| 1. |
- Tolmachev, Vladimir, et al.
(författare)
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Affibody molecules for epidermal growth factor receptor targeting in vivo : aspects of dimerization and labeling chemistry
- 2009
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Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 50:2, s. 274-283
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Tidskriftsartikel (refereegranskat)abstract
- Noninvasive detection of epidermal growth factor receptor (EGFR) expression in malignant tumors by radionuclide molecular imaging may provide diagnostic information influencing patient management. The aim of this study was to evaluate a novel EGFR-targeting protein, the ZEGFR:1907 Affibody molecule, for radionuclide imaging of EGFR expression, to determine a suitable tracer format (dimer or monomer) and optimal label. METHODS: An EGFR-specific Affibody molecule, ZEGFR:1907, and its dimeric form, (ZEGFR:1907)2, were labeled with 111In using benzyl-diethylenetriaminepentaacetic acid and with 125I using p-iodobenzoate. Affinity and cellular retention of conjugates were evaluated in vitro. Biodistribution of radiolabeled Affibody molecules was compared in mice bearing EGFR-expressing A431 xenografts. Specificity of EGFR targeting was confirmed by comparison with biodistribution of non-EGFR-specific counterparts. RESULTS: Head-to-tail dimerization of the Affibody molecule improved the dissociation rate. In vitro, dimeric forms demonstrated superior cellular retention of radioactivity. For both molecular set-ups, retention was better for the 111In-labeled tracer than for the radioiodinated counterpart. In vivo, all conjugates accumulated specifically in xenografts and in EGFR-expressing tissues. The retention of radioactivity in tumors was better in vivo for dimeric forms; however, the absolute uptake values were higher for monomeric tracers. The best tracer, 111In-labeled ZEGFR:1907, provided a tumor-to-blood ratio of 100 (24 h after injection). CONCLUSION: The radiometal-labeled monomeric Affibody molecule ZEGFR:1907 has a potential for radionuclide molecular imaging of EGFR expression in malignant tumors.
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| 2. |
- Tran, Thuy A., 1980-, et al.
(författare)
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Design, synthesis and biological evaluation of a multifunctional HER2-specific Affibody molecule for molecular imaging
- 2009
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Ingår i: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer Science and Business Media LLC. - 1619-7070 .- 1619-7089. ; 36:11, s. 1864-1873
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: The purpose of this study was to design and evaluate a novel platform for labelling of Affibody molecules, enabling for both recombinant and synthetic production and for site-specific labelling with 99mTc or trivalent radiometals. Methods: The HER2-specific Affibody molecule PEP05352 was made by peptide synthesis. The chelator sequence SECG (serine-glutamic acid-cysteine-glycine) was anchored on the C-terminal to allow 99mTc-labelling. The cysteine can alternatively serve as a conjugation site of the chelator DOTA for indium-labelling. The resulting 99mTc- and 111In-labelled Affibody molecules were evaluated both in vitro and in vivo. Results: Both conjugates retained their capacity to bind to HER2 receptors in vitro and in vivo. The tumour-to-blood ratio in LS174T xenografts was 30 at 4 h p.i. for both conjugates. Biodistribution data showed that 99mTc-labelled Affibody molecule had 4-fold lower kidney accumulation compared with 111In-labelled Affibody molecule while the accumulation in other organs was similar. Gamma-camera imaging of the conjugates could clearly visualise the tumours 4 h after injection. Conclusions: Incorporation of C-terminal SECG sequence in Affibody molecules provides a general multifunctional platform for site-specific labelling with different nuclides (technetium, indium, gallium, cobalt, or yttrium) and for a flexible production (chemical synthesis or recombinant).
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| 3. |
- Rosik, Daniel, et al.
