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

• Published in: Immunology letters 2010-11, Vol.134 (1), p.55-61
• Main Authors: Hur, Byung-ung, Yoon, Jae-bong, Liu, Li-Kun, Cha, Sang-hoon
• Format: Article
• Language: English
• Subjects: Allergy and Immunology; Animals; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - isolation & purification; Antibodies, Monoclonal - pharmacology; Antibody Affinity - immunology; Antibody library; Antibody Specificity - immunology; Biotinylation; Blotting, Western; Cell Line, Tumor; Dual-vector system; EGFR; Escherichia coli - genetics; Ex12 helper phage; Humans; Immunoglobulin Fab Fragments - genetics; Immunoglobulin Fab Fragments - immunology; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Heavy Chains - immunology; Immunoglobulin Light Chains - genetics; Immunoglobulin Light Chains - immunology; Jurkat Cells; Mice; NIH 3T3 Cells; Peptide Library; Phage display technology; Phosphorylation - drug effects; Receptor, Epidermal Growth Factor - genetics; Receptor, Epidermal Growth Factor - immunology; Recombinant Proteins - immunology; Tyrosine - metabolism
• ISSN: 0165-2478; 1879-0542
• DOI: 10.1016/j.imlet.2010.08.009

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 × 1017 ) 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 × 109 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 × 108 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 KD 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.