) such as bromo, chloro, fluoro and iodo deoxyuridine (BrdU, CldU, FdU and IdU), whether these are free in solution or incorporated into single stranded DNA in whole cells. The antibodies do not react with naturally occurring free nucleic acids or with deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) polymers. These antibodies are suitable for use in enzyme immunoassays for free CldU, FdU, IdU and BrdU and for detecting cells with these nucleotides incorporated into them. The monoclonal antibodies are useful in the detection of the sensitivity of tumor cells to specific chemotherapeutic agents, in the measurement of the rate of cellular DNA synthesis, in the measurement of the rate of proliferation of normal and malignant cells and in the detection of HPRT deficiency in cells. 1 tab.

  20. Monoclonal antibodies that detect live salmonellae.

    PubMed Central

    Torensma, R; Visser, M J; Aarsman, C J; Poppelier, M J; van Beurden, R; Fluit, A C; Verhoef, J

    1992-01-01

    Nine immunoglobulin G and nine immunoglobulin M murine monoclonal antibody-producing hybridomas reactive with live Salmonella bacteria were obtained from several fusions of immune spleen cells and Sp2/0 myeloma cells. The antibodies were selected by the magnetic immunoluminescence assay. The monoclonal antibodies were reactive with serogroups A, B, C1, C2, D, E, and K and Salmonella choleraesuis subsp. diarizonae. Each monoclonal antibody proved to be reactive with a distinct serotype. Clinical isolates belonging to these Salmonella serogroups could be detected. Reactivity with non-Salmonella bacteria proved to be minor. Images PMID:1476430

  1. Monoclonal Antibodies for the Treatment of Cancer

    PubMed Central

    Shuptrine, Casey; Surana, Rishi; Weiner, Louis M.

    2012-01-01

    Over the past decade, the clinical utility of monoclonal antibodies has been realized and antibodies are now a mainstay for the treatment of cancer. Antibodies have the unique capacity to target and kill tumor cells while simultaneously activating immune effectors to kill tumor cells through the complement cascade or antibody-dependent cellular cytotoxicity (ADCC). This multifaceted mechanism of action combined with target specificity underlies the capacity of antibodies to elicit anti-tumor responses while minimizing the frequency and magnitude of adverse events. This review will focus on mechanisms of action, clinical applications and putative mechanisms of resistance to monoclonal antibody therapy in the context of cancer. PMID:22245472

  2. Multimodal Cancer Therapy Involving Oncolytic Newcastle Disease Virus, Autologous Immune Cells, and Bi-Specific Antibodies

    PubMed Central

    Schirrmacher, Volker; Fournier, Philippe

    2014-01-01

    This paper focuses on oncolytic Newcastle disease virus (NDV). This paper summarizes (i) the peculiarities of this virus as an anti-cancer and immune stimulatory agent and (ii) the approaches to further harness this virus as a vector to combat cancer. Special emphasis is given on combining virus therapy with cell therapy and on improving tumor targeting. The review will include some of the authors work on NDV, bi-specific antibodies, and cell therapy as building blocks for a new perspective of multimodal cancer therapy. The broad anti-tumor immune reactivation includes innate and adaptive, tumor antigen (TA) specific and TA independent activities PMID:25309868

  3. Immunologic and pharmacologic concepts of monoclonal antibodies.

    PubMed

    Zuckier, L S; Rodriguez, L D; Scharff, M D

    1989-07-01

    While monoclonal antibodies have solved many of the difficulties of using immunologic reagents for radioimmunodiagnosis and therapy, in the 13 years since their introduction a number of persistent problems remain, most notably a low yield of antibody-producing cells from the fusion process, difficulty in obtaining high-affinity antibodies, and the potential immunogenicity of murine immunoglobulins (Igs). Several solutions are under development, including fusion techniques that enrich for cells producing desired antibodies, production of human-mouse chimeric antibodies by recombinant DNA technology, and the generation of human monoclonal antibodies by promising new approaches. Until these upcoming methodologies are established, and to better direct their development and application, a sound understanding of the pharmacology of presently available native and modified monoclonal antibodies is crucial. Although much has been already determined in this area, a great deal of further clarification remains necessary. PMID:2669128

  4. A novel, native-format bispecific antibody triggering T-cell killing of B-cells is robustly active in mouse tumor models and cynomolgus monkeys

    PubMed Central

    Smith, Eric J.; Olson, Kara; Haber, Lauric J.; Varghese, Bindu; Duramad, Paurene; Tustian, Andrew D.; Oyejide, Adelekan; Kirshner, Jessica R.; Canova, Lauren; Menon, Jayanthi; Principio, Jennifer; MacDonald, Douglas; Kantrowitz, Joel; Papadopoulos, Nicholas; Stahl, Neil; Yancopoulos, George D.; Thurston, Gavin; Davis, Samuel

    2015-01-01

    Bispecific antibodies, while showing great therapeutic potential, pose formidable challenges with respect to their assembly, stability, immunogenicity, and pharmacodynamics. Here we describe a novel class of bispecific antibodies with native human immunoglobulin format. The design exploits differences in the affinities of the immunoglobulin isotypes for Protein A, allowing efficient large-scale purification. Using this format, we generated a bispecific antibody, REGN1979, targeting the B cell marker, CD20, and the CD3 component of the T cell receptor, which triggers redirected killing of B cells. In mice, this antibody prevented growth of B cell tumors and also caused regression of large established tumors. In cynomolgus monkeys, low doses of REGN1979 caused prolonged depletion of B cells in peripheral blood with a serum half-life of approximately 14 days. Further, the antibody induced a deeper depletion of B cells in lymphoid organs than rituximab. This format has broad applicability for development of clinical bispecific antibodies. PMID:26659273

  5. Monoclonal antibodies in the treatment of cancer

    SciTech Connect

    Dillman, R.O.

    1984-01-01

    Potential uses of monoclonal antibodies in anti-cancer treatment include passive serotherapy, radioisotope conjugates, toxin-linked conjugates, and chemotherapy-monoclonal antibody conjugates. The bases for these applications have been founded in research with heterologous antisera, and in some cases with monoclonal antibodies in animal tumor models. Human trials with passive serotherapy have already begun in both hematopoietic and solid tumor malignancies. Promising results have been reported in cutaneous T cell lymphoma with anti-T cell monoclonal antibody, and in nodular lymphoma with anti-idiotype monoclonal antibody. Radioisotope conjugate work appears promising for imaging in both animals and humans, and this work will lay the foundation for possible therapeutic application of radio-immunotherapy. Toxin-linked conjugates are promising in vitro and may have application in autologous bone marrow transplantation. Research with chemotherapy conjugates is also underway. Preliminary results suggest that murine monoclonal antibodies will be well tolerated clinically except in the setting of circulating cells which bear the target antigen, where rapid infusions may be associated with intolerable side effects. In certain diseases, production of endogenous anti-mouse antibodies may also limit application. Advances in the technology for human-human hybridoma production may help solve some of these problems. 132 references.

  6. Monoclonal Antibody That Defines Human Myoepithelium

    NASA Astrophysics Data System (ADS)

    Dairkee, Shahnaz Hashmi; Blayney, Carlene; Smith, Helene S.; Hackett, Adeline J.

    1985-11-01

    We have isolated a mouse monoclonal antibody that, upon immunohistochemical localization in frozen sections, displays specificity for human myoepithelial cells in the resting mammary gland, sweat glands, and salivary glands. Furthermore, this antibody was strongly and homogeneously reactive with frozen sections of 3 of 60 breast carcinoma specimens. Using immunolocalization techniques in conjunction with polyacrylamide gel electrophoresis, we have determined that the reactivity of this monoclonal antibody is directed toward a 51,000-dalton keratin polypeptide. The potential uses of this antibody in the prognosis of human mammary carcinoma and in understanding the role of the myoepithelium in development and differentiation are discussed.

  7. Preparation of astatine-labeled monoclonal antibodies

    SciTech Connect

    Milesz, S.; Norseev, Yu.V.; Szucs, Z. |

    1995-07-01

    In the cationic state astatine forms a stable complex with diethylenetriaminepentaacetic acid. Thanks to this complex, astatine can be bound to monoclonal antibodies of the RYa{sub 1} type. The most favorable conditions for preparing astatine-labeled antibodies are established. The chromatographic analysis and electromigration experiments showed that astatine is firmly linked to a biomolecule in vitro and it did not escape from labeled monoclonal antibodies even under treatment with such highly effective astatine-complexing agent as thiourea. The immune activity of astatine-labeled antibodies did not change even after 20 h.

  8. Fab-based bispecific antibody formats with robust biophysical properties and biological activity.

    PubMed

    Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

    2015-01-01

    A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity. PMID:25774965

  9. Monoclonal Antibody Therapy for Advanced Neuroblastoma

    Cancer.gov

    NCI is sponsoring two clinical trials of a monoclonal antibody called ch14.18, in combination with other drugs, to see if the antibody may be helpful for children or young adults (up to age 21) with relapsed or refractory neuroblastoma.

  10. Rational Design and Characterization of the Novel, Broad and Potent Bispecific HIV-1 Neutralizing Antibody iMabm36

    PubMed Central

    Sun, Ming; Pace, Craig S.; Yao, Xin; Yu, Faye; Padte, Neal N.; Huang, Yaoxing; Seaman, Michael S.; Li, Qihan; Ho, David D.

    2014-01-01

    While broadly neutralizing monoclonal antibodies (bNAbs) have always been considered potential therapeutic options for the prophylactic and treatment of HIV infection, their lack of breadth against all HIV variants has been one of the limiting factors. To provide sufficient neutralization breadth and potency against diverse viruses, including neutralization escape variants, strategies to combine different bNAbs have been explored recently. We rationally designed and engineered a novel bispecific HIV-1 neutralizing antibody (bibNAb), iMabm36, for high potency and breadth against HIV. iMabm36 is composed of the anti-CD4 Ab ibalizumab (iMab) linked to two copies of the single-domain Ab m36 which targets a highly conserved CD4-induced epitope. iMabm36 neutralizes a majority of a large, multi-clade panel of pseudoviruses (96%, n=118) at an IC50 concentration of less than 10 ?g/mL, with 83% neutralized at an IC50 concentration of less than 0.1?g/ml. In addition, iMabm36 neutralizes six replication-competent transmitted-founder viruses to 100% inhibition at a concentration of less than 0.1?g/ml in a PBMC-based neutralizing assay. Mechanistically, improved antiviral activity of iMabm36 is dependent on both CD4 binding activity of iMab component and CD4i binding activity of the m36 component. After characterizing viral resistance to iMabm36 neutralization was due to mutations residing in the bridging sheet of gp120, an optimized m36 variant was engineered that, when fused to iMab, improved antiviral activity significantly. Together inter-dependency of this dual mechanism of action enables iMabm36 to potently inhibit HIV-1 entry. These results demonstrate that mechanistic-based design of bibNAbs could generate potential preventive and therapeutic candidates for HIV/AIDS. PMID:24853313

  11. Immunocytokines and bispecific antibodies: two complementary strategies for the selective activation of immune cells at the tumor site.

    PubMed

    Kiefer, Jonathan D; Neri, Dario

    2016-03-01

    The activation of the immune system for a selective removal of tumor cells represents an attractive strategy for the treatment of metastatic malignancies, which cannot be cured by existing methodologies. In this review, we examine the design and therapeutic potential of immunocytokines and bispecific antibodies, two classes of bifunctional products which can selectively activate the immune system at the tumor site. Certain protein engineering aspects, such as the choice of the antibody format, are common to both classes of therapeutic agents and can have a profound impact on tumor homing performance invivo of individual products. However, immunocytokines and bispecific antibodies display different mechanisms of action. Future research activities will reveal whether an additive of even synergistic benefit can be obtained from the judicious combination of these two types of biopharmaceutical agents. PMID:26864112

  12. Noninvasive brain cancer imaging with a bispecific antibody fragment, generated via click chemistry.

    PubMed

    Luo, Haiming; Hernandez, Reinier; Hong, Hao; Graves, Stephen A; Yang, Yunan; England, Christopher G; Theuer, Charles P; Nickles, Robert J; Cai, Weibo

    2015-10-13

    Early diagnosis remains a task of upmost importance for reducing cancer morbidity and mortality. Successful development of highly specific companion diagnostics targeting aberrant molecular pathways of cancer is needed for sensitive detection, accurate diagnosis, and opportune therapeutic intervention. Herein, we generated a bispecific immunoconjugate [denoted as Bs-F(ab)2] by linking two antibody Fab fragments, an anti-epidermal growth factor receptor (EGFR) Fab and an anti-CD105 Fab, via bioorthogonal "click" ligation of trans-cyclooctene and tetrazine. PET imaging of mice bearing U87MG (EGFR/CD105(+/+)) tumors with (64)Cu-labeled Bs-F(ab)2 revealed a significantly enhanced tumor uptake [42.9 9.5 percentage injected dose per gram (%ID/g); n = 4] and tumor-to-background ratio (tumor/muscle ratio of 120.2 44.4 at 36 h postinjection; n = 4) compared with each monospecific Fab tracer. Thus, we demonstrated that dual targeting of EGFR and CD105 provides a synergistic improvement on both affinity and specificity of (64)Cu-NOTA-Bs-F(ab)2. (64)Cu-NOTA-Bs-F(ab)2 was able to visualize small U87MG tumor nodules (<5 mm in diameter), owing to high tumor uptake (31.4 10.8%ID/g at 36 h postinjection) and a tumor/muscle ratio of 76.4 52.3, which provided excellent sensitivity for early detection. Finally, we successfully confirmed the feasibility of a ZW800-1-labeled Bs-F(ab)2 for near-infrared fluorescence imaging and image-guided surgical resection of U87MG tumors. More importantly, our rationale can be used in the construction of other disease-targeting bispecific antibody fragments for early detection and diagnosis of small malignant lesions. PMID:26417085

  13. Construction of a diabody (small recombinant bispecific antibody) using a refolding system.

    PubMed

    Takemura, S; Asano, R; Tsumoto, K; Ebara, S; Sakurai, N; Katayose, Y; Kodama, H; Yoshida, H; Suzuki, M; Imai, K; Matsuno, S; Kudo, T; Kumagai, I

    2000-08-01

    Diabodies are the recombinant bispecific antibodies (BsAbs), constructed from heterogeneous single-chain antibodies. Usually, diabodies have been prepared from bacterial periplasmic fraction using a co-expression vector (i.e. genes encoding two chains were tandemly located under the same promoter). Some diabodies, however, cannot be expressed as a soluble material owing to inclusion body formation, which limits the utilization of diabodies in various fields. Here we report an improved method for the construction of diabodies using a refolding system. As a model, a bispecific diabody binding to adenocarcinoma-associated antigen MUC1 and to CD3 on T cells was studied. One chain consisted of a VH specific for MUC1 linked to a VL specific for CD3 with a short polypeptide linker (GGGGS). The second was composed of a VL specific for MUC1 linked to a VH specific for CD3. The two hetero scFvs were independently obtained from intracellular insoluble fractions of Escherichia coli, purified, mixed stoichiometrically (at an equivalent molar ratio of 1:1) and refolded. The refolded two hetero scFv has a hetero-dimeric structure, with complete specificity for both target cells [i.e. MUC1 positive cells and CD3 positive lymphokine-activated killer cells with a T cell phenotype (T-LAK)]. Evaluation of the in vitro efficacy of T-LAK with the diabody by growth inhibition assay of cancer cells demonstrated maximum growth inhibition of cancer cells to reach approximately 98% at an effector:target ratio (E:T ratio) of 10, almost identical with that with anti-MUC1xanti-CD3 chemically synthesized BsAbs (c-BsAbs). This is the first report of the construction of a diabody using a refolding system. PMID:10964988

  14. Monoclonal Antibodies as Diagnostics; an Appraisal

    PubMed Central

    Siddiqui, M. Z.

    2010-01-01

    Ever since the development of Hybridoma Technology in 1975 by Kohler and Milstein, our vision for antibodies as tools for research for prevention, detection and treatment of diseases, vaccine production, antigenic characterization of pathogens and in the study of genetic regulation of immune responses and disease susceptibility has been revolutionized. The monoclonal antibodies being directed against single epitopes are homogeneous, highly specific and can be produced in unlimited quantities. In animal disease diagnosis, they are very useful for identification and antigenic characterization of pathogens. Monoclonal antibodies have tremendous applications in the field of diagnostics, therapeutics and targeted drug delivery systems, not only for infectious diseases caused by bacteria, viruses and protozoa but also for cancer, metabolic and hormonal disorders. They are also used in the diagnosis of lymphoid and myeloid malignancies, tissue typing, enzyme linked immunosorbent assay, radio immunoassay, serotyping of microorganisms, immunological intervention with passive antibody, antiidiotype inhibition, or magic bullet therapy with cytotoxic agents coupled with anti mouse specific antibody. Recombinant deoxyribonucleic acid technology through genetic engineering has successfully led to the possibility of reconstruction of monoclonal antibodies viz. chimeric antibodies, humanized antibodies and complementarily determining region grafted antibodies and their enormous therapeutic use. PMID:20582184

  15. Immunocytochemistry with internally labeled monoclonal antibodies

    SciTech Connect

    Cuello, A.C.; Priestley, J.V.; Milstein, C.

    1982-01-01

    One of the advantages of the production of monoclonal antibodies by tissue culture methods is that they can be internally labeled by using appropriate radioactive amino acid in the culture fluid. Thus, radioactive immunological probes of high specific activity can be prepared. Here we report applications of these internally labeled monoclonal antibodies for the direct localization of immunoreactive sites in the central nervous system of the rat at both light and electron microscopic levels (radioimmunocytochemistry). We explored the combined use of radioimmunocytochemistry with immunoenzymatic methods for the simultaneous detection of two antigenic sites: substance P and serotonin or substance P and enkephalin. In neurobiology this procedure could help to clarify certain aspects of transmitter-specific synaptic interactions and the coexistence of neuroactive substances in single neuronal cell bodies or nerve terminals. We also describe the application of radioimmunocytochemistry with internally labeled monoclonal antibodies to quantify the immunoreactions in discrete microscopic areas.

  16. Guiding the Killer and Bringing in Accomplices: Bispecific Antibody Treatment for Malignant Melanoma.

    PubMed

    Szegezdi, Eva; Leverkus, Martin

    2016-02-01

    Discovery of oncogene and immune checkpoint targeting has transformed melanoma therapy in the last 5 years. However, treatment of primary or secondary drug-resistant melanoma remains a challenge. Agents designed to activate the cell death machinery directly, for example by activating the death receptors expressed by melanoma cells, could break drug resistance, and they may achieve long-lasting therapeutic success. He etal. report their studies of an MCSPxDR5 bispecific, tetravalent antibody that can simultaneously target death receptor 5 (DR5, TRAIL-R2) and melanoma-associated chondroitin sulfate proteoglycan (MCSP). This antibody can exert strong and selective DR5-dependent cytotoxic activity against MCSP-expressing melanoma cells. Crosslinking of the antibody with Fc?-receptors increased the cytotoxic potential further, without compromising its selectivity. This approach offers a novel immunotherapeutic tool via coupling of three cooperating processes: delivering the death receptor agonist to the malignant cell population, potent activation of DR5-mediated cell death signaling, and recruitment of Fc?-receptor-carrying immune cells that can mount an immune response against the tumor cells. PMID:26802233

  17. Chemically Programmed Bispecific Antibody Targeting Legumain Protease and ?v?3 Integrin Mediates Strong Antitumor Effects.

    PubMed

    Liu, Yuan; Goswami, Rajib K; Liu, Cheng; Sinha, Subhash C

    2015-07-01

    A chemically programmed bispecific antibody (cp-bsAb) that targeted cysteine protease legumain and ?v?3 integrin has been prepared using the aldolase antibody chemical programming (AACP) strategy. In vitro evaluation of the anti-legumain, anti-integrin cp-bsAb and its comparison with cpAbs targeting either integrin or legumain have shown that the former possesses superior functions, including receptor binding and inhibitory effects on cell proliferation as well as capillary tube formation, among all three cpAbs. The anti-legumain, anti-integrin cp-bsAb also inhibited growth of primary tumor more effectively than either anti-legumain or anti-integrin cpAb as observed in the MDA-MB-231 human breast cancer mouse model. The AACP-based cp-bsAb, which contains a generic aldolase antibody, can also serve as a suitable platform for combination therapy, where two equally potent compounds are used to target extracellular receptors. PMID:26024761

  18. [Current situations and the future prospect of monoclonal antibody products].

    PubMed

    Yamaguchi, Teruhide

    2014-01-01

    Monoclonal antibody products and monoclonal antibody-based biopharmaceuticals have shown considerable effectiveness in the treatment for variety of diseases; cancer, auto-immune/auto-inflammation diseases and so on. Significant advance in monoclonal antibody products for cancer treatments was made with antibody-drug conjugates (ADC), and antibodies for blockade of immune checkpoints. Already 3 ADCs and 2 anti-immune-checkpoint antibodies products have been approved, and these monoclonal antibody-related product pipelines reach over 30. On the other hand, EU approved first monoclonal-antibody biosimilar, RemsimaTM (infliximab), suggesting that other monoclonal-antibody biosmilars will follow to the market. In this paper, several new issues about monoclonal antibody products will be discussed. PMID:25707201

  19. Hapten-Binding Bispecific Antibodies for the Targeted Delivery of SiRNA and SiRNA-Containing Nanoparticles.

    PubMed

    Thorey, Irmgard S; Grote, Michael; Mayer, Klaus; Brinkmann, Ulrich

    2016-01-01

    Hapten-binding bispecific antibodies (bsAbs) are effective and versatile tools for targeting diverse payloads, including siRNAs, to specific cells and tissues. In this chapter, we provide examples for successful SiRNA delivery using this powerful targeting platform. We further provide protocols for designing and producing bsAbs, for combining bsAbs with SiRNA into functional complexes, and achieving specific mRNA knockdown in cells by using these functional complexes. PMID:26472454

  20. MT110: a novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors.

    PubMed

    Brischwein, Klaus; Schlereth, Bernd; Guller, Benjamin; Steiger, Carola; Wolf, Andreas; Lutterbuese, Ralf; Offner, Sonja; Locher, Mathias; Urbig, Thomas; Raum, Tobias; Kleindienst, Petra; Wimberger, Pauline; Kimmig, Rainer; Fichtner, Iduna; Kufer, Peter; Hofmeister, Robert; da Silva, Antonio J; Baeuerle, Patrick A

    2006-03-01

    We have developed a novel single-chain Ep-CAM-/CD3-bispecific single-chain antibody construct designated MT110. MT110 redirected unstimulated human peripheral T cells to induce the specific lysis of every Ep-CAM-expressing tumor cell line tested. MT110 induced a costimulation independent polyclonal activation of CD4- and CD8-positive T cells as seen by de novo expression of CD69 and CD25, and secretion of interferon gamma, tumor necrosis factor alpha, and interleukins 2, 4 and 10. CD8-positive T cells made the major contribution to redirected tumor cell lysis by MT110. With a delay, CD4-positive cells could also contribute presumably as consequence of a dramatic upregulation of granzyme B expression. MT110 was highly efficacious in a NOD/SCID mouse model with subcutaneously growing SW480 human colon cancer cells. Five daily doses of 1 microg MT110 on days 0-4 completely prevented tumor outgrowth in all mice treated. The bispecific antibody construct also led to a durable eradication of established tumors in all mice treated with 1 microg doses of MT110 on days 8-12 after tumor inoculation. Finally, MT110 could eradicate patient-derived metastatic ovarian cancer tissue growing under the skin of NOD/SCID mice. MT110 appears as an attractive bispecific antibody candidate for treatment of human Ep-CAM-overexpressing carcinomas. PMID:16139892

  1. Cytosine arabinoside promotes cytotoxic effect of T cells on leukemia cells mediated by bispecific antibody.

    PubMed

    Li, Wei; Fan, DongMei; Yang, Ming; Yan, Yan; Shi, RuiZan; Cheng, JunPing; Li, ZhenZhen; Zhang, MengNan; Wang, JianXiang; Xiong, Dongsheng

    2013-08-01

    Chemotherapeutic drugs can enhance an immune response of the host against the tumor in addition to killing cancer cells by direct cytotoxicity. Therefore, the combination of chemotherapy and immunotherapy is a promising approach for eliminating tumors, particularly in advanced stages. A strategic medication is to use a bispecific antibody format that is capable of recruiting polyclonal T cells around antibody-target-expressing tumor cells. Recently, we have constructed a bispecific antibody, anti-CD3anti-CD19, in a diabody configuration. In this study, we measured B7 family members B7.1 (CD80) and B7.2 (CD86) expressed on a CD19(+) human leukemia cell line, Nalm-6, stimulated by cytosine arabinoside (Ara-C). We found that a low concentration of Ara-C could upregulate CD80 expressed on CD19(+) Nalm-6 cells. The cytotoxicity of T lymphocytes against Nalm-6 cells in vitro and in vivo mediated by the anti-CD3anti-CD19 diabody with or without a low dose of Ara-C was compared. The combination of the anti-CD3anti-CD19 diabody and Ara-C showed the greatest effectiveness in enhancing the cytotoxicity of T cells against the tumor cells in vitro and in vivo. Activated T cells expressed higher levels of CD25 and CD69 and released more interleukin 2. Both perforin/granzyme B system and Fas/FasL pathway were involved in the diabody-induced T-cell cytotoxicity. Moreover, the activated T cells could upregulate ICAM-3 expression on Nalm-6 cells, and inhibition of LFA-1-ICAM-3 interaction impaired cytotoxicity of T cells. It was noted that Ara-C could upregulate CD80 expressed on two of five specimens of acute B lymphoblastic leukemia patient-derived cells. Cytotoxicity of T cells against these two patient-derived cells was enhanced in the presence of the anti-CD3anti-CD19 diabody. These findings indicate that treatment strategy using both cytotoxic lymphocyte-based immunotherapy and chemotherapy may have synergistic effects. PMID:23879717

  2. Monoclonal antibodies reactive with chicken interleukin-17

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In our previous study chicken interleukin -17 (chIL-17) gene was cloned from the expressed sequence tag (EST) cDNA library and initially analyzed. To further investigate biological properties of chicken IL-17, six monoclonal antibodies (mAbs) against bacterially expressed protein were produced and c...

  3. Monoclonal antibodies against chicken interleukin-6

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Monoclonal antibodies (mAb) were produced against a recombinant (r) chicken interleukin-6 (IL-6). Eight mAbs that were produced were tested for isotype; ability to inhibit recombinant forms of chicken (ch), human (h) and murine (m) IL-6; and recognition of rchIL-6 by Western immunoblotting. The mA...

  4. Subcutaneous Administration of Monoclonal Antibodies in Oncology

    PubMed Central

    Jackisch, C.; Mller, V.; Maintz, C.; Hell, S.; Ataseven, B.

    2014-01-01

    Treatment with monoclonal antibodies (mabs) has become an established component of oncological therapy. The monoclonal antibodies available for this purpose are mainly administered intravenously in individually adapted doses according to body weight over longer treatment times. For other chronic diseases such as, for example, diabetes mellitus, the subcutaneous administration of drugs is an established therapy option. For the subcutaneous administration of larger volumes as needed for mab solutions the extracellular matrix of the subcutaneous tissue represents a problem. The co-formulation with recombinant human hyaluronidase makes the relatively pain-free administration of larger fluid volumes and thus the subcutaneous administration of monoclonal antibodies possible, as illustrated by the development of a subcutaneous formulation of trastuzumab. This constitutes a less invasive, time-optimised and flexible form of administration for patients with HER2-positive breast cancer that, with its fixed dosing possibilities, contributes to therapeutic safety. The example of trastuzumab shows that the subcutaneous administration of monoclonal antibodies can simplify oncological long-term therapy not only for the patients but also for the medical personnel. PMID:25076790

  5. Human derived dimerization tag enhances tumor killing potency of a T-cell engaging bispecific antibody

    PubMed Central

    Ahmed, Mahiuddin; Cheng, Ming; Cheung, Irene Y; Cheung, Nai-Kong V

    2015-01-01

    Bispecific antibodies (BsAbs) have proven highly efficient T cell recruiters for cancer immunotherapy by virtue of one tumor antigen-reactive single chain variable fragment (scFv) and another that binds CD3. In order to enhance the antitumor potency of these tandem scFv BsAbs (tsc-BsAbs), we exploited the dimerization domain of the human transcription factor HNF1? to enhance the avidity of a tsc-BsAb to the tumor antigen disialoganglioside GD2 while maintaining functional monovalency to CD3 to limit potential toxicity. The dimeric tsc-BsAb showed increased avidity to GD2, enhanced T cell mediated killing of neuroblastoma and melanoma cell lines in vitro (3237 fold), exhibited a near 4-fold improvement in serum half-life, and enhanced tumor ablation in mouse xenograft models. We propose that the use of this HNF1?-derived dimerization tag may be a novel and effective strategy to increase the potency of T-cell engaging antibodies for clinical cancer immunotherapy. PMID:26137406

  6. Microencapsulation of therapeutic bispecific antibodies producing cells: immunotherapeutic organoids for cancer management.

    PubMed

    Saenz del Burgo, Laura; Compte, Marta; Aceves, Mnica; Hernndez, Rosa Mara; Sanz, Laura; lvarez-Vallina, Luis; Pedraz, Jose Luis

    2015-02-01

    Regardless of the important therapeutic advances developed over the last years for the management of cancer, the fact is that many patients still suffer from a tremendous reduction on their quality of life due to lack of complete selectivity of conventionally administered chemotherapeutic drugs. In the search of more efficacious tumor-targeted therapies, the use of bispecific antibodies (bsAbs) capable of simultaneous binding to tumor-associated antigens and to an activating receptor, such as CD3, has emerged as a promising approach. With the intention to complementing and improving this cancer immunotherapy, human HEK-293 cells have been genetically modified ex vivo to secrete a recombinant anti-CEA (carcinoembryonic antigen)??anti-CD3 bsAb. After encapsulation in alginate-poly-l-lysine microcapsules, bsAb-secreting HEK-293 cells were monitorized for several weeks. This system has proved to be feasible for the maintenance of cell growth and recombinant antibody production giving proof-of-concept of its use as immunotherapeutic organoids in cancer treatment. PMID:25338126

  7. SPECT assay of radiolabeled monoclonal antibodies

    SciTech Connect

    Jaszczak, R.J.

    1991-05-01

    The accurate determination of the biodistribution of radiolabeled monoclonal antibodies (MoAbs) is important for calculation of dosimetry and evaluation of pharmacokinetic variables such as antibody dose and route of administration. A major long-term objective of this proposal is to determine the utility of single photon emission computed tomography (SPECT) for quantifying the biodistribution of monoclonal antibodies labeled with the clinically relevant radionuclide iodine-123 (I-123). The pharmacokinetics of I-123 labeled MoAbs will be determined by the SPECT in non-human primates. The errors associated with the SPECT measurements will be assessed with Monte Carlo simulations and by scanning phantoms containing I-123 activity in regions of uniform and nonuniform attenuation. The ability of SPECT to quantify I-123 distributions will be assessed, and new acquisition geometries and reconstruction algorithms for improved quantification will be evaluated. 33 refs.

  8. A novel bispecific EGFR/Met antibody blocks tumor-promoting phenotypic effects induced by resistance to EGFR inhibition and has potent antitumor activity

    PubMed Central

    Castoldi, R; Ecker, V; Wiehle, L; Majety, M; Busl-Schuller, R; Asmussen, M; Nopora, A; Jucknischke, U; Osl, F; Kobold, S; Scheuer, W; Venturi, M; Klein, C; Niederfellner, G; Sustmann, C

    2013-01-01

    Simultaneous targeting of epidermal growth factor receptor (EGFR) and Met in cancer therapy is under pre-clinical and clinical evaluation. Here, we report the finding that treatment with EGFR inhibitors of various tumor cells, when stimulated with hepatocyte growth factor (HGF) and EGF, results in transient upregulation of phosphorylated AKT. Furthermore, EGFR inhibition in this setting stimulates a pro-invasive phenotype as assessed in Matrigel-based assays. Simultaneous treatment with AKT and EGFR inhibitors abrogates this invasive growth, hence functionally linking signaling and phenotype. This observation implies that during treatment of tumors a balanced ratio of EGFR and Met inhibition is required. To address this, we designed a bispecific antibody targeting EGFR and Met, which has the advantage of a fixed 2:1 stoichiometry. This bispecific antibody inhibits proliferation in tumor cell cultures and co-cultures with fibroblasts in an additive manner compared with treatment with both single agents. In addition, cell migration assays reveal a higher potency of the bispecific antibody in comparison with the antibodies' combination at low doses. We demonstrate that the bispecific antibody inhibits invasive growth, which is specifically observed with cetuximab. Finally, the bispecific antibody potently inhibits tumor growth in a non-small cell lung cancer xenograft model bearing a strong autocrine HGF-loop. Together, our findings strongly support a combination treatment of EGFR and Met inhibitors and further evaluation of resistance mechanisms to EGFR inhibition in the context of active Met signaling. PMID:23812422

  9. Serovar determination of Chlamydia trachomatis isolates by using type-specific monoclonal antibodies.

    PubMed Central

    Newhall, W J; Terho, P; Wilde, C E; Batteiger, B E; Jones, R B

    1986-01-01

    A panel of 15 monoclonal antibodies was prepared that could distinguish among the 15 serovars of Chlamydia trachomatis. Twelve of these antibodies were specific for a single serovar (A, B, C, D, E, F, G, H, I, K, L1, and L2) and three were specific for two serovars (B/Ba, C/J, and C/L3). Ten of the serovar-specific and two of the bispecific antibodies were shown by immunoblotting to recognize epitopes on the major outer membrane protein. These data provide evidence that such epitopes are closely correlated with and may be partly responsible for the antigenic variations detected by microimmunofluorescence that distinguish the currently recognized serovars. When used in a radioimmunoassay, these antibodies correctly identified the serovar of 17 strains that had been serotyped by the microimmunofluorescence test. In addition, we found that the chlamydial antigen derived from 1.0 cm2 of an infected HeLa cell monolayer was sufficient to allow serotyping with these antibodies. Thus, these monoclonal antibodies may provide a rapid and reliable alternative to mouse immunization and microimmunofluorescence for serotyping of clinical isolates. Images PMID:2422202

  10. In vitro and in vivo antitumor effects of recombinant bispecific antibodies based on humanized anti-EGFR antibody.

    PubMed

    Watanabe, Yasuhiro; Asano, Ryutaro; Arai, Kyoko; Shimomura, Ippei; Ogata, Hiromi; Kawaguchi, Hiroko; Hayashi, Hiroki; Ohtsuka, Hideo; Yoshida, Hiroshi; Katayose, Yu; Egawa, Shinichi; Nakanishi, Takeshi; Umetsu, Mitsuo; Yasui, Hiroshi; Ishida, Tadao; Imai, Kohzoh; Kudo, Toshio; Unno, Michiaki; Kumagai, Izumi

    2011-10-01

    We performed in vitro and in vivo experiments of the anti-epidermal growth factor receptor (EGFR) x anti-CD3 bispecific diabody (hEx3-Db) with the IgG-like bispecific antibodies (BsAbs) (hEx3-scFv-Fc and hEx3-scDb-Fc) and the anti-EGFR therapeutic antibody cetuximab to assess the effect of BsAbs on cancer growth inhibition. In vitro, efficacy of the BsAbs and cetuximab were compared by growth inhibition assays of human cell lines of bile duct (TFK-1, HuCC-T1, OCUCh-LM1), epidermoid (A431), gastric (Kato-III), colon (DLD-1, SW480), and breast (SK-BR-3, MCF-7) cancer. In vivo, in three mouse models, we evaluated the anti-tumor activity of hEx3-Db and cetuximab, assessed the effect of hEx3-Db alone, and compared the antitumor activity of hEx3-Db with the IgG-like BsAbs. In vitro, hEx3-scFv-Fc showed nearly 100% killing activity for all cell lines. Both in vitro and in vivo, hEx3-Db needed CD3-positive phenotypes to induce a growth inhibitory effect. In contrast, IgG-like BsAbs showed monotherapeutic effects in vivo by inducing antibody-dependent cellular cytotoxicity (ADCC) similar to cetuximab. However, enhancement was not observed when lymphokine-activated killer cells with the T-cell phenotype were co-injected. Results suggest that IgG-like BsAbs could not efficiently direct T lymphocytes toward tumor cells to induce ADCC due to steric hindrance on binding to CD3- and Fc-receptor-positive phenotypes. Although hEx3-scFv-Fc showed high cytotoxicity in vitro, its high molecular weight limits its usefulness. With an in vivo effect comparable to hEx3-scFv-Fc and its realistic molecular weight, hEx3-scDb-Fc shows promise as a novel recombinant therapeutic antibody and may be modified to enhance its potency by prevention of steric hindrance. PMID:21743971

  11. SPECT assay of radiolabeled monoclonal antibodies

    SciTech Connect

    Jaszczak, R.J.

    1992-02-01

    The accurate determination of the biodistribution of radiolabeled monoclonal antibodies (MoAbs) is important for calculation of dosimetry and evaluation of pharmacokinetic variables such as antibody dose and route of administration. The hypothesis of this application is that the biodistribution of radiolabeled monoclonal antibodies (MoAbs) can be quantitatively determined using single photon emission computed tomography (SPECT). The major thrusts during the third year include the continued development and evaluation of improved 3D SPECT acquisition and reconstruction approaches to improve quantitative imaging of radiolabeled monoclonal antibodies (MoAbs), and the implementation and evaluation of algorithms to register serial SPECT image data sets, or to register 3D SPECT images with 3D image data sets acquired from positron emission tomography (PEI) and magnetic resonance images (MRI). The research has involved the investigation of statistical models and iterative reconstruction algorithms that accurately account for the physical characteristics of the SPECT acquisition system. It is our belief that SPECT quantification can be improved by accurately modeling the physical processes such as attenuation, scatter, geometric collimator response, and other factors that affect the measured projection data.

  12. Antitumor efficacy of a bispecific antibody that targets HER2 and activates T cells.

    PubMed

    Junttila, Teemu T; Li, Ji; Johnston, Jennifer; Hristopoulos, Maria; Clark, Robyn; Ellerman, Diego; Wang, Bu-Er; Li, Yijin; Mathieu, Mary; Li, Guangmin; Young, Judy; Luis, Elizabeth; Lewis Phillips, Gail; Stefanich, Eric; Spiess, Christoph; Polson, Andrew; Irving, Bryan; Scheer, Justin M; Junttila, Melissa R; Dennis, Mark S; Kelley, Robert; Totpal, Klara; Ebens, Allen

    2014-10-01

    Clinical results from the latest strategies for T-cell activation in cancer have fired interest in combination immunotherapies that can fully engage T-cell immunity. In this study, we describe a trastuzumab-based bispecific antibody, HER2-TDB, which targets HER2 and conditionally activates T cells. HER2-TDB specifically killed HER2-expressing cancer cells at low picomolar concentrations. Because of its unique mechanism of action, which is independent of HER2 signaling or chemotherapeutic sensitivity, HER2-TDB eliminated cells refractory to currently approved HER2 therapies. HER2-TDB exhibited potent antitumor activity in four preclinical model systems, including MMTV-huHER2 and huCD3 transgenic mice. PD-L1 expression in tumors limited HER2-TDB activity, but this resistance could be reversed by anti-PD-L1 treatment. Thus, combining HER2-TDB with anti-PD-L1 yielded a combination immunotherapy that enhanced tumor growth inhibition, increasing the rates and durability of therapeutic response. PMID:25228655

  13. Recent developments in monoclonal antibody radiolabeling techniques

    SciTech Connect

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

    1989-01-01

    Monoclonal antibodies (MAbs) have shown the potential to serve as selective carriers of radionuclides to specific in vivo antigens. Accordingly, there has been an intense surge of research activity in an effort to develop and evaluate MAb-based radiopharmaceuticals for tumor imaging (radioimmunoscintigraphy) and therapy (radioimmunotherapy), as well as for diagnosing nonmalignant diseases. A number of problems have recently been identified, related to the MAbs themselves and to radiolabeling techniques, that comprise both the selectivity and the specificity of the in vivo distribution of radiolabeled MAbs. This paper will address some of these issues and primarily discuss recent developments in the techniques for radiolabeling monoclonal antibodies that may help resolve problems related to the poor in vivo stability of the radiolabel and may thus produce improved biodistribution. Even though many issues are identical with therapeutic radionuclides, the discussion will focus mainly on radioimmunoscintigraphic labels. 78 refs., 6 tabs.

  14. Identification of Streptococcus sobrinus with monoclonal antibodies.

    PubMed Central

    de Soet, J J; van Dalen, P J; Appelmelk, B J; de Graaff, J

    1987-01-01

    Identification of Streptococcus sobrinus is often difficult to perform because of the great resemblance of the organism to other oral streptococcal species. Therefore, monoclonal antibodies were prepared which were shown to be highly specific for S. sobrinus. Cross-reactivity with other oral microorganisms has not been observed in an enzyme-linked immunosorbent assay and an immunofluorescence assay. These monoclonal antibodies belonged to the subclass immunoglobulin G2b. To be certain that the strains used in cross-reactivity tests were S. sobrinus, their DNA base composition was measured as a golden standard. Additional tests like colony morphology and sugar fermentation with the API 20 Strep system (Analytab Products, Montalieu-Vercieu, France) were performed. These additional tests turned out to be necessary because 100% correct identification could not be obtained by separate tests. Immunological characterization with the clones OMVU10 and OMVU11 proved to be discriminative between S. sobrinus and other streptococcal species. PMID:3323224

  15. Next generation and biosimilar monoclonal antibodies

    PubMed Central

    2011-01-01

    The Next Generation and Biosimilar Monoclonal Antibodies: Essential Considerations Towards Regulatory Acceptance in Europe workshop, organized by the European Centre of Regulatory Affairs Freiburg (EUCRAF), was held February 34, 2011 in Freiburg, Germany. The workshop attracted over 100 attendees from 15 countries, including regulators from 11 agencies, who interacted over the course of two days. The speakers presented their authoritative views on monoclonal antibodies (mAbs) as attractive targets for development, the experience to date with the regulatory process for biosimilar medicinal products, the European Medicines Agency draft guideline on biosimilar mAbs, as well as key elements in the development of mAbs. Participants engaged in many lively discussions, and much speculation on the nature of the quality, non-clinical and clinical requirements for authorization of biosimilar mAbs. PMID:21487235

  16. Process analytics for purification of monoclonal antibodies.

    PubMed

    Flatman, Stephen; Alam, Imtiaz; Gerard, Jeffery; Mussa, Nesredin

    2007-03-15

    The application of appropriate analytical methods is an essential requirement for the purification of therapeutic antibodies. A range of analytical methods need to be employed to effectively determine the purity, identity, integrity and activity of these important class of pharmaceuticals. These include notably electrophoresis, high performance liquid chromatography and immunoassays. Regulatory and industry demands in recent years have brought the need for improvements and many have been successfully implemented. This article reviews the current analytical methods applied to support the purification of monoclonal antibodies. PMID:17161664

  17. Microbials for the production of monoclonal antibodies and antibody fragments

    PubMed Central

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

    2014-01-01

    Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. PMID:24183828

  18. Retargeting T cells to GD2 pentasaccharide on human tumors using Bispecific humanized antibody.

    PubMed

    Xu, Hong; Cheng, Ming; Guo, Hongfen; Chen, Yuedan; Huse, Morgan; Cheung, Nai-Kong V

    2015-03-01

    Anti-disialoganglioside GD2 IgG antibodies have shown clinical efficacy in solid tumors that lack human leukocyte antigens (e.g., neuroblastoma) by relying on Fc-dependent cytotoxicity. However, there are pain side effects secondary to complement activation. T-cell retargeting bispecific antibodies (BsAb) also have clinical potential, but it is thus far only effective against liquid tumors. In this study, a fully humanized hu3F8-BsAb was developed, in which the anti-CD3 huOKT3 single-chain Fv fragment (ScFv) was linked to the carboxyl end of the anti-GD2 hu3F8 IgG1 light chain, and was aglycosylated at N297 of Fc to prevent complement activation and cytokine storm. In vitro, hu3F8-BsAb activated T cells through classic immunologic synapses, inducing GD2-specific tumor cytotoxicity at femtomolar EC50 with >10?-fold selectivity over normal tissues, releasing Th1 cytokines (TNF?, IFN?, and IL2) when GD2? tumors were present. In separate murine neuroblastoma and melanoma xenograft models, intravenous hu3F8-BsAb activated T cells in situ and recruited intravenous T cells for tumor ablation, significantly prolonging survival from local recurrence or from metastatic disease. Hu3F8-BsAb, but not control BsAb, drove T cells and monocytes to infiltrate tumor stroma. These monocytes were necessary for sustained T-cell proliferation and/or survival and contributed significantly to the antitumor effect. The in vitro and in vivo antitumor properties of hu3F8-BsAb and its safety profile support its further clinical development as a cancer therapeutic, and provide the rationale for exploring aglycosylated IgG-scFv as a structural platform for retargeting human T cells. PMID:25542634

  19. Retargeting T cells to GD2 pentasaccharide on human tumors using bispecific humanized antibody

    PubMed Central

    Xu, Hong; Cheng, Ming; Guo, Hongfen; Chen, Yuedan; Huse, Morgan; Cheung, Nai-Kong V.

    2015-01-01

    Anti-disialoganglioside GD2 IgG antibodies have shown clinical efficacy in solid tumors that lack human leukocyte antigens (e.g. neuroblastoma) by relying on Fc-dependent cytotoxicity. However, there are pain side effects secondary to complement activation. T-cell retargeting bispecific antibodies (BsAb) also have clinical potential, but it is thus far only effective against liquid tumors. In this study, a fully humanized hu3F8-BsAb was developed, in which the anti-CD3 huOKT3 single chain Fv fragment (ScFv) was linked to the carboxyl end of the anti-GD2 hu3F8 IgG1 light chain, and was aglycosylated at N297 of Fc to prevent complement activation and cytokine storm. In vitro, hu3F8-BsAb activated T cells through classic immunological synapses, inducing GD2-specific tumor cytotoxicity at femtomolar EC50 with >105-fold selectivity over normal tissues, releasing Th1 cytokines (TNF?, IFN? and IL2) when GD2(+) tumors were present. In separate murine neuroblastoma and melanoma xenograft models, intravenous hu3F8-BsAb activated T cells in situ and recruited intravenous T cells for tumor ablation, significantly prolonging survival from local recurrence or from metastatic disease. Hu3F8-BsAb, but not control BsAb, drove T cells and monocytes to infiltrate tumor stroma. These monocytes were necessary for sustained T-cell proliferation and/or survival and contributed significantly to the antitumor effect. The in vitro and in vivo antitumor properties of hu3F8-BsAb and its safety profile support its further clinical development as a cancer therapeutic, and provide the rationale for exploring aglycosylated IgG-scFv as a structural platform for retargeting human T cells. PMID:25542634

  20. The bi-specific CD3 NCAM antibody: a model to preactivate T cells prior to tumour cell lysis

    PubMed Central

    JENSEN, M; ERNESTUS, K; KEMSHEAD, J; KLEHR, M; VON BERGWELT-BAILDON, M S; SCHINKTHE, T; SCHULTZE, J L; BERTHOLD, F

    2003-01-01

    To target the neural cell adhesion molecule (NCAM, CD56) on neuroblastoma by T cell-based immunotherapy we have generated a bi-specific CD3 NCAM antibody (OE-1). This antibody can be used to redirect T cells to NCAM+ cells. Expectedly, the antibody binds specifically to NCAM+ neuroblastoma cells and CD3+ T cells. OE-1 induces T cell activation, expansion and effector function in peripheral blood mononuclear cell (PBMC)-derived CD4+ and CD8+ T cells. T cell activation was shown to depend on the presence of normal natural killer (NK) cells in the culture. Interestingly, while PBMC- derived T cells were activated by OE-1, NK cells were almost completely depleted, suggesting that T cells activated by OE-1 deleted the NK cells. Activated CD4+ and CD8+ T cells differentiate into a larger CCR7+ central memory and a smaller CCR7 effector memory cell population. Most importantly, preactivated T cells were highly cytotoxic for neuroblastoma cells. In eight of 11 experiments tumour-directed cytotoxicity was enhanced when NK cells were present during preactivation with OE-1. These data strongly support a bi-phasic therapeutic concept of primarily stimulating T cells with the bi-specific antibody in the presence of normal NCAM+ cells to induce T cell activation, migratory capacity and finally tumour cell lysis. PMID:14616785

  1. Bispecific Antibody Affords Complete Post-Exposure Protection of Mice from Both Ebola (Zaire) and Sudan Viruses.

    PubMed

    Frei, Julia C; Nyakatura, Elisabeth K; Zak, Samantha E; Bakken, Russell R; Chandran, Kartik; Dye, John M; Lai, Jonathan R

    2016-01-01

    Filoviruses (Ebola and Marburg) cause severe hemorrhagic fever. There are five species of ebolavirus; among these, the Ebola (Zaire) and Sudan viruses (EBOV and SUDV, respectively) are highly pathogenic and have both caused recurring, large outbreaks. However, the EBOV and SUDV glycoprotein (GP) sequences are 45% divergent and thus antigenically distinct. Few antibodies with cross-neutralizing properties have been described to date. We used antibody engineering to develop novel bispecific antibodies (Bis-mAbs) that are cross-reactive toward base epitopes on GP from EBOV and SUDV. These Bis-mAbs exhibit potent neutralization against EBOV and SUDV GP pseudotyped viruses as well as authentic pathogens, and confer a high degree (in one case 100%) post-exposure protection of mice from both viruses. Our studies show that a single agent that targets the GP base epitopes is sufficient for protection in mice; such agents could be included in panfilovirus therapeutic antibody cocktails. PMID:26758505

  2. Bispecific Antibody Affords Complete Post-Exposure Protection of Mice from Both Ebola (Zaire) and Sudan Viruses

    PubMed Central

    Frei, Julia C.; Nyakatura, Elisabeth K.; Zak, Samantha E.; Bakken, Russell R.; Chandran, Kartik; Dye, John M.; Lai, Jonathan R.

    2016-01-01

    Filoviruses (Ebola and Marburg) cause severe hemorrhagic fever. There are five species of ebolavirus; among these, the Ebola (Zaire) and Sudan viruses (EBOV and SUDV, respectively) are highly pathogenic and have both caused recurring, large outbreaks. However, the EBOV and SUDV glycoprotein (GP) sequences are 45% divergent and thus antigenically distinct. Few antibodies with cross-neutralizing properties have been described to date. We used antibody engineering to develop novel bispecific antibodies (Bis-mAbs) that are cross-reactive toward base epitopes on GP from EBOV and SUDV. These Bis-mAbs exhibit potent neutralization against EBOV and SUDV GP pseudotyped viruses as well as authentic pathogens, and confer a high degree (in one case 100%) post-exposure protection of mice from both viruses. Our studies show that a single agent that targets the GP base epitopes is sufficient for protection in mice; such agents could be included in panfilovirus therapeutic antibody cocktails. PMID:26758505

  3. Monoclonal antibodies specific for sickle cell hemoglobin

    SciTech Connect

    Jensen, R.H.; Vanderlaan, M.; Grabske, R.J.; Branscomb, E.W.; Bigbee, W.L.; Stanker, L.H.

    1985-01-01

    Two mouse hybridoma cell lines were isolated which produce monoclonal antibodies that bind hemoglobin S. The mice were immunized with peptide-protein conjugates to stimulate a response to the amino terminal peptide of the beta chain of hemoglobin S, where the single amino acid difference between A and S occurs. Immunocharacterization of the antibodies shows that they bind specifically to the immunogen peptide and to hemoglobin S. The specificity for S is high enough that one AS cell in a mixture with a million AA cells is labeled by antibody, and such cells can be analyzed by flow cytometry. Immunoblotting of electrophoretic gels allows definitive identification of hemoglobin S as compared with other hemoglobins with similar electrophoretic mobility. 12 references, 4 figures.

  4. Monoclonal antibodies and method for detecting dioxins and dibenzofurans

    DOEpatents

    Vanderlaan, Martin (San Ramon, CA); Stanker, Larry H. (Livermore, CA); Watkins, Bruce E. (Livermore, CA); Bailey, Nina R. (Berkley, CA)

    1989-01-01

    Compositions of matter are described which include five monoclonal antibodies that react with dioxins and dibenzofurans, and the five hybridomas that produce these monoclonal antibodies. In addition, a method for the use of these antibodies in a sensitive immunoassay for dioxins and dibenzofurans is given, which permits detection of these pollutants in samples at concentrations in the range of a few parts per billion.

  5. Bi-specific antibodies with high antigen-binding affinity identified by flow cytometry.

    PubMed

    Xu, Liming; Zhang, Yu; Wang, Qiuying; Zhao, Jingzhuang; Liu, Miao; Guo, Mo; Jiang, Yuanyuan; Cao, Hongwei; Li, Qingcui; Ren, Guiping; Li, Deshan

    2015-02-01

    Using conventional approaches, the antigen-binding affinity of a novel format of bi-specific antibody (BsAb) cannot be determined until purified BsAb is obtained. Here, we show that new lipoprotein A (NlpA)-based bacteria display technology, combined with flow cytometry (FCM), can be used to detect antigen-binding affinity of BsAbs, in the absence of expression and purification work. Two formats of BsAb, scFv2-CH/CL and Diabody-CH/CL, specific for human interleukin 1? (hIL-1?) and human interleukin 17A (hIL-17A), were constructed and displayed in Escherichia coli using NlpA-based bacteria display technology. Conversion of these cells to spheroplasts, and their incubation with fluorescently conjugated antigens resulted in the selective labeling of spheroplasts expressing BsAb; enabling their antigen-binding affinity to be analyzed with FCM. The association and dissociation of BsAbs for binding to hIL-1? and hIL-17A were analyzed using FCM-based assays. The results showed that antigen-binding affinity of Diabody-CH/CL was significantly higher than that of scFv2-CH/CL. To confirm these results of FCM-based assays, BsAbs were expressed, purified and subjected to relative affinity measurements, in vitro and in vivo bioactivity analysis. The results showed that Diabody-CH/CL had greater relative affinities for both antigens, resulting in better blocking bioactivities on cellular level and effects on alleviating joint inflammation, and cartilage destruction and bone damage in collagen induced arthritis (CIA) mice model. These results indicate that BsAbs with good antigen-binding affinity can be identified by FCM-based assays without expression and purification work, and the indentified BsAb can serve as a lead compound for further drug development. PMID:25526913

  6. Neutralizing monoclonal antibodies against three serotypes of porcine rotavirus.

    PubMed Central

    Nagesha, H S; Brown, L E; Holmes, I H

    1989-01-01

    Using three serotypes (four strains) of cultivable porcine rotavirus as immunizing antigens, 10 neutralizing monoclonal antibodies were characterized. One VP4-specific monoclonal antibody directed against porcine rotavirus BEN-144 (serotype G4) neutralized human rotavirus strain ST-3 in addition to the homologous porcine virus. All nine VP7-specific monoclonal antibodies were highly specific for viruses of the same serotype as the immunizing rotavirus strain. One exception was the VP7-specific monoclonal antibody C3/1, which neutralized both serotype G3 and G5 rotaviruses. However, this monoclonal antibody did not neutralize the porcine rotavirus AT/76, also of serotype G3, nor mutants of SA-11 virus (serotype G3) which were selected with monoclonal antibody A10/N3 and are known to have mutations affecting the C antigenic region. Images PMID:2545925

  7. Labeling of monoclonal antibodies with radionuclides.

    PubMed

    Bhargava, K K; Acharya, S A

    1989-07-01

    Antibodies, specifically monoclonal antibodies, are potentially very useful and powerful carriers of therapeutic agents to target tissues and diagnostic agents. The loading or charging of antibodies with agents, especially radiotracers, is reviewed here. The choice of radioisotope for immunodetection and/or immunotherapy is based on its availability, half-life, nature of the radiation emitted, and the metabolic pathways of the radionuclide in the body. Most important of all are the derivatization techniques available for labeling the antibody with the given radionuclide. Isotopes of iodine and divalent metal ions are the most commonly used radionuclides. Antibodies labeled with iodine at tyrosine residues are metabolized rapidly in vivo. This leads to the incorporation of metabolized radioactive iodine into various tissues, mainly the thyroid gland and stomach, and to the accumulation of high levels of circulating iodine in the blood, which masks tumor uptake considerably. To overcome these limitations, the use of iodohippurate as an iodine-anchoring molecule to the protein should be considered. When divalent or multivalent metal ions are used as the preferred radionuclide, bifunctional chelating reagents such as ethylenediaminepentaacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA) are first coupled to the protein or antibody. These chelating molecules are attached to the protein by formation of an isopeptide linkage between the carboxylate of the chelating reagent and the amino group of the protein. Several procedures are available to generate the isopeptide linkage. When the anchoring of the chelating agent through isopeptide linkage results in the inactivation of the antibody, periodate oxidation of the carbohydrate moiety of the antibody, followed by reductive coupling of chelator, could be considered as an alternative. There is still a need for better, simpler, and more direct methods for labeling antibodies with radionuclides. PMID:2503873

  8. Labeling of monoclonal antibodies with radionuclides

    SciTech Connect

    Bhargava, K.K.; Acharya, S.A. )

    1989-07-01

    Antibodies, specifically monoclonal antibodies, are potentially very useful and powerful carriers of therapeutic agents to target tissues and diagnostic agents. The loading or charging of antibodies with agents, especially radiotracers, is reviewed here. The choice of radioisotope for immunodetection and/or immunotherapy is based on its availability, half-life, nature of the radiation emitted, and the metabolic pathways of the radionuclide in the body. Most important of all are the derivatization techniques available for labeling the antibody with the given radionuclide. Isotopes of iodine and divalent metal ions are the most commonly used radionuclides. Antibodies labeled with iodine at tyrosine residues are metabolized rapidly in vivo. This leads to the incorporation of metabolized radioactive iodine into various tissues, mainly the thyroid gland and stomach, and to the accumulation of high levels of circulating iodine in the blood, which masks tumor uptake considerably. To overcome these limitations, the use of iodohippurate as an iodine-anchoring molecule to the protein should be considered. When divalent or multivalent metal ions are used as the preferred radionuclide, bifunctional chelating reagents such as EDTA or DTPA are first coupled to the protein or antibody. These chelating molecules are attached to the protein by formation of an isopeptide linkage between the carboxylate of the chelating reagent and the amino group of the protein. Several procedures are available to generate the isopeptide linkage. When the anchoring of the chelating agent through isopeptide linkage results in the inactivation of the antibody, periodate oxidation of the carbohydrate moiety of the antibody, followed by reductive coupling of chelator, could be considered as an alternative. There is still a need for better, simpler, and more direct methods for labeling antibodies with radionuclides. 78 references.

  9. Taxonomic investigation of Legionella pneumophila using monoclonal antibodies.

    PubMed

    Brindle, R J; Bryant, T N; Draper, P W

    1989-03-01

    A panel of 19 monoclonal antibodies was used to produce patterns of immunofluorescent staining of 468 isolates of Legionella pneumophila. Twelve monoclonal antibodies were selected that divided L. pneumophila into 17 phenons which, in the majority of cases, conform to serogroup divisions. These phenons are more easily defined than the present serogroups, and isolates can be placed in them with little ambiguity. The standardized set of monoclonal antibodies was also used to define the subgroups of serogroup 1. PMID:2654183

  10. Monoclonal antibodies recognizing L-thyroxine

    SciTech Connect

    Siebert, G.R.; Armstrong, J.

    1987-01-13

    A method is described for the radioimmunoassay of thyroxine comprising: (a) affixing to a solid phase particular mouse monoclonal antibodies produced by hybridoma cell lines designated as ATCC HB 8499 and ATCC HB 8500, the antibodies being specific for thyroxine and having a cross reactivity to triiodothyronine of no more than 0.12%; (b) reacting the monoclonal antibodies with a composition comprising thyroxine and a predetermined amount of radiolabeled thyroxine to cause binding therebetween; (c) separating the solid phase from unreacted thyroxine and radiolabeled thyroxine; (d) determining the amount of radiolabeled thyroxine bound on the solid phase; (e) repeating steps (a) to (d) with other compositions comprising predetermined and different amounts of thyroxine; (f) repeating steps (a) to (d) with a composition comprising an unknown amount of thyroxine; and (g) determining the unknown amount of thyroxine by ascertaining which of the solid phases reacted with a predetermined amount of thyroxine has the same amount of bound radiolabeled thyroxine as the solid phase reacted with the unknown amount of thyroxine.

  11. Therapeutic Monoclonal Antibodies and Fragments: Ranibizumab.

    PubMed

    Smith, Adiel G; Kaiser, Peter K

    2016-01-01

    Ranibizumab is a recombinant, humanized, affinity-matured, monoclonal antibody Fab fragment against all isoforms of vascular endothelial growth factor-A, which was developed specifically for intraocular use. Ranibizumab has been extensively investigated in clinical trials on choroidal neovascularization from wet age-related macular degeneration and pathologic myopia, as well as macular edema due to diabetic retinopathy and retinal vein occlusion. Numerous randomized, controlled clinical trials have shown this medication to be effective in improving both vision as well as anatomical outcomes, and the medication has repeatedly shown to have an acceptable safety profile. PMID:26501149

  12. Functional comparison of single-chain and two-chain anti-CD3-based bispecific antibodies in gene immunotherapy applications

    PubMed Central

    Compte, Marta; Álvarez-Cienfuegos, Ana; Nuñez-Prado, Natalia; Sainz-Pastor, Noelia; Blanco-Toribio, Ana; Pescador, Nuria; Sanz, Laura; Álvarez-Vallina, Luis

    2014-01-01

    Gene therapy to achieve in vivo secretion of recombinant anti-CD3 x anti-tumor bispecific antibodies in cancer patients is being explored as a strategy to counterbalance rapid renal elimination, thereby sustaining levels of bispecific antibodies in the therapeutic range. Here, we performed a comparative analysis between single- and two-chain configurations for anti-CD3 x anti-CEA (carcinoembryonic antigen) bispecific antibodies secreted by genetically-modified human cells. We demonstrate that tandem single-chain variable fragment (scFv) antibodies and two-chain diabodies are expressed as soluble secreted proteins with similar yields. However, we found significant differences in their biological functionality (i.e., antigen binding) and in their ability to induce non-specific T cell activation. Whereas single-chain tandem scFvs induced human T cell activation and proliferation in an antigen-independent manner, secreted two-chain diabodies exerted almost no proliferative stimulus when human T cells were cultured alone or in co-cultures with CEA negative cells. Thus, our data suggest that two-chain diabodies are preferable to single-chain tandem scFvs for immunotherapeutic strategies comprising in vivo secretion of bispecific antibodies aiming to recruit and activate anticancer specific lymphocytic effector T cells. PMID:25057445

  13. Functional comparison of single-chain and two-chain anti-CD3-based bispecific antibodies in gene immunotherapy applications.

    PubMed

    Compte, Marta; Alvarez-Cienfuegos, Ana; Nuez-Prado, Natalia; Sainz-Pastor, Noelia; Blanco-Toribio, Ana; Pescador, Nuria; Sanz, Laura; Alvarez-Vallina, Luis

    2014-01-01

    Gene therapy to achieve in vivo secretion of recombinant anti-CD3 x anti-tumor bispecific antibodies in cancer patients is being explored as a strategy to counterbalance rapid renal elimination, thereby sustaining levels of bispecific antibodies in the therapeutic range. Here, we performed a comparative analysis between single- and two-chain configurations for anti-CD3 x anti-CEA (carcinoembryonic antigen) bispecific antibodies secreted by genetically-modified human cells. We demonstrate that tandem single-chain variable fragment (scFv) antibodies and two-chain diabodies are expressed as soluble secreted proteins with similar yields. However, we found significant differences in their biological functionality (i.e., antigen binding) and in their ability to induce non-specific T cell activation. Whereas single-chain tandem scFvs induced human T cell activation and proliferation in an antigen-independent manner, secreted two-chain diabodies exerted almost no proliferative stimulus when human T cells were cultured alone or in co-cultures with CEA negative cells. Thus, our data suggest that two-chain diabodies are preferable to single-chain tandem scFvs for immunotherapeutic strategies comprising in vivo secretion of bispecific antibodies aiming to recruit and activate anticancer specific lymphocytic effector T cells. PMID:25057445

  14. Monoclonal antibody disulfide reduction during manufacturing

    PubMed Central

    Hutterer, Katariina M.; Hong, Robert W.; Lull, Jonathon; Zhao, Xiaoyang; Wang, Tian; Pei, Rex; Le, M. Eleanor; Borisov, Oleg; Piper, Rob; Liu, Yaoqing Diana; Petty, Krista; Apostol, Izydor; Flynn, Gregory C.

