Gallium-68-Labeled Affibody Molecule for PET Imaging of PDGFRÎ²

Article pubs.acs.org/molecularpharmaceutics

Gallium-68-Labeled Affibody Molecule for PET Imaging of PDGFRβ Expression in Vivo Joanna Strand,† Zohreh Varasteh,‡ Olof Eriksson,‡ Lars Abrahmsen,§ Anna Orlova,‡ and Vladimir Tolmachev*,† †

Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden Preclinical PET Platform, Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden § KDev Oncology, Karolinska Science Park, Solna, Sweden ‡

ABSTRACT: Platelet-derived growth factor receptor β (PDGFRβ) is a transmembrane tyrosine kinase receptor involved, for example, in angiogenesis. Overexpression and excessive signaling of PDGFRβ has been observed in multiple malignant tumors and fibrotic diseases, making this receptor a pharmaceutical target for monoclonal antibodies and tyrosine kinase inhibitors. Successful targeted therapy requires identification of responding patients. Radionuclide molecular imaging would enable determination of the PDGFRβ status in all lesions using a single noninvasive repeatable procedure. Recently, we have demonstrated that the affibody molecule Z09591 labeled with 111In can specifically target PDGFRβ-expressing tumors in vivo. The use of positron emission tomography (PET) as an imaging technique would provide superior resolution, sensitivity, and quantitation accuracy. In this study, a DOTAconjugated Z09591 was labeled with the generator-produced positron emitting radionuclide 68Ga (T1/2 = 67.6 min, Eβ + max = 1899 keV, 89% β+). 68Ga-DOTA-Z09591 retained the capacity to specifically bind to PDGFRβ-expressing U-87 MG glioma cells. The half-maximum inhibition concentration (IC50) of 68Ga-DOTA-Z09591 (6.6 ± 1.4 nM) was somewhat higher than that of 111 In-DOTA-Z09591 (1.4 ± 1.2 nM). 68Ga-DOTA-Z09591 demonstrated specific (saturable) targeting of U-87 MG xenografts in immunodeficient mice. The tumor uptake at 2 h after injection was 3.7 ± 1.7% IA/g, which provided a tumor-to-blood ratio of 8.0 ± 3.1. The only organ with higher accumulation of radioactivity was the kidney. MicroPET imaging provided high-contrast imaging of U-87 MG xenografts. In conclusion, the 68Ga-labeled affibody molecule Z09591 is a promising candidate for further development as a probe for imaging PDGFRβ expression in vivo using PET. KEYWORDS: PDGFR beta, PET, affibody molecule, gallium-68, DPTA

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various tumors, including chordoma,10 large granular lymphocyte leukemia,11 and prostate cancer.12 There is a growing amount of evidence that expression of PDGFRβ in tumorassociated pericytes and cancer associated fibroblasts is important for promoting of neoangiogenesis, invasion, and metastasis.13 In breast and prostate cancers, stromal PDGFRβreceptor expression significantly correlates with shorter survival.14,15 Thus, there is a rationale for targeting of PDGFRβ in both malignant tumors5,16 and nonmalignant diseases.6 Both PDGFRβ-specific monoclonal antibodies8,9,17,18 and PDGFRtargeting tyrosine kinase inhibitors (TKIs)12,19 are under investigation. Optimal targeted therapy requires identification of patients who would most likely benefit from PDGFRβ-targeting to

INTRODUCTION Platelet-derived growth factor receptor β (PDGFRβ) is a transmembrane receptor tyrosine kinase. PDGFRβ signaling is activated by binding of members of the platelet-derived growth factor (PDGF) family, such as PDGF-BB and PDGF-DD, and results in increased mitogenic and antiapoptotic activity as well as actin reorganization and cell migration.1 PDGFRβ plays an important role in angiogenesis and in embryonic development, in the formation of blood vessels, kidneys, and adipocytes.2 In adults, PDGFRβ is expressed mainly in vascular smooth muscle cells and pericytes. PDGFRβ signaling is essential for the formation of a connective tissue during wound healing and in regulation of the interstitial fluid pressure in tissues.3,4 Abnormal expression and signaling of the PDGFRβ has been shown in a variety of disorders, such as cancer, atherosclerosis, and fibrotic diseases.5,6 For example, paracrine activation of PDGFRβ has been identified as a key factor in development of glomerulosclerosis.7 PDGFRβ is a pharmaceutical target in liver fibrosis8 and atherosclerosis.9 Overexpression of PDGFRβ and its auto- and paracrine stimulation has been documented for © XXXX American Chemical Society

Special Issue: Positron Emission Tomography: State of the Art Received: April 17, 2014 Revised: June 16, 2014 Accepted: June 27, 2014

