| 1. |
- 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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| 2. |
- Deyev, Sergey M., et al.
(författare)
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Influence of the Position and Composition of Radiometals and Radioiodine Labels on Imaging of Epcam Expression in Prostate Cancer Model Using the DARPin Ec1
- 2021
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 13:14
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Tidskriftsartikel (refereegranskat)abstract
- Simple Summary Metastasis-targeting therapy might improve outcomes in oligometastatic prostate cancer. Epithelial cell adhesion molecule (EpCAM) is overexpressed in 40-60% of prostate cancer cases and might be used as a target for specific delivery of toxins and drugs. Radionuclide molecular imaging could enable non-invasive detection of EpCAM and stratification of patients for targeted therapy. Designed ankyrin repeat proteins (DARPins) are scaffold proteins, which can be selected for specific binding to different targets. The DARPin Ec1 binds strongly to EpCAM. To determine an optimal design of Ec1-based probes, we labeled Ec1 at two different positions with four different nuclides (Ga-68, In-111, Co-57 and I-125) and investigated the impact on Ec1 biodistribution. We found that the C-terminus is the best position for labeling and that In-111 and I-125 provide the best imaging contrast. This study might be helpful for scientists developing imaging probes based on scaffold proteins. The epithelial cell adhesion molecule (EpCAM) is intensively overexpressed in 40-60% of prostate cancer (PCa) cases and can be used as a target for the delivery of drugs and toxins. The designed ankyrin repeat protein (DARPin) Ec1 has a high affinity to EpCAM (68 pM) and a small size (18 kDa). Radiolabeled Ec1 might be used as a companion diagnostic for the selection of PCa patients for therapy. The study aimed to investigate the influence of radiolabel position (N- or C-terminal) and composition on the targeting and imaging properties of Ec1. Two variants, having an N- or C-terminal cysteine, were produced, site-specifically conjugated to a DOTA chelator and labeled with cobalt-57, gallium-68 or indium-111. Site-specific radioiodination was performed using ((4-hydroxyphenyl)-ethyl)maleimide (HPEM). Biodistribution of eight radiolabeled Ec1-probes was measured in nude mice bearing PCa DU145 xenografts. In all cases, positioning of a label at the C-terminus provided the best tumor-to-organ ratios. The non-residualizing [I-125]I-HPEM label provided the highest tumor-to-muscle and tumor-to-bone ratios and is more suitable for EpCAM imaging in early-stage PCa. Among the radiometals, indium-111 provided the highest tumor-to-blood, tumor-to-lung and tumor-to-liver ratios and could be used at late-stage PCa. In conclusion, label position and composition are important for the DARPin Ec1.
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| 3. |
- Deyev, Sergey M., et al.
(författare)
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Preclinical Evaluation of HER2-Targeting DARPin G3: Impact of Albumin-Binding Domain (ABD) Fusion
- 2024
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Ingår i: International Journal of Molecular Sciences. - : Multidisciplinary Digital Publishing Institute (MDPI). - 1661-6596 .- 1422-0067. ; 25:8
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Tidskriftsartikel (refereegranskat)abstract
- Designed ankyrin repeat protein (DARPin) G3 is an engineered scaffold protein. This small (14.5 kDa) targeting protein binds with high affinity to human epidermal growth factor receptor 2 (HER2). HER2 is overexpressed in several cancers. The use of the DARPin G3 for radionuclide therapy is complicated by its high renal reabsorption after clearance via the glomeruli. We tested the hypothesis that a fusion of the DARPin G3 with an albumin-binding domain (ABD) would prevent rapid renal excretion and high renal reabsorption resulting in better tumour targeting. Two fusion proteins were produced, one with the ABD at the C-terminus (G3-ABD) and another at the N-terminus (ABD-G3). Both variants were labelled with 177Lu. The binding properties of the novel constructs were evaluated in vitro and their biodistribution was compared in mice with implanted human HER2-expressing tumours. Fusion with the ABD increased the retention time of both constructs in blood compared with the non-ABD-fused control. The effect of fusion with the ABD depended strongly on the order of the domains in the constructs, resulting in appreciably better targeting properties of [177Lu]Lu-G3-ABD. Our data suggest that the order of domains is critical for the design of targeting constructs based on scaffold proteins.
