Autocrine activity of engineered IL-33 mRNA enhances adoptive T-cell therapy for peritoneal carcinomatosis and synergizes with IL-12 mRNA.

Rationale: Peritoneal carcinomatosis (PC) remains a major clinical challenge with limited therapeutic options across tumor types. Adoptive cell therapy (ACT) with tumor-specific T cells offers promise, but its efficacy is often impaired by the immunosuppressive tumor microenvironment (TME). Intraperitoneal ACT is under investigation to improve its effectiveness against metastases within the peritoneal cavity. IL-33, a cytokine of the IL-1 family, plays dual roles in immunity and inflammation and may enhance antitumor responses. We evaluated whether IL-33 mRNA-engineered T cells improve ACT efficacy in murine PC models and assessed potential synergy with IL-12 mRNA. Methods: OT.I, PMEL-1, and CEA-specific CAR T cells were electroporated with mRNA encoding IL-33, IL-12, or an IL-33 mutein. In vitro assays measured cytokine production and cytotoxicity. RNA-seq was performed to analyze transcriptomic changes following IL-33 mRNA electroporation. ST2(-/-) T cells were used to evaluate the role of IL-33 receptor expression on transferred T cells versus host cells. In vivo studies in murine PC models assessed survival and immune responses using ELISA, ELISpot, and flow cytometry. Results: IL-33 mRNA-electroporated OT.I T cells exhibited enhanced IFN-γ expression in a ST2-dependent, T cell-intrinsic manner. In vivo, IL-33-engineered T cells significantly improved survival in PC models. IL-33 reshaped the TME by increasing infiltration of innate lymphoid cells and eosinophils while reducing neutrophils. Engineering T cells with a stabilized IL-33 mutein further enhanced antitumor activity. Co-electroporation of IL-33 mutein and IL-12 mRNA in PMEL-1 T cells led to synergistic increases in IFN-γ production, cytotoxicity, and long-term memory, resulting in superior tumor control and protection upon rechallenge. These findings were confirmed using IL-33 mutein/IL-12 mRNA-electroporated CEA CAR T cells in peritoneal tumor models. Conclusions: IL-33 enhances ACT efficacy by promoting IFN-γ expression via autocrine ST2 signaling and by modulating the TME. The IL-33 mutein improves cytokine stability and antitumor activity, while combination with IL-12 yields synergistic effects. This strategy holds promise for enhancing ACT in peritoneal carcinomatosis.