Characterization and structural determination of a new anti-MET function-blocking antibody with binding epitope distinct from the ligand binding domain

Abstract

The growth and motility factor Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor, the product of the MET proto-oncogene, promote invasion and metastasis of tumor cells and have been considered potential targets for cancer therapy. We generated a new Met-blocking antibody which binds outside the ligand-binding site, and determined the crystal structure of the Fab in complex with its target, which identifies the binding site as the Met Ig1 domain. The antibody, 107_A07, inhibited HGF/SF-induced cell migration and proliferation in vitro and inhibited growth of tumor xenografts in vivo. In biochemical assays, 107_A07 competes with both HGF/SF and its truncated splice variant NK1 for MET binding, despite the location of the antibody epitope on a domain (Ig1) not reported to bind NK1 or HGF/SF. Overlay of the Fab-MET crystal structure with the InternalinB-MET crystal structure shows that the 107_A07 Fab comes into close proximity with the HGF/SF-binding SEMA domain when MET is in the “compact”, InternalinB-bound conformation, but not when MET is in the “open” conformation. These findings provide further support for the importance of the “compact” conformation of the MET extracellular domain, and the relevance of this conformation to HGF/SF binding and signaling.

A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing

“…In fact, in many affinity maturation campaigns much of the original amino acid sequence is restored in the high affinity "solutions". 21,23 Thus, the smaller library size required by the directed mutagenic approach used here scales well with the potential size of mammalian display libraries. Such a targeted approach is possible by novel synthesis technology enabling the economic synthesis of 10,000s oligonucleotides.…”

“…In many cases, it is found that improved clones are related in sequence to the parental sequence. 23 A more efficient and parsimonious route for mutagenesis therefore is to retain sequence information while exploring the surrounding sequence space. To achieve this, oligonucleotides were designed to a continuous stretch of eight amino acids in the CDR3 of VH and VLs such that every possible 2-mer and 1-mer variant, using all amino acids (with the exception of cysteine) were included (Figure 4(a,b)).…”

Hypoxia leads to decreased autophosphorylation of the MET receptor but promotes its resistance to tyrosine kinase inhibitors

“…To our knowledge it is the first time that such alteration of HGF/SF-MET interaction is evidenced according to the cellular context and thus could represent a novel molecular mechanism to regulate the MET receptor downstream signaling. Interestingly, recent structural data suggest the existence of two distinct states of MET receptor (open/close conformation) allowing or not ligand binding [ 66 ]. It is worth noticing that potential action of phosphatases and decrease of the HGF/SF-MET interaction could synergically contribute to decrease MET phosphorylation observed during hypoxia.…”

Attempts at Repeated Cell Re-Constitution as Integral Developmental Dynamics of Cell Proliferation in Oncogenesis

“…The promotion of pathway cascades allows particular definitions of oncogenesis to emerge as dynamics of an initial cell response; autonomous inhibitory biology systems may be viewed as being well-delineated morphologically, and also genetically, in terms of mutability and impaired DNA repair systems. The "compact" conformation of the MET extracellular domain is important and relevant to HGF/Scatter factor binding and signaling [23].…”