uPAR Targeted Radionuclide Therapy with 177Lu-DOTA-AE105

Jun 23, 2014 - Finsen Laboratory, Rigshospitalet & BRIC, Copenhagen Biocenter, Ole Maaloesvej 5, Copenhagen Ø 2200, Denmark. ⊥. Hevesy Laboratory ...
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uPAR Targeted Radionuclide Therapy with 177Lu-DOTA-AE105 Inhibits Dissemination of Metastatic Prostate Cancer Morten Persson,†,‡,§,∥ Karina Juhl,‡,§ Palle Rasmussen,⊥ Malene Brandt-Larsen,‡ Jacob Madsen,‡ Michael Ploug,†,∥ and Andreas Kjaer*,†,‡,§ †

The Danish-Chinese Center for Proteases and Cancer Department of Clinical Physiology, Nuclear Medicine and PET, Rigshospitalet, 4012 Blegdamsvej 9, Copenhagen Ø 2100, Denmark § Cluster for Molecular Imaging, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, Copenhagen Ø 2200, Denmark ∥ Finsen Laboratory, Rigshospitalet & BRIC, Copenhagen Biocenter, Ole Maaloesvej 5, Copenhagen Ø 2200, Denmark ⊥ Hevesy Laboratory, DTU Nutech, Technical University of Denmark, Building 202, Frederiksborgvej 399, Roskilde 4000, Denmark ‡

ABSTRACT: The urokinase-type plasminogen activator receptor (uPAR) is implicated in cancer invasion and metastatic development in prostate cancer and provides therefore an attractive molecular target for both imaging and therapy. In this study, we provide the first in vivo data on an antimetastatic effect of uPAR radionuclide targeted therapy in such lesions and show the potential of uPAR positron emission tomography (PET) imaging for identifying small foci of metastatic cells in a mouse model of disseminating human prostate cancer. Two radiolabeled ligands were generated in high purity and specific activity: a uPARtargeting probe (177Lu-DOTA-AE105) and a nonbinding control (177Lu-DOTA-AE105mut). Both uPAR flow cytometry and ELISA confirmed high expression levels of the target uPAR in PC-3M-LUC2.luc cells, and cell binding studies using 177LuDOTA-AE105 resulted in a specific binding with an IC50 value of 100 nM in a competitive binding experiment. In vivo, uPAR targeted radionuclide therapy significantly reduced the number of metastatic lesions in the disseminated metastatic prostate cancer model, when compared to vehicle and nontargeted 177Lu groups (p < 0.05) using bioluminescence imaging. Moreover, we found a significantly longer metastatic-free survival, with 65% of all mice without any disseminated metastatic lesions present at 65 days after first treatment dose (p = 0.047). In contrast, only 30% of all mice in the combined control groups treated with 177 Lu-DOTA-AE105mut or vehicle were without metastatic lesions. No treatment-induced toxicity was observed during the study as evaluated by observing animal weight and H&E staining of kidney tissue (dose-limiting organ). Finally, uPAR PET imaging using 64Cu-DOTA-AE105 detected all small, disseminated metastatic foci when compared with bioluminescence imaging in a cohort of animals during the treatment study. In conclusion, uPAR targeted radiotherapy resulted in a significant reduction in the number of metastatic lesions in a human metastatic prostate cancer model. Furthermore, we have provided the first evidence of the potential for identification of small metastatic lesions using uPAR PET imaging in disseminated prostate cancer, illustrating the promising strategy of uPAR theranostics in prostate cancer. KEYWORDS: cancer, metastases, urokinse-type plasminogen activator receptor, CD87, radionuclide therapy, radiation therapy, positron emission tomography, prostate cancer, theranostics

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INTRODUCTION The correct diagnosis and treatment management of prostate cancer (PC) remains particularly difficult due to the high heterogeneity of the disease. If diagnosed while the cancer is still localized, combined surgery and radiotherapy yield a disease-specific survival rate of more than 90%.1 However, once © 2014 American Chemical Society

Received: Revised: Accepted: Published: 2796

March 4, 2014 June 17, 2014 June 23, 2014 June 23, 2014 dx.doi.org/10.1021/mp500177c | Mol. Pharmaceutics 2014, 11, 2796−2806

