Sinteza i karakterizacija afinitetnih protutijela za detekciju stanica raka

master's thesis

• Title (english): Assembly and characterization of di-affibodies for cancer cell recognition and imaging
• Author: Edina Numanović
• Mentor: Vladimir Mrša (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2021-09-06, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Affibody molecules (AffB) are small (~ 6 kDa) and robust non-immunoglobulin affinity ligands capable of binding to a wide range of protein targets. Because of many advantages, they can be used as tools for molecular recognition in diagnostic and therapeutic applications. The human epidermal growth factor receptor 2 (HER2) (ErbB2/neu) receptor tyrosine kinase is often overexpressed in several different carcinomas, and it has attracted attention for use as a tumour marker in bioimaging and drug delivery systems. Therefore, binding molecules with the ability to target Her2 have been developed, for instance ZHER₂:₂₈₉₁, which comprises a cysteine residue (C-terminus). Three affibody conjugates (monoaffibody AffB-1MI and two diaffibodies AffB₂-2MI and AffB₂-3MI) were obtained by covalent coupling with bispecific linkers based on flexible PEG chains (maleimide-thiol conjugation), combined with a fluorescent pattern. Flow cytometry was performed to show the cell binding ability of the affibody conjugates to HER2-overexpressing cell line, SKOV3 cells, and HER2 low-expressing cell line, HeLa cells. It was found that all three conjugates could bind specifically to Her2, but AffB-1MI could bind more effectively to HER2-expressing cells. This study also showed that only anti-proliferative effect of AffB-1MI has some significance. Since AffB-1MI shows best results, it could be also used for imaging purposes. Optimization of all three conjugates might be further considered for developing tumour-targeting applications and for diagnostics and immunotherapy of adenocarcinomas such as breast and ovarian cancers.

Abstract (english)

Afinitetna protutijela su mali (~ 6 kDa) i robusni neimunoglobulinski afinitetni ligandi sa sposobnošću vezanja širokog raspona proteina. Zbog mnogih prednosti, mogu se koristiti kao alati za molekularno prepoznavanje tijekom dijagnosticiranja i terapijske svrhe. Humani epidermalni faktora rasta receptor 2 (HER2) (ErbB2/neu) često je prekomjerno eksprimiran u nekoliko različitih karcinoma i privlači pozornost za upotrebu kao tumorski marker za detekciju (eng. « Bioimaging ») i isporuku lijekova. Stoga su razvijene molekule sa sposobnošću vezanja na Her2 receptor, na primjer ZHER₂:₂₈₉₁, koji sadrži cisteinski ostatak (C-kraj). Tri konjugata afinitetnih protutijela (monoaffinitetno protutijelo AffB-1MI i dva diafitijela AffB₂-2MI i AffB₂-3MI) dobivena su kovalentnim spajanjem s bispecifičnim fleksibilnim PEG lancima (konjugacija maleimid-tiol), označenih fluorescentnom molekulom. Provedena je protočna citometrija kako bi se pokazala sposobnost vezanja konjugata afinitetnih protutijela na stanice koja prekomjerno eksprimiraju HER2, SKOV3 i stanice s niskom ekspresijom HER2, HeLa. Utvrđeno je da se sva tri konjugata mogu specifično vezati za Her2, ali se AffB-1MI veže učinkovitije za stanice koje eksprimiraju HER2. Ova studija je također pokazala da samo antiproliferativni učinak AffB-1MI ima određeni značaj. Budući da AffB-1MI pokazuje najbolje rezultate, mogao bi se koristiti za dijagnosticiranje. Optimizacija sva tri konjugata mogla bi se dalje razmatrati za detekciju tumora te za dijagnostiku i imunoterapiju adenokarcinoma, poput raka dojke i jajnika.

• Keywords: affibodies; HER2; maleimide-thiol conjugation; tumour targeting; immunotherapy
• Keywords (english): afinitetna protutijela; HER2; konjugacija maleimid-tiol; detekcija tumora; imunoterapija
• Language: croatian
• URN:NBN: urn:nbn:hr:159:069848
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2021-10-04 12:33:58