64 Cu-Labeled Affibody Molecules for Imaging of HER2 Expressing Tumors (original) (raw)
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Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 2006
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 (approximately 7 kDa) phage-display-selected 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. Z(HER2:342) was labeled with (111)In via isothiocyanate-benzyl-DTPA (DTPA is diethylenetriaminepentaacetic acid) and the conjugate was characterized in vitro and in vivo. (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 ...
Journal of Nuclear Medicine, 2009
The detection of human epidermal growth factor receptor type 2 (HER2) expression in malignant tumors provides important information influencing patient management. Radionuclide in vivo imaging of HER2 may permit the detection of HER2 in both primary tumors and metastases by a single noninvasive procedure. Small (7 kDa) high-affinity anti-HER2 Affibody molecules may be suitable tracers for SPECT visualization of HER2-expressing tumors. The use of generator-produced 99m Tc as a label would facilitate the prompt translation of anti-HER2 Affibody molecules into use in clinics. Methods: A C-terminal cysteine was introduced into the Affibody molecule Z HER2:342 to enable sitespecific labeling with 99m Tc. Two recombinant variants, His 6 -Z HER2:342 -Cys (dissociation constant [K D ], 29 pM) and Z HER2:2395 -Cys, lacking a His tag (K D , 27 pM), were labeled with 99m Tc in yields exceeding 90%. The binding specificity and the cellular processing of Affibody molecules were studied in vitro. Biodistribution and g-camera imaging studies were performed in mice bearing HER2-expressing xenografts. Results: 99m Tc-His 6 -Z HER2:342 -Cys was capable of targeting HER2-expressing SKOV-3 xenografts in SCID mice, but the liver radioactivity uptake was high. A series of comparative biodistribution experiments indicated that the presence of the His tag caused elevated accumulation in the liver. 99m Tc-Z HER2:2395 -Cys, not containing a His tag, showed low uptake in the liver and high and specific uptake in HER2-expressing xenografts. Four hours after injection, the radioactivity uptake values (percentage of injected activity per gram of tissue [%IA/g]) were 6.9 6 2.5 (mean 6 SD) %IA/g in LS174T xenografts (moderate level of HER2 expression) and 15 6 3 %IA/g in SKOV-3 xenografts (high level of HER2 expression). The corresponding tumor-to-blood ratios were 88 6 24 and 121 6 24, respectively. Both LS174T and SKOV-3 xenografts were clearly visualized with a clinical g-camera 1 h after injection of 99m Tc-Z HER2:2395 -Cys. Conclusion: The Affibody molecule 99m Tc-Z HER2:2395 -Cys is a promising tracer for SPECT visualization of HER2-expressing tumors.
European Journal of Nuclear Medicine and Molecular Imaging, 2007
Purpose Expression of human epidermal growth factor receptor type 2 (HER2) in malignant tumours possesses well-documented prognostic and predictive value. Noninvasive imaging of expression can provide valuable diagnostic information, thereby influencing patient management. Previously, we reported a phage display selection of a small (about 7 kDa) protein, the Affibody molecule Z HER2:342 , which binds HER2 with subnanomolar affinity, and demonstrated the feasibility of targeting of HER2expressing xenografts using radioiodinated Z HER2:342 . The goal of this study was to develop a method for 99m Tc labelling of Z HER2:342 using the MAG3 chelator, which was incorporated into Z HER2:342 using peptide synthesis, and evaluate the targeting properties of the labelled conjugate. Methods MAG3-Z HER2:342 was assembled using Fmoc/tBu solid phase peptide synthesis. Biochemical characterisation of the agent was performed using RP-HPLC, ESI-MS, biosensor studies and circular dichroism. A procedure for 99m Tc labelling in the presence of sodium/potassium tartrate was established. Tumour targeting was evaluated by biodistribution study and gamma camera imaging in xenograft-bearing mice. Biodistribution of 99m Tc-MAG3-Z HER2:342 and 125 I-para-iodobenzoate -Z HER2:342 was compared 6 h p.i. Results Synthetic MAG3-Z HER2:342 possessed an affinity of 0.2 nM for HER2 receptors. The peptide was labelled with 99m Tc with an efficiency of about 75-80%. Labelled 99m Tc-MAG3-Z HER2:342 retained capacity to bind specifically HER2-expressing SKOV-3 cells in vitro. 99m Tc-MAG3-Z HER2:342 showed specific tumour targeting with a contrast similar to a radioiodinated analogue in mice bearing LS174T xenografts. Gamma camera imaging demonstrated clear and specific visualisation of HER2 expression. Conclusion Incorporation of a mercaptoacetyl-containing chelating sequence during chemical synthesis enabled sitespecific 99m Tc labelling of the Z HER2:342 Affibody molecule with preserved targeting capacity.
