Anti-MSLN CAR-T (Zhujiang Hospital of Southern Medical University)

Mesothelin (MSLN), overexpressed in >90 % of malignant pleural mesotheliomas and pancreatic adenocarcinomas, with minimal expression in normal tissue, represents an ideal immunotherapeutic target. Here, we generated universal umbilical cord blood (UCB)-derived anti-MSLN CAR-Ts using a CAR construct comprising a single-chain variable fragment linked to CD8α H/TM, 4-1BB and CD3ζ signaling domain. CAR expression, T cell subsets, and exhaustion markers were characterized via flow cytometry. Cytotoxicity against MSLN-positive NCI-H2452 and MSLN-negative A431 cells was assessed via alamarBlue assay (effector-to-target ratio, 0.5:1-5:1; for 6-24 h), and cytokine secretion (IL-2, IFN-γ, TNF-α, IL-6) was quantified using enzyme-linked immunosorbent assay. In vivo anti-tumor activity was evaluated in female NOD/SCID/γc^-/^- xenograft models, with tumor burden monitored via bioluminescence imaging and survival through the Kaplan-Meier method. Experiments were performed in triplicate, with data analyzed using GraphPad Prism. CAR transduction efficiency in UCB mononuclear cells reached 88.27 %± 7.29 % by day 12. Compared to day 3 cells, they retained higher memory cell proportions (Tscm: 28.16 %→21.39 %), and lower exhaustion marker expression than peripheral blood T (PB-T) cells (APC anti-human CD269: 8.41 %±0.64 % vs 4.42 %±1.25 %; PE antihuman CD366: 5.48 %±0.76 % vs 3.58 %±0.89 %; p < 0.05). UCB-derived anti-MSLN CAR-T cells exhibited dose- and time-dependent cytotoxicity against NCI-H2452 cells, with minimal activity against A431 controls. Moreover, cytokine secretion was significantly elevated compared to that in PB-T co-cultures. In xenograft models, CAR-T cells significantly prolonged median survival. Thus, UCB-derived anti-MSLN CAR-Ts showed reduced exhaustion, increased memory T cell proportions, and enhanced anti-tumor efficacy in vitro and in vivo.