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J Biol Chem, Vol. 274, Issue 26, 18218-18230, June 25, 1999

A Large Non-immunized Human Fab Fragment Phage Library That Permits Rapid Isolation and Kinetic Analysis of High Affinity Antibodies

Hans J. de Haard, Nicole van Neer, Anneke Reurs^¶, Simon E. Hufton, Rob C. Roovers^¶, Paula Henderikx^¶, Adriaan P. de Bruïne^¶, Jan-Willem Arends^¶, and Hennie R. Hoogenboom^¶

From  Target Quest B.V. and the ^¶ Department of Pathology, Maastricht University and University Hospital Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands

We report the design, construction, and use of the first very large non-immunized phage antibody library in Fab format, which allows the rapid isolation and affinity analysis of antigen-specific human antibody fragments. Individually cloned heavy and light chain variable region libraries were combined in an efficient two-step cloning procedure, permitting the cloning of a total of 3.7 × 10¹⁰ independent Fab clones. The performance of the library was determined by the successful selection of on average 14 different Fabs against 6 antigens tested. These include tetanus toxoid, the hapten phenyl-oxazolone, the breast cancer-associated MUC1 antigen, and three highly related glycoprotein hormones: human chorionic gonadotropin, human luteinizing hormone, and human follicle-stimulating hormone. In the latter category, a panel of either homone-specific or cross-reactive antibodies were identified. The design of the library permits the monitoring of selections with polyclonal phage preparations and to carry out large scale screening of antibody off-rates with unpurified Fab fragments on BIAcore. Antibodies with off-rates in the order of 10² to 10⁴ s¹ and affinities up to 2.7 nM were recovered. The kinetics of these phage antibodies are of the same order of magnitude as antibodies associated with a secondary immune response. This new phage antibody library is set to become a valuable source of antibodies to many different targets, and to play a vital role in target discovery and validation in the area of functional genomics.

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