Chemically Modified Interleukin-6 Aptamer Inhibits Development of Collagen-Induced Arthritis in Cynomolgus Monkeys

Abstract

Interleukin-6 (IL-6) is a potent mediator of inflammatory and immune responses, and a validated target for therapeutic intervention of inflammatory diseases. Previous studies have shown that SL1026, a slow off-rate modified aptamer (SOMAmer) antagonist of IL-6, neutralizes IL-6 signaling in vitro. In the present study, we show that SL1026 delays the onset and reduces the severity of rheumatoid symptoms in a collagen-induced arthritis model in cynomolgus monkeys. SL1026 (1 and 10 mg/kg), administered q.i.d., delayed the progression of arthritis and the concomitant increase in serum IL-6 levels compared to the untreated control group. Furthermore, SL1026 inhibited IL-6-induced STAT3 phosphorylation ex vivo in T lymphocytes from human blood and IL-6-induced C-reactive protein and serum amyloid A production in human primary hepatocytes. Importantly, SOMAmer treatment did not elicit an immune response, as evidenced by the absence of anti-SOMAmer antibodies in plasma of treated monkeys. These results demonstrate that SOMAmer antagonists of IL-6 may be attractive agents for the treatment of IL-6-mediated diseases, including rheumatoid arthritis.

FIG. 1.

SL1025 occludes binding sites of IL-6R and gp130. (A) Sequences of SL1025 and SL1026 with 5-dU modifications indicated (Z = benzyl, P = naphthyl, E = phenethyl) and 2′-methoxy positions highlighted gray. SL1026 is comprised of SL1025 with a 40 kDa polyethylene glycol (PEG) conjugated to its 5′ terminus. Both sequences have a 3′ inverted dT (idT). (B) X-ray crystal structure of the IL-6:SL1025 complex in a cartoon and transparent surface rendering representation (PDB ID: 4NI9) [31]. IL-6 is colored blue, and SL1025 is colored green. Hydrophobic modifications in SL1025 that make direct contact with IL-6 are highlighted green. (C) X-ray crystal structure of the IL-6:IL-6R:gp130 complex in a cartoon and transparent surface rendering representation (PDB ID: 1P9M) [67]. IL-6 is colored blue, IL-6R is colored brown, and gp130 is colored pink.

FIG. 2.

SL1026 inhibits IL-6-induced STAT3 phosphorylation in human lymphocytes. Cells were induced with IL-6 and STAT3 phosphorylation was determined by FACS using a fluorescent anti-p-STAT3 antibody. Percent inhibition values (relative to no IL-6 and no inhibitor control samples) are plotted as the mean ± SEM of 10 samples at each concentration. A statistically significant increase of inhibition was observed compared to no-inhibitor control (0.0 ± 2.8%) for all SL1026 and tocilizumab groups (Dunnett's test, two-tailed, P < 0.01).

FIG. 3.

SL1026 inhibits IL-6-induced C-reactive protein (CRP) and serum amyloid A (SAA) expression in human primary hepatocytes. CRP (A) and SAA (B) concentrations were measured in hepatocyte supernatants by ELISA after IL-6 treatment and plotted as a function of SL1026 or tocilizumab concentration. Values are plotted as the mean ± SEM of three determinations.

FIG. 4.

Plasma concentration–time profiles of SL1026 in monkeys. Plasma SL1026 concentrations after administrations of 1 (○), 10 (●), or 30 mg/kg (△) were measured. Values are plotted as the mean ± SD of four monkeys. Values measured during the first 8 h after administration are shown in the inset plot.

FIG. 5.

Effect of SL1026 on plasma IL-6 levels and clinical assessments in collagen-induced arthritis (CIA) monkeys. (A) Animals were sensitized on day 0 and 21 with collagen and dosed with slow off-rate modified aptamer (SOMAmer) every 6 h on days 0–11. Plasma was collected and measurements of arthritis score and general condition score were made on days indicated with a dot. Arthritis score (B) and general condition score (C) were evaluated at days 6, 13, 20, 27, and 34 after the first sensitization. Values are plotted as the mean ± SEM (n = 4 in each group) for groups administered 0 (○), 1 (●), or 10 (▲) mg/kg SL1026. A statistically significant decrease of arthritis score was noted in the 10 mg/kg group (*P < 0.05, 10 mg/kg group vs. 0 mg/kg group) and of general condition score in both the 1 and 10 mg/kg groups (*P < 0.05, 1 and 10 mg/kg groups vs. 0 mg/kg group) by mixed effect model for repeated measures method in overall mean. (D) IL-6 concentration was measured in plasma samples collected at various times after the first immunization. Values are plotted as the median and interquartile range (n = 4 in each group) for groups administered 0 (○), 1 (●), or 10 (▲) mg/kg SL1026.

FIG. 6.

Anti-SL1026 antibody levels in CIA monkeys. Antibodies against the DNA component of SL1026 (A) and the PEG component of SL1026 (B) were measured in plasma collected from all monkeys 6 days before the first SL1026 dose (P), and on days 0, 6, 13, 20, 27, and 34 after the first SL1026 dose. Values are plotted as median and range (n = 4 in each group).