Anticancer Therapy by Tumor Vessel Infarction with Polyethylene

Article pubs.acs.org/jmc

Anticancer Therapy by Tumor Vessel Infarction with Polyethylene Glycol Conjugated Retargeted Tissue Factor Christian Schwöppe,*,† Caroline Zerbst,† Max Fröhlich,† Christoph Schliemann,† Torsten Kessler,† Ruediger Liersch,† Laura Overkamp,† Richard Holtmeier,§ Jörg Stypmann,‡,§ Alena Dreiling,∥ Simone König,∥ Carsten Höltke,⊥ Martin Lücke,# Carsten Müller-Tidow,† Rolf M. Mesters,† and Wolfgang E. Berdel*,† †

Department of Medicine A, Hematology, Oncology and Pneumology, ‡Interdisciplinary Center of Clinical Research (IZKF Muenster), Preclinical Imaging Experts Echocardiography (PIX ECHO), §Department of Cardiovascular Medicine, Division of Cardiology, ∥Integrated Functional Genomics (IZKF Muenster), ⊥Department of Clinical Radiology, and #Central Institute for Animal Experimentation, University of Muenster, Albert-Schweitzer-Campus 1, D-48129 Muenster, Germany S Supporting Information *

ABSTRACT: tTF-NGR consists of the extracellular domain of tissue factor and the peptide GNGRAHA, a ligand of the surface protein aminopeptidase N and of integrin αvβ3. Both surface proteins are upregulated on endothelial cells of tumor vessels. tTF-NGR shows antitumor activity in xenografts and inhibition of tumor blood flow in cancer patients. We performed random TMS(PEG)12 PEGylation of tTF-NGR to improve the antitumor profile of the molecule. PEGylation resulted in an approximately 2-log step decreased procoagulatory activity of the molecule. Pharmacokinetic studies in mice showed a more than 1-log step higher mean area under the curve. Comparison of the LD10 values for both compounds and their lowest effective antitumor dose against human tumor xenografts showed an improved therapeutic range (active/toxic dose in mg/kg body weight) of 1/5 mg/kg for tTF-NGR and 3/>160 mg/kg for TMS(PEG)12 tTF-NGR. Results demonstrate that PEGylation can significantly improve the therapeutic range of tTF-NGR.

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INTRODUCTION Surgery, radiation, and chemotherapy for more advanced stages have caused progress in cancer therapy during the past decades. However, the majority of patients with metastasized solid tumors still succumb to their disease. Besides targets for molecular therapy in cancer-driving pathways within the tumor cells, tumor stroma hosts a plethora of possible targets for cancer therapy. Some of those are related to tumor blood vessels. The formation of new blood vessels is essential for the spread and metastasis of solid tumors.1 This supports the supply of oxygen and nutrients as well as the removal of metabolic waste products. Attacking tumor vasculature can be conceptually divided into antiangiogenesis,2−4 vascular disruption,5,6 and vascular targeting of antitumor molecules.7,8 Direct contact to blood flow makes tumor vessel wall cells, such as endothelial cells and pericytes, easy to reach. Denekamp was the first to propose tumor vessels and endothelial cells as a targets for antitumor therapy.7 Markers with preferential expression in tumor vessel walls, such as the vascular endothelial growth factor-receptor 2,9 endosialin,10 the matrix metalloproteinases,11 several integrins,12−14 NG2 proteoglycan,15 and others are promising targets for specific ligands16−20 coupled with cytotoxic and proapoptotic substances and targeting these effector molecules to the tumor vessel wall. © 2013 American Chemical Society

Within the concept of vascular targeting, use of coagulation factors to induce tumor vessel infarction is a separate approach, which was initiated by Thorpe et al.21 Tissue factor (TF) represents a central initiator of the extrinsic coagulation pathway in vivo. The relative lack of coagulation inducing activity of a soluble form of TF missing its transmembrane domain (truncated TF: tTF)22 can partly be recovered by relocalizing tTF into the proximity of a phospholipid membrane.21 Accordingly, targeting tTF via peptides and antibodies to different specific tumor vessel markers has led to rapid induction of thrombosis in tumor vessels.23−27 Studies by Pasqualini et al.28 revealed that small peptides containing the NGR motif (asparagine-glycine-arginine) bind to aminopeptidase N (APN; CD13). CD13 is a cell surface molecule with upregulated expression on endothelial cells in tumors and tissues that undergo angiogenesis. This molecule is of specific interest, since its expression is not only restricted to tumor vessel endothelial cells but it can also be found on tumor cells, and both cooperate to promote tumor growth and metastasis.29 NGR binds differentially to CD13 in tumor vasculature but not to CD13 in normal kidney and myeloid Received: November 12, 2012 Published: March 15, 2013 2337

dx.doi.org/10.1021/jm301669z | J. Med. Chem. 2013, 56, 2337−2347

Journal of Medicinal Chemistry

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Figure 1. Purification of the TMS(PEG)12 reaction batches by gel filtration and control of PEGylated tTF-NGR by SDS-PAGE, western blotting, and MALDI-TOF MS. (A) Preparative gel-filtration chromatography removes the NHS-leaving group evolved during the successful PEGylation as well as remaining TMS(PEG)12 substrate (see arrows) from the fractionated protein peak (red bars). Both, protein and NHS(−ester) was measured at a UV wavelength of 280 nm. (B) For SDS-PAGE, proteins were stained with Coomassie Brilliant Blue R250. Shown are standard protein size marker (lane 1), four representative batches of TMS(PEG)12-PEGylated tTF-NGR (lanes 3−6), and non-PEGylated tTF-NGR (lane 2). (C) Western blotting using anti-PEG (methoxy group) antibodies (diluted 1:1000) as described of PEGylated tTF-NGR (lane 1) and non-PEGylated tTF-NGR as a negative control (lane 2). (D) MALDI-TOF MS spectra of PEGylated tTF-NGR. The distribution of modified protein forms peaking at 8 and showing up to 11 TMS(PEG)12 units is indicated (m/z, mass-to-charge ratio). For MS analysis, PEGylated tTF-NGR was dialyzed versus water; determination of free TMS(PEG)12 yields approximately 0.45 nmol per mg of PEGylated tTF-NGR protein.

