The thesis is focused on site-specific labeling of affinity molecules for different applications where two types of binding proteins, Affibody molecules and antibodies, have been used. For the purpose of improving the properties of Affibody molecules for in vivo imaging, novel bi-functional chelators for radiolabeling using the radionuclide 111In were evaluated. In a first study, two chelators denoted NOTA and DOTA, respectively, were separately conjugated via maleimide chemistry to a C-terminal cysteine residue in a HER2-binding Affibody molecule (ZHER2:2395). In vivo evaluation using mice with prostate carcinoma cell line xenografts showed that the 111In-NOTA-MMA-ZHER2:2395 tracer exhibited faster clearance from blood than the 111In-DOTA-MMA-ZHER2:2395 counterpart,resulting in improved tumor-to-organ ratios. In a second study the in vivo imaging properties of a third tracer, 111In-NODAGA-MMA-ZHER2:2395, was investigated in tumor-bearing mice. While the tumor uptake was lower than seen for the 111In-DOTA-MMA-ZHER2:2395 tracer, a low uptake in non-targeted organs and a fast clearance from blood resulted in higher tumor-to-organ ratios for 111In-NODAGA-MMA-ZHER2:2395 compared to the DOTA variant. In a following study, a synthetically produced HER2-targeting affibody variant, denoted ZHER2:S1, was used where NODAGA, NOTA and DOTA chelators instead were conjugated via an amide bond to the N-terminus. In vivo evaluation in mice showed an unfavorable uptake in liver for 111In-NOTA-ZHER2:S1, resulting in a discontinuation. The study showed faster clearance of 111In-NODAGA-ZHER2:S1 from blood, but also an increased uptake in bone in comparison to 111In-DOTA-ZHER2:S1. As bone is a common metastatic site in prostate cancer, the favorable tumor-to-bone ratio for 111In-DOTA-ZHER2:S1 suggests it as the tracer of choice for prostate cancer. Further, the DOTA chelator was also evaluated as conjugated to either N- or C-terminus or to the back of helix 3 via an amide bond, where the in vivo evaluation showed that that C-terminal conjugation resulted in the highest contrast. Site specificity is also of great importance for labeling antibodies, as conjugation in the antigen-binding regions might influence the affinity. A method for site-specific labeling of antibodies using an IgG-binding domain that becomes covalently attached to the Fc-region of an antibody by photoconjugation was optimized. By investigation of positions most suitable for incorporation of the photoreactive probe, the conjugation efficiencies were increased for antibody subclasses important for both diagnostic and therapeutic applications. In addition, optimized variants were used in combination with an incorporated click-reactive handle for selective labeling of the antibody with a detection molecule. / <p>QC 20140929</p>

We study multi-photon ionization of rare gas atoms and small molecules by infrared femtosecond laser pulses. We demonstrate that ionization is accurately described by a tunnelling model when many infrared photons are absorbed. By measuring photo-electron and photo-ion spectra, we show how the sub-Ångstrom spatial resolution of tunnelling gives information about electron densities in the valence shell of atoms and molecules. The photo-electron and photo-ion momentum distributions are recorded with a velocity map imaging (VMI) spectrometer. We describe a tomographic method for imaging a 3-D momentum distribution of arbitrary symmetry using a 2-D VMI detector. We apply the method to measure the 3-D photo-electron distribution in elliptically polarized light. Using circularly polarized light, we show how the photo-electron momentum distribution can be used to measure the focused laser intensity with high precision. We demonstrate that the gradient of intensities present in a focused femtosecond pulse can be replaced by a single average intensity for a highly nonlinear process like multi-photon ionization. By studying photo-electron angular distributions over a range of laser parameters, we determine experimentally how the photon linear momentum is shared between the photo-electron, photo-ion and light field. We find the photo-electron carries only a portion of the total linear momentum absorbed. In addition we consider how angular momentum is shared in multi-photon ionization, and find the photo-electron receives all of the angular momentum absorbed. Our results demonstrate how optical and material properties influence the photo-electron spectrum in multi-photon ionization. These will have implications for molecular imaging using femtosecond laser pulses, and controlling the initial conditions of laser generated plasmas.

