Covalent dimerization of camelidae anti-human TNF-alpha single domain antibodies by the constant kappa light chain domain improves neutralizing activity

The tumor necrosis factor-alpha (TNFα) plays an important role in a number of chronic inflammatory disorders. Monoclonal camelidae variable heavy chain domain-only antibodies (VHH) have been developed to antagonize the action of human TNFα (anti-TNF-VHH). Here we describe a strategy to obtain functional dimeric anti-TNF-VHH molecules, based on the C-terminal fusion of a κ light chain domain to the anti-TNF-VHH. The resulting fusion protein was transiently expressed by use of viral vectors in Nicotiana benthamiana⁽Nb⁾ leaves and purified. Competitive ELISA and cell cytotoxicity assays revealed that the dimerized anti-NbTNF-VHHCκ proteins blocked TNFα-activity more effectively than either the monomeric Escherichia coli⁽Ec⁾ produced anti-EcTNF-VHH or the monomeric anti-NbTNF-VHHCκ. We suggest that enhanced inhibition shown by dimeric anti-NbTNF-VHHCκ proteins is achieved by an increase in avidity. Biotechnol. Bioeng. 2010; 106: 161-166.