A human synthetic combinatorial library of arrayable single-chain antibodies based on shuffling in vivo formed CDRs into general framework regions

被引:41
作者
Azriel-Rosenfeld, R [1 ]
Valensi, M [1 ]
Benhar, I [1 ]
机构
[1] Tel Aviv Univ, George S Wise Fac Life Sci, Dept Mol Microbiol & Biotechnol, IL-69978 Ramat Aviv, Israel
基金
以色列科学基金会;
关键词
arrayable scFvs; cellulose-binding domain; phage display; colony lift screen;
D O I
10.1016/j.jmb.2003.10.053
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We describe a novel approach for high-throughput screening of recombinant antibodies, based on their immobilization on solid cellulose-based supports. We constructed a large human synthetic single-chain Fv antibody library where in vivo formed complementarity determining regions were shuffled combinatorially onto germline-derived human variable-region frameworks. The arraying of library-derived scFvs was facilitated by our unique display/ expression system, where scFvs are expressed as fusion proteins with a cellulose-binding domain (CBD). Escherichia coli cells expressing library-derived scFv-CBDs are grown on a porous master filter on top of a second cellulose-based filter that captures the antibodies secreted by the bacteria. The cellulose filter is probed with labeled antigen allowing the identification of specific binders and the recovery of the original bacterial clones from the master filter. These filters may be simultaneously probed with a number of antigens allowing the isolation of a number of binding specificities and the validation of specificity of binders. We screened the library against a number of cancer-related peptides, proteins, and peptide-protein complexes and yielded antibody fragments exhibiting dissociation constants in the low nanomolar range. We expect our new antibody phage library to become a valuable source of antibodies to many different targets, and to play a vital role in facilitating high-throughput target discovery and validation in the area of functional cancer genomics. (C) 2003 Elsevier Ltd. All rights reserved.
引用
收藏
页码:177 / 192
页数:16
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