1. |
- Abouzayed, Ayman, et al.
(författare)
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177Lu-labeled PSMA targeting therapeutic with optimized linker for treatment of disseminated prostate cancer; evaluation of biodistribution and dosimetry
- 2023
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Ingår i: Frontiers in Oncology. - : Frontiers Media S.A.. - 2234-943X. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Prostate specific membrane antigen (PSMA), highly expressed in metastatic castration-resistant prostate cancer (mCRPC), is an established therapeutic target. Theranostic PSMA-targeting agents are widely used in patient management and has shown improved outcomes for mCRPC patients. Earlier, we optimized a urea-based probe for radionuclide visualization of PSMA-expression in vivo using computer modeling. With the purpose to develop a targeting agent equally suitable for radionuclide imaging and therapy, the agent containing DOTA chelator was designed (BQ7876). The aim of the study was to test the hypothesis that Lu-177-labeled BQ7876 possesses target binding and biodistribution properties potentially enabling its use for radiotherapy.Methods: BQ7876 was synthesized and labeled with Lu-177. Specificity and affinity of [Lu-177]Lu-BQ7876 to PSMA-expressing PC3-pip cells was evaluated and its processing after binding to cells was studied. Animal studies in mice were performed to assess its biodistribution in vivo, target specificity and dosimetry. [Lu-177]Lu-PSMA-617 was simultaneously evaluated for comparison.Results: BQ7876 was labeled with Lu-177 with radiochemical yield >99%. Its binding to PSMA was specific in vitro and in vivo when tested in antigen saturation conditions as well as in PSMA-negative PC-3 tumors. The binding of [Lu-177]Lu-BQ7876 to living cells was characterized by rapid association, while the dissociation included a rapid and a slow phase with affinities K-D1 = 3.8 nM and K-D2 = 25 nM. The half-maximal inhibitory concentration for Lu-nat-BQ7876 was 59 nM that is equal to 61 nM for Lu-nat-PSMA-617. Cellular processing of [Lu-177]Lu-BQ7876 was accompanied by slow internalization. [Lu-177]Lu-BQ7876 was cleared from blood and normal tissues rapidly. Initial elevated uptake in kidneys decreased rapidly, and by 3 h post injection, the renal uptake (13 +/- 3%ID/g) did not differ significantly from tumor uptake (9 +/- 3%ID/g). Tumor uptake was stable between 1 and 3 h followed by a slow decline. The highest absorbed dose was in kidneys, followed by organs and tissues in abdomen.Discussion: Biodistribution studies in mice demonstrated that targeting properties of [Lu-177]Lu-BQ7876 are not inferior to properties of [Lu-177]Lu-PSMA-617, but do not offer any decisive advantages.
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2. |
- Bezverkhniaia, Ekaterina, et al.
(författare)
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Preclinical Evaluation of a Novel High-Affinity Radioligand [99mTc]Tc-BQ0413 Targeting Prostate-Specific Membrane Antigen (PSMA)
- 2023
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 24:24
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Tidskriftsartikel (refereegranskat)abstract
- Radionuclide imaging using radiolabeled inhibitors of prostate-specific membrane antigen (PSMA) can be used for the staging of prostate cancer. Previously, we optimized the Glu-urea-Lys binding moiety using a linker structure containing 2-napththyl-L-alanine and L-tyrosine. We have now designed a molecule that contains mercaptoacetyl-triglutamate chelator for labeling with Tc-99m (designated as BQ0413). The purpose of this study was to evaluate the imaging properties of [Tc-99m]Tc-BQ0413. PSMA-transfected PC3-pip cells were used to evaluate the specificity and affinity of [Tc-99m]Tc-BQ0413 binding in vitro. PC3-pip tumor-bearing BALB/C nu/nu mice were used as an in vivo model. [Tc-99m]Tc-BQ0413 bound specifically to PC3-pip cells with an affinity of 33 +/- 15 pM. In tumor-bearing mice, the tumor uptake of [Tc-99m]Tc-BQ0413 (38 +/- 6 %IA/g in PC3-pip 3 h after the injection of 40 pmol) was dependent on PSMA expression (3 +/- 2 %IA/g and 0.9 +/- 0.3 %IA/g in PSMA-negative PC-3 and SKOV-3 tumors, respectively). We show that both unlabeled BQ0413 and the commonly used binder PSMA-11 enable the blocking of [Tc-99m]Tc-BQ0413 uptake in normal PSMA-expressing tissues without blocking the uptake in tumors. This resulted in an appreciable increase in tumor-to-organ ratios. At the same injected mass (5 nmol), the use of BQ0413 was more efficient in suppressing renal uptake than the use of PSMA-11. In conclusion, [Tc-99m]Tc-BQ0413 is a promising probe for the visualization of PSMA-positive lesions using single-photon emission computed tomography (SPECT).
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3. |
- Bragina, Olga, et al.
(författare)
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Phase I clinical evaluation of 99mTc-labeled Affibody molecule for imaging HER2 expression in breast cancer
- 2023
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Ingår i: Theranostics. - : Ivyspring International Publisher. - 1838-7640. ; 13:14, s. 4858-4871
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Tidskriftsartikel (refereegranskat)abstract
- The determination of tumor human epidermal growth factor receptor type 2 (HER2) status is of increasing importance with the recent approval of more efficacious HER2-targeted treatments. There is a lack of suitable methods for clinical in vivo HER2 expression assessment. Affibody molecules are small affinity proteins ideal for imaging detection of receptors, which are engineered using a small (molecular weight 6.5 kDa) nonimmunoglobulin scaffold. Labeling of Affibody molecules with positron emitters enabled the development of sensitive and specific agents for molecular imaging. The development of probes for SPECT would permit the use of Affibody-based imaging in regions where PET is not available. In this first-in-human study, we evaluated the safety, biodistribution, and dosimetry of the Tc-99m-ZHER2:41071 Affibody molecule developed for SPECT/CT imaging of HER2 expression.Methods: Thirty-one patients with primary breast cancer were enrolled and divided into three cohorts (injected with 500, 1000, or 1500 mu g ZHER2:41071) comprising at least five patients with high (positive) HER2 tumor expression (IHC score 3+ or 2+ and ISH positive) and five patients with low (IHC score 2+ or 1+ and ISH negative) or absent HER2 tumor expression. Patients were injected with 451 +/- 71 MBq Tc-99m-ZHER2:4107. Planar scintigraphy was performed after 2, 4, 6 and 24 h, and SPECT/CT imaging followed planar imaging 2, 4 and 6 h after injection.Results: Injections of Tc-99m-ZHER2:41071 were well tolerated and not associated with adverse events. Normal organs with the highest accumulation were the kidney and liver. The effective dose was 0.019 +/- 0.004 mSv/MBq. Injection of 1000 mu g provided the best standard discrimination between HER2-positive and HER2-low or HER2-negative tumors 2 h after injection (SUVmax 16.9 +/- 7.6 vs. 3.6 +/- 1.4, p < 0.005). The Tc-99m-ZHER2:41071 uptake in HER2-positive lymph node metastases (SUVmax 6.9 +/- 2.4, n = 5) was significantly (p < 0.05) higher than that in HER2-low/negative lymph nodes (SUVmax 3.5 +/- 1.2, n = 4). Tc-99m-ZHER2:41071 visualized hepatic metastases in a patient with liver involvement.Conclusions: Injections of Tc-99m-ZHER2:41071 appear safe and exhibit favorable dosimetry. The protein dose of 1000 mu g provides the best discrimination between HER2-positive and HER2-low/negative expression of HER2 according to the definition used for current HER2-targeting drugs.
