CAR T cells engineered via mRNA exhibit enhanced targeting of colorectal cancer

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematologic malignancies, achieving unprecedented remission rates in patients with refractory leukemias, lymphomas, and multiple myeloma. However, translating CAR T therapy to solid tumors remains challenging due to intratumoral heterogeneity, an immunosuppressive tumor microenvironment, and limited tumor-specific antigens. In this work, we employed a lipid nanoparticle (LNP)-based mRNA delivery platform to transiently generate CAR T cells targeting Claudin-6 (CLDN6), an oncofetal tight junction protein aberrantly expressed in multiple tumors, including colorectal cancers. To enhance antitumor efficacy, we combined CAR T cell therapy with localized LNP-mediated delivery of TRAIL mRNA (LNP-TRAIL). We evaluated this dual approach in a subcutaneous xenograft model using Colo 205-GFP+Luc+ human colorectal cancer cells in NSG mice. Tumor growth was monitored by quantitative bioluminescence imaging and caliper-based tumor volume measurements. Tumor-infiltrating lymphocytes (TILs) were profiled for activation markers, proliferation, cytotoxic mediators (IFN-y, Granzyme B), and immune checkpoint expression (PD-1, CTLA-4) to assess T cell exhaustion. To examine translational potential, we performed ex vivo assays using freshly resected human colorectal tumor fragments treated with CAR-T cells. The resulting CAR-T cells generated via LNP-mRNA exhibited robust activation, proliferation, and cytotoxicity against CLDN6+ colorectal cancer cells both in vitro and in vivo. Combined CAR-T and intratumoral LNP-TRAIL therapy significantly reduced tumor burden, enhanced T cell infiltration and effector function, and reversed markers of T cell exhaustion. Ex vivo co-culture with primary human tumor samples further confirmed the improved cytotoxic and immunostimulatory effects of the dual strategy. These results establish a modular mRNA nanoplatform that integrates transient CAR expression with LNP-TRAIL delivery to overcome major barriers to CAR-T efficacy in solid tumors, highlighting its translational potential for CLDN6+ malignancies.