SELECTIVE CELL-DEATH-INDUCING BINARY ENZYME SYSTEM

Abstract

The invention relates to a combination preparation containing a selective cell death-inducing binary enzyme system for use in the therapy and/or treatment of cancer and tumors in humans and animals, a process, and its use.

Claims

1. A drug for use in the treatment and/or prophylaxis of cancer or tumor diseases containing an inactive form of granzyme B comprising the sequence set forth in SEQ ID NO: 2 or a nucleic acid encoding it, or a functional variant of either, and tobacco etch virus protease comprising the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5 or a nucleic acid encoding the tobacco etch virus protease or a functional variant of either, wherein the tobacco etch virus protease releases an active form of granzyme B comprising the sequence set forth in SEQ ID NO: 2.

2. The drug of claim 1, wherein the inactive form of granzyme B is a pre-granzyme B comprising the sequence set forth in SEQ ID NO: 3 or a nucleic acid encoding the pre-granzyme B, or a functional variant of either.

3. The drug of claim 1, wherein the inactive form of granzyme B is a fusion protein comprising the sequence set forth in SEQ ID NO: 2 or a nucleic acid encoding the sequence set forth in SEQ ID NO: 2, or a functional variant of either, wherein SEQ ID NO: 2 or a functional variant thereof is obtained by means of cleavage by tobacco etch virus protease comprising the sequence set forth in either SEQ ID NO: 4 or SEQ ID NO: 5 or a functional variant thereof.

4. A drug containing: i) an inactive form of granzyme B, wherein the inactive form of granzyme B is a fusion protein comprising the sequence set forth in SEQ ID NO: 2 or a nucleic acid encoding it, or a functional variant of either, wherein ENLYFQ (SEQ ID NO: 6) or ENLYFQG (SEQ ID NO: 7) is ligated with the N-terminus of SEQ ID NO: 2 and SEQ ID NO: 2 or a functional variant thereof is obtained or released by means of cleavage by tobacco etch virus protease at ENLYFQ (SEQ ID NO: 6) or ENLYFQG (SEQ ID NO: 7), and ii) a tobacco etch virus protease or a nucleic acid encoding the tobacco etch virus protease or a functional variant of either.

5. A combination preparation containing the drug of claim 1, and optionally excipients and additives.

6. The combination preparation of claim 5 for use in the treatment and/or prophylaxis of cancer or tumor diseases in humans and animals.

7. A process comprising administering the drug of claim 1 or a combination preparation comprising said drug by a gene therapy process.

8. A process comprising carrying out a gene therapy process by a vehicle comprising the drug of claim 1 or a combination preparation comprising said drug.

9. The process of claim 8, wherein the vehicle is selected from the group consisting of liposomes, nanoparticles, microparticles, viruses, and lipoplexes.

10. The process of claim 9, wherein the vehicle further comprises ligands that recognize tumor markers.

11. A process for introducing a drug or a combination preparation, comprising: i) introducing an inactive form of granzyme B comprising a nucleic acid encoding a polypeptide having the sequence set forth in SEQ ID NO: 2, or a functional variant thereof, and a nucleic acid encoding tobacco etch virus protease or a functional variant thereof, together or separately from one another, in at least one vehicle, ii) introducing the vehicle into a tumor cell, wherein the inactive form of granzyme B is expressed in the tumor cell, and iii) producing an active form of granzyme B in the tumor cell and inducing cell death in the tumor cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 describes the inventive therapeutic strategy to induce cell death of tumor cells.

[0022] FIG. 2.1 and FIG. 2.2 show tumor markers for certain cancer diseases.

[0023] FIG. 3 shows TEV cleavage results from Example 1.

DETAILED DESCRIPTION OF INVENTION

[0024] According to the invention, TEV recognizes the recognition site (recognition sequence) ENLYFQ (SEQ ID NO: 6) or ENLYFQG (SEQ ID NO: 7) in the inactive form, which is linked (ligated) with the N-terminus of SEQ ID NO: 2. However, according to the invention, it is preferable for ENLYFQ (SEQ ID NO: 6) to be ligated with the N-terminus of SEQ ID NO: 2, which surprisingly gives improved cleavage.

[0025] Therefore, the goal is achieved in its full scope by the claims that have been drawn up.

[0026] As soon as the inactive form of granzyme B and TEV are introduced, either together or separately from one another, into a tumor cell and expressed (if applicable), TEV releases the active form of granzyme B comprising SEQ ID NO: 2, inducing cell death through apoptosis or programmed cell death.

[0027] The inventive selection of the serine protease used in the invention and the means used, namely TEV, to unmask an inactive form of granzyme B into an active form, are especially advantageous. As soon as these two polypeptides are present in a tumor cell, the unmasking proceeds in a completely specific and efficient manner. Here it is especially advantageous that neither pre-granzyme B nor TEV occur in humans or mammals.

