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Vorobyeva, AnzhelikaORCID iD iconorcid.org/0000-0002-4778-3909
Publications (10 of 30) Show all publications
Deyev, S. M., Vorobyeva, A., Schulga, A., Abouzayed, A., Günther, T., Garousi, J., . . . Tolmachev, V. (2020). Effect of a radiolabel biochemical nature on tumor-targeting properties of EpCAM-binding engineered scaffold protein DARPin Ec1. International Journal of Biological Macromolecules, 145, 216-225
Open this publication in new window or tab >>Effect of a radiolabel biochemical nature on tumor-targeting properties of EpCAM-binding engineered scaffold protein DARPin Ec1
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2020 (English)In: International Journal of Biological Macromolecules, ISSN 0141-8130, E-ISSN 1879-0003, Vol. 145, p. 216-225Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
ELSEVIER, 2020
Keywords
Designed ankyrin repeat protein, EpCAM, Labeling, Structure-property relationship, Biodistribution
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-407362 (URN)10.1016/j.ijbiomac.2019.12.147 (DOI)000515196500023 ()31863835 (PubMedID)
Funder
Swedish Cancer Society, CAN 2015/350Swedish Cancer Society, 2017/425Swedish Research Council, 2015-02353Swedish Research Council, 2015-02509Knut and Alice Wallenberg Foundation
Available from: 2020-05-29 Created: 2020-05-29 Last updated: 2020-05-29Bibliographically approved
Altai, M., Garousi, J., Rinne, S. S., Schulga, A., Deyev, S. & Vorobyeva, A. (2020). On the prevention of kidney uptake of radiolabeled DARPins. EJNMMI Research, 10, Article ID 7.
Open this publication in new window or tab >>On the prevention of kidney uptake of radiolabeled DARPins
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2020 (English)In: EJNMMI Research, ISSN 2191-219X, E-ISSN 2191-219X, Vol. 10, article id 7Article in journal (Refereed) Published
Abstract [en]

Background: Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins (14-18 kDa) that demonstrated promising tumor-targeting properties in preclinical studies. However, high renal accumulation of activity for DARPins labeled with residualizing labels is a limitation for targeted radionuclide therapy. A better understanding of the mechanisms behind the kidney uptake of DARPins could aid the development of strategies to reduce it. In this study, we have investigated whether the renal uptake of [Tc-99m]Tc(CO)(3)-G3 DARPin could be reduced by administration of compounds that act on various parts of the reabsorption system in the kidney.

Results: Co-injection of lysine or Gelofusine was not effective for the reduction of kidney uptake of [Tc-99m]Tc(CO)(3)-G3. Administration of sodium maleate before the injection of [Tc-99m]Tc(CO)(3)-G3 reduced the kidney-associated activity by 60.4 +/- 10.3%, while administration of fructose reduced it by 46.9 +/- 7.6% compared with the control. The decrease in the kidney uptake provided by sodium maleate was also observed for [Tc-99m]Tc(CO)(3)-9_29 DARPin. Preinjection of colchicine, probenecid, mannitol, or furosemide had no effect on the kidney uptake of [Tc-99m]Tc(CO)(3)-G3. Kidney autoradiography showed mainly cortical accumulation of activity for all studied groups.

Conclusion: Common clinical strategies were not effective for the reduction of kidney uptake of [Tc-99m]Tc(CO)(3)-G3. Both fructose and maleate lower the cellular ATP level in the proximal tubule cells and their reduction of the kidney reuptake indicates the involvement of an ATP-driven uptake mechanism. The decrease provided by maleate for both G3 and 9_29 DARPins indicates that their uptake proceeds through a mechanism independent of DARPin structure and binding site composition.

