To produce antibodies that interfere with the binding of scatter factor to its receptor MET and mutant forms of the growth factor and receptor with therapeutic potential.
We have produced: (i) antibodies that inhibit the binding of scatter factor to the MET receptor, (ii) scatter factor mutants lacking heparin-binding but retaining full biological activity, (iii) MET receptor mutants with altered ability to transduce the signal and (iv) transgenic mice lacking a functional scatter factor gene.
MAJOR SCIENTIFIC BREAKTHROUGHS: AND/OR INDUSTRIAL APPLICATIONS:
Scatter factor is the first member of a new family of growth factors that share the domain organisation and mechanism of activation of the blood proteinase plasminogen. The factor is produced by fibroblast and other mesenchymal cells and acts on epithelia and endothelia through a tyrosine kinase receptor encoded by the protooncogene c-MET. The aim of this project was the engineering of antibodies and growth factor/receptor mutants which could find applications in therapy. The main achievements of the project are summarised below.
Blocking antibodies. Phage-derived antibodies have been produced that modulate the activity of scattter factor on target cells. These are human antibodies derived from large phage libraries which bind the growth factor with affinity in the nanomolar range. The affinity of these antibodies can now be improved further using protein engineering techniques such as successive rounds of mutagenesis and phage-selection.
Scatter factor mutants. We have constructed three-dimensional models of teh six individual domains of scatter factor and generated a number of mutants of several of these domains. Several mutants in which clusters of basic amino acids present in the hairpin loop of the N-terminal domain have been replaced with negatively charged residues appeared to be of considerable interest. These amino acid substitutions reduced the heparin-binding activity of scatter factor but not its ability to bind the MET receptor and transduce the growth factor signal. These mutants could find potential applications in therapy.
MET receptor mutants. A number of mutants of the tyrosine kinase domain of the MET receptor were generated which allowed the precise mapping of the tyrosine residues involved in signal transduction. Two tyrosines (Y1349 and Y1356) appeared critical for binding to SH2-containing molecules involved in intracellular signalling (such as PI 3-kinase, phospho-lipase-C-gamma, pp60c-src and the GRB-2/SoS complex). Other tyrosines, however, were found to be involved in the MET signal transduction pathway: mutations of the C-terminal tyrosines Y1311, Y1347, Y1354 and Y1363 reduced or abolished scatter factor-induced cell movement and morphogenesis whereas mutation of the justamembrane tyrosine Y1001 generated a constitutively activated form of c-MET.
Scatter factor knock-out mice. Transgenic lines carrying mutations in the scatter factor or MET genes were generated and were embryonic lethal. The scatter factor and MET mutations affected the development of the liver (hepatocytes), placenta (trophoblast) and limb bud (limb muscle). Thus scatter factor and its receptor are essential for the development of several epithelial lineages and a subset of skeletal muscles. These transgenic lines will prove valuable for assessing the activity of scatter factor mutants and analogues in vivo through their ability to rescue the lethal phenotype of the scatter factor mutant mice.
Conclusions. The network project aimed at the derivation of antibodies, scatter factor mutants and MET receptor mutants that could enable the development of therapeutic peptides and proteins. The project has achieved these goals and has yielded valuable results for the determination of the three-dimensional structure of scatter factor and MET domains. These results are of significant interest in the light of the increasing realisation that scatter factor and MET may constitute main targets for cancer therapy and should assist the development of such therapies.
NUMBER OF PUBLICATIONS:Total: 43Joint: 04
Funding SchemeCSC - Cost-sharing contracts