Anna Orlova | Uppsala University (original) (raw)
Papers by Anna Orlova
ChemistryOpen, 2015
Affibody molecules are small scaffold-based affinity proteins with promising properties as probes... more Affibody molecules are small scaffold-based affinity proteins with promising properties as probes for radionuclide-based molecular imaging. However, a high reabsorption of radiolabeled Affibody molecules in kidneys is an issue. We have shown that the use of (125)I-3-iodo-((4-hydroxyphenyl)ethyl)maleimide (IHPEM) for site-specific labeling of cysteine-containing Affibody molecules provides high tumor uptake but low radioactivity retention in kidneys. We hypothesized that the use of 4-iodophenethylmaleimide (IPEM) would further reduce renal retention of radioactivity because of higher lipophilicity of radiometabolites. An anti-human epidermal growth factor receptor type 2 (HER2) Affibody molecule (ZHER2:2395) was labeled using (125)I-IPEM with an overall yield of 45±3 %. (125)I-IPEM-ZHER2:2395 bound specifically to HER2-expressing human ovarian carcinoma cells (SKOV-3 cell line). In NMRI mice, the renal uptake of (125)I-IPEM-ZHER2:2395 (24±2 and 5.7±0.3 % IA g(-1)at 1 and 4 h after injection, respectively) was significantly lower than uptake of (125)I-IHPEM-ZHER2:2395 (50±8 and 12±2 % IA g(-1)at 1 and 4 h after injection, respectively). In conclusion, the use of a more lipophilic linker for the radioiodination of Affibody molecules reduces renal radioactivity.
European Journal of Nuclear Medicine and Molecular Imaging, 2010
Purpose Overexpression of HER2 receptors is a prognostic and predictive biomarker in breast cance... more Purpose Overexpression of HER2 receptors is a prognostic and predictive biomarker in breast cancer and a number of other malignancies. Radionuclide molecular imaging of HER2 overexpression may influence patient management making treatment more personalized. Earlier, 111 In-DOTA-Z HER2:342-pep2 (ABY-002) Affibody molecule demonstrated excellent imaging of HER2-expressing xenografts in mice shortly after injection. The use of the positronemitting nuclide 68 Ga instead of 111 In might increase both the sensitivity of HER2 imaging and accuracy of expression quantification. The goal of this study was to prepare and characterize 68 Ga-labelled ABY-002.
Oncology reports, Jan 9, 2015
Expression of human epidermal growth factor receptor type 3 (HER3) in malignant tumors has been a... more Expression of human epidermal growth factor receptor type 3 (HER3) in malignant tumors has been associated with resistance to a variety of anticancer therapies. Several anti-HER3 monoclonal antibodies are currently under pre-clinical and clinical development aiming to overcome HER3-mediated resistance. Radionuclide molecular imaging of HER3 expression may improve treatment by allowing the selection of suitable patients for HER3-targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. In a recent study, we selected affibody molecules with affinity to HER3 at a low picomolar range. The aim of the present study was to develop an anti-HER3 affibody molecule suitable for labeling with radiometals. The HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA HER3-specific affibody molecules were labeled with indium‑111 (111In) and assessed in vitro and in vivo for imaging properties using single photon emis...
Radiolabelled Affibody molecules have demonstrated a potential for visualization of tumour-associ... more Radiolabelled Affibody molecules have demonstrated a potential for visualization of tumour-associated molecular targets. Affibody molecules (7 kDa) are composed of three alpha-helices. Recently, a smaller two-helix variant of Affibody molecules (5.1 kDa) was developed. The aim of this study was to compare two- and three-helix HER2-targeting Affibody molecules directly in vivo. The three-helix Affibody molecule ABY-002 and the two-helix Affibody molecule PEP09239 were labelled with (111)In at the N-termini via DOTA chelator. Tumour-targeting properties were directly compared at 1 and 4 h after injection in mice bearing SKOV-3 xenografts with high HER2 expression and LS174T xenografts with low HER2 expression. The dissociation constants (K(D)) for HER2 binding were 78 pM for the three-helix Affibody molecule and 2.1 nM for the two-helix Affibody molecule. (111)In-PEP09239 cleared more rapidly from the blood. In xenografts with high HER2 expression, the uptake of (111)In-ABY-002 was significantly higher than that of (111)In-PEP09239. The tumour-to-blood ratio was higher for (111)In-PEP09239 at 4 h after injection, while there was no significant difference in other tumour-to-organ ratios. The tumour uptake of (111)In-ABY-002 was eightfold higher than that of (111)In-PEP09239 in xenografts with low expression. Tumour-to-blood ratios were equal in this case, but other tumour-to-organ ratios were appreciably higher for the three-helix variant. For tumours with high HER2 expression, two-helix HER2-targeting Affibody molecules can provide higher tumour-to-blood ratio at the cost of lower tumour uptake. In the case of low expression, both tumour uptake and tumour-to-organ ratios are appreciably higher for three-helix than for two-helix HER2-targeting Affibody molecules.
