WO2015092756A1 - Recombinant fusion protein prostat and uses thereof - Google Patents

Abstract

The invention provides recombinant fusion protein ProSTAT, comprising a transporting cassette which enables the protein to enter into the cell, and an active cassette comprising the sequence of the human BID protein, which selectively sensitizes cancer cells, in particular of prostate cancer, cervix carcinoma, non-small cell lung cancer, as well as composition comprising it and their use as anticancer medicaments or agents sensitizing to apoptosis or inducing apoptosis.

Description

Recombinant fusion protein ProSTAT and uses thereof

Description

Technical Field

The present invention provides a recombinant fusion protein ProSTAT comprising a transporting cassette which enables the protein to enter into the cell, more particularly, a TAT cassette, and an active cassette comprising an amino acid sequence of human BID protein, which selectively sensitizes cancer cells, more particularly, cells of prostate cancer, cervix carcinoma and non- small cell lung cancer, to anticancer drugs which induce apoptosis. The present invention also provides the use of recombinant fusion protein ProSTAT comprising a transporting cassette that enables the protein to enter the cell, more particularly, a TAT cassette, and an active cassette comprising an amino acid sequence of human BID protein, which sensitizes some cancer cells, as an anticancer drug which supports the induction of apoptosis in cancer cells, more particularly, in cells of prostate cancer, cervix carcinoma and non-small cell lung cancer.

The present invention also provides a pharmaceutical composition comprising the recombinant fusion protein ProSTAT according to the invention, more particularly, further comprising pharmaceutically acceptable substances lowering the recombinant protein ProSTAT sensitivity to proteolytic degradation, and/or its immunogenicity, and alternatively one or more pharmaceutically acceptable excipients and/or carriers.

The present invention also provides use of the recombinant fusion protein ProSTAT or the pharmaceutical composition comprising the recombinant fusion protein ProSTAT in the treatment of cancer, particularly prostate cancer, cervix carcinoma and non-small cell lung cancer.

The present invention also provides an expression vector comprising a nucleotide sequence encoding the recombinant fusion protein ProSTAT, allowing for the production and purification of the recombinant fusion protein ProSTAT.

Background Art

Prostate cancer is the third most commonly diagnosed cancer in males accounting for about mortality for males after lung cancer. According to recent estimates, approximately 1 ,750,000 men were diagnosed with prostate cancer in 2010 in seven countries which are the main market for anticancer drugs (The Cancer Market Outlook to 2016, 201 1). There were 8300 new prostate cancer cases in Poland, with approximately 4000 estimated attributable deaths (Jassem et al. (eds), 2013). According to WHO estimates, prostate cancer rates are ten times higher in high-income than in low- or middle-income countries (Global Health Observatory Data Repository, WHO). The highest morbidity from prostate cancer in 27 countries of UE (UE-27) is in Ireland (183.2 new cases per 100,000 population), and the lowest in Greece (27.9 new cases per 100,000 population) while the average is 105.6 new cases per 100,000 population (European Age-Standardized Rates, 2011). Prostate cancer rates in seven countries which are the main market are expected to moderately increase up to 2016, and the increase is expected to remain high compared to other types of cancer.

Treatment of prostate cancer depends on the risk of disease progression, the age of the patient and comorbidity (Heidenreich et al., 2008). In the case of low and moderate risk of disease progression, a standard treatment of prostate cancer is radical prostatectomy, radiotherapy and hormonal therapy. The latter includes blocking androgen receptors with anti-androgens (flutamide, bicalutamide), or inhibition of androgen secretion with analogs of luteinising hormone-releasing hormone (goserelin, leuprolide). Due to their mechanism of activity, these medicaments are targeted at hormone sensitive cancer cells. However, most of the patients responding well to hormonal therapy therapy develop a resistance to such treatment, called castration-resistant prostate cancer (CRPC). Chemotherapeutic approach employed in such a case includes only a small group of effective cytotoxic agents, first of all taxanes (docetaxel (Heidenreich et al., 2008), cabazitaxel (Jassem et al. (eds), 2013), and mitoxantrone (Heidenreich et al., 2008; Jassem et al. (eds), 2013). Therefore, despite some encouraging results of certain combined chemotherapies utilizing cytotoxic agents, new drugs are necessary, particularly for the treatment of advanced prostate cancer.

