Tumor Growth Inhibition via Occlusion of Tumor Vasculature Induced

Article pubs.acs.org/molecularpharmaceutics

Tumor Growth Inhibition via Occlusion of Tumor Vasculature Induced by N‑Terminally PEGylated Retargeted Tissue Factor tTFNGR Caroline Brand,† Max Fröhlich,† Janine Ring,† Christoph Schliemann,† Torsten Kessler,† Verena Mantke,† Simone König,‡ Martin Lücke,§ Rolf M. Mesters,† Wolfgang E. Berdel,*,†,∥ and Christian Schwöppe*,†,∥ †

Department of Medicine A, Hematology, Oncology and Pneumology, ‡Integrated Functional Genomics (IZKF Muenster), and Central Institute for Animal Experimentation, University of Muenster, Albert-Schweitzer-Campus 1, D-48129 Muenster, Germany

Downloaded by UNIV OF NEBRASKA-LINCOLN on September 1, 2015 | http://pubs.acs.org Publication Date (Web): August 31, 2015 | doi: 10.1021/acs.molpharmaceut.5b00508

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ABSTRACT: tTF-NGR retargets the extracellular domain of tissue factor via a Cterminal peptide GNGRAHA, a ligand of the surface protein aminopeptidase N (CD13) and upon deamidation of integrin αvβ3, to tumor vasculature. tTF-NGR induces tumor vascular infarction with consecutive antitumor activity against xenografts and selectively inhibits tumor blood flow in cancer patients. Since random PEGylation resulted in favorable pharmacodynamics of tTF-NGR, we performed site-directed PEGylation of PEG units to the N-terminus of tTF-NGR to further improve the antitumor profile of the molecule. Mono-PEGylation to the Nterminus did not change the procoagulatory activity of the tTF-NGR molecule as measured by Factor X activation. Experiments to characterize pharmacokinetics in mice showed a more than 1 log step higher mean area under the curve of PEG20ktTF-NGR over tTF-NGR. Acute (24 h) tolerability upon intravenous application for the mono-PEGylated versus non-PEGylated tTF-NGR compounds was comparable. PEG20k-tTF-NGR showed clear antitumor efficacy in vivo against human tumor xenografts when systemically applied. However, site-directed mono-PEGylation to the N-terminus does not unequivocally improve the therapeutic profile of tTF-NGR. KEYWORDS: vascular targeted tissue factor, site-directed PEGylation, vascular infarction, experimental cancer therapy

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transmembrane domain (truncated TF, tTF)17 can partly be reconstituted by targeting tTF into the proximity of a phospholipid membrane.18 We thus have designed and constructed fusion proteins consisting of various short NGRpeptides coupled to the C-terminus of tTF.19−22 Among others, HIStag-tTF-NGR1−218-GNGRAHA (tTF-NGR) retains its procoagulatory activity in vitro, it binds to both targets, CD13 and αvβ3 expressed on stimulated endothelial cells, and upon intravenous (iv) infusion it induces infarction of blood vessels in several human xenografts with subsequent growth retardation and regression of the xenograft tumors. A 1-h infusion of tTF-NGR via central venous access in cancer patients at doses between 1 and 4 mg/m2 body surface area caused no side effects, but was shown to reduce tumor blood flow in situ as measured by magnetic resonance imaging (MRI).20 Today, two CD13-targeted, NGR-coupled therapeutic antitumor molecules are in clinical studies. Advanced clinical studies are on the way using NGR-tumor necrosis factor (NGR-hTNF) in different tumor entities including malignant pleural mesothelioma.23

