Spelling suggestions: "subject:"radionuclides molecular imaging""
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Bombesin Antagonists for Targeting Gastrin-Releasing Peptide Receptor-Positive Tumors : Design, Synthesis, Preclinical Evaluation and Optimization of Imaging AgentsVarasteh, Zohreh January 2014 (has links)
This thesis is focused on the development, preclinical evaluation, and optimization of radiotracers for the detection of gastrin-releasing peptide receptor (GRPR)-expressing tumors. The work is divided into three distinct parts: (1) the development of bombesin (BN) antagonist (RM26)-based imaging radiotracers for the detection of GRPR-expressing tumors using different positron emission tomography (PET) and single photon emission computed tomography (SPECT) radionuclides (68Ga, 18F and 111In), (2) the establishment of a method to monitor the ligand-G protein-coupled receptor (GPCR) interaction in real time without requiring purification and stabilization of the receptors, and (3) the evaluation of radiopeptide structure-related factors (length of mini-PEG linker and composition of chelator for metal labeling) affecting the in vitro and in vivo characteristics of RM26-based tracers. We demonstrated the possibility of high-contrast in vivo imaging of GRPR-expressing xenografts despite the physiological expression of GRPR in abdominal organs. Fast radioactivity clearance from the blood and healthy organs, including receptor-positive organs, and long retention in the tumors resulted in high tumor-to-background ratios. A novel real-time assay for measuring the kinetics of the radiotracers targeting GPCR was evaluated. Living cells were used instead of purified receptors in this technology, bringing the developmental work one step closer to the true target environment (imaging in living systems). The comparative study of 68Ga-labeled NOTA-PEGn-RM26 with di-, tri-, tetra- and hexaethylene glycol chains demonstrated that the addition of only a few units of ethylene glycol to the spacer is insufficient to appreciably affect the biodistribution of the radiopeptide. Finally, a comparative study of 68Ga-labeled PEG2-RM26 analogs N-terminally conjugated to NOTA, NODAGA, DOTA or DOTAGA highlighted the influence of the chelator on the targeting properties of the radiopeptide. The main conclusion that can be drawn from this thesis is that 68Ga-NOTA-PEG2-RM26 has favorable biodistribution properties, such as rapid clearance from blood and tissues with physiological GRPR expression levels and long retention in GRPR-expressing tumors, and that this radiopeptide is potentially suitable for initial clinical investigation.
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Development of Affibody molecules for radionuclide molecular imaging and therapy of cancerHonarvar, Hadis January 2016 (has links)
Affibody molecules are a promising class of scaffold-based targeting proteins for radionuclide-based imaging and therapy of cancer. This thesis work is based on 5 original research articles (papers I-V), which focus on optimization of molecular design of HER2-binding Affibody variants for high contrast imaging of this predictive biomarker as well as development of Affibody molecules suitable for radionuclide-based targeted therapies. Papers I and II were dedicated to evaluation of the influence of the macrocyclic chelator DOTA positioning at N-terminus, in the middle of helix-3 and at C terminus of a synthetic Affibody molecule, ZHER2:S1. These synthetic variants were labelled with different radionuclides i.e. 111In and 68Ga to study also the effect of different labels on their biodistribution properties. In paper III a 2-helix variant, Z342min, was developed using native ligation cyclization to cross-link helices one and two resulting in a stable 2-helix scaffold and characterized in vivo. This study was performed with the aim to obtain structure-properties relationship for development of smaller Affibody molecules. Papers IV and V were devoted to development of therapeutic strategies. In paper IV, a series of peptide based chelators was investigated for labelling of Affibody molecules with 188Re to provide low renal retention. In paper V, a pretargeting approach using peptide nucleic acid was investigated. These studies were performed with the aim to overcome the high renal retention of Affibody molecules when labelled with residualizing therapeutic radionuclides. Otherwise, the particle emitting radiometals could damage the kidneys more than the tumours. The results obtained for anti-HER2 Affibody molecules summarized in this thesis might be of importance for the development of other scaffold protein based targeting agents.
