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Mitran, B., Andersson, K. G., Lindström, E., Garousi, J., Rosestedt, M., Tolmachev, V., . . . Löfblom, J. (2019). Affibody-mediated imaging of EGFR expression in prostate cancer using radiocobalt-labeled DOTA-Z(EGFR:2377). Oncology Reports, 41(1), 534-542
Open this publication in new window or tab >>Affibody-mediated imaging of EGFR expression in prostate cancer using radiocobalt-labeled DOTA-Z(EGFR:2377)
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2019 (English)In: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 41, no 1, p. 534-542Article in journal (Refereed) Published
Abstract [en]

The epidermal growth factor receptor (EGFR) is often overexpressed during prostate cancer (PCa) progression towards androgen-independence after hormone therapy, but the overexpression is lower than in other types of cancers. Despite the low expression, EGFR has emerged as a promising therapeutic target for patients with castration-resistant PCa. Non-invasive methods for determination of EGFR expression in PCa can serve for patient stratification and therapy response monitoring. Radionuclide imaging probes based on affibody molecules (7 kDa) provide high contrast imaging of cancer-associated molecular targets. We hypothesized that the anti-EGFR affibody molecule DOTA-Z(EGFR:2377) labeled with Co-55 (positron-emitter, T1/2=17.5 h) would enable imaging of EGFR expression in PCa xenografts. The human PCa cell line DU-145 was used for in vitro and in vivo experiments and Co-57 was used as a surrogate for Co-55 in the present study. Binding of Co-57-DOTA-Z(EGFR:2377) to EGFR-expressing xenografts was saturable with anti-EGFR monoclonal antibody cetuximab, which would motivate the use of this tracer for monitoring the receptor occupancy during treatment. A significant dose-dependent difference in radioactivity accumulation in tumors and normal organs was observed when the biodistribution was studied 3 h after the injection of 10 and 35 mu g of Co-57-DOTA-Z(EGFR:2377): At lower doses the tumor uptake was 2-fold higher although tumor-to-organ ratios were not altered. For clinically relevant organs for PCa, tumor-to-organ ratios increased with time, and at 24 h pi were 2.2 +/- 0.5 for colon, 7 +/- 2 for muscle, and 4.0 +/- 0.7 for bones. Small animal SPECT/CT images confirmed the capacity of radiocobalt labeled DOTA-Z(EGFR:2377) to visualize EGFR expression in PCa. In conclusion, the present study demonstrated the feasibility of using the radiocobalt labeled anti-EGFR affibody conjugate Z(EGFR:2377) as an imaging agent for in vivo visualization of low EGFR-expressing tumors, like PCa, and for monitoring of receptor occupancy during cetuximab therapy as well as the importance of optimal dosing in order to achieve higher sensitivity molecular imaging.

Keywords
prostate cancer, molecular imaging, cobalt, affibody molecule, HER1, EGFR
National Category
Cancer and Oncology Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-372751 (URN)10.3892/or.2018.6792 (DOI)000452152300051 ()30320363 (PubMedID)
Funder
Swedish Research Council, 621-2012-5236Swedish Research Council, 2015-02509Swedish Research Council, 2015-02353VINNOVA, 2016-04060VINNOVA, 2017-02015Knut and Alice Wallenberg Foundation
Available from: 2019-01-15 Created: 2019-01-15 Last updated: 2019-01-15Bibliographically approved
Mitran, B., Varasteh, Z., Puuvuori, E., Abousayed, A., Rinne, S. S., Larhed, M., . . . Orlova, A. (2019). Bispecific GRPR-antagonistic anti-PSMA/GRPR heterodimer for PET and SPECT diagnostic imaging of prostate cancer. Cancers, 11(9), Article ID 1371.
Open this publication in new window or tab >>Bispecific GRPR-antagonistic anti-PSMA/GRPR heterodimer for PET and SPECT diagnostic imaging of prostate cancer
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2019 (English)In: Cancers, ISSN 2072-6694, Vol. 11, no 9, article id 1371Article in journal (Refereed) Published
Abstract [en]

Prostate specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) are wellvalidated molecular targets that are overexpressed in most prostate cancers (PCa). Given thecomplexity and heterogeneity of PCa, targeting both receptors using bispecific radiotracers couldimprove the diagnostic accuracy and therapeutic outcome. The aim of this study was to develop aPSMA/GRPR-targeting bispecific heterodimer for SPECT and PET diagnostic imaging of PCa.Bispecific anti-GRPR/PSMA dimer NOTA-DUPA-RM26 was produced using a combination of solidphase and manual peptide synthesis. The heterodimer was successfully labeled with111In for SPECTand 68Ga for PET with radiochemical yields exceeding 99% for 111In and 98% for 68Ga. Theradiolabeled heterodimers demonstrated high label stability and retained binding specificity to PSMAand GRPR when tested using PC3-PIP cell line expressing both PSMA and GRPR. IC50 values fornatIn-NOTA-DUPA-RM26 were 4±1 nM towards GRPR and 350±240 nM towards PSMA. Cellularprocessing assay revealed a low degree of internalization for 111In-NOTA-DUPA-RM26. In vivobinding specificity tests in PC3-PIP xenografted mice 1 h pi of 111In-NOTA-DUPA-RM26demonstrated partially blockable tumor uptake when co-injected with excess of either PSMA- orGRPR-targeting agents. A pronounced blocking effect was observed for 111In and 68Ga-labeledheterodimer when co-injected simultaneously with excess of PSMA- and GRPR-targeting agents 1 hpi. Biodistribution was studied 1, 3 and 24 h pi for 111In-NOTA-DUPA-RM26, and 1 and 3 h pi for68Ga-NOTA-DUPA-RM26 and revealed a fast clearance of radioprobes from blood and normal organsvia renal excretion. Tumor uptake exceeded the uptake in all normal organs including excretory organsfor both 111In and 68Ga-labeled heterodimers 1 h pi. 68Ga-NOTA-DUPA-RM26 had a significantlylower tumor uptake (8±2%ID/g) compared to 111In-NOTA-DUPA-RM26 (12±2%ID/g), but a two-foldhigher uptake in liver 1h pi. The faster clearance of radioactivity from normal tissues compared totumor lead to an overall increase in tumor-to-organ ratios for both 111In and 68Ga-labeled heterodimers3 h pi. At 24 h pi, tumor-to-organ ratios decreased for 111In-NOTA-DUPA-RM26. MicroPET/CT andmicroSPECT/CT scans confirmed the ex vivo data and suggested that anti-GRPR/PSMA heterodimerNOTA-DUPA-RM26 labeled with galium-68 (for PET) and indium-111 (for SPECT) is a suitablecandidate for imaging of GRPR and PSMA expression in PCa shortly after administration.

