OC 58.3 - Allosteric Effects of Antibodies in Immune Thrombotic Thrombocytopenic Purpura

Background: Immune thrombotic thrombocytopenic purpura (iTTP) is caused by anti-ADAMTS13 antibodies which prevent cleavage of von Willebrand factor (VWF). Improving iTTP care requires deeper understanding of antibody mechanism(s) of action. Recent work has shown inhibitory anti-spacer human antibodies primarily influence kcat, or catalytic turnover, of ADAMTS13. Inhibitors appear to allosterically modify the S3 pocket of the metalloprotease domain, which interacts with the L1603 residue of the VWF A2 domain. Similarly, mouse anti-human CUB antibodies also affect catalytic turnover more than VWF binding. Anti-CUB antibodies appear to be important to the disease process as they can induce an "open" ADAMTS13 conformation, which predominates in acute iTTP.

Aims: We aim to characterize the mechanistic effects of anti-CUB human antibodies, explore whether allosteric effects depend upon S3-L1603 interaction, and further define their binding epitope(s).

Methods: Single chain fragments of the variable loop (scFv3-3 and scFv4-41) derived from iTTP patients that target ADAMTS13 CUB domains were expressed and purified. Normal human plasma (NHP) containing ADAMTS13 was titrated with FRETS-VWF73. Kcat and K0.5, substrate concentration when rate is half-maximal, were determined with and without scFv's. K0.5 ratios were used to quantify thermodynamic coupling. Experiments were repeated using variant FRETS-VWF73 (L1603A). Hydrogen-deuterium exchange mass spectrometry (HX-MS) was used to map the binding epitope of scFv4-41.

Results: ScFv3-3 and scFv4-41 increased kcat (41.1±7.6% and 38.5±16.9%, respectively) compared to no scFv with wild-type FRETS-VWF73, but not with L1603A (9.8±17.6% and 3.1±10.2%, respectively). Thermodynamic coupling in the presence of scFv's was negligible (ratio of K0.5±scFv range 0.88±0.04 to 1.12±0.10) . Peptide fragments corresponding to CUB2 residues 1360-1371, 1360-1377, and 1358-1371 demonstrated decreased deuterium uptake in the presence of scFv4-41.

Conclusion(s): Anti-CUB human antibodies allosterically affect catalytic turnover of ADAMTS13, which depends at least in part on the S3-L1603 interaction. This work provides valuable insight into the conformational environment of ADAMTS13 in iTTP.