Serum Bcma May Interfere with Anti-Bcma-CAR-Transduced T Cells or Other Anti-Bcma Antibody-Based Immunotherapy in Multiple Myeloma

Introduction: CAR T cell-based immunotherapy has shown clinical activity in a variety of B-cell malignancies. Specifically, the engineered CAR-transduced T cells targeting B-cell maturation antigen (BCMA) on multiple myeloma (MM) have shown promising activity for the treatment of relapsed/refractory (RR) MM patients. However, MM patients have high levels of solubilized BCMA (sBCMA) in the blood especially those with RR disease. Due to these findings, we hypothesized that circulating levels of sBCMA may interfere with the efficacy of CAR T cell immunotherapy or other anti-BCMA antibody (Ab)-based therapeutic approaches for treating MM patients.

Study design: Sera were collected from MM patients and healthy subjects after obtaining institutional review board approval (IRB). Serum levels of human BCMA were determined using an ELISA kit (R&D Systems, Minneapolis, MN). For the BCMA antibody binding assay, 50 µL of human BCMA fluorescein-conjugated polyclonal Ab or monoclonal Ab was incubated with 100 µL of diluent buffer and 100 µL of serum from human subjects with varying sBCMA concentrations. To demonstrate the effects of sBCMA interference with anti-BCMA CAR T function, we utilized a wide range of concentrations (0ng/ml, 3ng/ml, 18 ng/mL, 32ng/ml, 37ng/ml, 110ng/ml, 156ng/ml, 390ng/ml, 530ng/ml and 2167 ng/mL); recombinant BCMA (0ng/ml, 65ng/ml, 130ng/ml, 400ng/ml, 1200ng/ml). After one hour of incubation at room temperature (RT), each sample was added to primary tumor cells from MM patients with progressive disease. These cells were incubated for two hours at RT away from light. Following incubation, cells were washed three times using PBS. Flow cytometric analysis was performed using a Beckman Coulter Cytomics FC 500 with Cytomics CXP software. The same procedure was followed to observe the effects of recombinant human BCMA. To assess immunofluorescent (IFC) analysis of anti-BCMA binding to MM tumor cells, IFC staining was completed following a standard IFC assay protocol and the BCMA binding markers were identified under the microscope (Olympus BX51).

Results and discussion: The experimental results demonstrate a concentration dependent response of anti-BCMA antibody binding to primary MM tumor cells. At sBCMA levels lower than 150ng/mL, there was no significant difference in anti-BCMA antibody binding to tumor cells. However, at concentrations greater than 150 ng/mL, a significant decrease in binding was observed. At 390ng/ml, serum sBCMA blocked 50% of anti-BCMA antibody binding to the tumor cells. At 2000ng/ml, serum sBCMA interfered with anti-BCMA antibody binding to tumor cells by more than 80%. When using recombinant BCMA, we observed the same concentration dependent effects on anti-BCMA binding to MM cells. As the levels of recombinant BCMA increased, the antibody binding to MM cells decreased. These results suggest that higher BCMA levels that are present in patients with MM interfere with binding to MM tumor cells; and, thus, could reduce the efficacy of CAR T cell and other immunotherapy approaches targeting BCMA. The fluorescent image further demonstrated serum sBCMA blocked anti-BCMA antibody binding to primary MM tumor cells in a concentration dependent fashion. In 2013 Carpenter et al . showed that sBCMA levels do not interfere with CAR-T-cells. However, the sBCMA concentrations used in those experiments are based on low sBCMA levels (10ng/ml). We have consistently detected concentrations much higher than 150ng/ml of sBCMA in MM patients. Our experimental data showed 30-35% serum BCMA level was above 150ng/ml in 500 MM serum samples and 50% serum BCMA level was above 150ng/ml in relapsed/refractory patients.

Conclusion: The results demonstrated that higher serum sBCMA concentrations do interfere with anti-BCMA antibody directed CAR T cells immunotherapy therapies or any anti-BCMA antibody therapeutic agents in MM. We suggest determining serum sBCMA level before anti-BCMA antibody directed CAR T cells immunotherapy therapy or use of any anti-BCMA antibody therapeutic agents.