    2013-01-01

    Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production. PMID:23751615

  15. Preclinical evaluation of multistep targeting of diasialoganglioside GD2 using an IgG-scFv bispecific antibody with high affinity for GD2 and DOTA metal complex.

    PubMed

    Cheal, Sarah M; Xu, Hong; Guo, Hong-fen; Zanzonico, Pat B; Larson, Steven M; Cheung, Nai-Kong

    2014-07-01

    Bispecific antibodies (BsAb) have proven to be useful targeting vectors for pretargeted radioimmunotherapy (PRIT). We sought to overcome key PRIT limitations such as high renal radiation exposure and immunogenicity (e.g., of streptavidin-antibody fusions), to advance clinical translation of this PRIT strategy for diasialoganglioside GD2-positive [GD2(+)] tumors. For this purpose, an IgG-scFv BsAb was engineered using the sequences for the anti-GD2 humanized monoclonal antibody hu3F8 and C825, a murine scFv antibody with high affinity for the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexed with ?-particle-emitting radiometals such as (177)Lu and (90)Y. A three-step regimen, including hu3F8-C825, a dextran-based clearing agent, and p-aminobenzyl-DOTA radiolabeled with (177)Lu (as (177)Lu-DOTA-Bn; t1/2 = 6.71 days), was optimized in immunocompromised mice carrying subcutaneous human GD2(+) neuroblastoma (NB) xenografts. Absorbed doses for tumor and normal tissues were approximately 85 cGy/MBq and ?3.7 cGy/MBq, respectively, with therapeutic indices (TI) of 142 for blood and 23 for kidney. A therapy study (n = 5/group; tumor volume, 240 160 mm(3)) with three successive PRIT cycles (total (177)Lu: ?33 MBq; tumor dose ?3,400 cGy), revealed complete tumor response in 5 of 5 animals, with no recurrence up to 28 days after treatment. Tumor ablation was confirmed histologically in 4 of 5 mice, and normal organs showed minimal overall toxicities. All nontreated mice required sacrifice within 12 days (>1.0-cm(3) tumor volume). We conclude that this novel anti-GD2 PRIT approach has sufficient TI to successfully ablate subcutaneous GD2(+)-NB in mice while sparing kidney and bone marrow. PMID:24944121

  16. NCI Requests Cancer Targets for Monoclonal Antibody Production and Characterization

    Cancer.gov

    In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution.

  17. Monoclonal antibodies to Pseudomonas aeruginosa ferripyochelin-binding protein.

    PubMed Central

    Sokol, P A; Woods, D E

    1986-01-01

    Hybridomas secreting specific monoclonal antibodies against the Pseudomonas aeruginosa ferripyochelin-binding protein (FBP) were isolated. These monoclonal antibodies reacted with FBP in immunoblots of outer membrane preparations from all serotypes of P. aeruginosa. Two of the monoclonal antibodies also reacted with FBP in strains of P. putida, P. fluorescens, and P. stutzeri. These antibodies did not react with outer membranes of P. cepacia, "P. multivorans," P. maltophilia, or other gram-negative organisms. The monoclonal antibodies were opsonophagocytic and blocked the binding of [59Fe]ferripyochelin to isolated outer membranes of strain PAO. By indirect immunofluorescence techniques, the monoclonal antibodies were used to demonstrate that FBP is present on the cell surface of P. aeruginosa cells grown in low-iron but not high-iron medium. These observations were confirmed by using 125I in surface-labeling techniques. Images PMID:3091506

  18. DETECTION OF ROTAVIRUS IN HUMAN STOOLS BY USING MONOCLONAL ANTIBODY

    EPA Science Inventory

    A monoclonal antibody, 3F7, which reacts with the common rotavirus antigen on the sixth viral gene product was prepared. It was used in a direct monoclonal antibody radioimmunoassay (RIA) as a diagnostic reagent for detection in 3.5 hours of rotavirus in human pediatric stool spe...

  19. Effective introduction of T cell costimulatory molecules into virus modified tumor cell vaccines by modification with bispecific antibodies.

    PubMed

    Haas, C; Schirrmacher, V

    1997-11-01

    This report describes the generation of bispecific antibodies which bind with one arm to virus modified tumor cell vaccines and introduce with the other arm anti-murine CD28 T cell costimulatory molecules. This is an effective alternative to somatic gene therapy strategies using genes coding for ligands of CD28 such as CD80 (B7-1) or CD86 (B7-2). While these B7 molecules interact not only with CD28 but also with CLTA-4, thereby generating a negative signal, agonistic anti CD28 antibodies only bind to CD28 and therefore deliver only positive costimulatory signals. The new bispecific antibody (bsAb) HN x CD28 allows the introduction of anti-CD28 antibodies into the tumor cell vaccine ATV-NDV, an autologous tumor cell vaccine already modified by infection with Newcastle Disease Virus (NDV). The bsAb HN x CD28 attaches with its anti-HN binding site to the NDV derived hemagglutinin-neuraminidase (HN) molecule which serves as a common foreign anchoring molecule in the vaccine. NDV infected tumor cells which were further modified with HN x CD28 on their cell surface (bs-vaccine), showed increased T cell stimulatory capacity in vitro. This was revealed by augmented proliferation as well as augmented CTL activity. When syngeneic mice were injected with aggressive murine ESb lymphoma cells which were infected with NDV and further modified with the bsAb HN x CD28, delayed tumor development and prolonged survival was observed in comparison to respective controls. PMID:21528289

  20. Preparation and characterization of monoclonal antibodies to swine lymphocyte antigens

    SciTech Connect

    Lie, W.R.; Rothschild, M.F.; Warner, C.M.

    1986-03-05

    A panel of hybridoma lines were produced by the fusion of Sp2/0 myeloma cells and spleen cells from mice immunized with pig peripheral blood lymphocytes. Thirty-three stable hybridomas were produced which secreted monoclonal antibodies that reacted with pig lymphocytes, as determined by an ELISA screening procedure. These monoclonal antibodies were characterized by a complement-mediated cytotoxicity test and by flow cytometric analysis. The molecular weights of the antigens recognized by the monoclonal antibodies were determined by immunoprecipitation of /sup 125/I surface labeled lymphocytes, followed by SDS-PAGE. One monoclonal antibody, 7-34-1 (IgG2a), which reacted to all peripheral blood lymphocytes, precipitated a MHC class I molecule composed of a 50 kd heavy chain and a 12 kd light chain (..beta../sub 2/ microglobulin). This monoclonal antibody may prove to be an important reagent for the detection of SLA antigens on pig cells.

  1. A new tool for monoclonal antibody analysis

    PubMed Central

    An, Yan; Zhang, Ying; Mueller, Hans-Martin; Shameem, Mohammed; Chen, Xiaoyu

    2014-01-01

    Monoclonal antibody (mAb) products are extraordinarily heterogeneous due to the presence of a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, glycation, and terminal cyclization. The modifications in different domains of the antibody molecule can result in different biological consequences. Therefore, characterization and routine monitoring of domain-specific modifications are essential to ensure the quality of the therapeutic antibody products. For this purpose, a rapid and informative methodology was developed to examine the heterogeneity of individual domains in mAb products. A recently discovered endopeptidase, IdeS, cleaves heavy chains below the hinge region, producing F(ab')2 and Fc fragments. Following reduction of disulfide bonds, three antibody domains (LC, Fd, and Fc/2) can be released for further characterization. Subsequent analyses by liquid chromatography/mass spectrometry, capillary isoelectric focusing, and glycan mapping enable domain-specific profiling of oxidation, charge heterogeneity, and glycoform distribution. When coupled with reversed phase chromatography, the unique chromatographic profile of each molecule offers a simple strategy for an identity test, which is an important formal test for biopharmaceutical quality control purposes. This methodology is demonstrated for a number of IgGs of different subclasses (IgG1, IgG2, IgG4), as well as an Fc fusion protein. The presented technique provides a convenient platform approach for scientific and formal therapeutic mAb product characterization. It can also be applied in regulated drug substance batch release and stability testing of antibody and Fc fusion protein products, in particular for identity and routine monitoring of domain-specific modifications. PMID:24927271

  2. Monoclonal antibodies against Xenopus greatwall kinase.

    PubMed

    Wang, Ling; Fisher, Laura A; Wahl, James K; Peng, Aimin

    2011-10-01

    Mitosis is known to be regulated by protein kinases, including MPF, Plk1, Aurora kinases, and so on, which become active in M-phase and phosphorylate a wide range of substrates to control multiple aspects of mitotic entry, progression, and exit. Mechanistic investigations of these kinases not only provide key insights into cell cycle regulation, but also hold great promise for cancer therapy. Recent studies, largely in Xenopus, characterized a new mitotic kinase named Greatwall (Gwl) that plays essential roles in both mitotic entry and maintenance. In this study, we generated a panel of mouse monoclonal antibodies (MAbs) specific for Xenopus Gwl and characterized these antibodies for their utility in immunoblotting, immunoprecipitation, and immunodepletion in Xenopus egg extracts. Importantly, we generated an MAb that is capable of neutralizing endogenous Gwl. The addition of this antibody into M-phase extracts results in loss of mitotic phosphorylation of Gwl, Plk1, and Cdk1 substrates. These results illustrate a new tool to study loss-of-function of Gwl, and support its essential role in mitosis. Finally, we demonstrated the usefulness of the MAb against human Gwl/MASTL. PMID:22008075

  3. Chitinase 3-like 2 protein monoclonal antibodies.

    PubMed

    Avdieiev, Stanislav; Savinska, Liliia; Filonenko, Valeriy; Kavsan, Vadym

    2012-02-01

    Chitinase 3-like 2 (CHI3L2) is one of the most overexpressed genes in glioblastoma. Despite this, both the CHI3L2 gene and its protein product CHI3L2 are poorly characterized. Here we report the generation and characterization of monoclonal antibodies to CHI3L2 protein (CHI3L2 MAbs). Bacterially expressed 6 His-tagged full-length CHI3L2 was used as antigen. Spleen cells from immunized mice were collected and fused with SP2/0 myeloma cells. Hybridoma clones 2D3 and 4D2 producing high titer CHI3L2 MAbs were identified by enzyme-linked immunosorbent assay (ELISA) and further examined for their activity with the CHI3L2 protein by Western blot analysis and immunoprecipitation. The 2D3 clone was chosen for mouse inoculation and ascites formation. Antibodies derived from the ascitic fluid specifically recognized the recombinant CHI3L2 protein and strongly interacted with CHI3L2 in glioblastoma tissue lysate, as determined by Western blot analysis. The antibodies generated may be useful as a tool in various aspects of CHI3L2 investigation. PMID:22316483

  4. Detection of enterovirus 70 with monoclonal antibodies.

    PubMed Central

    Anderson, L J; Hatch, M H; Flemister, M R; Marchetti, G E

    1984-01-01

    To improve the ability to identify enterovirus-70 (EV-70) from patients with acute hemorrhagic conjunctivitis, we developed four monoclonal antibodies (MAbs) to EV-70. We reacted the four MAbs against nine previously characterized strains of EV-70 and heterologous viruses by virus neutralization, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Two of the MAbs neutralized all nine strains of EV-70 and none of the other enterovirus types tested. Two of the MAbs gave a positive reaction with all nine strains by indirect immunofluorescence, and three reacted with all nine strains by ELISA. None of the MAbs gave a positive reaction with heterologous viruses, including those associated with eye disease, by indirect immunofluorescence or ELISA. The two neutralizing MAbs failed to give a positive reaction with some of the strains of EV-70 by indirect immunofluorescence and ELISA, yet they neutralized these viruses. By ELISA with a polyclonal serum as capture antibody and a mixture of MAbs as detector antibody, we were able to detect from 10(2.2) to 10(5.8) 50% tissue culture infective doses of virus and to type lyophilized isolates of EV-70 sent from Taiwan from which we could not recover infectious virus. By choosing the appropriate MAb, or mixture of MAbs, we could construct a test which had the type specificity and strain sensitivity needed to type isolates of EV-70. PMID:6092426

  5. Complement in Monoclonal Antibody Therapy of Cancer

    PubMed Central

    Rogers, Laura M.; Veeramani, Suresh; Weiner, George J.

    2015-01-01

    Monoclonal antibodies (mAb) have been used as targeted treatments against cancer for more than a decade, with mixed results. Research is needed to understand mAb mechanisms of action with the goal of improving the efficacy of currently used mAbs, and guiding the design of novel mAbs. While some mAb-induced tumor cell killing is a result of direct effects on tumor cell signaling, mAb opsonization of tumor cells also triggers activation of immune responses due to complement activation and engagement of antibody receptors on immune effector cells. In fact, complement has been shown to play an important role in modulating the anti-tumor activity of many mAb through complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), and through indirect effects by modulating the tumor microenvironment. Complement activity can have both agonistic and antagonistic effects on these processes, and which mechanisms are most responsible for effective elimination of malignant cells remain unclear. In this review, we discuss the mAbs currently approved for cancer treatment, and examine how complement can impact their efficacy with a focus on how this information might be used to improve the clinical efficacy of mAb treatment. PMID:24906530

  6. Use of monoclonal antibodies for expression cloning.

    PubMed

    Hollenbaugh, Diane; Aruffo, Alejandro; Jones, Bryan; Linsley, Peter

    2003-05-01

    This unit details the use of transient expression in mammalian cells to screen cDNA libraries with monoclonal antibodies (MAb) to isolate cDNA clones encoding cell-surface and intracellular proteins. The first protocol in this unit describes the cloning of cDNAs encoding cell-surface antigens. Several steps in this protocol involve transfection procedures that are described in greater detail elsewhere in this volume. The second protocol is a modification that facilitates isolation of cDNAs encoding antigens that are expressed intracellularly. Both protocols are designed for use with the expression vector CDM8, which contains a polylinker for subcloning double-stranded cDNA. PMID:18265331

  7. SPECT assay of radiolabeled monoclonal antibodies

    SciTech Connect

    Jaszczak, R.J.

    1992-02-01

    The long-term goal of this research project is to develop methods to improve the utility of single photon emission computed tomography (SPECI) to quantify the biodistribution of monoclonal antibodies (MoAbs) labeled with clinically relevant radionuclides ({sup 123}I, {sup 131}I, and {sup 111}In) and with another radionuclide,{sup 211}At, recently used in therapy. We describe here our progress in developing quantitative SPECT methodology for {sup 111}In and {sup 123}I. We have focused our recent research thrusts on the following aspects of SPECT: (1) The development of improved SPECT hardware, such as improved acquisition geometries. (2) The development of better reconstruction methods that provide accurate compensation for the physical factors that affect SPECT quantification. (3) The application of carefully designed simulations and experiments to validate our hardware and software approaches.

  8. Building better monoclonal antibody-based therapeutics

    PubMed Central

    Weiner, George J.

    2015-01-01

    For 20 years, monoclonal antibodies (mAbs) have been a standard component of cancer therapy, yet there is still much room for improvement. Efforts continue to build better cancer therapeutics based on mAbs. Anti-cancer mAbs function via a variety of mechanisms including directly targeting the malignant cells, modifying the host response to the malignant cells, delivering cytotoxic moieties to the malignant cells or retargeting cellular immunity towards the malignant cells. Characteristics of mAbs that affect their efficacy include antigen specificity, overall structure, affinity for the target antigen and how a mAb component is incorporated into a construct that can trigger target cell death. This article reviews the various approaches to using mAb-based therapeutics to treat cancer, the strategies used to take advantage of the unique potential of each approach, and provides examples of current mAb-based treatments. PMID:25998715

  9. The birth pangs of monoclonal antibody therapeutics

    PubMed Central

    2012-01-01

    This paper examines the development and termination of nebacumab (Centoxin), a human IgM monoclonal antibody (mAb) drug frequently cited as one of the notable failures of the early biopharmaceutical industry. The non-approval of Centoxin in the United States in 1992 generated major concerns at the time about the future viability of any mAb therapeutics. For Centocor, the biotechnology company that developed Centoxin, the drug posed formidable challenges in terms of safety, clinical efficacy, patient selection, the overall economic costs of health care, as well as financial backing. Indeed, Centocor's development of the drug brought it to the brink of bankruptcy. This article shows how many of the experiences learned with Centoxin paved the way for the current successes in therapeutic mAb development. PMID:22531443

  10. Monitoring therapeutic monoclonal antibodies in brain tumor

    PubMed Central

    Ait-Belkacem, Rima; Berenguer, Caroline; Villard, Claude; Ouafik, L’Houcine; Figarella-Branger, Dominique; Beck, Alain; Chinot, Olivier; Lafitte, Daniel

    2014-01-01

    Bevacizumab induces normalization of abnormal blood vessels, making them less leaky. By binding to vascular endothelial growth factor, it indirectly attacks the vascular tumor mass. The optimal delivery of targeted therapies including monoclonal antibodies or anti-angiogenesis drugs to the target tissue highly depends on the blood-brain barrier permeability. It is therefore critical to investigate how drugs effectively reach the tumor. In situ investigation of drug distribution could provide a better understanding of pharmacological agent action and optimize chemotherapies for solid tumors. We developed an imaging method coupled to protein identification using matrix-assisted laser desorption/ionization mass spectrometry. This approach monitored bevacizumab distribution within the brain structures, and especially within the tumor, without any labeling. PMID:25484065

  11. Monoclonal antibodies in treatment of multiple sclerosis

    PubMed Central

    Rommer, P S; Dudesek, A; Stüve, O; Zettl, UK

    2014-01-01

    Monoclonal antibodies (mAbs) are used as therapeutics in a number of disciplines in medicine, such as oncology, rheumatology, gastroenterology, dermatology and transplant rejection prevention. Since the introduction and reintroduction of the anti-alpha4-integrin mAb natalizumab in 2004 and 2006, mAbs have gained relevance in the treatment of multiple sclerosis (MS). At present, numerous mAbs have been tested in clinical trials in relapsing–remitting MS, and in progressive forms of MS. One of the agents that might soon be approved for very active forms of relapsing–remitting MS is alemtuzumab, a humanized mAb against CD52. This review provides insights into clinical studies with the mAbs natalizumab, alemtuzumab, daclizumab, rituximab, ocrelizumab and ofatumumab. PMID:24001305

  12. Phenotyping cytochromes P450 with monoclonal antibodies.

    PubMed

    Friedman, F K; Park, S S; Fujino, T; Song, B J; Robinson, R C; West, D; Radkowsky, A K; Miller, H; Gelboin, H V

    1984-01-01

    Monoclonal antibodies (MAbs) to cytochrome P-450 isozymes can be used to phenotype tissues for epitope-specific cytochrome P-450 content. MAbs that inhibit specific cytochrome P-450 dependent drug or carcinogen reactions are useful tools for quantitative measurement of the individual or classes of cytochromes P-450 that catalyze these reactions. This method has been applied successfully to animal as well as human tissues. Radioimmunoassays based on MAbs have been developed and provide a rapid and efficient means for detecting cytochromes P-450 independent of functional enzyme activity. In addition, MAbs coupled to a Sepharose support can be used to immunopurify cytochromes P-450 in a procedure that is more rapid and efficient than conventional purification schemes. MAbs add a new dimension to analyses of cytochrome P-450 multiplicity and will find numerous applications in elucidation of the relationship between cytochrome P-450 phenotype and carcinogen or drug metabolism. PMID:11478317

  13. Monoclonal antibodies in treatment of multiple sclerosis.

    PubMed

    Rommer, P S; Dudesek, A; Stüve, O; Zettl, U K

    2014-03-01

    Monoclonal antibodies (mAbs) are used as therapeutics in a number of disciplines in medicine, such as oncology, rheumatology, gastroenterology, dermatology and transplant rejection prevention. Since the introduction and reintroduction of the anti-alpha4-integrin mAb natalizumab in 2004 and 2006, mAbs have gained relevance in the treatment of multiple sclerosis (MS). At present, numerous mAbs have been tested in clinical trials in relapsing-remitting MS, and in progressive forms of MS. One of the agents that might soon be approved for very active forms of relapsing-remitting MS is alemtuzumab, a humanized mAb against CD52. This review provides insights into clinical studies with the mAbs natalizumab, alemtuzumab, daclizumab, rituximab, ocrelizumab and ofatumumab. PMID:24001305

  14. Rat monoclonal antibodies against Aspergillus galactomannan.

    PubMed Central

    Stynen, D; Sarfati, J; Goris, A; Prvost, M C; Lesourd, M; Kamphuis, H; Darras, V; Latg, J P

    1992-01-01

    Monoclonal antibodies (MAbs) against Aspergillus fumigatus galactomannan were produced in rats. Seven of them, EB-A1 through EB-A7, were characterized in more detail. They were all immunoglobulin M antibodies, reacting in an indirect enzyme-linked immunosorbent assay with purified A. fumigatus galactomannan, with avidity constants of between 2 x 10(9) and 5 x 10(9)/M. Enzyme-linked immunosorbent assay inhibition experiments with modified galactomannan and synthetic oligomers of beta (1----5)galactofuranose demonstrated that the MAbs bound to an epitope located on the beta(1----5)galactofuranose-containing side chains of the galactomannan molecule. An identical or similar epitope also seemed to be present in other fungi. Immunofluorescence and immunoelectron microscopy experiments with EB-A2 revealed the presence of the antigen in the fungal wall and inside the cell. Immunoblotting experiments demonstrated that the epitope recognized by the MAbs was a common oligosaccharide moiety of a wide range of intracellular and extracellular glycoproteins in A. fumigatus. The characteristics of the MAbs justify their use in the diagnosis of invasive aspergillosis by antigen detection. Images PMID:1375195

  15. Clinical laboratory applications of monoclonal antibodies.

    PubMed Central

    Payne, W J; Marshall, D L; Shockley, R K; Martin, W J

    1988-01-01

    Monoclonal antibody (MAb) technology is well recognized as a significant development for producing specific serologic reagents to a wide variety of antigens in unlimited amounts. These reagents have provided the means for developing a number of highly specific and reproducible immunological assays for rapid and accurate diagnosis of an extensive list of diseases, including infectious diseases. The impact that MAbs have had in characterizing infectious disease pathogens, as well as their current and future applications for use in clinical microbiology laboratories, is reviewed. In addition, the advantages (and disadvantages) of the use of MAbs in a number of immunoassays, such as particle agglutination, radioimmunoassays, enzyme-linked immunosorbent assays, immunofluorescent-antibody assays, and immunohistology, are explored, including the use of these reagents in novel test system assays. Also, nucleic acid probe technology is compared with the use of MAbs from the perspective of their respective applications in the diagnosis of infectious disease agents. There is no question that hybridoma technology has the potential to alter significantly the methods currently used in most clinical microbiology laboratories. PMID:3058298

  16. Autoantibody potential of cancer therapeutic monoclonal antibodies.

    PubMed

    McIntyre, John A; Faulk, And W Page

    2010-07-15

    We and others have reported that multiple autoantibodies are unmasked in human polyclonal antibody preparations after exposure to physiological oxidizing agents (hemin) or electromotive force. We now have asked if oxidation unmasks autoantibody reactivities in monoclonal antibodies (mAb). To do this, we have studied 9 FDA approved mAb used therapeutically, including 4 chimeric, 4 humanized and 1 chemically modified chimeric Fab that were exposed to the physiological oxidizing agent hemin at 36 degrees C for 20 hr. These mAb were studied for autoantibody activity to phospholipids and DNA before and after oxidation with hemin and found to develop autoantibody activities after oxidation, while retaining their original specificity as measured by mAb anti-glycophorin A binding of erythrocytes, CD 19 binding to B lymphocytes and anti-HLA-A29 binding to A29-positive lymphocytes. The finding that certain mAb have the potential to unmask autoantibody activities as a consequence of exposure to physiological redox reactions in vitro gives pause to our present understanding of the immunological basis of tolerance and concern for potential autoimmune side effects in patients receiving mAb for diagnosis or treatment. PMID:19904753

  17. Production and characterization of monospecific and bispecific antibodies against dengue virus NS1 protein.

    PubMed

    Ganguly, Advaita; Malabadi, Ravindra B; Bhatnagar, Pravin K; Tang, Xinli; Das, Dipankar; Loebenberg, Raimer; Suresh, Mavanur R; Sunwoo, Hoon H

    2015-08-01

    Dengue is a mosquito borne infection, which in recent years has become a major international public health concern. Annually, 100 million dengue virus infections are reported worldwide. The nonstructural protein 1 (NS1) of dengue virus is a useful target for diagnostics of dengue infection since the protein is abundantly circulating in the blood during acute phase of the disease, in both primary and secondary infections. This research paper highlights the development of a panel of Mab and bsMab for dengue NS1 detection. The P148 series of Mabs showed high specificity for recombinant dengue NS1 antigen. These antibodies showed no cross reactivity with recombinant dengue envelope protein and other viral proteins. The hybrid-hybridoma approach to generate the P156.1 and P156.2 bsMabs from the P148 monoclonal antibody method was used during this study. Furthermore, the affinity purification provided good yields of quadromas associated with HRPO in two steps. Direct detection method involved coating of plates with different concentrations of recombinant antigen and detecting with bsMab. Sensitive sandwich assay with Mabs and bsMabs was also done. Detection of nonstructural dengue antigens may be of benefit for early and rapid diagnosis of dengue infection due to their long half-life in the blood. PMID:25869657

  18. Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies

    PubMed Central

    Hutchinson, Alistair P.; Nicklin, Stephen

    2015-01-01

    Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites. PMID:26252765

  19. Xenogeneic monoclonal antibodies in the management of cancer: control of their in vivo immunogenicity and induction of specific unresponsiveness using an antibody-drug immunoconjugate.

    PubMed Central

    Sivolapenko, G. B.; Moreno, C.; Smith, W.; Corvlan, J.; Ritter, M. A.; Epenetos, A. A.

    1991-01-01

    A bispecific mouse monoclonal antibody (mAb) that recognises carcinoembryonic antigen (CEA) with one binding site and vinblastine (VLB) with the other was used, and its in vivo immunosuppressive effect specific for anti-mouse immunoglobulin (Ig) was studied. The antibody was incubated with VLB at a molar ratio (MR) of 1:1, and administered i.v. to rabbits. Control animals received either the MAb alone, or the MAb with VLB covalently linked (MR 1:1), or the parental anti-CEA with equimolar amount of VLB. Seven days later, the rabbit anti-mouse Ig primary response was measured, and found to be almost 55% reduced in the animals that received the VLB 'loaded' MAb. In vivo kinetics and stability experiments revealed that the T1/2 of the MAb was 68 +/- 5 h, whereas free VLB disappeared within minutes. It was concluded that as soon as the drug dissociates from the antibody's binding site, it is rapidly removed. This problem was overcome by subcutaneously implanting osmotic mini-pumps containing VLB. The pumps released the drug at a constant rate for a period greater than 1 week, saturating the antibody's binding site. Under these conditions rabbits developed 80% less anti-mouse Ig antibodies when the bispecific antibody was administered (compared with the parental anti-CEA). The immunosuppression observed was specific for the mouse Ig, under conditions compatible with the full clinical therapeutic potential of the MAb. In conclusion, these experiments show, that it is possible to develop hybrid antibodies that can act as a 'lethal bait' to any specific lymphocyte in vivo, thus preventing undesirable responses against the xenogeneic MAb. PMID:1892756

  20. Development and characterization of monoclonal antibodies to Pneumocystis carinii.

    PubMed Central

    Graves, D C; McNabb, S J; Ivey, M H; Worley, M A

    1986-01-01

    Hybridoma-producing monoclonal antibodies against Pneumocystis carinii were produced by the fusion of nonsecreting mouse myeloma cells (P3X63-Ag8.653) with splenocytes from BALB/c mice that had been immunized with partially purified preparations of P. carinii. Of 227 hybridoma clones producing antibodies against P. carinii, as measured by an enzyme-linked immunosorbent assay, 12 monoclonal antibodies showing the highest reactivity in the enzyme-linked immunosorbent assay were further characterized. The majority (11 of 12) of the monoclonal antibodies did not cross-react with Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, or Mycobacterium avium as determined by absorption experiments. By using the indirect immunofluorescence assay, serological reactivity was shown for these antibodies with titers ranging from 1:40 to 1:10,240. By using a competitive binding assay, these 12 monoclonal antibodies could be divided into seven groups, each group reacting with a different antigenic determinant of P. carinii. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis of P. carinii, followed by Western immunoblot analysis, allowed the identification of one major antigen with an apparent molecular weight of 110,000 by all 12 monoclonal antibodies. Other minor bands with molecular weights of approximately 116,000, 90,000, 55,000, and 35,000 were recognized by several of the monoclonal antibodies. Images PMID:3510163

  1. Monoclonal antibody specific for a pigmentation associated antigen

    SciTech Connect

    Thomson, T.M.; Mattes, M.J.; Old, L.J.; Lloyd, K.O

    1989-01-17

    Monoclonal antibody TA99, which specifically binds to a pigmentation associated antigen present on melanoma cells is described. Additionally, the hybridoma cell line deposited with the ATCC under Accession Number HB 8704 from which the antibody is derived, as well as methods for using the antibody are described.

  2. Monoclonal AntibodiesTherapeutic and Diagnostic Uses in Malignancy

    PubMed Central

    Lowder, James N.; Levy, Ronald

    1985-01-01

    Murine monoclonal antibodies represent an attractive type of antitumor therapy because of their potential for exquisite specificity, production in large, pure quantities and mediation of in vivo cytotoxic effects. With maturing monoclonal antibody technology has come the use of these antibodies in clinical studies in patients with malignancy. These trials have established that monoclonal antibodies can be safely administered in large doses, that their pharmacokinetics and tissue penetration can be predicted and that in some instances a therapeutic effect can be produced by their infusion. A number of problems have also been identified by these studies, including antigenic heterogeneity of the tumor, the presence of free serum antigen, the immunogenicity of the xenogeneic antibody, modulation of the surface antigen by the antibody and a finite capacity of human effector mechanisms to mediate cytotoxicity directed by murine antibodies. Other workers are concurrently investigating the use of monoclonal antibodies in the ex vivo elimination of cells from bone marrow, as probes for serum tumor marker antigens and as carriers for radioimaging agents or toxins. Although most of these endeavors are at the earliest stages, promising preliminary results presage an important role for native and altered monoclonal antibodies in the diagnosis and treatment of malignant conditions. ImagesFigure 1.Figure 1. PMID:3911594

  3. Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle

    PubMed Central

    Spiesberger, Katrin; Paulfranz, Florian; Egger, Anton; Reiser, Judith; Vogl, Claus; Rudolf-Scholik, Judith; Mayrhofer, Corina; Grosse-Hovest, Ludger; Brem, Gottfried

    2015-01-01

    Background 30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested—mostly from the milk—of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4). Results With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody’s activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M. Conclusion Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma. PMID:26469402

  4. Future prospects of monoclonal antibodies as magic bullets in immunotherapy.

    PubMed

    Maleki, Leili Aghebati; Baradaran, Behzad; Majidi, Jafar; Mohammadian, Mozhdeh; Shahneh, Fatemeh Zare

    2013-01-01

    Monoclonal antibody therapy has become a critical component of clinical treatment procedure for a variety of indications. Therapeutic antibodies have made the transition from conception to clinical reality over the past two decades. Now, many of mAbs are being tested as adjuvant or first-line therapies to determine their efficacy in improving survival. In the future, the information drawn from genomemedical science and genome-informatics, that list the disease-related antigens useful for medical treatment, should be essential to develop the therapy using mAbs. Currently, the more attention is getting paid toward monoclonal antibody therapy. Several monoclonal antibodies, alone and in combination with other conventional therapies, are being tested in phase I and phase II clinical trials at the moment. Monoclonal antibody therapy can be done by using antibody fragments, antibody fusions with effector proteins and intrabodies. The large size and the long half-life of full-length antibody make them an inappropriate tool for radioimmunotherapy. Therefore, scientists produced some antibody fragments including scFv, Diabody and Nanobodies (sdAbs) which have smaller size besides maintaining the binding activity of the full-length molecule. Immunotoxin and Immunocytokines are consisting of toxin and cytokines fused to antibody fragments. An intrabody is produced by entering antibody into the cell and act against intracellular compartments. PMID:24284304

  5. Cation-exchange chromatography of monoclonal antibodies

    PubMed Central

    Urmann, Marina; Graalfs, Heiner; Joehnck, Matthias; Jacob, Lothar R

    2010-01-01

    A novel cation-exchange resin, Eshmuno™ S, was compared to Fractogel® SO3− (M) and Toyopearl GigaCap S-650M. The stationary phases have different base matrices and carry specific types of polymeric surface modifications. Three monoclonal antibodies (mAbs) were used as model proteins to characterize these chromatographic resins. Results from gradient elutions, stirred batch adsorptions and confocal laser scanning microscopic investigations were used to elucidate binding behavior of mAbs onto Eshmuno™ S and Fractogel® SO3− and the corresponding transport mechanisms on these two resins. The number of charges involved in mAb binding for Eshmuno™ S is lower than for Fractogel® SO3−, indicating a slightly weaker electrostatic interaction. Kinetics from batch uptake experiments are compared to kinetic data obtained from confocal laser scanning microscopy images. Both experimental approaches show an accelerated protein adsorption for the novel stationary phase. The influence of pH, salt concentrations and residence times on dynamic binding capacities was determined. A higher dynamic binding capacity for Eshmuno™ S over a wider range of pH values and residence times was found compared to Fractogel® SO3− and Toyopearl GigaCap S-650M. The capture of antibodies from cell culture supernatant, as well as post-protein A eluates, were analyzed with respect to their host cell protein (hcp) removal capabilities. Comparable or even better hcp clearance was observed at much higher protein loading for Eshmuno™ S than Fractogel® SO3− or Toyopearl GigaCap S-650M. PMID:20559022

  6. [Preparation of monoclonal antibodies to barley yellow mosaic virus].

    PubMed

    Zhao, X; Ma, H; We, X; Xu, A; Huang, C

    1997-10-01

    Using hybridoma techniques, six hybridoma celllines: 4D6, 4D4, 4F10, 3H6, 3H8 and 4E9 secreting monoclonal antibodies to Shanghai Isolate of Barley yellow mosaic virus (BaYMV) were prepared by fusion between myeloma cells NS-1 and spleen cells of BALB/C mouse immunized with BaYMV. The subclasses of monoclonal antibodies obtained belong to IgG2a. The cross-reaction test with different viruses (SMV, TMV and three isolates of TuMV) showed that six monoclonal antibodies were specific to BaYMV. The titers of six monoclonal antibodies to BaYMV in their cell cultured supernatauts were 1:1-5 x 10(4), and that of 4F10 in ascitic fluid was 1:6.4 x 10(5) by using indirected ELISA test respectively. PMID:11189368

  7. Anti-Mesothelin Monoclonal Antibodies for the Treatment of Cancer

    Cancer.gov

    The National Cancer Institute, Laboratory of Molecular Biology seeks parties interested in collaborative research to further co-develop monoclonal antibodies for the treatment of mesothelin-expressing cancers.

  8. Culturing hybridoma cell lines for monoclonal antibody production.

    PubMed

    Winzeler, Alissa; Wang, Jack T

    2013-07-01

    This protocol describes how to culture hybridoma cell lines (e.g., Thy1.1) for monoclonal antibody production. Supernatants harvested from such cultures can be used to purify various rodent neural cell types by immunopanning. PMID:23818668

  9. Orientation and density control of bispecific anti-HER2 antibody on functionalized carbon nanotubes for amplifying effective binding reactivity to cancer cells

    NASA Astrophysics Data System (ADS)

    Kim, Hye-In; Hwang, Dobeen; Jeon, Su-Ji; Lee, Sangyeop; Park, Jung Hyun; Yim, Dabin; Yang, Jin-Kyoung; Kang, Homan; Choo, Jaebum; Lee, Yoon-Sik; Chung, Junho; Kim, Jong-Ho

    2015-03-01

    Nanomaterial bioconjugates have gained unabated interest in the field of sensing, imaging and therapy. As a conjugation process significantly affects the biological functions of proteins, it is crucial to attach them to nanomaterials with control over their orientation and the nanomaterial-to-protein ratio in order to amplify the binding efficiency of nanomaterial bioconjugates to targets. Here, we describe a targeting nanomaterial platform utilizing carbon nanotubes functionalized with a cotinine-modified dextran polymer and a bispecific anti-HER2 × cotinine tandem antibody. This new approach provides an effective control over antibody orientation and density on the surface of carbon nanotubes through site-specific binding between the anti-cotinine domain of the bispecific tandem antibody and the cotinine group of the functionalized carbon nanotubes. The developed synthetic carbon nanotube/bispecific tandem antibody conjugates (denoted as SNAs) show an effective binding affinity against HER2 that is three orders of magnitude higher than that of the carbon nanotubes bearing a randomly conjugated tandem antibody prepared by carbodiimide chemistry. As the density of a tandem antibody on SNAs increases, their effective binding affinity to HER2 increases as well. SNAs exhibit strong resonance Raman signals for signal transduction, and are successfully applied to the selective detection of HER2-overexpressing cancer cells.Nanomaterial bioconjugates have gained unabated interest in the field of sensing, imaging and therapy. As a conjugation process significantly affects the biological functions of proteins, it is crucial to attach them to nanomaterials with control over their orientation and the nanomaterial-to-protein ratio in order to amplify the binding efficiency of nanomaterial bioconjugates to targets. Here, we describe a targeting nanomaterial platform utilizing carbon nanotubes functionalized with a cotinine-modified dextran polymer and a bispecific anti-HER2 × cotinine tandem antibody. This new approach provides an effective control over antibody orientation and density on the surface of carbon nanotubes through site-specific binding between the anti-cotinine domain of the bispecific tandem antibody and the cotinine group of the functionalized carbon nanotubes. The developed synthetic carbon nanotube/bispecific tandem antibody conjugates (denoted as SNAs) show an effective binding affinity against HER2 that is three orders of magnitude higher than that of the carbon nanotubes bearing a randomly conjugated tandem antibody prepared by carbodiimide chemistry. As the density of a tandem antibody on SNAs increases, their effective binding affinity to HER2 increases as well. SNAs exhibit strong resonance Raman signals for signal transduction, and are successfully applied to the selective detection of HER2-overexpressing cancer cells. Electronic supplementary information (ESI) available: Materials; synthesis of carboxymethylated phenoxy dextran (CM-PhO-dex); synthesis of the SWNT bioconjugate prepared by EDC coupling; NMR results; Raman Instrument for detection of cancer cells with SNAs; NIR fluorescence spectrophotometer; quantification of the bispecific tandem antibody bound to the SWNT; all supplementary figures, table and scheme. This material is available from the Wiley Online Library or from the author. See DOI: 10.1039/c4nr07305c

  10. Monoclonal antibodies against plant cell wall polysaccharides

    SciTech Connect

    Hahn, M.G.; Bucheli, E.; Darvill, A.; Albersheim, P. )

    1989-04-01

    Monoclonal antibodies (McAbs) are useful tools to probe the structure of plant cell wall polysaccharides and to localize these polysaccharides in plant cells and tissues. Murine McAbs were generated against the pectic polysaccharide, rhamnogalacturonan I (RG-I), isolated from suspension-cultured sycamore cells. The McAbs that were obtained were grouped into three classes based upon their reactivities with a variety of plant polysaccharides and membrane glycoproteins. Eleven McAbs (Class I) recognize epitope(s) that appear to be immunodominant and are found in RG-I from sycamore and maize, citrus pectin, polygalacturonic acid, and membrane glycoproteins from suspension-cultured cells of sycamore, maize, tobacco, parsley, and soybean. A second group of five McAbs (Class II) recognize epitope(s) present in sycamore RG-I, but do not bind to any of the other polysaccharides or glycoproteins recognized by Class I. Lastly, one McAb (Class III) reacts with sycamore RG-I, sycamore and tamarind xyloglucan, and sycamore and rice glucuronoarabinoxylan, but does not bind to maize RG-I, polygalacturonic acid or the plant membrane glycoproteins recognized by Class I. McAbs in Classes II and III are likely to be useful in studies of the structure, biosynthesis and localization of plant cell wall polysaccharides.

  11. Monoclonal antibodies as therapeutics in human malignancies.

    PubMed

    Pandey, Manjari; Mahadevan, Daruka

    2014-03-01

    Monoclonal antibodies (mAbs) are a proven effective therapeutic modality in human malignancy. Several mAbs are approved to targets critical in aberrant oncogenic signaling within tumors and their microenvironment. These targets include secreted ligands (e.g., VEGF and HGH), their receptors (e.g., HER2 and VEGFR2), cell surface counter receptors and their receptor-bound ligands (e.g., PD1 and PD1L, respectively). The ability to genetically engineer the structure and/or functions of mAbs has significantly improved their effectiveness. Furthermore, advances in gene expression profiling, proteomics, deep sequencing and deciphering of complex signaling networks have revealed novel therapeutic targets. We review target selection, approved indications and the rationale for mAb utilization in solid and hematologic malignancies. We also discuss novel mAbs in early- and late-phase clinical trials that are likely to change the natural history of disease and improve survival. The future challenge is to design mAb-based novel trial designs for diagnostics and therapeutics for human malignancies. PMID:24754592

  12. Therapeutic Monoclonal Antibodies and Fragments: Bevacizumab.

    PubMed

    Klein, Ainat; Loewenstein, Anat

    2016-01-01

    Bevacizumab (Avastin) is a recombinant humanized monoclonal immunoglobulin antibody that has two antigen-binding domains and blocks all active forms of vascular endothelial growth factor-A. It was originally designed and is still in use as antitumor agent (for colorectal and non-small cell lung cancers). Besides inhibiting vessel growth and neovascularization, the drug promotes the regression of existing microvessels and induces 'normalization' of surviving mature vasculature, stabilizes vessels and prevents leakage. Its molecular weight is 149 kDa and its estimated terminal half-life is approximately 20 days for both men and women. The effectiveness and safety of bevacizumab was proven in retrospective and prospective controlled clinical trials for the treatment of neovascular age-related macular degeneration, neovascularization in proliferative diabetic retinopathy, diabetic macular edema, retinal vein occlusion and retinopathy of prematurity, especially for zone I. Uncontrolled trials have shown its effectiveness in various other conditions as myopic and uveitic choroidal neovascularization and neovascular glaucoma. There are no absolute contraindications to intravitreal injection though it is recommended to withhold treatment in patients who have recently suffered from a cardiovascular or cerebrovascular event and during pregnancy. Ocular complications from intravitreal use are usually mild and transient (corneal abrasion, chemosis, subconjunctival hemorrhage and vitreous hemorrhage). Bacterial endophthalmitis is rare (about 0.1%). New or progressive subretinal hemorrhages, tears of the retinal pigment epithelium and an increased incidence of geographic atrophy have also been reported. PMID:26502311

  13. Monoclonal Antibodies for Systemic Lupus Erythematosus (SLE)

    PubMed Central

    Ponticelli, Claudio; Moroni, Gabriella

    2010-01-01

    A number of monoclonal antibodies (mAb) are now under investigation in clinical trials to assess their potential role in Systemic Lupus Erythematosus (SLE). The most frequently used mAb is rituximab, which is directed against CD20, a membrane protein expressed on B lymphocytes. Uncontrolled trials reported an improvement of SLE activity in non-renal patients and other studies even reported an improvement of severe lupus nephritis unresponsive to conventional treatments. However two randomized trials failed to show the superiority of rituximab over conventional treatment in non renal SLE and in lupus nephritis. Preliminary trials reported promising results with epratuzumab, a humanized mAb directed against CD22, and with belimumab, a human mAb that specifically recognizes and inhibits the biological activity of BLyS a cytokine of the tumor-necrosis-factor (TNF) ligand superfamily. Other clinical trials with mAb directed against TNF-alpha, interleukin-10 (Il-10), Il-6, CD154, CD40 ligand, IL-18 or complement component C5 are under way. At present, however, in spite of good results reported by some studies, no firm conclusion on the risk-benefit profile of these mAbs in patients with SLE can be drawn from the available studies.

  14. [Monoclonal antibodies from neurological and neuropsychological perspective].

    PubMed

    Piusińska-Macoch, Renata

    2013-05-01

    The role of monoclonal antibodies and other proinflammatory cytokines in the regulatory processes of the central and peripheral nervous system is not yet fully understood. Clinical studies show that they are involved in the pathogenesis of Alzheimer's disease, Parkinson's disease or other neurodegenerative disabilities with cognitive impairments. Genetic basis of these disorders is still in research. In the past few years it has been shown that increased levels of TNF-alpha and IL-6 in plasma play role in patients with ischemic stroke in the acute phase as well as transient ischemic episodes. Also the negative impact of TNF-alpha has been demonstrated on neck and coronary vessels, including the composition of plaques in the carotid arteries. A few reports indicate the involvement of tumor necrosis factor in such complex processes such as emotions, behavior or personality. Recent studies point to the important role of proinflammatory cytokines in the pathogenesis of sleep disorders such as narcolepsy, cataplexy and sleep paralysis. TNF-alpha can also activate nociceptive pathways, causing the intensity of neuropathic pain. However discloses asymmetric subtypes share TNF-1, TNF-2 in the induction and the maintenance of pain. The phenomenon of complex neurohormonal control mechanism support the proinflammatory cytokines is not fully understood and needs further empirical verification. PMID:23894773

  15. Monoclonal antibodies: new agents for cancer detection and targeted therapy

    SciTech Connect

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

    1991-01-01

    Antibodies directed against markers on cancer cells are gaining in importance for the purpose of targeting diagnostic and therapeutic agents. In the past, this approach has had very limited success principally because the classical methods for producing antibodies from blood serum of animals immunized with cancer cells or extracts were unsatisfactory. The situation has changed dramatically since 1975 following the design of procedures for 'immortalizing' antibody-producing cells (lymphocytes) by fusing them with cultured myeloma cells to form hybridomas which continuously secrete antibodies. Since these hybridomas produce antibodies coded for by a single antibody-producing cell, the antibodies are called monoclonal. Building on these advances in biomedical research, it is now possible to reproducibly manufacture monoclonal antibodies on a scale suitable for use in cancer detection and therapy.

  16. Serial killing of tumor cells by cytotoxic T cells redirected with a CD19-/CD3-bispecific single-chain antibody construct.

    PubMed

    Hoffmann, Patrick; Hofmeister, Robert; Brischwein, Klaus; Brandl, Christian; Crommer, Sandrine; Bargou, Ralf; Itin, Christian; Prang, Nadja; Baeuerle, Patrick A

    2005-05-20

    Certain bispecific antibodies exhibit an extraordinary potency and efficacy for target cell lysis by eliciting a polyclonal T-cell response. One example is a CD19-/CD3-bispecific single-chain antibody construct (bscCD19xCD3), which at femtomolar concentrations can redirect cytotoxic T cells to eliminate human B lymphocytes, B lymphoma cell lines and patient-derived malignant B cells. Here we have further explored the basis for this high potency. Using video-assisted microscopy, bscCD19xCD3 was found to alter the motility and activity of T cells from a scanning to a killing mode. Individual T cells could eliminate multiple target cells within a 9 hr time period, resulting in nuclear fragmentation and membrane blebbing of target cells. Complete target cell elimination was observed within 24 hr at effector-to-target cell ratios as low as 1:5. Under optimal conditions, cell killing started within minutes after addition of bscCD19xCD3, suggesting that the rate of serial killing was mostly determined by T-cell movement and target cell scanning and lysis. At all times, T cells remained highly motile, and no clusters of T and target cells were induced by the bispecific antibody. Bystanding target-negative cells were not detectably affected. Repeated target cell lysis by bscCD19xCD3-activated T cells increased the proportion of CD19/CD3 double-positive T cells, which was most likely a consequence of transfer of CD19 from B to T cells during cytolytic synapse formation. To our knowledge, this is the first study showing that a bispecific antibody can sustain multiple rounds of target cell lysis by T cells. PMID:15688411

  17. Preclinical evaluation of multistep targeting of diasialoganglioside GD2 using a IgG-scFv bispecific antibody with high affinity for GD2 and DOTA metal complex

    PubMed Central

    Cheal, Sarah M.; Xu, Hong; Guo, Hong-fen; Zanzonico, Pat B.; Larson, Steven M.; Cheung, Nai-Kong

    2014-01-01

    Bispecific antibodies (BsAb) have proven to be useful targeting vectors for pretargeted radioimmunotherapy (PRIT). We sought to overcome key PRIT limitations such as high renal radiation exposure and immunogenicity (e.g. of streptavidin-antibody fusions), to advance clinical translation of this PRIT strategy for diasialoganglioside GD2-positive (GD2(+)) tumors. For this purpose, a IgG-scFv BsAb was engineered using the sequences for the anti-GD2 humanized monoclonal antibody hu3F8 (1) and C825, a murine scFv antibody with high affinity for the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexed with beta-particle emitting radiometals such as 177Lu and 90Y (2, 3). A three-step regimen including hu3F8-C825, a dextran-based clearing agent, and p-aminobenzyl-DOTA radiolabeled with 177Lu (as 177Lu-DOTA-Bn; t1/2 = 6.71 days (d)) was optimized in immunocompromised mice carrying subcutaneous (s.c.) human GD2(+) neuroblastoma (NB) xenografts. Absorbed doses for tumor and normal tissues were ?85 cGy/MBq and ?3.7 cGy/MBq, respectively, with therapeutic indicies (TI) of 142 for blood and 23 for kidney. A therapy study (n = 5 per group; tumor volume: 240 160 mm3) with three successive PRIT cycles (total 177Lu: ?33 MBq; tumor dose ?3400 cGy), revealed complete tumor response in 5/5 animals, with no recurrence up to 28 d post-treatment. Tumor ablation was confirmed histologically in 4/5 mice, and normal organs showed minimal overall toxicities. All non-treated mice required sacrifice within 12 d (>1.0 cm3 tumor volume). We conclude that this novel anti-GD2 PRIT approach has sufficient TI to successfully ablate s.c. GD2(+)NB in mice while sparing kidney and bone marrow. PMID:24944121

  18. Characterization of a monoclonal antibody to bovine xanthine oxidase.

    PubMed Central

    Kaetzel, C S; Mather, I H; Bruder, G; Madara, P J

    1984-01-01

    The isolation of a hybridoma cell line, C-41, secreting monoclonal antibody to bovine xanthine oxidase (EC 1.2.3.2), is described. The specificity of this antibody was determined by solid-phase immunoassay, immunoblotting procedures, affinity chromatography, immunoelectrophoresis and precipitation techniques. The results are compared with those obtained in similar specificity studies on a previously described monoclonal antibody secreted by hybridoma cell line A-94 [Mather, Nace, Johnson & Goldsby (1980) Biochem. J. 188, 925-928]. This latter antibody appears to bind to xanthine oxidase only when the enzyme is immobilized on a solid support such as a plastic plate or nitrocellulose paper. Potential problems in the determination of the specificity of monoclonal antibodies, especially towards membrane proteins of unknown biological activity, are discussed. Images Fig. 2. Fig. 3. Fig. 6. Fig. 7. PMID:6378181

  19. Characterization and utilization of a monoclonal antibody against pancreatic carcinoma

    SciTech Connect

    Kurtzman, S.H.; Sindelar, W.F.; Atcher, R.W.; Mitchell, J.B.; DeGraff, W.G.; Gamson, J.; Russo, A.; Friedman, A.M.; Hines, J.J.

    1994-10-01

    A monoclonal antibody was produced against a human pancreatic adenocarcinoma line and was found to react with several different human carcinomas by immunoperoxidase staining of fixed tissues. The original cells used to generate the monoclonal antibody were treated with detergent to lyse the cell membrane. A membrane associated protein of molecular weight 35kD was isolated from this detergent lysed preparation and found to be recognized by the monoclonal antibody. The binding constant of the antigen antibody reaction on the cells is 5 x 10{sup {minus}5}. It was further determined that there are 700,000 binding sites per cell. Kinetics of the antigen-antibody reaction under several conditions were also explored.

  20. Bispecific antibodies targeting tumor-associated antigens and neutralizing complement regulators increase the efficacy of antibody-based immunotherapy in mice.

    PubMed

    Macor, P; Secco, E; Mezzaroba, N; Zorzet, S; Durigutto, P; Gaiotto, T; De Maso, L; Biffi, S; Garrovo, C; Capolla, S; Tripodo, C; Gattei, V; Marzari, R; Tedesco, F; Sblattero, D

    2015-02-01

    The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4-25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3-4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, polymorphonuclear and natural killer cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications. PMID:24903480

  1. Monoclonal antibody therapy in multiple sclerosis

    PubMed Central

    2010-01-01

    Therapeutic approaches to multiple sclerosis (MS) are based on altering the functions of the immune system, either by using broad immunosuppressive drugs used for transplantation rejection and rheumatology, or by modulating them more discreetly with beta interferon and synthetic amino-acid copolymers. These strategies are only partially successful, have important safety and tolerability limitations, and have shown to be mostly effective in earlier stages of the disease, in which acute relapses dominate the clinical picture. For progressive phenotypes of MS there are currently no effective therapeutic options. As very specific and potent immunosuppressive agents, monoclonal antibodies (mAbs) may offer considerable advantages over other therapies for MS. During the last decade, anti-a4 integrin natalizumab became the first approved mAb for treatment of relapsing MS, after convincingly demonstrating clinically significant effects on two large Phase 3 trials. Moreover, the concept of disease remission was introduced for the first time to describe patients who show no signs of clinical or imaging markers of disease activity during therapy with natalizumab. Of the mAbs under development for MS, alemtuzumab and rituximab have also shown promising evidence of effectiveness and potentially expanded the therapeutic horizon to reversal of disease progression in early relapsing patients and progressive patients who previously had not been studied. However, the appearance of progressive multifocal leukoencephalopathy (PML) in natalizumab-treated MS patients, as well as in patients with lymphoma, lupus and rheumatoid arthritis, treated with rituximab and autoimmune-type complications in alemtuzumab-treated MS patients underlines the fact that extended efficacy comes with significant clinical risks. The challenge is then how best to utilize therapies that have evidently superior efficacy in a chronic disease of young adults to obtain the best benefit-risk ratio and how to monitor and prevent emergent safety concerns. PMID:21124072

  2. Monoclonal antibodies specific for Campylobacter fetus lipopolysaccharides.

    PubMed

    Brooks, B W; Robertson, R H; Lutze-Wallace, C L; Pfahler, W

    2002-06-01

    Four monoclonal antibodies (mAbs) (M1357, M1360, M1823 and M1825) which reacted with Campylobacter fetus lipopolysaccharide (LPS) core region epitopes were produced and characterized. Reactivity of these mAbs with C. fetus core LPS epitopes was determined by enzyme-linked immunosorbent assay (ELISA) with whole cell proteinase K digests and phenol-water extracted LPS, and by immunoblotting with proteinase K digests. The specificities of the four mAbs were evaluated using an indirect ELISA. One of the mAbs reacted with 42 and three of the mAbs reacted with 41 of the 42 C. fetus strains examined. No reaction was observed between the four mAbs and 32 non-C. fetus bacteria tested, with the exception of one mAb with one organism. The four mAbs reacted with serotype A and B strains indicating the presence of shared epitopes in C. fetus LPS core oligosaccharides. The specificities of three mAbs previously produced to C. fetus LPS O-antigens (M1177, M1183 and M1194) were also evaluated and no reaction was observed with these mAbs and the 32 non-C. fetus bacteria tested. Strong immunofluorescence reactions were observed with the anti-O chain mAbs and selected C. fetus strains of the homologous serotype. These anti-LPS core oligosaccharide and anti-LPS O chain mAbs are highly specific for C. fetus and are potentially useful as immunodiagnostic reagents for detection, identification and characterization of C. fetus. PMID:12079745

  3. Structural design of disialoganglioside GD2 and CD3-bispecific antibodies to redirect T cells for tumor therapy

    PubMed Central

    Cheng, Ming; Ahmed, Mahiuddin; Xu, Hong; Cheung, Nai-Kong V.

    2015-01-01

    Antibody based immunotherapy has proven efficacy for patients with high risk neuroblastoma. However, despite being the most efficient tumoricidal effectors, T cells are underutilized because they lack Fc receptors. Using a monovalent single chain fragment (ScFv) platform, we engineered tandem scFv bispecific antibodies (BsAbs) that specifically target disialoganglioside (GD2) on tumor cells and CD3 on T cells. Structural variants of BsAbs were constructed and ranked based on binding to GD2, and on competency in inducing T cell mediated tumor cytotoxicity. In vitro thermal stability and binding measurements were used to characterize each of the constructs, and in silico molecular modeling was used to show how the orientation of the variable region heavy (VH) and light (VL) chains of the anti-GD2 ScFv could alter the conformations of key residues responsible for high affinity binding. We showed that the VH-VL orientation, the (GGGGS)3 linker, disulfide bond stabilization of scFv, when combined with an affinity matured mutation provided the most efficient BsAb to direct T cells to lyse GD2 positive tumor cells. In vivo, the optimized BsAb could efficiently inhibit melanoma and neuroblastoma xenograft growth. These findings provide preclinical validation of a structure-based method to assist in designing BsAb for T-cell mediated therapy. PMID:24895182

  4. A monoclonal antibody for the specific diagnosis of plague*

    PubMed Central

    Williams, J. E.; Gentry, M. K.; Braden, C. A.; Tyndal, G. L.; Altieri, P. L.; Berman, S.; Robinson, D. M.

    1988-01-01

    A stable mouse-cell hybridoma was obtained that secretes an IgA monoclonal antibody reactive with the fraction 1 (F1) envelope antigen of Yersinia pestis. Titres of the antibody typically ranged from 1:32 768 to 1:65 536 in mouse ascitic fluids. The monoclonal antibody formed a line of precipitation when run against F1 antigen in Ouchterlony gel diffusion tests. In tests of 235 strains of Y. pestis, lines of identity occurred between the precipitates formed with a solution of purified F1 antigen and the F1 antigen produced by the plague strains. No precipitates formed for 65 strains that were incapable of elaborating F1 antigen. Specificity of the monoclonal antibody for strains of Y. pestis producing F1 was also indicated by negative results for 50 yersinia strains other than Y. pestis tested by an ELISA that used the antibody to capture antigen. Experiments to determine the shelf-life of the antibody were conducted over 3-4 years. When the monoclonal antibody was freeze-dried in vials, titre was retained for three years when the vials were stored at -70 C but only for two months when they were stored at ambient temperatures. When the antibody was freeze-dried in wells of ELISA plates, sensitivity of the plates for capture of F1 antigen was preserved for four years when the plates were stored at -70 C compared with two weeks for plates stored at room temperature. When a solution of the antibody was sealed in wells of ELISA plates and refrigerated at 4 C, reactivity of the antibody and sensitivity of the plates were retained for a year. Alternatives for the application of this monoclonal antibody in ELISA and other plague diagnostic procedures are discussed. PMID:3260145

  5. MONOCLONAL ANTIBODY TO FENBENDAZOLE: UTILITY IN RESIDUE STUDIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A monoclonal antibody-based ELISA was developed for fenbendazole, a widely used benzimidazole anthelmintic, with approved uses in cattle and other food animals. The antibody was elicited using as hapten 2-succinamido-5(6)-phenylthiobenzimidazole, which was conjugated with bovine serum albumin to pro...

  6. Palladium-109 labeled anti-melanoma monoclonal antibodies

    DOEpatents

    Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

    1984-04-30

    The invention consists of new monoclonal antibodies labelled with Palladium 109, a beta-emitting radionuclide, the method of preparing this material, and its use in the radiotherapy of melanoma. The antibodies are chelate-conjugated and demonstrate a high uptake in melanomas. (ACR)

  7. [Monoclonal antibodies to the human group-specific antigen].

    PubMed

    Deriugina, E I; Drize, N I; Lemeneva, L N; Sadovnikova, E Iu; Udalov, G A

    1989-01-01

    Serologic description of murine monoclonal antibodies (H-86/44 and H-86/50) which are capable to detect a membrane-connected form of human H antigen is given. The antibodies are notable for ability (H-86/44) to be absorbed by salivary H substance of ABH secretors i.e. to detect a soluble form of H antigen. Highly specific, highly active and standard anti-H reagents may be produced on the basis of the anti-H monoclonal antibodies obtained. PMID:2482553

  8. A perspective of monoclonal antibodies: Past, present, and future

    SciTech Connect

    DeLand, F.H. )

    1989-07-01

    In 1975, the development of the technique to produce monoclonal antibodies revolutionized the approach to cancer detection and therapy. Hundreds of monoclonal antibodies to the epitopes of tumor cells have been produced, providing more specific tools for probing the cellular elements of cancer. At the same time, these tools have disclosed greater complexity in the character of these cells and stimulated further investigation. Although there are antibodies to specific epitopes of neoplastic cells, this purity has not provided the improved detection and therapy of cancer first expected. Technical manipulations have provided limited improvement in results, but more sophisticated techniques, such as biologic response modifiers, may be required to attain clinical results that can be universally applied. The intense research in monoclonal antibodies and their application does offer promise that the goal of improved cancer detection and therapy will be forthcoming. 58 references.

  9. Monoclonal antibodies to human kidney gamma-glutamyltransferase.

    PubMed

    Donati, R; Sabolovic, N; Wellman, M; Artur, Y; Siest, G

    1988-05-31

    Eight hybridoma clones secreting large amounts of monoclonal antibodies against purified human kidney gamma-glutamyltransferase (GGT) were isolated and produced in ascites. None of them inhibits the catalytic activity of GGT. They all bind to the heavy subunit of this dimeric enzyme. Immunoblot analysis showed that these antibodies react with the catalytically active GGT. The monoclonal antibodies also recognize the heavy subunit of the human liver enzyme. This is of interest, as serum GGT is known to originate from the liver. None of the monoclonals reacts with GGTs from rat or pig kidney. After identification of epitopes specificities, the antibodies will be used for the development of immunoassays of GGT especially in human serum. PMID:2898310

  10. Polymorphism of normal factor IX detected by mouse monoclonal antibodies.

    PubMed Central

    Wallmark, A; Ljung, R; Nilsson, I M; Holmberg, L; Hedner, U; Lindvall, M; Sjgren, H O

    1985-01-01

    Hemophilia B is an X-chromosomal recessive disease due to deficiency of coagulation factor IX. Three monoclonal antibodies against factor IX were prepared and used to develop immunoradiometric assays (IRMAs) of factor IX antigen (IX-Ag). IX-Ag was measured in 65 normal individuals with one IRMA based on polyclonal anti-IX antibodies and two IRMAs based on three monoclonal anti-IX antibodies. One of the monoclonal antibodies differed in specificity since it neutralized less than 50% of the clotting activity of factor IX (IX-C), whereas the other two monoclonal antibodies neutralized 80-95%. When the former antibody was used as the solid phase in IRMA, two groups of normal individuals were distinguished: group A with measurable IX-Ag, and group B without demonstrable IX-Ag. There were no differences between the groups either in IX-C or in IX-Ag measured with polyclonal antibodies. A subgroup comprising only women could be distinguished in group A, in whom intermediate IX-Ag concentrations were found. Family studies showed the group B variant of normal factor IX to be transmitted according to the pattern of X-linked recessive inheritance. The allelic frequency of group A was 0.66, and that of group B was 0.34. PMID:3873655

  11. Characterization of Candida albicans cell wall antigens with monoclonal antibodies.

    PubMed Central

    Ponton, J; Marot-Leblond, A; Ezkurra, P A; Barturen, B; Robert, R; Senet, J M

    1993-01-01

    The antigenic composition of Candida albicans is very complex. In order to study the antigenic relationship between blastoconidia and germ tubes of C. albicans, we produced several monoclonal antibodies and analyzed their reactivity against cell wall antigens either in intact cells or in cells treated with dithiothreitol. Overall, four types of reactivity were found. Monoclonal antibodies 3D9 and 15C9 stained the germ tubes only when tested by indirect immunofluorescence. However, they showed a different reactivity by immunoblotting. Monoclonal antibody 3D9 reacted with antigens with molecular masses of > 200 and 180 kDa specifically expressed in the germ tube. Monoclonal antibody 15C9 reacted with antigens of 87, 50, and 34 kDa present in the germ tube extract and with antigens of 92, 50, 34, and 32 kDa present in the blastoconidium extract. The reactivity of blastoconidia treated for different times with dithiothreitol with these monoclonal antibodies was also studied by enzyme-linked immunosorbent assay. The reactivity of monoclonal antibody 3D9 did not significantly change during the cell wall extraction. However, the reactivity of monoclonal antibody 15C9 was increased for blastoconidia extracted for 60 min and decreased markedly for blastocondia extracted for 120 min. Monoclonal antibody G3B was nonreactive by indirect immunofluoresence but reacted with antigens of 47 and 38 kDa present in the germ tube extract and with an antigen of 47 kDa present in the blastoconidium extract. Monoclonal antibody B9E stained both morphological phases by indirect immunofluorescence. By immunoblotting, it reacted with antigens of > 70 kDa present in the germ tube extract and with antigens of > 63, 56, 47, and 38 kDa present in the blastoconidium extract. Based on the results presented in this study, four types of antigens are described. Type I antigens are expressed on the outermost layers of the germ tube cell wall only. Type II antigens are expressed both on the germ tube cell wall surface and within the blastoconidium cell wall. Type III antigens are found within the cell wall of both blastoconidia and germ tubes. Type IV antigens are expressed on both the blastoconidium and germ tube surface. Two types more can be hypothesized for antigens expressed on the blastoconidium cell surface and within the germ tube cell wall (type V) and for those expressed on the blastoconidium surface only (type VI). Images PMID:8406886

  12. Targeting Cytomegalovirus-Infected Cells Using T Cells Armed with Anti-CD3 Anti-CMV Bispecific Antibody

    PubMed Central

    Lum, Lawrence G.; Ramesh, Mayur; Thakur, Archana; Mitra, Subhashis; Deol, Abhinav; Uberti, Joseph P.; Pellett, Philip E.