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4. |
- 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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5. |
- Ding, Haozhong, et al.
(författare)
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Affibody-Derived Drug Conjugates Targeting HER2 : Effect of Drug Load on Cytotoxicity and Biodistribution
- 2021
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 13:3
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules hold great promise as carriers of cytotoxic drugs for cancer therapy due to their typically high affinity, easy production, and inherent control of the drug molecules' loading and spatial arrangement. Here, the impact of increasing the drug load from one to three on the properties of an affibody drug conjugate targeting the human epidermal growth factor receptor 2 (HER2) was investigated. The affibody carrier was recombinantly expressed as a fusion to an albumin-binding domain (ABD) for plasma half-life extension. One or three cysteine amino acids were placed at the C-terminus to which cytotoxic mcDM1 molecules were conjugated. The resulting drug conjugates, Z(HER2)-ABD-mcDM1 and Z(HER2)-ABD-mcDM1(3), were characterized in vitro, and their biodistribution in mice carrying HER2-overexpressing SKOV3 xenografts was determined. Increasing the drug load from one to three led to a decrease in affinity for HER2, but a significantly more potent cytotoxic effect on SKOV3 cells with high HER2 expression. The difference in cytotoxic effect on other cell lines with high HER2 expression was not significant. In vivo, an increase in drug load led to a 1.45-fold higher amount of cytotoxic mcDM1 delivered to the tumors. The increase in drug load also led to more rapid hepatic clearance, warranting further optimization of the molecular design.
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6. |
- Garousi, Javad, et al.
(författare)
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Comparative evaluation of dimeric and monomeric forms of ADAPT scaffold protein for targeting of HER2-expressing tumours
- 2019
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Ingår i: European journal of pharmaceutics and biopharmaceutics. - : ELSEVIER SCIENCE BV. - 0939-6411 .- 1873-3441. ; 134, s. 37-48
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Tidskriftsartikel (refereegranskat)abstract
- ADAPTs are small engineered non-immunoglobulin scaffold proteins, which have demonstrated very promising features as vectors for radionuclide tumour targeting. Radionuclide imaging of human epidermal growth factor 2 (HER2) expression in vivo might be used for stratification of patients for HER2-targeting therapies. ADAPT6, which specifically binds to HER2, has earlier been shown to have very promising features for in vivo targeting of HER2 expressing tumours. In this study we tested the hypothesis that dimerization of ADAPT6 would increase the apparent affinity to HER2 and accordingly improve tumour targeting. To find an optimal molecular design of dimers, a series of ADAPT dimers with different linkers, -SSSG- (DiADAPT6L1), -(SSSG)(2)- (DiADAPT6L2), and -(SSSG)(3)- (DiADAPT6L3) was evaluated. Dimers in combination with optimal linker lengths demonstrated increased apparent affinity to HER2. The best variants, DiADAPT6L2 and DiADAPT6L3 were site-specifically labelled with In-111 and I-125, and compared with a monomeric ADAPT6 in mice bearing HER2-expressing tumours. Despite higher affinity, both dimers had lower tumour uptake and lower tumour-to-organ ratios compared to the monomer. We conclude that improved affinity of a dimeric form of ADAPT does not compensate the disadvantage of increased size. Therefore, increase of affinity should be obtained by affinity maturation and not by dimerization.
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7. |
- Garousi, Javad, et al.
(författare)
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Radionuclide therapy using ABD-fused ADAPT scaffold protein : Proof of Principle
- 2021
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Ingår i: Biomaterials. - : Elsevier. - 0142-9612 .- 1878-5905. ; 266
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Tidskriftsartikel (refereegranskat)abstract
- Molecular recognition in targeted therapeutics is typically based on immunoglobulins. Development of engineered scaffold proteins (ESPs) has provided additional opportunities for the development of targeted therapies. ESPs offer inexpensive production in prokaryotic hosts, high stability and convenient approaches to modify their biodistribution. In this study, we demonstrated successful modification of the biodistribution of an ESP known as ADAPT (Albumin-binding domain Derived Affinity ProTein). ADAPTs are selected from a library based on the scaffold of ABD (Albumin Binding Domain) of protein G. A particular ADAPT, the ADAPT6, binds to human epidermal growth factor receptor type 2 (HER2) with high affinity. Preclinical and early clinical studies have demonstrated that radiolabeled ADAPT6 can image HER2-expression in tumors with high contrast. However, its rapid glomerular filtration and high renal reabsorption have prevented its use in radionuclide therapy. To modify the biodistribution, ADAPT6 was genetically fused to an ABD. The non-covalent binding to the host's albumin resulted in a 14-fold reduction of renal uptake and appreciable increase of tumor uptake for the best variant, 177Lu-DOTA-ADAPT6-ABD035. Experimental therapy in mice bearing HER2-expressing xenografts demonstrated more than two-fold increase of median survival even after a single injection of 18 MBq 177Lu-DOTA-ADAPT6-ABD035. Thus, a fusion with ABD and optimization of the molecular design provides ADAPT derivatives with attractive targeting properties for radionuclide therapy.
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8. |
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9. |
- 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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10. |
- Leitao, Charles Dahlsson, 1992-, et al.
(författare)
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Conditionally activated affibody-based prodrug targeting EGFR demonstrates improved tumour selectivity
- 2023
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Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 357, s. 185-195
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Tidskriftsartikel (refereegranskat)abstract
- Safety and efficacy of cancer-targeting treatments can be improved by conditional activation enabled by the distinct milieu of the tumour microenvironment. Proteases are intricately involved in tumourigenesis and commonly dysregulated with elevated expression and activity. Design of prodrug molecules with protease -dependent activation has the potential to increase tumour-selective targeting while decreasing exposure to healthy tissues, thus improving the safety profile for patients. Higher selectivity could also allow for adminis-tration of higher doses or use of more aggressive treatment options, leading to higher therapeutic efficacy. We have previously developed an affibody-based prodrug with conditional targeting of EGFR conferred by an anti-idiotypic affibody masking domain (ZB05). We could show that binding to endogenous EGFR on cancer cells in vitro was restored following proteolytic removal of ZB05. In this study we evaluate a novel affibody-based pro -drug design, which incorporates a protease substrate sequence recognized by cancer-associated proteases and demonstrate the potential of this approach for selective tumour-targeting and shielded uptake in healthy tissues in vivo using tumour-bearing mice. This may widen the therapeutic index of cytotoxic EGFR-targeted thera-peutics by decreasing side effects, improving selectivity of drug delivery, and enabling the use of more potent cytotoxic drugs.