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dx.doi.org/10.1021/mp500284t | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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Positron emission tomography (PET) as a radionuclide imaging technique offers several advantages in comparison with single photon emission computer tomography (SPECT), including superior sensitivity, spatial and temporal resolution, and quantification accuracy.36 Currently, PET is predominantly associated with the use of small organic molecules labeled with short-lived biogenic radionuclides. However, there is a growing interest in PET as a modality for imaging using antibodies and peptides.37,38 Typically, long-lived positron-emitting nuclides, such as 64Cu (T1/2 = 12.7 h), 76Br (T1/2 = 16.2 h), 55Co (T1/2 = 17.5 h), 86Y (T1/2 = 14.7 h), 89Zr (T1/2 = 78.4 h), and 124I (T1/2 = 100 h), are considered as labels of antibodies and peptides. However, the rapid localization of DOTA-Z09591 enables the use of short-lived positron emitters, such as 68Ga (T1/2 = 67.6 min, Eβ + max = 1899 keV, 89% β+). Generator production of 68 Ga provides potential advantages in terms of availability and low cost.39 A good match between optimal imaging time and half-life of the nuclide would also reduce the radiation dose burden to the patient. The DOTA chelator in DOTA-Z09591 suits well for labeling with 68Ga.39 The aim of this study was to evaluate if labeling of DOTAZ09591 with 68Ga would provide an imaging agent suitable for PDGFRβ visualization using PET. The secondary goal was to compare the biodistribution profile for 111In -and 68Ga-labeled DOTA-Z09591 to assess the potential influence of different nuclides on the targeting properties.

avoid overtreatment. This raises the issue of detection of PDGFRβ in lesions. Currently, the predominant way to determine relevant molecular targets in tumors is based on taking a biopsy (e.g., to perform immunohistochemistry). This invasive procedure is associated with certain morbidity and requires a patient’s consent. Therefore, only a limited number of samples with limited volume can be taken from a few locations. This might cause false-negative findings due to intraand intertumor and temporal heterogeneity of target expression.20 An alternative to biopsies is radionuclide molecular imaging allowing visualization of a molecular target expression in all lesions simultaneously and repeatedly by a noninvasive procedure.21 This permits to follow changes in a molecular target expression during the course of disease and/or in response to a therapy. Radiolabeling of TKIs is a possible approach for development of imaging agents for visualization of molecular targets. For example, 11C-labeled imatinib has been synthesized and evaluated in vivo.22 A major issue with this kind of tracer is that the target structure is situated intracellularly, requiring that the tracer penetrates the cellular membrane. This ability is associated with noticeable lipophilicity. However, lipophilicity is generally associated with high liver uptake, complicating visualization of hepatic metastases that are frequent in many types of cancers.23 Another approach would be targeting of the extracellular domain of PDGFRβ. Unfortunately, the natural ligand PDGFBB is unsuitable as a tracer because of binding to proteins in serum.24 Phage-display technology has been used for selection of peptides and small proteins, which bind different proteins. The reported affinity of selected PDGFRβ-binding peptides is modest (KD in the range 100 to 1000 nM)25,26 and insufficient for in vivo imaging. In contrast, some small binding proteins have an inherent scaffold, a robust constant amino acids framework keeping variegated amino acids in defined positions and decreasing entropic penalty upon target binding.27 This is exemplified by affibody molecules, small robust nonimmunoglobulin affinity ligands derived from B-domain in immunoglobulin-binding region of staphylococcal protein A.28 These proteins lack cysteines and fold into a three-helix bundle domain containing 58 amino acids (7 kDa). The high affinity (in low nanomolar or subnanomolar range) and small size makes affibody molecules suitable as molecular imaging probes, as shown with binders specific for HER2,29 IGF-1R,30 and HER3.31 Favorable in vivo kinetics of affibody molecules has been shown to enable high contrast imaging only a few hours after injections, both in preclinical23 and clinical studies.32,33 Previously, our group reported that the 111In-labeled PDGFRβ targeting affibody molecule Z09591 could be used to visualize PDGFRβ in vivo. Z09591 was selected to bind with high affinity to both human and murine PDGFRβ, making the mouse a suitable in vivo model for imaging studies using this tracer.34 A unique cysteine was engineered at the C-terminus to enable site-specific conjugation and used for attachment of a maleimido derivative of the versatile DOTA chelator. The 111In labeled tracer, 111In-DOTA-Z09591, was bound to PDGFRβexpressing U-87 MG glioblastoma cells with high affinity (apparent KD 92 ± 10 pM). 111In-DOTA-Z09591 accumulated specifically in PDGFRβ-expressing U-87 MG glioblastoma xenografts in mice. The study demonstrated significant increase of the tumor-to-organ ratios between 1 and 2 h after injection.35 However, further increase of tumor-to-organ ratios was only marginal.

