The NF-Κb Inhibitor SN50 Enhances the Efficacy of BCMA CAR-T Cell Therapy

【Background】The introduction of chimeric antigen receptor T cell immunotherapy (CAR-T) marked a major breakthrough in cancer treatment. The B cell maturation antigen (BCMA) is selectively expressed in multiple myeloma (MM). A BCMA CAR-T has demonstrated outstanding efficacy against relapsed or refractory multiple myeloma (RRMM). However, in some patients, despite the continued presence of these cells after receiving BCMA CAR-T cell infusion for a period, the therapy remains ineffective in preventing the recurrence of MM. Therefore, a considerable knowledge gap remains in the understanding of how to enhance the effectiveness of BCMA CAR-T cell killing of MM cells and the associated mechanisms. Cilta-cel, a CAR-T cell therapy with dual-targeting BCMA nanobodies, has demonstrated outstanding therapeutic efficacy in clinical practice due to its unique bivalent BCMA CAR structure. Nevertheless, considering the high construction efficiency of a monovalent structure, we contemplate whether adopting a novel monovalent design in the manufacturing process would be more convenient and potentially reduce production costs. In pursuit of construction efficiency to ensure more significant success in the field of CAR-T cell therapy, this study constructed monovalent and bivalent BCMA CARs based on the Cilta-cel structure. Surprisingly, the bivalent BCMA CAR-T exhibited superior killing efficacy compared to the monovalent BCMA CAR-T. This research is aimed to dissect the functions and the specific mechanisms of the bivalent BCMA CAR-T on the MM cells.

【Objective】Two types of BCMA CAR-T cells, monovalent BCMA CAR-T (353T and 917T) and bivalent BCMA CAR-T (353/917T) were designed for this study. By comparing the differences in killing efficacy between these two types of CAR-T cells, we aimed to explore the functions and molecular mechanisms underlying such distinctions. We also tried to design optimized CAR molecular structures and formulate combination therapy strategies to maximize the efficacy of BCMA CAR-T treatment and improve clinical outcomes for MM patients.

【Methods】The monovalent and bivalent BCMA CAR recombinant expression plasmids were constructed, followed by lentivirus packaging, primary T cell infection with the virus, and detection of the expression levels of CAR by flow cytometry. The BCMA CAR-T cells were co-cultured with MM cells, MM cell apoptosis was determined by flow cytometry 16 hours later, and the assessment of activation or inhibition of downstream BCMA pathways was by Western Blotting.The BCMA CAR-T cells, treated the with NF-κB pathway inhibitor SN50, were co-cultured with MM cells and MM cell apoptosis was assessed by flow cytometry 16 hours later. An ARP-1 mouse model was constructed for the in vivo function validation.

【Results】The monovalent and bivalent BCMA CAR-T stable cell lines were successfully established, showing positive expression rates of over 80% in flow cytometry assays.The monovalent and bivalent BCMA CAR-T cells were successfully constructed, yielding cells with a positive expression rate of approximately 40% in a flow cytometry assay.The 353/917T cells exhibited the strongest killing effect on MM cell lines, followed by the 917T cells with the 353T cells having the weakest killing function of MM cells as determined by flow cytometry. The NF-κB pathway in MM cells was activated during the co-culture of BCMA CAR-T and MM cells as determined by Western blotting. The 353T cells exhibited the strongest activation followed by the 917 T cells, while the 353/917T cells exhibited the weakest activation. The activation of the NF-κB pathway resulted in increasing the expression of the Bcl-2 protein. NF-κB pathway inhibitor SN50 had synergistic effect of killing ability of BCMA CAR-T cells on the MM cells as assayed by flow cytometry. The 353T cells exhibited the most significant improvement in therapeutic effectiveness in the mouse tumor model.

【Conclusion】The bivalent BCMA CAR-T cells exhibit superior cytotoxic activity in vitro compared to monovalent BCMA CAR-T cells.The bivalent BCMA CAR-T cells demonstrate outstanding killing efficacy on MM cells, especially when correlated with its inhibition of the NF-κB pathway activation.The NF-κB pathway inhibitor SN50 enhances the killing efficacy of BCMA CAR-T cells on MM cells.

【Keywords】 Multiple Myeloma, BCMA CAR-T, NF-κB, SN50

No relevant conflicts of interest to declare.