ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 2. Pathogenicity in an animal model

Abstract

BACKGROUND Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease in which ultra-large von Willebrand Factor (UL-VWF) multimers accumulate as a result of autoantibody inhibition of the VWF protease, ADAMTS13. Current treatment is not specifically directed at the responsible autoantibodies and in some cases is ineffective or of transient benefit. More rationale, reliable and durable therapies are needed, and a human autoantibody-mediated animal model would be useful for their development. Previously, TTP patient anti-ADAMTS13 variable region fragments (scFv’s) were cloned that inhibited ADAMTS13 proteolytic activity in vitro and expressed features in common with inhibitory IgG in patient plasma. Here, pathogenicity of these scFv’s is explored in vivo by transfecting mice with inhibitory antibody cDNA. STUDY DESIGN AND METHODS Hydrodynamic tail vein injection of naked DNA encoding human anti-ADAMTS13 scFv was used to create sustained ADAMTS13 inhibition in mice. Accumulation of UL-VWF multimers was measured and formation of platelet thrombi following focal or systemic vascular injury was examined. RESULTS Transfected mice expressed physiological plasma levels of human scFv and developed sustained ADAMTS13 inhibition and accumulation of unprocessed UL-VWF multimers. Induced focal endothelial injury generated platelet thrombi extending well beyond the site of initial injury, and systemic endothelial injury induced thrombocytopenia, schistocyte formation, platelet thrombi, and death. CONCLUSIONS These results demonstrate for the first time the ability of human recombinant monovalent anti-ADAMTS13 antibody fragments to recapitulate key pathologic features of untreated acquired TTP in vivo, validating their clinical significance and providing an animal model for testing novel targeted therapeutic approaches.

Plasma levels of S100A8/A9, histone/DNA complexes, and cell‐free DNA predict adverse outcomes of immune thrombotic thrombocytopenic purpura

“…Plasma ADAMTS13 activity and inhibitors were determined in the reference laboratory (Versiti, Milwaukee, WI) or an in‐house FRETS‐VWF73 assay as previously described 6 …”

A New Classification System for IgG4 Autoantibodies

“…IgG4 (but also IgG1) antibodies cloned from TTP patients by EBV immortalization demonstrated pathogenicity in vitro in assays measuring ADAMTS13 activity ( 69 ). Also, several patient-derived scFv were shown to be pathogenic when expressed in mice, albeit the original subclass of the patient antibodies is unknown ( 59 ). There is also additional circumstantial evidence that pathogenic antibodies of IgG4 subclass exist in TTP as IgG4 levels are associated with relapse and some patients have exclusively IgG4 autoantibodies ( 235 ).…”

ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 1. Structural and functional characterization in vitro

“…56 The idiotypic relatedness of our set of inhibitory antibodies to patient IgG supports their clinical relevance and suggests that they might serve as useful targets for the design of therapeutic agents that block IgG binding. In the second manuscript in this series 57 , we explore antibody pathogenicity in vivo by transfecting mice with cDNA encoding these inhibitory antibodies and visualizing the effects of sustained antibody-mediated ADAMTS13 inhibition.…”

“…Anti-idiotypic antibodies were raised to a subset of these monoclonal ADAMTS13 autoantibodies to explore relatedness to one another and to those in patient sera. In an accompanying manuscript 57 , we test the pathogenicity of these antibodies in a novel murine model of human autoantibody-mediated TTP.…”