cells. 30 NGR peptides undergo a rapid nonenzymatic asparagine deamidation to isoaspartate-glycine-arginine (isoDGR) generating a further ligand for the integrin αvβ3,31,32 which is also upregulated on tumor endothelial cells.12,13,26,31 To date, two NGR-coupled molecules are in clinical studies. NGR-tumor necrosis factor (NGR-hTNF) is in early clinical trials in a variety of tumors including malignant pleural mesothelioma.33 We have constructed fusion proteins consisting of short NGR-peptide sequences coupled to the C-terminal end of tTF.34−36 Several of these fusion proteins including tTF-

NGR retain their thrombogenic activity in vitro, bind to their respective targets on endothelial cells, and upon intravenous (iv) infusion, induce thrombosis in blood vessels in several human solid tumors growing in athymic mice with subsequent tumor growth retardation or regression. Intravenous infusion of tTF-NGR in cancer patients at dose levels without side effects was shown to reduce tumor blood flow in situ.35 However, during toxicology studies, we occasionally observed mouse tail tip necrosis upon iv injection, and when choosing routes of application other than iv infusion, such as subcutaneous injection, systemic toxicity with pulmonary 2338



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Figure 2. Activation of FX by non-PEGylated tTF-NGR versus TMS(PEG)12 tTF-NGR. (A) Ability of non-PEGylated tTF-NGR and TMS(PEG)12 tTF-NGR to enhance the specific proteolytic activation of FX by FVIIa in the presence of phospholipids was evaluated by Michaelis−Menten analysis. (B) Michaelis constants (Km) of the FX activation of the tTF fusion proteins were calculated by hyperbolic regression analysis according to Hanes et al.46 Mean Km values of 46 (non-PEGylated tTF-NGR) and 12 (PEGylated tTF-NGR) assays are given in the inset of part B: 0.15 ± 0.01 nM for tTF-NGR and 10.59 ± 1.59 nM for PEGylated tTF-NGR. V, reaction velocity; mAU, milliabsorbance units; c/v, ratio of the initial substrate concentration to the reaction velocity; c, substrate concentration.

obtained a uniform peak of PEGylated protein upon gel filtration (Figure 1A) and, since we have performed random PEGylation, multiple bands with reproducible mass distribution in SDS-PAGE (Figure 1B) and western blotting (Figure 1C). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) showed that the protein distribution had its maximum at 8 TMS(PEG)12 per protein (equivalent to approximately 49 kDa; see also the abstract figure), but signals for protein species containing up to 11 TMS(PEG)12 were detected (Figure 1D). It is likely that protein forms containing even more of the reagent were present and that only the top of the Gaussian protein species distribution curve was visualized. Residual TMS(PEG)12 considerably hampered MS analysis and was very difficult to remove by both dialysis and filtration methods. Therefore, MS signals were not as intense as had been expected from an expressed protein. However, the amount of free PEG was approximately 0.45 nmol per mg of PEGylated tTF-NGR protein, which represents a >2000-fold decrease of free TMS(PEG)12 compared to the initial PEGylation reaction. Activation of Factor X by Non-PEGylated tTF-NGR and TMS(PEG)12 PEGylated tTF-NGR. tTF-NGR is a bifunctional protein with a C-terminal binding domain for CD13 and after deamidation to αvβ3, and a TF domain for binding FVIIa with consecutive enhancement of the specific proteolytic activity of factor X (FX). PEGylation theoretically can interfere with both functions. Thus, we assayed the activation of FX induced by

embolism and skin bleeding with laboratory signs of disseminated intravasal coagulation (DIC) occurred within the therapeutic dose range.37 Hypothesizing that this toxicity is connected to the pharmacokinetics including blood peak levels of active tTF-NGR and to formation of larger aggregates, we have undertaken studies with the aim to improve the safety and activity profile of tTF-NGR including conjugating the molecule to polyethylene glycol (PEG). PEGylation is an established method for conferring improved activity/toxicity profiles to high-molecular pharmacologic agents such as proteins by increasing half-life, reducing protein aggregation, immunogenicity, and uptake by the reticulo-histiocytic system (RHS), and an increasing number of PEGylated drugs are used in clinical oncology. 38−40 Here, we report random TMS(PEG) 12 PEGylation of the retargeted truncated tissue factor tTFNGR as a first proof-of-principle study leading to an improved anticancer profile in the experimental in vivo models used in this report.

