title: A new helper phage for improved monovalent display of Fab molecules.
abstract: Phage display technology is a powerful tool for the identification of novel antibodies for drug discovery. Phage display libraries have been constructed with massive diversity, but their use may be hindered by limited antibody display levels when rescued with the M13KO7 helper phage. Variants of M13KO7 have been constructed previously that increase the levels of display of rescued phage, but all produce phage that display multiple copies of the antibody fragment on their surface and have reduced titer and infectivity. In this study, we describe a new helper phage, XP5, which increased the display level of Fab molecules more than two-fold compared to phage rescued with M13KO7. XP5 uses a combination of ribosome binding site spacing alterations and rare codon clusters to reduce the expression of pIII from the helper phage. This reduction in pIII expression leads to an increase in the incorporation of pIII-Fab fusions during phage rescue. The rescued phage displayed a single copy of the Fab molecule, preventing any avidity effects during the selection process. This also suggests that the percentage of the population of phage displaying a Fab molecule is increased when rescued with XP5. Additionally, the phage titers and infectivity are comparable to libraries rescued with M13KO7. After two rounds of panning we observed a nearly 5-fold increase in the number of antigen binding Fab molecules compared to panning conducted with the same library rescued with M13KO7. The nature of the mutations in XP5 makes it a universal substitute for M13KO7 in pIII-based phage display, compatible with most phagemids and bacterial strains.
authors: Preclinical Research and Development, XOMA (US) LLC, Berkeley, CA 94710, United States. beaber@xoma.com
authors: John W
authors: JW
authors: Beaber
authors: Eric M
authors: EM
authors: Tam
authors: Llewelyn S
authors: LS
authors: Lao
authors: Isaac J
authors: IJ
authors: Rondon
mesh_terms: D017104:Bacteriophage M13 / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D007140:Immunoglobulin Fab Fragments / Q000276:immunology* / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed / Q000379:methods; D019151:Peptide Library
chemical_list: D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D004247:DNA