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11. |
- 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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12. |
- Liu, Yongsheng, et al.
(författare)
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Preclinical Evaluation of a New Format of Ga-68- and In-111-Labeled Affibody Molecule Z(IGF-1R:4551) for the Visualization of IGF-1R Expression in Malignant Tumors Using PET and SPECT
- 2022
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 14:7
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Tidskriftsartikel (refereegranskat)abstract
- The Insulin-like growth factor-1 receptor (IGF-1R) is a molecular target for several monoclonal antibodies undergoing clinical evaluation as anticancer therapeutics. The non-invasive detection of IGF-1R expression in tumors might enable stratification of patients for specific treatment and improve the outcome of both clinical trials and routine treatment. The affibody molecule Z(IGF-1R:4551) binds specifically to IGF-1R with subnanomolar affinity. The goal of this study was to evaluate the Ga-68 and In-111-labeled affibody construct NODAGA-(HE)(3)-Z(IGF-1R:4551) for the imaging of IGF-1R expression, using PET and SPECT. The labeling was efficient and provided stable coupling of both radionuclides. The two imaging probes, [Ga-68]Ga-NODAGA-(HE)(3)-Z(IGF-1R:4551) and [In-111]In-NODAGA-(HE)(3)-Z(IGF-1R:4551), demonstrated specific binding to IGF-1R-expressing human cancer cell lines in vitro and to IGF-1R-expressing xenografts in mice. Preclinical PET and SPECT/CT imaging demonstrated visualization of IGF-1R-expressing xenografts already one hour after injection. The tumor-to-blood ratios at 3 h after injection were 7.8 +/- 0.2 and 8.0 +/- 0.6 for [Ga-68]Ga-NODAGA-(HE)(3)-Z(IGF-1R:4551) and [In-111]In-NODAGA-(HE)(3)-Z(IGF-1R:4551), respectively. In conclusion, a molecular design of the Z(IGF-1R:4551) affibody molecule, including placement of a (HE)(3)-tag on the N-terminus and site-specific coupling of a NODAGA chelator on the C-terminus, provides a tracer with improved imaging properties for visualization of IGF-1R in malignant tumors, using PET and SPECT.
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13. |
- Lundmark, Fanny, et al.
(författare)
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Reduction of renal activity retention of radiolabeled albumin binding domain-derived affinity proteins using a non-residualizing label strategy compared with a cleavable glycine-leucine-glycine-lysine-linker
- 2024
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Ingår i: Molecular Medicine Reports. - : Spandidos Publications. - 1791-2997 .- 1791-3004. ; 29:2
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Tidskriftsartikel (refereegranskat)abstract
- The feasibility of targeted imaging and therapy using radiolabeled albumin-binding domain-derived affinity proteins (ADAPTs) has been demonstrated. However, high renal uptake of radioactivity limits the maximum tolerated dose. Successful reduction of renal retention of radiolabeled Fab fragments has been demonstrated by incorporating a cleavable linker between the targeting agent and the radiometal chelator. The present study investigated if the introduction of a glycine-leucine-glycine-lysine (GLGK)-linker would reduce the kidney uptake of radiolabeled ADAPT6 and also compared it with the non-residualizing [125I]I-[(4-hydroxyphenyl)ethyl]maleimide ([125I]I-HPEM) labeling strategy. GLGK was site-specifically coupled to human epidermal growth factor receptor 2 (HER2)-targeting ADAPT6. Conjugates without the cleavable linker were used as controls and all constructs were labeled with lutetium-177 (177Lu). [125I]I-HPEM was coupled to ADAPT6 at the C-terminus. Biodistribution of all constructs was evaluated in NMRI mice 4 h after injection. Specific binding to HER2-expressing cells in vitro was demonstrated for all constructs. No significant difference in kidney uptake was observed between the [177Lu]Lu-2,2 ',2",2"'-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid-GLGK-conjugates and the controls. The renal activity of [125I]I-HPEM-ADAPT6 was significantly lower compared with all other constructs. In conclusion, the incorporation of the cleavable GLGK-linker did not result in lower renal retention. Therefore, the present study emphasized that, in order to achieve a reduction of renal retention, alternative molecular design strategies may be required for different targeting agents.
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14. |
- Oroujeni, Maryam, PhD, 1982-, et al.
(författare)
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Affibody-Mediated PNA-Based Pretargeted Cotreatment Improves Survival of Trastuzumab-Treated Mice Bearing HER2-Expressing Xenografts
- 2022
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Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 63:7, s. 1046-1051
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Tidskriftsartikel (refereegranskat)abstract
- Treatment of patients with human epidermal growth factor receptor 2 (HER2)-expressing tumors using the monoclonal antibody trastuzumab increases survival. The Affibody-based peptide nucleic acid (PNA)-mediated pretargeted radionuclide therapy has demonstrated efficacy against HER2-expressing xenografts in mice. Structural studies suggest that Affibody molecules and trastuzumab bind to different epitopes on HER2. The aim of this study was to test the hypothesis that a combination of PNA-mediated pretargeted radionuclide therapy and trastuzumab treatment of HER2-expressing xenografts can extend survival compared with monotherapies. Methods: Mutual interference of the primary pretargeting probe Z(HER2:342)-SR-HP1 and trastuzumab in binding to HER2-expressing cell lines was investigated in vitro. Experimental therapy evaluated the survival of mice bearing HER2-expressing SKOV-3 xenografts after treatment with vehicle, trastuzumab only, pretargeting using Affibody-PNA chimera Z(HER2:342)-SR-HP1 and complementary probe Lu-177-HP2, and combination of trastuzumab and pretargeting. The ethical permit limited the study to 90 d. The animals'weightsweremonitored during the study. After study termination, samples of liver and kidneys were evaluated by a veterinary pathologist for toxicity signs. Results: The presence of a large molar excess of trastuzumab had no influence on the affinity of Z(HER2:342)-SR-HP1 binding to HER2-expressing cells in vitro. The affinity of trastuzumab was not affected by a large excess of Z(HER2:342)-SR-HP1. Themedian survival of mice treated with trastuzumab (75.5 d) was significantly longer than the survival of mice treated with a vehicle (59.5 d). Median survival of mice treated with pretargeting was not reached by day 90. Six mice of 10 in this group survived, and 2 had complete remission. All mice in the combination treatment group survived, and tumors in 7 mice had disappeared at study termination. There was no significant difference between animal weights in the different treatment groups. No significant pathologic alterations were detected in livers and kidneys of treated animals. Conclusion: Treatment of mice bearing HER2-expressing xenografts with the combination of trastuzumab and Affibody-mediated PNA-based radionuclide pretargeting significantly increased survival compared with monotherapies. Cotreatment was not toxic for normal tissues.
