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.

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).

Tumor Growth Inhibition via Occlusion of Tumor Vasculature Induced by N-Terminally PEGylated Retargeted Tissue Factor 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 αvß3, 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.