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(v/v) water with 0.1% (v/v) TFA and 95% (v/v) acetonitrile/ 5% (v/v) water with 0.1% (v/v) TFA. The TLC was performed with a Raytest MiniGita Star (Straubenhardt, Germany) TLCscanner equipped with a Beta-detector. The TLC eluent was ammonium acetate (0.65M) in 50% (v/v) methanol in water and the TLC-plate was a Silica60 on Al foil (Sigma-Aldrich Denmark A/S). For 64Cu, a Dionex Ultimate 3000 HPLC was used with a Kinetex 2.6 μm C18 100A 50 × 4.6 mm column. The mobile phase was Eluent A: 10% MeCN in H2O with 0.1% TFA. Eluent B: 10% H2O in MeCN with 0.1% TFA. Recombinant human uPAR was produced and purified as described.29,30 A polyclonal rabbit anti-uPAR antibody was prepared in-house using purified recombinant uPAR expressed in Chinese Hamster Ovary cells as antigen.31 2-(4,7,10-tris(2tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclo-dodecan-1-yl)acetic acid (DOTA-tris(tBu)ester was purchased from CheMatech (Dijon, France). Peptide Synthesis and Radiolabeling. Two 9-mer peptides were conjugated to DOTA as described previously,20 that is, DOTA-AE105 (DOTA-Asp-Cha-Phe-(D)Ser-(D)ArgTyr-Leu-Trp-Ser-CONH2) (Figure 1A) and DOTA-AE105mut (DOTA-Asp-Cha-Glu-(D)Ser-(D)Arg-Tyr-Leu-Glu-SerCONH2) (Figure 1A). Radiolabeling of DOTA-AE105 with 177 Lu was performed as previously reported.20,26 Radiolabeling of DOTA-AE105 with 64Cu for PET imaging was performed by adding 64CuCl2 (≈150 MBq) to a vial containing 0.1 M ammonium acetate buffer containing gentisic acid (5 mg/mL) (pH = 5.2) and peptide (2 nmol), with a temperature of 80 °C for 5 min. The labeling of DOTA-AE105 took less than 30 min and resulted in greater than 95% yield. No additional radiochemical purification step was required. The amount of unlabeled 64Cu in the product was less than 1%, as demonstrated by radio-TLC. The specific activity was approximately 40 MBq/nmol. uPAR ELISA. The uPAR content in PC-3 and PC-3MLUC2.luc cells were determined by ELISA as outlined.20 Confluent cells were harvested using 0.25% tryosin-ethylenediaminetetraacetic acid (EDTA), and cells were collected by centrifugation. The pellet was lysed using cell lysis buffer (Invitrogen). All measurements were performed in duplicate. uPAR Flow Cytometry. Confluent PC-3M-LUC2.luc cells were collected and washed by phosphate-buffered saline (PBS) and then kept in PBS supplemented with 1% bovine serum albumin and 0.03% sodium azide. Cells were then incubated with in-house-made rabbit antihuman uPAR antibody as previously described,20 at 4 °C for 60 min. After we washed the cells with PBS in the presence of 1% bovine serum albumin, the cells were incubated with FITC-conjugated goat antirabbit IgG (BD Biosciences no. 554020) at 4 °C for 60 min. After the cells were washed, they were resuspended in PBS and analyzed using a FACSCanto (Becton Dickson, Oxford, England) with FlowLogic analysis software (Inivai). Rabbit IgG isotype was used as negative control. Cell Binding Assays. PC-3M-LUC2-Luc cells were cultured in MEM standard medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin at 37 °C and 5% CO2. Confluent cells were detached with 0.5% trypsin-EDTA), 0.01 M PBS (pH 7.4), and plated in a 96-well plate (2 × 105 cells/wells). After 48 h of incubation, a cell binding assay (Figure 2B) was performed as a competition assay. Briefly, each well was incubated with approximately 100 KBq 177Lu-DOTA-AE105 and varying concentrations of AE105 peptide in binding buffer (25 mM Tris pH 7.4 and 0.1% BSA)

metastatic lesions are present, medical castration and chemotherapy remains ineffective, and most patients progress to hormone-refractory metastatic PC with high morbidity and mortality.1,2 The urokinase plasminogen activator receptor (uPAR) is involved in regulating tissue remodeling, which occurs frequently during tumor invasion and metastasis.3−6 Numerous studies have shown that uPAR is elevated in PC and high levels of uPAR are associated with an aggressive disease leading to poor prognosis.7−15 uPAR is therefore considered an attractive target for both therapy and imaging in PC, with the potential to overcome the difficulties in diagnosing, treatment stratification, and treatment of PC. The application of a single agent for both noninvasive quantitative positron emission tomography (PET) imaging and radionuclide-based therapy (i.e., theranostics) thus provides an intervention platform for the tailoring patient management.16,17 We have previously developed and characterized a small linear 9-mer uPAR-binding peptide18 and conjugated this to different metal chelators and radionuclides for PET imaging of uPAR positive cancers with promising results,19−24 including a PC model.25 In one study, this peptide conjugate was also radiolabeled with 177Lu for uPAR targeted radionuclide therapy and characterized in a human colorectal cancer mouse model.26 A specific depletion of uPAR-positive cancers cells was indeed accomplished, thus providing the first proof-of-concept for uPAR targeted radionuclide therapy in vivo. This subcutaneous colon cancer xenograft exhibited, nonetheless, neither invasion nor metastasis, which renders this mouse model a rather imperfect surrogate for clinically malignant, disseminating cancer. Prior to clinical translation, it therefore remains to be established whether eradication of uPAR positive cancer cells, using peptide-based targeted radionuclide therapy, translates into a reduction of metastatic burden in vivo. Targeted radionuclide therapy has shown promising results in several cancers, with somatostatin-based targeting of neuroendocrine tumors (e.g., 177Lu-DOTA-TATE, 90 YDOTA-TOC) still being the most successful in clinical use.27 Besides radiolabeled peptides, radiolabeled antibodies such as 131 I-tositumomab (Baxxar) and 90Y-Ibritumomab (Zevalin) are used for the treatment of non-Hodgkins lymphoma.28 The dual aim of the present study was first to investigate the impact of uPAR targeted radionuclide therapy on a mouse model of human disseminated prostate cancer, with particular emphasis on its antimetastatic effect. Second, to use uPAR PET imaging for monitoring efficacy of the above-mentioned treatment by visualizing small metastatic foci in the PC model, thus exploiting the full theranostic potential of DOTAAE105 in a PC model.

