The Investigation of Antitumor Effect of Humanized Bispecific BCMA/CD38 CAR-T Cells on Multiple Myeloma

Background Multiple myeloma (MM) is an incurable disease that will eventually deteriorate. Chimeric antigen receptor (CAR) T cell therapy has been widely used in the treatment of hematological malignancy and showed a promising effect. BCMA (CD269) is exclusively expressed on the MM cells, making it an ideal target for immunotherapy. CD38 is highly expressed on the surface of MM cells, but is also expressed in immune cells and a minority of hematopoietic stem/ progenitor cells. Humanization reshaped antibody may reduce the potential risk of immunogenicity associated with the animal-derived antibodies. In this study, we constructed humanized BCMA CAR-T, CD38 CAR-T and bispecific BCMA/CD38 CAR-T, and explored their killing efficiency towards MM cells, providing the basis for clinical application.

Methods Mouse anti-human BCMA and CD38 single chain fragment variable (scFv) sequences were obtained from previously established monoclonal antibody hybridoma cells (69G8 and HIT2) in our institution, respectively. The sequences were then humanized. Single-target CAR (BCMA CAR, CD38 CAR) and dual-target BCMA/CD38 CAR (LoopBC38 CAR) plasmids were constructed. Phenotype of CAR-T cells was detected. CAR-T cells and tumor cell lines were co-cultured in vitro, followed by the detection of the percentage of residual tumor cells, the degranulation and cytokine release level of T cells. Bone marrow mononuclear cells (BMMNC) from MM patients were co-cultured with CAR-T cells. MM cells were intravenously injected into immunodeficient mice, followed by CAR-T cell therapy. After co-culturing with peripheral blood mononuclear cells (PBMC) from healthy donors and CD34⁺ umbilical cord blood (UCB) stem cells, the proportion, cell subsets of residual cells and colony formation ability were assessed to verify the safety of CAR-T cells.

Results The proliferation ability of CD38 CAR-T and LoopBC38 CAR-T was weaker than that of VEC-T and BCMA CAR-T. The residual H929 cells (BCMA⁺CD38⁺) after 48h cocultured with VEC-T and LoopBC38 CAR-T at E:T ratio of 1:1 was (20.82±9.773) % vs. (0±0) % (p<0.0001). After 48h at E:T ratio of 1:1, residual K562-BCMA cells (BCMA⁺CD38^-) cocultured with CD38 CAR-T were (49.53±9.773) %, while that of LoopBC38 CAR-T were (8.49±4,766) % (p<0.0001). After 48h at E:T ratio of 1:4, residual Raji cells (BCMA^-CD38⁺) were (62.69±22.2) % in BCMA CAR-T and (19.81±12.22) % in LoopBC38 group (p<0.01). The secretion level of cytokines such as IFN-γ, TNF-α, IL-2 was significantly higher in CAR-T cells than that of VEC-T cells. After co-cultured with H929, the IL-6 secretion level of LoopBC38 CAR-T cells was (7.28±0.4558) %, which was lower than that of CD38 CAR-T cells [(259.4±3.039) %, p<0.0001]. When cocultured with BMMNC derived from MM patients, BCMA CAR-T could kill BCMA⁺ cells, CD38 CAR T could kill CD38⁺ cells, while LoopBC38 CAR-T has both effects. In H929 xenograft NSG mice, the median survival time of MM mice was 35 days for VEC-T, 82 days for CD38 CAR-T. Until now, more than 50% mice are alive in BCMA CAR-T group and LoopBC38 CAR-T group, so they didn't reach the median survival time yet. LoopBC38 CAR-T did not significantly kill PBMC form healthy donors, with the residual PBMC cells after 24h co-culturing was (48.26±1.708) % in VEC-T and (49.0±2.517%) in LoopBC38 CAR-T (p>0.05), but had killing efficiency towards mononuclear-macrophages and NK cell. A slight killing effect on UCB CD34⁺ cells was observed in LoopBC38 CAR-T. The residual CD34⁺ cells in LoopBC38 CAR-T were (29.47±4.077) %, while that of VEC-T was (48.5±11.88) % (p>0.05). The number of CFU-GEMM in LoopBC38 CAR-T was (3.0±1.512) %, which was lower than that of VEC-T [(4.667±3.386) %, (p<0.001)].

Conclusion All CAR-T cells showed strong killing efficiency toward MM cells both in vitro and in vivo. Humanized BCMA CAR-T and CD38 CAR-T can specifically kill multiple myeloma cell lines and primary cells of patients. Bispecific CAR-T was able to target both antigens. CD38 CAR-T and bispecific CAR-T cells showed self-killing ability and altered their phenotypes. They can kill NK cells, mononuclear-macrophages in peripheral blood of healthy donor and have certain effects on the formation of CFU-GEMM, which may be a safety problem that requires further improvement for the application of CD38 CAR-T and bispecific BCMA/CD38 CAR-T cells.

Wang:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy.