Presence of soluble and cell‐surface B‐cell maturation antigen in systemic light‐chain amyloidosis and its modulation by gamma‐secretase inhibition
Authors: Amandeep Godara, Ping Zhou, Adin Kugelmass, Xun Ma, Benjamin Rosenthal, Denis Toskic, Teresa Fogaren, Cindy Varga, Raymond L. Comenzo
Journal: American Journal of Hematology (2020)

To the Editor: B-cell maturation antigen (BCMA), a cell surface glycoprotein belonging to the tumor necrosis factor receptor (TNFR) superfamily, is expressed on plasma cells and some mature B-cells.1 BCMA is the receptor of a proliferation-inducing ligand (APRIL) and a B cell-activating factor of the TNF family (BAFF).2 It is shed from its membrane-bound form by the protease activity of γ-secretase and its soluble form (sBCMA) is elevated in the blood of patients with multiple myeloma.3 Systemic light-chain (AL) amyloidosis and multiple myeloma are overlapping plasma cell disorders but the mechanism of disease in AL is not tumor cell proliferation but rather toxicity and tissue deposition of the immunoglobulin light chains (LCs) produced by AL plasma cells; moreover, LC variable region germline gene utilization in AL is biased.4 Like myeloma, clonal AL plasma cells have cell surface glycoproteins such as CD38 and CD319, which have been harnessed for their therapeutic potential.5 BCMA has also become an important target for immunotherapy in myeloma forming the basis for chimeric antigen receptor T cell therapy (CAR-T) and other monoclonal antibody approaches.6, 7 The presence and modification of cell-surface and sBCMA in AL patients has not been previously reported. We report on the expression of membrane-bound and soluble BCMA in NOD scid gamma mouse (NSG) mice xenografted with the AL cell line, ALMC-1 and in AL patients, and show the clinical correlations of sBCMA levels with plasma cell disease burden in patients and ALMC-1 mice. Furthermore, we also test the effect of gamma-secretase inhibition (GSI) on sBCMA and mBCMA levels in patient samples. To evaluate the expression of BCMA in the ALMC-1 cells, cells were grown for 7 days and flow cytometry was then performed (Supplementary Methods) using APC conjugated anti-CD269 antibody and 84.6% of the cells exhibited mBCMA expression. The level of sBCMA in the cell culture supernatant was analyzed by sandwich ELISA (Supplementary Methods) and was 3.8 ng/mL per million cells. We then analyzed sBCMA levels in an in-vivo mouse xenograft model after injecting ALMC-1 cells into the flank of NSG mice (Supplementary Methods).8 Tumor uptake on bioluminescent imaging after 3 mg luciferin injection at 10 weeks was seen in seven out of nine mice (Figure 1A), with a total FLUX (photons per second) readout of 3.9 × 1010 (range, 2.02 × 109 to1.20 × 1011). Median sBCMA level in the mouse sera was 3.8 ng/mL (0.9-23.6) and the median λ-LC level was 949.1 mg/L (341.1-3460.5). sBCMA levels correlated with tumor burden measured as FLUX approaching statistical significance (Figure 1B, P = .07), and correlated significantly with λ-LC levels (Figure 1C, P << .01), indicating that sBCMA may be useful as a marker of tumor activity in AL-cell line xenograft model. We then evaluated mBCMA and sBCMA in paired bone marrow and peripheral blood samples from 20 patients with AL, of whom eight were newly diagnosed, four had progression of disease after prior therapy and eight were in hematologic response (≥ very good partial response-VGPR). Median age was 65 years (48-77) and 50% were male. Median plasma cells in the marrow aspirates and involved free LC (iFLC) levels were 5% (1-20%) and 33 mg/L (6.6-2220) respectively. After CD138 selection (Supplementary Methods), 12 patients had adequate bone marrow mononuclear cells available for assessment of mBCMA by flow cytometry. Amongst the CD138 selected cells, a median of 38.5% (4-83) cells were CD138/BCMA double