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16. |
- Oroujeni, Maryam, PhD, 1982-, et al.
(författare)
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Comparison of approaches for increasing affinity of affibody molecules for imaging of B7-H3 : dimerization and affinity maturation
- 2024
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Ingår i: EJNMMI Radiopharmacy and Chemistry. - : Springer. - 2365-421X. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundRadionuclide molecular imaging can be used to visualize the expression levels of molecular targets. Affibody molecules, small and high affinity non-immunoglobulin scaffold-based proteins, have demonstrated promising properties as targeting vectors for radionuclide tumour imaging of different molecular targets. B7-H3 (CD276), an immune checkpoint protein belonging to the B7 family, is overexpressed in different types of human malignancies. Visualization of overexpression of B7-H3 in malignancies enables stratification of patients for personalized therapies. Affinity maturation of anti-B7-H3 Affibody molecules as an approach to improve the binding affinity and targeting properties was recently investigated. In this study, we tested the hypothesis that a dimeric format may be an alternative option to increase the apparent affinity of Affibody molecules to B7-H3 and accordingly improve imaging contrast.ResultsTwo dimeric variants of anti-B7-H3 Affibody molecules were produced (designated ZAC12*-ZAC12*-GGGC and ZAC12*-ZTaq_3-GGGC). Both variants were labelled with Tc-99m (99mTc) and demonstrated specific binding to B7-H3-expressing cells in vitro. [99mTc]Tc-ZAC12*-ZAC12*-GGGC showed subnanomolar affinity (KD1=0.28 ± 0.10 nM, weight = 68%), which was 7.6-fold higher than for [99mTc]Tc-ZAC12*-ZTaq_3-GGGC (KD=2.1 ± 0.9 nM). Head-to-head biodistribution of both dimeric variants of Affibody molecules compared with monomeric affinity matured SYNT-179 (all labelled with 99mTc) in mice bearing B7-H3-expressing SKOV-3 xenografts demonstrates that both dimers have lower tumour uptake and lower tumour-to-organ ratios compared to the SYNT-179 Affibody molecule.ConclusionThe improved functional affinity by dimerization does not compensate the disadvantage of increased molecular size for imaging purposes.
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17. |
- Oroujeni, Maryam, PhD, 1982-, et al.
(författare)
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Conjugation of cyclodextrin to magnetic Fe3O4 nanoparticles via polydopamine coating for drug delivery
- 2018
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Ingår i: Progress in organic coatings. - : Elsevier BV. - 0300-9440 .- 1873-331X. ; 114, s. 154-161
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Tidskriftsartikel (refereegranskat)abstract
- AbstractIn this study, a novel magnetic nanocarrier for hydrophobic drugs (β-CD–PDA–MNPs) was fabricated using surface coating of Fe3O4 nanoparticles with polydopamine (PDA) followed by functionalization with 6-thio-β-cyclodextrin (6-thio-β-CD). The obtained magnetic nanoparticles were employed to investigate their interactions with diclofenac (DCF) as a model hydrophobic drug. The resulting β-CD–PDA–MNPs were characterized by various methods including transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA), and vibrating sample magnetometry (VSM). The newly fabricated magnetic nanocarrier exhibited considerably higher drug loading capacity as compared for its analogue lacking CD ligands. Moreover, the release profile of DCF from β-CD–PDA–MNPs showed a burst release during the initial 8 h followed by the drug sustained release. Facile coating of magnetic nanoparticles with PDA was therefore a robust synthetic procedure for the conversion of the nanoparticles into a drug vehicle.
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18. |
- Oroujeni, Maryam, PhD, 1982-, et al.
(författare)
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Evaluation of affinity matured Affibody molecules for imaging of the immune checkpoint protein B7-H3
- 2023
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Ingår i: Nuclear Medicine and Biology. - : Elsevier BV. - 0969-8051 .- 1872-9614. ; 124-125
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Tidskriftsartikel (refereegranskat)abstract
- B7-H3 (CD276), an immune checkpoint protein, is a promising molecular target for immune therapy of malignant tumours. Sufficient B7-H3 expression level is a precondition for successful therapy. Radionuclide molecular imaging is a powerful technique for visualization of expression levels of molecular targets in vivo. Use of small radiolabelled targeting proteins would enable high-contrast radionuclide imaging of molecular targets if adequate binding affinity and specificity of an imaging probe could be provided. Affibody molecules, small engineered affinity proteins based on a non-immunoglobulin scaffold, have demonstrated an appreciable potential in radionuclide imaging. Proof-of principle of radionuclide visualization of expression levels of B7-H3 in vivo was demonstrated using the [99mTc]Tc-AC12-GGGC Affibody molecule. We performed an affinity maturation of AC12, enabling selection of clones with higher affinity. Three most promising clones were expressed with a -GGGC (triglycine-cysteine) chelating sequence at the C-terminus and labelled with technetium-99m (99mTc). 99mTc-labelled conjugates bound to B7-H3-expressing cells specifically in vitro and in vivo. Biodistribution in mice bearing B7-H3-expressing SKOV-3 xenografts demonstrated improved imaging properties of the new conjugates compared with the parental variant [99mTc]Tc-AC12-GGGC. [99mTc]Tc-SYNT-179 provided the strongest improvement of tumour-to-organ ratios. Thus, affinity maturation of B7-H3 Affibody molecules could improve biodistribution and targeting properties for imaging of B7-H3-expressing tumours.
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19. |
- Oroujeni, Maryam, PhD, 1982-, et al.
(författare)
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Evaluation of an Affibody-Based Binder for Imaging of Immune Check-Point Molecule B7-H3
- 2022
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Ingår i: Pharmaceutics. - : MDPI. - 1999-4923. ; 14:9
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Tidskriftsartikel (refereegranskat)abstract
- Radionuclide molecular imaging could provide an accurate assessment of the expression of molecular targets in disseminated cancers enabling stratification of patients for specific therapies. B7-H3 (CD276) is a transmembrane protein belonging to the B7 superfamily. This protein is overexpressed in different types of human malignancies and such upregulation is generally associated with a poor clinical prognosis. In this study, targeting properties of an Affibody-based probe, AC12, containing a -GGGC amino acid sequence as a chelator (designated as AC12-GGGC) labelled with technetium-99m (Tc-99m) were evaluated for imaging of B7-H3-expressing tumours. AC12-GGGC was efficiently labelled with Tc-99m. [Tc-99m]Tc-AC12-GGGC bound specifically to B7-H3 expressing cells in vitro with affinities in nanomolar range. In mice bearing B7-H3-expressing xenografts, [Tc-99m]Tc-AC12-GGGC showed tumour uptake of 2.1 +/- 0.5 %ID/g at 2 h after injection. Its clearance from blood, normal organs and tissues was very rapid. This new targeting agent, [Tc-99m]Tc-AC12-GGGC, provided high tumour-to-blood ratio already at 2 h (8.2 +/- 1.9), which increased to 11.0 +/- 0.5 at 4 h after injection. Significantly (p < 0.05) higher tumour-to-liver and higher tumour-to-bone ratios at 2 h in comparison with 4 h after injection were observed. Thus, [Tc-99m]Tc-AC12-GGGC could be a promising candidate for further development.
