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Title: The use of a novel TIMP3 peptide to specifically target therapeutic drugs to tumours
Author: Aldughaim, Mohammed
ISNI:       0000 0004 7225 6064
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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Background: Tissue inhibitor of metalloproteinases-3 (TIMP3) is an extracellular matrix protein with a number of novel properties such as inhibition of matrix metalloproteinases. Another of its ability is to inhibit vascular endothelial growth factor receptor 2 (VEGFR2), a key mediator of angiogenesis. Our laboratory has begun to investigate the interaction sites between TIMP3 and VEGFR2 and have identified several regions within the C-terminal domain of TIMP3 that play a role in this interaction including a short sixteen amino-acid peptide (p700) inhibits VEGFR2 and other receptors which are upregulated in either tumour vasculature or tumour cells in several cancers. The aims of this study were to utilize p700 as a novel delivery vehicle for cytotoxic drugs to treat cancer and to engineer identified residues from the TIMP3 C-terminal into a chimeric protein of TIMP3 and TIMP4 for VEGFR2 inhibition. Results: P700 retained its receptor binding after successful coupling to Doxil®. In contrast to Doxil alone, P700-Doxil was rapidly internalized by H5V cells, mouse (4T1) and human (MCF7) breast cancer cells but not HUVEC cells, showing approximately 100-fold increase in uptake relative to Doxil alone. Furthermore, this enhanced uptake significantly increased the sensitivity of breast cancer cells to Doxil in vitro but not in vivo. Recombinant CPG2 was highly expressed in E. coli and successfully purified by nickel affinity chromatography. Biolayer interferometry showed that CPG2-p700 had a 100-fold increase in binding affinity for VEGFR2 compared with CPG2 alone. Recombinant CGP2-p700 retained its catalytic activity for MTX. In the presence of CPG2-p700, the ZD2676P pro-drug showed significant cytotoxicity for 4T1 cells compared with prodrug alone or CPG2 alone. Finally, we successfully transfected H5V endothelial cell line with TIMP3-TIMP4 chimeras for stable expression. Initial phosphorylation assays showed T3T4V inhibited VEGFR2 phosphorylation in H5V cells transfected while T3T4 that lacks these residues did not.
Supervisor: Barker, Mike Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Thesis
EThOS ID:  DOI: Not available