A radiolabeled anti-HER2 Affibody molecule (ZHER2:342) targets HER2-expressing xenografts with hi... more A radiolabeled anti-HER2 Affibody molecule (ZHER2:342) targets HER2-expressing xenografts with high selectivity and gives good imaging contrast. However, the small size (f7 kDa) results in rapid glomerular filtration and high renal accumu- lation of radiometals, thus excluding targeted therapy. Here, we report that reversible binding to albumin efficiently reduces the renal excretion and uptake, enabling radio- metal-based nuclide therapy. The
Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 2006
In vivo diagnosis with cancer-specific targeting agents that have optimal characteristics for ima... more In vivo diagnosis with cancer-specific targeting agents that have optimal characteristics for imaging is an important development in treatment planning for cancer patients. Overexpression of the HER2 antigen is high in several types of carcinomas and has predictive and prognostic value, especially for breast cancer. A new type of targeting agent, the Affibody molecule, was described recently. An Affibody dimer, His6-(ZHER2:4)2 (15.4 kDa), binds to HER2 with an affinity of 3 nmol/L and might be used for the imaging of HER2 expression. The use of 99mTc might improve the availability of the labeled conjugate, and Tc(I)-carbonyl chemistry enables the site-specific labeling of the histidine tag on the Affibody molecule. The goals of the present study were to prepare 99mTc-labeled His6-(ZHER2:4)2 and to evaluate its targeting properties compared with the targeting properties of 125I-4-iodobenzoate-His6-(ZHER2:4)2 [125I-His6-(ZHER2:4)2]. The labeling of His6-(ZHER2:4)2 with 99mTc was perfo...
Anticancer research
Negatively-charged polyhedral boron clusters can be easily halogenated with highly stable boron-h... more Negatively-charged polyhedral boron clusters can be easily halogenated with highly stable boron-halogen bonds and are promising in radionuclide diagnostics and cancer therapy. The radio-iodination conditions for the closo-dodecaborate anion and for the conjugation of its labeled isothiocyanatobenzylammonio derivative to the monoclonal antibody (mAb) were optimized. The labeling yield was about 90% and the overall conjugation yield was 55-60%. The in vitro stability of the radio-iodinated mAb was good under physiological and non-physiological conditions. The immunoreactivity of the labeled mAb (SK-BR-3 cells) was retained in the one-pot two-step labeling. Negatively-charged polyhedral boron clusters can be used for indirect radio-iodination of mAbs.
European journal of nuclear medicine and molecular imaging, 2013
Radionuclide imaging of insulin-like growth factor type 1 receptor (IGF-1R) expression in tumours... more Radionuclide imaging of insulin-like growth factor type 1 receptor (IGF-1R) expression in tumours might be used for selection of patients who would benefit from IGF-1R-targeted therapy. We have previously shown the feasibility of IGF-1R imaging using the Affibody molecule (111)In-DOTA-His(6)-Z(IGF1R:4551). The use of (99m)Tc instead of (111)In should improve sensitivity and resolution of imaging, and reduce the dose burden to patients. We hypothesized that inclusion of a HEHEHE tag instead of a His(6) tag in Z(IGF1R:4551) would permit its convenient purification using IMAC, enable labelling with [(99m)Tc(CO)(3)](+), and improve its biodistribution. Z(IGF1R:4551) was expressed with a HEHEHE tag in the N terminus. The resulting (HE)(3)-Z(IGF1R:4551) construct was labelled with [(99m)Tc(CO)(3)](+). Targeting of IGF-1R-expressing cells using [(99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) was evaluated in vitro and in vivo. (HE)(3)-Z(IGF1R:4551) was stably labelled with (99m)Tc with preserved...
International journal of oncology, 2014
Correct staging of prostate cancer is an unmet clinical need. Radionuclide targeting of prostate-... more Correct staging of prostate cancer is an unmet clinical need. Radionuclide targeting of prostate-specific membrane antigen (PSMA) with 111In-labeled capromab pendetide (ProstaScint) is a clinical option for prostate cancer staging. We propose the use of 124I-labeled capromab to decrease the retention of radioactivity in healthy organs (due to the non-residualizing properties of the radiolabel). The use of 124I as a label should increase imaging sensitivity due to the advantages of PET as an imaging modality. Capromab targets the intracellular domain of PSMA; accumulation of radioactivity in the tumor should not depend on internalization of the antigen/antibody complex. Capromab was iodinated, and its targeting properties were compared with indium labeled counterpart in LNCaP xenografts in dual isotope mode. PSMA-negative xenografts (PC3) were used as a negative control. Radioiodinated capromab bound to PSMA specifically. Biodistribution of 125I/111In-capromab showed a more rapid cle...
International journal of oncology, 2014
Prostate cancer (PCa) is the most common cancer type among men. Treatments against advanced PCa a... more Prostate cancer (PCa) is the most common cancer type among men. Treatments against advanced PCa are limited and in many cases only palliative. In a later, androgent independent, stage of PCa androgen receptors can be activated without interaction with ligand, i.e., by receptors of tyrosine kinase (RTK) family in the outlaw pathway. Human epidermal growth factor receptors HER2 and EGFR belong to RTK-family. HER2 is one of the main actors in the outlaw pathway with EGFR as the preferable heterodimerizing partner. We hypothesized that information on HER2 expression in advanced PCa could be useful for selection of patients for anti-RTK therapy and monitoring of therapy response. A panel of PCa cell lines (LNCap, PC3, DU-145) was subjected to a 8-week treatment using drugs influencing the RTK: trastuzumab (anti‑HER2), 17-DMAG (Hsp90 inhibitor), alone or in combination, and their HER2 and EGFR expressions were compared with non-treated cells. Treatment with trastuzumab decreased prolifera...
