Poster Session I: Bleeding disorders (congenital and acquired)
Recently a range of alternative novel therapies have been developed to improve treatment options for patients with Hemophilia A. One approach is to generate a Factor VIII (F.VIII) mimetic molecule using a humanised bispecific antibody, as was done for Hemlibra. Taking advantage of Kymab's fully human antibody discovery platform, we describe the selection and optimisation of an FVIII mimetic common light chain (CLC) bispecific antibody which can similarly catalyse the generation of Factor Xa (FXase) and normalise the activated partial thromboplastin time (aPTT).
To generate a functionally active F.VIII mimetic bispecific antibody for Haemophilia A treatment
F.IX and F.X. binding antibodies were generated by immunizing Kymouse™, which contain the full human immunoglobin repertoire, with F.IX or F.X, respectively. Isolated F.IX and F.X specific arms were co-expressed as 2-heavy-2-light-chain (2H2L) bispecific antibodies. Purified 2H2L bispecifics were screened using a high-throughput chromogenic FXase assay. The light chain of a promiscuous F.IX arm was chosen to generate transgenic mice expressing this bespoke common light chain (CLC) in the Kymouse™ background. By immunizing these transgenic mice with F.X, F.X binding antibodies containing the CLC were recovered. The heavy chains of these F.X binding antibodies were co-expressed with the heavy/light chains of the chosen F.IX arm as CLC bispecific antibodies. One biologically active CLC bispecific antibody was identified by functional assays, and chosen for further optimization. The optimization of the lead bispecific antibody, KY1049, was achieved by data mining of next generation sequencing information using Kymab's IntelliSelect™ bioinformatic platform, coupled with site-specific mutagenesis.
More than 8,000 2H2L bispecifics were screened by a chromogenic FXase assay to select the most active and versatile F.IX arm (Figure 1A). More than 400 F.X heavy chains subsequently isolated from the bespoke CLC Kymouse™ were screened to identify a highly active CLC bispecific (Figure 1B). Further optimisation of the molecule was carried out to iteratively increase FVIII mimetic activity by deep data-mining of heavy chain NGS sequence data, or site-specific mutagenesis. The combinatorial optimization process resulted in a highly functional CLC bispecific, KY1049 with comparable F.VIII mimetic activities to a sequence-identical analogue of Hemlibra (Figure 1C).
Our bispecific antibody discovery platform consisting of four-chain matrix screening, common light chain transgenic mouse technology, B cell network analysis and site-specific mutagenesis was applied to develop KY1049, a potent FV.III mimetic bispecific antibody, which shows equivalent activity to Hemlibra in vitro and holds promise as a future therapeutic in Haemophilia A.