Fig. 1.
Generation of humanized Zt/g4 antibody and characterization of RON-targeted antibody-drug conjugates: (a) Modeling of CDRs from mouse Zt/g4 in the variable regions of human IgG heavy chain and light chain. The framework of human IgG1 molecule was used for Zt/g4 humanization. The models of Zt/g4 CDRs grafted in the variable regions of human IgG1 heavy chain and light chain were generated by using the software PIGS from Automatic Predictions of Immunoglobulin Structures (Tramontano at University of Rome, Italy). (b) Binding of subclone H-Zt/g4 molecules to human RON. Different amounts of individual H-Zt/g4 s were incubated with NIH-3 T3 cells expressing human RON followed by addition of goat anti-human IgG1 antibody coupled with FITC. (c) Kinetic characterization of H-Zt/g4 interaction with human RON proteins by Octet RED96 system. Pure RON proteins from lysates of NIH3T3 cells expressing RON were immobilized onto the amine reactive sensor and assayed against individual H-Zt/g4 molecules in duplicate. The data set is analyzed with global fitting to produce the antibody-receptor binding affinity (KD). Blue curves represent experimental data and red curves represent the statistical fitting of curves. (d) Interaction of H-Zt/g4 H1L3 with RONs from different species. NIH3T3 cells expressing human, monkey, or mouse RON were incubated with H-Zt/g4 H1L3 followed by goat anti-human IgG coupled with FITC. Immunofluorescent intensities from individual samples were determined by flow cytometric analysis. (e) Schematic representation of H-Zt/g4-MMAE structure. MMAE was conjugated to H-Zt/g4 by the valine-citruline dipeptide linker according to the manufacturer’s instruction (www.concortis.com). (f) HIC analysis of MMAE conjugated to H-Zt/g4: Individual Zt/g4-MMAEs with different numbers of MMAE (0 to 8) are marked as P0 to P8. A DAR combining P2, P4, and P6 at 3.77:1 was achieved. (g) Free MMAE dissociated from H-Zt/g4-MMAE in human plasma. H-Zt/g4-MMAE at 10 μg per ml was incubated with fresh human plasma at 37 °C for 20 days. The amount of free MMAE in plasma was determined using the LC-MS/MS method with slight modifications. (h) Samples from (g) were used also for measuring MMAE conjugated H-Zt/g4 as detailed in Materials and Methods. A ratio from free MMAE to the total MMAE in H-Zt/g4-MMAE was calculated to determine the percentages of MMAE dissociated from H-Zt/g4-MMAE