Development of FAP-targeted theranostics discovered by next-generation sequencing-augmented mining of a novel immunized VNAR library

Cancer-associated fibroblasts (CAFs) in the stroma of solid tumors promote an immunosuppressive tumor microenvironment (TME) that drives resistance to therapies. The expression of the protease fibroblast activation protein (FAP) on the surface of CAFs has made FAP a target for development of therapies to dampen immunosuppression. Relatively few biologics have been developed for FAP and none have been developed that exploit the unique engagement properties of Variable New Antigen Receptors (VNARs) from shark antibodies. As the smallest binding domain in nature, VNARs cleverage unique geometries and recognize epitopes conventional antibodies cannot. By directly immunizing a nurse shark with FAP, we created a large anti-FAP VNAR phage display library. This library allowed us to identify a suite of anti-FAP VNARs through traditional biopanning and also by an in silico approach that did not require any prior affinity-based enrichment in vitro. We investigated four VNAR-Fc fusion proteins for theranostic properties and found that all four recognized FAP with high affinity and were rapidly internalized by FAP-positive cells. As a result, the VNAR-Fc constructs were effective antibody-drug conjugates in vitro and were able to localize to FAP-positive xenografts in vivo. Our findings establish VNAR-Fc constructs as a versatile platform for theranostic development that could yield innovative cancer therapies targeting the TME.