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 MSLNHigh/PD-L1High autologous PDOs.

However, only anti-MSLN-CAR5-T cells were able to sustain potent killing activity against PD-L1High 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.