European journal of medicinal chemistry, Jan 24, 2014
Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered t... more Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of (188)Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all (188)Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The (188)Re-ZHER2:V2 affibody molecule with a...
PLoS ONE, 2014
Histidine-rich glycoprotein (HRG) is implicated in tumor growth and metastasis by regulation of a... more Histidine-rich glycoprotein (HRG) is implicated in tumor growth and metastasis by regulation of angiogenesis and inflammation. HRG is produced by hepatocytes and carried to tissues via the circulation. We hypothesized that HRG's tissue distribution and turnover may be mediated by inflammatory cells. Biodistribution parameters were analyzed by injection of radiolabeled, bioactive HRG in the circulation of healthy and tumor-bearing mice. 125 I-HRG was cleared rapidly from the blood and taken up in tissues of healthy and tumor-bearing mice, followed by degradation, to an increased extent in the tumor-bearing mice. Steady state levels of HRG in the circulation were unaffected by the tumor disease both in murine tumor models and in colorectal cancer (CRC) patients. Importantly, stromal pools of HRG, detected in human CRC microarrays, were associated with inflammatory cells. In agreement, microautoradiography identified 125 I-HRG in blood vessels and on CD45-positive leukocytes in mouse tissues. Moreover, radiolabeled HRG bound in a specific, heparan sulfateindependent manner, to differentiated human monocytic U937 cells in vitro. Suppression of monocyte differentiation by systemic treatment of mice with anti-colony stimulating factor-1 neutralizing antibodies led to reduced blood clearance of radiolabeled HRG and to accumulation of endogenous HRG in the blood. Combined, our data show that mononuclear phagocytes have specific binding sites for HRG and that these cells are essential for uptake of HRG from blood and distribution of HRG in tissues. Thereby, we confirm and extend our previous report that inflammatory cells mediate the effect of HRG on tumor growth and metastatic spread.
Molecules, 2014
The overexpression of gastrin-releasing peptide receptor (GRPR) in cancer can be used for peptide... more The overexpression of gastrin-releasing peptide receptor (GRPR) in cancer can be used for peptide-receptor mediated radionuclide imaging and therapy. We have previously shown that an antagonist analog of bombesin RM26 conjugated to 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) via a diethyleneglycol (PEG 2 ) spacer (NOTA-PEG 2 -RM26) and labeled with 68 Ga can be used for imaging of GRPR-expressing tumors. In this study, we evaluated if a variation of mini-PEG spacer length can be used for optimization of targeting properties of the NOTA-conjugated RM26. A series of analogs with different PEG-length (n = 2, 3, 4, 6) was synthesized, radiolabeled and evaluated in vitro and in vivo. The IC 50 values of nat Ga-NOTA-PEG n -RM26 (n = 2, 3, 4, 6) were 3.1 ± 0.2, 3.9 ± 0.3, 5.4 ± 0.4 and 5.8 ± 0.3 nM, respectively. In normal mice all conjugates demonstrated similar biodistribution pattern, however 68 Ga-NOTA-PEG 3 -RM26 showed lower liver uptake. Biodistribution of 68 Ga-NOTA-PEG 3 -RM26 was evaluated in
Nuclear Medicine and Biology, 2014
Introduction: Overexpression of gastrin-releasing peptide receptors (GRPR) has been reported in s... more Introduction: Overexpression of gastrin-releasing peptide receptors (GRPR) has been reported in several cancers. Bombesin (BN) analogs are short peptides with a high affinity for GRPR. Different BN analogs were evaluated for radionuclide imaging and therapy of GRPR-expressing tumors. We have previously investigated an antagonistic analog of BN (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH 2 , RM26) conjugated to NOTA via a PEG 2 spacer (NOTA-PEG 2 -RM26) labeled with 68 Ga, 111 In and Al 18 F. 68 Ga-labeled NOTA-PEG 2 -RM26 showed high tumorto-organ ratios. Methods: The influence of different macrocyclic chelators (NOTA, NODAGA, DOTA and DOTAGA) on the targeting properties of 68 Ga-labeled PEG 2 -RM26 was studied in vitro and in vivo. Results: All conjugates were labeled with generator-produced 68 Ga with high yields and demonstrated high stability and specific binding to GRPR. The IC 50 values of nat Ga-X-PEG 2 -RM26 (X = NOTA, DOTA, NODAGA, DOTAGA) were 2.3 ± 0.2, 3.0 ± 0.3, 2.9 ± 0.3 and 10.0 ± 0.6 nM, respectively. The internalization of the conjugates by PC-3 cells was low. However, the DOTA-conjugated analog demonstrated a higher internalization rate compared to other analogs. GRPR-specific uptake was found in receptor-positive normal tissues and PC-3 xenografts for all conjugates. The biodistribution of the conjugates was influenced by the choice of the chelator moiety. Although all radiotracers cleared rapidly from the blood, [ 68 Ga]Ga-NOTA-PEG 2 -RM26 showed significantly lower uptake in lung, muscle and bone compared to the other analogs. The uptake in tumors (5.40 ± 1.04 %ID/g at 2 h p.i.) and the tumor-to-organ ratios (25 ± 3, 157 ± 23 and 39 ± 4 for blood, muscle and bone, respectively) were significantly higher for the NOTA-conjugate than the other analogs. Conclusions: Chelators had a clear influence on the biodistribution and targeting properties of 68 Ga-labeled antagonistic BN analogs. Positively charged [ 68 Ga]Ga-NOTA-PEG 2 -RM26 provided a low kidney radioactivity uptake, high affinity, high tumor uptake and high image contrast.
