Science.gov

Sample records for bispecific monoclonal antibody

  1. Anti-human CD138 monoclonal antibodies and their bispecific formats: generation and characterization.

    PubMed

    Chen, Dan; Zou, Jianxuan; Zong, Yunhui; Meng, Huimin; An, Gangli; Yang, Lin

    2016-06-01

    Syndecan-1 (CD138), a heparan sulfate proteoglycan, acts as a co-receptor for growth factors and chemokines and is a molecular marker associated with the epithelial-mesenchymal transition during development and carcinogenesis. In this study, we generated two specific mouse anti-human CD138 monoclonal antibodies (mAbs, clone ID: 480CT5.4.3, 587CT7.3.6.5) using hybridoma technology and identified their immunological characteristics. After hybridoma sequencing, the single-chain variable fragments (ScFvs) cloned from two hybridoma cells were combined with anti-CD3 OKT-3 ScFv to generate two recombinant bispecific antibodies (h-STL002, m-STL002) against CD138 and CD3 molecules, respectively. The bispecific antibodies were able to specifically target CD138 + multiple myeloma (MM) cells and CD3 + T cells, and showed the potent cytotoxicity against MM RPMI-8226 cell line through T cell activation. However, these bispecific antibodies without T cells did not cause toxic side effect on MM cells. Overall, the two hybridoma clones and their bispecific formats have great potential to promote diagnosis and immunotherapy of plasma cell malignancy. PMID:26954291

  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

    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 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

  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. Biodistribution Analyses of a Near-Infrared, Fluorescently Labeled, Bispecific Monoclonal Antibody Using Optical Imaging.

    PubMed

    Peterson, Norman C; Wilson, George G; Huang, Qihui; Dimasi, Nazzareno; Sachsenmeier, Kris F

    2016-01-01

    In recent years, biodistribution analyses of pharmaceutical compounds in preclinical animal models have become an integral part of drug development. Here we report on the use of optical imaging biodistribution analyses in a mouse xenograft model to identify tissues that nonspecifically retained a bispecific antibody under development. Although our bispecific antibody bound both the epidermal growth factor receptor and insulin growth factor 1 receptor are expressed on H358, nonsmall-cell lung carcinoma cells, the fluorescence from labeled bispecific antibody was less intense than expected in xenografted tumors. Imaging analyses of live mice and major organs revealed that the majority of the Alexa Fluor 750 labeled bispecific antibody was sequestered in the liver within 2 h of injection. However, results varied depending on which near-infrared fluorophore was used, and fluorescence from the livers of mice injected with bispecific antibody labeled with Alexa Fluor 680 was less pronounced than those labeled with Alexa Fluor 750. The tissue distribution of control antibodies remained unaffected by label and suggests that the retention of fluorophores in the liver may differ. Given these precautions, these results support the incorporation of optical imaging biodistribution analyses in biotherapeutic development strategies. PMID:27053562

  7. 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

  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. World Bispecific Antibody Summit, September 27–28, 2011, Boston, MA

    PubMed Central

    Dhimolea, Eugen

    2012-01-01

    With more than 30 therapeutic monoclonal antibodies (mAbs) approved and annual global sales of the products at ∼$50 billion in 2010, these products have proven to be successful in many ways. Nevertheless, there is room for improvement in performance, and substantial unmet medical needs remain. As a consequence, numerous organizations are devoting resources to engineering novel mAbs such as bispecific antibodies that have increased functionality compared with unmodified IgG molecules. The World Bispecific Antibody Summit, organized by Hanson Wade, drew over 100 participants to Boston to discuss engineering novel bispecific antibodies, generating lead candidates and clinical study and commercialization of the molecules. Approaches such as the trifunctional antibody (TRION), dual variable domain-Ig (Abbott), two-in-one (Genentech), dual affinity retargeting (MacroGenics), kappa-lambda body (NovImmune), bispecific T-cell engager (Micromet) and chemical generation (CovX/Pfizer) were discussed in detail. In addition, posters describing bispecific Affibody® molecules for targeting of EGFR and HER2 (Affibody), T-cell receptor based bi-specifics that target HLA-peptides (Immunocore), a novel mAb-Fv bispecific antibody format utilizing Fc region (Xencore), generation of a tetravalent bispecific antibody against IL4 and IL13 for the treatment of idiopathic pulmonary fibrosis (Sanofi), Combining Affibody® molecules and the AlbumodTM technology to create long acting multispecific protein therapeutics (Royal Institute of Technology, Affibody) and COVA301 as a highly potent bispecific inhibitor of IL-17A and TNFα (Covagen) were presented. PMID:22327426

  12. World Bispecific Antibody Summit, September 27-28, 2011, Boston, MA.

    PubMed

    Dhimolea, Eugen; Reichert, Janice M

    2012-01-01

    With more than 30 therapeutic monoclonal antibodies (mAbs) approved and annual global sales of the products at ~$50 billion in 2010, these products have proven to be successful in many ways. Nevertheless, there is room for improvement in performance, and substantial unmet medical needs remain. As a consequence, numerous organizations are devoting resources to engineering novel mAbs such as bispecific antibodies that have increased functionality compared with unmodified IgG molecules. The World Bispecific Antibody Summit, organized by Hanson Wade, drew over 100 participants to Boston to discuss engineering novel bispecific antibodies, generating lead candidates and clinical study and commercialization of the molecules. Approaches such as the trifunctional antibody (TRION), dual variable domain-Ig (Abbott), two-in-one (Genentech), dual affinity retargeting (MacroGenics), kappa-lambda body (NovImmune), bispecific T-cell engager (Micromet) and chemical generation (CovX/Pfizer) were discussed in detail. In addition, posters describing bispecific Affibody® molecules for targeting of EGFR and HER2 (Affibody), T-cell receptor based bi-specifics that target HLA-peptides (Immunocore), a novel mAb-Fv bispecific antibody format utilizing Fc region (Xencore), generation of a tetravalent bispecific antibody against IL4 and IL13 for the treatment of idiopathic pulmonary fibrosis (Sanofi), Combining Affibody® molecules and the Albumod™ technology to create long acting multispecific protein therapeutics (Royal Institute of Technology, Affibody) and COVA301 as a highly potent bispecific inhibitor of IL-17A and TNF-α (Covagen) were presented. PMID:22327426

  13. Bispecific antibody platforms for cancer immunotherapy.

    PubMed

    Lameris, Roeland; de Bruin, Renée C G; Schneiders, Famke L; van Bergen en Henegouwen, Paul M P; Verheul, Henk M W; de Gruijl, Tanja D; van der Vliet, Hans J

    2014-12-01

    Over the past decades advances in bioengineering and expanded insight in tumor immunology have resulted in the emergence of novel bispecific antibody (bsAb) constructs that are capable of redirecting immune effector cells to the tumor microenvironment. (Pre-) clinical studies of various bsAb constructs have shown impressive results in terms of immune effector cell retargeting, target dependent activation and the induction of anti-tumor responses. This review summarizes recent advances in the field of bsAb-therapy and limitations that were encountered. Furthermore, we will discuss potential future developments that can be expected to take the bsAb approach successfully forward. PMID:25195094

  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. 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

  16. 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

  17. 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

  18. 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; Böhne, 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

  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. 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…

  1. 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

  2. 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

  3. 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.

  4. Engineering of stable bispecific antibodies targeting IL-17A and IL-23.

    PubMed

    Mabry, Robert; Lewis, Katherine E; Moore, Margaret; McKernan, Patricia A; Bukowski, Thomas R; Bontadelli, Kristen; Brender, Ty; Okada, Shannon; Lum, Karen; West, James; Kuijper, Joseph L; Ardourel, Dan; Franke, Secil; Lockwood, Luann; Vu, Tuyen; Frank, Amanda; Appleby, Mark W; Wolf, Anitra; Reardon, Brian; Hamacher, Nels B; Stevens, Brenda; Lewis, Patsy; Lewis, Kenneth B; Gilbertson, Debra G; Lantry, Megan; Julien, Susan H; Ostrander, Craig; Chan, Chung; Byrnes-Blake, Kelly; Brody, Jennifer; Presnell, Scott; Meengs, Brent; Levin, Steven D; Snavely, Mark

    2010-03-01

    Bispecific antibodies (bsAbs) present an attractive opportunity to combine the additive and potentially synergistic effects exhibited by combinations of monoclonal antibodies (mAbs). Current challenges for engineering bsAbs include retention of the binding affinity of the parent mAb or antibody fragment, the ability to bind both targets simultaneously, and matching valency with biology. Other factors to consider include structural stability and expression of the recombinant molecule, both of which may have significant impact on its development as a therapeutic. Here, we incorporate selection of stable, potent single-chain variable fragments (scFvs) early in the engineering process to assemble bsAbs for therapeutic applications targeting the cytokines IL-17A/A and IL-23. Stable scFvs directed against human cytokines IL-23p19 and IL-17A/A were isolated from a human Fab phage display library via batch conversion of panning output from Fabs to scFvs. This strategy integrated a step for shuffling V regions during the conversion and permitted the rescue of scFv molecules in both the V(H)V(L) and the V(L)V(H) orientations. Stable scFvs were identified and assembled into several bispecific formats as fusions to the Fc domain of human IgG1. The engineered bsAbs are potent neutralizers of the biological activity of both cytokines (IC(50) < 1 nM), demonstrate the ability to bind both target ligands simultaneously and display stability and productivity advantageous for successful manufacture of a therapeutic molecule. Pharmacokinetic analysis of the bsAbs in mice revealed serum half-lives similar to human mAbs. Assembly of bispecific molecules using stable antibody fragments offers an alternative to reformatting mAbs and minimizes subsequent structure-related and manufacturing concerns. PMID:20022918

  5. A bispecific antibody effectively neutralizes all four serotypes of dengue virus by simultaneous blocking virus attachment and fusion.

    PubMed

    Shi, Xin; Deng, Yongqiang; Wang, Huajing; Ji, Guanghui; Tan, Wenlong; Jiang, Tao; Li, Xiaofeng; Zhao, Hui; Xia, Tian; Meng, Yanchun; Wang, Chao; Yu, Xiaojie; Yang, Yang; Li, Bohua; Qin, E-De; Dai, Jianxin; Qin, Cheng-Feng; Guo, Yajun

    2016-04-01

    Although dengue virus (DENV) infection severely threatens the health of humans, no specific antiviral drugs are currently approved for clinical use against DENV infection. Attachment and fusion are 2 critical steps for the flavivirus infection, and the corresponding functional epitopes are located at E protein domain III (E-DIII) and domain II (E-DII), respectively. Here, we constructed a bispecific antibody (DVD-1A1D-2A10) based on the 2 well-characterized anti-DENV monoclonal antibodies 1A1D-2 (1A1D) and 2A10G6 (2A10). The 1A1D antibody binds E-DIII and can block the virus attaching to the cell surface, while the 2A10 antibody binds E-DII and is able to prevent the virus from fusing with the endosomal membrane. Our data showed that DVD-1A1D-2A10 retained the antigen-binding activity of both parental antibodies. Importantly, it was demonstrated to be significantly more effective at neutralizing DENV than its parental antibodies both in vitro and in vivo, even better than the combination of them. To eliminate the potential antibody-dependent enhancement (ADE) effect, this bispecific antibody was successfully engineered to prevent Fc-γ-R interaction. Overall, we generated a bispecific anti-DENV antibody targeting both attachment and fusion stages, and this bispecific antibody broadly neutralized all 4 serotypes of DENV without risk of ADE, suggesting that it has great potential as a novel antiviral strategy against DENV. PMID:26905804

  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. Improved assembly of bispecific antibodies from knob and hole half-antibodies.

    PubMed

    Williams, Ambrose Jon; Giese, Glen; Persson, Josefine

    2015-01-01

    A process was developed for large-scale assembly of IgG1 and IgG4 bispecific antibodies from knob and hole half-antibodies. We optimized assembly conditions such as pH, temperature, stabilizers, and reducing agent. We also identified and exploited structural changes unique to knob and hole half-antibodies with the result of improving assembly outcome, specifically storing half-antibodies at higher pH will condition them to assemble more rapidly and produce fewer high molecular-weight species (HMWS). Application of heat to the assemblies resulted in an acceleration of assembly rate, with optimal formation of bispecific achieved at 37°C. IgG4 half-antibodies were unusually sensitive to temperature-dependent formation of HMWS in pre-assembly conditioning as well as during assembly. We selected l-histidine and Polyvinylpyrrolidone (PVP) as stabilizers to prevent HMWS formation in IgG4 , and achieved rapid and high-efficiency assemblies. Using optimized assembly conditions, we developed and scaled up a method for assembling bispecific antibody with 90% assembly efficiency over 6 h with minimal impact to product quality, generating a pool with bispecific antibody for downstream processing. PMID:26097217

  8. Ipilimumab augments antitumor activity of bispecific antibody-armed T cells

    PubMed Central

    2014-01-01

    Background Ipilimumab is an antagonistic monoclonal antibody against cytotoxic T-lymphocyte antigen-4 (CTLA-4) that enhances antitumor immunity by inhibiting immunosuppressive activity of regulatory T cells (Treg). In this study, we investigated whether inhibiting Treg activity with ipilimumab during ex vivo T cell expansion could augment anti-CD3-driven T cell proliferation and enhance bispecific antibody (BiAb)-redirected antitumor cytotoxicity of activated T cells (ATC). Methods PBMC from healthy individuals were stimulated with anti-CD3 monoclonal antibody with or without ipilimumab and expanded for 10-14 days. ATC were harvested and armed with anti-CD3 x anti-EGFR BiAb (EGFRBi) or anti-CD3 x anti-CD20 BiAb (CD20Bi) to test for redirected cytotoxicity against COLO356/FG pancreatic cancer cell line or Burkitt’s lymphoma cell line (Daudi). Results In PBMC from healthy individuals, the addition of ipilimumab at the initiation of culture significantly enhanced T cell proliferation (p = 0.0029). ATC grown in the presence of ipilimumab showed significantly increased mean tumor-specific cytotoxicity at effector:target (E:T) ratio of 25:1 directed at COLO356/FG and Daudi by 37.71% (p < 0.0004) and 27.5% (p < 0.0004), respectively, and increased the secretion of chemokines (CCL2, CCL3, CCL4,CCL5, CXCL9, and granulocyte-macrophage colony stimulating factor(GM-CSF)) and cytokines (IFN-γ, IL-2R, IL-12, and IL-13), while reducing IL-10 secretion. Conclusions Expansion of ATC in the presence of ipilimumab significantly improves not only the T cell proliferation but it also enhances cytokine secretion and the specific cytotoxicity of T cells armed with bispecific antibodies. PMID:25008236

  9. A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells.

    PubMed

    Li, Aifen; Xing, Jieyu; Li, Li; Zhou, Changhua; Dong, Bin; He, Ping; Li, Qing; Wang, Zhong

    2016-12-01

    Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy. PMID:27112931

  10. Development of a Human IgG4 Bispecific Antibody for Dual Targeting of Interleukin-4 (IL-4) and Interleukin-13 (IL-13) Cytokines*

    PubMed Central

    Spiess, Christoph; Bevers, Jack; Jackman, Janet; Chiang, Nancy; Nakamura, Gerald; Dillon, Michael; Liu, Hongbin; Molina, Patricia; Elliott, J. Michael; Shatz, Whitney; Scheer, Justin M.; Giese, Glen; Persson, Josefine; Zhang, Yin; Dennis, Mark S.; Giulianotti, James; Gupta, Prateek; Reilly, Dorothea; Palma, Enzo; Wang, Jianyong; Stefanich, Eric; Scheerens, Heleen; Fuh, Germaine; Wu, Lawren C.

    2013-01-01

    Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes. PMID:23880771

  11. 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

  12. 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

  13. Therapeutic bispecific T-cell engager antibody targeting the intracellular oncoprotein WT1

    PubMed Central

    Dao, Tao; Pankov, Dmitry; Scott, Andrew; Korontsvit, Tatyana; Zakhaleva, Victoriya; Xu, Yiyang; Xiang, Jingyi; Yan, Su; de Morais Guerreiro, Manuel Direito; Veomett, Nicholas; Dubrovsky, Leonid; Curcio, Michael; Doubrovina, Ekaterina; Ponomarev, Vladimir; Liu, Cheng; O’Reilly, Richard J; Scheinberg, David A

    2015-01-01

    Intracellular tumor antigens presented on the cell surface in the context of human leukocyte antigen (HLA) molecules have been targeted by T cell–based therapies, but there has been little progress in developing small-molecule drugs or antibodies directed to these antigens. Here we describe a bispecific T-cell engager (BiTE) antibody derived from a T-cell receptor (TCR)-mimic monoclonal antibody (mAb) ESK1, which binds a peptide derived from the intracellular oncoprotein WT1 presented on HLA-A*02:01. Despite the very low density of the complexes at the cell surface, ESK1-BiTE selectively activated and induced proliferation of cytolytic human T cells that killed cells from multiple leukemias and solid tumors in vitro and in mice. We also discovered that in an autologous in vitro setting, ESK1-BiTE induced a robust secondary CD8 T-cell response specific for tumor-associated antigens other than WT1. Our study provides an approach that targets tumor-specific intracellular antigens without using cell therapy and suggests that epitope spreading could contribute to the therapeutic efficacy of this BiTE. PMID:26389576

  14. 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.

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

    PubMed

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

    2013-09-30

    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

  16. Structural understanding of stabilization patterns in engineered bispecific Ig-like antibody molecules

    SciTech Connect

    Jordan, Jacob L.; Arndt, Joseph W.; Hanf, Karl; Li, Guohui; Hall, Janine; Demarest, Stephen; Huang, Flora; Wu, Xiufeng; Miller, Brian; Glaser, Scott; Fernandez, Erik J.; Wang, Deping; Lugovskoy, Alexey

    2010-01-12

    Bispecific immunoglobulin-like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin-beta receptor (LT{beta}R) binding Fv domain of an anti-LT{beta}R/anti-TNF-related apoptosis inducing ligand receptor-2 (TRAIL-R2) bispecific immunoglobulin-like antibody. We further describe a new hierarchical structure-guided approach toward engineering of antibody-like molecules to enhance their thermal and chemical stability.

  17. Immunotoxicity of monoclonal antibodies

    PubMed Central

    2009-01-01

    Monoclonal antibodies (mAbs) are large molecules intended to bind to specific targets often expressed on the immune system, and to treat various immunopathological conditions. Therefore, mAbs can be considered to have a high potential for immunotoxicity, which is reflected in the clinical experience accumulated on mAbs-induced adverse effects related to immunosuppression, immunostimulation and hypersensitivity (immunogenicity). So far, non clinical immunotoxicity studies have been inadequate to address all safety issues in relation to the possible immunotoxicity of mAbs, because they are fraught with limitations and pitfalls primarily related to the lack of relevant animal species. In addition, clinical studies rarely include validated end-points dedicated to the prediction of immunotoxicity. With the ongoing development of mAbs as novel therapeutic strategies for a wide variety of diseases, efforts should be paid to improve our understanding of mAbs-induced immunotoxic effects and design dedicated strategies to assess their immunological safety, both non clinically and clinically. PMID:20061816

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

    PubMed Central

    Laventie, Benoît-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; Prévost, Gilles; Grosveld, Frank; Drabek, Dubravka

    2011-01-01

    Panton–Valentine 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 anti–LukS-PV HCAb, three anti–LukF-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. Anti–LukS-PV HCAb also binds to γ-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

  19. Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies.

    PubMed Central

    Wickham, T J; Segal, D M; Roelvink, P W; Carrion, M E; Lizonova, A; Lee, G M; Kovesdi, I

    1996-01-01

    A major hurdle to adenovirus (Ad)-mediated gene transfer is that the target issue lacks sufficient levels of receptors to mediate vector attachment via its fiber coat protein. Endothelial and smooth muscle cells are primary targets in gene therapy approaches to prevent restenosis following angioplasty or to promote or inhibit angiogenesis. However, Ad poorly binds and transduces these cells because of their low or undetectable levels of functional Ad fiber receptor. The Ad-binding deficiency of these cells was overcome by targeting Ad binding to alpha v integrin receptors that are sufficiently expressed by these cells. In order to target alpha v integrins, a bispecific antibody (bsAb) that comprised a monoclonal Ab to the FLAG peptide epitope, DYKDDDDK, and a monoclonal Ab to alpha v integrins was constructed. In conjunction with the bsAb, a new vector, AdFLAG, which incorporated the FLAG peptide epitope into its penton base protein was constructed. Complexing AdFLAG with the bsAb increased the beta-glucuronidase transduction of human venule endothelial cells and human intestinal smooth muscle cells by seven- to ninefold compared with transduction by AdFLAG alone. The increased transduction efficiency was shown to occur through the specific interaction of the complex with alpha v integrins. These results demonstrate that bsAbs can be successfully used to target Ad to a specific cellular receptor and thereby increase the efficiency of gene transfer. PMID:8794324

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. Plant biopharming of monoclonal antibodies.

    PubMed

    Ko, Kisung; Koprowski, Hilary

    2005-07-01

    Recent advances in molecular biology and plant biotechnology have shifted the concept of growing crops as a food source to serving as a bioreactor for the production of therapeutic recombinant proteins. Plants are potential biopharming factories because they are capable of producing unlimited numbers and amounts of recombinant proteins safely and inexpensively. In the last two decades, plant production systems have been developed for monoclonal antibody production, which has been useful in passive immunization of viral or bacterial diseases. Recently, a recombinant monoclonal antibody for rabies prophylaxis was produced in transgenic plants. Rabies virus epidemics remain still problematic throughout the world, and adequate treatment has been hampered by the worldwide shortage and high cost of prophylactic antibodies such as HRIG. Successful mass production of this monoclonal antibody in plants might help to overcome these problems. An effective plant production system for recombinant biologicals requires the appropriate heterologous plant expression system, the optimal combination of gene expression regulatory elements, control of post-translational processing of recombinant products, and efficient purification methods for product recovery. This review discusses recent biotechnology developments for plant-derived monoclonal antibodies and discusses these products as a promising approach to rabies prophylaxis and the consequence for global health benefits. PMID:15896408

  9. 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.

  10. Biosensing made easy with PEG-targeted bi-specific antibodies.

    PubMed

    Raftery, Lyndon J; Grewal, Yadveer S; Howard, Christopher B; Jones, Martina L; Shiddiky, Muhammad J A; Carrascosa, Laura G; Thurecht, Kristofer J; Mahler, Stephen M; Trau, Matt

    2016-04-14

    Whilst recent advances in nanotechnology have yielded many new biosensing capabilities, innovative biological attachment and detection modalities remain relatively underdeveloped. Bi-specific antibodies (bsAbs) - which exhibit binding capability for two separate targets - offer an inherent advantage over conventional antibody reagents by significantly simplifying sensor surface preparation. Herein, we report the deployment of bsAbs for simultaneous attachment to a polymer-coated transducer and label-free, electrochemical (EC) detection of target antigens. PMID:27045479

  11. 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.

  12. Advances in monoclonal antibody application in myocarditis*

    PubMed Central

    Han, Li-na; He, Shuang; Wang, Yu-tang; Yang, Li-ming; Liu, Si-yu; Zhang, Ting

    2013-01-01

    Monoclonal antibodies have become a part of daily preparation technologies in many laboratories. Attempts have been made to apply monoclonal antibodies to open a new train of thought for clinical treatments of autoimmune diseases, inflammatory diseases, cancer, and other immune-associated diseases. This paper is a prospective review to anticipate that monoclonal antibody application in the treatment of myocarditis, an inflammatory disease of the heart, could be a novel approach in the future. In order to better understand the current state of the art in monoclonal antibody techniques and advance applications in myocarditis, we, through a significant amount of literature research both domestic and abroad, developed a systematic elaboration of monoclonal antibodies, pathogenesis of myocarditis, and application of monoclonal antibodies in myocarditis. This paper presents review of the literature of some therapeutic aspects of monoclonal antibodies in myocarditis and dilated cardiomyopathy to demonstrate the advance of monoclonal antibody application in myocarditis and a strong anticipation that monoclonal antibody application may supply an effective therapeutic approach to relieve the severity of myocarditis in the future. Under conventional therapy, myocarditis is typically associated with congestive heart failure as a progressive outcome, indicating the need for alternative therapeutic strategies to improve long-term results. Reviewing some therapeutic aspects of monoclonal antibodies in myocarditis, we recently found that monoclonal antibodies with high purity and strong specificity can accurately act on target and achieve definite progress in the treatment of viral myocarditis in rat model and may meet the need above. However, several issues remain. The technology on how to make a higher homologous and weak immunogenic humanized or human source antibody and the treatment mechanism of monoclonal antibodies may provide solutions for these open issues. If we are to further stimulate progress in the area of clinical decision support, we must continue to develop and refine our understanding and use of monoclonal antibodies in myocarditis. PMID:23897786

  13. 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 Fcγ receptor III (FcγRIII 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

  14. LC-MS characterization and purity assessment of a prototype bispecific antibody

    PubMed Central

    Woods, R. Jeremy; Xie, Michael Hongwei; Von Kreudenstein, Thomas Spreter; Ng, Gordon Y.; Dixit, Surjit B.

    2013-01-01

    Bispecific IgG asymmetric (heterodimeric) antibodies offer enhanced therapeutic efficacy, but present unique challenges for drug development. These challenges are related to the proper assembly of heavy and light chains. Impurities such as symmetric (homodimeric) antibodies can arise with improper assembly. A new method to assess heterodimer purity of such bispecific antibody products is needed because traditional separation-based purity assays are unable to separate or quantify homodimer impurities. This paper presents a liquid chromatography-mass spectrometry (LC-MS)-based method for evaluating heterodimeric purity of a prototype asymmetric antibody containing two different heavy chains and two identical light chains. The heterodimer and independently expressed homodimeric standards were characterized by two complementary LC-MS techniques: Intact protein mass measurement of deglycosylated antibody and peptide map analyses. Intact protein mass analysis was used to check molecular integrity and composition. LC-MSE peptide mapping of Lys-C digests was used to verify protein sequences and characterize post-translational modifications, including C-terminal truncation species. Guided by the characterization results, a heterodimer purity assay was demonstrated by intact protein mass analysis of pure deglycosylated heterodimer spiked with each deglycosylated homodimeric standard. The assay was capable of detecting low levels (2%) of spiked homodimers in conjunction with co-eluting half antibodies and multiple mass species present in the homodimer standards and providing relative purity differences between samples. Detection of minor homodimer and half-antibody C-terminal truncation species at levels as low as 0.6% demonstrates the sensitivity of the method. This method is suitable for purity assessment of heterodimer samples during process and purification development of bispecific antibodies, e.g., clone selection. PMID:23884083

  15. 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

  16. 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

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

    PubMed

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

    2015-01-01

    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

  18. Monoclonal Antibodies for Lipid Management.

    PubMed

    Feinstein, Matthew J; Lloyd-Jones, Donald M

    2016-07-01

    In recent years, biochemical and genetic studies have identified proprotein convertase subtilisin/kexin type 9 (PCSK9) as a major mediator of low-density lipoprotein cholesterol (LDL-c) levels and thereby a potential novel target for reducing risk of coronary heart disease (CHD). These observations led to the development of PCSK9 inhibitors, which lower LDL-c levels more than any other non-invasive lipid-lowering therapy presently available. The PCSK9 inhibitors furthest along in clinical trials are subcutaneously injected monoclonal antibodies. These PCSK9 inhibitors have demonstrated LDL-c-lowering efficacy with acceptable safety in phase III clinical trials and may offer a useful therapy in addition to maximally tolerated HMG-CoA reductase inhibitors (statins) in certain patient groups. Longer-term data are required to ensure sustained efficacy and safety of this new class of medications. This review provides an overview of the biology, genetics, development, and clinical trials of monoclonal antibodies designed to inhibit PCSK9. PMID:27221501

  19. Antigen protection of monoclonal antibodies undergoing labelling.

    PubMed

    Ramjeesingh, M; Zywulko, M; Rothstein, A; Whyte, R; Shami, E Y

    1990-10-19

    The effectiveness of a methodology designed to protect the antigen binding capacity of monoclonal antibodies undergoing labelling with a number of reagents was examined. The antigen binding sites of monoclonal antibodies were protected by complexing them with their antigen. Chemical modification with 6 mM of the water soluble Bolton-Hunter reagent of site protected monoclonal antibodies to glucoamylase resulted in antibodies that could tolerate a four-fold increase in reagent incorporation, without any loss of antigen binding capacity. Iodination of these antibodies (modified under site protected conditions) yielded over 70% increase in radioactivity incorporated in the active antibody fraction, compared with the incorporation into unprotected antibodies. Site protected labeling was found to be effective in retaining the antigen binding capacity of monoclonal antibodies modified with all reagents tested with the exception of chloramine-T. PMID:2230135

  20. 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.

  1. 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.

  2. Monoclonal antibodies against Vibrio cholerae lipopolysaccharide.

    PubMed Central

    Gustafsson, B; Rosén, A; Holme, T

    1982-01-01

    A cell line producing monoclonal antibodies directed against the core region of Vibrio cholerae lipopolysaccharide has been established. These antibodies were inhibited by lipopolysaccharide preparations of both O-group 1 vibrios and some non-O-group 1 vibrios as detected in enzyme-linked immunosorbent assay-inhibition experiments. Coagglutination experiments with monoclonal and polyclonal antibodies adsorbed to protein A-carrying staphylococci were performed. All V. cholerae strains tested, regardless of serotype, were agglutinated when mixed with staphylococci coated with the monoclonal antibodies, whereas staphylococci coated with group-specific (O1) polyclonal antibodies only agglutinated with O-group 1 vibrios. Images PMID:6183214

  3. 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

  4. Tetravalent anti-CD20/CD3 bispecific antibody for the treatment of B cell lymphoma.

    PubMed

    Lu, Chia-Yen; Chen, Gregory J; Tai, Pei-Han; Yang, Yu-Chen; Hsu, Yu-Shen; Chang, Mingi; Hsu, Chuan-Lung

    2016-05-13

    Bispecific antibodies (bsAbs) are second generation antibodies for therapeutic application in immunotherapy. One of the major strategies of the bsAb platform is the recruitment of immune effector T cells by incorporating an anti-CD3 domain. A bispecific T-cell engager (BiTE), with one end having an affinity for CD3 and the other end with affinity for CD19, has been approved in the US and Europe for the treatment of acute lymphoblastic leukemia. However, due to their small size and lack of Fc region, these single-chain variable fragment (scFv) bsAbs have short half-lives in vivo. Additionally, poor solubility, structural instability, and low production yields have also become major challenges in the bulk production process. To overcome these challenges, we have engineered a tetravalent bsAb with bivalent binding specificity for the CD20 and CD3 antigen in an immunoglobulin G (IgG) format. The fusion of the anti-CD3 scFvs to the CD20 antibody via a linker-hinge domain (LHD) results in improved antibody stabilization and properties. Here we demonstrate this antibody's highly efficient cancer cell elimination in a dose-dependent manner in a CD20-expressing B lymphoblastoid cell line in vitro. Our data suggest the potential clinical application of this bsAb for the treatment of CD20-expressing B cell malignancies. PMID:27040766

  5. 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.