    2013-01-01

    Human cytomegalovirus (CMV) reactivation and infection can lead to poor outcomes after allogeneic stem cell transplantation. We hypothesized that anti-CD3 activated T cells (ATCs) armed with chemically heteroconjugated anti-CD3 polyclonal anti-CMV bispecific antibody (CMVBi) will target and eliminate CMV-infected cells. Arming doses of CMVBi as low as 0.01 ng/106 ATCs was able to mediate specific cytotoxicity (SC) directed at CMV-infected target cells significant above unarmed ATCs at mutiplicities of infection (MOI) between 0.01 and 1. At effector-to-target ratios (E:T) of 25:1, 12.5:1, 6.25:1, and 3.125:1, armed ATCs significantly enhanced killing of CMV-infected targets compared with unarmed ATCs. At an MOI of 1.0, the mean % SC directed at CMV-infected targets cells for CMVBi-armed ATCs at E:T of 3.12, 6.25, and 12.5 were 79%, 81%, and 82%, respectively; whereas the mean % SC for unarmed ATCs at the same E:T were all <20%. ATCs, Cytogam, or CMVBi alone did not lyse uninfected or CMV-infected targets. Co-cultures of CMVBi-armed ATCs with CMV-infected targets induced cytokine and chemokine release from armed ATCs. This nonmajor histocompatibility complex restricted strategy for targeting CMV could be used to prevent or treat CMV infections after allogeneic stem cell transplantation or organ transplantation. PMID:22313635

  13. Effect of small-molecule-binding affinity on tumor uptake in vivo: a systematic study using a pretargeted bispecific antibody.

    PubMed

    Orcutt, Kelly Davis; Rhoden, John J; Ruiz-Yi, Benjamin; Frangioni, John V; Wittrup, K Dane

    2012-06-01

    Small-molecule ligands specific for tumor-associated surface receptors have wide applications in cancer diagnosis and therapy. Achieving high-affinity binding to the desired target is important for improving detection limits and for increasing therapeutic efficacy. However, the affinity required for maximal binding and retention remains unknown. Here, we present a systematic study of the effect of small-molecule affinity on tumor uptake in vivo with affinities spanning a range of three orders of magnitude. A pretargeted bispecific antibody with different binding affinities to different DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)-based small molecules is used as a receptor proxy. In this particular system targeting carcinoembryonic antigen, a small-molecule-binding affinity of 400 pmol/L was sufficient to achieve maximal tumor targeting, and an improvement in affinity to 10 pmol/L showed no significant improvement in tumor uptake at 24 hours postinjection. We derive a simple mathematical model of tumor targeting using measurable parameters that correlates well with experimental observations. We use relations derived from the model to develop design criteria for the future development of small-molecule agents for targeted cancer therapeutics. PMID:22491799

  14. Bispecific antibody releasing-mesenchymal stromal cell machinery for retargeting T cells towards acute myeloid leukemia blasts

    PubMed Central

    Aliperta, R; Cartellieri, M; Feldmann, A; Arndt, C; Koristka, S; Michalk, I; von Bonin, M; Ehninger, A; Bachmann, J; Ehninger, G; Bornhäuser, M; Bachmann, M P

    2015-01-01

    Bispecific antibodies (bsAbs) engaging T cells are emerging as a promising immunotherapeutic tool for the treatment of hematologic malignancies. Because their low molecular mass, bsAbs have short half-lives. To achieve clinical responses, they have to be infused into patients continously, for a long period of time. As a valid alternative we examined the use of mesenchymal stromal cells (MSCs) as autonomous cellular machines for the constant production of a recently described, fully humanized anti-CD33-anti-CD3 bsAb, which is capable of redirecting human T cells against CD33-expressing leukemic cells. The immortalized human MSC line SCP-1 was genetically modified into expressing bsAb at sufficient amounts to redirect T cells efficiently against CD33 presenting target cells, both in vitro and in an immunodeficient mouse model. Moreover, T cells of patients suffering from acute myeloid leukemia (AML) in blast crisis eliminated autologous leukemic cells in the presence of the bsAb secreting MSCs over time. The immune response against AML cells could be enhanced further by providing T cells an additional co-stimulus via the CD137-CD137 ligand axis through CD137L expression on MSCs. This study demonstrates that MSCs have the potential to be used as cellular production machines for bsAb-based tumor immunotherapy in the future. PMID:26383821

  15. TriFabsTrivalent IgG-Shaped Bispecific Antibody Derivatives: Design, Generation, Characterization and Application for Targeted Payload Delivery

    PubMed Central

    Mayer, Klaus; Baumann, Anna-Lena; Grote, Michael; Seeber, Stefan; Kettenberger, Hubert; Breuer, Sebastian; Killian, Tobias; Schfer, Wolfgang; Brinkmann, Ulrich

    2015-01-01

    TriFabs are IgG-shaped bispecific antibodies (bsAbs) composed of two regular Fab arms fused via flexible linker peptides to one asymmetric third Fab-sized binding module. This third module replaces the IgG Fc region and is composed of the variable region of the heavy chain (VH) fused to CH3 with knob-mutations, and the variable region of the light chain (VL) fused to CH3 with matching holes. The hinge region does not contain disulfides to facilitate antigen access to the third binding site. To compensate for the loss of hinge-disulfides between heavy chains, CH3 knob-hole heterodimers are linked by S354C-Y349C disulphides, and VH and VL of the stem region may be linked via VH44C-VL100C disulphides. TriFabs which bind one antigen bivalent in the same manner as IgGs and the second antigen monovalent in between these Fabs can be applied to simultaneously engage two antigens, or for targeted delivery of small and large (fluorescent or cytotoxic) payloads. PMID:26593903

  16. TriFabs-Trivalent IgG-Shaped Bispecific Antibody Derivatives: Design, Generation, Characterization and Application for Targeted Payload Delivery.

    PubMed

    Mayer, Klaus; Baumann, Anna-Lena; Grote, Michael; Seeber, Stefan; Kettenberger, Hubert; Breuer, Sebastian; Killian, Tobias; Schfer, Wolfgang; Brinkmann, Ulrich

    2015-01-01

    TriFabs are IgG-shaped bispecific antibodies (bsAbs) composed of two regular Fab arms fused via flexible linker peptides to one asymmetric third Fab-sized binding module. This third module replaces the IgG Fc region and is composed of the variable region of the heavy chain (VH) fused to CH3 with "knob"-mutations, and the variable region of the light chain (VL) fused to CH3 with matching "holes". The hinge region does not contain disulfides to facilitate antigen access to the third binding site. To compensate for the loss of hinge-disulfides between heavy chains, CH3 knob-hole heterodimers are linked by S354C-Y349C disulphides, and VH and VL of the stem region may be linked via VH44C-VL100C disulphides. TriFabs which bind one antigen bivalent in the same manner as IgGs and the second antigen monovalent "in between" these Fabs can be applied to simultaneously engage two antigens, or for targeted delivery of small and large (fluorescent or cytotoxic) payloads. PMID:26593903

  17. Humanization and simultaneous optimization of monoclonal antibody.

    PubMed

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

    2014-01-01

    Antibody humanization is an essential technology for reducing the potential risk of immunogenicity associated with animal-derived antibodies and has been applied to a majority of the therapeutic antibodies on the market. For developing an antibody molecule as a pharmaceutical at the current biotechnology level, however, other properties also have to be considered in parallel with humanization in antibody generation and optimization. This section describes the critical properties of therapeutic antibodies that should be sufficiently qualified, including immunogenicity, binding affinity, physiochemical stability, expression in host cells and pharmacokinetics, and the basic methodologies of antibody engineering involved. By simultaneously optimizing the antibody molecule in the light of these properties, it should prove possible to shorten the research and development period necessary to identify a highly qualified clinical candidate and consequently accelerate the start of the clinical trial. PMID:24037839

  18. Immunohistochemical characterization of monoclonal antibodies directed against the TSH receptor.

    PubMed

    Kohnert, K D; Krabbe, S; Meng, W

    1994-06-01

    Monoclonal antibodies have been obtained by fusing mouse myeloma cells with spleen cells of mice immunized with crude thyroid membranes. Among the antibodies reactive with different thyroid antigenic components, three were found to specifically react with TSH receptor molecules. These antibodies displayed characteristic staining patterns on frozen sections of thyroid tissue from patients with various thyroid diseases upon identification of antibody binding by indirect peroxidase staining. No specific reactivity was detected with tissue from other human organs, such as pancreas, liver, fat, and muscle. The results demonstrate that the immunoperoxidase technique and the specificity of the monoclonal antibodies produced permitted the identification of cellular constituents that might be important antigens in autoimmune thyroid disease. PMID:7976127

  19. Identification and typing of herpes simplex viruses with monoclonal antibodies.

    PubMed Central

    Balachandran, N; Frame, B; Chernesky, M; Kraiselburd, E; Kouri, Y; Garcia, D; Lavery, C; Rawls, W E

    1982-01-01

    Monoclonal antibodies which reacted with type-specific antigens of herpes simplex virus type 2 or with antigens shared by herpes simplex virus types 1 and 2 were used in an indirect immunofluorescence assay to type virus isolates and to detect viral antigens in cells obtained from herpetic lesions. Complete concordance was obtained for 42 isolates typed by endonuclease restriction analysis of viral DNA and by indirect immunofluorescence with monoclonal antibodies. Examination of a limited number of ulcerative lesions revealed that indirect immunofluorescence and virus isolation were comparable in detecting herpes simplex virus. The results indicate that monoclonal antibodies can be used to accurately identify and type isolates of herpes simplex virus. PMID:6286719

  20. [Regulatory consequences for the use of monoclonal antibodies].

    PubMed

    Lackner, Friedrich; Behr-Gross, Marie-Emmanuelle

    2009-12-01

    Khler and Milstein published a method for the manufacture of mouse monoclonal antibodies of predefined specificity 1975 [1], a work rewarded with the Nobel Prize 1984. Since then, the field has developed rapidly with new production methods such as recombinant DNA technology, phage display and genetically engineered animals. Following the first clinical applications with a mouse monoclonal antibody, new classes as chimaeric, humanized and human monoclonal antibodies appeared, with the advantages of less adverse reactions and better efficacy. The development over more than 30 years resulted in more than 25 approved products on the market for various therapeutic applications, e.g. for the treatment of cancer, inflammatory diseases, heart disease and transplantation, and medicines for many more applications are currently under development. PMID:20035703

  1. Monoclonal antibodies and Fc fragments for treating solid tumors

    PubMed Central

    Eisenbeis, Andrea M; Grau, Stefan J

    2012-01-01

    Advances in biotechnology, better understanding of pathophysiological processes, as well as the identification of an increasing number of molecular markers have facilitated the use of monoclonal antibodies and Fc fragments in various fields in medicine. In this context, a rapidly growing number of these substances have also emerged in the field of oncology. This review will summarize the currently approved monoclonal antibodies used for the treatment of solid tumors with a focus on their clinical application, biological background, and currently ongoing trials. PMID:22291463

  2. Targeting of cancer stem cell marker EpCAM by bispecific antibody EpCAMxCD3 inhibits pancreatic carcinoma

    PubMed Central

    Salnikov, Alexei V; Groth, Ariane; Apel, Anja; Kallifatidis, Georgios; Beckermann, Benjamin M; Khamidjanov, Akmal; Ryschich, Eduard; Büchler, Markus W; Herr, Ingrid; Moldenhauer, Gerhard

    2009-01-01

    Patients with pancreatic cancer have a poor survival rate, and new therapeutic strategies are needed. Epithelial cell adhesion molecule (EpCAM), suggested as a marker for cancer stem cells, is over-expressed on most pancreatic tumour cells but not on normal cells and may be an ideal therapeutic target. We evaluated the anti-tumour efficiency of bispecific EpCAMxCD3 antibody linking tumour cells and T lymphocytes. In NOD SCID mice, EpCAMxCD3 had a long serum half-life (t1/2∼ 7 days). EpCAMxCD3 significantly retarded growth of BxPC-3 pancreatic carcinoma xenografts. For mimicking a pancreatic cancer microenvironment in vitro, we used a three-dimensional tumour reconstruct system, in which lymphocytes were co-cultured with tumour cells and fibroblasts in a collagen matrix. In this in vivo–like system, EpCAMxCD3 potently stimulated production of the effector cytokines IFN-γ and TNF-α by extracorporally pre-activated lymphocytes. Moreover, compared with a bivalent anti-CD3 antibody, EpCAMxCD3 more efficiently activated the production of TNF-α and IFN-γ by non-stimulated peripheral blood mononuclear cells. Most excitingly, we demonstrate for the first time that EpCAMxCD3 induces prolonged contacts between lymphocytes and tumour cells, which may be the main reason for the observed anti-tumour effects. As an important prerequisite for future use in patients, EpCAMxCD3 did not alter lymphocyte migration as measured by time-lapse video microscopy. Our data may open a way to improve the immune response and treatment outcome in patients with pancreatic cancer. PMID:20196789

  3. Monoclonal antibody-defined human endothelial antigens as vascular markers.

    PubMed

    Ruiter, D J; Schlingemann, R O; Rietveld, F J; de Waal, R M

    1989-08-01

    A review is given of human endothelial antigens recognized by monoclonal antibodies and used as vascular markers. These antigens can be classified tentatively into two categories that partly overlap: 1) differentiation markers and 2) antigens involved in specific cellular functions. Monoclonal antibodies recognizing endothelial differentiation markers reacting with all types of human endothelium can be regarded as constitutive endothelial markers. Other differentiation markers have a restricted distribution that is associated with a subtype of endothelium. Although sensitivity of the markers is high in general, specificity for endothelium is not absolute, based on distribution studies in tissues or in cell lines. With the exception of PAL-E and EN-3/EN-4, it is not clear from the literature whether the antibodies also react with lymphatic endothelium. Immunohistochemical examination of other species indicate that only BW 200 is restricted to humans. Immunoelectron microscopy of microvascular cells in tissue specimens has revealed that the monoclonal antibodies recognizing differentiation antigens show different subcellular distribution patterns. PAL-E and BW 200 react with the luminal endothelial surface, in a local and diffuse pattern, respectively. Anti-Von Willebrand factor (i.e., Factor VIII-related ag) antibodies react with Weibel-Palade bodies but also with subendothelial structures. Applications of immunohistochemistry using monoclonal antibodies in diagnostic pathology include assessment of vascular invasion by cancer cells, and identification of endothelial neoplasms and related disorders. Because anti-Factor VIII-related antigen and BW 200 are applicable on formaldehyde-fixed and paraplast-embedded tissue, they are most suitable for histodiagnostic application. Immunohistochemistry using monoclonal antibodies recognizing endothelial antigens involved in specific cellular functions also may contribute to pathobiologic research on the characterization of blood-tissue barriers, e.g., in the tumor vascular bed. PMID:2666520

  4. Hexavalent bispecific antibodies represent a new class of anticancer therapeutics: 1. Properties of anti-CD20/CD22 antibodies in lymphoma

    PubMed Central

    Cardillo, Thomas M.; Stein, Rhona; Chang, Chien-Hsing

    2009-01-01

    The dock and lock (DNL) method is a new technology for generating multivalent antibodies. Here, we report in vitro and in vivo characterizations of 20-22 and 22-20, a pair of humanized hexavalent anti-CD20/22 bispecific antibodies (bsAbs) derived from veltuzumab (v-mab) and epratuzumab (e-mab). The 22-20 was made by site-specific conjugation of e-mab to 4 Fabs of v-mab; 20-22 is of the opposite configuration, composing v-mab and 4 Fabs of e-mab. Each bsAb translocates both CD22 and CD20 into lipid rafts, induces apoptosis and growth inhibition without second-antibody crosslinking, and is significantly more potent in killing lymphoma cells in vitro than their parental antibodies. Although both bsAbs triggered antibody-dependent cellular toxicity, neither displayed complement-dependent cytotoxicity. Intriguingly, 22-20 and 20-22 killed human lymphoma cells in preference to normal B cells ex vivo, whereas the parental v-mab depleted malignant and normal B cells equally. In vivo studies in Daudi tumors revealed 20-22, despite having a shorter serum half-life, had antitumor efficacy comparable with equimolar v-mab; 22-20 was less potent than 20-22 but more effective than e-mab and control bsAbs. These results indicate multiple advantages of hexavalent anti-CD20/22 bsAbs over the individual parental antibodies and suggest that these may represent a new class of cancer therapeutics. PMID:19372261

  5. Improved serological diagnosis of Poplar mosaic virus with monoclonal antibodies.

    PubMed

    Carra, A; Brocchi, E; Simone, F De; Luisoni, E

    2005-05-01

    Poplar mosaic virus (PopMV) is widespread in all countries where poplar is grown, and causes severe economic losses in terms of quantity and quality of wood production. Control is based on indexing, aimed at the production of healthy propagation material. The currently used diagnostic method is double antibody sandwich (DAS) ELISA with polyclonal antibodies, which is relatively simple and inexpensive and more reliable than visual inspection of symptoms in the nurseries. However, this method also has disadvantages, mainly low sensitivity in relation to low concentration and irregular distribution of the virus in the plant. In this study, a new diagnostic method for PopMV based on production and use of a monoclonal antibody (Mab) in a triple antibody sandwich (TAS) ELISA, is presented. The TAS-ELISA with monoclonal antibodies was optimised by testing a range of reagent combinations and concentrations. PopMV was detected by the optimised TAS-ELISA with sensitivity more than 100 times higher than by DAS-ELISA with polyclonal antibodies. Six PopMV isolates from four European countries were detected with the same efficiency, indicating that no limitations to the practical use of the TAS-ELISA arise due to excessive epitope-specificity of the monoclonal antibody employed. PMID:15794987

  6. Localisation of metastatic carcinoma by a radiolabelled monoclonal antibody.

    PubMed Central

    Smedley, H. M.; Finan, P.; Lennox, E. S.; Ritson, A.; Takei, F.; Wraight, P.; Sikora, K.

    1983-01-01

    Rat monoclonal antibodies were prepared by immunising rats with human colorectal carcinoma cell membranes and fusing splenic lymphocytes with a rat myeloma. Hybridoma supernatants were screened by binding assays on membranes prepared from colorectal carcinoma tissue. One hybridoma supernatant, containing a monoclonal antibody with high binding activity on malignant compared to normal colon sections, was grown in large quantities in serum-free medium. After ammonium sulphate precipitation the antibody was purified by ion-exchange chromatography and labelled with 131I. Radiolabelled antibody was administered i.v. to 27 patients with colonic and other tumours. Scintigrams were obtained at 48 h. Computerised subtraction of the blood pool image revealed localised areas of uptake corresponding with areas of known disease in 13/16 patients with colorectal carcinoma and 3/4 patients with breast cancer. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 Figure 7 PMID:6337613

  7. MONOCLONAL ANTIBODIES IDENTIFY CONSERVED EPITOPES ON THE POLYHEDRIN OF 'HELIOTHIS ZEA' NUCLEAR POLYHEDROSIS VIRUS

    EPA Science Inventory

    Recent advances in monoclonal antibody techniques have provided an opportunity to simplify the procedures of serological identification of microorganisms. Because monoclonal antibodies are raised against individual antigenic determinants (epitopes), they can be used to screen wit...

  8. Generation of monoclonal antibodies to recombinant vascular endothelial growth factor.

    PubMed

    Shein, S A; Gurina, O I; Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Grinenko, N F; Ivanova, N V; Volgina, N E; Ryabukhin, I A; Chekhonin, V P

    2012-05-01

    Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells. Subsequent cloning yielded hybridoma stably expressing monoclonal anti-VEGF antibodies recognizing recombinant and native VEGF. PMID:22808513

  9. Mechanisms of monoclonal antibody stabilization and release from silk biomaterials

    PubMed Central

    Guziewicz, Nicholas A.; Massetti, Andrew J.; Perez-Ramirez, Bernardo J.; Kaplan, David L.

    2013-01-01

    The availability of stabilization and sustained delivery systems for antibody therapeutics remains a major clinical challenge, despite the growing development of antibodies for a wide range of therapeutic applications due to their specificity and efficacy. A mechanistic understanding of protein-matrix interactions is critical for the development of such systems and is currently lacking as a mode to guide the field. We report mechanistic insight to address this need by using well-defined matrices based on silk gels, in combination with a monoclonal antibody. Variables including antibody loading, matrix density, charge interactions, hydrophobicity and water access were assessed to clarify mechanisms involved in the release of antibody from the biomaterial matrix. The results indicate that antibody release is primarily governed by hydrophobic interactions and hydration resistance, which are controlled by silk matrix chemistry, peptide domain distribution and protein density. Secondary ionic repulsions are also critical in antibody stabilization and release. Matrix modification by free methionine incorporation was found to be an effective strategy for mitigating encapsulation induced antibody oxidation. Additionally, these studies highlight a characterization approach to improve the understanding and development of other protein sustained delivery systems, with broad applicability to the rapidly developing monoclonal antibody field. PMID:23859659

  10. A Novel Glycoengineered Bispecific Antibody Format for Targeted Inhibition of Epidermal Growth Factor Receptor (EGFR) and Insulin-like Growth Factor Receptor Type I (IGF-1R) Demonstrating Unique Molecular Properties*

    PubMed Central

    Schanzer, Juergen M.; Wartha, Katharina; Croasdale, Rebecca; Moser, Samuel; Knkele, Klaus-Peter; Ries, Carola; Scheuer, Werner; Duerr, Harald; Pompiati, Sandra; Pollman, Jan; Stracke, Jan; Lau, Wilma; Ries, Stefan; Brinkmann, Ulrich; Klein, Christian; Umana, Pablo

    2014-01-01

    In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the knob-into-hole technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats. PMID:24841203

  11. Limitations of radiolabeled monoclonal antibodies for localization of human neoplasms

    SciTech Connect

    Epenetos, A.A.; Snook, D.; Durbin, H.; Johnson, P.M.; Taylor-Papadimitriou, J.

    1986-06-01

    Tumor-associated monoclonal antibodies were radiolabeled with /sup 125/I and /sup 131/I and given i.v. in pairs to 19 patients 1-26 days prior to surgical excision of primary and metastatic breast, ovarian, and gastrointestinal tumors. For individual patients each monoclonal antibody was designated as specific or nonspecific according to prior immunoperoxidase staining results on the appropriate target neoplastic tissues. Quantitation of antibody uptake was performed on resected normal and neoplastic tissues. Although good tumor:non-tumor ratios were obtained with the specific antibodies (maximal tumor:blood ratio, 35.8:1 at 12 days postadministration), the absolute amount of radiolabel detected in tumors was small (mean value of 0.015% of total injected amount per g of tumor occurring 1 day postadministration). Furthermore, both specific and nonspecific antibodies accumulated in normal lymph nodes to a significant extent (mean value of 0.0026% of total injected amount per g of tissue occurring 1 day postadministration). Knowledge of such data is essential prior to considering therapeutic uses of radiolabeled monoclonal antibodies.

  12. A photosensitizer delivered by bispecific antibody redirected T lymphocytes enhances cytotoxicity against EpCAM-expressing carcinoma cells upon light irradiation.

    PubMed

    Blaudszun, Andr-Ren; Moldenhauer, Gerhard; Schneider, Marc; Philippi, Anja

    2015-01-10

    Recently conducted clinical trials have provided impressive evidence that chemotherapy resistant metastatic melanoma and several hematological malignancies can be cured using adoptive T cell therapy or T cell-recruiting bispecific antibodies. However, a significant fraction of patients did not benefit from these treatments. Here we have evaluated the feasibility of a novel combination therapy which aims to further enhance the killing potential of bispecific antibody-redirected T lymphocytes by using these cells as targeted delivery system for photosensitizing agents. For a first in vitro proof-of-concept study, ex vivo activated human donor T cells were loaded with a poly(styrene sulfonate) (PSS)-complex of the model photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP). In the absence of light and when loading with the water-soluble PSS/mTHPP-complex occurred at a tolerable concentration, viability and cytotoxic function of loaded T lymphocytes were not impaired. When "drug-enhanced" T cells were co-cultivated with EpCAM-expressing human carcinoma cells, mTHPP was transferred to target cells. Notably, in the presence of a bispecific antibody, which cross-links effector and target cells thereby inducing the cytolytic activity of cytotoxic T lymphocytes, significantly more photosensitizer was transferred. Consequently, upon irradiation of co-cultures, redirected drug-loaded T cells were more effective in killing A549 lung and SKOV-3 ovarian carcinoma cells than retargeted unloaded T lymphocytes. Particularly, the additive approach using redirected unloaded T cells in combination with appropriate amounts of separately applied PSS/mTHPP was less efficient as well. Thus, by loading T lymphocytes with a stimulus-sensitive anti-cancer drug, we were able to enhance the cytotoxic capacity of carrier cells. Photosensitizer boosted T cells could open new perspectives for adoptive T cell therapy as well as targeted photodynamic therapy. PMID:25449805

  13. Indium-111 labeled anti-melanoma monoclonal antibodies

    DOEpatents

    Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

    1984-04-30

    A monoclonal antibody to a high molecular weight melanoma-associated antigen was chelated and radiolabeled with indium-111. This material shows high affinity for melanoma and thus can be used in the detection, localization and imaging of melanoma. 1 figure.

  14. Characterization of monoclonal antibodies produced against Avian metapneumovirus Sybtype C

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Monoclonal antibodies (MAbs) were prepared against avian metapneumovirus (aMPV) subtype C (aMPV/Minnesota/turkey/1a/97). Six MAbs were selected based on ELISA activities and characterized by isotyping, neutralization test, Western blot analysis, and immunohistochemistry (IHC) assay. The results show...

  15. Development and evaluation of monoclonal antibodies for paxilline

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrati...

  16. A mouse monoclonal antibody against Alexa Fluor 647.

    PubMed

    Wuethrich, Irene; Guillen, Eduardo; Ploegh, Hidde L

    2014-04-01

    Fluorophores are essential tools in molecular and cell biology. However, their application is mostly confined to the singular exploitation of their fluorescent properties. To enhance the versatility and expand the use of the fluorophore Alexa Fluor 647 (AF647), we generated a mouse monoclonal antibody against it. We demonstrate its use of AF647 for immunoblot, immunoprecipitation, and cytofluorimetry. PMID:24746152

  17. Salmonella-specific monoclonal antibodies against recombinant Salmonella typhi 36-kilodalton porin.

    PubMed Central

    Kissel, V; Gonzalez, C; Astudillo, M; Godard, A; Wachman, B; Cabello, F C

    1994-01-01

    Mouse monoclonal antibodies were raised against recombinant Salmonella typhi 36-kDa porin monomer. Specificities of 16 monoclonal antibodies were analyzed as reactivity patterns in dot immunobinding and Western blot (immunoblot) assays using isolated outer membrane proteins of gram-negative bacteria and cloned purified S. typhi porin monomers and trimers. Four monoclonal antibodies were specific for Salmonella spp. Images PMID:7496957

  18. A Novel Bispecific Antibody against Human CD3 and Ephrin Receptor A10 for Breast Cancer Therapy.

    PubMed

    Taki, Shintaro; Kamada, Haruhiko; Inoue, Masaki; Nagano, Kazuya; Mukai, Yohei; Higashisaka, Kazuma; Yoshioka, Yasuo; Tsutsumi, Yasuo; Tsunoda, Shin-Ichi

    2015-01-01

    Ephrin receptor A10 (EphA10), a transmembrane receptor that binds to ephrin, is a newly identified breast cancer marker protein that has also been detected in HER2-negative tissue. In this study, we report creation of a novel bispecific antibody (BsAb) binding both EphA10 and CD3, thereby forming a bridge between antigens expressed on both tumor and immune cells and promoting recognition of tumor cells by immune cells and redirection of cytotoxic T cells (CTL). This BsAb (EphA10/CD3) was expressed in supernatants of BsAb gene-transfected cells as monomeric and dimeric molecules. Redirected T-cell lysis was observed when monomeric and dimeric BsAb were added to EphA10-overexpressing tumor cells in vitro. Furthermore, dimeric BsAb (EphA10/CD3) was more cytotoxic than monomeric BsAb, with efficient tumor cell lysis elicited by lower concentrations (?10-1 ?g/mL) and a lower effector to target (E/T) cell ratio (E/T = 2.5). Dimeric BsAb (EphA10/CD3) also showed significant anti-tumor effects in human xenograft mouse models. Together, these results revealed opportunities to redirect the activity of CTL towards tumor cells that express EphA10 using the BsAb (EphA10/CD3), which could be tested in future clinical trials as a novel and potent therapeutic for breast cancer tumors. PMID:26678395

  19. A Novel Bispecific Antibody against Human CD3 and Ephrin Receptor A10 for Breast Cancer Therapy

    PubMed Central

    Inoue, Masaki; Nagano, Kazuya; Mukai, Yohei; Higashisaka, Kazuma; Yoshioka, Yasuo; Tsutsumi, Yasuo; Tsunoda, Shin-ichi

    2015-01-01

    Ephrin receptor A10 (EphA10), a transmembrane receptor that binds to ephrin, is a newly identified breast cancer marker protein that has also been detected in HER2-negative tissue. In this study, we report creation of a novel bispecific antibody (BsAb) binding both EphA10 and CD3, thereby forming a bridge between antigens expressed on both tumor and immune cells and promoting recognition of tumor cells by immune cells and redirection of cytotoxic T cells (CTL). This BsAb (EphA10/CD3) was expressed in supernatants of BsAb gene-transfected cells as monomeric and dimeric molecules. Redirected T-cell lysis was observed when monomeric and dimeric BsAb were added to EphA10-overexpressing tumor cells in vitro. Furthermore, dimeric BsAb (EphA10/CD3) was more cytotoxic than monomeric BsAb, with efficient tumor cell lysis elicited by lower concentrations (≤10−1 μg/mL) and a lower effector to target (E/T) cell ratio (E/T = 2.5). Dimeric BsAb (EphA10/CD3) also showed significant anti-tumor effects in human xenograft mouse models. Together, these results revealed opportunities to redirect the activity of CTL towards tumor cells that express EphA10 using the BsAb (EphA10/CD3), which could be tested in future clinical trials as a novel and potent therapeutic for breast cancer tumors. PMID:26678395

  20. Delivery of the ribosome-inactivating protein, gelonin, to lymphoma cells via CD22 and CD38 using bispecific antibodies.

    PubMed Central

    French, R. R.; Penney, C. A.; Browning, A. C.; Stirpe, F.; George, A. J.; Glennie, M. J.

    1995-01-01

    It is well established that bispecific antibodies (BsAbs) can be used effectively in targeting the ribosome-inactivating protein (RIP), saporin, against neoplastic B cells. We have now extended this delivery system for use with gelonin. By measuring antigen-binding characteristics and epitope mapping a panel of anti-gelonin MAbs using the IAsys resonant mirror bisensor, we were able to rapidly select the most suitable for making BaAbs. The Fab' fragments from these MAbs were chemically conjugated with Fab' from either anti-CD22 or anti-CD38. Cytotoxicity assays showed that BsAbs were highly efficient at delivering gelonin to cultured Daudi cells and achieved levels of toxicity which correlated closely with the affinity of the BsAbs. Using pairs of anti-CD22 BsAbs we were able to generate bivalent BsAb-gelonin complexes which achieved IC50 values of 2 x 10(-11) M gelonin, a potency which is equivalent to that reached by saporin in this targeting system. However, because gelonin is 5-10 times less toxic than saporin, the therapeutic ratio for gelonin is superior, making it potentially a more useful agent for human treatment. Cytotoxicity assays and kinetic analysis showed that targeting gelonin via CD38 was 2-5 times less effective than delivery through CD22. However, with a pair of BsAbs designed to co-target gelonin via CD22 and CD38, the cytotoxicity achieved equalled that obtained with a pair of anti-CD22 BsAbs (IC50 = 1 x 10(-11) M). This important result suggests that the anti-CD38 helps bind the gelonin to the cell and is then 'dragged' or 'piggy-backed' into the cell by the anti-CD22 BsAb. The implication of these findings for cancer therapy is discussed. PMID:7734325

  1. Anti-CD3 Anti-GD2 Bispecific Antibody Redirects T-Cell Cytolytic Activity to Neuroblastoma Targets

    PubMed Central

    Yankelevich, Maxim; Kondadasula, Sri Vidya; Thakur, Archana; Buck, Steven; Cheung, Nai-Kong V.; Lum, Lawrence G.

    2013-01-01

    Background The ganglioside GD2 is an attractive target for immunotherapy of neuroectodermal tumors. We tested a unique bispecific antibody anti-CD3 anti-GD2 (3F8BiAb) for its ability to redirect activated T cells (ATC) to target GD2-positive neuroblastomas. Procedure ATC were generated from normal human peripheral blood mononuclear cells (PBMC) by stimulating the PBMC with OKT3 and expanding the T cells in the presence of interleukin 2 (IL-2) for 14 days. ATC were armed with 3F8BiAb (100 ng/106 cells) or Her2BiAb (50 ng/106 cells) prior to use. 3F8 BiAb were tested for its dual-binding specificity to GD2 expressed on cancer cell lines and CD3 expressed on ATC. 3F8BiAb-armed ATC were further tested ex vivo for their cytotoxicity against GD2 positive tumor targets and its ability to induce cytokine response upon binding to targets. Results GD2 expression in neuroblastoma cells was confirmed by FACS analysis. Specific binding of 3F8BiAb to the tumor targets as well as to ATC was confirmed by FACS analysis. 3F8BiAb-armed ATC exhibited specific killing of GD2 positive neuroblastoma cell lines significantly above unarmed ATC (P < 0.001). GD2BiAb-armed ATC secreted significantly higher levels of Th1 cytokines and chemokines compared to unarmed ATC (P < 0.001). Conclusions These preclinical findings support the potential of a novel immunotherapeutic approach to target T cells to neuroblastoma. PMID:22707078

  2. The use of combinations of monoclonal antibodies in clinical oncology.

    PubMed

    Henricks, Linda M; Schellens, Jan H M; Huitema, Alwin D R; Beijnen, Jos H

    2015-12-01

    Treatment with monoclonal antibodies is becoming increasingly important in clinical oncology. These antibodies specifically inhibit signaling pathways in tumor growth and/or induce immunological responses against tumor cells. By combining monoclonal antibodies several pathways may be targeted simultaneously, potentially leading to additive or synergistic effects. Theoretically, antibodies are very suitable for use in combination therapy, because of limited overlapping toxicity and lack of pharmacokinetic interactions. In this article an overview is given of preclinical and clinical data on twenty-five different combinations of antibodies in oncology. Some of these combinations have proven clinical benefit, for example the combination of trastuzumab and pertuzumab in HER2-positive breast cancer, which exemplifies an additive or synergistic effect on antitumor activity in clinical studies and the combination of nivolumab and ipilimumab, which results in significant increases in progression-free and overall survival in patients with advanced melanoma. However, other combinations may lead to unfavorable results, such as bevacizumab with cetuximab or panitumumab in advanced colorectal cancer. These combinations result in shorter progression-free survival and increased toxicity compared to therapy with a single antibody. In summary, the different published studies showed widely varying results, depending on the combination of antibodies, indication and patient population. More preclinical and clinical studies are necessary to unravel the mechanisms behind synergistic or antagonistic effects of combining monoclonal antibodies. Most research on combination therapies is still in an early stage, but it is expected that for several tumor types the use of combination therapy of antibodies will become standard of care in the near future. PMID:26547132

  3. Production and characterization of monoclonal antibodies to oxidized LDL.

    PubMed

    Choi, K; Lee, H S; Chung, H K

    1998-03-31

    Oxidized low density lipoprotein (LDL) seems to take a part in atherogenesis through direct interactions with macrophages, endothelial cells, and smooth muscle cells, and is thought to participate in renal glomerular injury. For the purpose of illustrating the role of oxidized LDL in the human diseases, monoclonal antibodies were developed and characterized, recognizing oxidized LDL-specific epitopes that do not exist on native LDL. LDL was oxidized by the incubation with CuSO4, and used as immunogen. Splenocytes from the immunized mouse and mouse myeloma cells were fused to produce hybridomas, which were screened for the secretion of oxidized LDL-specific antibodies. Immunoblot analysis and binding affinity assay showed that these monoclonal antibodies recognize malondialdehyde-conjugated peptide epitopes. PMID:9873821

  4. Monoclonal antibodies to the alternative oxidase of higher plant mitochondria

    SciTech Connect

    Elthon, T.E.; Nickels, R.L.; McIntosh, L. )

    1989-04-01

    The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain which terminates with cytochrome oxidase, an alternative pathway that terminates with an alternative oxidase. The alternative oxidase of Sauromatum guttatum Schott has recently been identified as a cluster of proteins with apparent M{sub r} of 37, 36, and 35 kilodaltons (kD). Monoclonal antibodies have now been prepared to these proteins and designated as AOA (binding all three proteins of the alternative oxidase cluster), AOU (binding the upper or 37 kD protein), and AOL (binding the lower or 36 and 35 kD proteins). All three antibodies bind to their respective alternative oxidase proteins whether the proteins are in their native or denatured states. AOA and AOU inhibit alternative oxidase activity around 49%, whereas AOL inhibits activity only 14%. When coupled individually to Sepharose 4B, all three monoclonal resins were capable of retaining the entire cluster of alternative oxidase proteins, suggesting that these proteins are physically associated in some manner. The monoclonals were capable of binding similar mitochondrial proteins in a number of thermogenic and nonthermogenic species, indicating that they will be useful in characterizing and purifying the alternative oxidase of different systems. The ability of the monoclonal-Sepharose 4B resins to retain the cluster of previously identified alternative oxidase proteins, along with the inhibition of alternative oxidase activity by these monoclonals, supports the role of these proteins in constituting the alternative oxidase.

  5. Monoclonal antibody radioimmunodetection of human-derived colon cancer.

    PubMed

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

    1983-01-01

    This study was designed to determine whether monoclonal antibody directed against carcinoembryonic antigen could successfully be used in the scintigraphic localization of a human-derived colon carcinoma in a hamster model. An immunoglobulin G (IgG)-1 kappa monoclonal antibody, prepared in this laboratory, against carcinoembryonic antigen (CEA) was radiolabeled with iodine-131 (131I). Four Syrian hamsters bearing GW-39 human colon cancers received intracardiac injections of 50 mu Ci of 131I (14 micrograms of antibody). Gamma camera images were obtained at 24-hour intervals. Animals were sacrificed at 11 days, and the tumors and entire animals were counted. A double-label antibody experiment was conducted with 131I anti-CEA and nonspecific MOPC 21 IgG iodine-125 (125I) to assess localization specificity. The scintiphotos clearly showed the tumor at 24 hours, but there was significant background (blood-pool activity). Later images at six and 11 days showed a gradual decrease in background activity and more clear definition of the tumor. Animals sacrificed at 11 days showed 48-80% of residual whole body radioactivity to be present in the tumor. However, these tumors were large at sacrifice, weighing 8.9 to 12.4 g. Specific localization was confirmed by the double-label experiments where specific localization was twice nonspecific accretion of IgG in the tumor. This study has shown that a specific monoclonal antibody can successfully be used to scintigraphically localize a colon tumor of human origin. Although clearance of background activity is a gradual process, eventually most radioactivity left in the animal is localized in the tumor. This study illustrates that the potential radiolabeled monoclonal antibodies hold as immunodiagnostic agents. PMID:6832932

  6. Radioimmunodetection of human melanoma with indium-111-labeled monoclonal antibody

    SciTech Connect

    Taylor, A. Jr.; Milton, W.; Eyre, H.P.; Christian, P.; Wu, F.; Hagan, P.; Alazraki, N.; Datz, F.L.; Unger, M.

    1988-03-01

    The purpose of the study was threefold: (1) to evaluate the efficacy of an /sup 111/In-labeled murine monoclonal antibody (ZME-018) directed against a heavy molecular weight melanoma associated glycoprotein in localizing metastatic disease; (2) to determine the effect of unlabeled antibody mass (2.5, 5, 10, 20, and 40 mg) on labeled antibody blood clearance, biodistribution and lesion detection; (3) to estimate radiation dosimetry. Twenty-five patients with previously documented disease received an intravenous infusion of 2.5 to 40 mg of monoclonal antibody with 1 mg of the antibody labeled with 5 mCi of /sup 111/In. There were no acute reactions. Patients were scanned without computer enhancement or background subtraction techniques at 24 and 72 hr after injection. Imaging detected tumor in 14/18 (78%) patients with active disease, identified 24/44 (77%) of lesions greater than 1 cm and changed or specifically directed patient management in 22% (4/18) patients with tumor. There was a prolongation in blood clearance associated with decreased liver and spleen activity following administration of 20 and 40 mg of antibody compared to the three lower antibody dose levels. Assuming a biodistribution similar to (/sup 111/In)ZME-018, the radiation dose delivered to normal tissues by (90Y)ZME-018 would restrict its use as a routine vehicle for radioimmunotherapy; however, it may be possible to deliver substantial tumor doses in selected patients.

  7. Sperm-immobilizing monoclonal antibody to human seminal plasma antigens.

    PubMed Central

    Shigeta, M; Watanabe, T; Maruyama, S; Koyama, K; Isojima, S

    1980-01-01

    Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6783353

  8. Treating multiple sclerosis with monoclonal antibodies: a 2013 update.

    PubMed

    Deiß, Annika; Brecht, Isabel; Haarmann, Axel; Buttmann, Mathias

    2013-03-01

    The third part of this in-depth review series on the treatment of multiple sclerosis (MS) with monoclonal antibodies covers the years 2010-2012. The natalizumab section gives a progressive multifocal leukoencephalopathy update, focusing on clinically relevant aspects. Furthermore, it outlines problems around natalizumab cessation and current evidence on therapeutic strategies thereafter. Finally, it reviews evidence on Janus-faced modes of natalizumab action besides anti-inflammatory effects, including proinflammatory effects. The section on alemtuzumab critically analyzes recent Phase III results and discusses which patients might be best suited for alemtuzumab treatment, and reviews the long-term immunological impact of this anti-CD52 antibody. The daclizumab section critically summarizes results from the Phase IIb SELECT/SELECTION trial and introduces the Phase III program. The section on anti-CD20 antibodies reviews Phase II results on ocrelizumab and ofatumumab, and discusses current perspectives of these antibodies for MS therapy. Promising recent Phase II results on the anti-IL-17A antibody secukinumab (AIN457) are outlined and a short update on tabalumab (LY2127399) is given. Other highlighted antibodies currently being tested in MS patients include GNbAC1, BIIB033, MOR103 and MEDI-551. Finally, the authors give an update on the role monoclonal antibodies could play in the therapeutic armamentarium for MS in the medium term. PMID:23448220

  9. Advances in Antibody Design.

    PubMed

    Tiller, Kathryn E; Tessier, Peter M

    2015-12-01

    The use of monoclonal antibodies as therapeutics requires optimizing several of their key attributes. These include binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector functions, and compatibility with the attachment of additional antibody domains (bispecific antibodies) and cytotoxic drugs (antibody-drug conjugates). Addressing these and other challenges requires the use of systematic design methods that complement powerful immunization and in vitro screening methods. We review advances in designing the binding loops, scaffolds, domain interfaces, constant regions, post-translational and chemical modifications, and bispecific architectures of antibodies and fragments thereof to improve their bioactivity. We also highlight unmet challenges in antibody design that must be overcome to generate potent antibody therapeutics. PMID:26274600

  10. Production and preliminary characterization of monoclonal antibodies directed at two surface proteins of rhesus rotavirus.

    PubMed

    Greenberg, H B; Valdesuso, J; van Wyke, K; Midthun, K; Walsh, M; McAuliffe, V; Wyatt, R G; Kalica, A R; Flores, J; Hoshino, Y

    1983-08-01

    A series of monoclonal antibodies was isolated which reacted with one of two major surface proteins of rhesus rotavirus. Thirty-six monoclonal antibodies immunoprecipitated the 82-kilodalton outer capsid protein, the product of the fourth gene, the viral hemagglutinin. These monoclonal antibodies exhibited hemagglutination inhibition activity and neutralized rhesus rotavirus to moderate or high titer. Three monoclonal antibodies immunoprecipitated the 38-kilodalton outer capsid glycoprotein, the eighth or ninth gene product. These three monoclonal antibodies neutralized rhesus rotavirus to high titer and also inhibited viral hemagglutination. PMID:6312065

  11. Production and preliminary characterization of monoclonal antibodies directed at two surface proteins of rhesus rotavirus.

    PubMed Central

    Greenberg, H B; Valdesuso, J; van Wyke, K; Midthun, K; Walsh, M; McAuliffe, V; Wyatt, R G; Kalica, A R; Flores, J; Hoshino, Y

    1983-01-01

    A series of monoclonal antibodies was isolated which reacted with one of two major surface proteins of rhesus rotavirus. Thirty-six monoclonal antibodies immunoprecipitated the 82-kilodalton outer capsid protein, the product of the fourth gene, the viral hemagglutinin. These monoclonal antibodies exhibited hemagglutination inhibition activity and neutralized rhesus rotavirus to moderate or high titer. Three monoclonal antibodies immunoprecipitated the 38-kilodalton outer capsid glycoprotein, the eighth or ninth gene product. These three monoclonal antibodies neutralized rhesus rotavirus to high titer and also inhibited viral hemagglutination. Images PMID:6312065

  12. A First-Time-In-Human Phase I Clinical Trial of Bispecific Antibody-Targeted, Paclitaxel-Packaged Bacterial Minicells

    PubMed Central

    Rosenthal, Mark; McArthur, Grant A.; Pattison, Scott T.; Pattison, Stacey L.; MacDiarmid, Jennifer; Brahmbhatt, Himanshu; Scott, Andrew M.

    2015-01-01

    Background We have harnessed a novel biological system, the bacterial minicell, to deliver cancer therapeutics to cancer cells. Preclinical studies showed that epidermal growth factor receptor (EGFR)-targeted, paclitaxel-loaded minicells (EGFRminicellsPac) have antitumor effects in xenograft models. To examine the safety of the minicell delivery system, we initiated a first-time-in-human, open-label, phase I clinical study of EGFRminicellsPac in patients with advanced solid tumors. Methodology Patients received 5 weekly infusions followed by a treatment free week. Seven dose levels (1x108, 1x109, 3x109, 1x1010, 1.5x1010, 2x1010, 5x1010) were evaluated using a 3+3 dose-escalation design. Primary objectives were safety, tolerability and determination of the maximum tolerated dose. Secondary objectives were assessment of immune/inflammatory responses and antitumor activity. Principal Findings Twenty eight patients were enrolled, 22 patients completed at least one cycle of EGFRminicellsPac; 6 patients did not complete a cycle due to rapidly progressive disease. A total of 236 doses was delivered over 42 cycles, with a maximum of 45 doses administered to a single patient. Most common treatment-related adverse events were rigors and pyrexia. No deaths resulted from treatment-related adverse events and the maximum tolerated dose was defined as 1x1010 EGFRminicellsPac. Surprisingly, only a mild self-limiting elevation in the inflammatory cytokines IL-6, IL-8 and TNFα and anti-inflammatory IL-10 was observed. Anti-LPS antibody titers peaked by dose 3 and were maintained at that level despite repeat dosing with the bacterially derived minicells. Ten patients (45%; n = 22) achieved stable disease as their best response. Conclusions/Significance This is the first study in humans of a novel biological system that can provide targeted delivery of a range of chemotherapeutic drugs to solid tumor cells. Bispecific antibody-targeted minicells, packaged with the chemotherapeutic paclitaxel, were shown to be safe in patients with advanced solid tumors with modest clinical efficacy observed. Further study in Phase II trials is planned. Trial Registration Australian New Zealand Clinical Trials Registry ACTRN12609000672257 PMID:26659127

  13. Stimuli-responsive magnetic nanoparticles for monoclonal antibody purification.

    PubMed

    Borlido, Lus; Moura, Leila; Azevedo, Ana M; Roque, Ana C A; Aires-Barros, Maria R; Farinha, Jos Paulo S

    2013-06-01

    Monoclonal antibodies (mAbs) are important therapeutic proteins. One of the challenges facing large-scale production of monoclonal antibodies is the capacity bottleneck in downstream processing, which can be circumvented by using magnetic stimuli-responsive polymer nanoparticles. In this work, stimuli-responsive magnetic particles composed of a magnetic poly(methyl methacrylate) core with a poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAM-co-AA)) shell cross-linked with N, N'-methylenebisacrylamide were prepared by miniemulsion polymerization. The particles were shown to have an average hydrodynamic diameter of 317 nm at 18C, which decreased to 277 nm at 41C due to the collapse of the thermo-responsive shell. The particles were superparamagnetic in behavior and exhibited a saturation magnetization of 12.6 emu/g. Subsequently, we evaluated the potential of these negatively charged stimuli-responsive magnetic particles in the purification of a monoclonal antibody from a diafiltered CHO cell culture supernatant by cation exchange. The adsorption of antibodies onto P(NIPAM-co-AA)-coated nanoparticles was highly selective and allowed for the recovery of approximately 94% of the mAb. Different elution strategies were employed providing highly pure mAb fractions with host cell protein (HCP) removal greater than 98%. By exploring the stimuli-responsive properties of the particles, shorter magnetic separation times were possible without significant differences in product yield and purity. PMID:23420794

  14. Bispecific single-chain antibodies as effective tools for eliminating epithelial cancer cells from human stem cell preparations by redirected cell cytotoxicity.

    PubMed

    Maletz, K; Kufer, P; Mack, M; Raum, T; Pantel, K; Riethmller, G; Gruber, R

    2001-08-01

    High-dose chemotherapy (HDC) with autologous bone marrow or peripheral stem cell transplantation is discussed as one option to treat the extensive stage of a variety of tumors. Effective methods to eliminate contaminating tumor cells from human bone marrow or stem cell grafts may improve the outcome of the patients. We investigated 3 recombinant bispecific single-chain antibodies (bscAbs) directed against 17-1A (EpCAM), c-erbB-2 (HER-2/neu) and LeY on the one and CD3 on the other binding site for their ability to induce lysis of epithelial tumor cells by retargeting autochthonous T lymphocytes present in bone marrow mononuclear cells (BMMC) and in peripheral stem cell mononuclear cells (PSMC). The bscAbs showed remarkable specific lysis of different epithelial tumor cell lines with BMMCs as well as with PSMCs as effector cells. Investigation of the alpha 17-1A-alpha CD3 bscAb revealed a significant correlation between the percentage of CD3(+) cells present in the BMMCs and the rate of lysis as well as the absence of detrimental effects on the viability of hematopoietic progenitor cells as determined by colony-forming unit assays (CFUs). Our results indicate that recombinant bispecific single-chain antibodies could be new tools for purging of human bone marrow and peripheral stem cell grafts from contaminating epithelial cancer cells for patients receiving autologous stem cell transplantation after HDC. PMID:11433407

  15. Monoclonal antibodies to Ki-67 protein suitable for immunohistochemical analysis.

    PubMed

    Khoruzhenko, Antonina; Kukharchuk, Viktoriya; Cherednyk, Olga; Tykhonkova, Iryna; Ovcharenko, Galina; Malanchuk, Oksana; Filonenko, Valeriy

    2010-08-01

    Detection of cell proliferation index is widely used in experimental and clinical research. Earlier it was shown that nuclear Ki-67 protein expression is strictly related to cell proliferation. It was revealed during all active phases of the cell cycle in mammals but was absent in G0 phase, so Ki-67 presence in cell nuclei reflects a potential growth fraction of whole cell population. The main area of Ki-67 antibody application is in immunocytochemical and immunohistochemical analyses. The aim of our work was to generate mouse monoclonal antibodies for Ki-67 antigen detection in mammalian tissues and in cultured cells. His-tagged fragment of Ki-67 expressed in bacteria was used as an antigen. Antibody-producing hybridoma cells were generated by standard procedure by fusing SP2/0 myeloma cells with splenocytes of immunized mice. Monoclonal antibodies were analyzed using paraffin-embedded human melanoma tissue samples and breast cancer cell line MCF-7. It was shown that generated anti-Ki-67 antibodies revealed proliferating cells in MCF-7 culture and after heat-induced epitope retrieval on paraffin sections of human melanoma tissue. In summary, generated antibodies could be useful for detection of proliferating cells in immunohistochemical and immunofluorescence studies of mammalian cells and tissues. PMID:20795306

  16. [Batch release of immunoglobulin and monoclonal antibody products].

    PubMed

    Gross, S

    2014-10-01

    The Paul-Ehrlich Institute (PEI) is an independent institution of the Federal Republic of Germany responsible for performing official experimental batch testing of sera. The institute decides about the release of each batch and performs experimental research in the field. The experimental quality control ensures the potency of the product and also the absence of harmful impurities. For release of an immunoglobulin batch the marketing authorization holder has to submit the documentation of the manufacture and the results of quality control measures together with samples of the batch to the PEI. Experimental testing is performed according to the approved specifications regarding the efficacy and safety. Since implementation of the 15th German drug law amendment, the source of antibody is not defined anymore. According to 32 German drug law, all batches of sera need to be released by an official control laboratory. Sera are medicinal products, which contain antibodies, antibody fragments or fusion proteins with a functional antibody portion. Therefore, all batches of monoclonal antibodies and derivatives must also be released by the PEI and the marketing authorization holder has to submit a batch release application. Under certain circumstances a waiver for certain products can be issued with regard to batch release. The conditions for such a waiver apply to the majority of monoclonal antibodies. PMID:25200488

  17. [Monoclonal immunotherapy with human monoclonal antibody(CLN-IgG) in glioma patients].

    PubMed

    Kubo, Osami; Takakura, Kintomo

    2002-03-01

    It is well recognized that malignant gliomas escape an immune response by hiding behind the blood-brain barrier and by producing proteins that suppress systemic immunity. However, if gliomas can be made to be more immunogenic or if a tumor vaccine can be produced, then access to all tumor cells including those that infiltrate into the brain can be achieved through the patient's immune response. Several strategies have been investigated for immunotherapy. Laboratory studies and animal models have shown that these immune cells will attack the tumor cell, reduce the size of implanted tumors, and that the immune memory is sufficient to suppress tumor growth when the animal is rechallenges with a tumor implant. Since the development of hybridoma technology, monoclonal antibodies against human cancer cells have been produced and antigens have been identified. Hagiwara reported the production of a human monoclonal antibody, CLN-IgG, made by fusing UC 729-6, human lymphoblastoid B-cell line, with lymphocytes obtained from a patient with the cervical carcinoma. It has been reported that CLN-IgG recognized the antigen expressed in various histological types of human cancers including malignant gliomas. The effect of human monoclonal antibody(CLN-IgG) on malignant brain tumors was evaluated in patients with malignant glioma. Early phase II study was concluded that this specific immunotherapy with CLN-IgG is safe and effective therapy in patients with malignant glioma. We treated 10 cases of malignant gliomas with CLN-IgG. All patients had received radiotherapy and chemotherapy before this immunotherapy using the human monoclonal antibody. The human monoclonal antibody(CLN-IgG) was administered intravenously once or twice/week during 24 weeks. Six cases of glioblastoma, 1 medulloblastoma and 3 cases of potine glioma histologically unverified, were treated. Five cases of 6 glioblastomas died 4 to 12 months after this treatment, 3 cases of pontine glioma showed good responses, 2 cases showed marked decrease of tumor size and 1 case showed no regrowth of tumor on MRI imaging. For the above reasons, Human monoclonal antibody(CLN-IgG) might be useful as an immunotherapy of malignant gliomas. PMID:11904965

  18. Analysis of T-cell-dependent and -independent antigens of Rickettsia conorii with monoclonal antibodies.

    PubMed Central

    Feng, H M; Walker, D H; Wang, J G

    1987-01-01

    Four monoclonal antibodies from euthymic mice and two monoclonal antibodies from athymic mice were directed against antigens of Rickettsia conorii, as shown by both indirect immunofluorescence and an enzyme immunoassay. There was extensive cross-reactivity with other spotted fever group rickettsiae. Euthymic monoclonal antibodies 3-2 and 9-2 (immunoglobulin G2a [IgG2a]) and 27-10 (IgG1) distinctly outlined the acetone-fixed rickettsial surface, as determined by indirect immunofluorescence; only monoclonal antibody 3-2 reacted with the intact rickettsial surface, as determined by colloidal gold-protein A negative-stain electron microscopy. Athymic monoclonal antibodies 32-2 and 35-3 (IgM) and euthymic monoclonal antibody 31-15 (IgG3) all demonstrated an irregular, extrarickettsial morphology, as determined by immunofluorescence, and ultrastructural cell wall blebs that were readily shed from the rickettsial surface. Monoclonal antibody 3-2, the only antibody to confer protection in lethally challenged mice, reacted with a high-molecular-weight protein in Western immunoblots. Monoclonal antibodies 31-15, 32-2, and 35-3 reacted with a "ladder" of proteinase K-resistant, lipopolysaccharidelike antigens. None of the monoclonal antibodies stabilized the ultrastructural rickettsial slime layer, but both athymic and euthymic polyclonal antibodies to R. conorii did. This is, to the best of our knowledge, the first report of the production of monoclonal antibodies to R. conorii and their use for antigenic analysis. Images PMID:3793235

  19. [Increases in pharmaceutical expenditures of PHI by monoclonal antibodies].

    PubMed

    Wild, F

    2013-06-01

    The dynamics of one of the most innovative segments of health care and its impact on pharmaceutical expenditure of private health insurance (PHI) is examined on the basis of drug prescription data from private health insurance companies. The study shows that the increase in pharmaceutical expenditure can be explained partly by the new treatment possibilities available with monoclonal antibodies. The per capita expenditure on drugs with monoclonal antibodies increased by 255% from 2006 to 2010 in private health insurance, while the corresponding expenditure of all pharmaceuticals has risen by only 19% in the same period. In the coming years, growth on this scale will be a challenge for all payers in the health system. PMID:23926705

  20. Biosimilar monoclonal antibodies in lymphoma: a critical appraisal.

    PubMed

    Rioufol, Catherine; Salles, Gilles

    2015-05-01

    Rituximab, an anti-CD20 monoclonal antibody, revolutionized the treatment of lymphoma. Although newer generation anti-CD20 monoclonal antibodies are being examined, patent expiries and patient demand have fueled the development of rituximab biosimilars. The development of such agents is both an important and difficult undertaking. By definition, although they aim to have safety and efficacy comparable with their reference agents, biosimilars are not exact replicas of those agents, and small changes in nonclinical and preclinical properties may ultimately affect in vivo activity. Consideration must be given to the complex mechanisms of action, sensitive patient populations that may be treated, and appropriate clinical trial endpoints. Furthermore, extrapolation of indications is multifaceted, deserving close examination. This review represents a critical look at biosimilars in lymphoma and their safety, efficacy and long-term effects on patient outcomes. PMID:25818308

  1. Adverse Events of Monoclonal Antibodies Used for Cancer Therapy

    PubMed Central

    Guan, Mei; Zhou, Yan-Ping; Sun, Jin-Lu; Chen, Shu-Chang

    2015-01-01

    In 1997, the first monoclonal antibody (MoAb), the chimeric anti-CD20 molecule rituximab, was approved by the US Food and Drug administration for use in cancer patients. Since then, the panel of MoAbs that are approved by international regulatory agencies for the treatment of hematopoietic and solid malignancies has continued to expand, currently encompassing a stunning amount of 20 distinct molecules for 11 targets. We provide a brief scientific background on the use of MoAbs in cancer therapy, review all types of monoclonal antibodies-related adverse events (e.g., allergy, immune-related adverse events, cardiovascular adverse events, and pulmonary adverse events), and discuss the mechanism and treatment of adverse events. PMID:26075239

  2. Rhenium-186-labeled monoclonal antibodies for radioimmunotherapy: preparation and evaluation.

    PubMed

    John, E; Thakur, M L; DeFulvio, J; McDevitt, M R; Damjanov, I

    1993-02-01

    Rhenium-186 has been determined to be a leading radionuclide for radioimmunotherapy. However, the use of 186Re has been limited due to the lack of a convenient and efficient method by which the radionuclide can be bound to monoclonal antibodies. We have developed a simple technique to label IgM, IgG, fragmented antibodies and tumor necrosis factor-alpha with 186Re. This technique uses ascorbic acid (AA) for controlled reduction of antibody disulfide groups to sulfhydryls and SnCl2 in citric acid for the reduction of 186ReO4-. The labeling yields as determined by instant thin-layer chromatography, molecular filtration and gel filtration were greater than 95% and the colloid formation was less than 5%. The labeled antibodies were stable when challenged with 100 and 250 molar excess of DTPA and HSA for 24 hr at 37 degrees C. SDS-PAGE analysis and autoradiography of labeled IgM, IgG and F(ab')2 monoclonal antibodies indicated uniform labeling and that no fragmentation of the monoclonal antibodies had taken place during the labeling procedure. Immunospecificity of 186Re-labeled human neutrophil specific IgM, as determined by in vitro antigen excess assay, was comparable to that of indium-111-labeled c-DTPA-IgM and technetium-99m-labeled-IgM. A nuclear histone specific 186Re-TNT-1-F(ab')2 was evaluated in mice bearing experimental tumors. The tumor/muscle ratios at 4 and 24 hr were 5.9 +/- 0.21 and 13.8 +/- 6.7, respectively compared to that of 2.4 +/- 0.3 at 4 hr p.i. with a nonspecific protein. The labeling technique is simple, reliable and has already been adapted to a single-vial kit preparation. PMID:8429345

  3. Positron emission tomographic imaging of tumors using monoclonal antibodies

    SciTech Connect

    Zalutsky, M.R.

    1992-08-01

    This research project is developing methods for utilizing positron emission tomography (PET) to increase the clinical potential of radiolabeled monoclonal antibodies (MAbs). This report describes the development of methods for labeling MAbs and their fragments with positron-emitting halogen nuclides, fluorine-18 and iodine-124. These nulides were selected because of the widespread availability of F-18 and because of our extensive experience in the development of new protein radiohalogenation methods.

  4. Monoclonal antibodies to the two most basic papaya proteinases.

    PubMed

    Goodenough, P W; Kilshaw, P J; McEwan, F; Owen, A J

    1986-08-01

    The proteinases from Carica papaya include papain, isoenzymes of chymopapain and two proteinases A and B distinguished by their unusually high pI. The identity of one of the most basic proteinases has been questioned. The present report describes the preparation and characterisation of two monoclonal antibodies that react specifically with papaya proteinases A and B respectively and a third that identifies a common structural feature found in papain and proteinase A. PMID:3545314

  5. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1993-01-01

    The objective of this project is to generate a library of monoclonal antibodies (MAbs) to surface molecules of mammalian tumor and transformed cells grown as multicell spheroids (MCS). These MCS are highly organized, three dimensional multicellular structures which exhibit many characteristics of in vivo organized tissues not found in conventional monolayer or suspension culture; therefore, MCS make better in vitro model systems to study the interactions of mammalian cells. Additionally, they provide a functional assay for surface adhesion molecules.