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| 4. |
- 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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| 5. |
- Garousi, Javad, et al.
(författare)
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Experimental HER2-Targeted Therapy Using ADAPT6-ABD-mcDM1 in Mice Bearing SKOV3 Ovarian Cancer Xenografts : Efficacy and Selection of Companion Imaging Counterpart
- 2022
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Ingår i: Pharmaceutics. - : MDPI. - 1999-4923. ; 14:8
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Tidskriftsartikel (refereegranskat)abstract
- Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast and gastric cancer is exploited for targeted therapy using monoclonal antibodies and antibody-drug conjugates. Small engineered scaffold proteins, such as the albumin binding domain (ABD) derived affinity proteins (ADAPTs), are a promising new format of targeting probes for development of drug conjugates with well-defined structure and tunable pharmacokinetics. Radiolabeled ADAPT6 has shown excellent tumor-targeting properties in clinical trials. Recently, we developed a drug conjugate based on the HER2-targeting ADAPT6 fused to an albumin binding domain (ABD) for increased bioavailability and conjugated to DM1 for cytotoxic action, designated as ADAPT6-ABD-mcDM1. In this study, we investigated the therapeutic efficacy of this conjugate in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts. A secondary aim was to evaluate several formats of imaging probes for visualization of HER2 expression in tumors. Administration of ADAPT6-ABD-mcDM1 provided a significant delay of tumor growth and increased the median survival of the mice, in comparison with both a non-targeting homologous construct (ADAPT(Neg)-ABD-mcDM1) and the vehicle-treated groups, without inducing toxicity to liver or kidneys. Moreover, the evaluation of imaging probes showed that small scaffold proteins, such as Tc-99m(CO)(3)-ADAPT6 or the affibody molecule Tc-99m-Z(HER2:41071), are well suited as diagnostic companions for potential stratification of patients for ADAPT6-ABD-mcDM1-based therapy.
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| 6. |
- Garousi, Javad, et al.
(författare)
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Targeting HER2 Expressing Tumors with a Potent Drug Conjugate Based on an Albumin Binding Domain-Derived Affinity Protein
- 2021
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 13:11, s. 1847-
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Tidskriftsartikel (refereegranskat)abstract
- Albumin binding domain derived affinity proteins (ADAPTs) are a class of small and folded engineered scaffold proteins that holds great promise for targeting cancer tumors. Here, we have extended the in vivo half-life of an ADAPT, targeting the human epidermal growth factor receptor 2 (HER2) by fusion with an albumin binding domain (ABD), and armed it with the highly cytotoxic payload mertansine (DM1) for an investigation of its properties in vitro and in vivo. The resulting drug conjugate, ADAPT6-ABD-mcDM1, retained binding to its intended targets, namely HER2 and serum albumins. Further, it was able to specifically bind to cells with high HER2 expression, get internalized, and showed potent toxicity, with IC50 values ranging from 5 to 80 nM. Conversely, no toxic effect was found for cells with low HER2 expression. In vivo, ADAPT6-ABD-mcDM1, radiolabeled with Tc-99m, was characterized by low uptake in most normal organs, and the main excretion route was shown to be through the kidneys. The tumor uptake was 5.5% ID/g after 24 h, which was higher than the uptake in all normal organs at this time point except for the kidneys. The uptake in the tumors was blockable by pre-injection of an excess of the monoclonal antibody trastuzumab (having an overlapping epitope on the HER2 receptor). In conclusion, half-life extended drug conjugates based on the ADAPT platform of affinity proteins holds promise for further development towards targeted cancer therapy.
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| 7. |
- Larkina, Mariia, et al.