  6. 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

  7. 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

  8. 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

  9. Characterisation of Stress-Induced Aggregate Size Distributions and Morphological Changes of a Bi-Specific Antibody Using Orthogonal Techniques.

    PubMed

    Hamrang, Zahra; Hussain, Maryam; Tingey, Katie; Tracka, Malgorzata; Casas-Finet, José R; Uddin, Shahid; van der Walle, Christopher F; Pluen, Alain

    2015-08-01

    A critical step in monoclonal antibody (mAb) screening and formulation selection is the ability of the mAb to resist aggregation following exposure to environmental stresses. Regulatory authorities welcome not only information on the presence of micron-sized particles, but often any information on sub-visible particles in the size range obtained by orthogonal sizing techniques. The present study demonstrates the power of combining established techniques such as dynamic light scattering (DLS) and micro-flow imaging (MFI), with novel analyses such as raster image correlation spectroscopy (RICS) that offer to bridge existent particle sizing gaps in this area. The influence of thermal and freeze-thaw stress treatments on particle size and morphology was assessed for a bi-specific antibody (mAb2). Aggregation of mAb2 was confirmed to be concentration- and treatment-dependent following thermal stress and freeze-thaw cycling. Particle size and count data show concentration- and treatment-dependent behaviour of aggregate counts, morphological descriptors and particle size distributions. Complementarity in particle size output was observed between all approaches utilised, where RICS bridged the analytical size gap (∼0.5-5 μm) between DLS and MFI. Overall, this study highlights the potential of orthogonal image analyses such as RICS (analytical size gap) and MFI (particle morphology) for formulation screening. PMID:26053418

  10. Production of monoclonal antibodies against avidin.

    PubMed

    Ashorn, R; Ashorn, P; Kulomaa, M; Tuohimaa, P; Krohn, K

    1985-01-01

    Monoclonal antibodies of the IgG1 subclass were generated against chicken avidin. These antibodies were shown to be as sensitive as polyclonal antiserum in detecting avidin by radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) methods. Furthermore, the monoclonal antibodies were considerably more specific. Our results with a monoclonal anti-avidin RIA support previous findings that in inflammatory conditions avidin is synthesized also in other organs than the oviduct, although in the liver a major part of the activity detected by polyclonal anti-avidin RIA or biotin-bentonite assay was not due to avidin. PMID:4053566

  11. Targeting CD133high Colorectal Cancer Cells In Vitro and In Vivo With an Asymmetric Bispecific Antibody.

    PubMed

    Zhao, Lei; Yang, Yudan; Zhou, Pengfei; Ma, Hong; Zhao, Xiaolai; He, Xin; Wang, Tao; Zhang, Jing; Liu, Yang; Zhang, Tao

    2015-01-01

    A critical obstacle in advanced colorectal cancer (CRC) treatment is the insufficient improvement on survival of conventional chemotherapy. Cancer stem cells are reported to be one of the crucial explanations. CD133 has been identified as a surface marker of CRC stem cells. Bispecific antibodies (BiAbs) targeting tumor-specific antigens are promising therapeutics for malignant diseases, yet that targeting CD133 produced by genetic engineering has not been published. In the current research, CD133 expression in primary CRC was detected by immunohistochemistry, and an asymmetric BiAb consisting of monomer of chimeric AC133 (mouse anti-human CD133 monoclonal antibody) and single chain of humanized OKT3 was developed to eradicate CD133-expressing tumor cells by arming activated T cells in vitro and in vivo. In immunohistochemical examination, CD133 overexpression (>50% of stained cells) frequency was significantly correlated with lymphatic invasion and clinical stage. The new molecular revealed dual-antigen-binding specificity to CD133 and CD3, its distinct structure not only facilitated the purification procedure but also conferred the antibody to ensure a longer and stronger cytotoxic activity. By arming activated T cells, the new antibody displayed impressive cytotoxicity toward CD133(high) but not CD133(low) CRC cells in vitro, produced amounts of cytokines (interferon-γ and granulocyte-macrophage colony-stimulating factor), and could inhibit tumor growth and retard tumor development in nonobese diabetic-severe combined immunodeficient mice without apparent toxicity. Taken together, the new BiAb possesses prosperities that support that the molecule has the potential of being a promising candidate of new therapeutics for CRC therapy. PMID:26049545

  12. 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.

  13. Monoclonal antibody production techniques and applications

    SciTech Connect

    Schook, L.B.

    1987-01-01

    This book consists of three parts, each containing several papers. The part headings are: Techniques of Immunization and Hybrid Construction, Production and Modification of Antibodies and Lymphokines, and Applications of Monoclonal Lymphoid Products.

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

    PubMed

    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

  15. 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

  16. 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.

  17. Polyclonal and monoclonal antibodies in clinic.

    PubMed

    Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

    2014-01-01

    Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic. PMID:24037837

  18. An Alternative Chemical Redox Method for the Production of Bispecific Antibodies: Implication in Rapid Detection of Food Borne Pathogens

    PubMed Central

    Owais, Mohammad; Kazmi, Shadab; Tufail, Saba; Zubair, Swaleha

    2014-01-01

    Bi-functional antibodies with the ability to bind two unrelated epitopes have remarkable potential in diagnostic and bio-sensing applications. In the present study, bispecific antibodies that recognize human red blood cell (RBC) and the food borne pathogen Listeria monocytogenes (L. monocytogenes) were engineered. The procedure involves initial reduction of a mixture of anti-RBC and anti-Listeria antibodies followed by gradual re-oxidation of the reduced disulphides. This facilitates association of the separated antibody chains and formation of hybrid immunoglobulins with affinity for the L. monocytogenes and human RBC. The bispecific antibodies caused the agglutination of the RBCs only in the presence of L. monocytogenes cells. The agglutination process necessitated the specific presence of L. monocytogenes and the red colored clumps formed were readily visible with naked eyes. The RBC agglutination assay described here provides a remarkably simple approach for the rapid and highly specific screening of various pathogens in their biological niches. PMID:24637674

  19. 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

  20. Supramolecular assembly of porphyrins and monoclonal antibodies

    SciTech Connect

    Harada, A.; Shiotsuki, K.; Fukushima, H.; Yamaguchi, H.; Kamachi, M.

    1995-03-01

    Assemblies of porphyrins on DNA are of potential catalytic interest. Hence, the authors studied the interactions of metalloporphyrins and porphyrins with the monoclonal antibody for meso-tetrakis(carboxyphenyl)porphine (TCPP). UV-vis absorption spectroscopy, emission spectroscopy, and circular dichroism (CD) spectroscopy were used for this investigation. These techniques suggest higher than one-to-one porphyrin-to-antibody ratios.

  1. 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.

  2. 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

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

    PubMed Central

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

    2015-01-01

    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 64Cu-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 64Cu-NOTA-Bs-F(ab)2. 64Cu-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

  4. 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

  5. [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

  6. 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 et al. 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 Fcγ-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 Fcγ-receptor-carrying immune cells that can mount an immune response against the tumor cells. PMID:26802233

  7. 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...

  8. 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...

  9. Phase Separation in Solutions of Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Benedek, George; Wang, Ying; Lomakin, Aleksey; Latypov, Ramil

    2012-02-01

    We report the observation of liquid-liquid phase separation (LLPS) in a solution of humanized monoclonal antibodies, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective inter-protein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable protein condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.

  10. 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 HNF1α 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 (32–37 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 HNF1α-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

  11. 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

  12. 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

  13. 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

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

    PubMed

    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

    2013-12-12

    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

  15. Monoclonal antibodies reacting with murine teratocarcinoma cells.

    PubMed Central

    Goodfellow, P N; Levinson, J R; Williams, V E; McDevitt, H O

    1979-01-01

    Monoclonal antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a rat immunized with the C3H mouse teratocarcinoma C86-S1. After the fusion two clones were chosen for further analysis. The first clone, 3C4-10, produced an antibody recognizing an antigen with a distribution restricted to teratocarcinoma cell lines, an endoderm cell line, and a neuroblastoma. The second clone, 4A1-9, produced an antibody that reacted with all cultured murine cells tested and adult brain. Neither antibody reacted with preimplantation embryos. The 3C4-10 antibody recognized an antigen associated with proteins. The apparent molecular weight of the 3C4-10 antigen was greater than 100,000. PMID:284353

  16. Microheterogeneity of mouse antidextran monoclonal antibodies.

    PubMed

    Mimura, Y; Kabat, E A; Tanaka, T; Fujimoto, M; Takeo, K; Nakamura, K

    1995-01-01

    Mouse antidextran monoclonal antibodies showed microheterogeneity which was analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Not only the heavy (H) chains but also the light (L) chains were heterogeneous in terms of isoelectric point (pI). The higher the pI, the more prominent the H chain spots. To demonstrate the cause of the microheterogeneity an IgG1 monoclonal antibody (mAb 35.8.2H) was examined especially for involvement of the sugar moiety in the microheterogeneity. The glycosylated region was determined in the Fc portion from serine 239 to methionine 309 by a glycan detection method using mild periodate oxidation, which confirms that the sugar chain is attached to the conserved glycosylation site of asparagine 297. However, charge heterogeneity of the H chain was not entirely attributed to the Fc because the papain digest of the antibody was separated into two Fc spots, a few Fd spots and two L chain spots by 2-D PAGE. This indicates that factors other than the sugar moiety are responsible for charge heterogeneity of IgG monoclonal antibody. On the other hand, the H chain isoforms of lower pI were shown to be more susceptible to V8 protease by peptide mapping. This result strongly suggests the occurrence of deamidation at glutamine or asparagine residues. PMID:7537656

  17. Chemoenzymatic glyco-engineering of monoclonal antibodies

    PubMed Central

    Giddens, John P.; Wang, Lai-Xi

    2016-01-01

    Summary Monoclonal antibodies (mAbs) are an important class of therapeutic glycoproteins widely used for the treatment of cancer, inflammation, and infectious diseases. Compelling data have shown that the presence and fine structures of the conserved N-glycans at the Fc domain can profoundly affect the effector functions of antibodies. However, mAbs are usually produced as mixtures of Fc glycoforms and the control of glycosylation to a favorable, homogeneous status in various host expression systems is still a challenging task. In this chapter, we describe a detailed procedure of chemoenzymatic glyco-engineering of monoclonal antibodies, using rituximab (a therapeutic monoclonal antibody) as a model system. The protocol includes the deglycosylation of a mAb by an endoglycosidase (such as wild type EndoS) to remove the heterogeneous Fc N-glycans, leaving only the innermost GlcNAc or the core-fucosylated GlcNAc at the glycosylation site. Then the deglycosylated IgG serves as an acceptor for an endoglycosidase-catalyzed transglycosylation to add a desired N-glycan to the GlcNAc acceptor to reconstitute a defined, homogeneous natural glycoform of IgG, using a glycosynthase mutant as the enzyme and activated glycan oxazoline as the donor substrate. A semi-synthesis of sialylated and asialylated biantennary N-glycan oxazolines is also described. This detailed procedure can be used for the Fc glycosylation remodeling of other mAbs to provide homogeneous Fc glycoforms for various applications. PMID:26082235

  18. 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.

  19. Monoclonal Antibody Recognition of Abscisic Acid Analogs

    PubMed Central

    Walker-Simmons, Mary K.; Reaney, Martin J. T.; Quarrie, Stephen A.; Perata, Pierdomenico; Vernieri, Paolo; Abrams, Suzanne R.

    1991-01-01

    Specificities of three monoclonal antibodies (15-I-C5, DBPA 1, and MAC 62) raised against the plant hormone (S)-(+)-abscisic acid (ABA) have been compared. Immunological cross-reactivities against fifteen biologically active analogs of ABA were measured. The ABA analogs were altered at one or more of four positions: the double bonds in the ring, at C-2 C-3 and at C-4 C-5, and in the oxidation level at C-1. Several analogs were optically active with chiral centers at C-1′ and C-2′. For cross-reactivity, all three monoclonal antibodies required the carboxylic acid group, and the cis configuration of the double bond at C-2 C-3 of the ABA molecule. Monoclonals 15-I-C5 and DBPA 1 required the entire ABA sidechain from the C-1 to C-1′, but these monoclonals did cross-react with analogs with the ring double bond reduced and the C-2′ methyl cis to the sidechain. Only MAC 62 recognized analogs containing an acetylene at C-4 C-5. MAC 62 had more strict requirements for the ring double bond, but gave some cross-reactivity with acetylenic analogs having a saturated ring. All three monoclonals had higher specificity for analogs having the same absolute configuration at C-1′ as (S)-(+)-ABA. This work provides new information about the spatial regions of the ABA molecule that elicit immunological recognition, and serves as a basis for future investigations of the ABA receptor using ABA analogs and anti-idiotypic antibodies. PMID:16667979

  20. 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

  1. 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 3–4, 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

  2. 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.

  3. 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

  4. 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 >10⁵-fold selectivity over normal tissues, releasing Th1 cytokines (TNFα, IFNγ, 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. Monoclonal antibodies and method for detecting dioxins and dibenzofurans

    DOEpatents

    Vanderlaan, Martin; Stanker, Larry H.; Watkins, Bruce E.; Bailey, Nina R.

    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.

  10. 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.

  11. 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

  12. 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

  13. Monoclonal antibodies and immune therapies for adult precursor B-acute lymphoblastic leukemia.

    PubMed

    Sikaria, Swati; Aldoss, Ibrahim; Akhtari, Mojtaba

    2016-04-01

    Adult B-acute lymphoblastic leukemia (B-ALL) does not share the favorable prognosis seen in pediatric patients with the same disease. Less than 50% of patients experience long-term survival and for those adults over age 60, long-term survival is only 10%. At time of relapse, 5-year prognosis is a dismal 7%. Novel and less toxic agents are urgently needed. The last few years have seen a surge in immune therapies for B-ALL. These agents may target CD19, CD20, CD22, and less frequently CD52. Expression of these surface markers and the drugs which target them are discussed. Some immune therapies are simple monoclonal antibodies against B lymphocyte markers such as rituximab, ofatumumab, and epratuzumab. Others are in a class of antibody-drug conjugates which link a highly toxic chemotherapy to a monoclonal antibody for targeted delivery, such as inotuzumab and denintuzumab. Finally, novel immune therapies recruit (in the case of bispecific T cell engager [BiTE]) or modify (in the case of chimeric antigen receptor [CAR] T cells) one's own T cells to fight leukemic cells. This article reviews the rationale, clinical data, and toxicity profiles of immune therapies approved or in late stages of development for B-ALL. PMID:26921100

  14. Zinc oxide-coated plasmonic chip modified with a bispecific antibody for sensitive detection of a fluorescent labeled-antigen.

    PubMed

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

    2011-08-01

    A plasmonic biosensor chip of silver-coated PMMA grating with a zinc oxide (ZnO) overlayer is fabricated for surface plasmon field-enhanced fluorescence (SPF) detection of Cy5-labeled green fluorescent protein (GFP). A bispecific antibody (anti-GFP x anti-ZnO antibody) prepared in our lab is densely immobilized on the sensor chip for GFP detection. The sensitivity of the plasmonic biosensors is improved due to densely packed antibodies and ZnO-coating that suppresses nonspecific protein adsorption and fluorescent quenching. With the ZnO-coated plasmonic chip, Cy5-labeled GFP of 10 pM can be detected through SPF. This sensitivity is 100 higher compared with the normal fluorescent detection on a ZnO-coated glass slide. PMID:21692512

  15. Monoclonal antibody to growth hormone receptors

    SciTech Connect

    Simpson, J.S.A.; Friesen, H.G.

    1985-01-01

    Using hybridoma technology, monoclonal antibodies to growth hormone receptors can be prepared in large quantities with only a few micrograms of purified antigen by in vitro immunization or by immunization of larger quantities of less pure material. The discussion centers on areas most pertinent to receptors such as receptor preparation, immunization procedure, fusion method, screening assay, and identification of the immunoglobulin class. The specificity of the antibody for growth hormone receptor was examined by testing the ability of the ascitic fluid to inhibit binding of (/sup 125/I) growth hormone to the prolactin receptors on rabbit mammary gland membranes and the inhibition of /sup 125/I-labelled rat growth hormone binding to rabbit liver membrane.

  16. 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

  17. 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...

  18. 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

  19. 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

  20. 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.

  1. 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

  2. Building better monoclonal antibody-based therapeutics.

    PubMed

    Weiner, George J

    2015-06-01

    For 20 years, monoclonal antibodies (mAbs) have been a standard component of cancer therapy, but there is still much room for improvement. Efforts continue to build better cancer therapeutics based on mAbs. Anticancer mAbs function through various mechanisms, including directly targeting the malignant cells, modifying the host response, delivering cytotoxic moieties and 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 Review discusses the various approaches to using mAb-based therapeutics to treat cancer and the strategies used to take advantage of the unique potential of each approach, and provides examples of current mAb-based treatments. PMID:25998715

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. Monoclonal antibody therapy for Junin virus infection.

    PubMed

    Zeitlin, Larry; Geisbert, Joan B; Deer, Daniel J; Fenton, Karla A; Bohorov, Ognian; Bohorova, Natasha; Goodman, Charles; Kim, Do; Hiatt, Andrew; Pauly, Michael H; Velasco, Jesus; Whaley, Kevin J; Altmann, Friedrich; Gruber, Clemens; Steinkellner, Herta; Honko, Anna N; Kuehne, Ana I; Aman, M Javad; Sahandi, Sara; Enterlein, Sven; Zhan, Xiaoguo; Enria, Delia; Geisbert, Thomas W

    2016-04-19

    Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic. PMID:27044104

  9. Monoclonal antibody therapy for Junin virus infection

    PubMed Central

    Zeitlin, Larry; Geisbert, Joan B.; Deer, Daniel J.; Fenton, Karla A.; Bohorov, Ognian; Bohorova, Natasha; Goodman, Charles; Kim, Do; Hiatt, Andrew; Pauly, Michael H.; Velasco, Jesus; Whaley, Kevin J.; Altmann, Friedrich; Gruber, Clemens; Steinkellner, Herta; Honko, Anna N.; Kuehne, Ana I.; Aman, M. Javad; Sahandi, Sara; Enterlein, Sven; Zhan, Xiaoguo; Enria, Delia; Geisbert, Thomas W.

    2016-01-01

    Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic. PMID:27044104

  10. Characterization of monoclonal antibodies against fowl poxvirus.

    PubMed

    Singh, P; Tripathy, D N

    2000-01-01

    Vaccines for the prevention of fowl pox in chickens and turkeys have been available for more than five decades. However, in recent years outbreaks have occurred in several previously vaccinated chicken flocks. Presumably, fowl poxviruses (FPVs) antigenically different from the attenuated vaccine strains are responsible for such occurrences. In support of this concept, we previously detected minor antigenic changes in field isolates based on comparative immunoblotting with polyclonal anti-FPV serum. Realizing the need for antibodies specific against the dominant antigens of FPV, monoclonal antibodies (MAbs) were produced by immunizing mice with either a field strain of FPV or a pigeon poxvirus, currently used for vaccination. Three hybridoma clones producing MAbs reacting with a specific FPV protein were selected from a total of 83 clones. In immunoblots, two of the MAbs, P1D9 and P2H10, recognized an antigen with an apparent molecular weight varying from 39 to 46 kD, depending on the FPV strain. The third MAb, P2D4, reacted with an approximately 80-kD protein, regardless of which FPV isolate was tested. Immunofluorescent staining with P1D9 and P2D4 revealed that these MAbs react with intracytoplasmic antigens in FPV-infected cells. PMID:10879917

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

    PubMed

    Ulaeto, David O; Hutchinson, Alistair P; Nicklin, Stephen

    2015-08-01

    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

  12. 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

  13. 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

  14. 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.

  15. Aged venous thrombi: radioimmunoimaging with fibrin-specific monoclonal antibody

    SciTech Connect

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

    1987-02-01

    Radioimmunoimaging of fresh canine venous thrombi with a murine monoclonal antibody specific for human and dog fibrin has been reported. Successful imaging of canine deep venous thrombi 1, 3, and 5 days old at the time of antibody injection is reported. Images were positive in all dogs, and the uptake of fibrin-specific antibody was equivalent to that of fresh thrombi.

  16. 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

  17. 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

  18. DEVELOPMENT OF MONOCLONAL ANTIBODIES AGAINST FATHEAD MINNOW (PIMEPHALES PROMELAS) VITELLOGENIN

    EPA Science Inventory

    We have obtained a panel of monoclonal antibodies directed against fathead minnow vitellogenin (Vtg) for use in sensitive ELISAs to quantify the response of exposure in vivo to estrogen or estrogen mimics.

  19. 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.

  20. Preparation of Monoclonal Antibodies Against Bovine Haptoglobin

    PubMed Central

    Wang, Caihong; Gu, Cheng; Guo, Donghua; Gao, Jing; Li, Chunqiu; Liu, Na; Geng, Yufei; Su, Mingjun; Wang, Xinyu

    2014-01-01

    Female, 8-week-old BALB/c mice were immunized with purified recombinant proteins of the predicted immunodominant region of bovine haptoglobin (pirBoHp). Two monoclonal antibodies (MAbs), named 1B3 and 6D6, were prepared by conventional B lymphocyte hybridoma technique. Titers of ascitic fluid and cell culture supernatant of MAb 1B3 were 1:9.6×108 and 1:8.2×104, respectively, and that of MAb 6D6 were 1:4.4×105 and 1:1.0×104, respectively. The subtype of MAbs 1B3 and 6D6 was IgG1κ. In Western blot analysis, MAbs 1B3 and 6D6 could recognize the α-chain of native BoHp from plasma of dairy cows. These data indicated that MAbs 1B3 and 6D6 have a potential use for developing diagnostic reagents of BoHp. PMID:25358005

  1. [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

  2. Murine monoclonal antibodies specific for virulent Treponema pallidum (Nichols).

    PubMed Central

    Robertson, S M; Kettman, J R; Miller, J N; Norgard, M V

    1982-01-01

    Murine anti-Treponema pallidum (Nichols) lymphocyte hybridoma cell lines secreting monoclonal antibodies against a variety of treponemal antigens have been generated. Hybridomas isolated were of three major types: those that were directed specifically against T. pallidum antigens, those that were directed against treponemal group antigens (as evidenced by their cross-reactivity with T. phagedenis biotype Reiter antigens), and those that cross-reacted with both treponemal as well as rabbit host testicular tissue antigens. The majority (31 of 39 clones) of these anti-T. pallidum hybridomas, which produced monoclonal antibodies of mouse isotypes immunoglobulin G1 (IgG1), IgG2a, IgG2b, IgG3 or IgM, were directed specifically against T. pallidum and not other treponemal or rabbit antigens tested by radioimmunoassay. Four of these T. pallidum-specific hybridomas secreted monoclonal antibodies with greater binding affinity for "aged" rather than freshly isolated intact T. pallidum cells, suggesting a possible specificity for "unmasked" surface antigens of T. pallidum. Six anti-T. pallidum hybridomas produced complement-fixing monoclonal antibodies (IgG2a, IgG2b, or IgM) that were capable of immobilizing virulent treponemes in the T. pallidum immobilization (TPI) test; these may represent biologically active monoclonal antibodies against treponemal surface antigens. Three other hybridomas secreted monoclonal antibodies which bound to both T. pallidum and T. phagedenis biotype Reiter antigens, thus demonstrating a possible specificity for treponemal group antigens. Five hybridoma cell lines were also isolated which produced IgM monoclonal antibodies that cross-reacted with all treponemal and rabbit host testicular tissue antigens employed in the radioimmunoassays. This report describes the construction and characteristics of these hybridoma cell lines. The potential applications of the anti-T. pallidum monoclonal antibodies are discussed. PMID:7047388

  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. XGFR*, a novel affinity-matured bispecific antibody targeting IGF-1R and EGFR with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer.

    PubMed

    Schanzer, Juergen M; Wartha, Katharina; Moessner, Ekkehard; Hosse, Ralf J; Moser, Samuel; Croasdale, Rebecca; Trochanowska, Halina; Shao, Cuiying; Wang, Peng; Shi, Lei; Weinzierl, Tina; Rieder, Natascha; Bacac, Marina; Ries, Carola H; Kettenberger, Hubert; Schlothauer, Tilman; Friess, Thomas; Umana, Pablo; Klein, Christian

    2016-01-01

    The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the "knobs-into-holes" technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2-3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer. PMID:26984378

  5. 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.

  6. 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

  7. 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.

  8. Brain penetration, target engagement, and disposition of the blood-brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1.

    PubMed

    Webster, Carl I; Caram-Salas, Nadia; Haqqani, Arsalan S; Thom, George; Brown, Lee; Rennie, Kerry; Yogi, Alvaro; Costain, Willard; Brunette, Eric; Stanimirovic, Danica B

    2016-05-01

    Receptor mediated transcytosis harnessing the cellular uptake and transport of natural ligands across the blood-brain barrier (BBB) has been identified as a means for antibody delivery to the CNS. In this study, we characterized bispecific antibodies in which a BBB-crossing antibody fragment FC5 was used as a BBB carrier. Cargo antibodies were either a high-affinity, selective antibody antagonist of the metabotropic glutamate receptor-1 (BBB-mGluR1), a widely abundant CNS target, or an IgG that does not bind the CNS target (BBB-NiP). Both BBB-NiP and BBB-mGluR1 demonstrated a similar 20-fold enhanced rate of transcytosis across an in vitro BBB model compared with mGluR1 IgG fused to a control antibody fragment. All 3 bispecific antibodies exhibited identical pharmacokinetics in vivo Comparative assessment of BBB-NiP and BBB-mGluR1 revealed that, whereas their serum pharmacokinetics and BBB penetration were identical, their central disposition (brain levels) and elimination (cerebrospinal fluid levels) were widely different, due to central target-mediated removal of the mGluR1-engaging antibody. Central mGluR1 target engagement after systemic administration was demonstrated by a dose-dependent inhibition of mGluR-1-mediated thermal hyperalgesia and by colocalization of the antibody with thalamic neurons involved in mGluR1-mediated pain processing. We demonstrate the feasibility of targeting central G-protein-coupled receptors using a BBB-crossing bispecific antibody approach and emerging principles that govern brain distribution and disposition of these antibodies. These data will be important for designing safe and selective CNS antibody therapeutics.-Webster, C. I., Caram-Salas, N., Haqqani, A. S., Thom, G., Brown, L., Rennie, K., Yogi, A., Costain, W., Brunette, E., Stanimirovic, D. B. Brain penetration, target engagement, and disposition of the blood-brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1. PMID:26839377

  9. 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

  10. 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

  11. Syngeneic anti-idiotypic monoclonal antibodies to an anti-NeuGc-containing ganglioside monoclonal antibody.

    PubMed

    Vázquez, A M; Pérez, A; Hernández, A M; Macías, A; Alfonso, M; Bombino, G; Pérez, R

    1998-12-01

    An IgM monoclonal antibody (MAb), named P3, has the characteristic to react specifically with a broad battery of N-glycolyl containing-gangliosides and with antigens expressed on breast tumors. When this MAb was administered alone in syngeneic mice, an specific IgG anti-idiotypic antibody (Ab2) response was induced, this Ab2 response was increased when P3 MAb was injected coupled to a carrier protein and in the presence of Freund's adjuvant. Spleen cells from these mice were used in somatic-cell hybridization experiments, using the murine myeloma cell line P3-X63-Ag8.653 as fusion partner. Five Ab2 MAbs specific to P3 MAb were selected. These IgG1 Ab2 MAbs were able to block the binding of P3 MAb to GM3(NeuGc) ganglioside and to a human breast carcinoma cell line. Cross-blocking experiments demonstrated that these Ab2 MAbs are recognizing the same or very close sites on the Abl MAb. The five Ab2 MAbs were injected into syngeneic mice and four of them produced strong anti-anti-idiotypic antibody (Ab3) response. While these Ab2 MAbs were unable to generate Ab3 antibodies with the same antigenic specificity than P3 MAb, three of them induced antibodies bearing P3 MAb idiotopes (Ag-Id+ Ab3). These results demonstrated that these Ab2 MAbs are not "internal image" antibodies, but they could define "regulatory idiotopes." PMID:9890708

  12. 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...

  13. 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)

  14. 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

  15. 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.

  16. Coarse grained modeling of transport properties in monoclonal antibody solution

    NASA Astrophysics Data System (ADS)

    Swan, James; Wang, Gang

    Monoclonal antibodies and their derivatives represent the fastest growing segment of the bio pharmaceutical industry. For many applications such as novel cancer therapies, high concentration, sub-cutaneous injections of these protein solutions are desired. However, depending on the peptide sequence within the antibody, such high concentration formulations can be too viscous to inject via human derived force alone. Understanding how heterogenous charge distribution and hydrophobicity within the antibodies leads to high viscosities is crucial to their future application. In this talk, we explore a coarse grained computational model of therapeutically relevant monoclonal antibodies that accounts for electrostatic, dispersion and hydrodynamic interactions between suspended antibodies to predict assembly and transport properties in concentrated antibody solutions. We explain the high viscosities observed in many experimental studies of the same biologics.