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RESULTS TMS(PEG)12 PEGylation of tTF-NGR. tTF-NGR was conjugated to TMS(PEG)12 by standard methods. The PEG reaction batches were subsequently purified by gel filtration and controlled by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting using antiPEG antibodies. Figure 1 visualizes representative results. We 2339



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Figure 3. Comparative pharmacokinetic analysis of non-PEGylated versus TMS(PEG)12 PEGylated tTF-NGR. Blood peak levels and wash-out kinetics upon 1 mg/kg of bw iv injection of non-PEGylated (empty triangels) versus PEGylated (filled triangels) tTF-NGR. Shown are mean values obtained from four mice per group in a nonlogarithmic (A) and a logarithmic (B) way. Standard errors (selection; error bars are too small to illustrate in the figure): 10 min values: 639.1 (PEGylated) and 750.7 (non-PEGylated); 2 h values: 283.7 (PEGylated) and 45.0 (non-PEGylated); 24 h values: 39.0 (PEGylated) and 0.71 (non-PEGylated); all in ng/mL. Half-life was approximately 7 min for non-PEGylated and approximately 57 min for PEGylated tTF-NGR. (C) Surface integral (AUC) for non-PEGylated versus PEGylated tTF-NGR. Mean AUC as measured until 24 h after injection was 4.5 ng/(mL·h) for non-PEGylated tTF-NGR and 82.1 ng/(mL·h) for PEGylated tTF-NGR.

coagulation. Representative data are shown in Figure 2. The percentage deviation of the calculated mean values of the Michaelis constants of PEGylated tTF-NGR compared to those of non-PEGylated tTF-NGR revealed no wider variation in the FX activity. The smallest Km value of PEGylated tTF-NGR represents 23%, and the highest Km value represents 194% of

PEGylated versus non-PEGylated tTF-NGR. Michaelis− Menten analysis indeed revealed 2-log steps lower calculated Michaelis constants (Km) for tTF-NGR (0.15 ± 0.01 nM) versus PEGylated tTF-NGR (10.59 ± 1.59 nM) based on the respective protein mass, indicating a 2 orders of magnitude lower activity of TMS(PEG)12 PEGylated tTF-NGR to activate 2340



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Figure 4. Antitumor activity of TMS(PEG)12 tTF-NGR. Growth inhibition of (A) human fibrosarcoma HT1080 and of (B) human adenocarcinoma of the lung A549, xenotransplanted into athymic CD-1 mice, by iv administration of 1, 3, 5, and 7 mg/kg bw TMS(PEG)12 tTF-NGR, respectively, compared to the administration of 0.9% saline (control). Arrows indicate the time points of injection. Data are presented as means. Asterisks denote statistical significance (p < 0.05). (A) Standard error values expressed as range values for all days of observation in mm3: 76.0−153.0 (control), 87.8− 106.3 (1 mg/kg bw), 67.6−90.0 (3 mg/kg bw), 73.2−85.6 (5 mg/kg bw), 72.2−80.1 (7 mg/kg bw). (B) Standard error values expressed as range values for all days of observation in mm3: 51.3−68.8 (control), 42.6−83.0 (1 mg/kg bw), 57.8−111.3 (3 mg/kg bw), 38.3−55.1 (5 mg/kg bw), 27.5−36.5 (7 mg/kg bw).

the mean value; the smallest Km value of non-PEGylated tTFNGR represents 31%, and the highest Km value represents 218% of the mean value. Thus, concerning reproducibility of the PEGylation batches, a similar homogeneity of the bioactivity profile of PEGylated tTF-NGR compared to those of non-PEGylated tTF-NGR has been accomplished. Comparative Pharmacokinetics. The comparatively low specific activity of tTF-NGR in its PEGylated form to activate FX strongly argued against further development of this molecule for experimental cancer therapy. However, next, we performed pharmacokinetic studies upon iv injection of tTFNGR PEGylated with TMS(PEG)12 versus non-PEGylated tTF-NGR. Cohorts of CD-1 mice were iv injected with 1 mg/ kg of body weight (bw) of protein, neglecting the additional mass of the TMS(PEG)12, and blood samples collected after 1, 10, 30, and 60 min and 2, 4, 8, and 24 h were subjected to enzyme-linked immunosorbent assay (ELISA) as described below. We have taken an anti-TF antibody for analysis after observing that this antibody was able to detect the PEGylated form of tTF-NGR. As shown in Figure 3, peak levels upon iv injection did not much differ between PEGylated tTF-NGR (17 333.0 ng/mL) and non-PEGylated tTF-NGR (18 829.0 ng/mL). Taking into account the lower procoagulatory activity of PEGylated tTF-NGR as compared to the non-PEGylated form, the peak levels of active tTF-NGR upon an iv injection are considerably lower for the PEGylated form of the molecule. On the other hand, the half-life of PEGylated tTF-NGR was approximately 57 min compared to only approximately 7 min for non-PEGylated tTF-NGR, the wash-out time was approximately 20 h longer, and the blood levels after 24 h were approximately 500-fold higher for PEGylated tTF-NGR over non-PEGylated tTF-NGR (Figure 3A,B). This resulted in a more than 1-log step higher area under the curve (AUC) for PEGylated tTF-NGR (Figure 3C). Mean AUC as measured

