Isolation of a human anti-epidermal growth factor receptor Fab antibody, EG-19-11, with subnanomolar affinity from naïve immunoglobulin repertoires using a hierarchical antibody library system.
Specific antibodies that possess a subnanomolar affinity are very difficult to obtain from human naïve immunoglobulin repertoires without the use of lengthy affinity optimization procedures. Here, we designed a hierarchical phage-displayed antibody library system to generate an enormous diversity of combinatorial Fab fragments (6×10(17)) and attempted to isolate high-affinity Fabs against the human epidermal growth factor receptor (EGFR). A primary antibody library, designated HuDVFab-8L, comprising 4.5×10(9) human naïve heavy chains and eight unspecified human naïve light chains was selected against the EGFR-Fc protein by biopanning, and four anti-EGFR Fab clones were isolated. Because one of the Fab clones, denoted EG-L2-11, recognized a native EGFR expressed on A431 cells, the heavy chain of the Fab was shuffled with a human naïve light chain repertoire with a diversity of 1.4×10(8) and selected a second time against the EGFR-Fc protein again. One EG-L2-11 variant, denoted EG-19-11, recognized an EGFR epitope that was almost the same as that bound by cetuximab and had a K(D) of approximately 540 pM for soluble EGFR, which is about 7-fold higher than that of the FabC225 derived from cetuximab. This variant was also internalized by A431 cells, likely via receptor-mediated endocytosis, and it efficiently inhibited EGF-mediated tyrosine phosphorylation of the EGFR. These results demonstrate that the use of our hierarchical antibody library system is advantageous in generating fully human antibodies especially with a therapeutic purpose.
Hur
Byung-ung
BU
Department of Systems Immunology, College of Biomedical Science, Kangwon National University, Chuncheon, Gangwon 200-701, South Korea.
Yoon
Jae-bong
JB
Liu
Li-Kun
LK
Cha
Sang-hoon
SH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D019921:Biotinylation; D015153:Blotting, Western; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D019169:Jurkat Cells; D051379:Mice; D041681:NIH 3T3 Cells; D019151:Peptide Library; D010766:Phosphorylation / Q000187:drug effects; D011994:Recombinant Proteins / Q000276:immunology; D014443:Tyrosine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; C556215:EG-19-11; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019151:Peptide Library; D011994:Recombinant Proteins; D014443:Tyrosine; D066246:ErbB Receptors