  17. Monoclonal antibodies directed against different antigenic determinants of rotavirus.

    PubMed Central

    Lambert, J P; Marbehant, P; Marissens, D; Zissis, G

    1984-01-01

    We have developed a series of monoclonal antibodies against the calf strain RIT 4237 (subgroup 1) and the human strain 82-561 (subgroup 3) of rotavirus, both grown in tissue culture, and also against the human rotavirus 81-2162 (subgroup 2), extracted from a fecal specimen. A variety of different specificities was observed among these antibodies, namely, antibodies against group and subgroup determinants, as well as neutralizing antibodies. By using monoclonal antibodies against the subgroup antigen in an enzyme-linked immunoassay system, the constant predominance of subgroup 2 viruses in humans was confirmed in 74 stools collected from children in Brussels who suffered a diarrheal illness between July 1981 and June 1983. The availability of these antibodies also made it possible to improve the sensitivity and the specificity of the test system. PMID:6202884

  18. 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

  19. 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

  20. Monoclonal antibodies against human placental glutathione transferase (class pi).

    PubMed

    Massoud, R; Lo Bello, M; De Stefano, E; Molino, A; Zelaschi, D; Federici, G

    1991-02-01

    Five monoclonal antibodies (MAbs) were produced in a mouse hybridoma system against human placental glutathione transferase (GST pi). Four of these monoclonal antibodies, named 461 to 464, were of immunoglobulin G class, whereas the monoclonal antibody 465 was of IgA class. All these MAbs specifically recognized the glutathione transferase from human placenta (class pi) showing no cross reactivity against the basic and the neutral forms of GST from human liver. When each MAb was incubated with the GST pi, no inhibition of enzymatic activity towards 1-chloro-2,4-dinitrobenzene was observed except for MAb 465 which showed a slight inhibition to a serial dilution of 1:128. PMID:1709614

  1. 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

  2. Monoclonal antibodies to bovine parathyroid hormone: production and characterization.

    PubMed

    Vieira, J G; Federico, P; Matsueda, G; Neer, R M

    1988-01-01

    1. This paper describes the production and characterization of monoclonal antibodies against bovine parathyroid hormone (bPTH)-(1-84). 2. Spleen cells from A/J mice successfully immunized with bPTH-(1-84) were fused with SP2/O myeloma cells using PEG 4000 as fusogen. The screening method employed microtiter plates coated with sheep antimouse IgG and the presence of specific monoclonal antibodies was demonstrated by the binding of 125I-bPTH-(1-84). 3. A detailed study of the specificity of the three viable monoclonals with highest affinity showed that two (6FH6 and 6CD4) were amino-terminal specific and the other (5BG9) carboxyl-terminal specific. The two amino-terminal monoclonal antibodies appear to recognize the same antigenic site. 4. The monoclonal antibodies produced are potentially useful reagents for the development of new methods for the measurement of PTH in biological fluids, studies on the interaction of PTH with its receptor, as well as localization of PTH producing cells. PMID:3248228

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

    PubMed

    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

  4. 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; Schäfer, 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

  5. TriFabs—Trivalent 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; Schäfer, 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

  6. 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

  7. Monoclonal antibodies and Fc fragments for treating solid tumors.

    PubMed

    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

  8. 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

  9. 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

  10. Antibody discovery: sourcing of monoclonal antibody variable domains.

    PubMed

    Strohl, William R

    2014-03-01

    Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described. PMID:24168292

  11. 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

  12. 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...

  13. Monoclonal Antibodies Attached to Carbon Nanotube Transistors for Paclitaxel Detection

    NASA Astrophysics Data System (ADS)

    Lee, Wonbae; Lau, Calvin; Richardson, Mark; Rajapakse, Arith; Weiss, Gregory; Collins, Philip; UCI, Molecular Biology; Biochemistry Collaboration; UCI, Departments of Physics; Astronomy Collaboration

    Paclitaxel is a naturally-occurring pharmaceutical used in numerous cancer treatments, despite its toxic side effects. Partial inhibition of this toxicity has been demonstrated using weakly interacting monoclonal antibodies (3C6 and 8A10), but accurate monitoring of antibody and paclitaxel concentrations remains challenging. Here, single-molecule studies of the kinetics of antibody-paclitaxel interactions have been performed using single-walled carbon nanotube field-effect transistors. The devices were sensitized with single antibody attachments to record the single-molecule binding dynamics of paclitaxel. This label-free technique recorded a range of dynamic interactions between the antibody and paclitaxel, and it provided sensitive paclitaxel detection for pM to nM concentrations. Measurements with two different antibodies suggest ways of extending this working range and uncovering the mechanistic differences among different antibodies.

  14. Cooperative Immunoassays: Ultrasensitive Assays with Mixed Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Ehrlich, Paul H.; Moyle, William R.

    1983-07-01

    Mixtures of certain monoclonal antibodies appear to bind human chorionic gonadotropin in a ``cooperative'' fashion because they form circular complexes with the hormone. Experiments illustrate how this property might be exploited to develop very sensitive immunoassays for human chorionic gonadotropin or any other antigen. Since the assays are not based on competitive inhibition between radiolabeled and unlabeled antigen, they are much more sensitive than a traditional radioimmunoassay in which either one of the same antibodies is used alone.

  15. 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

  16. Humanization of a chicken anti-IL-12 monoclonal antibody.

    PubMed

    Tsurushita, Naoya; Park, Minha; Pakabunto, Kanokwan; Ong, Kelly; Avdalovic, Anamarija; Fu, Helen; Jia, Audrey; Vásquez, Max; Kumar, Shankar

    2004-12-01

    Chicken anti-IL-12 monoclonal antibodies were isolated by phage display using spleen cells from a chicken immunized with human and mouse IL-12 as a source for library construction. One of the chicken monoclonal antibodies, DD2, exhibited binding to both human and mouse IL-12 in the single-chain Fv form and also after conversion to chicken-human chimeric IgG1/lambda antibody. The chicken DD2 variable regions were humanized by transferring their CDRs and several framework amino acids onto human acceptor variable regions. In the Vlambda, six chicken framework amino acids were identified to be important for the conformation of the CDR structure by computer modeling and therefore were retained in the humanized form; likewise, five chicken amino acids in the VH framework regions were retained in the humanized VH. The affinities of humanized DD2 IgG1/lambda to human and mouse IL-12 measured by competitive binding were nearly identical to those of chicken-human chimeric DD2 IgG1/lambda. This work demonstrates that humanization of chicken monoclonal antibodies assisted by computer modeling is possible, leading to a new way to generate therapeutic humanized antibodies against antigens to which the rodent immune system may fail to efficiently raise high affinity antibodies. PMID:15627607

  17. 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...

  18. 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...

  19. 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.

  20. 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

  1. 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; Künkele, 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

  2. Development of Biodegradable Nanocarriers Loaded with a Monoclonal Antibody

    PubMed Central

    Gdowski, Andrew; Ranjan, Amalendu; Mukerjee, Anindita; Vishwanatha, Jamboor

    2015-01-01

    Treatments utilizing monoclonal antibody therapeutics against intracellular protein-protein interactions in cancer cells have been hampered by several factors, including poor intracellular uptake and rapid lysosomal degradation. Our current work examines the feasibility of encapsulating monoclonal antibodies within poly(lactic-co-glycolic acid) (PLGA) nanoparticles using a water/oil/water double emulsion solvent evaporation technique. This method can be used to prepare protective polymeric nanoparticles for transporting functional antibodies to the cytoplasmic compartment of cancer cells. Nanoparticles were formulated and then characterized using a number of physical and biological parameters. The average nanoparticle size ranged from 221 to 252 nm with a low polydispersity index. Encapsulation efficiency of 16%–22% and antibody loading of 0.3%–1.12% were observed. The antibody molecules were released from the nanoparticles in a sustained manner and upon release maintained functionality. Our studies achieved successful formulation of antibody loaded polymeric nanoparticles, thus indicating that a PLGA-based antibody nanoformulation is a promising intracellular delivery vehicle for a large number of new intracellular antibody targets in cancer cells. PMID:25690029

  3. Development of biodegradable nanocarriers loaded with a monoclonal antibody.

    PubMed

    Gdowski, Andrew; Ranjan, Amalendu; Mukerjee, Anindita; Vishwanatha, Jamboor

    2015-01-01

    Treatments utilizing monoclonal antibody therapeutics against intracellular protein-protein interactions in cancer cells have been hampered by several factors, including poor intracellular uptake and rapid lysosomal degradation. Our current work examines the feasibility of encapsulating monoclonal antibodies within poly(lactic-co-glycolic acid) (PLGA) nanoparticles using a water/oil/water double emulsion solvent evaporation technique. This method can be used to prepare protective polymeric nanoparticles for transporting functional antibodies to the cytoplasmic compartment of cancer cells. Nanoparticles were formulated and then characterized using a number of physical and biological parameters. The average nanoparticle size ranged from 221 to 252 nm with a low polydispersity index. Encapsulation efficiency of 16%-22% and antibody loading of 0.3%-1.12% were observed. The antibody molecules were released from the nanoparticles in a sustained manner and upon release maintained functionality. Our studies achieved successful formulation of antibody loaded polymeric nanoparticles, thus indicating that a PLGA-based antibody nanoformulation is a promising intracellular delivery vehicle for a large number of new intracellular antibody targets in cancer cells. PMID:25690029

  4. Library of monoclonal antibodies against brush border membrane epithelial antigens

    SciTech Connect

    Behar, M.; Katz, A.; Silverman, M.

    1986-03-01

    A purified fraction of proximal tubule brush border membranes (BBM) was prepared from dog kidney and used to immunize mice. The standard technique of hybridoma production was followed as described by Kohler and Milstein. Production of antibodies was detected by indirect immunofluorescence on dog kidney slices and by immunodot against the purified fraction on nitrocellulose. Five hybrids exhibited anti BBM activity. These were cloned twice and yielded stable cell lines producing IgG type monoclonal antibodies against BBM. They were designated A/sub 1/, C/sub 7/, D/sub 3/, D/sub 7/ and H/sub 4/. As a family these five monoclonals have broad tissue specificity, i.e. positive staining of the surface mucosa of intestinal kidney proximal tubules. D/sub 3/ exhibits even broader specificity for epithelium reacting with bile canaliculi and choroid plexus. The authors have verified that at least 4/5 antibodies are directed against BBM protein as revealed by immunoprecipitation of solubilized BBM and detected by Coomassie blue staining or autoradiography of lactoperoxidase labelled BBM. Most interestingly all antibodies bind to the surface of LL CPK/sub 1/ cells, a continuous pig kidney cell line of undefined origin but exhibiting many characteristics of proximal tubule cells. The library of monoclonal antibodies obtained provide important probes with which to study membrane biogenesis and polarization in epithelial cells.

  5. Specificity analysis of monoclonal anti-DNA antibodies.

    PubMed Central

    Karounos, D G; Pisetsky, D S

    1987-01-01

    The specificity of a panel of murine monoclonal anti-DNA antibodies for DNA antigenic determinants was evaluated by testing their relative binding to various animal and bacterial DNAs. The antibody panel consisted of six monoclonal anti-DNAs of MRL-lpr/lpr and B6-lpr/lpr origin, while the antigens tested were calf thymus (CT), salmon testes (ST), E. coli (EC) and Micrococcus (MC) DNA. While all antibodies bound to CT, ST, and EC DNA to a similar extent by direct ELISA, only one showed an equivalent level of interaction with MC DNA. The relationship of antigenic sites recognized by the antibodies was evaluated further by competition ELISA, assessing the ability of the anti-DNAs to block the interaction of a biotinylated anti-DNA with solid-phase DNA antigen. For each of the DNAs tested, two patterns of DNA interaction could be distinguished on the basis of the relative inhibitory activity of the different monoclonals. These results suggest that anti-DNA antibodies can be characterized using naturally occurring DNAs, with the observed patterns of binding suggesting recognition of unique antigenic sites, some of which are discrete and non-overlapping. PMID:3495481

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. Monoclonal IgA Antibodies for Aflatoxin Immunoassays

    PubMed Central

    Ertekin, Özlem; Pirinçci, Şerife Şeyda; Öztürk, Selma

    2016-01-01

    Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort. PMID:27187470

  12. Cysteinylation of a monoclonal antibody leads to its inactivation.

    PubMed

    McSherry, Troy; McSherry, Jennifer; Ozaeta, Panfilo; Longenecker, Kenton; Ramsay, Carol; Fishpaugh, Jeffrey; Allen, Steven

    2016-01-01

    Post-translational modifications can have a signification effect on antibody stability. A comprehensive approach is often required to best understand the underlying reasons the modification affects the antibody's potency or aggregation state. Monoclonal antibody 001 displayed significant variation in terms of potency, as defined by surface plasmon resonance testing (Biacore), from lot to lot independent of any observable aggregation or degradation, suggesting that a post-translational modification could be driving this variability. Analysis of different antibody lots using analytical hydrophobic interaction chromatography (HIC) uncovered multiple peaks of varying size. Electrospray ionization mass spectrometry (ESI-MS) indicated that the antibody contained a cysteinylation post-translational modification in complementarity-determining region (CDR) 3 of the antibody light chain. Fractionation of the antibody by HIC followed by ESI-MS and Biacore showed that the different peaks were antibody containing zero, one, or two cysteinylation modifications, and that the modification interferes with the ability of the modified antibody arm to bind antigen. Molecular modeling of the modified region shows that this oxidation of an unpaired cysteine in the antibody CDR would block a potential antigen binding pocket, suggesting an inhibition mechanism. PMID:27050640

  13. The Use of Monoclonal Antibodies in Human Prion Disease

    NASA Astrophysics Data System (ADS)

    Bodemer, Walter

    Detection of PrP and its pathological isoform(s) is the key to understanding the etiology and pathogenesis of transmissible spongiform encephalopathy. There is ample evidence that PrP isoforms constitute a major component of an unknown and perhaps unconventional infectious agent. An etiological relationship between human and zoonotic transmissible spongiform encephalopathies may be revealed with monoclonal antibodies. Knowledge of the conformational transition rendering a nonpathogenic, almost ubiquitous cellular protein into a pathogenic one is crucial to defining pathomechanisms. The stepwise or even continuous formation of pathogenic molecules can be monitored. Any improvement in the early diagnosis could help to conceive new therapeutic measures which are not currently available. Determination of PrP isoforms in tissue, cells, or body fluids may be of prognostic value. Many experimental approaches in molecular medicine and molecular biology of the prion protein already rely on monoclonal antibodies. Recombinant antibodies such as the single-chain Fv may soon replace traditional hybridoma techniques. Binding affinity can easily be manipulated by a number of techniques, including in vitro mutagenesis - a step which could never be carried out using the traditional hybridoma technology. Monoclonal antibodies are and will remain an essential support for ongoing research on the prion protein in general and on the unconventional infectious prions.

  14. Monoclonal antibodies to HLA-DP-transfected mouse L cells.

    PubMed Central

    Heyes, J; Austin, P; Bodmer, J; Bodmer, W; Madrigal, A; Mazzilli, M C; Trowsdale, J

    1986-01-01

    Mouse L cells transfected with human HLA-DP (DPw4) alpha and beta genes were used to make monoclonal antibodies in C3H mice. A polymorphic antibody, DP11.1, was obtained, as well as several monomorphic antibodies. In ELISAs, DP11.1 bound to DPw4 cells and, more weakly, to DPw2, but not DPw1, -3, -5, or -6, using HLA homozygous cells. It also bound to L-cell transfectants expressing either DPw2 or DPw4 products. From B lymphoblastoid cell lysates labeled with [35S]methionine, the antibody immunoprecipitated alpha and beta chains of a similar size to those precipitated by a well-characterized DP monoclonal antibody, B7/21.2. Immunoblotting indicated that the DP11.1 antibody was directed against the alpha chain. This result confirms partial sequence data that showed that the DP alpha chain, as well as DP beta, is polymorphic, and that DPw2 and -4 alpha chains are very similar, if not identical. Images PMID:3517859

  15. 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

  16. A FcγRIII-engaging bispecific antibody expands the range of HER2-expressing breast tumors eligible to antibody therapy

    PubMed Central

    Turini, Marc; Chames, Patrick; Bruhns, Pierre

    2014-01-01

    Trastuzumab is established as treatment of HER2high metastatic breast cancers but many limitations impair its efficacy. Here, we report the design of a Fab-like bispecific antibody (HER2bsFab) that displays a moderate affinity for HER2 and a unique, specific and high affinity for FcγRIII. In vitro characterization showed that ADCC was the major mechanism of action of HER2bsFab as no significant HER2-driven effect was observed. HER2bsFab mediated ADCC at picomolar concentration against HER2high, HER2low as well as trastuzumab-refractive cell lines. In vivo HER2bsFab potently inhibited HER2high tumor growth by recruitment of mouse FcγRIII and IV-positive resident effector cells and more importantly, exhibited a net superiority over trastuzumab at inhibiting HER2low tumor growth. Moreover, FcγRIIIA-engagement by HER2bsFab was independent of V/F158 polymorphism and induced a stronger NK cells activation in response to target cell recognition. Thus, taking advantage of its epitope specificity and affinity for HER2 and FcγRIIIA, HER2bsFab exhibits potent anti-tumor activity against HER2low tumors while evading most of trastuzumab Fc-linked limitations thereby potentially enlarging the number of patients eligible for breast cancer immunotherapy. PMID:24979648

  17. 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; Riethmüller, 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

  18. Bispecific antibodies: an innovative arsenal to hunt, grab and destroy cancer cells.

    PubMed

    Grandjenette, Cindy; Dicato, Mario; Diederich, Marc

    2015-01-01

    Targeted cellular immunotherapy with bifunctional antibodies (bsAbs) has emerged as a promising therapeutic approach for cancer over the last two decades. Progress in antibody engineering has led to the generation of many different types of antibody-derived entities that display at least two binding specificities. Most bsAbs consist of large IgG-like proteins with multiple antigen-binding regions containing Fc parts or smaller entities without Fc. BsAbs have the potential to engage effector cells of the immune system, thereby overcoming some of the immune response escape mechanisms of tumor cells. Preclinical and clinical trials of various bsAb constructs have demonstrated impressive results in terms of immune effector cell retargeting and induction of efficient anti-tumor responses. This review provides an overview of the established bsAbs focusing on improvements in format and design as well as the mechanisms of action of the most promising candidates and describes the results of the most recent clinical studies. PMID:25941884

  19. 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

  20. Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

    PubMed Central

    Hameed, A.; Olsen, K. J.; Cheng, L.; Fox, W. M.; Hruban, R. H.; Podack, E. R.

    1992-01-01

    Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue. Images Figure 1 PMID:1374586

  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. Human tumor antigens identified with monoclonal antibodies

    SciTech Connect

    AlSedairy, S.T.

    1987-01-01

    MoAbLc1 (IgM) and MoAbLc2 (IgG/sub 2a/) were produced against human lung carcinoma cell line (ChaGo). Lc1 recognizes a approx. = 330-kd/approx. = 310-kd glycoprotein complexes, and Lc2 recognizes a approx. = 60-kd/approx. = 47-kd protein complex. With a panel of cell lines of different tissue origin, Lc1 showed a more restricted reactivity to ChaGo; it cross-reacted with another lung carcinoma cell line (SK-Lc-2) and two breast carcinoma cell lines, but failed to react with cell lines of fetal lung, of colon, esophageal, prostate, stomach, and ovarian carcinomas, of B and T lymphoblastoid cells, neuroblastomas, glioblastoma, astrocytoma, and human peripheral blood lymphocytes. New and improved methods were developed for the production of indium-111-labeled MoAbs for tumor imaging. To facilitate the application of bicyclic anhydride diethylenetriaminepentaacetic acid (BADTPA) to In-111 labeling of antibodies, we have modified the original method by using C-14-labeled BADTPA, which allows precise quantitation of DTPA molecules incorporated. A new heterobifunctional reagent, 2,6-dioxo-N-(carboxyl)morpholine (DCM) was synthesized for chelating In-111 to MoAbs, and demonstrated higher retention of immunoreactivity of the labeled antibody.

  3. 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

  4. 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.

  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. Current status of tumor imaging with monoclonal antibodies.

    PubMed

    Blend, M J

    1991-12-01

    Although the full potential of MoAb imaging has yet to be realized, technologic advances continue with great intensity at a number of academic and industrial research institutions. Continuous production of MoAbs will eventually yield a variety of highly specific antibodies and novel approaches for improving cancer detection. As new diagnostic and therapeutic methods continue to be developed, MoAbs will begin to play a major role as targeted carriers, provided adequate funding from industry and government can be readily obtained. At present, the future of monoclonal antibodies in diagnosis and therapy for cancer patients appears promising. PMID:1790666

  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. Monoclonal antibodies for medical oncology: a few critical perspectives.

    PubMed

    Belda-Iniesta, Cristóbal; Ibáñez de Cáceres, Inmaculada; de Castro, Javier

    2011-02-01

    Incorporation of antibodies as weapons for cancer therapy has meant a turning point in the survival, clinical and radiological response of many oncology patients. These drugs are effective, well designed missiles that either alone or in combination with chemotherapy are unavoidable weapons for breast, lung and colon cancer as well as for haematological tumours. In addition, incoming monoclonal antibodies (mAbs) and folder-like proteins will be incorporated into clinical practice in the near future. This review aims to discuss a few imminent indications of current mAbs that are used for solid tumours and to briefly introduce future mAbs to the reader. PMID:21324795

  11. 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

  12. Murine monoclonal antibodies neutralizing the cytotoxic activity of diphtheria toxin.

    PubMed

    Usuwanthim, Kanchana; Pootong, Anek; Chaisri, Urai; Tongtawe, Pongsri; Tapchaisri, Pramuan; Chongsa-Nguan, Manas; Chaicumpa, Wanpen

    2008-03-01

    In this study, murine monoclonal antibodies that specifically bound to the A and B subunits of diphtheria toxin (DT) were produced by conventional hybridoma technology using the spleens of BALB/c mice immunized with diphtheria DTP vaccine and CRM197. Monoclonal antibodies specific to the A subunit, i.e. clone AC5, as well as those specific to the B subunit, i.e. clone BB7, could neutralize the DT-mediated cytotoxicity to Vero cells in microcultures. The DT neutralizing mechanisms have yet to be determined. The MAbBB7 is hypothesized to either interfere with the DT receptor binding or with the pore forming function of the T domain of the B subunit. The MAbAC5 could neutralize the DT mediated cytotoxicity when mixed with the DT before adding to the Vero cell culture thus suggesting that the antibody interfered with the translocation of the A subunit. The A subunit-antibody complex might be too large to pass through the membrane channel formed by the T domain and thus prevent the accessibility of the A subunit to the cytosolic target. It is also possible that the MAb AC5 blocked the enzymatic active site of the enzyme catalytic subunit. While further experiments are needed to localize the epitopes of the two MAbs on the holo-DT in order to reveal the DT neutralizing mechanisms, both MAbs in their humanized forms have a high potential as human therapeutic antibodies for diphtheria. PMID:18595529

  13. Production and characterization of monoclonal antibodies to Rickettsia rickettsii.

    PubMed

    Lange, J V; Walker, D H

    1984-11-01

    Five mouse ascitic fluids (MAFs) containing monoclonal antibody to Rickettsia rickettsii were produced from three original fusions by murine hybridoma technology. The five MAFs were fractionated and purified; each contained monoclonal antibody of the immunoglobulin G2a subclass. Each monoclonal antibody-containing MAF was titrated by indirect immunofluorescence against three R. rickettsii isolates from humans and four other spotted fever group rickettsiae. Each MAF was also titrated in the complement fixation, latex agglutination, microagglutination, and indirect hemagglutination tests. Two of the MAFs were examined for their ability to prevent fever and rickettsemia in susceptible guinea pigs after a 1:100 dilution of each was mixed with viable R. rickettsii, and all five MAFs were titrated in the mouse toxicity phenomenon assay. All MAFs had high indirect immunofluorescence titers to the three strains of R. rickettsii (1:200,000 to 1:800,000), reduced indirect immunofluorescence titers to R. montana, and were nonreactive with R. akari, R. sibirica, and R. conorii. Each MAF was able to fix complement in the presence of spotted fever group antigen reagent and agglutinate a suspension of purified R. rickettsii, and each was negative in both the latex agglutination and the indirect hemagglutination tests. The two MAFs which were tested proved to be capable of preventing rickettsemia and death in guinea pigs, and each MAF was able to prevent death in mice at dilutions ranging from 1:40 to 1:80. PMID:6209219

  14. 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

  15. 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.

  16. 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

  17. Efficient generation of human IgA monoclonal antibodies.

    PubMed

    Lorin, Valérie; 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

  18. Isolation of human monoclonal antibodies from peripheral blood B cells

    PubMed Central

    Huang, Jinghe; Doria-Rose, Nicole A; Longo, Nancy S; Laub, Leo; Lin, Chien-Li; Turk, Ellen; Kang, Byong H; Migueles, Stephen A; Bailer, Robert T; Mascola, John R; Connors, Mark

    2016-01-01

    Isolation of monoclonal antibodies is an important technique for understanding the specificities and characteristics of antibodies that underlie the humoral immune response to a given antigen. Here we describe a technique for isolating monoclonal antibodies from human peripheral blood mononuclear cells. The protocol includes strategies for the isolation of switch-memory B cells from peripheral blood, the culture of B cells, the removal of the supernatant for screening and the lysis of B cells in preparation for immunoglobulin heavy-chain and light-chain amplification and cloning. We have observed that the addition of cytokines IL-2, IL-21 and irradiated 3T3-msCD40L feeder cells can successfully stimulate switch-memory B cells to produce high concentrations of IgG in the supernatant. The supernatant may then be screened by appropriate assays for binding or for other functions. This protocol can be completed in 2 weeks. It is adaptable to use in other species and enables the efficient isolation of antibodies with a desired functional characteristic without prior knowledge of specificity. PMID:24030440

  19. Automated radiolabelling of monoclonal antibodies with the Modular Lab system.

    PubMed

    Ellison, David; Kaufman, Jens; Mather, Stephen J

    2010-02-01

    Automation of radiopharmaceutical preparation represents a way of reducing radiation doses to operators and providing good manufacturing practice-compliant manufacture of radiopharmaceuticals. Although widely adopted for PET radiochemical synthesis, such methods have not widely been used for the preparation of therapeutic radiopharmaceuticals. Here we describe an evaluation of an automated system for the preparation of radiolabelled antibodies. A labelling method based on the Modular Lab system was developed and applied to the radiolabelling of the monoclonal antibody conjugate, YAML568-CHX'A-DTPA, with indium-111. Antibodies were labelled using both manual and automated means and analysed by high-performance liquid chromatography and thin-layer chromatography methods. Radiochemical purities achieved using automation (mean 98%) were slightly lower than those achieved manually (mean 99%); however, the radiochemical purity obtained was reproducible and well within the set specification of greater than 95%. The Modular Lab system can be used to automate the radiolabelling of monoclonal antibody-chelate conjugates with metallic isotopes such as indium for the potential application for therapeutic radionuclide therapy. PMID:19952855

  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. 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

  2. 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

  3. 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

  4. Circadian variation of lymphocyte subpopulations: a study with monoclonal antibodies.

    PubMed Central

    Ritchie, A W; Oswald, I; Micklem, H S; Boyd, J E; Elton, R A; Jazwinska, E; James, K

    1983-01-01

    Use of monoclonal antibodies to identify subpopulations of circulating lymphocytes in healthy adults showed pronounced circadian variations in total T cells, the two major T cell subsets, and HLA-DR+ lymphocytes. When the results for the T cell subsets were expressed as a ratio (helper:suppressor) no significant rhythmic variation was observed. Lymphocytes bearing a surface antigen identified by the HNK-1 antibody (a population containing the natural killer and antibody dependent killer activity) did not show significant rhythmic variation. There was an inverse relation between plasma cortisol concentration and numbers of T and B cells. These observations have therapeutic implications and should be considered in the course of immunological monitoring. PMID:6407561

  5. Monoclonal antibody against bovine Lactoferricin and its epitopic site.

    PubMed

    Shimazaki, K; Nam, M S; Harakawa, S; Tanaka, T; Omata, Y; Saito, A; Kumura, H; Mikawa, K; Igarashi, I; Suzuki, N

    1996-12-01

    Bovine lactoferricin (LFcin B) is a strong antimicrobial peptide derived from N-lobe of lactoferrin. To study the immunochemical and structural properties of LFcin B, monoclonal antibody (mAb) was prepared and the amino acid sequence concerning with the binding to mAb has been identified. Mice injected with LFcin B showed no production of antibody specific to this peptide, whereas those with LFcin B-KLH conjugate produced anti-LFcin B antibodies. None of the mAb reacted with bovine lactoferrin C-lobe, human lactoferrin or LFcin H. By the reactivity of the mAb against the peptides synthesized on cellulose membranes using SPOTs and against chemically modified derivatives of LFcin B, the antigenic determinant of LFcin B was identified to be the sequence of "QWR". PMID:8996707

  6. 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

  7. [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

  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. Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies

    PubMed Central

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

    2015-01-01

    ABSTRACT Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28. IMPORTANCE Human parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more targeted treatment, we have searched for human monoclonal antibodies that would neutralize human parechoviruses 1 and 2, associated with mild infections such as gastroenteritis and severe infections of the central nervous system, and thus allow safe treatment. In the current study, we show how two such promising antibodies interact with the virus, modeling the atomic interactions between the virus and the antibody to propose how neutralization occurs. Both antibodies can cause aggregation; in addition, one antibody interferes with the virus recognizing its target cell, while the other, recognizing only the whole virus, inhibits the genome uncoating and replication in the cell. PMID:26157123

  10. 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

  11. Precipitation of a monoclonal antibody by soluble tungsten.

    PubMed

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

    2009-09-01

    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

  12. 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

  13. 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

  14. Characterization of oxidative carbonylation on recombinant monoclonal antibodies.

    PubMed

    Yang, Yi; Stella, Cinzia; Wang, Weiru; Schöneich, Christian; Gennaro, Lynn

    2014-05-20

    In the biotechnology industry, oxidative carbonylation as a post-translational modification of protein pharmaceuticals has not been studied in detail. Using Quality by Design (QbD) principles, understanding the impact of oxidative carbonylation on product quality of protein pharmaceuticals, particularly from a site-specific perspective, is critical. However, comprehensive identification of carbonylation sites has so far remained a very difficult analytical challenge for the industry. In this paper, we report for the first time the identification of specific carbonylation sites on recombinant monoclonal antibodies with a new analytical approach via derivatization with Girard's Reagent T (GRT) and subsequent peptide mapping with high-resolution mass spectrometry. Enhanced ionization efficiency and high quality MS(2) data resulted from GRT derivatization were observed as key benefits of this approach, which enabled direct identification of carbonylation sites without any fractionation or affinity enrichment steps. A simple data filtering process was also incorporated to significantly reduce false positive assignments. Sensitivity and efficiency of this approach were demonstrated by identification of carbonylation sites on both unstressed and oxidized antibody bulk drug substances. The applicability of this approach was further demonstrated by identification of 14 common carbonylation sites on three highly similar IgG1s. Our approach represents a significant improvement to the existing analytical methodologies and facilitates extended characterization of oxidative carbonylation on recombinant monoclonal antibodies and potentially other protein pharmaceuticals in the biotechnology industry. PMID:24731230

  15. 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.

