title: Obligate multivalent recognition of cell surface tomoregulin following selection from a multivalent phage antibody library.
abstract: A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.
authors.affiliation: Berlex Biosciences, Richmond, California 94806, USA.
authors.forename: Tara
authors.initials: T
authors.lastname: Heitner
authors.forename: Noboru
authors.initials: N
authors.lastname: Satozawa
authors.forename: Kirk
authors.initials: K
authors.lastname: McLean
authors.forename: David
authors.initials: D
authors.lastname: Vogel
authors.forename: Ronald R
authors.initials: RR
authors.lastname: Cobb
authors.forename: Bing
authors.initials: B
authors.lastname: Liu
authors.forename: Mithra
authors.initials: M
authors.lastname: Mahmoudi
authors.forename: Silke
authors.initials: S
authors.lastname: Finster
authors.forename: Brent
authors.initials: B
authors.lastname: Larsen
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhu
authors.forename: Hongxing
authors.initials: H
authors.lastname: Zhou
authors.forename: Beate
authors.initials: B
authors.lastname: Müller-Tiemann
authors.forename: Felipe
authors.initials: F
authors.lastname: Monteclaro
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Zhao
authors.forename: David R
authors.initials: DR
authors.lastname: Light
mesh_terms: D000818:Animals; D000906:Antibodies; D000954:Antigens, Surface / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics / Q000276:immunology*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D008565:Membrane Proteins / Q000494:pharmacology*; D009363:Neoplasm Proteins / Q000494:pharmacology*; D019151:Peptide Library; D020539:Sequence Analysis, Protein; D014162:Transfection; D019562:Viral Load
chemical_list: D000906:Antibodies; D000954:Antigens, Surface; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D019151:Peptide Library; C423567:TMEFF2 protein, human