Anti-Human Met(Onartuzumab)

(A) Schematic of MET and HGF domain architecture. Functional domains are indicated: Sema domain; PSI domain; Ig-like fold shared by plexins and transcriptional factors (IPT) domain; juxtamembrane (JM) domain; N-terminal (N) domain; kringle (K) domain; serine protease-like (SP-like) domain. The disulfide between the HGF α-and β-chains is shown as a yellow line. Colors used for individual domains in A match those used in D. (B) Competition-binding assays with MET ECD-Fc (encoding MET residues 25-929, which encode the MET Sema domain, the PSI domain, and all four IPT domains) immobilized on plates indicates that onartuzumab blocks HGF-α and NK1, but not HGF-β, binding to MET. (C) Analytical size-exclusion chromatography analysis of onartuzumab, MET Sema-PSI, and HGF-β mixed at 1:2:4 ratio. The three peaks in the chromatogram were visualized by SDS/ PAGE and correspond to the onartuzumab/MET Sema-PSI/HGF-β ternary complex, onartuzumab Fab/HGF-β binary complex, and excess HGF-β. (D) Cartoon representation of the 2.8-Å crystal structure of the onartuzumab Fab/MET Sema-PSI/HGF-β ternary complex (PDB ID code 4K3J) showing the Fab heavy chains (deep purple), Fab light chains (pink), MET Sema (light green) and PSI (deep green) domains, and HGF-β (cyan). The Sema β-propeller is viewed from the "bottom" face and the blades are numbered. (E) Openbook view of the interface between onartuzumab Fab (Left) and human MET Sema-PSI (Right). Residues in the interface were colored according to their percentage of reduction in accessible surface area upon complex formation (15-44%, yellow; 45-74%, orange;>75%, red). Interface analysis was performed using the protein interfaces, surfaces and assemblies (PISA) server (43).

Merchant, M., Ma, X., Maun, H. R., Zheng, Z., Peng, J., Romero, M., ... & Santell, L. (2013). Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent. Proceedings of the National Academy of Sciences, 110(32), E2987-E2996.

Immunoprecipitation-immunoblot analysis of p-MET (IP MET and IB p-Try) and total MET (IP and IB MET) in KP4 tumors collected at indicated time points following a single intraperitoneal dose of onartuzumab at 15 mg/kg. N 1⁄4 4/time; pMET 1⁄4 p-MET; tMET 1⁄4 total MET.

Xiang, H., Reyes, A. E., Bender, B. C., Merchant, M., Jumbe, N. L., Romero, M., ... & Peterson, A. (2013). Onartuzumab (MetMAb): using nonclinical pharmacokinetic and concentration-effect data to support clinical development. Clinical Cancer Research, clincanres-0260.

A, tumor volume – time pro fi le and estimated serum concentration using pharmacokinetic parameters obtained from pharmacokinetic study in nontumor-bearing mice following a total intravenous onartuzumab dose at 2.5 mg/kg with Q1W, Q2W, or Q3W dosing. B, tumor volume – time pro fi le and estimated serum concentration – time pro fi le using pharmacokinetic parameters obtained from pharmacokinetic study in nontumor-bearing mice following a total intravenous onartuzumab dose at 7.5 mg/kg with Q1W, Q2W, or Q3W dosing. The vehicle group is the same as in A. C, tumor volume – time pro fi le and estimated serum concentration – time pro fi le using pharmacokinetic parameters obtained from pharmacokinetic study in nontumor-bearing mice following a total intravenous onartuzumab dose at 30 mg/kg with Q1W, Q2W, or Q3W dosing. The vehicle group is the same as in A. The pharmacokinetics parameters used for above simulations are: CL 1⁄4 21.6 mL/d/kg, CL d 1⁄4 190 mL/d/kg, V 1 1⁄4 48.8 mL/kg, and V 2 1⁄4 90.7 mL/kg. Tumor volume was measured on indicated days and is reported as the mean ( Æ SE) for each group. Animals were dosed on day 0 for Q3W dosing; days 0 and 14 for Q2W dosing; and days 0, 7, and 14 for Q1W dosing. n 1⁄4 10 per group. One animal from vehicle control and one from 1.25 mg/kg Q2W groups had tumors that grew too large and the animals were taken off study on day 19. The tumor volume on day 19 was carried forward to day 21 to calculate group mean tumor volume.

Xiang, H., Reyes, A. E., Bender, B. C., Merchant, M., Jumbe, N. L., Romero, M., ... & Peterson, A. (2013). Onartuzumab (MetMAb): using nonclinical pharmacokinetic and concentration-effect data to support clinical development. Clinical Cancer Research, clincanres-0260.

Onartuzumab inhibits HGF-dependent MET phosphorylation and downstream activation of GAB1, AKT, ERK1/2, and P70-S6 in a dose-dependent fashion but does not induce any MET signaling in the absence of HGF.

Merchant, M., Ma, X., Maun, H. R., Zheng, Z., Peng, J., Romero, M., ... & Santell, L. (2013). Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent. Proceedings of the National Academy of Sciences, 110(32), E2987-E2996.

Onartuzumab inhibits HGF-dependent migration and wound closure in HGF-treated A549 cells with an IC 50 of 20 nM. All plots reflect group mean plus and minus the SEM (n = 3).

Merchant, M., Ma, X., Maun, H. R., Zheng, Z., Peng, J., Romero, M., ... & Santell, L. (2013). Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent. Proceedings of the National Academy of Sciences, 110(32), E2987-E2996.

(A) or onartuzumab (B). Cells were kept on ice (blue lines) or shifted to 37°C for 4 hours without (red lines) or with (green lines) exposure to 500 ng/mL human HGF, stained with PE-conjugated anti-murine IgG (3D6) or anti-human IgG (onartuzumab), and analyzed by flow cytometry.

Mai, E., Zheng, Z., Chen, Y., Peng, J., Severin, C., Filvaroff, E., ... & Nijem, I. (2014). Nonclinical evaluation of the serum pharmacodynamic biomarkers HGF and shed MET following dosing with the anti-MET monovalent monoclonal antibody onartuzumab. Molecular cancer therapeutics, 13(2), 540-552.

HeLa cells were incubated for 4 hours at 37°C with 3D6 without (C) or with 100 μg/mL HGF (D) or onartuzumab without (E) or with HGF (F) and analyzed by immunofluorescence microscopy.

Mai, E., Zheng, Z., Chen, Y., Peng, J., Severin, C., Filvaroff, E., ... & Nijem, I. (2014). Nonclinical evaluation of the serum pharmacodynamic biomarkers HGF and shed MET following dosing with the anti-MET monovalent monoclonal antibody onartuzumab. Molecular cancer therapeutics, 13(2), 540-552.