NUGC-4

Background

This study establishes a preclinical in vivo model that replicates the on-target off-tumor toxicity of CLDN18.2-directed CAR-T cells. Leveraging shared CLDN18.2 epitopes and expression between humans and mice, the authors show that CLDN18.2 CAR-T cells recognize healthy gastric mucosa, causing mucosal destruction and lethal toxicity in mice, independent of CAR binder, co-stimulatory domain, design, or tumor model. They further demonstrate that a Boolean-logic AND-gate strategy targeting CLDN18.2 and mesothelin mitigates toxicity while preserving anti-tumor efficacy, offering insights into balancing efficacy and safety in gastric cancer immunotherapy.

Key Findings

CLDN18.2-directed CAR-T cells caused rapid tumor regression but also severe, lethal on-target off-tumor toxicity in mice due to recognition of CLDN18.2 in healthy gastric glandular epithelium, with extensive CD3+ T cell infiltration and loss of glandular mucosa. Toxicity was independent of CAR binder (Zolbetuximab or hu8e5), co-stimulatory domain (CD28 or 4-1BB), scFv orientation, transmembrane domain, diet, and tumor model, and correlated with peripheral CAR-T cell expansion rather than tumor burden. A narrow therapeutic dose window was observed: 3x10^4 cells were ineffective, 3x10^5 cells gave partial tumor control with minimal toxicity, while >=1x10^6 cells were lethal. A LINK Boolean-logic AND-gate CAR targeting CLDN18.2 and mesothelin selectively killed double-positive tumor cells in vitro and in vivo, avoiding gastric toxicity and enabling durable tumor clearance in B2mKO NSG mice.

Contribution

Creative Bioarray supplied the GSU and NUGC-4 gastric cancer cell lines used in this study. These CLDN18.2-expressing lines were central to in vitro cytotoxicity and epitope-mapping assays and to establishing the subcutaneous xenograft mouse models in which CAR-T cell efficacy and on-target off-tumor gastric toxicity were evaluated.

Background

This study identifies MUC17, a membrane-bound mucin overexpressed in gastric and pancreatic cancers with limited normal tissue expression, as a promising CAR-T target. Researchers developed MUC17-specific CAR-T cells with 4-1BB/CD3ζ signaling and two scFv orientations. In vitro, these CAR-T cells showed potent antigen-specific cytotoxicity, cytokine secretion, and enriched central memory phenotypes. In NSG xenograft models, MUC17 CAR-T cells suppressed GSU gastric and ASPC-1 pancreatic tumor growth without detectable toxicity. The findings support MUC17 as a clinically relevant target for solid tumor immunotherapy.

Key Findings

MUC17 expression was largely restricted to small intestine and upregulated in gastric, colon, and pancreatic cancers; gastric cancer cell lines GSU and NUGC4 expressed high MUC17. MUC17-specific CAR-T cells with either VH-VL or VL-VH scFv and 4-1BB/CD3ζ showed potent cytotoxicity against GSU, NUGC4, and KATO-3, with killing abolished in CRISPR MUC17-knockout targets. Compared with mesothelin CAR-T cells, MUC17 CAR-T cells displayed comparable or superior cytotoxicity, increased central memory subsets, reduced TEMRA, and robust TNF-α, IL-2, IFN-γ, and Granzyme B secretion. In 3D NUGC4 spheroids, MUC17 CAR-T cells outperformed mesothelin CAR-T cells. In NSG mice, MUC17 CAR-T cells controlled GSU tumors, improved survival and body weight, and at lower dose sustained tumor control up to 90 days. They also controlled ASPC-1 pancreatic tumors better than mesothelin CAR-T cells. Histology indicated MUC17 apical localization in small intestine, spatially separated from circulating CAR-T cells, suggesting limited off-tumor toxicity.

Contribution

Creative Bioarray provided GSU and NUGC4 gastric cancer cell lines, which were used as MUC17-expressing target models for in vitro cytotoxicity, cytokine, phenotype, spheroid, and in vivo xenograft experiments, supporting preclinical validation of MUC17 CAR-T cells. These authenticated cell lines enabled robust target validation and efficacy testing for MUC17-directed immunotherapy.