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.
Department of Medicine / Hematology and Oncology, University of Muenster, Albert-Schweitzer-Strasse 33, D-48129 Muenster, Germany.
Torsten
T
Kessler
Christian
C
Schwöppe
Ruediger
R
Liersch
Christoph
C
Schliemann
Heike
H
Hintelmann
Ralf
R
Bieker
Wolfgang E
WE
Berdel
Rolf M
RM
Mesters
D000230:Adenocarcinoma / Q000098:blood supply / Q000188:drug therapy; D020533:Angiogenesis Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D001943:Breast Neoplasms / Q000098:blood supply / Q000188:drug therapy; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000187:drug effects; D005170:Factor X / Q000378:metabolism; D005260:Female; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000235:genetics; D016023:Integrins / Q000235:genetics; D008546:Melanoma, Experimental / Q000098:blood supply / Q000188:drug therapy; D051379:Mice; D009368:Neoplasm Transplantation; D009374:Neoplasms, Experimental / Q000098:blood supply* / Q000188:drug therapy*; D019030:Receptors, Vitronectin / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology*; D012039:Regional Blood Flow; D019809:Streptavidin / Q000494:pharmacology
D020533:Angiogenesis Inhibitors; D039302:Integrin alphaVbeta3; D016023:Integrins; D019030:Receptors, Vitronectin; D011993:Recombinant Fusion Proteins; C064660:integrin alphaVbeta5; D005170:Factor X; D019809:Streptavidin