INTRODUCTION CD13, a transmembrane aminopeptidase N, is an attractive molecule for targeting therapeutically active compounds to tumors. It represents a molecule with upregulated expression on the cell surface of endothelial cells in tumors and tissues with angiogenic activity. This molecule is of specific interest, since it is expressed not only on tumor vessel endothelial cells but also on tumor cells of different histology, and can influence tumor growth and metastasis.1 Pasqualini et al.2 were the first to report that small peptides containing the NGR motif (asparagine-glycine-arginine) bind to aminopeptidase N (APN; CD13). NGR binds to CD13 in tumor vasculature, but not to CD13 on normal kidney and myeloid cells.3 NGR peptides undergo nonenzymatic spontaneous deamidation of asparagine to isoaspartate-glycine-arginine (isoDGR) generating a ligand for the integrin αvβ3.4,5 This integrin is also reported to be upregulated on tumor endothelial cells.6,7 Because of the preferential expression of CD13 in tumors and tumor vasculature, NGR-containing radioisotopes and paramagnetic quantum dots are successfully used for experimental tumor imaging.8−16 Tissue factor (TF) is the central molecule activating the extrinsic coagulation in vivo. Thus, among other possible structures, we and others have chosen derivatives of this molecule to attempt tumor vascular infarction. The relative lack of procoagulatory activity of the soluble form of TF without the © XXXX American Chemical Society

Received: June 27, 2015 Revised: August 25, 2015 Accepted: August 27, 2015

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DOI: 10.1021/acs.molpharmaceut.5b00508 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Elution fractions having an absorbance at 280 nm were collected and analyzed by SDS−PAGE. Fractions with the mono-PEGylated protein were pooled, and buffer was exchanged to phosphate-buffered saline (PBS) by a HPLCbased gel filtration with Sephadex G-25 medium (GE Healthcare). The final mono-PEGylated-protein solution was adjusted to a concentration of 1 mg/mL and stored at −80 °C. The efficiency of the reaction and the purity of the product have 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 which detects the methoxy groups of the polyethylene glycols (PEG-B-47 rabbit monoclonal antibody; Epitomics, Burlingame, CA, USA). To optimize the PEGylation efficiency, reaction conditions were varied regarding reaction time (4, 16, 32 h) and reaction temperature (4 °C, 21 °C), and the reaction mixture was directly analyzed by SDS−PAGE to evaluate the ratio of nonPEGylated/mono-PEGylated protein. Mass Spectrometry. Mass spectrometric analysis of the intact protein and its tryptic digest was performed as described before:24 Matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF-MS) of desalted analyte was run using a MALDImicro MX instrument. Samples were also interrogated using reversed-phase liquid chromatography coupled to MS (Agilent HP1100Esquire 3000 ion trap (Bruker Daltonics) or nanoAcquityQ-TOF Premier (Waters Corp.). Factor X (FX) Coagulation Assay. Ability of the tTFNGR fusion proteins (PEGylated and non-PEGylated) to enhance the specific proteolytic activation of factor (F) X by FVIIa was assessed by Michaelis−Menten analysis as described by Ruf et al.27 This assay is appropriate to describe activation of the extrinsic coagulation cascade by TF and derivatives. In brief, 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 tTF-NGR protein or 75−500 pM mono-PEGylated tTF-NGR protein, respectively, in TBS-BSA; (c) 25 mM CaCl2 and 500 μM phospholipids (phosphatidylcholine/phosphatidylserine, 70/30, MM; Sigma, München, Germany). After 10 min at room temperature, the substrate FX (Enzyme Research Laboratories, Swansea, U.K.) was added (final concentration 1 μM). After an additional 10 min, the reaction was stopped in 100 mM 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). Michaelis constants (Km) of the FX activation of the PEGylated and nonPEGylated tTF-NGR proteins were calculated by hyperbolic regression analysis as described by Hanes.28 Pharmacokinetic Analyses with the IMUBIND Tissue Factor ELISA. Plasma levels of the proteins were assayed as published before with some modifications.24 The initial concentration/time curve of iv applied PEGylated and nonPEGylated tTF-NGR proteins within the blood of non-tumorbearing CD-1 mice was analyzed with the IMUBIND tissue factor ELISA (American Diagnostica, Stamford, CT, USA). In each case, 1 mg of the protein per kg body weight (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

We have reported that random PEGylation improves activity/toxicity profiles of tTF-NGR.24 Here we report experiments with 2 kDa and 20 kDa PEG units, respectively, conjugated site-directed to the N-terminal end of tTF-NGR. We obtained a lead compound with 20 kDa PEG units (PEG20k-tTF-NGR). Although HPLC-purified PEG20k-tTFNGR clearly shows antitumor activity in xenografts, this sitedirected PEGylation did not unequivocally improve pharmacological characteristics of tTF-NGR.


