Phage versus phagemid libraries for generation of human monoclonal antibodies.
Non-immune (naïve) phage antibody libraries have become an important source of antibodies for reagent, diagnostic, and therapeutic use. To date, reported naïve libraries have been constructed in phagemid vectors as fusions to pIII, yielding primarily single copy (monovalent) display of antibody fragments. For this work, we subcloned the single chain Fv (scFv) gene repertoire from a naïve phagemid antibody library into a true phage vector to create a multivalently displayed scFv phage library. Compared to monovalently displayed scFv, multivalent phage display resulted in improved efficiency of display as well as antibody selection. A greater number of antibodies were obtained and at earlier rounds of selection. Such increased efficiency allows the screening for binding antibodies after a single round of selection, greatly facilitating automation. Expression levels of antigen-binding scFv were also higher than from the phagemid library. In contrast, the affinities of scFv from the phage library were lower than from the phagemid library. This could be overcome by utilizing the scFv in a multivalent format, by affinity maturation, or by converting the library to monovalent display after the first round of selection.
Department of Anesthesia and Pharmaceutical Chemistry, University of California, San Francisco, Rm 3C-38, San Francisco General Hospital, 1001 Potrero Ave, 94110, USA.
David
D
O'Connell
Baltazar
B
Becerril
Arup
A
Roy-Burman
Mike
M
Daws
James D
JD
Marks
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D001331:Automation; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007700:Kinetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics*
D000911:Antibodies, Monoclonal; D000941:Antigens; D007135:Immunoglobulin Variable Region; D019151:Peptide Library