Glycoengineering CAR-T cells to overcome galectin-3-mediated immunosuppression

Chimeric antigen receptor (CAR)-T cell therapy has transformed treatment for relapsed or refractory B-cell malignancies; however, limited <i>in vivo</i> persistence and treatment-limiting toxicities continue to constrain durable efficacy. Because T cell glycan signatures and related galectin-binding properties impact their effector function, we postulated that CAR-T cells similarly displayed signature glycan features that govern their vulnerability to immunosuppressive galectins. In this report, public data mining, galectin-binding and glycosyltransferase expression assessments and glycomics showed that galectin (Gal)-3 was elevated in lymphoma-associated microenvironments and that anti-CD19 CAR-T cells displayed abundant Gal-3-binding glycans, reduced expression of the Gal-3-inhibitory enzyme α2,6-sialyltransferase 1 (<i>ST6GAL1</i>), and heightened susceptibility to Gal-3-mediated immunoregulation. To further explore this association, we enforced <i>ST6GAL1</i> expression in anti-CD19 CAR-T cells and found that Gal-3-binding was obstructed and Gal-3-mediated cell death and IL-5-induction were reversed. Enforcing <i>ST6GAL1</i> in CAR-T cells did not weaken tumoricidal activity and significantly improved anti-tumor responses and <i>in vivo</i> persistence. Collectively, this study identifies Gal-3 as a key extrinsic suppressor of CAR-T cell function and establish targeted cell surface α2,6 sialylation as a strategy to enhance CAR-T cell resistance to galectin-rich immunosuppressive microenvironments.