| 1. |
- Altai, Mohamed, et al.
(författare)
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On the prevention of kidney uptake of radiolabeled DARPins
- 2020
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Ingår i: EJNMMI Research. - : SPRINGEROPEN. - 2191-219X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Background: Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins (14-18 kDa) that demonstrated promising tumor-targeting properties in preclinical studies. However, high renal accumulation of activity for DARPins labeled with residualizing labels is a limitation for targeted radionuclide therapy. A better understanding of the mechanisms behind the kidney uptake of DARPins could aid the development of strategies to reduce it. In this study, we have investigated whether the renal uptake of [Tc-99m]Tc(CO)(3)-G3 DARPin could be reduced by administration of compounds that act on various parts of the reabsorption system in the kidney.Results: Co-injection of lysine or Gelofusine was not effective for the reduction of kidney uptake of [Tc-99m]Tc(CO)(3)-G3. Administration of sodium maleate before the injection of [Tc-99m]Tc(CO)(3)-G3 reduced the kidney-associated activity by 60.4 +/- 10.3%, while administration of fructose reduced it by 46.9 +/- 7.6% compared with the control. The decrease in the kidney uptake provided by sodium maleate was also observed for [Tc-99m]Tc(CO)(3)-9_29 DARPin. Preinjection of colchicine, probenecid, mannitol, or furosemide had no effect on the kidney uptake of [Tc-99m]Tc(CO)(3)-G3. Kidney autoradiography showed mainly cortical accumulation of activity for all studied groups.Conclusion: Common clinical strategies were not effective for the reduction of kidney uptake of [Tc-99m]Tc(CO)(3)-G3. Both fructose and maleate lower the cellular ATP level in the proximal tubule cells and their reduction of the kidney reuptake indicates the involvement of an ATP-driven uptake mechanism. The decrease provided by maleate for both G3 and 9_29 DARPins indicates that their uptake proceeds through a mechanism independent of DARPin structure and binding site composition.
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| 2. |
- Deyev, Sergey M., et al.
(författare)
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Effect of a radiolabel biochemical nature on tumor-targeting properties of EpCAM-binding engineered scaffold protein DARPin Ec1
- 2020
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Ingår i: International Journal of Biological Macromolecules. - : ELSEVIER. - 0141-8130 .- 1879-0003. ; 145, s. 216-225
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Tidskriftsartikel (refereegranskat)abstract
- Radionuclide-based imaging of molecular therapeutic targets might facilitate stratifying patients for specific biotherapeutics. New type of imaging probes, based on designed ankyrin repeat proteins (DARPins), have demonstrated excellent contrast of imaging of human epidermal growth factor type 2 (HER2) expression in preclinical models. We hypothesized that labeling approaches, which result in lipophilic radiometabolites (non-residualizing labels), would provide the best imaging contrast for DARPins that internalize slowly after binding to cancer cells. The hypothesis was tested using DARPin Ec1 that binds to epithelial cell adhesion molecule (EpCAM). EpCAM is a promising therapeutic target. Ec1 was labeled with I-125 using two methods to obtain the non-residualizing labels, while residualizing labels were obtained by labeling it with Tc-99m. All labeled Ec1 variants preserved target specificity and picomolar binding affinity to EpCAM-expressing pancreatic adenocarcinoma BxPC-3 cells. In murine models, all the variants provided similar tumor uptake. However, I-125-PIB-H-6-Ec1 had noticeably lower retention in normal tissues, which provided appreciably higher tumor-to-organ ratios. Furthermore, I-125-PIB-H-6-Ec1 demonstrated the highest imaging contrast in preclinical models than any other EpCAM-imaging agent tested so far. In conclusion, DARPin Ec1 in combination with a non-residualizing label is a promising probe for imaging EpCAM expression a few hours after injection.
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| 3. |
- Ding, Haozhong, et al.
(författare)
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HER2-Specific Pseudomonas Exotoxin A PE25 Based Fusions : Influence of Targeting Domain on Target Binding, Toxicity, and In Vivo Biodistribution
- 2020
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 12:4
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Tidskriftsartikel (refereegranskat)abstract
- The human epidermal growth factor receptor 2 (HER2) is a clinically validated target for cancer therapy, and targeted therapies are often used in regimens for patients with a high HER2 expression level. Despite the success of current drugs, a number of patients succumb to their disease, which motivates development of novel drugs with other modes of action. We have previously shown that an albumin binding domain-derived affinity protein with specific affinity for HER2, ADAPT(6), can be used to deliver the highly cytotoxic protein domain PE25, a derivative of Pseudomonas exotoxin A, to HER2 overexpressing malignant cells, leading to potent and specific cell killing. In this study we expanded the investigation for an optimal targeting domain and constructed two fusion toxins where a HER2-binding affibody molecule, Z(HER2:2891), or the dual-HER2-binding hybrid Z(HER2:2891)-ADAPT(6) were used for cancer cell targeting. We found that both targeting domains conferred strong binding to HER2; both to the purified extracellular domain and to the HER2 overexpressing cell line SKOV3. This resulted in fusion toxins with high cytotoxic potency toward cell lines with high expression levels of HER2, with EC50 values between 10 and 100 pM. For extension of the plasma half-life, an albumin binding domain was also included. Intravenous injection of the fusion toxins into mice showed a profound influence of the targeting domain on biodistribution. Compared to previous results, with ADAPT(6) as targeting domain, Z(HER2:2891) gave rise to further extension of the plasma half-life and also shifted the clearance route of the fusion toxin from the liver to the kidneys. Collectively, the results show that the targeting domain has a major impact on uptake of PE25-based fusion toxins in different organs. The results also show that PE25-based fusion toxins with high affinity to HER2 do not necessarily increase the cytotoxicity beyond a certain point in affinity. In conclusion, Z(HER2:2891) has the most favorable characteristics as targeting domain for PE25.