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EXPERIMENTAL SECTION Cloning, expression, and purification of proteins has been described in detail earlier.19−21,24 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 (tTFNGR), was amplified by polymerase chain reaction (PCR) and cloned into the expression vector pET30a(+) (Novagen). The generated tTF construct has an N-terminal histidine tag for the subsequent purification of the protein by using immobilized metal-chelate affinity chromatography (HIStag-tTF-NGR1−218GNGRAHA). Vector transformation into competent Escherichia coli (BL21 DE3) and biotechnological production were performed as described.19−21,24 To upscale the fabrication procedure, a multistep HPLC-based purification process has been established (HPLC unit: Ä KTA purifier system, GE healthcare, Uppsala, Sweden).21 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 anti-human tissue factor antibody (American Diagnostica, Pfungstadt, Germany; dilution 1:1000). In addition, every batch was analyzed by the Factor X coagulation assay (see below). Analysis of the bacterial endotoxin content of the tTF fusion protein batches has routinely been accomplished by a certified external laboratory (BioChem, Karlsruhe, Germany). The used kinetic chromogenic LAL (Limulus Amebocyte Lysate) assay was performed in compliance with the European Pharmacopoeia (sensitivity: 0.01 IU/mL). The determined values always were below an endotoxin level of 10 IU/mg protein (1.4−7.1 IU/mg protein). tTF-NGR solutions were microfiltrated with 22-μm filters before further use. Site-Directed Mono-PEGylation. Site-directed coupling of 2 or 20 kDa PEG units, respectively, to the N-terminus of tTF-NGR was performed by reductive alkylation according the protocol of PEGylation of granulocyte-colony stimulating factor G-CSF.25,26 In brief, a solution of tTF-NGR at 1 mg/mL in 0.1 M NaH2PO4, pH 5.0, was incubated with an 8-fold molar excess of 20 kDa-methoxy polyethylene glycol aldehyde (e.g., mPEG-CHO20kDa, Nanocs, New York, NY, USA) and sodium cyanoborohydride as reductant (NaBH3CN: 40 μmol per 0.1 μmol of protein). After slightly mixing for 16 h at room temperature, the reaction was stopped by adding a 10-fold volume excess of 1 mM HCl and the pH was lowered to 3.5 with 1 M HCl. The solution was filtered sterile and loaded onto a HiTrap SP XL cation exchange column (GE Healthcare, München, Germany; column volume 10 mL), pre-equilibrated with 20 mM sodium acetate, pH 4.0 (buffer A) using an Ä KTA purifier UPC system (GE Healthcare). After sample loading, the column was washed with three column volumes of buffer A, followed by a linear gradient to 45% buffer B (buffer A + 1 M NaCl) over 20 column volumes. Subsequently, a second gradient to 80% buffer B was applied over 8 column volumes. B


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Figure 1. Site-directed PEGylation of tTF-NGR. tTF-NGR was conjugated site-directed to 20 kDa PEG and 2 kDa PEG units, respectively, by reductive alkylation. (A) Mono-PEGylated tTF-NGR was isolated from the reaction mixture by cation exchange chromatography (CIEX): gradient elution revealed two distinguishable peaks that were further analyzed by SDS−PAGE. (B) PEGylation efficiency at different reaction conditions was analyzed by SDS−PAGE (lane 1, standard protein size marker; lane 2, non-PEGylated tTF-NGR; lanes 3−7, 8-fold excess of PEG and following variation of reaction temperatures and times (lane 3, 4 °C, 16 h; lane 4, 4 °C, 32 h; lane 5, 21 °C, 4 h; lane 6, 21 °C, 16 h; lane 7, 21 °C, 32 h; see Experimental Section]). (C) Elution fractions with the mono-PEGylated protein as visualized by SDS−PAGE are contained in the first peak (lanes 4−6), whereas intermediates and non-PEGylated protein are found in the second peak (see lanes 7−10).

experiments, tumor growth was allowed to a mean volume of approximately 200−300 mm3. Mice were randomly assigned to different experimental groups. The mono-PEGylated tTF-NGR protein was slowly applied intravenously (iv) via the tail veins. Injections were repeated every second day for at least 10 times (0.2 mg/kg bw, and 0.5 mg/kg bw) or every fourth day for 7 times (0.2 mg/kg bw and 0.3 mg/kg bw; see also figure legends). 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 mono-PEGylated tTFNGR. 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. 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.

