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Comparative evaluation of Affibody molecules for radionuclide imaging of in vivo expression of carbonic anhydrase IX Javad Garousi, Hadis Honarvar, Ken G Andersson, Bogdan Mitran, Anna Orlova, Jos Buijs, John Löfblom, Fredrik Y. Frejd, and Vladimir Tolmachev Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00502 • Publication Date (Web): 16 Aug 2016 Downloaded from http://pubs.acs.org on September 20, 2016
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Molecular Pharmaceutics
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Comparative evaluation of Affibody molecules for radionuclide imaging of in vivo expression of carbonic anhydrase IX
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Javad Garousi ‡, Hadis Honarvar‡, Ken G. Andersson¥, Bogdan Mitran †, Anna Orlova †, Jos Buijs‡,§, John Löfblom¥, Fredrik Y. Frejd‡,║ *,Vladimir Tolmachev1‡.
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‡
Department of Immunology, Genetics and Pathology, Uppsala University, SE-75285 Uppsala, Sweden;
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¥
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106 91 Stockholm, Sweden;
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Division of Protein Technology, School of Biotechnology, KTH-Royal Institute of Technology, SE-
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Division of Molecular Imaging, Department of Medicinal Chemistry, Uppsala University, SE-751 83 Uppsala, Sweden;
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§
Ridgeview Instruments AB, SE-74020 Vänge, Sweden.
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║
Affibody AB, SE-171 63 Stockholm, Sweden
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*Corresponding author: Prof. Fredrik Y. Frejd Department of Immunology, Genetics and Pathology, Uppsala University, SE-75185, Uppsala, Sweden Phone: +46 70 7225189 Fax: +46 18 471 34 32 e-mail:
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Abstract
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Overexpression of the enzyme carbonic anhydrase IX (CAIX) is documented for chronically
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hypoxic malignant tumors as well as for normoxic renal cell carcinoma. Radionuclide
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molecular imaging of CAIX would be useful for detection of hypoxic areas in malignant
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tumors, for patients’ stratification of CAIX-targeted therapies and for discrimination of
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primary malignant and benign renal tumors. Earlier, we have reported feasibility of in vivo
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radionuclide based imaging of CAIX expressing tumors using Affibody molecules, small
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affinity proteins based on a non-immunoglobulin scaffold. In this study, we compared
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imaging properties of several anti-CAIX Affibody molecules having identical scaffold parts
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and competing for the same epitope on CAIX, but having different binding paratopes. Four
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variants were labeled using residualizing 99mTc and non-residualizing 125I labels. All
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radiolabeled variants demonstrated high-affinity detection of CAIX-expressing cell line SK-
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RC-52 in vitro and specific accumulation in SK-RC-52 xenografts in vivo. 125I-labeled
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conjugates demonstrated much lower radioactivity uptake in kidneys but higher radioactivity
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concentration in blood compared with 99mTc-labed counterparts. Although all variants cleared
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rapidly from blood and non-specific compartments, there was noticeably difference in their
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biodistribution. The best variant for imaging of expression of CAIX- in disseminated cancer
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was 99mTc-(HE)3-ZCAIX:2 providing tumor uptake of 16.3±0.9 %ID/g and tumor-to-blood
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ratio of 44±7 at 4 h after injection. For primary renal cell carcinoma, the most promising
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imaging candidate was 125I-ZCAIX:4 providing tumor-kidney ratio of 2.1±0.5. In conclusion,
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several clones of scaffold proteins should be evaluated to select the best variant for
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development of an imaging probe with optimal sensitivity for the intended application.
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Key Words: CAIX, Affibody molecule, imaging, radionuclide
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INTRODUCTION
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Abnormal expression of cell-surface proteins often accompanies cancerogenesis.
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Understanding of the role and pattern of such aberrant expression creates a potential for
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improved diagnostics and treatment of disseminated cancer. Molecular recognition of such
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abnormally expressed proteins, for example, receptor tyrosine kinases, may be utilized for
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selective systemic tumor therapy1 and imaging of somatostatin receptor overexpression is
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applied to stratify patients for radionuclide therapy of neuroendocrine tumors.2 Carbonic
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anhydrase IX (CAIX) is an aberrantly expressed protein that could potentially be used for
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improved tumor therapy.3-5
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CAIX is a cellular membrane-bound enzyme catalyzing the conversion of carbon dioxide to
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hydrogen carbonate.6 Two extracellular catalytic domains of CAIX form a disulfide bond-
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stabilized dimer.7 In normal human tissues, CAIX is expressed only in proliferating crypt
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enterocytes of the intestinal mucosa.8 In hypoxic conditions of a tumor, CAIX is strongly
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upregulated through the hypoxia inducible factor 1 (HIF-1) cascade and in well oxygenated
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cells, the expression of HIF-1α is downregulated by the von Hippel-Lindau protein (pVHL).