Keywords
PSMA, GRPR, molecular imaging, prostate cancer
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-389562 (URN)10.3390/cancers11091371 (DOI)000489719000156 ()31540122 (PubMedID)
Funder
Swedish Cancer Society, CAN 2017/425Swedish Research Council, 2015-02509
Available from: 2019-07-17 Created: 2019-11-08 Last updated: 2019-08-15Bibliographically 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
Oroujeni, M., Abouzayed, A., Lundmark, F., Mitran, B., Orlova, A., Tolmachev, V. & Rosenström, U. (2019). Evaluation of Tumor-Targeting Properties of an Antagonistic Bombesin Analogue RM26 Conjugated with a Non-Residualizing Radioiodine Label Comparison with a Radiometal-Labelled Counterpart. Pharmaceutics, 11(8), Article ID 380.
Open this publication in new window or tab >>Evaluation of Tumor-Targeting Properties of an Antagonistic Bombesin Analogue RM26 Conjugated with a Non-Residualizing Radioiodine Label Comparison with a Radiometal-Labelled Counterpart
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2019 (English)In: Pharmaceutics, ISSN 1999-4923, E-ISSN 1999-4923, Vol. 11, no 8, article id 380Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
MDPI, 2019
Keywords
prostate cancer, bombesin antagonistic analogue, GRPR, RM26, tyrosine, PC-3 xenografts
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-394645 (URN)10.3390/pharmaceutics11080380 (DOI)000484515100013 ()31382362 (PubMedID)
Funder
Swedish Research Council, 2015-02509Swedish Research Council, 2015-02353Swedish Cancer Society, CAN 2017/425Swedish Cancer Society, CAN 2018/436
Available from: 2019-10-17 Created: 2019-10-17 Last updated: 2019-10-17Bibliographically 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
Rinne, S. S., Leitao, C. D., Gentry, J., Mitran, B., Abouzayed, A., Tolmachev, V., . . . Orlova, A. (2019). Increase in negative charge of 68Ga/chelator complex reduces unspecific hepatic uptake but does not improve imaging properties of HER3-targeting affibody molecules. Scientific Reports, 9, Article ID 17710.
Open this publication in new window or tab >>Increase in negative charge of 68Ga/chelator complex reduces unspecific hepatic uptake but does not improve imaging properties of HER3-targeting affibody molecules
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 17710Article in journal (Refereed) Published
Abstract [en]

Upregulation of the human epidermal growth factor receptor type 3 (HER3) is a common mechanism to bypass HER-targeted cancer therapy. Affibody-based molecular imaging has the potential for detecting and monitoring HER3 expression during treatment. In this study, we compared the imaging properties of newly generated Ga-68-labeled anti-HER3 affibody molecules (HE)(3)-Z(HER3)-DOTA and (HE)(3)-Z(HER3)-DOTAGA with previously reported [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA. We hypothesized that increasing the negative charge of the gallium-68/chelator complex would reduce hepatic uptake, which could lead to improved contrast of anti-HER3 affibody-based PET-imaging of HER3 expression. (HE)(3)-Z(HER3)-X (X = DOTA, DOTAGA) were produced and labeled with gallium-68. Binding of the new conjugates was specific in HER3 expressing BxPC-3 and DU145 human cancer cells. Biodistribution and in vivo specificity was studied in BxPC-3 xenograft bearing Balb/c nu/nu mice 3 h pi. DOTA- and DOTAGA-containing conjugates had significantly higher concentration in blood than [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA. Presence of the negatively charged Ga-68-DOTAGA complex reduced the unspecific hepatic uptake, but did not improve overall biodistribution of the conjugate. [Ga-68]Ga-(HE)(3)-Z(HER3)-DOTAGA and [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA had similar tumor-to-liver ratios, but [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA had the highest tumor uptake and tumor-to-blood ratio among the tested conjugates. In conclusion, [Ga-68] Ga-(HE)(3)-Z(HER3)-NODAGA remains the favorable variant for PET imaging of HER3 expression.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
National Category
Radiology, Nuclear Medicine and Medical Imaging Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-400004 (URN)10.1038/s41598-019-54149-3 (DOI)000499205900001 ()31776413 (PubMedID)
Funder
Swedish Cancer Society, CAN 2017/425Swedish Cancer Society, CAN 2018/436Swedish Cancer Society, CAN2017/649Swedish Cancer Society, CAN2016/463Swedish Research Council, 2015-02509Swedish Research Council, 2015-02353Vinnova, 2016/04060Vinnova, 2019/00104
Available from: 2019-12-19 Created: 2019-12-19 Last updated: 2019-12-19Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-6120-2683

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