Exploring Glycomic Strategies to Enhance CAR-T Cell Efficacy Against Diffuse Large B Cell Lymphoma
Abstract
Introduction and Objective. Diffuse large B cell lymphoma (DLBCL) is the most common and fast-growing B cell lymphoma and, while treatments continue to improve, many patients still remain unresponsive to therapy. Patients who do not respond to treatment or have disease relapse typically survive less than 6- 12 months. Scientists worldwide are striving to develop safer and more effective therapies. Chimeric antigen receptor (CAR)-T cell therapy offers a promising option for such patients, involving gene-modified T cells produced outside the body for longer-lasting anti-tumor effectiveness when reintroduced into the patient. However, CAR-T cell therapy can be improved by enhancing tumor kill efficiency and in vivo persistence. T cells express surface carbohydrate chains (glycans) that can bind to glycan-binding proteins (lectins), hindering their function and survival. Experimental evidence suggests that ß-galactoside-binding galectins are elevated in lymphoma patients and, when bound to T cell surface glycans, can suppress their anti-tumor activity and/or lifespan. We hypothesize that galectins significantly compromise CAR-T cell therapy effectiveness and modifying CAR-T cell surface glycans to evade galectin binding could enhance their persistence and activity in vivo. Methods. We analyzed Gal-1, -3, and -9 levels in DLBCL patient serum compared to healthy controls. We investigated the binding ability of common immunosuppressive galectins (rhGal-1, -3, -9) on control naïve T cells, on control and on CAR-T cells. We examined CAR-T cell glycans via MALDI-TOF MS analysis and assessed glycosyltransferase expression involved in synthesizing galectin-binding glycans using RT-qPCR. Validation was done via flow cytometry with plant lectins and compared to CAR-T cell controls, evaluating Gal-1 and -3 binding and cell death in ST6Gal1 overexpressing CAR-T cells. All methods were conducted at least 3 times and analyzed for statistical significance using Student’s paired t-test (*P ≤ .05, **P ≤ .001). Results. We found that Gal-3 levels were higher in patients with DLBCL compared to healthy individuals. Our binding assay showed strong binding of CAR-T cells to Gal-1, -3, and -9, correlating with increased susceptibility to Gal-3-induced apoptosis. Furthermore, we observed suppressed ST6Gal1 gene expression in CAR-T cells that is inversely related to Gal-3-binding in flow cytometry assessments. MALDI-TOF MS N-glycan analysis revealed elevated levels of tri-antennary N-glycan species, known to enhance Gal-3-binding. Overexpression of ST6Gal1 in CAR-T cells reduced Gal-3-binding and blocked Gal-3-induced apoptosis compared to control CAR-T cells, suggesting that ST6Gal1 expression and α2,6 sialylation of surface glycans may prolong CAR-T cell longevity. Conclusions-Implications. The data suggest that understanding a human T cell's surface glycome can provide strategies to evade immunosuppression and boost CAR-T cell persistence and/or anti-tumor functional activity.
Keywords
CAR-T cell, DLBCL, Galectin-3
Presentation Type
Oral Presentation
Exploring Glycomic Strategies to Enhance CAR-T Cell Efficacy Against Diffuse Large B Cell Lymphoma
Introduction and Objective. Diffuse large B cell lymphoma (DLBCL) is the most common and fast-growing B cell lymphoma and, while treatments continue to improve, many patients still remain unresponsive to therapy. Patients who do not respond to treatment or have disease relapse typically survive less than 6- 12 months. Scientists worldwide are striving to develop safer and more effective therapies. Chimeric antigen receptor (CAR)-T cell therapy offers a promising option for such patients, involving gene-modified T cells produced outside the body for longer-lasting anti-tumor effectiveness when reintroduced into the patient. However, CAR-T cell therapy can be improved by enhancing tumor kill efficiency and in vivo persistence. T cells express surface carbohydrate chains (glycans) that can bind to glycan-binding proteins (lectins), hindering their function and survival. Experimental evidence suggests that ß-galactoside-binding galectins are elevated in lymphoma patients and, when bound to T cell surface glycans, can suppress their anti-tumor activity and/or lifespan. We hypothesize that galectins significantly compromise CAR-T cell therapy effectiveness and modifying CAR-T cell surface glycans to evade galectin binding could enhance their persistence and activity in vivo. Methods. We analyzed Gal-1, -3, and -9 levels in DLBCL patient serum compared to healthy controls. We investigated the binding ability of common immunosuppressive galectins (rhGal-1, -3, -9) on control naïve T cells, on control and on CAR-T cells. We examined CAR-T cell glycans via MALDI-TOF MS analysis and assessed glycosyltransferase expression involved in synthesizing galectin-binding glycans using RT-qPCR. Validation was done via flow cytometry with plant lectins and compared to CAR-T cell controls, evaluating Gal-1 and -3 binding and cell death in ST6Gal1 overexpressing CAR-T cells. All methods were conducted at least 3 times and analyzed for statistical significance using Student’s paired t-test (*P ≤ .05, **P ≤ .001). Results. We found that Gal-3 levels were higher in patients with DLBCL compared to healthy individuals. Our binding assay showed strong binding of CAR-T cells to Gal-1, -3, and -9, correlating with increased susceptibility to Gal-3-induced apoptosis. Furthermore, we observed suppressed ST6Gal1 gene expression in CAR-T cells that is inversely related to Gal-3-binding in flow cytometry assessments. MALDI-TOF MS N-glycan analysis revealed elevated levels of tri-antennary N-glycan species, known to enhance Gal-3-binding. Overexpression of ST6Gal1 in CAR-T cells reduced Gal-3-binding and blocked Gal-3-induced apoptosis compared to control CAR-T cells, suggesting that ST6Gal1 expression and α2,6 sialylation of surface glycans may prolong CAR-T cell longevity. Conclusions-Implications. The data suggest that understanding a human T cell's surface glycome can provide strategies to evade immunosuppression and boost CAR-T cell persistence and/or anti-tumor functional activity.