TUNING IMMUNE FUNCTION WITH REVERSIBLE COVALENT CHEMISTRY

Abstract

Small molecules that form irreversible covalent bonds to proteins have great utility as tools for cancer immunotherapy. For bifunctional molecules that redirect the immune system against cancer, covalent engagement of immune or tumor proteins stabilizes the resulting cell-cell complex and provides a large efficacy advantage over non-covalent analogs. However, the efficacy of these irreversibly reactive covalent molecules is hindered by challenges, including their inactivation through hydrolysis and non-specific reactivity with nucleophiles throughout the proteome. Reversible covalent chemistry strikes a balance between these two paradigms, providing the required kinetic stabilization for productive immune complex formation while also avoiding permanent deactivation through promiscuous reactivity. This work applies reversible covalent chemistry to bifunctional immunotherapeutic small molecules, with a focus on T cell engagement. We apply a library of reversible, lysine-reactive covalent warheads with varying residence times to build bifunctionals that covalently engage synthetic immune receptors and direct universal synthetic antigen receptor (SAR)-T cells towards tumor targets. rCIRs show enhanced in vitro function when compared to non-covalent or irreversible covalent analogs and potentiate T cell-mediated killing of target tumors at sub-nanomolar concentrations. Due to their lack of promiscuous reactivity, rCIRs persist in media for an extended time. In vivo, rCIRs in combination with SAR-T cells reduce cell-derived xenograft tumor growth. We also leverage reversible covalency to build dual covalent bifunctionals, which display greater ternary complex formation than irreversible analogs. This work introduces reversible covalency as a powerful tool for building bifunctional molecules that induce cell-cell proximity.