HK1087418B - Fusion polypeptides, and use thereof in antivascular tumor therapy - Google Patents

Description

The present invention relates to fusion polypeptides composed of at least two peptides. One peptide consists of 3 to 30 amino acids and allows the fusion polypeptide to bind selectively to endothelial cells in tumour vessels. The other peptide consists of tissue factor TF or a fragment thereof, the tissue factor and fragment being characterised by the ability to activate blood clotting when the fusion polypeptide binds to endothelial cells in tumour vessels. The peptides may be linked together either directly or via a linker with up to 15 amino acids. The invention also relates to the use of these fusion proteins in the treatment of tumour diseases and their use in the manufacture of drugs for the treatment of tumour diseases.

Background to the invention

Adequate neovascularization is a prerequisite for progressive tumour growth (1). Neoangiogenesis is particularly necessary for maintaining expansive tumour growth, as it is the only way to ensure adequate oxygenation, the supply of nutrients to the tumour and the transport of tumour breakdown products.

Therefore, in order to combat tumours, in addition to antiangiogenic therapeutic strategies, which intervene in the complex process of blood vessel growth and differentiation, antivascular therapeutic strategies have been developed in the state of the art, which aim at destroying tumour blood vessels and a related tumour infarction.

These strategies require the identification of target structures in the tumor's vascular endothelial endothelium that are not found on resting endothelial cells in normal tissue. Such specific target structures could be used to deliver cytostatics or certain toxins to the tumor's vascular endothelial cells and less to the tumor cells themselves. Target structures that could be used for this purpose are bFGF (basic fibroblast growth factor), VEGF (vascular endothelial growth factor) and VEGF receptor 2 (VEGFR-2), endoglin, endosialin, a fibronectin isoform (ED-B), the integrin αβ3, αβv1, αβv5, αβ1 and αβ12, the matrix aminopeptidase, which was used for the production of the protein n-GMP2 (e.g. methotropeptide 9 and methotropeptide 9), and the enhancement of the action of the protein in the animal (2, 9) and the combination of the proteins n-GMP2 and n-GMP2 (e.g.

An alternative antivascular therapy approach is to selectively activate blood clotting in tumor vessels to induce tumor necrosis. For example, a bispecific F ((ab') 2) antibody fragment was generated that targets truncated tissue factor (tTF) and an MHC class II antigen. Following experimental induction of the antigen in tumor endothelial cells, an antivascular therapy by administration of the antibody in a murine neuroblastoma model was demonstrated (14 and 47). A second study in the same group used an immune conjugate that coupled tTF to a naturally occurring marker of tumor endothelial adhesion molecule, VAMC-1 (C-1), a tumor adhesion cell (15).

In a very similar approach, an antibody fragment (scFv) specific to the oncopetal ED-B domain was fused with tTF. The fusion proteins generated, scFv-tTF, resulted in complete and selective infarction in various tumours in the mouse model (16).

Alternatively, tTF was coupled to an inhibitor of prostate-specific membrane antigen (17). This fusion protein induced selective infarct necrosis in a rat prostate model following intravenous administration. Administration of this fusion protein in combination with a low-dose cytotoxic agent (doxorubicin) led to massive tumour regression up to complete tumour eradication (17). Other tTF fusion proteins consisting of antibody fragments against VEGFR, endoglin and VCAM-1 have been recently described (18).

However, the molecules produced in the state of the art for the anti-vascular tumor therapy have disadvantages, in particular the assumption that these molecules are immunogenic due to their size, and therefore the treatment of mammals with these molecules will trigger an immune reaction against the molecules, making it impossible to repeat the administration of the molecules.

The size of the coupling partner, by which the peptide component that can activate blood clotting is to be directed to the tumour tissue, may also sterically inhibit the formation of the macromolecular enzyme and substrate complex factor VIIa/FX, which is essential for blood clotting.

As of now (WO 03/035688), fusion polypeptides are still known in which a selective binding domain, e.g. an integrin binding domain from fibronectin, which includes e.g. RGD peptides, or the dipeptide D-β-E, which binds to PSMA (prostate specific membrane antigen), is coupled to the N-terminus of a tissue factor polypeptide. Although amyloidolytic and proteolytic effects were demonstrated in vitro, the constructs in vivo, even in combination with factor VIIa, showed only extremely weak anti-tumor effects.

Hu et al. (46) describe various fusion proteins and their use to generate thromboses in tumour vessels, including a fusion protein from a 9-amino acid oligopeptide containing the sequence RGD coupled to the shortened form of tissue factor. Again, the RGD peptides were linked to the N-terminus from tTF to RGD-tTF. Functional analyses showed that the fusion protein containing RGD did not cause significant inhibition of tumour growth.

The currently known constructs were therefore constructed to link the selective binding domain to the N-terminus of the tissue factor polypeptide, and it was even emphasized that this structure had to be chosen because the N-terminus was a particularly favourable site for a linkage which did not inhibit the initiation of thrombosis due to structural models.

