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- Altai, Mohamed, et al.
(författare)
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Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs
- 2018
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Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 7:10
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Tidskriftsartikel (refereegranskat)abstract
- Overexpression of human epidermal growth factor receptor type 3 (HER3) is associated with tumour cell resistance to HER-targeted therapies. Monoclonal antibodies (mAbs) targeting HER3 are currently being investigated for treatment of various types of cancers. Cumulative evidence suggests that affibody molecules may be appropriate alternatives to mAbs. We previously reported a fusion construct (3A3) containing two HER3-targeting affibody molecules flanking an engineered albumin-binding domain (ABD 035) included for the extension of half-life in circulation. The 3A3 fusion protein (19.7 kDa) was shown to delay tumour growth in mice bearing HER3-expressing xenografts and was equipotent to the mAb seribantumab. Here, we have designed and explored a series of novel formats of anti-HER3 affibody molecules fused to the ABD in different orientations. All constructs inhibited heregulin-induced phosphorylation in HER3-expressing BxPC-3 and DU-145 cell lines. Biodistribution studies demonstrated extended the half-life of all ABD-fused constructs, although at different levels. The capacity of our ABD-fused proteins to accumulate in HER3-expressing tumours was demonstrated in nude mice bearing BxPC-3 xenografts. Formats where the ABD was located on the C-terminus of affibody binding domains (3A, 33A, and 3A3) provided the best tumour targeting properties in vivo. Further development of these promising candidates for treatment of HER3-overexpressing tumours is therefore justified.
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| 2. |
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| 3. |
- Lindbo, Sarah, et al.
(författare)
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Optimized Molecular Design of ADAPT-Based HER2-Imaging Probes Labeled with 111In and 68Ga
- 2018
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Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 15:7, s. 2674-2683
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Tidskriftsartikel (refereegranskat)abstract
- Radionuclide molecular imaging is a promising tool for visualization of cancer associated molecular abnormalities in vivo and stratification of patients for specific therapies. ADAPT is a new type of small engineered proteins based on the scaffold of an albumin binding domain of protein G. ADAPTs have been utilized to select and develop high affinity binders to different proteinaceous targets. ADAPT6 binds to human epidermal growth factor 2 (HER2) with low nanomolar affinity and can be used for its in vivo visualization. Molecular design of 111In-labeled anti-HER2 ADAPT has been optimized in several earlier studies. In this study, we made a direct comparison of two of the most promising variants, having either a DEAVDANS or a (HE)3DANS sequence at the N-terminus, conjugated with a maleimido derivative of DOTA to a GSSC amino acids sequence at the C-terminus. The variants (designated DOTA-C59-DEAVDANS-ADAPT6-GSSC and DOTA-C61-(HE)3DANS-ADAPT6-GSSC) were stably labeled with 111In for SPECT and 68Ga for PET. Biodistribution of labeled ADAPT variants was evaluated in nude mice bearing human tumor xenografts with different levels of HER2 expression. Both variants enabled clear discrimination between tumors with high and low levels of HER2 expression. 111In-labeled ADAPT6 derivatives provided higher tumor-to-organ ratios compared to 68Ga-labeled counterparts. The best performing variant was DOTA-C61-(HE)3DANS-ADAPT6-GSSC, which provided tumor-to-blood ratios of 208 ± 36 and 109 ± 17 at 3 h for 111In and 68Ga labels, respectively.
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| 6. |
- Oroujeni, Maryam, PhD, 1982-, et al.
(författare)
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Influence of composition of cysteine-containing peptide-based chelators on biodistribution of Tc-99m-labeled anti-EGFR affibody molecules
- 2018
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Ingår i: Amino Acids. - : Springer. - 0939-4451 .- 1438-2199. ; 50:8, s. 981-994
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Tidskriftsartikel (refereegranskat)abstract
- Epidermal growth factor receptor (EGFR) is overexpressed in a number of cancers and is the molecular target for several anti-cancer therapeutics. Radionuclide molecular imaging of EGFR expression should enable personalization of anti-cancer treatment. Affibody molecule is a promising type of high-affinity imaging probes based on a non-immunoglobulin scaffold. A series of derivatives of the anti-EGFR affibody molecule ZEGFR:2377, having peptide-based cysteine-containing chelators for conjugation of Tc-99m, was designed and evaluated. It was found that glutamate-containing chelators Gly-Gly-Glu-Cys (GGEC), Gly-Glu-Glu-Cys (GEEC) and Glu-Glu-Glu-Cys (EEEC) provide the best labeling stability. The glutamate containing conjugates bound to EGFR-expressing cells specifically and with high affinity. Specific targeting of EGFR-expressing xenografts in mice was demonstrated. The number of glutamate residues in the chelator had strong influence on biodistribution of radiolabeled affibody molecules. Increase of glutamate content was associated with lower uptake in normal tissues. The Tc-99m-labeled variant containing the EEEC chelator provided the highest tumor-to-organ ratios. In conclusion, optimizing the composition of peptide-based chelators enhances contrast of imaging of EGFR-expression using affibody molecules.
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| 7. |
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| 8. |
- Summer, D., et al.