until 24 h after injection was 4.5 ng/(mL·h) for non-PEGylated tTF-NGR and 82.1 ng/(mL·h) for PEGylated tTF-NGR. The possibility of underestimating levels of PEGylated tTF-NGR, in case TMS(PEG)12 hampered with the anti-TF antibody binding to the PEGylated molecule in the ELISA, was excluded in these experiments by using the own ELISA calibration curves for both molecules. However, possible underestimating of the AUC difference, since for practicability reasons, we stopped taking blood and measuring at 24 h after injection when TMS(PEG)12 tTF-NGR was still measurable at a much higher concentration than tTF-NGR, was neglected in this analysis. Tolerability Comparison. After obtaining these promising pharmacokinetic results, we performed comparative studies on tolerability and acute toxicology to establish the lethal dose for 10% of the animals after one iv injection within 24 h (LD10). Figure S1 (Supporting Information) shows a dose−lethality curve with LD10 of approximately 5 mg/kg bw for tTF-NGR. For TMS(PEG)12 tTF-NGR, an LD10 could not be reached due to limits of applicability (protein solubility, injection volume) with the highest injected dose being 160 mg/kg bw without visible toxicity. Effect of TMS(PEG)12 tTF-NGR on Growth of HT1080 and A549 Xenotransplants. Subsequently, the in vivo antitumor activity of tTF-NGR in its non-PEGylated and PEGylated forms was determined in athymic CD-1 nude mice bearing human HT1080 fibrosarcoma or A459 lung adenocarcinoma xenografts. Drugs and controls (saline) were slowly injected iv at the doses and time intervals as shown in Figure 4. Injections of 1, 3, 5, or 7 mg of PEGylated tTF-NGR/kg bw induced tumor growth delay for a longer period of time when compared with saline controls in both tumor models. Furthermore, there was a clear dose−activity relation revealing higher doses as being more effective (Figure 4A,B). In summary, we have observed therapeutic antitumor effects 2341



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Figure 5. Molecular imaging was performed with U87 xenotransplant-bearing CD-1 nude mice. CEUS was performed with mice that received 5 mg/ kg bw TMS(PEG)12 tTF-NGR as daily iv application for 4 days (n = 4). (A) Relative tumor blood perfusion of the treated mice was significantly decreased as compared with untreated control tumors (n = 6). Asterisk denotes statistical significance, p < 0.05, Student’s t test). The contrast agent microbubbles in vivo revealed a well-perfused pattern in an untreated tumor exemplar (B, left), while the treated one showed considerably decreased contrast agent distribution (B, right). Fluorescence reflectance imaging (FRI) was performed with mice treated 3 times within 24 h (at 0, 8, 24 h) with TMS(PEG)12 tTF-NGR (10 mg/kg bw, iv). One hour after the last application, treated (n = 4) and untreated (n = 5) mice received 2 nmol (150 μL, iv) of the fluorescent in vivo blood pool and tumor-imaging agent AngioSense680. After further 24 h, the AngioSense680 fluorescence intensities of the explanted tumors were quantified by FRI. The fluorescence intensities of the explanted tumors showed a significant difference between treated and untreated control tumors (C; background intensity was subtracted; asterisk denotes statistical significance, p < 0.05, Student’s t test), which is also observable by directly comparing the fluorescence of the TMS(PEG)12 tTF-NGR-treated with the untreated control tumors (D; left panel, five control tumors; right panel, four treated tumors).

perfusion of the treated mice was significantly decreased in comparison to untreated control tumors (Figure 5A,B). While the contrast agent microbubbles revealed a well-perfused pattern of the untreated tumors (see the arrows in Figure 5B, left), the treated ones showed considerably decreased contrast agent distribution (Figure 5B, right). The perfusion signals of the control organ (kidney) showed no difference between treated and untreated mice (data not shown). Fluorescence Reflectance Imaging. The optical imaging of the in vivo blood pool- and tumor-imaging agent AngioSense680 also enabled us to monitor the restriction of tumor blood flow induced by TMS(PEG)12 tTF-NGR treatment. U87 xenotransplant-bearing mice received AngioSense680 1 h after the third application of TMS(PEG)12 tTFNGR. After further 24 h, the AngioSense680 fluorescence intensities of the explanted tumors showed a significant decrease of treated as compared with untreated control tumors (Figure 5C,D). Due to the thrombotic occlusions triggered by the TMS(PEG)12 tTF-NGR therapy, the accumulation of the tumor-imaging fluorescence agent was blocked; additionally, the hemorrhagic bleeding induced by the stop of blood flow within the treated tumors masked the fluorescence, which both led to reduced signal intensities (Figure 5 D). Histopathology and Immunofluorescence Staining of the Tumors and Normal Organs upon Treatment with TMS(PEG)12 tTF-NGR. To directly test for thrombotic occlusion of tumor vessels, tumors were excised in some experiments after treatment with TMS(PEG)12 tTF-NGR and

with PEGylated tTF-NGR, inhibiting tumor growth independent from tumor histology, being clearly visible with doses ≥3 mg/kg bw. However, as with non-PEGylated tTF-NGR, after termination of TMS(PEG)12 tTF-NGR treatment, tumor regrowth could be observed. Often, tumors showed blue and brown coloration and necrotic areas within some hours after injection of tTF-NGR, as previously published.26,35 This was only rarely observed upon injection of the PEGylated tTF-NGR and in a more delayed fashion occurring some days after injection (details not shown). In contrast to tTF-NGR, repeated iv application of PEGylated tTF-NGR via the tail veins of the mice did not result in local toxicity, such as tail tip necrosis. This may be due to the lower local peak concentration of active tTF-NGR, when given as a PEGylated molecule. Comparing the LD10 values for both compounds and their lowest effective antitumor dose, we obtained a safe therapeutic range (active/toxic dose in mg/kg bw) of 1/5 mg/kg bw for tTF-NGR and 3/>160 mg/kg bw for TMS(PEG)12 PEGylated tTF-NGR, characterizing the PEGylated form of the molecule as a preferred therapeutic preparation in these experimental models. Contrast-Enhanced Ultrasound. To study the mode of action for the in vivo drug activity by an easy-to-apply “bedside” method, contrast-enhanced ultrasound (CEUS) imaging was performed with xenotransplant-bearing mice (U87 glioblastoma) receiving 5 mg/kg bw TMS(PEG)12 tTF-NGR as daily iv application for 4 days. The relative tumor blood 2342



