B lymphocyte stimulator (BLyS; trademark of Human Genome Sciences, Rockville, MD) protein (also known as BAFF, THANK, TALL-1, TNFSF13B, and zTNF4), a member of the tumor necrosis factor (TNF) ligand superfamily, is synthesized as a 285–amino acid type II membrane protein and exists in both membrane and cleaved 152–amino acid soluble forms (1–6). BLyS is expressed on monocytes, macrophages, and monocyte-derived dendritic cells, and is up-regulated in response to interferon-γ and interleukin-10 (IL-10) (7). In vitro, recombinant human BLyS enhances B cell proliferation and Ig secretion through interaction with receptors expressed predominantly on B cells (1). In vivo, recombinant human BLyS causes splenic hyperplasia in mice, primarily due to increases in the number of mature B cells. BLyS administration to mice also causes increases in the total serum Ig concentration and enhanced humoral responses to both T cell–independent and T cell–dependent antigens (1, 2, 8).
BLyS has been shown to bind with high affinity to 3 receptors, all of which are members of the TNF receptor family (6, 9, 10). Two of the receptors, BCMA and TACI, also bind APRIL, another TNF family member that is the most homologous to BLyS (11–13). The precise function of these two receptors is not well understood, but studies in knockout mice suggest that BCMA is functionally redundant, since BCMA-deficient mice have a normal B cell phenotype (14). TACI has been shown to have an inhibitory role in B cell development, since TACI knockout animals exhibit increased peripheral B cells, reduced responses to T cell–independent antigens (15, 16), and lymphoproliferative disorders and autoimmune disease (17). In contrast, BLyS-deficient mice show a phenotype of severe loss of mature B cells in the spleen, peripheral blood, and lymph nodes (18, 19). A third receptor, BLyS receptor 3 (BR-3; BAFF-R), is specific for BLyS (9, 10). The A/WySnJ mouse strain, which harbors a naturally occurring truncation of BR-3, exhibits a phenotype similar to that of BLyS-deficient mice (9, 10, 20), suggesting that BR-3 is the primary mediator of the effects of BLyS on B cell survival and maturation.
Several lines of evidence suggest that elevated levels of BLyS may be involved in the pathogenesis of B cell–mediated autoimmune diseases. First, constitutive overexpression of BLyS in transgenic animals results in manifestations of autoimmune-like symptoms, including anti-DNA antibodies, rheumatoid factor, circulating immune complexes, and deposition of immune complexes in the kidney leading to glomerulonephritis. These symptoms resemble those of systemic lupus erythematosus (SLE) and some aspects of rheumatoid arthritis (RA) (21, 22). Second, elevated levels of BLyS have been found in other murine models of SLE, including MRL-lpr/lpr and (NZB × NZW)F1 strains (6). Finally, elevated levels of BLyS have been observed in the serum of patients with SLE and RA (23, 24) as well as Sjögren's syndrome (25, 26). Significant correlations between BLyS levels and autoantibody production were shown in these studies.
The association of BLyS overproduction with manifestations of several autoimmune diseases suggests that modulation of BLyS levels could be a novel therapeutic approach to the treatment of such diseases. Indeed, studies with soluble BLyS receptors as antagonists have shown efficacy in reducing disease manifestations in murine models of both SLE and RA (6, 27, 28). With the aim of developing a therapeutic agent for autoimmune disease, LymphoStat-B (Human Genome Sciences) antibody, a human, neutralizing monoclonal antibody against human BLyS, was generated. We describe herein the generation of human antibodies against BLyS using phage display, as well as the in vitro and in vivo characterization of LymphoStat-B.
