Expression of the CXCR4 S338X Variant Improves Anti‐Leukemia Efficacy of Anti‐ CD19 CAR ‐T Cells

ABSTRACT 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.