  6. A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities.

    PubMed Central

    Babcook, J S; Leslie, K B; Olsen, O A; Salmon, R A; Schrader, J W

    1996-01-01

    We report a novel approach to the generation of monoclonal antibodies based on the molecular cloning and expression of immunoglobulin variable region cDNAs generated from single rabbit or murine lymphocytes that were selected for the production of specific antibodies. Single cells secreting antibodies for a specific peptide either from gp116 of the human cytomegalovirus or from gp120 of HIV-1 or for sheep red blood cells were selected using antigen-specific hemolytic plaque assays. Sheep red blood cells were coated with specific peptides in a procedure applicable to any antigen that can be biotinylated. Heavy- and light-chain variable region cDNAs were rescued from single cells by reverse transcription-PCR and expressed in the context of human immunoglobulin constant regions. These chimeric murine and rabbit monoclonal antibodies replicated the target specificities of the original antibody-forming cells. The selected lymphocyte antibody method exploits the in vivo mechanisms that generate high-affinity antibodies. This method can use lymphocytes from peripheral blood, can exploit a variety of procedures that identify individual lymphocytes producing a particular antibody, and is applicable to the generation of monoclonal antibodies from many species, including humans. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8755564

  7. Recovery and purification process development for monoclonal antibody production

    PubMed Central

    Ma, Junfen; Winter, Charles; Bayer, Robert

    2010-01-01

    Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs. PMID:20647768

  8. Production of Monoclonal Antibodies in Plants for Cancer Immunotherapy

    PubMed Central

    Moussavou, Ghislain; Ko, Kisung; Lee, Jeong-Hwan; Choo, Young-Kug

    2015-01-01

    Plants are considered as an alternative platform for recombinant monoclonal antibody (mAb) production due to the improvement and diversification of transgenic techniques. The diversity of plant species offers a multitude of possibilities for the valorization of genetic resources. Moreover, plants can be propagated indefinitely, providing cheap biomass production on a large scale in controlled conditions. Thus, recent studies have shown the successful development of plant systems for the production of mAbs for cancer immunotherapy. However, their several limitations have to be resolved for efficient antibody production in plants. PMID:26550566

  9. [Targeted therapies including monoclonal antibodies for connective tissue diseases].

    PubMed

    Hayashi, Taichi; Sumida, Takayuki

    2009-03-01

    Recent advance of targeted therapies including monoclonal antibodies and fusion proteins has allowed effective strategies in the treatment of rheumatoid arthritis. And now, TNF inhibitors are broadly used for rheumatoid arthritis and prevent the disease progression. Meanwhile, B cell targeted therapies and anti-interleukin-6 receptor antibody treatment are not only used for second line biological agents for rheumatoid arthritis, but also expected for the treatments of various autoimmune diseases. Recent year, some of novel small molecules, which inhibit the signal transduction of various surface receptors of immune cells, are in clinical trials. These drugs will be a breakthrough for the treatment of some autoimmune disorders. PMID:19280937

  10. Radioimmunodetection of human melanoma with indium-111-labeled monoclonal antibodies

    SciTech Connect

    Taylor, A. Jr.; Milton, W.; Eyre, H.; Wu, F.P.; Christian, P.; Alazraki, N.; Datz, F.; Unger, M.W.

    1987-01-01

    Our purpose in conducting this study was to evaluate the efficacy of an /sup 111/In-labeled murine monoclonal antibody directed against a high-molecular-weight glycoprotein in localizing metastatic melanoma in 15 patients with previously documented disease and to determine the effect of antibody mass (2.5, 5.0, and 10.0 mg) on blood clearance, biodistribution, and lesion detection. Five mCi of /sup 111/In-antibody were infused over 1 hour, and patients were scanned at 24 and 72 hours after injection without computer enhancement or background subtraction techniques. No significant differences in the organ distribution, urine excretion, or plasma disappearance curves were noted at the three antibody dose levels. There were no acute reactions. The scan detected tumor in 9 of 12 (75%) patients with active disease, and 26 of 33 (79%) lesions greater than 1 cm. Patient management in 3 of 15 (20%) of patients studied was changed as a result.

  11. Epitope mapping of monoclonal antibodies for the Deinococcus radiodurans bacteriophytochome.

    PubMed

    Kim, Tae-Lim; Yoo, Jihey; Sangsawang, Kanidta; Cho, Man-Ho; Yang, Seung Hwan; Suh, Joo-Won; Hahn, Tae-Ryong; Bhoo, Seong Hee

    2014-06-01

    Bacteriophytochromes (BphP) are phytochrome-like light sensing proteins in bacteria, which use biliverdin as a chromophore. In order to study the biochemical properties of the DrBphP protein, five (2B8, 2C11, 3B2, 3D2, and 3H7) anti-DrBphP monoclonal antibodies were produced through the immunization of mice with purified full-length DrBphP and DrBphN (1-321 amino acid) proteins, and epitope mapping was then carried out. Among the five antibodies, 2B8 and 2C11 preferentially recognized the N-terminal region of BphP whereas 3B2, 3D2, and 3H7 showed preference for the C-terminal region. We performed further epitope mapping using recombinant truncated BphP proteins to narrow down their target sequences. The results demonstrated that each of the five monoclonal antibodies recognized different regions on the DrBphP protein. Additionally, epitopes of 2B8 and 3H7 antibodies were discovered to be shorter than 10 amino acids (2B8: RDPLPFFPP, 3H7: PGEIEEA). These two antibodies with such specific recognition epitopes could be especially valuable for developing new peptide tags for protein detection and purification. PMID:24677487

  12. Monoclonal antibodies in the treatment of pancreatic cancer

    PubMed Central

    Huang, Zhi-Qiang; Buchsbaum, Donald J

    2009-01-01

    Human pancreatic cancer is a malignant disease with almost equal incidence and mortality. Effective diagnostic and therapeutic strategies are still urgently needed to improve its survival rate. With advances in structural and functional genomics, recent work has focused on targeted molecular therapy using monoclonal antibodies. This review summarizes the target molecules on the tumor cell surface and normal tissue stroma, which are related to pancreatic cancer oncogenesis, tumor growth or resistance to chemotherapy, as well as molecules involved in regulating inflammation and host immunoresponses. Targeted molecules include cell-surface receptors, such as the EGF receptor, HER2, death receptor 5 and IGF-1 receptor. Effects of monoclonal antibodies against these target molecules alone or in combination with chemotherapy, small-molecule signal transduction inhibitors, or radiation therapy are also discussed. Also discussed are the use of toxin or radioisotope conjugates, and information relating to the use of these targeting agents in pancreatic cancer clinical trials. Although targeted molecular therapy with monoclonal antibodies has made some progress in pancreatic cancer treatment, especially in preclinical studies, its clinical application to improve the survival rate of pancreatic cancer patients requires further investigation. PMID:20046965

  13. Human monoclonal antibody and vaccine approaches to prevent human rabies.

    PubMed

    Nagarajan, T; Rupprecht, Charles E; Dessain, Scott K; Rangarajan, P N; Thiagarajan, D; Srinivasan, V A

    2008-01-01

    Rabies, being a major zoonotic disease, significantly impacts global public health. It is invariably fatal once clinical signs are apparent. The majority of human rabies deaths occur in developing countries. India alone reports more than 50% of the global rabies deaths. Although it is a vaccine-preventable disease, effective rabies prevention in humans with category III bites requires the combined administration of rabies immunoglobulin (RIG) and vaccine. Cell culture rabies vaccines have become widely available in developing countries, virtually replacing the inferior and unsafe nerve tissue vaccines. Limitations inherent to the conventional RIG of either equine or human origin have prompted scientists to look for monoclonal antibody-based human RIG as an alternative. Fully human monoclonal antibodies have been found to be safer and equally efficacious than conventional RIG when tested in mice and hamsters. In this chapter, rabies epidemiology, reservoir control measures, post-exposure prophylaxis of human rabies, and combination therapy for rabies are discussed. Novel human monoclonal antibodies, their production, and the significance of plants as expression platforms are emphasized. PMID:17990790

  14. Radiocurability by Targeting Tumor Necrosis Factor-{alpha} Using a Bispecific Antibody in Carcinoembryonic Antigen Transgenic Mice

    SciTech Connect

    Larbouret, Christel; Robert, Bruno; Linard, Christine; Teulon, Isabelle; Gourgou, Sophie M.Sc.; Bibeau, Frederic; Martineau, Pierre; Santoro, Lore; Pouget, Jean-Pierre; Pelegrin, Andre; Azria, David

    2007-11-15

    Purpose: Tumor necrosis factor-{alpha} (TNF-{alpha}) enhances radiotherapy (RT) killing of tumor cells in vitro and in vivo. To overcome systemic side effects, we used a bispecific antibody (BsAb) directed against carcinoembryonic antigen (CEA) and TNF-{alpha} to target this cytokine in a CEA-expressing colon carcinoma. We report the evaluation of this strategy in immunocompetent CEA-transgenic mice. Methods and Materials: The murine CEA-transfected colon carcinoma MC-38 was used for all experiments. In vitro, clonogenic assays were performed after RT alone, TNF-{alpha} alone, and RT plus TNF-{alpha}. In vivo, the mice were randomly assigned to treatment groups: control, TNF-{alpha}, BsAb, BsAb plus TNF-{alpha}, RT, RT plus TNF-{alpha}, and RT plus BsAb plus TNF-{alpha}. Measurements of endogenous TNF-{alpha} mRNA levels and evaluation of necrosis (histologic evaluation) were assessed per treatment group. Results: In vitro, combined RT plus TNF-{alpha} resulted in a significant decrease in the survival fraction at 2 Gy compared with RT alone (p < 0.00001). In vivo, we observed a complete response in 5 (50%) of 10, 2 (20%) of 10, 2 (18.2%) of 11, and 0 (0%) of 12 treated mice in the RT plus BsAb plus TNF-{alpha}, RT plus TNF-{alpha}, RT alone, and control groups, respectively. This difference was statistically significant when TNF-{alpha} was targeted with the BsAb (p = 0.03). The addition of exogenous TNF-{alpha} to RT significantly increased the endogenous TNF-{alpha} mRNA level, particularly when TNF-{alpha} was targeted with BsAb (p < 0.01). The percentages of necrotic area were significantly augmented in the RT plus BsAb plus TNF-{alpha} group. Conclusion: These results suggest that targeting TNF-{alpha} with the BsAb provokes RT curability in a CEA-expressing digestive tumor syngenic model and could be considered as a solid rationale for clinical trials.

  15. Impact of Cell-surface Antigen Expression on Target Engagement and Function of an Epidermal Growth Factor Receptor c-MET Bispecific Antibody.

    PubMed

    Jarantow, Stephen W; Bushey, Barbara S; Pardinas, Jose R; Boakye, Ken; Lacy, Eilyn R; Sanders, Renouard; Sepulveda, Manuel A; Moores, Sheri L; Chiu, Mark L

    2015-10-01

    The efficacy of engaging multiple drug targets using bispecific antibodies (BsAbs) is affected by the relative cell-surface protein levels of the respective targets. In this work, the receptor density values were correlated to the in vitro activity of a BsAb (JNJ-61186372) targeting epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-MET). Simultaneous binding of the BsAb to both receptors was confirmed in vitro. By using controlled Fab-arm exchange, a set of BsAbs targeting EGFR and c-MET was generated to establish an accurate receptor quantitation of a panel of lung and gastric cancer cell lines expressing heterogeneous levels of EGFR and c-MET. EGFR and c-MET receptor density levels were correlated to the respective gene expression levels as well as to the respective receptor phosphorylation inhibition values. We observed a bias in BsAb binding toward the more highly expressed of the two receptors, EGFR or c-MET, which resulted in the enhanced in vitro potency of JNJ-61186372 against the less highly expressed target. On the basis of these observations, we propose an avidity model of how JNJ-61186372 engages EGFR and c-MET with potentially broad implications for bispecific drug efficacy and design. PMID:26260789

  16. Impact of Cell-surface Antigen Expression on Target Engagement and Function of an Epidermal Growth Factor Receptor c-MET Bispecific Antibody*

    PubMed Central

    Jarantow, Stephen W.; Bushey, Barbara S.; Pardinas, Jose R.; Boakye, Ken; Lacy, Eilyn R.; Sanders, Renouard; Sepulveda, Manuel A.; Moores, Sheri L.; Chiu, Mark L.

    2015-01-01

    The efficacy of engaging multiple drug targets using bispecific antibodies (BsAbs) is affected by the relative cell-surface protein levels of the respective targets. In this work, the receptor density values were correlated to the in vitro activity of a BsAb (JNJ-61186372) targeting epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-MET). Simultaneous binding of the BsAb to both receptors was confirmed in vitro. By using controlled Fab-arm exchange, a set of BsAbs targeting EGFR and c-MET was generated to establish an accurate receptor quantitation of a panel of lung and gastric cancer cell lines expressing heterogeneous levels of EGFR and c-MET. EGFR and c-MET receptor density levels were correlated to the respective gene expression levels as well as to the respective receptor phosphorylation inhibition values. We observed a bias in BsAb binding toward the more highly expressed of the two receptors, EGFR or c-MET, which resulted in the enhanced in vitro potency of JNJ-61186372 against the less highly expressed target. On the basis of these observations, we propose an avidity model of how JNJ-61186372 engages EGFR and c-MET with potentially broad implications for bispecific drug efficacy and design. PMID:26260789

  17. Dissection of the human antigammaglobulin idiotype system with monoclonal antibodies.

    PubMed

    Posnett, D N; Wisniewolski, R; Pernis, B; Kunkel, H G

    1986-02-01

    Murine monoclonal antibodies (MoAb) were prepared by immunizing mice with human monoclonal rheumatoid factors from patients with mixed cryoglobulinaemia. Indirect solid phase radioimmunoassay and haemagglutination inhibition were used to screen the MoAb. Reactivity patterns of MoAb were determined using (a) polyclonal and monoclonal IgM proteins, (b) monoclonal IgM proteins from patients with neuropathy, (c) monoclonal and polyclonal IgM antigammaglobulins, and (d) various isolated VkIIIb-positive immunoglobulins. Several patterns were obtained: MoAb reacting with private idiotypic determinants, with VkIIIb-related determinants, and with cross-reactive idiotypes (CRI). Two MoAb of the second type reacted with VkIIIb-positive immunoglobulins and light chains regardless of their antigenic activity. Another MoAb reacted with VkIII light chains only when in association with mu heavy chains. MoAb of the third type defined distinct CRI systems. One of these was restricted to antigammaglobulins and another also involved neuropathy-associated monoclonal IgM proteins. All MoAb clearly reacted with a minor population of normal polyclonal IgM, except for the MoAb detecting private idiotypic determinants. Studies using inhibition of binding by enzyme-linked immunosorbent assay showed that polyclonal IgM antigammaglobulins may carry a CRI determinant detected by one of the MoAb. This CRI system, defined by the MoAb Glo 86.3, was similar to but not identical with the previously described Wa CRI, which is widely prevalent among IgM antigammaglobulins of rheumatoid arthritis. PMID:2419964

  18. Detection of chromogranin in neuroendocrine cells with a monoclonal antibody.

    PubMed Central

    Wilson, B. S.; Lloyd, R. V.

    1984-01-01

    A monoclonal antibody ( LK2H10 ) produced against a human pheochromocytoma reacted immunohistochemically with 126 normal and neoplastic endocrine tissues with secretory granules which were formalin-fixed and paraffin-embedded. Antibody LK2H10 did not react with 46 other endocrine tissues or tumors without secretory granules nor with 113 normal and neoplastic nonendocrine cells and tumors. Tumors with abundant secretory granules showed intense and diffuse staining, and tumors with few granules, such as Merkel cell carcinomas, neuroblastomas, and small cell carcinomas of lung, showed focal staining. Antibody LK2H10 did not react with melanomas, nevi, posterior pituitary, peripheral nerve tissues, or neurons. The target structure of LK2H10 was identified as human chromogranin, of which the major fraction was chromogranin A (mol wt 68,000 daltons). Preabsorption with purified chromogranin A blocked immunoperoxidase staining by LK2H10 in normal adrenal medulla, in the anterior pituitary, and in a pheochromocytoma. Ultrastructural immunohistochemistry with LK2H10 showed that chromogranin was present in cytoplasmic secretory granules. These results indicate that chromogranin is widely distributed in the secretory granules of most polypeptide-producing endocrine tissues, and it is readily detected with the use of monoclonal antibody LK2H10 . The detection of this marker can be very helpful as a diagnostic aid for neuroendocrine cells and tumors. Images Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 1 Figure 2 Figure 3 Figure 10 Figure 11 PMID:6375394

  19. Efficient generation of human IgA monoclonal antibodies.

    PubMed

    Lorin, Valrie; Mouquet, Hugo

    2015-07-01

    Immunoglobulin A (IgA) is the most abundant antibody isotype produced in humans. IgA antibodies primarily ensure immune protection of mucosal surfaces against invading pathogens, but also circulate and are present in large quantities in blood. IgAs are heterogeneous at a molecular level, with two IgA subtypes and the capacity to form multimers by interacting with the joining (J) chain. Here, we have developed an efficient strategy to rapidly generate human IgA1 and IgA2 monoclonal antibodies in their monomeric and dimeric forms. Recombinant monomeric and dimeric IgA1/IgA2 counterparts of a prototypical IgG1 monoclonal antibody, 10-1074, targeting the HIV-1 envelope protein, were produced in large amounts after expression cloning and transient transfection of 293-F cells. 10-1074 IgAs were FPLC-purified using a novel affinity-based resin engrafted with anti-IgA chimeric Fabs, followed by a monomers/multimers separation using size exclusion-based FPLC. ELISA binding experiments confirmed that the artificial IgA class switching of 10-1074 did not alter its antigen recognition. In summary, our technical approach allows the very efficient production of various forms of purified recombinant human IgA molecules, which are precious tools in dissecting IgA B-cell responses in physiological and pathophysiological conditions, and studying the biology, function and therapeutic potential of IgAs. PMID:25910833

  20. Monkey-derived monoclonal antibodies against Plasmodium falciparum

    SciTech Connect

    Stanley, H.A.; Reese, R.T.

    1985-09-01

    A system has been developed that allows efficient production of monkey monoclonal antibodies from owl monkeys. Splenocytes or peripheral blood lymphocytes from monkeys immune to the human malarial parasite, Plasmodium falciparum, were fused with P3X63 Ag8.653 mouse myelomas. The resulting hybridomas were screened by an indirect fluorescent antibody test for the production of monkey monoclonal antibodies (mAb) reactive with P. falciparum. Most of the mAb reacted with the P. falciparum merozoites and immunoprecipitated a parasite-derived glycoprotein having a relative molecular weight of 185,000. These mAb gave a minimum of five different immunoprecipitation patterns, thus demonstrating that a large number of polypeptides obtained when parasitized erythrocytes are solubilized share epitopes with this large glycoprotein. In addition, mAb were obtained that reacted with antigens associated with the infected erythrocyte membrane. One of these mAb bound a M/sub r/ 95,000 antigen. Radioimmunoprecipitation assays using /sup 125/T-antibodies were done.

  1. Transformation-Related Antigens Identified by Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Strand, Mette

    1980-06-01

    Tumor-cell proteins that were antigenic in a syngeneic animal were identified by immunoprecipitation with monoclonal antibodies. Spleen cells of BALB/c mice immunized with plasma membranes of Kirsten RNA sarcoma virus-transformed BALB/3T3 cells were fused with NS-l myeloma cells. Antibodies secreted into the culture fluid from these hybridomas were distinguished by their reactivity against proteins of different target cells. A total of 191 cultures were established; 143 produced antibodies that bound to BALB/3T3 cells transformed by the RNA sarcoma virus, of which antibodies from 82 bound to BALB/3T3 transformed with simian virus 40, and antibodies from 56 bound to BALB/3T3 cells. Thus, more than 50% of the cultures produced antibodies that possibly were specific to antigens of the transformed cell. Twenty different hybridomas have been cloned, and antibodies from eight of these were found to immunoprecipitate five different proteins. A protein of approximately 32,000 daltons was precipitated from BALB/3T3 cells transformed by the RNA sarcoma virus, simian virus 40, or methylcholanthrene but not from untransformed BALB/3T3 cells. A protein of about 300,000 daltons was precipitated from all four cell lines; precipitation was enhanced in the viral transformed cells. Proteins of approximately 57,000, 54,000, and 8500 daltons were immunoprecipitated from all four cell lines.

  2. Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110

    PubMed Central

    Deisting, Wibke; Raum, Tobias; Kufer, Peter; Baeuerle, Patrick A.; Mnz, Markus

    2015-01-01

    Background Bispecific T cell engager (BiTE) are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They can elicit a polyclonal cytotoxic T cell response that is not restricted by T cell receptor (TCR) specificity, and surface expression of MHC class I/peptide antigen complexes. Using human EpCAM/CD3-bispecific BiTE antibody construct AMG 110, we here assessed to what extent surface expression of PD-L1, cytoplasmic expression of indoleamine-2,3-deoxygenase type 1, Bcl-2 and serpin PI-9, and the presence of transforming growth factor beta (TGF-?), interleukin-10 (IL-10) and adenosine in culture medium can impact redirected lysis by AMG 110-engaged T cells. Methods The seven factors, which are all involved in inhibiting T cell functions by cancer cells, were tested with human EpCAM-expressing Chinese hamster ovary (CHO) target cells at levels that in most cases exceeded those observed in a number of human cancer cell lines. Co-culture experiments were used to determine the impact of the evasion mechanisms on EC50 values and amplitude of redirected lysis by AMG 110, and on BiTE-induced proliferation of previously resting human peripheral T cells. Findings An inhibitory effect on redirected lysis by AMG 110-engaged T cells was seen upon overexpression of serpin PI-9, Bcl-2, TGF-?and PD-L1. An inhibitory effect on induction of T cell proliferation was only seen with CHO cells overexpressing IDO. In no case, a single evasion mechanism rendered target cells completely resistant to BiTE-induced lysis, and even various combinations could not. Conclusions Our data suggest that diverse mechanisms employed by cancer cells to fend off T cells cannot inactivate AMG 110-engaged T cells, and that inhibitory effects observed in vitro may be overcome by increased concentrations of the BiTE antibody construct. PMID:26510188

  3. Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells

    PubMed Central

    Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

    2015-01-01

    Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs. PMID:25996084

  4. Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

    PubMed Central

    Safran, A; Neumann, D; Fuchs, S

    1986-01-01

    Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms. Images Fig. 2. Fig. 4. Fig. 5. PMID:3816758

  5. [Preparation and characterization of specific monoclonal antibodies against mercury ions].

    PubMed

    Zhao, Li; Wang, Fenglong; Yang, Hui; Li, Peng; Liu, Manxing; Li, Xia

    2010-06-01

    The environmental pollution by heavy metals such as mercury, cadmium and lead has become a worldwide public health hazard. To rapidly and inexpensively monitor environmental heavy metals is a prerequisite for minimizing human and animal exposure. The development of immunoassays to detect mercury ion residues has been a promising trend with the advantage of rapid and cheap operation. We reported the isolation and characterization of mercury-specific monoclonal antibodies. Because Hg2+ ions are too small to elicit an immune response, the metal was coupled to protein carrier (keyhole limpet, KLH) using a chelator (diethylenetriamine pentaacetic acid, DTPA). After the synthesis of antigen and characterization, monoclonal antibodies against mercury ions were generated by immunizing BALB/c mice with mercury conjugated antigen (Hg-DTPA-KLH). The stable hybridoma cell lines were produced by fusion of murine splenocytes and SP2/0 myeloma cells. The hybridoma cells were subcloned by the limiting dilution and screened by ELISA, two hybridoma cell lines producing stably specific monoclonal antibodies (MAbs) against mercury ions were obtained, named H2H5 and H1H8. The ascites fluid was produced in BABL/c mice by intraperitoneal injection of 1 x 10(7) H2H5 and H1H8 cells, respectively. The titers of ascites were all above 1:51 200. The isotyping of secrete antibodies from two hybridoma cell lines was IgG1, kappa type. These data laid a potency of establishing immunoassays methods of determining Hg2+ ion residues and had the realistic significance for improving the efficiency and quality of risk assessment. PMID:20815254

  6. Improved monoclonal antibody tumor/background ratios with exchange transfusions.

    PubMed

    Henry, C A; Clavo, A C; Wahl, R L

    1991-01-01

    Blood exchange transfusions were performed in nude rats with subcutaneous HTB77 human ovarian carcinoma xenografts in an attempt to improve specific monoclonal antibody (MoAb) tumor/non-tumor uptake ratios. Animals were injected intravenously with both 131I-5G6.4 specific and 125I-UPC-10 non-specific MoAb. Twenty-four hours later 65-80% of the original blood was exchanged with normal heparinized rat blood and then these rodents were sacrificed. Exchange transfusion significantly (P less than 0.05) decreased normal tissue activities of 131I (except for muscle) by 63-85%, while tumor activity decreased only 5%. Tumor to background ratios increased from 0.1-0.8 to 2.3-6.3. Exchange transfusions substantially enhance tumor/normal tissue antibody uptake ratios and, along with plasmapheresis, may be useful in enhancing antibody localization in vivo, particularly for therapy. PMID:1917528

  7. Recent advances in the generation of human monoclonal antibody

    PubMed Central

    Katakura, Yoshinroi; Shirahata, Sanetaka

    2007-01-01

    The use of monoclonal antibodies (mAbs) has now gained a niche as an epochal breakthrough in medicine. Engineered antibodies (Abs) currently account for over 30% of biopharmaceuticals in clinical trials. Several methods to generate human mAbs have evolved, such as (1) immortalization of antigen-specific human B cell hybridoma technology, (2) generation of chimeric and humanized antibody (Ab) from mouse Ab by genetic engineering, (3) acquisition of antigen-specific human B cells by the phage display method, and (4) development of transgenic mice for producing human mAbs. Besides these technologies, we have independently developed a method to generate human mAbs by combining the method of invitro immunization using peripheral blood mononuclear cells and the phage display method. In this paper, we review the developments in these technologies for generating human mAbs. PMID:19002994

  8. Use of heteropolymeric monoclonal antibodies to attach antigens to the C3b receptor of human erythrocytes: A potential therapeutic treatment

    SciTech Connect

    Taylor, R.P.; Sutherland, W.M.; Reist, C.J.; Webb, D.J.; Wright, E.L.; Labuguen, R.H. )

    1991-04-15

    The authors prepared bispecific, cross-linked monoclonal antibodies (heteropolymers) with specificity for both targeted antigens and the human erythrocyte (RBC) complement receptor. These heteropolymers facilitate binding of target antigens (human IgG and dinitrophenylated bovine {gamma} globulin) to human RBCs under conditions that either allow or preclude complement activation. Radioimmuno-assay analyses of this binding agree well with the number of complement receptors per RBC. In vitro whole-blood model experiments indicate heteropolymer-facilitated binding of antigens to RBCs is rapid and stable at 37C. It may be possible to extend these prototype experiments to the in vivo situation and use heteropolymer-attached RBCs for the safe and rapid binding, neutralization, and removal from the circulation of pathogenic antigens associated with infectious disease.

  9. Clinical development of immunostimulatory monoclonal antibodies and opportunities for combination.

    PubMed

    Melero, Ignacio; Grimaldi, Antonio M; Perez-Gracia, Jose L; Ascierto, Paolo A

    2013-03-01

    Immune system responses are under the control of extracellular biomolecules, which express functions in receptors present on the surface of cells of the immune system, and thus are amenable to be functionally modulated by monoclonal antibodies. Some of these mechanisms are activating and dictate whether the response ensues, while others play the role of powerful repressors. Antagonist antibodies acting on such repressors result in enhanced immune responses, a goal that is also achieved with agonist antibodies acting on the activating receptors. With these simple logics, a series of therapeutic agents are under clinical development and one of them directed at the CTL-associated antigen 4 (CTLA-4) inhibitory receptor (ipilimumab) has been approved for the treatment of metastatic melanoma. The list of antagonist agents acting on repressors under development includes anti-CTLA-4, anti-PD-1, anti-PD-L1 (B7-H1), anti-KIR, and anti-TGF-β. Agonist antibodies currently being investigated in clinical trials target CD40, CD137 (4-1BB), CD134 (OX40), and glucocorticoid-induced TNF receptor (GITR). A blossoming preclinical pipeline suggests that other active targets will also be tested in patients in the near future. All of these antibodies are being developed as conventional monoclonal immunoglobulins, but other engineered antibody formats or RNA aptamers are under preclinical scrutiny. The "dark side" of these immune interventions is that they elicit autoimmune/inflammatory reactions that can be severe in some patients. A critical and, largely, pending subject is to identify reliable predictive biomarkers both for efficacy and immune toxicity. Preclinical and early clinical studies indicate a tremendous potential to further improve efficacy, using combinations from among these new agents that frequently act in a synergistic fashion. Combinations with other more conventional means of treatment such as radiotherapy, chemotherapy, or cancer vaccines also hold much promise. PMID:23460531

  10. [Progress in preparation of small monoclonal antibodies of knock out technique].

    PubMed

    Liu, Jing; Mao, Xin-min; Li, Lin-lin; Li, Xin-xia; Wang, Ye; Lan, Yi

    2015-10-01

    With the application of monoclonal antibody technology more and more widely, its production technology is becoming more and more perfect. Small molecule monoclonal antibody technology is becoming a hot research topic for people. The application of traditional Chinese medicine small molecule monoclonal antibody technology has been more and more widely, the technology for effective Chinese medicine component knockout provide strong technical support. The preparation of monoclonal antibodies and small molecule knockout technology are reviewed in this paper. The preparation of several steps, such as: in the process of preparation of antigen, hapten carrier coupling, coupling ratio determination and identification of artificial antigen and establishment of animal immunization and hybridoma cell lines of monoclonal antibody, the large-scale preparation; small molecule monoclonal antibody on Immune in affinity chromatography column method is discussed in detail. The author believes that this technology will make the traditional Chinese medicine research on a higher level, and improve the level of internationalization of Chinese medicine research. PMID:26975094

  11. Blood-brain barrier protein recognized by monoclonal antibody.

    PubMed Central

    Sternberger, N H; Sternberger, L A

    1987-01-01

    An IgG1 mouse monoclonal antibody produced in response to immunization with rat brain homogenate reacted with endothelial cells in the central and peripheral nervous system. Because antibody reactivity was associated with endothelia that have a selective permeability barrier, the antibody was called anti-endothelial-barrier antigen (anti-EBA). Paraffin sections of Bouins'-fixed rat tissue were used for initial screening and subsequent characterization of antibody reactivity. The antibody was generally unreactive with endothelial cells in other organs and with nonendothelial cells in or outside of the nervous system. Antibody binding was greatly reduced or absent in endothelia of the area postrema and choroid plexus, sites known to possess fenestrated blood vessels. In developing rat brain, anti-EBA binding to some microvessels was seen at 3 days postnatally. Anti-EBA reactivity outside the nervous system occurred in spleen and skin. Patchy reaction with portions of some spleen blood vessels and binding to some cells in the spleen were observed. In the skin, small cells, tentatively identified as Langerhans cells, which participate in Ia presentation, were stained. On immunoblots of rat brain microvessel preparations electrophoresed in Na-DodSO4/polyacrylamide gels, anti-EBA reacted with a protein triplet of Mr 30,000, 25,000, and 23,500 components. Images PMID:3500474

  12. Trends in capacity utilization for therapeutic monoclonal antibody production.

    PubMed

    Langer, Eric S

    2009-01-01

    The administration of high doses of therapeutic antibodies requires large-scale, efficient, cost effective manufacturing processes. An understanding of how the industry is using its available production capacity is important for production planning, and facility expansion analysis. Inaccurate production planning for therapeutic antibodies can have serious financial ramifications. In the recent 5(th) Annual Report and Survey of Biopharmaceutical Manufacturing Capacity and Production, 434 qualified respondents from 39 countries were asked to indicate, among other manufacturing issues, their current trends and future predictions with respect to the production capacity utilization of monoclonal antibodies in mammalian cell culture systems. While overall production of monoclonals has expanded dramatically since 2003, the average capacity utilization for mammalian cell culture systems, has decreased each year since 2003. Biomanufacturers aggressively attempt to avoid unanticipated high production demands that can create a capacity crunch. We summarize trends associated with capacity utilization and capacity constraints which indicate that biopharmaceutical manufacturers are doing a better job planning for capacity. The results have been a smoothing of capacity use shifts and an improved ability to forecast capacity and outsourcing needs. Despite these data, today, the instability and financial constraints caused by the current global economic crisis are likely to create unforeseen shifts in our capacity utilization and capacity expansion trends. These shifts will need to be measured in subsequent studies. PMID:20061821

  13. Preparation and Identification of Monoclonal Antibodies Against ?-Conotoxin MVIIA

    PubMed Central

    Yang, Yanling; Ma, Yanling; Li, Heng; Wang, Shihua

    2014-01-01

    ?-Conotoxins MVIIA (?-CTX MVIIA) is a peptide with 25 amino acid residues. It is a selective and reversible N-type voltage-gated calcium channel blocker, which could be used as an analgesic for pain. To date, there are no monoclonal antibodies (MAb) for immunoassay against ?-conotoxin MVIIA. In this study, an MAb against ?-conotoxin MVIIA was prepared. The conotoxin-coding DNA sequence was chemically synthesized and cloned into expression vector pGEX-6p-1 and pET32a (+), respectively. The fusion protein GST-CTX was expressed and purified, and was used to immunize BALB/c mice for preparing the anti-CTX antibody. The spleen cells were fused with SP2/0 myeloma cells after the titer of antiserum was detected and qualified. After being screened by indirect ELISA and cloned by limiting dilution, a hybridoma named 4A12, which produces monoclonal antibody specifically against ?-CTX MVIIA, was successfully obtained. It was found that there are 102 chromosomes in the 4A12 cell, and the subclass for the MAb is IgM. The MAb affinity against ?-CTX MVIIA was 7.33109 L/mol, and the cross-reaction test showed that the MAb specifically bound ?-CTX MVIIA. The MAb could be used as a specific antagonist for ?-CTX MVIIA in the physiological study on the CaV channels in the nervous system. PMID:25171005

  14. Intraoperative probe-directed immunodetection using a monoclonal antibody

    SciTech Connect

    O'Dwyer, P.J.; Mojzisik, C.M.; Hinkle, G.H.; Rousseau, M.; Olsen, J.; Tuttle, S.E.; Barth, R.F.; Thurston, M.O.; McCabe, D.P.; Farrar, W.B.

    1986-12-01

    To assess monoclonal antibody (MAb) 17-1A and its F(ab')2 fragment in intraoperative radioimmunodetection and to evaluate further the clinical usefulness of a hand-held gamma-detecting probe (GDP), we injected radiolabeled monoclonal antibody 17-1A three to six days preoperatively or its F(ab')2 fragment two to three days preoperatively into 18 patients with colorectal cancer. Intraoperative GDP counts with tumor-tissue ratios of 1.5:1 or greater were obtained from 15 (75%) of 20 tumor sites, with ratios averaging 2.3:1 for fragments and 3.4:1 for whole antibody. The GDP counts contributed to intraoperative decision making in three patients, either by localization of tumor not identified by inspection or palpation or by mapping margins of resection with histologic confirmation of a local/regional recurrence. These preliminary data demonstrate that probe-directed, intraoperative radioimmunodetection can assist the surgeon in detecting subclinical tumor deposits and thus better evaluate the extent of primary or recurrent colorectal cancers intraoperatively.

  15. Precipitation of a Monoclonal Antibody by Soluble Tungsten

    PubMed Central

    Bee, Jared S.; Nelson, Stephanie A.; Freund, Erwin; Carpenter, John F.; Randolph, Theodore W.

    2009-01-01

    Tungsten microparticles may be introduced into some pre-filled syringes during the creation of the needle hole. In turn, these microcontaminants may interact with protein therapeutics to produce visible particles. We found that soluble tungsten polyanions formed in acidic buffer below pH 6.0 can precipitate a monoclonal antibody within seconds. Soluble tungsten in pH 5.0 buffer at about 3 ppm was enough to cause precipitation of a mAb formulated at 0.02 mg/mL. The secondary structure of the protein was near-native in the collected precipitate. Our observations are consistent with the coagulation of a monoclonal antibody by tungsten polyanions. Tungsten-induced precipitation should only be a concern for proteins formulated below about pH 6.0 since tungsten polyanions are not formed at higher pHs. We speculate that the heterogenous nature of particle contamination within the poorly mixed syringe tip volume could mean that a specification for tungsten contamination based on the entire syringe volume is not appropriate. The potential potency of tungsten metal contamination is highlighted by the small number of particles that would be required to generate soluble tungsten levels needed to coagulate this antibody at pH 5.0. PMID:19230018

  16. Development and application of a monoclonal antibody against Thiothrix spp.

    PubMed Central

    Brigmon, R L; Bitton, G; Zam, S G; O'Brien, B

    1995-01-01

    Historically, methods used to identify Thiothrix spp. in environmental samples have been inadequate because isolation and identification procedures are time-consuming and often fail to separate Thiothrix spp. from other filamentous microorganisms. We described a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) procedure which was used to identify Thiothrix spp. in wastewater, artesian springs, groundwater, and underwater subterranean samples. The ELISA utilized monoclonal antibody T3511 to a species-specific carbohydrate epitope of Thiothrix spp. No cross-reactions were observed among non-Thiothrix strains consisting of 12 species and nine genera. In field trials, the ELISA identified 100% of 20 biochemically and cytologically confirmed Thiothrix spp.-containing samples with no false positives. Indirect immunofluorescent microscopy utilizing T3511 was effective for wastewater samples but not for those from natural spring water because of background fluorescence in the latter. In addition, electron micrographs of Thiothrix spp. labeled with T3511-biotin-anti-mouse antibody-gold showed that epitope T3511 was intracellular both in laboratory strains and environmental isolates. The minimum level of detection of the ELISA was 0.10 microgram/ml. PMID:7887596

  17. Immunolocalization of neuroblastoma using radiolabeled monoclonal antibody UJ13A

    SciTech Connect

    Goldman, A.; Vivian, G.; Gordon, I.; Pritchard, J.; Kemshead, J.

    1984-08-01

    The monoclonal antibody UJ13A, raised after immunization of mice with human fetal brain, recognized an antigen expressed on human neuroblastoma cell lines and fresh tumors. Antibody was purified and radiolabeled with iodine isotopes using chloramine-T. In preclinical studies, 125I-labeled UJ13A was injected intravenously into nude mice bearing xenografts of human neuroblastoma. Radiolabeled UJ13A uptake by the tumors was four to 23 times greater than that by blood. In control animals, injected with a similar quantity of a monoclonal antibody known not to bind to neuroblastoma cells in vitro (FD44), there was no selective tumor uptake. Nine patients with histologically confirmed neuroblastoma each received 100 to 300 micrograms UJ13A radiolabeled with 1 to 2.8 mCi 123I or 131I. Sixteen positive sites were visible on gamma scans 1 to 7 days after injection: 15 were primary or secondary tumor sites, and one was a false positive; there were two false negatives. In two of the 15 positive sites, tumor had not been demonstrated by other imaging techniques; these were later confirmed as areas of malignant infiltration. No toxicity was encountered.

  18. Trends in capacity utilization for therapeutic monoclonal antibody production

    PubMed Central

    2009-01-01

    The administration of high doses of therapeutic antibodies requires large-scale, efficient, cost effective manufacturing processes. An understanding of how the industry is using its available production capacity is important for production planning, and facility expansion analysis. Inaccurate production planning for therapeutic antibodies can have serious financial ramifications. In the recent 5th Annual Report and Survey of Biopharmaceutical Manufacturing Capacity and Production, 434 qualified respondents from 39 countries were asked to indicate, among other manufacturing issues, their current trends and future predictions with respect to the production capacity utilization of monoclonal antibodies in mammalian cell culture systems. While overall production of monoclonals has expanded dramatically since 2003, the average capacity utilization for mammalian cell culture systems, has decreased each year since 2003. Biomanufacturers aggressively attempt to avoid unanticipated high production demands that can create a capacity crunch. We summarize trends associated with capacity utilization and capacity constraints which indicate that biopharmaceutical manufacturers are doing a better job planning for capacity. The results have been a smoothing of capacity use shifts and an improved ability to forecast capacity and outsourcing needs. Despite these data, today, the instability and financial constraints caused by the current global economic crisis are likely to create unforeseen shifts in our capacity utilization and capacity expansion trends. These shifts will need to be measured in subsequent studies. PMID:20061821

  19. Secretory expression of a bispecific antibody targeting tumor necrosis factor and ED-B fibronectin in Pichia pastoris and its functional analysis.

    PubMed

    Liu, Meng-yuan; Hu, Xue-ping; Xie, Mian; Jiang, Si-jing; Li, Lu-jun; Liu, Dong-xu; Yang, Xiao-song

    2014-12-01

    Specific targeting of tumor necrosis factor (TNF)-? antagonist to the inflamed site could increase its efficacy and reduce side-effects. Here, we constructed a bispecific diabody (BsDb) that targets TNF-? and ED-B-containing fibronectin, a fibronectin isoform specifically expressed in the pannus of the inflamed synovium in rheumatoid arthritis. BsDb was secreted from Pichia pastoris as functional protein and was purified to homogeneity. BsDb could simultaneously bind to human TNF-? and B-FN and neutralize TNF-? action. Additionally, BsDb showed a significant gain both in the antigen-binding affinity and in TNF-?-neutralizing ability as compared to its original antibodies, L19 and anti-TNF-? scFv, which were produced in E. coli. BsDb was constructed and was endowed with enhanced bioactivities and improved production processing. Therefore, it holds great potential for in vivo applications. PMID:25129049

  20. Detecting low level sequence variantsin recombinant monoclonal antibodies

    PubMed Central

    Yang, Yi; Strahan, Alex; Li, Charlene; Shen, Amy; Liu, Hongbin; Ouyang, Jun; Katta, Viswanatham; Francissen, Kathleen

    2010-01-01

    A systematic analytical approach combining tryptic and chymotryptic peptide mapping with a Mascot Error Tolerant Search (ETS) has been developed to detect and identify low level protein sequence variants, i.e., amino acid substitutions, in recombinant monoclonal antibodies. The reversed-phase HPLC separation with ultraviolet (UV) detection and mass spectral acquisition parameters of the peptide mapping methods were optimized by using a series of model samples that contained low levels (0.55.0%) of recombinant humanized anti-HER2 antibody (rhumAb HER2) along with another unrelated recombinant humanized monoclonal antibody (rhumAb A). This systematic approachs application in protein sequence variant analysis depends upon time and sensitivity constraints. An example of using this approach as a rapid screening assay is described in the first case study. For stable CHO clone selection for an early stage antibody project, comparison of peptide map UV profiles from the top four clone-derived rhumAb B samples quickly detected two sequence variants (M83R at 5% and P274Tat 42% protein levels) from two clones among the four. The second case study described in this work demonstrates how this approach can be applied to late stage antibody projects. A sequence variant, L413Q, present at 0.3% relative to the expected sequence of rhumAb C was identified by a Mascot-ETS for one out of four top producers. The incorporation of this systematic sequence variant analysis into clone selection and the peptide mapping procedure described herein have practical applications for the biotechnology industry, including possible detection of polymorphisms in endogenous proteins. PMID:20400866

  1. Monoclonal antibody that preferentially binds polylysine, polyarginine, and histones and selectively decorates nuclei and chromosomes.

    PubMed Central

    Morgan, J L; Dennis, D D

    1984-01-01

    A monoclonal antibody, designated J-57, selectively and uniformly decorates the interphase nuclei and mitotic chromosomes of a variety of eucaryotic cells as determined by indirect immunofluorescence. As determined by enzyme-linked immunosorbent assay, however, this monoclonal antibody is not monospecific. It reacts weakly with cytochrome c, RNase A, and brain tubulin. By these tests monoclonal antibody J-57 has broad cross-reactivity similar to that of antisera directed against polylysine. The differential reactions of this monoclonal antibody suggest that it may be a useful immunohistochemical probe for nuclei and chromosomes in whole cells. Images PMID:6490815

  2. The use of monoclonal antibodies and related agents in the treatment of respiratory disease.

    PubMed

    Schachter, E Neil; Neuman, Tzvi

    2009-07-01

    Monoclonal antibodies were first developed in 1975. Since their introduction they have had a progressively increasing impact on different areas of medical diagnostics and therapeutics. Currently, many monoclonal antibodies are under investigation for their potential role in pulmonary medicine. This review discusses a brief history of monoclonal antibodies; the manufacture, use, biosafety and nomenclature of these agents; the inflammatory response in asthma; and the use of omalizumab, an FDA-approved monoclonal antibody for use in asthma, will be described in detail, as well as its associated adverse effects. Additionally, other related biologic agents that may have a role in respiratory diseases are presented. PMID:19834630

  3. Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats.

    PubMed

    Byrnes-Blake, Kelly A; Laurenzana, Elizabeth M; Carroll, F Ivy; Abraham, Philip; Gentry, W Brooks; Landes, Reid D; Owens, S Michael

    2003-02-14

    Our studies examined pharmacokinetic mechanisms involved in high-affinity (K(d) approximately 11 nM) monoclonal antibody-based antagonism of (+)-methamphetamine-induced locomotor effects. Male rats received (+)-methamphetamine (0.3, 1, or 3 mg/kg i.v.) followed 30 min later by saline or anti-(+)-methamphetamine monoclonal antibody. All groups received a constant dose of monoclonal antibody that was equimolar in binding sites to the body burden of a 1 mg/kg i.v. (+)-methamphetamine dose 30 min after administration. The monoclonal antibody antagonized locomotor effects due to 0.3 and 1 mg/kg (+)-methamphetamine. In contrast, monoclonal antibody treatment increased locomotor activity due to 3 mg/kg (+)-methamphetamine. We also investigated the serum and brain pharmacokinetics of (+)-methamphetamine without and with the monoclonal antibody. Rats received (+)-methamphetamine (1 mg/kg i.v.) followed by saline or monoclonal antibody treatment at 30 min. The monoclonal antibody significantly increased serum methamphetamine concentrations and significantly decreased brain methamphetamine concentrations. These data indicate that anti-(+)-methamphetamine monoclonal antibody-induced pharmacodynamics are complex, but are related to time-dependent changes in (+)-methamphetamine brain distribution. PMID:12586207

  4. Monoclonal Antibodies in Cancer Therapy: Mechanisms, Successes and Limitations

    PubMed Central

    Coulson, A; Levy, A; Gossell-Williams, M

    2014-01-01

    ABSTRACT Rituximab was the first chemotherapeutic monoclonal antibody (CmAb) approved for clinical use in cancer therapeutics in 1997 and has significantly improved the clinical outcomes in non-Hodgkin's lymphoma. Since then, numerous CmAbs have been developed and approved for the treatment of various haematologic and solid human cancers. In this review, the classification, efficacy and significantly reduced toxicity of CmAbs available for use in the United States of America are presented. Finally, the limitations of CmAbs and future considerations are explored. PMID:25803383

  5. [Phylogenic distribution of human renal antigens defined by monoclonal antibodies].

    PubMed

    Candelier, J J; Couillin, P; Pluvinage, S; Vissault, M C; Bou, A

    1986-01-01

    The preparation fo five monoclonal antibodies specific of important human renal histologic structures both functionally and organogenetically has permitted to identify the repartition of the corresponding antigens in the vertebrate phylum. For three of them, appeared a clear cut histologic identity in intensity and localization between the mammals studied and man. For the two others a phylogenic and histologic dispersion was observed. It may be supposed, in the latter case, that the evolution and the biotope have acted in different manners on renal function and organogenesis according to the vertebrate classes or species investigated. PMID:2428454

  6. Antiepidermal Growth Factor Receptor Monoclonal Antibodies: Applications in Colorectal Cancer

    PubMed Central

    Azizi, Efat; Kittai, Adam; Kozuch, Peter

    2012-01-01

    Patients with metastatic colorectal cancer have a poor prognosis and present a challenge to clinicians. The role of the antiepidermal growth factor receptor (EGFR) pathway in tumorogenesis and tumor progression has been well defined. This paper will review the use of anti-EGFR monoclonal antibodies in the treatment of operable, as well as metastatic colorectal cancer both in the setting of KRAS mutation unselected patients and later in KRAS wild-type patients. Active investigations designed to further identify predictive biomarkers that may be potentially druggable are reviewed as well. PMID:23091721

  7. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1993-01-01

    The objective of this project is to generate a library of monoclonal antibodies (MAb's) to surface molecules involved in the cell-cell interactions of mammalian cells grown as multicell spheroids (MCS). MCS are highly organized 3-dimensional multicellular structures which exhibit many characteristics in vivo tissues not found in conventional monolayer or suspension culture. They also provide a functional assay for surface adhesion molecules. In brief, MCS combine the relevance of organized tissues with the accuracy of in vitro methodology. Further, one can manipulate these MCS experimentally to discern important information about their biology.

  8. Development of Monoclonal Antibodies in China: Overview and Prospects

    PubMed Central

    Zhang, Mao-Yu; Lu, Jin-Jian; Wang, Liang; Gao, Zi-Chao; Ung, Carolina Oi Lam; Wang, Yi-Tao

    2015-01-01

    Monoclonal antibodies (mAbs) have become increasingly important as human therapeutic agents. Yet, current research concentrates on technology itself and pays attention to developed countries. This paper aims to provide a comprehensive review of mAbs development in China through systematic analysis of drug registry, patent applications, clinical trials, academic publication, and ongoing R&D projects. The trends in therapeutic areas and industrialization process are also highlighted. Development and research trends of mAbs are analyzed to provide a future perspective of mAbs as therapeutic agents in China. PMID:25811022

  9. Positron emission tomographic imaging of tumors using monoclonal antibodies

    SciTech Connect

    Zalutsky, M.R. . Dept. of Radiology)

    1989-12-01

    The overall objective of this research project is to develop methods for utilizing positron emission tomography (PET) to increase the clinical potential of radiolabeled monoclonal antibodies (MAbs). Both diagnostic and therapeutic applications of labeled MAbs could be improved as a result of knowledge obtained through the exploitation of the advantageous imaging characteristics associated with PET. By labeling MAbs with positron-emitting nuclides, it should be possible to quantitate the dynamics of their three-dimensional distribution in vivo. Our long-term goals are to apply this approach. 3 tabs.

  10. Monoclonal antibody based immunoassays for cooking-induced meat mutagens

    SciTech Connect

    Vanderlaan, M.; Hwang, M.; Knize, M.G.; Watkins, E.; Felton, J.S.

    1989-06-29

    We report here new monoclonal antibodies (Mabs) numbered AIA-8 through AIA-12 produced using the same methods used to produce AIA-1, and a new Mab, IQ-7, produced with the same methods used for IQ-1. Our motivation in seeking these new clones was to increase the repertoire of available Mabs to insure adequate coverage of all known AIAs. Also, the mice used to produce these new hybridoma clones had been immunized about six months longer than those used to generate the first clones. Longer immunization is often associated with higher affinity Mabs and therefore more sensitive competition immunoassays. 15 refs., 2 figs., 2 tabs.

  11. Monoclonal antibody against membrane protein of transmissible gastroenteritis virus.

    PubMed

    Sun, Xuejiao; Ren, Yudong; Li, Yu; Zhu, Jiayi; Zhu, Weijuan; Ding, Fan; Li, Guangxing; Wang, Chunfeng; Gao, Ming; Gao, Yunhang; Cao, Liyan; Ren, Xiaofeng

    2013-02-01

    Transmissible gastroenteritis virus (TGEV) is a porcine coronavirus that can cause piglet diarrhea with high mortality rates. TGEV membrane (M) protein not only plays a vital role in the process of virus assembly and budding, but also induces the production of interferon-? during infection. In this study, a monoclonal antibody (MAb) designated 7G7, against the TGEV M protein was generated by inoculating BALB/c mice with TGEV followed by hybridoma technique. Immunofluorescence assays indicated that MAb 7G7 was capable of detecting cell infection by TGEV. Virus-based ELISA demonstrated that MAb 7G7 can be used as a highly specific diagnostic reagent for TGEV. PMID:23600504

  12. Generation and characterization of monoclonal antibodies to TDRD7 protein.

    PubMed

    Skorokhod, Oleksandr; Nemazanyy, Ivan; Breus, Oksana; Filonenko, Valeriy; Panasyuk, Ganna

    2008-06-01

    TDRD7 is a scaffold protein whose specific function is unknown. It has been identified in complexes with proteins that regulate cytoskeleton dynamics and centrosomal movements, mRNA transport, and protein translation apparatus. Here we report the generation and characterization of monoclonal antibodies against TDRD7 protein. Bacterially expressed His-tagged fragments of TDRD7 were used as antigens. Spleen cells from immunized mice were collected and fused with SP2/0 myeloma cells using PEG 2000. High titer anti-TDRD7 antibody-producing hybridoma cell lines were identified by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution. Antibodies produced by E6 clone were further tested for their reactivity with the TDRD7 recombinant proteins. The results obtained clearly indicate that E6 anti-TDRD7 antibodies recognize specifically recombinant 6His-tagged TDRD7 proteins and endogenous TDRD7 in Western blotting, immunoprecipitation, and immunocytochemistry. In summary, these antibodies will be useful for researchers investigating TDRD7 and molecular complexes involving this protein. PMID:18582216

  13. Internal radiation dosimetry for clinical testing of radiolabeled monoclonal antibodies

    SciTech Connect

    Fisher, D.R.; Durham, J.S.; Hui, T.E.; Hill, R.L.

    1990-11-01

    In gauging the efficacy of radiolabeled monoclonal antibodies in cancer treatment, it is important to know the amount of radiation energy absorbed by tumors and normal tissue per unit administered activity. This paper describes methods for estimating absorbed doses to human tumors and normal tissues, including intraperitoneal tissue surfaces, red marrow, and the intestinal tract from incorporated radionuclides. These methods use the Medical Internal Radiation Dose (MIRD) scheme; however, they also incorporate enhancements designed to solve specific dosimetry problems encountered during clinical studies, such as patient-specific organ masses obtained from computerized tomography (CT) volumetrics, estimates of the dose to tumor masses within normal organs, and multicellular dosimetry for studying dose inhomogeneities in solid tumors. Realistic estimates of absorbed dose are provided within the short time requirements of physicians so that decisions can be made with regard to patient treatment and procurement of radiolabeled antibodies. Some areas in which further research could improve dose assessment are also discussed. 16 refs., 3 figs.

  14. Discovery and characterization of hydroxylysine in recombinant monoclonal antibodies.

    PubMed

    Xie, Qing; Moore, Benjamin; Beardsley, Richard L

    2016-01-01

    Tryptic peptide mapping analysis of a Chinese hamster ovary (CHO)-expressed, recombinant IgG1 monoclonal antibody revealed a previously unreported +16 Da modification. Through a combination of MS(n) experiments, and preparation and analysis of known synthetic peptides, the possibility of a sequence variant (Ala to Ser) was ruled out and the presence of hydroxylysine was confirmed. Post-translational hydroxylation of lysine was found in a consensus sequence (XKG) known to be the site of modification in other proteins such as collagen, and was therefore presumed to result from the activity of the CHO homolog of the lysyl hydroxylase complex. Although this consensus sequence was present in several locations in the antibody sequence, only a single site on the heavy-chain Fab was found to be modified. PMID:26651858

  15. Glycoengineered Pichia-based expression of monoclonal antibodies.

    PubMed

    Zha, Dongxing

    2013-01-01

    Currently, mammalian cells are the most commonly used hosts for the production of therapeutic monoclonal antibodies (mAbs). These hosts not only secrete mAbs with properly assembled two heavy and two light chains but also deliver mAbs with a glycosylation profile that is compatible with administration into humans. GlycoFi, a wholly owned subsidiary of Merck & Co., Inc., humanized the Pichia glycosylation pathway which allows it to express glycoproteins with a human-like glycan profile. This offers an alternative mAb production platform similar to mammalian hosts and in some cases it even provides more homogenous product and better efficacy, such as enhanced effector function. This chapter describes a protocol for using glycoengineered Pichia to produce full-length mAbs. It covers a broad spectrum of mAb expression technologies in yeast including expression vector construction, yeast transformation, high-throughput strain selection to fermentation, and antibody purification. PMID:23475712

  16. A polymorphic monoclonal anti-BoLA class I antibody.

    PubMed

    Simon, M; Dusinsk, R; Horn, P; Duraj, J; Bilka, F; Tomskov, J

    1994-10-01

    Cytotoxic monoclonal antibody IVA 44 was generated after the intraperitoneal immunization with peripheral blood mononuclear (PBM) cells and the boost by the intrasplenic inoculation of skin graft. The detected membrane antigen isolated by immunoprecipitation appears to be composed of two subunits characteristic for the MHC class I molecules. The antibody IVA 44 exhibited a different reactivity: it recognized the BoLA A14 (A8) specificity in animals typed in the Fifth BoLA workshop, while it reacted with all A8 positive animals including subtypes A14 and A15 in Czech and Slovak cattle. It is concluded that mAb IVA 44 might detect the broad subtype of A8 covering A14 and certain A15 split(s). The diverse A15 reactivity of this mAb in the workshop and our population could be explained by the different occurrence of A15 splits in both populations. PMID:7818171

  17. Design and Validation of a Novel Generic Platform for the Production of Tetravalent IgG1-like Bispecific Antibodies.

    PubMed

    Golay, Josée; Choblet, Sylvie; Iwaszkiewicz, Justyna; Cérutti, Pierre; Ozil, Annick; Loisel, Séverine; Pugnière, Martine; Ubiali, Greta; Zoete, Vincent; Michielin, Olivier; Berthou, Christian; Kadouche, Jean; Mach, Jean-Pierre; Duonor-Cérutti, Martine

    2016-04-01

    We have designed and validated a novel generic platform for production of tetravalent IgG1-like chimeric bispecific Abs. The VH-CH1-hinge domains of mAb2 are fused through a peptidic linker to the N terminus of mAb1 H chain, and paired mutations at the CH1-CL interface mAb1 are introduced that force the correct pairing of the two different free L chains. Two different sets of these CH1-CL interface mutations, called CR3 and MUT4, were designed and tested, and prototypic bispecific Abs directed against CD5 and HLA-DR were produced (CD5xDR). Two different hinge sequences between mAb1 and mAb2 were also tested in the CD5xDR-CR3 or -MUT4 background, leading to bispecific Ab (BsAbs) with a more rigid or flexible structure. All four Abs produced bound with good specificity and affinity to CD5 and HLA-DR present either on the same target or on different cells. Indeed, the BsAbs were able to efficiently redirect killing of HLA-DR(+) leukemic cells by human CD5(+) cytokine-induced killer T cells. Finally, all BsAbs had a functional Fc, as shown by their capacity to activate human complement and NK cells and to mediate phagocytosis. CD5xDR-CR3 was chosen as the best format because it had overall the highest functional activity and was very stable in vitro in both neutral buffer and in serum. In vivo, CD5xDR-CR3 was shown to have significant therapeutic activity in a xenograft model of human leukemia. PMID:26921308

  18. Effects of the orientation of anti-BMP2 monoclonal antibody immobilized on scaffold in antibody-mediated osseous regeneration

    PubMed Central

    Ansari, Sahar; Freire, Marcelo; Choi, Moon G; Tavari, Azadeh; Almohaimeed, Mohammad; Moshaverinia, Alireza; Zadeh, Homayoun H

    2015-01-01

    Recently, we have shown that anti-BMP2 monoclonal antibodies (mAbs) can trap endogenous osteogenic BMP ligands, which can in turn mediate osteodifferentiation of progenitor cells. The effectiveness of this strategy requires the availability of the anti-BMP-2 monoclonal antibodies antigen-binding sites for anti-BMP-2 monoclonal antibodies to bind to the scaffold through a domain that will leave its antigen-binding region exposed and available for binding to an osteogenic ligand. We examined whether antibodies bound to a scaffold by passive adsorption versus through Protein G as a linker will exhibit differences in mediating bone formation. In vitro anti-BMP-2 monoclonal antibodies was immobilized on absorbable collagen sponge (ACS) with Protein G as a linker to bind the antibody through its Fc region and implanted into rat calvarial defects. The biomechanical strength of bone regenerated by absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies immune complex was compared to ACS/anti-BMP-2 monoclonal antibodies or ACS/Protein G/isotype mAb control group. Results demonstrated higher binding of anti-BMP-2 monoclonal antibodies/BMPs to C2C12 cells, when the mAb was initially attached to recombinant Protein G or Protein G-coupled microbeads. After eight weeks, micro-CT and histomorphometric analyses revealed increased bone formation within defects implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies compared with defects implanted with absorbable collagen sponge/anti-BMP-2 monoclonal antibodies (p < 0.05). Confocal laser scanning microscopy (CLSM) confirmed increased BMP-2, -4, and -7 detection in sites implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies in vivo. Biomechanical analysis revealed the regenerated bone in sites with Protein G/anti-BMP-2 monoclonal antibodies had higher mechanical strength in comparison to anti-BMP-2 monoclonal antibodies. The negative control group, Protein G/isotype mAb, did not promote bone regeneration and exhibited significantly lower mechanical properties (p < 0.05). Altogether, our results demonstrated that application of Protein G as a linker to adsorb anti-BMP-2 monoclonal antibodies onto the scaffold was accompanied by increased in vitro binding of the anti-BMP-2 mAb/BMP immune complex to BMP-receptor positive cell, as well as increased volume and strength of de novo bone formation in vivo. PMID:26184354

  19. Generation of rat monoclonal antibodies against murine LAIR-1.

    PubMed

    Wang, Chun-Yan; Zhang, Yuan; Wang, Dong-Lin; Song, Chao-Jun; Han, Wei-Ning; Yang, Kun; Jin, Bo-Quan

    2007-10-01

    The leukocyte-associated Ig-like receptor 1 (LAIR-1), an inhibitory receptor bearing two immunoreceptor tyrosine-based inhibitory motifs (ITIM), is expressed on the majority of peripheral leukocytes, including NK cells, T cells, B cells, monocytes, dendritic cells, granulocytes, and thymocytes and is involved in immunologic regulation and hematopoiesis. Murine LAIR-1 (mLAIR-1) is the homolog molecule of human LAIR-1. Using mLAIR-1-Fc as the immunogen and the technique of rat B lymphocyte hybridoma, we raised three hybridoma cell lines secreting monoclonal antibodies (MAb) to mLAIR-1, designated FMU-mLAIR-1.1, -1.2, and -1.3. Rat immunoglobulin class and subclass of the MAb FMU-mLAIR-1.1 approximately 3 were determined to be IgM, IgG1, and IgM, respectively. All these MAbs can bind the mLAIR-1 in immunocytochemistry and immunohistochemistry. FMU-m LAIR-1.2 worked well not only in Western blot assay but also in recognizing natural LAIR-1 molecules on the surface of P388D1, J774, and WEHI3 cells, and mLAIR-1 cDNA-transfected CHO cells detected by FCM. Thus, successful production of rat anti-murine LAIR-1 monoclonal antibodies provides a new powerful tool for investigation of murine LAIR-1 function in mouse model, both in vitro and in vivo. PMID:17979548

  20. Monoclonal Antibodies Directed to Fucoidan Preparations from Brown Algae

    PubMed Central

    Torode, Thomas A.; Marcus, Susan E.; Jam, Murielle; Tonon, Thierry; Blackburn, Richard S.; Hervé, Cécile; Knox, J. Paul

    2015-01-01

    Cell walls of the brown algae contain a diverse range of polysaccharides with useful bioactivities. The precise structures of the sulfated fucan/fucoidan group of polysaccharides and their roles in generating cell wall architectures and cell properties are not known in detail. Four rat monoclonal antibodies, BAM1 to BAM4, directed to sulfated fucan preparations, have been generated and used to dissect the heterogeneity of brown algal cell wall polysaccharides. BAM1 and BAM4, respectively, bind to a non-sulfated epitope and a sulfated epitope present in the sulfated fucan preparations. BAM2 and BAM3 identified additional distinct epitopes present in the fucoidan preparations. All four epitopes, not yet fully characterised, occur widely within the major brown algal taxonomic groups and show divergent distribution patterns in tissues. The analysis of cell wall extractions and fluorescence imaging reveal differences in the occurrence of the BAM1 to BAM4 epitopes in various tissues of Fucus vesiculosus. In Ectocarpus subulatus, a species closely related to the brown algal model Ectocarpus siliculosus, the BAM4 sulfated epitope was modulated in relation to salinity levels. This new set of monoclonal antibodies will be useful for the dissection of the highly complex and yet poorly resolved sulfated polysaccharides in the brown algae in relation to their ecological and economic significance. PMID:25692870

  1. Active Lymphocytic Myocarditis Treated with Murine OKT3 Monoclonal Antibody

    PubMed Central

    Bilinska, Zofia T.; Grzybowski, Jacek; Szajewski, Tomasz; Stepinska, Janina; Michalak, Ewa; Walczak, Ewa; Wagner, Teresa; Kwiatkowska, Barbara; Ruzyllo, Witold

    2002-01-01

    This report describes the case of a 33-year-old woman with biopsy-proven, active lymphocytic myocarditis manifested by intractable ventricular tachycardia, nonspecific intraventricular block, and myocardial dysfunction. We treated her successfully with OKT3 monoclonal antibody and antiarrhythmic agents. Immunosuppression is not recommended in patients with infectious or postinfectious myocarditis. However, it may have an important role in autoimmune myocarditis. In the few reports in the medical literature that we were able to find, OKT3 monoclonal antibody was administered early, in the setting of acute, fulminant autoimmune myocarditis. Our patient received OKT3 therapy in a later phase of the disease, when inflammatory infiltrates were accompanied by extensive fibrosis and severe damage of cardiomyocytes. Our patient had concomitant Helicobacter pylori infection and a strong positive family history of gastric cancer, a disease often associated with H. pylori. We discuss the possibility of a causal relationship between H. pylori infection and autoimmune myocarditis. (Tex Heart Inst J 2002;29:1137) PMID:12075867

  2. Bothropic antivenom based on monoclonal antibodies, is it possible?

    PubMed

    Frauches, Thiago S; Petretski, Jorge H; Arnholdt, Andrea C V; Lasunskaia, Elena B; de Carvalho, Eulgio C Q; Kipnis, Thereza L; da Silva, Wilmar D; Kanashiro, Milton M

    2013-09-01

    Neutralizing monoclonal antibodies against three major toxic components of Bothrops atrox venom were produced and tested. The mAbs against phospholipase A2, hemorrhagic metalloprotease, and thrombin-like enzymes were produced in large amounts and purified with caprylic acid followed by ammonium sulfate precipitation. Purified mAbs were analyzed by SDS-PAGE and their ability to neutralize the respective toxins was tested. Five Swiss mice were injected i.p. with 13.5mg of pooled mAbs and challenged via s.c. route with venom. Survival rate was recorded for the next 48h. All mice treated and challenged with venom survived, whereas only one mouse in the control group survived. Bleeding time in mice treated with mAbs was similar to that observed in control mice. Our results show that monoclonal antibodies neutralized the lethal toxicity of Bothrops venom and indicate that there is a reasonable possibility of developing antivenoms based on humanized mAbs to treat victims of venomous animals in the future. PMID:23732123

  3. NCI Requests Targets for Monoclonal Antibody Production and Characterization - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution.

