Nanobody-Directed CEA-Targeting CAR T Cells Eliminate Gastrointestinal Cancer Xenografts

Gastrointestinal cancers (GIC), including gastric cancers and colorectal cancers, are among the leading causes of cancer-related deaths worldwide. Metastatic gastric cancers and colorectal cancers often develop resistance or fail to respond to current therapies. Adoptive T-cell immunotherapy, especially with T cells expressing chimeric antigen receptors (CAR) targeting CD19, has revolutionized leukemia treatment. However, the development of CAR T-cell therapy for GICs is still in progress. In this study, we used a sequentially tumor-selected antibody and antigen retrieval system to isolate a nanobody that directs CAR T cells to attack gastrointestinal tumor cells in preclinical mouse models. The nanobody VHHB30 specifically binds to the N-terminal (nonglycosylated) domain of carcinoembryonic antigens (CEA). The resulting VHHB30-CAR T cells (CEACAR T cells) exhibited cytotoxicity against both colorectal cancer and gastric cancer cell lines in vitro in a CEA-dependent manner. Moreover, third-generation CEACAR T cells showed enhanced antitumor activity compared with second-generation CEACAR T cells. Furthermore, in vivo studies demonstrated that the CEACAR T cells eradicated various colorectal and gastric tumor xenografts in preclinical mouse models, highlighting a promising approach for CAR T-cell therapy development in GICs through unbiased in vivo selection of potent VHH binders.