Binding affinity of the mAbs, fluorescent-tagged mAbs, and ADCs to human or rat MET extracellular domain (ECD) were determined using surface plasmon resonance (SPR) on a Biacore T200 instrument (Biacore Life Sciences, Pittsburgh, PA, USA). All MET-ECD were made at Eli Lilly and Company (proprietary reagents). Except as noted, all reagents and materials were from GE Healthcare Bio-sciences Corp. Biacore experiments were performed at 37°C in HBS-EP+ buffer (GE Healthcare, 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% PS20, and 3 mM EDTA). A CM5 sensor chip containing immobilized protein A, generated using standard NHS-EDC amine coupling on all four flow cells (Fc), was employed as capture methodology. The mAb, fluorescent-tagged mAb, or ADC was captured by Protein A chip to yield about 100 response units. Multiple cycles of different concentrations of MET-ECD (6.25-200 nM) were then injected over flow cells. Each cycle includes a mAb, fluorescent-tagged mAb, or ADC capture step followed by the injection of MET-ECD at a single concentration with a 20-minute dissociation period, followed by a regeneration step using glycine HCl (pH 1.5). Bulk refractive index differences were corrected for by subtracting the response obtained in a reference flow cell, where no anti-MET antibody was captured. Data were fit to a 1:1 binding model using Biacore T200 Evaluation software version3.1 to determine the association rate (kon, M-1 s-1 units), dissociation rate (koff, s-1 units), and Rmax (RU units). The equilibrium dissociation constant (KD) was calculated from the relationship KD = koff / kon.

Binding affinity of the mAbs, fluorescent-tagged mAbs, and ADCs to human or rat MET extracellular domain (ECD) were determined using surface plasmon resonance (SPR) on a Biacore T200 instrument (Biacore Life Sciences, Pittsburgh, PA, USA). All MET-ECD were made at Eli Lilly and Company (proprietary reagents). Except as noted, all reagents and materials were from GE Healthcare Bio-sciences Corp. Biacore experiments were performed at 37°C in HBS-EP+ buffer (GE Healthcare, 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% PS20, and 3 mM EDTA). A CM5 sensor chip containing immobilized protein A, generated using standard NHS-EDC amine coupling on all four flow cells (Fc), was employed as capture methodology. The mAb, fluorescent-tagged mAb, or ADC was captured by Protein A chip to yield about 100 response units. Multiple cycles of different concentrations of MET-ECD (6.25-200 nM) were then injected over flow cells. Each cycle includes a mAb, fluorescent-tagged mAb, or ADC capture step followed by the injection of MET-ECD at a single concentration with a 20-minute dissociation period, followed by a regeneration step using glycine HCl (pH 1.5). Bulk refractive index differences were corrected for by subtracting the response obtained in a reference flow cell, where no anti-MET antibody was captured. Data were fit to a 1:1 binding model using Biacore T200 Evaluation software version3.1 to determine the association rate (kon, M-1 s-1 units), dissociation rate (koff, s-1 units), and Rmax (RU units). The equilibrium dissociation constant (KD) was calculated from the relationship KD = koff / kon.