Engineering CD33.CAR-CIK cells with CXCR2 to improve bone marrow homing and anti-leukemic activity in Acute Myeloid Leukemia

Chimeric antigen receptor (CAR)-T cell therapy has achieved remarkable success in treating B-cell malignancies, but its efficacy in acute myeloid leukemia (AML) remains limited by several challenges, including the poor accumulation of intravenously administered CAR-T cells in the leukemia-perturbed bone marrow (BM) niche. This is a critical limitation in AML, where durable remission depends on eradicating leukemic stem cells that reside within the BM microenvironment. One strategy to improve BM homing involves engineering CAR-T cells to express chemokine receptors specific for chemokines enriched in the altered microenvironment of leukemic BM niche. C-X-C motif chemokine ligand 8 (CXCL8), a chemokine upregulated in the AML microenvironment and associated with poor prognosis, is further elevated following chemotherapy. In this study, we detected significantly increased CXCL8 levels in BM plasma from pediatric AML patients at diagnosis. To exploit this chemokine axis, we engineered CD33-directed CAR Cytokine-Induced Killer (CD33.CAR-CIK) cells to co-express the CXCL8 receptors CXCR1 or CXCR2. These modified cells exhibited CXCL8-specific migration without altering their phenotype, differentiation or anti-leukemic function. Moreover, chemotherapeutic agents commonly used for lymphodepletion before CAR-T cell administration, such as fludarabine and cyclophosphamide, further enhanced CXCL8 production both in vitro in AML cells and in vivo in AML xenograft models. Notably, this chemotherapy-induced CXCL8 upregulation enhanced BM infiltration of CXCR2+ CD33.CAR-CIK cells. In humanized AML bone marrow organoids, CD33.CAR-CXCR2-CIK cells demonstrated accelerated and increased homing to CXCL8-rich niches. These findings support the CXCL8-CXCR1/2 axis as a promising strategy to improve the homing of CD33.CAR-CIK cells to the AML BM niche, ensuring further evaluation of their anti-leukemic activity in AML.