Next-generation of mesothelin-targeted CAR-T cells secreting anti-PD-L1 scFv for potent immunotherapy against 3D patient-derived colorectal cancer organoids

Chimeric antigen receptor (CAR)-T cell therapies have revolutionized the landscape of cancer treatment, particularly in hematological malignancies. However, their successful translation to solid tumors remains limited by several barriers, including immunosuppressive tumor microenvironment and on-target/off-tumor toxicity. One promising strategy to enhance efficacy of CAR-T cells is the rational selection of tumor-specific antigens coupled with engineering strategies that incorporate localized immune modulation, such as CAR-T cells secreting immune checkpoint-blocking anti-PD-L1 scFv. To better model therapeutic responses, we established a dynamic real-time autologous co-culture platform integrating colorectal cancer (CRC) patient-derived organoids (PDOs) and CAR-T cells to assess infiltration, persistence, and cytotoxicity ex vivo . Although early clinical trials of mesothelin (MSLN)-directed CAR-T cells have demonstrated high safety, their anti-tumor efficacy remains modest, highlighting the need for improved constructs. Therefore, we engineered anti-MSLN-CAR4-T cells using fully human anti-MSLN scFv linked to a triple costimulatory backbone (CD28, 4-1BB, and CD27) fused to CD3ζ, and anti-MSLN-CAR5-T cells, incorporating an additional anti-PD-L1 scFv. Both CAR4- and CAR5-T cells exhibited comparable cytotoxic efficacy against MSLN High /PD-L1 High autologous PDOs. However, only anti-MSLN-CAR5-T cells were able to sustain potent killing activity against PD-L1 High PDOs with high IFN-γ/cytolytic cytokine production at a low effector-to-target ratio (5:1), reflecting improved resilience to PD-L1-mediated suppression. Finally, our 14-day ex vivo CRC-PDOs/CAR-T platform provides a promising rapid and translational tool for tumor-associated antigen validation, streamlined PDO isolation, autologous CAR-T cytotoxicity testing, and personalized immunotherapy optimization in solid tumors. Schematic overview of the experimental strategy for personalized evaluation of anti-mesothelin (MSLN)-specific CAR-T cells using patient-derived colorectal cancer organoids (CRC-PDOs). Surgically resected tumor and matched normal tissues are processed to establish 3D-PDO cultures that retain the architectural complexity, cellular heterogeneity, and tumor-associated antigen (TAA) landscape of the original tumor. In parallel, autologous T cells are isolated from patient blood, activated, and engineered via lentiviral transduction to express MSLN-targeting CAR4 or CAR5 with additionally secretes an anti-PD-L1 scFv. CAR-T cells are subsequently co-cultured with patient-matched PDOs to assess tumor killing dynamics, cytokine secretion, and persistence/exhaustion profiles under both short-term and long-term immune pressure. This integrated autologous PDO-CAR-T platform models patient-specific antigen burden and immune resistance mechanisms, providing a translational system for preclinical efficacy testing and functional stratification of individuals who may benefit from MSLN-directed cell therapy.