| 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. |
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| 3. |
- Altai, Mohamed, et al.
(författare)
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Influence of Nuclides and Chelators on Imaging Using Affibody Molecules : Comparative Evaluation of Recombinant Affibody Molecules Site-Specifically Labeled with Ga-68 and In-111 via Maleimido Derivatives of DOTA and NODAGA
- 2013
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 24:6, s. 1102-1109
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Tidskriftsartikel (refereegranskat)abstract
- Accurate detection of cancer-associated molecular abnormalities in tumors could make cancer treatment more of personalized. Affibody molecules enable high contrast imaging of tumor-associated protein expression shortly after injection. The use should increase sensitivity of HER2 imaging. The chemical nature of the generator-produced positron-emitting radionuclide Ga-68 of radionuclides and chelators influences the biodistribution of Affibody molecules, providing an opportunity to further increase the imaging contrast. The aim of the study was to compare maleimido derivatives of DOTA and NODAGA for site-specific labeling of a recombinant Z(HER2:2395) HER2-binding Affibody molecule with Ga-68. DOTA and NODAGA were site-specifically conjugated to the Z(HER2:2395) Affibody molecule having a C-terminal cysteine and labeled with Ga-68 and In-111. All labeled conjugates retained specificity to HER2 in vitro. Most of the cell-associated activity was membrane-bound with a minor difference in internalization rate. All variants demonstrated specific targeting of xenografts and a high tumor uptake. The xenografts were dearly visualized using all conjugates. The influence of chelator on the biodistribution and targeting properties was much less pronounced for Ga-68 than for In-111. The tumor uptake of Ga-68-NODAGA-Z(HER2:2395) and Ga-68-NODAGA-Z(HER2:2395) and tumor-to-blood ratios at 2 h p.i. did not differ significantly. However, the tumor-to-liver ratio was significantly higher for Ga-68-NODAGA- Z(HER2:2395) (8 +/- 2 vs 5.0 +/- 0.3) offering the advantage of better liver metastases visualization. In conclusion, influence of chelators on biodistribution of Affibody molecules depends on the radionuclides and reoptimization of labeling chemistry is required when a radionuclide label is changed.
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| 4. |
- Feldwisch, Joachim, et al.
(författare)
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Design of an optimized scaffold for affibody molecules.
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 398:2, s. 232-247
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are non-immunoglobulin-derived affinity proteins based on a three-helical bundle protein domain. Here, we describe the design process of an optimized Affibody molecule scaffold with improved properties and a surface distinctly different from that of the parental scaffold. The improvement was achieved by applying an iterative process of amino acid substitutions in the context of the human epidermal growth factor receptor 2 (HER2)-specific Affibody molecule Z(HER2:342). Replacements in the N-terminal region, loop 1, helix 2 and helix 3 were guided by extensive structural modeling using the available structures of the parent Z domain and Affibody molecules. The effect of several single substitutions was analyzed followed by combination of up to 11 different substitutions. The two amino acid substitutions N23T and S33K accounted for the most dramatic improvements, including increased thermal stability with elevated melting temperatures of up to +12 degrees C. The optimized scaffold contains 11 amino acid substitutions in the nonbinding surface and is characterized by improved thermal and chemical stability, as well as increased hydrophilicity, and enables generation of identical Affibody molecules both by chemical peptide synthesis and by recombinant bacterial expression. A HER2-specific Affibody tracer, [MMA-DOTA-Cys61]-Z(HER2:2891)-Cys (ABY-025), was produced by conjugating MMA-DOTA (maleimide-monoamide-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to the peptide produced either chemically or in Escherichia coli. ABY-025 showed high affinity and specificity for HER2 (equilibrium dissociation constant, K(D), of 76 pM) and detected HER2 in tissue sections of SKOV-3 xenograft and human breast tumors. The HER2-binding capacity was fully retained after three cycles of heating to 90 degrees C followed by cooling to room temperature. Furthermore, the binding surfaces of five Affibody molecules targeting other proteins (tumor necrosis factor alpha, insulin, Taq polymerase, epidermal growth factor receptor or platelet-derived growth factor receptor beta) were grafted onto the optimized scaffold, resulting in molecules with improved thermal stability and a more hydrophilic nonbinding surface.
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| 5. |
- Heskamp, Sandra, et al.