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Figure 6. Histology and immunofluorescence (IF) studies of tumor and organ tissue sections upon treatment with TMS(PEG)12 tTF-NGR. Thrombotic hemorrhage is shown in an H&E-stained section of a TMS(PEG)12 tTF-NGR-treated tumor (A), and examples of thrombotic material inside tumor vessels are shown at higher magnification in B and C. For IF, tumors and organs were resected 24−48 h after the last treatment and immunostained with anti-PEG antibody (green staining; see the Experimental Section for details); the respective magnification is shown in the picture; nuclei are DAPI-stained (blue). Exemplary tumor tissue sections of HT1080 fibrosarcoma xenografts upon repeated treatment with TMS(PEG)12 tTF-NGR (7 mg/kg bw, I) are compared with untreated tumor tissue (D). Exemplary organ tissue sections of an untreated mouse are shown in E−H, tissue sections of identical organs of the TMS(PEG)12 tTF-NGR-treated mice are shown in J−M (lung: E, J; kidney: F, K; liver: G, L; brain: H, M).

specific autofluorescence, tumor tissues upon treatment showed clear accumulation of PEGylated tTF-NGR in the tumor (Figure 6I). These observations again underline the mode of antitumor activity of PEGylated tTF-NGR as being due to accumulation in tumor vessels with subsequent tumor vessel infarction, inhibition of tumor vessel blood flow with hemorrhage, and tumor cell death as the final consequence.

subjected to histological studies. The H&E-stained histology in Figure 6A shows areas of hemorrhage in the tumor tissue, due to vascular disruption and blood pooling as signs of inhibited blood flow caused by thromboembolism. Figure 6B,C shows thrombotic material inside tumor vessels upon treatment at a higher magnification. This was in contrast to the vital appearance of tumor tissue and tumor vessels within the tumors excised upon treatment with the saline control (details not shown). In addition, as reported before for non-PEGylated tTF-fusion proteins,26,35 sections of normal organs, such as liver, lung, heart, or kidney, explanted from identical tumorbearing animals after therapy and studied histologically showed no signs of thromboembolism or other toxicity (details not shown). We further performed immunofluorescence studies of snapfrozen tumor and organ sections upon treatment with TMS(PEG)12 tTF-NGR using an anti-PEG antibody as the primary antibody followed by an Alexa Fluor488-labeled goat antirabbit IgG antibody as described. In contrast to tissue from normal organs such as liver, heart, lung, and kidneys (Figure 6E−M), or untreated tumors (Figure 6D), which showed no immunofluorescence staining upon treatment or some non-

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DISCUSSION Important limitations of antibodies or larger antibody fragments as targeting moieties for tTF might be their low tumor penetration,41 nonspecific accumulation and uptake in the RHS, and the immunogenicity of the large, complex molecules. Thus, we developed a series of fusion proteins consisting of small peptides, including NGR-motifs, coupled to the C terminus of tTF.26,34−37 The tTF moiety is part of the tTF:VIIa complex,42 a potent initiator of coagulation. The NGR motif was chosen for retargeting tTF to tumor vessels, since it binds to aminopeptidase N (CD13), which is upregulated on tumor vessel endothelial cells and some tumor cells.28−30 The tTF-NGR fusion protein adopts an orientation perpendicular to 2343



