| 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. |
- 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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| 5. |
- 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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| 6. |
- 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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| 7. |
- 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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| 8. |
- 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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| 9. |
- Tolmachev, Vladimir, et al.
(författare)
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111In-benzyl-DTPA-ZHER2:342, an affibody-based conjugate for in vivo imaging of HER2 expression in malignant tumors
- 2006
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Ingår i: Journal of Nuclear Medicine. - 0161-5505 .- 1535-5667. ; 47:5, s. 846-53
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Tidskriftsartikel (refereegranskat)abstract
- Data on expression of the HER2 (erbB-2) receptor in breast carcinoma make it possible to select the most efficient treatment. There are strong indications that HER2 expression possesses prognostic and predictive values in ovarian, prostate, and lung carcinomas as well. Visualization of HER2 expression using radionuclide targeting can provide important diagnostic information. The Affibody Z(HER2:342) is a short (approximately 7 kDa) phage-display-selected protein that binds HER2 with an affinity of 22 pmol/L. The goal of this study was to evaluate whether (111)In-labeled HER2:342 can be used for imaging of HER2 overexpression in vivo. METHODS: Z(HER2:342) was labeled with (111)In via isothiocyanate-benzyl-DTPA (DTPA is diethylenetriaminepentaacetic acid) and the conjugate was characterized in vitro and in vivo. RESULTS: (111)In-Benzyl-DTPA-Z(HER2:342) preserved the capacity to bind living HER2-expressing cells specifically. The affinity of In-benzyl-DTPA-Z(HER2:342) to HER2 was 21 pmol/L according to surface plasmon resonance measurements. In nude mice bearing HER2-expressing SKOV-3 xenografts, a tumor uptake of 12% +/- 3% injected activity per gram and a tumor-to-blood ratio of about 100 were obtained 4 h after injection. Tumor uptake in vivo was receptor specific, as it could be blocked with an excess of nonlabeled Z(HER2:342). HER2-expressing xenografts were clearly imaged 4 h after injection using a gamma-camera. CONCLUSION: (111)In-Benzyl-DTPA-Z(HER2:342) is a promising candidate for visualization of HER2 expression in carcinomas, using the single-photon detection technique.
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| 10. |
- 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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