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Dennis R Burton - One of the best experts on this subject based on the ideXlab platform.
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anti acetylcholine receptor fab fragments isolated from thymus derived phage display libraries from myasthenia gravis patients reflect predominant specificities in serum and block the action of pathogenic serum antibodies
Immunology Letters, 1997Co-Authors: Yvo M F Graus, Marc H De Baets, Peter J C Van Breda Vriesman, Dennis R BurtonAbstract:Abstract Myasthenia gravis (MG) is a prototype antibody-mediated autoimmune disease in which antibodies against nicotinic acetylcholine receptors (AChR) induce loss of functional receptors at the neuromuscular junction. Germinal centers present in MG hyperplastic thymus contain activated B-cells spontaneously producing anti-human AChR (huAChR) Ab in vitro. In order to access the anti-huAChR repertoire phage display Fab libraries of thymic lymphocytes were constructed from two MG patients. A total of four Fabs highly specific for huAChR were isolated that bind to determinants in or near the main immunogenic region (MIR). These anti-huAChR Fabs showed evidence of significant somatic mutations supporting the notion that the anti-huAChR Ab response in MG patients is driven by antigen. A total of two Fabs were able to inhibit up to 90% of donor serum anti-huAChR antibodies. Competition with serum anti-huAChR Ab was also observed in unrelated MG patients and indicate that anti-huAChR Fabs bind to epitopes on huAChR recognized by the majority of MG patients. In vitro Antigenic Modulation studies demonstrated that anti-huAChR Fabs were able to induce AChR loss when cross-linked by an anti-Fab antibody but not as monovalent Fab. Moreover, anti-huAChR Fabs were able to protect against AChR loss by Antigenic Modulation induced by MG serum antibodies suggesting a potential therapeutic role for these recombinant Fabs in patients with a myasthenic crisis.
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human anti nicotinic acetylcholine receptor recombinant fab fragments isolated from thymus derived phage display libraries from myasthenia gravis patients reflect predominant specificities in serum and block the action of pathogenic serum antibodies
Journal of Immunology, 1997Co-Authors: Yvo Graus, Peter J C Van Breda Vriesman, M De Baets, Paul W H I Parren, Sonia Berrihaknin, John H J Wokke, Dennis R BurtonAbstract:Myasthenia gravis (MG) is a prototype Ab-mediated autoimmune disease in which Abs against nicotinic acetylcholine receptors (AChR) induce loss of functional receptors at the neuromuscular junction. Germinal centers present in MG hyperplastic thymus contain activated B cells spontaneously producing anti-human AChR (anti-huAChR) Ab in vitro. To access the anti-huAChR repertoire, phage display Fab libraries of thymic lymphocytes were constructed from two MG patients. Four Fabs highly specific for huAChR were isolated that bind to determinants in or near the main immunogenic region. These anti-huAChR Fabs showed evidence of significant somatic mutations, supporting the idea that the anti-huAChR Ab response in MG patients is driven by Ag. Two Fabs were able to inhibit up to 90% of donor serum anti-huAChR Abs. Competition with serum anti-huAChR Ab was also observed in unrelated MG patients and indicate that anti-huAChR Fabs bind to epitopes on huAChR recognized by the majority of MG patients. In vitro Antigenic Modulation studies demonstrated that anti-huAChR Fabs were able to induce AChR loss when cross-linked by an anti-Fab Ab but not as monovalent Fab. Moreover, anti-huAChR Fabs were able to protect against AChR loss by Antigenic Modulation induced by MG serum Abs, suggesting a potential therapeutic role for these recombinant Fabs in patients with a myasthenic crisis.
Yvo M F Graus - One of the best experts on this subject based on the ideXlab platform.