(författare)
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Comparative Preclinical Evaluation of Peptide-Based Chelators for the Labeling of DARPin G3 with 99mTc for Radionuclide Imaging of HER2 Expression in Cancer
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:21, s. 13443-
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Tidskriftsartikel (refereegranskat)abstract
- Non-invasive radionuclide imaging of human epidermal growth factor receptor type 2 (HER2) expression in breast, gastroesophageal, and ovarian cancers may stratify patients for treatment using HER2-targeted therapeutics. Designed ankyrin repeat proteins (DARPins) are a promising type of targeting probe for radionuclide imaging. In clinical studies, the DARPin [Tc-99m]Tc-(HE)(3)-G3 labeled using a peptide-based chelator His-Glu-His-Glu-His-Glu ((HE)(3)), provided clear imaging of HER2 expressing breast cancer 2-4 h after injection. The goal of this study was to evaluate if the use of cysteine-containing peptide-based chelators Glu-Glu-Glu-Cys (E3C), Gly-Gly-Gly-Cys (G(3)C), and Gly-Gly-Gly-Ser-Cys connected via a (Gly-Gly-Gly-Ser)(3)-linker (designated as G3-(G(3)S)(3)C) would further improve the contrast of imaging using Tc-99m-labeled derivatives of G3. The labeling of the new variants of G3 provided a radiochemical yield of over 95%. Labeled G3 variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 1.9-5 nM. Biodistribution of [Tc-99m]Tc-G3-G(3)C, [Tc-99m]Tc-G3-(G(3)S)(3)C, and [Tc-99m]Tc-G3-E3C in mice was compared with the biodistribution of [Tc-99m]Tc-(HE)(3)-G3. It was found that the novel variants provide specific accumulation in HER2-expressing human xenografts and enable discrimination between tumors with high and low HER2 expression. However, [Tc-99m]Tc-(HE)(3)-G3 provided better contrast between tumors and the most frequent metastatic sites of HER2-expressing cancers and is therefore more suitable for clinical applications.
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| 8. |
- Liu, Hao, et al.
(författare)
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Potent and specific fusion toxins consisting of a HER2‑binding, ABD‑derived affinity protein, fused to truncated versions of Pseudomonas exotoxin A
- 2019
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Ingår i: International Journal of Oncology. - : Spandidos Publications. - 1019-6439 .- 1791-2423. ; 55:1, s. 309-319
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Tidskriftsartikel (refereegranskat)abstract
- Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain-derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA-derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half-life extension, can be used for specific killing of HER2-expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity. All constructs had strong affinity for HER2 (K-D 10 to 26 nM) and no tendency for aggregation could be detected. The fusion toxins including the ABD showed strong interaction with human and mouse serum albumin [equilibrium dissociation constant (K-D) 1 to 3 nM and 2 to 10 nM, respectively]. The in vitro investigation of the cytotoxic potential revealed IC50-values in the picomolar range for cells expressing high levels of HER2. The specificity was also demonstrated, by showing that free HER2 receptors on the target cells are required for fusion toxin activity. In mice, the fusion toxins containing the ABD exhibited an appreciably longer time in circulation. The uptake was highest in liver and kidney. Fusion with PE25 was associated with the highest hepatic uptake. Collectively, the results suggest that fusion toxins consisting of ADAPTs and ETA-derivatives are promising agents for targeted cancer therapy.
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| 9. |
- Pinto, Soraia, et al.
(författare)
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Nanoparticles targeting the intestinal Fc receptor enhance intestinal cellular trafficking of semaglutide
- 2024
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Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 366, s. 621-636
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Tidskriftsartikel (refereegranskat)abstract
- Semaglutide is the first oral glucagon-like peptide-1 (GLP-1) analog commercially available for the treatment of type 2 diabetes. In this work, semaglutide was incorporated into poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles (NPs) to improve its delivery across the intestinal barrier. The nanocarriers were surface-decorated with either a peptide or an affibody that target the human neonatal Fc receptor (hFcRn), located on the luminal cell surface of the enterocytes. Both ligands were successfully conjugated with the PLGA-PEG via maleimide-thiol chemistry and thereafter, the functionalized polymers were used to produce semaglutide-loaded NPs. Monodisperse NPs with an average size of 170 nm, neutral surface charge and 3% of semaglutide loading were obtained. Both FcRn-targeted NPs exhibited improved interaction and association with Caco-2 cells (cells that endogenously express the hFcRn), compared to non-targeted NPs. Additionally, the uptake of FcRn-targeted NPs was also observed to occur in human intestinal organoids (HIOs) expressing hFcRn through microinjection into the lumen of HIOs, resulting in potential increase of semaglutide permeability for both ligand-functionalized nanocarriers. Herein, our study demonstrates valuable data and insights that the FcRn-targeted NPs has the capacity to promote intestinal absorption of therapeutic peptides.
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| 10. |
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