  16. Identification of Vibrio vulnificus O serovars with antilipopolysaccharide monoclonal antibody.

    PubMed Central

    Martin, S J; Siebeling, R J

    1991-01-01

    A serotyping scheme for Vibrio vulnificus predicated on the detection of lipopolysaccharide (LPS) antigens is proposed. The serovar O typing scheme used to type V. vulnificus employs polyclonal antisera raised in rabbits immunized with heat-killed whole-cell vaccines. Polyclonal typing sera produced in this manner cross-react with heterologous strains. Affinity purification of polyclonal antisera with LPS affinity columns resolved some of these cross-reactions; however, affinity-purified polyclonal antisera still showed cross-reactions that were nonreciprocal. On the basis of the serological patterns that were obtained with affinity-purified polyclonal antisera, V. vulnificus strains were selected as vaccine strains for production of monoclonal antibody. Spleen cells harvested from BALB/c mice immunized with formalin-killed V. vulnificus cells were fused with SP2/O-Ag 14 myeloma cells. Hybridomas were screened by using LPS and whole-cell enzyme-linked immunosorbent assay to identify clones secreting LPS-specific antibodies. Monoclonal antibodies identified five LPS serological varieties of V. vulnificus and a single serovar each for Vibrio damsela and Vibrio hollisae. No cross-reactions between V. vulnificus and V. hollisae or V. damsela were observed. PMID:1761690

  17. 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

  18. Endotoxin reduction in monoclonal antibody preparations using arginine.

    PubMed

    Ritzén, Ulrika; Rotticci-Mulder, Joke; Strömberg, Patrik; Schmidt, Stefan R

    2007-09-01

    A monoclonal antibody preparation was found to be contaminated with endotoxin. Several commercial endotoxin removal steps were attempted but failed to produce a significant reduction due to the fact that the endotoxin was associated with the antibody. Here, several methods for endotoxin removal based on immobilizing monoclonal antibodies to chromatographic media have been evaluated. A crucial step in this process was to dissociate the endotoxin from the protein surface for subsequent removal. This was accomplished by introducing different buffer additives in the mobile phase. In agreement with previous reports, non-ionic detergents efficiently removed endotoxin, but it was also found that 0.5M arginine performed equally well. Since arginine is a non-toxic common amino acid that can be readily removed, it was selected and successfully used in large-scale experiments. With this method, endotoxin could be reduced to <0.2 EU mg(-1) with recovery of the target protein being >95%. Since this procedure is easily integrated into the existing processes of mAb purification, it offers advantages in speed, cost and effort. PMID:17644450

  19. 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

  20. A human fetal monoclonal DNA-binding antibody shares idiotypes with fetal and adult murine monoclonal DNA-binding antibodies.

    PubMed Central

    Watts, R A; Ravirajan, C T; Staines, N A; Isenberg, D A

    1990-01-01

    A human DNA-binding monoclonal antibody was produced by fusing the hepatocytes from a 12-week-old human fetus with the lymphoblastoid cell line GM 4672 using polyethylene glycol. This antibody, designated BEG 2, binds to single-stranded (ss) DNA but also binds to double-stranded (ds) DNA, poly(dT), polyI and poly(ADP-ribose), but not to RNA, cardiolipin or K-30. The binding of BEG 2 to these polynucleotides can be inhibited by incubation with polynucleotides in the fluid phase. A rabbit polyclonal anti-idiotype was raised, and using this reagent it was shown that the BEG 2 idiotype is present in normal human serum (7%), systemic lupus erythematosus (SLE) sera (8%) and rheumatoid arthritis sera (23%). The extent of idiotypic sharing between BEG 2 and murine monoclonal DNA-binding antibodies, in particular monoclonal antibody (mAb) 423 (derived from a 15-day-old fetal MRL/Mp-lpr/lpr mouse) and mAb 402 (derived from an adult MRL/lpr mouse), was also investigated. Using a competition ELISA, it was shown that preincubation of BEG 2 with rabbit anti-423 and rabbit anti-402 inhibits the binding of BEG 2 to DNA, and the binding of 402 to DNA by anti-BEG 2 and anti-423. These data suggest that mAb BEG 2, 423 and 402 share common idiotypes, that autoreactivity is present in early fetal life, and that autoantibodies may be encoded for by germline genes, which have been conserved through evolution. Images Figure 2 Figure 3 Figure 7 PMID:2312159

  1. 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

  2. Comparison of type 2 and type 6 fimbriae of Bordetella pertussis by using agglutinating monoclonal antibodies.

    PubMed

    Li, Z M; Brennan, M J; David, J L; Carter, P H; Cowell, J L; Manclark, C R

    1988-12-01

    Two types of fimbriae have been identified on the pathogenic gram-negative organism Bordetella pertussis. Monoclonal antibodies to these fimbriae were produced to better understand the role of fimbriae as serotype-specific agglutinogens and to investigate the antigenic relationship between these fimbriae. Three monoclonal antibodies were identified that specifically agglutinated B. pertussis cells containing the U.S. Reference Factor 2 agglutinogen, and six monoclonal antibodies were produced that agglutinated only those strains containing the U.S. Reference Factor 6 agglutinogen. Indirect immunofluorescence studies and immunogold electron microscopy demonstrated that these monoclonal antibodies bind to an outer membrane component on serotype-specific strains of B. pertussis. All of the monoclonal antibodies reacted with native or partially assembled type-specific fimbriae but not with monomeric fimbrial subunits as indicated by Western blot (immunoblot) analysis. The fimbrial agglutinogens recognized by the monoclonal antibodies were also uniquely reactive with either U.S. Reference Factor 2 or 6 antiserum (Eldering agglutinogen 2 or 6 polyclonal antiserum) in an indirect ELISA. No cross-reactivity of the monoclonal antibodies with the unrelated fimbriae was observed in any of the comparative immunological studies. Some of the monoclonal antibodies agglutinated certain strains of B. bronchiseptica, suggesting that this closely related species can contain antigenically similar fimbriae. These monoclonal antibodies should prove useful for further structural and functional analysis of Bordetella fimbriae and for studies on the role that these antigens play in prevention of infection and disease. PMID:2903125

  3. Comparison of type 2 and type 6 fimbriae of Bordetella pertussis by using agglutinating monoclonal antibodies.

    PubMed Central

    Li, Z M; Brennan, M J; David, J L; Carter, P H; Cowell, J L; Manclark, C R

    1988-01-01

    Two types of fimbriae have been identified on the pathogenic gram-negative organism Bordetella pertussis. Monoclonal antibodies to these fimbriae were produced to better understand the role of fimbriae as serotype-specific agglutinogens and to investigate the antigenic relationship between these fimbriae. Three monoclonal antibodies were identified that specifically agglutinated B. pertussis cells containing the U.S. Reference Factor 2 agglutinogen, and six monoclonal antibodies were produced that agglutinated only those strains containing the U.S. Reference Factor 6 agglutinogen. Indirect immunofluorescence studies and immunogold electron microscopy demonstrated that these monoclonal antibodies bind to an outer membrane component on serotype-specific strains of B. pertussis. All of the monoclonal antibodies reacted with native or partially assembled type-specific fimbriae but not with monomeric fimbrial subunits as indicated by Western blot (immunoblot) analysis. The fimbrial agglutinogens recognized by the monoclonal antibodies were also uniquely reactive with either U.S. Reference Factor 2 or 6 antiserum (Eldering agglutinogen 2 or 6 polyclonal antiserum) in an indirect ELISA. No cross-reactivity of the monoclonal antibodies with the unrelated fimbriae was observed in any of the comparative immunological studies. Some of the monoclonal antibodies agglutinated certain strains of B. bronchiseptica, suggesting that this closely related species can contain antigenically similar fimbriae. These monoclonal antibodies should prove useful for further structural and functional analysis of Bordetella fimbriae and for studies on the role that these antigens play in prevention of infection and disease. Images PMID:2903125

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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

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

    Cancer.gov

    A monoclonal antibody has proven effective in preventing Middle Eastern Respiratory Syndrome (MERS) in lab animals, suggesting further development as a potential intervention for the deadly disease in humans, according to new research. MERS is a newly emerged coronavirus first detected in humans in 2012. Most cases have occurred in the Middle East, but the disease has appeared elsewhere. In all, MERS has infected more than 1,700 individuals and killed more than 600, according to the World Health Organization. No vaccines or antiviral therapies currently exist. Several candidate vaccines are being developed, and some have been tested in animal models, a prerequisite to human clinical trials.

  11. 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

  12. 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

  13. Moving through three-dimensional phase diagrams of monoclonal antibodies.

    PubMed

    Rakel, Natalie; Baum, Miriam; Hubbuch, Jürgen

    2014-01-01

    Protein phase behavior characterization is a multivariate problem due to the high amount of influencing parameters and the diversity of the proteins. Single influences on the protein are not understood and fundamental knowledge remains to be obtained. For this purpose, a systematic screening method was developed to characterize the influence of fluid phase conditions on the phase behavior of proteins in three-dimensional phase diagrams. This approach was applied to three monoclonal antibodies to investigate influences of pH, protein and salt concentrations, with five different salts being tested. Although differences exist between the antibodies, this extensive study confirmed the general applicability of the Hofmeister series over the broad parameter range analyzed. The influence of the different salts on the aggregation (crystallization and precipitation) probability was described qualitatively using this Hofmeister series, with a differentiation between crystallization and precipitation being impossible, however. PMID:25044865

  14. 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.

  15. Characterization of monoclonal antibodies against a human chondrocyte surface antigen.

    PubMed

    Chanmee, Theerawut; Phothacharoen, Peraphan; Thongboonkerd, Visith; Kasinrerk, Watchara; Kongtawelert, Prachya

    2013-06-01

    Chondrocytes express a number of cell-surface molecules that mediate cell-cell or cell-matrix interactions. Identification and full characterization of new chondrocyte surface molecules will lead to a better understanding of the function of the chondrocyte. Researchers used primary human chondrocytes as an immunogen, and various monoclonal antibodies (MAbs) were generated using standard hybridoma technology. A monoclonal antibody named 5D2 was selected for further characterization. The antigen recognized by 5D2 MAb is expressed by primary human chondrocytes, primary synovial fibroblasts, synovial fibroblast cell lines (SW982), primary skin fibroblasts, and osteoblasts, but not expressed in blood cells. Biochemical analysis revealed that the 5D2 antigen is a protein with a molecular weight of approximately 25-35 kDa. Protein identification by mass spectrometry and molecular cloning revealed that 5D2 antigen is identical to the Thy-1 molecule. Furthermore we confirmed this specificity of the antibody by the isolated and cloned Thy-1 gene to the COS-7 and probed it with the 5D2 antibody using Western blot analysis. We examined the role of the Thy-1 molecule in arthritis models and tissue; one was papain-induced rat arthritis, the other was immunohistological staining of osteoarthritic (OA) human articular cartilage. OA cartilage showed a higher expression of Thy-1 as compared with normal tissue in all experimental approaches. The in vitro studies showed that the inflammatory cytokine interleukin-1β up-regulated Thy-1 molecule expression in the cartilage tissue. It can be concluded that the Thy-1 might be a potential biomarker for cartilage pathogenesis, degradation, and metabolic turnover. PMID:23750475

  16. 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

  17. Humanization of murine monoclonal antibodies through variable domain resurfacing.

    PubMed

    Roguska, M A; Pedersen, J T; Keddy, C A; Henry, A H; Searle, S J; Lambert, J M; Goldmacher, V S; Blättler, W A; Rees, A R; Guild, B C

    1994-02-01

    Two murine monoclonal antibodies, N901 (anti-CD56) and anti-B4 (anti-CD19), were humanized by a process we call "resurfacing." A systematic analysis of known antibody structures has been used to determine the relative solvent accessibility distributions of amino acid residues in murine and human antibody variable (Fv) regions and has shown that the sequence alignment positions of surface amino acids for human and murine variable region heavy (VH) and light (VL) chains are conserved with 98% fidelity across species. While the amino acid usage at these surface positions creates surface residue patterns that are conserved within species, there are no identical patterns across species. However, surprisingly few amino acid changes need to be made to convert a murine Fv surface pattern to that characteristic of a human surface. Resurfacing was used to change the patterns of surface accessible residues in the Fv regions of the N901 and anti-B4 antibodies to resemble those found on the Fv regions of human antibody sequences. Two different procedures for selecting a human sequence were compared. For anti-B4, a data base of clonally derived human VL-VH sequence pairs was used, while for N901, sequences for VL and VH were independently selected from the Kabat et al. data base [Kabat, E. A., Wu, T. T., Reid-Miller, M., Perry, H. M. & Gottesman, K. S. (1991) Sequences of Proteins of Immunological Interest (DHHS, Washington, DC), 5th Ed.]. Resurfaced N901 and anti-B4 antibodies had apparent affinities for their cell surface ligands that were identical to those of their respective parent murine antibodies. These data provide evidence that, despite the differences in the surfaces of mouse and human Fv regions, it is possible to substitute one for the other while retaining full antigen binding affinity. PMID:8302875

  18. Monoclonal antibodies: a target therapy for multiple sclerosis.

    PubMed

    Lorefice, Lorena; Fenu, Giuseppe; Frau, Jessica; Coghe, Giancarlo; Marrosu, Maria Giovanna

    2014-01-01

    Multiple sclerosis (MS) is a complex autoimmune disease of the central nervous system. It is characterized by a proinflammatory and neurodegenerative process that results in neuroaxonal damage. Over the last two decades, a wide range of immunomodulatory and immunosuppressive treatments have been used for the management of MS. Several treatments have been developed or are under evaluation for reducing relapses, disease progression and long-term MS-related disability. Recently, a growing interest has emerged for therapeutics with very selective actions, particularly monoclonal antibodies, to target several biological pathways involved in MS. To date, only Natalizumab (Tysabri(®)) has been approved for the treatment of active MS forms. Its therapeutic mechanism is the blockade of the a4-integrin molecule of many leukocytes, which leads to a decrease of immune cells migration, in particular of lymphocytes, across the blood-brain barrier. Furthermore, other promising molecules are under study in clinical trials. In this review, we summarize and discuss the history, pharmacodynamics and safety of monoclonal antibodies that have been approved or are under evaluation for the selective treatment of MS. PMID:24479836

  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 antibody-directed radioimmunoassay of specific cytochromes P-450

    SciTech Connect

    Song, B.J.; Fujino, T.; Park, S.S.; Friedman, F.K.; Gelboin, H.V.

    1984-02-10

    A rapid solid phase radioimmunoassay (RIA) for cytochromes P-450 has been developed utilizing specific monoclonal antibodies to major forms of rat liver cytochrome P-450 that are induced by 3-methylcholanthrene (MC-P-450) and phenobarbital (PB-P-450). Monoclonal antibodies (MAbs) that were endogenously labeled with (/sup 35/S)methionine were used to detect MAb-specific cytochromes P-450 in liver microsomes from untreated rats and rats pretreated with 3-methylcholanthrene (MC) or phenobarbital. The competitive binding assays are rapid and can detect cytochrome P-450 in less than 100 ng of microsomal protein. Tthe RIA was used to examine the distribution of MAb-specific cytochromes P-450 in extrahepatic tissues of MC-treated rats; an approximately 30- to 50-fold greater amount of MC-P-450 in liver relative to lung and kidney was observed, which corresponds well with aryl hydrocarbon hydroxylase activity in these tissues. The inducibility of MAb-specific cytochromes P-450 were observed in MC-treated rats, guinea pigs, and C57BL/6 mice, all highly inducible for aryl hydrocarbon hydroxylase; little increase was observed for the relatively noninducible DBA/2 mouse strain.

  1. Monoclonal antibodies in conditioning regimens for hematopoietic cell transplantation.

    PubMed

    Kharfan-Dabaja, Mohamed A; Nishihori, Taiga; Otrock, Zaher K; Haidar, Nour; Mohty, Mohamad; Hamadani, Mehdi

    2013-09-01

    Monoclonal antibodies are increasingly being incorporated in conditioning regimens for autologous or allogeneic hematopoietic cell transplantation (HCT). The benefit of adding rituximab to autologous HCT regimens is purportedly related to in vivo purging of clonal B cells. Randomized trials comparing the addition (or not) of rituximab to high-dose therapy regimens are lacking. No benefit of standard-dose radioimmunotherapy-based regimens for autografting in aggressive lymphomas was seen in a randomized controlled study. The incorporation of rituximab into allogeneic HCT regimens aims to improve responses while reducing nonrelapse mortality resulting from acute graft-versus-host disease. The optimal dose and administration schedule of rituximab in this setting are unknown, and potentially serious complications from increased infections owing to prolonged (and profound) cytopenias or persistent hypogammaglobulinemia are of concern. Radioimmunotherapy-based conditioning for allografting holds promise as a modality to optimize tumor control and synergize adoptive immunotherapy effects, but it remains experimental at this time. The addition of alemtuzumab to allogeneic HCT regimens is associated with prolonged lymphopenia and impaired immune reconstitution, high relapse rates, and serious infections. The optimal dose and schedule of alemtuzumab to avoid prolonged immune paresis remain elusive. It is anticipated that additional monoclonal antibodies will soon become available that can be incorporated into HCT regimens after safety and clinical efficacy are demonstrated. PMID:23618718

  2. 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

  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. Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus 1.

    PubMed Central

    Collins, J K; Butcher, A C; Riegel, C A; McGrane, V; Blair, C D; Teramoto, Y A; Winston, S

    1984-01-01

    Monoclonal antibodies were used to study neutralizing determinants on polypeptides of bovine herpesvirus 1. Two of three monoclonal antibodies which recognized nonoverlapping epitopes on a glycoprotein of 82,000 daltons were found to neutralize. A second group of monoclonal antibodies that individually precipitated five viral glycopolypeptides ranging in size from 102,000 to 55,000 daltons also neutralized. Two monoclonal antibodies which were the most efficient in neutralization recognized a non-glycosylated protein of 115,000 daltons which was the major polypeptide on the virus. A fourth group of monoclonal antibodies precipitated a non-glycosylated polypeptide of 91,000 daltons and several smaller polypeptides, but these antibodies demonstrated only limited neutralizing activity. Images PMID:6208375

  5. 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

  6. 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 1–3 d (9–12 d including the steps for preparing arrays of microwells). PMID:19528952

  7. Polyclonal and monoclonal antibody therapy for experimental Pseudomonas aeruginosa pneumonia.

    PubMed Central

    Pennington, J E; Small, G J; Lostrom, M E; Pier, G B

    1986-01-01

    A human immunoglobulin G preparation, enriched in antibodies to lipopolysaccharide (LPS) Pseudomonas aeruginosa antigens (PA-IGIV) and murine monoclonal antibodies (MAb) to P. aeruginosa Fisher immunotype-1 (IT-1) LPS antigen and outer membrane protein F (porin), were evaluated for therapeutic efficacy in a guinea pig model of P. aeruginosa pneumonia. The concentration of antibodies to IT-1 LPS was 7.6 micrograms/ml in PA-IGIV and 478 micrograms/ml in the IT-1 MAb preparation. No antibody to IT-1 was detected in MAb to porin. For study, animals were infected by intratracheal instillation of IT-1 P. aeruginosa and then treated 2 h later with intravenous infusions of PA-IGIV, IT-1 MAb, or porin MAb. Control groups received intravenous albumin, and routinely died from pneumonia. Both PA-IGIV (500 mg/kg) and IT-1 MAb (greater than or equal to 2.5 mg/kg) treatment resulted in increased survival (P less than 0.01 to 0.001), and also improved intrapulmonary killing of bacteria. Porin MAb failed to protect from fatal pneumonia. IT-1 MAb treatment produced more survivals than did PA-IGIV treatment but only at dosages of MAb resulting in serum antibody concentrations greater than those achieved with PA-IGIV. PA-IGIV and IT-1 MAb demonstrated in vitro and in vivo (posttreatment guinea pig serum) opsonophagocytic activity for the IT-1 challenge strain. However, the polyclonal preparation required complement, whereas the MAb did not. We conclude that passive immunization with polyclonal hyperimmune P. aeruginosa globulin or with MAb to LPS antigens may be useful in the treatment of acute P. aeruginosa pneumonia. The relative efficacies of such preparations may be limited, however, by their type-specific LPS antibody concentrations. PMID:3093385

  8. 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 C(H)2 domain of the molecule, which is likely due to conjugation of DM1 on lysine residues in the C(H)2 domain. As a result, methionine at position 258 of the heavy chain, which is located in the C(H)2 domain of the antibody, is more susceptible to oxidation in thermally stressed ADC samples when compared to that of the naked antibody. PMID:26629796

  9. Cytotoxicity of monoclonal antibodies against individually immunophenotyped human leukemic cells.

    PubMed

    Beksac, M; Porwit-Ksiazek, A; Hast, R; Biberfeld, P; Reizenstein, P

    1985-01-01

    Peripheral blood and bone marrow mononuclear cells from 12 patients with acute myeloid leukemia (AML), 2 patients with acute lymphatic leukemia, and 1 patient with chronic myeloid leukemia in blastic crisis were taken at diagnosis or in relapse. Cells were immunophenotyped with a panel of monoclonal antibodies (Moab) (OKIa, Leu M1, Leu M2, Leu M3, Leu M4, B1, Okt 11, J5) and the same antibodies were used in an in vitro cytotoxicity test. Of the 14 patients, 10 had antibody-binding cells, and the percentage of lysed cells was almost equal to that of blasts. The other 4 patients had few binding cells and little lysis. Acute leukemia with and without preceding myelodysplastic features did not differ in immunophenotype. Mean spontaneous release of 51Cr was 12.7% and complement alone caused an additional average release of 11.8%. Four single antibodies together with complement showed a mean 51Cr release of 0.7-32.4% above that found with complement alone. Combinations of Moabs resulted in 51Cr release at least 10% above the single most efficient Moab in 8 out of 12 patients. Not all blast cells showed antibody binding, nor were all antibody-binding cells susceptible to cytotoxicity. Normal bone marrow growth in vitro seemed to be stimulated by factors in complement and in the Moab. When this stimulation was compensated for by adding fetal calf serum, cytotoxicity tests prior to CFUc assays resulted in a mean decrease of 46% of colonies and 25% of clusters in normal bone marrow. CFUc are thus sensitive to the cytotoxicity, although CFU may also be resistant. PMID:3859364

  10. Tau Monoclonal Antibody Generation Based on Humanized Yeast Models

    PubMed Central

    Rosseels, Joëlle; Van den Brande, Jeff; Violet, Marie; Jacobs, Dirk; Grognet, Pierre; Lopez, Juan; Huvent, Isabelle; Caldara, Marina; Swinnen, Erwin; Papegaey, Anthony; Caillierez, Raphaëlle; Buée-Scherrer, Valerie; Engelborghs, Sebastiaan; Lippens, Guy; Colin, Morvane; Buée, 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. Identification by monoclonal antibodies and characterization of human platelet caldesmon.

    PubMed

    Dingus, J; Hwo, S; Bryan, J

    1986-05-01

    Actin-based gels were prepared from clarified high-salt extracts of human platelets by dialysis against physiological salt buffers. The gel was partially solubilized with 0.3 M KCl. Mice were immunized with the 0.3 M KCl extract of the actin gel, and hybridomas were produced by fusion of spleen cells with myeloma cells. Three hybridomas were generated that secrete antibodies against an 80-kD protein. These monoclonal antibodies stained stress fibers in cultured cells and cross-reacted with proteins in several tissue types, including smooth muscle. The cross-reacting protein in chicken gizzard smooth muscle had an apparent molecular weight of 140,000 and was demonstrated to be caldesmon, a calmodulin and actin-binding protein (Sobue, K., Y. Muramoto, M. Fujita, and S. Kakiuchi, Proc. Natl. Acad. Sci. USA, 78:5652-5655). No proteins of molecular weight greater than 80 kD were detectable in platelets by immunoblotting using the monoclonal antibodies. The 80-kD protein is heat stable and was purified using modifications of the procedure reported by Bretscher for the rapid purification of smooth muscle caldesmon (Bretscher, A., 1985, J. Biol. Chem., 259:12873-12880). The 80-kD protein bound to calmodulin-Sepharose in a Ca++-dependent manner and sedimented with actin filaments, but did not greatly increase the viscosity of F-actin solutions. The actin-binding activity was inhibited by calmodulin in the presence of calcium. Except for the molecular weight difference, the 80-kD platelet protein appears functionally similar to 140-kD smooth muscle caldesmon. We propose that the 80-kD protein is platelet caldesmon. PMID:3517005

  13. Identification by monoclonal antibodies and characterization of human platelet caldesmon

    PubMed Central

    1986-01-01

    Actin-based gels were prepared from clarified high-salt extracts of human platelets by dialysis against physiological salt buffers. The gel was partially solubilized with 0.3 M KCl. Mice were immunized with the 0.3 M KCl extract of the actin gel, and hybridomas were produced by fusion of spleen cells with myeloma cells. Three hybridomas were generated that secrete antibodies against an 80-kD protein. These monoclonal antibodies stained stress fibers in cultured cells and cross- reacted with proteins in several tissue types, including smooth muscle. The cross-reacting protein in chicken gizzard smooth muscle had an apparent molecular weight of 140,000 and was demonstrated to be caldesmon, a calmodulin and actin-binding protein (Sobue, K., Y. Muramoto, M. Fujita, and S. Kakiuchi, Proc. Natl. Acad. Sci. USA, 78:5652-5655). No proteins of molecular weight greater than 80 kD were detectable in platelets by immunoblotting using the monoclonal antibodies. The 80-kD protein is heat stable and was purified using modifications of the procedure reported by Bretscher for the rapid purification of smooth muscle caldesmon (Bretscher, A., 1985, J. Biol. Chem., 259:12873-12880). The 80-kD protein bound to calmodulin- Sepharose in a Ca++-dependent manner and sedimented with actin filaments, but did not greatly increase the viscosity of F-actin solutions. The actin-binding activity was inhibited by calmodulin in the presence of calcium. Except for the molecular weight difference, the 80-kD platelet protein appears functionally similar to 140-kD smooth muscle caldesmon. We propose that the 80-kD protein is platelet caldesmon. PMID:3517005

  14. 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 ...

  15. Agglutinating monoclonal antibodies that specifically recognize lipooligosaccharide A of Bordetella pertussis.

    PubMed Central

    Li, Z M; Cowell, J L; Brennan, M J; Burns, D L; Manclark, C R

    1988-01-01

    Monoclonal antibodies that specifically agglutinate strains of Bordetella pertussis having serotype 1 agglutinogen were uniquely reactive with the electrophoretically slow-migrating A form of lipooligosaccharide. These monoclonal antibodies should be useful for the structural analysis of B. pertussis lipooligosaccharide and for the establishment of a better-defined serogroup for Bordetella species. Images PMID:2893776

  16. Agglutinating monoclonal antibodies that specifically recognize lipooligosaccharide A of Bordetella pertussis.

    PubMed

    Li, Z M; Cowell, J L; Brennan, M J; Burns, D L; Manclark, C R

    1988-03-01

    Monoclonal antibodies that specifically agglutinate strains of Bordetella pertussis having serotype 1 agglutinogen were uniquely reactive with the electrophoretically slow-migrating A form of lipooligosaccharide. These monoclonal antibodies should be useful for the structural analysis of B. pertussis lipooligosaccharide and for the establishment of a better-defined serogroup for Bordetella species. PMID:2893776

  17. 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.

  18. 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.

  19. 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.

  20. Bispecific antibodies directed to CD4 domain 2 and HIV envelope exhibit exceptional breadth and picomolar potency against HIV-1

    PubMed Central

    Pace, Craig S.; Song, Ruijiang; Ochsenbauer, Christina; Andrews, Chasity D.; Franco, David; Yu, Jian; Oren, Deena A.; Seaman, Michael S.; Ho, David D.

    2013-01-01

    In the absence of an effective HIV-1 vaccine, passive immunization using broadly neutralizing Abs or Ab-like molecules could provide an alternative to the daily administration of oral antiretroviral agents that has recently shown promise as preexposure prophylaxis. Currently, no single broadly neutralizing Ab (bNAb) or combination of bNAbs neutralizes all HIV-1 strains at practically achievable concentrations in vivo. To address this problem, we created bispecific Abs that combine the HIV-1 inhibitory activity of ibalizumab (iMab), a humanized mAb directed to domain 2 of human CD4, with that of anti-gp120 bNAbs. These bispecific bNAbs (BibNAbs) exploit iMab’s potent anti–HIV-1 activity and demonstrated clinical efficacy and safety to anchor and thereby concentrate a second broadly neutralizing agent at the site of viral entry. Two BibNabs, PG9-iMab and PG16-iMab, exhibit exceptional breadth and potency, neutralizing 100% of the 118 viruses tested at low picomolar concentrations, including viruses resistant to both parental mAbs. The enhanced potency of these BibNAbs was entirely dependent on CD4 anchoring, not on membrane anchoring per se, and required optimal Ab geometry and linker length. We propose that iMab-based BibNAbs, such as PG9-iMab and PG16-iMab, are promising candidates for passive immunization to prevent HIV-1 infection. PMID:23878231

  1. [Use of monoclonal antibodies for the immuno-imaging of cancers].

