CAR-T Cells Based on Novel BCMA Monoclonal Antibody Block Multiple Myeloma Cell Growth

Abstract

The cell-surface protein B cell maturation antigen (BCMA, CD269) has emerged as a promising target for CAR-T cell therapy for multiple myeloma. In order to create a novel BCMA CAR, we generated a new BCMA monoclonal antibody, clone 4C8A. This antibody exhibited strong and selective binding to human BCMA. BCMA CAR-T cells containing the 4C8A scFv were readily detected with recombinant BCMA protein by flow cytometry. The cells were cytolytic for RPMI8226, H929, and MM1S multiple myeloma cells and secreted high levels of IFN-γ in vitro. BCMA-dependent cytotoxicity and IFN-γ secretion were also observed in response to CHO (Chinese Hamster Ovary)-BCMA cells but not to parental CHO cells. In a mouse subcutaneous tumor model, BCMA CAR-T cells significantly blocked RPMI8226 tumor formation. When BCMA CAR-T cells were given to mice with established RPMI8226 tumors, the tumors experienced significant shrinkage due to CAR-T cell activity and tumor cell apoptosis. The same effect was observed with 3 humanized BCMA-CAR-T cells in vivo. These data indicate that novel CAR-T cells utilizing the BCMA 4C8A scFv are effective against multiple myeloma and warrant future clinical development.

DCLK1 Monoclonal Antibody-Based CAR-T Cells as a Novel Treatment Strategy against Human Colorectal Cancers

“…These data support our hypothesis that the TSC population can be targeted using a DCLK1-specific CAR-T. Here, we report that, in collaboration with ProMab Inc., we have developed a novel CAR-T based on the DCLK1 ScFv containing a CD28 transmembrane and co-stimulatory domain and CD3ζ activation domain [39][40][41][42][43][44][45][46]. CAR-T cells generated using DCLK1 ScFv (CBT-511) demonstrated~20% CAR expression and significantly induced CRC cell cytotoxicity.…”

“…Using the ScFv of the hCBT-15 DCLK1 mAb, we created a second-generation DCLK1-based CAR-T cell (CBT-511; ProMab biotechnologies, Richmond, CA, USA) ( Figure 3A) [39,[41][42][43]. CBT-511 has DCLK1 ScFv fused to a CD28 transmembrane domain and co-stimulatory domain with a CD8 hinge.…”

“…In real-time cytotoxicity assays (RTCA), CRC cells grown in 2D were co-cultured with effector cells (DCLK1 CAR-T cells or mock CAR-T cells) at dilutions of 10:1 and 20:1 (effector cells: CRC cells). Additionally, based on our previous experience and recent published reports an E:T 10:1 and 20:1 are optimal [39,42,43]. The CRC cells were monitored for another one to two days with the RTCA xCELLigence system (Acea Biosciences, San Diego, CA, USA), and impedance were plotted over time and cytolysis were calculated.…”

“…DNA encoding the DCLK1 CAR was synthesized and subcloned into a third-generation lentiviral vector, Lenti CMV-MCS-EF1a-puro by Syno Biological (Beijing, China) [39,42]. Ten million growth-arrested HEK293FT cells (ThermoFisher) were seeded into T75 flasks.…”

“…This personalized approach to cancer therapy enables a patient's own T cells to be programmed to attack and eliminate their specific cancer. Typically, the tumor antigen specific targeting region is a single-chain antibody variable fragment (ScFv) fused to a hinge, transmembrane domain, and co-stimulatory domains (CD28, 4-1BB, CD27 or others) to stimulate the immune response, as well as a CD3ζ activation domain [38][39][40][41][42][43].…”