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BamHI and NcoI sites of the vector. The generated tTF construct possesses an N-terminal histidine tag for subsequent purification of the protein by using immobilized metal affinity chromatography (IMAC). The vectors were introduced into competent Escherichia coli (BL21 DE3) according to the manufacturer’s protocol (Novagen). After stimulating with IPTG (Novagen), the cells were harvested, and 5−7 mL of lysis buffer (10 mM Tris−HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 10 μg/mL aprotinin, 2 mg/mL lysozyme) per gram of wet weight and 500 units of benzonase (Novagen) per gram of wet weight were added to the pellet. Then, the cells were incubated for 90 min at room temperature (RT) and centrifuged at 12 000g for 20 min at 4 °C. The pellet was resuspended and homogenized by sonicating in washing buffer (10 mM Tris−HCl, pH 7.5, 1 mM ethylenediaminetetraacetic acid (EDTA), 3% Triton X-100). To solubilize the inclusion bodies, 2−4 mL of guanidinium buffer (6 M GuCl, 0.5 M NaCl, 20 mM Tris−HCL, pH 7.5, and 1 mM dithiothreitol (DTT)) per gram of wet weight was added. After incubation overnight at RT, the suspension was centrifuged at 10 000g for 15 min at 4 °C. The supernatant was filtered through a 0.22 μm filter and stored at −25 °C. To upscale the fabrication procedure, a multistep high-performance liquid chromatography (HPLC)-based purification process has been established (HPLC unit: Ä KTA purifier 100 system, GE healthcare, Uppsala, Sweden).36 The first capture step consists of IMAC in which the immunogenic nickel is replaced by copper (IMAC Sepharose 6 FF, GE healthcare, München, Germany). The histidine-tagged (N terminus) tTF-NGR fusion protein binds to the immobilized copper ions so that the complete refolding (urea gradient from 6 to 0 M) and washing processes are performed on the column, from which the tTFNGR protein is eluted by applying 300 mM imidazole. During the subsequent gel filtration, the IMAC eluate is conditioned by a bufferexchanging step using Sephadex G-25 (GE healthcare) in order to prepare for the following intermediate purification step. This anionexchange chromatography step (AIEX; Q Sepharose HP, GE healthcare) allows further separation of the eluted proteins according to differences in their net charges. Moreover, it removes most of the remaining impurities such as other proteins, nucleic acids, endotoxin, and so forth. The concluding polishing step again comprises a gel filtration using Sephadex G-25 in order to remove any remaining trace impurities and to exchange the buffer to phosphate-buffered saline (PBS). The final protein solutions are stored at −80 °C. The tTF-NGR protein was analyzed under denaturing conditions on SDS-PAGE and western blot using mouse monoclonal antihuman tissue factor antibody (American Diagnostica, Pfungstadt, Germany; dilution 1:1000). Besides, every charge was analyzed by the FX coagulation assay (see below). Analyses of the bacterial endotoxin content of the tTF fusion protein charges have routinely been accomplished by a certified external laboratory (Bioassay, Heidelberg, Germany). The used kinetic chromogenic Limulus amebocyte lysate (LAL) assay was performed in compliance with the European Pharmacopoeia (sensitivity: 0.005 IU/mL). The determined values always were below an endotoxin level of 10 IU/mg of protein (1.4−7.1 IU/mg of protein). tTF-NGR solutions were microfiltrated with 22 μm filters before further use. TMS(PEG)12 PEGylation. Random TMS(PEG)12 PEGylation of the tTF-NGR protein was performed according to the manufacturer’s protocol (Thermo Scientific/Pierce, Bonn, Germany; see also the internet protocol by Sorina Morar et al.44). In brief, the protein was incubated for 2 h at 5 °C in PBS with a 30-fold excess of TMS(PEG)12 (a trimethyl succinimidyl polyethylene glycol ester; molecular weight: 2420.8 Da), which reacts with primary amino groups (such as lysines) within the tTF-NGR protein releasing NHS. Afterward, the PEGylated protein was purified by a HPLC-based gel filtration with Sephadex G25 medium (GE Healthcare, München, Deutschland) to remove NHS and excess TMS(PEG)12. The efficiency of the reaction has been verified by SDS-PAGE and western blotting according to standard protocols and as described above. For western blotting, an anti-PEG antibody was used that detects the methoxy groups of the polyethylene glycols (PEG-B-47 rabbit monoclonal antibody; Epitomics, Burlingame, CA, USA).

the phospholipid membrane of the endothelial cell providing a basis for coagulation. By connecting the peptide to the C terminus of tTF, sterical hindrance of the interaction of tTF, FVII, and FX should be eliminated.42 In addition, higher phosphatidylserine expression within the tumor vasculature might provide additional selectivity for procoagulant activities induced by tTF-NGR.30 Coagulation inducing activity of the iv application of tTF-NGR in tumor vasculature with subsequent tumor infarction and growth delay could be shown in several tumor mouse models. First-in-man experiences with low doses of tTF-NGR (1−4 mg/m2 by iv infusion) showed inhibition of tumor perfusion without any side effects as “proof-ofprinciple”.35 Furthermore, our retargeted tTF molecules could be successfully used in amplifying tumor targeting by communicating nanoparticles.43 However, we sometimes observed mouse tail tip necrosis upon iv injection, and when choosing routes of application other than iv infusion, such as subcutaneous injection, systemic toxicity with pulmonary embolism and skin bleeding with laboratory signs of DIC occurred within the therapeutic dose range.37 Hypothesizing that this toxicity is connected to the pharmacokinetics including blood peak levels of tTF-NGR and to formation of larger aggregates, we have undertaken studies with the aim to improve the safety and activity profile of tTFNGR. PEGylation is an established method for conferring improved activity/toxicity profiles to high-molecular pharmacologic agents such as proteins by increasing half-life, reducing protein aggregation, immunogenicity, and uptake by the RHS. Thus, an increasing number of PEGylated drugs are used in clinical oncology.38−40 Here, we report that random (TMS)PEG12 PEGylation of tTF-NGR is indeed leading to an improved anticancer profile in the experimental in vivo models used. Although approximately 3−5-fold single doses per kilogram of bw were necessary to induce tumor growth retardation, PEGylated tTF-NGR with the low toxicity observed has a considerably better therapeutic range than non-PEGylated tTF-NGR. It will be interesting and necessary to widen this approach by comparing PEG molecules approved for human use and of higher molecular mass and in particular site-directed PEGylation of the molecule especially with regard to the intended transfer to clinical application.

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CONCLUSION Random TMS(PEG)12 PEGylation of the retargeted truncated tissue factor tTF-NGR decreased the procoagulant activity of the molecule but showed an improved pharmacokinetic behavior and anticancer profile of the molecule in vivo. Further experiments have to verify whether PEGylation with PEG molecules of different molecular mass or site-directed PEGylation can further improve on these results.

