ST6GAL1 enhanced CAR-T cells improve persistence and antitumor efficacy in DLBCL

Diffuse large B cell lymphoma (DLBCL) is the most common and aggressive B cell lymphoma, with poor outcomes for patients who relapse or are refractory to treatment. Chimeric antigen receptor (CAR)-T cell therapy offers a promising therapeutic option, yet limited persistence and functional impairment remain major barriers to durable responses. Galectin (Gal)-3, a carbohydrate-binding protein highly expressed in the DLBCL microenvironment, has been implicated in T cell dysfunction, but its role in CAR-T cell impairment has not been fully defined. We hypothesized that Gal-3 binds to CAR-T cell surface glycans, promoting apoptosis and loss of effector function. ELISA revealed significantly elevated Gal-3 levels in serum from DLBCL patients versus healthy controls ( p <0.001). Flow cytometry confirmed strong Gal-3 binding to CAR-T cells, which correlated with increased apoptosis ( p <0.01). To overcome this, we engineered CAR-T cells to overexpress the alpha2,6 sialyltransferase ST6GAL1 (ST6 OE )CAR-T cells, which adds alpha2,6-sialic acids known to mask Gal-3-binding glycans. ST6 OE CAR T cells displayed markedly reduced Gal-3 binding, lower apoptosis ( p <0.001), and improved viability and cytotoxicity in Gal-3 high DLBCL co-cultures. Cytokine profiling further revealed that control CAR-T cells secreted elevated levels of interleukin-5 (IL-5),a Th2-associated cytokine linked to reduced antitumor activity. In contrast, ST6 OE CAR-T cells exhibited significantly lower IL-5 production ( p <0.01), indicating a shift toward amore cytotoxic, Th1-like functional profile. In a DLBCL xenograft model, ST6 OE CAR-T treatment significantly reduced tumor burden and improved survival compared to control CAR-T cells ( p <0.001). These findings demonstrate that ST6GAL1 overexpression mitigates apoptosis and IL-5-associated dysfunction, enhancing CAR-T cell persistence and antitumor efficacy. Glycoengineering CAR-T cells via ST6GAL1 provides a novel strategy to improve therapeutic outcomes for patients with DLBCL.