(författare)
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Direct comparison of In-111-labelled two-helix and three-helix Affibody molecules for in vivo molecular imaging
- 2012
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Ingår i: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer Science and Business Media LLC. - 1619-7070 .- 1619-7089. ; 39:4, s. 693-702
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Tidskriftsartikel (refereegranskat)abstract
- Radiolabelled Affibody molecules have demonstrated a potential for visualization of tumour-associated molecular targets. Affibody molecules (7 kDa) are composed of three alpha-helices. Recently, a smaller two-helix variant of Affibody molecules (5.1 kDa) was developed. The aim of this study was to compare two- and three-helix HER2-targeting Affibody molecules directly in vivo. The three-helix Affibody molecule ABY-002 and the two-helix Affibody molecule PEP09239 were labelled with In-111 at the N-termini via DOTA chelator. Tumour-targeting properties were directly compared at 1 and 4 h after injection in mice bearing SKOV-3 xenografts with high HER2 expression and LS174T xenografts with low HER2 expression. The dissociation constants (K (D)) for HER2 binding were 78 pM for the three-helix Affibody molecule and 2.1 nM for the two-helix Affibody molecule. In-111-PEP09239 cleared more rapidly from the blood. In xenografts with high HER2 expression, the uptake of In-111-ABY-002 was significantly higher than that of In-111-PEP09239. The tumour-to-blood ratio was higher for In-111-PEP09239 at 4 h after injection, while there was no significant difference in other tumour-to-organ ratios. The tumour uptake of In-111-ABY-002 was eightfold higher than that of In-111-PEP09239 in xenografts with low expression. Tumour-to-blood ratios were equal in this case, but other tumour-to-organ ratios were appreciably higher for the three-helix variant. For tumours with high HER2 expression, two-helix HER2-targeting Affibody molecules can provide higher tumour-to-blood ratio at the cost of lower tumour uptake. In the case of low expression, both tumour uptake and tumour-to-organ ratios are appreciably higher for three-helix than for two-helix HER2-targeting Affibody molecules.
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| 4. |
- Rosik, Daniel, et al.
(författare)
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Incorporation of a Triglutamyl Spacer Improves the Biodistribution of Synthetic Affibody Molecules Radiofluorinated at the N-Terminus via Oxime Formation with F-18-4-Fluorobenzaldehyde
- 2014
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 25:1, s. 82-92
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are a class of affinity agents for molecular imaging based on a non-immunoglobulin protein scaffold. Previous studies have demonstrated high contrast for in vivo imaging of cancer-associated molecular abnormalities using Affibody molecules. Using the radionuclide F-18 for labeling and PET as the imaging modality, the sensitivity of molecular imaging using Affibody molecules can be further increased. The use of oxime formation between an aminooxy-functionalized peptide and F-18-fluorobenzaldehyde (F-18-FBA) is a promising way of radiolabeling of targeting peptides. However, previous studies demonstrated that application of this method to Affibody molecules is associated with high liver uptake. We hypothesized that incorporation of a triglutamyl spacer between the aminooxy moiety and the N-terminus of a synthetic Affibody molecule would decrease the hepatic uptake of the F-18-N-(4-fluorobenzylidine)oxime) (F-18-FBO)-labeled tracer. To verify this, we have produced two variants of the HER2-targeting Z(HER2:342) Affibody molecule by peptide synthesis: OA-PEP4313, where aminooxyacetic acid was conjugated directly to the N-terminal alanine, and OA-E-3-PEP4313, where a triglutamyl spacer was introduced between the aminooxy moiety and the N-terminus. We have found that the use of the spacer is associated with a minor decrease of affinity, from K-D = 49 pM to K-D = 180 pM. Radiolabeled F-18-FBO-E-3-PEP4313 demonstrated specific binding to HER2-expressing ovarian carcinoma SKOV-3 cells and slow internalization. Biodistribution studies in mice demonstrated that the use of a triglutamyl linker decreased uptake of radioactivity in liver 2.7-fold at 2 h after injection. Interestingly, radioactivity uptake in kidneys was also reduced (2.4-fold). Experiments in BALB/C nu/nu mice bearing SKOV-3 xenografts demonstrated HER2-specific uptake of F-18-FBO-E-3-PEP4313 in tumors. At 2 h pi, the tumor uptake (20 +/- 2% ID/g) exceeded uptake in liver 5-fold and uptake in kidneys 3.6-fold. The tumor-to-blood ratio was 21 +/- 3. The microPET/CT imaging experiment confirmed the biodistribution data. In conclusion, the use of a triglutamyl spacer is a convenient way to improve the biodistribution profile of Affibody molecules labeled at the N-terminus using F-18-FBA. It provides a tracer capable of producing high-contrast images of HER2-expressing tumors.
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