Obligate multivalent recognition of cell surface tomoregulin following selection from a multivalent phage antibody library.
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.
Heitner
Tara
T
Berlex Biosciences, Richmond, California 94806, USA.
Satozawa
Noboru
N
McLean
Kirk
K
Vogel
David
D
Cobb
Ronald R
RR
Liu
Bing
B
Mahmoudi
Mithra
M
Finster
Silke
S
Larsen
Brent
B
Zhu
Ying
Y
Zhou
Hongxing
H
Müller-Tiemann
Beate
B
Monteclaro
Felipe
F
Zhao
Xiao-Yan
XY
Light
David R
DR
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
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