The growing understanding of tumor biology and the identification of tumor specificgenetic and molecular alterations, such as the overexpression of human epidermal growthfactor receptor 2 (HER2), opens up for personalization of patient management using targeted therapies. However, this puts stringent demands on the diagnostic tools usedto identify patients that are likely to respond to a particular treatment. Radionuclide molecular imaging is a promising non-invasive method to visualize and characterize the expression of such targets. This thesis, based on five papers, is focused on the development of radiolabeled Affibody molecules for imaging of HER2-expression in malignant tumors. Affibody molecules, which represent a rather novel class of affinity proteins developed by combinatorial protein engineering of the protein A derived Z-domain, display manyfeatures that make them promising tracers for molecular imaging applications. The aim of the work presented here was to further develop the tracer format for improved in vivo properties and flexibility in the choice of radionuclide. In paper I, the development of an assay that enables quantitative studies of the internalization rate and cellular processing of high affinity Affibody molecules is described. The assay was applied to a HER2-binding Affibody variant that was efficiently retained by HER2-expressing cells, although characterized by a slow internalization rate. This may have implications for the choice of label for Affibody molecules since high affinity to the target may be equally, or more, important for good imaging quality than residualizing properties of the radiolabel. In paper II, a HER2-binding Affibody molecule and the monoclonal antibody trastuzumab were labeled with positron emitting 124I, for a head-to-head in vivocomparison of the two tracer formats. The effects of tracer size and presence of an Fc region on the biodistribution profile were investigated. In paper III, a HER2-binding Affibody molecule was site-specifically labeled with radiocobalt and evaluated in vitro and in vivo.A head-to-head in vivo comparison with the well-studied 111In-labeled counterpart was performed, revealing promising potential for the cobalt-labeled molecule as a PET-tracerfor visualization of HER2. Paper IV describes the in vitro and in vivo evaluation of a panel of Affibody molecules with different C-terminal peptide-based chelators for the coordination of 99mTc. Even small changes in the C-terminal sequence had appreciable impact on the biodistribution of the Affibody molecules and by optimizing the design of the chelator, the kidney uptake of 99mTc could be significantly reduced. Finally, in paper V we describe the development of a HER2-targeting Affibody variant equipped with a Sel-tag for site-specific labeling with the short-lived positron emitter 11C. This novel Affibody tracer could be used to image HER2-expressing tumors in vivo within one hour after injection. Taken together, Affibody molecules show great promise as targeting tracers for radionuclide molecular imaging of HER2. Careful design and optimization of the tracer protein is important and can be used to improve the biodistribution and targeting properties of Affibody molecules. / QC 20110922

The images in positron emission tomography (PET) represent three dimensional dynamic distributions of biologically interesting molecules labelled with positron emitting radionuclides (radiotracers). Spatial localisation of the radio-tracers is achieved by detecting in coincidence two collinear photons which are emitted when the positron annihilates with an ordinary electron. In order to obtain quantitatively accurate images in PET, it is necessary to correct for the effects of photon attenuation within the subject being imaged. These corrections can be obtained using singles-mode photon transmission scanning. Although suitable for small animal PET, these scans are subject to high amounts of contamination from scattered photons. Currently, no accurate correction exists to account for scatter in these data. The primary purpose of this work was to implement and validate an analytical scatter correction for PET transmission scanning. In order to isolate the effects of scatter, we developed a simulation tool which was validated using experimental transmission data. We then presented an analytical scatter correction for singles-mode transmission data in PET. We compared our scatter correction data with the previously validated simulation data for uniform and non-uniform phantoms and for two different transmission source radionuclides. Our scatter calculation correctly predicted the contribution from scattered photons to the simulated data for all phantoms and both transmission sources. We then applied our scatter correction as part of an iterative reconstruction algorithm for simulated and experimental PET transmission data for uniform and non-uniform phantoms. We also tested our reconstruction and scatter correction procedure using transmission data for several animal studies (mice, rats and primates). For all studies considered, we found that the average reconstructed linear attenuation coefficients for water or soft-tissue regions of interest agreed with expected values to within 4%. Using a 2.2 GHz processor, the scatter correction required between 6 to 27 minutes of CPU time (without any code optimisation) depending on the phantom size and source used. This extra calculation time does not seem unreasonable considering that, without scatter corrections, errors in the reconstructed attenuation coefficients were between 18 to 45% depending on the phantom size and transmission source used.