Summary of the invention

The challenge, therefore, is to provide alternative thrombogenic agents that can effectively inhibit tumour growth in vivo.

This problem has now been solved by fusion polypeptides, which contain a peptide of 3-30 amino acids that allows the fusion polypeptide to bind selectively to tumour vascular endothelial cells and tissue factor TF or a fragment thereof, the tissue factor and fragment being characterised by the ability to activate blood clotting when the fusion polypeptide binds to tumour vascular endothelial cells, these peptides being coupled either directly or via a linker of up to 15 amino acids, the peptide containing a selective binding of the fusion polypeptide to tumour cells, which allows the activation of the blood polypeptide, which may be used in the treatment of tumour tumours, and the peptide containing a selective binding of the fusion polypeptide to tumour tumour cells, which may be used in the treatment of tumour tumours, as described in the present article.

Description of the figures

Err1:Expecting ',' delimiter: line 1 column 789 (char 788)The identity of the proteins was verified by Western blot using a monoclonal anti-tissue factor antibody (clone VIC7, American Diagnostics). Occupancy of the individual pathways: 1=tTF; 2=tTF-RGD; 3=tTF-NGR; 4=tTF-cycloNGR1; 5=tTF-cycloNGR2; 6=tTF-cycloNGR3; 7=tTF-GALNGRSHAG; M= molecular weight marker.Fig.3: Determination of the Michael constants (Km) for the diagnosis of FX by FVISAa/tTF1-218 and FVIIa/tTFTF1-218 fusion proteins respectively. The results of the Michaelis antibody particle quarantaine were calculated by the following method (4b): BTF-RGD was quantified in a range between 0,1 μg and tTF-RGD. The results of the BTF-RGD were calculated by the BTF-RGD. The BTF-RGD was quantified in a range between tTF-RGD and tTF-RGD. The BTF-RGD was activated by a tTF-RGD and tTF-RGD. The results were quantified in a range of 0,1 μg and tTF-RGD.The binding of tTF-RGD (0.1μM) to immobilized αvβ3 was significantly inhibited by competitive inhibition with the synthetic peptide GRGDSP (1-10μM) (p<0.001, Mann-Whitney test for both RGD peptide concentrations).Fig.6:Binding of tTF-RGD to endothelial cells.A: FACS analysis of endothelial cells with tTF-RGD (0.1μM (2) or 0.1μM tTF-RGD (3) incubated for 60 min. at 4°C. Reduction of the competition between the inhibition of TTF-RGD and the negative control was demonstrated in 75% of the BDSP.Fig.7:Inhibition of human lung cancer (CCL185) growing as a xenotransplant in thymus-free naked mice by intravenous therapy with tTF fusion proteins (tTF-RGD, n=6; tTF-NGR, n=6) compared to tumour growth by infusion of physiological saline (NaCl, n=8) or tTF (n=1).The vertical arrows indicate the times of injections with the respective substances.Fig.8:Inhibition and partial sub-remission of human lung cancer growing as a xenotransplant in thymus-free naked mice by intravenous therapy with malignant melanoma (MTF21) by infusion of tTF-Fusion proteins (TTF-GD, n=3; tTF-N=3) compared to the time of infusion of a physiological tumor (TTF21, tTF23, tTF23, tTF3 or tTF4) The growth points of the tumour growths in vivo are shown in the following table:Injecting the tTF-NGR fusion protein (A, left half of the image) or NaCl (A, right half of the image). The macroscopic image with a bluish-livid discoloration of the tumor after injection of tTF-NGR indicates tumor necrosis. After 60 min. both mice were exsanguinated, the tumor was exsanguinated in toto and examined histologically. In B, hemorrhagic imbibition of the tumor treated with tTF-NGR is visible as a sign of secondary hemorrhage due to the onset of the tumor. In contrast, the tumor treated with NaCl appears to be vital (C).F.10: Histology of the T-cell tumor 1 hour after intravenous injection of tTF-GD (A), T-GD (C), and T-GD (F), and the contrast between the T-cell and T-cell in the tumor (FGD) and the tumor (FGD) and the tumor (FGD) is seen in the contrast.In the area of supply of the vessel closed by a blood clot, extensive tumour necrosis can be observed (A-D). Representative areas of the tumours were photographed (A, C and E: 200-fold enlargement, B, D and F: 400-fold enlargement); HE staining (staining e.g. described in H.C. Burck, Histological Techniques - Guidelines for the Understanding of Microscopic Preparations in the Design and Practice of Microscopic Preparations, 5th Edition, Thiogem Verlag, Stuttgart, 1982, pp. 109 ff.F.11: Representative histologies of the heart (A), liver (B), kidney (C) and liver (D) were not detectable after 1 hour of injection of L-GNG or 4 mg/kg of this thrombosis.(HE staining; 200-fold magnification).Fig.12:Amino acid sequence of the human tissue factor (TFG) (also referred to as tTFNG-NGR).Fig.13:Amino acid sequence of the truncated human tissue factor (tTF1-218 (also referred to as tTFTF for the purposes of this application).Fig.14:Amino acid sequence of the fusion polypeptide (tTF-GRGDSP) (also referred to as tTF-RGD for the purposes of this application).Fig.15:Amino acid sequence of the fusion polypeptide (tTF-GNGRAHA) (also referred to as tTF-NGR).Fig.16:Amino acid sequence of the truncated human tissue factor (tTFTF1-218 (also referred to as tTFTF-GNGR for the purposes of this application).Fig.17:Amino acid sequence of the fusion polypeptide (tTF-GRGDSP) (also referred to as tTF-GRNGR).Fig.15:Amino acid sequence of the fusion polypeptide (tTF-GNGRGD) (also referred to as FGNGRGD) (also referred to as the human tTF-NGR).Fig.20:FGNGRGNGR2 (also called the tTF-NGR-NGR).The following is the nucleotide sequence of the fusion polypeptide tTF-GCNGNGRCG (also referred to as tTF-cycloNGR1).Fig.25:Nucleotide sequence of the fusion polypeptide tTF-GCNGRCVSGCAGRC (also referred to as tTF-cycloNGR2).Fig.26:Nucleotide sequence of the fusion polypeptide tTF-GCVLNGRMEC (also referred to as tTF-cycloNGR3).Fig.27:Nucleotide sequence of the Oligonucleotide Therapy to produce tTF-GCNGR1 (also referred to as tTF-GCNGR1 for short).Fig.25:Nucleotide sequence of the fusion polypeptide tTF-GCNGRCVSGCAGRC (also referred to as tTF-GCNGRCVSGCAGRC for short).Fig.26:Nucleotide sequence of the fusion polypeptide tTF-GCNGR2 (also referred to as tTF-GCNGR2 for short).Fig.26:Nucleotide sequence of the fusion polypeptide tTF-GCVLNGRMEC (also referred to as tTF-GCNGR3 for short).Fig.26:Nucleotide sequence of the fusion polypeptide tTF-GCVLNGRMEC (also referred to as tTF-GCNGR2 for short).Fig.30:Oligonucleotide Oligonucleotide B.Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:Oligon:OThe vertical arrows indicate the times of injections of the respective substances. b: Inhibition of human fibrosarcoma growing as a xenotransplant in thymus-free naked mice (HT1080) by intravenous therapy with t-fusion proteins (tTF-RGD, n=12) compared to tumor growth in physiological saline infusion (NaCl, n=15) or tTF (n=11).The statistical significance was assessed using the Mann-Whitney test for independent groups, where P values below 0.05 were considered significant. * indicates the statistical significance of the difference between tTF-RGD and buffer.Fig.35:Macroscopic image of a mouse carrying an M21 tumour after the end of treatment (day 7) with tTF-RGD fusion protein (A, C) or NaCl (B, D).The difference in size and appearance of the tTF-RGD treated tumours, as opposed to apparently clear signs of tumour necrosis, is clearly visible in the tumour, and is clearly detectable in the tumour control cells treated with TTF-RGD.B: 400x). Arrows show examples of thrombosis in blood vessels of the tumor. In animals treated with saline, no apparent thrombosis or necrosis occurs (C:200x, D: 400x). Arrows show intact blood vessels of the tumor with some erythrocytes. Heart (E), lung (F), liver (G), and kidney of animals treated with tTF-RGD showed no visible thrombosis or necrosis.Fig.37:Effects of tTF-NGR in a fibrosar model of fibrosar (HT1080) mice carrying were examined without (pre-tTF-NGR) or 6 hours after (compost-tTF-NGR) i.v. of tTF-NGR by MRI.

