Affinity maturation of Fab antibody fragments by fluorescent‐activated cell sorting of yeast‐displayed libraries

FEBS Letters

Hoogenboom, Hennie R.¹  Pieters, Henk¹  van den Beucken, Twan¹  Steukers, Mieke¹  Hufton, Simon E.¹  van der Vaart, Marcel³  Ladner, Robert Charles² 

[1]Dyax s.a., Boulevard du Rectorat 27, Sart Tilman, 4000 Liège 1, Belgium

[2]Dyax Corp., 300 Technology Square, Cambridge, MA 02139, USA

[3]Unilever Research Vlaardingen, O.V. Noortlaan 120, 3133 AT Vlaardingen, The Netherlands

关键词: Yeast display;    Fab antibody;    Affinity maturation;    CDR;    complementarity determining region;    VH;    variable region of an antibody heavy chain;    VL;    variable region of an antibody light chain;    FACS;    fluorescent-activated cell sorting;    LC;    light chain;    HC;    heavy chain;   

DOI  :  10.1016/S0014-5793(03)00602-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We report for the first time the affinity maturation of Fab antibody fragments using fluorescent-activated cell sorting (FACS) of yeast-displayed repertoires. A single yeast display vector which enables the inducible expression of an anchored heavy chain and a soluble light chain has been constructed. The assembly and functional display on the yeast cell surface of Fab antibodies specific for different protein targets has been demonstrated by flow cytometry and immunofluorescence microscopy. We have affinity matured a Fab antibody specific for the tetravalent antigen streptavidin using FACS of yeast-displayed repertoires diversified by error-prone polymerase chain reaction. A panel of variants with up to 10.7-fold improvement in affinity was obtained after selection. Two leading clones, R2H10 (3.2 nM) and R3B1 (5.5 nM), had mutations in light chain complementarity determining region 1 LC-CDR1 (H34R) and LC-CDR3 (Y96H or Y96F) and gave a 10.7-fold and 6.3-fold affinity improvement over the starting antibody, respectively. The ability to efficiently affinity mature Fab antibodies is an important component of the antibody development pipeline and we have shown that yeast display is an efficient method for this purpose.

【 授权许可】

Unknown