Offsetting Low-Affinity Carbohydrate Binding with Covalency to Engage Sugar-Specific Proteins for Tumor-Immune Proximity Induction

Abstract

Carbohydrate-binding receptors are often used by the innate immune system to potentiate inflammation, target endocytosis/destruction, and adaptive immunity (e.g., CD206, DC-SIGN, MBL, and anticarbohydrate antibodies). To access this class of receptors for cancer immunotherapy, a growing repertoire of bifunctional proximity-inducing therapeutics use high-avidity multivalent carbohydrate binding domains to offset the intrinsically low affinity associated with monomeric carbohydrate–protein binding interactions ( K _d ≈ 10 ^–3–10 ^–6 M). For applications aimed at recruiting anticarbohydrate antibodies to tumor cells, large synthetic scaffolds are used that contain both a tumor-binding domain (TBD) and a multivalent antibody-binding domain (ABD) comprising multiple l-rhamnose monosaccharides. This allows for stable bridging between tumor cells and antibodies, which activates tumoricidal immune function. Problematically, such multivalent macromolecules can face limitations including synthetic and/or structural complexity and the potential for off-target immune engagement. We envisioned that small bifunctional “proximity-inducing” molecules containing a low-affinity monovalent ABD could efficiently engage carbohydrate-binding receptors for tumor-immune proximity by coupling weak binding with covalent engagement. Typical covalent drugs and electrophilic chimeras use high-affinity ligands to promote the fast covalent engagement of target proteins (i.e., large k _inact/ K _I), driven by a favorably small K _I for binding. We hypothesized the much less favorable K _I associated with carbohydrate–protein binding interactions can be offset by a favorably large k _inact for the covalent labeling step. In the current study, we test this hypothesis in the context of a model system that uses rhamnose-specific antibodies to induce tumor-immune proximity and tumoricidal function. We discovered that synthetic chimeric molecules capable of preorganizing an optimal electrophile (i.e., SuFEx vs activated ester) for protein engagement can rapidly covalently engage natural sources of antirhamnose antibody using only a single low-affinity rhamnose monosaccharide ABD. Strikingly, we observe chimeric molecules lacking an electrophile, which can only noncovalently bind the antibody, completely lack tumoricidal function. This is in stark contrast to previous work targeting small molecule hapten and peptide-specific antibodies. Our findings underscore the utility of covalency as a strategy to engage low-affinity carbohydrate-specific proteins for tumor-immune proximity induction.

Abstract

We demonstate that heterobifunctional electrophilic molecules can covalently engage carbohydrate-specific proteins for tumor immunotherapy, leveraging only simple low-affinity sugars.