Modifying ADAMTS13 to modulate binding of pathogenic autoantibodies of patients with acquired thrombotic thrombocytopenic purpura

Abstract

Antibodies that develop in patients with immune thrombotic thrombocytopenic purpura (iTTP) commonly target the spacer epitope R568/F592/R660/Y661/Y665 (RFRYY). In this study we present a detailed contribution of each residue in this epitope for autoantibody binding. Different panels of mutations were introduced here to create a large collection of full-length ADAMTS13 variants comprising conservative (Y←→F), semi-conservative (Y/F→L), non-conservative (Y/F→N) or alanine (Y/F/R→A) substitutions. Previously reported Gain-of-Function (GoF, KYKFF) and truncated ‘MDTCS’ variants were also included. Sera of 18 patients were screened against all variants. Conservative mutations of the aromatic residues did not reduce the binding of autoantibodies. Moderate resistance was achieved by replacing R568 and R660 by lysines or alanines. Semi-conservative mutations of aromatic residues show a moderate effectiveness in autoantibody resistance. Non-conservative asparagine or alanine mutations of aromatic residues are the most effective. In the mixtures of autoantibodies from the majority (89%) of patients screened, autoantibodies targeting the spacer RFRYY epitope have preponderance compared to other epitopes. Reductions in ADAMTS13 proteolytic activity were observed for all full-length mutant variants, in varying degrees. The greatest activity reductions were observed in the most autoantibody-resistant variants (15-35% residual activity in FRETS-VWF73). Among these, a triple-alanine mutant RARAA showed activity in a VWF multimer assay. This study shows that non-conservative and alanine modifications of residues within the exosite-3 spacer RFRYY epitope in full-length ADAMTS13 resist the binding of autoantibodies from iTTP patients, while retaining residual proteolytic activity. Our study provides a framework for the design of autoantibody-resistant ADAMTS13 variants for further therapeutic development.

Should all patients with immune‐mediated thrombotic thrombocytopenic purpura receive caplacizumab?

“…iTTP, immune-mediated thrombotic thrombocytopenic purpura. TPE, therapeutic plasma exchange [Colour figure can be viewed at wileyonlinelibrary.com] of ADAMTS13 [38][39][40][41][42] that should further enrich our therapeutic arsenal, we could assume that the most exciting chapters of iTTP treatment history are still to be written. In the light of these transformative changes, iTTP illustrates the strength of translational medicine when basic science and clinical research combine efficiently for the benefit of patients.…”

Current and Future Perspectives on ADAMTS13 and Thrombotic Thrombocytopenic Purpura

“…Therefore, it is under investigation whether circumventing anti-ADAMTS13 autoantibody binding to the spacer domain, by making an ADAMTS13 spacer variant, could be a novel therapeutic strategy. 65,197,210,211 Therefore, also large epitope mapping studies and further characterization of the anti-ADAMTS13 autoantibodies are needed, since the functional significance of autoantibodies targeting domains outside the spacer domain has been largely unexplored. The potential of this strategy will depend on the diversity of (harmful) anti-ADAMTS13 autoantibodies outside the spacer domain in the iTTP patients.…”

Transforming the major autoantibody site on ADAMTS13: spacer domain variants retaining von Willebrand factor cleavage activity

“…In the current issue of Haematologica, Graça and colleagues present a detailed description of the influence of the epitope (R568/F592/R660/Y661/Y665 [RFRYY]), 9 within the spacer domain, the predominant site for autoantibody binding. The capacity for ADAMTS13 to cleave VWF with full-length mutants was reduced in varying amounts.…”

“…The greatest influence was noted with cumulative mutations of the aromatic residues, demonstrating the maximum effect in preventing ADAMTS13 autoantibody binding, which is achieved by re-presenting epitope loops, lowering the surface charge and reducing surface size. 9 Current therapy for TTP aims to replace ADAMTS13, via plasma exchange and immunosuppression to remove autoantibodies to ADAMTS13. The main therapeutic modalities used are steroids and rituximab.…”

“…On one hand, a humanized single-variable domain immunoglobulin (Nanobody ® ) 6,7 has recently been approved and commercialized as caplacizumab (Cablivi TM ) by Ablynx, a Sanofi company (Sanofi-Aventis, Paris, France) for the treatment of acute acquired TTP in adults, on the basis of positive results in phase II and III trials. 8,9 On the other hand, several aptamers, consisting of single-stranded DNA or RNA oligonucleotides with a specific and stable three-dimensional shape able to recognize their target with high affinity and specificity, were developed and tested in animal models. [10][11][12] However, only the historical anti-VWF-A1 aptamer, ARC1779, developed by Archemix (Cambridge, MA, USA), was investigated in ACD [13][14][15][16] and TTP 17-21 including limited phase II studies.…”