Place, publisher, year, edition, pages
SPRINGEROPEN, 2020
Keywords
DARPin, Radiolabel, Kidney, Reabsorption, Renal uptake
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-407141 (URN)10.1186/s13550-020-0599-1 (DOI)000511990100001 ()32020413 (PubMedID)
Funder
Swedish Society for Medical Research (SSMF)
Available from: 2020-03-19 Created: 2020-03-19 Last updated: 2020-03-19Bibliographically approved
Garousi, J., Huizing, F. J., Vorobyeva, A., Mitran, B., Andersson, K. G., Leitao, C. D., . . . Tolmachev, V. (2019). Comparative evaluation of affibody- and antibody fragments-based CAIX imaging probes in mice bearing renal cell carcinoma xenografts. Scientific Reports, 9, Article ID 14907.
Open this publication in new window or tab >>Comparative evaluation of affibody- and antibody fragments-based CAIX imaging probes in mice bearing renal cell carcinoma xenografts
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 14907Article in journal (Refereed) Published
Abstract [en]

Carbonic anhydrase IX (CAIX) is a cancer-associated molecular target for several classes of therapeutics. CAIX is overexpressed in a large fraction of renal cell carcinomas (RCC). Radionuclide molecular imaging of CAIX-expression might offer a non-invasive methodology for stratification of patients with disseminated RCC for CAIX-targeting therapeutics. Radiolabeled monoclonal antibodies and their fragments are actively investigated for imaging of CAIX expression. Promising alternatives are small non-immunoglobulin scaffold proteins, such as affibody molecules. A CAIX-targeting affibody ZCAIX:2 was re-designed with the aim to decrease off-target interactions and increase imaging contrast. The new tracer, DOTA-HE3-ZCAIX:2, was labeled with In-111 and characterized in vitro. Tumor-targeting properties of [In-111]In-DOTA-HE3-ZCAIX:2 were compared head-to-head with properties of the parental variant, [(99)mTc]Tc(CO)(3)-HE3-ZCAIX:2, and the most promising antibody fragment-based tracer, [In-111]In-DTPA-G250(Fab')(2), in the same batch of nude mice bearing CAIX-expressing RCC xenografts. Compared to the (99)mTc-labeled parental variant, [In-111]In-DOTA-HE3-ZCAIX:2 provides significantly higher tumor-to-lung, tumor-to-bone and tumor-to-liver ratios, which is essential for imaging of CAIX expression in the major metastatic sites of RCC. [In-111]In-DOTA-HE3-ZCAIX:2 offers significantly higher tumor-to-organ ratios compared with [In-111]In-G250(Fab']2. In conclusion, [In-111]In-DOTA-HE3-ZCAIX:2 can be considered as a highly promising tracer for imaging of CAIX expression in RCC metastases based on our results and literature data.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-396709 (URN)10.1038/s41598-019-51445-w (DOI)000490702200022 ()31624303 (PubMedID)
Funder
Swedish Cancer Society, CAN 2018/436Swedish Cancer Society, 2017/425Swedish Research Council, 2015-02353Swedish Research Council, 2015-02509
Available from: 2019-11-08 Created: 2019-11-08 Last updated: 2019-11-08Bibliographically approved
Garousi, J., Lindbo, S., Borin, J., von Witting, E., Vorobyeva, A., Oroujeni, M., . . . Hober, S. (2019). Comparative evaluation of dimeric and monomeric forms of ADAPT scaffold protein for targeting of HER2-expressing tumours. European journal of pharmaceutics and biopharmaceutics, 134, 37-48
Open this publication in new window or tab >>Comparative evaluation of dimeric and monomeric forms of ADAPT scaffold protein for targeting of HER2-expressing tumours
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2019 (English)In: European journal of pharmaceutics and biopharmaceutics, ISSN 0939-6411, E-ISSN 1873-3441, Vol. 134, p. 37-48Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2019
Keywords
ADAPT, HER2, Dimer, Radionuclide molecular imaging, Indium-111, Iodine-125
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-376893 (URN)10.1016/j.ejpb.2018.11.004 (DOI)000456225000004 ()30408518 (PubMedID)
Funder
Swedish Research Council, 2015-02353Swedish Research Council, 2015-02509VINNOVA, 2015-02509Swedish Cancer Society, CAN 2015/350Swedish Cancer Society, 2017/425
Available from: 2019-02-13 Created: 2019-02-13 Last updated: 2019-02-13Bibliographically approved
Deyev, S., Vorobyeva, A., Schulga, A., Proshkina, G., Guler, R., Lofblom, J., . . . Tolmachev, V. (2019). Comparative Evaluation of Two DARPin Variants: Effect of Affinity, Size, and Label on Tumor Targeting Properties. Molecular Pharmaceutics, 16(3), 995-1008
Open this publication in new window or tab >>Comparative Evaluation of Two DARPin Variants: Effect of Affinity, Size, and Label on Tumor Targeting Properties
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2019 (English)In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 16, no 3, p. 995-1008Article in journal (Refereed) Published
Abstract [en]

Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins that can be selected for binding to desirable molecular targets. High affinity and small size of DARPins render them promising probes for radionuclide molecular imaging. However, detailed knowledge on many factors influencing their imaging properties is still lacking. We have evaluated two human epidermal growth factor 2 (HER2)-specific DARPins with different size and binding properties. DARPins 9_29-H-6 and G3-H-6 were radiolabeled with iodine-125 and tricarbonyl technetium-99m and evaluated in vitro. A side-by-side comparison of biodistribution and tumor targeting was performed. HER2-specific tumor accumulation of G3-H-6 was demonstrated. A combination of smaller size and higher affinity resulted in a higher tumor uptake of G3-H-6 in comparison to 9_29-H6. Technetium-99m labeled G3-H-6 demonstrated a better biodistribution profile than 9_29-H-6, with several-fold lower uptake in liver. Radioiodinated G3-H-6 showed the best tumor-to-organ ratios. The combined effect of affinity, molecular weight, scaffold composition, and nonresidualizing properties of iodine label provided radioiodinated G3-H-6 with high clinical potential for imaging of HER2.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2019
Keywords
DARPin, targeting, radionuclide, imaging, I-12S, Tc-99m
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-380491 (URN)10.1021/acs.molpharmaceut.8b00922 (DOI)000460600400008 ()30608701 (PubMedID)
Funder
Swedish Research Council, 2015-02353Swedish Research Council, 2015-02509Vinnova, 2016-04060Swedish Cancer Society, CAN 2015/350Swedish Cancer Society, 2017/425Swedish Society for Medical Research (SSMF)
Available from: 2019-03-28 Created: 2019-03-28 Last updated: 2019-03-28Bibliographically approved
Vorobyeva, A., Schulga, A., Konovalova, E., Güler, R., Mitran, B., Garousi, J., . . . Tolmachev, V. (2019). Comparison of tumor-targeting properties of directly and indirectly radioiodinated designed ankyrin repeat protein (DARPin) G3 variants for molecular imaging of HER2. International Journal of Oncology, 54(4), 1209-1220
Open this publication in new window or tab >>Comparison of tumor-targeting properties of directly and indirectly radioiodinated designed ankyrin repeat protein (DARPin) G3 variants for molecular imaging of HER2
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2019 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 54, no 4, p. 1209-1220Article in journal (Refereed) Published
Abstract [en]

Evaluation of human epidermal growth factor receptor 2 (HER2) expression levels in breast and gastroesophageal cancer is used for the stratification of patients for HER2-targeting therapies. The use of radionuclide molecular imaging may facilitate such evaluation in a non-invasive way. Designed ankyrin repeat proteins (DARPins) are engineered scaffold proteins with high potential as probes for radionuclide molecular imaging. DARPin G3 binds with high affinity to HER2 and may be used to visualize this important therapeutic target. Studies on other engineered scaffold proteins have demonstrated that selection of the optimal labeling approach improves the sensitivity and specificity of radionuclide imaging. The present study compared two methods of labeling G3, direct and indirect radioiodination, to select an approach providing the best imaging contrast. G3-H-6 was labeled with iodine-124, iodine-125 and iodine-131 using a direct method. A novel construct bearing a C-terminal cysteine, G3-GGGC, was site-specifically labeled using [I-125]I-iodo-[(4-hydroxyphenyl)ethyl]maleimide (HPEM). The two radiolabeled G3 variants preserved binding specificity and high affinity to HER2-expressing cells. The specificity of tumor targeting in vivo was demonstrated. Biodistribution comparison of [I-131]I-G3-H-6 and [I-125]I-HPEM-G3-GGGC in mice, bearing HER2-expressing SKOV3 xenografts, demonstrated an appreciable contribution of hepatobiliary excretion to the clearance of [I-125]I-HPEM-G3-GGGC and a decreased tumor uptake compared to [I-131]I-G3-H-6. The direct label provided higher tumor-to-blood and tumor-to-organ ratios compared with the indirect label at 4 h post-injection. The feasibility of high contrast PET/CT imaging of HER2 expression in SKOV3 xenografts in mice using [I-124]I-G3-H-6 was demonstrated. In conclusion, direct radioiodination is the preferable approach for labeling DARPin G3 with iodine-123 and iodine-124 for clinical single photon emission computed tomography and positron emission tomography imaging.

