Inhibition of cMet activation by a one-armed antibody

1424 The proto-oncogene cMet is a receptor tyrosine kinase that is expressed in many epithelial cancers. On binding by its ligand, HGF, cMet activates cellular pathways that promote proliferation and invasive growth, characteristics of aggressive tumor behavior. We have produced a panel of monoclonal antibodies to the extracellular domain of cMet, one of which, 5D5, inhibits binding by HGF. As a normal, bivalent antibody, 5D5 is a potent cMet activator, but the monovalent Fab is a potent HGF antagonist that inhibits HGF-induced proliferation and migration. However, the Fab has a very short half-life in vivo, which limits its utility in animal models. To address that issue, we have produced a one-armed version of 5D5 by co-expressing 3 polypeptides in E coli. The first is a chimeric light chain consisting of the VL domain from the 5D5 murine hybridoma fused to human kappa constant domain. The second is a chimeric heavy chain consisting of the VH domain from the 5D5 murine hybridoma fused to the constant domains of human IgG1. And the third is an Fc domain, beginning at the hinge and ending with the normal carboxy terminus. The heavy chain covalently associates with both the light chain, through the normal heavy-light disulfide bond, and the truncated Fc, through the normal heavy-heavy disulfide bonds at the hinge. The result is a dimeric Fc from which emanates a single antigen-binding arm. Also produced are smaller amounts of bivalent antibody and dimers of truncated Fc. To further promote formation of the desired product, a “knob” (T366W) was introduced into the truncated Fc construct and a corresponding “hole” (T366S, L368A, Y407V) into the heavy chain. With this modification, >95% of the protein produced is the one-armed antibody. The one-armed version of 5D5 binds cMet with a Kd ∼4 nM and retains the ability to displace HGF binding (IC50 ∼ 5 nM). Like the Fab, it inhibits HGF-stimulated tyrosine phosphorylation of cMet, and inhibits HGF-induced proliferation and migration. Unlike the Fab, which has a half-life in mice of ∼15 minutes, the one-armed 5D5 has a half-life of 4.3 days, approaching that for a normal human IgG1 (∼7 days). As judged by SDS PAGE, the one-armed 5D5 remained intact in circulation for the entire 14 days of the PK experiment. Treatment of nude mice with the one-armed 5D5 (10 mg/kg weekly x 4) at the same time that the mice were inoculated with U87 glioma cells, which express cMet and produce HGF in an autocrine manner, completely prevented tumor development. Moreover, the one-armed 5D5 caused complete, dose-dependent regression of U87 glioma tumors when treatment was delayed until tumors were established. These results suggest that the one-armed 5D5 could have therapeutic utility in HGF-dependent neoplasias.