Detailed description of the invention

These state-of-the-art problems have now been solved by fusion polypeptides, which include the following peptides: (b) tissue factor TF or a fragment thereof, the tissue factor and the fragment being characterised by the ability to activate blood clotting when the fusion polypeptide is bound to tumour vascular endothelial cells; wherein the peptides (a) and (b) are either directly or via a linker of up to 15 amino acids coupled and the peptide allowing selective binding of the fusion polypeptide to tumor vascular endothelial cells is coupled to the C-terminus of the peptide which can activate blood clotting when the fusion polypeptide binds to tumor vascular endothelial cells.

The fusion polypeptides of the invention may include sequences other than the sequences (a) and (b) provided that they do not affect the steric conformation of the fusion polypeptide and do not prevent the formation of the enzyme and substrate complex that causes blood clotting. For example, the fusion polypeptides of the invention may contain the sequences of a His-tag that facilitates the recombinant expression and purification of the peptide (see examples). However, the presence of these sequences is not necessary. According to a preferred embodiment of the invention, the fusion polypeptide therefore consists of: (a) a peptide of 3 to 30 amino acids which allows the fusion polypeptide to bind selectively to tumour vascular endothelial cells; and (b) tissue factor TF or a fragment thereof, the tissue factor and the fragment being so designated;that they can activate blood clotting when the fusion polypeptide binds to tumour vascular endothelial cells, wherein the peptides (a) and (b) are either directly or through a linker of up to 15 amino acids linked together. (b) tissue factor TF or a fragment thereof, the tissue factor and the fragment being characterised by the ability to activate blood clotting when the fusion polypeptide is bound to tumour vascular endothelial cells; wherein the peptides (a) and (b) are coupled.

