Construction and functional analysis of a bispecific antibody that targets TNF-α and ED-B / 药学学报

In order to enhance the specificity of TNF-α monoclonal antibody to inflamed site, a bispecific antibody BsDb that targets TNF-α and the extra-domain B (ED-B) of fibronectin (FN) was constructed by covalently linking the anti-TNF-α single chain Fv antibody (TNF-scFv) and the anti-ED-B scFv L19 via a flexible peptide linker deriving from human serum albumin (HSA). ED-B is an antigen specifically expressed at the inflamed site. BsDb is expressed in E. coli, identified by immunoblot, and purified with affinity chromatography. This was followed by further examination of its bioactivities and pharmacokinetics. We demonstrated that BsDb retained the immunoreactivity of its original antibodies as it could simultaneously bind to TNF-α and ED-B and neutralize the biological action of TNF-α. In the collagen-induced arthritis mice model, BsDb selectively accumulate in the inflamed joint with a maximal uptake of (12.2 ± 1.50)% ID/g in a single inflamed paw and retain in the inflamed paw for at least 72 h. In contrast, BsDb showed a short serum half-life of (0.50 ± 0.05) h and a rapid clearance from normal tissues. The findings reported herein indicate that BsDb has good specificity to the inflamed site and low toxicity to normal tissues. BsDb is therefore likely to have greater clinical applications in the treatment of rheumatoid arthritis and other autoimmune diseases. This laid a stable basis for its preclinical study.

Construction of IL-1Ra-HSA fusion protein and analysis of its bioactivity and pharmacokinetics / 药学学报

In order to increase the plasma half-life and tissue specificity of IL-1 receptor antagonist, a recombinant fusion protein IL-1Ra-HSA, linked by a rigid peptide linker PAPAP, was engineered and expressed by the Pichia pastoris host cells. The fusion protein was secreted to the host cells culture, identified by Western blot, and purified by affinity chromatography. This was followed by a further examination of its bioactivity and pharmacokinetics. Our results demonstrated that the fusion protein retained the antagonist activity of IL-1Ra, capable of binding specifically to the IL-1 receptor on human melanoma A375.S2 cells, and inhibits the cytolytic effect of IL-1beta to A375.S2 cells. Albumin fusion dramatically extended the half-life of IL-1Ra and resulted in a specific accumulation of IL-1Ra in the arthritic paws and a lower distribution of IL-1Ra in other organs such as liver, kidney, spleen and lung in mice with collagen-induced arthritis. The findings reported herein indicate that the fusion protein is likely to have greater clinical applications in areas such as the treatment of rheumatoid arthritis.