Expression of the CXCR4 S338X Variant Improves Anti‐Leukemia Efficacy of Anti‐ <scp>CD19 CAR</scp> ‐T Cells

Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated remarkable success in treating hematological malignancies; however, antigen-positive relapse remains a significant obstacle to achieving sustained remission in B-cell acute lymphoblastic leukemia (B-ALL). To enhance the therapeutic efficacy of anti-CD19 CAR (CAR19)-T cells, we overexpressed CXCR4 in CAR19-T cells to improve their trafficking to bone marrow (BM), a key sanctuary for minimal residual disease. We engineered CAR19-T cells with overexpression of wild-type CXCR4 (CAR19/CXCR4^WT-T) or gain-of-function CXCR4 S338X mutant (CAR19/CXCR4^S338X-T). Both CAR19/CXCR4^WT-T and CAR19/CXCR4^S338X-T cells exhibited enhanced CXCR4 surface expression in vitro and in vivo compared to control CAR19-T cells, with the latter showing significantly superior improvements under all tested conditions, including engagement with CAR-specific antigens or CXCR4 ligand CXCL12. Upon engagement with CXCL12, CAR19/CXCR4^S338X-T cells, but not CAR19/CXCR4^WT-T cells, displayed significantly increased activation of ERK1/2 and AKT signaling pathways, as well as elevated transcription of TNF-α, IFN-γ, granzyme B, CDK6, and BCL2A1, along with strengthened effector functions, chemotaxis, and activation of anti-apoptotic pathways. Furthermore, CAR19/CXCR4^S338X-T cells demonstrated significantly improved migration to and retention in the BM accompanied by increased CD45RA⁺CCR7⁺ memory T cell populations, which correlated with enhanced anti-leukemic effects following injection into B-ALL-bearing mice. This study offers a potentially effective strategy to improve the functionality and durability of CAR-T cell responses in hematological malignancies.