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20. |
- Oroujeni, Maryam, PhD, 1982-, et al.
(författare)
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Evaluation of Tumor-Targeting Properties of an Antagonistic Bombesin Analogue RM26 Conjugated with a Non-Residualizing Radioiodine Label Comparison with a Radiometal-Labelled Counterpart
- 2019
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Ingår i: Pharmaceutics. - : MDPI. - 1999-4923. ; 11:8
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Tidskriftsartikel (refereegranskat)abstract
- Radiolabelled antagonistic bombesin analogues are successfully used for targeting of gastrin-releasing peptide receptors (GRPR) that are overexpressed in prostate cancer. Internalization of antagonistic bombesin analogues is slow. We hypothesized that the use of a non-residualizing radioiodine label might not affect the tumour uptake but would reduce the retention in normal organs, where radiopharmaceutical would be internalized. To test this hypothesis, tyrosine was conjugated via diethylene glycol linker to N-terminus of an antagonistic bombesin analogue RM26 to form Tyr-PEG(2)-RM26. [In-111]In-DOTA-PEG(2)-RM26 was used as a control with a residualizing label. Tyr-PEG(2)-RM26 was labelled with I-125 with 95% radiochemical purity and retained binding specificity to GRPR. The IC50 values for Tyr-PEG(2)-RM26 and DOTA-PEG(2)-RM26 were 1.7 +/- 0.3 nM and 3.3 +/- 0.5 nM, respectively. The cellular processing of [I-125]I-Tyr-PEG(2)-RM26 by PC-3 cells showed unusually fast internalization. Biodistribution showed that uptake in pancreas and tumour was GRPR-specific for both radioconjugates. Blood clearance of [I-125]I-Tyr-PEG(2)-RM26 was appreciably slower and activity accumulation in all organs was significantly higher than for [In-111]In-DOTA-PEG(2)-RM26. Tumor uptake of [In-111]In-DOTA-PEG(2)-RM26 was significantly higher than for [I-125]I-Tyr-PEG(2)-RM26, resulting in higher tumour-to-organ ratio for [In-111]In-DOTA-PEG(2)-RM26 at studied time points. Incorporation of amino acids with hydrophilic side-chains next to tyrosine might overcome the problems associated with the use of tyrosine as a prosthetic group for radioiodination.
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21. |
- 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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22. |
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23. |
- Oroujeni, Maryam, PhD, 1982-, et al.
(författare)
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Preclinical Evaluation of [Ga-68]Ga-DFO-ZEGFR:2377 : A Promising Affibody-Based Probe for Noninvasive PET Imaging of EGFR Expression in Tumors
- 2018
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Ingår i: Cells. - : MDPI. - 2073-4409. ; 7:9
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Tidskriftsartikel (refereegranskat)abstract
- Radionuclide imaging of epidermal growth factor receptor (EGFR) expression in tumors may stratify patients for EGFR-targeting therapies and predict response or resistance to certain treatments. Affibody molecules, which are nonimmunoglobulin scaffold proteins, have a high potential as probes for molecular imaging. In this study, maleimido derivative of desferrioxamine B (DFO) chelator was site-specifically coupled to the C-terminal cysteine of the anti-EGFR affibody molecule ZEGFR:2377, and the DFO-ZEGFR:2377 conjugate was labeled with the generator-produced positron-emitting radionuclide Ga-68. Stability, specificity of binding to EGFR-expressing cells, and processing of [Ga-68]Ga-DFO-ZEGFR:2377 by cancer cells after binding were evaluated in vitro. In vivo studies were performed in nude mice bearing human EGFR-expressing A431 epidermoid cancer xenografts. The biodistribution of [Ga-68]Ga-DFO-ZEGFR:2377 was directly compared with the biodistribution of [Zr-89]Zr-DFO-ZEGFR:2377. DFO-ZEGFR:2377 was efficiently (isolated yield of 73 +/- 3%) and stably labeled with Ga-68. Binding of [Ga-68]Ga-DFO-ZEGFR:2377 to EGFR-expressing cells in vitro was receptor-specific and proportional to the EGFR expression level. In vivo saturation experiment demonstrated EGFR-specific accumulation of [Ga-68]Ga-DFO-ZEGFR:2377 in A431 xenografts. Compared to [Zr-89]Zr-DFO-ZEGFR:2377, [Ga-68]Ga-DFO-ZEGFR:2377 demonstrated significantly (p < 0.05) higher uptake in tumors and lower uptake in spleen and bones. This resulted in significantly higher tumor-to-organ ratios for [Ga-68]Ga-DFO-ZEGFR:2377. In conclusion, [Ga-68]Ga-DFO-ZEGFR:2377 is a promising probe for imaging of EGFR expression.
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24. |
- Oroujeni, Maryam, PhD, 1982-, et al.
(författare)
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Preclinical Evaluation of Tc-99m-ZHER2:41071, a Second-Generation Affibody-Based HER2-Visualizing Imaging Probe with a Low Renal Uptake
- 2021
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:5
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Tidskriftsartikel (refereegranskat)abstract
- Radionuclide imaging of HER2 expression in tumours may enable stratification of patients with breast, ovarian, and gastroesophageal cancers for HER2-targeting therapies. A first-generation HER2-binding affibody molecule [Tc-99m]Tc-ZHER2:V2 demonstrated favorable imaging properties in preclinical studies. Thereafter, the affibody scaffold has been extensively modified, which increased its melting point, improved storage stability, and increased hydrophilicity of the surface. In this study, a second-generation affibody molecule (designated ZHER2:41071) with a new improved scaffold has been prepared and characterized. HER2-binding, biodistribution, and tumour-targeting properties of [Tc-99m]Tc-labelled ZHER2:41071 were investigated. These properties were compared with properties of the first-generation affibody molecules, [Tc-99m]Tc-ZHER2:V2 and [Tc-99m]Tc-ZHER2:2395. [Tc-99m]Tc-ZHER2:41071 bound specifically to HER2 expressing cells with an affinity of 58 +/- 2 pM. The renal uptake for [Tc-99m]Tc-ZHER2:41071 and [Tc-99m]Tc-ZHER2:V2 was 25-30 fold lower when compared with [Tc-99m]Tc-ZHER2:2395. The uptake in tumour and kidney for [Tc-99m]Tc-ZHER2:41071 and [Tc-99m]Tc-ZHER2:V2 in SKOV-3 xenografts was similar. In conclusion, an extensive re-engineering of the scaffold did not compromise imaging properties of the affibody molecule labelled with Tc-99m using a GGGC chelator. The new probe, [Tc-99m]Tc-ZHER2:41071 provided the best tumour-to-blood ratio compared to HER2-imaging probes for single photon emission computed tomography (SPECT) described in the literature so far. [Tc-99m]Tc-ZHER2:41071 is a promising candidate for further clinical translation studies.
