GLOBO H targeting CAR T Cells: generation and preclinical activity in gastrointestinal cancer cell lines

Background Chimeric Antigen Receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies, but its efficacy against solid tumors remains limited by several barriers, including the difficulty of identifying tumor-specific antigens that minimize on-target, off-tumor toxicities. Globo H is a tumor-associated glycosphingolipid expressed in several epithelial malignancies, including gastrointestinal cancers, and has limited expression in normal tissues. We investigated the preclinical activity of MBr1-derived Globo H-specific CAR T cells. Methods Peripheral blood T cells from healthy donors were engineered using SFG retroviral vectors encoding second-generation CARs comprising an MBr1-derived single-chain variable fragment, a FLAG tag, a CD8α hinge and transmembrane region, a CD3ζ signaling domain, and either CD28 (CAR CD28) or 41BB (CAR 41BB) costimulatory domain. Following ex-vivo expansion with IL-7 and IL-15, the products were characterized for antigen-dependent cytotoxicity, differentiation and inhibitory-receptor expression, including during six serial NCI-N87 tumor-cell challenges. Antitumor activity was evaluated in NSG mice bearing NCI-N87 gastric cancer or OE33 esophageal adenocarcinoma xenografts. Results Both CAR constructs recognized and killed Globo H–positive tumor cell lines, and maintained cytotoxic activity over six serial NCI-N87 challenges. Despite comparable cytotoxicity, the constructs exhibited different phenotypic profiles: CAR 41BB T cells were enriched for less differentiated populations, whereas CAR CD28 showed greater effector differentiation and more pronounced inhibitory-receptor co-expression, particularly among CD4 + CAR + cells. CAR 41BB T cells delayed tumor growth in NCI-N87 and OE33 xenografts without overt toxicity under the conditions tested. CAR CD28 administration was associated with severe systemic toxicity in the NCI-N87 model. Conclusion These findings provide preclinical evidence of antigen-dependent antitumor activity of Globo H-targeted CAR T cells in gastrointestinal cancer models. While the 4-1BB-based construct showed a favorable phenotypic profile and antitumor activity without overt toxicity in the models tested, the severe systemic toxicity observed with the CD28-based construct highlights the need for further investigation into the biological determinants of toxicity.