Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2016/07/19/jpet.116.236075.DC1 1521-0103/359/1/37–44$25.00 http://dx.doi.org/10.1124/jpet.116.236075 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 359:37–44, October 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Unexpected Potency Differences between B-Cell–Activating Factor (BAFF) Antagonist Antibodies against Various Forms of BAFF: Trimer, 60-Mer, and Membrane-Bound s Amy M. Nicoletti, Cynthia Hess Kenny, Ashraf M. Khalil, Qi Pan, Kerry L. M. Ralph, Julie Ritchie, Sathyadevi Venkataramani, David H. Presky, Scott M. DeWire, and Scott R. Brodeur Immune Modulation and Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut Received June 20, 2016; accepted July 18, 2016 Downloaded from ABSTRACT Therapeutic agents antagonizing B-cell–activating factor/B- human B-cell proliferation assay and in nuclear factor kB reporter lymphocyte stimulator (BAFF/BLyS) are currently in clinical assay systems in Chinese hamster ovary cells expressing BAFF development for autoimmune diseases; belimumab is the first receptors and transmembrane activator and calcium-modulator Food and Drug Administration–approved drug in more than and cyclophilin ligand interactor (TACI). In contrast to the mouse jpet.aspetjournals.org 50 years for the treatment of lupus. As a member of the tumor system, we find that BAFF trimer activates the human TACI necrosis factor superfamily, BAFF promotes B-cell survival and receptor. Further, we profiled the activities of two clinically ad- homeostasis and is overexpressed in patients with systemic vanced BAFF antagonist antibodies, belimumab and tabalumab. lupus erythematosus and other autoimmune diseases. BAFF Unexpectedly, we revealed differences in inhibitory potencies exists in three recognized forms: membrane-bound and two against the various BAFF forms, in particular that belimumab secreted, soluble forms of either trimeric or 60-mer oligomeric does not potently inhibit BAFF 60-mer. Through this increased states. To date, most in vitro pharmacology studies of BAFF understanding of the activity of BAFF antagonists against at ASPET Journals on September 26, 2021 neglect one or more of these forms. Here, we report a different forms of BAFF, we hope to influence the discovery of comprehensive in vitro cell-based analysis of BAFF in assay BAFF antagonist antibodies with distinct therapeutic mecha- systems that measure all forms of BAFF-mediated activation. We nisms for improvement in the treatment of lupus or other related demonstrate the effects of these BAFF forms in both a primary autoimmune pathologies. Introduction factor (TNF) SF13B provided clinical benefit in SLE, resulting in Food and Drug Administration (FDA) approval of the selective Systemic lupus erythematosus (SLE) is a chronic multisys- BAFF antagonist, belimumab (Benlysta) (Furie et al., 2011; Liu tem autoimmune disease characterized by the generation of and Davidson, 2011; Navarra et al., 2011). autoantibodies directed against a variety of self-antigens BAFF is a highly conserved member of the TNF superfamily (Tsokos, 2011). These autoantibodies form pathogenic im- of cytokines, primarily expressed by innate immune cells mune complexes that deposit into organs, ultimately inducing including monocytes, macrophages, dendritic cells, and other inflammation and tissue damage (Cancro et al., 2009; Sang nonhematopoetic cells (Shu et al., 1999; Tribouley et al., 1999; et al., 2014). Many of the current therapeutic options for the Nardelli et al., 2001). Early reports on BAFF function in- treatment of SLE include antimalarial agents, nonsteroidal anti-inflammatory drugs, glucocorticoids, and immunosup- dicated a potential critical role for determining B-cell fate and pressive drugs, which all seek to reduce the autoimmune the establishment of tolerance (Moore et al., 1999). The inflammatory process but also can result in general immuno- physiologic activity of BAFF is reportedly mediated by suppression and toxicity (Tsokos, 2011). Opportunities for new signaling via three receptors: B-cell activating factor receptor therapies exist among patients with the most severe disease (BAFFR), transmembrane activator and calcium-modulator as well as for steroid-sparing treatments. In recent years, and cyclophilin ligand interactor (TACI), and, in some reports, inhibition of the cytokine B-cell–activating factor [BAFF, also B-cell maturation antigen (BCMA). These three receptors are known as B-lymphocyte stimulator (BLyS)], tumor necrosis primarily expressed on B cells at different developmental stages (Gross et al., 2000; Marsters et al., 2000; Wu et al., dx.doi.org/10.1124/jpet.116.236075. 