[0028] TEV is referred to in the document Kapust et al, The P1 specificity of tobacco etch virus protease, Biochemical and Biophysical Research Communications, 294 (2002) 949-955.

[0029] Therefore, the invention relates to a drug or combination preparation comprising an inactive form of granzyme B comprising SEQ ID NO: 2, or a nucleic acid encoding it, and TEV (e.g., SEQ ID NO: 4 or SEQ ID NO: 5), or a nucleic acid encoding it. TEV recognizes the recognition site (recognition sequence) ENLYFQ (SEQ ID NO: 6) or ENLYFQG (SEQ ID NO: 7) in the inactive form of granzyme B.

[0030] In a preferred embodiment of the invention, the inactive form of granzyme B is a pre-granzyme B (SEQ ID NO: 3) or a nucleic acid encoding it (Zhinan Xia et al, Expression and Purification of Enzymatically Active Recombinant Granzyme B in a Baculovirus System, Biochemical and Biophysical Research Communications, 243 (1998) 384-389). An example of such an encoding nucleic acid is SEQ ID NO: 8.

[0031] In another preferred embodiment of the invention, the inactive form of granzyme B is a fusion protein comprising SEQ ID NO: 2 or a nucleic acid encoding it, wherein SEQ ID NO: 2 is obtained or released through cleavage by TEV (e.g., SEQ ID NO: 4 or SEQ ID NO: 5) at ENLYFQ (SEQ ID NO: 6) or ENLYFQG (SEQ ID NO: 7).

[0032] Therefore, the invention relates to an inactive form of granzyme B, namely a fusion protein comprising SEQ ID NO: 2 or a nucleic acid encoding it, wherein SEQ ID NO: 2 is released through cleavage by TEV (e.g., SEQ ID NO: 4 or SEQ ID NO: 5) at the recognition sequence ENLYFQ (SEQ ID NO: 6) or ENLYFQG (SEQ ID NO: 7).

[0033] Therefore, the invention relates to an inactive form of granzyme B, namely a fusion protein comprising SEQ ID NO: 2 and ENLYFQ (SEQ ID NO: 6) or ENLYFQG (SEQ ID NO: 7), or a nucleic acid encoding it, and possibly other sequences, wherein ENLYFQ (SEQ ID NO: 6) or ENLYFQG (SEQ ID NO: 7) is linked with the N-terminus of SEQ ID. NO: 2. A corresponding embodiment is given in SEQ ID NO: 3, namely of the type: FLAG-Tag-ENLYFQ-SEQ ID NO: 2. Any other fusion proteins can be prepared in a corresponding manner (e.g., by means of an HIS-tag, and others), wherein the sample FLAG-tag can be replaced by any peptide, for example, 50 to 100 amino acids.

[0034] The person skilled in the art is able to produce and design suitable fusion proteins (Ausubel et al. (ed.), (1989). Preparation of Genomic DNA from Mammalian Tissue. In: Short Protocols in Molecular Biology: A Compendium of Methods from CURRENT PROTOCOLS IN MOLECULAR BIOLOGY. John Wiley & Sons).

[0035] The inventive combination preparations and drugs can have suitable excipients and additives added to them. Examples of suitable additives and/or excipients are, e.g., physiological saline solution, stabilizers, proteinase inhibitors, nuclease inhibitors, etc.

[0036] Therefore, the invention also relates to a combination preparation or drug as described above for application or use in the treatment and/or prophylaxis of cancer or tumor diseases in humans and animals, especially mammals.

[0037] In another preferred embodiment, the inventive combination preparations or drugs are administered by means of a gene therapy process.

[0038] Gene therapy processes can be obtained, e.g., by complexing the inventive nucleic acids with liposomes. Lipid mixtures suitable for this purpose are described by Felgner, P. L. et al. (1987) Proc. Natl. Acad. Sci, USA 84, 7413; Behr, J. P. et al. (1989) Proc. Natl. Acad. Sci. USA 86, 6982; Felgner, J. H. et al. (1994) J. Biol. Chem. 269, 2550, or Gao, X. & Huang, L. (1991) Biochim. Biophys. Acta 1189, 195. When the liposomes are produced, the DNA is ionically bound to the surface of the liposomes, and in such a ratio that a positive net charge remains, and the DNA is completely complexed by the liposomes. Sterically stabilized liposomes with a polyethylene glycol (PEG) shell exhibit clearly reduced ingestion through the mononuclear phagocyte system (MPS), and also have greatly prolonged blood circulation times, reduced aggregation of PEGylated vesicles, and improved stability of the liposomal formulations. Analogous to PEG, linear and hyperbranched polyglycerol (lPG and hbPG) show excellent biocompatibility, but allow further derivatives to be formed by the addition of functional groups. Novel lipids based on hyperbranched polyglycerol, linear-hyperbranched PEG-hbPG-block copolymers and statistical PEG-PG-copolymers were produced through combined anionic polymerizations of various epoxide monomers using lipophilic initiators such as cholesterol or 1,2-bis-n-alkyl glyceryl ethers. The novel amphiphilic structures were successfully introduced into liposomal membranes using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) as a colipid.