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EXPERIMENTAL SECTION Buffers including 0.1 M phosphate buffered saline (PBS), pH 7.5, 0.2 M ammonium acetate, pH 5.5, 1.25 M sodium acetate buffer, pH 3.6, and 0.2 M citric acid were prepared using common methods from chemicals supplied by Merck (Darmstadt, Germany). Buffers, which were used for labeling, were purified from metal contamination using Chelex 100 resin (Bio-Rad Laboratories, Richmond, USA). High-quality Milli-Q water (resistance higher than 18 ΩM cm) was used for preparing the solutions. 68Ge/68Ga generator (50 mCi) was from Eckert and Ziegler. 111In-indium chloride (in 0.05 M HCl) was purchased from Covidien. Labeling yield and radiochemical purity of the labeled affibody molecule was determined by radio-ITLC (150−771 DARK GREEN strips, Biodex Medical Systems New York, US) cross-validated by SDS-PAGE as described earlier.40 The distribution of radioactivity along the thin layer chromatography strips and SDS-PAGE gels was measured on a Cyclone Storage Phosphor System and analyzed using the OptiQuant software (PerkinElmer Wellesey, MA, USA). Ketalar (ketamine, 50 mg/mL, Pfizer), Rompun (xylazin, 20 mg/mL, Bayer), and heparin (5000 IE/mL, Leo Pharma) were obtained commercially. The radioactivity uptake from cellular processing and the biodistribution studies was measured using an automated gamma-counter with a 3 inch NaI(TI) detector (1480 WIZARD, Wallac). The data were analyzed by unpaired, two-tailed t-test using GraphPad Prism (version 4.00 for Windows GraphPad Software San Diego, US) in order to determine significant differences (P < 0.05). A paired t-test was used in the dual label study. Affibody Molecule. The affibody molecule Z09591-Cys was expressed in E. coli and purified essentially as described earlier.35 The maleimido derivative of DOTA chelator (Macrocyclics) was conjugated to the cysteine thiol of Z09591, as described earlier.41 The identity of the conjugate (designated as DOTA-Z09591) was confirmed, and the purity was evaluated B



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compound (protein concentration of 0.5 nM) was added to 12 Petri dishes containing approximately 106 cells/dish. The cells were incubated at 37 °C, in a humidified incubator. At predetermined time points (0.5, 1, 2, and 3 h after incubation start), the medium from a set of three dishes was removed. The cells were washed twice with 1 mL of ice-cold medium. To collect the membrane-bound radioactivity, the cells were treated with 0.5 mL of 0.2 M glycine buffer containing 4 M urea, pH 2.0, for 5 min on ice. Dishes were additionally washed with 0.5 mL acidic buffer followed by 1 mL PBS, and the fractions were pooled. To collect radioactivity internalized by the cells, treatment with 0.5 mL of 1 M NaOH at 37 °C for 0.5 h was performed. Dishes were additionally washed with 0.5 mL of NaOH solution followed by 1 mL of PBS, and the alkaline fractions were pooled. The percentage of internalized radioactivity was calculated for each time point. In Vivo Evaluation of 68Ga-DOTA-Z09591 and 111InDOTA-Z09591. All animal experiments were performed in accordance with national legislation on laboratory animal’s protection and have been approved by the ethics committee for Animal Research in Uppsala. Euthanasia was performed under Rompun−Ketalar anesthesia, and all efforts were made to minimize suffering. To minimize the number of animals and to improve statistical power, comparison of 68Ga- and 111Inlabeled DOTA-Z09591 was performed as a dual label study.42 Female BALB/C nu/nu mice (6−8 weeks old at arrival) were purchased from Taconic M&B. At the time of experiment the average weight was 20.6 ± 0.9 g. For xenografting, 2 × 106 U87 MG cells in PBS were subcutaneously implanted in the right hind leg. The average tumor weight at the time of experiment was 0.32 ± 0.28 g. A mixture of 10 kBq 111In-DOTA-Z09591 and 380 kBq 68Ga-DOTA-Z09591 in 100 μL of PBS were injected in three groups of mice (four mice each). The total injected amount of protein dose was adjusted to 1 μg/animal by adding unlabeled protein. In order to saturate PDGFRβ receptors in tumors, one group of mice was preinjected with 70 μg of unlabeled Z09591 affibody molecules. The mice were euthanized at 1 h p.i. and 2 h p.i. by an intraperitoneal injection of Ketalar−Rompun solution (20 μL of solution/g body weight: Ketalar, 10 mg/mL; Rompun, 1 mg/mL) followed by heart puncture with a syringe rinsed with heparin (5000 IE/mL) and exsanguinated. Blood and organ samples, lung, liver, spleen, stomach, colon, kidneys, tumor, muscle, bone, and gastrointestinal tract (with content), were collected and weighed. The animals in the control group were euthanized at 1 h after injection. The whole spectra for each sample as well as spectra of injected solution standards were recorded immediately after dissection and 20 h later (after complete decay of 68Ga). The data were corrected for background radiation, gamma spectrometer dead time during each measurement, and decay during measurement. On the basis of the second measurement, organ uptake values for 111In were calculated as percent of injected activity per gram of tissue (% IA/g), except for the gastrointestinal tract and the remaining carcass, which were calculated as % IA per whole sample. Thereafter, indium counts were corrected for decay (using an 111In sample as standard) and subtracted from counts obtained during the first measurement (immediately after dissection). The corrected values showing radioactivity in all investigated organs during the first measurement were used to calculate the biodistribution of 68Ga-labeled tracer. Imaging. One U87 xenografted mouse was injected with 1.5 MBq of 68Ga-DOTA-Z09591. Immediately before imaging (2 h