Cervix carcinoma is the third most commonly diagnosed cancer in females, accounting for 9% of all new cancer cases and 8% of all cancer deaths among females in 2008 (Gizzo et al., 2013; Salomon-Perzyhska et al., 2014). In the majority of cases, it is diagnosed at advanced stages (Salomon-Perzyhska et al., 2014). The treatment is based on radical surgery, often with subsequent radiotherapy, and on chemotherapy (Gizzo et al., 2013). Chemotherapy is mainly based on platinium-containing drugs (cisplatin), taxanes (paclitaxel) and camptothecin derivatives (topotecan), recently complemented with specific monoclonal antibodies (bevacizumab) (Markman, 2014; Salomon-Perzyhska et al., 2014).

Lung cancer is the most common (18.2%) cause of all cancer related deaths (Ferlay et al., 2010). The great majority (85-90%) of lung cancer cases are non-small cell lung cancer. Surgery and radiotherapy are combined with chemotherapy based on cisplatin, docetaxel, antimetabolites (pemetrexed) and on specific kinase inhibitors (erlotinib, ceritinib) (Melosky, 2014). Similarly as in the case of advanced prostate cancer new drugs are also required in the treatment of advanced cervix carcinoma and non-small lung cancer, more effective than the ones used to date.

Decreased sensitivity to apoptotic death inducing agents is a common characteristic of cancer cells. Typically it is associated with reduced levels of an active pro-apoptotic protein or proteins, or with increased levels of anti-apoptotic proteins. This is the case in cancer cell lines derived from prostate cancer and cervix carcinoma which contain i.a. very low amounts of a key apoptotic protein BID (Orzechowska et al., 2014). The BID protein is used by type II cells in signaling between death receptors and mitochondria (Kantari & Walczak, 201 1), as well as in the DNA damage response (Liu et al., 2011). In the first case, the BID protein is cleaved by caspase 8 giving an active form that triggers next stages of apoptosis. Previous attempts of increasing BID in cancer cells were based on overexpression thereof from DNA vectors provided to the cells (Tsuno et al., 2012; Yi et al., 2003; Song et al, 2008) or adenoviruses introduced into the cells (Miao et al., 2006; Fukazawa et al., 2003). A drawback of such an approach is that it does not provide any control of the amount of BID supplied to the cell. An excess of BID in the cell may directly induce its apoptotic death itself (Fukazawa et al., 2003). It poses a risk of leading to excessively high BID levels also in non-cancer cells, which may result in their death. Attempts have been made to use expression vectors allowing for pro-apoptotic protein expression, controlled by promoters specific to cancer cells, e.g. the hTERT promoter of telomerase (e.g. Wirth et al., 2003), and for the BID protein as well (Kazhdan et al., 2006), however, the activity of such a promoter may also be high in many types of stem cells or hepatocytes of cirrhotic patients (Kotoula et al., 2002). New, more specific promoter systems have been established for some cancers, e.g. the TTS system, which was used i.a. to obtain specific expression of the introduced BID protein encoding gene in lung cancer (Fukazawa et al., 2009). However, such systems are not established for majority of cancer types, including prostate cancer and cervix carcinoma. Therefore, there is a need for new anticancer drugs which might be used for treatment of cancers that are poorly responsive to apoptotic death inducing agents, for example conventional cytostatics or proteins which induce apoptotic death, particularly drugs for treatment or for supporting of a treatment of prostate cancer, cervix carcinoma and non-small cell lung cancer, especially drugs which allow to specifically sensitize cancer cells to apoptotic death inducing agents, for example to conventional cytostatics or proteins inducing apoptotic death, without being highly cytotoxic to normal cells.

The above description of the background art applies particularly in regard to prostate cancer, and less particularly in regard to cervix carcinoma and non-small cell lung cancer. They are the three types of cancer used for isolation of the cell lines against which the recombinant fusion protein ProSTAT, advantageously ODD-ProSTAT according to the invention, has been found effective in experiments made by the inventors and presented below.

Disclosure of the Invention

The invention provides a recombinant fusion protein ProSTAT (short for: Protein Selectively Triggering Apoptosis in Tumor cells) considered as a potential therapeutic agent which selectively sensitizes cancer cells, particularly cells of prostate cancer, cervix carcinoma and non-small lung cancer, to anticancer drugs which induce apoptosis, and uses thereof.