[0039] Therefore, the invention also relates to a gene therapy process involving delivery into a target cell, preferably a tumor cell, by using a vehicle.

[0040] In another embodiment, this vehicle can be selected from the group of liposomes, nano- or microparticles, viruses, lipoplexes, etc. (Gene delivery by lipoplexes and polyplexes. Tros de Ilarduya C, Sun Y, Dzgne N. Eur J Pharm Sci. 2010 Jun. 14; 40(3):159-70. doi: 10.1016/j.ejps.2010.03.019. Epub 2010 Mar. 30; Efficient gene delivery by EGF-lipoplexes in vitro and in vivo, Bunuales M, Dzgne N, Zalba S, Garrido M J, de Ilarduya C T. Nanomedicine (Lond). 2011 January; 6(1):89-98. doi: 10.2217/nnm.10.100; Genetic nanomedicine: gene delivery by targeted lipoplexes, Dzgne N, de Ilarduya CT. Methods Enzymol. 2012; 509:355-67. doi: 10.1016/B978-0-12-391858-1.00018-6) .

[0041] In an especially preferred embodiment, the inventive vehicles have ligands on the surface that recognize tumor markers. Examples of such ligands are polyclonal or monoclonal antibodies or covalent binders (aptamers) that are able to bind to tumor markers.

[0042] Finally, such presenting tumor markers cannot be:

[0043] Carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), carbohydrate antigen 19-9 (CA19-9), cancer antigen 72-4 (CA 72-4), cancer antigen 125, cancer antigen 15-3 (CA 15-3), neuron-specific enolase (NSE), squamous cell carcinoma antigen (SCC), cytokeratin fragment (CYFRA), human chorionic gonadotropin (HCG), prostate-specific antigen (PSA), human thyroglobulin (HTG), mucin-like cancer associated antigen (MCA), etc. FIG. 2.1 and FIG. 2.2 show examples of tumor markers and the cancers for which they are suitable.

[0044] Therefore, the invention also relates to a process for introducing an inventive drug or combination preparation, wherein an inactive form of granzyme B comprising a nucleic acid encoding SEQ ID NO: 2, and a nucleic acid encoding tobacco etch virus protease (e.g., SEQ ID NO: 4 or SEQ ID NO: 5), especially an inactive form of granzyme B comprising a nucleic acid encoding a fusion protein comprising SEQ ID NO: 2 and ENLYFQ (SEQ ID NO: 6) or ENLYFQG (SEQ ID NO: 7) and a nucleic acid encoding tobacco etch virus protease,

[0045] i.) are introduced, together or separately from one another, in at least one vehicle,

[0046] ii.) into a tumor cell and expressed there,

[0047] iii.) producing an active form of granzyme B and inducing cell death in the tumor cell.

[0048] The process can be correspondingly adapted by other previously mentioned embodiments. The inventive drugs, combination preparations, and especially their vehicles can preferably be locally administered to humans and animals, e.g., subcutaneously administered. Of course the invention comprises all applications in tumor treatment.

[0049] As defined in this invention, the term functional variant is understood to mean polypeptides or nucleic acids that are functionally related with the inventive peptide. The term variants is also understood to mean allelic variants or polypeptides and nucleic acids that are derived from other organisms, cells, or tissues.

[0050] More broadly, it is also understood to mean polypeptides or nucleic acids that have a sequence homology, especially a sequence identity, of about 70%, preferably about 80%, especially preferably about 90%, most preferably about 95% with the designated SEQ ID.

[0051] This also includes polypeptide deletion in the range of about 1-50, preferably about 1-30, especially preferably about 1-15, most preferably about 1-6 amino acids. For example, the first amino acid can lack methionine, without substantially changing the function of the polypeptide.

[0052] In addition, this also includes fusion proteins that contain the above-described inventive polypeptides, the fusion proteins themselves already having the function of the respective SEQ ID or only being able to acquire the specific function after elimination of the fusion moiety. Above all, this includes fusion proteins whose component especially of non-human sequences is about 1-50, preferably about 1-30 amino acids. Examples of non-human peptide sequences are prokaryotic peptide sequences, e.g., from E. coli galactosidase or [those with] a so-called histidine tag, e.g., a Met-Ala-His6-Tag. An especially advantageous application for which fusion proteins with a so-called histidine tag are suitable is to purify the expressed protein through metal ion-containing columns, for example through a Ni.sup.2+-NTA column. Here NTA stands for the chelating agent nitrilotriacetic acid (Qiagen GmbH, Hilden).

