High-Throughput Screening of a Large Combinatorial Library of Small Proteins Against Human Spleen-Derived Monoclonal Anti-ADAMTS13 Antibodies Yields Specific Anti-Idiotypic Molecules – Potential Tools for New Treatment Strategy in Acquired TTP

Acquired Thrombotic Thrombocytopenic Purpura (TTP) is the result of autoantibodies (Abs) neutralizing and/or accelerating clearance of ADAMTS13. Despite the first-line treatment with daily plasma exchange (PEX), acute TTP is still associated with a mortality rate of ∼20% leaving survivors at high risk of relapse. Availability of molecules for neutralization of pathogenic anti-ADAMTS13 Abs during acute bouts as well as for the targeted elimination of anti-ADAMTS13 specific memory B- and plasma-cells to prevent relapses are thus highly desirable tools to improve patient care and/or treatment of TTP.

The primary epitope of pathological relevant, inhibitory anti-ADAMTS13 Abs was identified to lay in the ADAMTS13 spacer domain and to be conformational compromising amino acid residues R568, F592, Y660, Y661 and Y665. We have previously generated 29 specific human monoclonal anti-ADAMTS13 Abs from 2 patients (A and B) splenectomized for frequently relapsing TTP using spleen-derived mononuclear cells (ASH 2011, abstract #194). These anti-ADAMTS13 Abs, characterized by a 5–6 fold increased inhibitory capacity than previously reported as well as eight unique CDR3 motifs in their variable heavy chain genes, four of which are common to both acquired TTP patients, are useful tools to select small anti-idiotypic molecules, mimicking the conformational epitope on ADAMTS13. For this purpose we chose the large combinatorial small protein library of Designed-Ankryin-Repeat-Protein (DARPins), characterized by: (1) a high diversity with a library size of 10^15–23, allowing to find molecules to any target including conformational epitopes, (2) expressing molecules with a high stability, and (3) being immune tolerant when administered.

Two DARPin libraries, N2C (diversity of 10¹⁵) and N3C (10²³) coding for two or three randomized ankyrin repeat modules respectively, provided by Molecular Partners AG (Schlieren, Switzerland), were screened against three biotinylated inhibitory anti-ADAMTS13 Abs from patient A belonging to one CDR3 motif (CDR3 motif-1) and pooled in equimolar ratio as targets. Selected DARPins from the fourth round of ribosomal display were cloned, and single clones producing anti-ADAMTS13 idiotypes were purified, tested separately for their specificity towards 29 single, spleen-derived monoclonal anti-ADAMTS13 Abs by ELISA and their DNA sequence analyzed.

Seven of 192 (N2C-library) and 2/192 (N3C) single DARPin clones selected against a pool of three inhibitory anti-ADAMTS13 Abs, of a the single CDR3 motif-1, were highly specific for their target. Detailed DNA sequence analysis showed that 4/7 (N2C) and 2/2 (N3C) of the anti-ADAMTS13 specific DARPins were unique whereas 3/7 N2C DARPins consisted of repeats of the unique clones. So far, four (3 N2C and 1 N3C) unique DARPins were purified and their reactivity at equimolar concentration tested towards 200 nM of each of the 29 anti-ADAMTS13 Abs, individually coated on a microtiter plate by ELISA assay. All four DARPins were highly specific for the pool of Abs used for selection as well as other Abs belonging to the same CDR3 motif-1 (n=5). Anti-ADAMTS13 Abs containing the remaining three of the four common (patient A and B) CDR3 motifs, not used for selection, were recognized by 3/4 unique DARPins although to a lesser extent and more anti-ADAMTS13 Abs originating from patient A, were recognized than from patient B, namely 94%, 69% and 56% for CDR3 motif1-3 compared to only 31 %, 54% and 15% from patient B, respectively.

Using inhibitory anti-ADAMTS13 of one patient (A) and one CDR3 motif, we were able to select a number of highly specific anti-idiotypic DARPins binding to different monoclonal anti-ADAMTS13 Abs of two genetically and geographically unrelated acquired TTP patients. The fact that several although similar (belonging to the same CDR3 motif) antibodies can be recognized by one anti-idiotypic molecule is promising and hints at a limited number of different anti-idiotypic molecules necessary to neutralize inhibitory anti-ADAMTS13 Abs in the plasma of acquired TTP patients. The analysis of their affinity and the neutralizing potential are ongoing.

No relevant conflicts of interest to declare.