  4. Antigen-specific in vitro immunization: a source for human monoclonal antibodies.

    PubMed

    Tomimatsu, Kosuke; Shirahata, Sanetaka

    2014-01-01

    Human monoclonal antibody has great potential for treatment of various diseases utilizing their specificity against antigens. We have shown an in vitro immunization (IVI) protocol inducing antigen-specific immune responses in human peripheral blood mononuclear cells (PBMCs) for efficient production of human monoclonal antibodies. By using IVI method antigen specific antibody genes can be efficiently obtained because of increasing production of antigen-specific antibodies from in vitro immunized PBMCs. This IVI protocol will be widely applied for combination with several display methods and enhance the production of human monoclonal antibodies. PMID:24037847

  5. Screening individual hybridomas by microengraving to discover monoclonal antibodies

    PubMed Central

    Ogunniyi, Adebola O; Story, Craig M; Papa, Eliseo; Guillen, Eduardo; Love, J Christopher

    2014-01-01

    The demand for monoclonal antibodies (mAbs) in biomedical research is significant, but the current methodologies used to discover them are both lengthy and costly. Consequently, the diversity of antibodies available for any particular antigen remains limited. Microengraving is a soft lithographic technique that provides a rapid and efficient alternative for discovering new mAbs. This protocol describes how to use microengraving to screen mouse hybridomas to establish new cell lines producing unique mAbs. Single cells from a polyclonal population are isolated into an array of microscale wells (~105 cells per screen). The array is then used to print a protein microarray, where each element contains the antibodies captured from individual wells. The antibodies on the microarray are screened with antigens of interest, and mapped to the corresponding cells, which are then recovered from their microwells by micromanipulation. Screening and retrieval require approximately 13 d (912 d including the steps for preparing arrays of microwells). PMID:19528952

  6. Examination of HER3 targeting in cancer using monoclonal antibodies

    PubMed Central

    Gaborit, Nadge; Abdul-Hai, Ali; Mancini, Maicol; Lindzen, Moshit; Lavi, Sara; Leitner, Orith; Mounier, Lucile; Chentouf, Myriam; Dunoyer, Sai; Ghosh, Manjusha; Larbouret, Christel; Chards, Thierry; Bazin, Herv; Plegrin, Andr; Sela, Michael; Yarden, Yosef

    2015-01-01

    The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity. Nevertheless, in addition to serving as a dimerization partner of EGFR and HER2, HER3 acts as a key player in tumor cells ability to acquire resistance to cancer drugs. In this study, we generated several monoclonal antibodies to HER3. Comparisons of their ability to degrade HER3, decrease downstream signaling, and inhibit growth of cultured cells, as well as recruit immune effector cells, selected an antibody that later emerged as the most potent inhibitor of pancreatic cancer cells grown as tumors in animals. Our data predict that anti-HER3 antibodies able to intercept autocrine and stromatumor interactions might strongly inhibit tumor growth, in analogy to the mechanism of action of anti-EGFR antibodies routinely used now to treat colorectal cancer patients. PMID:25564668

  7. Generation and characterization of monoclonal antibodies against FABP4.

    PubMed

    Gorbenko, Olena; Filonenko, Valeriy; Gout, Ivan

    2006-04-01

    Fatty acid binding protein 4 (FABP4) is a key mediator of intracellular transport and metabolism of fatty acids in adipose tissues. FABP4 binds fatty acids with high affinity and transports them to various compartments in the cell. When in complex with fatty acids, FABP4 interacts with and modulates the activity of two important regulators of metabolism: hormone-sensitive lipase and peroxisome proliferator-activated receptor gamma. Genetic studies in mice clearly indicated that deregulation of FABP4 function may lead to the development of severe diseases such as diabetes II type and atherosclerosis. In this study, we report the production and detailed characterization of monoclonal antibodies (MAbs) against FABP4. Recombinant glutathione S-transferase (GST)-FABP4 or His-FABP4 was expressed in bacteria, affinity purified, and used for immunization of mice, enzyme-linked immunosorbent assay (ELISA) screening, and characterization of selected clones. We have isolated two hybridoma clones that produced antibodies specific for recombinant and native FABP4, as shown by Western blotting and immunoprecipitation. The specificity of generated antibodies was further tested in a cell-based model of adipogenesis. In this analysis, the accumulation of FABP4 during NIH 3T3-L1 differentiation into adipocytes was detected by generated antibodies, which correlates well with previously published data. Taken together, we produced MAbs that will be useful for the scientific community working on fatty acid-binding proteins and lipid metabolism. PMID:16704309

  8. Engineering fully human monoclonal antibodies from murine variable regions.

    PubMed

    Bernett, Matthew J; Karki, Sher; Moore, Gregory L; Leung, Irene W L; Chen, Hsing; Pong, Erik; Nguyen, Duc-Hanh T; Jacinto, Jonathan; Zalevsky, Jonathan; Muchhal, Umesh S; Desjarlais, John R; Lazar, Greg A

    2010-03-12

    Fully human monoclonal antibodies (mAbs) derived from transgenic mice or human antibody libraries are the current state of the art for reducing the immunogenicity risk of antibody drugs. Here, we describe a novel method for generating fully human mAbs from nonhuman variable regions using information from the human germline repertoire. Central to our strategy is the rational engineering of residues within and proximal to CDRs and the V(H)/V(L) interface by iteratively exploring substitutions to the closest human germline sequences using semi-automated computational methods. Starting from the parent murine variable regions of three currently marketed mAbs targeting CD25, vascular endothelial growth factor, and tumor necrosis factor alpha, we have generated fully human antibodies with 59, 46, and 45 substitutions, respectively, compared to the parent murine sequences. A large number of these substitutions were in the CDRs, which are typically avoided in humanization methods. Antigen affinities of the fully human variants were comparable to the chimeric mAbs in each case. Furthermore, in vitro functional characterization indicated that all retain potency of the chimeric mAbs and have comparable activity to their respective marketed drugs daclizumab, bevacizumab, and infliximab. Based on local and global sequence identity, the sequences of our engineered mAbs are indistinguishable from those of fully human mAbs isolated from transgenic mice or human antibody libraries. This work establishes a simple rational engineering methodology for generating fully human antibody therapeutics from murine mAbs produced from standard hybridoma technology. PMID:20045416

  9. Structural Characterization of a Monoclonal Antibody-Maytansinoid Immunoconjugate.

    PubMed

    Luo, Quanzhou; Chung, Hyo Helen; Borths, Christopher; Janson, Matthew; Wen, Jie; Joubert, Marisa K; Wypych, Jette

    2016-01-01

    Structural characterization was performed on an antibody-drug conjugate (ADC), composed of an IgG1 monoclonal antibody (mAb), mertansine drug (DM1), and a noncleavable linker. The DM1 molecules were conjugated through nonspecific modification of the mAb at solvent-exposed lysine residues. Due to the nature of the lysine conjugation process, the ADC molecules are heterogeneous, containing a range of species that differ with respect to the number of DM1 per antibody molecule. The DM1 distribution profile of the ADC was characterized by electrospray ionization mass spectrometry (ESI-MS) and capillary isoelectric focusing (cIEF), which showed that 0-8 DM1s were conjugated to an antibody molecule. By taking advantage of the high-quality MS/MS spectra and the accurate mass detection of diagnostic DM1 fragment ions generated from the higher-energy collisional dissociation (HCD) approach, we were able to identify 76 conjugation sites in the ADC, which covered approximately 83% of all the putative conjugation sites. The diagnostic DM1 fragment ions discovered in this study can be readily used for the characterization of other ADCs with maytansinoid derivatives as payload. Differential scanning calorimetric (DSC) analysis of the ADC indicated that the conjugation of DM1 destabilized the CH2 domain of the molecule, which is likely due to conjugation of DM1 on lysine residues in the CH2 domain. As a result, methionine at position 258 of the heavy chain, which is located in the CH2 domain of the antibody, is more susceptible to oxidation in thermally stressed ADC samples when compared to that of the naked antibody. PMID:26629796

  10. Tau Monoclonal Antibody Generation Based on Humanized Yeast Models

    PubMed Central

    Rosseels, Jolle; Van den Brande, Jeff; Violet, Marie; Jacobs, Dirk; Grognet, Pierre; Lopez, Juan; Huvent, Isabelle; Caldara, Marina; Swinnen, Erwin; Papegaey, Anthony; Caillierez, Raphalle; Bue-Scherrer, Valerie; Engelborghs, Sebastiaan; Lippens, Guy; Colin, Morvane; Bue, Luc; Galas, Marie-Christine; Vanmechelen, Eugeen; Winderickx, Joris

    2015-01-01

    A link between Tau phosphorylation and aggregation has been shown in different models for Alzheimer disease, including yeast. We used human Tau purified from yeast models to generate new monoclonal antibodies, of which three were further characterized. The first antibody, ADx201, binds the Tau proline-rich region independently of the phosphorylation status, whereas the second, ADx215, detects an epitope formed by the Tau N terminus when Tau is not phosphorylated at Tyr18. For the third antibody, ADx210, the binding site could not be determined because its epitope is probably conformational. All three antibodies stained tangle-like structures in different brain sections of THY-Tau22 transgenic mice and Alzheimer patients, and ADx201 and ADx210 also detected neuritic plaques in the cortex of the patient brains. In hippocampal homogenates from THY-Tau22 mice and cortex homogenates obtained from Alzheimer patients, ADx215 consistently stained specific low order Tau oligomers in diseased brain, which in size correspond to Tau dimers. ADx201 and ADx210 additionally reacted to higher order Tau oligomers and presumed prefibrillar structures in the patient samples. Our data further suggest that formation of the low order Tau oligomers marks an early disease stage that is initiated by Tau phosphorylation at N-terminal sites. Formation of higher order oligomers appears to require additional phosphorylation in the C terminus of Tau. When used to assess Tau levels in human cerebrospinal fluid, the antibodies permitted us to discriminate patients with Alzheimer disease or other dementia like vascular dementia, indicative that these antibodies hold promising diagnostic potential. PMID:25540200

  11. Using monoclonal antibodies as an international standard for the measurement of anti-adalimumab antibodies.

    PubMed

    van Schouwenburg, Pauline A; Kruithof, Simone; Wolbink, Gertjan; Wouters, Diana; Rispens, Theo

    2016-02-20

    Comparing studies investigating anti-drug antibody (ADA) formation is hampered by the lack of comparability between study protocols, assay formats, and standardized reference materials. In this respect, the use of an international standard would mean a major step forward. Here we compared 11 fully human monoclonal antibodies against adalimumab in two assays commonly used for ADA measurement; the bridging ELISA and the antigen binding test (ABT). Our results show non-parallel titration of the monoclonal antibodies in both assays, which we also find for polyclonal ADA sources. Moreover, we observed that the output of the bridging ELISA depends to a large degree on the affinity of the monoclonal antibody. For the ABT, results reflect a combination of affinity and avidity. This suggests that rather than reporting ADA values in nanogram per milliliter, arbitrary units may be more appropriate. Together our data highlight the difficulty of ADA standardization by identifying several pitfalls that should be taken into account when selecting a standard for ADA testing. PMID:26748377

  12. Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies

    PubMed Central

    Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Zhang, Kai; Batt, Micheal; Fitchett, Jonathan R; He, Dongmei; Rick, Heather L; Conner, Elaine M; Demarest, Stephen J

    2015-01-01

    Immunoglobulins and T cell receptors (TCRs) share common sequences and structures. With the goal of creating novel bispecific antibodies (BsAbs), we generated chimeric molecules, denoted IgG_TCRs, where the Fv regions of several antibodies were fused to the constant domains of the ?/? TCR. Replacing CH1 with C? and CL with C?, respectively, was essential for achieving at least partial heavy chain/light chain assembly. Further optimization of the linker regions between the variable and constant domains, as well as replacement of the large FG loop of C? with a canonical ?-turn, was necessary to consistently obtain full heavy chain/light chain assembly. The optimized IgG_TCR molecules were evaluated biophysically and shown to maintain the binding properties of their parental antibodies. A few BsAbs were generated by co-expressing native Fabs and IgG_TCR Fabs within the same molecular construct. We demonstrate that the IgG_TCR designs steered each of the light chains within the constructs to specifically pair with their cognate heavy chain counterparts. We did find that even with complete constant domain specificity between the CH1/CL and C?/C? domains of the Fabs, strong variable domain interactions can dominate the pairing specificity and induce some mispairing. Overall, the IgG_TCR designs described here are a first step toward the generation of novel BsAbs that may be directed toward the treatment of multi-faceted and complex diseases. PMID:25611120

  13. Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies.

    PubMed

    Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Zhang, Kai; Batt, Micheal; Fitchett, Jonathan R; He, Dongmei; Rick, Heather L; Conner, Elaine M; Demarest, Stephen J

    2015-01-01

    Immunoglobulins and T cell receptors (TCRs) share common sequences and structures. With the goal of creating novel bispecific antibodies (BsAbs), we generated chimeric molecules, denoted IgG_TCRs, where the Fv regions of several antibodies were fused to the constant domains of the ?/? TCR. Replacing CH1 with C? and CL with C?, respectively, was essential for achieving at least partial heavy chain/light chain assembly. Further optimization of the linker regions between the variable and constant domains, as well as replacement of the large FG loop of C? with a canonical ?-turn, was necessary to consistently obtain full heavy chain/light chain assembly. The optimized IgG_TCR molecules were evaluated biophysically and shown to maintain the binding properties of their parental antibodies. A few BsAbs were generated by co-expressing native Fabs and IgG_TCR Fabs within the same molecular construct. We demonstrate that the IgG_TCR designs steered each of the light chains within the constructs to specifically pair with their cognate heavy chain counterparts. We did find that even with complete constant domain specificity between the CH1/CL and C?/C? domains of the Fabs, strong variable domain interactions can dominate the pairing specificity and induce some mispairing. Overall, the IgG_TCR designs described here are a first step toward the generation of novel BsAbs that may be directed toward the treatment of multi-faceted and complex diseases. PMID:25611120

  14. Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex

    PubMed Central

    Kolumam, Ganesh; Chen, Mark Z.; Tong, Raymond; Zavala-Solorio, Jose; Kates, Lance; van Bruggen, Nicholas; Ross, Jed; Wyatt, Shelby K.; Gandham, Vineela D.; Carano, Richard A.D.; Dunshee, Diana Ronai; Wu, Ai-Luen; Haley, Benjamin; Anderson, Keith; Warming, Søren; Rairdan, Xin Y.; Lewin-Koh, Nicholas; Zhang, Yingnan; Gutierrez, Johnny; Baruch, Amos; Gelzleichter, Thomas R.; Stevens, Dale; Rajan, Sharmila; Bainbridge, Travis W.; Vernes, Jean-Michel; Meng, Y. Gloria; Ziai, James; Soriano, Robert H.; Brauer, Matthew J.; Chen, Yongmei; Stawicki, Scott; Kim, Hok Seon; Comps-Agrar, Laëtitia; Luis, Elizabeth; Spiess, Christoph; Wu, Yan; Ernst, James A.; McGuinness, Owen P.; Peterson, Andrew S.; Sonoda, Junichiro

    2015-01-01

    Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin. PMID:26288846

  15. Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/?Klotho Complex.

    PubMed

    Kolumam, Ganesh; Chen, Mark Z; Tong, Raymond; Zavala-Solorio, Jose; Kates, Lance; van Bruggen, Nicholas; Ross, Jed; Wyatt, Shelby K; Gandham, Vineela D; Carano, Richard A D; Dunshee, Diana Ronai; Wu, Ai-Luen; Haley, Benjamin; Anderson, Keith; Warming, Sren; Rairdan, Xin Y; Lewin-Koh, Nicholas; Zhang, Yingnan; Gutierrez, Johnny; Baruch, Amos; Gelzleichter, Thomas R; Stevens, Dale; Rajan, Sharmila; Bainbridge, Travis W; Vernes, Jean-Michel; Meng, Y Gloria; Ziai, James; Soriano, Robert H; Brauer, Matthew J; Chen, Yongmei; Stawicki, Scott; Kim, Hok Seon; Comps-Agrar, Latitia; Luis, Elizabeth; Spiess, Christoph; Wu, Yan; Ernst, James A; McGuinness, Owen P; Peterson, Andrew S; Sonoda, Junichiro

    2015-07-01

    Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/?Klotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/?Klotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/?Klotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/?Klotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/?Klotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin. PMID:26288846

  16. Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function

    PubMed Central

    Chung, Amy W.; Crispin, Max; Pritchard, Laura; Robinson, Hannah; Gorny, Miroslaw K.; Yu, Xiaojie; Bailey-Kellogg, Chris; Ackerman, Margaret E.; Scanlan, Chris; Zolla-Pazner, Susan; Alter, Galit

    2015-01-01

    Objective To determine monoclonal antibody (mAb) features that predict fragment crystalizable (Fc)-mediated effector functions against HIV. Design Monoclonal antibodies, derived from Chinese hamster ovary cells or EpsteinBarr virus-immortalized mouse heteromyelomas, with specificity to key regions of the HIV envelope including gp120-V2, gp120-V3 loop, gp120-CD4+ binding site, and gp41-specific antibodies, were functionally profiled to determine the relative contribution of the variable and constant domain features of the antibodies in driving robust Fc-effector functions. Methods Each mAb was assayed for antibody-binding affinity to gp140SF162, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and for the ability to bind to FcgRIIa, FcgRIIb and FcgRIIIa receptors. Antibody glycan profiles were determined by HPLC. Results Neither the specificity nor the affinity of the mAbs determined the potency of Fc-effector function. FcgRIIIa binding strongly predicted ADCC and decreased galactose content inversely correlated with ADCP, whereas N-glycolylneuraminic acid-containing structures exhibited enhanced ADCP. Additionally, the bi-antenary glycan arm onto which galactose was added predicted enhanced binding to FcgRIIIa and ADCC activity, independent of the specificity of the mAb. Conclusions Our studies point to the specific Fc-glycan structures that can selectively promote Fc-effector functions independently of the antibody specificity. Furthermore, we demonstrated antibody glycan structures associated with enhanced ADCP activity, an emerging Fc-effector function that may aid in the control and clearance of HIV infection. PMID:25160934

  17. Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics.

    PubMed

    Asano, Ryutaro; Shimomura, Ippei; Konno, Shota; Ito, Akiko; Masakari, Yosuke; Orimo, Ryota; Taki, Shintaro; Arai, Kyoko; Ogata, Hiromi; Okada, Mai; Furumoto, Shozo; Onitsuka, Masayoshi; Omasa, Takeshi; Hayashi, Hiroki; Katayose, Yu; Unno, Michiaki; Kudo, Toshio; Umetsu, Mitsuo; Kumagai, Izumi

    2014-01-01

    One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)-variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH-VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies. PMID:25517309

  18. Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics

    PubMed Central

    Asano, Ryutaro; Shimomura, Ippei; Konno, Shota; Ito, Akiko; Masakari, Yosuke; Orimo, Ryota; Taki, Shintaro; Arai, Kyoko; Ogata, Hiromi; Okada, Mai; Furumoto, Shozo; Onitsuka, Masayoshi; Omasa, Takeshi; Hayashi, Hiroki; Katayose, Yu; Unno, Michiaki; Kudo, Toshio; Umetsu, Mitsuo; Kumagai, Izumi

    2014-01-01

    One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)–variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH–VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies. PMID:25517309

  19. An amphipathic sulphated glycoconjugate of Leishmania: characterization with monoclonal antibodies.

    PubMed Central

    Handman, E; Greenblatt, C L; Goding, J W

    1984-01-01

    A major glycoconjugate of Leishmania tropica major identified by two monoclonal antibodies was shown to be an externally oriented, amphipathic membrane antigen shed into the culture medium in which the parasites grow. This molecule could be labelled metabolically with [3H]glucose, [3H]galactose, [32P]phosphate and [35S]sulphate. It migrated as a polydisperse band upon electrophoresis in SDS-polyacrylamide gels, spanning the region of the gel corresponding to an apparent mol. wt. of 20 000-67 000 daltons. An apparently identical family of molecules could be labelled on the surface of living promastigotes using galactose oxidase and [3H]-sodium borohydride. This molecule was shown to be released into the supernatant over a period of several hours. Detection of the 3H- or 35S-labelled molecule required several days exposure of autoradiographs, but a novel blotting technique using nitrocellulose coated with monoclonal antibody allowed rapid detection of the molecule in charge shift electrophoresis, Western blotting and dot blotting. The electrophoretic mobility of the glycoconjugate in agarose relative to its mobility in Triton X-100 was increased in the presence of deoxycholate, and decreased in the presence of cetyl trimethyl-ammonium bromide, indicating amphipathic properties consistent with insertion into the lipid bilayer of the membrane. Using the dot-blotting technique the glycoconjugate was detected in all virulent and avirulent clones of LRC-L137 and in two additional isolates of L. tropica major (LRC-L287 and LRC-L251), but not in L. donovani or L. mexicana, consistent with the previously described specificity of the antibodies. However, the general approaches used in this paper showed that L. donovani (LRC-L52) and L. mexicana (LRC-L94) synthesize a similar, but antigenically distinct glycoconjugate. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6499830

  20. Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

    DOEpatents

    Stanker, Larry H.; Vanderlaan, Martin; Watkins, Bruce E.

    1994-01-01

    Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples.

  1. Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

    DOEpatents

    Stanker, L.H.; Vanderlaan, M.; Watkins, B.E.

    1994-08-02

    Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples. 13 figs.

  2. Development and characterization of mouse monoclonal antibodies specific for chicken interleukin 18

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Four mouse monoclonal antibodies (mAbs) which are specific for chicken interleukin 18 (chIL18) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting, quantitative real-time PCR and neutralization assays. Monoclonal antibodies specific for chIL18 identified a ...

  3. Scintigraphy of normal mouse ovaries with monoclonal antibodies to ZP-2, the major zona pellucida protein

    SciTech Connect

    East, I.J.; Keenan, A.M.; Larson, S.M.; Dean, J.

    1984-08-31

    The zona pellucida is an extracellular glycocalyx, made of three sulfated glycoproteins, that surrounds mammalian oocytes. Parenterally administered monoclonal antibodies specific for ZP-2, the most abundant zona protein, localize in the zona pellucida. When labeled with iodine-125, these monoclonal antibodies demonstrate a remarkably high target-to-nontarget tissue ratio and provide clear external radioimaging of ovarian tissue.

  4. Method of rapid production of hybridomas expressing monoclonal antibodies on the cell surface

    DOEpatents

    Meagher, Richard B.; Laterza, Vince

    2006-12-12

    The present invention relates to genetically altered hybridomas, myelomas and B cells. The invention also relates to utilizing genetically altered hybridomas, myelomas and B cells in methods of making monoclonal antibodies. The present invention also provides populations of hybridomas and B cells that can be utilized to make a monoclonal antibody of interest.

  5. Immunologic characterization and specificity of three monoclonal antibodies against the 58-kilodalton protein of Legionella pneumophila.

    PubMed Central

    Sampson, J S; Plikaytis, B B; Aloisio, C H; Carlone, G M; Pau, C P; Stinson, A R

    1991-01-01

    Three monoclonal antibodies against the Legionella pneumophila 58-kDa protein were produced. By using immunoblot analysis, the percentages of reactivity against 47 serogroups of Legionella representing 29 species were determined to be 80.9, 87.2, and 95.6 for monoclonal antibodies GB5BE8, GB5AF6, and CA4AF5, respectively. Specificities obtained from testing 63 heterologous organisms representing 22 genera and 46 species were 90.7, 92.2, and 95.3% for monoclonal antibodies GB5BE8, GB5AF6, and CA4AF5, respectively. No single heterologous strain was reactive with all three monoclonal antibodies. These monoclonal antibodies successfully identified all 10 clinical isolates of Legionella examined in a dot blot assay and should be excellent reagents for use in genuswide diagnostic immunoassays. Images PMID:1890189

  6. Therapy of a murine sarcoma using syngeneic monoclonal antibody

    SciTech Connect

    Kennel, S.J.; Lankford, T.; Flynn, K.M.

    1983-01-01

    Syngeneic monoclonal antibodies (MoAb) to Moloney sarcoma cells were produced by fusion of spleen cells from MSC regressor mice to myeloma SP2/0. MoAb 244-19A, an immunoglobulin G2b, bound to MSC cells and did not bind to two other sarcomas (K-BALB and Ha2), a carcinoma (Line 1), a fibroblast (A31) or a fibroblast infected with C-type virus (A31) or a fibroblast infected with C-type virus (A31-Moloney leukemia virus). In contrast, MoAb 271-1A bound to the MSC and Ha2 sarcoma and line 1 carcinoma as well as to the normal and infected fibroblast cultures. Antibodies were tested for therapeutic effect using three schedules of antibody injection. Injection i.p. of ascites fluid containing 244-19A MoAb given on Days -1, 0, and +1 relative to tumor cell injection increased life span significantly over that of control animals given injections (P3, immunoglobulin G, or MoAb 271-1A) and produced some seven of 19, one of five, and one of five long-term survivors in three separate experiments. Antibody given to animals with established tumors (4 days after implantation) also prolonged life span significantly and produced three of nine long-term survivors. Antibody given to animals with very large tumor burdens (10 days after implantation) did not prolong life span significantly. Optimal dose, schedule, and mechanism studies concerning this therapy are in progress.

  7. Selection of Ceratitis capitata (Diptera: Tephritidae) Specific Recombinant Monoclonal Phage Display Antibodies for Prey Detection Analysis

    PubMed Central

    Monz, Csar; Urbaneja, Alberto; Ximnez-Embn, Miguel; Garca-Fernndez, Julia; Garca, Jos Luis; Castaera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators. PMID:23272105

  8. Preparation and identification of anti-2, 4-dinitrophenyl monoclonal antibodies.

    PubMed

    Yang, Tangbin; Zhong, Ping; Qu, Lina; Wang, Chunyan; Yuan, Yanhong

    2006-06-30

    2, 4-Dinitrophenyl (DNP) is a widely used hapten in molecular biology and immunoassay fields. Considering that 2, 4-dintrophenylhydrazine (DNPH) could be used as DNA probe and bind with protein carbonyl to form a stable 2 4-dinitrophenyl (DNP) hydrazone product, on which the level of oxidative stress could be validated with a sensitive noncompetitive ELISA, we prepared DNP-aminocaproic acid and NHS-aminocaproic acid-dinitrobenzene and the conjugates between DNP and carrier proteins such as bovine thyroglobulin (BTG) and bovine serum albumin (BSA). High titer antibody producing spleen cells were removed and fused with myeloma cells of SP2/0 origin. Using a conventional immunization protocol, twenty stable murine monoclonal antibodies (MAbs) producing cell lines to DNP were generated. The donor mouse produced antiserum with a high titer of 1/1,280,000. Five MAbs were selected for further characterization as class and subclass. After four successive limiting dilutions, antibodies were produced by five clones with high affinities ranging from 10(10) to 10(11) M(-1). These clones were found to be of IgG(1) subclass with kappa and lambda light chain. Competitive ELISA and SPR-based sensing system for the detection of DNPH are both used to confirm the specificity of MAb (4D(9)A(9)C(2)C(2)). PMID:16765373

  9. Legionella micdadei and Legionella dumoffii monoclonal antibodies for laboratory diagnosis of Legionella infections.

    PubMed Central

    Cercenado, E; Edelstein, P H; Gosting, L H; Sturge, J C

    1987-01-01

    Two different monoclonal antibodies directed against Legionella micdadei and L. dumoffii (Genetic Systems Corp., Seattle, Wash.) were evaluated for their specificity and ability to detect L. micdadei and L. dumoffii in human and animal clinical samples and bacterial isolates in an indirect immunofluorescence assay. All three frozen sputum samples and all three Formalin-fixed sputum and liver samples from patients with culture-documented L. micdadei pneumonia were positive when tested with the L. micdadei monoclonal antibody. A Formalin-preserved lung sample from a patient with culture-documented L. dumoffii pneumonia was positive with its homologous monoclonal antibody. No cross-staining reactions were found with either monoclonal antibody on any of 25 human sputum samples tested from patients without Legionella infections. A total of 66 Legionella strains and 56 non-Legionella strains including 22 Pseudomonas strains and 34 other bacterial strains were studied. No cross-staining reactions were found except in Staphylococcus aureus Cowan 1 ATCC 12598. The lower limit of detection in seeded sputum samples was about 7 X 10(4) cells per ml for both monoclonal antibodies. Lung and tracheal lavage specimens from L. micdadei- or L. dumoffii-infected guinea pigs showed specific staining only with their respective monoclonal antibodies. The monoclonal antibodies stained homologous bacteria slightly less intensely than did the polyclonal antisera, but the signal-to-noise ratio was considerably higher for the monoclonal antibodies. No differences in sensitivity of staining of clinical specimens or bacterial isolates were noted between the monoclonal antibodies and the polyclonal reagents for L. micdadei and L. dumoffii (Centers for Disease Control, Atlanta, Ga., and BioDx, Denville, N.J. These monoclonal antibodies ae sensitive and specific, making them good candidates for laboratory diagnostic purposes. PMID:3320084

  10. Inhibitory, opsonic and cytotoxic activities of monoclonal antibodies against asexual erythrocytic stages of Plasmodium falciparum.

    PubMed

    Li, J L; Li, Y J

    1987-10-01

    A range of monoclonal antibodies specific for Plasmodium falciparum were tested in vitro for their abilities to inhibit the multiplication of a partially synchronized culture of P. falciparum, to augment the phagocytosis of the parasites by macrophages, and to enhance the killing of parasites by peritoneal cells depleted of adherent cells. Seven of 17 monoclonal antibodies, ranging from culture supernatant fluid and ascitic fluid to purified IgG, showed dose- and time-dependent inhibition of parasite growth in vitro. At a concentration of 0.6 mg/ml, the inhibitory capacity of these monoclonal IgGs was above 94% over a 3-day culture period, much higher than that of the relevant polyclonal IgG. Four of 6 monoclonal antibodies tested augmented the phagocytosis of the parasites by macrophages, which occurred as a result of opsonization of the parasites. Four of 7 monoclonal antibodies examined showed cytotoxic activity on malaria parasites. Peritoneal cells depleted of adherent cells were capable of killing the parasites in the presence of monoclonal antibodies. These results indicate that there may be 'monofunction', 'bifunction', and 'multifunction' types of monoclonal antibodies against P. falciparum. The putative protective antigen of malaria parasites purified by 'multifunctional monoclonal antibody' affinity chromatography may have potential interest as a vaccine against the parasite or as an immunodiagnostic reagent for human malaria. PMID:3320888

  11. Potential of palladium-109-labeled antimelanoma monoclonal antibody for tumor therapy

    SciTech Connect

    Fawwaz, R.A.; Wang, T.S.T.; Srivastava, S.C.; Rosen, J.M.; Ferrone, S.; Hardy, M.A.; Alderson, P.O.

    1984-07-01

    Palladium-109, a beta-emitting radionuclide, was chelated to the monoclonal antibody 225.28S to the high molecular weight antigen associated with human melanoma. Injection of the radiolabeled monoclonal antibody into nude mice bearing human melanoma resulted in significant accumulation of the radiolabel in the tumors: 19% injected dose/g; 38:1 and 61:1 tumor-to-blood ratios at 24 and 48 hr, respectively. The localization of the radiolabeled antibody in liver and kidney also was high, but appreciably lower than that achieved in tumor. These results suggest Pd-109-labeled monoclonal antibody to tumor-associated antigens may have potential applications in tumor immunotherapy.

  12. Modulation of catalysis and inhibition of fetal bovine serum acetylcholinesterase by monoclonal antibodies

    SciTech Connect

    Doctor, B.P.; Gentry, M.K.; Saxena, A.; Ashani, Y.

    1995-12-31

    Monoclonal antibodies have been raised against acetyicholinesterase isolated from a variety of sources and species. Although none of these antibodies bind to the esteratic site, some of them appear to interact with the region of the catalytic subunit referred to as the peripheral anionic site. We describe here the production and characterization of six inhibitory monoclonal antibodies against fetal bovine serum acetylcholinesterase. Results show that changes in the conformation of acetyicholinesterase caused by interaction with monoclonal antibodies at a site remote from the catalytic site result in the modulation of catalytic activity of acetylcholinesterase.

  13. Radioimmunoimaging of venous thrombi using iodine-131 monoclonal antibody. [Dogs

    SciTech Connect

    Rosebrough, S.F.; Kudryk, B.; Grossman, Z.D.; McAfee, J.G.; Subramanian, G.; Ritter-Hrncirik, C.A.; Witanowski, L.S.; Tillapaugh-Fay, G.

    1985-08-01

    Murine monoclonal antibody (Mab) specific for the NH/sub 2/-terminal region of human fibrin, but not cross-reactive with fibrinogen, was used in radioimmuno-imaging of fresh, induced venous thrombi in three dogs. Iodine-131-labeled Mab was injected intravenously, with iodine 125-labeled polyclonal murine gamma-G globulin (IgG) simultaneously injected as a control. Images were strongly positive at 24 and 48 hours in all three animals, with thrombus-to-blood and thrombus-to-muscle ratios of 8.4 and 228.0, respectively, for I-131-labeled Mab; these ratios for control IgG were 1.2 and 13.0. Radioimmunodetection of thrombi in vivo is feasible in dogs and may have clinical application since Mab is specific to human fibrin.

  14. A highly sensitive caffeine immunoassay based on a monoclonal antibody.

    PubMed

    Carvalho, Jos Joo; Weller, Michael G; Panne, Ulrich; Schneider, Rudolf J

    2010-04-01

    A new immunoassay has been developed based on a commercially available anti-caffeine monoclonal antibody and a de novo synthesized tracer, using horseradish peroxidase and UV-visible detection. Caffeine, which is frequently found in surface waters, can be quantified with a relative error lower than 20% for concentrations above 0.025 microg L(-1) (limit of quantitation, direct analysis). The limit of detection is 0.001 microg L(-1) and can be reduced by solid-phase extraction (SPE). Moreover, with minor adaptations, the assay can be used to quantify caffeine in several beverages, shampoo, and caffeine tablets. The results obtained by ELISA correlate well with those from liquid chromatography-tandem mass spectrometry (LC-MS-MS) for the tested matrices. Several surface waters from Berlin were analysed and all tested positive for caffeine, with concentrations higher than 0.030 microg L(-1). In one run 66 samples can be analysed within 2 h. PMID:20155491

  15. Infectious Complications Associated with Monoclonal Antibodies and Related Small Molecules

    PubMed Central

    Salvana, Edsel Maurice T.; Salata, Robert A.

    2009-01-01

    Summary: Biologics are increasingly becoming part of routine disease management. As more agents are developed, the challenge of keeping track of indications and side effects is growing. While biologics represent a milestone in targeted and specific therapy, they are not without drawbacks, and the judicious use of these magic bullets is essential if their full potential is to be realized. Infectious complications in particular are not an uncommon side effect of therapy, whether as a direct consequence of the agent or because of the underlying disease process. With this in mind, we have reviewed and summarized the risks of infection and the infectious disease-related complications for all FDA-approved monoclonal antibodies and some related small molecules, and we discuss the probable mechanisms involved in immunosuppression as well as recommendations for prophylaxis and treatment of specific disease entities. PMID:19366915

  16. A novel monoclonal antibody specific to peptide pdnaelvlltlgqawqg.

    PubMed

    Ji, Genlin

    2009-10-01

    Cis-aconitic acid decarboxylase (CAD) has been assumed to be a key enzyme in the production of itaconic acid. Here we aimed to efficiently generate the monoclonal antibody against the CAD protein. We synthesized the peptide "pdnaelvlltlgqawqg" based on the published CAD cDNA sequences. The peptide was chemically linked with the carrier protein keyhole limpet hemocyanin and then injected into Balb/c mice. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using either purified 6 x His-CAD fusion protein or the peptide. One MAb named K2 (IgG1), effective in detecting the native CAD protein, was characterized by ELISA and Western immunoblotting. By using the MAb, we found that the CAD protein was more highly expressed in poorly differentiated gastric cancer tissues than in well-differentiated and moderately differentiated tissues. Taken together, the MAb K2 would be helpful for understanding the functions of CAD in gastric carcinogenesis. PMID:19857120

  17. Monoclonal antibodies and the transformation of blood typing

    PubMed Central

    Marks, Lara

    2014-01-01

    Today, when monoclonal antibodies (mAbs) have become one of the most important classes of therapeutic drugs, it is easy to forget how much they have transformed our healthcare in other ways. One of the first clinical areas, as this paper shows, where mAbs made their mark was in the field of blood typing. The adoption of mAbs for this purpose was done with little public fanfare or funding. Nonetheless, it radically transformed the accuracy and cost of blood typing and shifted the procedure away from a dependence on reagents made from human blood donated by volunteers. This paper argues that the development of mAbs as reagents for blood typing laid the foundation for the first large-scale production of mAbs thereby paving the way to the advent of mAb diagnostics and therapeutics. PMID:25484059

  18. Novel CD20 monoclonal antibodies for lymphoma therapy

    PubMed Central

    2012-01-01

    Rituximab (RTX), a monoclonal antibody (mAb) against CD20, has been widely used for lymphoma therapy. RTX in combination with cyclophosphamide /doxorubicin /vincristine /prednisone (R-CHOP) remains the standard frontline regimen for diffuse large B-cell lymphoma. However, suboptimal response and /or resistance to rituximab have remained a challenge in the therapy of B-cell non-Hodgkins lymphoma (NHL). Novel agents are under active clinical trials. This review will summarize the latest development in new mAbs against CD20, which include second-generation mAbs, ofatumumab, veltuzumab (IMMU-106), ocrelizumab (PRO70769), and third-generation mAbs, AME-133v (ocaratuzumab), PRO131921 and GA101 (obinutumumab). PMID:23057966

  19. High-level iodination of monoclonal antibody fragments for radiotherapy

    SciTech Connect

    Ferens, J.M.; Krohn, K.A.; Beaumier, P.L.; Brown, J.P.; Hellstroem, I.; Hellstroem, K.E.; Carrasquillo, J.A.; Larson, S.M.

    1984-03-01

    Two different murine monoclonal antibody Fab fragments specific for p97, a melanoma-associated antigen, were labeled with I-131 at high activity levels without excessive chemical damage. Up to 20 mg of Fab were labeled with up to 300 mCi of I-131 using the chloramine-T method and large working volumes at room temperature. As much as 90% of the initial activity was recovered as labeled product. The labeled Fabs varied in their sensitivity to radioiodination damage, as measured by an in vitro cell-binding assay. Radioiodination was performed safely using a remote iodination apparatus. The final product was of radiopharmaceutical quality suitable for clinical diagnosis and experimental radiotherapy in humans.

  20. Monoclonal antibodies in infectious diseases: clinical pipeline in 2011.

    PubMed

    Ter Meulen, Jan

    2011-12-01

    Of the more than 20 monoclonal antibodies (mAbs) generated to combat infectious diseases (ID) that are in clinical development in 2011, most are in phase 1 or 2 and are directed against either viruses or bacterial toxins. Several high-profile anti-ID mAbs have recently failed in clinical trials. Despitethe advancement in recombinant engineering technologies, anti-ID mAbs have yet to deliver on their promise as "magic bullets," especially against nosocomial infections. A paradigm shift in favor of developing mAb combinations, which act synergistically with each other or with small molecule drugs, may be required to move the field forward. PMID:22054756

  1. Novel CD20 monoclonal antibodies for lymphoma therapy.

    PubMed

    Cang, Shundong; Mukhi, Nikhil; Wang, Kemeng; Liu, Delong

    2012-01-01

    Rituximab (RTX), a monoclonal antibody (mAb) against CD20, has been widely used for lymphoma therapy. RTX in combination with cyclophosphamide /doxorubicin /vincristine /prednisone (R-CHOP) remains the standard frontline regimen for diffuse large B-cell lymphoma. However, suboptimal response and /or resistance to rituximab have remained a challenge in the therapy of B-cell non-Hodgkin's lymphoma (NHL). Novel agents are under active clinical trials. This review will summarize the latest development in new mAbs against CD20, which include second-generation mAbs, ofatumumab, veltuzumab (IMMU-106), ocrelizumab (PRO70769), and third-generation mAbs, AME-133v (ocaratuzumab), PRO131921 and GA101 (obinutumumab). PMID:23057966

  2. [Monoclonal antibodies for the treatment of multiple sclerosis].

    PubMed

    Sánchez-Seco, Victoria Galán; Casanova Peño, Ignacio; Arroyo González, Rafael

    2014-12-01

    Until the mid 1990s, with the appearance of interferon beta and glatiramer acetate, there was no treatment for multiple sclerosis (MS). However, due to their moderate therapeutic potential in some patients, a broad search was continued to find new and more effective treatment strategies, largely concentrated on monoclonal antibodies (MOAB). Natalizumab, the first MOAB for the treatment of MS, was approved at the end of 2004, representing a major advance in the field of neuroimmunology. Today, there is broad experience with natalizumab and other MOAB (alemtuzumab, daclizumab, rituximab, ocrelizumab, ofatumumab and anti-lingo-1) that are pending commercialization or are under phase II or III of development with promising results. The present review analyzes the efficacy and safety results of all these drugs. PMID:25732947

  3. Maximizing productivity of chromatography steps for purification of monoclonal antibodies.

    PubMed

    Tugcu, Nihal; Roush, David J; Gklen, Kent E

    2008-02-15

    The large scale production of monoclonal antibodies presents a challenge to design efficient and cost effective downstream purification processes. We explored a two stage resin screening approach to identify the best candidates to be utilized for the platform purification of monoclonal antibodies. The study focused on commercially available affinity resins including Protein A, mimetic and mixed-mode interaction resins as well as ion exchangers used in polishing steps. An initial screening using pure proteins was followed by a final screening where selected resins were utilized for the purification of MAbs in complex mixtures. Initial screenings aimed to measure the theoretical upper limit for dynamic binding capacity (DBC) at 1% breakthrough and productivity. We confirmed that DBC of affinity, mimetic and mixed-mode resins was a strong function of the linear velocity used for loading. Productivities >27 g/(L-h), were obtained for rProtein A FF, Mabselect and Prosep rA Ultra at 2 min residence time. For the cation exchangers, we identified UNOsphere S and Fractogel SO(3) as the best candidates for our purification based on DBC. For anion exchangers operated in flowthrough mode, Q Sepharose XL and UNOsphere Q were selected from the initial screening based on DBC and resolution of IgG from BSA. Finally, a three step purification scheme was implemented using the selected affinity and ion exchangers for the purification of IgG from complex feedstocks. We found that Mabselect followed by UNOsphere Q and UNOsphere S provided the best purification scheme for our applications based on productivity. PMID:17680666

  4. Alkaline cation-exchange chromatography for the reduction of aggregate and a mis-formed disulfide variant in a bispecific antibody purification process.

    PubMed

    Hall, Troii; Wilson, Joseph J; Brownlee, Tammy J; Swartling, James R; Langan, Sarah E; Lambooy, Peter K

    2015-01-15

    During the purification development of a bispecific antibody, cation-exchange chromatography was screened for its ability to separate a prominently expressed (>12%) mis-formed disulfide bond variant, termed MAb-diabody, and aggregate from the product of interest. The influence of pH, product load (g of product per liter of resin) and linear velocity on the separations were evaluated for the strong cation-exchange resins SP Sepharose HP and POROS() HS50. Cation-exchange chromatography is commonly operated distant to the isoelectric point of a molecule, generally leading to acidic conditions for antibody purification. However, the results herein demonstrated improved removal of MAb-diabody with increasing pH, resulting in reduction of MAb-diabody content greater than 12-fold when operating near the alkaline pI of the product. This approach was successful over a range of linear velocities and g/L of resin loading. Aggregate removal was less affected by pH and was effectively reduced from 10.9% to less than 3% for each condition. Furthermore, this method was successfully scaled to a 60 cm diameter column using SP Sepharose HP resin. PMID:25462105

  5. Anti-factor IXa/X bispecific antibody ACE910 prevents joint bleeds in a long-term primate model of acquired hemophilia A

    PubMed Central

    Yoshihashi, Kazutaka; Takeda, Minako; Kitazawa, Takehisa; Soeda, Tetsuhiro; Igawa, Tomoyuki; Sampei, Zenjiro; Kuramochi, Taichi; Sakamoto, Akihisa; Haraya, Kenta; Adachi, Kenji; Kawabe, Yoshiki; Nogami, Keiji; Shima, Midori; Hattori, Kunihiro

    2014-01-01

    ACE910 is a humanized anti-factor IXa/X bispecific antibody mimicking the function of factor VIII (FVIII). We previously demonstrated in nonhuman primates that a single IV dose of ACE910 exerted hemostatic activity against hemophilic bleeds artificially induced in muscles and subcutis, and that a subcutaneous (SC) dose of ACE910 showed a 3-week half-life and nearly 100% bioavailability, offering support for effective prophylaxis for hemophilia A by user-friendly SC dosing. However, there was no direct evidence that such SC dosing of ACE910 would prevent spontaneous bleeds occurring in daily life. In this study, we newly established a long-term primate model of acquired hemophilia A by multiple IV injections of an anti-primate FVIII neutralizing antibody engineered in mouse-monkey chimeric form to reduce its antigenicity. The monkeys in the control group exhibited various spontaneous bleeding symptoms as well as continuous prolongation of activated partial thromboplastin time; notably, all exhibited joint bleeds, which are a hallmark of hemophilia. Weekly SC doses of ACE910 (initial 3.97 mg/kg followed by 1 mg/kg) significantly prevented these bleeding symptoms; notably, no joint bleeding symptoms were observed. ACE910 is expected to prevent spontaneous bleeds and joint damage in hemophilia A patients even with weekly SC dosing, although appropriate clinical investigation is required. PMID:25274508

  6. Anti-factor IXa/X bispecific antibody ACE910 prevents joint bleeds in a long-term primate model of acquired hemophilia A.

    PubMed

    Muto, Atsushi; Yoshihashi, Kazutaka; Takeda, Minako; Kitazawa, Takehisa; Soeda, Tetsuhiro; Igawa, Tomoyuki; Sampei, Zenjiro; Kuramochi, Taichi; Sakamoto, Akihisa; Haraya, Kenta; Adachi, Kenji; Kawabe, Yoshiki; Nogami, Keiji; Shima, Midori; Hattori, Kunihiro

    2014-11-13

    ACE910 is a humanized anti-factor IXa/X bispecific antibody mimicking the function of factor VIII (FVIII). We previously demonstrated in nonhuman primates that a single IV dose of ACE910 exerted hemostatic activity against hemophilic bleeds artificially induced in muscles and subcutis, and that a subcutaneous (SC) dose of ACE910 showed a 3-week half-life and nearly 100% bioavailability, offering support for effective prophylaxis for hemophilia A by user-friendly SC dosing. However, there was no direct evidence that such SC dosing of ACE910 would prevent spontaneous bleeds occurring in daily life. In this study, we newly established a long-term primate model of acquired hemophilia A by multiple IV injections of an anti-primate FVIII neutralizing antibody engineered in mouse-monkey chimeric form to reduce its antigenicity. The monkeys in the control group exhibited various spontaneous bleeding symptoms as well as continuous prolongation of activated partial thromboplastin time; notably, all exhibited joint bleeds, which are a hallmark of hemophilia. Weekly SC doses of ACE910 (initial 3.97 mg/kg followed by 1 mg/kg) significantly prevented these bleeding symptoms; notably, no joint bleeding symptoms were observed. ACE910 is expected to prevent spontaneous bleeds and joint damage in hemophilia A patients even with weekly SC dosing, although appropriate clinical investigation is required. PMID:25274508

  7. Murine lupus monoclonal antibodies define five epitopes on two different Sm polypeptides.

    PubMed Central

    Williams, D G; Stocks, M R; Smith, P R; Maini, R N

    1986-01-01

    Monoclonal anti-Sm (Smith) antibodies derived from the mouse strain MRL/lpr were isolated and characterized by binding to purified antigen, immunoprecipitating characteristic uridine-rich RNAs from Hela cell extracts, and by Western blot analysis using rabbit thymus extract. Five different Sm epitopes were demonstrated by epitope blockade and probing Western blots with the monoclonal antibodies. Human anti-Sm serum inhibited each monoclonal antibody from binding to antigen, indicating that both human and mouse antibodies bind to the same Sm epitopes. Human anti-Sm antibodies bound to 28,000 and 16,000 MW polypeptides, a small number also binding to a 14,000 MW polypeptide. The monoclonal antibodies also bound to the 28,000 and/or the 16,000 polypeptide, and provided evidence to suggest that these two Sm polypeptides bear some structural similarities, but are distinct molecules. Images Figure 2 Figure 3 PMID:2426187

  8. Comparison of the islet cell antibody pattern of monoclonal glutamic acid decarboxylase antibodies recognizing linear and conformational epitopes.

    PubMed

    Augstein, P; Schlosser, M; Ziegler, B; Hahmann, J; Mauch, L; Ziegler, M

    1996-04-01

    In order to compare the reactivity of glutamic acid decarboxylase (GAD) antibodies recognizing linear and conformational epitopes as islet cell cytoplasmic antibodies (ICA), monoclonal antibodies were generated. An ELISA displacement test using two biotinylated monoclonals recognizing a linear (M61/7E11) or a conformational GAD65 epitope (M65/6B12) was performed to identify epitope regions recognized by monoclonal GAD antibodies. The GAD binding by monoclonal GAD antibodies was tested by immunofluorescence on fixed and unfixed pancreatic sections of human, rat, and mouse, and by Dot-blot experiments. 16/23 (69.6%) of the monoclonals were specifically reactive with GAD65 and 7/23 (30.4%) were reactive with both GAD isoforms. 8/16 (50%) of monoclonal GAD65 antibodies recognized a linear GAD epitope located at the N-terminus (pattern 1). 5/16 (31.3%) displaced M65/6B12, indicating the recognition of a conformational GAD epitope (pattern 2). Monoclonals belonging to patterns 1 and 2 showed strong ICA binding. 3/16 (18.8%) of monoclonals specific for GAD65 with weak or no immunostaining of pancreatic islets (pattern 3) did not inhibit the binding of both biotinylated antibodies in the displacement test, indicating other epitope specificities. In conclusion, GAD antibodies recognizing both conformational and linear epitopes of the GAD65 molecule are involved in ICA binding with strong reactivity. Furthermore, results obtained with monoclonals of pattern 3 suggest the occurrence of GAD65 epitopes partly inaccessible on cryosections, which may result in an ICA-negative test of GAD65 autoantibody positive sera. PMID:8739307

  9. Generation of monoclonal antibody targeting fibroblast growth factor receptor 3.

    PubMed

    Gorbenko, Olena; Ovcharenko, Galyna; Klymenko, Tetyana; Zhyvoloup, Olexandr; Gaman, Nadia; Volkova, Darija; Gout, Ivan; Filonenko, Valeriy

    2009-08-01

    Fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR family of receptor tyrosine kinases, whose function has been implicated in diverse biological processes, including cell proliferation, differentiation, survival, and tumorigenesis. Deregulation of FGFR3 signaling has been implicated with human pathologies, including cancer. Activating mutations in FGFR3 gene are frequently detected in bladder cancer, multiple myeloma, and noninvasive papillary urothelial cell carcinomas, while the overexpression of the receptor is observed in thyroid lymphoma and bladder cancer. The main aim of this study was to generate hybridoma clones producing antibody that could specifically recognize FGFR3/S249C mutant, but not the wild-type FGFR. To achieve this, we used for immunization bacterially expressed fragment of FGFR3 corresponding to loops II-III of the extracellular domain (GST-His/FGFR3/S249C-LII-III), which possesses oncogenic mutation at Ser249 detected in at least 50% of bladder cancers. Primary ELISA screening allowed us to isolate several hybridoma clones that showed specificity towards FGFR3/S249C, but not FGFR3wt protein. Unfortunately, these clones were not stable during single-cell cloning and expansion and lost the ability to recognize specifically FGFR3/S249C. However, this study allowed us to generate several monoclonal antibodies specific towards both FGFR3wt and FGFR3/S249C recombinant proteins. Produced hybridomas secreted MAbs that were specific in Western blotting towards bacterially expressed FGFR3wt and FGFR3/S249C, as well as the full-length receptors ectopically expressed in Sf21 and HEK293 cells. Moreover, transiently expressed wild-type and oncogenic forms of FGFR were efficiently immunoprecipitated with selected antibodies from the lysates of infected Sf21 and transiently transfected HEK293. In summary, generated antibodies should be useful as tools for examining the expression pattern and biological functions of FGFR3 in normal and pathological cells and tissues. PMID:19663703

  10. Monoclonal gammopathy (Waldenstrm's macroglobulinaemia) producing specific red cell antibody.

    PubMed Central

    Oluboyede, O A; Bademosi, O; David-West, A; Thomas, C O; Francis, T I; Luzzatto, L

    1976-01-01

    Two cases of Waldenstrm's macroglobulinaemia have been seen at University College Hospital, Ibadan in the last four years, Case 1 was a 30-year-old soldier who presented with splenomegaly and anaemia, was treated with chlorambucil, and had a complete remission sustained for over two years. Case 2 was a 58-year-old retired civil servant who presented with very severe anaemia and also splenomegaly, and died within three weeks of admission. Both patients had most of the typical features of Waldenstrm's disease, including retinal changes and serum IgM levels of 4200 and 5500 mg/dl respectively. In both cases an atypical cold antibody was detected in the course of blood cross-matching procedures. In case 1, the antibody agglutinated all adult and cord red cells tested, including the patient's own cells, to a titre of 8000 and above at 4 degrees C. Suprisingly enough, when the patient went into remission and the serum IgM level had fallen to 400 mg/dl, this antibody was no longer detectable and has not reappeared two years later. In case 2, the antibody agglutinated all adult red cells tested to a titre of 2000 at 20 degrees C but not the patient's own red cells. Since cord cells were agglutinated only to a titre of 4 to 20 degrees C it was concluded that the patient had an alloantibody with I-specificity. Therefore in both these patients the monoclonal immunoglobulin produced by the neoplastic lymphoid cell clone had specific activity against red cell antigens. Images PMID:818128

  11. Computational design of a CNT carrier for a high affinity bispecific anti-HER2 antibody based on trastuzumab and pertuzumab Fabs.

    PubMed

    Salazar-Salinas, Karim; Kubli-Garfias, Carlos; Seminario, Jorge M

    2013-07-01

    This is a preliminary cross multidisciplinary theoretical-computational approach for the design of a drug delivery system based on immunoconjugated carbon nanotube against HER2- overexpressing cancer cells. This drug delivery system allows the release of an encapsulated cytotoxic cocktail in a controlled manner under pulsed radio frequency (RF) irradiation. Our effort is focused on the computational aided design of a high affinity bispecific anti-HER2 antibody and an opening mechanism of the carbon nanotube (CNT) based cytotoxic carrier for controlling multiple drug release. We study the main interactions between the antibody and the antigen by a computational scanning mutagenesis approach of trastuzumab and pertuzumab fragment antigen binding (Fab) structures in order to enhance their binding affinity. Then, each Fab fragments is joined by a polypeptide linker which should be stable enough to avoid the "open form" of antibody. On the other hand, we also conjugate the engineered antibody to functionalized CNTs (f-CNTs), which encapsulate the inhibitors of the HER2/PI3K/Akt/mTOR signaling pathway. We take advantage of the fact that f-CNT converts the RF radiation absorption into heat release. A pulsed laser at 13.45 MHz increments the temperature around 40 C for triggering the nano-caps destabilization, which allows the switching of the opening mechanism of the drug carrier. Nano-caps will be a dual pH/temperature responsive in order to take advantage of lysosome characteristic (acidic pH) and heat release from the carrier. Nano-caps are functionalized with organic amide moieties, which hydrolyze quickly at an acidic pH into primary amines, and protonated amines generate repulsion interactions with other charged species, which trigger the cytotoxics release. PMID:23143677

  12. Monoclonal antibodies against rabbit mammary prolactin receptors. Specific antibodies to the hormone binding domain

    SciTech Connect

    Katoh, M.; Djiane, J.; Kelly, P.A.

    1985-09-25

    Three monoclonal antibodies (M110, A82, and A917) were obtained by fusing myeloma cells and spleen cells from mice immunized with partially purified rabbit mammary gland prolactin (PRL) receptors. All 3 antibodies were capable of complete inhibition of SVI-ovine prolactin (oPRL) binding to rabbit mammary PRL receptors in either particulate or soluble form. M110 showed slightly greater potency than oPRL in competing for SVI-oPRL binding. These antibodies also inhibited PRL binding to microsomal fractions from rabbit liver, kidney, adrenal, ovary, and pig mammary gland, although A82 showed poor inhibition in pig mammary gland. There was no cross-reaction of any of the 3 monoclonal antibodies (mAbs) for the other species tested: human (T-47D breast cancer cells) and rat (liver, ovary). In order to confirm that these antibodies are specific to the binding domain, antibodies were purified, iodinated, and binding characteristics were investigated. SVI-M110 and SVI-A82 binding was completely inhibited by lactogenic hormones, whereas nonlactogenic hormones did not cross-react. Competition of 125I-M110 by oPRL was comparable to that of SVI-oPRL by unlabeled oPRL, while SVI-A917 binding was only partially competed (30-60%) by lactogenic hormones. Tissue and species specificity of labeled antibody binding paralleled results of binding inhibition experiments using 125I-oPRL. In addition, A82 and A917 completely inhibited 125I-M110 binding. In contrast, 125I-A82 binding was stimulated by A917 and 125I-A917 binding was stimulated by A82.

  13. Defining process design space for monoclonal antibody cell culture.

    PubMed

    Abu-Absi, Susan Fugett; Yang, LiYing; Thompson, Patrick; Jiang, Canping; Kandula, Sunitha; Schilling, Bernhard; Shukla, Abhinav A

    2010-08-15

    The concept of design space has been taking root as a foundation of in-process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non-key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product-related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified. PMID:20589669

  14. A monoclonal antibody specifically reactive with Ewing's sarcoma.

    PubMed Central

    Hara, S.; Ishii, E.; Tanaka, S.; Yokoyama, J.; Katsumata, K.; Fujimoto, J.; Hata, J.

    1989-01-01

    We have developed a mouse monoclonal antibody 5C11 (IgG2a) against cell surface antigen of Ewing's sarcoma (ES). 5C11 specifically reacted with ESs but not with other small round cell tumours in childhood, i.e. neuroblastomas, primitive neuroectodermal tumours (PNETs), rhabdomyosarcomas and malignant lymphomas. 5C11 did not react with any other tumours in children except for hepatoblastomas. No reactivity has been identified in normal tissues with the exception of fetal hepatocytes. Immunoelectron microscopically, 5C11 reactive antigen was located on cell membrane of ES cells. Biochemically, 5C11 immunoprecipitated a cell surface protein having molecular weight of 81,000 Da. 5C11 is the first antibody which can clearly distinguish ES from neurogenic tumours, especially from PNETs which were recently reported to have common features to ESs regarding chromosal abnormality and proto-oncogene expression but show evident differentiation into neurogenic direction. The results strongly indicate the usefulness of 5C11 not only for diagnostic purpose when no specific marker is available but also for studying the histogenesis of ES. In addition, no reactivity in normal tissue implies its potential application as a therapeutic reagent when the management of ES patients is still a great problem in clinical field. Images Figure 1 Figure 2 Figure 4 PMID:2605097

  15. Tregalizumab - A Monoclonal Antibody to Target Regulatory T Cells.

    PubMed

    Knig, Martin; Rharbaoui, Faiza; Aigner, Silke; Dlken, Benjamin; Schttrumpf, Jrg

    2016-01-01

    Regulatory T cells (Tregs) represent a subpopulation of CD4(+) T cells, which are essential for the maintenance of immunological tolerance. The absence or dysfunction of Tregs can lead to autoimmunity and allergies. The restoration of functional Tregs and/or Treg cell numbers represents a novel and attractive approach for the treatment of autoimmune diseases, e.g., rheumatoid arthritis (RA). The CD4 cell surface receptor is a target for modulation of T cell function. Monoclonal antibodies (mAbs) against CD4 have previously been tested for the treatment of autoimmune diseases, including RA. Furthermore, in model systems, anti-CD4 antibodies are able to induce tolerance and mediate immunomodulatory effects through a variety of mechanisms. Despite the availability of innovative and effective therapies for RA, many patients still have persistently active disease or experience adverse events that can limit use. A growing body of evidence suggests that Treg modulation could offer a new therapeutic strategy in RA and other autoimmune disorders. Here, we describe tregalizumab (BT-061), which is a novel, non-depleting IgG1 mAb that binds to a unique epitope of CD4. Tregalizumab represents the first humanized anti-CD4 mAb that selectively induces Treg activation. PMID:26834751

  16. A monoclonal antibody against a novel Sialomucin CD300LG.

    PubMed

    Jiang, Xiaomei; Wang, Haibin; Li, Zhiguang; Wei, Dapeng; Yang, Yuhui; Zheng, Xulei; Bi, Jianhong; Zhang, Chongjie

    2013-04-01

    CD300LG is a novel O-glycosylated member of the CD300 antigen-like family. Besides a classical mucin-like domain, it contains a V-type Ig domain. CD300LG binds lymphocyte L-selectin via its Ig domain and supports lymphocyte rolling via its mucin-like domain. The unique structure and function of CD300LG suggest it may play an important role in inflammation. For preparation of a monoclonal antibody (MAb) against human CD300LG, prokaryotic and eukaryotic expressing human CD300LG proteins were used as immunogen and detection antigen, respectively. One stable strain of hybridomas (3C7C5A6) was successfully established using the hybridoma technique. The Western blot and immunohistochemistry analyses demonstrated that the MAb was directed against human CD300LG with high specificity. This antibody could possibly facilitate studies on the pathomechanism of inflammation and may have the potential to be a means of effective anti-inflammation. PMID:23607343

  17. Monoclonal antibodies: pharmacokinetics as a basis for new dosage regimens?

    PubMed

    Azanza, J-R; Sdaba, B; Gmez-Guiu, A

    2015-10-01

    Complete monoclonal IgG antibodies which are in use in clinical practice share some pharmacological properties resulting in high concentrations in plasma. This fact is reflected in their low volumes of distribution, which can also be correlated with a high molecular weight and water solubility. This feature allows a novel approach to be applied to the dosing schedule for this group of drugs with fixed doses being used instead of the initially developed weight- or body surface-adjusted dosing schedules. In addition, the development of a new formulation containing hyaluronidase allows a subcutaneous route of administration to be used, because hyaluronidase creates a space in the subcutaneous tissue that helps antibody absorption. This method requires higher doses, but has allowed testing the feasibility of administering a fixed dose, with no individual dose adjustments based on weight or body surface. Moreover, loading doses are not needed, because the first dose results, within 3 weeks, in minimum concentrations that are higher than effective concentrations. PMID:24903270

  18. Tumor size: effect on monoclonal antibody uptake in tumor models

    SciTech Connect

    Hagan, P.L.; Halpern, S.E.; Dillman, R.O.; Shawler, D.L.; Johnson, D.E.; Chen, A.; Krishnan, L.; Frincke, J.; Bartholomew, R.M.; David, G.S.