ChemistryOpen, 2015
Affibody molecules are small scaffold-based affinity proteins with promising properties as probes... more Affibody molecules are small scaffold-based affinity proteins with promising properties as probes for radionuclide-based molecular imaging. However, a high reabsorption of radiolabeled Affibody molecules in kidneys is an issue. We have shown that the use of (125)I-3-iodo-((4-hydroxyphenyl)ethyl)maleimide (IHPEM) for site-specific labeling of cysteine-containing Affibody molecules provides high tumor uptake but low radioactivity retention in kidneys. We hypothesized that the use of 4-iodophenethylmaleimide (IPEM) would further reduce renal retention of radioactivity because of higher lipophilicity of radiometabolites. An anti-human epidermal growth factor receptor type 2 (HER2) Affibody molecule (ZHER2:2395) was labeled using (125)I-IPEM with an overall yield of 45±3 %. (125)I-IPEM-ZHER2:2395 bound specifically to HER2-expressing human ovarian carcinoma cells (SKOV-3 cell line). In NMRI mice, the renal uptake of (125)I-IPEM-ZHER2:2395 (24±2 and 5.7±0.3 % IA g(-1)at 1 and 4 h after in...
International journal of molecular medicine, 2007
Imaging of expression of human epidermal growth factor receptor type 2 (HER2) in breast carcinoma... more Imaging of expression of human epidermal growth factor receptor type 2 (HER2) in breast carcinomas may help to select patients eligible for trastuzumab therapy. The Affibody molecule Z(HER2:342) is a small (7-kDa) non-immunoglobulin affinity protein, which binds to HER2 with a picomolar affinity. Previously, a benzyl-DTPA conjugate of Z(HER2:342) was labeled with 111In and demonstrated good targeting in murine xenografts. We considered that the use of the macrocyclic chelator DOTA could increase the label stability and enhance a choice of nuclides, which could be used as a label for Z(HER2:342). The goal of this study was the preparation and pre-clinical evaluation of the indium-111- labeled DOTA-derivative of Z(HER2:342). Isothiocyanate-benzyl-DOTA was coupled to recombinant Z(HER2:342), and the conjugate was efficiently labeled with 111In at 60 degrees C. The specificity of 111In-benzyl-DOTA-Z(HER2:342) binding to HER2 was confirmed in vitro using HER2-expressing breast carcinoma ...
Data on expression of the HER2 (erbB-2) receptor in breast carcinoma make it possible to select t... more Data on expression of the HER2 (erbB-2) receptor in breast carcinoma make it possible to select the most efficient treatment. There are strong indications that HER2 expression possesses prognostic and predictive values in ovarian, prostate, and lung carcinomas as well. Visualization of HER2 expression using radionuclide targeting can provide important diagnostic information. The Affibody Z HER2:342 is a short (;7 kDa) phage-displayselected protein that binds HER2 with an affinity of 22 pmol/L. The goal of this study was to evaluate whether 111 In-labeled HER2: 342 can be used for imaging of HER2 overexpression in vivo. Methods: Z HER2:342 was labeled with 111 In via isothiocyanatebenzyl-DTPA (DTPA is diethylenetriaminepentaacetic acid) and the conjugate was characterized in vitro and in vivo. Results: 111 In-Benzyl-DTPA-Z HER2:342 preserved the capacity to bind living HER2-expressing cells specifically. The affinity of In-benzyl-DTPA-Z HER2:342 to HER2 was 21 pmol/L according to surface plasmon resonance measurements. In nude mice bearing HER2-expressing SKOV-3 xenografts, a tumor uptake of 12% 6 3% injected activity per gram and a tumor-to-blood ratio of about 100 were obtained 4 h after injection. Tumor uptake in vivo was receptor specific, as it could be blocked with an excess of nonlabeled Z HER2:342 . HER2-expressing xenografts were clearly imaged 4 h after injection using a g-camera.
