Abstract 1625 XCyTE: A novel protein therapeutic designed to eliminate specific B cell populations with cytotoxic T cells

An XCyTE is a cytotoxic T cell engager (CyTE) linked to another moiety (X), with the goal of crosslinking CD8-positive T cells to specific target cells. The XCyTE is constructed as a bispecific monoclonal antibody that interacts with both the CD8 coreceptor on cytotoxic T lymphocytes (CTLs) and a surface protein on the target cells. We hypothesize that bringing the CTLs into contact with the target cells will initiate CTL-mediated killing. We are exploring this mechanism to treat inhibitor formation in hemophilia A (HA) patients, who suffer from a deficiency of clotting factor VIII (FVIII). The standard treatment for HA is routine infusions with either plasma-derived or recombinant FVIII, but 20-30% of patients develop an antibody response to the treatment, known as inhibitor formation, that neutralizes its effect. To tolerize patients to FVIII, we have designed an XCyTE to eliminate FVIII-specific B lymphocytes. This XCyTE employs the FVIII C2 domain, one of the most immunogenic regions of FVIII, and an anti-human CD8 antibody Fab derived from OKT8. The two binding elements are recombinantly connected to immunoglobulin heavy chains with point mutations to introduce electrostatic interactions that favor a heterodimer configuration. We designed a single expression vector to produce the FVIII(C2)-CyTE and expressed it from HEK293 cells. Using protein G affinity and size exclusion chromatography, we purified the XCyTE and evaluated it using non-reducing SDSPAGE. The results show the expected profile of the XCyTE. We conclude that we were able to successfully design and produce a FVIII(C2)-CyTE. Our laboratory is currently testing its efficacy at inducing CTL-mediated killing of FVIII-specific B cells. We thank Grant R21 HL152318 "Bispecific Antibody to Target FVIII-specific B Cells." for funding our research.