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EXPERIMENTAL SECTION

Cloning, Expression, and Purification of Proteins. Cloning, expression, and purification of proteins have been described in detail earlier.35−37 Briefly, the cDNA coding for tTF containing amino acids 1−218 and GNGRAHA, in which the heptapeptide is linked to the C terminus of tTF (tTF-NGR), was amplified by polymerase chain reaction (PCR) using the primers 5′-CATGCCATGGGATCAGGCACTACAAATACTGTGGCAGCATATAAT-3′ (5′-primer) and 5′CGGGATCCTATTATGCATGTGCTCTTCCGTTACCTCTGAATTCCCC-3′ (3′-primer) for tTF-NGR. With the DNA-ligation kit (Novagen, Schwalbach am Taunus, Germany), the cDNA was cloned into the expression vector pET30a(+) (Novagen) using the 2344



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was allowed to a mean volume of approximately 500−600 mm3 (HT1080) and 200 mm3 (A549). Mice were randomly assigned to different experimental groups. The PEGylated tTF-NGR protein was slowly applied iv via the tail veins. Injections were repeated daily for 7 times (HT1080) or for 10 times (A549). Tumor size was evaluated using a standard caliper measuring tumor length and width; tumor volumes were calculated using the standardized formula (length × width2 × π/6). According to our project license, animals had to be sacrificed when tumors became too large, if mice lost >20% of body weight, or at signs of pain. In this case, mice were sacrificed by cervical dislocation in deep ketamine/xylazine anesthesia in agreement with standard regulations and the project license. Studies on tolerability and acute toxicity were performed in CD-1 mice with single iv injections of tTF-NGR and its PEGylated form. LD10 was defined as the dose leading to death of 10% of the injected animals within 24 h after application. Spontaneous deaths and mice sacrificed upon intolerable side effects were counted. Contrast-Enhanced Ultrasound. CEUS47,48 was performed using the small-animal ultrasound machine Vevo 2100 (Visualsonics, Toronto, Canada) equipped with a MS-250 transducer operating in a contrast-specific imaging. As the microbubbles contrast agent, we used the Vevo MicroMarker non-targeted contrast agent kit (Visualsonics) according to the manufacturer’s protocol. A 100 μL volume of the contrast agent was injected via a 26G-tail vein catheter prior to the ultrasound imaging of the tumor; the adjacent kidney served as control tissue. Imaging was gated by electrocardiography and respiration to ensure comparable conditions. CEUS was performed with U87 xenotransplant-bearing mice, which received 5 mg/kg bw TMS(PEG)12 tTF-NGR as daily iv application for 4 days, 1 day after the last treatment. Fluorescence Reflectance Imaging. CD-1 nude mice bearing U87 xenotransplants were treated three times within 24 h (at 0, 8, and 24 h) with TMS(PEG)12 tTF-NGR (10 mg/kg bw, iv). One hour after the last application, treated and untreated mice received 2 nmol (150 μL, iv) of the fluorescent in vivo blood pool- and tumor-imaging agent AngioSense680 (PerkinElmer, Rodgau, Germany). After a further 24 h, the AngioSense680 fluorescence intensities of the explanted tumors were quantified by FRI with the in vivo imaging system FX PRO (Carestream Health, Rochester, NY, USA). Histology and Immunofluorescence Imaging. Histological analyses of tumor tissues and normal organs were performed with O.C.T.-embedded and cryo-conserved tissues according to standard protocols. Briefly, tissues were embedded in Tissue-TEK O.C.T. (Sakura, Alphen aan den Rijn, Netherlands), snap-frozen in liquid nitrogen, and stored at −85 °C. Frozen tissue samples were cut to 4 μm sections and transferred onto glass slides. For identification of thrombosis and organ pathologies, H&E-stained sections were examined using conventional light microscopy. For immunofluorescence staining, excised and frozen tumor and organ tissue samples were serial sectioned (6 μm), transferred to glass slides, fixed with acetone/methanol, and dried according to standard protocols. Sections were incubated with an anti-PEG antibody as primary antibody (see above; dilution 1:100) followed by a AlexaFluor488-labeled goat antirabbit IgG antibody (Invitrogen, Darmstadt, Germany; dilution 1: 200); nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI). The accumulation of PEG within the organ sections was examined with a fluorescence microscope. Statistical Analyses. Statistical significance of differences in the treatment experiments was tested by the t test or by Mann−Whitney rank sum test for independent groups upon removal of few individual outliers. Two-tailed P values lower than 0.05 were considered as indicating significant differences.