According to the invention, it was thus surprisingly shown that fusion polypeptides from a particularly small peptide, which allows a selective binding of the fusion polypeptide to tumor vascular endothelial cells, and a peptide, which can activate blood clotting when binding the fusion polypeptide to tumor vascular endothelial cells, for which anti-vascular tumor therapy is particularly beneficial, can be shown to be of interest. The small size of the polypeptide, which allows binding to the tumor vascular endothelial cells, improves the orientation of the fusion protein phospholipid membrane of the terminal cell. The formation of the enzyme/substrate complex essential for blood clotting is not inhibited and the activation of the blood binding factor TF cannot be prevented, which causes a change in the blood formation.

A preferred embodiment of the present invention is the peptide that can activate blood clotting when the fusion polypeptide binds to tumor vascular endothelial cells, the tissue factor TF, with the amino acid sequence shown in SEQ ID NO:1 (Fig. 12). The invention also includes tissue factor sequences that have an amino acid homology of at least 70% or at least 80% to the SEQ ID NO:1 (Fig. 12), with sequences with a homology of at least 95% being particularly preferred. The degree of homology is determined by superimposing the two sequences, with gaps of four 100 amino acids in length, to achieve the closest possible correspondence.

The peptide that can activate blood clotting in tumor vessels when the fusion polypeptide binds to tumor vessel endothelial cells may also be a fragment of tissue factor TF or a fragment of a sequence homologous to TF. Preferably the fragment has the sequence shown in SEQ ID NO:2 (Fig. 13). The sequence shown in SEQ ID NO:2 (Fig. 13) (tTF1-218 or tTF for short) includes the N-terminals of 218 amino acids of TF. Furthermore, according to the invention, Fragments of tTF may be used in which tTF is missing up to several amino acids at the N-terminus or C-terminus. For example, Fragments of TTF may be used up to 10 amino acids at the N-terminus (Fig. 11-214), whereas Fragments of TTF may be missing up to 8 amino acids at the C-terminus (Fig. 11-214), whereas Fragments of TTF may be used up to 10 amino acids at the N-terminus (Fig. 11-214).

Err1:Expecting ',' delimiter: line 1 column 286 (char 285)

The peptide that allows the fusion polypeptide to bind selectively to tumor vascular endothelial cells can be any peptide with a length of 3-30 amino acids and binds tumor vascular endothelial cells with high specificity.

In accordance with one embodiment of the present invention, the peptides which enable the fusion polypeptide to bind selectively to tumor vascular endothelial cells include the amino acid sequence RGD or NGR. Both sequences were at the state of the art for their specific binding to integrins, in particular αvβ3 and αvβ5 integrins (RGD peptides), and are known as cell adhesion motifs (NGR peptides) (see (8)).

In particular, fusion polypeptides comprising these sequences and the sequence of the first 218 amino acids of human TF have been shown to be highly suitable for antitumor anti-vascular therapy. In particular, these fusion polypeptides have been shown to significantly reduce tumour growth or tumour size (see Figures 7 and 8). The induction of a large part of tumour hemorrhage (see Figures 8) is observed due to the high prognosis of positive results expected in human tumour therapy (42, 43, 44).

The invention also covers fusion proteins with cyclic RGD peptides, as cycling increases the affinity for integrins (as described, for example, in Publication 21).

The present invention also relates to fusion polypeptides having one of the sequences shown in SEQ ID NO:3-8 (Fig. 14-19).

In another embodiment, the present invention relates to nucleic acids that code for a fusion polypeptide as described above, e.g. corresponding nucleic acids may have one of the sequences shown in SEQ ID NO: 10-15 (Fig. 21-26).

In another respect, the present invention relates to vectors which include one of the nucleic acids mentioned above. Corresponding vectors usually also include regulatory sequences for the expression of the nucleic acid. Such vectors are comprehensively described in the state of the art and can be commercially acquired by a wide variety of companies.

In another embodiment, the present invention relates to cells containing one of the nucleic acids or vectors described. The cells are commonly used for nucleic acid expression and the recombinant production of the fusion polypeptide of the invention. A variety of cells can be used for this purpose, including E. coli, yeast cells and animal cell lines such as CHO or COS cells. The corresponding cells and their use are described in detail in the state of the art.

The polypeptides of the invention described in claim 1 can also be produced by other suitable methods, such as chemical coupling of individual peptides, so that individual peptides can be produced by techniques currently used, such as chemical synthesis or heterologous expression, and then joined together by coupling.

Finally, the present invention also applies to medicinal products which include the fusion polypeptides, nucleic acids, vectors or cells described above. The medicinal products may also include pharmaceutically compatible carriers, excipients or adjuvants. Furthermore, the polypeptides in such a medicinal product may be present in a modified state, e.g. pegylated, i.e. coupled to a polyethylene glycol molecule.

