Site directed modification of recombinant antibody fragments for in vivo fluorescence imaging and targeted drug delivery [Elektronische Ressource] / Florian Kampmeier

Florian Paris

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2011
• Nombre de lectures: 26
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Site Directed Modification of Recombinant Antibody
Fragments for In vivo Fluorescence Imaging and Targeted
Drug Delivery




Von der Fakultät für Mathematik, Informatik und Naturwissenschaften
der RWTH Aachen University zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften genehmigte Dissertation
vorgelegt von



Diplom Biologe
Florian Kampmeier
aus Aachen



Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer
Universitätsprofessor Dr. rer. nat. Dr. rer. medic. Stefan Barth



Tag der mündlichen Prüfung: 21. Juli 2010




Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.
Content I
1 Introduction ...................................................................................................................................... 1
1.1 Tumour targeting for diagnosis and therapy ............... 1
1.2 Affinity probes for molecular targeting ......................................................................................... 2
1.3 Targeted drug delivery ........................................................ 5
1.4 Molecular imaging ................................................................................................. 6
1.5 EGFR as target for imaging and therapy ..................... 10
1.6 Bioconjugation of proteins with effector molecules .............................. 12
1.7 SNAP-tag technology .......................................................................................................................... 13
1.8 Aims and Objectives ........................... 14
2 Materials and Methods .............................................................................................................. 16
2.1 Materials ................................................. 16 2.1.1 Equipment ....................................................................................................................................................... 16 2.1.2 Chemicals and Consumables.................................................................................................................... 17 2.1.3 Buffers and Media ........................................................................................................................................ 17 2.1.4 Kits and enzymes .......................................................................................................................................... 19 2.1.5 Antibodies ....................................................................................................................................................... 19 2.1.6 Engineered protein tags and source plasmids ................................................................................. 19 2.1.7 Synthetic oligonucleotides........................................................................................................................ 20 2.1.8 Bacterial strains ............................................................................................................................................ 20 2.1.9 Mammalian cell lines .................................................................................................................................. 21 2.1.10 Antibody fragments and protein ligands ......................................................................................... 21 2.1.11 Plasmid vectors .......................................................................................................................................... 21
2.2 Methods .................................................................................................................................................. 22 2.2.1 Molecular cloning and DNA work .......................................................................................................... 22 2.2.2 Cell culture ...................................................................................................................................................... 25 2.2.3 Protein biochemical methods.................................................................................................................. 26 2.2.4 Flow cytometry ............................................................................................................................................. 30 2.2.5 Cofocal microscopy ...................................................................................................................................... 31 2.2.6 Coupling of SNAP-tag fusion proteins to “nano-sized” particles .............................................. 31 2.2.7 Cell viability assay with polyglycerol-Doxorubicin conjugates ................................................. 33 2.2.8 Coupling of oligonucleotides to SNAP-tag fusion proteins ......................................................... 34 2.2.9 In vivo imaging methods ............................................................................................................................ 34



Content II
3 Results ............................................................................................................................................. 39
3.1 Cloning, expression and characterization of ligand-tag fusion proteins ........................ 39 3.1.1 Cloning of antibody fragment and protein ligands fusion proteins ........................................ 39 3.1.2 Expression and purification of tagged proteins............................................................................... 40 3.1.3 Storage and serum stability of SNAP-tag fusion proteins............................................................ 41 3.1.4 Labelling of SNAP fusion proteins with organic fluorophores .................................................. 41 3.1.5 Binding activity of fluorescently labelled SNAP fusion proteins .............................................. 42 3.1.6 Labelling and binding of CLIP/ACP fusion proteins ...................................................................... 42 3.1.7 Biotinylation via the SNAP-tag ................................................................................................................ 44 3.1.8 Competitive binding of SNAP-EGF and 425(scFv)SNAP .............................................................. 45
3.2 Internalization of 425(scFv)SNAP by L3.6pl cells ................................................................... 45
3.3 Coupling of oligonucleotides to SNAP-tag fusion proteins ................................................... 46
3.4 Coupling SNAP-tag fusion proteins to luminescent silica beads ........ 47
3.5 Coupling of SNAP-tag fusion proteins to Polyglycerol Doxorubicin Conjugates .......... 48
3.6 SNAP fusion proteins for in vivo optical imaging ..................................................................... 50 3.6.1 Experimental setup ..................................................................................................................................... 50 3.6.2 Verification of labelling and binding activity before administration in vivo ....................... 50 3.6.3 The L3.6pl-GFP subcutaneous tumour model .................................................................................. 51 3.6.4 Distribution and tumour accumulation of the imaging probes ................................................. 52 3.6.5 Tumour to background ratios and absolute signal intensities .................................................. 54 3.6.6 Comparison of 425(scFv)SNAP and C225 optical probes ........................................................... 55 3.6.7 Optical imaging using the LI-COR Pearl system ............................................................................... 58 3.6.8 Fluorescence molecular tomography................................................................................................... 59
4 Discussion ....................................................................................................................................... 61
4.1 Production and site-specific labelling of SNAP-tag fusion proteins.. 61
4.2 Conjugation of scFvSNAP with nucleic acids ............. 63
4.3 Coupling of scFvSNAP to silica nanoparticles ........................................................................... 64
4.4 Coupling of scFvSNAP to PG-Doxorubicin ................... 65
4.5 In vivo optical imaging ....................................................................................................................... 67 4.5.1 The subcutaneous pancreatic carcinoma model ............................................................................. 67 4.5.2 425(scFv)SNAP as in vivo optical imaging probe ............................................................................ 68 4.5.3 Comparison of 425(scFv)SNAP with C225 ........................................................................................ 69 4.5.4 In vivo optical imaging with the LI-COR Pearl and VisEn-FMT system .................................. 70 4.5.5 In vivo optical imaging: concluding remarks .................................................................................... 71
4.6 Outlook .................................................................................................................................................... 72
Content III
5 Summary ......................................................................................................................................... 74
6 Literature ........ 76
7 Appendix .