Covalent Proximity-inducing Molecules for Synthetic Immunotherapy

Abstract

The immune system is adept at the recognition and elimination of foreign entities. Immunotherapies may operate through proximity induction, creating new molecular recognition events between the immune system and foreign antigen. Previous work has demonstrated antibody recruiting molecules (ARMs), which are small molecule bifunctionals able to simultaneously bind endogenous serum antibodies, and cancer antigens overexpressed on cancer cells. These antibodies are then redirected towards cancer, leading to targeted clearance. However, ARMs are limited by rapid clearance, affinity and concentration of endogenous serum antibodies. In Chapter Two, to address ARM limitations we created covalent antibody recruiting molecules (cARMs), which are able to covalently pre-associate with endogenous serum antibodies and redirect them towards cancer receptors. We elucidate the kinetics of the covalent reaction, characterize potency of immune activation, and demonstrate that covalently stabilized immune complexes lead to enhanced immune activity in functional assays at biologically relevant concentrations of serum antibody. Uniquely, we find that this covalent enhancement in function is not due to increased immune complex/ternary complex formation but rather a result of the kinetic stability of these complexes which is “sensed” by the immune cell.

In Chapter Three, we expanded the covalent bifunctional strategy to target viral pathogens, specifically SARS-Cov-2. Neutralizing antibodies (nAbs) are an essential component of the immune response against CoVID-19. These operate by coating the viral particle and preventing adhesion to host cells. However, antibodies are sensitive to escape mutations. These mutations are highly prevalent on variants of concern (VOCs) and render monoclonal antibodies (mAbs) ineffective. As a complementary “mutation-proof” strategy, we devised a covalent labeling strategy to chemically opsonize viral particles with serum antibodies. Here we demonstrate the site-selective covalent labeling of SARS-CoV-2 Spike RBD, outside of the binding site with ACE2 with anti-hapten endogenous antibodies, can affect viral immune recognition and simultaneously block Spike RBD binding to ACE2. These molecules, named Covalent Viral Opsonizers (CVOs), enact neutralization and targeted antiviral phagocytosis with enhanced resistance to RBD mutations compared to conventional monoclonal antibodies.

In Chapter Four we devised a dual proximity labelling-chimeric molecular strategy to create covalent glue mimics (CGMs) which kinetically enforce protein-protein interactions. Molecular glues (MGs) use positive cooperativity to stabilize two terminal proteins in a ternary complex to enact a biological response. Despite their promise, they are challenging to rationally design and limited in scope. CGMs are chimeric molecules that mimic molecular glue stabilization by site selectively covalently cross linking both terminal proteins within the ternary complex. We demonstrate CGMs leads to significant functional enhancement in several distinct tumor immunotherapeutic model systems.