The invention is based on an unexpected finding that a recombinant protein comprising a transporting cassette, advantageously comprising a peptide deriving from the TAT protein, and an active cassette comprising the human BID protein, when entering the cells may enable controlled delivery of relatively small amounts of BID to the cells, not inducing apoptosis, but only sensitizing them to anticancer drugs which induce apoptosis. The inventors unexpectedly found that it is possible to introduce the BID protein in the form of the fusion protein ProSTAT which is delivered in such a dose so it would not spontaneously induce apoptosis but selectively sensitize the cancer cells, particularly cells of prostate cancer, cervix carcinoma and non-small cell lung cancer, to another agent which induces apoptosis, e.g. conventional anticancer drugs which induce apoptosis. Such a sensitization of cancer cells by administration of sensitizing amount of the recombinant fusion protein ProSTAT combined with other anticancer agent which induces apoptosis not only enhances the effects of the other anticancer agent due to synergistic action of both compounds, but also enables administering the other anticancer agent in smaller dose, and consequently minimalizing side effects of such therapy which is therefore less harmful for the patient. Such a sensitization of cancer cells by administration of sensitizing amount of the recombinant fusion protein ProSTAT enables inducing apoptosis in the cancer cells using an agent which was previously ineffective against these cells or had a very limited use, or reducing the doses of the previously used drugs thereby reducing the toxicity of the therapy or the observed side effects.

The essence of the invention is thus the recombinant fusion protein ProSTAT or a functional variant thereof, which is an artificial fusion protein comprising a transporting cassette which enables the protein to enter the cell, advantageously a transporting cassette TAT, advantageously comprising an amino acid sequence set forth in SEQ ID NO: 4 (encoded by a nucleotide sequence set forth in SEQ ID NO: 3), operationally linked with an active cassette which selectively sensitizes cancer cells, advantageously cells of prostate cancer, cervix carcinoma and non-small cell lung cancer, to anticancer drugs which induce apoptosis, and wherein the active cassette comprises an amino acid sequence of the human protein BID(L), isoform 1 , advantageously an amino acid sequence set forth in SEQ ID NO: 2 (encoded by a nucleotide sequence set forth in SEQ ID NO: 1).

The recombinant fusion protein ProSTAT according to the invention is advantageously a protein which comprises an amino acid sequence set forth in SEQ ID NO: 6 (encoded by a nucleotide sequence set forth in SEQ ID NO: 5) or a functional variant thereof, e.g. a variant protected against phosphorylation by the CK2 kinase, advantageously wherein two mutations in the active cassette are introduced: T58A and S75A (namely threonine and serine in positions 58 and 75, respectively, are substituted with alanine), advantageously the mutations T58A and S75A are introduced to the amino acid sequence set forth in SEQ ID NO: 2 as the amino acid sequence of the active cassette (residue positions in the active cassette mentioned here and below refer to SEQ ID NO: 2, which differs from the amino acid sequence of the human protein BID set forth in protein databases, e.g. in UniProtKB/SwissProt No. P55957, in that it lacks methionine, the first residue of the human protein BID).

This invention also provides a functional variant of the recombinant fusion protein ProSTAT being a recombinant protein with a highly homologous amino acid sequence, advantageously highly identical to SEQ ID NO: 6, or a functional variant of the fusion protein ProSTAT being a protein comprising the transporting cassette with a highly homologous amino acid sequence, advantageously highly identical to SEQ ID NO: 4, further comprising the active cassette, sequence operationally linked with the transporting cassette, wherein the amino acid sequence of the former is highly homologous, advantageously highly identical to SEQ ID NO: 2, or a fusion protein ODD-ProSTAT, with a highly homologous amino acid sequence, advantageously highly identical to SEQ ID NO: 7, (encoded by the nucleotide sequence set forth in SEQ ID NO: 8), wherein the homologous, advantageously highly identical sequence, comprises deletions, insertions and substitutions in the sequence, as long as the protein functions to sensitize the cells to anticancer drugs inducing apoptosis, as is defined herein. In this respect, especially advantageous substitutions will usually be conservative, i.e. advantageously it will be substitutions occurring within one amino acid family. For example, amino acids are usually divided into four families: (1) acidic - aspartate and glutamate; (2) basic - lysine, arginine and histidine; (3) non-polar - glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, cysteine, methionine and tryptophan; and (4) non-charged polar - asparagine, glutamine, serine, threonine and tyrosine. Phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids. It is reasonable to expect that an isolated substitution of leucine with isoleucine or valine, or vice versa; aspartate with glutamate or vice versa; threonine with serine or vice versa; or a similar conservative substitution of an amino acid residue with a structurally related amino acid residue will not substantially influence biological activity of the protein.

