Anti-ADAMTS13 Autoantibodies against Cryptic Epitopes in Immune-Mediated Thrombotic Thrombocytopenic Purpura

Abstract

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by severe ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13) deficiency, the presence of anti-ADAMTS13 autoantibodies and an open ADAMTS13 conformation with a cryptic epitope in the spacer domain exposed. A detailed knowledge of anti-ADAMTS13 autoantibodies will help identifying pathogenic antibodies and elucidating the cause of ADAMTS13 deficiency. We aimed at cloning anti-ADAMTS13 autoantibodies from iTTP patients to study their epitopes and inhibitory characteristics. We sorted anti-ADAMTS13 autoantibody expressing B cells from peripheral blood mononuclear cells of 13 iTTP patients to isolate anti-ADAMTS13 autoantibody sequences. Ninety-six B cell clones producing anti-ADAMTS13 autoantibodies were identified from which 30 immunoglobulin M (IgM) and 5 IgG sequences were obtained. For this study, we only cloned, expressed and purified the five IgG antibodies. In vitro characterization revealed that three of the five cloned IgG antibodies, TTP73–1, ELH2–1 and TR8C11, indeed recognize ADAMTS13. Epitope mapping showed that antibodies TTP73–1 and TR8C11 bind to the cysteine–spacer domains, while the antibody ELH2–1 recognizes the T2–T3 domains in ADAMTS13. None of the antibodies inhibited ADAMTS13 activity. Given the recent findings regarding the open ADAMTS13 conformation during acute iTTP, we studied if the cloned antibodies could recognize cryptic epitopes in ADAMTS13. Interestingly, all three antibodies recognize cryptic epitopes. In conclusion, we cloned three anti-ADAMTS13 autoantibodies from iTTP patients that recognize cryptic epitopes. Hence, these data nicely fit our recent finding that the conformation of ADAMTS13 is open during acute iTTP.

Generation of anti-idiotypic antibodies to detect anti-spacer antibody idiotopes in acute thrombotic thrombocytopenic purpura patients

“…To generate anti-idiotypic antibodies recognizing particular idiotopes in anti-spacer autoantibodies involved in ADAMTS13 binding, 3 cloned human anti-spacer autoantibodies with different epitopes and inhibitory characteristics were available: II-1, 40 TTP73 42 and I-9 41 (see Online Supplementary Methods ) and were used to immunize BALB/c mice. As the injected anti-spacer autoantibodies are full IgG antibodies in which the variable regions are grafted onto a human IgG1 constant region, 40,41 the mice developed antibodies that either recognized conserved regions (e.g.…”

Anti‐ADAMTS13 autoantibodies in immune‐mediated thrombotic thrombocytopenic purpura do not hamper ELISA‐based quantification of ADAMTS13 antigen

“…For this purpose, total IgGs from 19 acute iTTP patients (C2VN, Marseille) and 9 healthy donors (Belgian Red Cross‐Flanders, Ghent) were purified from plasma samples using Protein G Sepharose ® 4 Fast Flow column chromatography (GE Healthcare). Binding of these purified total IgGs to recombinant human (rh)ADAMTS13 was verified in our in‐house developed anti‐ADAMTS13 autoantibody ELISA . Briefly, microtiter plates were coated with rhADAMTS13 (2 µg/mL in phosphate buffered saline [PBS]) overnight and bound anti‐ADAMTS13 autoantibodies (200 µg/mL) were detected by HRP‐labeled anti‐human IgG (Fc specific) antibodies (Sigma Aldrich).…”

Current and Future Perspectives on ADAMTS13 and Thrombotic Thrombocytopenic Purpura

“…[72][73][74] Currently, also more than 90 monoclonal anti-ADAMTS13 autoantibodies isolated from iTTP patients have been cloned using phage display, Epstein-Barr virus immortalization, or B cell sorting followed by antibody cloning to further understand the immune response in iTTP patients. [76][77][78][79][80][81] Remarkably, almost all iTTP patients have VH1-69 encoded anti-ADAMTS13 autoantibodies and most of the them target the spacer domain. 57,76-80 However, the role and function of anti-ADAMTS13 autoantibodies outside the spacer domain is largely unexplored.…”

“…Recently it has been shown that iTTP patients have an open ADAMTS13 conformation and anti-ADAMTS13 autoantibodies against cryptic epitopes in ADAMTS13 have been identified. 70,81,190 It has been suggested that the development of anti-ADAMTS13 autoantibodies could be caused by exposure of cryptic epitopes due to interaction of ADAMTS13 with certain drugs, since several drugs (ticlopidine, clopidogrel) have been associated with the onset of iTTP. [46][47][48] However, also other mechanisms like posttranslational modifications (glycosylation, oxidation, citrullination…) could lead to the exposure of cryptic epitopes.…”