Table 1. Binding properties and neutralizing activity of human c-Met antibodies.

Assay	CE-310393	CE-310397	CE-310400	CE-310083
ECD binding EC50*	185 ± 12 pM10	377 ± 25 pM6	347 ± 89 pM6	281 ± 28 pM4
Epitope class ** (Biacore)	1	1	1	2
Biacore** KD	220 pM	530 pM	330 pM	820 pM
Biacore** koff (1/s)	1.5 × 10−4	1.5 × 10−4	6.1 × 10−5	6.8 × 10−5
flow cytometry EC50#	39 ± 7 pM4	26 ± 4 pM3	41 pM1	169 ± 46 pM3
Ligand Binding IC50 (pM)##	279 ± 21 pM10	652 ± 67 pM7	582 ± 113 pM6	304 ± 48 pM4
Cell pMet IC50 (pM)^	150 ± 13 pM10	336 ± 61 pM7	666 ± 183 pM6	155 ± 39 pM4
Soft Agar Growth IC50 (nM)^^	57 nM1	68 ± 2 nM2	167 nM1	47 ± 12 nM3

*Binding of lead antibodies to recombinant human c-Met ECD-Fc protein was evaluated by ELISA. **The binding kinetics of the four lead antibodies were determined using surface plasmon resonance (Biacore), and epitope class was determined in cross competition studies using Biacore. ^#The binding affinity (EC₅₀) to native c-Met expressed on A549 cells was estimated using flow cytometry. ^##Antibodies were assessed for their ability to inhibit binding of recombinant HGF to immobilized human c-Met ECD-Fc by ELISA. Antibodies were preincubated for 4 h followed by addition of HGF (100 ng/ml) for 15 min. HGF binding was detected with biotinylated anti-HGF antibodies and streptavidin-HRP. ^{^}Antibodies inhibit HGF-dependent activation of c-Met in A549 cells. A549 cells were preincubated with varying concentrations of c-Met antibodies for 4 h then stimulated with 200 ng/ml HGF for 15 min. The level of c-Met autophosphorylation was measured by capture ELISA using PY20-HRP for detection. ^{^^}Soft agar growth of S114 cells, which are NIH-3T3 cells engineered to overexpress human HGF and human c-Met, was evaluated 7–10 d after plating cells with varying concentrations of c-Met antibodies. Colony number and size of p-iodonitrotetrazolium violet stained colonies were determined. Values for the ECD binding, flow cytometry, ligand binding, cellular pMet, and soft agar growth assays represent means ± SEM of multiple dose titration experiments. IC₅₀ values were calculated using GraphPad Prism software. The number of experimental replicates is noted in superscript.