| 1. |
- Ahlgren, Sara, et al.
(författare)
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Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules
- 2008
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 19:1, s. 235-243
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in vivo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for 111 In-labeling of anti-HER2 Affibody molecules His 6-Z HER2:342-Cys and Z HER2:2395-Cys has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for His 6-Z HER2:342-Cys and 27 pM for Z HER2:2395-Cys, comparable with 22 pM for the parental Z HER2:342. MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with 111 In to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non-His-tagged variant 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys demonstrated appreciably lower liver uptake than its His-tag-containing counterpart. In mice bearing HER2-expressing LS174T xenografts, 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 +/- 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 +/- 8. Xenografts were clearly visualized already 1 h p.i.
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| 2. |
- Ahlgren, Sara, et al.
(författare)
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Targeting of HER2-expressing tumors with a site-specifically 99mTc-labeled recombinant affibody molecule, ZHER2:2395, with C-terminally engineered cysteine
- 2009
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Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 50:5, s. 781-789
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Tidskriftsartikel (refereegranskat)abstract
- The detection of human epidermal growth factor receptor type 2 (HER2) expression in malignant tumors provides important information influencing patient management. Radionuclide in vivo imaging of HER2 may permit the detection of HER2 in both primary tumors and metastases by a single noninvasive procedure. Small (7 kDa) high-affinity anti-HER2 Affibody molecules may be suitable tracers for SPECT visualization of HER2-expressing tumors. The use of generator-produced (99m)Tc as a label would facilitate the prompt translation of anti-HER2 Affibody molecules into use in clinics. METHODS: A C-terminal cysteine was introduced into the Affibody molecule Z(HER2:342) to enable site-specific labeling with (99m)Tc. Two recombinant variants, His(6)-Z(HER2:342)-Cys (dissociation constant [K(D)], 29 pM) and Z(HER2:2395)-Cys, lacking a His tag (K(D), 27 pM), were labeled with (99m)Tc in yields exceeding 90%. The binding specificity and the cellular processing of Affibody molecules were studied in vitro. Biodistribution and gamma-camera imaging studies were performed in mice bearing HER2-expressing xenografts. RESULTS: (99m)Tc-His(6)-Z(HER2:342)-Cys was capable of targeting HER2-expressing SKOV-3 xenografts in SCID mice, but the liver radioactivity uptake was high. A series of comparative biodistribution experiments indicated that the presence of the His tag caused elevated accumulation in the liver. (99m)Tc-Z(HER2:2395)-Cys, not containing a His tag, showed low uptake in the liver and high and specific uptake in HER2-expressing xenografts. Four hours after injection, the radioactivity uptake values (percentage of injected activity per gram of tissue [%IA/g]) were 6.9 +/- 2.5 (mean +/- SD) %IA/g in LS174T xenografts (moderate level of HER2 expression) and 15 +/- 3 %IA/g in SKOV-3 xenografts (high level of HER2 expression). The corresponding tumor-to-blood ratios were 88 +/- 24 and 121 +/- 24, respectively. Both LS174T and SKOV-3 xenografts were clearly visualized with a clinical gamma-camera 1 h after injection of (99m)Tc-Z(HER2:2395)-Cys. CONCLUSION: The Affibody molecule (99m)Tc-Z(HER2:2395)-Cys is a promising tracer for SPECT visualization of HER2-expressing tumors.
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| 3. |
- Ekblad, Torun, et al.
