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.

The fusion protein tTF-NGR consists of the extracellular domain of the thrombogenic human tissue factor (truncated tissue factor, tTF) and the peptide GNGRAHA (NGR), a ligand of the surface protein CD13 (aminopeptidase N), upregulated on endothelial cells of tumor vessels. tTF-NGR preferentially activates blood coagulation within tumor vasculature, resulting in tumor vessel infarction and subsequent tumor growth retardation/regression. The anti-vascular mechanism of the tTF-NGR therapy approach was verified by quantifying the reduced tumor blood-perfusion with contrast-enhanced ultrasound, the reduced relative tumor blood volume by ultrasmall superparamagnetic iron oxide-enhanced magnetic resonance imaging, and by in vivo-evaluation of hemorrhagic bleeding with fluorescent biomarkers (AngioSense(680)) in fluorescence reflectance imaging. The accumulation of tTF-NGR within the tumor was proven by visualizing the distribution of the iodine-123-labelled protein by single-photon emission computed tomography. Use of these multi-modal vascular and molecular imaging tools helped to assess the therapeutic effect even at real time and to detect non-responding tumors directly after the first tTF-NGR treatment. This emphasizes the importance of imaging within clinical studies with tTF-NGR. The imaging techniques as used here have applicability within a wider scope of therapeutic regimes interfering with tumor vasculature. Some even are useful to obtain predictive biosignals in personalized cancer treatment.

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Retargeted tissue factor can induce tumor vessel infarction as a new approach for tumor therapy via vascular targeting. To optimize this anti-vascular approach with retargeted tissue factor (tTF) and to simplify the characterization and batch-to-batch reproducibility, single polyethylene glycol (PEG) units were site-specifically linked to tTF proteins such as tTF-NGR and compared to randomly PEGylated tTF derivatives. Experimental procedures Site-directed (SD) coupling of PEG units (4 and 20 kDa, respectively) to the N-terminus of recombinant tTF-fusion proteins was performed by reductive alkylation according to the PEGylation of granulocyte-colony stimulating factor G-CSF (Kinstler et al., 1996 & 2002). Random PEGylation was accomplished by nucleophilic substitution of short, branched PEG units (2.4 kDa each) to primary amines such as lysine residues. Successful coupling and purification steps were verified by HPLC, SDS-PAGE, Western blotting and mass spectrometry. The biological ability of PEGylated tTF derivatives to induce coagulation was assessed by FX-activation assay according to Ruf et al. (1991). Pharmacokinetic analyses were performed with blood probes from CD-1 mice after intravenous application of the PEG-tTF proteins, using a tissue factor ELISA kit. For in vivo evaluation of tumor growth, immunodeficient CD-1 nude mice were xenotransplanted with human tumor cells; tolerability studies were carried out with non-tumor-bearing mice. Histological analyses of tumor tissues and normal organs were performed according to standard protocols using an anti-PEG antibody. The anti-vascular mechanism was further verified by molecular imaging methods such as MRI. Results SD-PEGylation revealed mono-PEGylated tTF proteins, clearly separable from non-PEGylated tTF by using cation-exchange HPLC, leading to a homogeneous protein solution with a high batch-to-batch reproducibility. The ability of the SD-PEGylated tTF-NGR to induce coagulation within the FX-activation assay was barely affected in comparison to the non-PEGylated tTF-NGR protein, while the pharmacokinetic profile of the mono-PEGylated tTF-NGR resembles the profile of the randomly PEGylated protein (area under the curve was increased more than 1-log step). In vivo studies of mono-PEGylated tTF-NGR revealed a markedly reduced effective dose for tumor growth inhibition compared to the non-PEGylated protein (0.2 vs. 1 mg/kg bw). Tolerability and molecular imaging studies are ongoing to study in vivo activity/safety of mono-PEGylated tTF-NGR. Conclusion Promising results have been achieved to 1. simplify the characterization and batch-to-batch reproducibility and 2. to optimize the activity/toxicity profile of tumor-vessel infarction by retargeted tTF. The therapeutic range of tTF-NGR fusion protein can be improved by using SD- and random-PEGylation techniques, respectively. Citation Format: Caroline Zerbst, Janine Ring, Max Fröhlich, Christoph Schliemann, Rolf M. Mesters, Wolfgang E. Berdel, Christian Schwöppe. Site-directed and random PEGylation of retargeted tissue factor can improve the activity/toxicity profile of the molecule. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2629. doi:10.1158/1538-7445.AM2015-2629

The purpose of this study is to improve the safety and activity profile of therapeutic tumor vascular infarction using retargeted tissue factor-(tTF-) by in vitro- and in-vivo analysis of 1. newly generated tTF fusion proteins, 2. site-specific and randomly PEGylated tTF-NGR protein, 3. combination therapy with radiation, cytotoxics, low-energy ultrasound, and by 4. using different routes of application. Experimental procedures Molecular cloning strategies and site-directed mutagenesis were used to generate new tTF constructs. Coupling of polyethylene glycol (PEG) units to the recombinant tTF proteins was performed by site-directed and random PEGylation and coupling of non-peptidic ligands was performed by maleimide-activated reagents. The biological ability of the tTF-fusion proteins to induce coagulation was assessed by a FX assay. The differential binding of fusion proteins to HUVECs and HuAoSMCs was analyzed by FACS. For the in-vivo evaluation of tumor growth, immunodeficient CD-1 nude mice were transplanted with various human tumor xenotransplants. The anti-vascular mechanism was verified by the molecular imaging methods SPECT, MRI, FRI, and CEUS. Results To improve the specificity of the therapy, novel fusion proteins were constructed which are supposed to selectively target the receptor protein NG2 on tumor vessel pericytes. The newly synthesized tTF-TAA/-LTL fusion proteins retained their thrombogenic activity and specific binding to the pericyte marker NG2 in vitro; first therapeutic trials with tumor-bearing mice revealed a comparatively small therapeutic range. The conjugation of tTF-NGR with low-molecular PEG-chains improved the therapeutic range of the tTF-fusion protein substantially, together with increased in-vivo tolerance. Moreover, site-directed PEGylated of cysteinated tTF molecules were constructed and revealed promising analytical and in-vitro thrombogenic effects. Cysteinated tTF (tTF-C) acts as fast and convenient “building block” for non-peptidic ligands of receptor proteins overexpressed in the tumor vasculature (such as CD13, αV-integrins etc.). Combination therapies of tTF-NGR with doxorubicin, irradiation, or low-energy-ultrasound, respectively, showed synergistic effects. Conclusion Promising results have been achieved to optimize the anticancer profile of tumor-vessel infarction by retargeted tTF: The therapeutic range of the tTF-NGR fusion protein has been improved by PEGylation and by combination with either radiation, chemotherapy with doxorubicin, or low-energy ultrasound, respectively. Ongoing experiments further optimize this anti-vascular therapy by using new (PEG) polymers. Citation Format: Christian Schwöppe, Caroline Zerbst, Christoph Schliemann, Rolf M. Mesters, Wolfgang E. Berdel. Optimization of antivascular tumor therapy with retargeted tissue factor proteins to improve the activity/toxicity profiles and therapeutic outcome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2675. doi:10.1158/1538-7445.AM2014-2675