positive (Figure 1D). Quantitative assessment of sBCMA was done in all 20 patients and the median level was 28.5 ng/mL (6.6-100.3, Figure 1E). The sBCMA correlated with the percentage of plasma cells in the marrow aspirate (Figure 1F, P << .01). There was also a significant correlation between sBCMA and iFLC (P < <.01, Figure 1G). In patients who were newly diagnosed, median BCMA level was 44.9 ng/mL (23.8-100.3) compared to 22.5 ng/mL (13.2-44.4) in patients with light-chain progression and 16.5 ng/mL (6.6-40.8) in patients with hematologic response. These three groups differed in their plasma cell and iFLC levels (Table S1). In patients who had a hematologic response and iFLC <20 mg/L, median BCMA level was 13.4 ng/mL (11.7-35.8) while it was 19.5 ng/mL (6.6-40.8) for those who had an iFLC >20 mg/L. These results suggest that sBCMA, facilitated by its short half-life (24-36 hours), may serve as a dynamic marker of disease state. γ-secretase is a four-protein complex responsible for the cleavage of membrane bound BCMA, an action that can be inhibited by the small molecule LY-411575. To investigate the effect of GSI on AL plasma cells, ALMC-1 cells were exposed to LY-411575 (0.1 nM) for 48 hours in culture. mBCMA expression of ALMC-1 cells increased from 84.6% to 99.9% (Figure S1A) compared to untreated controls (84.6% to 77.1%). In contrast, the sBCMA level in the cell-culture supernatant decreased from 3.76 ng/mL to 1.82 ng/mL (Figure S1B). We incubated CD138 selected cells from marrow aspirates of AL patients with LY-411575 (0.1 nM) for 48 hours and evaluated the mBCMA level by flow cytometry and sBCMA by sandwich ELISA. The population of cells that became CD138/BCMA double positive increased from 36% (6.5-83) to 68% (34-99.9, P < .01, Figure S1C). In conclusion, BCMA is a target for immunotherapy aimed at clonal AL plasma cells. The presence and modulation of the cell-surface BCMA of clonal AL plasma cells may also enhance its use as a target for immunotherapeutic approaches such as CAR-T cell, bispecific or drug-conjugated monoclonal antibody therapies for AL patients. Concurrent treatment with the γ-secretase inhibitor may enhance the therapeutic outcomes in AL patients because of a greater density of BCMA on the surface of the AL plasma cells. Also, the measurement of sBCMA in the blood correlates strongly with the active vector of disease in AL - the involved free LC - and the marrow plasma cell burden. It is worth mentioning that in multiple myeloma, sBCMA appears to be predictive of early treatment response and, as such, levels are not affected by renal impairment.7 Further studies are needed to investigate the role of BCMA as a marker of disease burden and treatment response in AL, especially in patients with underlying renal dysfunction or low level circulating amyloidogenic LCs. The authors thank the clinical research coordinators who contributed to this study. For their continued support we also thank the Division of Hematology-Oncology and Departments of Medicine and Pathology and Laboratory Medicine at Tufts, the Amyloidosis and Myeloma Research Fund at Tufts, the Cam Neely and John Davis Myeloma Research Fund, the John C Davis Program for Myeloma and Amyloid at Tufts, the Sidewater Family Fund, the Lavonne Horowitz Trust, the Werner and Elaine Dannheiser Fund for Research on the Biology of Aging of the Lymphoma Foundation, the Amyloidosis Foundation, David and Barbara Levine (in memoriam), and the Demarest Lloyd Jr Foundation. The authors have declared that no conflict of interest exists. P.Z., A.K., B.R., X.M. and A.G. performed experiments; A.G. and B.R. analyzed the results and made the figures; R.L.C. designed the research; A.G. and R.L.C wrote the paper. All authors edited the manuscript and approved the final version. Appendix S1. 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