The fusion polypeptides of the invention or the medicinal products containing them are used for the treatment of tumour diseases, in particular for the purpose of anti-vascular tumour therapy. Tumor diseases which can be treated with the fusion polypeptides of the invention or with medicinal products containing these fusion polypeptides include, for example, bronchial carcinomas and other tumours of the thorax and mediastinum, breast cancers and other gynaecological tumours, colorectal cancers, gastrointestinal cancers and other tumours, malignant melanoma and other tumours of the skin, head and neck cancers, promotromycin and other tumours, sarcomas, endogenous cancers, myelogenous cancers and myelogenous cancers, and non-melodic cancers in the head and neck, prostagland and other tumors, sarcomas, endogenous cancers, myelogenous cancers and myelogenous cancers in the lymphoma.

Benign tumours, such as haemangiomas, and the re-growth of blood vessels in diabetic retinopathy can also be treated.

In addition to intravenous administration, subcutaneous and intraperitoneal administration of the fusion polypeptides or medicinal products is also possible.

A combination of administration of the fusion polypeptides of the invention with other therapeutic approaches, e.g. cytotoxic chemotherapy or radiation, may still be advantageous.

The invention is described in more detail by the following examples:

Examples Example 1: Expression and purification of tTF and tTF fusion proteins

The cDNA encoding the N-terminal 218 amino acids of tissue factor TF (hereinafter tTF) was synthesised by polymerase chain reaction (PCR) using the expression vector pET-30a (Novagen) and cloned in the expression vector pET-30a (Novagen) using the techniques in SEQ ID NO:16 and SEQ ID NO:17 (Figure 27), and the recombinant plasmids were transformed, expressed and purified in E. coli (BL21) using the Qiagen Plasmid Kit.

In addition to the truncated tissue factor tTF, tTF peptide fusion proteins have been constructed in which the targeting peptides are first bound to the carboxyterminal end of the soluble tissue factor tTF. The test chemical is used to determine the concentration of the test chemical in the test chemical and the concentration of the test chemical in the test chemical.

In addition, the following cyclic fusion proteins have been synthesised: The test chemical is a mixture of two or more of the following: tTF-GCNGRCG (SEQ ID NO:6; Fig.17; hereinafter referred to as tTF-cycloNGR1; the PCR primers SEQ ID NO:22 and SEQ ID NO:23 (Fig.30) were used);tTF-GCNGRCVSGCAGRC (SEQ ID NO:7; Fig.18; hereinafter referred to as tTF-cycloNGR2; the PCR primers SEQ ID NO:24 and SEQ ID NO:25 (Fig.31) were used);tTF-GCVLNGRMEC (SEQ ID NO:8; Fig.19; hereinafter referred to as tTF-cycloNGR3; the PCR primers SEQ ID NO:26 and SEQ ID NO:27 (Fig.32) were used)

Err1:Expecting ',' delimiter: line 1 column 173 (char 172)

The constructs were selected so that, based on the known X-ray crystal structure of the tTF:FVIIa complex (19), a vertical orientation of the tTF fusion protein to the phospholipid membrane of the endothelial cell is ensured, corresponding to the orientation of the native TF. On the other hand, it was considered that the chosen structure should not create a steric impediment of tTF to interact with FVIIa and the macromolecular substrate FX. The specificity of the RGD sequence for the αvβ3 integrin and the NGR sequence for CD13 (aminopeptidase N) provides tumor selectivity, as these receptors are selective and specifically expressed in high-end tumour cells, but with few exceptions, not in the resting tumour (Figure 1).

Err1:Expecting ',' delimiter: line 1 column 706 (char 705)

SDS-PAGE, Western blot and mass spectroscopy analyses confirmed the identity of the proteins (see Figure 2).

Example 2: Functional characterisation of tTF and tTF fusion proteins

The functional activity of these fusion proteins in terms of cofactor activity in activation of factor X to factor Xa by factor Vlla was demonstrated in vitro by Michaelis-Menten analyses. The ability of tTF and tTF fusion polypeptides to enhance the specific proteolytic activation of FX by FVIIa in the presence of phospholipids was determined in a slight modification according to the method described by Ruf (45). For this purpose, 20 μl of each of the following reagents were pipetted in microtiter plates: (a) 50 nM recombinant FVIIa (Novo-Nisk) in TBS-BSA; (b) 0.16 nM - 1.6 μM Ca/tTF-F polypeptide in TBS-BSA; 25 μm/m2 phosphatidylserine (Mphospholipid) in TBS-BSA; and (c) 500 μm/m2 phosphatidylserine (Mphospholipid) in TBS-BSA.After 10 min incubation at room temperature, 20 μl of natural substrate FX (Enzyme Research Laboratories) were added at a concentration of 5 μM. A sample was then pipetted at minute intervals and the reaction was stopped by adding 100 mM EDTA solution. The amount of FXa formed was measured by adding the chromogenic substrate Spectrozym FXa in a microplate reader by determining the absorption change at 405 nm and the parameters for Michaelis-Menten kinetics were analysed using the method indicated by Ruf. The results show that both tTFTF and the tTFTF fusion polypeptides (Fig. 3) produced by Michaelis-Menten were functionally active under these conditions.The effect of the peptide fusion on the functional activity of tTF is not affected by the merger of tTF with the peptides.