    1986-03-01

    Studies were performed to determine the effect of tumor size on the incorporation of radiolabeled monoclonal antitumor antibodies (MoAbs) into human tumors growing in nude mice. The colon tumors ranged in size from 0.03-1.6 g, the melanoma from 0.1 to 6.7 g, and the lymphoma from 0.06 to 10.2 g. Indium-111 was primarily used as the radiolabel, however, both 125I and 111In were used as tracers for the MoAb in one experiment. The per g radiopharmaceutical uptake by tumors was inversely proportional to tumor size when tumor specific MoAb was administered. This finding was independent of the radiolabel and was demonstrable when the mice bore two tumors of differing size. When the MoAb was not specific for the tumor, the data were less well defined and a statistically significant correlation with size did not occur. These data are strong evidence for a decrease in per g uptake of labeled tumor specific antibodies as tumors increase in size.

  19. Profiling formulated monoclonal antibodies by (1)H NMR spectroscopy.

    PubMed

    Poppe, Leszek; Jordan, John B; Lawson, Ken; Jerums, Matthew; Apostol, Izydor; Schnier, Paul D

    2013-10-15

    Nuclear magnetic resonance (NMR) is arguably the most direct methodology for characterizing the higher-order structure of proteins in solution. Structural characterization of proteins by NMR typically utilizes heteronuclear experiments. However, for formulated monoclonal antibody (mAb) therapeutics, the use of these approaches is not currently tenable due to the requirements of isotope labeling, the large size of the proteins, and the restraints imposed by various formulations. Here, we present a new strategy to characterize formulated mAbs using (1)H NMR. This method, based on the pulsed field gradient stimulated echo (PGSTE) experiment, facilitates the use of (1)H NMR to generate highly resolved spectra of intact mAbs in their formulation buffers. This method of data acquisition, along with postacquisition signal processing, allows the generation of structural and hydrodynamic profiles of antibodies. We demonstrate how variation of the PGSTE pulse sequence parameters allows proton relaxation rates and relative diffusion coefficients to be obtained in a simple fashion. This new methodology can be used as a robust way to compare and characterize mAb therapeutics. PMID:24006877

  20. A Monoclonal Antibody Against the Oncogenic Mucin 1 Cytoplasmic Domain

    PubMed Central

    Panchamoorthy, Govind; Rehan, Hala; Kharbanda, Akriti; Ahmad, Rehan

    2011-01-01

    Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed in diverse human carcinomas and certain hematologic malignancies. The transmembrane MUC1-C subunit confers tumorigenicity and is a target for anti-cancer drug development. In this regard, the MUC1-C cytoplasmic domain interacts with multiple effectors that have been linked to transformation. Here we report on the generation of a mouse monoclonal antibody (MAb) against the human MUC1-C cytoplasmic domain (MUC1-CD). This IgG1 MAb, designated anti-MUC1-CD, reacts with the NYGQLDIFP epitope. We show that anti-MUC1-CD is useful in immunoblotting and immunoprecipitation experiments. In addition, anti-MUC1-CD can be used to detect expression of the MUC1-C subunit in formalin-fixed, paraffin-embedded tissues. The MUC1-C inhibitor has entered Phase I evaluation for patients with refractory solid tumors. The present results indicate that the anti-MUC1-CD antibody could be useful as a biomarker to identify patients with tumors that may be responsive to MUC1-C inhibitors. PMID:22149278

  1. A monoclonal antibody specific for cells of the melanocyte lineage.

    PubMed

    Vennegoor, C; Hageman, P; Van Nouhuijs, H; Ruiter, D J; Calafat, J; Ringens, P J; Rümke, P

    1988-01-01

    A monoclonal antibody, NKI/beteb, was prepared against membranes from a human melanoma metastasis, and in immunoprecipitates of melanoma cell lysates specific 100- and 7-kd glycoproteins were found. The large glycoproteins were also present in conditioned medium of melanoma cell lines. The antigen is located on the inner side of membranes of (pre)melanosomes and premelanosomelike vesicles. The antibody reacted in the immunoperoxidase test on frozen tissue sections with 27 of 28 nevocellular nevi (15/16 common, 12/12 dysplastic), 39/39 primary melanomas (3 intraepidermal, 24 cutaneous, 12 choroidal), 56/63 melanoma metastases, and 4/4 clear-cell sarcomas (melanoma of soft tissue). With sections of formalin-fixed paraffin-embedded tissues, the reaction was less sensitive. No reactivity was detected with frozen sections of 185 other tumors, except for 1 case of non-Hodgkin's lymphoma in which macrophages were positive. With the exception of melanocytes, all frozen sections of adult tissues that were tested were negative with NKI/beteb. On the basis of its tissue distribution so far, the antigen recognized by NKI/beteb seems to be a specific and sensitive diagnostic marker for cells of the melanocyte lineage. PMID:3276209

  2. A monoclonal antibody specific for cells of the melanocyte lineage.

    PubMed Central

    Vennegoor, C.; Hageman, P.; Van Nouhuijs, H.; Ruiter, D. J.; Calafat, J.; Ringens, P. J.; Rümke, P.

    1988-01-01

    A monoclonal antibody, NKI/beteb, was prepared against membranes from a human melanoma metastasis, and in immunoprecipitates of melanoma cell lysates specific 100- and 7-kd glycoproteins were found. The large glycoproteins were also present in conditioned medium of melanoma cell lines. The antigen is located on the inner side of membranes of (pre)melanosomes and premelanosomelike vesicles. The antibody reacted in the immunoperoxidase test on frozen tissue sections with 27 of 28 nevocellular nevi (15/16 common, 12/12 dysplastic), 39/39 primary melanomas (3 intraepidermal, 24 cutaneous, 12 choroidal), 56/63 melanoma metastases, and 4/4 clear-cell sarcomas (melanoma of soft tissue). With sections of formalin-fixed paraffin-embedded tissues, the reaction was less sensitive. No reactivity was detected with frozen sections of 185 other tumors, except for 1 case of non-Hodgkin's lymphoma in which macrophages were positive. With the exception of melanocytes, all frozen sections of adult tissues that were tested were negative with NKI/beteb. On the basis of its tissue distribution so far, the antigen recognized by NKI/beteb seems to be a specific and sensitive diagnostic marker for cells of the melanocyte lineage. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:3276209

  3. Tregalizumab – A Monoclonal Antibody to Target Regulatory T Cells

    PubMed Central

    König, Martin; Rharbaoui, Faiza; Aigner, Silke; Dälken, Benjamin; Schüttrumpf, Jörg

    2016-01-01

    Regulatory T cells (Tregs) represent a subpopulation of CD4+ T cells, which are essential for the maintenance of immunological tolerance. The absence or dysfunction of Tregs can lead to autoimmunity and allergies. The restoration of functional Tregs and/or Treg cell numbers represents a novel and attractive approach for the treatment of autoimmune diseases, e.g., rheumatoid arthritis (RA). The CD4 cell surface receptor is a target for modulation of T cell function. Monoclonal antibodies (mAbs) against CD4 have previously been tested for the treatment of autoimmune diseases, including RA. Furthermore, in model systems, anti-CD4 antibodies are able to induce tolerance and mediate immunomodulatory effects through a variety of mechanisms. Despite the availability of innovative and effective therapies for RA, many patients still have persistently active disease or experience adverse events that can limit use. A growing body of evidence suggests that Treg modulation could offer a new therapeutic strategy in RA and other autoimmune disorders. Here, we describe tregalizumab (BT-061), which is a novel, non-depleting IgG1 mAb that binds to a unique epitope of CD4. Tregalizumab represents the first humanized anti-CD4 mAb that selectively induces Treg activation. PMID:26834751

  4. Establishment of Novel Monoclonal Antibody PMab-32 Against Rabbit Podoplanin.

    PubMed

    Honma, Ryusuke; Fujii, Yuki; Ogasawara, Satoshi; Oki, Hiroharu; Liu, Xing; Nakamura, Takuro; Kaneko, Mika K; Takagi, Michiaki; Kato, Yukinari

    2016-02-01

    Podoplanin (PDPN) is a type I transmembrane O-glycoprotein, which is known as a specific lymphatic marker. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelet. PDPN is also expressed in several normal tissues, including podocytes and type I alveolar cells. Although many monoclonal antibodies (MAbs) against human PDPN (hPDPN), mouse PDPN (mPDPN), and rat PDPN (rPDPN) have been established, useful antibodies against rabbit PDPN (rabPDPN) have not been developed. In this study, we immunized mice with the recombinant proteins of rabPDPN, and developed a novel anti-rabPDPN MAb, named PMab-32. PMab-32 could detect endogenous and exogenous rabPDPN in flow cytometry and Western blot analysis. The KD of PMab-32 was determined to be 6.2??10(-8) M by flow cytometry. Immunohistochemical analysis showed that PMab-32 is useful for detecting podocytes, type I alveolar cells, and lymphatic endothelial cells in normal rabbit tissues. PMab-32 is expected to be useful for various rabbit experiments. PMID:26788987

  5. Production of monoclonal antibodies for detection of Vibrio harveyi.

    PubMed

    Phianphak, Wannipa; Rengpipat, Sirirat; Rukpratanporn, Sombat; Longyant, Siwaporn; Chaivisuthangkura, Parin; Sithigorngul, Weerawan; Sithigorngul, Paisarn

    2005-02-28

    Monoclonal antibodies (MAbs) against Vibrio harveyi were produced from mice immunized with heat-killed and SDS-mercaptoethanol-treated highly virulent V. harveyi 639. Fifteen MAbs were selected and sorted into 6 groups according to their specificity to various proteins of apparent molecular weight ranging from 8 to 49 kDa. Some antibodies were used for detection of V. harveyi at concentrations as low as 10(4) CFU ml(-1) using immunodot blots. Most of the selected MAbs did not show cross-reactivity to other Vibrio species and other gram-negative bacteria tested. Only 1 MAb (VH39-4E) showed slight cross-reactivity to Aeromonas hydrophila. Another MAb (VH24-8H) bound lightly to V. harveyi 1526 but strongly to V. harveyi 639, allowing rapid differentiation. Two of the MAb groups were used to localize V. harveyi in tissues of infected black tiger shrimp Penaeus monodon by immunohistochemistry. This study demonstrates the versatility of a highly specific immunological tool for the detection of V. harveyi in aquaculture and opens the way for further development of convenient test kits. PMID:15819431

  6. Protective effect of antilipopolysaccharide monoclonal antibody in experimental Klebsiella infection.

    PubMed Central

    Rukavina, T; Tcac, B; Susa, M; Jendrike, N; Jonjc, S; Lucin, P; Marre, R; Dorc, M; Trautmann, M

    1997-01-01

    An O-antigen-specific murine monoclonal antibody (MAb) directed against an immunodominant epitope expressed on Klebsiella O1, O6, and O8 lipopolysaccharides (LPS) was examined with respect to its binding to nonencapsulated and encapsulated bacterial cells and its ability to protect against lethal murine Klebsiella sepsis. While the MAb (clone Ru-O1, mouse immunoglobulin G2b) bound well to nonencapsulated organisms of the O1 serogroup, binding was significantly, but not completely, abolished by the presence of the K2 capsule. In a model of experimental Klebsiella peritonitis and sepsis induced by a virulent O1:K2 serogroup strain, higher doses of anti-LPS MAb Ru-O1 than of a previously described anticapsular MAb specific for the K2 capsular polysaccharide were needed to provide protection. However, high-dose (40 microg/g of body weight) pretreatment with anti-LPS MAb Ru-O1 significantly reduced bacterial dissemination to various organs as well as macroscopic and histologic pulmonary alterations. Thus, since the number of Klebsiella capsular antigens occurring in clinical material is too large to be completely "covered" by a K-antigen-specific hyperimmunoglobulin preparation, O-antigen-specific antibodies may supplement K-antigen-specific immunoprophylaxis and -therapy of clinical Klebsiella infection. PMID:9125558

  7. Isolation of Monoclonal Antibodies with Predetermined Conformational Epitope Specificity

    PubMed Central

    Humbert, Michael; Essono, Sosthne S.; Watkins, Jennifer D.; Vyas, Hemant K.; Shanmuganathan, Vivekanandan; Hemashettar, Girish; Kahn, Maria; Hu, Shiu-Lok; Montefiori, David C.; Polonis, Victoria R.; Schur, Peter H.; Ruprecht, Ruth M.

    2012-01-01

    Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs) from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes) that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs) that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs). To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents. PMID:22737224

  8. Monoclonal antibody-based therapies for microbial diseases

    PubMed Central

    Saylor, Carolyn; Dadachova, Ekaterina; Casadevall, Arturo

    2009-01-01

    The monoclonal antibody (mAb) revolution that currently provides many new options for the treatment of neoplastic and inflammatory diseases has largely bypassed the field of infectious diseases. Only one mAb is licensed for use against an infectious disease, although there are many in various stages of development. This situation is peculiar given that serum therapy was one of the first effective treatments for microbial diseases and that specific antibodies have numerous antimicrobial properties. The underdevelopment and underutilization of mAb therapies for microbial diseases has various complex explanations that include the current availability of antimicrobial drugs, small markets, high costs and microbial antigenic variation. However, there are signs that the climate for mAb therapeutics in infectious diseases is changing given increasing antibiotic drug resistance, the emergence of new pathogenic microbes for which no therapy is available, and development of mAb cocktail formulations. Currently, the major hurdle for the widespread introduction of mAb therapies for microbial diseases is economic, given the high costs of immunoglobulin preparations and relatively small markets. Despite these obstacles there are numerous opportunities for mAb development against microbial diseases and the development of radioimmunotherapy provides new options for enhancing the magic bullet. Hence, there is cautious optimism that the years ahead will see more mAbs in clinical use against microbial diseases. PMID:20006139

  9. Monoclonal Antibodies for the Diagnosis of Borrelia crocidurae.

    PubMed

    Fotso Fotso, Aurlien; Mediannikov, Oleg; Nappez, Claude; Azza, Sad; Raoult, Didier; Drancourt, Michel

    2016-01-01

    Relapsing fever borreliae, produced by ectoparasite-borne Borrelia species, cause mild to deadly bacteremia and miscarriage. In the perspective of developing inexpensive assays for the rapid detection of relapsing fever borreliae, we produced 12 monoclonal antibodies (MAbs) against Borrelia crocidurae and characterized the two exhibiting the highest titers. P3A10 MAb reacts with the 35.6-kDa flagellin B (flaB) of B. crocidurae while P6D9 MAb recognizes a 35.1-kDa variable-like protein (Vlp) in B. crocidurae and a 35.2-kDa Vlp in Borrelia duttonii. Indirect immunofluorescence assay incorporating relapsing fever and Lyme group borreliae and 11 blood-borne organisms responsible for fever in West Africa confirmed the reactivity of these two MAbs. Combining these two MAbs in indirect immunofluorescence assays detected relapsing fever borreliae including B. crocidurae in ticks and the blood of febrile Senegalese patients. Both antibodies could be incorporated into inexpensive and stable formats suited for the rapid point-of-care diagnosis of relapsing fever. These first-ever MAbs directed against African relapsing fever borreliae are available for the scientific community to promote research in this neglected field. PMID:26598566

  10. Serotyping of Chlamydia psittaci isolates using serovar-specific monoclonal antibodies with the microimmunofluorescence test.

    PubMed Central

    Andersen, A A

    1991-01-01

    A panel of 10 serovar-specific monoclonal antibodies that could distinguish 10 distinct serovars of Chlamydia psittaci was prepared. The panel included one monoclonal antibody to each of the 10 serovars. Monoclonal antibodies were selected for their specificity in the indirect microimmunofluorescence test. Each of the monoclonal antibodies had a titer of 1:1,280 or higher to the homologous strain, with only two showing any cross-reactivity at a dilution of 1:10. Chlamydial antigen derived from organisms growing in tissue culture of one well of a 96-well multiwell dish was usually sufficient for the serotyping of an isolate. Infected yolk sac preparations were also suitable for serotyping. The panel of monoclonal antibodies was used to serotype 55 mammalian and avian strains. All except five of the strains were successfully serotyped; these five strains are presumed to represent at least two additional serovars. The use of a panel of monoclonal antibodies in the indirect microimmunofluorescence test provides a rapid and reliable method for serotyping new isolates. Monoclonal antibodies to new serovars can easily be added to the panel. PMID:1890172

  11. Monoclonal antibodies that inhibit mitogenic activity of Mycoplasma pulmonis.

    PubMed Central

    Lapidot, Z; Siman-Tov, R; Naot, Y

    1995-01-01

    Previous studies have suggested a correlation between mitogenic, polyclonal activation of host lymphocytes and the respiratory tract inflammatory diseases induced by Mycoplasma pulmonis. This study describes the generation of monoclonal antibodies (MAbs) to M. pulmonis membrane antigens with different capacities to inhibit stimulation of cultured rat lymphocytes by mycoplasmal membranes and with variable effects on M. pulmonis growth. We show that the inhibitory effects exerted on mitogenesis by purified MAbs are inversely related to the effects of MAbs on M. pulmonis growth. Immunoblotting of electrophoretically separated membrane proteins, with both growth- and mitogenesis-inhibiting antibodies, revealed significant changes in the reactions obtained with both types of MAb following short exposure of membranes to heat. Growth-inhibiting MAbs strongly react with heat-labile antigenic complexes with molecular weights of 65,000 to 75,000. Inhibition of mitogenesis is mainly associated with recognition of membrane complexes of 84 to 113 kDa that exhibit disperse smears and variable heat sensitivities. Following brief heating of membranes, more distinct bands of 103, 90, and 84 kDa are obtained with MAbs that inhibit mitogenesis. Experiments with other mitogenic mycoplasma species and MAb 3.3.10.2, a potent inhibitor of mitogenesis reveal that whereas the antigenic epitope recognized by this antibody is present on unheated membranes from different mycoplasmas, with heated membranes the MAb yields reactions only with M. pulmonis and M. arthritidis. Our studies suggest that M. pulmonis mitogens are unique membrane complexes of variable molecular weights, highly susceptible to heat and less sensitive to reducing agents. PMID:7806349

  12. A monoclonal antibody against Echinococcus multilocularis Em2 antigen.

    PubMed

    Deplazes, P; Gottstein, B

    1991-08-01

    A monoclonal antibody (MAb G11) species-specific to the Em2 antigen of Echinococcus multilocularis was generated for (i) further biological characterization of the Em2 antigen, (ii) easy affinity-purification of Em2 antigen for immunodiagnostic and immunological investigations and (iii) development of a sandwich-ELISA for the detection of Em2 antigen in diagnostic samples and thus species-specific identification of E. multilocularis metacestode material. The MAb G11 was used in an antibody sandwich-ELISA to detect soluble Em2 antigen with a methodical sensitivity of 80 ng E. multilocularis antigen/ml of solution. MAb G11 specifically detected Em2 antigen in all of 15 E. multilocularis-isolates originating from various geographical areas and in none of other helminth isolates (e.g. Echinococcus granulosus, E. vogeli, and others). Further biological analysis by FITC-labelled MAb G11 demonstrated unique binding activity to the laminated layer of the metacestode. Also, oncospheres were binding FITC-labelled MAb G11 on an outer layer synthesized during cultivation in vitro for 13 days after hatching. Application of the MAb G11 antibody sandwich-ELISA for investigation of solubilized oncospheres confirmed the in vitro synthesis of Em2 antigen by oncospheres on day 13 p.i. Adult stages (somatic antigens) and freshly hatched oncospheres were always MAb G11 negative. Solid-phase MAb G11 was used for purification of the corresponding Em2 antigen by affinity chromatography. A preliminary serological evaluation of the Em2(G11) antigen by ELISA revealed identical immunodiagnostic characteristics, compared to Em2 obtained by classical means, thus suggesting the presented method for future isolation of large-scale Em2 antigen. PMID:1945524

  13. Tumor-associated hyaluronan limits efficacy of monoclonal antibody therapy.

    PubMed

    Singha, Netai C; Nekoroski, Tara; Zhao, Chunmei; Symons, Rebecca; Jiang, Ping; Frost, Gregory I; Huang, Zhongdong; Shepard, H Michael

    2015-02-01

    Despite tremendous progress in cancer immunotherapy for solid tumors, clinical success of monoclonal antibody (mAb) therapy is often limited by poorly understood mechanisms associated with the tumor microenvironment (TME). Accumulation of hyaluronan (HA), a major component of the TME, occurs in many solid tumor types, and is associated with poor prognosis and treatment resistance in multiple malignancies. In this study, we describe that a physical barrier associated with high levels of HA (HA(high)) in the TME restricts antibody and immune cell access to tumors, suggesting a novel mechanism of in vivo resistance to mAb therapy. We determined that approximately 60% of HER2(3+) primary breast tumors and approximately 40% of EGFR(+) head and neck squamous cell carcinomas are HA(high), and hypothesized that HA(high) tumors may be refractory to mAb therapy. We found that the pericellular matrix produced by HA(high) tumor cells inhibited both natural killer (NK) immune cell access to tumor cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. Depletion of HA by PEGPH20, a pegylated recombinant human PH20 hyaluronidase, resulted in increased NK cell access to HA(high) tumor cells, and greatly enhanced trastuzumab- or cetuximab-dependent ADCC in vitro. Furthermore, PEGPH20 treatment enhanced trastuzumab and NK cell access to HA(high) tumors, resulting in enhanced trastuzumab- and NK cell-mediated tumor growth inhibition in vivo. These results suggest that HA(high) matrix in vivo may form a barrier inhibiting access of both mAb and NK cells, and that PEGPH20 treatment in combination with anticancer mAbs may be an effective adjunctive therapy for HA(high) tumors. PMID:25512619

  14. Structure of solid tumors and their vasculature: Implications for therapy with monoclonal antibodies

    SciTech Connect

    Dvorak, H.F.; Nagy, J.A.; Dvorak, A.M. )

    1991-03-01

    Delivery of monoclonal antibodies to solid tumors is a vexing problem that must be solved if these antibodies are to realize their promise in therapy. Such success as has been achieved with monoclonal antibodies is attributable to the local hyperpermeability of the tumor vasculature, a property that favors antibody extravasation at tumor sites and that is mediated by a tumor-secreted vascular permeability factor. However, leaky tumor blood vessels are generally some distance removed from target tumor cells, separated by stroma and by other tumor cells that together represent significant barriers to penetration by extravasated monoclonal antibodies. For this reason, alternative approaches may be attractive. These include the use of antibody-linked cytotoxins, which are able to kill tumor cells without immediate contact, and direction of antibodies against nontumor cell targets, for example, antigens unique to the tumor vascular endothelium or to tumor stroma. 50 refs.

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

    SciTech Connect

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

    1986-01-01

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

  16. Protection of mice against Clostridium chauvoei infection by anti-idiotype antibody to a monoclonal antibody to flagella.

    PubMed

    Kijima-Tanaka, M; Nakamura, M; Nagamine, N; Takahashi, T; Aoki, A; Tamura, Y

    1994-03-01

    Polyclonal rabbit anti-idiotypic antibody (anti-Id) against the protective monoclonal antibody specific to the flagella of Clostridium chauvoei was produced, purified, and characterized. Anti-Id inhibited the binding of its related monoclonal antibody to the flagellar antigen, suggesting that the anti-Id bore an internal image of the flagellar antigen. When mice were immunized with anti-Id intraperitoneally, the survival rate increased significantly, compared with mice immunized with normal rabbit IgG (P < 0.01), and specific anti-flagellar antibodies were induced. PMID:8004054

  17. Development of monoclonal antibodies suitable for use in antigen quantification potency tests for clostridial veterinary vaccines.

    PubMed

    Hauer, P J; Clough, N E

    1999-01-01

    The quality control testing of clostridial veterinary vaccines currently requires large numbers of animals. Alternative in vitro test methods are being investigated by researchers in industry and by regulatory authorities in many countries. Monoclonal antibodies that neutralize Clostridium perfringens alpha toxin, C. perfringens beta toxin, C. perfringens epsilon toxin, and C. sordellii lethal toxin as well as a monoclonal antibody directed against C. chauvoei flagellar antigen have been developed by the Center for Veterinary Biologics-Laboratory for use in antigen quantification assays. A proposal to create an international standard collection of clostridial-specific monoclonal antibodies is made. PMID:10566780

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

    PubMed Central

    Arcuri, Santo; Galletti, Silvia; Faldella, Giacomo

    2013-01-01

    Monoclonal antibodies are widely used both in infants and in adults for several indications. Humanized monoclonal antibodies (palivizumab) have been used for many years for the prevention of respiratory syncytial virus infection in pediatric populations (preterm infants, infants with chronic lung disease or congenital heart disease) at high risk of severe and potentially lethal course of the infection. This drug was reported to be safe, well tolerated and effective to decrease the hospitalization rate and mortality in these groups of infants by several clinical trials. In the present paper we report the development and the current use of monoclonal antibodies for prophylaxis against respiratory syncytial virus. PMID:23840240

  19. Reactivities of serotyping monoclonal antibodies with culture-adapted human rotaviruses.

    PubMed Central

    Ward, R L; McNeal, M M; Clemens, J D; Sack, D A; Rao, M; Huda, N; Green, K Y; Kapikian, A Z; Coulson, B S; Bishop, R F

    1991-01-01

    Rotaviruses collected in Bangladesh during 1985 to 1986 were culture adapted and used in a comparative serotyping study with three groups of monoclonal antibodies, all of which reacted with the major neutralization protein (VP7) of serotype 1, 2, 3, or 4. The goals were to determine which monoclonal antibodies most accurately predicted the serotype and why large variations in serotyping efficiencies have occurred with these monoclonal antibodies in previous studies. The 143 rotavirus isolates used in this study belonged to 69 different electropherotypes; and 44, 23, 21, and 55 isolates were identified as serotype 1 through 4, respectively, by neutralization with serotype-specific hyperimmune antisera. Serotyping specificity by enzyme-linked immunosorbent assay with monoclonal antibodies was 100% consistent with results found by neutralization with polyclonal antisera, but large differences were observed in the sensitivities of the different monoclonal antibodies. Monoclonal antibodies 5E8 (serotype 1), 1C10 (serotype 2), 159 (serotype 3), RV3:1 (serotype 3), ST-3:1 (serotype 4), and ST-2G7 (serotype 4) reacted with all the isolates of the corresponding serotype for which there were sufficient infectious particles. Monoclonal antibody 2F1 (serotype 2) was much less sensitive and reacted with only five serotype 2 isolates, but these were among those with the highest titers. Monoclonal antibodies RV4:2 (serotype 1), KU6BG (serotype 1), RV5:3 (serotype 2), and S2-2G10 (serotype 2), on the other hand, failed to react with between one and three isolates of the corresponding serotypes which had high titers, apparently because of epitope changes in these isolates. Effects of epitope variation were, however, most apparent with monoclonal antibodies 2C9 (serotype 1) and YO-1E2 (serotype 3), which reacted with one and no isolates of the corresponding serotypes, respectively. Cross-neutralization of escape mutants indicated that the serotype 1 monoclonal antibodies 5E8, 2C9, and RV4:2 reacted with different but probably overlapping epitopes, as did serotype 2 monoclonal antibodies 2F1, 1C10, and RV5:3, finding that were consistent with the enzyme-linked immunosorbent assay data. Because of epitope variations between rotavirus strains, serotyping with several monoclonal antibodies directed at different epitopes may increase the sensitivity of the method. PMID:1709945

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

    NASA Astrophysics Data System (ADS)

    Minkoff, Marjorie Sue

    A monoclonal antibody to an eosinophil specific determinant is very important in assessing eosinophil function during helminthic infection. Eosinophils induced by Schistosoma mansoni infection in BALB/c mice were used to induce C57B1/6 immunocytes for production of hybridomas secreting eosinophil monoclonal antibodies. These antibodies were shown to react with an eosinophil surface epitope but not with neutrophils or macrophages as determined by ELISA, immunodiffusion, immunofluorescence, and immunoblot assay. Affinity chromatography with eosinophil chemotactic factor-sepharose consistently selected out a { rm M_ R} 67,000 protein from solubilized eosinophil membrane antigens but not from neutrophil and macrophage antigens. In vitro studies showed that the eosinophil-specific monoclonal antibodies abrogated antibody-dependent eosinophil -mediated killing of S. mansoni schistosomula using mouse, rat or human eosinophils. Neutrophil and macrophage killing activities were unaffected. The monoclonal antibodies effected complement-dependent lysis of mouse and rat eosinophils but not of human eosinophils. ECF-treated eosinophils showed enhanced killing of schistosomula which was blocked by the monoclonal antibody. Murine and human eosinophils preincubated with monoclonal antibody exhibited decreased chemotaxis to ECF at optimal chemotactic concentrations. The monoclonal antibody also blocked eosinophil binding to ECF- sepharose beads. In vivo induction of peripheral blood eosinophilia by injection of S. mansoni eggs was suppressed by injections of monoclonal antibodies 2CD13 and 2QD45 in mouse and rat experimental models. Eosinophilia induced by keyhole limpet hemocyanin- cyclophosphamide treatment was also suppressed by monoclonal antibody in both murine and rat systems. Pulmonary granulomas in mice given egg injection and monoclonal antibody were smaller and contained fewer eosinophils than those granulomas from mice given eggs only. In immuno-biochemical studies, the monoclonal antibody 2QD45 specifically immunoprecipitated the {rm M_ R} 67,000 ECF-binding protein from ^{125}{rm I}-labeled mouse, rat, and human eosinophils as assessed by SDS-PAGE and autoradiography. Two-dimensional gel electrophoresis showed that this ECF-binding protein has a lower PI point than either mouse or bovine albumin.

  1. PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES DIRECTED AGAINST THE AH RECEPTOR

    EPA Science Inventory

    Six hybridomas secreting monoclonal antibodies that are specific for the N-terminal peptide sequence of the murine Ah receptor were isolated. hese antibodies bind with high specificity to the Al receptor on protein blots of Hepa 1c1c7 cytosol. hree IgG1 antibodies (Rpt1, 2, and 3...

  2. Phase I Study of Anti-CD3 x Anti-Her2 Bispecific Antibody in Metastatic Castrate Resistant Prostate Cancer Patients

    PubMed Central

    Thakur, Archana; Rathore, Ritesh; Kouttab, Nicola; Lum, Lawrence G.

    2015-01-01

    Background. New nontoxic targeted approaches are needed for patients with castrate resistant prostate cancer (CRPC). Our preclinical studies show that activated T cells (ATC) armed with anti-CD3 x anti-Her2 bispecific antibody (Her2Bi) kill prostate cancer cells lines, induce a Th1 cytokine pattern upon engagement of tumor cells, prevent the development of prostate tumors, and retard tumor growth in immunodeficient mice. These studies provided strong rationale for our phase I dose-escalation pilot study to test ATC armed with Her2Bi (aATC) for safety in men with CRPC. Methods. Seven of 8 men with CRPC were evaluable after receiving two infusions per week for 4 weeks. The men received 2.5, 5 or 10 × 109 aATC per infusion with low dose interleukin-2 and granulocyte-macrophage colony stimulating factor. Results. There were no dose limiting toxicities, and there was 1 partial responder and 3 of 7 patients had significant decreases in their PSA levels and pain scores. Immune evaluations of peripheral blood mononuclear cells in 2 patients before and after immunotherapy showed increases in IFN-γ EliSpot responses and Th1 serum cytokines. Conclusions. These results provide a strong rationale for developing phase II trials to determine whether aATC are effective for treating CRPC. PMID:25802762

  3. A phase 1 study of the bispecific anti-CD30/CD16A antibody construct AFM13 in patients with relapsed or refractory Hodgkin lymphoma

    PubMed Central

    Rothe, Achim; Sasse, Stephanie; Topp, Max S.; Eichenauer, Dennis A.; Hummel, Horst; Reiners, Katrin S.; Dietlein, Markus; Kuhnert, Georg; Kessler, Joerg; Buerkle, Carolin; Ravic, Miroslav; Knackmuss, Stefan; Marschner, Jens-Peter; Pogge von Strandmann, Elke; Borchmann, Peter

    2015-01-01

    AFM13 is a bispecific, tetravalent chimeric antibody construct (TandAb) designed for the treatment of CD30-expressing malignancies. AFM13 recruits natural killer (NK) cells via binding to CD16A as immune effector cells. In this phase 1 dose-escalation study, 28 patients with heavily pretreated relapsed or refractory Hodgkin lymphoma received AFM13 at doses of 0.01 to 7 mg/kg body weight. Primary objectives were safety and tolerability. Secondary objectives included pharmacokinetics, antitumor activity, and pharmacodynamics. Adverse events were generally mild to moderate. The maximum tolerated dose was not reached. Pharmacokinetics assessment revealed a half-life of up to 19 hours. Three of 26 evaluable patients achieved partial remission (11.5%) and 13 patients achieved stable disease (50%), with an overall disease control rate of 61.5%. AFM13 was also active in brentuximab vedotin–refractory patients. In 13 patients who received doses of ≥1.5 mg/kg AFM13, the overall response rate was 23% and the disease control rate was 77%. AFM13 treatment resulted in a significant NK-cell activation and a decrease of soluble CD30 in peripheral blood. In conclusion, AFM13 represents a well-tolerated, safe, and active targeted immunotherapy of Hodgkin lymphoma. A phase 2 study is currently planned to optimize the dosing schedule in order to further improve the therapeutic efficacy. This phase 1 study was registered at www.clinicaltrials.gov as #NCT01221571. PMID:25887777

  4. Phase I Study of Anti-CD3 x Anti-Her2 Bispecific Antibody in Metastatic Castrate Resistant Prostate Cancer Patients.

    PubMed

    Vaishampayan, Ulka; Thakur, Archana; Rathore, Ritesh; Kouttab, Nicola; Lum, Lawrence G

    2015-01-01

    Background. New nontoxic targeted approaches are needed for patients with castrate resistant prostate cancer (CRPC). Our preclinical studies show that activated T cells (ATC) armed with anti-CD3 x anti-Her2 bispecific antibody (Her2Bi) kill prostate cancer cells lines, induce a Th1 cytokine pattern upon engagement of tumor cells, prevent the development of prostate tumors, and retard tumor growth in immunodeficient mice. These studies provided strong rationale for our phase I dose-escalation pilot study to test ATC armed with Her2Bi (aATC) for safety in men with CRPC. Methods. Seven of 8 men with CRPC were evaluable after receiving two infusions per week for 4 weeks. The men received 2.5, 5 or 10 10(9) aATC per infusion with low dose interleukin-2 and granulocyte-macrophage colony stimulating factor. Results. There were no dose limiting toxicities, and there was 1 partial responder and 3 of 7 patients had significant decreases in their PSA levels and pain scores. Immune evaluations of peripheral blood mononuclear cells in 2 patients before and after immunotherapy showed increases in IFN-? EliSpot responses and Th1 serum cytokines. Conclusions. These results provide a strong rationale for developing phase II trials to determine whether aATC are effective for treating CRPC. PMID:25802762

  5. Production and characterization of monoclonal antibodies against dog immunoglobulin isotypes.

    PubMed

    Arce, C; Moreno, A; Milln, Y; Martn de las Mulas, J; Llanes, D

    2002-09-01

    A panel of six monoclonal antibodies (mAbs) recognizing antigenic determinants on canine immunoglobulin (Ig) heavy or light chains was produced and characterized. All monoclonals recognized the IgG(2) subclass, although only two were subclass-specific (CA3H1 and CA4F1). The CA3B8 mAb was found to be specific for an epitope on canine immunoglobulin G heavy chain, (IgG(1) and IgG(2) subclasses). Two mAbs (CA2E9 and CA5B2) reacted with an epitope on the heavy chain of canine IgG and IgM and another, CA4E7, bound to canine IgA, IgG and IgM isotypes; CA4E7 recognized an epitope on canine immunoglobulin light chain. CA4E7, CA4F1 and CA5B2 recognized an epitope in the Fab region. Three mAbs, CA3B8, CA4E7 and CA5B2, showed much lower reactivity with canine IgG by ELISA when IgG was periodate-treated, suggesting that they recognized a carbohydrate determinant. Cross-reactivity analysis of these mAbs with sera from horse, goat, cow, sheep, pig, cat, rabbit, hamster, rat, mouse and human indicated that two mAbs, CA3B8 and CA5B2, recognized a canine IgG-specific epitope; two others, CA3H1 and CA4E7, recognized an epitope also present in rabbit and sheep immunoglobulin respectively; and the remaining two (CA2E9 and CA4F1) recognized an epitope broadly present on the Igs of the species analyzed. This panel of antibodies will be a useful tool for future canine immunodiagnosis tests. With the exception of CA2E9, all mAbs were able to recognize plasma cells on paraffin-embedded tissues, and will thus be useful for immunohistochemical assays. PMID:12088642

  6. Monoclonal antibodies against soman: Characterization of soman stereoisomers. (Reannouncement with new availability information)

    SciTech Connect

    Lenz, D.E.; Yourick, J.J.; Dawson, J.S.; Scott, J.

    1992-12-31

    Hybridomas were produced which expressed monoclonal anti-soman antibodies as determined by microtiter enzyme-linked-antibody immunoassay (EIA). Each of these antibodies was titrated using a competitive inhibition enzyme immunoassay (CIEIA) with a variety of test ligands. The ligands used included soman (a racemic mixture), sarin, tabun, and each of the four stereoisomers of soman(C+P+, C+P-, C-P+ and C-P-). In all cases the antibodies tested exhibited IC50 values of 10 - 4 - 5 X 10 - 6 M for soman. When sarin or tabun was used as a ligand, the antibodies exhibited no cross reactivity. All of the antibodies cross reacted with the four soman stereoisomers. A second group of hybridomas were produced which expressed monoclonal antibodies against CsPs-soman. These antibodies were used to make preliminary absolute chiral assignments to the four soman stereoisomers. Soman; Antibodies; Stereoisomers; Absolute configuration.

  7. How does mild hypothermia affect monoclonal antibody glycosylation?

    PubMed

    Sou, Si Nga; Sellick, Christopher; Lee, Ken; Mason, Alison; Kyriakopoulos, Sarantos; Polizzi, Karen M; Kontoravdi, Cleo

    2015-06-01

    The application of mild hypothermic conditions to cell culture is a routine industrial practice used to improve recombinant protein production. However, a thorough understanding of the regulation of dynamic cellular processes at lower temperatures is necessary to enhance bioprocess design and optimization. In this study, we investigated the impact of mild hypothermia on protein glycosylation. Chinese hamster ovary (CHO) cells expressing a monoclonal antibody (mAb) were cultured at 36.5C and with a temperature shift to 32C during late exponential/early stationary phase. Experimental results showed higher cell viability with decreased metabolic rates. The specific antibody productivity increased by 25% at 32C and was accompanied by a reduction in intracellular nucleotide sugar donor (NSD) concentrations and a decreased proportion of the more processed glycan structures on the mAb constant region. To better understand CHO cell metabolism at 32C, flux balance analysis (FBA) was carried out and constrained with exometabolite data from stationary phase of cultures with or without a temperature shift. Estimated fluxomes suggested reduced fluxes of carbon species towards nucleotide and NSD synthesis and more energy was used for product formation. Expression of the glycosyltransferases that are responsible for N-linked glycan branching and elongation were significantly lower at 32C. As a result of mild hypothermia, mAb glycosylation was shown to be affected by both NSD availability and glycosyltransferase expression. The combined experimental/FBA approach generated insight as to how product glycosylation can be impacted by changes in culture temperature. Better feeding strategies can be developed based on the understanding of the metabolic flux distribution. PMID:25545631

  8. Clinical Application of Anti-CCR4 Monoclonal Antibody.

    PubMed

    Ueda, Ryuzo

    2015-11-01

    Mogamulizumab (KW-0761) is a humanized anti-CCR4 monoclonal antibody with a defucosylated Fc region (Potelligent Technology), which markedly enhances antibody-dependent cellular cytotoxicity by increasing its binding affinity to the Fcx03B3; receptor expressed on effector cells. It is an effective agent for patients with CCR4-positive adult T-cell leukemia and peripheral T-cell lymphoma, for which no standard therapy exists, and it has an acceptable toxicity profile. In addition, because CCR4 is expressed on CD45RA-FOXP3highCD4+ effector regulatory T (Treg) cells, it is an even more attractive target, because Treg cells involved in the tumor escape from host immunity in the tumor microenvironment. Based on this concept, we conducted a clinical study of mogamulizumab for the treatment of CCR4-negative advanced or recurrent solid cancer, with the aim of depleting effector Treg cells and thus boosting anti-cancer immune responses. In this study, mogamulizumab infusion at doses ranging from 0.1 to 1.0 mg/kg was safe and well tolerated. Four of 10 patients showed stable disease during treatment and showed long-term survival. Mogamulizumab efficiently depleted effector Treg cells even at the lowest dose of 0.1 mg/kg, and an augmentation or induction of specific immune responses to cancer/testis antigens was observed in some patients. In the near future, a novel immunotherapy targeting Treg cells with mogamulizumab will be offered to patients with different types of cancer. PMID:26550987

  9. Identification of a microsporidian polar tube protein reactive monoclonal antibody.

    PubMed

    Keohane, E M; Takvorian, P M; Cali, A; Tanowitz, H B; Wittner, M; Weiss, L M

    1996-01-01

    The microsporidia are characterized by spores containing a single polar tube that coils around the sporoplasm. When triggered by appropriate stimuli, the polar tube rapidly discharges out of the spore forming a hollow tube. The sporoplasm passes out of the spore through this tube serving as a unique vehicle of infection. Due to the unusual functional and solubility properties of the polar tube, the proteins comprising it are likely to be members of a protein family with a highly conserved amino acid composition among the various microsporidia. Polar tube proteins were separated from the majority of other proteins in glass bead disrupted spores of Glugea americanus using sequential 1% sodium dodecyl sulfate (SDS) and 9M urea extractions. The resultant spore pellet demonstrated broken, empty spore coats and numerous polar tubes in straight and twisted formations by negative stain transmission electron microscopy. After subsequent incubation of the pellet with 2% dithiothreitol (DTT), empty spore coats were still observed but the polar tubes were no longer present in the pellet. The DTT supernatant demonstrated four major protein bands by SDS-PAGE: 23, 27, 34 and 43 kDa. Monoclonal antibodies were produced to these proteins using Hunter's Titermax adjuvant. Mab 3C8.23.1 which cross-reacted with a 43-kDa antigen by immunoblot analysis, demonstrated strong reactivity with the polar tube of G. americanus spores by immunogold electron microscopy. This antibody will be useful in further characterization of polar tube proteins and may lead to novel diagnostic and therapeutic reagents. PMID:8563706

  10. Heterobifunctional reagents: A new approach to radiolabeling of monoclonal antibodies

    SciTech Connect

    Wang, T.S.T.; Ng, A.K.; Fawwaz, R.A.; Liu, Z.; Alderson, P.O.

    1985-05-01

    The use of bifunctional chelate such as the cyclic anhydride of DTPA for radiolabeling antibodies (Abs) may lead to homopolymerization, and intra- or intermolecular cross-linking, with resulting denaturation and decrease immunoreactivity of Abs. The authors, therefore, investigated the use of heterobifunctional reagents, whereby one group selectively couples to the amino group of the Ab and the other group to the radiometal for Ab labeling. One such reagent, 2,6-Dioxo-N-(carboxymethyl)morphine (DCM) was synthesized by reacting nitrilotriacetic acid with acetic anhydride. The other agent tested was commercially available N-Succinimidyl-3-(2-pyridyldithio) propionate (SPDP). These agents were evaluated independently for their ability to label a monoclonal antibody (MoAb) to a melanoma associated antigen (Ag). Labeling proceeded at a 2mg/ml concentration of the Ab, at HEPES pH 8.2, and 7.0, respectively, at room temperature for 30 min. The conjugate subsequently was labeled with Tc-99m or In-111. For comparison, the same labeled Abs also were prepared by using the cyclic anhydride of DTPA. Binding of the Ab to melanoma cells and control cells then was assayed. The results of cell binding experiments (N=3 per agent) in the region of Ag excess (X+-SD) were as follows: 62.6 +- 2.83% for Tc-99m-DCM-MoAb and 41.3+-1.84% for Tc-99m-SPDP-MoAb vs. 28.6 +- 1.16% for Tc-99m-DTPA-MoAb (p<0.01); 56.2 +- 2.97% for In-111-DCM-MoAb vs. 28.6 +- 1.16% for In-111-DTPA-M0Ab. Binding of all agents to the control lymphoid cell line was less than 3%. These results suggest that heterobifunctional reagents can reduce the loss of immunoreactivity of labeled MoAbs.

  11. DEMONSTRATION OF MULTIPLE ANTIGENIC DETERMINANTS ON 'MYCOPLASMA PNEUMONIAE' ATTACHMENT PROTEIN BY MONOCLONAL ANTIBODIES

    EPA Science Inventory

    Distinct multiple antigenic determinants of the attachment protein of Mycoplasma pneumoniae have been identified by limited proteolytic cleavage using specific monoclonal antibodies. Western blots prepared from the gels containing the cleaved fragments were probed with antiserum ...

  12. Emerging monoclonal antibodies as targeted innovative therapeutic approaches to asthma.

    PubMed

    Mitchell, P D; El-Gammal, A I; O'Byrne, P M

    2016-01-01

    Asthma is characterized by discordant responses among cells of the adaptive and innate immune systems. This interplay involves a complex pattern of cytokine-driven processes resulting in cell migration and recruitment, inflammation, and proliferative states. The significant majority of asthmatic patients respond well to conventional inhaled treatments. However, about 5% of asthmatics have severe refractory asthma and account for 50% of the health expenditure on asthma. Human(ized) monoclonal antibodies (hMabs) targeting inflammatory pathways are promising therapeutic agents in asthma management. The anti-IgE hMab omalizumab was the first biologic treatment approved for the treatment of allergic asthma. Potential future strategies and targets include interleukin (IL)-5, IL-4, and IL-13, anti-TSLP, IL-25, and IL-33. hMabs targeting IL-5 have shown great promise in severe refractory asthma with a persisting eosinophilia, and clinical trials with hMabs against IL-13 and IL4Rα have also shown clinical benefit. Studies of hMabs against other cytokines in severe asthma are under way. PMID:26502193

  13. Trial Watch: Tumor-targeting monoclonal antibodies in cancer therapy.

    PubMed

    Vacchelli, Erika; Aranda, Fernando; Eggermont, Alexander; Galon, Jrme; Sauts-Fridman, Catherine; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2014-01-01

    In 1997, for the first time in history, a monoclonal antibody (mAb), i.e., the chimeric anti-CD20 molecule rituximab, was approved by the US Food and Drug Administration for use in cancer patients. Since then, the panel of mAbs that are approved by international regulatory agencies for the treatment of hematopoietic and solid malignancies has not stopped to expand, nowadays encompassing a stunning amount of 15 distinct molecules. This therapeutic armamentarium includes mAbs that target tumor-associated antigens, as well as molecules that interfere with tumor-stroma interactions or exert direct immunostimulatory effects. These three classes of mAbs exert antineoplastic activity via distinct mechanisms, which may or may not involve immune effectors other than the mAbs themselves. In previous issues of OncoImmunology, we provided a brief scientific background to the use of mAbs, all types confounded, in cancer therapy, and discussed the results of recent clinical trials investigating the safety and efficacy of this approach. Here, we focus on mAbs that primarily target malignant cells or their interactions with stromal components, as opposed to mAbs that mediate antineoplastic effects by activating the immune system. In particular, we discuss relevant clinical findings that have been published during the last 13 months as well as clinical trials that have been launched in the same period to investigate the therapeutic profile of hitherto investigational tumor-targeting mAbs. PMID:24605265

  14. Characterization of a new monoclonal antibody against mercury (II)

    SciTech Connect

    Marx, A.; Hock, B.

    1998-07-01

    Monoclonal antibodies (mabs) were produced against mercury (II) and an enzyme immunoassay was developed for the detection of mercury (II) in water. Since mercury (II) ions are too small to elicit an immune response, they were coupled via glutathione (GSH) to the immunogenic carrier protein keyhole limpet hemocyanin (KLH). Several mice were immunized with this KLH-GSH-Hg immunoconjugate. Spleen cells of immunized mice were fused with myeloma cells. The resulting hybridoma cells were screened for the production of specific anti-Hg mabs. Five positive cells were detected. The hybridoma cell line K3C6 was adjusted to protein free medium; it produced mabs with high selectivity and sensitivity. A detection limit of 2.8 {micro}g/L HgCl{sub 2} (= 2.1 {micro}g/L Hg{sup 2+}) was achieved with a non-competitive enzyme immunoassay (EIA). Cross-reactivities with other metals were below 1%. Measurement of spiked water samples with this EIA showed good correlation with results obtained by mass spectrometry with inductive coupled plasma (ICP-MS).

  15. Production of a Chaetomium globosum enolase monoclonal antibody.

    PubMed

    Green, Brett J; Nayak, Ajay P; Lemons, Angela R; Rittenour, William R; Hettick, Justin M; Beezhold, Donald H

    2014-12-01

    Chaetomium globosum is a hydrophilic fungal species and a contaminant of water-damaged building materials in North America. Methods to detect Chaetomium species include subjective identification of ascospores, viable culture, or molecular-based detection methods. In this study, we describe the production and initial characterization of a monoclonal antibody (MAb) for C. globosum enolase. MAb 1C7, a murine IgG1 isotype MAb, was produced and reacted with recombinant C. globosum enolase (rCgEno) in an enzyme-linked immunosorbent assay and with a putative C. globosum enolase in a Western blot. Epitope mapping showed MAb 1C7 specific reactivity to an enolase decapeptide, LTYEELANLY, that is highly conserved within the fungal class Sordariomycetes. Cross-reactivity studies showed MAb 1C7 reactivity to C. atrobrunneum but not C. indicum. MAb 1C7 did not react with enolase from Aspergillus fumigatus, which is divergent in only two amino acids within this epitope. The results of this study suggest potential utility of MAb 1C7 in Western blot applications for the detection of Chaetomium and other Sordariomycetes species. PMID:25495488

  16. Monoclonal antibody production and immunochemical detection of polyether antibiotics.

    PubMed

    Lee, Hyo Jeong; Cho, Seung Sik; Simkhada, Jaya Ram; Yoo, Jin Cheol

    2009-03-01

    Polyether antibiotics such as monensin and salinomycin have been primarily used as coccidiostat and growth promoter. Since residues of these antibiotic in food may pose a health risk for sensitive individuals, their use should be carefully monitored. An immunochemical method was developed for the determination of polyether antibiotic using monoclonal antibody (Mab) produced by immunized mice. Conjugates of monensin, salinomycin and laidlomycin were prepared with bovine serum albumin (BSA), keyhole limpet haemocyanine (KLH) and ovalbumin (OVA) by mixed anhydride method and then used as immunogene to produce Mab. Eight hybridoma cell lines were isolated that produced Mabs that competed with polyether antibiotic-protein conjugates in BALB/c-SP2/0 fusion system. Two hybridoma with higher sensitivity, designated as 4G11F and 1C8F1F, were cultured for mass production and then purified from ascites fluid. Antibiotic-protein conjugates were quantitavely analyzed by using the purified Mabs through a competitive enzyme-linked immunosorbent assay (ELISA). PMID:19387589

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

    PubMed

    Momp, Susana Magadn; Gonzlez-Fernndez, Africa

    2014-01-01

    Due to the difficulties found when generating fully human monoclonal antibodies (mAbs) by the traditional method, several efforts have attempted to overcome these problems, with varying levels of success. One approach has been the development of transgenic mice carrying immunoglobulin (Ig) genes in germ line configuration. The engineered mouse genome can undergo productive rearrangement in the B cell population, with the generation of mouse B lymphocytes expressing human Ig (hIg) chains. To avoid the expression of mouse heavy or light chains, the endogenous mouse Ig (mIg) loci must be silenced by gene-targeting techniques. Subsequently, to obtain antigen-specific mAbs, conventional immunization protocols can be followed and the mAb technique used (fusion of activated B cells with mouse myeloma cells, screening, cloning, freezing, and testing) with these animals expressing human Ig genes. This chapter describes the type of transgenic knockout mice generated for various research groups, provides examples of human mAbs developed by research groups and companies, and includes protocols of immunization, generation, production, and purification of human mAbs from such mice. In addition, it also addresses the problems detected, and includes some of the methods that can be used to analyze functional activities with human mAbs. PMID:24037845

  18. Development of Sensitive Monoclonal Antibody PMab-2 Against Rat Podoplanin.

    PubMed

    Oki, Hiroharu; Honma, Ryusuke; Ogasawara, Satoshi; Fujii, Yuki; Liu, Xing; Takagi, Michiaki; Kaneko, Mika K; Kato, Yukinari

    2015-12-01

    Podoplanin (PDPN) is a platelet aggregation-inducing factor, which is known as an endogenous ligand of C-type lectin-like receptor-2 (CLEC-2). PDPN is also expressed in several normal tissues, such as lung type I alveolar cells and kidney podocytes. Although many monoclonal antibodies (MAbs) against human PDPN (hPDPN) or mouse PDPN (mPDPN) have been established, anti-rat PDPN (rPDPN) MAbs, especially against platelet aggregation-stimulating (PLAG) domain (29-54 amino acids) of rPDPN, have not been developed. Therefore, functional analysis of rPDPN in normal tissues has been limited. Here, we immunized mice with rPDPN peptides (38-51 amino acids) and developed a novel mouse anti-rPDPN MAb, PMab-2 (IgG1, kappa), which possesses high affinity compared with anti-hPDPN or mPDPN MAbs. The KD of PMab-2 was determined to be 5.9??10(-10) M. PMab-2 is useful, not only in flow cytometry and Western blot analysis against endogenous rPDPN, which is expressed in rat dermal fibroblast, but also in immunohistochemistry against normal tissues. PMab-2 showed extraordinarily high sensitivity in immunohistochemistry, indicating that PMab-2 is very advantageous for functional analysis of rPDPN. PMID:26683179

  19. Reversible cluster formation in concentrated monoclonal antibody solutions

    NASA Astrophysics Data System (ADS)

    Godfrin, P. Douglas; Porcar, Lionel; Falus, Peter; Zarraga, Isidro; Wagner, Norm; Liu, Yun

    2015-03-01

    Protein cluster formation in solution is of fundamental interest for both academic research and industrial applications. Recently, industrial scientists are also exploring the effect of reversible cluster formation on biopharmaceutical processing and delivery. However, despite of its importance, the understanding of protein clusters at concentrated solutions remains scientifically very challenging. Using the neutron spin echo technique to study the short time dynamics of proteins in solutions, we have recently systematically studied cluster formation in a few monoclonal antibody (mAb) solutions and their relation with solution viscosity. We show that the existence of anisotropic attraction can cause the formation of finite sized clusters, which increases the solution viscosity. Interestingly, once clusters form at relatively low concentrations, the average size of clusters in solutions remains almost constant over a wide range of concentrations similar to that of micelle formation. For a different mAb we have also investigated, the attraction is mostly induced by hydrophobic patches. As a result, these mAbs form large clusters with loosely linked proteins. In both cases, the formation of clusters all increases the solution viscosity substantially. However, due to different physics origins of cluster formation, solutions viscosities for these two different types of mAbs need to be controlled by different ways.

  20. Trial watch: Tumor-targeting monoclonal antibodies for oncological indications

    PubMed Central

    Vacchelli, Erika; Pol, Jonathan; Bloy, Norma; Eggermont, Alexander; Cremer, Isabelle; Fridman, Wolf Hervé; Galon, Jérôme; Marabelle, Aurélien; Kohrt, Holbrook; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2015-01-01

    An expanding panel of monoclonal antibodies (mAbs) that specifically target malignant cells or intercept trophic factors delivered by the tumor stroma is now available for cancer therapy. These mAbs can exert direct antiproliferative/cytotoxic effects as they inhibit pro-survival signal transduction cascades or activate lethal receptors at the plasma membrane of cancer cells, they can opsonize neoplastic cells to initiate a tumor-targeting immune response, or they can be harnessed to specifically deliver toxins or radionuclides to transformed cells. As an indication of the success of this immunotherapeutic paradigm, international regulatory agencies approve new tumor-targeting mAbs for use in cancer patients every year. Moreover, the list of indications for previously licensed molecules is frequently expanded to other neoplastic disorders as the results of large, randomized clinical trials become available. Here, we discuss recent advances in the preclinical and clinical development of tumor-targeting mAbs for oncological indications. PMID:25949870

  1. Kinase inhibitors and monoclonal antibodies in oncology: clinical implications.

    PubMed

    Gharwan, Helen; Groninger, Hunter

    2016-04-01

    Molecularly targeted cancer therapies, such as small-molecule kinase inhibitors and monoclonal antibodies, constitute a rapidly growing and an important part of the oncology armamentarium. Unlike conventional (cytotoxic) chemotherapeutics, targeted therapies were designed to disrupt cancer cell pathogenesis at specific biological points essential for the development and progression of the tumour. These agents were developed to disrupt specific targets with the aim of minimizing treatment burden compared with conventional chemotherapy. Nevertheless the increasingly common use of targeted therapies has revealed some unanticipated, often clinically significant toxic effects, as well as compromising effective palliative and end-of-life management approaches. Although patients and clinicians welcome improvements in cancer prognosis, these changes can also impact patient quality-of-life. Therefore, as demand for oncology expertise increases, physicians need to apprise themselves of targeted therapies and their clinical implications, including drug-specific side effects, impact on quality of life, and cost issues, especially in relation to end-of-life care. This Review provides a useful summary and guide for professionals treating patients with malignant diseases. PMID:26718105

  2. Monoclonal antibodies which identify a genus-specific Listeria antigen.

    PubMed Central

    Butman, B T; Plank, M C; Durham, R J; Mattingly, J A

    1988-01-01

    Fifteen murine monoclonal antibodies (MAbs) which react specifically with a protein antigen found in all species of Listeria were developed and characterized. These MAbs were tested extensively by both enzyme-linked immunosorbent assay and Western blot (immunoblot) analyses for cross-reaction with non-Listeria organisms, such as Staphylococcus, Streptococcus, Citrobacter, Pseudomonas, and Salmonella species, and were found to be nonreactive. The genus-specific antigen was identified as a heat-stable protein with a molecular weight in the range of 30,000 to 38,000 (under both reducing and nonreducing conditions), depending on the species of Listeria tested. In Listeria monocytogenes, L. innocua, L. ivanovii, and L. seeligeri the antigen has a molecular weight of approximately 30,000 to 34,000. In L. grayi and L. murrayi it has a molecular weight of approximately 35,000 to 38,000. In addition, several of the MAbs recognize lower-molecular-weight protein bands. There appear to be at least two groups of Listeria-specific MAbs based upon isotype and results of enzyme-linked immunosorbent assay and Western blot analyses. These MAbs have proven to be useful in the development of a diagnostic assay for Listeria species in food products. Images PMID:3137865

  3. Adverse events to monoclonal antibodies used for cancer therapy

    PubMed Central

    Baldo, Brian A

    2013-01-01

    Fifteen monoclonal antibodies (mAbs) are currently registered and approved for the treatment of a range of different cancers. These mAbs are specific for a limited number of targets (9 in all). Four of these molecules are indeed directed against the B-lymphocyte antigen CD20; 3 against human epidermal growth factor receptor 2 (HER2 or ErbB2), 2 against the epidermal growth factor receptor (EGFR), and 1 each against epithelial cell adhesion molecule (EpCAM), CD30, CD52, vascular endothelial growth factor (VEGF), tumor necrosis factor (ligand) superfamily, member 11 (TNFSF11, best known as RANKL), and cytotoxic T lymphocyte-associated protein 4 (CTLA4). Collectively, the mAbs provoke a wide variety of systemic and cutaneous adverse events including the full range of true hypersensitivities: Type I immediate reactions (anaphylaxis, urticaria); Type II reactions (immune thrombocytopenia, neutopenia, hemolytic anemia); Type III responses (vasculitis, serum sickness; some pulmonary adverse events); and Type IV delayed mucocutaneous reactions as well as infusion reactions/cytokine release syndrome (IRs/CRS), tumor lysis syndrome (TLS), progressive multifocal leukoencephalopathy (PML) and cardiac events. Although the term “hypersensitivity” is widely used, no common definition has been adopted within and between disciplines and the requirement of an immunological basis for a true hypersensitivity reaction is sometimes overlooked. Consequently, some drug-induced adverse events are sometimes incorrectly described as “hypersensitivities” while others that should be described are not. PMID:24251081

  4. Development and Evaluation of Monoclonal Antibodies for Paxilline

    PubMed Central

    Maragos, Chris M.

    2015-01-01

    Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrations of PAX required to inhibit signal development by 50% (IC50s) ranged from 1.2 to 2.5 ng/mL. One mAb (2-9) was applied to the detection of PAX in maize silage. The assay was sensitive to the effects of solvents, with 5% acetonitrile or 20% methanol causing a two-fold or greater increase in IC50. For analysis of silage samples, extracts were cleaned up by adsorbing potential matrix interferences onto a solid phase extraction column. The non-retained extract was then diluted with buffer to reduce solvent content prior to assay. Using this method, the limit of detection for PAX in dried silage was 15 µg/kg and the limit of quantification was 90 µg/kg. Recovery from samples spiked over the range of 100 to 1000 µg/kg averaged 106% ± 18%. The assay was applied to 86 maize silage samples, with many having detectable, but none having quantifiable, levels of PAX. The results suggest the CI-ELISA can be applied as a sensitive technique for the screening of PAX in maize silage. PMID:26426046

  5. Trial Watch: Immunomodulatory monoclonal antibodies for oncological indications

    PubMed Central

    Buqué, Aitziber; Bloy, Norma; Aranda, Fernando; Castoldi, Francesca; Eggermont, Alexander; Cremer, Isabelle; Fridman, Wolf Hervé; Fucikova, Jitka; Galon, Jérôme; Marabelle, Aurélien; Spisek, Radek; Tartour, Eric; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2015-01-01

    Immunomodulatory monoclonal antibodies (mAbs) differ from their tumor-targeting counterparts because they exert therapeutic effects by directly interacting with soluble or (most often) cellular components of the immune system. Besides holding promise for the treatment of autoimmune and inflammatory disorders, immunomodulatory mAbs have recently been shown to constitute a potent therapeutic weapon against neoplastic conditions. One class of immunomodulatory mAbs operates by inhibiting safeguard systems that are frequently harnessed by cancer cells to establish immunological tolerance, the so-called “immune checkpoints.” No less than 3 checkpoint-blocking mAbs have been approved worldwide for use in oncological indications, 2 of which during the past 12 months. These molecules not only mediate single-agent clinical activity in patients affected by specific neoplasms, but also significantly boost the efficacy of several anticancer chemo-, radio- or immunotherapies. Here, we summarize recent advances in the development of checkpoint-blocking mAbs, as well as of immunomodulatory mAbs with distinct mechanisms of action. PMID:26137403

  6. Production of a Chaetomium globosum Enolase Monoclonal Antibody

    PubMed Central

    Nayak, Ajay P.; Lemons, Angela R.; Rittenour, William R.; Hettick, Justin M.; Beezhold, Donald H.

    2014-01-01

    Chaetomium globosum is a hydrophilic fungal species and a contaminant of water-damaged building materials in North America. Methods to detect Chaetomium species include subjective identification of ascospores, viable culture, or molecular-based detection methods. In this study, we describe the production and initial characterization of a monoclonal antibody (MAb) for C. globosum enolase. MAb 1C7, a murine IgG1 isotype MAb, was produced and reacted with recombinant C. globosum enolase (rCgEno) in an enzyme-linked immunosorbent assay and with a putative C. globosum enolase in a Western blot. Epitope mapping showed MAb 1C7 specific reactivity to an enolase decapeptide, LTYEELANLY, that is highly conserved within the fungal class Sordariomycetes. Cross-reactivity studies showed MAb 1C7 reactivity to C. atrobrunneum but not C. indicum. MAb 1C7 did not react with enolase from Aspergillus fumigatus, which is divergent in only two amino acids within this epitope. The results of this study suggest potential utility of MAb 1C7 in Western blot applications for the detection of Chaetomium and other Sordariomycetes species. PMID:25495488

  7. Monoclonal antibodies directed against surface molecules of multicell spheroids

    NASA Technical Reports Server (NTRS)

    Martinez, Andrew O.

