title: Selection and maturation of antibodies by phage display through fusion to pIX.
abstract: Antibody discovery and optimization by M13 phage display have evolved significantly over the past twenty years. Multiple methods of antibody display and selection have been developed - direct display on pIII or indirect display through a Cysteine disulfide linkage or a coiled-coil adapter protein. Here we describe display of Fab libraries on the smaller pIX protein at the opposite end of the virion and its application to discovery of novel antibodies from naive libraries. Antibody selection based on pIX-mediated display produces results comparable to other in vitro methods and uses an efficient direct infection of antigen-bound phages, eliminating any chemical dissociation step(s). Additionally, some evidence suggests that pIX-mediated display can be more efficient than pIII-mediated display in affinity selections. Functional assessment of phage-derived antibodies can be hindered by insufficient affinities or lack of epitopic diversity. Here we describe an approach to managing primary hits from our Fab phage libraries into epitope bins and subsequent high-throughput maturation of clones to isolate epitope- and sequence-diverse panels of high affinity binders. Use of the Octet biosensor was done to examine Fab binding in a facile label-free method and determine epitope competition groups. A receptor extracellular domain and chemokine were subjected to this method of binning and affinity maturation. Parental clones demonstrated improvement in affinity from 1-100nM to 10-500pM.
authors: Janssen Pharmaceutical Companies of Johnson and Johnson, 145 King of Prussia Rd. Radnor, PA 19089, United States.
authors: Mark
authors: M
authors: Tornetta
authors: Ramachandra
authors: R
authors: Reddy
authors: John C
authors: JC
authors: Wheeler
mesh_terms: D017104:Bacteriophage M13 / Q000235:genetics; D036022:Capsid Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D061505:Cell Surface Display Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D004926:Escherichia coli; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D019151:Peptide Library; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012641:Selection, Genetic
chemical_list: D036022:Capsid Proteins; D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019151:Peptide Library; D011993:Recombinant Fusion Proteins