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Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular TargetingHofström, Camilla January 2013 (has links)
Affibody molecules are small (7 kDa) affinity proteins of non-immunoglobulin origin that have been generated to specifically interact with a large number of clinically important molecular targets. In this thesis, Affibody molecules have been employed as tracers for radionuclide molecular imaging of HER2- and IGF-1R-expressing tumors, paper I-IV, and for surface knock-down of EGFR, paper V. In paper I, a tag with the amino acid sequence HEHEHE was fused to the N-terminus of a HER2-specific Affibody molecule, (ZHER2), and was shown to enable facile IMAC purification and efficient tri-carbonyl 99mTc-labeling. In vivo evaluation of radioactivity uptake in different organs showed an improved biodistribution, including a 10-fold lower radioactivity uptake in liver, compared to the same construct with a H6-tag. In paper II, it was further shown that an N-terminally placed HEHEHE-tag on ZHER2 provided lower unspecific uptake of radioactivity in liver compared to its H6-tagged counterpart even when radiolabeling was at the C-terminus using alternative chemistries to attach 99mTc, 111In or 125I. In paper III, the H6-tag’s composition and position was varied with regards to charge, hydrophobicity and its C- or N-terminal placement on ZHER2. Among the ten variants investigated, it was found that an N-terminal HEHEHE-tag provided the most favorable overall biodistribution profile and that introduction of hydrophobic and positively charged amino acids provoked liver uptake of radioactivity. In paper IV, the HEHEHE-tag was shown to enable IMAC purification and tri-carbonyl 99mTc-labeling of an IGF-1R-specific Affibody molecule and improved its overall biodistribution when compared to the same construct with a H6-tag. In paper V, the aim was to develop an intracellular receptor-entrapment system to reduce the surface levels of EGFR. An EGFR-specific Affibody molecule was expressed as a fusion to different mutants of an intracellular transport protein in SKOV-3 cells, resulting in a collection of cell lines with 50%, 60%, 80% and 96% reduced surface level of EGFR. Analysis of the proliferation rate of these cell lines showed that a modest reduction (15%) in proliferation occurs between 60% and 80% reduction of the surface level of EGFR. / <p>QC 20130129</p>
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On the Design of Affibody Molecules for Radiolabeling and In Vivo Molecular ImagingRosik, Daniel January 2013 (has links)
Affibody molecules have lately shown great potential as tools for in vivo molecular imaging. These small, 3-helical bundles, with their highly stable protein scaffold, are well suited for the often harsh conditions of radiolabeling. Their small size allows for rapid clearance from the blood circulation which permits the collection of images already within hours after injection. This thesis includes four papers aimed at engineering different variants of a HER2-binding Affibody molecule to enable effective and flexible radiolabeling and enhancing the molecular imaging in terms of imaging contrast and resolution. In paper I an Affibody molecule was engineered to function as a multifunctional platform for site-specific labeling with different nuclides for radionuclide imaging. This was done using only natural amino acids, thereby allowing for both synthetic and recombinant production. By grafting the amino acid sequence -GSECG to the C-terminal of our model-protein, a HER2-binding Affibody molecule, we enabled site specific labeling with both trivalent radiometals and with 99m Tc. Maleim-ide-DOTA was conjugated to the cysteine residue for labeling with 111 In, while the peptide sequence was able to chelate 99m Tc directly. This approach can also be used for site-specific labeling with other probes available for thiol-chemistry, and is applicable also to other protein scaffolds. In paper II we investigated the impact of size and affinity of radiolabeled Affibody molecules on tumor targeting and image contrast. Two HER2-targeting Affibody molecules, a two-helix (~5 kDa) and a three-helix (~7 kDa) counterpart, were synthetically produced, labeled with 111 In via chelation by DOTA and directly compared in terms of biodistribution and targeting properties. Results showed that the smaller variant can provide higher contrast images, at the cost of lower tumor uptake, in high-expressing HER2-tumors. However, neither the tumor uptake nor the contrast of the two-helix variant is sufficient to compete with the three-helix molecule in tumors with low expression of HER2. In paper III and IV we were aiming to find methods to improve the labeling of Affibody molecules with 18 F for PET imaging. Current methods are either complex, time-consuming or generate heavily lipophilic conjugates. This results in low yields of radiolabeled tracer, low specific activity left for imaging, undesirable biodistribution or a combination thereof. In paper III we demonstrate a swift and efficient 2-step, 1-pot method for labeling HER2-binding Affibody molecules by the formation of aluminum 18 F-fluoride (Al 18 F) and its chelation by NOTA, all in 30 min. The results show that the 18 F-NOTA-approach is a very promising method of labeling Affibody molecules with 18 F and further investigation of this scheme is highly motivated. In the last paper we pursued the possibility of decreasing the high kidney retention that is common among small radiotracers with residual-izing radiometabolites. In this work 18 F-4-fluorobenzaldehyde (FBA) was conjugated to a synthetic HER2-targeting Affibody molecule via oxime ligation. However, to avoid elevated liver retention, as seen in previous studies with this kind of label, a hydrophilic triglutamyl spacer between the aminooxy moiety and the N-terminal was introduced. A comparison of the two constructs (with and without the triglutamyl spacer) showed a clear reduction of retention in both kidney and liver in NMRI mice at 2 h p.i. when the spacer was included. In the light of these promising results, further studies including tumor-bearing mice, are in preparation. / <p>QC 20130203</p>
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Design and Evaluation of Radiolabeled Affibody Tracers for Imaging of HER2-expressing TumorsWållberg, Helena January 2011 (has links)
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
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Development of ADAPT-based tracers for radionuclide molecular imaging of cancerGarousi, Javad January 2017 (has links)
ABD-Derived Affinity Proteins (ADAPTs) is a novel class of small engineered scaffold proteins based on albumin-binding domain (ABD) of streptococcal protein G. High affinity ADAPT binders against various therapeutic targets can be selected. In this thesis, we report a development of ADAPT-based radionuclide imaging agents providing high sensitivity and specificity of molecular imaging of HER2 expression in disseminated cancers. We investigated the feasibility of the use of ADAPTs as imaging agents and influence of molecular design and radiolabeling chemistry on in vivo targeting and biodistribution properties of the tracers. In Paper I we demonstrated the feasibility of the use of anti-HER2 ADAPT6 molecule as a high contrast imaging agent; In Paper II we evaluated the influence of composition of histidine-containing tag on in vivo biodistribution of ADAPT-based tracers labeled with 99mTc using 99mTc(CO)3 binding to histidine-containing tags and 111In using DOTA chelator at N-terminus; In Paper III we evaluated the influence of different aspects of N-terminus leading sequence on targeting including effect of sequence size on clearance rate and effect of the composition of the sequence on biodistribution profile; In Paper IV, we evaluated the influence of residualizing properties and positioning of the label on biodistribution and targeting; and In Paper V, we compared tumor-targeting properties of the ADAPT6 labeled at C-terminus with 99mTc using N3S chelator and 111In using DOTA chelator. In conclusion, ADAPTs constitute a very promising class of targeting probes for molecular imaging providing high contrast. Molecular design of the ADAPT proteins and chelators/linkers for labeling has an appreciable effect on their imaging properties.
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