by high-performance liquid chromatography and in-line mass spectrometry (HPLC-MS) using an Agilent 1100 HPLC/MSD equipped with electrospray ionization (ESI) and a single quadrupole as described earlier.41 According to the mass spectrometry analysis, 95% of the affibody molecules were conjugated with a single DOTA chelator. Five percent remained unconjugated and exist in dimeric form. A stock solution of DOTA-Z09591 (1.32 mg/mL in 0.2 M sodium acetate, pH 5.4) was stored frozen. Labeling. The 68Ge/68Ga generator was eluted with 0.1 M hydrochloric acid as eluent (prepared from 30% ultrapure HCl from Merck), and fractions containing 500 μL were collected. The third fraction containing the maximum radioactivity was used for labeling. The stock solution of DOTA-Z09591 (40 μL, 52.8 μg protein) was diluted with 100 μL of 1.25 M sodium acetate buffer, pH 3.6. Gallium-68 containing eluate, 2 MBq per microgram of affibody molecules, was added to the mixture. The mixture was incubated in 95 °C for 15 min. To measure the labeling yield, a small aliquot (1.6 μL) was analyzed by radio-ITLC eluted with 0.2 M citric acid. In this system free 68 Ga migrates with the solvent front, and the affibody molecule stays on the application point. After labeling, the conjugate was purified using NAP-5 size-exclusion column, pre-equilibrated with PBS. A small fraction of the purified mixture was analyzed using ITLC to determine purity. To evaluate the stability of the labeling, 2 aliquots of the labeled conjugate were incubated with a 500-fold molar excess of ethylenediaminetetraacetic acid (EDTA) for 4 h. The control aliquots were incubated with equal volume of PBS. Thereafter, the samples were analyzed using ITLC. Labeling of DOTA-Z09591 with 111In was performed as described earlier.35 In Vitro Cell Binding and Processing of 68Ga-Labeled Affibody Molecules. Binding specificity and cellular processing of 68Ga-DOTA- Z09591 was studied using PDGFRβexpressing U-87 MG glioma cells (American Type Culture Collection, ATCC) (ca. 3.6 × 104 receptors/cells35) using the method validated earlier.35 To test the binding specificity, 68Ga-DOTA-Z09591 with a protein concentrations of 27 pM was added to 6 dishes (106 cells/dish). A 100-fold excess of nonlabeled conjugate was added to three of these Petri dishes 5 min before the labeled conjugate to saturate the receptors. The dishes were incubated at 37 °C for 1 h in a humidified incubator. The media was collected, the cells were detached using trypsin−EDTA solution, and radioactivity was measured both in the media and cell suspension. Percent of cell-bound radioactivity was calculated for both the presaturated and unsaturated cells. To evaluate relative binding strength of gallium- and indiumlabeled DOTA-Z09591, the half-maximum inhibition concentration (IC50) was measured using 111In-DOTA-Z09591. Monolayers of U-87 MG cells were incubated for 4 h at 4 °C with natGa- or natIn-DOTA-Z09591 (0−500 nM) in the presence of 0.5 nmol 111In-DOTA-Z09591. After incubation, the cells were washed with 3 mL of media and treated with 1 mL of trypsin−EDTA solution (0.25% trypsin, 0.02% EDTA in buffer; Biochrom AG). The detached cells were collected, and the cell-associated radioactivity was measured. The IC50 values were determined using GraphPad Prism software. The rate of internalization of 68Ga-DOTA-Z09591 by U-87 MG glioma cells during continuous incubation was studied according to a method described and validated for 111InDOTA-Z09591 by Tolmachev and co-workers.35 The labeled C



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p.i.), the animal was sacrificed and the urine bladder was dissected to avoid interfering activity in urine close to the tumor xenograft. The microPET/CT imaging was performed in the Triumph Trimodality system (Gamma Medica, Inc.), a fully integrated SPECT/PET/CT hardware and software platform optimized for small animals. The acquisition time was 30 min. The PET data was reconstructed into a static image using a MLEM 2D algorithm (10 iterations). The CT raw file was reconstructed using Filter Back Projection (FBP). PET and CT dicom files were analyzed using PMOD v 3.12 software (PMOD Technologies Ltd., Zurich, Switzerland).

