Our investigations have confirmed that anti-c-MET antibodies with receptor antagonistic activity can be generated by converting intact and bivalent antibodies to a monovalent format, a strategy employed in order to generate the MetMAb antibody 
. This approach also proved necessary in order to remove the partial agonistic activity of the DN-30 antibody 
More importantly, the data presented here highlights two novel classes of c-MET-targeting antibodies. The first is the generation of three antibodies (LMH 80–82) that specifically recognize the 170 kDa c-MET precursor at the cell surface. Neither of the two epitopes recognized by these antibodies overlaps with the furin cleavage site itself (i.e. the loop connecting strands 4d and 5a). LMH 80 and LMH 81 bind the α-helix of the CRD while LMH 82 binds the loop connecting strands 2a-2b on the bottom face of the MET β-propeller domain (). This information, and the fact that these antibodies fail to bind mature c-MET by IP, strongly suggest that furin cleavage of the c-MET precursor is accompanied by a major conformational change. Using LMH 80 and non-permeating immunofluorescence, we could definitively establish that p170 c-MET is expressed on cell surface of human cancer cells, in agreement with previous studies in human SkHep1 and LoVo cell lines 
. Additionally, there is convincing evidence from murine derived cancer cells that p170 c-MET is exposed at the cell surface, and can be activated by HGF 
. Significantly, non-permeating analyses of primary rat hepatocytes suggested that p170 c-MET was unavailable for 125
I-HGF/SF binding at the cell surface 
. The lack of p170 c-MET on the surface of normal cells strongly suggests that its cell surface localization is specific for cancerous cells. The punctate c-MET staining described here for LMH 85 most likely represents non-covalent clusters of c-MET on the cell surface as reported previously 
. As this pattern was also observed for LMH 80, it indicates that p170 c-MET is probably contained within these non-covalent clusters on the cell surface. LMH 80–82 may be useful for targeting radiotherapeutic and chemotherapeutic agents to tumors while preventing toxic exposure to normal tissues such as liver. Furthermore, as nanoparticle technology develops, these antibodies could be extremely useful tools for promoting the retention of such particles within the tumor mass.
The second novel class of antibody (LMH 87 and LMH 88) is one that promotes c-MET internalization and degradation and/or interferes with receptor recycling to the cell surface (). Alterations of RTK degradation and trafficking have emerged in recent years as common and important features of tumor cells as well as a rational target for therapeutic intervention 
. Binding of the LMH 87 and LMH 88 antibodies to an epitope in the α-chain portion of the c-MET β-propeller promoted receptor degradation independent of HGF/SF, leading to reduced levels of surface c-MET in tumor cells. An initial animal experiment showed that LMH 87 inhibited the growth of U87MG xenografts, confirming that this mAb was effective in vivo
even in the presence of autocrine HGF/SF. The LMH 87 epitope is distinct from the HGF/SF binding site, suggesting that the intact, bivalent form of this mAb should lack agonistic activity; a hypothesis we confirmed experimentally. Therapeutic inhibition of c-MET with intact, bivalent LMH 87 would have clear advantages over the Fab or scFv formats in terms of stability, half-life and the potential to mediate immune-effector functions. LMH 87's partial antagonistic activity may be advantageous when used in combination with other targeted therapies, especially those targeting EGFR, as it may improve the therapeutic window without significantly increasing toxicity; a potential problem when combining such agents.
The established role of c-MET and HGF/SF in the initiation and progression of a variety of human cancers 
has stimulated an extraordinary effort into the development of c-MET specific TKIs, with many of these currently under investigation in patients 
. However, the potential for off-target effects and/or the emergence of resistant populations of tumor cells have already been observed. Thus, despite the difficulties encountered, the drive to develop anti-HGF/SF and anti-c-MET targeted mAbs has a solid and rational foundation 
. We expect that the two novel classes of anti-c-MET antibodies described here (LMH 80–82 and LMH 87–88) are potential therapeutic candidates for the treatment of human cancer.