Doctor of Philosophy(PhD) / The aims of this project were to contribute to the elucidation of the role of the cholinergic system in attention and memory, two cognitive processes severely compromised in Alzheimer’s disease (AD), and to evaluate and develop tools for the functional molecular imaging of this system with a view to improving knowledge of AD and other neurological disorders. Towards the first aim, the specific anti-cholinergic toxin 192 IgG-saporin (SAP) was administered to female Sprague-Dawley rats via either an intracerebroventricular (icv) or an intracortical route and animals were tested with a vibrissal-stimulation reaction-time task and an object recognition task to evaluate their attentional and mnemonic function, respectively. The second aim was approached in two ways. Firstly, relative neuronal densities from animals with icv lesions were assessed with both ex vivo and in vitro autoradiography with the specific cholinergic radiopharmaceuticals [123I]iodobenzovesamicol (123IBVM) and 125I-A-85380, ligands for the vesicular acetylcholine transporter and the nicotinic acetylcholine receptor, respectively. Secondly, a number of in vivo and in vitro studies were performed on a novel and unique molecular imaging system (TOHR), with which it had been hoped initially to image eventually SAP-lesioned animals, with a view to measuring and ameliorating its performance characteristics and assessing its in-principle suitability for small-animal molecular imaging. The behavioural studies support a critical role for the cholinergic system in normal attentional function. Additionally, in accord with literature evidence, no significant impairment was observed in mnemonic function. It is postulated however that the results observed in the intracortically-lesioned animals support the published hypothesis that cholinergic projections to the perirhinal cortex are critical for object-recognition memory. In autoradiographic studies, SAP-lesioned animals demonstrated reduced uptake of 123IBVM in multiple regions. A reduction of nicotinic receptors was also seen in SAP-lesioned animals, a novel finding supportive of the excellent characteristics of radioiodinated I-A-85380. Examination of the performance characteristics of the TOHR support in principle its utility for targeted small-animal molecular imaging studies.

Στόχος της διπλωματικής εργασίας ήταν η εφαρμογή της Μοριακής Απεικόνισης για την λήψη εικόνων παθολογικής αγγειογένεσης σε πειραματικό μοντέλο λευκού κόνικλου Νέας Ζηλανδίας με καρκίνωμα VX2. Στη πρώτη φάση της διπλωματικής εργασίας προσδιορίσαμε ως κατάλληλο μοριακό στόχο για την απεικόνιση της αγγειογένεσης, την ιντεγκρίνη ανβ3. Η επιλογή του στόχου βασίστηκε στο γεγονός ότι το επίπεδο έκφρασης της ιντεγκρίνης ανβ3 αυξάνεται σημαντικά κατά την αγγειογένεση, η οποία έχει διαπιστωθεί ότι συμμετέχει στην ανάπτυξη ενός όγκου και επάγεται από χημικούς παράγοντες σε καταστάσεις ισχαιμίας, ενώ σχετίζεται και με πολλές άλλες ασθένειες. Στη δεύτερη φάση στόχος μας ήταν να γίνει εμφύτευση καρκινικών κυττάρων της σειράς VX2 σε κόνικλο και η δημιουργία ενός carrier animal με όγκο VX2. Η δημιουργία ενός δότη κυττάρων VX2 χρησιμεύει για την επίτευξη συνεχών μετεμφυτεύσεων του όγκου σε κόνικλους που προορίζονται για απεικόνιση του επιλεγμένου μοριακού στόχου. Για τη μελέτη του επιπέδου έκφρασης της ιντεγκρίνης ανβ3 επιλέχθηκαν ως μόρια προς επισήμανση πεπτίδια τα οποία φέρουν την αλληλουχία RGD (Arginine-Glycine-Aspartic acid) μέσω της οποίας αντιδρούν με το μόριο της ιντεγκρίνης ανβ3 και ως κατάλληλο σύστημα απεικόνισης περιστρεφόμενη γ κάμερα. Τελικός στόχος είναι μέσω του δείκτη ανβ3 να μελετήσουμε πως εξελίσσεται χρονικά και χωρικά σε σχέση με τον όγκο η αγγειογένεση. Μελλοντικές προοπτικές είναι η ακριβής παρακολούθηση σε κλινικό επίπεδο της διαδικασίας της αγγειογένεσης μετά από χορήγηση προ-αγγειογενετικών ή αντι-αγγειογενετικών παραγόντων σε περιπτώσεις ισχαιμίας ή καρκίνου αντίστοιχα. / The aim of the study was the aquisition of images of tumor angiogenesis by implementing Molecular Imaging techniques. The small animal experimental