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(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.

• Authors: Ingo Plagmann, Athena Chalaris, Andrei A Kruglov, Sergei Nedospasov, Philip Rosenstiel, Stefan Rose-John, Jürgen Scheller
• Journal: Journal of biotechnology (J Biotechnol) Vol. 142 Issue 2 Pg. 170-8 (Jun 15 2009) ISSN: 1873-4863 [Electronic] Netherlands
• PMID: 19439388 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

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

Topics

• Animals
• Bacterial Proteins (genetics, metabolism)
• Cell Line
• Cytotoxicity Tests, Immunologic
• Escherichia coli (genetics)
• Etanercept
• Humans
• Immunoglobulin G (metabolism)
• Immunoglobulin Heavy Chains (genetics, 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, immunology)