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.