Debra Gardner

Anti‐interleukin‐6 monoclonal antibody inhibits autoimmune responses in a murine model of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease resulting from dysregulation of the immune system. Interleukin‐6 (IL‐6) is a multifunctional cytokine produced by macrophages, monocytes and T and B cells. It stimulates B‐cell differentiation/maturation, immunoglobulin secretion, and T‐cell functions. Elevated levels of IL‐6 in serum, urine and renal glomeruli were detected in patients with active SLE and in murine models of SLE. Our study investigated the role of IL‐6 in an SLE‐like disease in New Zealand Black/White (NZB/W) F1 mice by administration of an anti‐murine IL‐6 monoclonal antibody (mAb). Intraperitoneal administration of the anti‐IL‐6 mAb suppressed the production of anti‐dsDNA autoantibody. B‐cell proliferation induced by anti‐IgM and anti‐CD40 was lower in the anti‐IL‐6 mAb‐treated mice, ex vivo studies demonstrated that anti‐IL‐6 mAb treatment inhibited anti‐dsDNA production. Anti‐CD3‐induced T‐cell proliferation and mixed lymphocyte reactions were inhibited by anti‐IL‐6 mAb treatment, indicating a partial down‐regulation of T cells. Histological analysis showed that treatment with anti‐IL‐6 mAb prevented the development of severe kidney disease. These results suggest that treatment with anti‐IL‐6 mAb has a beneficial effect on autoimmunity in murine SLE and that autoreactive B cells may be the primary target for anti‐IL‐6 mAb treatment; its effect on autoreactive T cells is also indicated.

Evaluation of anti-IL-6 monoclonal antibody therapy using murine type II collagen-induced arthritis.

Interleukin-6 is a multifunctional cytokine that is critical for T/B-cell differentiation and maturation, immunoglobulin secretion, acute-phase protein production, and macrophage/monocyte functions. Extensive research into the biology of IL-6 has implicated IL-6 in the pathophysiology and pathogenesis of RA. An anti-murine IL-6 mAb that neutralizes mouse IL-6 activities was tested in animal model of collagen-induced arthritis. Prophylactic treatment with anti-IL-6 mAb significantly reduced the incidence and severity of arthritis compared to control mAb treated mice. The mitogenic response of B and T cells isolated from the lymph nodes of anti-IL-6 treated mice was significantly reduced compared to cells isolated from control mAb treated mice. The overall histopathology score for paws from the anti-IL-6 treated mice was significantly reduced when compared to paws from mice treated with control mAb, including both inflammatory (synovitis and pannus) and erosive (erosions and architecture) parameters. Reduced loss of cartilage matrix components was also observed in the anti-IL-6 treated mice. Collectively, these data suggest that IL-6 plays a major role in the pathophysiology of rheumatoid arthritis, and thus support the potential benefit of anti-IL-6 mAb treatment in rheumatoid arthritis patients.

THU0042 Preclinical Characterization of Sirukumab, a Human Monoclonal Antibody that Targets Human Interleukin-6 Signaling

