Collagen-binding IL-12-armored STEAP1 CAR-T cells for advanced prostate cancer

Metastatic castration-resistant prostate cancer (mCRPC) remains an incurable and immunologically cold solid malignancy. Six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is highly expressed in over 85% of mCRPC tumors and represents an attractive therapeutic target. Although chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematologic cancers, its efficacy in solid tumors, including prostate cancer, has been limited by the immunosuppressive tumor microenvironment (TME) and heterogeneous antigen expression. Interleukin-12 (IL-12) has the potential to overcome these barriers by activating and recruiting immune cells into tumors and promoting epitope spreading to counter antigen heterogeneity. While armored CAR-T cells engineered to produce IL-12 have been developed, further refinement is needed to optimize both potency and safety. Autologous IL-12-producing T cell therapy has previously shown clinical activity in melanoma, but systemic toxicity constrained its use, indicating that IL-12 secreted by CAR-T cells, though locally produced, can still diffuse into circulation. Here, we present STEAP1-directed CAR-T cells engineered to conditionally secrete a collagen-binding domain-IL-12 fusion protein (CBD-IL-12) upon antigen engagement. We demonstrate that fusing IL-12 to a CBD markedly enhances its retention within prostate tumors while limiting systemic spread in mice. As a result, intra-tumoral levels of IFN-gamma, CXCL9, and GM-CSF remained comparably high to those induced by IL-12, yet without associated elevations in serum alanine aminotransferase (ALT) or off-target T cell infiltration in healthy organs. Flow cytometry revealed increased infiltration of T cells, NK cells, and cross-presenting dendritic cells, together with reduced monocytic myeloid-derived suppressor cells, following treatment with CBD-IL-12-expressing STEAP1 CAR-T cells. Immunohistochemistry and spatial transcriptomic analysis confirmed increased immune infiltrates and activation of IL-12 pathway and antigen processing and presentation by major histocompatibility complex-I in the CBD-IL-12 CAR-T treated tumor. Further, tertiary lymphoid structure-related chemokine and chemokine receptors including cxcr4, cxcr5, cxcl12 and cxcl13 as well as co-stimulatory molecules such as cd80, cd86, cd40 and tnfsf4 were upregulated in the tumor. When combined with anti-PD-1 and anti-CTLA-4 antibodies, CBD-IL-12 armored CAR-T cells eradicated established prostate tumors in mice without preconditioning. The CAR-T therapy generated durable anti-tumor immune memory to STEAP1 and other antigens. Our findings suggest that CBD fusion can localize potent but toxic immunomodulators such as IL-12 to the tumor site, offering a promising strategy to improve the safety and effectiveness of CAR-T therapies for solid tumors.