SCIENCE UP THE WORLD
15TH INTERNATIONAL STUDENT CONGRESS OF MEDICAL SCIENCES
JUNE 3RD - 6TH 2008
Abstract Mandeep Kang
Use of phage display to identify novel antagonists for angiogenic receptor tyrosine kinases1Miss Mandeep Kang, 1Dr Nick Brindle
1Department of Cardiovascular Sciences, University of Leicester, UK (United Kingdom)
Introduction
Angiogenesis is required for normal vascular development and aberrant angiogenesis contributes to several diseases, including cancer, diabetes and tissue ischaemia. Molecular studies have identified the Tie-receptor pathway as an endothelial-cell-specific proangiogenic system which has a critical role in angiogenesis.
Tie1 and Tie2 are receptor tyrosine-kinases expressed predominately on endothelial cells. They are required for blood vessel formation and maintenance of adult vasculature. Ligands (angiopoietins) have been identified for Tie2 which regulates endothelial migration, survival, permeability and suppression of vascular inflammation.
The availability of Tie2 antagonists could be promising for the treatment of various angiogenic disorders. Therefore, in this study, we attempted to identify small molecule inhibitors using the method of phage display, which were able to block the binding of angiopoietins to Tie2. This could represent a good lead for the development of anti-angiogenic drugs against angiogenic diseases, and would also be a useful tool to clarify the mechanisms of angiopoietin/Tie2 signalling.
Material and Methods
Peptides specifically binding Tie2-ectodomain were identified by using the Ph.D.-7 Phage Display Peptide Library. It consists of random peptide 7-mers fused to a minor coat protein (pIII) of M13 [bacterio]phage comprising 1.28 x 109 primary clones. Biopanning is carried out by incubating the phage displayed peptides with immobilised Tie2 Fc, washing away unbound phage, and eluting specifically bound phage. Eluted phage is amplified in vivo and the process repeated, resulting in stepwise enrichment of the phage pool in favour of the tightest binding sequence. Binding clones were characterised and sequenced. Specific binding was tested with immobilised Tie2 ectodomains, in the form of ELISAs, and on HMEC/Tie2 cells. Clones showing highest specific interaction were chosen for further experiments.
Results
At the end of 2 screens and 3 rounds of selection - in the format of solid ELISAs and in solution - 20 clones were isolated and sequenced, showing that 13 different clones were represented. Each selected clone was assayed by ELISA for binding to Tie2 Fc. All tested clones gave a significant ELISA signal, demonstrating specific binding to Tie2. Clone 1 (1st screen) and clone 2 and 10 (2nd screen) were utilised in cellular angiogenesis assays.
Conclusion
Our data shows that the 3 clones identified all bind specifically to the Tie2 receptor and so we present evidence of specific peptides (other than the angiopoietins) interacting with Tie2. These specific peptides, due to their small size, could represent compounds for the development of therapeutic agents against angiogenesis. In addition, these peptides will be useful to dissect the signal transduction mechanisms involving the Tie2 receptor in endothelial cells.
Keyword(s): receptor tyrosine kinases, phage display, Tie receptors, angiogenesis
