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

J Biotechnol. 2009 Jun 15;142(2):170-8. doi: 10.1016/j.jbiotec.2009.04.002. Epub 2009 Apr 14.


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(H)H) were developed to antagonize the action of human TNF (hTNF). Here, we show that hTNF-V(H)H does not interfere with hTNF trimerization, but competes with hTNF for hTNF-receptor binding. Moreover, we describe posttranslational dimerization and multimerization of hTNF-V(H)H 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(H)H and the fusion protein was expressed and purified from Escherichia coli culture supernatants. hTNF-V(H)H-S-tag fusion proteins were efficiently dimerized and multimerized by MTG whereas hTNF-V(H)H was not susceptible to protein cross-linking. Cell cytotoxicity assays, using hTNF as apoptosis inducing cytokine, revealed that dimerized and multimerized hTNF-V(H)H proteins were much more active than the monomeric hTNF-V(H)H. We hypothesize that improved inhibition by dimeric and multimeric single chain hTNF-V(H)H proteins is caused by avidity effects.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Cell Line
  • Cytotoxicity Tests, Immunologic
  • Escherichia coli / genetics
  • Etanercept
  • Humans
  • Immunoglobulin G / metabolism
  • Immunoglobulin Heavy Chains / genetics
  • Immunoglobulin Heavy Chains / metabolism*
  • Protein Binding
  • Protein Multimerization*
  • Receptors, Tumor Necrosis Factor / metabolism
  • Recombinant Fusion Proteins / metabolism*
  • Ruminants / immunology
  • Spectrometry, Fluorescence
  • Streptomyces / enzymology
  • Transglutaminases / metabolism*
  • Tumor Necrosis Factor-alpha* / antagonists & inhibitors
  • Tumor Necrosis Factor-alpha* / immunology


  • Bacterial Proteins
  • Immunoglobulin G
  • Immunoglobulin Heavy Chains
  • Receptors, Tumor Necrosis Factor
  • Recombinant Fusion Proteins
  • TNF protein, human
  • Tumor Necrosis Factor-alpha
  • Transglutaminases
  • Etanercept