On the Design of Affibody Molecules for Radiolabeling and In Vivo Molecular Imaging
Abstract: 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-?uoride (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.
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