Abstract
Abstract 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
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