Transglutaminase-catalyzed covalent multimerization of camelidae anti-human TNF single domain antibodies improves neutralizing activity

Abstract

Tumor necrosis factor (TNF) plays an important role in chronic inflammatory disorders, such as Rheumatoid Arthritis and Crohn's disease. Recently, monoclonal Camelidae variable heavy-chain domain-only antibodies (V HH) were developed to antagonize the action of human TNF (hTNF). Here, we show that hTNF–V HH does not interfere with hTNF trimerization, but competes with hTNF for hTNF-receptor binding. Moreover, we describe posttranslational dimerization and multimerization of hTNF–V HH molecules in vitro catalyzed by microbial transglutaminases (MTG). The ribonuclease S-tag-peptide was shown to act as a peptidyl substrate in covalent protein cross-linking reactions catalyzed by MTG from Streptomyces mobaraensis. The S-tag sequence was C-terminally fused to the hTNF–V HH and the fusion protein was expressed and purified from Escherichia coli culture supernatants. hTNF–V HH-S-tag fusion proteins were efficiently dimerized and multimerized by MTG whereas hTNF–V HH was not susceptible to protein cross-linking. Cell cytotoxicity assays, using hTNF as apoptosis inducing cytokine, revealed that dimerized and multimerized hTNF–V HH proteins were much more active than the monomeric hTNF–V HH. We hypothesize that improved inhibition by dimeric and multimeric single chain hTNF–V HH proteins is caused by avidity effects.