European Journal of Nuclear Medicine and Molecular Imaging, 2009
Purpose The purpose of this study was to design and evaluate a novel platform for labelling of A... more Purpose The purpose of this study was to design and evaluate a novel platform for labelling of Affibody molecules, enabling both recombinant and synthetic production and site-specific labelling with 99mTc or trivalent radiometals. Methods The HER2-specific Affibody molecule PEP05352 was made by peptide synthesis. The chelator sequence SECG (serine-glutamic acid-cysteine-glycine) was anchored on the C-terminal to allow 99mTc labelling. The cysteine can
ChemistryOpen, 2015
Affibody molecules are small scaffold-based affinity proteins with promising properties as probes... more Affibody molecules are small scaffold-based affinity proteins with promising properties as probes for radionuclide-based molecular imaging. However, a high reabsorption of radiolabeled Affibody molecules in kidneys is an issue. We have shown that the use of (125)I-3-iodo-((4-hydroxyphenyl)ethyl)maleimide (IHPEM) for site-specific labeling of cysteine-containing Affibody molecules provides high tumor uptake but low radioactivity retention in kidneys. We hypothesized that the use of 4-iodophenethylmaleimide (IPEM) would further reduce renal retention of radioactivity because of higher lipophilicity of radiometabolites. An anti-human epidermal growth factor receptor type 2 (HER2) Affibody molecule (ZHER2:2395) was labeled using (125)I-IPEM with an overall yield of 45±3 %. (125)I-IPEM-ZHER2:2395 bound specifically to HER2-expressing human ovarian carcinoma cells (SKOV-3 cell line). In NMRI mice, the renal uptake of (125)I-IPEM-ZHER2:2395 (24±2 and 5.7±0.3 % IA g(-1)at 1 and 4 h after injection, respectively) was significantly lower than uptake of (125)I-IHPEM-ZHER2:2395 (50±8 and 12±2 % IA g(-1)at 1 and 4 h after injection, respectively). In conclusion, the use of a more lipophilic linker for the radioiodination of Affibody molecules reduces renal radioactivity.
European Journal of Nuclear Medicine and Molecular Imaging, 2010
Purpose Overexpression of HER2 receptors is a prognostic and predictive biomarker in breast cance... more Purpose Overexpression of HER2 receptors is a prognostic and predictive biomarker in breast cancer and a number of other malignancies. Radionuclide molecular imaging of HER2 overexpression may influence patient management making treatment more personalized. Earlier, 111 In-DOTA-Z HER2:342-pep2 (ABY-002) Affibody molecule demonstrated excellent imaging of HER2-expressing xenografts in mice shortly after injection. The use of the positronemitting nuclide 68 Ga instead of 111 In might increase both the sensitivity of HER2 imaging and accuracy of expression quantification. The goal of this study was to prepare and characterize 68 Ga-labelled ABY-002.
Oncology reports, Jan 9, 2015
Expression of human epidermal growth factor receptor type 3 (HER3) in malignant tumors has been a... more Expression of human epidermal growth factor receptor type 3 (HER3) in malignant tumors has been associated with resistance to a variety of anticancer therapies. Several anti-HER3 monoclonal antibodies are currently under pre-clinical and clinical development aiming to overcome HER3-mediated resistance. Radionuclide molecular imaging of HER3 expression may improve treatment by allowing the selection of suitable patients for HER3-targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. In a recent study, we selected affibody molecules with affinity to HER3 at a low picomolar range. The aim of the present study was to develop an anti-HER3 affibody molecule suitable for labeling with radiometals. The HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA HER3-specific affibody molecules were labeled with indium‑111 (111In) and assessed in vitro and in vivo for imaging properties using single photon emis...
Radiolabelled Affibody molecules have demonstrated a potential for visualization of tumour-associ... more Radiolabelled Affibody molecules have demonstrated a potential for visualization of tumour-associated molecular targets. Affibody molecules (7 kDa) are composed of three alpha-helices. Recently, a smaller two-helix variant of Affibody molecules (5.1 kDa) was developed. The aim of this study was to compare two- and three-helix HER2-targeting Affibody molecules directly in vivo. The three-helix Affibody molecule ABY-002 and the two-helix Affibody molecule PEP09239 were labelled with (111)In at the N-termini via DOTA chelator. Tumour-targeting properties were directly compared at 1 and 4 h after injection in mice bearing SKOV-3 xenografts with high HER2 expression and LS174T xenografts with low HER2 expression. The dissociation constants (K(D)) for HER2 binding were 78 pM for the three-helix Affibody molecule and 2.1 nM for the two-helix Affibody molecule. (111)In-PEP09239 cleared more rapidly from the blood. In xenografts with high HER2 expression, the uptake of (111)In-ABY-002 was significantly higher than that of (111)In-PEP09239. The tumour-to-blood ratio was higher for (111)In-PEP09239 at 4 h after injection, while there was no significant difference in other tumour-to-organ ratios. The tumour uptake of (111)In-ABY-002 was eightfold higher than that of (111)In-PEP09239 in xenografts with low expression. Tumour-to-blood ratios were equal in this case, but other tumour-to-organ ratios were appreciably higher for the three-helix variant. For tumours with high HER2 expression, two-helix HER2-targeting Affibody molecules can provide higher tumour-to-blood ratio at the cost of lower tumour uptake. In the case of low expression, both tumour uptake and tumour-to-organ ratios are appreciably higher for three-helix than for two-helix HER2-targeting Affibody molecules.
