Covalently dimerized Camelidae antihuman TNFa single-domain antibodies expressed in yeast Pichia pastoris show superior neutralizing activity.

Antagonists of tumor necrosis factor alpha (TNFa) have revolutionized the treatment of selected inflammatory diseases. Recombination Camelidae variable heavy-chain domain-only TNFa antibodies (anti-TNF-VHH) have been developed to antagonize the action of human and murine TNFa. Here, we describe a strategy to obtain functional covalent dimer anti-TNF-VHH molecules with the C-terminal fusion of human IgG1 Fc domain named anti-TNF-VHH-Fc. The resulting fusion proteins were separately expressed by use of the pET28a vector in Escherichia coli ((Ec)) strain BL21 and the pPICZaA vector in Pichia pastoris ((Pp)) strain GS115, then purified by protein A affinity resin. Fc-engineered anti-(Ec)TNF-VHH-Fc was about 40 kDa and anti-(Pp)TNF-VHH-Fc was about 43 kDa. Monomeric VHH was also cloned and expressed in E. coli strain BL21, with the molecular weight of about 18 kDa. Enzyme-linked immunosorbent assay and L929 cell cytotoxicity assay demonstrated that the fusion protein anti-(Pp)TNF-VHH-Fc blocked TNFa activity more effectively than either anti-(Ec)TNF-VHH-Fc or monomeric anti-(Ec)TNF-VHH protein. We suggest that efficient disulfide bond formation using the P. pastoris expression system improves the covalent dimer anti-TNF-VHH-Fc neutralizing activity.