Abstract
Tumor necrosis factor-alpha and interleukin-6 are proinflammatory cytokines involved in several autoimmune diseases, including rheumatoid arthritis. Although monoclonal antibodies targeting these cytokines or their receptors are successful treatment approaches for autoimmune diseases, approximately 30% of treated patients fail to respond. Therefore, the designation of more effective and versatile therapeutics for simultaneously targeting multiple inflammatory pathways is pivotal. This study aimed to design a recombinant bispecific antibody (BisAb) targeting TNF-α and IL-6 receptor based on the complementarity-determining regions (CDRs) of adalimumab and tocilizumab antibodies. The physicochemical properties and tertiary structure of the modeled bispecific antibody proteins were studied through bioinformatics. The interaction of the designed bispecific antibody with human TNF-α and IL-6R was computed by molecular docking and molecular dynamics. The recombinant BisAb (60kDa) was expressed in Escherichia coli. The ELISA results demonstrated that the affinity of BisAb and its Ada-scFv moiety to TNF-α was quite similar with K(aff) values of 7.7 and 9.4 × 10(-13) M, respectively. The recombinant BisAb and Toci-scFv also represented similar affinities towards IL-6R (K(aff) values of 1.65 and 1.87 × 10(-12) M), closely comparable with the parental tocilizumab antibody. A dose-dependent neutralization of TNF-mediated cytotoxicity was also observed on L929 cells. Decreased phosphorylation of signal transducer and activator of transcription 3 (STAT3) was determined through western blotting with specific antibodies, indicating the blockade of IL-6 receptors before the preincubation with IL-6. The designed BisAb, a minimized biotherapeutic molecule, could successfully target two main ligands (IL-6R and TNF-α) involved in rheumatoid arthritis and many inflammatory/infectious diseases. KEY POINTS: • A new bispecific antibody was designed to target TNF-α and IL-6R. • In silico studies confirmed the physicochemical and structural properties of BisAb. • The best-modeled protein was recombinantly expressed in Escherichia coli. • BisAb significantly inhibited TNF-α-mediated cytotoxicity. • BisAb could efficiently suppress the IL-6-related phosphorylation of STAT3 protein.