Particularly, the present invention provides a functional variant of the fusion protein ProSTAT which is protected against phosphorylation by the CK2 kinase, advantageously wherein threonine and serine in positions 58 and 75, respectively, advantageously in positions corresponding to 58 and 75 in SEQ ID NO: 2, are substituted with alanine, hereinafter referred to as ProSTAT^{T58A S75A} (residue in the active cassette positions mentioned here and below refer to SEQ ID NO: 2, which differs from the amino acid sequence of the human protein BID in protein databases, e.g. in UniProtKB/SwissProt No. P55957, in that it lacks the first residue of the BID protein - methionine).

Thus, proteins having an amino acid sequence substantially the same as the reference sequence, but containing inconsiderable amino acid substitutions, not significantly affecting the protein sensitizing activity to anticancer drugs, fall within the scope of the definition for the recombinant fusion protein ProSTAT or a functional variant thereof according to the invention. The highly homologous sequence means that the sequence is similar, advantageously identical, at least in 70%, advantageously in 80%, more advantageously in 90%, the most advantageously in at least 95%. It will be apparent to one skilled in the art that the above changes also refer to the nucleotide sequences encoding as a whole or comprising in part such recombinant fusion proteins ProSTAT according to the invention, or the cassettes they are composed of (e.g. SEQ ID NO: 3, SEQ ID NO: 1 , SEQ ID NO: 5, SEQ ID NO: 8), or the functional variants of the recombinant proteins according to the invention (e.g. a functional variant of the fusion protein ProSTAT protected against phosphorylation by the CK2 kinase, or ODD-ProSTAT), and that they are also encompassed by the invention.

In one advantageous embodiment, the recombinant fusion protein ProSTAT further comprises additional cassettes, for example those enabling the isolation of the recombinant fusion protein ProSTAT, such as a 6xHis cassette, or the identification of the recombinant fusion protein ProSTAT using immunological methods, such as a HA cassette or cassettes, or using microscopy, such as an EGFP cassette.

Additional cassettes in the amino acid sequence of the recombinant fusion protein ProSTAT can also advantageously comprise sequences encoding elements increasing selectivity of the protein towards selected cancer cells. An example of such an additional cassette is a selectivity enhancing ODD cassette (oxygen destruction domain) which advantageously increases specificity of the fusion protein ProSTAT towards tumor forming cancers, wherein oxygen concentration is reduced (hypoxic conditions). The ODD cassette added to the protein causes its degradation under normoxia conditions present within the majority of normal non-cancerous cells, whereas the protein remains un-degraded under hypoxia conditions, present within tumors (Huang et al., 1998). In particular, oxygen concentration inside prostate cancer tumors is more than four-fold lower than in untransformed prostate cells (Vaupel & Kelleher, 2013). Therefore, the addition of the ODD cassette solves the problem of increasing selectivity of the ProSTAT protein which, when fused with the ODD cassette, sensitizes cancer cells without influencing the sensitivity of untransformed cells. Therefore, the invention provides the ProSTAT fusion protein linked to any additional selectivity increasing cassette, the so-called targeting cassette, advantageously with the ODD cassette, namely the ODD-ProSTAT fusion protein, the amino acid sequence thereof set forth in SEQ ID NO: 7, and the nucleotide sequence encoding it in SEQ ID NO: 8.

