Activation-Inducible Bispecific BCMA-CD24 CAR-T Cells Precisely and Safely Control Multiple Myeloma Growth

Introduction: B cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T-cell therapies have emerged as a promising treatment approach for patients with multiple myeloma (MM). However, responses are not durable in the great majority of cases and sustained remission following BCMA CAR-T cell therapy is rare. CD24-positive myeloma cells are critical factors for relapse. We reported previously that dual-targeted BCMA-CD24 CAR-T cells exhibit enhanced efficacy and prolonged response duration compared to monospecific BCMA CAR-T cells (Sun et al. Nature Communications, 2024). However, the circumstance that CD24 is broadly expressed on various cell types, including hematopoietic, neural, epithelial, and pancreatic cells, raises significant safety concerns for CD24-targeted CAR-T therapy. To mitigate on-target, off-tumor toxicity, we have engineered activation-inducible bispecific BCMA-CD24 CAR-T (Aib-CAR-T) cells that express the CD24-targeted CAR in BCMA-dependent fashion exclusively in the myeloma microenvironment. Our results demonstrate that these engineered CAR-T cells can precisely and safely target MM cells.

Methods: The Aib-CAR vector comprises a BCMA-CAR expression cassette and an inducible nuclear factor of activated T cells (NFAT)-driven CD24-CAR expression cassette integrated into a single lentiviral vector backbone. This design enables ubiquitous BCMA-CAR expression in all transduced T cells and inducible CD24-CAR expression via NFAT promoters activated by binding of the BCMA-CAR to its ligand on myeloma cells. To evaluate the performance of Aib-CAR-T cells, we established a panel of isogeneic myeloma cell lines derived from MM1.S (97% and 12% of these cells express BCMA and CD24, respectively). Derivative cell lines included a BCMA knockout (MM1.S-BCMA^KO), a BCMA knockout with high CD24 expression (MM1.S-BCMA^KO/CD24^OE), and a BCMA / CD24 double knockout (MM1.S-BCMA^KO/CD24^KO). HEK293T kidney cells, genetically modified in the same way as the myeloma cells, were used as controls. CAR-T cells were co-cultured with myeloma cells at a 5:1 ratio for 24 hours. T-cell activation was assessed by measurements of both CD69 expression and release of cytokines IFN-γ, IL-2, and TNF-α into the cell co-culture supernatant.

Results: Co-culture of Aib-CAR-T effector cells with unmodified, parental MM1.S cells resulted in 89.5% target lysis after 24 hours. There was no difference in killing when Aib-CAR-T cells were compared to traditional dual BCMA-CD24 CAR-T cells. MM1.S-BCMA^KO and MM1.S-BCMA^KO/CD24^OE cells resisted Aib-CAR-T cytotoxicity, which was in line with the finding that Aib-CAR-T cells were not activated (based on CD69 expression and production of IFN-γ, IL-2 and TNF-α) in the presence of these targets. In contrast, in the traditional dual BCMA-CD24 CAR-T cells efficiently lysed MM1.S-BCMA^KO (62.3%) and MM1.S-BCMA^KO/CD24^OE (89.2%) cells, and increased both CD69 expression and cytokine production (IFN-γ, IL-2, and TNF-α) as one would have expected. In the case of MM1.S-BCMA^KO/CD24^KO target cells, neither Aib-CAR-T cells nor dual CAR-T cells exhibited significant cytotoxicity, thus demonstrating the specificity of the experimental model system employed. The same findings were obtained using gene-edited HEK293T cells, including the observation that Aib-CAR-T cells did not kill targets devoid of BCMA.

Conclusion: The results demonstrate that traditional dual-targeted BCMA-CD24 CAR-T cells can attack BCMA^-CD24⁺ cells, potentially leading to on-target, off-tumor toxicity in vivo. In contrast, Aib-CAR-T cells must first be activated by binding to BCMA on the surface of myeloma cells before CD24⁺ targets, such as stem cell-like myeloma cells, can be recognized and killed. This design confines the activity of CD24-CAR to the myeloma microenvironment. Given that multiple myeloma cells grow in large clusters within the bone marrow, the design of Aib-CAR-T cells could significantly enhance safety by reducing on-target, off-tumor toxicity, thus suggesting their potential as a promising therapeutic approach for multiple myeloma. Importantly, inducible NFAT-driven CAR expression can be widely used for various target combinations in liquid and solid tumors, thus enhancing the safety and precision of dual-targeted CAR-T therapies.