Isolation of picomolar affinity Anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site

被引:312
作者
Schier, R
McCall, A
Adams, GP
Marshall, KW
Merritt, H
Yim, M
Crawford, RS
Weiner, LM
Marks, C
Marks, JD
机构
[1] UNIV CALIF SAN FRANCISCO, DEPT PHARMACEUT CHEM, SAN FRANCISCO, CA 94110 USA
[2] FOX CHASE CANC CTR, DEPT MED ONCOL, PHILADELPHIA, PA 19111 USA
关键词
c-erbB-2; single-chain Fv; antibody phage display; affinity maturation; BIAcore;
D O I
10.1006/jmbi.1996.0598
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 determined the extent to which additional binding energy could be achieved by diversifying the complementarity determining regions (CDRs) located in the center of the antibody combining site of C6.5, a human single-chain Fv (scFv) isolated from a non-immune phage library which binds the tumor antigen c-erbB-2. CDR3 of the-light (V-L) and heavy (V-H) chain variable region of C6.5 were sequentially mutated, the mutant scFv displayed on phage, and higher affinity mutants selected on antigen. Mutation of V-L CDR3 yielded a scFv (C6ML3-9) with a 16-fold lower K-d (1.0 x 10(-9) M) than C6.5. Due to its length of 20 amino acids, four V-H CDR3 libraries of C6ML3-9 were constructed. The greatest increase in affinity from a single library was ninefold (K-d = 1.1 x 10(-10) M). Combination of mutations isolated from separate V-H CDR3 libraries yielded additional ninefold decreases in K-d, resulting in a scFv with a 1230-fold increase in affinity from wild-type C6.5 (K-d = 1.3 x 10(-11) M). The increase in affinity, and its absolute value, are comparable to the largest values observed for antibody affinity maturation in vivo or in vitro and indicate that mutation of V-L. and V-H CDR3 may be a particularly efficient means to increase antibody affinity. This result, combined with the location of amino acid conservation and substitution, suggests an overall strategy for in vitro antibody affinity maturation. In addition, the affinities and binding kinetics of the single-chain Fv provide reagents with potential tumor targeting abilities not previously available. (C) 1996 Academic Press Limited
引用
收藏
页码:551 / 567
页数:17
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