Inhibitory ADAMTS13 Monoclonal Autoantibodies Cloned from TTP Patients Show Restricted Gene Usage, Inhibit Murine ADAMTS13, and Are Blocked by Rabbit Anti-Idiotypic Antibodies.

Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disorder often associated with autoantibody inhibition of ADAMTS13, a VWF-cleaving protease. Autoantibodies decrease ADAMTS13 activity resulting in accumulation of “unusually” large VWF multimers that mediate platelet thrombosis. To better understand the role autoantibodies play in disease pathogenesis, as well as to develop more specific methods for diagnosis and therapy, it is necessary to characterize pathogenic antibodies on a molecular level, something not possible through analysis of polyclonal patient antisera. The ability to clone large repertoires of patient monoclonal autoantibodies (mAbs) using phage display offers a unique opportunity to address this issue. Three patient (Pt) antibody phage display libraries were created from either splenocytes (Pt1) or peripheral blood lymphocytes (Pt2, Pt3) of individuals with acquired TTP. ADAMTS13-specific mAbs were isolated by panning against recombinant ADAMTS13. Unique clones were identified by DNA sequencing, and their ability to interact with ADAMTS13 was characterized. After antigen selection of Pt1 library, 56 mAbs were randomly-selected from panning rounds 2 through 4 and 68% were found to comprise heavy chains encoded by VH1-69 paired with a VL3 family lambda light chain (3h or 3m). The remaining mAbs comprised heavy chains from the VH1, 3, or 4 families usually paired with kappa light chains. For Pt2 and Pt3 libraries, there was an identical pattern of genetic restriction in immune response to ADAMTS13, i.e. 16 of 24 mAbs (Pt2) and 27 of 27 mAbs (Pt3) were encoded by VH1-69 heavy chains and VL3 family lambda light chains. Though nearly all mAbs were unique, common CDR3 regions among some of the mAbs provided evidence of B-cell clonal expansion and somatic mutation. Though all mAbs bound to ADAMTS13 irrespective of genetic origin, mAbs comprising a VH1-69 heavy chain paired with a VL3 light chain inhibited ADAMTS13 using the FRET-VW73 assay while mAbs comprising a VH1-69 paired with a kappa light chain or comprising non-VH1-69 heavy chains did not inhibit ADAMTS13, with only two exceptions. MAb binding to ADAMTS13 was blocked by preincubation with normal human or murine plasma, but much less so by plasma from TTP patients or ADAMTS13 knockout mice suggesting crossreactivity with mouse ADAMTS13. Certain human mAbs inhibited cleavage of FRET-VWF73 by mouse ADAMTS13 and also inhibited ADAMTS13 in vivo after injection into the internal jugular vein of mice. Rabbit anti-idiotypic antibodies raised against mAb 416, a prototypical VH1-69-encoded mAb, blocked 416’s ability to inhibit human ADAMTS13. Taken together, the cloning and analyses of a large cohort of ADAMTS13 inhibitory autoantibodies derived from 3 unrelated individuals with acquired TTP revealed a genetically restricted immune response. This feature, if common among TTP patients, offers a potential therapeutic target for treatment of TTP, e.g. selective deletion of B-cells utilizing the VH1-69 heavy chain gene. Furthermore, crossreactivity of some human mAbs with murine ADAMTS13 provides a mouse model of acquired ADAMTS13 deficiency that may prove useful for determining the role of autoantibodies in the pathogenesis of TTP, particularly in the context of additional factors (e.g. environmental) that may be required to trigger the disease. Finally, anti-idiotypic mAbs, currently being cloned from rabbit phage display libraries, may help identify pathogenic antibodies in patient plasma and/or lead to novel therapeutic approaches.