(författare)
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Cyclic versus Noncyclic Chelating Scaffold for 89Zr-Labeled ZEGFR:2377 Affibody Bioconjugates Targeting Epidermal Growth Factor Receptor Overexpression
- 2018
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Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 15:1, s. 175-185
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Tidskriftsartikel (refereegranskat)abstract
- Zirconium-89 is an emerging radionuclide for positron emission tomography (PET) especially for biomolecules with slow pharmacokinetics as due to its longer half-life, in comparison to fluorine-18 and gallium-68, imaging at late time points is feasible. Desferrioxamine B (DFO), a linear bifunctional chelator (BFC) is mostly used for this radionuclide so far but shows limitations regarding stability. Our group recently reported on fusarinine C (FSC) with similar zirconium-89 complexing properties but potentially higher stability related to its cyclic structure. This study was designed to compare FSC and DFO head-to-head as bifunctional chelators for 89Zr-radiolabeled EGFR-targeting ZEGFR:2377 affibody bioconjugates. FSC-ZEGFR:2377 and DFO-ZEGFR:2377 were evaluated regarding radiolabeling, in vitro stability, specificity, cell uptake, receptor affinity, biodistribution, and microPET-CT imaging. Both conjugates were efficiently labeled with zirconium-89 at room temperature but radiochemical yields increased substantially at elevated temperature, 85 °C. Both 89Zr-FSC-ZEGFR:2377 and 89Zr-DFO-ZEGFR:2377 revealed remarkable specificity, affinity and slow cell-line dependent internalization. Radiolabeling at 85 °C showed comparable results in A431 tumor xenografted mice with minor differences regarding blood clearance, tumor and liver uptake. In comparison 89Zr-DFO-ZEGFR:2377, radiolabeled at room temperature, showed a significant difference regarding tumor-to-organ ratios. MicroPET-CT imaging studies of 89Zr-FSC-ZEGFR:2377 as well as 89Zr-DFO-ZEGFR:2377 confirmed these findings. In summary we were able to show that FSC is a suitable alternative to DFO for radiolabeling of biomolecules with zirconium-89. Furthermore, our findings indicate that 89Zr-radiolabeling of DFO conjugates at higher temperature reduces off-chelate binding leading to significantly improved tumor-to-organ ratios and therefore enhancing image contrast.
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| 9. |
- Summer, D, et al.
(författare)
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PP15 89Zr-Siderophore-Affibody conjugates for imaging EGFR expression
- 2018
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Ingår i: EJNMMI Research. - : Springer Science and Business Media LLC. - 2191-219X. ; 8:S1
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Aim: Zirconium-89 has gained great interest for PET, when imaging at late time points is required. Desferrioxamine B (DFO), is mostly used for this radionuclide as bifunctional chelator (BFC) and we recently reported on fusarinine C (FSC) with similar zirconium-89 complexing properties but potentially higher stability related to its cyclic structure. This study reports on the comparison of FSC and DFO as BFCs for 89Zr labelling of the affibody ZEGFR:2377 targeting Epidermal Growth Factor Receptors (EGFR).Methods: FSC-ZEGFR:2377 and DFO-ZEGFR:2377 were evaluated regarding labeling, in vitro stability, specificity, cell uptake, receptor affinity, biodistribution and microPET-CT imaging.Results: Both conjugates showed increased labelling yields at elevated temperature (85°C). Both conjugates revealed remarkable specificity, affinity and slow cell-line dependent internalisation. Labeling at 85°C showed comparable results in A431 tumor xenografted mice with minor differences regarding blood clearance, tumor and liver uptake but clear improvement as compared to 89Zr-DFO-ZEGFR:2377, labeled at room temperature, which was confirmed by MicroPET-CT imaging.Conclusion: We were able to show that FSC is a suitable alternative to DFO for labeling of biomolecules with zirconium-89. Furthermore our findings indicate that 89Zr- labeling of DFO conjugates at higher temperature reduces off-chelate binding leading to significantly improved tumor-to-organ ratios and therefore enhancing image contrast.
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| 10. |
- Vorobyeva, Anzhelika, et al.
(författare)
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Comparative Evaluation of Radioiodine and Technetium-Labeled DARPin 9_29 for Radionuclide Molecular Imaging of HER2 Expression in Malignant Tumors
- 2018
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Ingår i: Contrast Media & Molecular Imaging. - : WILEY-HINDAWI. - 1555-4309 .- 1555-4317.
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Tidskriftsartikel (refereegranskat)abstract
- High expression of human epidermal growth factor receptor 2 (HER2) in breast and gastroesophageal carcinomas is a predictive biomarker for treatment using HER2-targeted therapeutics (antibodies trastuzumab and pertuzumab, antibody-drug conjugate trastuzumab DM1, and tyrosine kinase inhibitor lapatinib). Radionuclide molecular imaging of HER2 expression might permit stratification of patients for HER2-targeting therapies. In this study, we evaluated a new HER2-imaging probe based on the designed ankyrin repeat protein (DARPin) 9_29. DARPin 9_29 was labeled with iodine-125 by direct radioiodination and with [Tc-99m] Tc(CO)(3) using the C-terminal hexahistidine tag. DARPin 9_29 preserved high specificity and affinity of binding to HER2-expressing cells after labeling. Uptake of [I-125] I-DARPin 9_29 and [Tc-99m] Tc(CO)(3)-DARPin 9_29 in HER2-positive SKOV-3 xenografts in mice at 6 h after injection was 3.4 +/- 0.7 % ID/g and 2.9 +/- 0.7 % ID/g, respectively. This was significantly (p < 0.00005) higher than the uptake of the same probes in HER2-negative Ramos lymphoma xenografts, 0.22 +/- 0.09 % ID/g and 0.30 +/- 0.05 % ID/g, respectively. Retention of [I-125] I-DARPin 9_29 in the lung, liver, spleen, and kidneys was appreciably lower compared with [Tc-99m] Tc(CO)(3)-DARPin 9_29, which resulted in significantly (p < 0.05) higher tumor-to-organ ratios. The biodistribution data were confirmed by SPECT/CT imaging. In conclusion, radioiodine is a preferable label for DARPin 9_29.
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