scholarly journals MuSK myasthenia gravis monoclonal antibodies

2019 ◽  
Vol 6 (3) ◽  
pp. e547 ◽  
Author(s):  
Maartje G. Huijbers ◽  
Dana L. Vergoossen ◽  
Yvonne E. Fillié-Grijpma ◽  
Inge E. van Es ◽  
Marvyn T. Koning ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Myasthenia Gravis ◽  
Binding Sites ◽  
Neuromuscular Junctions ◽  
Affinity Maturation ◽  
Fab Fragments ◽  
Serum Igg4 ◽  
Muscle Specific Kinase ◽  
Achr Clustering

ObjectiveTo isolate and characterize muscle-specific kinase (MuSK) monoclonal antibodies from patients with MuSK myasthenia gravis (MG) on a genetic and functional level.MethodsWe generated recombinant MuSK antibodies from patient-derived clonal MuSK-specific B cells and produced monovalent Fab fragments from them. Both the antibodies and Fab fragments were tested for their effects on neural agrin-induced MuSK phosphorylation and acetylcholine receptor (AChR) clustering in myotube cultures.ResultsThe isolated MuSK monoclonal antibody sequences included IgG1, IgG3, and IgG4 that had undergone high levels of affinity maturation, consistent with antigenic selection. We confirmed their specificity for the MuSK Ig-like 1 domain and binding to neuromuscular junctions. Monovalent MuSK Fab, mimicking functionally monovalent MuSK MG patient Fab-arm exchanged serum IgG4, abolished agrin-induced MuSK phosphorylation and AChR clustering. Surprisingly, bivalent monospecific MuSK antibodies instead activated MuSK phosphorylation and partially induced AChR clustering, independent of agrin.ConclusionsPatient-derived MuSK antibodies can act either as MuSK agonist or MuSK antagonist, depending on the number of MuSK binding sites. Functional monovalency, induced by Fab-arm exchange in patient serum, makes MuSK IgG4 antibodies pathogenic.

scholarly journals Evaluation of E-rosetting human lymphocytes with OKT11 and other monoclonal antibodies

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 795-799 ◽  
Author(s):  
SH Ip ◽  
CW Rittershaus ◽  
CC Struzziero ◽  
JA Hoxie ◽  
RA Hoffman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Fluorescence Intensity ◽  
Binding Sites ◽  
Blood Cells ◽  
Human Lymphocytes ◽  
Immunofluorescent Staining ◽  
Intensity Data ◽  
Blood Samples ◽  
Staining Methods

Abstract Monoclonal antibody OKT11 was found to compete with sheep red blood cells for binding sites on human lymphocytes. Preincubation of lymphocytes with OKT11 eliminated E-rosette formation. In a study of 142 peripheral blood samples ranging from 1% to over 90% E-rosette- positive cells, comparison to the percent OKT11-positive cells yielded a correlation coefficient of 0.93. In normal donors, subsets of OKT11+ cells were identified using two-color immunofluorescent staining methods with OKT3, OKT4, and OKT8. On the average, approximately 13% of OKT11+ lymphocytes were OKT3- and 13% of OKT11+ lymphocytes were OKT4- and OKT8-. Based on our double antibody fluorescence intensity data, low antigen density OKT11+ lymphocytes were OKT3-. OKT4+ and OKT8+ lymphocytes in normal peripheral lymphocytes have similar OKT11 antigen density.

scholarly journals Functional monovalency amplifies the pathogenicity of anti-MuSK IgG4 in myasthenia gravis

2021 ◽  
Vol 118 (13) ◽  
pp. e2020635118
Author(s):  
Dana L. E. Vergoossen ◽  
Jaap J. Plomp ◽  
Christoph Gstöttner ◽  
Yvonne E. Fillié-Grijpma ◽  
Roy Augustinus ◽  
...  
Keyword(s):  
Stochastic Process ◽  
Autoimmune Disease ◽  
Myasthenia Gravis ◽  
Muscle Weakness ◽  
Binding Sites ◽  
Antiinflammatory Activity ◽  
Autoimmune Response ◽  
Cross Link ◽  
Muscle Specific Kinase ◽  
Opposing Effects

