Transglutaminase-based antibody-drug conjugation: antibody site-specific mutation and identification
10.16438/j.0513-4870.2016-0962
- VernacularTitle:基于酶催化抗体药物偶联物中抗体的定点突变及鉴定
- Author:
Zhao-xiong MA
1
;
Yao XU
1
;
Hong ZHAO
2
;
Fu-mou SUN
1
;
Xin-rong ZHANG
1
;
Min WANG
1
;
Juan ZHANG
1
Author Information
1. College of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
2. Zhejiang Province Hospital of Traditional Chinese Medicine, Hangzhou 310006, China
- Publication Type:ORIGINAL ARTICLES
- Keywords:
antibody;
site-specific mutagenesis;
transglutaminase;
antibody-drug conjugate
- From:
Acta Pharmaceutica Sinica
2017;52(3):403-408
- CountryChina
- Language:Chinese
-
Abstract:
Transglutaminase (TG) posttranslational modification of antibody permits more precisely conjugating. Based on the amino acid sequence of an anti-CD24 antibody (cG7), this article is aimed to generate a deglycosylated cG7 mutant (cG7Q). Firstly, we introduced additional glutamines at position 297 (N297Q) by site-directed mutagenesis, and then transfected the recombinant plasmids into CHO-s cells via electroporation method and screened by Dot blot assay. Subsequently, cG7Q was expressed and purified through Protein A affinity chromatography, further identified by SDS-PAGE electrophoresis and Western blot. Its affinity was detected with surface plasmon resonance and flow cytometry assay, and ADCC effect was determined by lactate dehydrogenase (LDH) release. Eventually, a cG7 mutant, cG7Q was successfully expressed with sequence-specific conjugation sites for further study.
