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Development of new radiolabelled PET-ligands for a cancer specific epidermal growth factor receptor and their applications for cancer diagnosis

Charlotte Lund Denholt  


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Accepted by: Faculty of Health Sciences University of Copenhagen
Defended on: September 17, 2008
Official opponents: Prof. Knud Jensen , Dr. Scient. Gunnar Houen , Prof. Philip Elsinga
Tutors: Prof. Andreas Kjær , Assoc. Prof. Paul R Hansen , Dr. Nic Gillings

Published in the PhD Database: October 2, 2008


English abstract
Early diagnosis and staging is of critical importance for correct treatment of cancer. Positron Emission Tomography (PET) is a functional imaging modality, which due to its high sensitivity can detect very small malignant foci, making it a useful method for staging, treatment monitoring and detection of recurrence of cancer. Currently, only non-specific cancer ligands, such as fluorodeoxyglucose, (18F-FDG), are available. Therefore, the identification of novel, cancer specific targets and receptor ligands for PET imaging is of great interest. Most of the receptors, which are expressed on cancer cells, are also expressed on normal tissue. One of the few known cancer specific surface markers is the epidermal growth factor tyrosine kinase receptor mutation (EGFRvIII), which is expressed by many cancer forms; glioblatsomas, breast and ovarian cancer. EGFRvIII has never been identified in normal tissues, making it the obvious choice as a target for development of new cancer specific ligands.
The aim of the project was to develop radiolabelled peptides, which bind specifically to EGFRvIII, and subsequently label these peptides with positron emitting isotopes for PET studies.
Two strategies were used to achieve this aim. The first strategy was performing an alanine-scan on PEPHC1 (HFLIIGFMRRALCGA), a peptide which previously been shown to bind selectively to EGFRvIII, to identify the amino acids residues of importance of the binding of the peptide to the mutated receptor. The binding of peptides were tested using N6RM, N6RW-A and N6R cell lines, expressing EGFRvIII, wildtype EGFR and neither of the receptors, respectively, using a biotin-streptavidin binding assay. The results indicated that the amino acid residues at the N-terminus of PEPHC1 are essential for the binding to the mutated receptor. One analogue, [Ala12]PEPHC1, showed higher binding to EGFRvIII than PEPHC1. On the basis of alanine-scan results, eight truncated peptide analogues derived from the N-terminus of PEPHC1 were synthesized. Six of the truncated peptides were fluorine-18 labelled using 1-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione. Only one, H-CHIILGF-NH2, bound selectively to EGFRvIII and showed two-fold higher binding to the mutated receptor than PEPHC1.
The second strategy was using positional scanning synthetic combinatorial library. The library consists of hexa-peptides with six independent positional libraries, giving 114 sublibraries and a total theoretical number of 196 peptides. The binding studies were performed on N6RM, N6RW-A and N6R cell lines using a biotin-streptavidin strategy. Of the most active amino-acid-building blocks from the six sublibraries, eight new peptides were synthesized. However, only two of the peptides showed selective binding to the mutated receptor when re-tested, where FALGEA-NH2, showed the highest and most selective bind to EGFRvIII.
H-FALGEA-NH2 was fluorine-18 labelled using 4-[18F]-fluorobenzoic acid ([18F]FBA) for binding assay and PET investigation on a nude mouse xenografted subcutaneously with genuine human glioblastomas multiform tumours expressing the mutated receptor in this native form. [18F]FALGEA-NH2 showed some binding to EGFRvIII when tested in vitro and no binding when tested in vivo. [18F]FALGEA-NH2 was rapidly eliminated from the mouse, probably due to rapid proteolysis of the peptide.
Two promesing peptides were identified, PEPHC1c1 and H-FALGEA-NH2, which could be used as a starting-point for further work in the development of potential EGFRvIII targeting ligands.



Danish abstract
Positron Emissions Tomografi (PET) er en funktionel billeddiagnostisk metode, som i tiltagende grad anvendes til diagnostik, stadieinddeling og vurdering af behandlingsrespons ved cancer. Aktuelt findes kun uspecifikke PET-ligander, som for eksempel fluorodeoxyglucose, (18F-FDG). Udvikling af cancerspecifikke PET-ligander vil derfor være af stor klinisk betydning. De fleste receptorer der udtrykkes i cancerceller udtrykkes også i normalt væv. En af de få kendte cancerspecifikke overflademarkører er epidermal vækst faktor receptor mutationen (EGFRvIII). EGFRvIII er aldrig blevet identificeret i normalt væv og er derfor en potentiel specifik markør for forskellige cancerformer, bl.a. glioblastomer, bryst- og ovariankræft.
Formålet med nærværende projektet var at udvikle cancerspecifikke ligander, i form af peptider der binder specifikt til EGFRvIII og efterfølgende radiomærke dem til brug i PET studier. To strategier blev anvendt til at opnået dette formål. Den første strategi var at udføre et ala-scan på PEPHC1 (HFLIIGFMRRALCGA), et peptid som tidligere er blevet identificeret og som binder specifikt til EGFRvIII, for at identificere de aminosyrer som er vigtige for binding af peptidet til den muteret receptor. Binding af peptiderne blev undersøgt på NR6M, N6RW-A and NR6 celleliner, som udtrykker henholdsvis EGFRvIII, wildtypen EGFR og ingen af receptorerne, med en biotin-streptavidin strategi. Resultatet af Ala-scannet indikerede at aminosyrerne i N-terminalen af peptidet var vigtige for bindingen til EGFRvIII. En analog, [Ala12]PEPHC1 viste en mere selektiv binding til den muteret receptor end PEPHC1. På grundlag af resultaterne fra ala-scannet blev otte nye trunkeret peptider syntetiseret indeholdende aminosyrerne fra N-terminalen af PEPHC1. Seks af peptiderne blev fluor-18 mærket med 1-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione. Kun et af de trunkerede peptider, H-CHIILGF-NH2, bandt selektivt til EGFRvIII og havde en to gange højere binding til den muteret receptor end PEPHC1.
Den anden strategi var at anvende positional scanning synthetic kombinatorisk bibliotek. Biblioteket bestod af hexa-peptider med seks uafhængige positional biblioteker indeholdende, teoretisk set 196 peptider fordelt i 114 underbiblioteker. Disse blandinger blev testet for deres bindingsevne til den muteret receptor, ved at anvende NR6M, NR6W-A og NR6 cellelinier og en biotin-streptavidin strategi. De aminosyrer med størst binding i de 6 positioner blev udvalgt, og otte peptider, indeholdende disse aminosyrer, blev syntetiseret. To af disse peptider udvist selektiv binding til den muteret receptor, hvor, FALGEA-NH2, viste den højeste selektive binding.
H-FALGEA-NH2 blev fluor-18 mærket med 4-[18F]-Fluorobenzosyre ([18F]FBA) og binding af det radiomærket peptid blev undersøgt in vitro og in vivo. In vivo studierne blev testet på nøgen mus inkuberet subkutant med ægte glioblastomer tumorer fra mennesker, som udtrykker den muteret receptor. [18F]FALGEA-NH2 viste en svag selektiv binding og ingen binding, når bindingen af peptidet til den muteret receptor blev testet henholdsvis in vitro og in vivo. [18F]FALGEA-NH2 blev hurtigt elimineret fra musen, hvilket indikerer at peptidet blev proteolyseret i musen.
To lovende peptider blev identificeret, PEPHCc1 og H-FALGEA-NH2, som kan blive anvendt som udgangspunkt til videreudvikling af EGFRvIII specifikke ligander.