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MATERIALS AND METHODS Chemical and Biological Reagents. All chemicals were purchased from Sigma-Aldrich Denmark A/S unless specified otherwise. 177Lu was purchased from PerkinElmer (Boston, MA, U.S.A.). All solutions were made using ultrapure water (40 MBq/nmol. uPAR Expression in Prostate Cancer Cell Lines. uPAR expression levels were confirmed in the human prostate cancer

at room temperature for 2 h. The cell-bound radioactivity remaining after washing was determined by gamma counting. Wells without cells were used as background, and wells with no added AE105 peptide added served as 100% binding reference. IC50 values were determined by nonliniar regression using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, U.S.A.). Experiments were performed in triplicate. To define specificity (Figure 2C), PC-3M-LUC2.Luc cells were incubated 2798

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Figure 2. uPAR expression and cell binding studies. uPAR ELISA assay (A) revealed uPAR expression in both PC cell lines, with the highest level in the metastatic variant (PC-3M.LUC2.Luc cell line). (B) uPAR flow cytometry confirmed extracellular uPAR expression in PC-3M.LUC2.Luc cancer cells. (C) In vitro binding specificity of 177Lu-DOTA-AE105 in PC-3M.LUC2.Luc cell line. (D) In vitro competitive cellular binding assay using PC3M.LUC2.Luc cell line resulted in an IC50 value of 108 ± 1.2 nM, with the core peptide AE105 as competitor ligand.

human uPAR (p < 0.001) (Figure 2C). In a competition cellbinding assay, an IC50 value of 108 nM was found using PC3M-LUC2 cells (Figure 2D) with the unconjugated peptide AE105 as competitor. Efficacy of uPAR Targeted Radiotherapy on Metastatic Development. The protocol designed to address the in vivo efficacy is shown in Figure 3A. PC-3M-LUC2 cells were inoculated by intracardiac injection to mimic intravascular dissemination and subsequent systemic establishment of metastatic disease. As the PC-3M-LUC2 cell-line is stably transfected with luciferase, the formation of small metastatic lesions can be followed with BLI in all treatment groups. Representative images for each group are shown in Figure 3B. A clear tendency toward increased numbers of metastatic lesions was observed for both vehicle and 177Lu-DOTAAE105mut groups, respectively, as seen in Figure 3C. After day 35, a general decrease in total number of tumor lesions were observed in all three treatment groups due to the death of high-tumor-burden animals. The mean numbers of lesions before treatment initiation was 0.50, 1.00, and 1.33, with a total number of tumor lesion of 193, 134, and 72 in control, 177LuDOTA-AE105mut, and 177Lu-DOTA-AE105 treatment group, respectively. The mean number of tumor lesion at time of death were significantly lower in the 177Lu-DOTA-AE105 group compared with control (p = 0.013) and 177Lu-DOTA-

cell lines PC-3 (parental) and its subline (PC-3M-LUC2). PC-3 is derived from a lumbar vertebral metastasis in a 62-year-old white man with PC. To generate a more aggressive metastatic variant, PC-3 cells have been xenografted in nude mice and a cell line from a metastatic lesion from the primary tumor has been established and denoted PC-3M-LUC2,32 which has been found to have increased uPAR expression.33 In line with this, we also observed an increased uPAR expression in PC-3MLUC2 cells compared to PC-3 when analyzing whole cell extract, as shown in Figure 2A. uPAR flow cytometry confirmed extracellular uPAR expression in PC-3M-LUC2 cells (Figure 2B), necessary for optimal targeted uPAR radionuclide therapy with 177Lu-DOTA-AE105. AE105·uPAR Interaction in Vitro Assessed by Cell Binding. The specific high-affinity binding of DOTA-AE105 to uPAR has previously been published.18,22 In the present study, IC50 values were found to be 6.7 nM for both the unconjugated peptide AE105 and the DOTA-conjugated peptide (DOTAAE105) as shown in Table 1. By substituting two amino acids in AE105, known to be important for the binding toward uPAR (i.e., Phe3 → Glu and Trp8 → Glu), a significant reduction in the IC50 value was observed (IC50 > 103 nM), generating a nonbinding control ligand (i.e., 177Lu-DOTA-AE105mut). Cellbinding experiments in vitro using PC-3M-LUC2 confirmed the specific high-affinity binding of 177Lu-DOTA-AE105 to 2799

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Figure 3. In vivo efficacy. (A) Treatment and imaging scheme of the in vivo therapy study. (B) Representative BLI for each treatment group during the study. Depicts a clear tendency for an increased metastatic burden in both control groups (vehicle + 177Lu-DOTA-AE105mut) compared with the uPAR targeted treatment group (177Lu-DOTA-AE105). (C) Total number of tumor lesions in each treatment group during the entire study. After day 37, high-tumor-burden animals started to died, resulting in a decreased number of mean total tumor lesions in all three treatments group. (D) Comparison of the mean number of tumor lesion at time of death in each treatment group confirmed a significant antimetastatic effect of 177LuDOTA-AE105 compared with both 177Lu-DOTA-AE105mut (p = 0.048) and control (p = 0.013).

Table 1. Binding Properties uPAR Targeting Peptidea ligand

amino acid sequence

IC50 (nM)

KD (nM)

AE105 DOTA-AE105 DOTA-AE105mut

Asp1-Cha2-Phe3-ser4-arg5-Tyr6-Leu7-Trp8-Ser9 DOTA-Asp1-Cha2-Phe3-ser4-arg5-Tyr6-Leu7-Trp8-Ser9 DOTA-Asp1-Cha2-Glu3-ser4-arg5-Tyr6-Leu7-Glu8-Ser9

6.7 ± 1.6 6.7 ± 1.0 ≫ 103

12 9.4

a

Note: residues in bold are hot spots for the interaction with uPAR. Cha is cyclohexyl-(L)-alanine. Ser and Arg are both present in the Dconfiguration. Values from ref 22

after first treatment dose. Similar performance was only reached by 24% and 33% of the mice included in the 177Lu-DOTAAE105mut (control) and vehicle control group, respectively. The median for metastatic-free status was accordingly 12.5, 16, and >65 days for vehicle, 177Lu-DOTA-AE105mut, and 177LuDOTA-AE105, respectively. These parameters are summarized in Table 2. Due to the relatively low numbers of animals included, the inherent biological variance, and the similar metastatic-free survival time in each of the two control groups, we combined the two control groups (i.e., vehicle + 177LuDOTA-AE105mut) and compared them to the treatment

