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

TLDR

The study reports the design, construction, and use of the first very large non‑immunized phage antibody library in Fab format, enabling rapid isolation and affinity analysis of antigen‑specific human antibody fragments. The library was built by combining individually cloned heavy and light chain variable region libraries in a two‑step cloning procedure, yielding 3.7 × 10¹⁰ independent Fab clones and allowing large‑scale screening of antibody off‑rates with unpurified Fab fragments on BIAcore. The library successfully yielded an average of 14 distinct Fabs against six antigens—including tetanus toxoid, phenyl‑oxazolone, MUC1, hCG, LH, and FSH—producing antibodies with off‑rates of 10⁻²–10⁻⁴ s⁻¹, affinities up to 2.7 nM, and kinetics comparable to secondary immune responses, positioning it as a valuable resource for functional genomics target discovery and validation.

Abstract

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 10⁻² to 10⁻⁴s⁻¹ 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.