[Construction and expression of disulphide stabilized anti-CD3/anti-Pgp diabody].
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.
Su
Ye
Y
State Key Laboratory of Experimental Hematology, Institute of Hematology & Hospital of Blood Diseases, Chinese Academic of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.
Liu
Juanni
J
Gao
Yingdai
Y
Qin
Li
L
Yang
Ming
M
Wang
Jinhong
J
Xu
Yuanfu
Y
Shao
Xiaofeng
X
Ji
Qing
Q
Xiong
Dongsheng
D
Yang
Chunzheng
C
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000276:immunology*; D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001667:Binding, Competitive; D017252:CD3 Complex / Q000276:immunology*; D002460:Cell Line; D004220:Disulfides / Q000737:chemistry*; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans
D018435:ATP Binding Cassette Transporter, Subfamily B; D018033:Antibodies, Bispecific; D017252:CD3 Complex; D004220:Disulfides