Anti-EGFR Fibronectin Bispecific Chemically Self-Assembling Nanorings (CSANs) Induce Potent T Cell-Mediated Antitumor Responses and Downregulation of EGFR Signaling and PD-1/PD-L1 Expression

Abstract

Overexpression of Epidermal Growth Factor Receptor (EGFR) on various cancers makes it an important target for cancer immunotherapy. We recently demonstrated that single-chain variable fragment-based bispecific chemically self-assembled nanorings (CSANs) can succesfully modify T cell surfaces and function as prosthetic antigen receptors (PARs) allowing selective targeting of tumor antigens while incorporating a dissociation mechanism of the rings. Here, we report generation of anti-EGFR fibronectin (FN3)-based PARs with high yield, rapid protein production, predicted low immunogenicity and increased protein stability. We demonstrated the cytotoxicity of FN3-PARs successfully while evaluating FN3 affinities, CSANs valencies and antigen expression levels. Using an orthotopic breast cancer model we showed that FN3-PARs can suppress tumor growth with no adverse effects and FN3-PARs reduced immunosuppressive PD-L1 expression by downregulating EGFR signaling. These results demonstrate the potential of FN3-PARs to direct selective T cell-targeted tumor killing and to enhance anti-tumor T cell efficacy by modulating the tumor microenvironment.