Comparison of Soluble Decoy IgG Fusion Proteins of BAFF-R and BCMA as Antagonists for BAFF

Abstract

BAFF is considered a therapeutic target because dysregulated production of BAFF can induce systemic lupus erythematosus-like phenotype in mice, and elevated levels of BAFF are associated with disease severity in systemic lupus erythematosus and rheumatoid arthritis patients. Fc fusion decoy receptors, BCMA-Fc and BAFF-R-Fc, are therapeutic candidates for blocking BAFF. While studying their interactions with BAFF, we found that BAFF-R-Fc is more effective than BCMA-Fc for blocking BAFF binding to its receptors. We also found that a trimeric BAFF can bind more than one BAFF-R-Fc but only one BCMA-Fc. Moreover, we show that, in contrast to monovalent BAFF-R-Fc, monovalent BCMA does not form stable complexes with BAFF. Differences in their interaction with BAFF predict BAFF-R-Fc would be a better inhibitor. Indeed, we show BAFF-R-Fc is 10-fold more efficacious than BCMA-Fc for blocking BAFF-induced B cell proliferation in vitro and for blocking BAFF-mediated survival of mouse splenic B lymphocytes in vivo. Members of the tumor necrosis factor (TNF)1 superfamily are type II membrane proteins, each containing a shared TNF homologous extracellular domain of ϳ150 amino acids which is responsible for trimer formation of these proteins (1, 2). The TNF homologous regions can be proteolytically released as soluble trimeric proteins that, in many cases, are fully capable of interacting with their receptors. BAFF was identified as a TNF family member by searching the expressed sequence tag data base for homology in amino acid sequence to the extracellular domain of TNF (3-5).

Constitutive NF-κB and NFAT activation leads to stimulation of the BLyS survival pathway in aggressive B-cell lymphomas

“…[1][2][3][4][5]10,19 Abnormally expressed BLyS protein has been observed in a variety Various studies also have shown that inhibiting BLyS through use of decoy receptors or blocking antibodies restores the apoptotic process in malignant B cells. 6,9,12,13 However, it has been unclear as to whether constitutive BLyS expression is directly involved in malignant B-cell survival. We have shown that BLyS protein is constitutively expressed in aggressive NHL-B cell lines (LBCL and MCL), as well as in tumor cells from NHL-B patient biopsies, but, as expected, the expression level of BLyS in the tumor cells varies quantitatively.…”

“…32,33 Several groups have reported that in normal mature murine and human B cells, BLyS signaling stimulates both the NF-B1 and NF-B2 pathways through one or more of the 3 BLyS receptors: BAFF-R, TACI, and BCMA. [6][7][8]27,28,35,38 However, whether both the NF-B1 and NF-B2 pathways are involved individually or together in BLySmediated survival in NHL-B cells has not been addressed. Our studies have shown that all 3 BLyS receptors are expressed in NHL-B cells at various levels (data not shown).…”

“…In particular, the BLyS/BAFF-R pathway is crucial to the survival of peripheral B cells. [6][7][8] BLyS is expressed in a cell membrane bound or soluble form [1][2][3][4] by various cell types, including dendritic cells, monocytes, macrophages, activated T cells, neutrophils, and antigen-presenting cells. [1][2][3][4][9][10][11][12][13][14][15][16][17][18][19] Cytokines such as interleukin 10 (IL-10), interferon ␥ (IFN-␥), and IFN-␣ augment BLyS expression in monocytes, macrophages, and dendritic cells, while CD40 ligand stimulates BLyS secretion from dendritic cells.…”

B Cell-Activating Factor Belonging to the TNF Family (BAFF)-R Is the Principal BAFF Receptor Facilitating BAFF Costimulation of Circulating T and B Cells

“…CD3 ϩ cells were then isolated in a magnetic field, to Ͼ98% purity. For human B cell stimulation, PBLs were incubated in 96-well plates (10 5 cells/well in 100 l RPMI 1640 supplemented with 10% FBS) for 48 h with 75 ng/ml soluble BAFF in the presence of 5 g/ml goat F(abЈ) 2 anti-human -chain Ab (Jackson ImmunoResearch Laboratories), and with different concentrations of antih.BAFF-R mAb, h.BAFF-R-Fc, or human IgG (hIgG) control (29).…”

“…For mouse B cell proliferation assays, B cells were isolated from spleens of 2-mo old C57BL/6 mice using B cell recovery columns (Accurate Chemical & Scientific, Westbury, NY). Mouse B cells were incubated in 96-well plates (10 5 cells/well in 50 l RPMI 1640 supplemented with 10% FBS) for 72 h with 75 ng/ml BAFF (Biogen Idec, Cambridge, MA), in the presence of 2 g/ml goat anti-mouse -chain Ab (Jackson ImmunoResearch Laboratories), and with the indicated concentrations of previously described reagents, including polyclonal hIgG (Novartis Pharmaceuticals, East Hanover, NJ), h.BCMA-Fc, or antimouse BAFF-R (m.BAFF-R) mAbs (29). For Ag-specific T cell assays, splenic T cells from DO11.10 TCR transgenic mice were purified by magnetic separation, and 5 ϫ 10 4 cells/well were cultured with 1 ϫ 10 5 cells/ well of mitomycin C-inactivated APC, together with OVA peptide in 96-well U-bottom plates.…”