Abstract A117: Design of EGFR and TCR targeted bispecific protease-activatable chemically self-assembled nanorings with reduced off-tumor toxicity for treatment of solid tumors

Abstract Overexpression of cell surface antigens such as epidermal growth factor receptor (EGFR) on a variety of solid tumors makes EGFR an attractive target for developing immunotherapies. However, EGFR is also widely expressed on healthy tissues such as skin, liver, and gastrointestinal tract. EGFR expression on healthy tissue limits the clinical utility of T-cell based immunotherapies due to significant on-target, off-tumor toxicities. To address this, we designed and developed a tumor-microenvironment (TME) specific T-cell receptor (TCR) targeting dihydrofolate reductase (DHFR2) fusion protein. This engineered protein – HINT1-anti-TCR-DHFR2 possesses a TCR targeting nanobody linked to histidine-triad nucleotide binding protein-1 (HINT1) through a matrix metalloproteinase-2 (MMP-2) sensitive linker. HINT1 was hypothesized to serve as a steric protein mask that will be cleaved by MMP-2 upregulated in solid tumors. Combining HINT1-anti-TCR-DHFR2 and anti-EGFR-DHFR2 fusion proteins in the presence of a chemical dimerizer bis-methotrexate leads to the formation of bispecific protease-activatable chemically self-assembled nanorings (pro-CSANs). Once the pro-CSANs enter the TME and bind to EGFR, cleavage by MMP-2 will lead to unmasking of the T cell binder, thereby crosslinking and activating T cells, causing targeted cancer cell death. To this end, HINT1-anti-TCR-DHFR2 fusion protein was successfully cloned and characterized. HINT1-anti-TCR-DHFR2 was completely unmasked in vitro using recombinant MMP-2 and the newly designed fusion protein was stable in human serum for up to three days. 2D cytotoxicity assays with bispecific nanorings comprised of anti-TCR-DHFR2 and anti-EGFR-DHFR2 showed low nanomolar potencies against a panel of EGFR+ solid tumor cell lines such as A431 (skin), A549 (lung), MDA-MB-231 (breast), HT29 (colon) and DU145 (prostate). T cell activation markers CD69 and CD25 were significantly upregulated and high levels of the cytokines IFN-gamma and IL-2 were secreted. T cell mediated killing and T cell activation was completely attenuated in the presence of HINT1 mask, thereby establishing proof-of-concept for our pro-CSANs platform. The in vivo efficacy and safety of the pro-CSANs is currently being evaluated in a previously established human tumor xenograft mouse model. Overall, the pro-CSANs platform serves as a promising strategy to enhance the safety of EGFR-targeted T cell-based cancer immunotherapies. Citation Format: Abhishek S Kulkarni, Debasmita Paul, Kostana Ligori, Freddys Rodriguez, Lakmal Rozumalski, Carston R Wagner. Design of EGFR and TCR targeted bispecific protease-activatable chemically self-assembled nanorings with reduced off-tumor toxicity for treatment of solid tumors [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A117.