Nature uses multivalency to govern many biological processes. The development of macromolecular and cellular therapies has largely been dependent on engineering similar polyvalent interactions to enable effective targeting. Such therapeutics typically utilize high-affinity binding domains that have the propensity to recognize both antigen-overexpressing tumors and normal-expressing tissues, leading to "on-target, off-tumor" toxicities. One strategy to improve these agents' selectivity is to reduce the binding affinity, such that biologically relevant interactions between the therapeutic and target cell will only exist under conditions of high avidity. Preclinical studies have validated this principle of avidity optimization in the context of chimeric antigen receptor (CAR) T cells; however, a rigorous analysis of this approach in the context of soluble multivalent targeting scaffolds has yet to be undertaken. Using a modular protein nanoring capable of displaying ≤8 fibronectin domains with engineered specificity for a model antigen, epithelial cell adhesion molecule (EpCAM), this study demonstrates that binding affinity and ligand valency can be optimized to afford discrimination between EpCAM High (2.8-3.8 × 10 6 antigens/cell) and EpCAM Low (5.2 × 10 4 to 2.2 × 10 5 antigens/cell) tissues both in vitro and in vivo.
Bibliographical noteFunding Information:
This work was supported by the National Institutes of Health (NIH) R21 CA185627 (C.R.W.), F30 CA210345 (C.M.C.), T32 GM008244 (C.M.C.), and the University of Minnesota, including an endowed fellowship on behalf of Dr. and Mrs. Bighley (C.M.C.) and the Academic Health Center Faculty Development Grant Program (C.R.W.). Flow cytometry work was performed in the University Flow Cytometry Resource core facility at the University of Minnesota. The biodistribu-tion work was performed in the University Imaging Center at the University of Minnesota.