Figure 2. Cell-associated radioactivity as a function of time during continuous incubation of PDGFRβ-expressing U87 MG cells with the 68 Ga-labeled affibody molecule. Data are presented as mean from three dishes ± SD and normalized to the maximum uptake. Because of small variability, some error bars are hidden behind the symbols.

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RESULTS Labeling. Efficient labeling resulted in an average radiochemical yield of 92.3 ± 0.6% 68Ga-DOTA-Z09591 and a specific activity over 1.5 MBq/μg. After purification using disposable size-exclusion column, the radiochemical purity was 99.4 ± 0.3%. Stability of 68Ga-DOTA-Z09591 was evaluated by a challenge with 500-fold molar excess EDTA at room temperature with subsequent ITLC analysis. The radiochemical purity of EDTAtreated samples was 95.3 ± 0.7%, while the purity of the untreated controls was 98.5 ± 0.1%, i.e., the difference was close to the accuracy of the analytical method. This indicates high stability of the radiolabel. In Vitro Binding and Processing of 68Ga-Labeled Z09591. Binding of 68Ga-DOTA-Z09591 to the PDGFRβexpressing U-87 MG glioma cells was significantly lower (p < 0.001) for cells treated with a large excess of the nonlabeled affibody molecule. This demonstrates saturability of binding and suggests receptor-mediated binding of 68Ga-DOTAZ09591 to PDGFRβ-expressing cells. The results of competitive inhibition assay are presented in Figure 1. The IC50 values of natGa -DOTA-Z09591 and natIn-

Figure 3. In vivo targeting specificity of 68Ga-DOTA-Z09591 and 111 In-DOTA-Z09591 in mice bearing U87 MG xenografts at 1 h p.i. The blocked group was subcutaneously preinjected with an excess amount of nonlabeled affibody molecule. Results are presented as percentage of injected activity per gram of tissue (% IA/g).

without saturation, 3.6 ± 1.2 and 4.1 ± 1.3% IA/g, respectively. This suggests that the tumor accumulation of the conjugates in PDGFRβ expressing tumors is saturable and hence specific. There was also significantly lower uptake in normal tissues (lung, liver, spleen, and muscles) after preinjection of a saturating amount of nonlabeled DOTA-Z09591. Both 68Ga- and 111In-labeled conjugates demonstrated rapid clearance from blood and low uptake in normal organs except for kidney (Table 1). Uptake of radioactivity in the gastrointestinal tract (with content) was low, indicating that hepatobiliary excretion played a minor role in the clearance. The high nonsaturable uptake in kidney suggests that the conjugates were cleared by glomerular filtration with subsequent reabsorption and accumulation. There was no significant difference between tumor uptake of 68Ga-DOTAZ09591 (3.6 ± 1.2 and 3.7 ± 1.7 %IA/g at 1 and 2 h p.i., respectively) and 111In-DOTA-Z09591 (4.1 ± 1.3 and 4.2 ± 2.0% IA/g at 1 and 2 h p.i., respectively). The increase in tumor uptake from 1 to 2 h after injection was not statistically significant for either of the conjugates. The blood clearance of 111In-DOTA-Z09591 was somewhat faster than for 68Ga-DOTA-Z09591. The fraction of the radioactivity found in blood was significantly (p < 0.05 in the paired t-test) lower with 111In-DOTA-Z09591 that with 68GaDOTA-Z09591, both at 1 and at 2 h after injection (0.6 ± 0.3% IA/g vs 0.8 ± 0.3% IA/g and 0.23 ± 0.03% IA/g vs 0.46 ± 0.06% IA/g, respectively). The renal accumulation of the label

Figure 1. Inhibition of 111In-DOTA-Z09591 binding to U-87 MG cells with natGa -DOTA-Z09591 and natIn-DOTA-Z09591. Data presented as mean ± SD of three culture dishes.

