Fig. 1

Characterization of mouse mAb specific to the RON PSI domain and its humanization: a Schematic representation of RON, RON isoforms, and MET extracellular structure. Mature RON contains a 35 kDa α-chain and a 145 kDa β-chain linked by a disulfide bond. SEMA, PSI and IPT domains are located in the β-chain. The listed four RON isoforms are shown with unique truncations and deletions either by alternative initiation or by mRNA splicing [8]. The MET extracellular structure is similar that of RON. b Direct ELISA for PCM5B14 reactive to the RON PSI domain. RON, RON isoforms, RON PSI peptides, and MET extracellular protein were coated at 150 ng per well in triplicate in a 96-well plate. Goat-anti-mouse IgG coupled with HRP was used as the detecting antibody. Results are shown as the percentage of antibody-binding activity. PCM5B14 reactive to RON was set as 100%. c Modeling of CDRs from PCM5B14 in human IgG heavy chain and light chain. The framework of human IgG1 molecule was used for PCM5B14 humanization. The models of PCM5B14 CDRs grafted in the variable regions of human IgG1 heavy chain and light chain were generated using the software Automatic Predictions of Immunoglobulin Structures (Tramontano at University of Rome, Italy). d Binding of humanized antibody subclones to RON expressed by HT-29 cells. Humanized IgG subclones at 1.5 μg per ml were incubated with HT-29 cells followed by addition of goat anti-human IgG1 antibody coupled with FITC. Immunofluorescence intensity from individual samples was determined by flow cytometric analysis. e and f Analysis of binding affinities of humanized IgG subclones to human RON. Different amounts of humanized PCM5B14 subclones were incubated with NIH-3 T3 cells expressing human RON followed by addition of goat anti-human IgG1 antibody coupled with fluorescein isothiocyanate (FITC). Antibody-binding affinity was calculated as previously described [6]