platform that was required for the study was the New Zealand White rabbit with VX2 carcinoma. The molecular target for imaging angiogenesis was integrin ανβ3, which is found to be overexpressed in endothelial cells participating in angiogenesis. Angiogenesis is found to be crucial for the development of solid tumors (excessive angiogenesis) and other pathological conditions (insufficient angiogenesis). Tracers that interact with ανβ3 are RGD (Arginine-Glycine-Aspartic acid residues)peptides labeled with radionuclides and suitable imaging modality is rotating gamma camera. Targeted molecular imaging may visualize the early activation of the angiogenic type of endothelial cells.To that end, we hope to gain further insights into the specific temporal and spatial characteristics of the onset of angiogenesis.

La maladie coronaire représente un problème majeur de santé publique. Sa manifestation la plus sévère, le syndrome coronarien aigu, est en grande majorité lié à la rupture de plaques d'athérome vulnérables. Les travaux effectués dans le cadre de cette thèse portent sur l'étude pré-clinique de la plaque d'athérome dans un modèle murin : la souris déficiente en apolipoprotéine E (apoE-/-).La rupture des plaques d'athérome vulnérables survient à la suite d'une élévation des contrainte mécaniques au niveau de la capsule fibreuse. Une première partie de ce travail de thèse porte sur l'évaluation de la contrainte présente dans la paroi des vaisseaux de souris apoE-/- porteuses de lésions athéromateuses. Ces lésions présentent une faible rigidité et en conséquence de faibles contraintes. Ces observations donnent un élément d'explication de l'absence de rupture dans ce modèle animal.Les plaques d'athérome vulnérables présentent une inflammation importante ; l'imagerie de l'inflammation vasculaire est une approche pertinente pour l'identification des plaques d'athérome vulnérables. Une seconde partie de ce travail de thèse porte sur l'imagerie de l'athérosclérose en ciblant le processus inflammatoire chez la souris apoE-/- avec d'une part l'évaluation de radiotraceurs peptidiques développés au sein du laboratoire et décrits comme ligands de la molécule d'adhésion VCAM-1, surexprimée dans le contexte inflammatoire, et d'autre part l'évaluation du [18F]-fluorodésoxyglucose (FDG), décrit pour s'accumuler dans les cellules inflammatoires et le tissu adipeux brun. L'évaluation poussée des peptides originaux a mis en évidence un manque de robustesse dans leur comportement biologique. D'autre part, une captation similaire de FDG a été observée dans les lésions athéromateuses et du tissu le brun péri-aortique se trouvant à proximité chez la souris apoE-/- . Cette limitation souligne la nécessité de l'optimisation des procédés expérimentaux destinés à l'évaluation de l'inflammation des lésions d'athérosclérose avec le FDG dans ce modèle animal. / Coronary artery disease is a major healthcare issue. Acute coronary syndrome is mainly caused by the rupture of vulnerable coronary plaques. This thesis focused on the preclinical study of atherosclerosis in a mouse model: the apolipoprotein E-deficient mouse (apoE-/-).Vulnerable plaque rupture is caused by an elevation of mechanical stress in the fibrous cap of atherosclerotic plaques. The first part of this thesis focused on the parietal stress evaluation in atheromatous lesions from apoE-/- mouse vessels. Atherosclerotic tissue showed low stiffness resulting in low levels of mechanical stress in atheromatous lesions. The low level of mechanical stress might account for the atherosclerotic plaque stability in this animal model.Rupture-prone vulnerable atheromatous plaques are highly inflammatory; vascular inflammation imaging appears therefore as a relevant strategy for vulnerable plaque imaging. The second part of this thesis dealt with atherosclerosis imaging in the apoE-/- mouse model, firstly with the evaluation of original peptidic radiotracers described as a VCAM-1 binders, an adhesion molecule over-expressed during inflammation, and secondly with the evaluation of [18F]-fluorodeoxyglucose (FDG), which was shown to accumulate in inflammatory cells and in the brown adipose tissue (BAT). Advanced evaluation of peptides revealed a lack of robustness in their biological behavior. A similar FDG uptake was observed in the atherosclerotic lesions and in the periaortic brown adipose tissue of apoE-/- mouse found in the vicinity. This potential confounding factor emphasizes the need to carefully design preclinical studies using FDG for the evaluation of lesion inflammation in this animal model.