Human immunoglobulin (Ig) G4 usually displays antiinflammatory activity, and observations of IgG4 autoantibodies causing severe autoimmune disorders are therefore poorly understood. In blood, IgG4 naturally engages in a stochastic process termed “Fab-arm exchange” in which unrelated IgG4s exchange half-molecules continuously. The resulting IgG4 antibodies are composed of two different binding sites, thereby acquiring monovalent binding and inability to cross-link for each antigen recognized. Here, we demonstrate that this process amplifies autoantibody pathogenicity in a classic IgG4-mediated autoimmune disease: muscle-specific kinase (MuSK) myasthenia gravis. In mice, monovalent anti-MuSK IgG4s caused rapid and severe myasthenic muscle weakness, whereas the same antibodies in their parental bivalent form were less potent or did not induce a phenotype. Mechanistically this could be explained by opposing effects on MuSK signaling. Isotype switching to IgG4 in an autoimmune response thereby may be a critical step in the development of disease. Our study establishes functional monovalency as a pathogenic mechanism in IgG4-mediated autoimmune disease and potentially other disorders.

scholarly journals Binding characteristics of antibodies to the TSH receptor

1998 ◽  
Vol 20 (2) ◽  
pp. 233-244 ◽  
Author(s):  
Y Oda ◽  
J Sanders ◽  
S Roberts ◽  
M Maruyama ◽  
R Kato ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Western Blotting ◽  
Tsh Receptor ◽  
Affinity Constant ◽  
Translation System ◽  
Scatchard Analysis ◽  
Facs Analysis ◽  
Binding Characteristics ◽  
Fab Fragments

We have used fragments of the TSH receptor (TSHR) expressed in E. coli as glutathione S-transferase fusion proteins to produce rabbit polyclonal antibodies and a panel (n=5) of monoclonal antibodies to the extracellular fragment of the TSHR. The binding characteristics of the antibodies to linear, conformational, glycosylated and unglycosylated forms of the receptor in different assay systems have been investigated. The reactivity of these antibodies with the TSHR was assessed by Western blotting with both native and recombinant human TSHR expressed in CHO cells, immunoprecipitation of 35S-labelled full-length TSHR produced in an in vitro transcription/ translation system, immunoprecipitation of 125I-TSH/TSHR complexes, inhibition of 125I-TSH binding to the TSHR and fluorescence activated cell sorter (FACS) analysis of binding to CHO-K1 cells expressing the TSHR on their cell surface. Fab fragments of monoclonal antibodies were isolated, labelled with 125I and used to determine the affinity constants of these antibodies with receptor, bound and free Fab being separated by polyethylene glycol (PEG) precipitation. Rabbit polyclonal and mouse monoclonal antibodies reacted with the TSHR in Western blotting and one monoclonal antibody (3C7) was able to inhibit 125I-TSH binding to native human TSHR (74% inhibition), recombinant human TSHR (84% inhibition) and porcine TSHR (65% inhibition). Affinity constant values for TSHR monoclonal antibody Fab fragments calculated using Scatchard analysis were about 10(7) M(-1). Four out of five monoclonal antibodies reacted in FACS analysis with TSHR expressed on the surface of CHO-K1 cells. The FACS unreactive monoclonal (3C7) bound well to detergent solubilised TSH receptors and this emphasised the importance of using a combination of FACS analysis and radioactively-labelled probes in analysis of the TSH receptor. The monoclonal antibodies produced in this study were found to be of relatively low affinity but proved useful for detection of the receptor by Western blotting and by FACS analysis.

scholarly journals Identification of Residues within Human Glycoprotein VI Involved in the Binding to Collagen

2004 ◽  
Vol 279 (50) ◽  
pp. 52293-52299 ◽  
Author(s):  
Christelle Lecut ◽  
Véronique Arocas ◽  
Hans Ulrichts ◽  
Anthony Elbaz ◽  
Jean-Luc Villeval ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Molecular Modeling ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binding Assays ◽  
Glycoprotein Vi ◽  
Ligand Binding Sites ◽  
Human Receptor