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25. |
- Oroujeni, Maryam, PhD, 1982-
(författare)
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Radiolabelled affibody molecules for imaging EGFR expression in tumours
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Affibody molecules are promising scaffold-based targeting proteins for radionuclide imaging and cancer therapy. This thesis is based on 5 original research articles (Papers I-V), with the primary focus being placed on the optimization of molecular design of EGFR-binding affibody variants for high contrast imaging of epidermal growth factor receptor (EGFR) expression in tumours. The goal of my studies was to investigate the effect of labelling chemistry on the targeting properties of the anti-EGFR affibody molecule ZEGFR:2377 labelled with technetium-99m (99mTc) for single-photon emission computed tomography (SPECT), gallium-68 (68Ga), zirconium-89 (89Zr) and gallium-66 (66Ga) for positron-emission tomography (PET) to select radiolabelled variants providing the best imaging contrast.In Paper I, we showed the feasibility of stably labelling the anti-EGFR affibody molecule ZEGFR:2377 with 99mTc using a peptide-based cysteine-containing chelator and evaluated the imaging of EGFR expression in tumours using the [99mTc]Tc-ZEGFR:2377 affibody molecule.In Paper II, the effect of the composition of cysteine-containing peptide-based chelators on the biodistribution of 99mTc-labelled anti-EGFR affibody molecules was investigated. We evaluated whether the use of glutamate-based chelators improved the imaging properties of 99mTc-labelled ZEGFR:2377.In Paper III, the use of cyclic (FSC) versus noncyclic chelators (DFO) as bifunctional chelators for radiolabelling the anti-EGFR affibody molecule ZEGFR:2377 with 89Zr was investigated. The in vitro and in vivo properties of the resulting DFO- and FSC-ZEGFR:2377 molecules labelled with 89Zr were studied.In Paper IV, the targeting properties of [68Ga]Ga-DFO-ZEGFR:2377 were evaluated and compared directly with the properties of [89Zr]Zr-DFO-ZEGFR:2377 at 3 h after injection.In Paper V, the targeting properties of [66Ga]Ga-DFO-ZEGFR:2377 were evaluated and compared directly with the properties of [68Ga]Ga-DFO-ZEGFR:2377 and [89Zr]Zr-DFO-ZEGFR:2377 at 3 and 24 h after injection, respectively.
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26. |
- Oroujeni, Maryam, PhD, 1982-, et al.
(författare)
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The Use of a Non-Conventional Long-Lived Gallium Radioisotope Ga-66 Improves Imaging Contrast of EGFR Expression in Malignant Tumours Using DFO-ZEGFR:2377 Affibody Molecule
- 2021
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 13:2
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Tidskriftsartikel (refereegranskat)abstract
- Epidermal growth factor receptor (EGFR) is overexpressed in many malignancies. EGFR-targeted therapy extends survival of patients with disseminated cancers. Radionuclide molecular imaging of EGFR expression would make EGFR-directed treatment more personalized and therefore more efficient. A previous study demonstrated that affibody molecule [Ga-68]Ga-DFO-ZEGFR:2377 permits specific positron-emission tomography (PET) imaging of EGFR expression in xenografts at 3 h after injection. We anticipated that imaging at 24 h after injection would provide higher contrast, but this is prevented by the short half-life of Ga-68 (67.6 min). Here, we therefore tested the hypothesis that the use of the non-conventional long-lived positron emitter Ga-66 (T-1/2 = 9.49 h, beta(+) = 56.5%) would permit imaging with higher contrast. Ga-66 was produced by the Zn-66(p,n)Ga-66 nuclear reaction and DFO-ZEGFR:2377 was efficiently labelled with Ga-66 with preserved binding specificity in vitro and in vivo. At 24 h after injection, [Ga-66]Ga-DFO-ZEGFR:2377 provided 3.9-fold higher tumor-to-blood ratio and 2.3-fold higher tumor-to-liver ratio than [Ga-68]Ga-DFO-ZEGFR:2377 at 3 h after injection. At the same time point, [Ga-66]Ga-DFO-ZEGFR:2377 provided 1.8-fold higher tumor-to-blood ratio, 3-fold higher tumor-to-liver ratio, 1.9-fold higher tumor-to-muscle ratio and 2.3-fold higher tumor-to-bone ratio than [Zr-89]Zr-DFO-ZEGFR:2377. Biodistribution data were confirmed by whole body PET combined with magnetic resonance imaging (PET/MRI). The use of the positron emitter Ga-66 for labelling of DFO-ZEGFR:2377 permits PET imaging of EGFR expression at 24 h after injection and improves imaging contrast.
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27. |
- 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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28. |
- Tano, Hanna, et al.
(författare)
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Comparative Evaluation of Novel Lu-177-Labeled PNA Probes for Affibody-Mediated PNA-Based Pretargeting
- 2021
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 13:3
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Tidskriftsartikel (refereegranskat)abstract
- Simple Summary Affibody molecules are small, engineered affinity proteins based on a nonimmunoglobulin scaffold. Affibody-based radionuclide imaging probes have demonstrated excellent tumor targeting. However, the renal clearance of affibody molecules is accompanied by high reabsorption and retention of activity in the kidney, which prevents their use for radionuclide therapy. We have previously shown the feasibility of overcoming the high renal uptake using a pretargeting approach for affibody-mediated therapy based on peptide nucleic acid (PNA) hybridization. In this study, we test the hypothesis that shortening the PNA pretargeting probes would further increase the difference between the accumulation of radiometals in tumor xenografts and in kidneys. A series of novel PNA probes has been designed and evaluated in vitro and in vivo. We have found that a variant containing 9 nucleobases enables a two-fold increase of the tumor-to-kidney dose ratio compared with a variant containing 15 nucleobases. This creates preconditions for more efficient therapy of cancer. Affibody-mediated PNA-based pretargeting is a promising approach to radionuclide therapy of HER2-expressing tumors. In this study, we test the hypothesis that shortening the PNA pretargeting probes would increase the tumor-to-kidney dose ratio. The primary probe Z(HER2:342)-SR-HP15 and the complementary secondary probes HP16, HP17, and HP18, containing 9, 12, and 15 nucleobases, respectively, and carrying a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator were designed, synthesized, characterized in vitro, and labeled with Lu-177. In vitro pretargeting was studied in HER2-expressing SKOV3 and BT474 cell lines. The biodistribution of these novel probes was evaluated in immunodeficient mice bearing SKOV3 xenografts and compared to the previously studied [Lu-177]Lu-HP2. Characterization confirmed the formation of high-affinity duplexes between HP15 and the secondary probes, with the affinity correlating with the length of the complementary PNA sequences. All the PNA-based probes were bound specifically to HER2-expressing cells in vitro. In vivo studies demonstrated HER2-specific uptake of all Lu-177-labeled probes in xenografts in a pretargeting setting. The ratio of cumulated radioactivity in the tumor to the radioactivity in kidneys was dependent on the secondary probe's size and decreased with an increased number of nucleobases. The shortest PNA probe, [Lu-177]Lu-HP16, showed the highest tumor-to-kidney ratio. [Lu-177]Lu-HP16 is the most promising secondary probe for affibody-mediated tumor pretargeting.