prevent coagulation. After centrifugation, the serum probes were retained and stored at −25 °C. For the analysis, 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, nonPEGylated tTF-NGR protein (for analyzing non-PEGylated tTF-NGR) or mono-PEGylated tTF-NGR protein (for analyzing mono-PEGylated tTF-NGR) was used as standard, respectively (the difference between both standards was only small). The IMUBIND tissue factor ELISA was performed according to the manufacturer’s protocol. The “area under the curve” (AUC) was determined by mathematical peak integration using Excel. Cell Culture. The HT1080-fibrosarcoma cell line was obtained from ATCC (Manassas, VA, USA), and identity was confirmed by short tandem repeat (STR) profiling. Cells were cultured in Dulbecco’s medium supplemented with 10% FCS and maintained at 37 °C in 5% CO2 and high humidity. Mice and Tumor Xenograft Models. All procedures on mice were performed in agreement with German regulations (Tierversuchsgesetz §8 Abs. 2) and specifically approved in the form of a project license. CD-1 mice 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 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) were injected subcutaneously (sc) into the right anterior flank of female CD-1 nude mice (9−12 weeks old). For therapeutic

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RESULTS Site-Directed PEGylation of tTF-NGR and Molecular Characterization. The N-terminus of tTF-NGR was conC


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Molecular Pharmaceutics jugated to PEG units with different molecular mass, preferentially PEG20k, as described in the Experimental Section. Resulting mono-PEGylated tTF-NGR (PEG20k-tTFNGR) was isolated from the reaction mixture by cation exchange chromatography (CIEX). Gradient elution revealed two distinguishable peaks (Figure 1A). PEGylation was further optimized by varying the reaction conditions regarding reaction temperature and time as well as PEG excess and analyzed by SDS−PAGE (Figure 1B). Best ratio of PEGylated tTFNGR:non-PEGylated tTF-NGR was achieved with 8-fold excess of PEG and 16 h reaction time at room temperature (21 °C; see arrow in lane 6). CIEX gradient elution revealed pure elution fractions with the mono-PEGylated protein, which are contained in the first peak and could be shown by SDS− PAGE (see lanes 4−6, Figure 1C), whereas intermediates and non-PEGylated protein are found in the second peak (lanes 7− 10, Figure 1C). In contrast to the product upon random PEGylation,24 the mono-PEGylated PEG20k-tTF-NGR was represented by one clear band in SDS−PAGE and Western blotting (Figure 2A,B). Additionally, we observed excellent batch-to-batch reproducibility (Figure 2B). Thus, we could obtain mono-PEGylated PEG20k-tTF-NGR with a good purity and batch-to-batch reproducibility. Next, we performed analysis of the intact protein and its tryptic digest by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) of desalted analyte. MALDI-TOF-MS spectrum of PEG20k-tTFNGR showed a mass increase of 20 kDa (Figure 3A), and the spectra obtained with PEG2k-tTF-NGR showed a mass increase of 2 kDa (inset), respectively, compared to control (MH+ 30 383 Da). PEGylation resulted in peak broadening due to the heterogeneity of the chemical. Furthermore, we could identify and confirm the PEGylation site by liquid chromatography and subsequent ion trap MS analysis using a 2 kDa PEG unit (Figure 3B, see also abstract figure). Activation of Factor X (FX) by Non-PEGylated tTFNGR and PEG20k-tTF-NGR. The tissue factor (TF) domain of tTF-NGR upon localization of the molecule into the phospholipid environment of the outer cell membrane binds FVIIa with consecutive enhancement of the specific proteolytic activity of FX. PEGylation can interfere with procoagularory activity, and we could indeed show this for random-PEGylated tTF-NGR.24 Thus, we tested for activation of FX induced by mono-PEGylated versus non-PEGylated tTF-NGR (Figure 4). Michaelis−Menten analysis revealed comparable Michaelis constants (Km) for tTF-NGR (248.0 ± 18.5 pM) versus mono-PEGylated tTF-NGR (373.0 ± 62.66 pM) based on the respective protein mass, indicating that mono-PEGylation sitedirected to the N-terminus of the tTF-NGR molecule does not interfere with the procoagulatory activity of the molecule. This is in contrast to our results with random PEGylation, since a 2 order of magnitude lower activity of the random-PEGylated TMS(PEG)12 tTF-NGR was found in comparison to nonPEGylated tTF-NGR (see ref 24 and Figure 4C). Furthermore, concerning reproducibility of different PEG20k-tTF-NGR batches, a similar homogeneity of the bioactivity profile of the mono-PEGylated tTF-NGR compared to the one of the non-PEGylated tTF-NGR has been accomplished (details not shown). Pharmacokinetics. For comparative pharmacokinetic studies upon iv injection of non-PEGylated tTF-NGR, monoPEGylated PEG20k-tTF-NGR, and random-PEGylated TMS(PEG)12 tTF-NGR groups of CD-1 mice were iv injected with