Glycoprotein VI (GPVI) has a crucial role in platelet responses to collagen. Still, little is known about its interaction with its ligands. In binding assays using soluble or cell-expressed human GPVI, we observed that (i) collagen, and the GPVI-specific ligands collagen-related peptides (CRP) and convulxin, competed with one another for the binding to GPVI and (ii) monoclonal antibodies directed against the extracellular part of the human receptor displayed selective inhibitory properties on GPVI interaction with its ligands. Monoclonal antibody 9E18 strongly reduced the binding of GPVI to collagen/CRP, 3F8 inhibited its interaction with convulxin, whereas 9O12 prevented all three interactions. These observations suggest that ligand-binding sites are distinct, exhibiting specific features but at the same time also sharing some common residues participating in the recognition of these ligands. The epitope of 9O12 was mapped by phage display, along with molecular modeling of human GPVI, which allowed the identification of residues within GPVI potentially involved in ligand recognition. Site-directed mutagenesis revealed that valine 34 and leucine 36 are critical for GPVI interaction with collagen and CRP. The loop might thus be part of a collagen/CRP-binding site.

scholarly journals Preferential Binding of Anti-Neutrophil Cytoplasmic Antibodies to an Unexpected Epitope of a Chimeric Proteinase 3 Mutant

2019 ◽  
Author(s):  
Marta Casal Moura ◽  
Gwen E. Thompson ◽  
Darlene A. Nelson ◽  
Lynn A. Fussner ◽  
Amber M. Hummel ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Binding Sites ◽  
Granulomatosis With Polyangiitis ◽  
Antigen Binding ◽  
Proteinase 3 ◽  
Necrotizing Vasculitis ◽  
Novel Treatment ◽  
Preferential Binding ◽  
Major Antigen

AbstractProteinase 3 (PR3) is the major antigen for anti-neutrophil cytoplasmic antibodies (ANCAs) in the systemic autoimmune vasculitis, granulomatosis with polyangiitis (GPA). PR3-targeting ANCAs (PR3-ANCAs) recognize different epitopes on PR3 and are thought to be pathogenic for the development of the necrotizing vasculitis. To identify epitopes recognized by PR3-ANCAs, we pursued a strategy based on human-murine chimeric PR3 mutants. Interestingly, rather than observing reduced binding of PR3-ANCAs to Epitope 5 on a PR3 mutant (iHm5-Val103) with chimeric mutations in Epitope 5, we found substantially increased binding of the majority of PR3-ANCAs to iHm5-Val103 compared with the PR3 mutant (iPR3-Val103) clinically used to detect PR3-ANCAs. More interestingly, using iHm5-Val103 we identified a monoclonal antibody (moANCA518) from a patient with GPA that bound selectively to iHm5-Val103. Inhibition experiments using epitope-specific monoclonal antibodies and their antigen-binding fragments mapped the binding sites of moANCA518 and PR3-ANCAs (from patients displaying preferential binding to iHm5-Val103 over iPR3-Val103) to Epitope 3 on iHm5-Val103, a mutation-free epitope located far from the mutation sites in Epitope 5. These results demonstrate that the selective binding of moANCA518 (and likely the preferential binding of PR3-ANCAs from patients) to iHm5-Val103 is conferred by increased antigenicity of Epitope 3 on iHm5-Val103 caused by distal mutations, indicating that PR3-ANCAs bind to epitopes of a folded antigen conducive to allosteric effects of mutations—a previously unrecognized characteristic with implications for studying antibody-mediated autoimmune diseases and novel treatment approaches.

scholarly journals Monoclonal antibodies against porcine platelet membrane glycoproteins Ib and IIb/IIIa

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1740-1747 ◽  
Author(s):  
H Takami ◽  
WL Nichols ◽  
SE Kaese ◽  
RS Miller ◽  
JA Katzmann ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Platelet Membrane ◽  
In Vivo Studies ◽  
Membrane Glycoproteins ◽  
Fab Fragments