AE105mut (p = 0.048) (Figure 3D). No significant difference was observed between the two control groups (p = 0.81). The mean number of tumor lesion were 3.6 ± 0.6, 3.3 ± 0.8, and 1.4 ± 0.5 in control, 177Lu-DOTA-AE105mut, and 177Lu-DOTAAE105 treatment groups, respectively. A clear tendency toward prolonged metastatic-free survival was furthermore found for the uPAR-targeted treatment group (177Lu-DOTA-AE105) by analyzing the time until the appearance of the first tumor lesion (excluding cardiac deposits) (Figure 4A). In 65% of the mice dosed with 177LuDOTA-AE105, no distant metastases were detected 65 days 2800

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the entire observation period (65 days). This included no wasting syndrome as indicated by uniform weight gain following treatment day 0, 7, and 14 for all mice enrolled in the study (Figure 5A). When tumor burden became more pronounced (after day 38), a decline in animal weight was generally observed until the animal had to be sacrificed. Test for equality (maximal claimed difference in weight of 1 g) was performed and confirmed for all pairwise comparison between each treatment group. Previous dosimetry calculations for 177 Lu-DOTA-AE105 have determined that the kidneys receive the highest dose of radioactivity,26 and the kidneys were consequently collected at the end of this study for pathology examination. H&E-stained kidney sections were examined, and no morphological signs of overt nephrotoxicity were observed in any of the mice (Figure 5B). uPAR PET Imaging for Identification of Occult Micrometastasis in Vivo. To demonstrate the applicability of PET imaging as a noninvasive imaging platform for the specific detection of small metastatic lesions expressing uPAR, we analyzed three mice from a nontreated cohort with 64CuDOTA-AE105 on study day 31. After a preceding BLI scan, these mice were subjected to a uPAR PET scan 1 h post injection of 64Cu-DOTA-AE105. By comparing the reconstructed PET/CT images (Figure 6) with BLI, all tumor lesions identified on BLI scanning were also identified on the subsequent PET scanning (9 out of 9) (white arrows). Actually, uPAR PET revealed two new lesions (X and Y) not detected using BLI This is because there are well-established limitations of BLI in areas of high tissue density. This proves the applicability of this clinically relevant imaging modality for detection of small occult tumor lesions expressing uPAR and also the limitations of BLI in treatment monitoring in mice, because relatively large tumor lesions located on the back of the mouse appear small using BLI due to high tissue absorption. On the contrary, the lesions are large when identified using uPAR PET imaging. As illustrated by the images in Figure 6, PET provided a superior resolution and three-dimensional reconstruction of the anatomy of these lesions.

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DISCUSSION Current medical tests including pathology assessment of random biopsies or plasma biomarkers (e.g., prostate-specific antigen (PSA) levels) cannot by themselves reliably identify those PC patients having a high probability of developing metastatic disease. Moreover, treatment options for patients with metastatic PC remain ineffective with an overall 5 year survival below 30%. There is accordingly a huge clinical demand for new noninvasive techniques capable of discriminating patients with aggressive PC (i.e., high metastatic potential) from those with a more indolent disease and therefore unlikely to progress. Moreover, new targeted treatment modalities for patients with aggressive metastatic disease is needed. The validity of uPAR as a target for both imaging and therapy in PC has been explored in a number of previous studies. Elevated plasma levels of uPAR have furthermore been associated with advanced PC stage and bone metastases, and in patients with localized PC, high preoperative plasma uPAR levels have been shown to correlate with early progression.11,14 uPAR therefore seems to be an attractive target for both PC therapy34 and molecular imaging.35 In line with this, a number of uPAR targeted therapies has also been reported in PC animal models. First, a monoclonal uPAR antibody (ATN-658) was

Figure 4. Metastatic-free survival. (A) A Kaplan−Meier plot shows the metastatic-free survival in each of the three treatment groups, (i.e., the time until first metastatic lesion was observed, excluding the heart, because this was the injection site). A high percentage (63%) of all animals had no metastatic lesion at day 65 post first dose; this was only the case in 33% and 24% in the two control groups, respectively. (B) A significantly longer metastatic-free survival (p = 0.0474) was found in this study, when pooling the two control groups. The median metastatic-free survival was 12.5, 16, and >65 days in vehicle, 177LuDOTA-AE105mut, and 177Lu-DOTA-AE105 treatment groups, respectively. Black arrows indicate dosing days.

group, which resulted in a significant prolonged metastatic-free survival for 177Lu-DOTA-AE105 groups (p = 0.0474) (Figure 4B). Evaluation of Potential Treatment-Induced Toxicity. All mice tolerated the treatment well and showed no signs of abnormal behavior and/or clinical symptoms of toxicity during 2801

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Table 2. Summary of Treatment Efficiency and Metastatic-Free Survival p value animals injected activity treatment efficiency stable disease/response progression control vs 177Lu-DOTA-AE105 control vs 177Lu-DOTA-AE105mut 177Lu-DOTA-AE105 vs 177Lu-DOTA-AE105mut median metastatic-free time (days) metastatic-free day 65

control

177Lu-DOTA-AE105mut (control)

177Lu-DOTA-AE105

n = 12

n = 11 3 × 18 MBq

n = 12 3 × 18 MBq

4 7

3 5

8 1

12.5 33%

16 24%

>65 65%

0.028 0.100 0.049

Figure 5. Histopathological examination of kidneys. All mice tolerated the treatment well. No mean weight difference between the treatment groups was observed (as shown in panel A). Histopathological examination of kidneys revealed no toxicity. Representative examples of H&E stains of kidney tissue from control group (vehicle, left), 177Lu-DOTA-AE105mut control group (middle) and uPAR targeted ligand 177Lu-DOTA-AE105 (right) are shown in panel B.