(författare)
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Imaging of Human Epidermal Growth Factor Receptor Type 2 Expression with (18)F-Labeled Affibody Molecule Z(HER2:2395) in a Mouse Model for Ovarian Cancer
- 2012
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Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 53:1, s. 146-153
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are small (7 kDa) proteins with subnanomolar targeting affinity. Previous SPECT studies in xenografts have shown that the Affibody molecule (111)In-DOTA-Z(HER2:2395) can discriminate between high and low human epidermal growth factor receptor type 2 (HER2)-expressing tumors, indicating that radiolabeled Affibody molecules have potential for patient selection for HER2-targeted therapy. Compared with SPECT, PET with positron-emitting radionuclides, such as (18)F, may improve imaging of HER2 expression because of higher sensitivity and improved quantification of PET. The aim of the present study was to determine whether the (18)F-labeled NOTA-conjugated Affibody molecule Z(HER2:2395) is a suitable agent for imaging of HER2 expression. The tumor-targeting properties of (18)F-labeled Z(HER2:2395) were compared with (111)In- and (68)Ga-labeled Z(HER2:2395) in mice with HER2-expressing SK-OV-3 xenografts. Methods: Z(HER2:2395) was conjugated with NOTA and radiolabeled with (18)F, (68)Ga, and (111)In. Radiolabeling with (18)F was based on the complexation of Al(18)F by NOTA. The 50% inhibitory concentration values for NOTA-Z(HER2:2395) labeled with (19)F, (69)Ga, and (115)In were determined in a competitive cell-binding assay using SK-OV-3 cells. Mice bearing subcutaneous SK-OV-3 xenografts were injected intravenously with radiolabeled NOTA-Z(HER2:2395). One and 4 h after injection, PET/CT or SPECT/CT images were acquired, and the biodistribution was determined by ex vivo measurement. Results: The 50% inhibitory concentration values for (19)F-, (69)Ga-, and (115)In-NOTA-Z(HER2:2395) were 5.0, 6.3, and 5.3 nM, respectively. One hour after injection, tumor uptake was 4.4 +/- 0.8 percentage injected dose per gram (% ID/g), 5.6 +/- 1.6 % ID/g, and 7.1 +/- 1.4 % ID/g for (18)F-, (68)Ga-, and (111)In-NOTA-Z(HER2:2395), respectively, and the respective tumor-to-blood ratios were 7.4 +/- 1.8, 8.0 +/- 1.3, and 4.8 +/- 1.3. Tumor uptake was specific, because uptake could be blocked efficiently by coinjection of an excess of unlabeled Z(HER2:2395). PET/CT and SPECT/CT images clearly visualized HER2-expressing SK-OV-3 xenografts. Conclusion: This study showed that (18)F-NOTA-Z(HER2:2395) is a promising new imaging agent for HER2 expression in tumors. Affibody molecules were successfully labeled with (18)F within 30 min, based on the complexation of Al(18)F by NOTA. Further research is needed to determine whether this technique can be used for patient selection for HER2-targeted therapy.
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| 6. |
- Honarvar, Hadis, et al.
(författare)
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Evaluation of backbone-cyclized HER2-binding 2-helix Affibody molecule for In Vivo molecular imaging
- 2013
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Ingår i: Nuclear Medicine and Biology. - : Elsevier BV. - 0969-8051 .- 1872-9614. ; 40:3, s. 378-386
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Affibody molecules, small scaffold proteins, have demonstrated an appreciable potential as imaging probes. Affibody molecules are composed of three alpha-helices. Helices 1 and 2 are involved in molecular recognition, while helix 3 provides stability. The size of Affibody molecules can be reduced by omitting the third alpha-helix and cross-linking the two remaining, providing a smaller molecule with better extravasation and quicker clearance of unbound tracer. The goal of this study was to develop a novel 2-helix Affibody molecule based on backbone cyclization by native chemical ligation (NCL). Methods: The HER2-targeting NCL-cyclized Affibody molecule Z(HER2:342min) has been designed, synthesized and site-specifically conjugated with a DOTA chelator. DOTA-Z(HER2:342min) was labeled with In-111 and (68) Ga. The binding affinity of DOTA-Z(HER2:342min) was evaluated in vitro. The targeting properties of In-111- and (68) Ga-DOTA-Z(HER2:342min) were evaluated in mice bearing SKOV-3 xenografts and compared with the properties of In-111- and (68) Ga-labeled PEP09239, a DOTA-conjugated 2-helix Affibody analogue cyclized by a homocysteine disulfide bridge. Results: The dissociation constant (K-D) for DOTA-Z(HER2:342min) binding to HER2 was 18 nM according to SPR measurements. DOTA-Z(HER2:342min) was labeled with In-111 and (68) Ga. Both conjugates demonstrated bi-phasic binding kinetics to HER2-expressing cells, with K-D1 in low nanbmolar range. Both variants demonstrated specific uptake in HER2-expressing xenografts. Tumor-to-blood ratios at 2 h p.i. were 6.1 +/- 1.3 for In-111-DOTA-Z(HER2:342min) and 4.6 +/- 0.7 for (68) Ga-DOTA-Z(HER2:342min). However, the uptake of DOTA-Z(HER2:342min) in lung, liver and spleen was appreciably higher than the uptake of PEP09239-based counterparts. Conclusions: Native chemical ligation enables production of a backbone-cyclized HER2-binding 2-helix Affibody molecule (Z(HER2:342min)) with low nanomolar target affinity and specific tumor uptake.