(författare)
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Development and preclinical characterisation of 99mTc-labelled Affibody molecules with reduced renal uptake
- 2008
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Ingår i: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer Science and Business Media LLC. - 1619-7070 .- 1619-7089. ; 35:12, s. 2245-2255
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Tidskriftsartikel (refereegranskat)abstract
- Purpose Affibody molecules are low molecular weight proteins (7 kDa), which can be selected to bind to tumour-associated target proteins with subnanomolar affinity. Because of rapid tumour localisation and clearance from nonspecific compartments, Affibody molecules are promising tracers for molecular imaging. Earlier, 99mTc-labelled Affibody molecules demonstrated specific targeting of tumour xenografts. However, the biodistribution was suboptimal either because of hepatobiliary excretion or high renal uptake of the radioactivity. The goal of this study was to optimise the biodistribution of Affibody molecules by chelator engineering. Materials and methods Anti-HER2 ZHER2:342 Affibody molecules, carrying the mercaptoacetyl-glutamyl-seryl-glutamyl (maESE), mercaptoacetyl-glutamyl-glutamyl-seryl (maEES) and mercaptoacetyl-seryl-glutamyl-glutamyl (maSEE) chelators, were prepared by peptide synthesis and labelled with 99mTc. The tumour-targeting capacity of these conjugates was compared with each other and with the best previously available conjugate, 99mTc-maEEE-ZHER2:342, in nude mice bearing SKOV-3 xenografts. The tumour-targeting capacity of the most promising conjugate, 99mTc-maESE-ZHER2:342, was compared with radioiodinated ZHER2:342. Results All novel conjugates demonstrated successful tumour targeting and a low degree of hepatobiliary excretion. The renal uptakes of serine-containing conjugates, 33 ± 5, 68 ± 21 and 71 ± 10%IA/g, for99mTc-maESE-ZHER2:342, 99mTc-maEES-ZHER2:342 and 99mTc-maSEE-ZHER2:342, respectively, were significantly reduced in comparison with 99mTc-maEEE-ZHER2:342 (102 ± 13%IA/g). For 99mTc-maESE-ZHER2:342, a tumour uptake of 9.6 ± 1.8%IA/g and a tumour-to-blood ratio of 58 ± 6 were reached at 4 h p.i. Conclusions A combination of serine and glutamic acid residues in the chelator sequence confers increased renal excretion and relatively low renal uptake of 99mTc-labelled Affibody molecules. In combination with preserved targeting capacity, this improved imaging of targets in abdominal area.
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| 4. |
- Ekblad, Torun, et al.
(författare)
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Positioning of Tc-99m-chelators influences radiolabeling, stability and biodistribution of Affibody molecules
- 2009
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Ingår i: Bioorganic & Medicinal Chemistry Letters. - : Elsevier BV. - 0960-894X .- 1464-3405. ; 19:14, s. 3912-3914
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules represent a novel class of affinity proteins with a high potential as tracers for radio-nuclide molecular imaging. In this comparative structure-property study, a series of Affibody molecules with the Tc-99m-chelators maGGG, maSSS, or maESE attached to the e-amine of the internally positioned K49 was prepared by peptide synthesis, for comparison to molecules with similar chelators positioned at the N-terminus. The conjugates were labeled with Tc-99m and evaluated in vitro and in vivo. It was found that both composition and position of the chelating moiety influence the label stability, biodistribution and targeting properties of HER2-binding Affibody molecules.
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| 5. |
- Engfeldt, Torun, et al.
(författare)
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99mTc-chelator engineering to improve tumour targeting properties of a HER2-specific Affibody molecule
- 2007
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Ingår i: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer Science and Business Media LLC. - 1619-7070 .- 1619-7089. ; 34:11, s. 1843-1853
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: Monitoring HER2 expression is crucial for selection of breast cancer patients amenable to HER2-targeting therapy. The Affibody molecule Z(HER2:342) binds to HER2 with picomolar affinity and enables specific imaging of HER2 expression. Previously, Z(HER2:342) with the additional N-terminal mercaptoacetyl-glycyl-glycyl-glycyl (maGGG) sequence was labelled with (99m)Tc and demonstrated specific targeting of HER2-expressing xenografts. However, hepatobiliary excretion caused high radioactivity accumulation in the abdomen. We investigated whether the biodistribution of Z(HER2:342) can be improved by substituting glycyl residues in the chelating sequence with more hydrophilic seryl residues. METHODS: The Affibody molecule Z(HER2:342), carrying the chelators mercaptoacetyl-glycyl-seryl-glycyl (maGSG), mercaptoacetyl-glycyl-D: -seryl-glycyl [maG(D-S)G] and mercaptoacetyl-seryl-seryl-seryl (maSSS), were prepared by peptide synthesis and labelled with (99m)Tc. The differences in the excretion pathways were evaluated in normal mice. The tumour targeting capacity of (99m)Tc-maSSS-Z(HER2:342) was studied in nude mice bearing SKOV-3 xenografts and compared with the capacity of radioiodinated Z(HER2:342). RESULTS: A shift towards renal excretion was obtained when glycine was substituted with serine in the chelating sequence. The radioactivity in the gastrointestinal tract was reduced threefold for the maSSS conjugate in comparison with the maGGG conjugate 4 h post injection (p.i.). The tumour uptake of (99m)Tc-maSSS-Z(HER2:342) was 11.5 +/- 0.5% IA/g 4 h p.i., and the tumour-to-blood ratio was 76. The pharmacokinetics and uptake characteristics of technetium-labelled Z(HER2:342) were better than those of radioiodinated Z(HER2:342). CONCLUSION: The introduction of serine residues in the chelator results in better tumour imaging properties of the Affibody molecule Z(HER2:342) compared with glycyl-containing chelators and is favourable for imaging of tumours and metastases in the abdominal area.
