Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen.
We have developed a strategy for guiding the selection of human antibody fragments from phage display repertoires to a single epitope of an antigen, using rodent monoclonal antibodies as a template. Thus the heavy chain of a rodent antibody (MAb32) directed against human tumor necrosis factor alpha (TNF alpha) was cloned and paired as a template chain with a repertoire of human light chains for display as Fab fragments on filamentous phage. The phage were selected by binding to the antigen. The selected human light chains were in turn paired with a repertoire of human heavy chains displayed on phage, and the phage selected again. The isolated phage displaying human antibody fragments binding to TNF alpha also bound to a peptide comprising the N-terminal region of TNF alpha as with MAb32. One of the human Fab fragments was recloned for expression as a glycosylated human antibody in mammalian cells: Binding to TNF alpha could be competed with MAb32 or with anti-serum to the peptide, indicating the same epitope. The human antibody was found to have a binding affinity (Kd = 15 nM) similar to MAb32 (Kd = 26 nM). The process contrasts with existing means of "humanizing" rodent monoclonal antibodies in that the antibodies derived are completely human.
Jespers
L S
LS
MRC Centre for Protein Engineering, MRC Centre, Cambridge, U.K.
Roberts
A
A
Mahler
S M
SM
Winter
G
G
Hoogenboom
H R
HR
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D000939:Epitopes / Q000276:immunology*; D005803:Genes, Immunoglobulin; D006031:Glycosylation; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis*; D007145:Immunoglobulin kappa-Chains / Q000096:biosynthesis*; D007700:Kinetics; D051379:Mice / Q000276:immunology; D008961:Models, Structural; D008969:Molecular Sequence Data; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000941:Antigens; D017931:DNA Primers; D000939:Epitopes; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D014409:Tumor Necrosis Factor-alpha