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| 4. |
- Garousi, Javad, et al.
(författare)
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Imaging using radiolabelled targeted proteins : radioimmunodetection and beyond.
- 2020
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Ingår i: EJNMMI Radiopharmacy and Chemistry. - : Springer Science and Business Media LLC. - 2365-421X. ; 5:1
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Tidskriftsartikel (refereegranskat)abstract
- The use of radiolabelled antibodies was proposed in 1970s for staging of malignant tumours. Intensive research established chemistry for radiolabelling of proteins and understanding of factors determining biodistribution and targeting properties. The use of radioimmunodetection for staging of cancer was not established as common practice due to approval and widespread use of [18F]-FDG, which provided a more general diagnostic use than antibodies or their fragments. Expanded application of antibody-based therapeutics renewed the interest in radiolabelled antibodies. RadioimmunoPET emerged as a powerful tool for evaluation of pharmacokinetics of and target engagement by biotherapeutics. In addition to monoclonal antibodies, new radiolabelled engineered proteins have recently appeared, offering high-contrast imaging of expression of therapeutic molecular targets in tumours shortly after injection. This creates preconditions for noninvasive determination of a target expression level and stratification of patients for targeted therapies. Radiolabelled proteins hold great promise to play an important role in development and implementation of personalised targeted treatment of malignant tumours. This article provides an overview of biodistribution and tumour-seeking features of major classes of targeting proteins currently utilized for molecular imaging. Such information might be useful for researchers entering the field of the protein-based radionuclide molecular imaging.
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| 5. |
- Garousi, Javad, et al.
(författare)
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Influence of Several Compounds and Drugs on the Renal Uptake of Radiolabeled Affibody Molecules
- 2020
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Ingår i: Molecules (Basel, Switzerland). - : MDPI AG. - 1420-3049 .- 1431-5157. ; 25:11
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are the most studied class of engineered scaffold proteins (ESPs) in radionuclide molecular imaging. Attempts to use affibody molecules directly labelled with radiometals for targeted radionuclide therapy were hampered by the high uptake and retention of radioactivity in kidneys. Several promising strategies have been implemented to circumvent this problem. Here, we investigated whether a pharmacological approach targeting different components of the reabsorption system could be used to lower the uptake of [99mTc]Tc-ZHER:2395 affibody molecule in kidneys. Pre-injection of probenecid, furosemide, mannitol or colchicine had no influence on activity uptake in kidneys compared to the control group. Mice pre-injected with maleate and fructose had 33% and 51% reduction in the kidney-associated activity, respectively, compared to the control group. Autoradiography images showed that the accumulation of activity after [99mTc]Tc-ZHER2:2395 injection was in the renal cortex and that both maleate and fructose could significantly reduce it. Results from this study demonstrate that pharmacological intervention with maleate and fructose was effective in reducing the kidney uptake of affibody molecules. A presumable mechanism is the disruption of ATP-mediated cellular uptake and endocytosis processes of affibody molecules by tubular cells.
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| 6. |
- Vorobyeva, Anzhelika, et al.
(författare)
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Feasibility of Imaging EpCAM Expression in Ovarian Cancer Using Radiolabeled DARPin Ec1
- 2020
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 21:9
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Tidskriftsartikel (refereegranskat)abstract
- Epithelial cell adhesion molecule (EpCAM) is overexpressed in 55%-75% of ovarian carcinomas (OC). EpCAM might be used as a target for a treatment of disseminated OC. Designed ankyrin repeats protein (DARPin) Ec1 is a small (18 kDa) protein, which binds to EpCAM with subnanomolar affinity. We tested a hypothesis that Ec1 labeled with a non-residualizing label might serve as a companion imaging diagnostic for stratification of patients for EpCAM-targeting therapy. Ec1 was labeled with I-125 using N-succinimidyl-para-iodobenzoate. Binding affinity, specificity, and cellular processing of [I-125]I-PIB-Ec1 were evaluated using SKOV-3 and OVCAR-3 ovarian carcinoma cell lines. Biodistribution and tumor-targeting properties of [I-125]I-PIB-Ec1 were studied in Balb/c nu/nu mice bearing SKOV-3 and OVCAR-3 xenografts. EpCAM-negative Ramos lymphoma xenografts served as specificity control. Binding of [I-125]I-PIB-Ec1 to ovarian carcinoma cell lines was highly specific and had affinity in picomolar range. Slow internalization of [I-125]I-PIB-Ec1 by OC cells confirmed utility of non-residualizing label for in vivo imaging. [I-125]I-PIB-Ec1 provided 6 h after injection tumor-to-blood ratios of 30 +/- 11 and 48 +/- 12 for OVCAR-3 and SKOV-3 xenografts, respectively, and high contrast to other organs. Tumor targeting was highly specific. Saturation of tumor uptake at a high dose of Ec1 in SKOV-3 model provided a rationale for dose selection in further studies using therapeutic conjugates of Ec1 for targeted therapy. In conclusion, [I-125]I-PIB-Ec1 is a promising agent for visualizing EpCAM expression in OC.
