VHH-based CAR-T cells targeting Claudin 18.2 show high efficacy in pancreatic cancer models.

Background: Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers, with a 5-year survival rate below 10%, largely due to late-stage diagnosis and the limited effectiveness of conventional therapies such as surgery, chemotherapy, and radiation. Claudin 18.2 (CLDN18.2)has emerged as a promising target for PDAC. While single-chain variable fragment (scFv)-based CAR-T cells targeting CLDN18.2 have demonstrated therapeutic potential, CAR-T cells engineered with variable heavy-chain-only domains (VHH) exhibit superior efficacy, highlighting the advantages of VHH-based constructs in targeting this antigen. However, the therapeutic efficacy of anti-CLDN18.2 VHH-CAR-T cells remains to be fully elucidated, as previous studies have not comprehensively characterized Methods: To characterize the therapeutic potential of anti-CLDN18.2 VHHs, we employed phage display technology to screen a VHH library, resulting in the identification of three positive clones. These candidates were further evaluated and ranked based on binding affinity and multi-round cytotoxicity in Chimeric antigen receptor T (CAR-T) cell models. To reduce immunogenicity, the lead VHH was humanized. VHH-CAR-T cells incorporating this humanized domain were assessed through Results: High-affinity anti-CLDN18.2 VHHs from phage libraries and engineered CAR-T cells using HM2, a humanized VHH, as the antigen-binding domain were successfully identified. Notably, HM2-CAR-T cells demonstrated potent and sustained cytokine secretion and cytotoxic activity against CLDN18.2-expressing tumor cells Conclusion: This study establishes an effective framework for developing CLDN18.2-specific VHHs and demonstrates their successful integration into CAR-T cell therapy. The humanized HM2-CAR-T cells not only maintain high antigen specificity but also exhibit strong effector functions and pronounced antitumor activity in preclinical models. These findings support the clinical promise of VHH-based CAR-T cells as a next-generation immunotherapy for CLDN18.2-expressing malignancies, particularly PDAC, where effective treatment options remain limited.