Example 3: Binding of tTF fusion proteins to αvβ3 in vitro and in vivo

The binding of tTF-RGD and tTF-NGR to the integrin αvβ3 was demonstrated in an ELISA (Enzyme Linked Immunosorbent Assay) by immobilizing purified αvβ3 on microtiter plates (see Figure 4). The specificity of the binding of tTF-RGD to αvβ3 is underlined by the fact that the synthetic peptide with the sequence GRGDSP (Fa. Gibco) competently inhibits the binding of tTF-RGD to αvβ3 in this test system (see Figure 5).

The specific binding of tTF-RGD to αvβ3 on endothelial cells was then evaluated by analysing the differential binding of biotinyllated tTF and tTF-RGD to endothelial cells in suspension by means of fluorescence activated cell sorting (FACS). The experimental advantage of the fact that all endothelial cells in tissue culture are activated, i.e. expressing αvβ3 molecules, was then used to evaluate this by various immunochemical methods. A cultured endothelial cell is sometimes similar in its expression pattern to that of a tumor cell in relation to αvβ3.

The measured fluorescence intensity was 8 times higher for tTF-RGD than for tTF (Figure 6A). Furthermore, the binding of 0.1 μM tTF-RGD to endothelial cells was competitively reduced by 75% by administration of 1 μM of the synthetic peptide GRGDSP (Figure 6B). This highlights the specificity of the binding of tTF-RGD to RGD-binding receptors on the endothelial cell surface such as αvβ3.

Example 4: Anti-tumor effect of tTF fusion proteins in the animal model

The fusion proteins tTF-RGD and tTF-NGR were evaluated for their effects and side effects on human tumor xenotransplants in thymus-free naked mice using the models established in our laboratory (33, 34). Cell lines CCL185 (human adenocarcinoma of the lung) and M-21 (human melanoma) were injected subcutaneously into the flanks of male BALB/c/naked mice (9-12 weeks of age) after reaching a tumor volume of approximately 50-100 mm3 (CCL185) and 400-600 mm3 (M-21) respectively. The mice were randomized into four groups. Group 1 received only physiological fusion saline (NaTF-1), group 2 received tTF-13, group 3 received tTF-3 and group 8 received tTF-3; each group received significantly reduced growth rate compared to their body weight and the protein levels in the females (FTF-2 and TTF-3), and the growth rate of the females (FTF-2 and TTF-3G) was significantly reduced compared to their body weight (FGGGGGG) or 1.5 mg/kg (GGGGGGGGGGG).

To demonstrate the mechanism of action of tumor vessel thrombosis, the following experiment was performed: human melanoma cell line was injected into the flank of two male balb/c/naked mice. When the tumor size was approximately 500 mm3, 2.0 mg/kg KG tTF-NGR or NaCl was injected into the tail vein. Figure 9A shows a macroscopic in vivo image of the tumor-bearing mouse 20 min after injection of the tTF-NGR fusion protein (left image) and right image (right image) respectively. The macroscopic image with a secondary blue-green inhibition of the tumor indicates a 60 min tumor-stimulating sign. In Figure 9 the tumor appears to have been excised in histology after treatment with excretion of the tumor (in contrast to the histological image shown in Figure 9B, where the tumor is treated with excretion of tTF-NGR).

The histological examination of the melanoma tumour shows microscopically visible thrombosis in blood vessels (Figure 10A-D). This finding demonstrates the assumed anti-tumor mechanism of action of tTF-NGR, i. e. the induction of thrombosis in blood vessels. The high selectivity of tTF-NGR for tumour vessels is demonstrated by the lack of histological evidence of clot and necrosis in normal tissues such as heart, kidney, liver and lung (Figure 11A-D). Even repeated high doses of tTF-NGR (4 mg/kg body weight) did not result in visible thrombosis or organ necrosis.

Example 5: Anti-tumor effect of tTF fusion proteins in the animal model of HT1080 tumour

The results of two experiments are summarized in Table 2 and Figure 34. After the second injection of tTF-RGD, significant inhibition of growth of HT1080 tumors was observed compared to control groups. This effect persisted until the end of the experiment on day 7 (P=0.021 for tTF-RGD versus buffer control (physiological saline), P=0.005 for tTF-RGD versus buffer control). Similar to previous experiments, a partial regress in tumor volume was observed in this model. Other Tabelle 1:

Wirkung von tTF-RGD auf das Wachstum von M21-Tumoren in Mäusen
Behandlung			P ggü. Puffer	P ggü. tTF	n
	Tag 0	Tag 7
Puffer	590 +/-77	994 +/-140		ns	9
tTF	558+/-47	931 +/-147	ns		11
tTF-RGD	585+/-85	514+/-81	<0,01	<0,05	7

