Abstract 2629: Site-directed and random PEGylation of retargeted tissue factor can improve the activity/toxicity profile of the molecule
Authors: Zerbst Caroline , Ring Janine , Fröhlich Max , Schliemann Christoph , Mesters Rolf M. , Berdel Wolfgang E. , Schwöppe Christian 
Published: August 2015

Abstract
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