2802

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Figure 6. uPAR PET imaging for identification of small metastatic lesions. A subgroup of animals was PET/CT scanned using 64Cu-DOTA-AE105 on day 31 post study initiation. Representative PET/CT images are shown with the corresponding BLI images for each mouse. uPAR PET imaging was able to detect all lesions (9 out of 9) found using BLI. Two additional tumor lesions (X and Y) not seen with BLI were detected using uPAR PET imaging. White arrow indicates tumor lesions.

disseminating breast cancer model using an 111In/177Luconjugated recombinant antibody.40 Unfortunately, this study used somewhat suboptimal controls, and the fact that the unlabeled antibody allegedly has a therapeutic impact in its unlabeled form complicates the definition of the added beneficial theranostic impact originating from the radionuclide targeting per se. In addition, the choice of SPECT imaging using an 111In-conjugated recombinant antibody does not in our view represent an optimal imaging platform for subsequent clinical translation due to the slow pharmacokinetics of the targeting probe and lower resolution of the acquired images. In the present study, we showed for the first time that uPAR PET imaging with 64Cu-DOTA-AE105 can detect small metastatic lesions (Figure 6). However, despite the high clinical translational potential of this modality, the use of 64Cu as isotope remains a challenge because of the limited number of production sites and cost. Therefore, for future large-scale clinical uPAR PET studies, other PET isotopes, such as generator-based 68Ga or generally widely produced 18F, seem more optimal. Preclinical characterizations of such uPAR PET ligands have been reported21,25 The amount of free 177Lu in the final product for injection was in all cases below 4%, as seen in Figure 1B,C. Any

reported to decrease tumor volume in mice transplanted with the PC cell line PC-3 subcutaneously or orthotopically in the tibia.36 Second, in vitro studies using ATF-coated nanoparticles for uPAR-targeted delivery of noscapine in PC-3 cells demonstrated a 6-fold stronger inhibitory effect on PC3-cell proliferation compared to the free, untargeted noscapine.37 However, no in vivo data was reported. In the present study, we now show that 177Lu-DOTA-AE105 treatment reduces the formation of metastatic foci and prolongs the median time until first appearance of metastatic lesions, with a median of 12.5 days for control group versus >65 days for treatment group. This efficacy is comparable to that reported for a folate-receptor-targeted radionuclide therapy using 177Lu-EC0800 in combination with pemetrexed, where an increased survival of 75%−100% of mice bearing KB and IGROV-1 tumor xenografts.38 Another study targeting bombesin receptors with four doses of 28 MBq 177Lu-DOTA-PESIN, improved survival 40% at day 63 post therapy initiation in a human prostate xenograft mouse model using the PC-3 cell line.39 Following our initial 177Lu-uPAR targeted therapy study in xenograft models of colorectal cancer,26 an independent group has also explored the theranostic targeting of uPAR in a 2803

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detect uPAR positive tumor lesions with high sensitivity and resolution, thus setting the stage for its theranostics application in clinical management of PC.

nonspecific treatment effect and/or toxicity caused by this residual 4% free 177Lu seems unlikely. Considering that the amount of free 177Lu was identical in the two 177Lu treatment groups (e.g., 177 Lu-DOTA-AE105 and 177 Lu-DOTAAE105mut) and no significant treatment difference between 177Lu-DOTA-AE105mut and vehicle was found (e.g., metastatic-free survival), any effect, if present, must be marginal. The kidney generally represents the dose-limiting organ for radionuclide-based therapy in humans, with possible nephrotoxicity caused by ionization-induced tissue damage. Our dosimetry calculations26 for 177Lu-DOTA-AE105-based therapy concur with this assumption. Nevertheless, we observed no gross histopathological abnormalities in kidneys exposed to three doses of 177Lu-DOTA-AE105 (this study and the study in ref 26). In accordance, two clinical studies found no significant long-term impairment of kidney functions in heavily treated patients receiving multiple doses of peptide receptor radionuclide therapy.41,42 The potential problem of nephotoxicity after peptide-based radionuclide therapy therefore seems to be limited. It should, nevertheless, be emphasized that toxicity caused by a specific targeting of the normal, baseline uPARexpression in nontarget organs, in particular kidney (podocytes) and bone marrow (leukocytes), will be underestimated in these preclinical studies using xenografted mouse models as a result of th especies-selectivity in the uPA·uPAR and AE105· uPAR interactions.18,30 Notwithstanding the above-mentioned limitations in toxicity assessment, the species-selectivity in uPAR targeting by 177Lu-DOTA-AE105 does in fact also underestimate the expected impact of the specific cytotoxic effect exerted on human primary tumor lesions, as well as their corresponding metastases. The rationale behind this prediction is related to the unique architecture of the invading front observed for most solid tumors, where the activated tumor− stromal microenvironment generally contains the highest density of uPAR expression, which is provided by a mixed population of cancer and stromal cells.4,19,43,44 In the present preclinical mouse model using PC cell xenografts, the radionuclide will thus only target cancer cells (human origin), leaving the stromal compartment essentially unharmed (mouse origin). In cancer patients, both compartments will be targeted with equal efficacy. Considering the importance of the activated tumor−stromal microenvironment in tumor growth and metastasis,45,46 it will be very interesting to explore the full impact of such uPAR targeted therapy in a human setting. Besides the successful treatment of neuroendocrine tumors with 177Lu-DOTA-TATE,47 the clinical potential of radionuclide-based therapy in PC has recently been provided, with the FDA approval of Xofigo (former Alpharadin), an alphaemitter for the treatment of castration-resistant prostate cancer with bone metastases based on its ability to extend overall survival as shown its pivotal phase III trial (the increase was from 11.3 months to 14.9 months, p < 0.001).48 Because Xofigo efficacy is based on the nonspecific uptake of 223RaCl2 in the bone as a result of the physical similarity between radium and calcium, future targeted radionuclide treatment options seem very intriguing. To summarize, uPAR targeted radionuclide therapy and imaging of disseminated PC has been demonstrated. A significant reduction in metastatic lesions and longer overall metastatic-free survival was found in mice dosed with 177LuDOTA-AE105 compared with control groups. uPAR PET using 64 Cu-DOTA-AE105 confirmed the ability for this ligand to