Background A significant fraction of patients with rheumatoid arthritis (RA) have an inadequate response to tumor necrosis factor α inhibitors as well as to biologics that target other pathways, such as anti-CD20 (rituximab), CTLA4-Ig (abatacept), or anti–interleukin-6 (IL-6) receptor (tocilizumab; TCZ). Due to the high concentrations of soluble IL-6 receptor (IL-6R) found in the blood and synovial fluid of patients with RA, we elected to examine whether targeting the cytokine IL-6 rather than IL-6R is a more efficient means to inhibit IL-6 signaling. Objectives To characterize sirukumab (SIR), a monoclonal human IgG1κ antibody specific for human IL-6, and compare neutralization of IL-6 signaling with TCZ, which targets the soluble and membrane forms of IL-6R. Methods Affinity and selectivity for human IL-6 were determined using kinetic exclusion, in vitro binding, and 7TD1 cell proliferation assays. The epitope recognized by SIR on human IL-6 was determined by protease protection and solution hydrogen-deuterium exchange, which was confirmed by alanine substitution. In vitro bioassays were used to demonstrate inhibition of IL-6 signaling and compare potency with TCZ. BALB/c mice challenged with human IL-6 produce serum amyloid A; this model was used to examine the potency of SIR in vivo. Results SIR bound to human IL-6 with high affinity (0.175 pM). Epitope mapping identified helix D, spatially proximal regions of helix A, and the loop before helix B as the binding sites for SIR on IL-6. These overlap with the site where soluble IL-6R binds to IL-6. SIR showed no binding of human IL-6 bound to the IL-6R/gp130 receptor complex on U937 cells. SIR did not recognize other ligands that signal through gp130, and species crossreactivity to IL-6 was limited to human and nonhuman primates. SIR, at doses of 5 and 0.5 mg/kg, significantly reduced expression of serum amyloid A in BALB/c mice challenged with human IL-6 compared to mice that received an isotype-matched, negative control antibody (P <0.05). In several bioassays utilizing U937, HepG2, and human endothelial cells, SIR ranged from 50-fold to 90-fold more potent than TCZ. Conclusions Current data suggests that SIR binds with high affinity and neutralizes the biological effects of human IL-6. In side-by-side bioassays, SIR appears to be more potent than TCZ. Phase 3 clinical trials are underway to establish the efficacy and safety of SIR in patients with RA. Acknowledgements Study sponsored by Janssen Research & Development, LLC, in collaboration with GlaxoSmithKline. Disclosure of Interest D. Gardner Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, E. Lacy Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, S. Wu Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, D. Shealy Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC

FRI0069 Neutralization of IL6 by Sirukumab (SIR) Inhibits Inflammation and Cellular Stress in a Human Vascular Surrogate System of Atherosclerosis

Background Rheumatoid arthritis (RA) and atherosclerosis are chronic inflammatory diseases that share pathologic and molecular features. RA doubles the risk of cardiovascular disease (CVD) compared to the non-RA population. Common RA treatments are anti-inflammatory by design, but effects on CVD are unclear. Previously we reported the effect of SIR on IL6 and tumor necrosis factor (TNF) α signaling using a human endothelial cell (EC) and smooth muscle cell (SMC) co-culture system. Here we report additional analyses further characterizing effects of SIR on atherosclerotic (ATH) and oxidative (OX) stress pathways. Objectives To examine if RA drugs decrease ATH signaling and cellular stress in vascular cells under CVD conditions. Methods An in vitro surrogate system that co-cultures human ECs and SMCs was used to assess effects of RA drugs on vascular cells. Fluid flow conditions that drive cardiovascular health and CVD were applied. Atheroprone flow conditions were based on human hemodynamics from the carotid bifurcation, a site prone to developing atherosclerosis. The culture medium contained atherogenic risk factors including in vivo concentrations of oxidized LDL (oxLDL), soluble IL6 receptor (sIL6R), and TNF. Using RNA sequencing and microarray, we performed transcriptomic and biologic pathway analyses of surrogate system response. We compared treatments targeting pathogenic RA pathways, including anti-IL6 (SIR), anti-IL6 receptor (tocilizumab; TCZ), anti-TNF (adalimumab; ADA), and a small molecule JAK inhibitor (tofacitinib; TOF). The magnitude of pathway response was calculated as the L2 norm of the log2 fold change of genes in the pathway. Results The combination of atheroprone flow, sIL6R, TNF, and oxLDL (RA-CVD conditions) induced a robust response of inflammatory and OX stress pathways compared to healthy conditions (atheroprone flow without TNF and with non-oxLDL and sIL6R). The anti-IL6/IL6R treatments (SIR, TCZ) improved RA-CVD conditions by inhibiting key pathogenic pathways, including ATH signaling and NRF2-mediated OX stress. SIR attenuated the magnitude of RA-CVD response in ATH and OX stress pathways vs IgG or vehicle control the most: by 17% (adj P=0.035; Wilcoxon signed-rank test) and 34% (adj P=0.094) in ECs; and 49% (adj P=1.4e-5) and 47% (adj P=1.1e-3) in SMCs, respectively. TCZ was similar to SIR in restoring ECs and SMCs to healthy conditions in both pathways; ADA showed a weaker, similar trend compared to IL6 inhibition. TOF was not effective in suppressing (and tended to exacerbate) ATH and OX stress pathways in ECs. Conclusions IL6 pathway inhibitors SIR and TCZ potently suppressed ATH and cellular stress in vitro. The degree of suppression suggests that these drugs may mitigate the effects of atherogenic factors sIL6R, TNF, and oxLDL. In contrast, TNF inhibitor ADA was a less effective inhibitor of key CVD pathways, while JAK inhibitor TOF tended to exacerbate CVD pathways. Collectively, the data suggest that IL6 inhibition may provide more CVD benefit compared to RA drugs targeting other pathways. Acknowledgements Study sponsored by Janssen Research & Development, LLC, in collaboration with GlaxoSmithKline. Disclosure of Interest R. Feaver Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, S. Collado Employee of: HemoShear, LLC, S. Hoang Employee of: HemoShear, LLC, E. Berzin Employee of: HemoShear, LLC, A. Armstrong Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, D. Gardner Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, H. Liu Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, A. Mackey Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, D. Manka Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC, D. Shealy Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, B. Blackman Shareholder of: HemoShear, LLC, Employee of: HemoShear, LLC