A radiolabeled anti-HER2 Affibody molecule (ZHER2:342) targets HER2-expressing xenografts with hi... more A radiolabeled anti-HER2 Affibody molecule (ZHER2:342) targets HER2-expressing xenografts with high selectivity and gives good imaging contrast. However, the small size (f7 kDa) results in rapid glomerular filtration and high renal accumu- lation of radiometals, thus excluding targeted therapy. Here, we report that reversible binding to albumin efficiently reduces the renal excretion and uptake, enabling radio- metal-based nuclide therapy. The
Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 2006
In vivo diagnosis with cancer-specific targeting agents that have optimal characteristics for ima... more In vivo diagnosis with cancer-specific targeting agents that have optimal characteristics for imaging is an important development in treatment planning for cancer patients. Overexpression of the HER2 antigen is high in several types of carcinomas and has predictive and prognostic value, especially for breast cancer. A new type of targeting agent, the Affibody molecule, was described recently. An Affibody dimer, His6-(ZHER2:4)2 (15.4 kDa), binds to HER2 with an affinity of 3 nmol/L and might be used for the imaging of HER2 expression. The use of 99mTc might improve the availability of the labeled conjugate, and Tc(I)-carbonyl chemistry enables the site-specific labeling of the histidine tag on the Affibody molecule. The goals of the present study were to prepare 99mTc-labeled His6-(ZHER2:4)2 and to evaluate its targeting properties compared with the targeting properties of 125I-4-iodobenzoate-His6-(ZHER2:4)2 [125I-His6-(ZHER2:4)2]. The labeling of His6-(ZHER2:4)2 with 99mTc was perfo...
Anticancer research
Negatively-charged polyhedral boron clusters can be easily halogenated with highly stable boron-h... more Negatively-charged polyhedral boron clusters can be easily halogenated with highly stable boron-halogen bonds and are promising in radionuclide diagnostics and cancer therapy. The radio-iodination conditions for the closo-dodecaborate anion and for the conjugation of its labeled isothiocyanatobenzylammonio derivative to the monoclonal antibody (mAb) were optimized. The labeling yield was about 90% and the overall conjugation yield was 55-60%. The in vitro stability of the radio-iodinated mAb was good under physiological and non-physiological conditions. The immunoreactivity of the labeled mAb (SK-BR-3 cells) was retained in the one-pot two-step labeling. Negatively-charged polyhedral boron clusters can be used for indirect radio-iodination of mAbs.
European journal of nuclear medicine and molecular imaging, 2013
Radionuclide imaging of insulin-like growth factor type 1 receptor (IGF-1R) expression in tumours... more Radionuclide imaging of insulin-like growth factor type 1 receptor (IGF-1R) expression in tumours might be used for selection of patients who would benefit from IGF-1R-targeted therapy. We have previously shown the feasibility of IGF-1R imaging using the Affibody molecule (111)In-DOTA-His(6)-Z(IGF1R:4551). The use of (99m)Tc instead of (111)In should improve sensitivity and resolution of imaging, and reduce the dose burden to patients. We hypothesized that inclusion of a HEHEHE tag instead of a His(6) tag in Z(IGF1R:4551) would permit its convenient purification using IMAC, enable labelling with [(99m)Tc(CO)(3)](+), and improve its biodistribution. Z(IGF1R:4551) was expressed with a HEHEHE tag in the N terminus. The resulting (HE)(3)-Z(IGF1R:4551) construct was labelled with [(99m)Tc(CO)(3)](+). Targeting of IGF-1R-expressing cells using [(99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) was evaluated in vitro and in vivo. (HE)(3)-Z(IGF1R:4551) was stably labelled with (99m)Tc with preserved...
International journal of oncology, 2014
Correct staging of prostate cancer is an unmet clinical need. Radionuclide targeting of prostate-... more Correct staging of prostate cancer is an unmet clinical need. Radionuclide targeting of prostate-specific membrane antigen (PSMA) with 111In-labeled capromab pendetide (ProstaScint) is a clinical option for prostate cancer staging. We propose the use of 124I-labeled capromab to decrease the retention of radioactivity in healthy organs (due to the non-residualizing properties of the radiolabel). The use of 124I as a label should increase imaging sensitivity due to the advantages of PET as an imaging modality. Capromab targets the intracellular domain of PSMA; accumulation of radioactivity in the tumor should not depend on internalization of the antigen/antibody complex. Capromab was iodinated, and its targeting properties were compared with indium labeled counterpart in LNCaP xenografts in dual isotope mode. PSMA-negative xenografts (PC3) were used as a negative control. Radioiodinated capromab bound to PSMA specifically. Biodistribution of 125I/111In-capromab showed a more rapid cle...
International journal of oncology, 2014
Prostate cancer (PCa) is the most common cancer type among men. Treatments against advanced PCa a... more Prostate cancer (PCa) is the most common cancer type among men. Treatments against advanced PCa are limited and in many cases only palliative. In a later, androgent independent, stage of PCa androgen receptors can be activated without interaction with ligand, i.e., by receptors of tyrosine kinase (RTK) family in the outlaw pathway. Human epidermal growth factor receptors HER2 and EGFR belong to RTK-family. HER2 is one of the main actors in the outlaw pathway with EGFR as the preferable heterodimerizing partner. We hypothesized that information on HER2 expression in advanced PCa could be useful for selection of patients for anti-RTK therapy and monitoring of therapy response. A panel of PCa cell lines (LNCap, PC3, DU-145) was subjected to a 8-week treatment using drugs influencing the RTK: trastuzumab (anti‑HER2), 17-DMAG (Hsp90 inhibitor), alone or in combination, and their HER2 and EGFR expressions were compared with non-treated cells. Treatment with trastuzumab decreased prolifera...