    1994-01-01

    The objective of this project is to generate a library of monoclonal antibodies (MAbs) directed against surface molecules of tumor and transformed cells grown as multicell spheroids (MCS). These MCS are highly organized, 3-dimensional multicellular structures which exhibit many characteristics of in vivo organized tissues not found in conventional monolayer or suspension culture. Therefore MCS make better in vitro model systems to study the interactions of mammalian cells, and provide a functional assay for surface adhesion molecules. This project also involves investigations of cell-cell interactions in a gravity-based environment. It will provide a base of scientific information necessary to expand the focus of the project in future years to microgravity and hypergravity-based environments. This project also has the potential to yield important materials (e.g., cellular products) which may prove useful in the diagnosis and/or treatment of certain human diseases. Moreover, this project supports the training of both undergraduate and graduate students; thus, it will assist in developing a pool of future scientists with research experience in an area (gravitational biology) of interest to NASA.

  8. Preparation and Characterization of a Monoclonal Antibody Against Morphine.

    PubMed

    Kashanian, Susan; Shams, Ali; Ghahremani, Hossein; Paknejad, Maliheh

    2015-08-01

    A monoclonal antibody (MAb) was produced by immunization of a BALB/c mouse with a conjugated morphine C6-hemisuccinated derivative (MHS) to cationized bovine serum albumin (cBSA). The hybridoma clones were screened by indirect ELISA using MHS-BSA. The best hybridoma clone was subcloned thrice by limiting dilution. This hybridoma was found to be of IgG2b class and subclass and contained lambda light chain. The affinity of the MAb to morphine was obtained 2.810(9) M(-1). The titer of the cell culture supernatant was at least 1:800. The MAb was cross-reacted with codeine (100%) and apomorphine (16.5%), but not with heroin, naloxone, naltrexone, or papaverine. Morphine was conjugated to HRP using a mixed anhydride method and a direct competitive ELISA was designed using anti-morphine MAb. The assay was sensitive over the 50?ng/mL to 5??g/mL concentration range. In conclusion, this MAb is useful for the development of immunoassays to measure morphine in urine. PMID:26301931

  9. Downstream processing of monoclonal antibodies--application of platform approaches.

    PubMed

    Shukla, Abhinav A; Hubbard, Brian; Tressel, Tim; Guhan, Sam; Low, Duncan

    2007-03-15

    This paper presents an overview of large-scale downstream processing of monoclonal antibodies and Fc fusion proteins (mAbs). This therapeutic modality has become increasingly important with the recent approval of several drugs from this product class for a range of critical illnesses. Taking advantage of the biochemical similarities in this product class, several templated purification schemes have emerged in the literature. In our experience, significant biochemical differences and the variety of challenges to downstream purification make the use of a completely generic downstream process impractical. Here, we describe the key elements of a flexible, generic downstream process platform for mAbs that we have adopted at Amgen. This platform consists of a well-defined sequence of unit operations with most operating parameters being pre-defined and a small subset of parameters requiring development effort. The platform hinges on the successful use of Protein A chromatography as a highly selective capture step for the process. Key elements of each type of unit operation are discussed along with data from 14 mAbs that have undergone process development. Aspects that can be readily templated as well as those that require focused development effort are identified for each unit operation. A brief description of process characterization and validation activities for these molecules is also provided. Finally, future directions in mAb processing are summarized. PMID:17046339

  10. Treating multiple sclerosis with monoclonal antibodies: a 2010 update.

    PubMed

    Buttmann, Mathias

    2010-05-01

    Treating multiple sclerosis (MS) with monoclonal antibodies (mAbs) has been marked by both progress and setbacks in the past 2 years, which are reviewed here. The natalizumab section of the article centers around progressive multifocal leukoencephalopathy (PML), and discusses PML risk in relation to treatment duration, bioassays for individual risk prediction, the concept of drug holidays, clinical course and treatment of PML, as well as safety-related regulatory actions. The rituximab section critically analyzes recent clinical trial results, discusses the clinical relevance of anti-idiotypic mAbs and makes a short excursion to neuromyelitis optica. Following this, the newer anti-CD20 mAbs ocrelizumab and ofatumumab, which are currently being tested in Phase II for MS, are reviewed and compared. The alemtuzumab section highlights novel data on mechanisms of action, potentially allowing individual risk prediction, and new results from the CAMMS223 trial, as well as the current status of the pivotal MS studies. The daclizumab section summarizes new open-label data, shedding more light on the adverse-effect profile of the drug in MS patients, and reports on its Phase III status. Subsequently, a failed ustekinumab trial and LY2127399 are reviewed. Taking into account late Phase II and III data on novel oral agents, the final section attempts to provide a detailed perspective on disease-modifying MS therapy in the medium term. PMID:20420497

  11. Production of monoclonal antibody to herbicide fenoxaprop-ethyl.

    PubMed

    Cui, Yongliang; Nan, Tiegui; Tan, Guiyu; Li, Qing X; Wang, Baomin; Liu, Shangzhong

    2011-10-01

    Fenoxaprop-ethyl is a selective aryloxyphenoxypropionate herbicide used widely to control annual and perennial grasses. A monoclonal antibody (MAb) against fenoxaprop-ethyl (FE), designated as 3E6B9C, was produced and had very low cross-reactivity with some of its structural analogs, such as clodinafop-propargyl, diclofop-methyl, lactofen, and quizalofop-p-ethyl. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. The concentration of R-(+)-fenoxaprop-ethyl (R-FE) producing 50% of inhibition (IC(50)) and the working range of icELISA were 3.1?ng/mL and 0.6-29?ng/mL, respectively. This assay is also sensitive to R-fenoxaprop, S-(-)-fenoxaprop-ethyl, and metamifop with IC(50) of 3.4, 2.7, and 3.5?ng/mL, respectively. The recoveries of R-FE in soil samples with the icELISA were 86-102%. PMID:22008074

  12. Quantitative Assessment of Antibody Internalization with Novel Monoclonal Antibodies against Alexa Fluorophores

    PubMed Central

    Liao-Chan, Sindy; Daine-Matsuoka, Barbara; Heald, Nathan; Wong, Tiffany; Lin, Tracey; Cai, Allen G.; Lai, Michelle; D’Alessio, Joseph A.; Theunissen, Jan-Willem

    2015-01-01

    Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface–bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor–labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology. PMID:25894652

  13. In vitro inhibition of Cryptosporidium parvum infection by human monoclonal antibodies.

    PubMed Central

    Elliot, B C; Wisnewski, A V; Johnson, J; Fenwick-Smith, D; Wiest, P; Hamer, D; Kresina, T; Flanigan, T P

    1997-01-01

    Cryptosporidium parvum infection of the small epithelial intestine causes unremitting diarrhea and malabsorption that can lead to chronic and sometimes fatal illness in patients with AIDS. The illness may be ameliorated by passive oral immunoglobulin therapy. The objective of this study was to produce anti-Cryptosporidium human monoclonal antibodies for evaluation as potential therapy. All human monoclonal cell lines that produced C. parvum antibodies were originally generated from the peripheral blood lymphocytes of a human immunodeficiency virus-seronegative woman. She had recovered from C. parvum infection and had a high specific antibody titer. Hybridization of these lymphocytes with a tumor cell line was accomplished by hypo-osmolar electrofusion. Twelve clones were identified by enzyme-linked immunosorbent assay (ELISA) as secreting anti-Cryptosporidium antibodies after the initial hybridization. From the 12 positive clones, two high antibody-secreting clones, 17A and 17B, were maintained in long-term culture. A second hybridization produced two other human monoclonal cell lines, EC5 and BB2. Human monoclonal antibody from the first two cell lines bound to C. parvum sporozoites and oocysts by immunofluorescence. The ability of human monoclonal antibodies to inhibit C. parvum infection in vitro was assessed by using a human enterocyte cell line, HT29.74. The antibodies of the four different human hybridomas inhibited infection by 35 to 68% (P < 0.05) compared to a control irrelevant human monoclonal antibody derived in a similar fashion. Human monoclonal antibodies are candidate molecules for immunotherapy of C. parvum infection. PMID:9284173

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

    PubMed

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

    2016-01-01

    Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immuno extract BChE from the plasma of humans, monkey and other animals. PMID:26585590

  15. Assignment of a fibronectin gene to human chromosome 2 using monoclonal antibodies

    SciTech Connect

    Koch, G.A.; Schoen, R.C.; Klebe, R.J.; Shows, T.B.

    1982-10-01

    The locus coding for the presumed structural gene for fibronectin has been mapped to human chromosome 2 using human-mouse somatic cell hybrids. The assignment of fibronectin has been made by testing man-mouse somatic cell hybrids with two anti-human fibronectin monoclonal antibodies which recognize different antigenic determinants of human, but not mouse, fibronectin. Both monoclonal antibodies demonstrate a highly concordant association between the presence of two different human fibronectin antigens and human chromosome 2.

  16. Identification of serotype 9 human rotavirus by enzyme-linked immunosorbent assay with monoclonal antibodies.

    PubMed

    Midthun, K; Valdesuso, J; Kapikian, A Z; Hoshino, Y; Green, K Y

    1989-09-01

    Hybridomas producing monoclonal antibodies to VP7, a major neutralizing protein of serotype 9 rotavirus (strain W161), were prepared. One monoclonal antibody, W161-6A1, was shown to neutralize only serotype 9 rotavirus strains and reacted specifically with serotype 9 rotaviruses in an enzyme-linked immunosorbent assay. The development of an immunoassay for detection of serotype 9 rotaviruses should facilitate epidemiologic studies. PMID:2550520

  17. Identification of serotype 9 human rotavirus by enzyme-linked immunosorbent assay with monoclonal antibodies.

    PubMed Central

    Midthun, K; Valdesuso, J; Kapikian, A Z; Hoshino, Y; Green, K Y

    1989-01-01

    Hybridomas producing monoclonal antibodies to VP7, a major neutralizing protein of serotype 9 rotavirus (strain W161), were prepared. One monoclonal antibody, W161-6A1, was shown to neutralize only serotype 9 rotavirus strains and reacted specifically with serotype 9 rotaviruses in an enzyme-linked immunosorbent assay. The development of an immunoassay for detection of serotype 9 rotaviruses should facilitate epidemiologic studies. PMID:2550520

  18. [Targeted therapy and progressive multifocal leukoencephalopathy (PML): PML in the era of monoclonal antibody therapies].

    PubMed

    Takao, Masaki

    2013-11-01

    Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system and is associated with John Cunningham (JC) virus infection in the oligodendrocytes. The number of patients with PML increased after the pandemic of acquired immunodeficiency syndrome. Thereafter, an association between PML and monoclonal antibody therapy has come into light. Thus far, several monoclonal antibodies have been reported to cause PML. Currently, according to the Barts and the London School of Medicine and Dentistry, the number of PML cases due to natalizumab treatment for multiple sclerosis is 395 (incidence is 3.28/1,000). Moreover, the number of individuals with PML due to rituximab treatment is increasing (over 100 cases). Efalizumab, infliximab, adalimumab, etanercept, ibritumomab tiuxetan, bevacizumab, alemtuzumab, cetuximab, and brentuximab are also reported as risk factors of PML. The diagnosis of PML is based on clinical, neuroradiological, pathological, and molecular analyses. In clinical setting, magnetic resonance imaging provides the most important information in the diagnosis of PML. Patients with PML due to monoclonal antibody treatment may present clinical symptoms different from that of the classic PML, such as sensory disturbance and seizure. Once PML is identified in an individual receiving monoclonal antibody therapy, the monoclonal antibody must be immediately discontinued and removed from the body by plasmapheresis. Because most patients may present immune reconstitution inflammatory syndrome (IRIS), steroid therapy must be considered immediately. However, the prognosis of PML is still worse in patients receiving monoclonal antibody therapy. To prevent PML development, sophisticated and well-organized strategies must be established for monoclonal antibody treatment. Besides neurologists, physicians from other fields must be aware of PML associated with resulted from monoclonal antibody therapy. PMID:24200614

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

    NASA Astrophysics Data System (ADS)

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

    1995-07-01

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

  20. Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

    NASA Technical Reports Server (NTRS)

    Hamilton, Robert G.; Rodkey, L. Scott; Reimer, Charles B.

    1987-01-01

    The quality control of murine hybridoma secretory products has been performed using two approaches for isoelectric focusing affinity immunoblot analysis: (1) a method in which antigen-coated nitrocellulose is placed on top of an acrylamide gel containing isoelectrically focused ascites to bind the antigen specific monoclonal antibody; and (2) a method in which focused ascite proteins were passively blotted onto nitrocellulose and specific monoclonal antibodies were detected with enzyme-conjugated antigen. Analysis by both methods of batches of ascites containing antihuman IgG antibodies that were produced by six hybridomas permitted effective monitoring of immunoreactive antibodies for pI microheterogeneity.

  1. Murine monoclonal antibody to a single protein neutralizes the infectivity of human cytomegalovirus.

    PubMed Central

    Rasmussen, L E; Nelson, R M; Kelsall, D C; Merigan, T C

    1984-01-01

    Murine monoclonal antibodies to human cytomegalovirus (CMV) strain AD169 were selected that neutralized virus infectivity. One monoclonal antibody-producing hybridoma, 1G6, was used to produce ascites fluid from which immunoglobulin was isolated. This antibody efficiently neutralized CMV AD169, other laboratory strains (Towne, Davis), and clinical isolates of CMV in early tissue culture passage (less than 10) in the absence of complement. The antibody immunoprecipitated a single 86,000-dalton protein from both laboratory and clinical strains. This viral protein was demonstrated by indirect immunofluorescence to be localized in the cytoplasm of CMV-infected cells. Images PMID:6199788

  2. Nonmitogenic Anti-CD3 Monoclonal Antibodies Deliver a Partial T Cell Receptor Signal and Induce Clonal Anergy

    PubMed Central

    Smith, Judith A.; Tso, J. Yun; Clark, Marcus R.; Cole, Michael S.; Bluestone, Jeffrey A.

    1997-01-01

    Anti-CD3 monoclonal antibodies (mAbs) are potent immunosuppressive agents used in clinical transplantation. However, the activation-related adverse side effects associated with these mAbs have prompted the development of less toxic nonmitogenic anti-CD3 mAb therapies. At present, the functional and biochemical consequences of T cell exposure to nonmitogenic anti-CD3 is unclear. In this study, we have examined the early signaling events triggered by a nonmitogenic anti-CD3 mAb. Like the mitogenic anti-CD3 mAb, nonmitogenic anti-CD3 triggered changes in the T cell receptor (TCR) complex, including ζ chain tyrosine phosphorylation and ZAP-70 association. However, unlike the mitogenic anti-CD3 stimulation, nonmitogenic anti-CD3 was ineffective at inducing the highly phosphorylated form of ζ (p23) and tyrosine phosphorylation of the associated ZAP-70 tyrosine kinase. This proximal signaling deficiency correlated with minimal phospholipase Cγ-1 phosphorylation and failure to mobilize detectable Ca2+. Not only did biochemical signals delivered by nonmitogenic anti-CD3 resemble altered peptide ligand signaling, but exposure of Th1 clones to nonmitogenic anti-CD3 also resulted in functional anergy. Finally, a bispecific anti-CD3 × anti-CD4 F(ab)′2 reconstituted early signal transduction events and induced proliferation, suggesting that defective association of lck with the TCR complex may underlie the observed signaling differences between the mitogenic and nonmitogenic anti-CD3. PMID:9126922

  3. Exploration of overloaded cation exchange chromatography for monoclonal antibody purification.

    PubMed

    Liu, Hui F; McCooey, Beth; Duarte, Tiago; Myers, Deanna E; Hudson, Terry; Amanullah, Ashraf; van Reis, Robert; Kelley, Brian D

    2011-09-28

    Cation exchange chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation exchange membrane (Mustang S) and monolith (SO(3) Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000 g/L of chromatographic matrix. The dynamic binding capacity of the cation exchange resins is typically below 100 g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3 mS/cm. HMW binding capacity was optimized at 5 mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb purification process employing protein A affinity chromatography, isocratic overloaded cation exchange chromatography using Poros 50HS and anion exchange chromatography using QSFF in flow through mode was compared with the MAb's commercial manufacturing process, which consisted of protein A affinity chromatography, cation exchange chromatography using SPSFF in bind-elute mode and anion exchange chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes. PMID:21871630

  4. Human monoclonal antibody with dual GM2/GD2 specificity derived from an immunized melanoma patient.

    PubMed Central

    Yamaguchi, H; Furukawa, K; Fortunato, S R; Livingston, P O; Lloyd, K O; Oettgen, H F; Old, L J

    1990-01-01

    GM2 ganglioside is a common cell surface constituent of human melanoma and other tumors of neuroectodermal origin, and vaccination with GM2 ganglioside results in high levels of anti-GM2 antibodies in patients with melanoma. Lymphocytes from a GM2-vaccinated patient (VS) were transformed by Epstein-Barr virus and tested for production of antibodies with reactivity for GM2-positive tumor cells. A high percentage of antibody-producing B cells was detected, but antibody reactivity was generally lost during culture expansion. Two cultures, however, remained stable for antibody productivity and one was used to develop a stable hybrid line with mouse myeloma. The monoclonal antibody (designated 3-207) derived from patient VS has dual specificity for GM2 and GD2, despite the fact that only GM2 antibody could be detected in the patient's serum. Monoclonal antibody 3-207 shows high-titered reactivity with a range of melanoma, astrocytoma, neuroblastoma, and leukemia cell lines, cells with prominent cell surface expression of GM2 and GD2. The cell surface reactivity of monoclonal antibody 3-207 was not abolished by treatment of target cells with neuraminidase, as the enzyme converted GD2 to GM2, which was still detected by monoclonal antibody 3-207. Images PMID:2159145

  5. The development of glioblastoma multiforme reactive monoclonal antibodies and their use in drug targeting

    SciTech Connect

    Klaich, G.M.

    1989-01-01

    The objectives of this project were to develop monoclonal antibodies reactive with the tumor glioblastoma multiforme and to use them to study and develop new treatment modalities for this disease. A tumor antigen enriched immunogen, prepared by immunoaffinity chromatography, was compared to a whole tumor homogenate immunogen with the difference in the yield of tumor reactive, normal brain unreactive monoclonal antibodies proving to be significant. Monoclonal antibody A7, reactive with tumor tissue but unreactive with normal tissue, was isotyped to be an IgG2a immunoglobulin and could be purified to electrophoretic homogeneity by using serum-free culture conditions and protein A sepharose chromatography. Monoclonal antibody A7 is noncytotoxic as measured by the {sup 3}H-nicotinamide release assay and binds to a 138 kd membrane antigen which is not internalized. Localization studies using {sup 14}C-labeled monoclonal antibody A7 and the U-87 MG nude mouse xenograft model resulted in a tumor:serum ratio of 1.25:1.0 as compared to 0.29:1.0 for the negative control. A monoclonal antibody A7-doxorubicin immunoconjugate proved to be more cytotoxic than free doxorubicin in vitro while lethality studies using Swiss mice demonstrated the lack of toxicity of the immunoconjugate as compared to free doxorubicin. In vivo chemotherapy studies using the U-87 MG nude mouse xenograft failed to demonstrate any immunoconjugate anti-tumor activity which may be attributable to the route of administration.

  6. Monoclonal antibodies to amoxicillin express different idiotypes determined by anti-idiotype antibodies production.

    PubMed

    Mayorga, C; Torres, M J; Romano, A; Moreno, F; Snchez-Sabater, E; Jurez, C; Blanca, M

    2002-01-01

    Penicillins are beta-lactam antibiotics able to generate several antigenic determinants that are recognized by the immune system. To study the differences in the antigen binding site of two monoclonal antibodies (Mab) specific to amoxicillin, polyclonal rabbit anti-idiotypic antibodies were produced. One Mab, AO3.2 (IgG2a), specific to a structure formed by the acyl-side chain structure and a part of the nuclear region of amoxicillin. The second one, AO6.2 (IgE), is specific to the side chain of amoxicillin, although it also recognizes the side chain of other penicillins (penicillin G and ampicillin). These antibodies were used to immunize rabbits in order to produce polyclonal anti-idiotypic antibodies, which were purified in several steps by affinity chromatography. The specificity and cross-reactivity studies were made by ELISA and ELISA inhibition. The results suggest that the anti-Id antibodies produced are the internal image of the antigen, since the binding to their specific idiotype is blocked mainly by the original hapten (amoxicillin): in 98% of the cases with anti-id-1 (induced against AO3.2) and in 59% with anti-id-2 (induced against AO6.2). The absence of cross-reactivity of each anti-idiotypic antibody with the different Mabs specific to amoxicillin shows that the idiotypes induced by the same hapten have differences that are reflected by the nonrecognition of these anti-idiotypes. We conclude that such a small molecule as amoxicillin can present several antigenic determinants that induce a panel of antibody specificities especially directed against the side chain. PMID:12144555

  7. The use of monoclonal antibodies for the antigenic analysis of influenza A viruses

    PubMed Central

    Pereira, M. S.; Chakraverty, P.; Cunningham, P.; Webster, R. G.

    1985-01-01

    Monoclonal antibodies have been found to provide useful additional information for the antigenic analysis of influenza A viruses of the H3N2 and H1N1 subtypes. They have been particularly useful in the interpandemic period when multiple variants circulate concurrently. Apparently heterogeneous isolates can be placed in fairly clear-cut groups on the basis of their reactivity with certain monoclonal antibody preparations. It is thought likely that variants reacting with the least number of monoclones are the most different antigenically from the fully reactive strains. PMID:2410156

  8. Phage display for the production of human monoclonal antibodies against human pathogens.

    PubMed

    Mancini, N; Carletti, S; Perotti, M; Canducci, F; Mammarella, M; Sampaolo, M; Burioni, R

    2004-10-01

    In the last decade an increasing number of antibodies have made their way from the research benchtops into the clinics and many more are currently under clinical trial. Among monoclonal antibody-producing techniques, phage-display is undoubtedly the most effective and versatile. Cloning of the entire humoral repertoire derived from an infected patients into a phage display vector allows not only the simple generation of monoclonal antibodies of desired specificity, but also the molecular dissection of the antibody response itself. Generation of large panels of human monoclonal antibodies against human pathogens could open new perspectives in understanding the interplay between the infectious agent and the infected host providing tools for the prevention and the therapy of human communicable diseases. In this paper the basic principles of the phage-display approach as well as its most recent applications are reviewed. PMID:15646045

  9. Rapid Identification of Monospecific Monoclonal Antibodies Using a Human Proteome Microarray*

    PubMed Central

    Jeong, Jun Seop; Jiang, Lizhi; Albino, Edisa; Marrero, Josean; Rho, Hee Sool; Hu, Jianfei; Hu, Shaohui; Vera, Carlos; Bayron-Poueymiroy, Diane; Rivera-Pacheco, Zully Ann; Ramos, Leonardo; Torres-Castro, Cecil; Qian, Jiang; Bonaventura, Joseph; Boeke, Jef D.; Yap, Wendy Y.; Pino, Ignacio; Eichinger, Daniel J.; Zhu, Heng; Blackshaw, Seth

    2012-01-01

    To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His6 fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity. PMID:22307071

  10. Differentiation of Naegleria fowleri from Acanthamoeba species by using monoclonal antibodies and flow cytometry.

    PubMed Central

    Flores, B M; Garcia, C A; Stamm, W E; Torian, B E

    1990-01-01

    Monoclonal antibodies to Naegleria fowleri and Acanthamoeba polyphaga were analyzed by enzyme-linked immunosorbent assay, indirect immunofluorescence microscopy, and fluorescence flow cytometry to assess specificity and cross-reactivity with axenically cultured N. fowleri and Acanthamoeba spp. Four monoclonal antibodies to N. fowleri were specific for N. fowleri and had no reactivity to A. polyphaga. Similarly, four monoclonal antibodies to A. polyphaga did not react with N. fowleri. Two of the four monoclonal antibodies to A. polyphaga did not react with other Acanthamoeba spp. tested, while two of the antibodies demonstrated a high degree of cross-reactivity with a putative Acanthamoeba castellanii strain by immunofluorescence microscopy; this was confirmed by fluorescence flow cytometry for one of the antibodies. These monoclonal antibodies were used to identify Acanthamoeba trophozoites in infected brain sections of a patient who died of suspected Acanthamoeba-caused granulomatous amoebic encephalitis, demonstrating potential utility in the direct identification of N. fowleri and Acanthamoeba spp. in clinical specimens. Images PMID:2229384

  11. Demonstration of two distinct antigenic determinants on hepatitis B e antigen by monoclonal antibodies

    SciTech Connect

    Imai, M.; Nomura, M.; Gotanda, T.; Sano, T.; Tachibana, K.; Miyamoto, H.; Takahashi, K.; Toyama, S.; Miyakawa, Y.; Mayumi, M.

    1982-01-01

    Mice were immunized against hepatitis B e antigen (HBeAg) isolated from sera of asymptomatic carriers of hepatitis B virus. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 5 clones of hybridoma cells secreting antibody against HBeAg (anti-HBe) were isolated. For the production of anti-HBe in large scale, cells were cultivated both in vitro and in the peritoneal cavity of ascitic mice. Although monoclonal antibodies produced by these clones showed a strong reactivity of anti-HBe in hemagglutination tests, individual monoclonal anti-HBe did not reveal any precipitin line in immunodiffusion. When 2 of the 5 monoclonal antibodies were mixed together, however, some combinations showed a precipitin line against HBeAg, whereas others did not. Utilizing solid-phase radioimmunoassay involving a number of combinations of monoclonal antibodies used for solid-phase and radiolabeling, the 5 antibodies were classified into 2 groups. Three of the anti-HBe antibodies were found to be directed to 1 determinant of HBeAg (determinant a); the remaining 2 to the other determinant (determinant b). Determinants a and b were detected on HBeAg in the serum, as well as on the polypeptide of 19,000 daltons (P19) derived from the nucleocapsid of hepatitis B virus. Monoclonal anti-HBe antibodies with different specificities may provide useful tools in delineating the antigenic structure of HBeAg and also in evaluating immune responses of the host directed to its subdeterminants.

  12. Development of an antigen microarray for high throughput monoclonal antibody selection.

    PubMed

    Staudt, Nicole; Mller-Sienerth, Nicole; Wright, Gavin J

    2014-03-21

    Monoclonal antibodies are valuable laboratory reagents and are increasingly being exploited as therapeutics to treat a range of diseases. Selecting new monoclonal antibodies that are validated to work in particular applications, despite the availability of several different techniques, can be resource intensive with uncertain outcomes. To address this, we have developed an approach that enables early screening of hybridoma supernatants generated from an animal immunised with up to five different antigens followed by cloning of the antibody into a single expression plasmid. While this approach relieved the cellular cloning bottleneck and had the desirable ability to screen antibody function prior to cloning, the small volume of hybridoma supernatant available for screening limited the number of antigens for pooled immunisation. Here, we report the development of an antigen microarray that significantly reduces the volume of supernatant required for functional screening. This approach permits a significant increase in the number of antigens for parallel monoclonal antibody selection from a single animal. Finally, we show the successful use of a convenient small-scale transfection method to rapidly identify plasmids that encode functional cloned antibodies, addressing another bottleneck in this approach. In summary, we show that a hybrid approach of combining established hybridoma antibody technology with refined screening and antibody cloning methods can be used to select monoclonal antibodies of desired functional properties against many different antigens from a single immunised host. PMID:24472540

  13. Perfusion of tumor-bearing kidneys as a model for scintigraphic screening of monoclonal antibodies

    SciTech Connect

    van Dijk, J.; Oosterwijk, E.; van Kroonenburgh, M.J.; Jonas, U.; Fleuren, G.J.; Pauwels, E.K.; Warnaar, S.O.

    1988-06-01

    Tumor-bearing human kidneys were used in an ex vivo perfusion model to screen monoclonal antibodies, recognizing renal cell carcinoma-associated antigens for diagnostic potential in vivo. Perfusion of tumor-bearing kidneys with /sup 99m/Tc-labeled G250 and RC38 antibody resulted in visualization of the tumor, whereas perfusion with two other monoclonal antibodies, RC2 and RC4, did not lead to tumor visualization. Uptake of radiolabel in normal kidney tissue was low for G250 and RC38 antibody. Tumor-to-kidney tissue ratios after perfusion with G250 and RC38 antibody were 2.7 and 2.2, respectively. After rinsing for 3 hr with unlabeled perfusion fluid the tumor-to-kidney tissue ratios increased to 8.6 for G250 antibody and to 2.7 for RC38 antibody. We conclude that perfusion of tumor-bearing human kidneys with radiolabeled monoclonal antibodies is a relatively simple way to evaluate renal cell carcinoma associated monoclonal antibodies as diagnostic agents in vivo.

  14. Radioimmunological imaging of metastatic prostatic cancer with 111indium-labeled monoclonal antibody PAY 276

    SciTech Connect

    Babaian, R.J.; Murray, J.L.; Lamki, L.M.; Haynie, T.P.; Hersh, E.M.; Rosenblum, M.G.; Glenn, H.J.; Unger, M.W.; Carlo, D.J.; von Eschenbach, A.C.

    1987-03-01

    A total of 25 patients with histologically proved adenocarcinoma of the prostate, whose disease was staged clinically as D2 by appropriate radiographic and nuclear medicine studies, received increasing doses of PAY 276, an antiprostatic acid phosphatase monoclonal antibody for radioimmunological imaging. The patients were divided into 5 groups of 5. Groups 1 through 5 received an infusion of 5, 10, 20, 40 or 80 mg. monoclonal antibody, respectively, 1 mg. of which was labeled to 5 mCi. of /sup 111/indium, while stable monoclonal antibody was added to achieve the desired antibody concentration. No patient had an allergic reaction, and no significant change in serial hemoglobin levels, platelet count, chemistry profile or results of urinalyses was noted. The monoclonal antibody scan visualized at least 1 lesion in 19 of 25 patients (76 per cent): 4 in groups 1 and 2, and all 15 in groups 3 to 5. With results of conventional radiography and bone scintigraphy considered definitive for metastases, monoclonal antibody scans detected 7 of 32 metastases (21.8 per cent) in group 3 (20 mg.), 31 of 58 (53.4 per cent) in group 4 (40 mg.) and 101 of 134 (75.4 per cent) in group 5 (80 mg). In group 5 the incidence of false positive and false negative scans was 2.3 per cent (3 of 132) and 24.6 per cent (33 of 134), respectively. The detection of metastatic lesions increased as the concentration of unlabeled monoclonal antibody increased. Radioimmunological imaging of prostatic cancer with antiprostatic acid phosphatase monoclonal antibody seems to be feasible.

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

    SciTech Connect

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

    2008-03-07

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

  16. Two monoclonal antibodies raised against different epitopes of chloroplast fructose-1. 6-bisphosphatase (FBPase)

    SciTech Connect

    Hermoso, R.; Fonolla, J.; Lopez-Gorge, J. ); Ruiz-Cabello, F.; Garrido, F. )

    1990-05-01

    Two monoclonal antibodies (GR-BP5 and GR-BP8) were obtained by fusion of spleen cells of mice immunized against pea photosynthetic FBPase with cells of myeloma NSI. Both mAbs showed by double immunodiffusion a {chi} light chain, and the GR-BP8 secreted an IgM. By Western-blotting and immunoprecipitation of the in vivo labelled pea FBPase, GR-BP5 and GR-BP8 showed specificity for the chloroplast enzyme. Competition binding of the {sup 125}I-labelled mAbs against pea FBPase showed specific binding sites to different epitopes of the enzyme molecule. Cross reaction assays between both monoclonal antibodies and pea and spinach chloroplast FBPases showed a 90-100% homology in the corresponding epitopes of both enzymes. Preliminary assays showed a moderate inhibition of FBPase by GR-BP5 monoclonal antibody, but a weak enhancement by the GR-BP8 monoclonal one.

  17. Monoclonal antibodies defining shared human macrophage-endothelial antigens.

    PubMed

    Koch, A E; Burrows, J C; Domer, P H; Ashmun, R A; Look, A T; Leibovich, S J

    1992-01-01

    We have selected several monoclonal antibodies (mAbs) producing using human rheumatoid arthritis (RA) synovial macrophages (m phi s) as immunogen. Of these, mAbs 8H2, 10G7 and 10G9 showed cross reactivity with endothelium, suggesting common antigens between these cell types. We have determined the spectrum of reactivity of these mAbs on hematopoietic cell lines, peripheral blood cells, and inflammatory and non-inflammatory tissues by immunohistochemistry. MAb 8H2 does not react with the myeloid cell lines HL60 (myelocytic), U937 (histiocytic lymphoma), and K562 (erythroleukemia), or with peripheral blood cells. In normal and inflamed tissue sections, mAb 8H2 reacts with m phi s and endothelial cells. In contrast, mAb 10G7 does not react with peripheral blood cells, but reacts with HL60, U937, and K562 cell lines, as well as with m phi s and endothelial cells in inflamed and noninflamed tissues. MAb 10G9 does not react with myeloid cell lines, but reacts with monocytes and platelets in peripheral blood. In both normal and inflamed tissues, mAb 10G9 reacts with m phi s and endothelial cells. The antigens identified by these three mAbs were characterized biochemically, by enzymatic digestion of RA synovial tissue m phi s followed by a cellular ELISA, as well as by reactivity of the mAbs with NIH-3T3 cells genetically engineered to express known myeloid antigens. These mAbs reacted with protein or glycoprotein antigens distinct from the known myeloid antigens CD13, CD14, CD33, CD34, CD36, and c-fms. These mAbs should prove to be a valuable tool for studying m phi s and endothelial cells and their shared antigenic determinants. PMID:1571092

  18. Mapping Broadly Reactive Norovirus Genogroup I and II Monoclonal Antibodies

    PubMed Central

    Crawford, Sue E.; Ajami, Nadim; Parker, Tracy Dewese; Kitamoto, Noritoshi; Natori, Katsuro; Takeda, Naokazu; Tanaka, Tomoyuki; Kou, Baijun; Atmar, Robert L.

    2014-01-01

    Noroviruses are responsible for most acute nonbacterial epidemic outbreaks of gastroenteritis worldwide. To develop cross-reactive monoclonal antibodies (MAbs) for rapid identification of genogroup I and II (GI and GII) noroviruses (NoVs) in field specimens, mice were immunized with baculovirus-expressed recombinant virus-like particles (VLPs) corresponding to NoVs. Nine MAbs against the capsid protein were identified that detected both GI and GII NoV VLPs. These MAbs were tested in competition enzyme-linked immunosorbent assays (ELISAs) to identify common epitope reactivities to GI and GII VLPs. Patterns of competitive reactivity placed these MAbs into two epitope groups (groups 1 and 2). Epitopes for MAbs NV23 and NS22 (group 1) and MAb F120 (group 2) were mapped to a continuous region in the C-terminal P1 subdomain of the capsid protein. This domain is within regions previously defined to contain cross-reactive epitopes in GI and GII viruses, suggesting that common epitopes are clustered within the P1 domain of the capsid protein. Further characterization in an accompanying paper (B. Kou et al., Clin Vaccine Immunol 22:160–167, 2015, http://dx.doi.org/10.1128/CVI.00519-14) revealed that MAb NV23 (epitope group 1) is able to detect GI and GII viruses in stool. Inclusion of the GI and GII cross-reactive MAb NV23 in antigen detection assays may facilitate the identification of GI and GII human noroviruses in stool samples as causative agents of outbreaks and sporadic cases of gastroenteritis worldwide. PMID:25428246

  19. Advective hydrogel membrane chromatography for monoclonal antibody purification in bioprocessing.

    PubMed

    Hou, Ying; Brower, Mark; Pollard, David; Kanani, Dharmesh; Jacquemart, Renaud; Kachuik, Bradley; Stout, James

    2015-01-01

    Protein A chromatography is widely employed for the capture and purification of monoclonal antibodies (mAbs). Because of the high cost of protein A resins, there is a significant economic driving force to seek new downstream processing strategies. Membrane chromatography has emerged as a promising alternative to conventional resin based column chromatography. However, to date, the application has been limited to mostly ion exchange flow through (FT) mode. Recently, significant advances in Natrix hydrogel membrane has resulted in increased dynamic binding capacities for proteins, which makes membrane chromatography much more attractive for bind/elute operations. The dominantly advective mass transport property of the hydrogel membrane has also enabled Natrix membrane to be run at faster volumetric flow rates with high dynamic binding capacities. In this work, the potential of using Natrix weak cation exchange membrane as a mAb capture step is assessed. A series of cycle studies was also performed in the pilot scale device (> 30 cycles) with good reproducibility in terms of yield and product purities, suggesting potential for improved manufacturing flexibility and productivity. In addition, anion exchange (AEX) hydrogel membranes were also evaluated with multiple mAb programs in FT mode. Significantly higher binding capacity for impurities (support mAb loads up to 10Kg/L) and 40X faster processing speed were observed compared with traditional AEX column chromatography. A proposed protein A free mAb purification process platform could meet the demand of a downstream purification process with high purity, yield, and throughput. PMID:26018631

  20. Monoclonal antibodies as probes for fungal wall structure during morphogenesis.

    PubMed

    Marshall, M; Gull, K; Jeffries, P

    1997-07-01

    Three monoclonal antibodies (mAbs), S4D1, S3B3 and S1E5, were produced from hybridoma cell lines raised from mice immunized with hyphal walls of Neurospora crassa and one (Pax-1) from mice immunized with hyphal walls of Paxillus involutus. In immunofluorescence studies, the three N. crassa mAbs recognized epitopes with different patterns of distribution at the hyphal surface of N. crassa. S4D1 recognized an epitope which was present on the surface of both conidia and hyphae; S3B3 recognized an epitope seen only at the ends of conidia or in the septal region of hyphae and conidial chains; and S1E5 recognized an epitope present on the surface of hyphae, but not on mature conidia. mAb Pax-1 reacted with hyphal wall fragments of Pax. involutus and with N. crassa conidia in a similar way to S3B3. S4D1 reacted with an epitope found in 1,3-alpha-glycan preparations from hyphal walls of different fungi. The surface distribution of this epitope varied: it was found on the surface of both conidia and hyphae of N. crassa and Aspergillus nidulans, on the basidiospore surface only of Amanita muscaria, and on the hyphae but not the conidia of Penicillium chrysogenum. Immunogold studies revealed that the epitope was present throughout the wall of conidia and hyphae of N. crassa. mAbs S3B3, S1E5 and Pax-1 also reacted with other fungi: for example Pax-1 cross-reacted with all fungi tested except for a member of the Zygomycota. Immunogold studies revealed that epitopes of these three mAbs were present within the inner layers of the walls of conidia and hyphae of N. crassa. PMID:9245814

  1. Safety and immunotoxicity assessment of immunomodulatory monoclonal antibodies

    PubMed Central

    Morton, Laura Dill; Spindeldreher, Sebastian; Kiessling, Andrea; Allenspach, Roy; Hey, Adam; Muller, Patrick Y; Frings, Werner; Sims, Jennifer

    2010-01-01

    Most therapeutic monoclonal antibodies (mAbs) licensed for human use or in clinical development are indicated for treatment of patients with cancer and inflammatory/autoimmune disease and as such, are designed to directly interact with the immune system. A major hurdle for the development and early clinical investigation of many of these immunomodulatory mAbs is their inherent risk for adverse immune-mediated drug reactions in humans such as infusion reactions, cytokine storms, immunosuppression and autoimmunity. A thorough understanding of the immunopharmacology of a mAb in humans and animals is required to both anticipate the clinical risk of adverse immunotoxicological events and to select a safe starting dose for first-in-human (FIH) clinical studies. This review summarizes the most common adverse immunotoxicological events occurring in humans with immunomodulatory mAbs and outlines non-clinical strategies to define their immunopharmacology and assess their immunotoxic potential, as well as reduce the risk of immunotoxicity through rational mAb design. Tests to assess the relative risk of mAb candidates for cytokine release syndrome, innate immune system (dendritic cell) activation and immunogenicity in humans are also described. The importance of selecting a relevant and sensitive toxicity species for human safety assessment in which the immunopharmacology of the mAb is similar to that expected in humans is highlighted, as is the importance of understanding the limitations of the species selected for human safety assessment and supplementation of in vivo safety assessment with appropriate in vitro human assays. A tiered approach to assess effects on immune status, immune function and risk of infection and cancer, governed by the mechanism of action and structural features of the mAb, is described. Finally, the use of immunopharmacology and immunotoxicity data in determining a minimum anticipated biologic effect Level (MABEL) and in the selection of safe human starting dose is discussed. PMID:20421713

  2. Characterization of Cross-Reactive Norovirus-Specific Monoclonal Antibodies

    PubMed Central

    Kou, Baijun; Crawford, Sue E.; Ajami, Nadim J.; Czakó, Rita; Neill, Frederick H.; Tanaka, Tomoyuki N.; Kitamoto, Noritoshi; Palzkill, Timothy G.; Estes, Mary K.

    2014-01-01

    Noroviruses (NoVs) commonly cause acute gastroenteritis outbreaks. Broadly reactive diagnostic assays are essential for rapid detection of NoV infections. We previously generated a panel of broadly reactive monoclonal antibodies (MAbs). We characterized MAb reactivities by use of virus-like particles (VLPs) from 16 different NoV genotypes (6 from genogroup I [GI], 9 from GII, and 1 from GIV) coating a microtiter plate (direct enzyme-linked immunosorbent assay [ELISA]) and by Western blotting. MAbs were genotype specific or recognized multiple genotypes within a genogroup and between genogroups. We next applied surface plasmon resonance (SPR) analysis to measure MAb dissociation constants (Kd) as a surrogate for binding affinity; a Kd level of <10 nM was regarded as indicating strong binding. Some MAbs did not interact with the VLPs by SPR analysis. To further assess this lack of MAb-VLP interaction, the MAbs were evaluated for the ability to identify NoV VLPs in a capture ELISA. Those MAbs for which a Kd could not be measured by SPR analysis also failed to capture the NoV VLPs; in contrast, those with a measurable Kd gave a positive signal in the capture ELISA. Thus, some broadly cross-reactive epitopes in the VP1 protruding domain may be partially masked on intact particles. One MAb, NV23, was able to detect genogroup I, II, and IV VLPs from 16 genotypes tested by sandwich ELISA, and it successfully detected NoVs in stool samples positive by real-time reverse transcription-PCR when the threshold cycle (CT) value was <31. Biochemical analyses of MAb reactivity, including SPR analysis, identified NV23 as a broadly reactive ligand for application in norovirus diagnostic assays. PMID:25428247

  3. Biotherapies in inflammatory ocular disorders: Interferons, immunoglobulins, monoclonal antibodies.

    PubMed

    Saadoun, D; Bodaghi, B; Bienvenu, B; Wechsler, B; Sene, D; Trad, S; Abad, S; Cacoub, P; Kodjikian, L; Sve, P

    2013-05-01

    Biotherapies used in clinical practice for the treatment of ophthalmologic manifestations of systemic diseases include interferons (IFN), intravenous immunoglobulins (IVIG) and monoclonal antibodies (anti-TNF, anakinra, tocilizumab and rituximab). Several open prospective studies have shown the effectiveness of IFN-? (78 to 98% complete remission) for the treatment of severe uveitis in Behcet's disease. IFN is capable of inducing prolonged remission and continued after his arrest, in 20-40% of patients. Side effects (flu-like, psychological effects) limit its use in practice. Anti-TNF? (infliximab and adalimumab) represents an attractive alternative therapeutic in severe uveitis refractory to immunosuppressants, especially in Behcet's disease. They are almost always (>90% of cases) and rapidly effective but their action is often suspensive. Anti-TNF? requires an extended prescription or takes over from another immunosuppressant once ocular inflammation has been controlled. IVIG are used for the treatment of Kawasaki disease and Birdshot disease. Several open or retrospective studies showed their effectiveness for the treatment of severe and refractory cicatricial pemphigoid. Tolerance of IVIG is good but their efficacy is transient. Rituximab showed an efficacy in few observations of various inflammatory eye diseases (uveitis, scleritis and idiopathic inflammatory pseudo-tumors or associated with granulomatosis with polyangiitis) and cicatricial pemphigoid. The risk of infection associated with this biotherapy limits its use in refractory diseases to conventional therapy. Anakinra (a soluble antagonist of IL-1R) showed interesting results in terms of efficiency in one small open study in Behcet's disease. Its safety profile is good and with a quick action that could be interesting for the treatment of severe uveitis. PMID:23470459

  4. Characterizing monoclonal antibody structure by carboxyl group footprinting.

    PubMed

    Kaur, Parminder; Tomechko, Sara E; Kiselar, Janna; Shi, Wuxian; Deperalta, Galahad; Wecksler, Aaron T; Gokulrangan, Giridharan; Ling, Victor; Chance, Mark R

    2015-01-01

    Structural characterization of proteins and their antigen complexes is essential to the development of new biologic-based medicines. Amino acid-specific covalent labeling (CL) is well suited to probe such structures, especially for cases that are difficult to examine by alternative means due to size, complexity, or instability. We present here a detailed account of carboxyl group labeling (with glycine ethyl ester (GEE) tagging) applied to a glycosylated monoclonal antibody therapeutic (mAb). The experiments were optimized to preserve the structural integrity of the mAb, and experimental conditions were varied and replicated to establish the reproducibility of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include aspartic acid (D), glutamic acid (E), and the C-terminus (i.e., the target probes), with the experimental data in order to understand the accuracy of the approach. Data from the mAb were compared to reactivity measures of several model peptides to explain observed variations in reactivity. Attenuation of reactivity in otherwise solvent accessible probes is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. A comparison of results with previously published data by Deperalta etal using hydroxyl radical footprinting showed that 55% (32/58) of target residues were GEE labeled in this study whereas the previous study reported 21% of the targets were labeled. Although the number of target residues in GEE labeling is fewer, the two approaches provide complementary information. The results highlight advantages of this approach, such as the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling, reproducibility of replicate experiments (<2% variation in modification extent), the similar reactivity of the three target probes, and significant correlation of reactivity and solvent accessible surface area. PMID:25933350

  5. Monoclonal antibodies against Neisseria gonorrhoeae: production of antibodies directed against a strain-specific cell surface antigen.

    PubMed Central

    Nachamkin, I; Cannon, J G; Mittler, R S

    1981-01-01

    Hybridomas secreting monoclonal antibodies against an apparent strain-specific cell surface antigen of Neisseria gonorrhoeae were produced. Spleen cells from BALB/c mice immunized with whole gonococci were fused with mouse myeloma cell line Sp2/0, and hybrid cells were selected in culture. One hybridoma that secreted antibodies reactive with the immunizing strain was cloned by limiting dilution to obtain cell lines secreting monoclonal antibodies. These antibodies reacted with purified outer membranes from the immunizing strain as well as with whole gonococci. Binding of antibodies to whole gonococci was highly strain specific, with most gonococcal strains showing less than 1% of the binding with the immunizing strain. Antibodies did not bind to the other Neisseria species tested. Binding of monoclonal antibodies to whole gonococci of the immunizing strain was not dependent on state of piliation. The extent of antibody binding did vary in different colonial variants of the immunizing strain. Antibody bound to cells from colonies that were transparent or of intermediate opacity, but did not bind to cells from deeply opaque colony variants. Images PMID:6166560

  6. The current status and prospects of antibody engineering for therapeutic use: focus on glycoengineering technology.

    PubMed

    Niwa, Rinpei; Satoh, Mitsuo

    2015-03-01

    Monoclonal antibodies have demonstrated enormous potential as new classes of drugs that confer great benefits to patients, and more than 40 therapeutic antibodies have already been approved for clinical use. In particular, the past 5 years might be recognized as the period guiding the new era for "engineered antibodies," with the successful approval of numerous antibody-drug conjugates, bispecific antibodies, and glyco-engineered antibodies for clinical applications. In this review, we summarize the development of antibody engineering technologies that are proving their concepts in the clinic, mainly focusing on the latest trends in defucosylated antibody technologies. PMID:25583555

  7. Phase 2 study of the bispecific T-cell engager (BiTE) antibody blinatumomab in relapsed/refractory diffuse large B-cell lymphoma

    PubMed Central

    Goebeler, Marie-Elisabeth; Hess, Georg; Neumann, Svenja; Pfreundschuh, Michael; Adrian, Nicole; Zettl, Florian; Libicher, Martin; Sayehli, Cyrus; Stieglmaier, Julia; Zhang, Alicia; Nagorsen, Dirk; Bargou, Ralf C.

    2016-01-01

    Few patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) achieve prolonged disease-free survival. Blinatumomab, a bispecific T-cell engaging antibody construct, transiently links CD3-positive T cells to CD19-positive B cells. This phase 2 study evaluated stepwise (9-28-112 μg/d with weekly dose increases; n = 23) or flat (112 μg/d; n = 2) dosing of blinatumomab by continuous infusion, with dexamethasone prophylaxis, in patients with relapsed/refractory DLBCL. Patients received a median of 3 prior lines of therapy. Median time since last regimen was 1.5 months. Seventeen patients ended treatment in cycle 1 (induction), 7 in cycle 2 (consolidation), and 1 in retreatment. Among 21 evaluable patients, the overall response rate after 1 blinatumomab cycle was 43%, including complete responses (CRs) in 19%. Three patients had late CR in follow-up without other treatment. The most common adverse events with stepwise dosing were tremor (48%), pyrexia (44%), fatigue (26%), and edema (26%). Grade 3 neurologic events with stepwise dosing were encephalopathy and aphasia (each 9%) and tremor, speech disorder, dizziness, somnolence, and disorientation (each 4%). Of 5 (22%) patients who discontinued stepwise dosing because of adverse events, 4 (17%) had neurologic events. Most neurologic events resolved. The flat-dose cohort was stopped because of grade 3 neurologic events in both patients. Blinatumomab monotherapy appears effective in patients with relapsed/refractory DLBCL, a heavily pretreated patient population with a high unmet medical need. Further studies need to define the optimal approach to achieve the target dose without early dropout. The study was registered at www.clinicaltrials.gov as #NCT01741792. PMID:26755709

  8. Phase 2 study of the bispecific T-cell engager (BiTE) antibody blinatumomab in relapsed/refractory diffuse large B-cell lymphoma.

    PubMed

    Viardot, Andreas; Goebeler, Marie-Elisabeth; Hess, Georg; Neumann, Svenja; Pfreundschuh, Michael; Adrian, Nicole; Zettl, Florian; Libicher, Martin; Sayehli, Cyrus; Stieglmaier, Julia; Zhang, Alicia; Nagorsen, Dirk; Bargou, Ralf C

    2016-03-17

    Few patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) achieve prolonged disease-free survival. Blinatumomab, a bispecific T-cell engaging antibody construct, transiently links CD3-positive T cells to CD19-positive B cells. This phase 2 study evaluated stepwise (9-28-112 μg/d with weekly dose increases; n = 23) or flat (112 μg/d; n = 2) dosing of blinatumomab by continuous infusion, with dexamethasone prophylaxis, in patients with relapsed/refractory DLBCL. Patients received a median of 3 prior lines of therapy. Median time since last regimen was 1.5 months. Seventeen patients ended treatment in cycle 1 (induction), 7 in cycle 2 (consolidation), and 1 in retreatment. Among 21 evaluable patients, the overall response rate after 1 blinatumomab cycle was 43%, including complete responses (CRs) in 19%. Three patients had late CR in follow-up without other treatment. The most common adverse events with stepwise dosing were tremor (48%), pyrexia (44%), fatigue (26%), and edema (26%). Grade 3 neurologic events with stepwise dosing were encephalopathy and aphasia (each 9%) and tremor, speech disorder, dizziness, somnolence, and disorientation (each 4%). Of 5 (22%) patients who discontinued stepwise dosing because of adverse events, 4 (17%) had neurologic events. Most neurologic events resolved. The flat-dose cohort was stopped because of grade 3 neurologic events in both patients. Blinatumomab monotherapy appears effective in patients with relapsed/refractory DLBCL, a heavily pretreated patient population with a high unmet medical need. Further studies need to define the optimal approach to achieve the target dose without early dropout. The study was registered at www.clinicaltrials.gov as #NCT01741792. PMID:26755709

  9. Development and Characterization of Monoclonal Antibodies and Aptamers Against Major Antigens of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Specific antibodies, available in unlimited quantities, have not been produced against Mycobacterium avium subsp. paratuberculosis, the bacterium that causes Johne’s disease (JD). To fill this gap in JD research, monoclonal antibodies (mAbs) against M. avium subsp. paratuberculosis were produced fr...

  10. New Stx2e monoclonal antibodies for immunological detection and distinction of Stx2 subtypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Stx2e is a primary virulence factor in STEC strains that cause edema disease in neonatal piglets. Though Stx2a and Stx2e are similar, most antibody-based Stx detection kits are designed to detect Stx2a and do not recognize the Stx2e subtype. Methods and Findings Four monoclonal antibodie...

  11. PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO ENTERIC ADENOVIRUS TYPES 40 AND 41

    EPA Science Inventory

    The authors have prepared monoclonal antibodies to each of the enteric adenoviruses types 40 and 41. Three different hybridoma cell lines were selected which produced antibody found to react by radioimmunoprecipitation with adenovirus (Ad) hexon antigens. One was specific for Ad4...

  12. Prophylaxis and therapy of influenza pneumonia in mice by intratracheal instillation of monoclonal antibody

    SciTech Connect

    Ratcliffe, D.R.

    1985-01-01

    This study on passive immunity dealt principally with the following topics: pathogenesis of the pneumonia produced by influenza virus (PR8) in CF-1 mice; the distribution and retention of monoclonal antibody instilled intratracheally (IT) into the lung; and prophylaxis and therapy of influenza pneumonia using specific monoclonal antibody (IgG 2a/k anti-HA). The fate of a single 50 ul bolus of antibody instilled IT was determined by monitoring the activity of /sup 125/I-labelled monoclonal IgG in the lungs and by lavage recovery of functional antibody.Antibody was demonstrated in high concentrations for the first 3 days and was present in the lungs for a period of 7 days. For prophylaxis several trials indicated that monoclonal antibody provided significant protection from lethal effects of the virus. Antibody given to clinically ill mice on day 3 produced a highly significant reduction in mortality (P < 0.001) when compared to control mice. The treatment reversed the weight loss and apparently arrested the development of lesions in most of the mice within 2 days following antibody administration.

  13. The generation of monoclonal antibodies and their use in rapid diagnostic tests

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibodies are the most important component of an immunoassay. In these proceedings we outline novel methods used to generate and select monoclonal antibodies that meet performance criteria for use in rapid lateral flow and microfluidic immunoassay tests for the detection of agricultural pathogens ...

  14. PRODUCTION OF MONOCLONAL ANTIBODIES TO 'LEGIONELLA PNEUMOPHILA' SEROGROUPS 1 AND 6

    EPA Science Inventory

    To better define the surface antigens of Legionella pneumophila for clinical and experimental purposes, were produced monoclonal antibodies to L. pneumophila serogroups 1 and 6. Two hybridomas were produced in serogroup 1. One antibody, LP-I-17, recognized a serogroup-common anti...

  15. Immunodiagnosis of human cysticercosis (Taenia solium) with antigens purified by monoclonal antibodies.

    PubMed Central

    Nascimento, E; Tavares, C A; Lopes, J D

    1987-01-01

    Monoclonal antibodies were generated from mice immunized with scolex protein antigen of Cysticercus cellulosae. Three monoclonal antibodies specific for cysticercal antigens, which did not show any cross-reactivity with Taenia solium or Taenia saginata antigens, were selected. Each monoclonal antibody coupled to Sepharose could purify one antigen, which appeared as a single band on polyacrylamide gel electrophoresis. When antigens purified by monoclonal antibodies were used to detect antibody in serum samples taken from patients with cysticercosis, taeniasis, and other parasitic infections in an enzyme-linked immunosorbent assay, cross-reactivity was observed until a serum dilution of 1:128 was reached. Since serum samples from unexposed subjects showed positive reactions until a dilution of 1:64 was reached, we chose a discriminative dilution (1:128) above which no cross-reaction was observed. The percent positive serum samples from cysticercosis patients was 100% by the enzyme-linked immunosorbent assay with any of the antigens purified by monoclonal antibodies. Images PMID:3611310

  16. A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells

    NASA Astrophysics Data System (ADS)

    Colcher, D.; Horan Hand, P.; Nuti, M.; Schlom, J.

    1981-05-01

    Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a ``pancarcinoma'' reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

  17. Optimized Expression and Purification of Humbug in Pichia pastoris and Its Monoclonal Antibody Preparation

    PubMed Central

    HUYAN, Ting; TANG, Ruihua; LI, Jing; LI, Qi; XUE, Xiaoping; YANG, Hui

    2015-01-01

    Background: The humbug gene is a truncated isoform of Aspartyl β-hydroxylase (ASPH) gene that is overexpressed in many human malignancies. In recent years, since humbug has received increasing attention, it is considered as a potential therapeutic molecular target. Therefore, it is necessary for preparing humbug protein and its monoclonal antibody to investigate its structure and function. Method: The optimized humbug gene, synthesized by Genscript in Nanjing, China on December 21st 2013, was expressed in Pichia pastoris cells that were cultured in a 10-L bioreactor. The recombinant protein was further obtained and purified by using ion exchange chromatography and Sephadex G75. The humbug protein was used to immunize Balb/c mice to generate the monoclonal antibodies. The specificity and sensitivity of the monoclonal antibodies were assessed by indirect enzyme-linked immunosorbent assay. Finally, the humbug monoclonal antibodies were used to detect the expression of humbug in several tumor cell lines via indirect immunofluorescence. Results: Firstly, the recombinant humbug was expressed in P. pastoris successfully and efficiently by using a gene-optimized strategy. Secondly, the purification process of humbug was established via multiple chromatography methods. In addition, four monoclonal antibodies against humbug were obtained from the immunized Balb/c mice, and the result of indirect immunofluorescence was indicated that the humbug monoclonal antibody showed the high affinity with humbug protein, which expressed in several tumor cell lines. Conclusion: The over-expression of recombinant humbug provides adequate sources for its structural study and the preparation of the humbug-specific monoclonal antibody can potentially be used in tumor initial diagnosis and immunotherapy. PMID:26811814

  18. Simple diagnosis of Encephalitozoon sp. microsporidial infections by using a panspecific antiexospore monoclonal antibody.

    PubMed

    Enriquez, F J; Ditrich, O; Palting, J D; Smith, K

    1997-03-01

    Microsporidia (phylum Microsproa) have recently become recognized as common opportunistic protozoans in the United States and worldwide, particularly affecting immunodeficient patients. Microsporidian organisms within the genus Encephalitozoon are the cause of nephrologic, ophthalmic, pneumologic, gastroenteric, and systemic infections. However, diagnosis of the small spores by light microscopy is difficult, even with newly developed and improved staining techniques. We have developed an anti-Encephalitozoon species monoclonal antibody-based immunoassay for easy diagnosis. A hybridoma was produced and selected following one main criterion: recognition by immunofluorescence of all known Encephalitozoon spores affecting humans. The selected monoclonal antibody-secreting hybridomas were characterized by enzyme-linked immunosorbent assay, immunofluorescence, Western blot, and immunoelectron microscopy using Encephalitozoon species from fresh and fixed samples from patients and from in vitro cultures. In the immunofluorescence assay, one monoclonal antibody, termed 3B6, strongly recognized Encephalitozoon cuniculi, E. hellem, and E. intestinalis. Monoclonal antibody 3B6 bound to other microsporidia (Nosema and Vairimorpha spp.) without cross-reacting with any other parasite, including Enterocytozoon bieneusi, fungus, or bacterium tested. In immunoelectron microscopy assays, monoclonal antibody 3B6 bound to the exospore of Encephalitozoon species, while in Western blot assays, it recognized three to seven antigens with molecular masses ranging from 34 to 117 kDa. We have developed a sensitive and specific monoclonal antibody-based immunoassay to diagnose common microsporidian infections, particularly with Encephalitozoon species. This is a new tool for identifying spores in bodily fluids and biopsy samples and is an efficient diagnostic test. Additionally, monoclonal antibody 3B6 can serve to assess the prevalence of microsporidial infections in immunodeficient and immunocompetent patients. PMID:9041420

  19. Diffusion and binding of monoclonal antibody TNT-1 in multicellular tumor spheroids

    SciTech Connect

    Cheng, F.M.; Hansen, E.B.; Taylor, C.R.; Epstein, A.L. )

    1991-02-06

    Tumor spheroids of HT-29 human colon adenocarcinoma and A375 melanoma were established to investigate the uptake and clearance kinetics of TNT-1, a monoclonal antibody that targets necrotic cells of tumors. Our data reveal that there was rapid uptake of TNT-1 and its F(ab')2 fragment in both spheroid models, whereas an antibody of irrelevant specificity, Lym-1, and its F(ab')2 fragment bound poorly to the spheroids. Unlike previously reported monoclonal antibodies to tumor cell-surface antigens, TNT-1 showed (1) a linear uptake that increased over time without saturation in tumor spheroids and (2) an unexpected uptake by a subpopulation of cells in the viable outer rim of the spheroids. These preclinical studies provide important information concerning the therapeutic potential of TNT monoclonal antibodies for the treatment of cancer and micrometastases.

  20. Virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions.

    PubMed Central

    Holland, J J; de la Torre, J C; Steinhauer, D A; Clarke, D; Duarte, E; Domingo, E

    1989-01-01

    Monoclonal antibody-resistant mutants have been widely used to estimate virus mutation frequencies. We demonstrate that standard virion neutralization inevitably underestimates monoclonal antibody-resistant mutant genome frequencies of vesicular stomatitis virus, due to phenotypic masking-mixing when wild-type (wt) virions are present in thousandsfold greater numbers. We show that incorporation of antibody into the plaque overlay medium (after virus penetration at 37 degrees C) can provide accurate estimates of genome frequencies of neutral monoclonal antibody-resistant mutant viruses in wt clones. By using this method, we have observed two adjacent G----A base transition frequencies in the I3 epitope to be of the order of 10(-4) in a wt glycine codon. This appears to be slightly lower than the frequencies observed at other sites for total (viable and nonviable) virus genomes when using a direct sequence approach. Images PMID:2479770

  1. Eradication of tumors from a human colon cancer cell line and from ovarian cancer metastases in immunodeficient mice by a single-chain Ep-CAM-/CD3-bispecific antibody construct.

    PubMed

    Schlereth, Bernd; Fichtner, Iduna; Lorenczewski, Grit; Kleindienst, Petra; Brischwein, Klaus; da Silva, Antonio; Kufer, Peter; Lutterbuese, Ralf; Junghahn, Ilse; Kasimir-Bauer, Sabine; Wimberger, Pauline; Kimmig, Rainer; Baeuerle, Patrick A

    2005-04-01

    Bispecific T-cell engager (BiTE) are a class of bispecific single-chain antibodies that can very effectively redirect cytotoxic T cells for killing of tumor target cells. Here, we have assessed the in vivo efficacy of one representative, called bscEp-CAMxCD3, with specificity for tumors overexpressing epithelial cell adhesion molecule (Ep-CAM) in human xenograft models. Cells of the human colon carcinoma line SW480 were mixed at a 1:1 ratio with unstimulated human peripheral mononuclear cells, s.c. injected in nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mice, and animals were treated with bscEp-CAMxCD3. Five daily i.v. injections of as little as 100 ng per mouse of bscEp-CAMxCD3 completely prevented tumor outgrowth when treatment was started at the day of tumor cell inoculation. BscEp-CAMxCD3 was also efficacious when administered up to 8 days after xenograft injection. Established tumors could be eradicated in all animals by five 10 microg doses given between days 8 and 12 after tumor cell inoculation. To test the efficacy of bscEp-CAMxCD3 in a more physiologic model, pieces of primary metastatic tumor tissue from ovarian cancer patients were implanted in NOD/SCID mice. Partial tumor engraftment and growth was observed with four of six patient samples. Treatment of established tumors with daily 5 microg doses led to a significant reduction and, in some cases, eradication of human tumor tissue. These effects obviously relied on the tumor-resident T cells reactivated by bscEp-CAMxCD3. Our data show that the class of single-chain bispecific antibodies has very high antitumor efficacy in vivo and can use previously unstimulated T cells at low effector-to-target ratios. PMID:15805290

  2. Improving monoclonal antibody selection and engineering using measurements of colloidal protein interactions

    PubMed Central

    Geng, Steven B.; Cheung, Jason K.; Narasimhan, Chakravarthy; Shameem, Mohammed; Tessier, Peter M.

