A native-like bispecific antibody suppresses the inflammatory cytokine response by simultaneously neutralizing tumor necrosis factor-alpha and interleukin-17A

Abstract

Anti-tumor necrosis factor (TNF) therapies are successful in the treatment of inflammatory disorders. However, some patients with rheumatoid arthritis (RA) fail to response anti-TNF drugs due to the compensation of other inflammatory signals. In this study, to reduce compensatory responses of interleukin-17A (IL-17A) during TNF-α inhibition, we generated an IgG-like bispecific antibodiy (bsAb) against TNF-α and IL-17A through a combination method of electrostatic Fc pairing and light chain crossover. This bsAb exhibited relatively high stability comparable to natural IgG antibodies, and retained the unaltered affinities to both of two targets. BsAb significantly decreased not only the expression level of neutrophil or Th17 chemokines, but also the secretion of IL-6/IL-8 on fibroblast-like synoviocytes (FLS) from a patient with RA. Meanwhile, TNF-α-mediated cellular cytotoxicity of fibroblasts was neutralized by bsAb. Importantly, we demonstrate that the combined blockade of TNF-α and IL-17A is more efficient than inhibition of either factor alone. Our results suggest the IgG-like anti-TNF-α/IL-17A bispecific molecule overcome the limited therapeutic responses using anti-TNF drugs. It may be a promising therapeutic agent for the treatment of autoimmune diseases.

Keywords: Fc heterodimerization; IL-17; Immune response; Immunity; Immunology and Microbiology Section; TNF-α; bispecific antibody; rheumatoid arthritis.

Figure 1. Generation and characterization of anti-TNF-α/IL-17A bispecific antibody (bsAb)

A. Schematic diagram of bsAb generated by fusion of anti-TNF-α antibody and anti-IL-17A antibody. BsAb consists of a crossed anti-IL-17A arm (CH1-C exchange) and an uncrossed anti-TNF-α arm. The parental monoclonal antibodies (mAb) are shown on the left. Fc parts of heavy chains are paired through an electrostatic steering effects. B. SDS-PAGE analysis of purified bsAb (lane 1), anti-IL-17A mAb (lane 2), and anti-TNF-α mAb (lane 3). C. Comparison of thermal stability of three antibodies by circular dichroism. D. The purity analysis of bsAb and parental mAbs by a size exclusion chromatography column.

Figure 2. Binding abilities of bsAb to TNF-α and IL-17A in ELISA

A. Immobilized TNF-α was bound with anti-TNF-α mAb or bsAb with serial dilutions. B. Immobilized IL-17A was bound with anti-IL-17A mAb or bsAb with serial dilutions. C. Specificity of bsAb were measure by detecting seven antigens, including TNF-α, IL-17A, insulin, IL-2, IGF-I, IGF-II, and INF-γ. Bound antibodies were detected with a horseradish peroxidase-conjugated anti-human Fc antibody and measured as optical densities (OD) at 450nm.The data are a representative of 3 separate experiments.

Figure 3. SPR analysis of antibodies against TNF-α and IL-17A

Anti-IL-17A mAb, ant-TNF-α mAb, or bispecific antibody was captured by anti-human Fc antibody on to a CM5 chip. A range of TNF-α (0 nM to 100 nM) and IL-17A (0 nM to 100 nM) were injected over all antibodies. Data was fitted to a 1:1 Langmuir binding model to determine binding parameters.

Figure 4. Neutralization of TNF-α-mediated cellular cytotoxicity by bsAb

L929 cells were incubated with 1 ng/ml of TNF-α and 4 μg/ml of actinomycin D. Then, cells were treated with various concentrations of bsAb, anti-TNFα mAb, anti-IL-17A mAb and anti-IGF-I/II mAb m708.5 as a control antibody. After 24 h, cell viabilities were determined by MTT method. The error bars represent mean ± SEM. Data shown are representatives of three independent experiments.

Figure 5. Addition and synergy of combined IL-17 and TNF-α to induce the production of IL-6 and IL-8 on FLS

A. After cells were stimulated with IL-17 and TNF-α for 12 h, mRNA expressions of cytokines (IL-6, IL-8) were determined by realtime-PCR. B. Secretions of cytokines (IL-6 and IL-8) in culture media were measured by ELISA. All results are representative of at least three independent experiments.

Figure 6. Inhibition of bsAb on the production of neutrophil and Th17 chemokines on FLS

A. FLS cells were incubated with IL-17 and TNF-α or in the presence of different, antibodies. The mRNA expression levels of CXCL1, CXCL2, CXCL6, and CCL20 were measured by realtime-PCR after stimulation for 12 h. B. After FLS were treated, the secretion of chemokines from RA-FLS were measured by ELISA. All results are representative of at least three independent experiments.

Figure 7. Inhibition of bsAb on the production of IL-6 and IL- 8 and proliferation, and migration of FLS

FLS cells were stimulated with TNF-α and IL-17A or in the presence of different, antibodies. A. The mRNA levels of IL-6 and IL- 8 were determined by realtime-PCR. B. Secretions of cytokines (IL-6 and IL-8) in culture media were measured by ELISA. C. Anti-proliferative effects of bsAb on FLS. After FLS cells were incubated with TNF-α and IL-17A, antibodies was added during the incubation. Cell proliferation was assessed using MTT assay. D. The anti-migration effects of bsAb on FLS were evaluated by the scratch assay. After wounds were formed, FLS were incubated with antibodies in the presence of TNF-α and IL-17A. Parallel lines represent the borders of the wounds.