The invention also provides a pharmaceutical composition comprising the recombinant fusion protein ProSTAT according to the invention, or a functional variant thereof, advantageously the fusion protein ODD-ProSTAT, advantageously also comprising one or more pharmaceutically acceptable excipients and/or carriers. Advantageously, the pharmaceutical composition according to the invention comprises pharmaceutically acceptable ingredients which decrease the sensitivity of the recombinant fusion protein ProSTAT, advantageously ODD-ProSTAT according to the invention, to proteolytic degradation and/or decrease immunogenicity of the recombinant protein ProSTAT, advantageously ODD-ProSTAT according to the invention. The above refers both to classical methods for reducing immunogenicity and increasing stability of protein drugs by glycosylation and pegylation (van Beers & Bardor, 2012), and to more recent procedures based on administration of protein drugs in nanoparticles built of biodegradable polymers (Danhier et al., 2012).

Moreover, an advantageous pharmaceutical composition according to the invention further comprises another anticancer drug which induces apoptosis, advantageously doxorubicin (adriamycin), TRAIL protein, camptothecin or its derivatives.

The invention also provides the recombinant fusion protein ProSTAT, advantageously ODD- ProSTAT according to the invention, or the pharmaceutical composition according to the invention, for use as a medicament. In one advantageous embodiment, the recombinant fusion protein ProSTAT, advantageously ODD-ProSTAT according to the invention, or the pharmaceutical composition according to the invention, are used in treatment of cancer, advantageously prostate cancer, cervix carcinoma, non-small cell lung cancer.

In one advantageous embodiment, the recombinant fusion protein ProSTAT, advantageously ODD-ProSTAT according to the invention, or the pharmaceutical composition according to the invention, are used in treatment of cancer, advantageously prostate cancer, cervix carcinoma, non-small cell lung cancer, in combination with at least one anticancer drug which induces apoptosis, advantageously doxorubicin, the TRAIL protein, camptothecin derivatives or other anticancer drugs.

In another advantageous embodiment, the recombinant fusion protein ProSTAT, advantageously ODD-ProSTAT according to the invention, or the pharmaceutical composition according to the invention, are used in treatment of cancer, advantageously prostate cancer, cervix carcinoma, non-small cell lung cancer, in combination with at least one anticancer drug such as an antibody or antibodies, or a low molecular weight anticancer compound, such as anti-metabolite, alkylating agent, topoisomerase inhibitor, inhibitor of microtubules polymerization, kinase inhibitor, inhibitor of protein synthesis, immunotherapeutic agent, hormone or its analogue, analogue of somatostatin, glucocorticoid, aromatase inhibitor, inhibitor of mTOR.

The recombinant fusion protein ProSTAT or a functional variant thereof, advantageously ODD- ProSTAT according to the invention, or the pharmaceutical composition according to the invention, can be used to sensitize cancer cells to induction of apoptosis, in different cancer therapies, advantageously against prostate cancer, cervix carcinoma, non-small cell lung cancer, and other cancer types.

In one advantageous embodiment, the recombinant fusion protein ProSTAT or a functional variant thereof, advantageously ODD-ProSTAT according to the invention, or the pharmaceutical composition according to the invention, are used by administration of a therapeutically effective dose of the recombinant fusion protein ProSTAT, advantageously ODD-ProSTAT or the pharmaceutical composition according to the invention, to a subject with cancer. The term "therapeutically effective dose" means a dose which brings about the effect for which it is used. An exact dose will depend on the aim of the treatment and it will be verifiable by a skilled person using known techniques. As is known in the field, corrections may be needed considering systemic or topical administration, age, bodyweight, general condition, sex, diet, time of administration, drug interactions and severity of the medical condition, and they will be verifiable with routine experiments by persons skilled in the field.

The recombinant fusion protein ProSTAT, advantageously ODD-ProSTAT according to the invention, or the pharmaceutical composition according to the invention, may be administered by one or more administration routes, using one or more of a variety of methods known in the art. A skilled person will appreciate that the administration routes and/or methods are diversified, depending on desired effects. Advantageous administration routes for the recombinant fusion protein ProSTAT, advantageously ODD-ProSTAT according to the invention, or the pharmaceutical composition according to the invention, include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intramedullary or other parenteral administration routes, for example injections or infusions. The term "parenteral administration" as used herein denotes the administration routes different than enteral or a topical, usually in the form of an injection, and includes, without limitations, intravenous, intramuscular, intraarterial, intra-articular, intradermal, intraperitoneal, subcutaneous, subepidermal, injections and infusions. Alternatively, the recombinant fusion protein ProSTAT, advantageously ODD- ProSTAT according to the invention, or the pharmaceutical composition according to the invention, may be administered by a non-parenteral administration route, such as topical or epidermal. The recombinant fusion protein ProSTAT, advantageously ODD-ProSTAT according to the invention, may be prepared together with carriers protecting the compound against rapid release, such as formulations for controlled release, including implants, transdermal patches and microcapsule drug delivery systems. Biodegradable, biocompatible polymers, such as ethylene with vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid, may be used. Numerous methods of producing such formulations have been patented or are generally known to skilled persons (see, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson ed., Marcel Dekker, Inc. New York, 1978).