    PubMed

    Endo, K; Oriuchi, N; Takenoshita, S

    1994-02-01

    Monoclonal antibodies are expected to carry radio-nuclides selectively to cancer tissues and to offer antigen-specific diagnosis and/or therapy of cancer. Monoclonal antibodies tagged with indium (111In) and technetium (99mTc) are clinically used for the localization of malignant melanoma, colorectal and ovarian cancer. They have detected about 80% of tumors, tumors missed by conventional diagnostic methods and tumors in patients with normal serum tumor marker levels. Acute or subacute toxicity was not observed. However, human anti-murine antibodies (HAMA) were detectable in sera of some cases after receiving radiolabeled monoclonal antibodies. Development of protein engineering has made it possible to produce humanized antibodies, which are expected to diminish the HAMA response. The same monoclonal antibodies are also employed for therapy of some cancers using iodine (131I), yttrium (90Y) and rhenium (186Re) labeled antibodies. PMID:8311487

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

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

    PubMed Central

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

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

  8. Monoclonal antibodies reactive with acute myelogenous leukemia cells.

    PubMed

    Linker-Israeli, M; Billing, R J; Foon, K A; Terasaki, P I

    1981-12-01

    Two cytotoxic monoclonal antibodies (D5D6 and C10H5) were raised to acute myelogenous leukemia (AML) cells. One of them, D5D6, reacted with 100% of the cells from 44/50 AML patients. The second, C10H5, reacted with 100% of the cells from 13/39 AML patients. D5D6 and C10H5 were cytotoxic to 100% of the monocytes from all healthy donors tested. Apart from monocytes, these antibodies did not react with any other peripheral blood cells from normal donors or with cancerous cells either from cell culture lines or from patients with chronic myelogenous leukemia (CML), lymphoid leukemias, or solid tumors. These antibodies inhibited the growth of the myeloid-monocytic stem cell, the CFU-C, and reacted with a minor population of normal bone marrow cells by immunofluorescence. Both D5D6 and CH5 have potential utility for the differential diagnosis of acute leukemia, for the detection of early leukemic relapse, and for enrichment of human stem cells. PMID:7028873

  9. Monoclonal antibodies reactive with acute myelogenous leukemia cells.

    TOXLINE Toxicology Bibliographic Information

    Linker-Israeli M; Billing RJ; Foon KA; Terasaki PI

    1981-12-01

    Two cytotoxic monoclonal antibodies (D5D6 and C10H5) were raised to acute myelogenous leukemia (AML) cells. One of them, D5D6, reacted with 100% of the cells from 44/50 AML patients. The second, C10H5, reacted with 100% of the cells from 13/39 AML patients. D5D6 and C10H5 were cytotoxic to 100% of the monocytes from all healthy donors tested. Apart from monocytes, these antibodies did not react with any other peripheral blood cells from normal donors or with cancerous cells either from cell culture lines or from patients with chronic myelogenous leukemia (CML), lymphoid leukemias, or solid tumors. These antibodies inhibited the growth of the myeloid-monocytic stem cell, the CFU-C, and reacted with a minor population of normal bone marrow cells by immunofluorescence. Both D5D6 and CH5 have potential utility for the differential diagnosis of acute leukemia, for the detection of early leukemic relapse, and for enrichment of human stem cells.

  10. Protective activities in mice of monoclonal antibodies against pertussis toxin.

    PubMed Central

    Sato, H; Sato, Y

    1990-01-01

    Pertussis toxin (PT) protein, which is the most important protective antigen of Bordetella pertussis, has a hexameric structure composed of five subunits, designated S1 through S5. Immunoprotective activity of 20 different mouse monoclonal antibodies (MAbs) against pertussis toxin, 10 anti-S1, 1 anti-S2, 2 anti-S3, 4 anti-S23, and 3 anti-S4 antibodies, were investigated by aerosol and intracerebral challenges with virulent B. pertussis organisms in mice. Four anti-S1, named 1B7, 1D7, 3F11, and 10D6, and three anti-S23 antibodies, named 11E6, 10B5, and 10C9, showed the highest, and almost complete, protectivity against the aerosol challenge. Mouse protectivity against the intracerebral challenge was significant for these four anti-S1 MAbs but not for any of the three anti-S23 MAbs. Four anti-S1 and two anti-S4 MAbs did not protect the mice against either challenge. The other seven MAbs also showed dose-dependent moderate but significant protection against the aerosol challenge. In the aerosol challenge system, bacterial numbers and amounts of PT detected in the lung and the number of peripheral leukocytes were lower in the mice given the protective MAbs. All mice surviving 5 weeks after the infection produced high titers of antibodies against PT, filamentous hemagglutinin (FHA), and agglutinogens from the challenge organisms. A combination of the protective MAbs 1B7 and 11E6 strongly suppressed the disease and mortality of the mice at smaller amounts than with the anti-PT polyclonal antibody. Although combinations of one of the protective MAb and anti-FHA or anti-agglutinogen 2 also showed extremely high mouse protection without development of symptoms of the disease, antibody titers of the survivors against PT, FHA, and agglutinogens were significantly low. The foregoing results suggest that some important protective epitopes should be in S1 and S2 and/or S3, although there are both differences and similarities in the protective roles between anti-S1 and anti-S23 antibodies and also in the pathogenic mechanisms between aerosol and intracerebral infections. Furthermore, it was suggested that although not only FHA and agglutinogen 2 but also PT have roles as attachment factors, the processes of infection and protection are different between mice immunized with antibody against FHA or agglutinogen 2 and that against PT because the latter mice are also able to neutralize toxicity of PT diffused into the mice. Images PMID:1698179

  11. Protective activities in mice of monoclonal antibodies against pertussis toxin.

    PubMed

    Sato, H; Sato, Y

    1990-10-01

    Pertussis toxin (PT) protein, which is the most important protective antigen of Bordetella pertussis, has a hexameric structure composed of five subunits, designated S1 through S5. Immunoprotective activity of 20 different mouse monoclonal antibodies (MAbs) against pertussis toxin, 10 anti-S1, 1 anti-S2, 2 anti-S3, 4 anti-S23, and 3 anti-S4 antibodies, were investigated by aerosol and intracerebral challenges with virulent B. pertussis organisms in mice. Four anti-S1, named 1B7, 1D7, 3F11, and 10D6, and three anti-S23 antibodies, named 11E6, 10B5, and 10C9, showed the highest, and almost complete, protectivity against the aerosol challenge. Mouse protectivity against the intracerebral challenge was significant for these four anti-S1 MAbs but not for any of the three anti-S23 MAbs. Four anti-S1 and two anti-S4 MAbs did not protect the mice against either challenge. The other seven MAbs also showed dose-dependent moderate but significant protection against the aerosol challenge. In the aerosol challenge system, bacterial numbers and amounts of PT detected in the lung and the number of peripheral leukocytes were lower in the mice given the protective MAbs. All mice surviving 5 weeks after the infection produced high titers of antibodies against PT, filamentous hemagglutinin (FHA), and agglutinogens from the challenge organisms. A combination of the protective MAbs 1B7 and 11E6 strongly suppressed the disease and mortality of the mice at smaller amounts than with the anti-PT polyclonal antibody. Although combinations of one of the protective MAb and anti-FHA or anti-agglutinogen 2 also showed extremely high mouse protection without development of symptoms of the disease, antibody titers of the survivors against PT, FHA, and agglutinogens were significantly low. The foregoing results suggest that some important protective epitopes should be in S1 and S2 and/or S3, although there are both differences and similarities in the protective roles between anti-S1 and anti-S23 antibodies and also in the pathogenic mechanisms between aerosol and intracerebral infections. Furthermore, it was suggested that although not only FHA and agglutinogen 2 but also PT have roles as attachment factors, the processes of infection and protection are different between mice immunized with antibody against FHA or agglutinogen 2 and that against PT because the latter mice are also able to neutralize toxicity of PT diffused into the mice. PMID:1698179

  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. 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.

  14. A monoclonal antibody capture ELISA to detect antibody to border disease virus in sheep serum.

    PubMed

    Fenton, A; Sinclair, J A; Entrican, G; Herring, J A; Malloy, C; Nettleton, P F

    1991-08-30

    An enzyme-linked immunosorbent assay (ELISA) has been developed to detect antibody to border disease virus (BDV) in sheep serum. A monoclonal antibody bound to 96-well microplates was used to capture antigen from detergent-solubilised BDV-infected cells. Single dilutions of test sera were then added to wells containing bound BDV antigen and control wells containing uninfected cell lysates. Specific antibody to BDV was detected by an anti-ovine IgG antiserum conjugated with horseradish peroxidase and the results expressed as ELISA units with reference to a standard curve. Sequential sera from 16 experimentally infected sheep and single sera from 103 sheep involved in a field outbreak were tested in the ELISA and for neutralising antibody. There was good qualitative correlation between the two tests. PMID:1659026

  15. Analysis of Campylobacter jejuni antigens with monoclonal antibodies.

    PubMed Central

    Kosunen, T U; Bång, B E; Hurme, M

    1984-01-01

    To develop monoclonal reagents for antigenic analysis and serotyping of Campylobacter spp., hybridoma cell lines were produced by fusion of mouse myeloma cells and spleen cells from mice immunized with Formalin-treated Campylobacter jejuni organisms. An enzyme immunoassay was used for preliminary screening of the cell culture supernatants and ascites. Twenty-nine clones which reacted with the immunogen were obtained. Seven of these clones were positive in passive hemagglutination tests with sheep erythrocytes coated with boiled saline extract of whole bacteria; four of these reacted with the purified polysaccharide preparation and with the autoclaved saline extract, but not with lipopolysaccharide prepared from the immunogen strain. Two of the antipolysaccharide clones agglutinated live bacteria in slide tests. Four additional clones gave positive slide agglutination tests with live bacteria, but in tube testing no clones agglutinated Formalin-treated bacteria. No cross-reactions with unrelated bacteria were seen, but several clones reacted in the enzyme immunoassay with many of the 24 Campylobacter strains studied. The clone which gave the highest mean enzyme immunoassay values with Campylobacter coli and C. jejuni strains also reacted with Campylobacter fetus subsp. veneralis and C. fetus subsp. fetus strains. This clone also gave the highest enzyme immunoassay value with an acid glycine extract of the immunogen, which indicates the presence of common antigens in the extract. The results suggest that monoclonal antibodies may be used to devise serotyping schemes for Campylobacter spp. PMID:6365954

  16. 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

  17. Parenteral Treatment of Multiple Sclerosis: The Advent of Monoclonal Antibodies.

    PubMed

    Singer, Barry A

    2016-04-01

    Improved disease control is critical for enhancing the lives of those living with multiple sclerosis. With specific immunologic targets, monoclonal antibody (mAb) treatments are highly effective options for relapsing forms of multiple sclerosis. The mechanism, efficacy, and current safety profiles are detailed for the two mAb therapies, natalizumab and alemtuzumab, with regulatory approval in multiple countries. Daclizumab, which targets the interleukin-2 receptor, and ocrelizumab, which depletes B cells, have convincing phase 3 clinical trial data and may very well provide new options in the near future. Trial results of other B-cell-directed therapies, ofatumumab and rituximab, are reviewed. Less-frequent dosing of glatiramer acetate and interferon β-1a highlight developments in the first generation of parenteral immunomodulatory therapy. Remyelination using mAbs has moved into clinical trials with the first agents, anti-LINGO-1, rHIgM22, and anti-SEMA 4D. PMID:27116720

  18. [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

  19. Positron emission tomographic imaging of tumors using monoclonal antibodies

    SciTech Connect

    Zalutsky, M.R.

    1990-12-01

    The overall objective for this research project is to develop methods for utilizing Positron Emission Tomography (PET) to increase the clinical potential of radiolabelled monoclonal antibodies (MAbs). 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 to investigate the following: normal tissue toxicity; radiation dose to the tumor; and early tumor imaging. The research plans of this proposal include the following specific aims: optimize labeling of MAbs with fluorine 18, bromine 76 and bromine 75; label MAb Mel-14 (reactive against human gliomas and melanomas) and its Fab and F(ab{prime}){sub 2} fragments while retaining immunoreactivity; determine the distribution of Mel-14 in athymic mice bearing human gliomas; determine pharmacokinetics of Mel-14 in nonhuman primates. Experiments with another MAb, TP-1, and iodine 124 and 131 are also planned. 8 figs.

  20. 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

  1. Generation of genetic engineering monoclonal antibodies against prion protein.

    PubMed

    Huang, Yin-Xia; Han, Jun; Dong, Chen-Fang; Sun, Li; Gao, Chen; Wang, Xiao-Fan; Han, Lu; Zhou, Wei; Zhang, Bao-Yun; Jiang, Hui-Ying; Liang, Mi-Fang; Dong, Xiao-Ping

    2007-12-01

    Two strains of Fab monoclonal antibodies (mAbs) against prion protein, designated as IV-66 and IV-78, were selected from the phage display libraries. The gene sequences encoding the light kappa chain and heavy Fd chain of IV-78 were inserted into a baculovirus expression cassette vector for mouse IgG expression. Western blot, Dot-ELISA and immunoprecipitation confirmed that these Fab and IgG mAbs reacted well with the recombinant hamster and human PrP proteins expressed in prokaryotic and in mammalian cells and PrP(Sc) from scrapie-infected hamsters. It demonstrates that mAbs against prion protein are successfully generated by phage-display technique. PMID:17486363

  2. 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

  3. [Development of HPLC analysis methods for therapeutic monoclonal antibodies].

    PubMed

    Todoroki, Kenichiro

    2015-01-01

    Therapeutic monoclonal antibody (mAb) preparations are produced from cultured cells; therefore, detailed and multidimensional analyses of their heterogeneities are required. We analyzed five commercially available mAb preparations by high-temperature reversed-phase LC using a wide-pore core-shell column for pluralistic quality assessment. At a highly elevated column temperature, isopropanol with high eluotropic strength coefficients and a wide-pore core-shell type octyl column showed good peak resolution of the investigated mAbs and their related constituents. We used this method to estimate the residual rate of intact mAbs after a heat aggregation treatment and conducted fragmentation analysis by analyzing their pepsin digests. Each peak component was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. All results were compared with those of reversed-phase and size exclusion analyses. PMID:25747215

  4. High levels of antibodies to streptococcal cell membrane antigens specifically bound to monoclonal antibodies in acute poststreptococcal glomerulonephritis.

    PubMed Central

    Yoshimoto, M; Hosoi, S; Fujisawa, S; Sudo, M; Okuda, R

    1987-01-01

    We produced 15 immunoglobulin G class monoclonal antibodies against antigens of the streptococcal cell membrane (SCM) of Streptococcus pyogenes (T type 12, Tanaka strain) and determined the levels in human sera of antibodies against Triton-X-extracted antigens specifically bound to each of these 15 monoclonal antibodies by enzyme-linked immunosorbent assay. Sample sera were obtained from 10 normal controls (group 1), 10 patients with streptococcal pharyngitis without sequelae (group 2), and 8 patients with acute poststreptococcal glomerulonephritis (APSGN) (group 3). Anti-streptolysin O (ASO) titers of the sera increased in the order of groups 1, 2, and 3. There was no relationship between ASO titer and the level of anti-SCM antibodies, and there was no significant difference in the level of anti-SCM antibodies determined with each of the 15 monoclonal antibodies between group 1 and group 2 sera. Group 3 sera had higher levels of antibodies to SCM antigens specifically bound to each of 14 of these 15 monoclonal antibodies than group 1 or group 2 sera did. Of these 14 monoclonal antibodies, 9 reacted with the four SCM antigens separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to a nitrocellulose sheet. These results suggest that high levels of antibodies to SCM antigens are related to the development of APSGN and that the systemic immune response to SCM antigens is involved in the pathogenesis of APSGN. Images PMID:3553232

  5. Cell line profiling to improve monoclonal antibody production.

    PubMed

    Kang, Sohye; Ren, Da; Xiao, Gang; Daris, Kristi; Buck, Lynette; Enyenihi, Atim A; Zubarev, Roman; Bondarenko, Pavel V; Deshpande, Rohini

    2014-04-01

    Mammalian cell culture performance is influenced by both intrinsic (genetic) and extrinsic (media and process) factors. In this study, intrinsic capacity of various monoclonal antibody-producing Chinese Hamster Ovary (CHO) cell lines was compared by exposing them to the same culture condition. Microarray-based transcriptomics and LC-MS/MS shotgun proteomics technologies were utilized to obtain expression landscape of different cell lines. Specific transcripts and proteins correlating with productivity, growth rate and cell size have been identified. The proteomics analysis results showed a strong correlation between the intracellular protein expression levels of the recombinant DHFR and productivity. In contrast, neither the light chain nor the heavy chain of the recombinant monoclonal antibody showed correlation to productivity. Other top ranked proteins which demonstrated positive correlation to productivity included the adaptor protein complex subunits AP3D1and AP2B2, DNA repair protein DDB1 and the ER translocation complex component, SRPR. The subunits of molecular chaperone T-complex protein 1 and the regulator of mitochondrial one-carbon metabolism MTHFD2 showed negative correlation to productivity. The transcriptomics analysis has identified the regulators of calcium signaling, Tmem20 and Rcan1, as the top ranked genes displaying positive and negative correlation to productivity, respectively. For the second part of the study, the principal component analysis (PCA) was generated to view the underlying global structure of the expression data. A clear division and expression polarity was observed between the two distinct clusters of cell lines, independent of link to productivity or any other traits examined. The primary component of the PCA generated from either transcriptomics or proteomics data displayed a strong correlation to cell size and doubling time, while none of the main principal components showed correlation to productivity. Our findings suggest that productivity is rather a minor feature in the context of global transcriptional or protein expression space. PMID:24249214

  6. 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

  7. Non-antigen-contacting region of an asymmetric bispecific antibody to factors IXa/X significantly affects factor VIII-mimetic activity.

    PubMed

    Sampei, Zenjiro; Igawa, Tomoyuki; Soeda, Tetsuhiro; Funaki, Miho; Yoshihashi, Kazutaka; Kitazawa, Takehisa; Muto, Atsushi; Kojima, Tetsuo; Nakamura, Satoshi; Hattori, Kunihiro

    2015-01-01

    While antibody engineering improves the properties of therapeutic antibodies, optimization of regions that do not contact antigens has been mainly focused on modifying the effector functions and pharmacokinetics of antibodies. We recently reported an asymmetric anti-FIXa/FX bispecific IgG4 antibody, ACE910, which mimics the cofactor function of FVIII by placing the two factors into spatial proximity for the treatment of hemophilia A. During the optimization process, we found that the activity was significantly affected by IgG subclass and by modifications to the inter-chain disulfide bonds, upper hinge region, elbow hinge region, and Fc glycan, even though these regions were unlikely to come into direct contact with the antigens. Of these non-antigen-contacting regions, the tertiary structure determined by the inter-chain disulfide bonds was found to strongly affect the FVIII-mimetic activity. Interestingly, IgG4-like disulfide bonds between Cys131 in the heavy chain and Cys114 in the light chain, and disulfide bonds between the two heavy chains at the hinge region were indispensable for the high FVIII-mimetic activity. Moreover, proline mutations in the upper hinge region and removal of the Fc glycan enhanced the FVIII-mimetic activity, suggesting that flexibility of the upper hinge region and the Fc portion structure are important for the FVIII-mimetic activity. This study suggests that these non-antigen-contacting regions can be engineered to improve the biological activity of IgG antibodies with functions similar to ACE910, such as placing two antigens into spatial proximity, retargeting effector cells to target cells, or co-ligating two identical or different antigens on the same cell. PMID:25524207

  8. Monoclonal Antibody-Directed Effector Cells Selectively Lyse Human Melanoma Cells in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Schulz, Gregor; Bumol, Thomas F.; Reisfeld, Ralph A.

    1983-09-01

    Monoclonal antibody 9.2.27 (mAb 9.2.27) directed to a chondroitin sulfate proteoglycan on human melanoma cells was able to suppress tumor growth in athymic (nu/nu) mice more effectively when bound with polyethylene glycol to murine effector cells than when injected alone. These ``armed'' effector cells also proved more effective than the monoclonal antibody in eliciting antibody-dependent cellular cytotoxicity against human melanoma target cells in vitro.

  9. 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

  10. 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.

  11. Monoclonal Antibodies for the Diagnosis of Borrelia crocidurae.

    PubMed

    Fotso Fotso, Aurélien; Mediannikov, Oleg; Nappez, Claude; Azza, Saïd; 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

  12. Hierarchical Cluster Formation in Concentrated Monoclonal Antibody Formulations

    NASA Astrophysics Data System (ADS)

    Godfrin, P. Douglas; Zarzar, Jonathan; Zarraga, Isidro Dan; Porcar, Lionel; Falus, Peter; Wagner, Norman; Liu, Yun

    Reversible cluster formation has been identified as an underlying cause of large solution viscosities observed in some concentrated monoclonal antibody (mAb) formulations. As high solution viscosity prevents the use of subcutaneous injection as a delivery method for some mAbs, a fundamental understanding of the interactions responsible for high viscosities in concentrated mAb solutions is of significant relevance to mAb applications in human health care as well as of intellectual interest. Here, we present a detailed investigation of a well-studied IgG1 based mAb to relate the short time dynamics and microstructure to significant viscosity changes over a range of pharmaceutically relevant physiochemical conditions. Using a combination of experimental techniques, it is found that upon adding Na2SO4, these antibodies dimerize in solution. Proteins form strongly bounded reversible dimers at dilute concentrations that, when concentrated, interact with each other to form loosely bounded, large, transient clusters. The combined effect of forming strongly bounded dimers and a large transient network is a significant increase in the solution viscosity. Strongly bounded, reversible dimers may exist in many IgG1 based mAb systems such that these results contribute to a more comprehensive understanding of the physical mechanisms producing high viscosities in concentrated protein solutions.

  13. 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

  14. 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.

  15. 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

  16. Thrombus radioimmunoscintigraphy: an approach using monoclonal antiplatelet antibody

    SciTech Connect

    Oster, Z.H.; Srivastava, S.C.; Som, P.; Meinken, G.E.; Scudder, L.E.; Yamamoto, K.; Atkins, H.L.; Brill, A.B.; Coller, B.S.

    1985-05-01

    An approach to thrombus imaging using a monoclonal antiplatelet antibody labeled with In or STI is described. The antibody (7E3) prepared against human platelets inhibits the interaction between fibrinogen-coated beads and both human and dog platelets. 7E3 is an IgG1 that binds to the complexed glycoprotein IIb/IIIa. Ninety percent of a tracer dose of radiolabeled 7E3 binds to human platelets and 50% binds to dog platelets. In vitro studies showed that virtually all of the platelet-bound radioactivity becomes incorporated into clots formed by adding thrombin to whole blood. 7E3 was labeled with In by the cyclic anhydride diethylenetriaminepentaacetic acid method or by radioiodination with STI. With el camera, visualization of venous and arterial thrombi as well as sites of initial injury without visible thrombi, could be observed 1-1.5 hr after injection. There was no need for delayed imaging because of the fast clearance of radioactivity from the circulation nor was there need for blood pool subtraction. Two to 10-hr thrombi could be imaged but 48-hr thrombi were not detectable with this method. No change in platelet counts before and after the injection of labeled 7E3 nor increased bleeding tendency occurred. The advantages of this method are a shorter preimaging preparation time, faster visualization after injection, and no need for delayed imaging or subtraction techniques. For these reasons human investigations seem to be warranted.

  17. Interfacial dilatational deformation accelerates particle formation in monoclonal antibody solutions.

    PubMed

    Lin, Gigi L; Pathak, Jai A; Kim, Dong Hyun; Carlson, Marcia; Riguero, Valeria; Kim, Yoen Joo; Buff, Jean S; Fuller, Gerald G

    2016-04-14

    Protein molecules are amphiphilic moieties that spontaneously adsorb at the air/solution (A/S) interface to lower the surface energy. Previous studies have shown that hydrodynamic disruptions to these A/S interfaces can result in the formation of protein aggregates that are of concern to the pharmaceutical industry. Interfacial hydrodynamic stresses encountered by protein therapeutic solutions under typical manufacturing, filling, and shipping conditions will impact protein stability, prompting a need to characterize the contribution of basic fluid kinematics to monoclonal antibody (mAb) destabilization. We demonstrate that dilatational surface deformations are more important to antibody stability when compared to constant-area shear of the A/S interface. We have constructed a dilatational interfacial rheometer that utilizes simultaneous pressure and bubble shape measurements to study the mechanical stability of mAbs under interfacial aging. It has a distinct advantage over methods utilizing the Young-Laplace equation, which incorrectly describes viscoelastic interfaces. We provide visual evidence of particle ejection from dilatated A/S interfaces and spectroscopic data of ejected mAb particles. These rheological studies frame a molecular understanding of the protein-protein interactions at the complex-fluid interface. PMID:26891116

  18. 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

  19. 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

  20. Production of monoclonal antibodies against chicken Pop1 (BVES).

    PubMed

    DiAngelo, J R; Vasavada, T K; Cain, W; Duncan, M K

    2001-01-01

    Pop1 (BVES) is a member of the Popeye gene family which was named for its high-level expression in the heart and other muscle lineages. However, these proteins have no sequence similarity to any other gene family and their function is unclear. Here we report the production of recombinant chicken Pop1/BVES protein and the generation of two Pop1/BVES specific monoclonal antibodies (MAbs) 3F11-D9-E8 and 1B3-G11-A8. These antibodies detect recombinant Pop1/BVES in ELISAs and endogenous chicken Pop1/BVES in chicken heart extracts by Western blotting. Further, both 3F11-D9-E8 and 1B3-G11-A8 detect Pop1/BVES specifically in the cardiomyocytes of 8-day-old embryonic chicken hearts by immunofluorescence. These MAbs will be useful in immunolocalization and immunoblotting experiments of different tissue types to determine the location and levels of Pop1/BVES expression throughout development, as well as further analysis of the biochemistry of this protein. PMID:11839256

  1. 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

  2. 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

  3. High-affinity lamprey VLRA and VLRB monoclonal antibodies.

    PubMed

    Tasumi, Satoshi; Velikovsky, C Alejandro; Xu, Gang; Gai, S Annie; Wittrup, K Dane; Flajnik, Martin F; Mariuzza, Roy A; Pancer, Zeev

    2009-08-01

    Lamprey are members of the ancestral vertebrate taxon (jawless fish), which evolved rearranging antigen receptors convergently with the jawed vertebrates. But instead of Ig superfamily domains, lamprey variable lymphocyte receptors (VLRs) consist of highly diverse leucine-rich repeats. Although VLRs represent the only known adaptive immune system not based on Ig, little is known about their antigen-binding properties. Here we report robust plasma VLRB responses of lamprey immunized with hen egg lysozyme and beta-galactosidase (beta-gal), demonstrating adaptive immune responses against soluble antigens. To isolate monoclonal VLRs, we constructed large VLR libraries from antigen-stimulated and naïve animals in a novel yeast surface-display vector, with the VLR C-terminally fused to the yeast Flo1p surface anchor. We cloned VLRB binders of lysozyme, beta-gal, cholera toxin subunit B, R-phycoerythrin, and B-trisaccharide antigen, with dissociation constants up to the single-digit picomolar range, equivalent to those of high-affinity IgG antibodies. We also isolated from a single lamprey 13 anti-lysozyme VLRA clones with affinities ranging from low nanomolar to mid-picomolar. All of these VLRA clones were closely related in sequence, differing at only 15 variable codon positions along the 244-residue VLR diversity region, which augmented antigen-binding affinity up to 100-fold. Thus, VLRs can provide a protective humoral antipathogen shield. Furthermore, the broad range of nominal antigens that VLRs can specifically bind, and the affinities achieved, indicate a functional parallelism between LRR-based and Ig-based antibodies. VLRs may be useful natural single-chain alternatives to conventional antibodies for biotechnology applications. PMID:19625627

  4. High-affinity lamprey VLRA and VLRB monoclonal antibodies

    PubMed Central

    Tasumi, Satoshi; Velikovsky, C. Alejandro; Xu, Gang; Gai, S. Annie; Wittrup, K. Dane; Flajnik, Martin F.; Mariuzza, Roy A.; Pancer, Zeev

    2009-01-01

    Lamprey are members of the ancestral vertebrate taxon (jawless fish), which evolved rearranging antigen receptors convergently with the jawed vertebrates. But instead of Ig superfamily domains, lamprey variable lymphocyte receptors (VLRs) consist of highly diverse leucine-rich repeats. Although VLRs represent the only known adaptive immune system not based on Ig, little is known about their antigen-binding properties. Here we report robust plasma VLRB responses of lamprey immunized with hen egg lysozyme and β-galactosidase (β-gal), demonstrating adaptive immune responses against soluble antigens. To isolate monoclonal VLRs, we constructed large VLR libraries from antigen-stimulated and naïve animals in a novel yeast surface-display vector, with the VLR C-terminally fused to the yeast Flo1p surface anchor. We cloned VLRB binders of lysozyme, β-gal, cholera toxin subunit B, R-phycoerythrin, and B-trisaccharide antigen, with dissociation constants up to the single-digit picomolar range, equivalent to those of high-affinity IgG antibodies. We also isolated from a single lamprey 13 anti-lysozyme VLRA clones with affinities ranging from low nanomolar to mid-picomolar. All of these VLRA clones were closely related in sequence, differing at only 15 variable codon positions along the 244-residue VLR diversity region, which augmented antigen-binding affinity up to 100-fold. Thus, VLRs can provide a protective humoral antipathogen shield. Furthermore, the broad range of nominal antigens that VLRs can specifically bind, and the affinities achieved, indicate a functional parallelism between LRR-based and Ig-based antibodies. VLRs may be useful natural single-chain alternatives to conventional antibodies for biotechnology applications. PMID:19625627

  5. Kinetic analysis of the multistep aggregation mechanism of monoclonal antibodies.

    PubMed

    Nicoud, Lucrèce; Arosio, Paolo; Sozo, Margaux; Yates, Andrew; Norrant, Edith; Morbidelli, Massimo

    2014-09-11

    We investigate by kinetic analysis the aggregation mechanism of two monoclonal antibodies belonging to the IgG1 and IgG2 subclass under thermal stress. For each IgG, we apply a combination of size exclusion chromatography and light scattering techniques to resolve the time evolution of the monomer, dimer, and trimer concentrations, as well as the average molecular weight and the average hydrodynamic radius of the aggregate distribution. By combining the detailed experimental characterization with a theoretical kinetic model based on population balance equations, we extract relevant information on the contribution of the individual elementary steps on the global aggregation process. The analysis shows that the two molecules follow different aggregation pathways under the same operating conditions. In particular, while the monomer depletion of the IgG1 is found to be rate-limited by monomeric conformational changes, bimolecular collision is identified as the rate-limiting step in the IgG2 aggregation process. The measurement of the microscopic rate constants by kinetic analysis allows the quantification of the protein-protein interaction potentials expressed in terms of the Fuchs stability ratio (W). It is found that the antibody solutions exhibit large W values, which are several orders of magnitude larger than the values computed in the frame of the DLVO theory. This indicates that, besides net electrostatic repulsion, additional effects delay the aggregation kinetics of the antibody solutions with respect to diffusion-limited conditions. These effects likely include the limited efficiency of the collision events due to the presence of a limited number of specific aggregation-prone patches on the heterogeneous protein surface, and the contribution of additional repulsive non-DLVO forces to the protein-protein interaction potential, such as hydration forces. PMID:25119992

  6. 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

  7. 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

  8. 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.''