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anti acetylcholine receptor fab fragments isolated from thymus derived phage display libraries from myasthenia gravis patients reflect predominant specificities in serum and block the action of pathogenic serum antibodies
Immunology Letters, 1997Co-Authors: Yvo M F Graus, Marc H De Baets, Peter J C Van Breda Vriesman, Dennis R BurtonAbstract:Abstract Myasthenia gravis (MG) is a prototype antibody-mediated autoimmune disease in which antibodies against nicotinic acetylcholine receptors (AChR) induce loss of functional receptors at the neuromuscular junction. Germinal centers present in MG hyperplastic thymus contain activated B-cells spontaneously producing anti-human AChR (huAChR) Ab in vitro. In order to access the anti-huAChR repertoire phage display Fab libraries of thymic lymphocytes were constructed from two MG patients. A total of four Fabs highly specific for huAChR were isolated that bind to determinants in or near the main immunogenic region (MIR). These anti-huAChR Fabs showed evidence of significant somatic mutations supporting the notion that the anti-huAChR Ab response in MG patients is driven by antigen. A total of two Fabs were able to inhibit up to 90% of donor serum anti-huAChR antibodies. Competition with serum anti-huAChR Ab was also observed in unrelated MG patients and indicate that anti-huAChR Fabs bind to epitopes on huAChR recognized by the majority of MG patients. In vitro Antigenic Modulation studies demonstrated that anti-huAChR Fabs were able to induce AChR loss when cross-linked by an anti-Fab antibody but not as monovalent Fab. Moreover, anti-huAChR Fabs were able to protect against AChR loss by Antigenic Modulation induced by MG serum antibodies suggesting a potential therapeutic role for these recombinant Fabs in patients with a myasthenic crisis.
Socrates J Tzartos - One of the best experts on this subject based on the ideXlab platform.
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a rapid fluorescence based assay for detecting Antigenic Modulation of the acetylcholine receptor on human cell lines
Cytometry Part B-clinical Cytometry, 2009Co-Authors: Dennis Keefe, Socrates J Tzartos, Donna Hess, Jennifer Bosco, Jan Powell, Justin Lamsa, Serene JosiahAbstract:Background: Myasthenia gravis (MG) is an autoimmune disease affecting 40,000 patients in the United States. One of the major mechanisms of disease pathology in MG is the binding, internalization, and eventual destruction of acetylcholine receptors (AChR) at the neuromuscular junction by cross-linking AChR-specific autoantibodies. This process, known as Antigenic Modulation, ultimately attenuates the ability of muscle cells to contract in response to signals from neurons, leading to muscle weakness and fatigue. For this reason, Antigenic Modulation of the AChR on cultured cells has become an important diagnostic tool for assessing the pathogenicity of AChR-specific autoantibodies. Traditionally, these assays have been done using radiolabeled AChR ligands such as 125 I alpha-bungarotoxin to determine relative AChR number. Here, we present a high-throughput immunofluorescent flow cytometry-based assay that can be used to quantify AChR levels on the cell surface and assess the efficacy of molecules designed to rescue Antigenic Modulation. Methods: AChR levels were quantified on human muscle cells before and after treatment with AChR antibodies via immunofluorescent labeling with the AChR monoclonal antibodies, mAb210 and mAb B3, followed by flow cytometry of EDTA-treated cells. Results: Using a novel, flow cytometry-based assay, Antigenic Modulation of the AChR was demonstrated on human cells using both AChR-specific monoclonal antibody and MG patient serum. The degree of Antigenic Modulation was dose responsive to antibody levels and could be reversed by preincubating antibodies with soluble AChR alpha subunit extracellular domain. Summary: A rapid, nonradioactive assay was developed to determine the potential of AChR-specific antibodies in the serum of MG patients to bind and down-regulate the AChR. This assay can be used to assess the ability of putative therapeutics that rescue Antigenic Modulation and could be developed for the treatment of MG. q 2008 Clinical Cytometry Society Key terms: myasthenia gravis; Antigenic Modulation; flow cytometry; acetylcholine receptor
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bacterial expression of a single chain fv fragment which efficiently protects the acetylcholine receptor against Antigenic Modulation caused by myasthenic antibodies
European Journal of Immunology, 1993Co-Authors: Avgi Mamalaki, Nikos Trakas And And, Socrates J TzartosAbstract:Monoclonal antibodies (mAb) against the main immunogenic region (MIR) of the acetylcholine receptor (AChR) are very potent in inducing Antigenic Modulation of the AChR in animals and in muscle cell cultures. A recombinant antibody fragment of the rat anti-MIR mAb198 was cloned by polymerase chain reaction and expressed as soluble single-chain Fv fragment (scFv198) in E. coli and affinity purified. DNA sequencing was used to define the VH (IB) and VL (K2) chain gene usage. scFv198 was found immunologically and biologically active. Its binding affinity for the Torpedo AChR (KD = 2 +/- 0.6 nM) was very similar with that of the intact mAb198 (KD = 1.8 +/- 0.6 nM) while for the human AChR (KD = 80.7 +/- 16.6 nM) it was about four times lower than that of the intact mAb198 (KD = 21.6 +/- 6.6 nM). This fragment was capable of efficiently protecting the AChR in human cell cultures, against Antigenic Modulation caused by the intact mAb198 or by the antibodies from a myasthenic patient. The produced scFv198 fragment is, therefore, potentially useful in therapeutic applications for myasthenia gravis after appropriate genetic manipulations.