2000; Thompson et al., 2001; Yan et al., 2001; Karpusas s This article has supplemental material available at jpet.aspetjournals.org. et al., 2002; Liu et al., 2003; Bossen and Schneider, 2006). In ABBREVIATIONS: BAFF, B-cell–activating factor; BAFFR, B-cell–activating factor receptor; BCMA, B-cell maturation antigen; belim-comp, belimumab-comparator; BLyS, B-lymphocyte stimulator; CHO, Chinese hamster ovary; FDA, Food and Drug Administration; NF, nuclear factor; SLE, systemic lupus erythematosus; tabal-comp, tabalumab-comparator; TACI, transmembrane activator and calcium-modulator and cyclophilin ligand interactor; TEV, tobacco etch virus; TNF, tumor necrosis factor. 37 38 Nicoletti et al. humans, BAFFR is widely expressed by all B cells, with the distinct oligomeric states remain unclear, and in vitro phar- exception of bone marrow plasma cells, and BAFF-BAFFR macology assays often neglect one of these forms. interactions have been proven critical to maintenance of We sought to characterize and compare the activities of primary B cells (Oren et al., 2002; Ng et al., 2004). BAFFR membrane-bound and secreted, soluble BAFF forms with selectively recruits TNF receptor–associated factor 3, leading trimer and 60-mer oligomeric states in a set of in vitro, cell- to downstream signaling cascades that can modulate B-cell based assay systems. Further,weprofiletwomonoclonal survival and differentiation, including activation of the nu- antibodies generated based on the published sequences of clear factor kB (NFkB) pathway (Mukhopadhyay et al., 1999; belimumab and tabalumab for their inhibitory capacity Claudio et al., 2002; Hatada et al., 2003; Bossen and against these various BAFF forms. Interestingly, we find Schneider, 2006; Mackay and Schneider, 2009). Transgenic significantly differing potencies at inhibiting the various mice that overexpress BAFF display phenotypic characteris- membrane and soluble forms of BAFF. Results reported here tics that closely mimic those observed in SLE and Sjögren’s provide insight on the biologic activity of membrane-bound syndrome, including increased numbers of anti-DNA anti- and soluble, secreted BAFF as well as the pharmacologic bodies, B-cell expansion, proteinuria, and glomerulonephritis mechanisms of BAFF antagonists. A better pharmacologic (Mackay et al., 1999; Yan et al., 2001; Groom et al., 2002). In understanding of the inhibitory capability of BAFF antago- humans, serum levels of BAFF are significantly elevated in nists against all forms of BAFF has the potential to impact patients with SLE, Sjögren’s syndrome, and rheumatoid the therapeutic success of these antibodies in the treatment arthritis compared with healthy controls (Cheema et al., of lupus and related pathologies. Downloaded from 2001; Mariette et al., 2003; Tan et al., 2003). In humans, BAFF exists in both a membrane-bound form (of 285 amino acids) and two soluble forms (Moore et al., 1999; Materials and Methods Schneider et al., 1999; Thompson et al., 2001). Soluble BAFF Antibodies. Amino acid sequence for the belimumab-comparator exists predominantly as a trimer or 60-mer (Liu et al., (belim-comp) antibody was derived from patent US20110293610 2002; Cachero et al., 2006); however, the oligomeric state of (Ruben et al., 2011). Belim-comp was produced on a human IgG1 jpet.aspetjournals.org soluble BAFF in vivo has been the subject of debate. Both wild-type framework with a l light chain to conform to the FDA- soluble and membrane-bound forms of BAFF are biologically approved molecule (Baker et al., 2003). Belimumab was also pur- active (Schneider et al., 1999; Liu et al., 2002; Liu et al., chased as the marketed product (GlaxoSmithKline, London, UK) and 2003; Zhukovsky et al., 2004), and data from transgenic mice showed identical inhibitory potency to the inhouse produced material indicate a role for soluble BAFF in homeostasis; membrane- in the Chinese hamster ovary (CHO)-BAFFR assay (data not shown). Amino acid sequence for the tabalumab-comparator (tabal-comp) bound BAFF plays an accessory role (Bossen et al., 2011). antibody was derived from World Health Organization INN publi- at ASPET Journals on September 26, 2021 Binding affinities of BAFF for BAFFR, TACI, and BCMA have cation vol. 25 (WHO, 2011). Tabal-comp was produced on a human been previously described (Wu et al., 2000; Yu et al., 2000; IgG4-Pro (S228P) framework with a k light chain to conform to the Bossen and Schneider, 2006), although the subsequent down- clinical-stage molecule (Manetta et al., 2014). Each antibody was stream signaling properties of the various BAFF forms have cloned in pTT5-Neo, expressed in CHO-E cells, and purified by not been well studied. The clinical consequences of blocking