Anti-Idiotypic-Designed-Ankyrin-Repeat-Molecules (DARPins) Universally Neutralize Plasma-Derived Autoantibodies in Acquired TTP Patients

Background. Autoantibodies (Abs) neutralizing and/or accelerating clearance of the von Willebrand factor-cleaving protease-ADAMTS13 are the major culprits in acquired Thrombotic Thrombocytopenic Purpura (aTTP). Despite the first-line treatment with daily plasma exchange (PEX), acute aTTP is still today associated with a mortality rate of ~20% leaving survivors at high risk of relapse. To improve patient care and/or treatment of aTTP we explored the capacity of anti-idiotypic small molecules to specifically bind and potentially neutralize pathogenic anti-ADAMTS13 autoantibodies thereby restoring plasma-derived ADAMTS13 activity in acquired TTP patients.

Method: As selecting tools we used previously generated spleen-derived inhibitory human monoclonal anti-ADAMTS13 antibodies consisting of the 4 antigen-binding CDR3 motifs shared by the two aTTP patients (Schaller et al, Blood 2014;124(23):3469-79). Either 3 mAbs representative for CDR3 Motif-1 or 3 mAbs for CDR3 Motif-3 or 2 mAbs representing CDR3 Motif-4 were pooled at equimolar concentrations by ribosomal display to screen for binders in two combinatorial small protein libraries (DARPins from Molecular Partners, Schlieren, Switzerland) containing either two (N2C) or three (N3C) randomized ankyrin repeat modules, respectively From each of the 6 libraries single anti-idiotypic DARPins were cloned into a pMPAG6 expression vector and purified by His-tag affinity chromatography. In total 22 unique anti-idiotypic DARPins, 4 N2C-DARPins and 2 N3C-DARPins selected by Motif-1, 9 N2C-DARPins by Motif-3 and 7 N2C-DARPins by Motif-4 mAbs were identified.

Inhibitor targeting Strategy: The neutralization potential towards plasma-derived anti-ADAMTS13 Abs, was tested by pre-incubating the DARPins prior measuring the residual activity by functional FRETS assay, in a cohort of 50 acute aTTP patients with pathological inhibitor titers >0.4 BU/ml (diagnosed at our Center 2006-2014). First, equimolar pools (1000 nM) of anti-idiotypic DARPins (Motif-1, Motif-3 or Motif-4) were tested in 11/50 patients, secondly the contribution of the single DARPins from the responsive pools was assessed in 18/50 patients to finally test the entire cohort with a combination of all neutralizing anti-idiotypic DARPins.

Results: The pool of all Motif-1 anti-idiotypic DARPins (n=6) neutralized inhibitor titers below the pathological threshold in 9/11 (82 %) aTTP patients tested, whereas only a slight reduction of the inhibitor titers (in average 15%) was observed for Motif-3 and Motif-4 DARPin pools, revealing the Motif-1 DARPins as the most universal neutralizers. To test the contribution of each Motif-1 anti-idiotypic DARPin to the observed inhibitor neutralization, single DARPins were tested in 18/50 aTTP revealing a reduction of the inhibitor of 20-50% (N2C-19) or 20% (N3C-74) in 5/18 patients tested. In contrast 90% (45/50) aTTP patients incubated with the pool of all 6 Motif-1 DARPins caused inhibitor titers to drop either completely or below the pathological threshold. The inhibitor titer was reduced below 1BU/ml in 3/50, but was irresponsive in 2/50 patients.

Conclusions: Our data clearly show that anti-idiotypic DARPins can potently and universally neutralize inhibitory anti-ADAMTS13 Abs of aTTP patients. However an effective neutralization, restoring ADAMTS13 activity above 10-15% in most patients was only achieved when pooling all Motif-1 DARPins. If a combination of Motif-3 and/or Motif-4 DARPins with Motif-1 DARPins can additionally increase neutralization capacity also in relapsing patients is currently under investigation.