[Construction and expression of disulphide stabilized anti-CD3/anti-Pgp diabody]

Sheng Wu Gong Cheng Xue Bao. 2009 Jul;25(7):1042-8.
[Article in Chinese]

Abstract

We constructed and expressed an anti-CD3/anti-Pgp (P-glycoprotein) diabody previously. However, the two chains of diabody are associated non-covalently, resulting in being capable of dissociating. The aim of this study is to enhance the stability of the diabody. We introduced cysteine residues into the CD3 or Pgp V-domain to covalently lock the two chains together. The disulphide crosslinked diabody were expressed by Escherichia coli (E. coli) 16C9 and purified by a cation exchange column and an anti-Etag affinity chromatography. The purified proteins were verified through SDS-PAGE. Flow cytometry (FCM) was used to analyse the binding properties, competitive binding capacity and stability in vitro. The dsPpg-diabody failed to form disulphide bond properly. The designed disulphide bridge between the different chains of dsCD3-diabody was formed correctly. FCM demonstrated the dsCD3-diabody has specific antigen binding activity, the same binding activity and competitive binding activity as its parent diabody. The dsCD3-diabody retained the full activity even after 72 h incubation at 37 degrees C in human serum, in contrast, the parent diabody began to lose activity after only 1 h and lose all its activity 24 hours later. The induced disulphide bond in the CD3 V-domain effectively enhanced the stability of anti-CD3/anti-Pgp diabody. The method of stabilizing a diabody by introducing a disulphide bond into is practical.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B / immunology*
  • Antibodies, Bispecific / biosynthesis*
  • Antibodies, Bispecific / chemistry
  • Antibodies, Bispecific / genetics
  • Antibodies, Bispecific / immunology
  • Binding, Competitive
  • CD3 Complex / immunology*
  • Cell Line
  • Disulfides / chemistry*
  • Drug Stability
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Humans

Substances

  • ATP Binding Cassette Transporter, Subfamily B
  • Antibodies, Bispecific
  • CD3 Complex
  • Disulfides