Collagen-binding IL-12 Expressing STEAP1 CAR-T Cells Reduce Toxicity and Eradicate Mouse Prostate Cancer in Combination with Checkpoint Inhibitors

Immunosuppressive microenvironments, the lack of immune infiltration, and antigen heterogeneity pose significant challenges for chimeric antigen receptor (CAR)-T cell therapies to tackle solid tumors. CAR-T cells were armed with immunostimulatory payloads, such as interleukin-12 (IL-12), to overcome this issue, but faced intolerable toxicity during clinical development. Here, we show that collagen-binding domain-fused IL-12 (CBD-IL-12) was retained within syngeneic murine prostate tumors, after secretion from CAR-T cells targeting human six transmembrane epithelial antigen of the prostate 1 (STEAP1). This led to equivalently high intratumoral interferon-γ levels without hepatotoxicity and infiltration of T cells into non-target organs, compared with unmodified IL-12. Both innate and adaptive immune compartments were dramatically activated and recognized diverse tumor antigens after CBD-IL-12 CAR-T cell treatment. Combination immunotherapy of CBD-IL-12 CAR-T cells and immune checkpoint inhibitors eradicated large tumors in an established prostate cancer model, without pre-conditioning chemotherapy. The therapy generated anti-tumor immunological memory. CBD-fusion to potent yet toxic payloads of CAR-T therapy may remove obstacles to their clinical translation towards elimination of solid tumors. ### Competing Interest Statement K.S., Y.A., A.G.C, J.K.L. and J.I. are inventors on a provisional patent application covering the technology described in this work. K.S. and J.I. are inventors on international patent applications covering CBD-IL-12 protein therapy. J.K.L. is an inventor on International Patent Applications related to STEAP1 CAR-T cells. J.K.L. holds equity in, serves on the scientific advisory board of, and receives research funding from PromiCell Therapeutics. J.K.L. is a consultant for Lyell Immunopharma. J.I. is a founder and shareholder in Arrow Immune Inc. J.I. is a scientific advisor of Libo Pharma Corp.