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

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.

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.

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.

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.