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| 7. |
- Kotowski, Krzysztof, et al.
(författare)
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Role of PFKFB3 and PFKFB4 in Cancer: Genetic Basis, Impact on Disease Development/Progression, and Potential as Therapeutic Targets
- 2021
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Ingår i: Cancers. - : MDPI. - 2072-6694. ; 13:4
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Forskningsöversikt (refereegranskat)abstract
- Glycolysis is a crucial metabolic process in rapidly proliferating cells such as cancer cells. Phosphofructokinase-1 (PFK-1) is a key rate-limiting enzyme of glycolysis. Its efficiency is allosterically regulated by numerous substances occurring in the cytoplasm. However, the most potent regulator of PFK-1 is fructose-2,6-bisphosphate (F-2,6-BP), the level of which is strongly associated with 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase activity (PFK-2/FBPase-2, PFKFB). PFK-2/FBPase-2 is a bifunctional enzyme responsible for F-2,6-BP synthesis and degradation. Four isozymes of PFKFB (PFKFB1, PFKFB2, PFKFB3, and PFKFB4) have been identified. Alterations in the levels of all PFK-2/FBPase-2 isozymes have been reported in different diseases. However, most recent studies have focused on an increased expression of PFKFB3 and PFKFB4 in cancer tissues and their role in carcinogenesis. In this review, we summarize our current knowledge on all PFKFB genes and protein structures, and emphasize important differences between the isoenzymes, which likely affect their kinase/phosphatase activities. The main focus is on the latest reports in this field of cancer research, and in particular the impact of PFKFB3 and PFKFB4 on tumor progression, metastasis, angiogenesis, and autophagy. We also present the most recent achievements in the development of new drugs targeting these isozymes. Finally, we discuss potential combination therapies using PFKFB3 inhibitors, which may represent important future cancer treatment options.
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| 8. |
- Myrhammar, Anders, et al.
(författare)
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Photocontrolled Reversible Binding between the Protein A-Derived Z Domain and Immunoglobulin G
- 2020
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 31:3, s. 622-630
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Tidskriftsartikel (refereegranskat)abstract
- Photoisomerization of the trans and cis isomers of azobenzene derivatives has been used to control the function of biomolecules in a reversible and nondestructive manner. In this study, affibody molecules, representing a class of small, helical proteins that can be engineered for binding to a wide range of target proteins, have been investigated by the incorporation of a photoswitchable azobenzene derivative in the molecule. Three different Z domain variants were produced by solid phase peptide synthesis and conjugated by thiol-directed chemistry to an azobenzene-based photoswitch. The proteins were screened for binding to and light elution from an IgG-sepharose affinity column. One of the tested Z variants, Z(C3), showed efficient binding to the column and could be eluted by irradiation with light at 400 nm. In a reverse affinity chromatography assay, where the Z(C3) variant was coupled to sepharose, human IgG1 could be captured to the column and partially eluted by light. Further studies of the azobenzene-conjugated Z(C3) domain by surface plasmon resonance (SPR) confirmed the high affinity binding to IgG, and circular dichroism (CD) spectroscopy showed that the protein has a high alpha-helical secondary structure content.
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| 9. |
- Orlova, Anna, et al.