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- MATERIALS AND METHODS
BLyS plays a critical role in the normal regulation of B cell development and immune response. This was demonstrated by the severely depleted B cell phenotype observed in BLyS-deficient animals (18, 19). As is the case for other cytokines, BLyS levels must be carefully regulated in order to maintain “immune homeostasis” (37). Consistent with this notion are findings that overproduction of BLyS is associated with increased immunoglobulin production and autoimmunity. Transgenic mice overexpressing either murine or human BLyS show increased numbers of mature B cells in the spleen and lymph nodes, elevated levels of total immunoglobulins, rheumatoid factor, anti–double-stranded DNA antibodies, and circulating immune complexes, all of which are manifestations associated with autoimmune disease. The mice eventually develop proteinuria and glomerulonephritis, symptoms that are specifically associated with SLE (21, 22). Elevated levels of BLyS have also been detected in the (NZB × NZW)F1 and MRL-lpr/lpr mouse models of lupus. BLyS levels in these animals increase with age, in parallel with increased autoantibody production, proteinuria, and other disease manifestations, again suggesting an important role for BLyS in the development of autoimmune disease in animals (6).
Evidence that BLyS is associated with human autoimmune disease comes from several studies demonstrating elevated levels of circulating BLyS in serum samples from patients with SLE and RA (23, 24). In RA patients, elevated levels of BLyS were also found in synovial fluid and were consistently greater than the serum levels in the same patient, possibly suggesting that there is local production of BLyS at sites of inflammation (38). Increased BLyS levels have also been found in sera from patients with Sjögren's syndrome, another autoimmune disease characterized by B cell hyperreactivity and autoantibody production (25, 26). Mice transgenic for BLyS also develop symptoms of Sjögren's syndrome (25), consistent with clinical observations of the association of Sjögren's syndrome in subsets of patients with SLE (39). Taken together, these observations suggest that elevated levels of BLyS may contribute to B cell hyperactivity and autoantibody production in multiple autoimmune diseases by enhancing the survival of B cells. Specific targeting of BLyS may therefore provide a novel treatment for these diseases. Toward that end, we have generated a human antibody specific for human BLyS that can neutralize the biologic functions of BLyS in vitro and in vivo.
LymphoStat-B was generated from a naive human Ig library using phage display technology. More than 1,200 antibodies with distinct amino acid sequences were isolated that specifically bound soluble human BLyS. Optimization of selected main candidates allowed further improvements in activity, leading to the selection of the clinical development candidate, LymphoStat-B.
Interactions between BLyS and its receptors are complex. At least 3 distinct receptors exist for BLyS. The major receptor for mediating BLyS-dependent B cell development appears to be the BLyS-specific receptor BR-3. This is based on data showing that the A/WySnJ mouse strain, which has a defect in BR-3, mirrors the severe deficits in peripheral mature B cell populations seen in BLyS-deficient mice (9, 10, 18–20). Although BLyS binds to 2 additional receptors, BCMA and TACI, analyses of mice deficient in these receptors show no B cell deficits in the BCMA knockout mice (14) and increased levels of B cells in TACI-deficient mice (15), suggesting a redundant role for BCMA and an inhibitory role for TACI in the regulation of B cell maturation (17). Importantly, we show by 2 independent methods that LymphoStat-B is able to neutralize BLyS interactions with all 3 BLyS receptors.
BLyS, like many TNF family members, exists in both soluble and membrane-bound forms. Most available data support the action of BLyS as a soluble cytokine (1), and elevated levels of soluble BLyS are correlated with autoimmune disease (23, 24). Any specific activity of membrane-bound BLyS remains to be determined. In fact, the exact nature of membrane-bound BLyS is not fully understood, but it has been shown that a panel of antibodies can differentially recognize membrane-bound BLyS (7). The BLyS antibodies described in the present study were selected specifically for their ability to recognize and inhibit the effects of soluble human BLyS. LymphoStat-B does not recognize membrane-bound BLyS and does not bind to the surface of human cells that bear membrane-bound BLyS. Thus, the specific activity of soluble BLyS is targeted, and any nonspecific effects that might be associated with antibody binding to BLyS-producing cells should be prevented.
