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.

tTF-NGR retargets the extracellular domain of tissue factor via a C-terminal 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 N-terminus 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 PEG20k-tTF-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.

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