In one advantageous embodiment, the recombinant fusion protein ProSTAT, advantageously ODD-ProSTAT according to the invention, or the pharmaceutical composition according to the invention, are administered in combination with one or more anticancer substances, wherein the administration may be separated in time or simultaneous.

The invention also provides an expression vector comprising a nucleotide sequence of the recombinant fusion protein ProSTAT, advantageously ODD-ProSTAT according to the invention, or a functional variant thereof, that enables production and purification of the recombinant fusion protein ProSTAT, advantageously ODD-ProSTAT. Advantageously, the expression vector comprises a nucleotide sequence of any vector used for protein expression in bacterial, yeast, insect, mammalian cell-based or other systems, wherein the sequence encoding the fusion protein ProSTAT has been inserted, advantageously of SEQ ID NO: 6, wherein the advantageous nucleotide coding sequence is SEQ ID NO: 5, and for the ODD- ProSTAT protein, advantageously of SEQ ID NO: 7, wherein the advantageous nucleotide coding sequence is SEQ ID NO: 8.

The invention also provides use of the recombinant fusion protein ProSTAT, advantageously ODD-ProSTAT or a functional variant thereof according to the invention, or the pharmaceutical composition according to the invention, as a medicament in the treatment of cancer, advantageously prostate cancer, cervix carcinoma, non-small cell lung cancer.

In particular, the invention provides use of the recombinant fusion protein ProSTAT, advantageously ODD-ProSTAT according to the invention, or a functional variant thereof according to the invention, or the pharmaceutical composition according to the invention, as an agent for selective sensitization of cancer cells to anticancer drugs which induce apoptosis, advantageously the cancer cells are prostate cancer, cervix carcinoma, non-small cell lung cancer cells.

Advantageously, the recombinant fusion protein ProSTAT, more advantageously ODD- ProSTAT according to the invention, or a functional variant thereof, or the pharmaceutical composition according to the invention, are used as agents for selective sensitization of cancer cells to anticancer drugs which induce apoptosis in combination with at least one anticancer drug which induces apoptosis, advantageously doxorubicin, TRAIL protein, camptothecin derivatives or other anticancer drugs.

The publications cited here and references therein are hereby incorporated herein in their entirety as reference.

In order to better understand the invention, it is illustrated with examples and the appended drawings as well as one table.

Brief description of drawings

Fig. 1 demonstrates the cassette organization of the fusion protein ProSTAT, in one of advantageous variants (diagram A) and the changes introduced into the active cassette (marked in capital letters) in the mutant protected against phosphorylation by the CK2 kinase (ProSTAT^{T58A S75A}; diagram B), and in the mutant not cleaved by caspase 8 (ProSTAT^D59E; diagram C).

Fig. 2 demonstrates the purity of protein preparations used in the tests: the fusion protein ProSTAT, the fusion protein ProSTAT mutated at the sites phosphorylated by the CK2 kinase (ProSTAT^{T58A S75A}), the fusion protein ProSTAT mutated at the site cleaved by caspase 8 (ProSTAT^D59E), and the TRAIL protein. Proteins were analyzed using SDS-PAGE. M denotes molecular weight standards in kDa.

Fig. 3 shows the dose-dependence (A) and administration time-dependence (B-E) for the level of the recombinant fusion protein ProSTAT in the cells. A, B. PC3 cells. C. LNCaP cells. D. A549 cells. E. HeLa cells. ProSTAT and endogenous BID content were identified in cell extracts after electrophoretic separation of proteins using anti-BID antibodies. A. PC3 cells were cultured for 120 min in the presence of different doses of the fusion protein ProSTAT. B, C. PC3 or LNCaP cells were cultured for different times (up to 120 min) in the presence of the fusion protein ProSTAT at the concentration of 30 μg/ml. D, E. A549 or HeLa cells were cultured for different times (up to 120 min) in the presence of the fusion protein ProSTAT at the concentration of 40 μg/ml. GAPDH protein present in the extract, its content identified using anti-GAPDH antibodies, was used as control for the amount of extract loaded onto a polyacrylamide gel. The amount of the fusion protein ProSTAT in the cells in relation to the amount of endogenous BID (with the endogenous BID level taken as 100%) is shown on the graph.