Figure 2. Analysis of mono-PEGylated tTF-NGR versus randomPEGylated tTF-NGR. (A) Purified PEGylated protein was analyzed by SDS−PAGE (stained with Coomassie; left) and Western blotting (right panel) using an anti-PEG antibody: For site-directed monoPEGylated tTF-NGR [PEG20k-tTF-NGR] we obtained one defined band (see lanes 4 (left panel) and 7 (right)), whereas randomPEGylated tTF-NGR [TMS(PEG)12 tTF-NGR] revealed multipleband patterns as published before24 with reproducible mass distribution (see lanes 3 (left) and 6 (right)); non-PEGylated tTFNGR, lanes 2 (left) and 5 (right); standard protein size marker, lane 1. (B) Batch-to-batch reproducibility of mono-PEGylated tTF-NGR: SDS−PAGE of 3 representative batches of PEG20k-tTF-NGR (lanes 2−4) and non-PEGylated tTF-NGR (lane 5).

1 mg/kg bw of protein, neglecting the additional mass of the PEG. Blood samples collected after 1, 10, 30, and 60 min and 2, 4, 8, and 24 h were subjected to ELISA as described using antiTF antibody for analysis. This anti-TF antibody was able to detect tTF-NGR and both PEGylated forms of tTF-NGR. As shown in Figure 5, peak levels (Cmax) upon iv injection did not much differ between the non-PEGylated tTF-NGR (18829.0 ng/mL) and both PEGylated forms (PEG20k-tTFNGR 14677.0 ng/mL; TMS(PEG)12 tTF-NGR 17333.0 ng/ mL, see also ref 24). As expected, the half-life of PEG20k-tTFNGR was approximately 61 min and similar to TMS(PEG)12 tTF-NGR (57 min), whereas non-PEGylated tTF-NGR was measured with only approximately 7 min (Figure 5A). The wash-out time was approximately 20 h longer and the blood levels after 24 h comparable to the random-PEGylated tTFNGR24 over the non-PEGylated tTF-NGR (Figure 5B), resulting in a more than 1 log step higher area under the curve (AUC) for both PEGylated tTF-NGR molecules (Figure D


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Figure 3. Mass spectrometric analysis of mono-PEGylated tTF-NGR. (A) MALDI-TOF-MS spectrum of PEG20k-tTF-NGR shows a mass increase of 20 kDa and MALDI-TOF-MS spectrum of PEG2k-tTF-NGR a mass increase of 2 kDa (inset), respectively, compared to control (MH+ 30383 Da). PEGylation causes peak broadening due to the heterogeneity of the chemical; m/z, mass-to-charge ratio. (B) Liquid chromatography (LC) with subsequent ion trap MS analysis of mono-PEGylated tTF-NGR (with a 2 kDa PEG unit) versus non-PEGylated tTF-NGR was performed to identify the PEGylation site: The LC-UV spectrum of PEG2k-tTF-NGR (blue) revealed a disappearance of the peptide peak containing the alpha amino acid methionine (MHHHHHHSSGLVPR) after the PEGylation reaction when compared to the LC−UV spectrum of non-PEGylated tTF-NGR (pink). The terminal peptide also disappeared from the MALDI-TOF peptide mass fingerprint after PEGylation. The molecular complexity of PEG causes the formation of an equally complex product population. E


