Characterization of a Human Monoclonal Antibody Directed Against the Spacer Domain of ADAMTS13; a Prototype of Antibodies Present in Patients with Acquired Thrombotic Thrombocytopenic Purpura.
Authors: Luken Brenda M. , Turenhout Ellen A.M. , Kaijen Paul H.P. , Kremer Hovinga Johanna A. , Van Mourik Jan A. , Fijnheer Rob , Voorberg Jan 
Published: April 2020

Abstract
Acquired thrombotic thrombocytopenic purpura (TTP) is often accompanied by the presence of (auto-) antibodies directed against the plasma metalloprotease ADAMTS13. The antibodies prevent efficient proteolytic cleavage of unusually large von Willebrand factor multimers by ADAMTS13, which is highly reactive with platelets. This may result in the formation of platelet aggregates that occlude the microvasculature of patients with TTP. In all presently characterized patients with acquired TTP and detectable anti-ADAMTS13 antibodies, antibodies directed to the (cysteine-rich) spacer domain were present. In order to study anti-ADAMTS13 antibodies at the molecular level, we have isolated single-chain Fv antibody fragments (scFv) from an immunoglobulin V-gene phage-display library derived from CD19+ B-cells of a patient with acquired TTP. From this library scFv I-9 was isolated, of which the variable heavy chain segment is homologous to members of the VH1-69 family. Using ELISA and immunoprecipitation analysis we confirmed that antibody I-9 also binds to the spacer domain. We converted the scFv antibody fragment into a full-length human antibody to better study its effect on proteolytic cleavage of VWF and substrate binding. Antibody I-9 was included into a modified FRETS-VWF73 assay and a dose-dependent inhibition of VWF73 proteolysis by ADAMTS13 was observed. In addition, antibody I-9 was able to inhibit the proteolysis of plasma-derived VWF multimers by ADAMTS13. In both assays inhibition was not very efficient; which is most likely due to the low affinity of antibody I-9 for ADAMTS13. Chimeric ADAMTS13 variants in which 5–10 amino acid residues of the ADAMTS13 spacer domain have been exchanged for the corresponding sequence of ADAMTS1 were used to determine the binding site for antibody I-9 in the spacer domain. Our findings show that amino acid regions 572–579 and 657–666 of the spacer domain are required for binding of antibody I-9, although several other regions are also involved in the interaction. We have previously shown that these amino acid regions are also crucial for binding of (polyclonal) antibodies present in plasma of patients with acquired TTP. Additionally, competition experiments were performed to address whether antibodies with similar properties as antibody I-9 are indeed present in plasma from patients with acquired TTP during acute disease. Plasma from 6 patients with acquired TTP was able to compete in a dose-dependent manner with antibody I-9 for binding to immobilized ADAMTS13, including plasma from the patient whose lymphocytes were used for construction of the phage-display library. These results show that antibodies with similar epitope-specificity as antibody I-9 are present in plasma of patients with acquired TTP. Since anti-spacer domain antibodies have been found in all presently characterized patients with anti-ADAMTS13 antibodies, the current data suggest that antibody I-9 is representative of the pathogenic antibodies present in plasma of the majority of patients with acquired TTP.