Place, publisher, year, edition, pages
SPANDIDOS PUBL LTD, 2019
Keywords
DARPin, HER2, imaging, radionuclide, iodine, radioiodination
National Category
Radiology, Nuclear Medicine and Medical Imaging Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-380422 (URN)10.3892/ijo.2019.4712 (DOI)000461097600006 ()
Funder
Swedish Research Council, 2015-02353Swedish Research Council, 2015-02509Vinnova, 2016-04060Swedish Cancer Society, 2015/350Swedish Cancer Society, 2017/425
Available from: 2019-04-02 Created: 2019-04-02 Last updated: 2019-04-02Bibliographically approved
Rosestedt, M., Andersson, K. G., Rinne, S. S., Leitao, C. D., Mitran, B., Vorobyeva, A., . . . Orlova, A. (2019). Improved contrast of affibody-mediated imaging of HER3 expression in mouse xenograft model through co-injection of a trivalent affibody for in vivo blocking of hepatic uptake. Scientific Reports, 9, Article ID 6779.
Open this publication in new window or tab >>Improved contrast of affibody-mediated imaging of HER3 expression in mouse xenograft model through co-injection of a trivalent affibody for in vivo blocking of hepatic uptake
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 6779Article in journal (Refereed) Published
Abstract [en]

Human epidermal growth factor receptor type 3 (HER3) plays a crucial role in the progression of many cancer types. In vivo radionuclide imaging could be a reliable method for repetitive detection of HER3-expression in tumors. The main challenge of HER3-imaging is the low expression in tumors together with endogenous receptor expression in normal tissues, particularly the liver. A HER3-targeting affibody molecule labeled with radiocobalt via a NOTA chelator [Co-57]Co-NOTA-Z(08699) has demonstrated the most favorable biodistribution profile with the lowest unspecific hepatic uptake and high activity uptake in tumors. We hypothesized that specific uptake of labeled affibody monomer might be selectively blocked in the liver but not in tumors by a co-injection of non-labeled corresponding trivalent affibody (Z(08699))(3). Biodistribution of [Co-57]Co-NOTA-Z(08699) and [In-111]ln-DOTA-(Z(08699))(3) was studied in BxPC-3 xenografted mice. [Co-57]Co-NOTA-Z(08699) was co-injected with unlabeled trivalent affibody DOTA-(Z(08699))(3) at different monomer:trimer molar ratios. HER3-expression in xenografts was imaged using [Co-57]Co-NOTA-Z(08699) and [Co-57]Co-NOTA-Z(08699): DOTA-(Z(08699))(3). Hepatic activity uptake of [Co-57] Co-NOTA-Z(08699): DOTA-(Z(08699))(3) decreased with increasing monomer:trimer molar ratio. The tumor activity uptake and tumor-to-liver ratios were the highest for the 1:3 ratio. SPECT/CT images confirmed the biodistribution data. Imaging of HER3 expression can be improved by co-injection of a radiolabeled monomeric affi body-based imaging probe together with a trivalent affibody.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-386176 (URN)10.1038/s41598-019-43145-2 (DOI)000466358700048 ()31043683 (PubMedID)
Funder
Swedish Research Council, 621-2012-5236Swedish Research Council, 2015-02509Swedish Research Council, 2015-02353Vinnova, 2016-04060Knut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Cancer Society, CAN2016-463Swedish Cancer Society, CAN2014-474Swedish Cancer Society, CAN 2017/425Swedish Cancer Society, CAN2015/350Swedish Cancer Society, CAN 2018/436
Available from: 2019-06-20 Created: 2019-06-20 Last updated: 2019-06-20Bibliographically approved
Ding, H., Altai, M., Rinne, S. S., Vorobyeva, A., Tolmachev, V., Gräslund, T. & Orlova, A. (2019). Incorporation of a Hydrophilic Spacer Reduces Hepatic Uptake of HER2-Targeting Affibody-DM1 Drug Conjugates. Cancers, 11(8), Article ID 1168.
Open this publication in new window or tab >>Incorporation of a Hydrophilic Spacer Reduces Hepatic Uptake of HER2-Targeting Affibody-DM1 Drug Conjugates
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2019 (English)In: Cancers, ISSN 2072-6694, Vol. 11, no 8, article id 1168Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are small affinity-engineered scaffold proteins which can be engineered to bind to desired targets. The therapeutic potential of using an affibody molecule targeting HER2, fused to an albumin-binding domain (ABD) and conjugated with the cytotoxic maytansine derivate MC-DM1 (AffiDC), has been validated. Biodistribution studies in mice revealed an elevated hepatic uptake of the AffiDC, but histopathological examination of livers showed no major signs of toxicity. However, previous clinical experience with antibody drug conjugates have revealed a moderateto high-grade hepatotoxicity in treated patients, which merits efforts to also minimize hepatic uptake of the AffiDCs. In this study, the aim was to reduce the hepatic uptake of AffiDCs and optimize their in vivo targeting properties. We have investigated if incorporation of hydrophilic glutamate-based spacers adjacent to MC-DM1 in the AffiDC, (Z(HER2:2891))(2) -ABD-MC-DM1, would counteract the hydrophobic nature of MC-DM1 and, hence, reduce hepatic uptake. Two new AffiDCs including either a triglutamate-spacer-, (Z(HER2:2891))(2)-ABD-E-3-MC-DM1, or a hexaglutamate-spacer-, (Z(HER2:2891))(2)-ABD-E-6-MC-DM1 next to the site of MC-DM1 conjugation were designed. We radiolabeled the hydrophilized AffiDCs and compared them, both in vitro and in vivo, with the previously investigated (Z(HER2:2891))(2)-ABD-MC-DM1 drug conjugate containing no glutamate spacer. All three AffiDCs demonstrated specific binding to HER2 and comparable in vitro cytotoxicity. A comparative biodistribution study of the three radiolabeled AffiDCs showed that the addition of glutamates reduced drug accumulation in the liver while preserving the tumor uptake. These results confirmed the relation between DM1 hydrophobicity and liver accumulation. We believe that the drug development approach described here may also be useful for other affinity protein-based drug conjugates to further improve their in vivo properties and facilitate their clinical translatability.

