Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast.
We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single-chain variable fragment (scFv) antibody library was constructed in a yeast two-hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin-8 (hIL8) into the yeast two-hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One clone was chosen for further improvement by a novel affinity maturation process using the error-prone PCR of the scFv sequence followed by additional rounds of yeast two-hybrid screening. The scFv antibodies of both primary and affinity-matured scFv clones were expressed in E. coli. All purified scFvs showed specific binding to hIL8 in reciprocal coimmunoprecipitation and ELISA assays. All scFvs, as well as a fully human IgG antibody converted from one of the scFv clones and expressed in the mammalian cells, were able to effectively inhibit hIL8 in neutrophil chemotaxis assays. The technology described can generate fully human antibodies with high efficiency and low cost.
Ding
Ling
L
Department of Medical Oncology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Azam
Mark
M
Lin
Yu-Huei
YH
Sheridan
James
J
Wei
Shuanghong
S
Gupta
Gigi
G
Singh
Rakesh K
RK
Pauling
Michelle H
MH
Chu
Waihei
W
Tran
Antares
A
Yu
Nai-Xuan
NX
Hu
Jiefeng
J
Wang
Wei
W
Long
Hao
H
Xiang
Dong
D
Zhu
Li
L
Hua
Shao-Bing
SB
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D000915:Antibody Affinity / Q000276:immunology; D002465:Cell Movement / Q000187:drug effects; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D019151:Peptide Library; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D016209:Interleukin-8; D019151:Peptide Library; D057127:Single-Chain Antibodies