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by The Danish National Research Foundation (Centre for Proteases and Cancer), Danish Medical Research Council, the Danish National Advanced Technology Foundation, the Novo Nordisk Foundation, the Lundbeck Foundation, the Arvid Nilsson Foundation, the A.P. Moeller Foundation and the Svend Andersen Foundation.

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ABBREVIATIONS DOTA,1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; EDTA,ethylenediaminetetraacetic acid; PC,prostate cancer; PET,positron emission tomography; SPECT,single positron emission computed tomography; uPAR,urokinase-type plasminogen activator receptor; RP-HPLC,reversed-phase high pressure liquid chromatography; Luc,luciferase; BLI,bioluminescence imaging; CT,computed tomography

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REFERENCES

(1) Walczak, J. R.; Carducci, M. A. Prostate cancer: a practical approach to current management of recurrent disease. Mayo Clin. Proc. 2007, 82 (2), 243−249. (2) Chi, K. N.; Bjartell, A.; Dearnaley, D.; Saad, F.; Schroder, F. H.; Sternberg, C.; Tombal, B.; Visakorpi, T. Castration-resistant prostate cancer: from new pathophysiology to new treatment targets. Eur. Urol 2009, 56 (4), 594−605. (3) Duffy, M. J. The urokinase plasminogen activator system: role in malignancy. Curr. Pharm. Des. 2004, 10 (1), 39−49. (4) Dano, K.; Behrendt, N.; Hoyer-Hansen, G.; Johnsen, M.; Lund, L. R.; Ploug, M.; Romer, J. Plasminogen activation and cancer. Thromb. Haemostasis 2005, 93 (4), 676−681. (5) Blasi, F.; Sidenius, N. The urokinase receptor: focused cell surface proteolysis, cell adhesion and signaling. FEBS Lett. 2010, 584 (9), 1923−1930. (6) Noh, H.; Hong, S.; Huang, S. Role of urokinase receptor in tumor progression and development. Theranostics 2013, 3 (7), 487−495. (7) Rabbani, S. A.; Xing, R. H. Role of urokinase (uPA) and its receptor (uPAR) in invasion and metastasis of hormone-dependent malignancies. Int. J. Oncol. 1998, 12 (4), 911−920. (8) Miyake, H.; Hara, I.; Yamanaka, K.; Arakawa, S.; Kamidono, S. Elevation of urokinase-type plasminogen activator and its receptor densities as new predictors of disease progression and prognosis in men with prostate cancer. Int. J. Oncol. 1999, 14 (3), 535−541. (9) Miyake, H.; Hara, I.; Yamanaka, K.; Gohji, K.; Arakawa, S.; Kamidono, S. Elevation of serum levels of urokinase-type plasminogen activator and its receptor is associated with disease progression and prognosis in patients with prostate cancer. Prostate 1999, 39 (2), 123− 129. (10) Piironen, T.; Haese, A.; Huland, H.; Steuber, T.; Christensen, I. J.; Brunner, N.; Dano, K.; Hoyer-Hansen, G.; Lilja, H. Enhanced discrimination of benign from malignant prostatic disease by selective measurements of cleaved forms of urokinase receptor in serum. Clin. Chem. 2006, 52 (5), 838−844. (11) Shariat, S. F.; Roehrborn, C. G.; McConnell, J. D.; Park, S.; Alam, N.; Wheeler, T. M.; Slawin, K. M. Association of the circulating levels of the urokinase system of plasminogen activation with the 2804

