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.