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29. |
- Tolmachev, Vladimir, et al.
(författare)
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Direct In Vivo Comparison of Tc-99m-Labeled Scaffold Proteins, DARPin G3 and ADAPT6, for Visualization of HER2 Expression and Monitoring of Early Response for Trastuzumab Therapy
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:23
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Tidskriftsartikel (refereegranskat)abstract
- Non-invasive radionuclide molecular visualization of human epidermal growth factor receptor type 2 (HER2) can provide stratification of patients for HER2-targeting therapy. This method can also enable monitoring of the response to such therapies, thereby making treatment personalized and more efficient. Clinical evaluation in a phase I study demonstrated that injections of two scaffold protein-based imaging probes, [Tc-99m]Tc-(HE)(3)-G3 and [Tc-99m]Tc-ADAPT6, are safe, well-tolerated and cause a low level of radioactivity in healthy tissue. The goal of this preclinical study was to select the best probe for stratification of patients and response monitoring. Biodistribution of both tracers was compared in mice bearing SKOV-3 xenografts with high HER2 expression or MDA-MB-468 xenografts with very low expression. Changes in accumulation of the probes in SKOV-3 tumors 24 h after injection of trastuzumab were evaluated. Both [Tc-99m]Tc-ADAPT6 and [Tc-99m]Tc-(HE)(3)-G3 permitted high contrast imaging of HER2-expressing tumors and a clear discrimination between tumors with high and low HER2 expression. However, [Tc-99m]Tc-ADAPT6 has better preconditions for higher sensitivity and specificity of stratification. On the other hand, [Tc-99m]Tc-(HE)(3)-G3 is capable of detecting the decrease of HER2 expression on response to trastuzumab therapy only 24 h after injection of the loading dose. This indicates that the [Tc-99m]Tc-(HE)(3)-G3 tracer would be better for monitoring early response to such treatment. The results of this study should be considered in planning of further clinical development of HER2 imaging probes.
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30. |
- von Witting, Emma, et al.
(författare)
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Selection of the optimal macrocyclic chelators for labeling with 111In and 68Ga improves contrast of HER2 imaging using engineered scaffold protein ADAPT6
- 2019
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Ingår i: European journal of pharmaceutics and biopharmaceutics. - : Elsevier BV. - 0939-6411 .- 1873-3441. ; 140, s. 109-120
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Tidskriftsartikel (refereegranskat)abstract
- Radionuclide molecular imaging is a promising tool that becomes increasingly important as targeted cancer therapies are developed. To ensure an effective treatment, a molecular stratification of the cancer is a necessity. To accomplish this, visualization of cancer associated molecular abnormalities in vivo by molecular imaging is the method of choice. ADAPTs, a novel type of small protein scaffold, have been utilized to select and develop high affinity binders to different proteinaceous targets. One of these binders, ADAPT6 selectively interacts with human epidermal growth factor 2 (HER2) with low nanomolar affinity and can therefore be used for its in vivo visualization. Molecular design and optimization of labeled anti-HER2 ADAPT has been explored in several earlier studies, showing that small changes in the scaffold affect the biodistribution of the domain. In this study, we evaluate how the biodistribution properties of ADAPT6 is affected by the commonly used maleimido derivatives of the macrocyclic chelators NOTA, NODAGA, DOTA and DOTAGA with the aim to select the best variants for SPECT and PET imaging. The different conjugates were labeled with 111In for SPECT and 68Ga for PET. The acquired data show that the combination of a radionuclide and a chelator for its conjugation has a strong influence on the uptake of ADAPT6 in normal tissues and thereby gives a significant variation in tumor-toorgan ratios. Hence, it was concluded that the best variant for SPECT imaging is 111In-(HE)3DANS-ADAPT6-GSSC-DOTA while the best variant for PET imaging is 68Ga-(HE)3DANS-ADAPT6-GSSC-NODAGA.
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31. |
- 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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32. |
- Westerlund, Kristina, et al.
(författare)
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Radionuclide Therapy of HER2-Expressing Human Xenografts Using Affibody-Based Peptide Nucleic Acid-Mediated Pretargeting : In Vivo Proof of Principle
- 2018
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Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 59:7, s. 1092-1098
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are small proteins engineered using a nonanti-body scaffold. Radiolabeled Affibody molecules are excellent imaging probes, but their application to radionuclide therapy has been prevented by high renal reabsorption. The aim of this study was to test the hypothesis that Affibody-based peptide nucleic acid (PNA)-mediated pretargeted therapy of human epidermal growth factor receptor 2 (HER2)-expressing cancer extends survival without accompanying renal toxicity.Methods: A HER2-targeting Affibody molecule ligated with an AGTCGTGATGTAGTC PNA hybridization probe (Z(HER2:342)-SR-HP1) was used as the primary pretargeting agent. A complementary AGTCGTGATGTAGTC PNA conjugated to the chelator DOTA and labeled with the radionuclide Lu-177 (Lu-177-HP2) was used as the secondary agent. The influence of different factors on pretargeting was investigated. Experimental radionuclide therapy in mice bearing SKOV-3 xenografts was performed in 6 cycles separated by 7 d.Results: Optimal tumor targeting was achieved when 16 MBq/3.5 mu g (0.65 nmol) of Lu-177-HP2 was injected 16 h after injection of 100 mu g (7.7 nmol) of Z(HER2:342)-SR-HP1. The calculated absorbed dose to tumors was 1,075 mGy/MBq, whereas the absorbed dose to kidneys was 206 mGy/MBq and the absorbed dose to blood (surrogate of bone marrow) was 4 mGy/MBq. Survival of mice was significantly longer (P < 0.05) in the treatment group (66 d) than in the control groups treated with the same amount of Z(HER2:342)-SR-HP1 only (37 d), the same amount and activity of Lu-177-HP2 only (32 d), or phosphate-buffered saline (37 d).Conclusion: The studied pretargeting system can deliver an absorbed dose to tumors appreciably exceeding absorbed doses to critical organs, making Affibody-based PNA-mediated pretargeted radionuclide therapy highly attractive.
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33. |
- Xu, Tianqi, et al.
