CD38BiTE-secretion overcomes antigen escape in BCMA-targeted CAR T cell therapy for multiple myeloma

Abstract Background: Chimeric antigen receptor (CAR) T cell immunotherapy has revolutionized cancer treatment, providing unprecedented outcomes in hematologic malignancies. Despite these advances, limited CAR T cell efficacy has been linked to antigen escape, limited CAR T cell persistence, and an immunosuppressive tumor microenvironment. For instance, B cell maturation antigen (BCMA) loss has been observed in 4-33% of multiple myeloma (MM) patients treated with BCMA-targeted CAR T cells; immunosuppressive cells, such as regulatory T cells (Tregs), regulatory B cells (Bregs), myeloid-derived suppressor cells (MDSCs), reside in the MM bone marrow niche, and may harm the overall function of CAR T cells. CD38 has been reported to be highly expressed on plasma cells from MM patients, but also on a subpopulation of immunosuppressive cells (Tregs, Bregs, MDSCs). Compared with CD38-negative counterparts, CD38-positive subsets of Tregs, Bregs, MDSCs are more immunosuppressive and secrete more cytokines, including IL-10, TGF-β, which can induce exhaustion of CAR T cells. Relapsed/refractory MM patients have significantly higher number of CD38+ Treg cells than non-relapsed/refractory MM patients and healthy donors. Objective: To engineer BCMA-targeted CAR T cells secreting CD38-targeting bispecific T cell engager (BiTE) to target MM cells and CD38-positive immunosuppressive cells, which will overcome BCMA-antigen escape and the negative impact of the immunosuppressive tumor microenvironment, thus overall increasing the efficacy of BCMA-targeted CAR T cell therapy. Methods: We constructed αBCMA-CAR.CD38BiTE T cells targeting both BCMA and CD38. αBCMA-CAR.CD19BiTE was constructed as control. The expression of the αBCMA-CAR was characterized using flow cytometry. The secretion of CD38BiTE by CAR T was validated using western blot and flow cytometry using purified and concentrated CD38BiTE, and the binding and function of CD38BiTE was characterized using flow cytometry. We evaluated cytotoxicity of the αBCMA-CAR.CD38BiTE cells for myeloma cells and immunosuppressive cells from normal donor microenvironment in vitro using co-culture and transwell assays by flow cytometry. We established immunodeficient mouse models with heterogenous target antigen expression mimicking mixed populations of BCMA-positive and BCMA-negative MM cells co-existing in the bone marrow niche, to test the functional advantage of αBCMA-CAR.CD38BiTE T cells over conventional αBCMA-CAR T cells. Results: We demonstrate that the MM cell lines MM1.S and OPM2 and CD38-postive Tregs have comparable and high levels of CD38 expression. We show that CD38BiTE is secreted by CAR T cells upon activation, and binds to MM cells and normal donor T cells. αBCMA-CAR.CD38BiTE and of αBCMA-CAR.CD19BiTE T cells induce up to 90% of specific cytotoxicity of BCMA-positive CD38-positive MM1.S cells at E:T ratio of 9:1 after 24h coculture. αBCMA-CAR.CD38BiTE T cells maintain the high cytotoxicity (70%) at E:T ratio of 1:9. By contrast, the cytotoxicity of αBCMA-CAR.CD19BiTE T cells drops to 30% at this E:T ratio. Secreted CD38BiTE also induces T cell-mediated cytotoxicity (around 40%, E:T ratios = 3:1, 1:1, 72h) of BCMA-negative CD38-positive MM cells (OPM2-BCMA-KO and MM1.S-BCMA-KO) and preferential killing of CD38-positive Treg cells within normal donor CD4+ T cells. When high cell numbers of αBCMA-CAR.CD38BiTE T cells are plated in co-culture assays (90,000 effector cells, E:T ratio = 9:1) we observe CD38BiTE induced fratricide of CAR T cells after 72h culture. Fratricide is overcome by plating low cell numbers of αBCMA-CAR.CD38BiTE T cells (10,000 effector cells, E:T ratio = 1:1), while maintaining cytotoxicity of BCMA-negative myeloma cells (18% vs 42% respectively). Conclusions: CD38BiTE-secreting BCMA-targeted CAR T cells enable the CAR T cells to target BCMA-positive and BCMA-negative CD38-positive MM cells, as well as CD38-positive immunosuppressive cells. This strategy may overcome BCMA antigen escape and the effects of immunosuppressive cells in the myeloma microenvironment, simultaneously, thus overall increasing the efficacy of CAR T cells for MM treatment.