Abstract We prepared murine monoclonal antibodies to porcine platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa for further study of the porcine hemostatic mechanism. One monoclonal antibody, designated PP3–4C, blocked Ristocetin-induced platelet agglutination and caused 80% inhibition of Ristocetin-induced 125I-von Willebrand factor (vWF) binding to porcine platelets at a concentration of greater than or equal to 12 micrograms IgG/mL. PP3–4C did not affect adenosine diphosphate (ADP)- or collagen-induced platelet aggregation. Binding of 125I-Fab fragments of PP3–4C to platelets was saturable at 3.7 x 10(4) +/- 0.8 x 10(4) molecules per platelet. Another monoclonal antibody, designated PP3–3A, blocked ADP- or collagen-induced platelet aggregation at 6 micrograms IgG/mL. At a concentration of 10 micrograms IgG/mL, PP3–3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated platelets. PP3–3A did not affect Ristocetin-induced platelet agglutination nor 125I-vWF binding to platelets in the presence of Ristocetin. Binding of 125I-Fab' fragments of PP3–3A to platelets was saturable at 9.8 x 10(4) +/- 1.2 x 10(4) molecules per platelet. PP3–4C antibody (anti-GP Ib) did not bind to human platelets; however, PP3–3A antibody (anti-GP IIb-IIIa) had partial cross-reactivity with human platelets. Immunoaffinity chromatography of solubilized surface-radiolabeled porcine platelets and subsequent sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis demonstrated that PP3–4C recognized a GP with an apparent molecular weight of 160,000 (nonreduced), and 140,000 (reduced). PP3–3A recognized GPs with apparent molecular weights of 130,000 and 80,000 (nonreduced), and 115,000 and 95,000 (reduced). These monoclonal antibodies to porcine platelet membrane GPs, which are structural and functional analogues of human GP Ib and GP IIb/IIIa, will be useful for in vitro and in vivo studies of the mammalian hemostatic mechanism.

scholarly journals Evaluation of E-rosetting human lymphocytes with OKT11 and other monoclonal antibodies

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 795-799
Author(s):  
SH Ip ◽  
CW Rittershaus ◽  
CC Struzziero ◽  
JA Hoxie ◽  
RA Hoffman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Fluorescence Intensity ◽  
Binding Sites ◽  
Blood Cells ◽  
Human Lymphocytes ◽  
Immunofluorescent Staining ◽  
Intensity Data ◽  
Blood Samples ◽  
Staining Methods

Monoclonal antibody OKT11 was found to compete with sheep red blood cells for binding sites on human lymphocytes. Preincubation of lymphocytes with OKT11 eliminated E-rosette formation. In a study of 142 peripheral blood samples ranging from 1% to over 90% E-rosette- positive cells, comparison to the percent OKT11-positive cells yielded a correlation coefficient of 0.93. In normal donors, subsets of OKT11+ cells were identified using two-color immunofluorescent staining methods with OKT3, OKT4, and OKT8. On the average, approximately 13% of OKT11+ lymphocytes were OKT3- and 13% of OKT11+ lymphocytes were OKT4- and OKT8-. Based on our double antibody fluorescence intensity data, low antigen density OKT11+ lymphocytes were OKT3-. OKT4+ and OKT8+ lymphocytes in normal peripheral lymphocytes have similar OKT11 antigen density.

scholarly journals Isolation and characterization of rare circulating autoantibody-producing cells from patients with muscle-specific kinase myasthenia gravis

2018 ◽  
Author(s):  
Kazushiro Takata ◽  
Panos Stathopoulos ◽  
Michelangelo Cao ◽  
Marina Mane-Damas ◽  
Miriam Fichtner ◽  
...  
Keyword(s):  
B Cells ◽  
Neuromuscular Junction ◽  
Tyrosine Kinase ◽  
Myasthenia Gravis ◽  
Neuromuscular Junctions ◽  
Ex Vivo ◽  
Autoimmune Disorder ◽  
C2c12 Myotubes ◽  
Isolation And Characterization ◽  
Achr Clustering