European journal of medicinal chemistry, Jan 24, 2014
Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered t... more Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of (188)Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all (188)Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The (188)Re-ZHER2:V2 affibody molecule with a...
PLoS ONE, 2014
Histidine-rich glycoprotein (HRG) is implicated in tumor growth and metastasis by regulation of a... more Histidine-rich glycoprotein (HRG) is implicated in tumor growth and metastasis by regulation of angiogenesis and inflammation. HRG is produced by hepatocytes and carried to tissues via the circulation. We hypothesized that HRG's tissue distribution and turnover may be mediated by inflammatory cells. Biodistribution parameters were analyzed by injection of radiolabeled, bioactive HRG in the circulation of healthy and tumor-bearing mice. 125 I-HRG was cleared rapidly from the blood and taken up in tissues of healthy and tumor-bearing mice, followed by degradation, to an increased extent in the tumor-bearing mice. Steady state levels of HRG in the circulation were unaffected by the tumor disease both in murine tumor models and in colorectal cancer (CRC) patients. Importantly, stromal pools of HRG, detected in human CRC microarrays, were associated with inflammatory cells. In agreement, microautoradiography identified 125 I-HRG in blood vessels and on CD45-positive leukocytes in mouse tissues. Moreover, radiolabeled HRG bound in a specific, heparan sulfateindependent manner, to differentiated human monocytic U937 cells in vitro. Suppression of monocyte differentiation by systemic treatment of mice with anti-colony stimulating factor-1 neutralizing antibodies led to reduced blood clearance of radiolabeled HRG and to accumulation of endogenous HRG in the blood. Combined, our data show that mononuclear phagocytes have specific binding sites for HRG and that these cells are essential for uptake of HRG from blood and distribution of HRG in tissues. Thereby, we confirm and extend our previous report that inflammatory cells mediate the effect of HRG on tumor growth and metastatic spread.
Molecules, 2014
The overexpression of gastrin-releasing peptide receptor (GRPR) in cancer can be used for peptide... more The overexpression of gastrin-releasing peptide receptor (GRPR) in cancer can be used for peptide-receptor mediated radionuclide imaging and therapy. We have previously shown that an antagonist analog of bombesin RM26 conjugated to 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) via a diethyleneglycol (PEG 2 ) spacer (NOTA-PEG 2 -RM26) and labeled with 68 Ga can be used for imaging of GRPR-expressing tumors. In this study, we evaluated if a variation of mini-PEG spacer length can be used for optimization of targeting properties of the NOTA-conjugated RM26. A series of analogs with different PEG-length (n = 2, 3, 4, 6) was synthesized, radiolabeled and evaluated in vitro and in vivo. The IC 50 values of nat Ga-NOTA-PEG n -RM26 (n = 2, 3, 4, 6) were 3.1 ± 0.2, 3.9 ± 0.3, 5.4 ± 0.4 and 5.8 ± 0.3 nM, respectively. In normal mice all conjugates demonstrated similar biodistribution pattern, however 68 Ga-NOTA-PEG 3 -RM26 showed lower liver uptake. Biodistribution of 68 Ga-NOTA-PEG 3 -RM26 was evaluated in
Nuclear Medicine and Biology, 2014
Introduction: Overexpression of gastrin-releasing peptide receptors (GRPR) has been reported in s... more Introduction: Overexpression of gastrin-releasing peptide receptors (GRPR) has been reported in several cancers. Bombesin (BN) analogs are short peptides with a high affinity for GRPR. Different BN analogs were evaluated for radionuclide imaging and therapy of GRPR-expressing tumors. We have previously investigated an antagonistic analog of BN (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH 2 , RM26) conjugated to NOTA via a PEG 2 spacer (NOTA-PEG 2 -RM26) labeled with 68 Ga, 111 In and Al 18 F. 68 Ga-labeled NOTA-PEG 2 -RM26 showed high tumorto-organ ratios. Methods: The influence of different macrocyclic chelators (NOTA, NODAGA, DOTA and DOTAGA) on the targeting properties of 68 Ga-labeled PEG 2 -RM26 was studied in vitro and in vivo. Results: All conjugates were labeled with generator-produced 68 Ga with high yields and demonstrated high stability and specific binding to GRPR. The IC 50 values of nat Ga-X-PEG 2 -RM26 (X = NOTA, DOTA, NODAGA, DOTAGA) were 2.3 ± 0.2, 3.0 ± 0.3, 2.9 ± 0.3 and 10.0 ± 0.6 nM, respectively. The internalization of the conjugates by PC-3 cells was low. However, the DOTA-conjugated analog demonstrated a higher internalization rate compared to other analogs. GRPR-specific uptake was found in receptor-positive normal tissues and PC-3 xenografts for all conjugates. The biodistribution of the conjugates was influenced by the choice of the chelator moiety. Although all radiotracers cleared rapidly from the blood, [ 68 Ga]Ga-NOTA-PEG 2 -RM26 showed significantly lower uptake in lung, muscle and bone compared to the other analogs. The uptake in tumors (5.40 ± 1.04 %ID/g at 2 h p.i.) and the tumor-to-organ ratios (25 ± 3, 157 ± 23 and 39 ± 4 for blood, muscle and bone, respectively) were significantly higher for the NOTA-conjugate than the other analogs. Conclusions: Chelators had a clear influence on the biodistribution and targeting properties of 68 Ga-labeled antagonistic BN analogs. Positively charged [ 68 Ga]Ga-NOTA-PEG 2 -RM26 provided a low kidney radioactivity uptake, high affinity, high tumor uptake and high image contrast.
