In situ Transglutaminase 2-mediated Crosslinking of Engineered IL-7 to the Tumor Matrix Enhances Immunotherapy and Anti-PD-1 Response 2308334

Abstract

Abstract Introduction Cytokine therapies in immuno-oncology have been limited by rapid clearance and systemic toxicity. We present a novel strategy to anchor cytokines to the tumor ECM via tissue transglutaminase 2 (TG2)—mediated transamidation, an enzymatic process that covalently crosslinks ECM proteins. Because the enzymatic activity of TG2 is restricted to the tumor microenvironment, our strategy enables tumor-specific conjugation of systemically administered cytokines, thereby enhancing their antitumor activity while limiting systemic exposure. Methods We engineered a TG2 substrate domain—fused interleukin-7 (TSD-IL-7) that undergoes TG2-mediated covalent conjugation to lysine-containing extracellular matrix components. Tumor retention and bioactivity were evaluated following intravenous administration across multiple murine solid tumor models. Therapeutic efficacy was assessed as monotherapy and in combination with αPD-1 immune checkpoint blockade, with immune phenotypes analyzed in tumors and tumor-draining lymph nodes. Results TSD-IL-7 exhibited potent antitumor activity across multiple murine solid tumor models including orthotopic and metastatic disease, and enhanced responses to αPD-1 immune checkpoint inhibition without systemic toxicity. Remarkably, it drove complete tumor clearance in most animals in a checkpoint-refractory ovarian cancer model. Mechanistically, TSD-IL-7 promoted T cell proliferation and antitumor cytokine and chemokine production in the tumor and tumor-draining lymph nodes, while reducing T cell exhaustion and promoting CD8+ central memory T cell development within the tumor. Conclusion We present a novel strategy for covalently anchoring therapeutics to the tumor matrix following systemic administration by harnessing tumor-specific transglutaminase-2 activity, amplifying the antitumor immunity of engineered IL-7 while synergizing with immune checkpoint blockade to clear otherwise therapy-resistant solid tumors. Funding Source National Cancer Institute grant R21-CA277414 (J.A.H.). National Cancer Institute grant R01-CA219304 (M.A.S.). National Institutes of Health Immunology Training Grant T32AI07090 (H.C.). National Research Foundation of Korea Basic Science Research Program, Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)