Heike Hintelmann

Generation of Fusion Proteins for Selective Occlusion of Tumor Vessels

Selective activation of blood coagulation in tumor vessels with subsequent thrombosis and tumor infarction is a promising strategy in cancer therapy. To this end, different fusion proteins consisting of the extracellular domain of tissue factor (truncated tissue factor, tTF) were fused to the peptides GRGDSP (abbr. RGD), GNGRAHA (abbr. NGR) or cyclic derivates of these peptides, which selectively target alpha(v)-integrins or aminopeptidase N (CD13), respectively. Rationale for this strategy is the fact that these surface receptors are preferentially expressed on tumor endothelial cells. The tTF constructs were expressed in Escherichia coli BL21 (DE3). The integrity of the fusion proteins was evaluated by SDS-PAGE, immunoblotting and mass spectrometry. The screening process for the activity contained coagulation assays as well as purified receptor binding assays. The fusion proteins which retained their thrombogenic and binding activity were evaluated further. In vivo studies in nude mice bearing established different malignant human tumors revealed that i.v. administration of tTF-RGD or tTF-NGR induced partial or complete thrombotic occlusion of tumor vessels, which was demonstrated by histological analysis. Furthermore, treatment studies showed that the targeted tTF fusion proteins but not untargeted tTF proteins induced significant tumor growth retardation in human adenocarcinoma of the breast in a nude mice model without apparent side effects such as thrombosis in liver, kidney, heart or lung at therapeutic dose levels. Finally, we illustrate the upscaling process of fusion protein fabrication in order to produce the amounts needed for clinical studies. Thus, generation and screening of active fusion proteins, which induce selective thrombosis in the tumor vasculature, may be a promising strategy for the development of new drugs as cancer therapeutics.

Tissue-factor fusion proteins induce occlusion of tumor vessels

A variety of fusion proteins consisting of the extracellular domain of tissue factor (truncated tissue factor, tTF) fused to the peptides GRGDSP (abbr. RGD), GNGRAHA (abbr. NGR) or derivates of these peptides, have been synthesized. These binding motif peptides target av-integrins or aminopeptidase N (CD13), respectively, on tumor endothelial cells. After expression and deposition as inclusion bodies in Escherichia coli BL21 (DE3), the tTF-fusion proteins were refolded and purified in a multi-step chromatography process. The upscaling process of fusion protein synthesis in order to produce amounts needed for clinical studies is presented. The proteins retained their specific proteolytic ability to activate FX by FVIIa and were able to bind to endothelial cells in vitro. Western blot analysis, analytic chromatography, FX coagulation assay and in vivo experiments have been performed to test for the in vitro stability of the tTF-NGR protein after long-term incubation at 5 degrees C or 25 degrees C, respectively. In vivo xenograft studies in nude mice bearing different malignant human tumors (mammary carcinoma SKBR3, adenocarcinoma of the lung A549) revealed that intravenous or subcutaneous administration of tTF-NGR or -RGD fusion proteins, but not the tTF protein without binding motif, induced thrombosis of tumor vessels which led to significant tumor growth retardation or regression. The anti-vascular mechanism of the tTF fusion proteins was verified by the molecular imaging methods such as magnetic resonance imaging (MRI) and fluorescence reflectance imaging (FRI); MRI showed a reduction of the relative tumor blood volume (BV) and FRI the formation of fibrin in the tTF-fusion protein treated tumors.

NG2 proteoglycan as a pericyte target for anticancer therapy by tumor vessel infarction with retargeted tissue factor

tTF-TAA and tTF-LTL are fusion proteins consisting of the extracellular domain of tissue factor (TF) and the peptides TAASGVRSMH and LTLRWVGLMS, respectively. These peptides represent ligands of NG2, a surface proteoglycan expressed on angiogenic pericytes and some tumor cells. Here we have expressed the model compound tTF-NGR, tTF-TAA, and tTF-LTL with different lengths in the TF domain in E. coli and used these fusion proteins for functional studies in anticancer therapy. We aimed to retarget TF to tumor vessels leading to tumor vessel infarction with two barriers of selectivity, a) the leaky endothelial lining in tumor vessels with the target NG2 being expressed on pericytes on the abluminal side of the endothelial cell barrier and b) the preferential expression of NG2 on angiogenic vessels such as in tumors. Chromatography-purified tTF-TAA showed identical Factor X (FX)-activating procoagulatory activity as the model compound tTF-NGR with Km values of approx. 0.15 nM in Michaelis-Menten kinetics. The procoagulatory activity of tTF-LTL varied with the chosen length of the TF part of the fusion protein. Flow cytometry revealed specific binding of tTF-TAA to NG2-expressing pericytes and tumor cells with low affinity and dissociation KD in the high nM range. In vivo and ex vivo fluorescence imaging of tumor xenograft-carrying animals and of the explanted tumors showed reduction of tumor blood flow upon tTF-TAA application. Therapeutic experiments showed a reproducible antitumor activity of tTF-TAA against NG2-expressing A549-tumor xenografts, however, with a rather small therapeutic window (active/toxic dose in mg/kg body weight).

Combinatorial effects of doxorubicin and retargeted tissue factor by intratumoral entrapment of doxorubicin and proapoptotic increase of tumor vascular infarction

Truncated tissue factor (tTF), retargeted to tumor vasculature by GNGRAHA peptide (tTF-NGR), and doxorubicin have therapeutic activity against a variety of tumors. We report on combination experiments of both drugs using different schedules. We have tested fluorescence- and HPLC-based intratumoral pharmacokinetics of doxorubicin, flow cytometry for cellular phosphatidylserine (PS) expression, and tumor xenograft studies for showing in vivo apoptosis, proliferation decrease, and tumor shrinkage upon combination therapy with doxorubicin and induced tumor vascular infarction. tTF-NGR given before doxorubicin inhibits the uptake of the drug into human fibrosarcoma xenografts in vivo. Reverse sequence does not influence the uptake of doxorubicin into tumor, but significantly inhibits the late wash-out phase, thus entrapping doxorubicin in tumor tissue by vascular occlusion. Incubation of endothelial and tumor cells with doxorubicin in vitro increases PS concentrations in the outer layer of the cell membrane as a sign of early apoptosis. Cells expressing increased PS concentrations show comparatively higher procoagulatory efficacy on the basis of equimolar tTF-NGR present in the Factor X assay. Experiments using human M21 melanoma and HT1080 fibrosarcoma xenografts in athymic nude mice indeed show a combinatorial tumor growth inhibition applying doxorubicin and tTF-NGR in sequence over single drug treatment. Combination of cytotoxic drugs such as doxorubicin with tTF-NGR-induced tumor vessel infarction can improve pharmacodynamics of the drugs by new mechanisms, entrapping a cytotoxic molecule inside tumor tissue and reciprocally improving procoagulatory activity of tTF-NGR in the tumor vasculature via apoptosis induction in tumor endothelial and tumor cells.

Low-Energy Ultrasound Treatment Improves Regional Tumor Vessel Infarction by Retargeted Tissue Factor.

To enhance the regional antitumor activity of the vascular‐targeting agent truncated tissue factor (tTF)‐NGR by combining the therapy with low‐energy ultrasound (US) treatment.