title: A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies.
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 x 10(10) 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(-4) s-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.
authors.affiliation: Target Quest B.V., Maastricht University and University Hospital Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands.
authors.forename: H J
authors.initials: HJ
authors.lastname: de Haard
authors.forename: N
authors.initials: N
authors.lastname: van Neer
authors.forename: A
authors.initials: A
authors.lastname: Reurs
authors.forename: S E
authors.initials: SE
authors.lastname: Hufton
authors.forename: R C
authors.initials: RC
authors.lastname: Roovers
authors.forename: P
authors.initials: P
authors.lastname: Henderikx
authors.forename: A P
authors.initials: AP
authors.lastname: de Bruïne
authors.forename: J W
authors.initials: JW
authors.lastname: Arends
authors.forename: H R
authors.initials: HR
authors.lastname: Hoogenboom
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000302:isolation & purification*; D001435:Bacteriophages; D001483:Base Sequence; D015374:Biosensing Techniques; D003429:Cross Reactions; D017931:DNA Primers; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics*; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D015183:Restriction Mapping; D013154:Spleen / Q000737:chemistry
chemical_list: D000906:Antibodies; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library