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Figure 4. Activation of FX by non-PEGylated tTF-NGR versus mono- and random-PEGylated tTF-NGR. (A) Ability of non-PEGylated tTF-NGR compared with PEG20k-tTF-NGR to enhance the specific proteolytic activation of FX 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. (details see Experimental Section). Mean Km values of non-, mono-, and random-PEGylated tTF-NGR (each with n = 12) are given in panel C: 248 ± 18.53 pM for tTF-NGR, 374 ± 62.66 pM for PEG20k-tTF-NGR, and 10595 ± 1584 pM for TMS(PEG)12 tTF-NGR. Data of TMS(PEG)12 tTF-NGR were published before24 and are reproduced with permission. v, reaction velocity; mAU, milliabsorbance units; c/v, ratio of the initial substrate concentration to the reaction velocity; c, substrate concentration.

Antitumor Activity of PEG20k-tTF-NGR in Vivo. The in vivo antitumor activity of PEG20k-tTF-NGR was tested in athymic CD-1 nude mice bearing human HT1080 fibrosarcoma xenografts. PEG20k-tTF-NGR or control saline was slowly injected iv at the doses and time intervals as shown in Figure 6A,B. Doses of 0.2 and 0.5 mg/kg bw given every second day (Figure 6A), as well as 0.2 and 0.3 mg/kg bw given every fourth day (Figure 6B) were used. Repeated application of 0.5 mg/kg bw showed antitumor activity, but the experiment was terminated due to limiting toxicity (Figure 6A). Doses of 0.2 mg/kg bw either every second or every fourth day induced tumor growth delay which reached statistical significance (p = 0.049; Figure 6A) when compared with saline controls. However, as with non-PEGylated tTF-NGR, we have observed regrowth of the tumors after termination of PEG20k-tTF-NGR treatment. As with non-PEGylated tTF-NGR, repeated iv application of PEG20k-tTF-NGR via the tail veins of the mice resulted in local toxicity manifested as tail tip necrosis.

5C). Mean AUC as measured until 24 h after injection was 4817 ng/mL × h for non-PEGylated tTF-NGR, 71021 ng/mL × h for PEG20k-tTF-NGR, the latter value almost identical to TMS(PEG)12 tTF-NGR (82107 ng/mL × h).24 To exclude the possibility of underestimating levels of PEGylated tTF-NGR, in case PEG20k-tTF-NGR interfered with the anti-TF antibody binding to the PEGylated molecule in the ELISA, we have established and used our own ELISA calibration curves for each molecule. We have taken blood for analysis for up to 24 h after injection of the proteins and then stopped the procedure for practicability reasons. At this time PEGylated tTF-NGR was still measurable at a much higher concentration than tTF-NGR. Possible underestimation of the AUC differences for this reason was neglected in this analysis. Taking into account the identical procoagulatory activity of the mono-PEGylated PEG20k-tTF-NGR as compared to the non-PEGylated tTF-NGR and the prolonged half-life of PEG20k-tTF-NGR, the antitumor activity and tolerability experiments with PEG20k-tTF-NGR were planned within lower dose frames. Tolerability Comparison. 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 6C shows dose−lethality curves with LD10 estimates of approximately 5 mg/kg bw for tTF-NGR as published before.24 The LD10 for PEG20k-tTF-NGR was approximately 7 mg/kg, i.e., similar to the one for nonPEGylated tTF-NGR, which can be partially explained with the identical peak levels and procoagulatory efficacy. For TMS(PEG)12 tTF-NGR, LD10 was not reached with 160 mg/kg bw; these values were published before24 and are shown again for comparability (with permission of Journal of Medicinal Chemistry).