dx.doi.org/10.1021/mp500177c | Mol. Pharmaceutics 2014, 11, 2796−2806

Molecular Pharmaceutics

Article

presence of prostate cancer and invasion, progression, and metastasis. J. Clin. Oncol. 2007, 25 (4), 349−355. (12) Gupta, A.; Lotan, Y.; Ashfaq, R.; Roehrborn, C. G.; Raj, G. V.; Aragaki, C. C.; Montorsi, F.; Shariat, S. F. Predictive value of the differential expression of the urokinase plasminogen activation axis in radical prostatectomy patients. Eur. Urol. 2009, 55 (5), 1124−1133. (13) Milanese, G.; Dellabella, M.; Fazioli, F.; Pierpaoli, E.; Polito, M.; Siednius, N.; Montironi, R.; Blasi, F.; Muzzonigro, G. Increased urokinase-type plasminogen activator receptor and epidermal growth factor receptor in serum of patients with prostate cancer. J. Urol. 2009, 181 (3), 1393−1400. (14) Almasi, C. E.; Brasso, K.; Iversen, P.; Pappot, H.; HoyerHansen, G.; Dano, K.; Christensen, I. J. Prognostic and predictive value of intact and cleaved forms of the urokinase plasminogen activator receptor in metastatic prostate cancer. Prostate 2011, 71 (8), 899−907. (15) Kjellman, A.; Akre, O.; Gustafsson, O.; Hoyer-Hansen, G.; Lilja, H.; Norming, U.; Piironen, T.; Tornblom, M. Soluble urokinase plasminogen activator receptor as a prognostic marker in men participating in prostate cancer screening. J. Intern. Med. 2011, 269 (3), 299−305. (16) Persson M, K. A. Urokinase-type plasminogen activator receptor (uPAR) as a promising new imaging target: potential clinical applications. Clin. Physiol. Funct. Imaging 2013, 33, 329−337. (17) Li, D.; Liu, S.; Shan, H.; Conti, P.; Li, Z. Urokinase plasminogen activator receptor (uPAR) targeted nuclear imaging and radionuclide therapy. Theranostics 2013, 3 (7), 507−515. (18) Ploug, M.; Ostergaard, S.; Gardsvoll, H.; Kovalski, K.; HolstHansen, C.; Holm, A.; Ossowski, L.; Dano, K. Peptide-derived antagonists of the urokinase receptor. Affinity maturation by combinatorial chemistry, identification of functional epitopes, and inhibitory effect on cancer cell intravasation. Biochemistry 2001, 40 (40), 12157−12168. (19) Kriegbaum, M. C.; Persson, M.; Haldager, L.; Alpizar-Alpizar, W.; Jacobsen, B.; Gardsvoll, H.; Kjaer, A.; Ploug, M. Rational targeting of the urokinase receptor (uPAR): development of antagonists and non-invasive imaging probes. Curr. Drug Targets 2011, 12 (12), 1711− 1728. (20) Persson, M.; Madsen, J.; Ostergaard, S.; Jensen, M. M.; Jorgensen, J. T.; Juhl, K.; Lehmann, C.; Ploug, M.; Kjaer, A. Quantitative PET of human urokinase-type plasminogen activator receptor with 64Cu-DOTA-AE105: implications for visualizing cancer invasion. J. Nucl. Med. 2012, 53 (1), 138−145. (21) Persson, M.; Madsen, J.; Ostergaard, S.; Ploug, M.; Kjaer, A. (68)Ga-labeling and in vivo evaluation of a uPAR binding DOTA- and NODAGA-conjugated peptide for PET imaging of invasive cancers. Nucl. Med. Biol. 2012, 39 (4), 560−569. (22) Persson M, H. M.; Madsen, J.; Jørgensen, T. J. D.; Jensen, K. J.; Kjaer, A.; Ploug, M. Improved PET Imaging of uPAR Expression Using new 64Cu-labeled Cross-Bridged Peptide Ligands: Comparative in vitro and in vivo Studies. Theranostics 2013, 3 (9), 618−632. (23) Ploug, M. Structure-driven design of radionuclide tracers for non-invasive imaging of uPAR and targeted radiotherapy. The tale of a synthetic peptide antagonist. Theranostics 2013, 3 (7), 467−476. (24) Li, Z. B.; Niu, G.; Wang, H.; He, L.; Yang, L.; Ploug, M.; Chen, X. Imaging of urokinase-type plasminogen activator receptor expression using a 64Cu-labeled linear peptide antagonist by microPET. Clin. Cancer Res. 2008, 14 (15), 4758−4766. (25) Persson, M.; Liu, H.; Madsen, J.; Cheng, Z.; Kjaer, A. First Flabeled ligand for PET imaging of uPAR: in vivo studies in human prostate cancer xenografts. Nucl. Med. Biol. 2013, 40, 618−624. (26) Persson, M.; Rasmussen, P.; Madsen, J.; Ploug, M.; Kjaer, A. New peptide receptor radionuclide therapy of invasive cancer cells: in vivo studies using (177)Lu-DOTA-AE105 targeting uPAR in human colorectal cancer xenografts. Nucl. Med. Biol. 2012, 39, 962−969. (27) Bergsma, H.; van Vliet, E. I.; Teunissen, J. J.; Kam, B. L.; de Herder, W. W.; Peeters, R. P.; Krenning, E. P.; Kwekkeboom, D. J. Peptide receptor radionuclide therapy (PRRT) for GEP-NETs. Best Pract. Res., Clin. Gastroenterol. 2012, 26 (6), 867−881.

