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

Lee Seng Lau¹Maria Suarez¹Brandon Fernandez¹Joseph Souchak¹Aristotelis Antonopoulos²Nan Hu³Maria M Abreu⁴Guenther Koehne⁴Anne Dell²Stuart Haslam²Avery D Posey, Jr.⁵Charles J Dimitroff^1*

• ¹Herbert Wertheim College of Medicine, Florida International University, Miami, United States
• ²Imperial College London, London, United Kingdom
• ³Florida International University Robert Stempel College of Public Health and Social Work, Miami, United States
• ⁴Baptist Health South Florida Inc, Coral Gables, United States
• ⁵University of Pennsylvania Perelman School of Medicine, Philadelphia, United States

Chimeric antigen receptor (CAR)-T cell therapy has transformed treatment for relapsed or refractory B-cell malignancies; however, limited in vivo 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 (ST6GAL1), and heightened susceptibility to Gal-3–mediated immunoregulation. To further explore this association, we enforced ST6GAL1 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 ST6GAL1 in CAR-T cells did not weaken tumoricidal activity and significantly improved anti-tumor responses and in vivo 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.