(författare)
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Evaluation of [(111/114m)In]CHX-A''-DTPA-ZHER2:342, an affibody ligand coniugate for targeting of HER2-expressing malignant tumors
- 2007
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Ingår i: The Quarterly Journal of Nuclear Medicine and Molecular Imaging. - 1824-4785. ; 51:4, s. 314-323
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Tidskriftsartikel (refereegranskat)abstract
- AIM: Radionuclide imaging of the HER2 receptor, which is a target for trastuzumab therapy, can provide important diagnostic information. Further, targeting radionuclide therapy might be an option for treatment of HER2 expressing tumors. The phage-display selected Affibody ligand Z(HER2:342), which binds to HER2 with an affinity of 22 pM, may here play an important role. The small size of the Z(HER2:342), 7.5 kDa, enables quick tumor localization and fast blood clearance. Earlier, successful targeting of HER2-expressing xenografts using Z(HER2:342) labeled using [(111)In]benzyl-DTPA was reported. By changing to the CHX-A''-DTPA chelator, the stability and labeling kinetics of the radiometal-Z(HER2:342) conjugate can be improved. The aim of this study was to evaluate the labeling of the CHX-A''-DTPA-Z(HER2:342) conjugate with (111)In for diagnostic imaging and with (114m)In for locoregional radionuclide therapy. METHODS: The isothiocyanate derivative of CHX-A''-DTPA was coupled to Z(HER2:342) in alkaline conditions at 37 degrees C. The conjugate was labeled with both (111)In and (114m)In and evaluated in vitro and in vivo. RESULTS: Labeling with (111)In and (114m)In provided >95% yield after 30 min at RT. Specific radioactivity was 0.5 and 12 MBq/nmol, for (114m)In and (111)In, respectively. The radiolabeled conjugates demonstrated specific binding to HER2 expressing SKOV-3 cells. In mice bearing SKOV-3 xenografts, the tumor uptake of [(111)In]CHX-A''-DTPA-Z(HER2:342) 4 h postinjection was 10.3+/-3.6% IA/g and tumor-to-blood ratio about 190. CONCLUSION: [(111)In]CHX-A''-DTPA-Z(HER2:342) is a promising candidate for the visualization of HER2 expression in malignant tumors. Labeled with (114m)In it could also be used for locoregional treatment of HER2 expressing tumors.
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| 10. |
- Orlova, Anna, et al.
(författare)
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Site-specific radiometal labeling and improved biodistribution using ABY-027, a novel HER2-targeting affibody molecule-albumin-binding domain fusion protein
- 2013
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Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 54:6, s. 961-968
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Tidskriftsartikel (refereegranskat)abstract
- Because of their better penetration, smaller targeting proteins may be superior to antibodies for radioimmunotherapy of solid tumors. Therefore, Affibody molecules (6.5 kDa) have a potential for being suitable as targeted moiety for radiolabeled therapeutic proteins. Previous studies have demonstrated that a fusion of an Affibody molecule with an albumin-binding domain (ABD) provides a strong noncovalent binding to albumin in vivo. This strong noncovalent binding can be used for reduction of the renal uptake of the Affibody molecule while maintaining a size smaller than that of an antibody, which is important when using residualizing radionuclide labels conjugated to Affibody molecules. The goal of this study was to design and evaluate a new targeting Affibody - ABD fusion protein with improved biodistribution properties for radionuclide therapy. Methods: A novel Affibody-based construct, Z HER2:2891-ABD035-DOTA (ABY-027), was created by fusion of the reengineered HER2-binding Affibody molecule ZHER2:2891 to the N terminus of the high-affinity ABD035, and a maleimido-derivative of DOTA was conjugated at the C terminus of the construct. Binding and processing of 177Lu-ABY-027 by HER2-expressing cells were evaluated in vitro. Targeting of HER2-expressing SKOV-3 xenografts was evaluated in BALB/C nu/nu mice and compared with targeting of previously reported ABD-(Z HER2:342)2. Results: The binding affinity (dissociation constant) of ABY-027 to HER2 (74 pM) was the same as for the parental Z HER2:2891 (76 pM). ABY-027 was stably labeled with 177Lu and 111In with preserved specific binding to HER2-expressing cells in vitro. In vivo receptor saturation experiments demonstrated that targeting of SKOV-3 xenografts in BALB/C nu/nu mice was HER2-specific. 177Lu-ABY- 027 demonstrated substantially (2- to 3-fold) lower renal and hepatic uptake than previously assessed HER2-specific Affibody-based albumin-binding agents. Tumor uptake of radiolabeled ABY-027 at 48 h after injection was 2-fold higher than that for previously reported ABD-(ZHER2:342)2. Conclusion: An optimized molecular design of an ABD fusion protein resulted in an Affibody molecule construct with better properties for therapy. Fully preserved in vivo targeting of the fusion protein was shown in xenografted mice. Site-specific coupling of DOTA provides a uniform conjugate and creates the potential for labeling with a broad range of therapeutic radionuclides. The biodistribution of 177Lu-ABY-027 in a murine model suggests it is more suitable for therapy than alternative approaches.
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