(28) Tomblyn, M. Radioimmunotherapy for B-cell non-hodgkin lymphomas. Cancer Control: Journal of the Moffitt Cancer Center 2012, 19 (3), 196−203. (29) Jacobsen, B.; Gardsvoll, H.; Juhl Funch, G.; Ostergaard, S.; Barkholt, V.; Ploug, M. One-step affinity purification of recombinant urokinase-type plasminogen activator receptor using a synthetic peptide developed by combinatorial chemistry. Protein Expr. Purif. 2007, 52 (2), 286−296. (30) Lin, L.; Gardsvoll, H.; Huai, Q.; Huang, M.; Ploug, M. Structure-based engineering of species selectivity in the interaction between urokinase and its receptor: implication for preclinical cancer therapy. J. Biol. Chem. 2010, 285 (14), 10982−10992. (31) Ronne, E.; Hoyer-Hansen, G.; Brunner, N.; Pedersen, H.; Rank, F.; Osborne, C. K.; Clark, G. M.; Dano, K.; Grondahl-Hansen, J. Urokinase receptor in breast cancer tissue extracts. Enzyme-linked immunosorbent assay with a combination of mono- and polyclonal antibodies. Breast Cancer Res. Treat. 1995, 33 (3), 199−207. (32) Kozlowski, J. M.; Fidler, I. J.; Campbell, D.; Xu, Z. L.; Kaighn, M. E.; Hart, I. R. Metastatic behavior of human tumor cell lines grown in the nude mouse. Cancer Res. 1984, 44 (8), 3522−3529. (33) Forbes, K.; Gillette, K.; Kelley, L. A.; Sehgal, I. Increased levels of urokinase plasminogen activator receptor in prostate cancer cells derived from repeated metastasis. World J. Urol. 2004, 22 (1), 67−71. (34) Li, Y.; Cozzi, P. J. Targeting uPA/uPAR in prostate cancer. Cancer Treat. Rev. 2007, 33 (6), 521−527. (35) Nogueira, L.; Corradi, R.; Eastham, J. A. Other biomarkers for detecting prostate cancer. BJU Int. 2010, 105 (2), 166−169. (36) Rabbani, S. A.; Ateeq, B.; Arakelian, A.; Valentino, M. L.; Shaw, D. E.; Dauffenbach, L. M.; Kerfoot, C. A.; Mazar, A. P. An antiurokinase plasminogen activator receptor antibody (ATN-658) blocks prostate cancer invasion, migration, growth, and experimental skeletal metastasis in vitro and in vivo. Neoplasia 2010, 12 (10), 778−788. (37) Abdalla, M. O.; Karna, P.; Sajja, H. K.; Mao, H.; Yates, C.; Turner, T.; Aneja, R. Enhanced noscapine delivery using uPARtargeted optical-MR imaging trackable nanoparticles for prostate cancer therapy. J. Controlled Release 2011, 149 (3), 314−322. (38) Reber, J.; Haller, S.; Leamon, C. P.; Muller, C. 177Lu-EC0800 Combined with the Antifolate Pemetrexed: Preclinical Pilot Study of Folate Receptor Targeted Radionuclide Tumor Therapy. Mol. Cancer Ther. 2013, 12 (11), 2436−45. (39) Wild, D.; Frischknecht, M.; Zhang, H.; Morgenstern, A.; Bruchertseifer, F.; Boisclair, J.; Provencher-Bolliger, A.; Reubi, J. C.; Maecke, H. R. Alpha- versus beta-particle radiopeptide therapy in a human prostate cancer model (213 Bi-DOTA-PESIN and 213 BiAMBA versus 177Lu-DOTA-PESIN). Cancer Res. 2011, 71 (3), 1009−1018. (40) LeBeau, A. M.; Duriseti, S.; Murphy, S. T.; Pepin, F.; Hann, B.; Gray, J. W.; VanBrocklin, H. F.; Craik, C. S. Targeting uPAR with antagonistic recombinant human antibodies in aggressive breast cancer. Cancer Res. 2013, 73 (7), 2070−2081. (41) Sabet, A.; Ezziddin, K.; Pape, U. F.; Reichman, K.; Haslerud, T.; Ahmadzadehfar, H.; Biersack, H. J.; Nagarajah, J.; Ezziddin, S. Accurate assessment of long-term nephrotoxicity after peptide receptor radionuclide therapy with Lu-octreotate. Eur. J. Nucl. Med. Mol. Imaging 2013, 41, 505−510. (42) Kunikowska, J.; Krolicki, L.; Sowa-Staszczak, A.; Pawlak, D.; Hubalewska-Dydejczyk, A.; Mikolajczak, R. Nephrotoxicity after PRRT - still a serious clinical problem? Renal toxicity after peptide receptor radionuclide therapy with 90Y-DOTATATE and 90Y/177LuDOTATATE. Endokrynologia Polska 2013, 64 (1), 13−20. (43) Pyke, C.; Graem, N.; Ralfkiaer, E.; Ronne, E.; Hoyer-Hansen, G.; Brunner, N.; Dano, K. Receptor for urokinase is present in tumorassociated macrophages in ductal breast carcinoma. Cancer Res. 1993, 53 (8), 1911−1915. (44) Plesner, T.; Ploug, M.; Ellis, V.; Ronne, E.; Hoyer-Hansen, G.; Wittrup, M.; Pedersen, T. L.; Tscherning, T.; Dano, K.; Hansen, N. E. The receptor for urokinase-type plasminogen activator and urokinase is translocated from two distinct intracellular compartments to the 2805

dx.doi.org/10.1021/mp500177c | Mol. Pharmaceutics 2014, 11, 2796−2806

Molecular Pharmaceutics

Article

plasma membrane on stimulation of human neutrophils. Blood 1994, 83 (3), 808−815. (45) Fang, H.; Declerck, Y. A. Targeting the tumor microenvironment: from understanding pathways to effective clinical trials. Cancer Res. 2013, 73 (16), 4965−4977. (46) Sleeman, J. P. The metastatic niche and stromal progression. Cancer Metastasis Rev. 2012, 31 (3−4), 429−440. (47) Kam, B. L.; Teunissen, J. J.; Krenning, E. P.; de Herder, W. W.; Khan, S.; van Vliet, E. I.; Kwekkeboom, D. J. Lutetium-labelled peptides for therapy of neuroendocrine tumours. Eur. J. Nucl. Med. Mol. Imaging 2012, 39 (Suppl 1), S103−S112. (48) Parker, C.; Nilsson, S.; Heinrich, D.; Helle, S. I.; O’Sullivan, J. M.; Fossa, S. D.; Chodacki, A.; Wiechno, P.; Logue, J.; Seke, M.; Widmark, A.; Johannessen, D. C.; Hoskin, P.; Bottomley, D.; James, N. D.; Solberg, A.; Syndikus, I.; Kliment, J.; Wedel, S.; Boehmer, S.; Dall’Oglio, M.; Franzen, L.; Coleman, R.; Vogelzang, N. J.; O’BryanTear, C. G.; Staudacher, K.; Garcia-Vargas, J.; Shan, M.; Bruland, O. S.; Sartor, O.; ALSYMPCA Investigators. Alpha emitter radium-223 and survival in metastatic prostate cancer. N. Engl. J. Med. 2013, 369 (3), 213−223.

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