Soluble Bcma in Myeloma Serum Binds Its Ligands BAFF and Prevents Normal Antibody Production in Multiple Myeloma Patients
Authors: Sanchez Eric , Gillespie Abigail , Manik Harutyunyan Nika , Tang George , Gottlieb Jillian , Vardanyan Suzie , Li Mingjie , Andreu-Vieyra Claudia , Ghermezi Michael , Wang Cathy , Ben-Zvi Joseph , Garzio Gigi , Chen Haiming , Berenson James 
Published: October 2019

Abstract
Introduction: A hallmark of multiple myeloma (MM) is the low levels of uninvolved immunoglobulin (Ig) levels. B-cell maturation antigen (BCMA) is a receptor expressed in mature non-malignant and malignant B lymphocytes, including plasma cells. Its ligands are B-cell activating factor (BAFF) and a proliferation inducing ligand (APRIL). We previously demonstrated that BCMA is present in the serum of MM patients (pts) and that its levels predict survival (Sanchez et al. Br J Haematol 2012). We hypothesized that circulating BCMA binds it ligands, preventing normal plasma cell development in MM patients which may explain their reduction in uninvolved Ig levels.
Methods: BCMA-Fc and control Ig were obtained and reconstituted in PBS (R&D Systems). Retro-orbital bleeds were performed on C57 Bl/6 and SCID mice implanted with the human MM xenografts LAGλ-1, LAGk-1A or LAGk-2. Human BCMA and mouse BAFF, IgM, IgA and IgG levels were measured with ELISA (R&D Systems & Bethyl Laboratories). The Raji B-cell line was obtained from the American Type Culture Collection (Rockville, MD, USA). Human IgA and IgG levels were determined in MM patients using nephelometry (Immage 800, Beckman Coulter). Hevylite® Assays (Binding Site) were used to quantify the levels of heavy-light chain isoform pairs in MM patients.
Results: We determined if mBAFF formed complexes with human BCMA (hBCMA) in the plasma from SCID mice implanted with LAGλ-1, LAGκ-2 or LAGκ-1A, and were able to identify mBAFF-hBCMA complexes in plasma samples from these mice. To determine what effect human BCMA had on Ig levels in immune competent mice, rhBCMA-Fc or control Ig-Fc (100 mg) was injected into C57 Bl/6 mice, and plasma IgA, IgM and IgG levels were measured. Decreases in IgA levels were observed following BCMA treatment when compared to baseline plasma IgA on days 4 and 6 (P = 0.0031 and P = 0.0064, respectively), and the control group (P = 0.0087 and P = 0.0221). Samples were also analyzed for mouse IgM levels with similar marked reductions when compared to the untreated (P = 0.0001) and Ig-Fc (P = 0.0088) groups. For plasma IgG levels, a marked decrease was observed on day 6 following rhBCMA-Fc administration when compared to its baseline levels (P = 0.0023), and also when compared to the Ig-Fc control protein (P = 0.0014) and the untreated control (P = 0.0129) groups. We then set out to determine if sera from MM patients contained BCMA-BAFF complexes, using ELISA plates coated with an anti-human BAFF antibody followed by exposure to a polyclonal anti-human BCMA antibody. A strong absorbance indicating the presence of BCMA-BAFF complexes was detected in serum samples from MM patients, whereas no antibody cross reactivity was observed in control samples. We also determined whether human MM serum or rhBCMA-Fc blocked BAFF from binding to Raji B-cells. Raji cells (B-cell line) were incubated with serum from a MM patient containing hBCMA (0.75μg/ml) or rhBCMA-Fc (3 μg/ml) in the presence of rhBAFF (500 ng/ml). Myeloma serum and rhBCMA-Fc decreased rhBAFF binding to Raji cells by 71% (from 96.8 to 25.6 %) and 74% (from 96.8 to 22.9 %), respectively. Next, we determined whether serum BCMA levels inversely correlated with uninvolved Ig levels in MM pts. For pts with IgA (n = 134) or IgG (n = 313) MM, higher BCMA levels (> 100 ng/ml) correlated with below normal levels of uninvolved IgG in IgA MM and uninvolved IgA in IgG MM, whereas lower BCMA levels (< 100 ng/ml) correlated with normal uninvolved levels (P < 0.0001). Using the Hevylite Assay, similar results were observed for the levels of BCMA compared to uninvolved IgG isoforms in both pts with involved IgG lambda (n = 62, P = 0.0006) and IgG kappa (n = 117, P < 0.0001) MM.
Conclusions: Our laboratory previously has reported that serum levels of BCMA are increased in the serum of MM patients, and correlates with response to treatment and predicts survival. We now demonstrate 1) the formation of complexes of BCMA with its B-cell ligand BAFF in the plasma of MM xenografts and sera of MM patients, 2) show that rhBCMA and MM patient serum blocks the binding of BAFF to human B-cells, 3) administration of rhBCMA to normal mice results in marked reductions in their antibody levels, and 4) show that BCMA levels inversely correlate with uninvolved Ig levels in MM pts. Thus, the lack of normal antibody production in MM pts results, in part, from circulating BCMA binding its ligands, preventing production of normal antibody-producing cells.

Disclosures
No relevant conflicts of interest to declare.