ChemistryOpen, 2015
Affibody molecules are small scaffold-based affinity proteins with promising properties as probes... more Affibody molecules are small scaffold-based affinity proteins with promising properties as probes for radionuclide-based molecular imaging. However, a high reabsorption of radiolabeled Affibody molecules in kidneys is an issue. We have shown that the use of (125)I-3-iodo-((4-hydroxyphenyl)ethyl)maleimide (IHPEM) for site-specific labeling of cysteine-containing Affibody molecules provides high tumor uptake but low radioactivity retention in kidneys. We hypothesized that the use of 4-iodophenethylmaleimide (IPEM) would further reduce renal retention of radioactivity because of higher lipophilicity of radiometabolites. An anti-human epidermal growth factor receptor type 2 (HER2) Affibody molecule (ZHER2:2395) was labeled using (125)I-IPEM with an overall yield of 45±3 %. (125)I-IPEM-ZHER2:2395 bound specifically to HER2-expressing human ovarian carcinoma cells (SKOV-3 cell line). In NMRI mice, the renal uptake of (125)I-IPEM-ZHER2:2395 (24±2 and 5.7±0.3 % IA g(-1)at 1 and 4 h after in...
International journal of molecular medicine, 2007
Imaging of expression of human epidermal growth factor receptor type 2 (HER2) in breast carcinoma... more Imaging of expression of human epidermal growth factor receptor type 2 (HER2) in breast carcinomas may help to select patients eligible for trastuzumab therapy. The Affibody molecule Z(HER2:342) is a small (7-kDa) non-immunoglobulin affinity protein, which binds to HER2 with a picomolar affinity. Previously, a benzyl-DTPA conjugate of Z(HER2:342) was labeled with 111In and demonstrated good targeting in murine xenografts. We considered that the use of the macrocyclic chelator DOTA could increase the label stability and enhance a choice of nuclides, which could be used as a label for Z(HER2:342). The goal of this study was the preparation and pre-clinical evaluation of the indium-111- labeled DOTA-derivative of Z(HER2:342). Isothiocyanate-benzyl-DOTA was coupled to recombinant Z(HER2:342), and the conjugate was efficiently labeled with 111In at 60 degrees C. The specificity of 111In-benzyl-DOTA-Z(HER2:342) binding to HER2 was confirmed in vitro using HER2-expressing breast carcinoma ...
Data on expression of the HER2 (erbB-2) receptor in breast carcinoma make it possible to select t... more Data on expression of the HER2 (erbB-2) receptor in breast carcinoma make it possible to select the most efficient treatment. There are strong indications that HER2 expression possesses prognostic and predictive values in ovarian, prostate, and lung carcinomas as well. Visualization of HER2 expression using radionuclide targeting can provide important diagnostic information. The Affibody Z HER2:342 is a short (;7 kDa) phage-displayselected protein that binds HER2 with an affinity of 22 pmol/L. The goal of this study was to evaluate whether 111 In-labeled HER2: 342 can be used for imaging of HER2 overexpression in vivo. Methods: Z HER2:342 was labeled with 111 In via isothiocyanatebenzyl-DTPA (DTPA is diethylenetriaminepentaacetic acid) and the conjugate was characterized in vitro and in vivo. Results: 111 In-Benzyl-DTPA-Z HER2:342 preserved the capacity to bind living HER2-expressing cells specifically. The affinity of In-benzyl-DTPA-Z HER2:342 to HER2 was 21 pmol/L according to surface plasmon resonance measurements. In nude mice bearing HER2-expressing SKOV-3 xenografts, a tumor uptake of 12% 6 3% injected activity per gram and a tumor-to-blood ratio of about 100 were obtained 4 h after injection. Tumor uptake in vivo was receptor specific, as it could be blocked with an excess of nonlabeled Z HER2:342 . HER2-expressing xenografts were clearly imaged 4 h after injection using a g-camera.
European Journal of Nuclear Medicine and Molecular Imaging, 2009
Purpose The purpose of this study was to design and evaluate a novel platform for labelling of A... more Purpose The purpose of this study was to design and evaluate a novel platform for labelling of Affibody molecules, enabling both recombinant and synthetic production and site-specific labelling with 99mTc or trivalent radiometals. Methods The HER2-specific Affibody molecule PEP05352 was made by peptide synthesis. The chelator sequence SECG (serine-glutamic acid-cysteine-glycine) was anchored on the C-terminal to allow 99mTc labelling. The cysteine can