Tabelle 1:

ns: nicht signifikant

Tabelle 2:

Wirkung von tTF-RGD auf das Wachstum von HT1080-Tumoren in Mäusen
Behandlung			P ggü. Puffer	P ggü. tTF	n
Tag 0	Tag 7
Puffer	1671+/-296	2431+/-559		ns	15
tTF	1751 +/-269	2335+/-398	ns		14
tTF-RGD	1725+/-197	1241+/-122	<0,05	<0,01	12

Tabelle 2:

ns: nicht signifikant

Tabelle 3:

Wirkung von tTF-RGD auf das Wachstum von CCL185-Tumoren in Mäusen
Behandlung			P ggü. Puffer	P ggü. tTF	n
Tag 0	Tag 7
Puffer	39+/-3	467+/-137		ns	9
tTF	44+/-8	764+/-148	ns		5
tTF-RGD	45+/-5	130+/-19	<0,01	<0,01	10

Tabelle 3:

ns: nicht signifikant

Other tTF fusion proteins can be easily constructed by the practitioner based on the disclosure of the present invention. Potential candidates are the peptides TAASGVRSMH and LTLRWVGLMS, which bind to NG2, the murine homologue of human melanoma proteoglycan (12). NG2 expression is limited to tumor cells and angiogenous vessels of a tumor (35). Another candidate is the synthetic peptide TTHWGFTL, which selectively and potently inhibits matrix metallo-proteinase-2 (MMP-2) (13). Since the integrin αvβ3 also appears to bind to MMP-2 in an RGD independent manner, the active enzyme is thus localized to the upper blood vessels of the tumor (31-26).

Other

The following is a list of the most commonly used and used antibodies for the treatment of cancer: 1.Rettig WJ, Garinchesa P, Healey JH, Su SL, Jaffe EA, Old LJ: Identification of endosialin, a cell surface glycoprotein of vascular endothelial cells in human cancer. Natl Acad Sciotti 89:32 108-10836, 1992 USA. Carnemolla B, Balza E, Siri A, Zotra L, Nicrardi, Big MR A, PG Natali: A tumor-associated fibrin isoform generated by splicing of alternative messenger precursors Biol 118, J1039-1148.The study was conducted in the laboratory of the University of California, Irvine, and was funded by the National Institutes of Health (NIH). The study was conducted in the laboratory of the University of California, Irvine, and was funded by the National Institutes of Health (NIH).The following is a list of the most commonly used antibodies in the treatment of cancer: cancer-causing antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antibodies, antigenic antigenic antibodies, antigenic antigenic antibodies, antigenic antigenic antibodies, antigenic antigenic antigenic antigenic antigenic antigenic antigenic antigen, antigenic antigenic antigen, antigenic antigenic antigen, antigenic antigenic antigen, antigenic antigenic antigen, antigenic antigen, antigenic antigen, antigenic antigen antigen antigen, antigen antigen antigen antigen antigen, antigen antigen antigen antigen antigen antigen antigen, antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antigen antiRote N, Thorpe PE: Infarction of solid Hodgkin's tumors in mice by antibody-directed targeting of tissue factor to tumor vasculature. Cancer Res 58: 4646-4653, 199816. Nilsson F, Kosmehl H, Zardi L, Neri D: Targeted delivery of tissue factor to the ED-B domain of fibronectin, a marker of angiogenesis, mediates the infarction of solid tumors in mice. Cancer 61: 711-716, 200117. Liu C, Huang H, Donate F, Dick C, Santucci R, EI-Sheikh A, Vessella R, Edgington TS. Prostate-specific hybrid antigen directed selective thrombotic infarction of tumors. Cancer Res 62: 5470-5475, 200218. Gottstein, C. B, W. W. Thor, B, W. W. B, W. PE: characterization and recombination of cells from vascular cells and recombinant agents.The chemical composition of the protein is determined by the presence of a protein called beta-agonist, which is a protein that is a protein that is a protein that is a protein.The following is a list of the most commonly used chemical names in the field of cancer immunotherapy: Respiratory chemotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, cancer immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherapy, immunotherPasqualini R, Koivunen E, Kain R, Lahdenranta J, Sakamoto M, Stryhn A, Ashmun RA, Shapiro LH, Arap W, Ruohslahti E. Aminopeptidase N is a receptor for tumor-homing peptides and a trajectory for inhibiting angiogenesis. Cancer Res Results 60: 722-727, 200028. Frigoni, Goni, Pasqualini, A, Sacchi, A, W, A, A, R, A, R, A, R, A, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R,The following is a list of the most commonly used and used antibodies for cancer: angiogenesis promoted by vascular endothelial growth factor: regulation through □1□1 and □2□1 integrins. Proc Natl Acad Sci USA 94: 13612-13617, 199730. Yun Z, Menter DG, Nicolson GL: Involvement of integrin αvβ3 in cell adhesion, motility and liver metastasis of murine RAW117 large cell lymphoma.The following is a list of the most commonly used and used antibodies for cancer: Topp MS, Koenigsmann M, Mire-Sluis A, Oberberg D, Eitelbach F, von Marschall Z, Notter M, Reufi B, Stein H, Thiel E, Berdel WE: Recombinant human interleukin-4 inhibits growth of some human lung tumor cell lines in vitro and in vivo. Blood 82: 2837-2844, 199334.Brooks PC, Silletti S, by Schalscha TL, Friedlander M, Cheresh DA: Disruption of angiogenesis by PEX, a noncatalytic metalloproteinase fragment with integrin binding activity. Cell 92: 391-400, 199838. Schnurch H, Risau W: Expression of tie2, a member of a novel family of receptor tyrosine kinase in the endothelial angial lineage. 1993 Development of 119: 957-968, Peters39.The following is a list of the active substances in the active substance, including the active substance, which may be used in the active substance, and the active substance, which may be used in the active substance, including the active substance, which may be used in the active substance, and the active substance, which may be used in the active substance, including the active substance, which may be used in the active substance, and the active substance, which may be used in the active substance, which may be used in the active substance, and the active substance, which may be used in the active substance, which may be used in the active substance, which may be used in the active substance, and the active substance, which may be used in the active substance, which may be used in the active substance, which may be used in the active substance, and which may be used in the active substance, which may be used in the active substance, which may be used in the active substance, which may be used in the active substance, and which may be used in the active substance.The study was conducted in the United States, Canada, and the United Kingdom, and was funded by the National Institutes of Health (NIH). The study was conducted in the United States, Canada, and the United States. The study was funded by the National Institutes of Health (NIH).The use of a combination of the two methods is recommended for the diagnosis of cancer of the liver and kidney.