Myasthenia gravis (MG) is a chronic autoimmune disorder characterized by muscle weakness and caused by autoantibodies that bind to functional membrane proteins at the neuromuscular junction. Most patients have autoantibodies to the acetylcholine receptor (AChR), but a subset of patients instead have autoantibodies to muscle specific tyrosine kinase (MuSK). MuSK is an essential component of the Agrin/Lipoprotein receptor-related protein 4(LRP4)/MuSK/downstream of tyrosine kinase 7 (DOK7) pathway that is responsible for synaptic differentiation, including clustering of AChRs at the neuromuscular junction, both during development and in adult muscle. Nerve-released Agrin binds to LRP4 which then binds to MuSK, stimulating autophosphorylation and recruitment of DOK7 to complete the membrane component of the pathway. Serum-derived IgG4 subclass MuSK autoantibodies prevent the binding of LRP4 to MuSK, subsequently impairing autophosphorylation, resulting in the loss of Agrin-induced AChR clustering in the mouse myotube-forming C2C12 line. Although this autoimmune mechanism appears well understood, MuSK autoantibodies from patients are polyclonal. Most are IgG4 but IgG1, 2 and 3 are also present and can achieve similar results on AChR clustering. In addition, most bind the first Ig-like domain in MuSK, however some patients harbor serum autoantibodies that recognize other domains in MuSK. We sought to establish individual MuSK IgG clones so that the disease mechanisms could be better understood. We isolated MuSK autoantibody-expressing B cells from MuSK MG patients with a fluorescent-tagged MuSK antigen multimer and generated a panel of human monoclonal autoantibodies (mAbs) from these cells. We produced 77 mAbs from single B cells collected from six MuSK MG patients. Here we focused on three highly specific mAbs that bound quantitatively to MuSK in solution, to MuSK-expressing HEK cells and at mouse neuromuscular junctions where they co-localized with AChRs. These three IgG isotype mAbs (two IgG4 and one IgG3 subclass) recognized the Ig-like domain 2 of MuSK. The three MuSK mAbs inhibited Agrin induced-AChR clustering in C2C12 myotubes, but intriguingly, they enhanced rather than inhibited MuSK phosphorylation. This approach for ex vivo isolation of MuSK MG autoantibody-producing cells and production of human recombinant mAbs has identified distinct autoantibody specificities and likely divergent effector mechanisms. Collectively, these findings will enable a better understanding of MuSK autoantibody-mediated pathology.

FAB FRAGMENTS OF MONOCLONAL ANTIBODIES SPECIFIC FOR PORCINE PLATELET MEMBRANE GLYCOPROTEINS GP IB ANDGP IIB/IIIA

1987 ◽  
Author(s):  
H Takami ◽  
W L Nichols ◽  
S E Kaese ◽  
R S Miller ◽  
J A Katzmann ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Platelet Membrane ◽  
In Vivo Studies ◽  
Membrane Glycoproteins ◽  
Fab Fragments ◽  
Igg1 Subclass ◽  
Von Willebrand

For further study of the porcine hemostatic mechanism, we have prepared murine monoclonal antibodies, and F(ab')2 and Fab fragments, specific for porcine platelet membrane glycoproteins GP lb and GP Ilb/IIIa. To avoid production of antibodies to von Willebrand factor (vWF), mice were immunized with platelets obtained from pigs with severe von Willebrand,s disease. One monoclonal antibody (PP3-4C), of IgG1 subclass, caused 85% inhibition of Ristocetin-induced platelet binding of 125I-vWF (porcine) at ≥12 µg IgG/ml. PP3-4C did not affect ADP or collagen-induced platelet aggregation nor inhibit 125I-fibrinogen (porcine) binding. Pepsin and papain digestion, respectively, were used to prepare PP3-4C F(ab')2 and Fab fragments. PP3-4C F(ab')2 at concentrations ≥12 µg/ml caused 80% inhibition of washed platelet agglutination in the presence of vWF and Ristocetin, whereas Fab fragments at concentrations ≥10 µg/ml caused 60% inhibition. Another monoclonal antibody (PP3-3A), of IgG1 subclass, completely inhibited ADP or collagen-induced platelet aggregation at an IgG concentration of 6 µg/ml. At 10 µg IgG/ml PP3-3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated porcine platelets. PP3-3A did not affect vWF-dependent Ristocetin-induced platelet agglutination, nor 125I-vWF binding to platelets in the presence of Ristocetin. PP3-3A did not bind to platelets which were treated with 10 mM EDTA at 37°C for 60 min. F(ab')2 and Fab fragments were isolated from PP3-3A pepsin or papain digests. Both types of PP3-3A fragments caused 100% inhibition of ADP-induced platelet aggregation, at concentrations ≥6 yg/ml. Immunoprecipitation of surface-radiolabeled porcine platelets and subsequent SDS-PAGE demonstrated that PP3-4C recognized a glycoprotein with molecular weight of 140,000 (under reducing conditions), and 165,000 (non-reduced). PP3-3A recognized glycoproteins with molecular weights of 115,000 and 100,000 (reduced), and 130,000 and 80,000 (non-reduced). Neither monoclonal antibody bound to human platelets. These monoclonal antibodies to porcine platelet membrane glycoproteins which are analogues of human GP lb and GP Ilb/IIIa will be useful for in vitro and in vivo studies to further understanding of mammalian hemostatic mechanisms.

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