Abstract 1351: IL-18 secreting CAR-T cells uncover divergent immune mechanisms of antigen-heterogeneous solid tumor clearance.

Abstract CAR-T therapy has improved the outcome for patients with refractory hematological malignancies, however this success has not translated to solid tumors, largely due to an immunosuppressive tumor microenvironment (TME) and heterogenous antigen expression. CAR-Ts capable of reshaping the TME and recruiting endogenous immune cells, could improve outcomes by eradicating cells with low or absent expression of the CAR targeted antigen. However, the biological mechanisms necessary to induce endogenous anti-tumor T cell responses remain unclear. Previous studies have reported that CAR-T cells engineered to secrete IL-18 induced immune priming and an endogenous anti-tumor response. We sought to study tumor clearance rates and endogenous immune responses induced by control and IL-18 secreting CAR-T cells by administering murine CAR-Ts targeting human CD19 (huCD19.28.z-CARs) to mice bearing a mixture of B16F10-Ova+huCD19+/- cells. We profiled endogenous lymphoid and myeloid compartments, as well as engineered CAR-Ts, during tumor clearance within lymphoid and tumor tissues at 14 and 26 days post CAR-T treatment. Additionally, we assessed the induction of CD8 T cells reactive to tumor-derived peptides using ELISPOT. IL-18 secreting CARs demonstrated robust antigen-heterogenous tumor control compared to the control. By day 26 post treatment, tumor measurements in IL-18 secreting CAR-T treated mice were undetectable, while control tumors averaged 680 mm3. Despite improved tumor control, IL-18 secreting CAR treated mice generated fewer CD8s reactive to Ova, a tumor neoantigen, compared to the CAR only control. Instead, IL-18 secretion induced a strong type II interferon response, skewing effector differentiation of lymphocytes and initiating expansion and activation of myeloid cells, including macrophages and neutrophils. This work reveals that enhanced clearance of heterogenous solid tumors by cytokine secreting CAR-Ts may occur through distinct immunological programs. CAR-Ts secreting IL-18 cleared mixed antigen expressing tumors most effectively, but induced a low/absent anti-tumor CD8 T cell response. In contrast, CAR-Ts alone failed to control tumors, but effectively induced priming of CD8s towards a tumor expressed neoantigen. These findings suggest activated innate myeloid cells as key mediators in eradicating antigen negative cells within a heterogeneous tumor and illustrate distinct biology between tumor clearance via innate immune responses versus induction of persistent adaptive immunity to tumor associated antigens. Taken together, this study can inform next-generation CAR-T design by underscoring the importance of balancing effector potency with durable endogenous anti-tumor immunity to engineer sustained efficacy in solid tumors. Citation Format: Audre May, Elena Sotillo, Peng Xu, Aarti Malhotra, Katie Ho, Crystal Mackall, . IL-18 secreting CAR-T cells uncover divergent immune mechanisms of antigen-heterogeneous solid tumor clearance abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1351.