Mass Spectrometry. MALDI-TOF MS has been performed using a MALDImicro MX instrument from Waters Corp. (Manchester, UK) and sinapinic acid as the matrix. To that end, the protein was further purified using ZipTips c4 (Millipore, Billerica, MA, USA). Free TMS(PEG)12 was determined using HP1100 C8 liquid chromatography coupled to an Esquire 3000 ion trap (Agilent, Bruker); UV peak areas at 214 nm were compared. Factor X Coagulation Assay. The ability of the tTF-NGR fusion proteins (PEGylated and non-PEGylated) to enhance the specific proteolytic activation of FX by FVIIa was assessed by Michaelis− Menten analyses as basically described by Ruf et al.45 Briefly, 20 μL of the following was added to each well in a microtiter plate: (a) 50 nM recombinant FVIIa (Novo−Nordisc, Bagsværd, Denmark) in TBS containing 0.1% bovine serum albumin (BSA); (b) 25−300 pM tTFNGR protein or 1−50 nM PEGylated tTF-NGR protein, respectively, in TBS−BSA; (c) 25 nM CaCl2 and (d) 500 μM phospholipids (phosphatidylcholine/phosphatidylserine, 70/30, MM; Sigma, München, Germany). After 10 min at RT, the substrate FX (Enzyme Research Laboratories, Swansea, UK) was added (final concentration 1 μM). After additional 10 min, the reaction was stopped in 100 nM EDTA, and Spectrozyme FXa (American Diagnostica, Greenwich, USA; final concentration 0.7 mM) was added. The rates of FXa generation were monitored by the development of color at 405 nm with a microplate reader (Bio-Rad, München, Germany). The Michaelis constants (Km) of the FX activation of the PEGylated and non-PEGylated tTF-NGR proteins were calculated by hyperbolic regression analysis according to Hanes.46 Pharmacokinetic Analyses with the IMUBIND Tissue Factor ELISA. The initial concentration/time curve of iv applied PEGylated and non-PEGylated tTF-NGR proteins within the blood of nontumor bearing CD-1 mice was analyzed with the IMUBIND TF ELISA (American Diagnostica, Stamford, USA). In each case, 1 mg of the protein per kg of bw was dissolved in PBS and injected via the tail vein (300−350 μL/mouse, depending on the animal weight). Blood samples were collected via the tail veins after 1, 10, 30, and 60 min and 2, 4, 8, and 24 h, along with immediate dilution in citrate buffer to prevent coagulation. After centrifugation, the serum probes were retained and stored at −25 °C. For the analyses, samples were diluted appropriately to provide TF concentrations in line with the linearity range of the TF ELISA (approximately 50−2000 pg/mL). Depending on the tested protein samples, non-PEGylated tTF-NGR protein (for analyzing non-PEGylated tTF-NGR) or PEGylated tTF-NGR protein (for analyzing PEGylated tTF-NGR) was used as the standard, respectively (the difference between both standards was only small). The IMUBIND tissue factor ELISA was performed according to the manufacturer’s protocol. The AUC was determined by mathematical peak integration using Excel. Mice and Tumor Xenograft Models. The HT1080-fibrosarcoma cell line was cultured in Dulbecco’s medium supplemented with 10% fetal calf serum (FCS), the A549 cell line (adenocarcinoma of the lung) was cultured in HAM’s F12 medium (GIBCO-BRL) supplemented with 10% FCS and 2 mM L-glutamine, and the U87 cell line (glioblastoma) was cultured in MEM medium (GIBCO-BRL) supplemented with 10% FCS, pyruvate (2 mM), and nonessential amino acids (2%). Cell lines were obtained from ATCC (Manassas, VA, USA), and the identity was confirmed by short tandem repeat (STR) profiling. All procedures on animals were performed in agreement with German regulations (Tierversuchsgesetz §8 Abs. 2) and specifically approved in the form of a project license. Mice (CD-1 and CD-1 nude mice) were purchased from Charles River Laboratories (Sulzfeld, Germany) and acclimated to our animal-experiment facility for at least 1 week before any experimentation. Mice were maintained in individually ventilated cages (IVC) on a 12:12 h light/dark cycle in a low-stress environment (22 °C, 50% humidity, low noise) and given food and water ad libitum. Single cell suspensions (4 × 106 in 100 μL for HT1080, 3 × 106 in 100 μL for A549, and 2 × 106 in 100 μL for U87) were injected subcutaneously (sc) into the right anterior flank of female CD-1 nude mice (9−12 weeks old). For therapeutic experiments, tumor growth

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S Supporting Information *

Figure showing the tolerability of PEGylated versus nonPEGylated tTF-NGR in vivo. This material is available free of charge via the Internet at http://pubs.acs.org. 2345



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AUTHOR INFORMATION

Corresponding Author

*(W.E.B.) Phone: + 49 251 83 47587; fax: + 49 251 83 47588; e-mail: [email protected]. (C.S.) Phone: + 49 251 83 56225; fax: + 49 251 83 56709; e-mail: christian.schwoeppe@ uni-muenster.de. Notes

The authors declare the following competing financial interest(s): R.M.M. and W.E.B. share a patent on vascular targeting with TF constructs. No potential conflict of interest was declared by the other authors.

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ACKNOWLEDGMENTS This work was supported by grants of the Deutsche Krebshilfe e.V. (109245 to W.E.B.), the Deutsche Forschungsgemeinschaft (SFB656, projects C8 and C3), the Sybille-HahneStiftung, and IZKF Münster PIX ECHO. C.Z. contributed experiments in partial fulfillment of the requirements to obtain a Ph.D. degree; M.F. contributed experiments in partial fulfillment of the requirements to obtain an M.D. degree.

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ABBREVIATIONS USED

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REFERENCES

APN, aminopeptidase N; AUC, area under the curve; BSA, bovine serum albumin; bw, body weight; ; CEUS, contrastenhanced ultrasound; DIC, disseminated intravascular coagulation; EDTA, ethylenediaminetetraacetic acid; ELISA, enzymelinked immunosorbent assay; IF, immunofluorescence; FRI, fluorescence reflectance imaging; LD10, lethal dose for 10% of animals treated upon one iv injection; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; NGR, peptide GNGRAHA; NHS, N-hydroxysuccinimide; PBS, phosphate-buffered saline; PEG, polyethylene glycol; RHS, reticulo-histiocytic system; STR, short tandem repeat; tTF, truncated tissue factor; TMS(PEG)12, trimethyl succinimidyl polyethylene glycol ester; RT, room temperature; TBS, Tris-based saline

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