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| 6. |
- Orlova, Anna, et al.
(författare)
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Synthetic affibody molecules : a novel class of affinity ligands for molecular imaging of HER2-expressing malignant tumors
- 2007
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Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 67:5, s. 2178-2186
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Tidskriftsartikel (refereegranskat)abstract
- The Affibody molecule Z(HER2:342-pep2), site-specifically and homogeneously conjugated with a 1,4,7,10-tetra-azacylododecane-N,N',N'',N'''-tetraacetic acid (DOTA) chelator, was produced in a single chemical process by peptide synthesis. DOTA-Z(HER2:342-pep2) folds spontaneously and binds HER2 with 65 pmol/L affinity. Efficient radiolabeling with >95% incorporation of (111)In was achieved within 30 min at low (room temperature) and high temperatures (up to 90 degrees C). Tumor uptake of (111)In-DOTA-Z(HER2:342-pep2) was specific for HER2-positive xenografts. A high tumor uptake of 23% injected activity per gram tissue, a tumor-to-blood ratio of >7.5, and high-contrast gamma camera images were obtained already 1 h after injection. Pretreatment with Herceptin did not interfere with tumor targeting, whereas degradation of HER2 using the heat shock protein 90 inhibitor 17-allylamino-geldanamycin before administration of (111)In-DOTA-Z(HER2:342-pep2) obliterated the tumor image. The present results show that radiolabeled synthetic DOTA-Z(HER2:342-pep2) has the potential to become a clinically useful radiopharmaceutical for in vivo molecular imaging of HER2-expressing carcinomas.
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| 7. |
- Orlova, Anna, et al.
(författare)
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Update : Affibody molecules for molecular imaging and therapy for cancer
- 2007
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Ingår i: Cancer Biotherapy and Radiopharmaceuticals. - 1084-9785 .- 1557-8852. ; 22:5, s. 573-584
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are scaffold proteins, having a common frame of amino acids determining the overall fold or tertiary structure, but with each member characterized by a unique amino acid composition in an exposed binding surface determining binding specificity and affinity for a certain target. Affibody molecules represent a new class of affinity proteins based on a 58-amino acid residue protein domain, derived from one of the IgG binding domains of staphylococcal protein A. They combine small size ( approximately 6.5 kDa) with high affinity and specificity. Affibody molecules with nanomolar affinities were selected from an initial library (3 x 10(9) members) and, after affinity maturation, picomolar binders were obtained. The small size and simple structure of affibody molecules allow their production by chemical synthesis with homogeneous site-specific incorporation of moieties for further labeling using a wide range of labeling chemistries. The robustness and the refolding properties of affibody molecules make them amenable to labeling conditions that denature most proteins, including incubation at pH 11 at 60 degrees C for up to 60 minutes. Affibody molecules meet the requirements which are key for successful clinical use as imaging agents: high-affinity binding to the chosen target; short plasma half-life time; rapid renal clearance for nonbound drug substance and, high, continuously increasing tumor-to-organ ratios, resulting in high-contrast in vivo images shortly after injection of the diagnostic agent.
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| 8. |
- Renberg, Björn, et al.
