Figure 1.

Phage display of Fab fragments derived from nimotuzumab. pCS1 phagemid vector is represented in (A). The vector contains pBR322 ori and f1 ori (replication origins for double and single strand DNA), an ampicillin resistance gene (amp^r), and a bicistronic expression cassette containing the LacZ promoter, two ribosome binding sites (RBS), two signal sequence-coding genes (SS), and the genes coding for human C_K and CH1 human antibody constant domains. The latter was fused to sequences coding for a 6-His tag, the c-myc tag peptide and full-length phage PIII protein. Nimotuzumab light chain variable region gene was cloned between ApaLI and XhoI restriction sites (downstream of the M13 gen III SS), and heavy chain variable region gene was cloned between SfiI and BstEII (downstream of the pelB SS). Purified phage particles displaying nimotuzumab-derived Fab fragments were tested by ELISA (B) on polyvinyl chloride microtiter plates coated with the anti-c-myc tag 9E10 mAb, a recombinant protein comprising the extracellular region of the human EGF receptor (erEGF-R), and the unrelated protein BSA. Bound phages were detected with an anti-M13 mAb labeled with horseradish peroxidase.