CAR-macrophages targets CD26 to eliminate chronic myeloid leukemia stem cells.

Abstract

Background: Chronic myeloid leukemia stem cells (CML-LSCs), which exhibit resistance to tyrosine kinase inhibitors (TKIs), are the leading cause of treatment failure and recurrence in chronic myeloid leukemia (CML). This highlights the urgent need for novel therapies aimed at eliminating these CML-LSCs. Chimeric antigen receptor macrophages (CAR-M) not only perform phagocytosis on target cells but also function as antigen-presenting cells, thereby activating the anti-tumor immune response.CD26 (dipeptidyl peptidase 4, DPP IV) is abundantly expressed in CML-LSCs and functions as a tumor-specific antigen (TSA) in CAR-M treatment. The purpose of this study is to evaluate CAR-M's efficacy in targeting CD26-positive CML cells and to develop a novel strategy for CML treatment.

Methods: CD26 CAR-M was constructed using mouse-derived macrophage Raw264.7 cells. CD26 was overexpressed in CML cell lines BP210 and BP210-T315I. The targeting phagocytosis of CAR-M was verified using confocal microscopy and flow cytometry. X-ray was used to eliminate the tumorigenicity of CAR-M, and the safety of CAR-M was verified through CCK-8, clone formation assays, and animal experiments. To assess the anti-leukemia ability of CAR-M in the CML mouse model, the survival, peripheral blood white blood cell counts, and CML cell infiltration in the liver, spleen, and bone marrow (BM) were measured. Additionally, CD26 CAR-THP1 was constructed, and its phagocytic ability against CD26-positive cells NCI-H2452 was confirmed by confocal microscopy.

Results: We successfully constructed CD26 CAR-M and validated its targeted phagocytosis of CD26-positive CML cells both in vitro and in vivo. The data indicate that CAR-M has higher phagocytic efficiency in CD26-positive CML cells than in CD26-negative cells. CAR-M-treated CML mice demonstrated extended survival and reduced CML invasion. In addition, CAR-THP1 demonstrated targeted phagocytosis of NCI-H2452 cells that normally express CD26.

Conclusion: This study demonstrates that CD26 CAR-M effectively targets and phagocytizes CD26-positive CML cells, implying that targeting CD26 with CAR-M could be a viable method for eradicating CML-LSCs. Furthermore, our discoveries illuminate the potential application of CAR-M in treating hematological malignancies.