(författare)
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Affibody molecules in protein capture microarrays : Evaluation of multidomain ligands and different detection formats
- 2007
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:1, s. 171-179
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Tidskriftsartikel (refereegranskat)abstract
- The importance of the ligand presentation format for the production of protein capture microarrays was evaluated using different Affibody molecules, produced either as single 6 kDa monomers or genetically linked head-to-tail multimers containing up to four domains. The performances in terms of selectivity and sensitivity of the monomeric and the multidomain Affibody molecules were compared by immobilization of the ligands on microarray slides, followed by incubation with fluorescent-labeled target protein. An increase in signal intensities for the multimers was demonstrated, with the most pronounced difference observed between monomers and dimers. A protein microarray containing six different dimeric Affibody ligands with specificity for IgA, IgE, IgG, TNF-alpha, insulin, or Taq DNA polymerase was characterized for direct detection of fluorescent-labeled analytes. No cross-reactivity was observed and the limits of detection were 600 fM for IgA, 20 pM for IgE, 70 fM for IgG, 20 pM for TNF-alpha, 60 pM for insulin, and 10 pM for Taq DNA polymerase. Also, different sandwich formats for detection of unlabeled protein were evaluated and used for selective detection of IgA or TNF-alpha in human serum or plasma samples, respectively. Finally, the presence of IgA was determined using detection of directly Cy5-labeled normal or IgA-deficient serum samples.
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| 9. |
- Tolmachev, Vladimir, et al.
(författare)
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Affibody molecules : potential for in vivo imaging of molecular targets for cancer therapy
- 2007
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Ingår i: Expert Opinion on Biological Therapy. - : Informa Healthcare. - 1471-2598 .- 1744-7682. ; 7:4, s. 555-568
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Forskningsöversikt (refereegranskat)abstract
- Targeting radionuclide imaging of tumor-associated antigens may help to select patients who will benefit from a particular biological therapy. Affibody molecules are a novel class of small (approximately 7 kDa) phage display-selected affinity proteins, based on the B-domain scaffold of staphylococcal protein A. A large library (3 x 10(9) variants) has enabled selection of high-affinity (up to 22 pM) binders for a variety of tumor-associated antigens. The small size of Affibody molecules provides rapid tumor localization and fast clearance from nonspecific compartments. Preclinical studies have demonstrated the potential of Affibody molecules for specific and high-contrast radionuclide imaging of HER2 in vivo, and pilot clinical data using indium-111 and gallium-68 labeled anti-HER2 Affibody tracer have confirmed its utility for radionuclide imaging in cancer patients.
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| 10. |
- Tran, Thuy, et al.
(författare)
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99mTc-maEEE-ZHER2:342, an Affibody Molecule-Based Tracer for the Detection of HER2 Expression in Malignant Tumors
- 2007
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 18:6, s. 1956-1964
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Tidskriftsartikel (refereegranskat)abstract
- Detection of HER2-overexpression in tumors and metastases is important for the selection of patients who will benefit from trastuzumab treatment. Earlier investigations showed successful imaging of HER2-positive tumors in patients using indium- or gallium-labeled Affibody molecules. The goal of this study was to evaluate the use of 99mTc-labeled Affibody molecules for the detection of HER2 expression. The Affibody molecule ZHER2:342 with the chelator sequences mercaptoacetyl-Gly-Glu-Gly (maGEG) and mercaptoacetyl-Glu-Glu-Glu (maEEE) was synthesized by peptide synthesis and labeled with technetium-99m. Binding specificity, cellular retention, and in vitro stability were investigated. The biodistribution of 99mTc-maGEG-ZHER2:342 and 99mTc-maEEE-ZHER2:342 was compared with 99mTc-maGGG-ZHER2:342 in normal mice, and the tumor targeting properties of 99mTc-maEEE-ZHER2:342 were determined in SKOV-3 xenografted nude mice. The results showed that the Affibody molecules were efficiently labeled with technetium-99m. The labeled conjugates were highly stable in vitro with preserved HER2-binding capacity. The use of glutamic acid in the chelator sequences for 99mTc-labeling of ZHER2:342 reduced the hepatobiliary excretion 3-fold with a single Gly-to-Glu substitution and 10-fold with three Gly-to-Glu substitutions. 99mTc-maEEE-ZHER2:342 showed a receptor-specific tumor uptake of 7.9 ± 1.0 %IA/g and a tumor-to-blood ratio of 38 at 4 h pi. Gamma-camera imaging with 99mTc-maEEE-ZHER2:342 could detect HER2-expressing tumors in xenografts already at 1 h pi. It was concluded that peptide synthesis for the coupling of chelator sequences to Affibody molecules for 99mTc labeling is an efficient way to modify the in vivo kinetics. Increased hydrophilicity, combined with improved stability of the mercaptoacetyl-triglutamyl chelator, resulted in favorable biodistribution, making 99mTc-maEEE-ZHER2:342 a promising tracer for clinical imaging of HER2 overexpression in tumors.
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