BLyS has also been shown to exist in a complex with APRIL to form heterotrimers. Although the biologic significance of these heterotrimers is not clearly defined, they have in vitro biologic activity and interact with BLyS receptors (40). While LymphoStat-B was raised against soluble “homotrimeric” BLyS, interactions with heterotrimers have been shown in in vitro assays (Smith R, Roschke V: unpublished observations), although the affinity of these interactions was 7–8-fold lower than that of the heterotrimer recognizing antibodies reported previously. The significance of this inhibition awaits a clearer delineation of the in vivo role of BLyS/APRIL heterotrimers in B cell regulation.
In an in vitro assay, LymphoStat-B was found to inhibit human BLyS-induced stimulation of B cells from both murine splenocytes and human tonsillar B cells (data not shown). In vivo, LymphoStat-B neutralizes the observable effects of exogenously administered human BLyS in mice. These effects include stimulation of B cell proliferation, as evidenced by increases in spleen weight, representation of mature marginal zone B cells, as demonstrated by increases in B220+/ThB+ B cells, and immunoglobulin secretion, as evidenced by increases in IgA levels. LymphoStat-B at dosages of 1–5 mg/kg completely prevented these BLyS-induced activities. In this model system, LymphoStat-B has similar efficiencies when administered via the intravenous or the subcutaneous route (Hilbert D: unpublished observations). It should also be noted that the dosing schedule of 4 daily injections of BLyS used in the present study has only modest effects on increasing IgM levels and little, if any, effects on IgG levels (35). Nevertheless, LymphoStat-B can reduce BLyS-induced changes in IgM (data not shown).
Administration of LymphoStat-B to normal cynomolgus monkeys resulted in tissue B cell depletion that was sustained for up to 4 weeks after the treatment period. Although no significant depletion of circulating B cells was observed at these time points, it has been suggested that the spleen and lymphoid tissue act as reservoirs for the supply of B cells into the bloodstream (41), and effects on circulating B cells may not be observed until reserves of B cells in lymphoid tissues are adequately suppressed. No significant changes were observed in serum immunoglobulin levels following LymphoStat-B administration. However, since the B cell–depleting drug rituximab results in severe (>90%) and prolonged B cell depletion, with only minor concomitant effects on serum immunoglobulin levels (42), it is perhaps not surprising that changes in serum immunoglobulins were not observed in the study reported here. Although the effects of LymphoStat-B administration over a short period of time might not be expected to fully reflect the phenotypes of BLyS knockout mice, one key similarity with the BLyS knockout mice, namely a reduction of tissue B cell populations, was observed following drug administration.
LymphoStat-B possesses many properties that make it ideally suited for use as a therapeutic agent. First, it binds with high affinity to soluble human BLyS and inhibits its activities both in vitro and in vivo. Second, it does not recognize other TNF family members, including its closest homolog human APRIL, and is therefore specific for the factor implicated in the pathogenesis of several autoimmune diseases. As an antibody, an additional advantage of LymphoStat-B is its long terminal half-life. In mice and monkeys, the half-life of LymphoStat-B is 2.5 days and 11–14 days, respectively (Riccobene T: unpublished observations).
Overproduction of BLyS has been associated with SLE, RA, and Sjögren's syndrome. The etiologies of these diseases are unknown, but they share the common features of B cell hyperactivity and autoantibody production, which are strongly implicated in the pathogenesis of the disease process. Support for the ability of an antagonist of BLyS to affect these diseases comes from several animal studies with soluble BLyS receptors. Administration of TACI-Fc or BCMA-Fc has been shown to inhibit antigen-specific antibody responses and decrease B cell numbers and germinal centers (43, 44). TACI-Fc and BR-3-Fc have also been shown to inhibit the development of SLE disease manifestations in the (NZB × NZW)F1 mouse (6, 28). TACI-Fc has also prevented disease onset in the collagen-induced arthritis model in mice (27).
The critical role BLyS plays in the regulation of B cell homeostasis suggests that deregulation of BLyS may underlie these as well as additional B cell–mediated diseases. LymphoStat-B is being developed as a novel treatment for autoimmune diseases and is currently being evaluated in a phase I clinical trial in SLE patients. Studies are also under way to identify additional diseases in which BLyS might play a role and LymphoStat-B may have therapeutic potential.