Place, publisher, year, edition, pages
MDPI, 2019
Keywords
affibody, drug conjugates, hepatic uptake, DM1
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-394647 (URN)10.3390/cancers11081168 (DOI)000484438000128 ()31416167 (PubMedID)
Funder
Swedish Research Council, 2015-02509Swedish Research Council, 2015-02353Swedish Cancer Society, CAN 2018/824Swedish Cancer Society, CAN 2017/425Swedish Cancer Society, CAN2015/350Vinnova, 2016-04060Vinnova, 2019-00104Swedish Society for Medical Research (SSMF)
Available from: 2019-10-17 Created: 2019-10-17 Last updated: 2019-10-17Bibliographically approved
Vorobyeva, A., Schulga, A., Rinne, S. S., Günther, T., Orlova, A., Deyev, S. & Tolmachev, V. (2019). Indirect Radioiodination of DARPin G3 Using N-succinimidyl-Para-Iodobenzoate Improves the Contrast of HER2 Molecular Imaging. International Journal of Molecular Sciences, 20(12), Article ID 3047.
Open this publication in new window or tab >>Indirect Radioiodination of DARPin G3 Using N-succinimidyl-Para-Iodobenzoate Improves the Contrast of HER2 Molecular Imaging
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2019 (English)In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, no 12, article id 3047Article in journal (Refereed) Published
Abstract [en]

Radionuclide molecular imaging of human epidermal growth factor receptor 2 (HER2) in breast and gastroesophageal cancer might be used to stratify patients for HER2-targeted therapy as well as monitor treatment response and disease progression. Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins with favorable properties for molecular imaging. Herein we compared two methods for labeling the anti-HER2 DARPin (HE)(3)-G3, direct and indirect radioiodination. We hypothesized that the use of N-succinimidyl-para-iodobenzoate (SPIB) for radioiodination would facilitate the clearance of radiometabolites and improve the contrast of imaging. Both radiolabeled (HE)(3)-G3 variants preserved their binding specificity and high affinity to HER2-expressing cells. The specificity of tumor targeting in vivo was also demonstrated. A biodistribution comparison of [I-125]I-(HE)(3)-G3 and [I-125]I-PIB-(HE)(3)-G3, in mice bearing HER2 expressing SKOV3 xenografts, showed rapid clearance of [I-125]I-PIB-(HE)(3)-G3 from normal organs and tissues and low accumulation of activity in organs with NaI-symporter expression. Both radiolabeled (HE)(3)-G3 variants had equal tumor uptake. Consequently, the indirect label provided higher tumor-to-blood and tumor-to-organ ratios compared with the direct label. Comparative Single Photon Emission Computed Tomography (SPECT)/CT imaging of HER2 expression in SKOV3 xenografts, using both radiolabeled DARPins, demonstrated the superior imaging contrast of the indirect label. Indirect radioiodination of (HE)(3)-G3 using SPIB could be further applied for SPECT and PET imaging with iodine-123 and iodine-124.

