Abstract LB-A007: Non-viral site-directed hYP218 CAR T cells for mesothelin-expressing solid tumors

Abstract CAR T cell therapy has emerged as a transformative modality in cancer immunotherapy. However, its efficacy in solid tumors remains limited due to challenges like an immunosuppressive tumor microenvironment (TME), high manufacturing costs, and toxicities such as cytokine release syndrome (CRS). Mesothelin, a tumor-associated antigen highly expressed in ovarian, pancreatic, and mesothelioma tumors, is a promising target for CAR T-cell therapy. However, traditional viral-based CAR T-cell production poses concerns, including insertional mutagenesis, excessive cytokine release, and T-cell exhaustion. We hypothesized that a non-viral, site-directed approach to generate mesothelin-specific CAR T cells would reduce exhaustion, improve cytotoxicity, and minimize risks like CRS. Using a CRISPR/Cas9 platform, we engineered non-viral hYP218 CAR T cells by inserting the CAR cassette into the AAVS1 and PD1 genomic loci. Peripheral blood mononuclear cells (PBMCs) from healthy donors were activated and nucleofected with linear DNA templates and Cas9-gRNA complexes. CAR expression, PD1 disruption, and functionality were analyzed. Non-viral PD1-integrated hYP218 CAR T cells demonstrated efficient CAR expression and robust PD1 knockout. Anti-tumor efficacy was evaluated in vitro using mesothelin-positive ovarian (OVCAR8), mesothelioma (RH63), and pancreatic (KLM) tumor models, as well as in xenografts in NSG mice. In vitro non-viral hYP218 CAR T cells achieved a near complete tumor lysis and produced pro-inflammatory cytokines, including IFN-γ and TNF-α. In vivo, non-viral PD1-hYP218 CAR T cells induced complete tumor regression in the OVCAR8 and RH63 models. In the aggressive KLM model, partial tumor regression was observed, comparable to that observed with lentiviral hYP218 CAR T cells. AAVS1-integrated hYP218 CAR T cells demonstrated slightly lower antitumor efficacy than PD1-integrated and lentiviral CAR T cells. Mechanistically, PD1-hYP218 CAR T cells maintained a naïve-like phenotype and reduced expression of exhaustion markers. This study highlights the potential of non-viral hYP218 CAR T cell therapy as a scalable, cost-effective, and safer strategy for treating mesothelin-expressing solid tumors. Citation Format: Sameer Mir, Abhilash A. Venugopalan, Yosef A. Charkatli, Raj Chari, Qun A. Jiang, Raffit A. Hassan. Non-viral site-directed hYP218 CAR T cells for mesothelin-expressing solid tumors abstract. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-A007.