A novel fourth generation of CAR-T cells: CD19 CAR-T cells engineered to express membrane-bound interleukin-15 and CXCR5 for the treatment of lymphoma.

The efficacy of CD19 CAR-T cells in B-cell lymphoma patients is not as good as that in B-cell acute lymphoblastic leukemia (B-ALL) patients. This might be attributed to the intricate tumor microenvironment of B-cell lymphoma, which leads to CAR-T cell exhaustion, inability to sustain function, and difficulty infiltrating into the tumor interior. We developed CD19 CAR structures that simultaneously produce membrane-bound IL-15 (mbIL-15) and CXCR5. This design aims to enhance the migration of CAR-T cells into CXCL13+ B-cell lymphomas and their long-term antitumor ability. Compared with CD19 CAR-T cells, CD19 mbIL15-CXCR5 CAR-T cells exhibited greater cytotoxicity against CD19+ tumor cell lines in vitro. In particular, when exposed to recurrent tumor antigen stimulation, CD19 mbIL15-CXCR5 CAR-T cells still exerted long-lasting antitumor effects. CD19 mbIL15-CXCR5 CAR-T cells had a greater proportion of central memory T (TCM) and effector memory T (TEM) cells, which allowed them to exhibit more long-lasting antitumor effects. Moreover, in Transwell assays and mouse models, compared with CD19 CAR-T cells, CD19 mbIL15-CXCR5-CAR-T cells exhibited significant chemotaxis toward CXCL13+ tumor cells and superior tumor infiltration ability. The Xenogram animal model demonstrated better and more persistent tumor suppression ability than did the CD19 CAR-T cells. We preliminarily demonstrated the safety of CD19 mbIL15-CXCR5-CAR-T cells in vivo by evaluating liver and kidney function and major organ morphology in mice. In summary, the use of CD19 mbIL15-CXCR5-CAR-T cells is a relatively safe and effective option for the treatment of B-cell malignancies.