VHH-based antigen recognition programs Claudin18.2 CAR-T cells for durable activity in low-antigen solid tumors

Abstract Background Claudin18.2 is a clinically validated target for gastric and gastroesophageal junction adenocarcinoma. However, the antigen heterogeneity, low surface density, and issues related to scFv-associated conformational instability and tonic signaling may limit the durability and therapeutic window of Claudin18.2-directed CAR-T cell therapy. Therefore, improving the design of CAR-T cells targeting Claudin18.2 could enhance their effectiveness in treating these cancers. Methods We generated third-generation Claudin18.2 CAR-T cells using either a humanized camelid single-domain antibody (VHH) or a conventional scFv as the antigen-recognition module, maintaining an identical CAR backbone for both constructs. To evaluate their performance, we used a gastric cancer cell-line panel with graded Claudin18.2 expression and corresponding xenograft models. We assessed CAR-T cell activity by measuring antigen-dependent cytotoxicity, cytokine secretion (IFN-γ and TNF-α), and reactivity toward antigen-negative targets. In vivo, we examined tumor control, T-cell infiltration, and CD8⁺ T-cell enrichment, and assessed toxicity. Additionally, we investigated the stability of CAR expression, T-cell expansion, and exhaustion-associated phenotypes. Structural modeling and molecular dynamics simulations were performed to evaluate the stability of the binding interface of the VHH–Claudin18.2 complex relative to the scFv–Claudin18.2 complex. Results VHH-based CAR-T cells exhibited superior antigen-dependent activity compared to scFv-based CAR-T cells. VHH-CAR-T cells maintained higher cytotoxicity and IFN-γ and TNF-α secretion even under low antigen density and displayed minimal reactivity against antigen-negative targets. In vivo, VHH-CAR-T cells achieved faster and more durable tumor control, with enhanced intratumoral T-cell infiltration and CD8⁺ T-cell enrichment, without any overt toxicity. Mechanistically, the use of a VHH recognition domain was associated with better preserved surface CAR expression after antigen engagement, expanded more robustly, displayed fewer exhaustion-associated phenotypes, had a higher proportion of central memory T cells, and showed transcriptional enrichment of proliferative and effector programs with restrained exhaustion signatures. Structural modeling and molecular dynamics simulations further confirmed that the VHH–Claudin18.2 binding interface was more stable than that of scFv–Claudin18.2. Conclusion Replacing scFv with VHH is associated with enhanced functional performance, persistence, and therapeutic efficacy of Claudin18.2-directed CAR-T. These findings support the further development and translation of VHH-based CAR-T strategies for solid tumor treatment.