L’imagerie en oncologie a fait des progrès considérables ces dernières années avec l’introduction du CT scan spiralé, de la résonance magnétique, de la mammographie digitalisée et du PET scan. Des combinaisons de différentes techniques ont vu le jour, telles que le PET/CT, et améliorent encore les possibilités de stadification de la maladie cancéreuse ainsi que le monitoring de son évolution dans le temps, et notamment sous traitement.Parallèlement, de grands progrès thérapeutiques ont étés réalisés en oncologie, en particulier le développement de médicaments « ciblés » dont l’efficacité dépend de l’expression par la cellule tumorale d’une molécule cible jouant un rôle important dans sa survie et/ou sa prolifération. L’expression de la molécule cible est une condition nécessaire mais pas suffisante pour observer une réponse au traitement ciblé :l’échec de ce dernier peut aussi s’expliquer par des altérations moléculaires en amont ou en aval de la « cible ».Le cancer du sein dit « HER2 positif » représente 20 à 25% des cancers du sein. Celui-ci est caractérisé par l’expression membranaire, en quantités importantes, d’une protéine, appelée HER2, qui lui confère une biologie agressive et un mauvais pronostic. L’expression de HER2 au niveau de la tumeur, déterminée en routine clinique par immunohistochimie et/ou par hybridation in situ en fluorescence, est le seul biomarqueur validé aujourd’hui dans le cancer du sein HER2 positif pour prédire l’efficacité des traitements ciblés anti-HER2. Cette prédiction est toutefois peu satisfaisante en termes de valeur prédictive positive (50% environ). Après une revue de la litérature sur les études d’imagerie fonctionnelle, peu nombreuses, réalisées dans le cancer du sein HER2 positif, nous avons décidé d’explorer le rôle de l’imagerie moléculaire avec la technologie PET/CT dans l’individualisation de la prise en charge du cancer du sein HER2 positif avec deux radio traceurs (FDG et zirconium89-trastuzumab), et ce, dans deux contextes cliniques distincts :dans la maladie précoce soumise à un traitement neoadjuvant et dans le contexte métastatique, en cas de traitement par le T-DM1. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

L’imagerie moléculaire est un nouveau domaine de la recherche biomédicale qui peut être défini comme l’observation non invasive d’un processus biologique au sein d’organismes vivants. Cette technique nécessite l’utilisation d’un agent de contraste, une molécule composée d’un vecteur se liant spécifiquement à un marqueur du processus biologique étudié et d’un contrastophore permettant l’acquisition du signal. L’objectif de cette thèse est de développer un agent de contraste permettant de diagnostiquer la présence de plaques d’athérome. L’athérosclérose est une pathologie inflammatoire chronique, caractérisée par l’accumulation de cholestérol, sous forme de plaque appelée « athérome », dans la paroi des moyennes et grosses artères. Les complications majeures de l’athérosclérose surviennent lorsque ces plaques obstruent le flux sanguin, localement ou lors de leurs ruptures. Ces phénomènes de thrombose sont à l’origine des infarctus et des accidents cardio- ou neuro-vasculaire, premières causes de mortalité dans les pays développés. Nous avons choisi le peptide comme outils de vectorisation. Mon travail a consisté en l’étude et la caractérisation de l’interaction de peptides envers les bio-marqueurs de la plaque d’athérome. Dans un second temps, j’ai effectué un travail de pharmacomodulation afin d’améliorer les propriétés physico-chimiques de ces peptides. Ainsi, nous avons synthétisé