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DISCUSSION Targeting tumor vasculature can be conceptually divided into antiangiogenesis,29−31 vascular disruption,32,33 and vascular targeting of antitumor molecules.34,35 Within the framework of vascular targeting we have constructed fusion proteins consisting of small peptides, including RGD36 and NGR motifs,19−22 coupled to the C-terminus of tTF. tTF, upon building the tTF:VIIa complex,37 is a central activator of coagulation when relocalized to a phospholipid membrane of endothelial cells. Among other constructs systemic application of tTF-NGR was shown to induce tumor growth inhibition and size reduction via tumor vascular infarction. The NGR motif was chosen for retargeting tTF to tumor vessels since it binds F


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delay or size reduction.20−22 First application of low doses of tTF-NGR (1−4 mg/m2 by 1-h infusion via a central venous access) to cancer patients showed inhibition of tumor blood perfusion with no side effects as proof of principle.20 PEGylation is an established method to improve pharmacokinetics and pharmacodynamics of high-molecular pharmacologic molecules such as proteins by increasing half-life, reducing protein aggregation, immunogenicity and uptake by the reticulohistiocytic system. Various drugs PEGylated by random or site-directed methods are used in clinical oncology.38−40 Indeed, advanced technology has been described using PEG block copolymers with cyclic NGR peptides to target cytotoxic docetaxel to endothelial and tumor cells expressing CD13.41 We have reported earlier that random (TMS)PEG 12 PEGylation of tTF-NGR results in somewhat improved anticancer properties of the molecule in experimental in vivo models.24 Here we report on our attempts to further improve pharmacodynamics of tTF-NGR by site-directed PEGylation. We have chosen monoPEGylation to the N-terminus of tTFNGR, since this location does neither interfere with the specific binding of the C-terminal NGR-motif to its target nor with the procoagulatory activity of the TF moiety, and since in contrast to random PEGylation one can easily obtain a single molecule of one molecular structure, which allows for better pharmacological characterization. In summary, site-directed PEGylation to the N-terminus of tTF-NGR of PEG molecules with different molecular size is possible and shows a high batch-to-batch stability. Initial screening did not reveal major advantages of using PEG molecules of a certain molecular mass. PEG20k-tTF-NGR was studied in detail and clearly showed therapeutic activity in human xenografts in vivo. Comparing the LD10 values and lowest effective antitumor doses with non-PEGylated tTFNGR, we obtained a therapeutic range (active/toxic dose in mg/kg bw) of 1/5 mg/kg bw for the non-PEGylated tTF-NGR versus 0.2/7 mg/kg bw for PEG20k-tTF-NGR, characterizing this mono-PEGylated form of tTF-NGR with a larger therapeutic window upon single application. On the other hand, there was a steep activity-to-toxicity curve when PEG20ktTF-NGR was repeatedly applied, and thus, monoPEGylation on the N-terminus did not unequivocally improve the therapeutic activity and pharmacodynamic profile of tTF-NGR. While non-PEGylated tTF-NGR enters early clinical trials in cancer patients, combination of tumor vessel infarction with cytotoxic anticancer drugs, which can possibly be entrapped inside the tumor tissue by vascular occlusion, and the search for alternative targets, are among the strategies to improve on the anticancer profile of tTF-fusion proteins that we further study.

Figure 5. Comparative pharmacokinetic analysis of non-PEGylated versus mono- and random-PEGylated tTF-NGR. (A, B) Blood peak levels and wash-out kinetics upon 1 mg/kg bw intravenous (iv) injection of non-PEGylated (gray) versus mono- (light blue) or random- (dark blue) PEGylated tTF-NGR. Shown are mean values obtained from four or five 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) for non-PEGylated/mono-PEGylated/ random-PEGylated tTF-NGR: 10 min values, 750.7/1339.0/639.1; 2 h values, 45.0/411.3/283.7; 24 h values, 0.71/55.9/39.0; all in ng/mL. Half-life was about 7 min for non-PEGylated tTF-NGR and approximately 61 min for PEG20k-tTF-NGR and 57 min for TMS(PEG)12 tTF-NGR, respectively. (C) The surface integral (AUC) as measured until 24 h after injection was 4817 ng/(mL × h) for non-PEGylated, 71021 ng/(mL × h) for mono-PEGylated, and 82107 ng/(mL × h) for random-PEGylated tTF-NGR. Data of tTFNGR and TMS(PEG)12 tTF-NGR were published before24 and are reproduced with permission (Journal of Medicinal Chemistry, American Chemical Society).