[0053] Especially the mentioned parts of the polypeptide can also be synthesized using classical peptide synthesis (Merrifield method). They are especially suitable for obtaining antisera, which can be used to search through suitable gene expression libraries to achieve other functional variants of the inventive polypeptides.

[0054] In a preferred embodiment, the inventive nucleic acid previously mentioned in each case is a DNA, cDNA, or RNA, preferably a double-stranded DNA, however a PNA or something similar is also conceivable.

[0055] The inventive nucleic acids can also be introduced into the tumor cell by means of (expression) vectors, for example, by means of the vector pcDNA3.1 (Invitrogen) with a constitutive CMV promoter, etc.

[0056] As defined in this invention, the terms tumor, cancer, cancer cells, and tumor cells should be read as synonyms, and comprise every benign or malignant tumor, especially a growth with a locally circumscribed increase in tissue volume, comprising every localized swelling due to edema, acute and chronic inflammation, aneurysmal enlargement (pulsating tumor) etc., and also inflammatory organ swelling (e.g., as in the case of a so-called splenic tumor) as well as a tissue neoplasm (growth, blastoma, neoplasia) in the form of a spontaneous, autonomous and irreversible excessive growth of the body's own tissue, disinhibited to different extents, which is, as a rule, connected with loss of specific cell and tissue functions of different severity (see Pschyrembel, (261st edition) 2007, de Gruyter, Berlin).

[0057] Examples and figures:

[0058] These examples serve exclusively to explain the invention, without limiting the it to these examples.

EXAMPLES

Example 1

[0059] Production of the pSTdna 1023 Plasmid

[0060] The tobacco etch virus protease sequence was ligated into the pcDNA3.1 vector (Invitrogen company). In addition, the FLAG sequence (MDYKDDDDKGDYKDDDDKGGGT; SEQ ID NO: 9) was cloned into the n-terminal region twice.

[0061] Production of pSTdna 1024 (GrzB WT)

[0062] An additional plasmid based on the vector pcDNA3.1 was cloned with the following sequence; this cloning was done twice FLAG sequence (MDYKDDDDKGDYKDDDDKGGGT; SEQ ID NO: 9) in combination with TEV recognition sequence (SEQ ID NO: 6) and granzyme B AA21-240.

[0063] Production of the pSTdna 1025 Plasmid

[0064] The amino acid serine 203 in plasmid pSTdna 1024 was replaced by alanine 203. This mutation specifically inhibits granzyme B activity (GrzB mut).

[0065] The results of cleavage with TEV are shown in FIG. 3. If the recognition site (SEQ ID NO: 6) has a G instead of Q, no cleavage by TEV takes place (not shown).

Example 2

[0066] Protocol

[0067] 1. HeLa cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) at 37 C. under 5% CO.sub.2 to 75% confluence.

[0068] 2. The medium was carefully aspirated and the cells were washed with phosphate buffered saline.

[0069] 3. The cells were digested for 5 minutes using trypsin EDTA (200 mL/L), so that the cells were detached from the Petri dish. The digestion was stopped by adding DMEM.

[0070] 4. The cells were centrifuged at 500 g for 5 minutes and resuspended with a 1 g plasmid in a medium (Lonza) for electroporation. Then, the cells were electroporated with an Amaxa Nucleofector II electroporator and resuspended in fresh DMEM.

[0071] 5. The cells were incubated at 37 C. under 5% CO.sub.2 for 4 h.

[0072] 6. Solution A: 1 pg of plasmid was mixed with 100 L of Opti-MEM in a 1.5 mL tube.

[0073] 7. Solution B: 8 L of Lipofectamine were mixed in a 1.5 mL tube and incubated at room temperature for 5 minutes.

[0074] 8. Solutions A and B were pipetted together and carefully mixed. The incubation time is 30 minutes at room temperature.

[0075] 9. The cell medium was carefully aspirated and the cells were washed twice with Opti-MEM. The cells are incubated with 3 mL of Opti-MEM at 37 C. under 5% CO.sub.2.

[0076] 10. The mixture of solutions A and B from step 8 was incubated for 30 minutes, and then AB was pipetted into the Opti-MEM and carefully mixed.

[0077] 11. The cells were incubated at 37 C. under 5% CO.sub.2 for 5 hours.

[0078] 12. After 5 h 5 mL of DMEM was added.