Anti-CXCL13 and Anti-TNFα Monoclonal Antibodies Combinatorial Treatment Inhibits Autoimmune Disease in a Murine Model of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the involvement of multiple organ systems with alternating clinical exacerbations and remissions. Circulating immune complexes and autoantibodies can cause tissue damage and organ dysfunction with manifestations involving the skin, serosal surfaces, central nervous system, and kidneys (Rahman & Isenberg, 2008). B cells are believed to play an important role in SLE. B cells can function as APCs, produce cytokines and chemokines contributing to lymphoid regulation, and can respond to stimuli in the microenvironment at local tissues (Ramanujam & Davidson, 2008). Pathogenic autoantibodies produced by autoreactive B cells are believed to play an important role in the pathogenesis of SLE. CXCL13 has been shown to be a key mediator of organization of lymphoid tissues. CXCL13 is a B cell chemoattractant that is expressed by peritoneal macrophages and follicular dendritic cells in secondary lymphoid organs, such as the follicles of Peyer’s patches, the spleen and lymph nodes. Through interaction with CXCR5, a G-protein coupled receptor, CXCL13 attracts B lymphocytes and promotes migration of small numbers of T helper follicular cells and macrophages (Gunn et al., 1998). CXCL13 is critical for B cell homing and follicle formation in lymph node and spleen, and it is required for the development of lymph nodes and Peyer’s patches (Ansel et al., 2000). CXCL13 protein level is elevated in ectopic B cell follicles formed in the inflamed tissues of multiple chronic diseases, and plays an important role in maintaining inflammation by actively recruiting B cells (Carlsen et al., 2004; Magliozzi et al., 2004; Salolonsson et al., 2002; Shi et al., 2001;). CXCL13 has been shown to have increased expression in the thymus and kidney of aged NZB/W F1 mice, and may play a role in breaking immune tolerance in the thymus of autoimmune prone mice (Ishikawa et al., 2001). Treatment with anti-CXCL13 has shown efficacy in animal models of RA and EAE (Bagaeva et al. 2006; Zheng et al., 2005). Because of its function and presence in various pathological conditions, CXCL13 and CXCL13 dependent pathways are thought to be instrumental in the pathogenesis of a variety of diseases where B cells may play a significant role, including RA, OA, UC, and SLE, and could be potential targets for autoimmune therapy (Table 1).