DOTA-Z09591 were determined to be 6.6 ± 1.4 and 1.4 ± 1.2 nM, respectively. This suggests somewhat weaker binding of Ga-containing conjugate to PDGFRβ-expressing cells. Data on internalization of 68Ga-DOTA-PDGFRβ by PDGFRβ-expressing U-87 MG glioma cells are presented in Figure 2. The internalization was slow, although the internalized fraction of the radioactivity increased over time. After 3 h of incubation, the internalized fraction was 26 ± 1% of total cell-bound activity. Biodistribution Study. Data obtained in the in vivo specificity test are presented in Figure 3. After saturation of the PDGFRβ receptors by preinjection of a large molar excess of nonlabeled Z09591, the tumor uptake of both 68Ga-DOTAZ09591 (0.8 ± 0.1%IA/g) and 111In-DOTA-Z09591 (0.8 ± 0.2% IA/g) was significantly (p < 0.0005) lower than uptake D



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kidney (Figure 5). There was no noticeable uptake of radioactivity in any other organ. On the basis of biodistribution

Table 1. Comparative Biodistribution of Z09591 Labeled with Gallium-68 and Indium-111 1 and 2 h after Intravenous Injection in BALB/C nu/nu Mice Bearing U-87 MG Xenograftsa 1 h after injection 68

Ga-DOTAZ09591

blood lung liver spleen stomach colon kidney tumor muscle bone GI tract carcass

0.8 ± 0.3b 1.0 ± 0.2 0.91 ± 0.01 2.1 ± 0.4 1.4 ± 0.3 1.7 ± 0.3 179 ± 53 3.6 ± 1.2 0.4 ± 0.04 0.7 ± 0.6 1.8 ± 0.8 14 ± 3

111

In-DOTAZ09591

0.6 ± 0.3 1.0 ± 0.3 0.95 ± 0.04 2.2 ± 0.4 1.5 ± 0.3 1.8 ± 0.4 181 ± 60 4.1 ± 1.3 0.49 ± 0.09 0.7 ± 0.6 1.9 ± 0.8 16 ± 4

2 h after injection 68

Ga-DOTAZ09591

0.46 ± 0.06b 0.8 ± 0.2 0.7 ± 0.1 1.5 ± 0.2 0.9 ± 0.2 1.1 ± 0.2 233 ± 25 3.7 ± 1.7 0.5 ± 0.2 0.59 ± 0.09 1.07 ± 0.18 10 ± 2

111

In-DOTAZ09591

0.23 ± 0.03 0.8 ± 0.3 0.69 ± 0.08 1.5 ± 0.2 0.78 ± 0.08 1.0 ± 0.1 241 ± 24 4.2 ± 2.0 0.5 ± 0.3 0.51 ± 0.09 1.1 ± 0.1 11 ± 2

Figure 5. Small-animal PET/CT of mice bearing U-87 MG xenografts at 2 h after injection of 68Ga-DOTA-Z09591. (A) Maximum intensity projection showing whole body distribution with only kidneys and tumor (white arrow) visible. (B) Transaxial projection showing tumor (white arrow) and contralateral leg (red arrow). (C) Coronal projection showing biodistribution at the plane covering both kidneys and tumor (white arrow). The color bar to the right indicates the scale for panels B and C in % IA/g (the MIP in panel A is not quantitative).

a

Data are presented as an average % IA/g and standard deviation for four mice. Data for gastrointestinal (GI) tract is presented as % IA per whole sample. bSignificant difference (p < 0.05 in paired t test) between 68Ga-DOTA-Z09591 and 111In-DOTA-Z09591.

data, one could also expect visualization of the spleen. However, the spleen could not be seen due to the proximity to the kidneys.

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was significantly lower with the 111In-labeled conjugate than with the 68Ga-labeled at 2 h after injection (233 ± 25% IA/g vs 241 ± 24% IA/g, respectively). There was no significant difference in uptake in any other organ. The tumor-to-organ ratios were similar using either label (Figure 4). The only statistically significant differences at 1 h

DISCUSSION A role of PDGFRβ in the development of multiple diseases renders it a target for specific therapies.5,6,16 An application of precision medicine requires rapid, repeatable, and noninvasive determination of PDGFRβ status in lesions. Radionuclide molecular imaging has a potential to meet these requirements on preconditions of sufficient sensitivity. Recently, we reported development of an affibody-based imaging agent, 111In-DOTAZ09591, which provides unparalleled contrast,35 and therefore sensitivity for in vivo imaging of PDGFRβ. Here we report on the construction and properties of a Z09591-based PET tracer, expected to provide enhanced sensitivity for PDGFRβ. The use of positron-emitting 68Ga instead of 111In as a label may be considered as a facile way for creation of an imaging agent for PET. However, the imaging sensitivity is determined by the combined properties of the imaging probe and the imaging modality. Although gallium and indium are both trivalent transition metals, they differ in coordination chemistry, resulting in different geometry of their DOTA complexes.43 This modifies the surface of the radiolabeled proteins and peptides, and may influence both their on-target and off-target interactions. Indeed, difference in affinity and biodistribution of 111 In- and 68Ga-labeled peptides is well documented.44,45 The properties of the radionuclide have been shown to also influence the biodistribution of affibody molecules despite their larger size.40,42 Thus, the imaging properties of affibodybased imaging probes have to be re-evaluated for each new radionuclide. The use of the site-specifically incorporated DOTA chelator enabled rapid (15 min) and efficient (radiochemical yield of 92.3 ± 0.6%) labeling of DOTA-Z09591 with 68Ga. Efficient labeling requires the use of high temperature (95 °C), which is not compatible with most proteins having tertiary structure. However, affibody molecules have been shown to refold after denaturing, which permits the use of extreme labeling conditions (pH in the range of 3.4 to 11.5; temperature up to 95 °C).46 Indeed, 68Ga-DOTA-Z09591 showed a retained

