OC 78.1 - Harnessing the co-inhibitory receptor G6b-B using cis-acting platelet receptor inhibitors (CAPRIs)

Background: All current anti-platelet therapies targeting activation pathways cause bleeding. In this study, we describe a novel approach of inhibiting immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor-mediated platelet activation through the use of bispecific single-chain variable fragment (bi-scFvs), termed cis-acting platelet receptor inhibitors (CAPRIs) that mediate co-clustering with the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing megakaryocyte platelet co-inhibitory receptor G6b-B (MPIG6B, G6B).

Aims: To develop a novel approach of inhibiting platelet activation with improved efficacy, specificity and fewer bleeding events than current competitive antagonists.

Methods: G6B-CD32A and G6B-GPVI CAPRIs were generated by recombining variable regions (VRs) of anti-G6B monoclonal antibody (mAb) 17-4 to VRs of either anti-CD32A mAb IV.3 or anti-GPVI mAb 3J24 using glycine-serine-rich linker peptides, respectively. CAPRIs were expressed in HEK293 cells, and purified by affinity and size exclusion chromatography. Effects on platelet activation, function and phosphorylation were tested using conventional approaches.

Results: The G6B-CD32A CAPRI inhibited platelet activation and aggregation in response to CD32A agonists, including heparin-induced thrombocytopenia (HIT) mAbs KKO and 5B9 in the presence of heparin, and vaccine-induced thrombotic thrombocytopenia (VITT) mAb 1E12. The G6B-CD32A CAPRI was also highly effective at inhibiting 1E12-mediated thrombus formation under venous shear on a von Willebrand factor-coated surface. The G6B-GPVI CAPRI inhibited platelet activation and aggregation in response to the GPVI-specific agonist collagen-related peptide (CRP) and collagen in suspension, and on a collagen-coated surface under arterial shear. Inhibitory effects were more robust than G6B, IV.3 and 3J24 scFvs. Further, CD32A- and GPVI-mediated Syk phosphorylation was inhibited with either the G6B-CD32A or G6B-GPVI CAPRI, respectively.

Conclusion(s): Findings provide proof-of-concept that CAPRI-mediated co-clustering of ITIM- and ITAM-containing receptors can be used to inhibit ITAM-mediated platelet activation and function, pioneering a promising novel class of anti-thrombotic therapeutic agents.