Chemical generation of checkpoint inhibitory T cell engagers for the treatment of cancer

Abstract

Bispecific T cell engagers (BiTEs), a subset of bispecific antibodies (bsAbs), can promote a targeted cancer cell’s death by bringing it close to a cytotoxic T cell. Checkpoint inhibitory T cell engagers (CiTEs) comprise a BiTE core with an added immunomodulatory protein, which serves to reverse cancer-cell immune-dampening strategies, improving efficacy. So far, protein engineering has been the main approach to generate bsAbs and CiTEs, but improved chemical methods for their generation have recently been developed. Homogeneous fragment-based bsAbs constructed from fragment antigen-binding regions (Fabs) can be generated using click chemistry. Here we describe a chemical method to generate biotin-functionalized three-protein conjugates, which include two CiTE molecules, one containing an anti-PD-1 Fab and the other containing an immunomodulatory enzyme, Salmonella typhimurium sialidase. The CiTEs’ efficacy was shown to be superior to that of the simpler BiTE scaffold, with the sialidase-containing CiTE inducing substantially enhanced T cell-mediated cytotoxicity in vitro. The chemical method described here, more generally, enables the generation of multi-protein constructs with further biological applications. [Figure not available: see fulltext.]