| 1. |
- Ahlgren, Sara, et al.
(författare)
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Targeting of HER2-Expressing Tumors Using 111In-ABY-025, a Second-Generation Affibody Molecule with a Fundamentally Reengineered Scaffold
- 2010
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Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 51:7, s. 1131-1138
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Tidskriftsartikel (refereegranskat)abstract
- Overexpression of HER2 in breast carcinomas predicts response to trastuzumab therapy. Affibody molecules based on a non-immunoglobulin scaffold have demon-strated high potential for in vivo molecular imaging of HER2-expressing tumors. Re-engineering of the molecular scaffold has led to a second generation of optimized Affibody molecules, having a surface distinctly different from the parental protein domain from staphylococcal protein A. The new tracer showed further increased melting point, stability and overall hydrophilicity compared to the parental molecule, and was shown to be more amenable for chemical peptide synthesis. The goal of this study was to assess potential effects of this extensive re-engineering on HER2 targeting, using ABY-025, a DOTA conjugated variant of the novel tracer. Methods: 111In-ABY-025 was compared with previously evaluated parent HER2-binding Affibody tracers in vitro and in vivo. The in vivo behavior was further evaluated in mice bearing SKOV-3 xenografts, in rats and in cynomolgus macaques. Results: 111In-ABY-025 bound specifically to HER2 in vitro and in vivo. Direct comparison with the previous generation of HER2-binding tracers showed that ABY-025 retained excellent targeting properties. Rapid blood clearance was shown in mice, rats and macaques. A highly specific tumor uptake of 16.7 ± 2.5 %IA/g was seen at 4 h after injection. The tumor-to-blood ratio was 6.3 at 0.5 h, 88 at 4 h, and increased up to 3 days after injection. Gamma camera imaging of tumors was already possible 0.5 h after injection. Furthermore, repeated i.v. administration of ABY-025 did not induce antibody formation in rats. Conclusions: The biodistribution of 111In-ABY-025 was in remarkably good agreement with the parent tracers, despite profound re-engineering of the non-binding surface. The molecule displayed rapid blood clearance in all species investigated and excellent targeting capacity in tumor bearing mice, leading to high tumor-to-organ-ratios and high contrast imaging shortly after injection.
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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. |
- Altai, Mohamed, et al.
(författare)
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188Re-ZHER2:V2, a promising affibody-based targeting agent against HER2-expressing tumors : preclinical assessment
- 2014
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Ingår i: Journal of nuclear medicine : official publication, Society of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 55:11, s. 8-1842
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Tidskriftsartikel (refereegranskat)abstract
- UNLABELLED: Affibody molecules are small (7 kDa) nonimmunoglobulin scaffold proteins with favorable tumor-targeting properties. Studies concerning the influence of chelators on biodistribution of (99m)Tc-labeled Affibody molecules demonstrated that the variant with a C-terminal glycyl-glycyl-glycyl-cysteine peptide-based chelator (designated ZHER2:V2) has the best biodistribution profile in vivo and the lowest renal retention of radioactivity. The aim of this study was to evaluate (188)Re-ZHER2:V2 as a potential candidate for radionuclide therapy of human epidermal growth factor receptor type 2 (HER2)-expressing tumors.METHODS: ZHER2:V2 was labeled with (188)Re using a gluconate-containing kit. Targeting of HER2-overexpressing SKOV-3 ovarian carcinoma xenografts in nude mice was studied for a dosimetry assessment.RESULTS: Binding of (188)Re-ZHER2:V2 to living SKOV-3 cells was demonstrated to be specific, with an affinity of 6.4 ± 0.4 pM. The biodistribution study showed a rapid blood clearance (1.4 ± 0.1 percentage injected activity per gram [%ID/g] at 1 h after injection). The tumor uptake was 14 ± 2, 12 ± 2, 5 ± 2, and 1.8 ± 0.5 %IA/g at 1, 4, 24, and 48 h after injection, respectively. The in vivo targeting of HER2-expressing xenografts was specific. Already at 4 h after injection, tumor uptake exceeded kidney uptake (2.1 ± 0.2 %IA/g). Scintillation-camera imaging showed that tumor xenografts were the only sites with prominent accumulation of radioactivity at 4 h after injection. Based on the biokinetics, a dosimetry evaluation for humans suggests that (188)Re-ZHER2:V2 would provide an absorbed dose to tumor of 79 Gy without exceeding absorbed doses of 23 Gy to kidneys and 2 Gy to bone marrow. This indicates that future human radiotherapy studies may be feasible.CONCLUSION: (188)Re-ZHER2:V2 can deliver high absorbed doses to tumors without exceeding kidney and bone marrow toxicity limits.
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| 4. |
- Altai, Mohamed, et al.
(författare)
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Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of Tc-99m-labeled recombinant Affibody molecules
- 2012
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Ingår i: Amino Acids. - : Springer Science and Business Media LLC. - 0939-4451 .- 1438-2199. ; 42:5, s. 1975-1985
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide Tc-99m. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of Tc-99m-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied Tc-99m-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of Tc-99m. The biodistribution of all Tc-99m-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of Tc-99m was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.
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| 5. |
- Altai, Mohamed, et al.
(författare)
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Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with (188)Re.