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| 7. |
- Vorobyeva, Anzhelika, et al.
(författare)
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Investigation of a Pharmacological Approach for Reduction of Renal Uptake of Radiolabeled ADAPT Scaffold Protein
- 2020
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Ingår i: Molecules. - : MDPI. - 1431-5157 .- 1420-3049. ; 25:19
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Tidskriftsartikel (refereegranskat)abstract
- Albumin binding domain-Derived Affinity ProTeins (ADAPTs) are small (5 kDa) engineered scaffold proteins that are promising targeting agents for radionuclide-based imaging. A recent clinical study has demonstrated that radiolabeled ADAPTs can efficiently visualize human epidermal growth factor receptor 2 (HER2) expression in breast cancer using SPECT imaging. However, the use of ADAPTs directly labeled with radiometals for targeted radionuclide therapy is limited by their high reabsorption and prolonged retention of activity in kidneys. In this study, we investigated whether a co-injection of lysine or gelofusin, commonly used for reduction of renal uptake of radiolabeled peptides in clinics, would reduce the renal uptake of [Tc-99m]Tc(CO)(3)-ADAPT6 in NMRI mice. In order to better understand the mechanism behind the reabsorption of [Tc-99m]Tc(CO)(3)-ADAPT6, we included several compounds that act on various parts of the reabsorption system in kidneys. Administration of gelofusine, lysine, probenecid, furosemide, mannitol, or colchicine did not change the uptake of [Tc-99m]Tc(CO)(3)-ADAPT6 in kidneys. Sodium maleate reduced the uptake of [Tc-99m]Tc(CO)(3)-ADAPT6 to ca. 25% of the uptake in the control, a high dose of fructose (50 mmol/kg) reduced the uptake by ca. two-fold. However, a lower dose (20 mmol/kg) had no effect. These results indicate that common clinical strategies are not effective for reduction of kidney uptake of [Tc-99m]Tc(CO)(3)-ADAPT6 and that other strategies for reduction of activity uptake or retention in kidneys should be investigated for ADAPT6.
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| 8. |
- Vorobyeva, Anzhelika, et al.
(författare)
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Radionuclide Molecular Imaging of EpCAM Expression in Triple-Negative Breast Cancer Using the Scaffold Protein DARPin Ec1
- 2020
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Ingår i: Molecules. - : MDPI. - 1431-5157 .- 1420-3049. ; 25:20
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Tidskriftsartikel (refereegranskat)abstract
- Efficient treatment of disseminated triple-negative breast cancer (TNBC) remains an unmet clinical need. The epithelial cell adhesion molecule (EpCAM) is often overexpressed on the surface of TNBC cells, which makes EpCAM a potential therapeutic target. Radionuclide molecular imaging of EpCAM expression might permit selection of patients for EpCAM-targeting therapies. In this study, we evaluated a scaffold protein, designed ankyrin repeat protein (DARPin) Ec1, for imaging of EpCAM in TNBC. DARPin Ec1 was labeled with a non-residualizing [I-125]I-para-iodobenzoate (PIB) label and a residualizing [Tc-99m]Tc(CO)(3) label. Both imaging probes retained high binding specificity and affinity to EpCAM-expressing MDA-MB-468 TNBC cells after labeling. Internalization studies showed that Ec1 was retained on the surface of MDA-MB-468 cells to a high degree up to 24 h. Biodistribution in Balb/c nu/nu mice bearing MDA-MB-468 xenografts demonstrated specific uptake of both [I-125]I-PIB-Ec1 and [Tc-99m]Tc(CO)(3)-Ec1 in TNBC tumors. [I-125]I-PIB-Ec1 had appreciably lower uptake in normal organs compared with [Tc-99m]Tc(CO)(3)-Ec1, which resulted in significantly (p < 0.05) higher tumor-to-organ ratios. The biodistribution data were confirmed by micro-Single-Photon Emission Computed Tomography/Computed Tomography (microSPECT/CT) imaging. In conclusion, an indirectly radioiodinated Ec1 is the preferable probe for imaging of EpCAM in TNBC.
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