The following shall be added:

The first two studies were carried out in the field of the use of polypeptides in the treatment of cancer.

Claims (16)

1. A fusion polypeptide, comprising

   a) a peptide of 3 to 30 amino acids capable of selectively binding the fusion polypeptide to tumor vessel endothelial cells; and

   b) a tissue factor (TF) or a fragment thereof, the tissue factor and the fragment being characterized in that they are able to activate blood clotting when the fusion polypeptide binds to tumor vessel endothelial cells,

   wherein the peptides a) and b) are coupled to one another either directly or via a linker having up to 15 amino acids, characterized in that the peptide capable of selectively binding the fusion polypeptide to tumor vessel endothelial cells is coupled to the C-terminus of the peptide capable of activating blood clotting upon binding of the fusion polypeptide to tumor vessel endothelial cells.
2. The fusion polypeptide according to claim 1 consisting of the peptides a) and b) and a linker having up to 15 amino acids.
3. The fusion polypeptide according to claim 1 wherein the peptides a) and b) are coupled to one another directly.
4. The fusion polypeptide according to one of claims 1 to 3, characterized in that the peptide capable of activating blood clotting upon binding of the fusion polypeptide to tumor vessel endothelial cells is the tissue factor TF, which has the sequence shown in SEQ ID NO:1.
5. The fusion polypeptide according to one of claims 1 to 3, characterized in that the peptide capable of activating blood clotting upon binding of the fusion polypeptide to tumor vessel endothelial cells is a fragment of the tissue factor TF, which preferably has the sequence shown in SEQ ID NO:2.
6. The fusion polypeptide according to one of claims 1 to 5, characterized in that the peptide of 3 to 30 amino acids capable of selectively binding the fusion polypeptide to tumor vessel endothelial cells, has a linear or cyclic structure.
7. The fusion polypeptide according to one of claims 1 to 6, characterized in that the peptide of 3 to 30 amino acids capable of selectively binding the fusion polypeptide to tumor vessel endothelial cells comprises the amino acid sequence RGD or NGR.
8. The fusion polypeptide according to claim 7, characterized in that the peptide capable of selectively binding the fusion polypeptide to tumor vessel endothelial cells is selected from the group comprising GRGDSP and GNGRAHA.
9. The fusion polypeptide according to claim 7, characterized in that the peptide capable of selectively binding the fusion polypeptide to tumor vessel endothelial cells is selected from the group comprising GCNGRCG, GCNGRCVSGCAGRC, GCVLNGRMEC and GALNGRSHAG.
10. The fusion polypeptide according to claims 1 to 9, characterized in that it has one of the sequences shown in SEQ ID NO:3-8.
11. A nucleic acid encoding a fusion polypeptide according to one of claims 1 to 10.
12. The nucleic acid according to claim 11, characterized in that it has one of the sequences shown in SEQ ID NO:10-15.
13. A vector comprising a nucleic acid according to claim 11 or 12.
14. A cell comprising a nucleic acid according to claim 11 or 12 or a vector according to claim 14.
15. A pharmaceutical composition comprising a fusion polypeptide according to one of the claims 1 to 10, a nucleic acid according to claim 11 or 12, a vector according to claim 13 or a cell according to claim 14.
16. The pharmaceutical composition according to claim 15, which further comprises pharmaceutically acceptable carriers, excipients or adjuvants.