Place, publisher, year, edition, pages
MDPI, 2019
Keywords
DARPin, HER2, imaging, radionuclide, iodine, radioiodination
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-390692 (URN)10.3390/ijms20123047 (DOI)000473756000197 ()31234471 (PubMedID)
Funder
Swedish Research Council, 2015-02353Swedish Research Council, 2015-02509Swedish Cancer Society, CAN 2015/350Swedish Cancer Society, 2017/425
Available from: 2019-08-14 Created: 2019-08-14 Last updated: 2019-08-14Bibliographically approved
Dahlsson Leitao, C., Rinne, S. S., Mitran, B., Vorobyeva, A., Andersson, K. G., Tolmachev, V., . . . Orlova, A. (2019). Molecular Design of HER3-Targeting Affibody Molecules: Influence of Chelator and Presence of HEHEHE-Tag on Biodistribution of 68Ga-Labeled Tracers. International Journal of Molecular Sciences, 20(5), Article ID 1080.
Open this publication in new window or tab >>Molecular Design of HER3-Targeting Affibody Molecules: Influence of Chelator and Presence of HEHEHE-Tag on Biodistribution of 68Ga-Labeled Tracers
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2019 (English)In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, no 5, article id 1080Article in journal (Refereed) Published
Abstract [en]

Affibody-based imaging of HER3 is a promising approach for patient stratification. We investigated the influence of a hydrophilic HEHEHE-tag ((HE)3-tag) and two different gallium-68/chelator-complexes on the biodistribution of Z08698 with the aim to improve the tracer for PET imaging. Affibody molecules (HE)3-Z08698-X and Z08698-X (X = NOTA, NODAGA) were produced and labeled with gallium-68. Binding specificity and cellular processing were studied in HER3-expressing human cancer cell lines BxPC-3 and DU145. Biodistribution was studied 3 h p.i. in Balb/c nu/nu mice bearing BxPC-3 xenografts. Mice were imaged 3 h p.i. using microPET/CT. Conjugates were stably labeled with gallium-68 and bound specifically to HER3 in vitro and in vivo. Association to cells was rapid but internalization was slow. Uptake in tissues, including tumors, was lower for (HE)3-Z08698-X than for non-tagged variants. The neutral [68Ga]Ga-NODAGA complex reduced the hepatic uptake of Z08698 compared to positively charged [68Ga]Ga-NOTA-conjugated variants. The influence of the chelator was more pronounced in variants without (HE)3-tag. In conclusion, hydrophilic (HE)3-tag and neutral charge of the [68Ga]Ga-NODAGA complex promoted blood clearance and lowered hepatic uptake of Z08698. [68Ga]Ga-(HE)3-Z08698-NODAGA was considered most promising, providing the lowest blood and hepatic uptake and the best imaging contrast among the tested variants.

Keywords
HER3, affibody, NOTA, NODAGA, molecular imaging, gallium-68, PET
National Category
Biochemistry and Molecular Biology Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-381825 (URN)10.3390/ijms20051080 (DOI)000462542300079 ()30832342 (PubMedID)
Funder
Swedish Research Council, 2015-02509Swedish Research Council, 2015-02353Swedish Research Council, 2012-05236Vinnova, 2016/04060Swedish Cancer Society, CAN2014/474Swedish Cancer Society, CAN 2017/425Swedish Cancer Society, CAN2015/350Swedish Cancer Society, CAN 2018/436Swedish Cancer Society, CAN2017/649Swedish Cancer Society, CAN2016/463
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Available from: 2019-04-18 Created: 2019-04-18 Last updated: 2019-04-18Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0002-4778-3909

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