  9. 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.

  10. 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

  11. Mouse monoclonal antibodies in biological research: strategies for high-throughput production.

    PubMed

    Chiarella, Pieranna; Fazio, Vito Michele

    2008-08-01

    Mouse monoclonal antibodies have become key components in basic research as well as in the clinical laboratory. Being invaluable tools in many biological assays, they continue to be the primary choice in the research field, although the conventional technology used for hybridoma generation and screening is a still lengthy, time-consuming and low-throughput process. With the advent of genetic immunisation and the application of automation and microarray to the traditional biological assays, the monoclonal antibody field has been revolutionised. Here, we will briefly review the most relevant strategies which have made the manufacture of murine monoclonal antibodies a faster and high-throughput technology. PMID:18418716

  12. Melioidosis in a patient on monoclonal antibody therapy for psoriatic arthritis.

    PubMed

    Commons, R J; Grivas, R; Currie, B J

    2014-12-01

    Melioidosis is caused by the environmental bacterium Burkholderia pseudomallei and can present with severe sepsis. Predisposing risk factors are present in 80% of cases. Monoclonal antibodies are increasingly prescribed for varied medical conditions. This report describes the first known case of melioidosis in a patient whose only risk factor for disease is treatment with a monoclonal antibody. Prescribers of monoclonal antibodies and other immunosuppressants should ensure that their patients are aware of the potential risk of melioidosis prior to travel and the precautions that should be taken. PMID:25442759

  13. Surface antigenic change during differentiation of a parasitic protozoan, Leishmania mexicana: Identification by monoclonal antibodies.

    PubMed Central

    Fong, D; Chang, K P

    1982-01-01

    The fusion of SP2/0 myeloma cells with spleen cells from mice immunized with Leishmania mexicana amazonensis promastigotes produced hybridoma clones. Indirect immunofluorescent antibody assay with live leishmanias showed that the monoclonal antibody 6H12 recognized only the antigens bound to the surface of L. mexicana amazonensis promastigotes. It also showed that the antibody bound to neither amastigotes of this species nor to other Leishmania species--i.e., L. braziliensis braziliensis, L. tropica, and L. donovani. Monoclonal antibodies from three other clones (4D11, 4H9, and 6A11) were found to compete with 6H12 for binding to L. mexicana promastigotes. With lysates of [35S]methionine-labeled promastigotes, all four monoclonal antibodies precipitated the same triplet set of protein bands at the approximately equal to 68,000-dalton region, whereas another monoclonal antibody (6G5) precipitated a different band at approximately equal to 90,000 daltons. During differentiation of L. mexicana amazonensis from amastigotes to promastigotes, there was a 4- to 8-fold increase above the initial level in the binding of 6H12 monoclonal antibody to leishmanias, as detected by enzyme-linked immunosorbent assay and quantitative fluorometric assay, respectively. Thus, we have demonstrated the use of monoclonal antibodies as probes for antigens that change during leishmanial differentiation. Images PMID:6961414

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

    PubMed

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

    2016-04-22

    High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. PMID:26979754

  15. Development of monoclonal antibodies suitable for rabies virus antibody and antigen detection.

    PubMed

    Chander, Vishal; Singh, R P; Verma, P C

    2012-12-01

    The control of an infectious viral disease as rabies is made easier by rapid and accurate diagnosis. Successful rabies prophylaxis is dependent upon the active immunization with vaccine along with passive administration of rabies virus neutralizing antibodies which together clear the virus before widespread infection of central nervous system occurs. The present study aimed at the development of monoclonal antibodies (MAbs) suitable for rabies virus antibody and antigen detection. For the production of rabies specific MAbs, immunization of Swiss albino mice with a commercially available vaccine was done and Polyethylene glycol mediated fusion of spleenocytes with myeloma cells was performed. The positive clones were selected on the basis of distinct reactivity by cell Enzyme linked immunosorbent assay and fluorescence in Indirect Fluorescent antibody test. The positive clones obtained were subjected to single cell cloning by limiting dilution method. The reactive clones were further titrated and employed for virus titration and virus neutralization. The neutralizing activity was evaluated using Fluorescence Activated Cell Sorter technique. Three MAb clones showed a distinct percent inhibition in the presence of positive serum. One of the MAb clone No. 5C3 was relatively more specific in detecting rabies antibodies and also found suitable for competitive ELISA to assess the antibody level in vaccinated subjects. PMID:24293819

  16. 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...

  17. Production and characterization of monoclonal antibodies against dog immunoglobulin isotypes.

    PubMed

    Arce, C; Moreno, A; Millán, Y; Martín 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

  18. A first-in-human phase 1 study of ACE910, a novel factor VIII–mimetic bispecific antibody, in healthy subjects

    PubMed Central

    Uchida, Naoki; Sambe, Takehiko; Yoneyama, Koichiro; Fukazawa, Naoki; Kawanishi, Takehiko; Kobayashi, Shinichi

    2016-01-01

    ACE910 is a recombinant humanized bispecific antibody that binds to activated factor IX and factor X and mimics the cofactor function of factor VIII (FVIII). This first-in-human study examined the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ACE910 in healthy male adults. A total of 40 Japanese and 24 white subjects were randomized to receive a single subcutaneous injection of ACE910 (Japanese: 0.001, 0.01, 0.1, 0.3, or 1 mg/kg; white: 0.1, 0.3, or 1 mg/kg; n = 6 per dose group) or placebo (n = 2 per dose group). ACE910 exhibited a linear PK profile and had a half-life of ∼4 to 5 weeks. In FVIII-neutralized plasma, ACE910 shortened activated partial thromboplastin time and increased peak height of thrombin generation in a dose-dependent manner. All adverse events were nonserious and did not lead to any subject’s withdrawal. Neither clinical findings nor laboratory abnormalities indicating hypercoagulability were observed. Two of 48 subjects receiving ACE910 (1 Japanese and 1 white) were positive for anti-ACE910 antibodies (anti-drug antibodies [ADAs]). One subject tested positive for ADAs both before and after ACE910 administration, whereas the other became ADA positive after receiving ACE910. The PK and PD profiles of ACE910 were similar in healthy Japanese and white subjects and suggest that ACE910 will be an effective and convenient prophylactic treatment of hemophilia A. This trial was registered at www.clinicaltrials.jp as #JapicCTI-121934. PMID:26626991

  19. A first-in-human phase 1 study of ACE910, a novel factor VIII-mimetic bispecific antibody, in healthy subjects.

    PubMed

    Uchida, Naoki; Sambe, Takehiko; Yoneyama, Koichiro; Fukazawa, Naoki; Kawanishi, Takehiko; Kobayashi, Shinichi; Shima, Midori

    2016-03-31

    ACE910 is a recombinant humanized bispecific antibody that binds to activated factor IX and factor X and mimics the cofactor function of factor VIII (FVIII). This first-in-human study examined the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ACE910 in healthy male adults. A total of 40 Japanese and 24 white subjects were randomized to receive a single subcutaneous injection of ACE910 (Japanese: 0.001, 0.01, 0.1, 0.3, or 1 mg/kg; white: 0.1, 0.3, or 1 mg/kg; n = 6 per dose group) or placebo (n = 2 per dose group). ACE910 exhibited a linear PK profile and had a half-life of ∼4 to 5 weeks. In FVIII-neutralized plasma, ACE910 shortened activated partial thromboplastin time and increased peak height of thrombin generation in a dose-dependent manner. All adverse events were nonserious and did not lead to any subject's withdrawal. Neither clinical findings nor laboratory abnormalities indicating hypercoagulability were observed. Two of 48 subjects receiving ACE910 (1 Japanese and 1 white) were positive for anti-ACE910 antibodies (anti-drug antibodies [ADAs]). One subject tested positive for ADAs both before and after ACE910 administration, whereas the other became ADA positive after receiving ACE910. The PK and PD profiles of ACE910 were similar in healthy Japanese and white subjects and suggest that ACE910 will be an effective and convenient prophylactic treatment of hemophilia A. This trial was registered atwww.clinicaltrials.jpas #JapicCTI-121934. PMID:26626991

  20. 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 α/β TCR. Replacing CH1 with Cα and CL with Cβ, 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 Cβ with a canonical β-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 Cα/Cβ 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

  1. Localization of a tumor cell adhesion domain of laminin by a monoclonal antibody

    SciTech Connect

    Skubitz, A.P.N.; Charonis, A.S.; Tsilibary, E.C.; Furcht, L.T. )

    1987-12-01

    Monoclonal antibodies were prepared to localize the domain(s) of laminin to which tumor cells adhere. Rat Y3-Ag 1.2.3 myeloma cells were fused with spleen cells from a rat immunized with a purified 440-kDa fragment of chymotrypsin-digested laminin. Three monoclonal antibodies (AL-1 to AL-3) that bound to intact laminin in a solid-phase radioimmunoassay were chosen for further analysis. The epitopes recognized by these antibodies were characterized by radioimmunoassays, immunoblotting, radioimmunoprecipitation, and immunoaffinity chromatography. In cell adhesion assays, monoclonal antibody AL-2 inhibited the highly metastatic melanoma cell line, K-1735-M4, to both intact laminin and the 440-kDa fragment of laminin. Electron microscopic examination of laminin-monoclonal antibody interactions showed that monoclonal antibody AL-2 reacted with the long arm of laminin directly below the cross-region. Two monoclonal antibodies that failed to inhibit tumor cell adhesion to laminin reacted with epitopes on the lateral short arms or cross-region of laminin as seen by electron microscopy. These results suggest that a new tumor cell binding domain of laminin may be located close to the cross-region on the long arm of laminin.

  2. Characterization of a novel single-chain bispecific antibody for retargeting of T cells to tumor cells via the TCR co-receptor CD8.

    PubMed

    Michalk, Irene; Feldmann, Anja; Koristka, Stefanie; Arndt, Claudia; Cartellieri, Marc; Ehninger, Armin; Ehninger, Gerhard; Bachmann, Michael P

    2014-01-01

    There is currently growing interest in retargeting of effector T cells to tumor cells via bispecific antibodies (bsAbs). Usually, bsAbs are directed on the one hand to the CD3 complex of T cells and on the other hand to a molecule expressed on the surface of the target cell. A bsAb-mediated cross-linkage via CD3 leads to an activation of CD8+ T cells and consequently to killing of the target cells. In parallel, CD4+ T cells including TH1, TH2, TH17 cells and even regulatory T cells (Tregs) will be activated as well. Cytokines produced by CD4+ T cells can contribute to severe side effects e. g. life-threatening cytokine storms and, thinking of the immunosupressive function of Tregs, can even be counterproductive. Therefore, we asked whether or not it is feasible to limit retargeting to CD8+ T cells e. g. via targeting of the co-receptor CD8 instead of CD3. In order to test for proof of concept, a novel bsAb with specificity for CD8 and a tumor-associated surface antigen was constructed. Interestingly, we found that pre-activated (but not freshly isolated) CD8+ T cells can be retargeted via CD8-engaging bsAbs leading to an efficient lysis of target cells. PMID:24751697

  3. 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

  4. Highly Efficient Elimination of Colorectal Tumor-Initiating Cells by an EpCAM/CD3-Bispecific Antibody Engaging Human T Cells

    PubMed Central

    Herrmann, Ines; Baeuerle, Patrick A.; Friedrich, Matthias; Murr, Alexander; Filusch, Susanne; Rüttinger, Dominik; Majdoub, Mariam W.; Sharma, Sherven; Kufer, Peter; Raum, Tobias; Münz, Markus

    2010-01-01

    With their resistance to genotoxic and anti-proliferative drugs and potential to grow tumors and metastases from very few cells, cancer stem or tumor-initiating cells (TICs) are a severe limitation for the treatment of cancer by conventional therapies. Here, we explored whether human T cells that are redirected via an EpCAM/CD3-bispecific antibody called MT110 can lyse colorectal TICs and prevent tumor growth from TICs. MT110 recognizes EpCAM, a cell adhesion molecule expressed on TICs from diverse human carcinoma, which was recently shown to promote tumor growth through engagement of elements of the wnt pathway. MT110 was highly potent in mediating complete redirected lysis of KRAS-, PI3 kinase- and BRAF-mutated colorectal TICs, as demonstrated in a soft agar assay. In immunodeficient mice, MT110 prevented growth of tumors from a 5,000-fold excess of a minimally tumorigenic TIC dose. T cells engaged by MT110 may provide a potent therapeutic means to eradicate TICs and bulk tumor cells derived thereof. PMID:20976159

  5. 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

  6. 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 36.5°C and with a temperature shift to 32°C during late exponential/early stationary phase. Experimental results showed higher cell viability with decreased metabolic rates. The specific antibody productivity increased by 25% at 32°C 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 32°C, 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 32°C. 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

  7. A monoclonal antibody specific for mouse dendritic cells.

    PubMed Central

    Nussenzweig, M C; Steinman, R M; Witmer, M D; Gutchinov, B

    1982-01-01

    Dendritic cells (DC) are a small subpopulation of lymphoid cells with distinctive cytologic features, surface properties, and functions. This report describes the DC-specific antibody (Ab) secreted by clone 33DI. Rat spleen cells immune to mouse DC were fused to the P3U myeloma. Hybrid culture supernatants were screened simultaneously against DC, a macrophage (M phi) cell line, and mitogen-stimulated lymphoblasts. 33DI Ab specifically killed 80-90% of DC from spleen and lymph node, but no other leukocytes, including Ia+ and Ia- M phi (Ia, I-region-associated antigen,). Quantitative binding studies with 5H-labeled 33D1 Ab showed that DC had an average of 14,000 binding sites per cell. Binding to DC was inhibited with Fab fragment of 33D1 Ab but not with a panel of other monoclonal antibodies, including anti-Ia Ab. Adherence and flotation procedures that enriched for DC enriched for 3H-labeled 33D1 Ab binding in parallel. 33D1 antigen was not detectable on: M phi from spleen, peritoneal cavity, and blood; three M phi cell lines; lymphocytes; granulocytes; platelets; and erythroid cells. DC continued to express the 33D1 antigen after 4 days in culture, whereas M phi and lymphocytes did not acquire it. Quantitative and autoradiographic studies confirmed that spleen and lymph node suspensions contain less than 1% DC. We conclude that 33D1 Ab detects a stable and specific DC antigen and can be used to monitor DC content in complex lymphoid mixtures. Images PMID:6948298

  8. 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.

  9. Production and characterization of monoclonal antibodies specific to sweet clover necrotic mosaic virus.

    PubMed

    Hiruki, C; Figueiredo, G; Inoue, M; Furuya, Y

    1984-07-01

    Monoclonal antibodies were produced in vitro by fusing mouse myeloma cells P3-X63-AgU1 with spleen cells derived from BALB/c mice immunized with purified sweet clover necrotic mosaic virus (SCNMV). Twenty-one out of 47 clones which secreted monoclonal antibodies of high titres against SCNMV were injected intraperitoneally into mice previously primed with Pristane. The ascites fluid harvested 10 to 14 days later showed a strong and specific anti-SCNMV activity in reverse passive haemagglutination inhibition, passive haemagglutination and indirect enzyme linked immunosorbent assay. The monoclonal antibodies of the 21 clones did not react with red clover necrotic mosaic virus (Swedish isolate). The class and subclass of immunoglobulins of the monoclonal antibodies secreted by established cultures were determined to be IgG2a for 15 clones and IgG3 for 6 clones. PMID:6470091

  10. 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 ...

  11. Anti-Mesothelin Monoclonal Antibodies for the Treatment of Cancer | NCI Technology Transfer Center | TTC

    Cancer.gov

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

  12. 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

  13. 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

  14. Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

    PubMed

    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

  15. Monoclonal antibody characterization of a leukoagglutinin produced by Renibacterium salmoninarum.

    PubMed

    Wiens, G D; Kaattari, S L

    1991-02-01

    Renibacterium salmoninarum causes a chronic disease of salmonid fish known as bacterial kidney disease. High concentrations of bacterially produced extracellular protein (ECP) are present in plasma, kidney, and spleen tissue of naturally and experimentally infected fish. ECP agglutinated salmonid leukocytes in vitro at concentrations which correspond to levels found in highly infected fish. Association of biological activity with the structure of the major protein constituent of ECP, p57, was accomplished by monoclonal antibody (MAb) analysis. Location of the antigenic binding sites recognized by the MAbs was determined by two-dimensional electrophoresis and Western immunoblotting of the proteolytic breakdown fragments of p57. Eight MAbs have been classified into three groups on the basis of their differential recognition of these proteolytic breakdown products. Group I MAbs bound a region proximal to the amino terminus of the protein. Two of these MAbs were also able to block leukoagglutinating activity. Group III MAbs bound to a region associated with the bacterial cell surface, while group II MAbs bound a region between group I and group III. These analyses have allowed the identification of potential structural and functional regions of p57. PMID:1987079

  16. Monoclonal antibody characterization of a leukoagglutinin produced by Renibacterium salmoninarum.

    PubMed Central

    Wiens, G D; Kaattari, S L

    1991-01-01

    Renibacterium salmoninarum causes a chronic disease of salmonid fish known as bacterial kidney disease. High concentrations of bacterially produced extracellular protein (ECP) are present in plasma, kidney, and spleen tissue of naturally and experimentally infected fish. ECP agglutinated salmonid leukocytes in vitro at concentrations which correspond to levels found in highly infected fish. Association of biological activity with the structure of the major protein constituent of ECP, p57, was accomplished by monoclonal antibody (MAb) analysis. Location of the antigenic binding sites recognized by the MAbs was determined by two-dimensional electrophoresis and Western immunoblotting of the proteolytic breakdown fragments of p57. Eight MAbs have been classified into three groups on the basis of their differential recognition of these proteolytic breakdown products. Group I MAbs bound a region proximal to the amino terminus of the protein. Two of these MAbs were also able to block leukoagglutinating activity. Group III MAbs bound to a region associated with the bacterial cell surface, while group II MAbs bound a region between group I and group III. These analyses have allowed the identification of potential structural and functional regions of p57. Images PMID:1987079

  17. 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

  18. [Study of plant lectins from Viscum album using monoclonal antibodies].

    PubMed

    Tonevitskiĭ, A G; Rakhmanova, V A; Shamshiev, A T; Usachaeva, E A; Agapov, I I; Prokov'ev, S A; Denisenko, O N; Pfuller, U; Eifler, R

    1995-01-01

    Monoclonal antibodies (monAT) against both native (TA5, TB12) and denatured (TB33, TB35) plant toxin ML1 from Viscum album have been obtained. The interaction of monAT against native toxin with its isoforms ML2 and ML3 was investigated. It was shown that monAT TA5 to A-chain of ML1 toxin cross-reacted with ML2 and ML3 isoforms. TA5 did not inhibit enzyme activity of A-chain in cell-free rabbit reticulocyte system. It was shown that monAT TB12 reacted with galactose-binding site of B-subunit. Both monAT had no cross-reactions with plant toxin ricin. The binding constants for TA5 with ML1, ML2, ML3 respectively were 4.3.10(7) M-1, 1.2.10(7) M-1, and 0.3.10(7) M-1. The binding constants for TB12 were 2.10(7) M-1 with ML1 toxin, and more than 10(6) M-1 with ML2 and ML3. The nature of heterogeneity in ML toxin family is discussed. Test-systems for ML1 determination in different V. album extracts are suggested. PMID:8552066

  19. 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

  20. Kinetics of Monoclonal Antibody Aggregation from Dilute toward Concentrated Conditions.

    PubMed

    Nicoud, Lucrèce; Jagielski, Jakub; Pfister, David; Lazzari, Stefano; Massant, Jan; Lattuada, Marco; Morbidelli, Massimo

    2016-04-01

    Gaining understanding on the aggregation behavior of proteins under concentrated conditions is of both fundamental and industrial relevance. Here, we study the aggregation kinetics of a model monoclonal antibody (mAb) under thermal stress over a wide range of protein concentrations in various buffer solutions. We follow experimentally the monomer depletion and the aggregate growth by size exclusion chromatography with inline light scattering. We describe the experimental results in the frame of a kinetic model based on population balance equations, which allows one to discriminate the contributions of the conformational and of the colloidal stabilities to the global aggregation rate. Finally, we propose an expression for the aggregation rate constant, which accounts for solution viscosity, protein-protein interactions, as well as aggregate compactness. All these effects can be quantified by light scattering techniques. It is found that the model describes well the experimental data under dilute conditions. Under concentrated conditions, good model predictions are obtained when the solution pH is far below the isoelectric point (pI) of the mAb. However, peculiar effects arise when the solution pH is increased toward the mAb pI, and possible explanations are discussed. PMID:27007829

  1. 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

  2. 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

  3. 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

  4. DNA immunization as a technology platform for monoclonal antibody induction

    PubMed Central

    Liu, Shuying; Wang, Shixia; Lu, Shan

    2016-01-01

    To combat the threat of many emerging infectious diseases, DNA immunization offers a unique and powerful approach to the production of high-quality monoclonal antibodies (mAbs) against various pathogens. Compared with traditional protein-based immunization approaches, DNA immunization is efficient for testing novel immunogen designs, does not require the production or purification of proteins from a pathogen or the use of recombinant protein technology and is effective at generating mAbs against conformation-sensitive targets. Although significant progress in the use of DNA immunization to generate mAbs has been made over the last two decades, the literature does not contain an updated summary of this experience. The current review provides a comprehensive analysis of the literature, including our own work, describing the use of DNA immunization to produce highly functional mAbs, in particular, those against emerging infectious diseases. Critical factors such as immunogen design, delivery approach, immunization schedule, use of immune modulators and the role of final boost immunization are discussed in detail. PMID:27048742

  5. 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

  6. 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

  7. 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.

  8. Polyelectrolyte Nanogels Decorated with Monoclonal Antibody for Targeted Drug Delivery

    PubMed Central

    Nukolova, Nataliya V.; Yang, Zigang; Kim, Jong Oh; Kabanov, Alexander V.; Bronich, Tatiana K.

    2010-01-01

    Novel surface-functionalized cross-linked nanogels were developed as a platform to allow conjugation of monoclonal antibodies (mAb) for targeted drug delivery. Well-defined diblock copolymers of poly(ethylene glycol)-b-poly(methacrylic acid) (PEG-b-PMA) with PEG terminal aldehyde functionality were synthesized by atom transfer radical polymerization (ATRP) and characterized by GPC and 1H NMR. These copolymers were used to prepare nanogels via condensation of PEG-b-PMA with Ca2+ ions into micelle-like aggregates, cross-linking of the PMA/Ca2+ cores and removal of Ca2+ ions. The resulting nanogels represent highly swollen spherical polyelectrolyte particles with free terminal aldehyde functionalities at the nonionic PEG chains. A reductive amination reaction between aldehyde groups and amino groups of mAb resulted in effective conjugation to the nanogels of mAb CC49 against tumor-associated glycoprotein 72 (TAG-72). The mAb retained the binding affinity to bovine submaxillary mucin after conjugation as shown by surface plasmon resonance (SPR). Therefore, aldehyde functionalized nanogels can be linked to mAb using a simple, one-step approach. They may have potential for targeted delivery of diagnostic and therapeutic agents to tumors. PMID:21503276

  9. Influence of macromolecular precipitants on phase behavior of monoclonal antibodies.

    PubMed

    Rakel, Natalie; Galm, Lara; Bauer, Katharina Christin; Hubbuch, Juergen

    2015-01-01

    For the successful application of protein crystallization as a downstream step, a profound knowledge of protein phase behavior in solutions is needed. Therefore, a systematic screening was conducted to analyze the influence of macromolecular precipitants in the form of polyethylene glycol (PEG). First, the influence of molecular weight and concentration of PEG at different pH-values were investigated and analyzed in three-dimensional (3-D) phase diagrams to find appropriate conditions in terms of a fast kinetic and crystal size for downstream processing. In comparison to the use of salts as precipitant, PEG was more suitable to obtain compact 3-D crystals over a broad range of conditions, whereby the molecular weight of PEG is, besides the pH-value, the most important parameter. Second, osmotic second virial coefficients as parameters for protein interactions are experimentally determined with static light scattering to gain a deep insight view in the phase behavior on a molecular basis. The PEG-protein solutions were analyzed as a pseudo-one-compartment system. As the precipitant is also a macromolecule, the new approach of analyzing cross-interactions between the protein and the macromolecule PEG in form of the osmotic second cross-virial coefficient (B23 ) was applied. Both parameters help to understand the protein phase behavior. However, a predictive description of protein phase behavior for systems consisting of monoclonal antibodies and PEG as precipitant is not possible, as kinetic phenomena and concentration dependencies were not taken into account. PMID:25504581

  10. 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

  11. 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

  12. 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

  13. Potent neutralizing monoclonal antibodies against Ebola virus infection

    PubMed Central

    Zhang, Qi; Gui, Miao; Niu, Xuefeng; He, Shihua; Wang, Ruoke; Feng, Yupeng; Kroeker, Andrea; Zuo, Yanan; Wang, Hua; Wang, Ying; Li, Jiade; Li, Chufang; Shi, Yi; Shi, Xuanling; Gao, George F.; Xiang, Ye; Qiu, Xiangguo; Chen, Ling; Zhang, Linqi

    2016-01-01

    Ebola virus infections cause a deadly hemorrhagic disease for which no vaccines or therapeutics has received regulatory approval. Here we show isolation of three (Q206, Q314 and Q411) neutralizing monoclonal antibodies (mAbs) against the surface glycoprotein (GP) of Ebola virus identified in West Africa in 2014 through sequential immunization of Chinese rhesus macaques and antigen-specific single B cell sorting. These mAbs demonstrated potent neutralizing activities against both pseudo and live Ebola virus independent of complement. Biochemical, single particle EM, and mutagenesis analysis suggested Q206 and Q411 recognized novel epitopes in the head while Q314 targeted the glycan cap in the GP1 subunit. Q206 and Q411 appeared to influence GP binding to its receptor NPC1. Treatment with these mAbs provided partial but significant protection against disease in a mouse model of Ebola virus infection. These novel mAbs could serve as promising candidates for prophylactic and therapeutic interventions against Ebola virus infection. PMID:27181584

  14. 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

  15. DNA immunization as a technology platform for monoclonal antibody induction.

    PubMed

    Liu, Shuying; Wang, Shixia; Lu, Shan

    2016-01-01

    To combat the threat of many emerging infectious diseases, DNA immunization offers a unique and powerful approach to the production of high-quality monoclonal antibodies (mAbs) against various pathogens. Compared with traditional protein-based immunization approaches, DNA immunization is efficient for testing novel immunogen designs, does not require the production or purification of proteins from a pathogen or the use of recombinant protein technology and is effective at generating mAbs against conformation-sensitive targets. Although significant progress in the use of DNA immunization to generate mAbs has been made over the last two decades, the literature does not contain an updated summary of this experience. The current review provides a comprehensive analysis of the literature, including our own work, describing the use of DNA immunization to produce highly functional mAbs, in particular, those against emerging infectious diseases. Critical factors such as immunogen design, delivery approach, immunization schedule, use of immune modulators and the role of final boost immunization are discussed in detail. PMID:27048742

  16. Development of a monoclonal antibody specific to cooked mammalian meats.

    PubMed

    Hsieh, Y H; Sheu, S C; Bridgman, R C

    1998-04-01

    Detection of species adulteration in ground meat products is important for consumer protection and food-labeling law enforcement. This study was conducted to develop monoclonal antibodies (MAbs) that can be used in an enzyme-linked immunosorbent assay (ELISA) for rapid detection of any cooked mammalian meats in cooked poultry products. Soluble muscle proteins extracted from cooked pork (heated at 100 degrees C for 15 min) were used as the antigen to immunized mice for developing the MAb. One that was developed, MAb 2F8 (IgG2b class), strongly reacted with cooked meat of five mammalian species (beef cattle, hogs, sheep, horse, and deer) but did not react with any cooked poultry (chicken, turkey, and duck) or raw meats. At least 0.5% by weight of pork, beef, lamb, and horse meats in a chicken-based mixture could not detect using the indirect ELISA with MAb 2F8. The MAb 2F8 is useful in a single initial screening test to detect the presence of five nonpoultry meat adulterants in cooked poultry products. PMID:9709213

  17. 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.