(författare)
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Drug Conjugates Based on a Monovalent Affibody Targeting Vector Can Efficiently Eradicate HER2 Positive Human Tumors in an Experimental Mouse Model
- 2021
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Ingår i: Cancers. - : MDPI. - 2072-6694. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- The human epidermal growth factor receptor 2 (HER2) is frequently overexpressed in a variety of cancers and therapies targeting HER2 are routinely used in the clinic. Recently, small engineered scaffold proteins, such as affibody molecules, have shown promise as carriers of cytotoxic drugs, and these drug conjugates may become complements or alternatives to the current HER2-targeting therapies. Here, we investigated if a monovalent HER2-binding affibody molecule, Z(HER2:2891), fused with a plasma half-life extending albumin binding domain (ABD), may be used as carrier of the cytotoxic maytansine derivate mcDM1. We found that the resulting drug conjugate, Z(HER2:2891)-ABD-E-3-mcDM1, had strong affinity for its cognate molecular targets: HER2 and serum albumin. Z(HER2:2891)-ABD-E-3-mcDM1 displayed potent cytotoxic activity towards cells with high HER2 expression, with IC50 values ranging from 0.6 to 33 nM. In vivo, an unspecific increase in uptake in the liver, imparted by the hydrophobic mcDM1, was counteracted by incorporation of hydrophilic and negatively charged glutamate residues near the site of mcDM1 conjugation. A dose-escalation experiment showed that increasing doses up to 15.1 mg/kg gave a proportional increase in uptake in xenografted HER2-overexpressing SKOV3 tumors, after which the tumors became saturated. Experimental therapy with four once-weekly injection of 10.3 or 15.1 mg/kg led to efficient regression of tumors in all animals and complete regression in some. Weight loss was detected for some animals in the group receiving the highest dose, suggesting that it was close to the maximum tolerated dose. In conclusion, the monovalent HER2-targeting affibody drug conjugate presented herein have potent anti-tumor activity in vivo.
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34. |
- Xu, Tianqi, et al.
(författare)
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Effect of Inter-Domain Linker Composition on Biodistribution of ABD-Fused Affibody-Drug Conjugates Targeting HER2
- 2022
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 14:3, s. 522-
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Tidskriftsartikel (refereegranskat)abstract
- Targeted drug conjugates based on Affibody molecules fused to an albumin-binding domain (ABD) for half-life extension have demonstrated potent anti-tumor activity in preclinical therapeutic studies. Furthermore, optimization of their molecular design might increase the cytotoxic effect on tumors and minimize systemic toxicity. This study aimed to investigate the influence of length and composition of a linker between the human epidermal growth factor receptor 2 (HER2)-targeted affibody molecule (Z(HER2:2891)) and the ABD domain on functionality and biodistribution of affibody-drug conjugates containing a microtubulin inhibitor mertansin (mcDM1) (AffiDCs). Two conjugates, having a trimeric (S(3)G)(3) linker or a trimeric (G(3)S)(3) linker were produced, radiolabeled with Tc-99m(CO)(3), and compared side-by-side in vitro and in vivo with the original Z(HER2:2891)-G(4)S-ABD-mcDM1 conjugate having a monomeric G(4)S linker. Both conjugates with longer linkers had a decreased affinity to HER2 and mouse and human serum albumin in vitro, however, no differences in blood retention were observed in NMRI mice up to 24 h post injection. The use of both (S(3)G)(3) and (G(3)S)(3) linkers reduced liver uptake of AffiDCs by approximately 1.2-fold compared with the use of a G(4)S linker. This finding provides important insights into the molecular design for the development of targeted drug conjugates with reduced hepatic uptake.
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35. |
- Yin, Wen, 1993-, et al.
(författare)
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Comparison of HER2-targeted affibody conjugates loaded with auristatin-and maytansine-derived drugs
- 2023
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Ingår i: Journal of Controlled Release. - : Elsevier. - 0168-3659 .- 1873-4995. ; 355, s. 515-527
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Tidskriftsartikel (refereegranskat)abstract
- Treatment with antibody drug conjugates targeting receptors over-expressed on cancer cells is well established for clinical use in several types of cancer, however, resistance often occurs motivating the development of novel drugs. We have recently investigated a drug conjugate consisting of an affibody molecule targeting the human epidermal growth factor receptor 2 (HER2), fused to an albumin-binding domain (ABD) for half-life extension, loaded with the cytotoxic maytansine derivative DM1. In this study, we investigated the impact of the cytotoxic payload on binding properties, cytotoxicity and biodistribution by comparing DM1 with the auristatins MMAE and MMAF, as part of the drug conjugate. All constructs had specific and high affinity binding to HER2, human and mouse albumins with values in the low- to sub-nM range. ZHER2-ABD-mcMMAF demonstrated the most potent cytotoxic effect on several HER2-over-expressing cell lines. In an experimental therapy study, the MMAFbased conjugate provided complete tumor regression in 50% of BALB/c nu/nu mice bearing HER2-overexpressing SKOV3 tumors at a 2.9 mg/kg dose, while the same dose of ZHER2-ABD-mcDM1 provided only a moderate anti-tumor effect. A comparison with the non-targeting ZTaq-ABD-mcMMAF control demonstrated HER2-targeting specificity. In conclusion, a combination of potent cytotoxicity in vitro, with minimal uptake in normal organs in vivo, and efficient delivery to tumors provided a superior anti-tumor effect of ZHER2-ABDmcMMAF, while maintaining a favorable toxicity profile with no observed adverse effects.
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36. |
- Yin, Wen, 1993-, et al.
(författare)
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The Influence of Domain Permutations of an Albumin-Binding Domain-Fused HER2-Targeting Affibody-Based Drug Conjugate on Tumor Cell Proliferation and Therapy Efficacy
- 2021
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 13:11, s. 1974-1974
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Tidskriftsartikel (refereegranskat)abstract
- Human epidermal growth factor receptor 2 (HER2) is a clinically validated target for breast cancer therapy. Previously, a drug-fused HER2-targeting affinity protein construct successfully extended the survival of mice bearing HER2-expressing xenografts. The aim of this study was to evaluate the influence of the number and positioning of the protein domains in the drug conjugate. Seven HER2-targeting affibody-based constructs, including one or two affibody molecules (Z) with or without an albumin-binding domain (ABD), namely Z, Z-ABD, ABD-Z, Z-Z, Z-Z-ABD, Z-ABD-Z, and ABD-Z-Z, were evaluated on their effects on cell growth, in vivo targeting, and biodistribution. The biodistribution study demonstrated that the monomeric constructs had longer blood retention and lower hepatic uptake than the dimeric ones. A dimeric construct, specifically ABD-Z-Z, could stimulate the proliferation of HER2 expressing SKOV-3 cells in vitro and the growth of tumors in vivo, whereas the monomeric construct Z-ABD could not. These two constructs demonstrated a therapeutic effect when coupled to mcDM1; however, the effect was more pronounced for the non-stimulating Z-ABD. The median survival of the mice treated with Z-ABD-mcDM1 was 63 days compared to the 37 days for those treated with ABD-Z-Z-mcDM1 or for the control animals. Domain permutation of an ABD-fused HER2-targeting affibody-based drug conjugate significantly influences tumor cell proliferation and therapy efficacy. The monomeric conjugate Z-ABD is the most promising format for targeted delivery of the cytotoxic drug DM1.
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