HGF or c-Met stimulation is required for 4MBr-5 cells proliferation. H1975, which expresses moderate levels of wild type c-Met without genomic amplification. By comparison, the c-Met TKI, PHA-665752, demonstrated modest tumor growth inhibition in MKN-45, and no inhibition at all in H1975. Taken together, these data suggest that P3D12-vc-MMAF may have a superior clinical profile in treating JMV 390-1 c-Met positive malignancies in contrast to c-Met pathway inhibitors. KEYWORDS: c-Met, ADC (antibody drug conjugate), MMAF, gastric cancer, non-agonistic antibody, lung cancer Introduction Many tumors depend on the continued expression and/or amplification JMV 390-1 of a single oncogene for the initiation and maintenance of a malignant state, and its product can be targeted with small-molecule drugs or biotherapeutics. c-Met, the receptor for hepatocyte growth factor (HGF), is a receptor tyrosine kinase overexpressed and activated in 75C90% of gastric cancers and 41C72% of lung cancers,1 and a constitutively active signaling pathway of c-Met has been linked to malignant cell growth.2 C-Met protein expression is also associated with poor prognosis in many solid tumor types.3,4 So far, several clinically available TKIs (tyrosine kinase inhibitors) have shown efficacy in a subset of patients with tumors exhibiting MET gene amplification (e.g., 6% of gastric cancers and 1% of lung cancers)5 or exon 14-skipping mutations.6 However, these tumors eventually acquire resistance, and long-term efficacy of the treatment is ineffective.7,8 Other therapeutic approaches using anti-c-Met antibodies or anti-HGF antibodies to target c-Met initially looked promising. Several anti-c-Met antibodies have been generated,9,10 however, some of them mimicked unwanted HGF agonism by inducing receptor dimerization. Others, like onartuzumab, which does not show agonistic activity of c-Met signaling, failed to demonstrate significant efficacy in a phase JMV 390-1 III clinical trial of NSCLC.11 We developed an antibody-based therapeutic that targets amplified and non-amplified c-Met-overexpressing JMV 390-1 tumors without activating c-Met signaling. We identified a high affinity, specific c-Met antibody (P3D12) that induces c-Met degradation with minimal activation of ERK, and no measurable mitogenic activity. By conjugating P3D12 to the cytotoxic drug vc-MMAF using maleimide-based conjugation, we achieved the specificity of a targeted therapy and the potency and efficacy of a chemotherapeutic agent in a single agent. The ADC, P3D12-vc-MMAF, showed a much higher potency than the c-Met TKI, PHA-665752, in cytotoxicity assays against a panel of gastric and lung cancer cell lines and xenograft models. Results Generation of anti-c-Met antibodies and identification of candidate P3D12 which induces c-Met degradation with minimal agonistic activity Generation of anti-c-Met antibodies to inhibit cancers with constitutive JMV 390-1 c-Met signaling has been challenging.12 Some antibodies had strong agonistic activity, including the bivalent 5D5 (Genentech, Inc) and DO-24 antibodies,13 which could lead to cancer cell proliferation. We developed a three-step screening method for generating anti-c-Met antibodies with DDIT1 high c-Met affinity, high c-Met internalizing and degrading properties while maintaining low to no agonistic activity. Mice were immunized with human c-Met-Fc recombinant protein. Twenty thousand hybridomas were produced and high-affinity antibodies were selected that bound specifically to human c-Met by ELISA and were rapidly internalized detected by FACS (data not shown). In the second step, about 500 selected antibodies were assayed for induction of c-Met degradation as a surrogate marker for delivery of the mAbs to the lysosomal compartment, as opposed to receptor recycling (Figure 1a). The tested antibodies showed different degrees of c-Met degradation, and one candidate, P3D12, showed the highest activity. In addition, selected hit antibodies were assessed in a c-Met agonist assay using ERK phosphorylation status as a surrogate marker for c-Met activation and downstream signaling (Figure 1b). Three c-Met antibody candidates (P1E2, P3D12, P1H5) demonstrated strongly reduced agonistic activity compared to c-Mets natural ligand, hepatocyte growth factor (HGF) and the bivalent 5D5 antibody. Open in a separate window Figure 1. Identification of the lead P3D12.