Antibody Fab display and selection through fusion to the pIX coat protein of filamentous phage.
Fab antibody display on filamentous phage is widely applied to de novo antibody discovery and engineering. Here we describe a phagemid system for the efficient display and affinity selection of Fabs through linkage to the minor coat protein pIX. Display was successful by fusion of either Fd or Lc through a short linker to the amino terminus of pIX and co-expression of the counter Lc or Fd as a secreted, soluble fragment. Assembly of functional Fab was confirmed by demonstration of antigen-specific binding using antibodies of known specificity. Phage displaying a Fab specific for RSV-F protein with Fd linked to pIX showed efficient, antigen-specific enrichment when mixed with phage displaying a different specificity. The functionality of this system for antibody engineering was evaluated in an optimization study. A RSV-F protein specific antibody with an affinity of about 2nM was randomized at 4 positions in light chain CDR1. Three rounds of selection with decreasing antigen concentration yielded Fabs with an affinity improvement up to 70-fold and showed a general correlation between enrichment frequency and affinity. We conclude that the pIX coat protein complements other display systems in filamentous phage as an efficient vehicle for low copy display and selection of Fab proteins.
Tornetta
Mark
M
Centocor Research and Development Inc., Radnor, PA 19087, USA. mtornett@its.jnj.com
Baker
Scott
S
Whitaker
Brian
B
Lu
Jin
J
Chen
Qiang
Q
Pisors
Eileen
E
Shi
Lei
L
Luo
Jinquan
J
Sweet
Raymond
R
Tsui
Ping
P
D000595:Amino Acid Sequence; D000915:Antibody Affinity / Q000235:genetics; D017104:Bacteriophage M13 / Q000235:genetics*; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D015202:Protein Engineering; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012136:Respiratory Syncytial Viruses / Q000276:immunology*; D014169:Transformation, Bacterial; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology; D014764:Viral Proteins / Q000276:immunology
D036022:Capsid Proteins; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D014764:Viral Proteins; C025668:coat protein, Bacteriophage M13