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passive transfer of experimental myasthenia gravis via Antigenic Modulation of acetylcholine receptor
European Journal of Immunology, 1992Co-Authors: Heleni Loutrari, Anna Kokla, Socrates J TzartosAbstract:Antigenic Modulation of acetylcholine receptor (AChR) is considered to contribute to the reduction of endplate AChR in myasthenia gravis (MG). Yet, the pathogenic significance of this mechanism is unclear. To investigate the in vivo role of AChR Antigenic Modulation we examined the ability of bivalent F(ab')2 and monovalent Fab fragments of monoclonal antibody (mAb) 35 to passively transfer experimental autoimmune MG (EAMG) in rats. mAb 35 which binds at the main immunogenic region (MIR) of the AChR causes severe EAMG without being involved in channel function. Compared to the intact mAb, F(ab')2 35 proved to be less potent but still capable of inducing moderate EAMG, whereas Fab 35 were totally ineffective. Furthermore, both intact and F(ab')2 35 induced mild EAMG in complement-depleted rats. These results (a) provide evidence that Antigenic Modulation of endplate AChR is sufficient to generate passive transfer of EAMG and (b) further support the pathogenic potential of the anti-MIR antibodies in MG.
Peter J C Van Breda Vriesman - One of the best experts on this subject based on the ideXlab platform.
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anti acetylcholine receptor fab fragments isolated from thymus derived phage display libraries from myasthenia gravis patients reflect predominant specificities in serum and block the action of pathogenic serum antibodies
Immunology Letters, 1997Co-Authors: Yvo M F Graus, Marc H De Baets, Peter J C Van Breda Vriesman, Dennis R BurtonAbstract:Abstract Myasthenia gravis (MG) is a prototype antibody-mediated autoimmune disease in which antibodies against nicotinic acetylcholine receptors (AChR) induce loss of functional receptors at the neuromuscular junction. Germinal centers present in MG hyperplastic thymus contain activated B-cells spontaneously producing anti-human AChR (huAChR) Ab in vitro. In order to access the anti-huAChR repertoire phage display Fab libraries of thymic lymphocytes were constructed from two MG patients. A total of four Fabs highly specific for huAChR were isolated that bind to determinants in or near the main immunogenic region (MIR). These anti-huAChR Fabs showed evidence of significant somatic mutations supporting the notion that the anti-huAChR Ab response in MG patients is driven by antigen. A total of two Fabs were able to inhibit up to 90% of donor serum anti-huAChR antibodies. Competition with serum anti-huAChR Ab was also observed in unrelated MG patients and indicate that anti-huAChR Fabs bind to epitopes on huAChR recognized by the majority of MG patients. In vitro Antigenic Modulation studies demonstrated that anti-huAChR Fabs were able to induce AChR loss when cross-linked by an anti-Fab antibody but not as monovalent Fab. Moreover, anti-huAChR Fabs were able to protect against AChR loss by Antigenic Modulation induced by MG serum antibodies suggesting a potential therapeutic role for these recombinant Fabs in patients with a myasthenic crisis.
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human anti nicotinic acetylcholine receptor recombinant fab fragments isolated from thymus derived phage display libraries from myasthenia gravis patients reflect predominant specificities in serum and block the action of pathogenic serum antibodies
Journal of Immunology, 1997Co-Authors: Yvo Graus, Peter J C Van Breda Vriesman, M De Baets, Paul W H I Parren, Sonia Berrihaknin, John H J Wokke, Dennis R BurtonAbstract:Myasthenia gravis (MG) is a prototype Ab-mediated autoimmune disease in which Abs against nicotinic acetylcholine receptors (AChR) induce loss of functional receptors at the neuromuscular junction. Germinal centers present in MG hyperplastic thymus contain activated B cells spontaneously producing anti-human AChR (anti-huAChR) Ab in vitro. To access the anti-huAChR repertoire, phage display Fab libraries of thymic lymphocytes were constructed from two MG patients. Four Fabs highly specific for huAChR were isolated that bind to determinants in or near the main immunogenic region. These anti-huAChR Fabs showed evidence of significant somatic mutations, supporting the idea that the anti-huAChR Ab response in MG patients is driven by Ag. Two Fabs were able to inhibit up to 90% of donor serum anti-huAChR Abs. Competition with serum anti-huAChR Ab was also observed in unrelated MG patients and indicate that anti-huAChR Fabs bind to epitopes on huAChR recognized by the majority of MG patients. In vitro Antigenic Modulation studies demonstrated that anti-huAChR Fabs were able to induce AChR loss when cross-linked by an anti-Fab Ab but not as monovalent Fab. Moreover, anti-huAChR Fabs were able to protect against AChR loss by Antigenic Modulation induced by MG serum Abs, suggesting a potential therapeutic role for these recombinant Fabs in patients with a myasthenic crisis.