Tumor Imaging Using a Picomolar Affinity HER2 Binding Affibody Molecule
Cancer Research, 2006
The detection of cell-bound proteins that are produced due to aberrant gene expression in malignant tumors can provide important diagnostic information influencing patient management. The use of small radiolabeled targeting proteins would enable high-contrast radionuclide imaging of cancers expressing such antigens if adequate binding affinity and specificity could be provided. Here, we describe a HER2specific 6 kDa Affibody molecule (hereinafter denoted Affibody molecule) with 22 pmol/L affinity that can be used for the visualization of HER2 expression in tumors in vivo using gamma camera. A library for affinity maturation was constructed by re-randomization of relevant positions identified after the alignment of first-generation variants of nanomolar affinity (50 nmol/L). One selected Affibody molecule, Z HER2:342 showed a >2,200-fold increase in affinity achieved through a single-library affinity maturation step. When radioiodinated, the affinity-matured Affibody molecule showed clear, high-contrast visualization of HER2-expressing xenografts in mice as early as 6 hours post-injection. The tumor uptake at 4 hours post-injection was improved 4-fold (due to increased affinity) with 9% of the injected dose per gram of tissue in the tumor. Affibody molecules represent a new class of affinity molecules that can provide small sized, high affinity cancer-specific ligands, which may be well suited for tumor imaging. (Cancer Res 2006; 66(8): 4339-48)
Conjugate for In Vivo Imaging of HER2 Expression in Malignant Tumors
an Affibody-Based Conjugate for In Vivo Imaging of HER2:342 Z − In-Benzyl-DTPA 111 http://jnm.snmjournals.org/content/47/5/846 This article and updated information are available at: http://jnm.snmjournals.org/site/subscriptions/online.xhtml Information about subscriptions to JNM can be found at: http://jnm.snmjournals.org/site/misc/permission.xhtml
Cancer Research, 2007
The Affibody molecule Z(HER2:342-pep2), site-specifically and homogeneously conjugated with a 1,4,7,10-tetra-azacylododecane-N,N',N'',N'''-tetraacetic acid (DOTA) chelator, was produced in a single chemical process by peptide synthesis. DOTA-Z(HER2:342-pep2) folds spontaneously and binds HER2 with 65 pmol/L affinity. Efficient radiolabeling with >95% incorporation of (111)In was achieved within 30 min at low (room temperature) and high temperatures (up to 90 degrees C). Tumor uptake of (111)In-DOTA-Z(HER2:342-pep2) was specific for HER2-positive xenografts. A high tumor uptake of 23% injected activity per gram tissue, a tumor-to-blood ratio of >7.5, and high-contrast gamma camera images were obtained already 1 h after injection. Pretreatment with Herceptin did not interfere with tumor targeting, whereas degradation of HER2 using the heat shock protein 90 inhibitor 17-allylamino-geldanamycin before administration of (111)In-DOTA-Z(HER2:342-pep2) obliterated the tumor image. The present results show that radiolabeled synthetic DOTA-Z(HER2:342-pep2) has the potential to become a clinically useful radiopharmaceutical for in vivo molecular imaging of HER2-expressing carcinomas.
European journal of nuclear medicine and molecular imaging, 2008
Purpose The expression of human epidermal growth factor receptor-2 (HER2) receptors in cancers is correlated with a poor prognosis. If assessed in vivo, it could be used for selection of appropriate therapy for individual patients and for monitoring of the tumor response to targeted therapies. We have radiolabeled a HER2-binding Affibody molecule with fluorine-18 for in vivo monitoring of the HER2 expression by positron emission tomography (PET). Materials and methods The HER2-binding Z HER2:342 -Cys Affibody molecule was conjugated with N-2-(4-[ 18 F]fluorobenzamido)ethyl]maleimide ([ 18 F]FBEM). The in vitro binding of the resulting radioconjugate was characterized by receptor saturation and competition assays. For in vivo studies, the radioconjugate was injected into the tail vein of mice bearing subcutaneous HER2-positive or HER2-negative tumors. Some of the mice were pre-treated with non-labeled Z HER2:342 −Cys. The animals were sacrificed at different times post-injection, and the radioactivity in selected tissues was measured. PET images were obtained using an animal PET scanner.
Bioconjugate Chemistry, 2007
Detection of HER2-overexpression in tumors and metastases is important for the selection of patients who will benefit from trastuzumab treatment. Earlier investigations showed successful imaging of HER2-positive tumors in patients using indium-or gallium-labeled Affibody molecules. The goal of this study was to evaluate the use of 99m Tc-labeled Affibody molecules for the detection of HER2 expression. The Affibody molecule Z HER2:342 with the chelator sequences mercaptoacetyl-Gly-Glu-Gly (maGEG) and mercaptoacetyl-Glu-Glu-Glu (maEEE) was synthesized by peptide synthesis and labeled with technetium-99m. Binding specificity, cellular retention, and in Vitro stability were investigated. The biodistribution of 99m Tc-maGEG-Z HER2:342 and 99m Tc-maEEE-Z HER2:342 was compared with 99m Tc-maGGG-Z HER2:342 in normal mice, and the tumor targeting properties of 99m Tc-maEEE-Z HER2:342 were determined in SKOV-3 xenografted nude mice. The results showed that the Affibody molecules were efficiently labeled with technetium-99m. The labeled conjugates were highly stable in Vitro with preserved HER2-binding capacity. The use of glutamic acid in the chelator sequences for 99m Tc-labeling of Z HER2:342 reduced the hepatobiliary excretion 3-fold with a single Gly-to-Glu substitution and 10-fold with three Gly-to-Glu substitutions. 99m Tc-maEEE-Z HER2:342 showed a receptor-specific tumor uptake of 7.9 (1.0 %IA/g and a tumor-to-blood ratio of 38 at 4 h pi. Gamma-camera imaging with 99m Tc-maEEE-Z HER2:342 could detect HER2-expressing tumors in xenografts already at 1 h pi. It was concluded that peptide synthesis for the coupling of chelator sequences to Affibody molecules for 99m Tc labeling is an efficient way to modify the in ViVo kinetics. Increased hydrophilicity, combined with improved stability of the mercaptoacetyl-triglutamyl chelator, resulted in favorable biodistribution, making 99m Tc-maEEE-Z HER2:342 a promising tracer for clinical imaging of HER2 overexpression in tumors.