  18. An anti-Cryptococcus neoformans monoclonal antibody directed against galactoxylomannan.

    PubMed

    van de Moer, A; Salhi, S L; Cherniak, R; Pau, B; Garrigues, M L; Bastide, J M

    1990-01-01

    Six monoclonal antibodies (mAb) were produced by immunizing mice with a Cryptococcus neoformans serotype A spheroplast lysate (CNSL). Two mAb were of the IgG1 isotype; the others were IgM. The results obtained with one IgM mAb (CN6) are reported herein. This mAb recognized the four serotypes of C. neoformans and no cross-reactions were observed with extracts from Cryptococcus melibiosum, Candida albicans, Saccharomyces cerevisiae, Torulopsis glabrata or Trichosporon beigelii. Fractionation of the CNSL by gel filtration revealed that mAb CN6 recognized high molecular weight substances as well as a range of smaller molecules. Indirect ELISA inhibition studies showed that this mAb recognized substances in a cryptococcal culture filtrate. Inhibition studies and agglutination tests using latex beads sensitized with purified CN6 showed that CN6 strongly reacted with the C. neoformans serotype A cell envelope galactoxylomannan-mannoprotein complex (GAlXM-MP) and only weakly with the glucuronoxylomannan (GXM) component. These tests also showed that purified galactoxylomannan (GalXM) from C. neoformans serotype A was more reactive than purified mannoprotein (MP). An anti-GalXM mAb might be a useful tool for monitoring the clinical course of cryptococcal infections. PMID:2189167

  19. 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

  20. 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

  1. 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).

  2. Potent neutralizing monoclonal antibodies against Ebola virus infection.

    PubMed

    Zhang, Qi; Gui, Miao; Niu, Xuefeng; He, Shihua; Wang, Ruoke; Feng, Yupeng; Kroeker, Andrea; Zuo, Yanan; Wang, Hua; Wang, Ying; Li, Jiade; Li, Chufang; Shi, Yi; Shi, Xuanling; Gao, George F; Xiang, Ye; Qiu, Xiangguo; Chen, Ling; Zhang, Linqi

    2016-01-01

    Ebola virus infections cause a deadly hemorrhagic disease for which no vaccines or therapeutics has received regulatory approval. Here we show isolation of three (Q206, Q314 and Q411) neutralizing monoclonal antibodies (mAbs) against the surface glycoprotein (GP) of Ebola virus identified in West Africa in 2014 through sequential immunization of Chinese rhesus macaques and antigen-specific single B cell sorting. These mAbs demonstrated potent neutralizing activities against both pseudo and live Ebola virus independent of complement. Biochemical, single particle EM, and mutagenesis analysis suggested Q206 and Q411 recognized novel epitopes in the head while Q314 targeted the glycan cap in the GP1 subunit. Q206 and Q411 appeared to influence GP binding to its receptor NPC1. Treatment with these mAbs provided partial but significant protection against disease in a mouse model of Ebola virus infection. These novel mAbs could serve as promising candidates for prophylactic and therapeutic interventions against Ebola virus infection. PMID:27181584

  3. Haptens and monoclonal antibodies for immunoassay of imidazolinone herbicides.

    PubMed

    Chin, Tina E; Wong, Rosie B; Pont, Joseph L; Karu, Alexander E

    2002-06-01

    A set of haptens structurally resembling the herbicide imazethapyr (PURSUIT) was synthesized and used to derive monoclonal antibodies (MAbs) and direct and indirect competition enzyme immunoassays (EIAs) which could detect imazethapyr, imazaquin (SCEPTER), imazapic (CADRE), and imazamox (RAPTOR) in the 3-30 ng/mL (parts per billion) range, and imazapyr (ARSENAL) and imazamethabenz-methyl (ASSERT) in the 300-500 ppb range. Two MAbs, 3A2 and 3A5, had affinities of 10-75 nM for imazethapyr. MAbs 1A5, 1D2, and 3A5 were specific for the S isomers of the herbicides. Some MAbs were stable in solutions containing up to 15% methanol and 5% acetonitrile in indirect EIAs. Plates coated with hapten conjugates for indirect EIA could be stored frozen. Selectivity for the imidazolinones by some MAbs varied with different coating conjugates. These MAbs and haptens should prove useful in immunochemical analysis and residue recovery methods for imazethapyr and other imidazolinone herbicides. PMID:12033799

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies.

    PubMed

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu

    2016-01-01

    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  10. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies

    PubMed Central

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu

    2016-01-01

    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  11. 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.

  12. 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

  13. 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

  14. 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.

  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 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

  18. Characterization of cross-reactive norovirus-specific monoclonal antibodies.

    PubMed

    Kou, Baijun; Crawford, Sue E; Ajami, Nadim J; Czakó, Rita; Neill, Frederick H; Tanaka, Tomoyuki N; Kitamoto, Noritoshi; Palzkill, Timothy G; Estes, Mary K; Atmar, Robert L

    2015-02-01

    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

  19. 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

  20. Lymphocyte phosphatase-associated phosphoprotein proteoforms analyzed using monoclonal antibodies

    PubMed Central

    Filatov, Alexander; Kruglova, Natalia; Meshkova, Tatiana; Mazurov, Dmitriy

    2015-01-01

    Phosphatase CD45 regulates the activation of lymphocytes by controlling the level of receptor and signal molecule phosphorylation. However, it remains unknown which molecules mediate the phosphatase activity of CD45. A candidate for such a molecule is a small transmembrane adapter protein called lymphocyte phosphatase-associated phosphoprotein (LPAP). LPAP forms a supramolecular complex that consists of not only CD45 molecule but also CD4 and Lck kinase. The function of LPAP has not been defined clearly. In our study, we determined the pattern of LPAP expression in various cell types and characterized its proteoforms using new monoclonal antibodies generated against the intracellular portion of the protein. We show that LPAP is a pan-lymphocyte marker, and its expression in cells correlates with the expression of CD45. The majority of T, B and NK cells express high levels of LPAP, whereas monocytes, granulocytes, monocyte-derived dendritic cells, platelets and red blood cells are negative for LPAP. Using one- and two-dimensional protein gel electrophoresis, we demonstrate that LPAP has at least four sites of phosphorylation. The resting cells express at least six different LPAP phosphoforms representing mono-, di- and tri-phosphorylated LPAP. T and B cells differ in the distribution of the protein between phosphoforms. The activation of lymphocytes with PMA reduces the diversity of phosphorylated forms. Our experiments on Lck-deficient Jurkat cells show that Lck kinase is not involved in LPAP phosphorylation. Thus, LPAP is a dynamically phosphorylated protein, the function of which can be understood, when all phosphosites and kinases involved in its phosphorylation will be identified. PMID:26682052

  1. 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

  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. Monoclonal antibodies: longitudinal prescribing information analysis of hypersensitivity reactions.

    PubMed

    Kleyman, Konstantin; Weintraub, Debra S

    2012-01-01

    Monoclonal antibodies (mAbs) are known to cause hypersensitivity reactions (HSRs). The reactions pose a significant challenge to investigators, regulators, and health providers. Because HSRs cannot be predicted through the pharmacological basis of a therapy, clinical data are often relied upon to detect the reactions. Unfortunately, clinical studies are often unable to adequately characterize HSRs especially in therapies for orphan diseases. HSRs can go undetected until post-marketing safety surveillance when a large number of patients have been exposed to the therapy. The presented data demonstrates how hypersensitivity reaction warnings have changed over time in the prescribing information (PI), i.e., the drug package insert, through August 1, 2011 for 28 US-marketed mAbs. Tracking all PI revisions for each mAb over time revealed that hypersensitivity warning statements were expanded to include more severe manifestations. Over the course of a mAb therapy's life cycle, the hypersensitivity warning is twice more likely to be upgraded than downgraded in priority. Approximately 85% of hypersensitivity-associated fatality warnings were added in PI revisions as a result of post-marketing experience. Over 60% (20/33) of revisions to hypersensitivity warnings occurred within 3-4 y of product approval. While HSRs are generally recognized and described in the initial PI of mAbs, fatal HSRs are most commonly observed in post-marketing surveillance. Results of this study suggest that initial product labeling information may not describe rare but clinically significant occurrences of severe or fatal HSRs, but subsequent label revisions include rare events observed during post-marketed product use. PMID:22531444

  4. 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

  5. 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

  6. Pharmacological effects of two anti-methamphetamine monoclonal antibodies

    PubMed Central

    Laurenzana, Elizabeth M; Stevens, Misty W; Frank, John C; Hambuchen, Michael D; Hendrickson, Howard P; White, Sarah J; Williams, D Keith; Owens, S Michael; Gentry, W Brooks

    2014-01-01

    This lead candidate selection study compared two anti-(+)-methamphetamine (METH) monoclonal antibodies (mAb) to determine their ability to reduce METH-induced locomotor effects and redistribute METH and (+)-amphetamine (AMP) in a preclinical overdose model. Both mAbs have high affinity for METH, but mAb4G9 has moderate and mAb7F9 has low affinity for AMP. In the placebo-controlled behavioral experiment, the effects of each mAb on the locomotor response to a single 1 mg/kg intravenous (IV) METH dose were determined in rats. The doses of mAb binding sites were administered such that they equaled 1, 0.56, 0.32, and 0.1 times the molar equivalent (mol-eq) of METH in the body 30 min after the METH dose. METH disposition was determined in separate animals that similarly received either a 1 or 0.32 mol-eq dose of mAb binding sites 30 min after a 1 mg/kg METH dose. Total METH-induced distance traveled was significantly reduced in rats that received the highest three doses of each mAb compared with saline. The duration of METH effects was also significantly reduced by mAb7F9 at the highest dose. The disposition of METH was altered dose-dependently by both mAbs as shown in reductions of volume of distribution and total clearance, and increases in elimination half-life. These data indicate that both mAbs are effective at reducing METH-induced behavior and favorably altering METH disposition. Both were therefore suitable for further preclinical testing as potential human medications for treating METH use; however, due to results reported here and in later studies, mAb7F9 was selected for clinical development. PMID:25483484

  7. 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

  8. Response of a concentrated monoclonal antibody formulation to high shear.

    PubMed

    Bee, Jared S; Stevenson, Jennifer L; Mehta, Bhavya; Svitel, Juraj; Pollastrini, Joey; Platz, Robert; Freund, Erwin; Carpenter, John F; Randolph, Theodore W

    2009-08-01

    There is concern that shear could cause protein unfolding or aggregation during commercial biopharmaceutical production. In this work we exposed two concentrated immunoglobulin-G1 (IgG1) monoclonal antibody (mAb, at >100 mg/mL) formulations to shear rates between 20,000 and 250,000 s(-1) for between 5 min and 30 ms using a parallel-plate and capillary rheometer, respectively. The maximum shear and force exposures were far in excess of those expected during normal processing operations (20,000 s(-1) and 0.06 pN, respectively). We used multiple characterization techniques to determine if there was any detectable aggregation. We found that shear alone did not cause aggregation, but that prolonged exposure to shear in the stainless steel parallel-plate rheometer caused a very minor reversible aggregation (<0.3%). Additionally, shear did not alter aggregate populations in formulations containing 17% preformed heat-induced aggregates of a mAb. We calculate that the forces applied to a protein by production shear exposures (<0.06 pN) are small when compared with the 140 pN force expected at the air-water interface or the 20-150 pN forces required to mechanically unfold proteins described in the atomic force microscope (AFM) literature. Therefore, we suggest that in many cases, air-bubble entrainment, adsorption to solid surfaces (with possible shear synergy), contamination by particulates, or pump cavitation stresses could be much more important causes of aggregation than shear exposure during production. PMID:19370772

  9. Characterizing monoclonal antibody structure by carboxyl group footprinting

    PubMed Central

    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 et al 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

  10. 77 FR 9678 - Prospective Grant of Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-17

    ... Human Anti-CD22 Monoclonal Antibodies for the Treatment of Human Cancers and Autoimmune Disease AGENCY... Antibodies Specific for CD22'' , PCT Application PCT/US2009/124109 entitled ``Human and Improved Murine Monoclonal Antibodies Against CD22'' , U.S. patent application 12/934,214 entitled ``Human...

  11. 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...

  12. 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...

  13. 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...

  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. 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 ...

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

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

    PubMed Central

    Bidlingmaier, Scott; Su, Yang; Liu, Bin

    2016-01-01

    Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties. PMID:26060069

  2. 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

  3. 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.

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

    PubMed

    Skowicki, Michał; Lipiński, Tomasz

    2016-01-01

    Monoclonal antibodies (mAbs) are biomolecules of great scientific and practical significance. In contrast to polyclonal antibodies from immune sera, they are homogeneous and monospecific, since they are produced by hybridoma cells representing a clone arising from a single cell. The successful technology was described for the first time in 1975; the inventors were later awarded the Nobel Prize. Currently, mAbs are broadly used as a research tool, in diagnostics and medicine in particular for the treatment of cancer or in transplantology. About 47 therapeutics based on monoclonal antibodies are now available in the US and Europe, and the number is still growing. Production of monoclonal antibodies is a multistage, time-consuming and costly process. Growing demand for these molecules creates space for research focused on improvements in hybridoma technology. Lower costs, human labor, and time are important goals of these attempts. In this article, a brief review of current methods and their advances is given. PMID:27117113

  5. 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

  6. 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

  7. 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

  8. 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.

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

    PubMed Central

    Caruana, Ignazio; Diaconu, Iulia; Dotti, Gianpietro

    2014-01-01

    Monoclonal antibodies (mAbs) and their directly derived cell-based application known as chimeric antigen receptors (CARs) ensue from the need to develop novel therapeutic strategies that retain high anti-tumor activity, but carry reduced toxicity compared to conventional chemo- and radio-therapies. In this concise review article we will summarize the application of antibodies designed to target antigens expressed by tumor cells, and the transition from these antibodies to the generation of CARs. PMID:25440610

  10. Combination of monoclonal antibodies improves immunohistochemical diagnosis of Neospora caninum.

    PubMed

    Uzêda, R S; Schares, G; Ortega-Mora, L M; Madruga, C R; Aguado-Martinez, A; Corbellini, L G; Driemeier, D; Gondim, L F P

    2013-11-01

    Histological analysis is commonly used for a conclusive diagnosis of neosporosis. Immunohistochemistry (IHC) using monoclonal (mAb) and polyclonal (pAb) antibodies can improve diagnosis; however, the use of pAb may induce cross-reactivity with other related parasites. The aims of this study were to compare the performance of mAbs and their combinations with that of pAb in IHC and evaluate the usefulness of mAb to identify Neospora caninum infection in aborted bovine fetal tissues. For this purpose, mAbs targeting NcSRS2 (4.15.15) or NcGRA7 (4.11.5 and 1/24-12) and one pAb collected from a rabbit inoculated with N. caninum tachyzoites were tested by IHC. Artificial standardized tissue sections were prepared as positive controls using homogenized bovine brain spiked with cultured tachyzoites of N. caninum. The numbers of labeled parasites were counted in each positive control section. In addition, four equal proportional combinations of the mAbs were also analyzed in the IHC. Finally, the pAb and the best combination of mAbs obtained in the positive control experiments were tested with tissue sections of naturally-infected cattle. To confirm analytical specificity, mAbs and a pAb were tested with Toxoplasma gondii and Besnoitia besnoiti positive control slides and tissues sections from naturally infected cattle containing Sarcocystis spp. and B. besnoiti antigens. The mAb 4.15.15 detected 57% of the total parasites in sections while 4.11.5 and 1/24-12 were able to detect 49% and 41%, respectively. For the mAb combinations (I: 1/24-12+4.11.5, II: 1/24-12+4.15.15, III: 4.15.15+4.11.5, IV: 1/24-12+4.11.5+4.15.15), the detection capacity was 32.4%, 79.4%, 66.6% and 60.7% for each combination, respectively. The best mAb combination (1/24-12 and 4.15.15) and the pAb serum detected 100% (18/18) of naturally-infected animals. Sarcocystis spp. or B. besnoiti were not detected by mAb combinations in IHC, however the pAb cross-reacted with Sarcocystis spp. cysts. These results confirm the usefulness of mAb application in IHC to N. caninum. PMID:23927916

  11. Characterization and applications of monoclonal antibodies to the prolactin receptor

    SciTech Connect

    Okamura, H.; Zachwieja, J.; Raguet, S.; Kelly, P.A.

    1989-05-01

    Monoclonal antibodies (mAbs) were produced in BALB/c mice immunized with partially purified PRL receptors from rat liver. Two mAbs (T1 and T6) were able to completely inhibit (125I)ovine PRL ((125I)oPRL) binding to solubilized rat liver PRL receptors, while two other mAbs (U5 and U6) showed only a small effect on PRL binding, but were able to precipitate hormone-receptor complexes. Scatchard analysis of (125I)oPRL binding to rat liver microsomes in the presence of mAbs resulted in a decrease in the number of sites without changing the affinity of PRL binding by T1 and T6, whereas U5 and U6 altered neither parameter. (125I)mAb binding to rat liver microsomes was performed in the presence of various concentrations of unlabeled mAbs or oPRL to examine the interaction between mAbs. Competition of binding to the receptor was observed, respectively, between T1 and T6, U5 and U6, and U5 and E21 (a mAb to the rat liver PRL receptor previously produced). Both (125I)T1 and (125I)T6 binding were inhibited by oPRL, although not completely (80% inhibition at the higher concentrations). When (125I)T1 binding was analyzed by Scatchard analysis, two classes of binding sites to rat liver microsomes were found, of which only the number of higher affinity sites was affected by the presence of oPRL in incubation. Similar results were observed for (125I)T6 binding. (125I)mAb binding to microsomes from other tissues and species was examined. All five mAbs were able to bind to microsomes from rat tissues (liver, ovary, adrenal, prostate, and Nb2 lymphoma cells), similar to the level of (125I)oPRL binding in these tissues. The binding characteristics of (125I)T6 or (125I)U5 were essentially identical in all rat tissues examined.

  12. West Nile virus: characterization and diagnostic applications of monoclonal antibodies

    PubMed Central

    2012-01-01

    Background Diagnosis of West Nile virus (WNV) infections is often difficult due to the extensive antigenic cross-reactivity among flaviviruses, especially in geographic regions where two or more of these viruses are present causing sequential infections. The purpose of this study was to characterize a panel of monoclonal antibodies (MAbs) produced against WNV to verify their applicability in WNV diagnosis and in mapping epitope targets of neutralizing MAbs. Methods Six MAbs were produced and characterized by isotyping, virus-neutralization, western blotting and MAb-epitope competition. The MAb reactivity against various WNVs belonging to lineage 1 and 2 and other related flaviviruses was also evaluated. The molecular basis of epitopes recognized by neutralizing MAbs was defined through the selection and sequencing of MAb escape mutants. Competitive binding assays between MAbs and experimental equine and chicken sera were designed to identify specific MAb reaction to epitopes with high immunogenicity. Results All MAbs showed stronger reactivity with all WNVs tested and good competition for antigen binding in ELISA tests with WNV-positive equine and chicken sera. Four MAbs (3B2, 3D6, 4D3, 1C3) resulted specific for WNV, while two MAbs (2A8, 4G9) showed cross-reaction with Usutu virus. Three MAbs (3B2, 3D6, 4D3) showed neutralizing activity. Sequence analysis of 3B2 and 3D6 escape mutants showed an amino acid change at E307 (Lys → Glu) in the E protein gene, whereas 4D3 variants identified mutations encoding amino acid changed at E276 (Ser → Ile) or E278 (Thr → Ile). 3B2 and 3D6 mapped to a region on the lateral surface of domain III of E protein, which is known to be a specific and strong neutralizing epitope for WNV, while MAb 4D3 recognized a novel specific neutralizing epitope on domain II of E protein that has not previously been described with WNV MAbs. Conclusions MAbs generated in this study can be applied to various analytical methods for virological and serological WNV diagnosis. A novel WNV-specific and neutralizing MAb (4D3) directed against the unknown epitope on domain II of E protein can be useful to better understand the role of E protein epitopes involved in the mechanism of WNV neutralization. PMID:22500562

  13. Evaluation of Ion Mobility-Mass Spectrometry for Comparative Analysis of Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Ferguson, Carly N.; Gucinski-Ruth, Ashley C.

    2016-03-01

    Analytical techniques capable of detecting changes in structure are necessary to monitor the quality of monoclonal antibody drug products. Ion mobility mass spectrometry offers an advanced mode of characterization of protein higher order structure. In this work, we evaluated the reproducibility of ion mobility mass spectrometry measurements and mobiligrams, as well as the suitability of this approach to differentiate between and/or characterize different monoclonal antibody drug products. Four mobiligram-derived metrics were identified to be reproducible across a multi-day window of analysis. These metrics were further applied to comparative studies of monoclonal antibody drug products representing different IgG subclasses, manufacturers, and lots. These comparisons resulted in some differences, based on the four metrics derived from ion mobility mass spectrometry mobiligrams. The use of collision-induced unfolding resulted in more observed differences. Use of summed charge state datasets and the analysis of metrics beyond drift time allowed for a more comprehensive comparative study between different monoclonal antibody drug products. Ion mobility mass spectrometry enabled detection of differences between monoclonal antibodies with the same target protein but different production techniques, as well as products with different targets. These differences were not always detectable by traditional collision cross section studies. Ion mobility mass spectrometry, and the added separation capability of collision-induced unfolding, was highly reproducible and remains a promising technique for advanced analytical characterization of protein therapeutics.

  14. Evaluation of Ion Mobility-Mass Spectrometry for Comparative Analysis of Monoclonal Antibodies.

    PubMed

    Ferguson, Carly N; Gucinski-Ruth, Ashley C

    2016-05-01

    Analytical techniques capable of detecting changes in structure are necessary to monitor the quality of monoclonal antibody drug products. Ion mobility mass spectrometry offers an advanced mode of characterization of protein higher order structure. In this work, we evaluated the reproducibility of ion mobility mass spectrometry measurements and mobiligrams, as well as the suitability of this approach to differentiate between and/or characterize different monoclonal antibody drug products. Four mobiligram-derived metrics were identified to be reproducible across a multi-day window of analysis. These metrics were further applied to comparative studies of monoclonal antibody drug products representing different IgG subclasses, manufacturers, and lots. These comparisons resulted in some differences, based on the four metrics derived from ion mobility mass spectrometry mobiligrams. The use of collision-induced unfolding resulted in more observed differences. Use of summed charge state datasets and the analysis of metrics beyond drift time allowed for a more comprehensive comparative study between different monoclonal antibody drug products. Ion mobility mass spectrometry enabled detection of differences between monoclonal antibodies with the same target protein but different production techniques, as well as products with different targets. These differences were not always detectable by traditional collision cross section studies. Ion mobility mass spectrometry, and the added separation capability of collision-induced unfolding, was highly reproducible and remains a promising technique for advanced analytical characterization of protein therapeutics. Graphical Abstract ᅟ. PMID:26988372

  15. Evaluation of Ion Mobility-Mass Spectrometry for Comparative Analysis of Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Ferguson, Carly N.; Gucinski-Ruth, Ashley C.

    2016-05-01

    Analytical techniques capable of detecting changes in structure are necessary to monitor the quality of monoclonal antibody drug products. Ion mobility mass spectrometry offers an advanced mode of characterization of protein higher order structure. In this work, we evaluated the reproducibility of ion mobility mass spectrometry measurements and mobiligrams, as well as the suitability of this approach to differentiate between and/or characterize different monoclonal antibody drug products. Four mobiligram-derived metrics were identified to be reproducible across a multi-day window of analysis. These metrics were further applied to comparative studies of monoclonal antibody drug products representing different IgG subclasses, manufacturers, and lots. These comparisons resulted in some differences, based on the four metrics derived from ion mobility mass spectrometry mobiligrams. The use of collision-induced unfolding resulted in more observed differences. Use of summed charge state datasets and the analysis of metrics beyond drift time allowed for a more comprehensive comparative study between different monoclonal antibody drug products. Ion mobility mass spectrometry enabled detection of differences between monoclonal antibodies with the same target protein but different production techniques, as well as products with different targets. These differences were not always detectable by traditional collision cross section studies. Ion mobility mass spectrometry, and the added separation capability of collision-induced unfolding, was highly reproducible and remains a promising technique for advanced analytical characterization of protein therapeutics.

  16. 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.

  17. 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

  18. 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

  19. 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

  20. Characterization of Norwalk virus GI specific monoclonal antibodies generated against Escherichia coli expressed capsid protein and the reactivity of two broadly reactive monoclonal antibodies generated against GII capsid towards GI recombinant fragments

    PubMed Central

    Yoda, Tomoko; Terano, Yoshitake; Suzuki, Yasuhiko; Yamazaki, Kenji; Oishi, Isao; Kuzuguchi, Tsuyoshi; Kawamoto, Hiroyoshi; Utagawa, Etsuko; Takino, Koichi; Oda, Hajime; Shibata, Tadayoshi

    2001-01-01

    Background Norwalk virus causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. In a previous study, the capsid protein of recombinant Norwalk virus genogroup II was expressed in an E. coli system and monoclonal antibodies were generated against it. The analysis of the reactivity of those monoclonal antibodies suggested that the N-terminal domain might contain more antigenic epitopes than the C-terminal domain. In the same study, two broadly reactive monoclonal antibodies were observed to react with genogroup I recombinant protein. Results In the present study, we used the recombinant capsid protein of genogroup I and characterized the obtained 17 monoclonal antibodies by using 19 overlapping fragments. Sixteen monoclonal antibodies recognized sequential epitopes on three antigenic regions, and the only exceptional monoclonal antibody recognized a conformational epitope. As for the two broadly reactive monoclonal antibodies generated against genogroup II, we indicated that they recognized fragment 2 of genogroup I. Furthermore, genogroup I antigen from a patient's stool was detected by sandwich enzyme-linked immunosorbent assay using genogroup I specific monoclonal antibody and biotinated broadly reactive monoclonal antibody. Conclusion The reactivity analysis of above monoclonal antibodies suggests that the N-terminal domain may contain more antigenic epitopes than the C-terminal domain as suggested in our previous study. The detection of genogroup I antigen from a patient's stool by our system suggested that the monoclonal antibodies generated against E. coli expressed capsid protein can be used to detect genogroup I antigens in clinical material. PMID:11710959

  1. 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.

  2. Monoclonal antibodies to human prostatic acid phosphatase: probes for antigenic study.

    PubMed Central

    Lillehoj, H S; Choe, B K; Rose, N R

    1982-01-01

    Hybrid cell lines producing monoclonal antibodies against human prostatic acid phosphatase [PAPase; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) were prepared by the fusion of mouse myeloma cells with the spleen cells of PAPase-immunized BALB/c mice. Approximately 23% of the hybrid cells initially plated after cell fusion produced specific antibodies: 34 microcultures were cloned, and 8 eventually yielded stable cell lines. The monoclonal antibodies produced by these eight hybridomas were characterized for their isotypes, isoelectric points, concentrations, and affinities. All of the eight monoclonal antibodies exhibited strict specificity for PAPase as determined by radioimmunoassay and immunohistochemical methods. These antibodies were used as probes for the antigenic mapping of this enzyme, and three nonoverlapping determinants were recognized. Further binding studies with PAPase fragments, generated by cleavage with a submaxillaris protease, showed that those three determinants are clustered on one fragment of PAPase. These monoclonal antibodies may be useful in refinement of clinical immunoassays of PAPase or immunohistological study of PAPase-synthesizing cells. Images PMID:6956915

  3. 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

  4. 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.

  5. 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...

  6. 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.

  7. The Cloning and Expression of Human Monoclonal Antibodies: Implications for Allergen Immunotherapy.

    PubMed

    James, Louisa K

    2016-02-01

    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

  8. Production of Vi monoclonal antibodies and their application as diagnostic reagents.

    PubMed Central

    Tsang, R S; Chau, P Y

    1987-01-01

    Serum antibodies to Vi antigen were detected in mice immunized with the purified antigen but not with Vi-bearing Salmonella typhi whole cells. Fusion of the spleen cells from one of the Vi antibody-producing mice with NSI myeloma cells produced four stable hybridomas that secreted antibodies to Vi. Monoclonal antibodies from these four clones were all of the immunoglobulin G class and, as determined by competition, appeared to have the same epitope specificity. Despite their immunoglobulin G nature, mouse ascitic fluids induced by one of the hybridomas strongly agglutinated the Vi-positive strains of S. typhi, S. dublin, and Citrobacter strain 5396/38. Thus, 10 clinical isolates of S. typhi but not 98 strains of other bacteria were reactive in slide agglutination tests with the monoclonal antibodies. PMID:3571457

  9. Stability of monoclonal IgM antibodies freeze-dried in the presence of trehalose.

    PubMed

    Dráber, P; Dráberová, E; Nováková, M

    1995-04-12

    We describe the use of the disaccharide trehalose for stabilization of mouse monoclonal IgM antibodies during freeze-drying and prolonged storage at elevated temperatures. Spent culture media, ascitic fluids and isolated immunoglobulins were freeze-dried in the presence of trehalose, stored at different temperatures, and tested after rehydration for their binding to their corresponding antigens. Antibodies, directed against various types of antigens, effectively recovered their binding efficiency as tested in enzyme-linked immunoassays, flow cytometry and immunofluorescence. Application of trehalose for freeze-drying of labile monoclonal IgM antibodies permits convenient long-term storage of large quantities of antibodies, facilitates their transport at ambient temperature and simplifies the construction of pre-aliquoted kits based on such antibodies. PMID:7730665

  10. Prolongation of rat corneal graft survival by treatment with anti-CD4 monoclonal antibody.

    PubMed Central

    Ayliffe, W; Alam, Y; Bell, E B; McLeod, D; Hutchinson, I V

    1992-01-01

    A rat model of orthotopic corneal graft rejection was used to investigate the effect of depletion of subpopulations of immune cells by treatment with monoclonal antibodies. Though CD4+ cells were not eliminated completely by anti-CD4 monoclonal antibodies there was a profound delay in the rejection times of orthotopic corneal allografts. Furthermore a third of the CD4+ depleted animals failed to reject corneal allografts by 100 days post grafting. Despite an almost complete depletion of circulating CD8+ cells, the anti-CD8 antibody treated animals rejected corneal allografts in a similar time course to allografted controls treated with a non-reactive control antibody OX21. These results demonstrate that CD8+ T-cells are not required for rejection of corneal allografts whereas CD4+ T-cells play a critical role in the rejection response. Treatment with anti-CD4 antibodies may have a useful clinical application. Images PMID:1358194

  11. 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

  12. Localization of human tumour xenografts after i.v. administration of radiolabeled monoclonal antibodies.

    PubMed

    Moshakis, V; McIlhinney, R A; Raghavan, D; Neville, A M

    1981-07-01

    A mouse monoclonal antibody (LICR-LON/HT13) has been developed to a cell-surface antigen carried on a human germ-cell tumour xenograft (HX39). After radioiodination, the antibody localized in vivo preferentially in xenografted tumours as opposed to normal mouse tissue, whereas tumor uptake did not occur with normal mouse IgG or nonspecific monoclonal IgG. This selective localization could be abolished by simultaneous injection of an excess of the unlabelled LICR-LON/HT13. The kinetics of and factors influencing localization have been examined. Tumour weight was important in that the smaller the tumour the better the localization. LICR-LON/HT13 was found to localize also in other xenografted germ-cell tumours, but not in non-germ-cell tumour xenografts. Thus monoclonal antibodies are capable of selective in vivo localization of human tumours in an animal model, and their clinical value should now be assessed. PMID:6789857

  13. 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.