Paul W H I Parren - One of the best experts on this subject based on the ideXlab platform.
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loss of cd20 and bound cd20 antibody from opsonized b cells occurs more rapidly because of trogocytosis mediated by fc receptor expressing effector cells than direct internalization by the b cells
Journal of Immunology, 2011Co-Authors: Paul V Beum, Margaret A Lindorfer, Paul W H I Parren, Elizabeth M Peek, Frank J Beurskens, Patrick J Engelberts, Jan G J Van De Winkel, Ronald P TaylorAbstract:We previously reported that 1 h after infusion of CD20 mAb rituximab in patients with chronic lymphocytic leukemia (CLL), >80% of CD20 was removed from circulating B cells, and we replicated this finding, based on in vitro models. This reaction occurs via an endocytic process called shaving/trogocytosis, mediated by FcγR on acceptor cells including monocytes/macrophages, which remove and internalize rituximab–CD20 immune complexes from B cells. Beers et al. reported that CD20 mAb-induced Antigenic Modulation occurs as a result of internalization of B cell-bound mAb–CD20 complexes by the B cells themselves, with internalization of ∼40% observed after 2 h at 37°C. These findings raise fundamental questions regarding the relative importance of shaving versus internalization in promoting CD20 loss and have substantial implications for the design of mAb-based cancer therapies. Therefore, we performed direct comparisons, based on flow cytometry, to determine the relative rates and extent of shaving versus internalization. B cells, from cell lines, from patients with CLL, and from normal donors, were opsonized with CD20 mAbs rituximab or ofatumumab and incubated for varying times and then reacted with acceptor THP-1 monocytes to promote shaving. We find that shaving induces considerably greater loss of CD20 and bound mAb from opsonized B cells in much shorter time periods (75–90% in <45 min) than is observed for internalization. Both shaving/trogocytosis and internalization could contribute to CD20 loss when CLL patients receive rituximab therapy, but shaving should occur more rapidly and is most likely to be the key mechanism of CD20 loss.
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human anti nicotinic acetylcholine receptor recombinant fab fragments isolated from thymus derived phage display libraries from myasthenia gravis patients reflect predominant specificities in serum and block the action of pathogenic serum antibodies
Journal of Immunology, 1997Co-Authors: Yvo Graus, Peter J C Van Breda Vriesman, M De Baets, Paul W H I Parren, Sonia Berrihaknin, John H J Wokke, Dennis R BurtonAbstract:Myasthenia gravis (MG) is a prototype Ab-mediated autoimmune disease in which Abs against nicotinic acetylcholine receptors (AChR) induce loss of functional receptors at the neuromuscular junction. Germinal centers present in MG hyperplastic thymus contain activated B cells spontaneously producing anti-human AChR (anti-huAChR) Ab in vitro. To access the anti-huAChR repertoire, phage display Fab libraries of thymic lymphocytes were constructed from two MG patients. Four Fabs highly specific for huAChR were isolated that bind to determinants in or near the main immunogenic region. These anti-huAChR Fabs showed evidence of significant somatic mutations, supporting the idea that the anti-huAChR Ab response in MG patients is driven by Ag. Two Fabs were able to inhibit up to 90% of donor serum anti-huAChR Abs. Competition with serum anti-huAChR Ab was also observed in unrelated MG patients and indicate that anti-huAChR Fabs bind to epitopes on huAChR recognized by the majority of MG patients. In vitro Antigenic Modulation studies demonstrated that anti-huAChR Fabs were able to induce AChR loss when cross-linked by an anti-Fab Ab but not as monovalent Fab. Moreover, anti-huAChR Fabs were able to protect against AChR loss by Antigenic Modulation induced by MG serum Abs, suggesting a potential therapeutic role for these recombinant Fabs in patients with a myasthenic crisis.