le traceur Ga-P04087 qui répond à tous les critères du cahier des charges et qui est actuellement en phase pré-clinique. / Molecular imaging is a new domain of biomedical research which can be defined as the non-invasive observation of a biological process within the living body. This technique requires the use of a contrast agent, a molecule composed of a vector targeting a biomolecule specifically expressed during the studied biological process, and a contrastophore allowing the acquisition of the signal. The aim of this work is to develop a contrast agent to diagnose the presence of atheromatous plaques. Indeed, atherosclerosis is an inflammatory chronic disease, characterized by the accumulation of cholesterol, in the form of plaques called "atheroma", in the wall of the average and broad arteries. The major complications of atherosclerosis arise when these plaques block the blood flow, locally or after they break free and are released in the artery. These phenomena of thrombosis are at the origin of myocardial and cerebral infarctions, first causes of death in all developed countries. We chose the peptide as tool of vectorisation, and my work consisted of studying and characterizing the interaction of these peptides to three different biomarkers of atherosclerosis. Secondly, I used various tools developed in our lab to conduct a pharmacomodulation campaign based on these compounds, in order to improve their physicochemical properties. We also achieved the synthesis of the tracer Ga-P04087 who answers all the criteria of the specifications and who is now in preclinical phase.

The images in positron emission tomography (PET) represent three dimensional dynamic distributions of biologically interesting molecules labelled with positron emitting radionuclides (radiotracers). Spatial localisation of the radio-tracers is achieved by detecting in coincidence two collinear photons which are emitted when the positron annihilates with an ordinary electron. In order to obtain quantitatively accurate images in PET, it is necessary to correct for the effects of photon attenuation within the subject being imaged. These corrections can be obtained using singles-mode photon transmission scanning. Although suitable for small animal PET, these scans are subject to high amounts of contamination from scattered photons. Currently, no accurate correction exists to account for scatter in these data. The primary purpose of this work was to implement and validate an analytical scatter correction for PET transmission scanning. In order to isolate the effects of scatter, we developed a simulation tool which was validated using experimental transmission data. We then presented an analytical scatter correction for singles-mode transmission data in PET. We compared our scatter correction data with the previously validated simulation data for uniform and non-uniform phantoms and for two different transmission source radionuclides. Our scatter calculation correctly predicted the contribution from scattered photons to the simulated data for all phantoms and both transmission sources. We then applied our scatter correction as part of an iterative reconstruction algorithm for simulated and experimental PET transmission data for uniform and non-uniform phantoms. We also tested our reconstruction and scatter correction procedure using transmission data for several animal studies (mice, rats and primates). For all studies considered, we found that the average reconstructed linear attenuation coefficients for water or soft-tissue regions of interest agreed with expected values to within 4%. Using a 2.2 GHz processor, the scatter correction required between 6 to 27 minutes of CPU time (without any code optimisation) depending on the phantom size and source used. This extra calculation time does not seem unreasonable considering that, without scatter corrections, errors in the reconstructed attenuation coefficients were between 18 to 45% depending on the phantom size and transmission source used. / Science, Faculty of / Physics and Astronomy, Department of / Graduate