  14. 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; Castañon, 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

  15. Monoclonal antibodies to DNA modified with cis- or trans-diamminedichloroplatinum(II)

    SciTech Connect

    Sundquist, W.I.; Lippard, S.J.; Stollar, B.D.

    1987-12-01

    Murine monoclonal antibodies that bind selectively to adducts formed on DNA by the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, or to the chemothrapeutically inactive trans isomer trans-DDP were elicited by immunization with calf thymus DNA modified with either cis- or trans-DDP at ratios of bound platinum per nucleotide, (D/N)/sub b/, of 0.06-0.08. The binding of two monoclonal antibodies to cis-DDP-modified DNA was competitively inhibited in an enzyme-linked immunosorbent assay (ELISA) by 4-6 nM concentrations of cis-DDP bound to DNA. Adducts formed by cis-DDP on other synthetic DNA polymers did not inhibit antibody binding to cis-DDP-DNA. The biologically active compounds (Pt(en)Cl/sub 2/), (Pt(dach)Cl/sub 2/), and (Pt(NH/sub 3/)/sub 2/(cbdca)) (carboplatin) all formed antibody-detectable adducts on DNA, whereas the inactive platinum complexes trans-DDP and (Pt(dien)Cl)Cl (dien, diethylenetriamine) did not. The monoclonal antibodies therefore recognize a bifunctional Pt-DNA adduct with cis stereochemistry in which platinum is coordinated by two adjacent guanines or, to a lesser degree, by adjacent adenine and guanine. A monoclonal antibody raised against trans-DDP-DNA was competitively inhibited in an ELISA by 40 nM trans-DDP bound to DNA. This antibody crossreacted with unmodified, denatured DNA. The recognition of cis- or trans-DDP-modified DNAs by monoclonal antibodies thus parallels the known modes of DNA binding of these compounds and may correlate with their biological activities.

  16. Rhizosecretion of a monoclonal antibody protein complex from transgenic tobacco roots.

    PubMed

    Drake, Pascal M W; Chargelegue, Daniel M; Vine, Nicholas D; van Dolleweerd, Craig J; Obregon, Patricia; Ma, Julian K C

    2003-05-01

    The secretion of a functional, full-length monoclonal antibody complex from transgenic Nicotiana tabacum roots has been demonstrated. Initially, seeds were germinated on nitrocellulose membranes and antibody secretion detected from the developing roots. Plants were then established in hydroponic culture and secretion into the growth medium measured over 25 days. Western blotting indicated that full-length antibody was present in the medium along with other fragments. Secreted antibody was shown to be functional by binding to antigen in ELISA studies. In contrast, no antibody could be detected from transgenic Nicotiana in which the same antibody was expressed as a membrane protein in the plasmalemma. These results indicate that antibody accumulation in the growth medium is genuinely caused by rhizosecretion and not cell damage. Addition of gelatin to plant growth medium markedly increased levels of antibody accumulation. The mean antibody yield per plant was calculated to be 11.7 microg per gram root dry weight per day. Rhizosecretion may be a viable alternative to agricultural production or cell culture for the generation of monoclonal antibodies in transgenic plants. It may also give rise to novel applications for antibodies expressed in plants such as removal or neutralisation of environmental pollutants and attenuation of pathogens which infect the plant via the rhizosphere. PMID:12825702

  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. Chemotherapy Combinations With Monoclonal Antibodies in Non-Hodgkin’s Lymphoma

    PubMed Central

    Kahl, Brad

    2010-01-01

    Although the use of monoclonal antibodies as single agents has had a tremendous impact on the care of patients with non-Hodgkin’s lymphoma (NHL), the greatest benefit has been generated by the addition of monoclonal antibodies to conventional cytotoxic chemotherapy. Rituximab is the monoclonal antibody responsible for all clinical improvement noted to date. The addition of rituximab to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy (R-CHOP regimen) improves the response rate, progression-free survival (PFS), and overall survival (OS) in diffuse large B-cell lymphoma (DLBCL). Adding rituximab to CHOP chemotherapy improves response rates and PFS in mantle cell lymphoma (MCL). Finally, the addition of rituximab to a variety of chemotherapy regimens improves the response rates, PFS, and OS in follicular lymphoma (FL). Several other (epratuzumab, bevacizumab, alemtuzumab) monoclonal antibody–chemotherapy combinations are currently under study in NHL. This review will summarize the data supporting the addition of rituximab to chemotherapy in NHL and discuss preliminary data regarding the use of other monoclonal antibodies in combination with chemotherapy. PMID:18381103

  19. Production and characterization of monoclonal antibody against 10S DNA polymerase alpha from calf thymus.

    PubMed Central

    Masaki, S; Shiku, H; Kaneda, T; Koiwai, O; Yoshida, S

    1982-01-01

    One hybridoma cell line that produces an antibody directed against 10S DNA polymerase alpha purified from calf thymus was obtained. The monoclonality of the antibody was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectrofocusing and antibody subclass determination. The antibody specifically recognized the 10S DNA polymerase alpha and 6.5S DNA polymerase alpha-2 from calf thymus, but not 6.5S DNA polymerase alpha-1. The antibody precipitated both polypeptides of 140-150,000 and 46-50,000 dalton of 10S DNA polymerase alpha. The antibody also recognized the DNA polymerase alpha purified from human cells, but did pig DNA polymerase alpha only partially. The antibody did not crossreact with rat DNA polymerase alpha, calf DNA polymerase beta, virus DNA polymerase and E. coli DNA polymerase I. This antibody will be a useful tool for studying the mechanism of DNA replication in eukaryotic cells. Images PMID:7133996

  20. 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.

  1. 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

  2. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

    PubMed Central

    Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

    2016-01-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

  3. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

    PubMed

    Hehle, Verena K; Paul, Matthew J; Roberts, Victoria A; van Dolleweerd, Craig J; Ma, Julian K-C

    2016-04-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformedNicotiana tabacum Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently inN. tabacumand demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

  4. 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

  5. Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins

    PubMed Central

    Gao, Jian-En; Tao, Qi-Min; Guo, Jian-Ping; Ji, He-Ping; Lang, Zheng-Wei; Ji, Ying; Feng, Bai-Fang

    1997-01-01

    AIM: To prepare hybridoma cell lines that secrete monoclonal antibodies against hepatitis C virus (HCV) recombinant proteins NS3 and NS5 and to evaluate their use in the study of HCV NS3 and NS5 antigen distribution in human liver tissue. METHODS: Hybridoma cell lines were generated using spleen cells from BALB/C mice immunized with recombinant NS3 and NS5 proteins, following conventional protocols. Antibody-secreting cells were screened by solid phase ELISA and cloned by limited dilution. The specificity of the monoclonal antibodies was determined by testing hybridoma culture supernatants by Western blots of E. coli expressing the recombinant HCV proteins and ELISA with HCV core and hepatitis B virus (HBV) antigens. The monoclonal antibodies were employed in immunohistochemistry studies to determine the distribution of HCV NS5 and NS3 antigens in 51 paraffin embedded human liver tissue samples. RESULTS: Eight hybridoma cell lines secreting monoclonal antibodies against HCV NS3 and NS5 proteins were generated and named 2B6, 2F3, 3D8, 3D9, 8B2, 6F11, 4C6 and 7D9. Only one of them, 2B6 (secreting antibodies against NS3 protein), cross-reacted with the C7 polypeptide, a different recombinant NS3 polypeptide. The rest of the cell lines showed no cross-reactivity with HCV core or HBV antigens. In addition, monoclonal antibodies against NS3 antigens did not cross-react with NS5 antigens, and vice versa. In immunohistochemistry studies, these monoclonal antibodies did not detect HCV antigens in specimens from patients infected only with HBV (n = 20). In HCV-infected specimens (n = 31), the rates of positive detection of NS3 and NS5 antigens were 51.6% (16/31) and 54.9% (17/31), respectively. Six of these 31 specimens were from patients infected only with HCV and half of them were positive for HCV NS3 and NS5 antigens. In specimens from patients co-infected with HBV and HCV (n = 25), the rates of NS3 and NS5 antigen positive detection were 52% (13/25) and 56% (14/25), respectively, which are similar to those obtained in samples from patients infected only with HCV. In specimens from chronic active cirrhosis patients, the rates of HCV NS3 and NS5 antigen detection were 70.6% (12/17) and 76.5% (13/17), respectively. CONCLUSION: We successfully prepared monoclonal antibodies that are specific against recombinant HCV NS3 and NS5 proteins and could be useful for clinical immunohistochemistry diagnosis.

  6. In-situ Detection of Squalane in Sedimentary Organic Matter Using Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Bailey, J. V.; Corsetti, F. A.; Moldowan, J. M.; Fago, F.; Caron, D.

    2008-12-01

    Sedimentary geolipids can serve as powerful tools for reconstructing ancient ecosystems, but only if investigators can demonstrate that the hydrocarbons are indigenous to their host rocks. The association of molecules with primary sedimentary fabrics could indicate a syngenetic relationship. However, traditional biomarker analyses require extraction from large quantities of powdered rock, confounding detailed spatial correlations. Biological studies commonly use antibodies as extremely sensitive molecular probes. When coupled with fluorescent labels, antibodies allow for the visual localization of molecules. Here we show that monoclonal antibodies that bind specifically to geolipid compounds can be used for in situ detection and labeling of such compounds in mineral-bound organic macerals. Monoclonal antibodies to squalene, produced for human health studies, also react with the geolipid, squalane. We show that squalene antibodies do not react with other common sedimentary hydrocarbons. We also show that squalane antibodies bind specifically to isolated organic-rich lamina in Eocene-age, squalane-containing rocks. These results suggest that squalane is confined to discrete organo-sedimentary fabrics within those rocks, providing evidence for its syngeneity. The chemical similarity of squalane to other sedimentary hydrocarbons hints at the potential for developing monoclonal antibodies to a variety of biomarkers that could then be localized in rocks, sediments, and extant cells.

  7. 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.

  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. 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.

  10. Characterization of Two Human Monoclonal Antibodies Neutralizing Influenza A H7N9 Viruses

    PubMed Central

    Wang, Jianmin; Chen, Zhe; Bao, Linlin; Zhang, Weijia; Xue, Ying; Pang, XingHuo; Zhang, Xi

    2015-01-01

    H7N9 was a cause of significant global health concern due to its severe infection and approximately 35% mortality in humans. By screening a Fab antibody phage library derived from patients who recovered from H7N9 infections, we characterized two human monoclonal antibodies (HuMAbs), HNIgGD5 and HNIgGH8. The epitope of these two antibodies was dependent on two residues in the receptor binding site at positions V186 and L226 of the hemagglutinin glycoprotein. Both antibodies possessed high neutralizing activity. PMID:26063436

  11. 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

  12. Pretargeted Radioimmunotherapy of Prostate Cancer with an Anti-TROP-2×Anti-HSG Bispecific Antibody and a 177Lu-Labeled Peptide

    PubMed Central

    Frielink, Cathelijne; Goldenberg, David M.; Sharkey, Robert M.; Lütje, 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-TROP2×anti-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

  13. 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

  14. [Recent progress in radioimmunodetection for cancer using radio-labeled monoclonal antibodies].

    PubMed

    Toshihiko, K; Nobuko, I; Shigenobu, N; Tatsuo, M; Keisuke, N

    1983-10-01

    The concept of injecting anti-tumor antibodies to localize tumors was first introduced in experimental systems by Pressman (1957). Since then, various trials for tumor detection have been performed using anti-tumor antibodies. In 1970's, the radioimmunodetection of cancer has rapidly developed by the use of radioantibodies to oncofetal proteins such as CEA, AFP and hCG. Recently, there are papers dealing with animal studies of external scanning by the monoclonal antibodies that bind selectively to tumor cells derived from murine teratocarcinoma, human malignant melanoma, human mammary carcinoma and human osteosarcoma. As clinical trials the radiolabeled monoclonal antibodies against CEA or AFP were also used for the radioimmunodetection in patients with CEA or AFP-producing tumors, and the positive rates of the scanning ranged from 40 to 94%. Various related problems are also discussed. PMID:6194758

  15. 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

  16. Measurement of free light chains with assays based on monoclonal antibodies.

    PubMed

    Te Velthuis, Henk; Drayson, Mark; Campbell, John P

    2016-06-01

    Recently, serum free light chain (FLC) assays incorporating anti-kappa (κ) and anti-lambda (λ) FLC monoclonal antibodies have become available: N Latex FLC assay (Siemens) and Seralite® (Abingdon Health). The purpose of this review is to provide an overview of these two new monoclonal antibody-based methods. In doing so, the review will outline the performance characteristics of each method, including a summary of: assay principles, antibody specificity, analytical performance and assay performance in disease. Additionally, the review will describe the potential user benefits of adopting these new generation FLC assays, which are designed to overcome the established limitations of existing polyclonal antibody based FLC assays. PMID:27010775

  17. Analysis of oligosaccharides involved in the asymmetrical glycosylation of IgG monoclonal antibodies.

    PubMed

    Morelli, L; Plotkin, L; Leoni, J; Fossati, C A; Margni, R A

    1993-05-01

    The aim of this study is to identify the oligosaccharide residues involved in the asymmetric glycosylation of immunoglobulins. We have studied two anti-DNP monoclonal antibodies of the IgG1 isotype. Results show both qualitative and quantitative differences in the carbohydrates of both monoclonal antibodies and their fragments F(ab')2, Fab' and Fd. One of the antibodies -112D5-, which appears to be homogeneous in the Scatchard plot, has oligosaccharide residues in the L chain and in the Fd of one of the Fab'. On the other hand, 112B2 mAb, which also appears to be asymmetrically glycosylated, shows the bimodal curve characteristic of antibodies with different combining site affinities. PMID:8487785

  18. Monoclonal antibody production in hollow fiber bioreactors using serum-free medium.

    PubMed

    Heifetz, A H; Braatz, J A; Wolfe, R A; Barry, R M; Miller, D A; Solomon, B A

    1989-02-01

    Murine hybridoma cells that produce monoclonal antibody directed against human fibronectin have been cultured in VITAFIBER II and VITAFIBER V hollow fiber bioreactors using defined, serum-free WRC 935 medium. During a two-week growth period, following inoculation of the bioreactors, the cells proliferated to an extent where the bioreactor was filled with cultured cells. Using a 5 sq. ft. VITAFIBER V bioreactor, over 15 grams of antibody were produced during the 40 days of the experiment. This antibody was greater than 95% IgG. During the production period, this packed mass of cells produced 579 +/- 15 mg IgG per day. Because the medium is formulated for air equilibration and high cell densities, WRC 935 medium is especially useful for production of gram quantities of monoclonal antibodies using continuous feed hollow fiber bioreactor cell culture systems. PMID:2629847

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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 visualized directly. Only binding of the secondary (fluorescent) antibodies to the constant region of the primary antibodies allows consecutively the visualization in a fluorescent microscope.Primary antibodies can be either of monoclonal (normally produced in mouse) or of polyclonal origin (normally produced in rabbit, goat, sheep, or donkey). Using (primary) monoclonal antibodies has the clear advantage that all antibodies used are identical in origin and behavior and should thus give a more clear-cut labeling result. On the other hand the demands towards labeling protocols might be concomitantly higher: Binding of primary antibodies will only occur if fixation and labeling protocols preserve the antigen sufficiently to keep its specific and unique target structure available. One could imagine that for polyclonal antibodies this demand is slightly lower as there is a pool of antibodies with varying specificities against multiple parts of their target antigens. Certain fractions of this pool might thus tolerate a larger variety of conditions, and consequently a larger variety of protocols might still result in successful labeling.Each step in a labeling protocol can be decisive for the outcome of an experiment especially if monoclonal antibodies are used. Especially critical are choice of buffer and fixation and permeabilization parameters of the protocol.In this chapter we discuss and detail proven protocols using monoclonal antibodies for two key targets within a cell, namely, (a) the cytoskeleton with emphasis on microtubules (Cramer and Mitchison, J Cell Biol 31:179-189, 1995; Traub et al., Biol Cell 90:319-337, 1998; Desai et al., Methods Cell Biol 61:385-412, 1999) and (b) the nuclear pore complex (Davis and Blobel, Cell 45:699-709, 1986; Kimura et al., Mol Cell Biol 23:1304-1315, 2003). The two protocols which differ substantially in using either a chemical fixation/permeabilization approach versus a one-step coagulation protocol are chosen to offer the reader tools to design their own procedures and adapt them accordingly to fit their individual experimental setup and the biological question asked. PMID:24515489

  4. Combination epigenetic and immunotherapy overcomes resistance to monoclonal antibodies in hematologic malignancies: A new therapeutic approach.

    PubMed

    Epner, Elliot M; Saroya, Bikramajit Singh; Hasanali, Zainul S; Loughran, Thomas P

    2016-03-01

    We recently reported that addition of epigenetic agents could overcome resistance of leukemic cells to monoclonal antibody-mediated anti-tumor effects in T-cell prolymphocytic leukemia. We also reported that epigenetic agents could induce expression of the CD30 gene, thus providing a therapeutic target for the antibody drug conjugate brentuximab vedotin. Here we discuss these findings and their generality to treatment of other hematologic and solid malignancies. PMID:26802532

  5. In vivo Therapy with Monoclonal Anti-I-A Antibody Suppresses Immune Responses to Acetylcholine Receptor

    NASA Astrophysics Data System (ADS)

    Waldor, Matthew K.; Sriram, Subramaniam; McDevitt, Hugh O.; Steinman, Lawrence

    1983-05-01

    A monoclonal antibody to I-A gene products of the immune response gene complex attenuates both humoral and cellular responses to acetylcholine receptor and appears to suppress clinical manifestations of experimental autoimmune myasthenia gravis. This demonstrates that use of antibodies against immune response gene products that are associated with susceptibility to disease may be feasible for therapy in autoimmune conditions such as myasthenia gravis.

  6. Isolation of Highly Active Monoclonal Antibodies against Multiresistant Gram-Positive Bacteria

    PubMed Central

    Rossmann, Friederike S.; Laverde, Diana; Kropec, Andrea; Romero-Saavedra, Felipe; Meyer-Buehn, Melanie; Huebner, Johannes

    2015-01-01

    Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL) of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA). At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium), a mouse peritonitis model (using S. aureus Newman and LAC) and a rat endocarditis model (using E. faecalis 12030) and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials. PMID:25706415

  7. Production and characterization of monoclonal antibodies to the macrocyclic trichothecene roridin A.

    PubMed Central

    Hack, R; Märtlbauer, E; Terplan, G

    1988-01-01

    Two murine monoclonal antibodies to the macrocyclic trichothecene roridin A are described. Screening for antibody production was performed on absorbed anti-mouse immunoglobulin serum as double-antibody solid phase, and further characterization was done on affinity-purified anti-mouse IgG serum. The antibodies, designated 5G11 and 4H10, had affinity constants for roridin A of 9.25 X 10(7) and 1.7 X 10(7) liters/mol, respectively. In monoclonal antibody-based direct enzyme immunoassays, these IgG1 antibodies had detection limits for roridin A of 0.4 ng/ml (0.02 ng per assay) and 1.8 ng/ml (0.09 ng per assay), respectively. Both antibodies were most specific for the tested macrocyclic trichothecenes. The relative cross-reactivities of antibody 5G11 with roridin A, roridin J, verrucarin A, satratoxin G, and satratoxin H were 100.0, 43.8, 16.7, 3.7, and 18.9%, respectively; for antibody 4H10 they were 100.0, 6.3, 64.0, 4.4, and 4.9%, respectively. PMID:3190229

  8. Isolation and characterization of monoclonal antibodies specific for chondroitin sulfate E.

    PubMed

    Watanabe, Ippei; Hikita, Tomoya; Mizuno, Haruka; Sekita, Risa; Minami, Akira; Ishii, Ami; Minamisawa, Yuka; Suzuki, Kiyoshi; Maeda, Hiroshi; Hidari, Kazuya I P J; Suzuki, Takashi

    2015-09-01

    Chondroitin sulfate E (CSE) is a polysaccharide containing mainly disaccharide units of D-glucuronic acid (GlcA) and 4,6-O-disulfated N-acetyl-D-galactosamine (GalNAc) residues (E-unit) in the amount of ∼ 60%. CSE is involved in many biological and pathological processes. In this study, we established new monoclonal antibodies, termed E-12C and E-18H, by using CSE that contained more than 70% of E-units as an immunogen. These antibodies recognized CSE but not other CSs isomers or dermatan sulfate (DS). We evaluated the reactivities of the antibodies to 6-O-sulfated CSA (6S-CSA) and DS (6S-DS) that possessed ∼ 60% of GalNAc (4S, 6S) moieties in their structures. Neither of the antibodies reacted with 6S-DS. The antibodies strictly distinguished the structural difference of GlcA and L-iduronic acid in the polysaccharide. Binding affinities of the antibodies were determined by a surface plasmon resonance assay using CSE and 6S-CSA. The binding affinities were strongly associated with the molecular weight of CSE and the E-unit content of 6S-CSA. Moreover, we demonstrated that the antibodies are applicable to histochemical analysis. In conclusion, the new anti-CSE monoclonal antibodies specifically recognize the E-unit of CSE. The antibodies will become useful tools for the investigation of the biological and pathological significance of CSE. PMID:26036195

  9. Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

    NASA Astrophysics Data System (ADS)

    Liu, Hongcheng; Gaza-Bulseco, Georgeen; Chumsae, Chris

    2009-12-01

    Size-exclusion chromatography (SEC) has been widely used to detect antibody aggregates, monomer, and fragments. SEC coupled to mass spectrometry has been reported to measure the molecular weights of antibody; antibody conjugates, and antibody light chain and heavy chain. In this study, separation of antibody light chain and heavy chain by SEC and direct coupling to a mass spectrometer was further studied. It was determined that employing mobile phases containing acetonitrile, trifluoroacetic acid, and formic acid allowed the separation of antibody light chain and heavy chain after reduction by SEC. In addition, this mobile phase allowed the coupling of SEC to a mass spectrometer to obtain a direct molecular weight measurement. The application of the SEC-MS method was demonstrated by the separation of the light chain and the heavy chain of multiple recombinant monoclonal antibodies. In addition, separation of a thioether linked light chain and heavy chain from the free light chain and the free heavy chain of a recombinant monoclonal antibody after reduction was also achieved. This optimized method provided a separation of antibody light chain and heavy chain based on size and allowed a direct measurement of molecular weights by mass spectrometry. In addition, this method may help to identify peaks eluting from SEC column directly.

  10. Review of Cisplatin and oxaliplatin in current immunogenic and monoclonal antibody treatments.

    PubMed

    Mehmood, Rao Khalid

    2014-09-23

    Platinum-based chemotherapy agents initially transformed cancer treatment. However their effectiveness peaked as combined regimes showed little additional benefit in trials. New research frontiers developed with the discovery that conventional chemotherapy can induce immunological cell death by recruiting high mobility group box 1 protein through T-cell immunity. Simultaneously monoclonal antibody agents (not effective as monotherapies) showed good results in combination with conventional chemotherapy. Some of these combinations are currently in use and researchers hope to develop regimes which can offer substantial benefits. Several resistance mechanisms against platinum compounds are known, but more knowledge is still needed to gain a full understanding. It seems reasonable therefore to revisit the pharmacology of these agents, which may also lead to identify rational combinations with monoclonal agents providing regimes with less toxicity and better efficacy. This article reviews the pharmacology of cisplatin and oxaliplatin and explores their possible association with monoclonal antibody treatments. PMID:25992242

  11. Review of Cisplatin and Oxaliplatin in Current Immunogenic and Monoclonal Antibody Treatments

    PubMed Central

    Mehmood, Rao Khalid

    2014-01-01

    Platinum-based chemotherapy agents initially transformed cancer treatment. However their effectiveness peaked as combined regimes showed little additional benefit in trials. New research frontiers developed with the discovery that conventional chemotherapy can induce immunological cell death by recruiting high mobility group box 1 protein through T-cell immunity. Simultaneously monoclonal antibody agents (not effective as monotherapies) showed good results in combination with conventional chemotherapy. Some of these combinations are currently in use and researchers hope to develop regimes which can offer substantial benefits. Several resistance mechanisms against platinum compounds are known, but more knowledge is still needed to gain a full understanding. It seems reasonable therefore to revisit the pharmacology of these agents, which may also lead to identify rational combinations with monoclonal agents providing regimes with less toxicity and better efficacy. This article reviews the pharmacology of cisplatin and oxaliplatin and explores their possible association with monoclonal antibody treatments. PMID:25992242

  12. Normal values for the different classes of venous blood mononuclear cells defined by monoclonal antibodies.

    PubMed Central

    Goff, L K; Habeshaw, J A; Rose, M L; Gracie, J A; Gregory, W

    1985-01-01

    We present the data obtained from routine quantitation of normal venous blood mononuclear cells using 19 monoclonal antibodies against defined mononuclear cell surface antigens. The results indicate different values for percentage and absolute numbers of T lymphocytes and of T lymphocyte subsets depending on the monoclonal antibodies used to quantify these populations. In most instances the total number of identified cells was significantly less than the total number of recovered blood mononuclear cells, which suggests the existence in blood of a null cell population. Evidence is advanced to support previous observations that at least a proportion of this population is of B cell lineage, expressing cytoplasmic immunoglobulin but lacking other class or lineage specific markers. The value of a diverse monoclonal panel in routine quantitation of peripheral blood mononuclear cells is discussed. PMID:3871442

  13. Production and Characterization of a Novel Monoclonal Antibody Against Human Sortilin.

    PubMed

    Ghaemimanesh, Fatemeh; Bayat, Ali Ahmad; Babaei, Sepideh; Ahmadian, Gholamreza; Zarnani, Amir-Hassan; Behmanesh, Mehrdad; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2015-12-01

    Sortilin, as a member of Vps10p-domain sorting receptor family, is overexpressed in a number of malignancies, including ovarian carcinoma. Antibodies against sortilin may contribute to further clarification of sortilin functional activities in signal transduction, intracellular sorting of proteins, and endocytosis. The aim of this study was to produce a monoclonal antibody against a synthetic peptide derived from extracellular N-terminal region of sortilin to be used as a tool for investigating sortilin characteristics in ovarian carcinoma. A synthetic peptide derived from the last 50 amino acids of extracellular domain of sortilin protein was selected and conjugated to keyhole limpet hemocyanin and used to immunize mice. The anti-sortilin monoclonal antibody (MAb), clone 2D8, was purified from supernatant of final hybridoma clone using peptide-affinity chromatography column. Reactivity of antibody with the immunizing peptide was assessed in ELISA. Furthermore, flow cytometry and Western blot analyses were used to investigate the reactivity of antibody with its target in a panel of ovarian carcinoma cell lines or tissues. MAb 2D8 was able to recognize the coated immunizing peptide in ELISA and detect its protein target, sortilin, in flow cytometry and Western blot analyses. The achieved data suggest that the developed monoclonal antibody may be applicable as a research tool for detection of sortilin protein in Western blot as well as flow cytometry tests. PMID:26683178

  14. Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival

    PubMed Central

    Kloepper, Jonas; Riedemann, Lars; Amoozgar, Zohreh; Seano, Giorgio; Susek, Katharina; Yu, Veronica; Dalvie, Nisha; Amelung, Robin L.; Datta, Meenal; Song, Jonathan W.; Askoxylakis, Vasileios; Taylor, Jennie W.; Lu-Emerson, Christine; Batista, Ana; Kirkpatrick, Nathaniel D.; Jung, Keehoon; Snuderl, Matija; Muzikansky, Alona; Stubenrauch, Kay G.; Krieter, Oliver; Wakimoto, Hiroaki; Xu, Lei; Munn, Lance L.; Duda, Dan G.; Fukumura, Dai; Batchelor, Tracy T.; Jain, Rakesh K.

    2016-01-01

    Inhibition of the vascular endothelial growth factor (VEGF) pathway has failed to improve overall survival of patients with glioblastoma (GBM). We previously showed that angiopoietin-2 (Ang-2) overexpression compromised the benefit from anti-VEGF therapy in a preclinical GBM model. Here we investigated whether dual Ang-2/VEGF inhibition could overcome resistance to anti-VEGF treatment. We treated mice bearing orthotopic syngeneic (Gl261) GBMs or human (MGG8) GBM xenografts with antibodies inhibiting VEGF (B20), or Ang-2/VEGF (CrossMab, A2V). We examined the effects of treatment on the tumor vasculature, immune cell populations, tumor growth, and survival in both the Gl261 and MGG8 tumor models. We found that in the Gl261 model, which displays a highly abnormal tumor vasculature, A2V decreased vessel density, delayed tumor growth, and prolonged survival compared with B20. In the MGG8 model, which displays a low degree of vessel abnormality, A2V induced no significant changes in the tumor vasculature but still prolonged survival. In both the Gl261 and MGG8 models A2V reprogrammed protumor M2 macrophages toward the antitumor M1 phenotype. Our findings indicate that A2V may prolong survival in mice with GBM by reprogramming the tumor immune microenvironment and delaying tumor growth. PMID:27044098

  15. Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival.

    PubMed

    Kloepper, Jonas; Riedemann, Lars; Amoozgar, Zohreh; Seano, Giorgio; Susek, Katharina; Yu, Veronica; Dalvie, Nisha; Amelung, Robin L; Datta, Meenal; Song, Jonathan W; Askoxylakis, Vasileios; Taylor, Jennie W; Lu-Emerson, Christine; Batista, Ana; Kirkpatr