Anti-BCMA Dual Epitope-Binding CAR-Marrow Infiltrating Lymphocytes (MILs) Could Offer a Potent Innovative Immunotherapeutic Tool Against Multiple Myeloma

Introduction: Chimeric antigen receptor (CAR) T cells are a promising new treatment for multiple myeloma (MM). Marrow-infiltrating lymphocytes (MILs) derived from MM patient's bone marrow are another potential innovative T cell source for CAR-based cell therapy, capable of robustly and specifically targeting myeloma cells through polyclonal targets. MILs possess polyclonal central memory T cell populations, which could be as effective MM killings via polyantigenic cytotoxic T cells with greater recognitions of broad range of tumor antigens via the polyclonal memory TCR as well as the CAR pathway with strong recognition of tumor antigens. Nanobodies are currently the smallest stable antibody molecules having high affinity for antigen, allowing them to strongly penetrate tumor tissue and even directly to the blood-brain barrier while remaining low toxicity and immunogenicity. With the peculiar properties, nanobodies have been widely used as the antigen biding domain of CAR-T cell. In this study, we investigated anti-B-cell maturation antigen (BCMA) dual epitope-binding CAR-MILs against MM.

Methods: CD3 ⁺ T cells isolated from bone marrow mononuclear cells of MM patients were stimulated with Ex Vivo CD3/CD28 in the presence of recombinant human IL-2. The nanobodies VHH01 and VHH02 were provided as dual epitope-binding for BCMA (Senlang, China). After expansion and activation for 2 days, anti-BCMA dual epitope-binding CAR was transduced to the activated MILs and, they were stimulated with Ex Vivo CD3/CD28 in the presence of recombinant human IL-2, IL-7, IL-15, and IL-21. Expanded and activated BCMA dual epitope-binding CAR-MILs (BCMA CAR-MILs) were harvested on days 7, 14, and 21 for further experiments.

Results: The transduction efficiency of BCMA CAR-MILs was approximately 34%, and the expansion of BCMA CAR-MILs was > 100 folds over 21-day culture period. Additionally, BCMA CAR-MILs showed a dramatically increased proportion of CD8 ⁺ T cells, while the proportion of CD4 ⁺ T cells decreased. Moreover, BCMA CAR-MILs were enriched with a higher proportion of central memory T cells (Tcm) (80%), while the proportions of regulatory T cells (Tregs) and checkpoint molecules (TIGIT, TIM3, and CD73) were not so high. The BCMA CAR-MILs showed potent cytotoxicity, particularly high levels of CD107a degranulation against BCMA-expressing target cancer cell lines, such as Daudi, Raji, RPMI8226, and U266B1 cells, and autologous CD138 ⁺ primary myeloma cells.

Conclusion: This study suggested that BCMA dual epitope-binding CAR-MILs were easily expanded and activated using bone marrow mononuclear cells from MM patients with high transduction efficiency and induced a potent anti-myeloma immune response, which may offer an alternative innovative immunotherapeutic approach against MM patients in the future.