Figure 4. Comparison of tumor-to-organ ratios 1 and 2 h p.i. for 68GaDOTA-Z09591 and 111In-DOTA-Z09591 in mice bearing U-87 MG xenografts. Data are presented as mean ± SD for four mice. *, Significant difference (p < 0.05) between 68Ga-DOTA-Z09591 and 111 In-DOTA-Z09591

after injection related to the liver and spleen, 111In-DOTAZ09591 and 68Ga-DOTA-Z09591, showing tumor-to-liver ratios 4.3 ± 1.2 vs 4.0 ± 1.3 and tumor-to-spleen ratios of 1.9 ± 0.3 vs 1.7 ± 0.3. At 2 h after injection, the 111In-labeled conjugate yielded higher values than the 68Ga-labeled conjugate for tumor-to-blood (18.4 ± 8.3 vs 8.0 ± 3.1), tumor-to-lung (5 ± 1 vs 4 ± 1.), tumor-to-liver (6 ± 3 vs 5 ± 2), and tumor-tospleen (2.8 ± 0.9 vs 2.5 ± 0.8) ratios. Imaging. MicroPET/CT imaging acquired after injecting 68 Ga-DOTA-Z9195 in a mouse bearing U-87 xenografts confirmed the ability to visualize the PDGFRβ expressing tumors. As predicted from the results of the biodistribution study, high accumulation of radioactivity was observed in the E



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kidneys. When using nonresidualizing radiohalogens for affibody labeling the radioactivity was rapidly cleared from the kidneys,52−55 which supports a rapid internalization and degradation (not to be mixed with the slow internalization and cellular processing of affibody molecules by tumor cells after a target mediated binding). Taken together, all assembled data support that renal reabsorption of affibody molecules is mediated by a high-capacity low-specificity mechanism associated with rapid internalization. Very high renal uptake might, in principle, have a negative impact on the clinical application of an imaging agent. The most obvious issues are high dose burden to kidneys and issues with imaging of tumors close to kidneys, due to reconstruction artifacts caused by renal radioactivity content. However, clinical data are available for the DOTA-Z09591-similar anti-HER2 ABY-025 affibody molecule labeled with 111In33 or 68Ga (Prof. J. Sörensen, unpublished results). HER2-expressing bone metastases in the lumbar area could be visualized using either label. In addition, an adrenal metastasis localized directly on a kidney could be visualized using 111In-labeled ABY-025.33 The dose to kidney for 68Ga-ABY-025 was 0.4 mGy/MBq, i.e., 0.08 Gy for a typical study (Prof. M. Lubberink, unpublished results). This suggests that the high renal accumulation of 68Ga-DOTAZ09591 observed in murine models may not indicate a major negative impact on clinical imaging. In vivo comparison of 68Ga-DOTA-Z09591 and 111InDOTA-Z09591 demonstrated that blood clearance of 111Inlabeled compound was more rapid. The difference in blood clearance of short peptides labeled with different nuclides has been noted earlier.45,56 It has been shown also with HER2targeting affibody molecules.42,57 As the size of imaging probes was the same, the difference was most likely due to off-target interactions, possibly transient binding to blood plasma proteins. This resulted in the reduction of tumor-to-blood ratio of 68Ga-DOTA-Z09591 (8.01 ± 3.15) in comparison with 111 In-DOTA-Z09591 (18.37 ± 8.32). Despite this decrease of tumor-to-blood ratio in comparison with 111In-DOTA-Z09591, microPET imaging using 68Ga-DOTA-Z09591 has clearly visualized PDGFRβ-expressing U-87 MG xenografts in mice at 2 h after injection (Figure 5). Furthermore, this level of contrast is higher than the level provided by radiolabeled monoclonal antibodies in tumors with a high antigen expression several days after injection.29 Our previous studies have demonstrated that the impact of nuclide on biodistribution and targeting properties of antiHER2 affibody molecules is influenced by the chelator used for labeling and by the chemistry for conjugating the chelator to the affibody molecule and the position of the chelator (C- or Ntreminus).40,42,57 For example, replacement of DOTA by NODAGA chelator at N-terminus of synthetic affibody molecules increased the tumor-to-blood ratio of 68Ga-labeled conjugates two-fold. This finding implies that optimization of targeting properties of affibody molecules should also involve the chelator moiety. In conclusion, the PDGFRβ-binding DOTA-Z09591 affibody molecule was efficiently and stably labeled with generatorproduced positron-emitting radionuclide 68Ga. The 68GaDOTA-Z09591 demonstrated rapid and specific targeting of PDGFRβ-expressing U-87 MG xenografts in immunodeficient mice and provided a high-contrast imaging using microPET at 2 h after injection. Further development of Z09591 may provide a sensitive and specific agent for imaging of PDGFRβexpression in vivo using PET.

