A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies

We report the design, construction, and use of the first very large non-imm unized phage antibody library in Fab format, which allows the rapid isolati on and affinity analysis of antigen-specific human antibody fragments. Indi vidually cloned heavy and light chain variable region libraries were combin ed in an efficient two-step cloning procedure, permitting the cloning of a total of 3.7 x 10(10) independent Fab clones. The performance of the librar y was determined by the successful selection of on average 14 different Fab s against 6 antigens tested. These include tetanus toroid, the hapten pheny l-oxazolone, the breast cancer-associated MUC1 antigen, and three highly re lated glycoprotein hormones: human chorionic gonadotropin, human luteinizin g hormone, and human follicle-stimulating hormone. In the latter category, a panel of either homone-specific or crossreactive antibodies were identifi ed. The design of the library permits the monitoring of selections with pol yclonal phage preparations and to carry out large scale screening of antibo dy off-rates with unpurified Fab fragments on BIAcore. Antibodies with off- rates in the order of 10(-2) to 10(-4) s(-1) and affinities up to 2.7 nM we re recovered. The kinetics of these phage antibodies are of the same order of magnitude as antibodies associated with a secondary immune response. Thi s new phage antibody library is set to become a valuable source of antibodi es to many different targets, and to play a vital role in target discovery and validation in the area of functional genomics.