(311b) Enhancing the Tumor Selectivity of Targeted Proteins through Avidity Optimization

Biologic immunotherapies that target tumor-associated antigens have become a mainstay of cancer treatment. Unfortunately, the target antigens are rarely exclusive to the neoplasm, leading to “on-target, off-tumor” toxicities that result from the recognition healthy cells that express the cognate antigen at physiologic levels.

One potential strategy to improve these agents’ selectivity is to reduce the binding affinity while increasing the interaction valency. In this manner, biologically relevant interactions between the therapeutic and target cell will only exist under conditions of high avidity. While this approach has been pursued for highly polyvalent nanoparticles and engineered T cells, comprehensive assessments of soluble targeting scaffolds with lower valencies are lacking.

To address this, we engineered a panel of protein ligands based upon the tenth type III fibronectin domain (Fn3) that bind to epithelial cell adhesion molecule (EpCAM), an overexpressed tumor antigen. We then incorporated these Fn3s into a multivalent chemically self-assembled nanoring (CSAN). We hypothesized that by varying the affinity and valency of the Fn3 domains in the modular CSAN, we could prepare a scaffold that binds specifically to cells overexpressing EpCAM and not to cells expressing low quantities of EpCAM.

Accordingly, we titrated avidity-modulated CSANs against various EpCAM⁺ cell lines. This enabled us to quantitate the apparent affinity of the scaffold in the context of variable Fn3 affinities (11 nM – 1.1 µM), valencies (1 – 8 ligands), and EpCAM-expression densities (5.2x10⁴ – 3.8x10⁶ antigens/cell). These results informed the design of CSANs capable of discriminating between EpCAM^High and EpCAM^Low cell lines in vitro.

Finally, we evaluated these principles in vivo using murine xenografts of EpCAM⁺ breast adenocarcinoma cell lines. The highest avidity CSANs accumulated in both EpCAM^High and EpCAM^Low tumors equivalently. In contrast, CSANs with reduced binding affinity and ligand valency accumulated specifically within the EpCAM^High tumor, indicating successful tissue discrimination.

These results demonstrate that avidity optimization is a viable strategy to enhance the specificity of soluble targeting scaffolds with limited valency. Thus, this work provides insight into the future design of multivalent scaffolds for a host of applications, including therapeutic drug conjugates and diagnostic imaging agents.