151. 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.
- Author
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Hur BU, Yoon JB, Liu LK, and Cha SH
- Subjects
- Animals, Antibodies, Monoclonal isolation & purification, Antibodies, Monoclonal pharmacology, Antibodies, Monoclonal, Humanized, Antibody Affinity immunology, Antibody Specificity immunology, Biotinylation, Blotting, Western, Cell Line, Tumor, ErbB Receptors genetics, Escherichia coli genetics, Humans, Immunoglobulin Fab Fragments genetics, Immunoglobulin Heavy Chains genetics, Immunoglobulin Heavy Chains immunology, Immunoglobulin Light Chains genetics, Immunoglobulin Light Chains immunology, Jurkat Cells, Mice, NIH 3T3 Cells, Phosphorylation drug effects, Recombinant Proteins immunology, Tyrosine metabolism, Antibodies, Monoclonal immunology, ErbB Receptors immunology, Immunoglobulin Fab Fragments immunology, Peptide Library
- Abstract
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., (Copyright © 2010 Elsevier B.V. All rights reserved.)
- Published
- 2010
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