    2014-01-01

    A limitation of using monoclonal antibodies as therapeutic molecules is their propensity to associate with themselves and/or with other molecules via non-affinity (colloidal) interactions. This can lead to a variety of problems ranging from low solubility and high viscosity to off-target binding and fast antibody clearance. Measuring such colloidal interactions is challenging given that they are weak and potentially involve diverse target molecules. Nevertheless, assessing these weak interactions especially during early antibody discovery and lead candidate optimization is critical to preventing problems that can arise later in the development process. Here we review advances in developing and implementing sensitive methods for measuring antibody colloidal interactions as well as using these measurements for guiding antibody selection and engineering. These systematic efforts to minimize non-affinity interactions are expected to yield more effective and stable monoclonal antibodies for diverse therapeutic applications. PMID:25209466

  3. Radioimmunoassay for detecting antibodies against murine malarial parasite antigens: monoclonal antibodies recognizing Plasmodium yoelii antigens

    SciTech Connect

    Kim, K.J.; Taylor, D.W.; Evans, C.B.; Asofsky, R.

    1980-12-01

    A solid-phase radioimmunoassay (SPRIA) in microtiter wells was established for detecting antibodies against Plasmodium yoelii Ag. The SPRIA was found (1) to require as little as 5 ..mu..g of crude parasite Ag per well, (2) to be able to detect 0.5 ng of monoclonal Ab, and (3) to be 10/sup 4/ times more sensitive than the indirect fluorescent Ab staining technique. In a modification of the above assay using intact RBC as an Ag, hyperimmune serum showed significant binding to the surface of erythrocytes of mice infected with P. yoelii parasites but not to RBC of normal mice. Hybridomas were prepared by fusing infected mouse spleen cells with myeloma cells. Using the SPRIA, hybrids secreting Ab against P. yoelii 17XL Ag were detected.

  4. A monoclonal antibody to human immunodeficiency virus type 1 which mediates cellular cytotoxicity and neutralization.

    PubMed Central

    Broliden, P A; Ljunggren, K; Hinkula, J; Norrby, E; Akerblom, L; Wahren, B

    1990-01-01

    Monoclonal antibodies (MAbs) were raised against human immunodeficiency virus type 1 gp120. One MAb, P4/D10, was found to mediate highly efficient antibody-dependent cellular cytotoxicity and virus neutralization. The reactivity was located to a major neutralizing region (amino acids 304 to 323) on gp120. Five other MAbs with a similar epitopic reactivity did not show any antibody-dependent cellulan cytotoxicity activity but had a virus-neutralizing capacity. PMID:2296090

  5. Molecular and structural analysis of a continuous birch profilin epitope defined by a monoclonal antibody.

    PubMed

    Wiedemann, P; Giehl, K; Almo, S C; Fedorov, A A; Girvin, M; Steinberger, P; Rdiger, M; Ortner, M; Sippl, M; Dolecek, C; Kraft, D; Jockusch, B; Valenta, R

    1996-11-22

    The interaction of a mouse monoclonal antibody (4A6) and birch profilin, a structurally well conserved actin- and phosphoinositide-binding protein and cross-reactive allergen, was characterized. In contrast to serum IgE from allergic patients, which shows cross-reactivity with most plants, monoclonal antibody 4A6 selectively reacted with tree pollen profilins. Using synthetic overlapping peptides, a continuous hexapeptide epitope was identified. The exchange of a single amino acid (Gln-47 --> Glu) within the epitope was found to abolish the binding of monoclonal antibody 4A6 to other plant profilins. The NMR analyses of the birch and the nonreactive timothy grass profilin peptides showed that the loss of binding was not due to major structural differences. Both peptides adopted extended conformations similar to that observed for the epitope in the x-ray crystal structure of the native birch profilin. Binding studies with peptides and birch profilin mutants generated by in vitro mutagenesis demonstrated that the change of Gln-47 to acidic amino acids (e.g. Glu or Asp) led to electrostatic repulsion of monoclonal antibody 4A6. In conclusion the molecular and structural analyses of the interaction of a monoclonal antibody with a continuous peptide epitope, recognized in a conformation similar to that displayed on the native protein, are presented. PMID:8939935

  6. Role of monoclonal antibodies in the treatment of immune-mediated glomerular diseases.

    PubMed

    Manrique, Joaquín; Cravedi, Paolo

    2014-05-21

    Non-specific immunosuppressants have represented for decades the only therapies for patients with immune-mediated glomerular diseases. These treatments, however, are associated with high rates of no-response and are burdened by toxicities that frequently offset the benefits of proteinuria reduction. Monoclonal antibodies targeting selective cell populations or mediators implicated in the pathophysiology of glomerular diseases have recently become available. Rituximab, a chimeric monoclonal antibody against the CD20 antigen on B cells, safely reduced proteinuria in patients with nephrotic syndrome secondary to membranous nephropathy, minimal change disease, or focal segmental glomerulosclerosis. Its ability to reduce auto-antibody formation has been instrumental to treat also ANCA-associated vasculitis, lupus nephritis, and mixed cryoglobulinemia. Many reports have also documented the efficacy of the anti-C5 humanized monoclonal antibody Eculizumab to treat atypical hemolytic uremic syndrome, C3 nephropathy, and membranoproliferative glomerulonephritis. Thanks to these encouraging findings, monoclonals are becoming very helpful tools to treat patients with glomerular diseases. Moreover, thanks to their specific mechanism of action, these and other monoclonal antibodies are important in improving our understanding of the pathophysiology of glomerular diseases. Their still high costs, however, might represent a major hurdle for their widespread implementation for all patients in need. PMID:24798567

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

    SciTech Connect

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

    1985-09-01

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

  8. Detection of antibodies to equine arteritis virus by a monoclonal antibody-based blocking ELISA.

    PubMed Central

    Cho, H J; Entz, S C; Deregt, D; Jordan, L T; Timoney, P J; McCollum, W H

    2000-01-01

    A potent ELISA antigen was prepared from equine arteritis virus (EAV) by differential centrifugation of EAV-infected cell culture fluid, followed by solubilization of the preparation by Triton X-100 treatment. Using this antigen and a mouse monoclonal antibody against the G(L) protein of EAV, a reliable blocking ELISA (bELISA) was developed for the detection of EAV antibodies in equine sera. The bELISA was evaluated using a total of 837 test serum samples. The relative sensitivity (n = 320) of the bELISA compared to the serum neutralization (SN) test was 99.4%. The bELISA appears to be a highly specific test, the specificity of which did not appear to be adversely affected by previous exposure of horses to non-EAV-containing biologicals. Of 119 serum samples, 21 from horses without any history of exposure to EAV and 98 from racetrack Thoroughbreds, 118 were negative in the SN test and bELISA. One sample was SN-negative but suspicious with the bELISA. Based on testing 465 SN-negative field samples and 52 SN-negative samples from experimental horses, and excluding any sera giving a suspicious reaction, the relative specificity of the bELISA was 97.7%. Samples should be examined undiluted and diluted 1/10 in the bELISA because the testing of sera of high neutralizing antibody titer may be affected by a prozone-like phenomenon. The bELISA is a more rapid and cost-efficient test than the SN test for the detection of EAV antibodies in equine sera. PMID:10680655

  9. Detection of microsporidia by indirect immunofluorescence antibody test using polyclonal and monoclonal antibodies.

    PubMed

    Aldras, A M; Orenstein, J M; Kotler, D P; Shadduck, J A; Didier, E S

    1994-03-01

    During a screening for monoclonal antibodies (MAbs) to the microsporidian Encephalitozoon hellem, three murine hybridoma cell lines producing strong enzyme-linked immunosorbent assay (ELISA) reactivities were cloned twice, were designated C12, E9, and E11, and were found to secrete MAbs to the immunoglobulin M isotype. On subsequent ELISAs, the three MAbs reacted most strongly to E. hellem, and they reacted somewhat less to Encephalitozoon cuniculi and least to Nosema corneum, two other microsporidian species. The MAbs produced values of absorbance against microsporidia that were at least three times greater than reactivities obtained with control hybridoma supernatants or with uninfected host cell proteins used as antigens. By Western blot immunodetection, the three MAbs detected three E. hellem antigens with relative molecular weights (M(r)s) of 62, 60, and 52 when assayed at the highest supernatant dilutions producing reactivity. At lower dilutions, the MAbs detected additional proteins with M(r)s of 55 and 53. By using indirect immunofluorescence antibody staining, the MAbs, as well as hyperimmune polyclonal murine antisera raised against E. cuniculi and E. hellem, were able to detect formalin-fixed, tissue culture-derived E. cuniculi and E. hellem and two other human microsporidia, Enterocytozoon bieneusi and Septata intestinalis, in formalin-fixed stool and urine, respectively. E. bieneusi, however, stained more intensely with the polyclonal antisera than with the MAbs. Neither the MAbs nor the hyperimmune murine polyclonal antibodies detected Cryptosporidium, Giardia, Trichomonas, or Isospora spp. At higher concentrations, the polyclonal antisera did stain N. corneum and yeast cells. The background staining could be absorbed with Candida albicans. These results demonstrate that polyclonal antisera to E. cuniculi and E. hellem, as well as MAbs raised against E. hellem, can be used for indirect immunofluorescence antibody staining to detect several species of microsporidia known to cause opportunistic infections in AIDS patients. PMID:8195366

  10. Detection of microsporidia by indirect immunofluorescence antibody test using polyclonal and monoclonal antibodies.

    PubMed Central

    Aldras, A M; Orenstein, J M; Kotler, D P; Shadduck, J A; Didier, E S

    1994-01-01

    During a screening for monoclonal antibodies (MAbs) to the microsporidian Encephalitozoon hellem, three murine hybridoma cell lines producing strong enzyme-linked immunosorbent assay (ELISA) reactivities were cloned twice, were designated C12, E9, and E11, and were found to secrete MAbs to the immunoglobulin M isotype. On subsequent ELISAs, the three MAbs reacted most strongly to E. hellem, and they reacted somewhat less to Encephalitozoon cuniculi and least to Nosema corneum, two other microsporidian species. The MAbs produced values of absorbance against microsporidia that were at least three times greater than reactivities obtained with control hybridoma supernatants or with uninfected host cell proteins used as antigens. By Western blot immunodetection, the three MAbs detected three E. hellem antigens with relative molecular weights (M(r)s) of 62, 60, and 52 when assayed at the highest supernatant dilutions producing reactivity. At lower dilutions, the MAbs detected additional proteins with M(r)s of 55 and 53. By using indirect immunofluorescence antibody staining, the MAbs, as well as hyperimmune polyclonal murine antisera raised against E. cuniculi and E. hellem, were able to detect formalin-fixed, tissue culture-derived E. cuniculi and E. hellem and two other human microsporidia, Enterocytozoon bieneusi and Septata intestinalis, in formalin-fixed stool and urine, respectively. E. bieneusi, however, stained more intensely with the polyclonal antisera than with the MAbs. Neither the MAbs nor the hyperimmune murine polyclonal antibodies detected Cryptosporidium, Giardia, Trichomonas, or Isospora spp. At higher concentrations, the polyclonal antisera did stain N. corneum and yeast cells. The background staining could be absorbed with Candida albicans. These results demonstrate that polyclonal antisera to E. cuniculi and E. hellem, as well as MAbs raised against E. hellem, can be used for indirect immunofluorescence antibody staining to detect several species of microsporidia known to cause opportunistic infections in AIDS patients. Images PMID:8195366

  11. Development, characterization, and use of monoclonal and polyclonal antibodies against the myxosporean, Ceratomyxa shasta

    USGS Publications Warehouse

    Bartholomew, J.L.; Rohovec, J.S.; Fryer, J.L.

    1989-01-01

    Both monoclonal and polyclonal antisera were produced against Ceratomyxa shasta. Ascites containing trophozoites of the parasite was collected from infected fish and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted specifically with trophozoite and sporoblast stages but did not react with C. shasta spores by either indirect fluorescent antibody techniques or in Western blots. This indicates that some C. shasta antigens are specific to certain life stages of the parasite. Polyclonal antiserum was produced in a rabbit by injecting a spore protein electro-eluted from an SDS-polyacrylamide gel. This antiserum reacted with both trophozoites and spores by indirect fluorescent antibody techniques and in Western blots. All antisera were tested for cross-reactivity to trout white blood cells, a contaminant of the ascites, and to other myxosporea. Two monoclonal antibodies reacted with white blood cells and myxosporea of the genera Sphaerospora and Myxobilatus. One hybridoma produced antibodies of high specificity for C. shasta pre-spore stages. This is the first report of a monoclonal antibody produced against a myxosporean parasite.

  12. Boronated monoclonal antibody 225. 28S for potential use in neutron capture therapy of malignant melanoma

    SciTech Connect

    Tamat, S.R.; Moore, D.E.; Patwardhan, A.; Hersey, P. )

    1989-07-01

    The concept of conjugating boron cluster compounds to monoclonal antibodies has been examined by several groups of research workers in boron neutron capture therapy (BNCT). The procedures reported to date for boronation of monoclonal antibodies resulted in either an inadequate level of boron incorporation, the precipitation of the conjugates, or a loss of immunological activity. The present report describes the conjugation of dicesium-mercapto-undecahydrododecaborate (Cs2B12H11SH) to 225.28S monoclonal antibody directed against high molecular weight melanoma-associated antigens (HMW-MAA), using poly-L-ornithine as a bridge to increase the carrying capacity of the antibody and to minimize change in the conformational structure of antibody. The method produces a boron content of 1,300 to 1,700 B atoms per molecule 225.28S while retaining the immunoreactivity. Characterization in terms of the homogeneity of the conjugation of the boron-monoclonal antibody conjugates has been studied by gel electrophoresis and ion-exchange HPLC.

  13. Immunological characterization of Rhizobium leguminosarum outer membrane antigens by use of polyclonal and monoclonal antibodies.

    PubMed Central

    de Maagd, R; de Rijk, R; Mulders, I H; Lugtenberg, B J

    1989-01-01

    Surface antigens of Rhizobium leguminosarum biovar viciae strain 248 were characterized by using polyclonal and monoclonal antibodies. With Western immunoblotting as the criterion, an antiserum raised against living whole cells recognized mainly flagellar antigens and the O-antigen-containing part of the lipopolysaccharide (LPS). Immunization of mice with a peptidoglycan-outer membrane complex yielded eight monoclonal antibodies, of which three reacted with LPS and five reacted with various sets of outer membrane protein antigens. The observation that individual monoclonal antibodies react with sets of related proteins is discussed. Studies of the influence of calcium deficiency and LPS alterations on surface antigenicity showed that in normally grown wild-type cells, the O-antigenic side chain of LPS blocks binding of an antibody to a deeper-lying antigen. This antigen is accessible to antibodies in cells grown under calcium limitation as well as in O-antigen-lacking mutant cells. Two of the antigen groups which can be distinguished in cell envelopes of free-living bacteria were depleted in cell envelopes of isolated bacteroids, indicating that the monoclonal antibodies could be useful tools for studying the differentiation process from free-living bacteria to bacteroids. Images PMID:2914865

  14. Monoclonal antibodies and recombinant immunoglobulins for the treatment of multiple sclerosis.

    PubMed

    Gensicke, Henrik; Leppert, David; Yaldizli, Özgür; Lindberg, Raija L P; Mehling, Matthias; Kappos, Ludwig; Kuhle, Jens

    2012-01-01

    Multiple sclerosis (MS) is an inflammatory and degenerative disease leading to demyelination and axonal damage in the CNS. Autoimmunity plays a central role in MS pathogenesis. Per definition, monoclonal antibodies are recombinant biological compounds with a well defined target, thus carrying the promise of targeting pathogenic cells or molecules with high specificity, avoiding undesired off-target effects. Natalizumab was the first monoclonal antibody to be approved for the treatment of MS. Several other monoclonal antibodies are in development and have demonstrated promising efficacy in phase II studies. They can be categorized according to their mode of action into compounds targeting (i) leukocyte migration into the CNS (natalizumab); (ii) cytolytic antibodies (rituximab, ocrelizumab, ofatumumab, alemtuzumab); or (iii) antibodies and recombinant proteins targeting cytokines and chemokines and their receptors (daclizumab, ustekinumab, atacicept, tabalumab [Ly-2127399], secukinumab [AIN457]). In this review, we discuss the specific molecular targets, clinical efficacy and safety of these compounds and discuss criteria to anticipate the position of monoclonal antibodies in the diversifying armamentarium of MS therapy in the coming years. PMID:22171583

  15. Monoclonal antibody capture enzyme immunoassay for detection of Paracoccidioides brasiliensis antibodies in paracoccidioidomycosis.

    PubMed Central

    Camargo, Z P; Gesztesi, J L; Saraiva, E C; Taborda, C P; Vicentini, A P; Lopes, J D

    1994-01-01

    Four murine monoclonal antibodies (MAbs 17C, 21A, 21F, and 32B) raised against the 43-kDa glycoprotein of Paracoccidioides brasiliensis were tested in a capture enzyme immunoassay (EIA) for the detection of specific human anti-gp43 immunoglobulin G in patients with paracoccidioidomycosis (PCM). All MAbs reacted similarly in the assay. These MAbs, which detected anti-gp43 at levels of as low as 500 pg/ml, were demonstrated to specifically recognize at least two different epitopes in gp43 binding assays. Specific antibodies in the sera of patients with active PCM were detected at dilutions of as high as 1:819,200, and the reactivities of patient sera, as measured by optical densities, were found to be significantly higher than those of control sera. The comparison between classical ELISA and our capture enzyme immunoassay showed that both sensitivity and specificity were greatly improved by the latter. These MAbs represent the first specific reagents to P. brasiliensis described for use in serological tests for PCM. Images PMID:7814469

  16. DETECTION OF ROTAVIRUS WITH A NEW POLYCLONAL ANTIBODY ENZYME IMMUNOASSAY (ROTAZYME 2) AND A COMMERCIAL LATEX AGGLUTINATION TEXT (ROTALEX): COMPARISON WITH A MONOCLONAL ANTIBODY ENZYME IMMUNOASSAY

    EPA Science Inventory

    A total of 176 human fecal specimens were examined for the presence of rotavirus using four different assays: a monoclonal antibody enzyme immunoassay; the original polyclonal antibody enzyme immunoassay marketed by Abbott Laboratories, Chicago, IL (Rotazyme I); a modification of...

  17. 8th Annual European Antibody Congress 2012

    PubMed Central

    Beck, Alain; Carter, Paul J.; Gerber, Hans-Peter; Lugovskoy, Alexey A.; Wurch, Thierry; Junutula, Jagath R.; Kontermann, Roland E; Mabry, Robert

    2013-01-01

    The 8th European Antibody Congress (EAC), organized by Terrapin Ltd., was again held in Geneva, Switzerland, following on the tradition established with the 4th EAC. The new agenda format for 2012 included three parallel tracks on: (1) naked antibodies; (2) antibody drug conjugates (ADCs); and (3) bispecific antibodies and alternative scaffolds. The meeting started and closed with three plenary lectures to give common background and to share the final panel discussion and conclusions. The two day event included case studies and networking for nearly 250 delegates who learned of the latest advances and trends in the global development of antibody-based therapeutics. The monoclonal antibody track was focused on understanding the structure-function relationships, optimization of antibody design and developability, and processes that allow better therapeutic candidates to move through the clinic. Discussions on novel target identification and validation were also included. The ADC track was dedicated to evaluation of the ongoing success of the established ADC formats alongside the rise of the next generation drug-conjugates. The bispecific and alternative scaffold track was focused on taking stock of the multitude of bispecific formats being investigated and gaining insight into recent innovations and advancements. Mechanistic understanding, progression into the clinic and the exploration of multispecifics, redirected T cell killing and alternative scaffolds were extensively discussed. In total, nearly 50 speakers provided updates of programs related to antibody research and development on-going in the academic, government and commercial sectors. PMID:23493119

  18. A monoclonal antibody suitable for the radioimmunoassay of prolactin in human serum.

    PubMed

    Stuart, M C; Underwood, P A; Boscato, L

    1982-05-01

    Monoclonal antibodies directed toward human PRL (hPRL) have been produced by fusion of mouse myeloma cells (Sp2/0-Ag 14) with spleen cells from mice immunized with hPRL. Total immunizing doses of 20 microgram and 64 microgram hPRL resulted in the production of three highly specific hPRL antibodies. The high affinity antibody, with a Ka value of 0.23 X 10(10) M-1, was used to establish a RIA highly suitable for the measurement of hPRL levels in human serum. The correlation of serum hPRL levels measured using the antibody and those in a conventional rabbit anti-hPRL assay was 0.99 (y = 1.16 - 7.2). These results demonstrate that using the mouse hybridoma technique, it is possible to produce high affinity monospecific monoclonal antibody suitable for the measurement of hPRL in human serum. PMID:7061699

  19. Development of monoclonal antibodies against parathyroid hormone: genetic control of the immune response to human PTH

    SciTech Connect

    Nussbaum, S.R.; Lin, C.S.; Potts, J.T. Jr.; Rosenthal, A.S.; Rosenblatt, M.

    1985-01-01

    Seventeen monocloanl antibodies against the aminoterminal portion of parathyroid hormone (PTH) were generated by using BALB/c mouse for immunization fully biologically active synthetic human PTH-(1-34) and bovine PTH-(1-84) as immunogens, monoclonal antibody methods, and a solid-phase screening assay. Isotypic analysis of these monoclonal antibodies was performed using affinity purified goat antimouse immunoglobulins specific for IgG heavy chains and ..mu..(IgM). All antibodies were IgM as evidenced by 40 times greater than background activity when 25,000 cpm of /sup 125/I-labelled goat anti-mouse IgM was used as second antibody in a radioimmunoassay.

  20. The Cloning and Expression of Human Monoclonal Antibodies: Implications for Allergen Immunotherapy.

    PubMed

    James, Louisa K

    2016-01-01

    Allergic responses are dependent on the highly specific effector functions of IgE antibodies. Conversely, antibodies that block the activity of IgE can mediate tolerance to allergen. Technologies that harness the unparalleled specificity of antibody responses have revolutionized the way that we diagnose and treat human disease. This area of research continues to advance at a rapid pace and has had a significant impact on our understanding of allergic disease. This review will present an overview of humoral responses and provide an up-to-date summary of technologies used in the generation of human monoclonal antibodies. The impact that monoclonal antibodies have on allergic disease will be discussed, with a particular focus on allergen immunotherapy, which remains the only form of treatment that can modulate the underlying immune mechanisms and induce long-term clinical tolerance. PMID:26780523

  1. Production of polyhedrin monoclonal antibodies for distinguishing two Orgyia pseudotsugata baculoviruses.

    PubMed Central

    Quant, R L; Pearson, M N; Rohrmann, G F; Beaudreau, G S

    1984-01-01

    Monoclonal antibodies were produced to polyhedrins from Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) and single-capsid nuclear polyhedrosis virus (OpSNPV). Although the polyhedrins are closely related, antibodies were selected which allowed differentiation between the two viruses. In an indirect enzyme-linked immunosorbent assay, purified OpMNPV and OpSNPV polyhedrins could be detected by specific monoclonal antibodies at concentrations as low as 2 and 5 ng/ml, respectively. The antibodies were also capable of identifying their homologous polyhedrin in extracts of infected insects. These antibodies would be useful for monitoring production of the viral insecticide, TM Biocontrol-1, which by license must contain only OpMNPV, and to confirm that insect mortality after aerial spraying with this insecticide is attributable to OpMNPV infection. Images PMID:6391377

  2. Australian consensus guidelines for the safe handling of monoclonal antibodies for cancer treatment by healthcare personnel.

    PubMed

    Alexander, M; King, J; Bajel, A; Doecke, C; Fox, P; Lingaratnam, S; Mellor, J D; Nicholson, L; Roos, I; Saunders, T; Wilkes, J; Zielinski, R; Byrne, J; MacMillan, K; Mollo, A; Kirsa, S; Green, M

    2014-10-01

    These consensus guidelines provide recommendations for the safe handling of monoclonal antibodies. Definitive recommendations are given for the minimum safe handling requirements to protect healthcare personnel. The seven recommendations cover: (i) appropriate determinants for evaluating occupational exposure risk; (ii) occupational risk level compared with other hazardous and non-hazardous drugs; (iii) stratification of risk based on healthcare personnel factors; (iv) waste products; (v) interventions and safeguards; (vi) operational and clinical factors and (vii) handling recommendations. The seventh recommendation includes a risk assessment model and flow chart for institutions to consider and evaluate clinical and operational factors unique to individual healthcare services. These guidelines specifically evaluated monoclonal antibodies used in the Australian cancer clinical practice setting; however, the principles may be applicable to monoclonal antibodies used in non-cancer settings. The guidelines are only applicable to parenterally administered agents. PMID:25302720

  3. Monoclonal antibody therapy in multiple myeloma: where do we stand and where are we going?

    PubMed

    Thanendrarajan, Sharmilan; Davies, Faith E; Morgan, Gareth J; Schinke, Carolina; Mathur, Pankaj; Heuck, Christoph J; Zangari, Maurizio; Epstein, Joshua; Yaccoby, Shmuel; Weinhold, Niels; Barlogie, Bart; van Rhee, Frits

    2016-03-01

    Multiple myeloma is a plasma cell malignancy that is characterized by refractory and relapsing course of disease. Despite the introduction of high-dose chemotherapy in combination with autologous stem cell transplantation and innovative agents such as proteasome inhibitors and immunomodulatory drugs, achieving cure in multiple myeloma is a challenging endeavor. In the last couple of years, enormous advances were made in implementing monoclonal antibody therapy in multiple myeloma. A large number of preclinical and clinical studies have been introduced successfully, demonstrating a safe and efficient administration of monoclonal antibodies in multiple myeloma. In particular, the application of monoclonal antibodies in combination with immunomodulatory drugs, proteasome inhibitors, corticosteroids or conventional chemotherapy seem to be promising and will expand the treatment arsenal for patients with multiple myeloma. PMID:26888183

  4. A monoclonal antibody for distinction of invasive and noninvasive clinical isolates of Entamoeba histolytica.

    PubMed Central

    Gonzalez-Ruiz, A; Haque, R; Rehman, T; Aguirre, A; Jaramillo, C; Castaon, G; Hall, A; Guhl, F; Ruiz-Palacios, G; Warhurst, D C

    1992-01-01

    Approximately 10% of the world population is infected with Entamoeba histolytica, but only 10% of the carriers develop symptomatic amebiasis. This discrepancy could be explained by the genotypic differences between the morphologically indistinguishable invasive and noninvasive strains of E. histolytica currently identified by zymodeme analysis, a technique that is unsuitable for routine diagnostic laboratories. Here we report the production of a monoclonal antibody against E. histolytica and its use in an immunofluorescence assay to identify invasive isolates cultured from stool samples of infected patients in several regions where amebiasis is endemic: Bangladesh, Colombia, and Mexico. After testing a total of 88 E. histolytica isolates, the correlation between zymodeme characterization and the immunofluorescence assay with the invasive isolate-specific monoclonal antibody was 100%. The epitope detected by the invasive isolate-specific monoclonal antibody resides in a previously undescribed internal protein with molecular masses of 84 and 81 kDa in axenic and polyxenic E. histolytica strains, respectively. Images PMID:1452651

  5. Simultaneous Raising of Rabbit Monoclonal Antibodies to Fluoroquinolones with Diverse Recognition Functionalities via Single Mixture Immunization.

    PubMed

    Liu, Na; Zhao, Zhiyong; Tan, Yanglan; Lu, Lei; Wang, Lin; Liao, Yucai; Beloglazova, Natalia; De Saeger, Sarah; Zheng, Xiaodong; Wu, Aibo

    2016-01-19

    Highly specific monoclonal and polyclonal antibodies are the key components in a diverse set of immunoassay applications, from research work to routine monitoring and analysis. In the current manuscript, combinatorial strategies for a single mixture immunization, screening and rabbit hybridoma cell technology were described. Fluoroquinolones (FQs) drugs were chosen as representative analytes. Six FQs were conjugated with bovine serum albumin and used as immunogens for subsequent immunization, while a mixture of all was injected for coimmunization. The hybridomas obtained against the individual and multiple FQs were used for the production of diverse varieties of rabbit monoclonal antibodies (RabMAbs) against the target analytes. As was proven by indirect competitive ELISA and quantitative lateral flow immunoassay, this approach opens a new way for simultaneously obtaining functional monoclonal antibodies which are capable of recognizing both individual and multiple analytes in a single preparation circle. This addresses various needs of different monitoring regulations as analytical methodology advances. PMID:26653330

  6. The effect of space flight on monoclonal antibody synthesis in a hybridoma mouse cell line

    NASA Technical Reports Server (NTRS)

    Smiley, S. A.; Gillock, E. T.; Black, M. C.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1997-01-01

    The hybridoma cell line, 3G10G5, producing a monoclonal antibody to the major capsid protein VP1 from the avian polyomavirus budgerigar fledgling disease virus, was produced from a Balb/C mouse. This cell line was used to test the effects of microgravity on cellular processes, specifically protein synthesis. A time course study utilizing incorporation of [35S]methionine into newly synthesized monoclonal antibody was performed on STS-77. After 5.5 days, it was observed that cell counts for the samples exposed to microgravity were lower than those of ground-based samples. However, radiolabel incorporation of the synthesized monoclonal antibody was similar in both orbiter and ground control samples. Overall, microgravity does not seem to have an effect on this cell line's ability to synthesize IgG protein.

  7. Engineered monoclonal antibody with novel antigen-sweeping activity in vivo.

    PubMed

    Igawa, Tomoyuki; Maeda, Atsuhiko; Haraya, Kenta; Tachibana, Tatsuhiko; Iwayanagi, Yuki; Mimoto, Futa; Higuchi, Yoshinobu; Ishii, Shinya; Tamba, Shigero; Hironiwa, Naoka; Nagano, Kozue; Wakabayashi, Tetsuya; Tsunoda, Hiroyuki; Hattori, Kunihiro

    2013-01-01

    Monoclonal antibodies are widely used to target disease-related antigens. However, because conventional antibody binds to the antigen but cannot eliminate the antigen from plasma, and rather increases the plasma antigen concentration by reducing the clearance of the antigen, some clinically important antigens are still difficult to target with monoclonal antibodies because of the huge dosages required. While conventional antibody can only bind to the antigen, some natural endocytic receptors not only bind to the ligands but also continuously eliminate them from plasma by pH-dependent dissociation of the ligands within the acidic endosome and subsequent receptor recycling to the cell surface. Here, we demonstrate that an engineered antibody, named sweeping antibody, having both pH-dependent antigen binding (to mimic the receptor-ligand interaction) and increased binding to cell surface neonatal Fc receptor (FcRn) at neutral pH (to mimic the cell-bound form of the receptor), selectively eliminated the antigen from plasma. With this novel antigen-sweeping activity, antibody without in vitro neutralizing activity exerted in vivo efficacy by directly eliminating the antigen from plasma. Moreover, conversion of conventional antibody with in vitro neutralizing activity into sweeping antibody further potentiated the in vivo efficacy. Depending on the binding affinity to FcRn at neutral pH, sweeping antibody reduced antigen concentration 50- to 1000-fold compared to conventional antibody. Thereby, sweeping antibody antagonized excess amounts of antigen in plasma against which conventional antibody was completely ineffective, and could afford marked reduction of dosage to a level that conventional antibody can never achieve. Thus, the novel mode of action of sweeping antibody provides potential advantages over conventional antibody and may allow access to the target antigens which were previously undruggable by conventional antibody. PMID:23667591

  8. Production and characterization of monoclonal antibodies to soluble rat lung guanylate cyclase.

    PubMed Central

    Brandwein, H; Lewicki, J; Murad, F

    1981-01-01

    Four monoclonal antibodies to rat lung soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing) EC 4.6.1.2] have been produced by fusing spleen cells from immunized BALB/c mice with SP-2/0 myeloma cells. The antibodies were detected by their ability to bind immobilized guanylate cyclase and by immunoprecipitation of purified enzyme in the presence of second (rabbit anti-mouse) antibody. After subcloning by limiting dilution, hybridomas were injected intraperitoneally into mice to produce ascitic fluid containing 2-5 mg of antibody per ml. The four antibodies obtained had titers of between 1:1580 and 1:3160 but were detectable at dilutions greater than 1:20,000. Soluble guanylate cyclase from several rat tissues were crossreactive with the four monoclonal antibodies, suggesting that the soluble enzyme from different rat tissues is antigenically similar. The antibodies also recognized soluble lung enzyme from rat, beef, and pig, while enzyme from rabbit was not crossreactive and mouse enzyme was recognized by only one of the antibodies. Particulate guanylate cyclase from a number of tissues had only minimal crossreactivity with the antibodies. Immunoprecipitated guanylate cyclase retained catalytic activity, could be activated with sodium nitroprusside, and was inhibited by cystamine. None of the antibodies were inhibitory under the conditions examined. These antibodies will be useful probes for the study of guanylate cyclase regulation and function under a variety of physiological conditions. PMID:6117073

  9. Production and Characterization of Monoclonal Antibodies against Aspartate Aminotransferase-P1 from Lupin Root Nodules.

    PubMed Central

    Jones, W. T.; Jones, S. D.; Harvey, D.; Rodber, K. R.; Ryan, G. B.; Reynolds, PHS.

    1994-01-01

    Six hybridoma clones were obtained that secreted monoclonal antibodies against the aspartate aminotransferase-P1 (AAT-P1) isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. This enzyme is found constitutively in the plant cytosol fraction. The monoclonal antibodies produced were all of the immunoglobulin G1 class, recognized two distinct epitopes on the protein, and represented the major paratopes found in the immunoglobulin fraction of sera taken from mice and rabbits immunized with the pure AAT-P1 protein. One of these epitopes was unique to lupin nodule AAT-P1. The other epitope was shown to be present on enzyme from lupin bean, white clover and tobacco leaves, lupin roots and nodules, and potato tubers. Both epitopes were recognized by the appropriate monoclonal antibodies in both their native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium lupini NZP2257, Escherichia coli extracts, or with the inducible aspartate aminotransferase-P2 (AAT-P2) isoform also found in root nodules. A sandwich enzyme-linked immunosorbent assay utilizing two monoclonal antibodies recognizing the two distinct epitopes was developed and was capable of quantitating AAT-P1 in plant extracts. The limit of detection of AAT-P1 was less than 15 pg/mL and AAT-P1 protein could be quantified in the range 80 to 1000 pg/mL. Using this assay, AAT-P1 protein was shown to remain relatively constant during nodule development. Use of an AAT-P2-specific monoclonal antibody that inhibits the enzyme activity of this isoform enabled the direct determination of AAT-P1 enzyme activity in nodule extracts. Using these assays, specific activities of the individual isoforms were calculated; that of the AAT-P1 isoform was shown to be 7.5-fold higher than that of the AAT-P2 isoform. PMID:12232065

  10. Development of a stable radioiodinating reagent to label monoclonal antibodies for radiotherapy of cancer

    SciTech Connect

    Wilbur, D.S.; Hadley, S.W.; Hylarides, M.D.; Abrams, P.G.; Beaumier, P.A.; Morgan, A.C.; Reno, J.M.; Fritzberg, A.R. )

    1989-02-01

    A method of radioiodinating monoclonal antibodies such that the labeled antibodies do not undergo in vivo deiodination has been studied. The method utilizes conjugation of succinimidyl para-iodobenzoate to the antibody. The iodobenzoate was radiolabeled by using an organometallic intermediate to facilitate the reaction. Thus, succinimidyl para-tri-n-butylstannylbenzoate was radiolabeled in 60-90% radiochemical yield and subsequently conjugated to the antibody in 80-90% yield. Animal biodistribution studies were carried out with two separate anti-melanoma antibodies (9.2.27 and NR-M1-05) labeled by this method, and examined in nude mice bearing human melanoma tumor xenografts. Very large differences in the localization of radioactivity were observed in the thyroids and stomachs of mice when the iodobenzoyl-labeled antibodies were compared with the same antibodies labeled using the chloramine-T method of radioiodination. Few other significant differences in the tissue distribution of the radioiodinated antibodies were seen.

  11. Monoclonal antibody, mAb 4C13, an effective detoxicant antibody against ricin poisoning.

    PubMed

    Dong, Na; Luo, Longlong; Wu, Junhua; Jia, Peiyuan; Li, Qian; Wang, Yuxia; Gao, Zhongcai; Peng, Hui; Lv, Ming; Huang, Chunqian; Feng, Jiannan; Li, Hua; Shan, Junjie; Han, Gang; Shen, Beifen

    2015-07-31

    Ricin is a glycoprotein produced in castor seeds and consists of two polypeptide chains named Ricin Toxin A Chain (RTA) and Ricin Toxin B Chain (RTB), linked via a disulfide bridge. Due to its high toxicity, ricin is regarded as a high terrorist risk for the public. However, antibodies can play a pivotal role in neutralizing the toxin. In this research, the anti-toxicant effect of mAb 4C13, a monoclonal antibody (mAb) established using detoxicated ricin as the immunized antigen, was evaluated. Compared with mAb 4F2 and mAb 5G6, the effective mechanism of mAb 4C13 was analyzed by experiments relating to its cytotoxicity, epitope on ricin, binding kinetics with the toxin, its blockage on the protein synthesis inhibition induced by ricin and the intracelluar tracing of its complex with ricin. Our result indicated that mAb 4C13 could recognize and bind to RTA, RTB and exert its high affinity to the holotoxin. Both cytotoxicity and animal toxicity of ricin were well blocked by pre-incubating the toxin with mAb 4C13. By intravenous injection, mAb 4C13 could rescue the mouse intraperitoneally (ip) injected with a lethal dose of ricin (20μg/kg) even at 6h after the intoxication and its efficacy was dependent on its dosage. This research indicated that mAb 4C13 could be an excellent candidate for therapeutic antibodies. Its potent antitoxic efficiency was related to its recognition on the specific epitope with very high affinity and its blockage of protein synthesis inhibition in cytoplasm followed by cellular internalization with ricin. PMID:26141013

  12. Neutralizing monoclonal antibodies specific for herpes simplex virus glycoprotein D inhibit virus penetration.

    PubMed Central

    Highlander, S L; Sutherland, S L; Gage, P J; Johnson, D C; Levine, M; Glorioso, J C

    1987-01-01

    Nine monoclonal antibodies specific for glycoprotein D (gD) of herpes simplex virus type 1 were selected for their ability to neutralize virus in the presence of complement. Four of these antibodies exhibited significant neutralization titers in the absence of complement, suggesting that their epitope specificities are localized to site(s) which contribute to the role of gD in virus infectivity. Each of these antibodies was shown to effectively neutralize virus after virion adsorption to cell surfaces, indicating that neutralization did not involve inhibition of virus attachment. Although some of the monoclonal antibodies partially inhibited adsorption of radiolabeled virions, this effect was only observed at concentrations much higher than that required to neutralize virus and did not correlate with complement-independent virus-neutralizing activity. All of the monoclonal antibodies slowed the rate at which virus entered cells, further suggesting that antibody binding of gD inhibits virus penetration. Experiments were carried out to determine the number of different epitopes recognized by the panel of monoclonal antibodies and to identify epitopes involved in complement-independent virus neutralization. Monoclonal antibody-resistant (mar) mutants were selected by escape from neutralization with individual gD-specific monoclonal antibodies. The reactivity patterns of the mutants and antibodies were then used to construct an operational antigenic map for gD. This analysis identified a minimum of six epitopes on gD that could be grouped into four antigenic sites. Antibodies recognizing four distinct epitopes contained in three antigenic sites were found to neutralize virus in a complement-independent fashion. Moreover, mar mutations in these sites did not affect the processing of gD, rate of virus penetration, or the ability of the virus to replicate at high temperature (39 degrees C). Taken together, these results (i) confirm that gD is a major target antigen for neutralizing antibody, (ii) indicate that the mechanism of neutralization can involve inhibition of virus penetration of the cell surface membrane, and (iii) strongly suggest that gD plays a direct role in the virus entry process. PMID:2444713

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

    SciTech Connect

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

    1987-11-01

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

  14. Onco-fetal specific monoclonal antibodies, methods of preparation and use

    SciTech Connect

    Coggin, J.H. Jr.; Payne, W.J. Jr.

    1987-08-11

    A process is described of preparing a hybridoma secreting oncofetal specific monoclonal antibodies, which comprises: (a) immunizing an animal with immunizing amounts of a non-proliferating syngeneic mid-gestation fetal cell preparation; (b) isolating immunized lymphocytes from the animal; and (c) fusing the lymphocytes under appropriate fusion conditions with an immortalizing cell line to thereby obtain the hybridoma. A hybridoma cell line produces a monoclonal antibody having the following specificity characteristics: (a) immune reactivity towards rodent mid-gestation antigens; (b) immune reactivity towards human onco-fetal tumor antigens; (c) substantially no immune reactivity towards rodent late gestational fetal tissue; and (d) substantially no immune reactivity towards human normal tissue.

  15. Pretargeted Radioimmunotherapy of Prostate Cancer with an Anti-TROP-2Anti-HSG Bispecific Antibody and a 177Lu-Labeled Peptide

    PubMed Central

    Frielink, Cathelijne; Goldenberg, David M.; Sharkey, Robert M.; Ltje, Susanne; McBride, William J.; Oyen, Wim J.G.; Boerman, Otto C.

    2014-01-01

    Abstract TROP-2 is a pancarcinoma marker that is expressed at high levels in many epithelial cancers, including prostate cancer (PC). The trivalent bispecific antibody TF12 (anti-TROP2anti-HSG [histamine-succinyl-glycine]) has shown to effectively target PC. In this study, the efficacy of pretargeted radioimmunotherapy (PRIT) with multiple cycles of TF12 and 177Lu-labeled diHSG-peptide (IMP288) in mice with s.c. PC3 tumors was investigated and compared with that of conventional RIT with 177Lu-labeled anti-TROP-2 mAb hRS7. Methods: The potential of one, two, and three cycles of PRIT using the TF12 pretargeted 177Lu-IMP288 (41?MBq per cycle) was determined in mice with s.c. PC3 tumors, and compared with the efficacy and toxicity of RIT with 177Lu-hRS7 dosed at the maximum tolerated dose (11?MBq). Results: PRIT of two and three cycles showed significantly higher median survival (>150 days) compared with PRIT of one cycle of TF12 and 177Lu-IMP288 (111 days, p<0.001) or the controls (76 days, p<0.0001). All mice treated with the mAb 177Lu-hRS7 survived at the end of the experiment (150 days), compared with 80% in the mice that were treated with three cycles of PRIT and 70% in the group that received two cycles of PRIT. Clinically significant hematologic toxicity was found only in the groups that received either three cycles of PRIT (p<0.0009) or RIT (p<0.0001). Conclusions: TROP-2-expressing PC can be targeted efficiently with TF12 and radiolabeled IMP288. 177Lu-IMP288 accumulated rapidly in the tumors. PRIT of multiple cycles inhibited the growth of s.c. PC3 tumors. Clinically relevant hematological toxicity was observed in the group that received three cycles of PRIT; however, conventional RIT with the parent mAb 177Lu-hRS7 was at least as effective with similar toxicity. PMID:25226447

  16. Bispecific Antibody Conjugated Manganese-Based Magnetic Engineered Iron Oxide for Imaging of HER2/neu- and EGFR-Expressing Tumors

    PubMed Central

    Wu, Shou-Cheng; Chen, Yu-Jen; Wang, Hsiang-Ching; Chou, Min-Yuan; Chang, Teng-Yuan; Yuan, Shyng-Shiou; Chen, Chiao-Yun; Hou, Ming-Feng; Hsu, John Tsu-An; Wang, Yun-Ming

    2016-01-01

    The overexpression of HER2/neu and EGFR receptors plays important roles in tumorigenesis and tumor progression. Targeting these two receptors simultaneously can have a more widespread application in early diagnosis of cancers. In this study, a new multifunctional nanoparticles (MnMEIO-CyTE777-(Bis)-mPEG NPs) comprising a manganese-doped iron oxide nanoparticle core (MnMEIO), a silane-amino functionalized poly(ethylene glycol) copolymer shell, a near infrared fluorescence dye (CyTE777), and a covalently conjugated anti-HER2/neu and anti-EGFR receptors bispecific antibody (Bis) were successfully developed. In vitro T2-weighted MR imaging studies in SKBR-3 and A431 tumor cells incubated with MnMEIO-CyTE777-(Bis)-mPEG NPs showed - 94.8 ± 3.8 and - 84.1 ± 2.8% negative contrast enhancement, respectively. Pharmacokinetics study showed that MnMEIO-CyTE777-(Bis)-mPEG NPs were eliminated from serum with the half-life of 21.3 mins. In vivo MR imaging showed that MnMEIO-CyTE777-(Bis)-mPEG NPs could specifically and effectively target to HER2/neu- and EGFR-expressing tumors in mice; the relative contrast enhancements were 11.8 (at 2 hrs post-injection) and 61.5 (at 24 hrs post-injection) fold higher in SKBR-3 tumors as compared to Colo-205 tumors. T2-weighted MR and optical imaging studies revealed that the new contrast agent (MnMEIO-CyTE777-(Bis)-mPEG NPs) could specifically and effectively target to HER2/neu- and/or EGFR-expressing tumors. Our results demonstrate that MnMEIO-CyTE777-(Bis)-mPEG NPs are able to recognize the tumors expressing both HER2/neu and/or EGFR, and may provide a novel molecular imaging tool for early diagnosis of cancers expressing HER2/neu and/or EGFR. PMID:26722378

  17. Bispecific Antibody Conjugated Manganese-Based Magnetic Engineered Iron Oxide for Imaging of HER2/neu- and EGFR-Expressing Tumors.

    PubMed

    Wu, Shou-Cheng; Chen, Yu-Jen; Wang, Hsiang-Ching; Chou, Min-Yuan; Chang, Teng-Yuan; Yuan, Shyng-Shiou; Chen, Chiao-Yun; Hou, Ming-Feng; Hsu, John Tsu-An; Wang, Yun-Ming

    2016-01-01

    The overexpression of HER2/neu and EGFR receptors plays important roles in tumorigenesis and tumor progression. Targeting these two receptors simultaneously can have a more widespread application in early diagnosis of cancers. In this study, a new multifunctional nanoparticles (MnMEIO-CyTE777-(Bis)-mPEG NPs) comprising a manganese-doped iron oxide nanoparticle core (MnMEIO), a silane-amino functionalized poly(ethylene glycol) copolymer shell, a near infrared fluorescence dye (CyTE777), and a covalently conjugated anti-HER2/neu and anti-EGFR receptors bispecific antibody (Bis) were successfully developed. In vitro T 2-weighted MR imaging studies in SKBR-3 and A431 tumor cells incubated with MnMEIO-CyTE777-(Bis)-mPEG NPs showed - 94.8 3.8 and - 84.1 2.8% negative contrast enhancement, respectively. Pharmacokinetics study showed that MnMEIO-CyTE777-(Bis)-mPEG NPs were eliminated from serum with the half-life of 21.3 mins. In vivo MR imaging showed that MnMEIO-CyTE777-(Bis)-mPEG NPs could specifically and effectively target to HER2/neu- and EGFR-expressing tumors in mice; the relative contrast enhancements were 11.8 (at 2 hrs post-injection) and 61.5 (at 24 hrs post-injection) fold higher in SKBR-3 tumors as compared to Colo-205 tumors. T 2-weighted MR and optical imaging studies revealed that the new contrast agent (MnMEIO-CyTE777-(Bis)-mPEG NPs) could specifically and effectively target to HER2/neu- and/or EGFR-expressing tumors. Our results demonstrate that MnMEIO-CyTE777-(Bis)-mPEG NPs are able to recognize the tumors expressing both HER2/neu and/or EGFR, and may provide a novel molecular imaging tool for early diagnosis of cancers expressing HER2/neu and/or EGFR. PMID:26722378

  18. RIA of thyroglobulin using monoclonal antibodies: Minimal interference by anti-thyroglobulin autoantibodies

    SciTech Connect

    Nakashima, T.; Koizumi, M.; Sakahara, H.; Ohta, H.; Kohsaka, T.; Misaki, T.; Iida, Y.; Kasagi, K.; Endo, K.; Konishi, J.

    1985-05-01

    Thyroglobulin (Tg) is considered to be secreted from the thyroid gland with the stimulation of TSH and/or thyroid stimulating immunoglobulins. However its use as a prognostic marker for Graves' disease is hampered by anti-Tg autoantibodies in patients' serum. In order to resolve this drawback, the authors have developed monoclonal antibodies to human Tg with very little cross-reactivities with autoantiobodies. Nine monoclonal antibodies were produced by the immunization with Tg prepared from Graves' thyroid and one of them (IgGl), designated as 59A, showed the highest affinity to Tg (3.6 x 10/sup 40/M/sup -1/) and the least cross-reactivity with anti-Tg autoantibodies. The binding of I-125 labeled 59A to beads coated with Tg was not inhibited by the addition of purified IgG obtained from various thyroid diseases except a few Hashimoto's patients with very high titer of anti-Tg antibodies, although the binding of other monoclonal antibodies to Tg was greatly influenced even in the presence of Graves' IgG. The sensitivity of the assay using 59A was enough to detect 20ng Tg/ml and Tg concentrations, in patients with no detectable anti-Tg antibodies, were comparable to those determined by the conventional RIA kit (Eiken), using radioiodinated Tg and polyclonal rabbit anti-Tg antiserum. Further, the shelf-life of I-125 labeled monoclonal antibody was much longer than the radioiodinated Tg. These results indicated that RIA of Tg using monoclonal antibodies would be useful for measuring Tg values not only in patients with thyroid cancer but also in Graves' disease with anti-Tg autoantibodies.

  19. Discovery of lung cancer biomarkers by profiling the plasma proteome with monoclonal antibody libraries.

    PubMed

    Guergova-Kuras, Mariana; Kurucz, Istvn; Hempel, William; Tardieu, Nadge; Kdas, Jnos; Malderez-Bloes, Carole; Jullien, Anne; Kieffer, Yann; Hincapie, Marina; Guttman, Andrs; Csnky, Eszter; Dezso, Balzs; Karger, Barry L; Takcs, Lszl

    2011-12-01

    A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ. The approach is capable of generating independent antibodies against different epitopes of the same proteins, allowing fast translation to multiplexed sandwich assays. Based on these results, we have verified in two independent clinical collections a panel of five biomarkers for classifying patient disease status with a diagnostics performance of 77% sensitivity and 87% specificity. Combining CYFRA, an established cancer marker, with the panel resulted in a performance of 83% sensitivity at 95% specificity for stage I NSCLC. PMID:21947365

  20. Production, characterization, and protective effect of monoclonal antibodies to Clostridium chauvoei flagella.

    PubMed Central

    Tanaka, M; Hirayama, N; Tamura, Y

    1987-01-01

    Monoclonal antibodies to flagella of Clostridium chauvoei were obtained by the fusion of murine myeloma cells (P3-X63-Ag8-U1) and spleen cells from BALB/c mice immunized with partially purified flagella of strain Okinawa. Enzyme-linked immunosorbent assay and Western blot analysis with partially purified flagella, flagellated cells, and nonflagellated mutants were used to show that five monoclonal antibodies are specific for the flagella. In the Western blot analysis, all five antiflagellar antibodies reacted strongly with the 56,000-molecular-weight protein, which corresponds to the flagellin. By using the ELISA-derived reactivity of monoclonal antibodies to the various clostridia and the competitive binding assay, we showed that the flagella of C. chauvoei had at least three epitopes. The three antiflagellar monoclonal antibodies (one immunoglobulin G and two immunoglobulin M) demonstrated passive protective effects in mice. These results strongly suggest that the flagella of C. chauvoei are important for protective immunity in mice. Images PMID:3301677

  1. Effect of FGF10 monoclonal antibody on psoriasis-like model in guinea pigs

    PubMed Central

    Xia, Jian-Xin; Mei, Xiang-Lin; Zhu, Wen-Jing; Li, Xue; Jin, Xian-Hua; Mou, Yan; Yu, Kai; Wang, Yi-Yu; Li, Fu-Qiu

    2014-01-01

    Objective: To investigate the therapeutical effect of topical application of FGF10 monoclonal antibody on the guinea pig model with psoriasis. Methods: Blank group, model group, hydrocortisone butyrate treatment group and high-dose (0.188mg/ml), middle-dose (0.094mg/ml) and low-dose (0.063mg/ml) FGF10 antibody group were set, respectively. After two-week treatment, pathological changes of psoriasis-like models were observed by HE staining, and the difference in VEGF and PCNA expression levels among different groups was observed by immunohistochemical staining. Results: All the test indicators of each treatment group were lower than those of the model group, and there was a significant difference (P<0.05). The inflammatory cell count of the high-dose FGF10 antibody group was not statistically different from those of the blank group (t=0.77, P=0.443), and the counts of the rest treatment groups were significantly higher than those of the blank group and the high-dose FGF10 antibody group (P<0.05). The epidermal thickness of each FGF10 antibody treatment group was significantly higher than that of hydrocortisone butyrate treatment group (P<0.05), while no statistical difference was found in the epidermal thickness among the FGF10 antibody treatment groups (P>0.05). FGF10 monoclonal antibodies can reduce the PCNA and VEGF expression in psoriasis-like model of guinea pig’s ear. Conclusion: FGF10 monoclonal antibodies can affect keratinocyte proliferation and division and can also significantly inhibit the inflammatory response in the psoriasis model. Meanwhile, FGF10 monoclonal antibodies can produce a therapeutic effect on psoriatic lesions by inhibiting the abnormal epidermis cell proliferation and neovascularization of the dermis in the psoriasis model. PMID:24966930

  2. Monoclonal antibodies to adenosine receptor by an auto-anti-idiotypic approach

    SciTech Connect

    Ku, Hsing-Hsu.

    1988-01-01

    BALB/c mice were immunized with adenosine 6-aminocaproyl-BSA. Hybridoma cell lines that secreted anti-idiotypic antibodies were identified by their binding to rabbit anti-adenosine antibodies, but not to normal rabbit immunoglobulins. Two such monoclonal antibodies, AA18 and AA21, also inhibited the binding of ({sup 3}H)adenosine to the rabbit anti-adenosine antibodies. Therefore, both appeared to recognize idiotypic determinants on the rabbit anti-adenosine antibodies. The monoclonal antibodies AA18 and AA21 were established as being directed at adenosine receptors by the following criteria: (1) they bound to both rat and bovine brain membranes, and binding could be inhibited by CHA, an adenosine receptor agonist, (2) they inhibited the binding of ({sup 3}H)R-PIA, an adenosine receptor agonist, to rat brain membranes; and (3) they inhibited the adenylate cyclase of rat brain membranes. The monoclonal antibodies were used to screen cDNA libraries in lambda gt11.

  3. Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies

    PubMed Central

    Duong, Minh Ngoc; Matera, Eva-Laure; Math, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clmenceau, Batrice; Dumontet, Charles

    2015-01-01

    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. PMID:25523586

  4. Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies.

    PubMed

    Duong, Minh Ngoc; Matera, Eva-Laure; Math, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clmenceau, Batrice; Dumontet, Charles

    2015-01-01

    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. PMID:25523586

  5. Identification of a novel group of Serpulina hyodysenteriae isolates by using a lipopolysaccharide-specific monoclonal antibody.

    PubMed Central

    Alderton, M R; Smith, S C; Coloe, P J

    1993-01-01

    A monoclonal antibody to Serpulina hyodysenteriae 8930 was produced and was used to probe pronase-treated cell lysates of S. hyodysenteriae isolates in immunblots. The results showed that the monoclonal antibody was specific for only five closely related S. hyodysenteriae isolates: 8930, 5380, 70A, RMIT 88, and RMIT 97. Images PMID:8501237

  6. A murine monoclonal antibody against blood group H type-1 and -2 structures.

    PubMed

    Doinel, C; Edelman, L; Rouger, P; LeBlanc, J; Reviron, J; Bach, J F; Salmon, C

    1983-10-01

    A murine anti-H monoclonal antibody was produced. This antibody, called sp115, is an IgM that does not agglutinate classical Bombay. Sp115 may differentiate the two categories of H-deficient phenotypes. It detects H substance in both plasma and fresh saliva. Tissue antigen is found when using this antibody. The specificity of sp115 antibody was determined by adsorption with, and elution from synthetic oligosaccharide immunoadsorbents. Sp115 does not distinguish H type-1 and H type 2-structures. PMID:6352465

  7. Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP

    NASA Technical Reports Server (NTRS)

    Fattaey, A.; Lenz, L.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.

  8. Monoclonal Antibodies in the Lymphatics: Selective Delivery to Lymph Node Metastases of a Solid Tumor

    NASA Astrophysics Data System (ADS)

    Weinstein, John N.; Steller, Michael A.; Keenan, Andrew M.; Covell, David G.; Key, Marc E.; Sieber, Susan M.; Oldham, Robert K.; Hwang, Kou M.; Parker, Robert J.

    1983-10-01

    After subcutaneous injection, monoclonal antibodies directed against a tumor can enter local lymphatic vessels, pass to the draining lymph nodes, and bind to metastases there. Lymphatic delivery of antibody to early metastases is more efficient than intravenous administration, and the lymphatic route can be used to image smaller metastatic deposits. Perhaps more important, the lymphatic route minimizes binding of antibodies to circulating tumor antigens and to cross-reactive antigens present on normal tissues. Antibodies inappropriate for intravenous use because of binding to normal tissues may therefore be useful against lymph node metastases when injected subcutaneously or directly into lymphatic vessels.

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

    DOEpatents

    Jensen, R.H.; Vanderlaan, M.; Bigbee, W.L.; Stanker, L.H.; Branscomb, E.W.; Grabske, R.J.

    1984-11-29

    The present invention provides monoclonal antibodies specific to and distinguishing between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype. 4 figs.

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

    DOEpatents

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

    1988-01-01

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

  11. Monoclonal antibodies against hepatitis B e antigen: production, characterization, and use for diagnosis.

    PubMed

    Korec, E; Dostlov, V; Korcov, J; Mancal, P; Knig, J; Borisova, G; Cibinogen, V; Pumpen, P; Gren, E; Hloznek, I

    1990-05-01

    Five different hybridoma clones secreting anti-HBeAg antibody were constructed by fusing cells of mouse myeloma line SP2/0 with splenocytes from BALB/c mice immunized with recombinant HBeAg. The monoclonal antibodies obtained were characterized immunologically and one was used to develop ELISA for detection of HBeAg and anti-HBeAg antibody. These monoclonal assays enabled the detection of 3 U HBeAg/ml and 1 U anti-HBeAg/ml with reference to standards of the Paul Ehrlich Institute, Frankfurt, F.R.G. Both assays compared well with a commercially available kit (Abbott Laboratory) and were used for detection of HBeAg and anti-HBeAg antibody in clinical serum samples. PMID:2370287

  12. Imaging of bone tumors using a monoclonal antibody raised against human osteosarcoma

    SciTech Connect

    Armitage, N.C.; Perkins, A.C.; Pimm, M.V.; Wastie, M.; Hopkins, J.S.; Dowling, F.; Baldwin, R.W.; Hardcastle, J.D.

    1986-07-01

    The radiolabeled monoclonal antibody 791T/36 raised against a human osteosarcoma was injected into 20 patients with known or suspected bone tumors. Gamma camera images were acquired at 48 or 72 hours after injection, and assessed for antibody localization. Positive images were obtained in all five osteosarcomas and four other primary malignant sarcomas. Two of the four other primary bone tumors gave positive images. Three patients with trauma had negative images as did one patient with Paget's disease. Two patients with suppurative disease gave positive images. The antibody localized in the majority of malignant sarcomas tested. In one tumor where tissue was available, a tumor:non-tumor ratio of 2.8:1 was measured. Repeat imaging was performed in five patients. Immunoscintigraphy using the monoclonal antibody 791T/36 has shown tumor localization in patients with bone and soft tissue sarcomas.

  13. Monoclonal 'internal image' anti-idiotypic antibodies of hepatitis B surface antigen.

    PubMed Central

    Thanavala, Y M; Bond, A; Tedder, R; Hay, F C; Roitt, I M

    1985-01-01

    The hypervariable regions of the immunoglobulin molecule which function as the antigen-combining site are, themselves, capable of provoking an antibody response. These antigenic determinants on the immunoglobulin are termed the 'idiotype', and antibodies directed against them 'anti-idiotype'. In circumstances where there is a close complementarity of shape between antigen and idiotype, and subsequently between idiotype and anti-idiotype, it would be predicted that anti-idiotype would be like an 'internal image' of the antigen. Starting with a monoclonal antibody (idiotype) to the protective a determinant of the hepatitis B surface antigen (HBsAg), we have succeeded in raising two monoclonal anti-idiotypes which mimic HBsAg in their ability to bind polyclonal antibodies to HBsAg produced in a variety of species. These internal image anti-idiotypes may provide a strategy for immunization without the need for antigen. Images Figure 2 PMID:2408999

  14. Novel immunohistochemical monoclonal antibody against human glucose-regulated protein 78.

    PubMed

    Yang, Liu; Su, Mingquan; Ma, Yueyun; Hao, Xiaoke

    2011-12-01

    Glucose-regulated protein (GRP78), an ER chaperone that belongs to the heat-shock protein (HSP) family, exist in all cells and plays important roles in maintaining cellular homeostasis. GRP78 participates in protein folding, transportation, and degradation. Lack of high affinity antibodies especially monoclonal antibodies (MAbs) suitable for Western blot and immunohistochemical staining has lagged. To gain further insight into its possible functions, we generated a novel MAb specific for hGRP78 in Western blot and immunohistochemistry and localized hGRP78 in some human cancer cell lines and cancer tissues. Immunoreactivity of GRP78 was prominent in Hela, Colo205, and A549 detected by 3F9 in Western blot analysis. 3F9 antibody recognized endogenous GRP78 in human cervical cancer, colonic cancer, esophageal cancer, and lung cancer. Thus, successful production of GRP78 monoclonal antibodies provides a new powerful tool for investigation of GRP78 function. PMID:22149283

  15. Immunological identification of Neisseria gonorrhoeae with monoclonal and polyclonal antibody coagglutination reagents.

    PubMed

    Young, H; Reid, K G

    1984-11-01

    The reliability of immunological identification of Neisseria gonorrhoeae using polyclonal and monoclonal antibody coagglutination reagents has been evaluated. When clinical isolates of neisseriae were tested in an "in use" trial the sensitivity and specificity of each reagent were similar and the overall agreement with carbohydrate utilisation was 97.9% (141/144) for the polyclonal antibody reagent and 97.2% (140/144) for the monoclonal reagent. When results of testing 13 stock cultures of N lactamica and five stock cultures of beta-lactamase producing Branhamella catarrhalis were combined with the results for clinical isolates of non-gonococcal neisseriae the agreement with carbohydrate utilisation was 86.5% (64/74) for the polyclonal reagent and 97.3% (72/74) for the monoclonal reagent: this difference is statistically significant at the 5% level. Calculation of positive and negative predictive values showed differences in the reliability of the coagglutination reagents when testing Gram negative diplococci isolated from various anatomical sites. The value and limitations of the polyclonal and monoclonal reagents were similar with respect to anogenital isolates: N gonorrhoeae was confirmed by a positive result but not excluded by a negative result. The monoclonal reagent was superior for testing throat isolates; although a negative result with either reagent confirmed Gram negative diplococci as non-gonococcal neisseriae, a positive result with the monoclonal reagent was more reliable (predictive value 93%) than a positive result with the polyclonal reagent (predictive value 86%). PMID:6438184

  16. Human Monoclonal Antibodies Against a Plethora of Viral Pathogens From Single Combinatorial Libraries

    NASA Astrophysics Data System (ADS)

    Williamson, R. Anthony; Burioni, Roberto; Sanna, Pietro P.; Partridge, Lynda J.; Barbas, Carlos F., III; Burton, Dennis R.

    1993-05-01

    Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro.

  17. Labeling and use of monoclonal antibodies in immunofluorescence: protocols for cytoskeletal and nuclear antigens.

    PubMed

    Bauer, Christoph R

    2014-01-01

    Antibodies are widely used to target and label specifically extra- or intracellular antigens within cells and tissues. Most protocols follow an indirect approach implying the successive incubation with primary and secondary antibodies. In these protocols the primary antibodies are specifically targeted against the antigen in question and are normally not labeled. The secondary antibodies come from a different species and are in contrast fluorescently labeled. The idea is that the primary antibodies specifically bind to their targets but cannot be