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CONCLUSION

Site-directed mono-PEGylation of the N-terminus of retargeted truncated tissue factor tTF-NGR is feasible; PEG20k-tTF-NGR retains the procoagulatory activity of the tTF-NGR molecule and shows therapeutic antitumor efficacy in vivo, but lacks an improved pharmacokinetic behavior and anticancer profile when compared with the non-PEGylated molecule. Further experiments may test whether site-directed PEGylation with PEG molecules of different molecular mass or to alternative sites of the tTF-NGR molecule can improve results.

to aminopeptidase N (CD13), which is upregulated on tumor vessel endothelial cells and some tumor cells.2−4,8−16 Induction of coagulation in tumor vasculature with subsequent tumor infarction could be directly visualized by different imaging methods as being the mechanism underlying tumor growth G


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Figure 6. Antitumor activity and comparative tolerability of mono-PEGylated tTF-NGR. Tumor growth inhibiting effect of intravenous (iv) PEG20k-tTF-NGR in human HT1080 fibrosarcoma, xenotransplanted into athymic CD-1 mice. (A) 0.2 and 0.5 mg/kg bw iv every second day or (B) 0.2 mg/kg iv every second day and 0.2 and 0.3 mg/kg every fourth day. 0.9% saline was administered iv as control every second day. Arrows indicate the time points of injection. Standard error values expressed as range values for all days of observation in mm3: (A) 61.1−243.0 (control), 30.3−179.0 (0.2 mg/kg bw), 38.6−84.4 (0.5 mg/kg bw); (B) 23.8−84.2 (control), 24.6−198.1 (0.2 mg/kg bw every second day), 19.4−113.3 (0.2 mg/kg bw every fourth day), 14.9−96.7 (0.3 mg/kg every fourth day). Due to limiting toxicity, the 0.5 mg/kg treatment had to be stopped after the fourth administration. Treatment with a lower dose (0.2 or 0.3 mg/kg) showed a tumor growth-retarding effect as compared to the saline controls reaching statistical significance (p = 0.049, t test). (C) Acute lethality (24 h) of non-tumor-bearing CD-1 mice after one iv injection of nonPEGylated tTF-NGR versus mono- and random-PEGylated protein, respectively: The approximately LD10 value is 5 mg/kg bw for tTF-NGR, 7 mg/ kg for PEG20k-tTF-NGR, and was not reached for TMS(PEG)12 tTF-NGR. Data of tTF-NGR and TMS(PEG)12 tTF-NGR were published before24 and are reproduced with permission (Journal of Medicinal Chemistry, American Chemical Society).

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AUTHOR INFORMATION

Sybille-Hahne-Stiftung support the laboratory of W. E. Berdel. The model of PEG6k-tTF-NGR was kindly provided by Dr. Carsten Höltke, Institute of Clinical Radiology, University Hospital Münster.

Corresponding Authors

*Phone: + 49 251 83 56225. Fax: + 49 251 83 56709. E-mail: [email protected]. *Phone: + 49 251 83 47587. Fax: + 49 251 83 47588. E-mail: [email protected].

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ABBREVIATIONS USED APN, aminopeptidase N; AUC, area under the curve; BSA, bovine serum albumin; bw, body weight; CIEX, cation exchange chromatography; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; LD10, lethal dose for 10% of animals treated upon one iv injection and an observation period of 24 h; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; NGR, peptide GNGRAHA; PBS, phosphate-buffered saline; PEG, polyethylene glycol; PEG20k-tTF-NGR, PEG with a molecular weight of 20 kDa conjugated site-directed to the Nterminus of tTF-NGR; STR, short tandem repeat; tTF, truncated tissue factor; TMS(PEG)12, trimethyl succinimidyl polyethylene glycol ester; RT, room temperature

Author Contributions ∥

Wolfgang E. Berdel and Christian Schwöppe contributed equally and share senior authorship.

Notes

The authors declare the following competing financial interest(s): R. M. Mesters and W. E. Berdel 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 M.F. contributed experiments in partial fulfillment of the requirements to obtain an M.D. title. This work was supported by grants of the Else Kröner-Fresenius-Stiftung (2013_A284 to C. Schwöppe), the Deutsche Krebshilfe (110886 to W. E. Berdel). The Deutsche Forschungsgemeinschaft (DFG EXC1003 Excellence Cluster “Cells in Motion”) and the

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Tumor Growth Inhibition via Occlusion of Tumor Vasculature Induced

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