Enhanced homing and infiltration capabilities of CI9 cells improve treatment efficacy for T-cell acute lymphoblastic leukemia with extramedullary involvement

Abstract Current CAR-T therapies targeting pan-T-cell antigens for T-cell acute lymphoblastic leukemia (T-ALL) are challenged by fratricide among CAR-T cells, off-target killing of normal T and NK cells, manufacturing difficulties, and poor efficacy against extramedullary disease (EMD), which frequently accompanies relapsed/refractory T-ALL. In addition, conventional CAR-T cells lack sufficient bone marrow (BM) homing ability, impairing their effectiveness in eliminating minimal residual disease. CCR9 is highly expressed in relapsed T-ALL (88.57% ± 3.5%) but is rarely found on normal T, NK, B cells, or CD34⁺ stem cells (<5%). We therefore hypothesized that co-expression of CXCR4 in CCR9-targeted CAR-T cells (CI9s)—whose ligand, CXCL12, is abundantly expressed by both T-ALL cells and BM stromal cells—could improve CAR-T cell homing to EMD and BM niches, thereby promoting interaction with leukemic cells and facilitating more effective disease eradication. Compared with conventional CCR9 CAR-T cells, CI9s expanded >150-fold by day 9 with >95% viability, showed no fratricide or off-target killing of T/NK cells, and exhibited superior chemotactic ability. Both CI9s and CCR9 CAR-Ts demonstrated potent cytotoxicity in vitro and in murine models via tail vein injection. However, in subcutaneous and combined subcutaneous and vein models, CI9s showed superior anti-leukemic efficacy, with enhanced infiltration into both BM niches and EMD sites. Complete remission was achieved in 100% (4/4) of CI9-treated mice, with only 25%(1/4) relapse by day 50. In contrast, the CCR9 CAR-T group achieved 50% complete remission. Half(2/4) of the mice achieved partial remission, but these mice exhibited disease progression by day 50. In vivo bioluminescent imaging further confirmed that CI9s exhibit enhanced tissue tropism and broader biodistribution, particularly with significantly improved BM homing. In summary, CXCR4-modified CCR9 CAR-T cells (CI9s) exhibit enhanced homing and infiltration capabilities, leading to improved control of EMD and more effective clearance of minimal residual disease in the BM. CI9s represent a promising strategy for the treatment of T-ALL with EMD, offering strong potential for clinical translation.