Intelligent bispecific BCMA-CD24 CAR-T cells for precision navigation and elimination of multiple myeloma Free

Introduction: The high relapse rate following BCMA CAR-T therapy remains a challenge in the treatment of multiple myeloma (MM). We previously reported that bispecific BCMA-CD24 CAR-T cells exhibit higher efficacy and longer response than BCMA CAR-T cells (Sun et al. Nature Communications, 2024). However, CD24 is widely expressed in various cell types, which poses a potential safety threat to anti-CD24 CAR-T therapy. To mitigate on-target, off-tumor toxicity, we have engineered IF/THEN logic-gated bispecific BCMA-CD24 CAR-T cells (intelligent CAR-T, iCAR-T) that autonomously navigate to BCMA⁺MM lesions, activate the CD24-CAR expression program via T-cell-induced nuclear factor (NFAT) promoters, and intelligently achieve the therapeutic goal of precise targeting of CD24⁺MM. Our results demonstrate that these iCAR-T can precisely and safely target different MM populations while circumventing off-tumor cytotoxicity.

Methods: The CAR vector includes a constitutive BCMA-CAR expression cassette and an inducible CD24-CAR expression cassette driven by activated NFAT integrated into a lentiviral vector backbone. This design forms an IF/THEN logic-gated program, inducing CD24-CAR expression via NFAT promoters activated by binding of the BCMA-CAR to its ligand. OPM2 models were constructed, including BCMA knockout (OPM2-BCMA^KO), BCMA knockout with elevated CD24 expression (OPM2-BCMA^KO/CD24^OE) and BCMA/CD24 double knockout (OPM2-BCMA^KO/CD24^KO). CAR-T cells were separately co-cultured with BCMA protein and MM cells for different durations to assess T-cell activation as indicated by CD24-CAR expression, CD69 expression and release of cytokines IL-2, IFN-γ, and TNF-α. The CAR-T cytotoxicity against OPM2 cells was assessed. To evaluate the tumor-inhibiting activity in vivo, NSG mice were engrafted 2×10⁶ OPM2 cells expressing luciferase by intravenous injection. On day 35 after receiving CAR-T cell infusion, the mice were euthanized to analyze the abundance and tissue distribution of CD24-CAR.

Results: To validate the specificity of BCMA CAR-induced CD24-CAR expression, the CAR-T cells were incubated on BCMA protein-coated plates. Exposed to BCMA protein or not, traditional BCMA-CD24 CAR-T expressed both BCMA-CAR (87.93% and 86.8%) and CD24-CAR (94.3% and 95.1%) as one would have expected. However, upon exposure to BCMA in culture, iCAR-T gradually upregulated CD24-CAR expression over time, reaching 58.3% at 12 hours and 87.7% at 24 hours. CD24-CAR expression peaked at 92.5% by 48 hours and was then maintained at high levels for ~7 days. This activation was accompanied by increased cytokine production, including IL-2, IFN-γ, and TNF-α. The co-culturing of iCAR-T with OPM2 cells resulted in a remarkable 83.0% lysis of OPM2 cells after 24 hours, with no significant difference in cytotoxicity observed between the iCAR-T and traditional CAR-T. OPM2-BCMA^KO, OPM2-BCMA^KO/CD24^OE and OPM2-BCMA^KO/CD24^KO cells resisted the iCAR-T cytotoxicity. This is consistent with the finding that iCAR-T was not activated in the absence of BCMA, as indicated by low CD69 expression and minimal production of IL-2, IFN-γ, and TNF-α. In contrast, in traditional CAR-T, lysis of OPM2-BCMA^KO (71.4%) and lysis of OPM2-BCMA^KO/CD24^OE (89.2%) cells were effectively increased.

Bioluminescence imaging showed that the iCAR-T exhibited excellent anti-tumor activity in vivo and almost completely cleared the tumor on day 21. Traditional and iCAR-T showed similar effects in inhibiting tumor growth. Although a strong CD24-CAR signal was detected at the MM lesion site in both of CAR-T groups, CD24-CAR also showed a positive intensity in other non-lesion tissues in the traditional CAR-T group. This demonstrates the effectiveness and safety of iCAR-T in treatment. The CD24 CAR-inducible circuit responds strongly to BCMA-CAR activation and can switch between ON and OFF states based on BCMA availability, helping reduce off-target cytotoxicity when cells circulate from BCMA⁺CD24⁺ tumors to BCMA^-CD24⁺ peripheral sites.

Conclusions: This study presents the next logical step in the development of BCMA-CD24 CAR-T cells for MM. The IF/THEN logic-gated CAR-T engineering strategy used afforded enhanced tumor-inhibiting activity in vitro and in vivo, while limiting on-target, off-tumor toxicity. Our design could be readily extended to a broad range of antigen combinations on target cells, offering a versatile platform to improve the safety and precision of dual-targeted CAR-T therapies.