(21d) Directed Evolution of Monoclonal Antibodies Against Tumor Associated Carbohydrate Antigens

Tumor associated carbohydrate antigens (TACAs) are a promising, yet difficult class of biomolecules to target for theragnostics. Overexpression of TACAs is a unique molecular signature that healthy cells lack. However, most are not recognized as non-self, causing low immunogenicity thereby hindering traditional antibody immunization techniques. This difficulty is increased by the massive, mostly unfunctional sequence space available. We address these challenges using a novel immunization strategy paired with large, rationally designed antibody libraries. Directed evolution of a rationally designed library based on immune-evolved antibodies decreases time and effort spent to achieve a high-affinity, high-specificity binder compared to a naïve library. Moreover, by utilizing a novel immunization technique involving the display of TACAs on multi-valent Qβ nanoparticles, a stronger immunogenic response is elicited from the animal being challenged, causing higher titers of immune-evolved antibodies. Yet even with improved titers, the resulting antibodies commonly show insufficient binding affinity. To overcome this challenge, we utilize dominant antibodies discovered from Qβ nanoparticles as a starting point for our library. In this study, we have generated immune-evolved antibodies which weakly bind NHAcGD2, a ganglioside implicated in the proliferation and invasion of multiple cancers (e.g., neuroblastoma and melanoma). We use this diverse panel of lead antibodies as starting points for cell surface display directed evolution campaigns. To navigate the mutational landscape of these promising monoclonal antibodies more efficiently, our cell surface display libraries are guided by single cell deep sequencing of the immunized animal’s B cell repertoire, allowing for native pairing of heavy and light chains. We also leverage in silico experiments to help determine promising candidates. From this, we can identify mutations to increase functionality and drive potent interactions with the TACA target. In this way, we provide a novel platform for discovering antibodies with clinical potential against a notoriously challenging class of biomarkers.