- 2014
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Ingår i: European Journal of Medicinal Chemistry. - : Elsevier BV. - 0223-5234 .- 1768-3254. ; 87, s. 519-28
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of (188)Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all (188)Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The (188)Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of (188)Re-ZHER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental (188)Re-ZHER2:2395 (172 ± 32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of (188)Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.
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| 6. |
- Altai, Mohamed, et al.
(författare)
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Selection of an optimal cysteine-containing peptide-based chelator for labeling of Affibody molecules with 188-Re
- 2013
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Ingår i: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer Science and Business Media LLC. - 1619-7070 .- 1619-7089. ; 40:Suppl. 2, s. S219-S220
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of 188Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all 188Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The 188Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of 188Re-ZHER2:V2 (3.1±0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental 188Re-ZHER2:2395 (172±32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of 188Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.
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| 7. |
- Ekerljung, Lina, 1980-, et al.
(författare)
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Generation and Evaluation of Bispecific Affibody Molecules for Simultaneous Targeting of EGFR and HER2
- 2012
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 23:9, s. 1802-1811
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Tidskriftsartikel (refereegranskat)abstract
- Co-expression of several ErbB receptors has been found in many cancers and has been linked with increased aggressiveness of tumors and a worse patient prognosis. This makes the simultaneous targeting of two surface receptors by using bispecific constructs an increasingly appreciated strategy. Here we have generated six such bispecific targeting proteins, which each comprising two monomeric affibody molecules with specific binding to either of the two human epidermal growth factor receptors, EGFR and HER2, respectively. The bispecific constructs were designed with (i) alternative positioning (N- or C-terminal) of the different affibody molecules, (ii) two alternative peptide linkers (Gly4Ser)3 or (Ser4Gly)3, and (iii) affibody molecules with different affinity (nanomolar or picomolar) for HER2. Using both Biacore technology and cell binding assays it was demonstrated that all six constructs could bind simultaneously to both their target proteins. N-terminal positioning of the monomeric affibody molecules was favorable to promote the binding to respective target. Interestingly, bispecific constructs containing the novel (Ser4Gly)3 linker displayed a higher affinity in cell binding, as compared to constructs containing the more conventional linker, (Gly4Ser)3. It could further be concluded that bispecific constructs (but not the monomeric affibody molecules) induced dimerization and phosphorylation of EGFR in SKBR3 cells, which express fairly high levels of both receptors. It was also investigated whether the bispecific binding would influence cell growth or sensitize cells for ionizing radiation, but no such effects were observed.
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| 8. |
- Jonsson, Andreas, et al.
(författare)
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Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro
- 2009
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Ingår i: Biotechnology and applied biochemistry. - : Wiley. - 0885-4513 .- 1470-8744. ; 54, s. 93-103
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For Z(TNF alpha:185), subnanomolar affinity (K-D = 0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the Z(TNF-alpha:185) affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dinners with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.
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| 9. |
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| 10. |
- Lindberg, Hanna, et al.
(författare)
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Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for Tc-99m-labelling at the C terminus
- 2012
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Ingår i: Tumor Biology. - : Springer. - 1010-4283 .- 1423-0380. ; 33:3, s. 641-651
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are a class of small (ca.7 kDa) robust scaffold proteins with high potential as tracers for radionuclide molecular imaging in vivo. Incorporation of a cysteine-containing peptide-based chelator at the C terminus provides an opportunity for stable labelling with the radionuclide Tc-99m. The use of a GGGC chelator at the C terminus has provided the lowest renal radioactivity retention of the previously investigated peptide-based chelators. Previously, it has also been demonstrated that replacement of the His(6)-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of affibody molecules by immobilized metal ion affinity chromatography (IMAC) and provides low hepatic accumulation of radioactivity of conjugates site-specifically labelled at the C terminus using several different nuclides. We hypothesized that the combination of a HEHEHE-tag at the N terminus and a GGGC chelator at the C terminus of an affibody molecule would be a favourable format permitting IMAC purification and providing low uptake in excretory organs. To investigate this hypothesis, a (HE)(3)-Z(HER2:342)-GGGC affibody molecule was generated. It could be efficiently purified by IMAC and stably labelled with Tc-99m. Tc-99m-(HE)(3)-Z(HER2:342)-GGGC preserved specific binding to HER2-expressing cells. In NMRI mice, hepatic uptake of Tc-99m-(HE)(3)-Z(HER2:342)-GGGC was lower than the uptake of the control affibody molecules, Tc-99m-Z(HER2:2395)-VDC and Tc-99m-Z(HER2:342)-GGGC. At 1 and 4 h after injection, the renal uptake of Tc-99m-(HE)(3)-Z(HER2:342)-GGGC was 2-3-fold lower than uptake of Tc-99m-Z(HER2:2395)-VDC, but it was substantially higher than uptake of Tc-99m-Z(HER2:342)-GGGC. Further investigation indicated that a fraction of Tc-99m was chelated by the HEHEHE-tag which caused a higher accumulation of radioactivity in the kidneys. Thus, a combination of a HEHEHE-tag and the GGGC chelator in targeting scaffold proteins was found to be undesirable in the case of Tc-99m labelling due to a partial loss of site-specificity of nuclide chelation.
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