Chimeric Antigen Receptor (CAR) T cell therapy has significantly improved outcomes for patients with B-cell Acute Lymphoblastic Leukaemia (B-ALL) and other B cell cancers, relapse remains a major clinical challenge, occurring in up to 60% of cases. Post-CD19 CAR T-cell relapse can present as either CD19-positive (CD19+/+) or CD19-negative (CD19-/-) disease. While the mechanisms underlying CD19-/- relapse are relatively well characterised, CD19+/+ relapses are less well understood and have largely been attributed to therapy-related factors (e.g. limited CAR T persistence or exhaustion). Through analysis of paediatric B-ALL patient samples across different stages of disease progression, our laboratory has identified Galectin-9, a β-galactoside-binding immunomodulatory lectin to be upregulated in CD19+/+ leukaemic cells following CAR T cell relapse.
Galectin-9, can be membrane bound or secreted, and interacts with T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), an inhibitory receptor expressed T cells. While the Galectin-9–TIM-3 pathway has been linked to immune evasion in other cancers, its role in CAR T-cell dysfunction in B-ALL has not been previously explored.
Using CRISPR activation (CRISPRa), we upregulated endogenous Galectin-9 in three CD19⁺ B-ALL cell lines. Western blot confirmed expression, and ELISA showed >10-fold increases in secreted Galectin-9. Interestingly, surface expression remained negligible, suggesting that leukemic cells primarily secrete Galectin-9 rather than present it on their surface. Supporting this, even brief exposure of CD19 CAR T cells to conditioned media from Galectin-9⁺ B-ALL cells significantly impaired their cytotoxic activity, independent of target cell Galectin-9 expression. The functional consequences of Galectin-9 on CAR T-cell activity are now being explored using apoptosis markers, degranulation assays, and live-cell imaging of CAR T cell–target cell synapses. These findings highlight a novel, leukemia-intrinsic mechanism of CAR T resistance and suggest that targeting the Galectin-9–TIM-3 axis could enhance CAR T cell efficacy in B-ALL.