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This control is however inactivated in hypoxic cells present in tumors.9 It is worth mentioning
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that overexpression of CAIX in tumors is not always associated with hypoxia. In clear cell
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renal carcinomas (CCRC), pVHL is inactivated, which causes high expression of HIF-1α
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even in normoxic conditions. 10 This is associated with overexpression of CAIX in over 90%
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of CCRC.11
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Overexpression of CAIX causes acidification of the tumor microenvironment resulting in
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reduced cell adhesion, increased motility and migration, and in activated proteases. This
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increases the potential for tumor invasion and metastasis.9 The acidification of the tumor
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extracellular space can influence cellular uptake of drugs by favoring weak acids, but reduce
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uptake of weak bases.12 This is in agreement with the results of clinical studies where
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expression of CAIX in tumors was correlated with chemoresistance of tumors and shorter
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relapse-free survival of breast cancer patients treated by(neo) adjuvant therapy.13-15 This
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created a rationale for development of CAIX-targeting anticancer drugs, such as small
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molecular inhibitors and monoclonal antibodies.16-18 CAIX is considered as a high-priority
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target by METOXIA, European Collaborative Project aiming at investigation of hypoxic
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microenvironment of tumors to develop new strategies for improved biomarkers for diagnosis
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and for more efficient chemo- and radiotherapy.19 The level of tumor-associated CAIX
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expression, which is determined by immunohistochemistry, correlates strongly with the level
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of hypoxia in e.g. cervical tumor,20 head and neck carcinoma,21 meningioma, glioblastoma
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multiforme, comedo type of breast carcinomas and anaplastic ependymoma.22 As hypoxic
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tumors are radioresistant,23 in vivo detection of CAIX might be a predictive biomarker for
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resistance to radiation therapy.24 For example, high expression of CAIX is a predictor of local
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recurrence after radiotherapy of glottic laryngeal carcinoma.25
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Radionuclide molecular detection of CAIX expression in vivo can be useful for stratifying
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cancer patients that would most likely respond to emerging CAIX-targeting therapies, and to
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select subjects with hypoxic tumors, that are not well addressed with conventional radiation
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therapy. In addition, CAIX-specific probes might be used for imaging primary renal cell
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carcinomas, where the use of 18F-FDG does not provide sufficient sensitivity due to low
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glucose utilization.26
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Current imaging approaches include the use of radiolabeled anti-CAIX monoclonal antibody
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G250,27 antibody fragments or small molecular CAIX inhibitors, e.g. tertiary
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benzenesulfonamides.28-30 The use of intact G250 provides high tumor uptake of
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radionuclides, but a relatively long time (ca. 7 days) is required high contrast (tumor-to-blood
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ratio of ca. 10).31 The use of smaller fragments enabled to reach a comparable contrast 4 ACS Paragon Plus Environment
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(tumor-to-blood ratio of 8.72 ± 3.45) within 24 hours after injection.28 This suggests that the
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reduction of the size of protein-based CAIX imaging agents could improve contrast and
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therefore sensitivity of imaging.
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The use of non-immunoglobulin scaffold proteins instead of antibody derivatives is an
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efficient way to create small high-affinity imaging probes.32-33 Scaffold proteins include a
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robust backbone scaffold providing structural stability and variable amino acids enabling
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creation of combinatorial libraries. The use of phage, bacterial, ribosome, or yeast display
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techniques enables selection of small binding proteins having high affinity to desirable
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molecular targets.34 Affibody molecules are small (6-7 kDa) high affinity protein binders that
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have been successfully used for development of probes for radionuclide imaging of cancer-
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related molecular targets, for example, HER2,35-37 EGFR,38 IGF-1R,39 and PDGFRβ.40,41
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Phase I/II trials with HER2-targeting Affibody molecules have demonstrated that this tracer
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can provide high contrast imaging in the clinic.42-43 We have recently demonstrated feasibility
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of radionuclide imaging of CAIX-expression in vivo using the radiolabeled ZCAIX:1
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Affibody molecule.44 Upon labelling with 99mTc(CO)3, that probe allowed for imaging of
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CAIX-expressing xenografts 4 h post injection. Tumor-to-blood ratio of 53±1 in a murine
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model has been achieved at 4 h after injection. This suggested that ZCAIX:1 Affibody
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molecule could become a useful tool for detection of CAIX expression in vivo. Still, three
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more clones, ZCAIX:2, ZCAIX:3 and ZCAIX:4 demonstrated similar binding strength during
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the selection process. Our previous studies have shown that target expression level, size and
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affinity of a probe are not the only factors influencing contrast of imaging using Affibody
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molecules. Minor variations in the binding site composition influences off-target interaction
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of Affibody molecules such as binding to blood proteins and uptake in normal tissues.45-46
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This may result in an appreciable difference in blood clearance rate and background, affecting
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the imaging contrast and therefore sensitivity of the imaging. Thus, evaluation of several
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candidates might enable selection of a probe with optimal imaging properties.
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This study aimed at a comparative assessment of the most promising CAIX-binding Affibody
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molecules to select the best candidate for molecular imaging probes. To perform the
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evaluation, the residualizing 99mTc(CO)3-HEHEHE and non-residualizing 125I-PIB labels
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(Figure 1) were used.
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Figure 1. Schematic presentation of 99mTc(CO)3-HEHEHE and 125I-PIB labels
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EXPERIMENTAL SECTION
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Reagents, equipment and statistics
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Affibody molecules have been provided by Affibody AB (Solna, Sweden). The CRS kits for
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production of technetium tricarbonyl were purchased from Center for Radiopharmaceutical
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sciences (Villigen, Switzerland). An automatic gamma-spectrometer with a NaI (Tl) detector
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(1480 WIZARDWallac Oy, Turku, Finland) was used for measurement of cell-associate
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radioactivity and for biodistribution measurements. Formulation of injection solutions was
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performed using VDC-405 ionization chamber (Veenstra Instruments BV, The Netherlands).
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Statistical analysis of biological data was performed using GraphPad Prism (version 6.00 for
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Windows GraphPad Software, San Diego CA) to find any significant differences (p