capacity for specific binding to PDGFRβ-expressing U-87 MG glioma cells. The binding of Ga-DOTA-Z09591 to PDGFRβexpressing cells was somewhat weaker than binding of InDOTA-Z09591 (IC50s of 6.6 ± 1.4 and 1.4 ± 1.2 nM, respectively). However, our earlier studies have demonstrated that a small difference in affinity in this range does not influence in vivo targeting shortly after injection.47 In addition, the gallium-labeled tracer was unexpectedly shown to internalize more rapidly: already after 3 h of incubation, 26 ± 1% of 68GaDOTA-Z09591 was internalized by U-87 MG glioma cells, whereas only 10% of 111In-DOTA-Z09591 was internalized after 24 h.35 A rapid internalization of an imaging agent with a residualizing radiometal label should contribute to better tumor retention of radioactivity despite lower affinity. Indeed, although there was a tendency to higher accumulation of 111 In-DOTA-Z09591 in tumors (Table 1), the difference with 68 Ga-DOTA-Z09591 was not significant. Thus, a higher internalization rate seems to have compensated for the lower affinity of the gallium-labeled Z09591. In vivo, 68Ga-DOTA-Z09591 demonstrated accumulation in PDGFRβ expressing tumors, which was higher than accumulation in any other organ or tissue except kidney. Saturable tumor uptake (Figure 3) suggested that this uptake was PDGFRβ-specific. Saturable uptake was also shown for lung, liver, spleen, and muscle, reflecting expression of PDGFRβ in the normal vasculature as we have shown and discussed in our previous study.35 Low uptake of 68Ga-DOTA-Z09591 in liver and gastrointestinal tract (with content) suggests that hepatobiliary clearance did not play an appreciable role in excretion of this affibody molecule. This feature is favorable for detection of hepatic metastases, as well as for imaging of PDGFRβ in liver fibrosis. The clearance was apparently renal and accompanied by reabsorption and accumulation in the kidney. Renal clearance is the main excretion route for the majority of proteins and peptides with molecular weight of less than 70 kDa, often stated as the cutoff for glomerular filtration.47 However, there are several mechanisms providing reabsorption in the proximal tubuli. The degree of the reabsorption of peptides and proteins depends on their affinity to endocytic scavenger receptors in the proximal tubuli and varies widely. Generally reabsorbed proteins and peptides are transferred into lysosomes and degraded. Radiocatabolites from peptides radioactively labeled with a radiometal are trapped inside the proximal tubuli cells.47 High renal reabsorption is characteristic for affibody molecules and has been observed for affibody-based agents specific to a range of targets, e.g., HER2,48 IGF-1R,30 HER3,31 and EGFR.49 Endocytosis mediated by the megalin receptor has been shown to be an important pathway for renal reabsorption for many proteins and peptides.50,47 Our studies have shown that this is not the case for affibody molecules, e.g., the renal uptake of affibody molecules was equal in both normal and megalin knockout mice.51 Target-specific binding in kidneys can be also excluded, as pre- or coinjection of large doses of nonlabeled affibody molecules has been shown to cause a significant reduction of uptake only in tumors and target-expressing tissues, but not in kidneys in any in vivo blocking experiments with any radiometal-labeled affibody molecule. On the contrary, blocking of target-specific uptake in murine tissues may cause an elevated uptake in kidneys, as seen with IGF-1R-, EGFR-, and HER3-targeting affibody molecules30,31,49 and in this study. Possibly, saturation of specific uptake in tissues leaves more radiolabeled conjugate to be excreted and reabsorbed by F



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AUTHOR INFORMATION

Corresponding Author

*(V.T.) Phone: +46 18 471 3414. Fax: +46 18 471 3432. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by grants from the Swedish Cancer Society (Cancerfonden) and Swedish Research Council (Vetenskapsrådet).

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