Isolation of a human anti-epidermal growth factor receptor Fab antibody, EG-19-11, with subnanomolar affinity from naive immunoglobulin repertoires using a hierarchical antibody library system

摘要

Specific antibodies that possess a subnanomolar affinity are very difficult to obtain from human naive 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 x 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 x 10(9) human naive heavy chains and eight unspecified human naive 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 naive light chain repertoire with a diversity of 1 4 x 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 (C) 2010 Elsevier B V All rights reserved