Fig. 4 shows the results of the analysis of viability of PC3 control cells and cells treated for 24 h with different doses of the fusion protein ProSTAT, measured by an MTT assay. The mean relative cell viability (± SD) is shown. Fig. 5 shows the results of the analysis of sensitization of different cell lines to TRAIL by the fusion protein ProSTAT. The mean relative cell viability (± SD) is shown, for the cells treated for 24 h with the fusion protein ProSTAT (30 vg/m\ for PC3 and LNCaP cells, and 40 g/ml for HeLa and A549 cells), TRAIL (100 ng/ml), or both proteins. The analysis was conducted by an MTT assay. Statistically significant differences are shown with p-value < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).

Fig. 6 shows the results of the analysis of sensitization of different cell lines to camptothecin (CPT) by the fusion protein ProSTAT. The mean relative cell viability (± SD) is shown, for the cells treated for 24 h with the fusion protein ProSTAT (30 μg/m\ for PC3 and LNCaP cells, and 40 μg/ml for HeLa and A549 cells), CPT (0.2 μΜ for HeLa cells and 1 μΜ for the remaining cell lines), or both compounds. The analysis was conducted by an MTT assay. Statistically significant differences are shown as on Fig. 5.

Fig. 7 shows the results of the analysis of sensitization of different cell lines to doxorubicin (DOX) by the fusion protein ProSTAT. The mean relative cell viability (± SD) is shown, for the cells treated for 48 h with the fusion protein ProSTAT (30 μg/m\ for PC3 and LNCaP cells, and 40 μg/ml for HeLa and A549 cells), DOX (1 μΜ), or both compounds. The analysis was conducted by an MTT assay. Statistically significant differences are shown as on Fig. 5.

Fig. 8 shows the results of the analysis of the effects of mutated variants of the fusion protein ProSTAT on the PC3 cells. A. Lack of sensitization of PC3 cells by the protein not cleaved by caspase 8 (ProSTAT^D59E), administered in 30 g/ml to TRAIL (100 ng/ml) or CPT (1 μΜ). B. Similarity of sensitization of PC3 cells by the wild type fusion protein ProSTAT and its variant not phosphorylated by the CK2 kinase (ProSTAT^{T58A S75A}), both administered at the dose of 30 μg/ml, to TRAI L. TRAIL concentration 100 ng/ml. The mean experimental results are shown on the figure, for the wild type ProSTAT (light bars); and for ProSTAT mutated at the sites phosphorylated by the CK2 kinase (ProSTAT^{T58A S75A}, dark bars). The analysis was conducted by an MTT assay. Relative cell viability (± SD) is shown.

Fig. 9 shows the results of the cytometric analysis of sensitization of PC3 cells by the fusion protein ProSTAT to apoptosis induction by TRAIL. A. Percentage of apoptotic cells (± SD). Cells were treated for 24 h with the fusion protein ProSTAT (30 Mg/ml), TRAIL (200 ng/ml), or both proteins. Apoptotic cells were detected by flow cytometry. Statistically significant differences are shown as on Fig. 5. B-E. Representative cytograms from one of the experiments with results presented on Fig. 9A.

Fig. 10 shows cytochrome c release from mitochondria of PC3 cells treated for 4 h with ProSTAT (30 μg/ml), TRAIL (100 ng/ml), or both proteins. Cytochrome c level in mitochondria is expressed relative to the GAPDH value. Cytochrome c and GAPDH were identified in mitochondrial extracts using specific antibodies. Fig. 11 shows representative microscopic photographs of the cells treated with ProSTAT (30 μg/ml for PC3 cells or 40 μg/ml for HeLa cells) and either TRAIL (200 ng/ml) or doxorubicin (1 μΜ). Cells were stained with Giemsa and May-Grunwald dyes. A. PC3 cells treated for 24 h with ProSTAT, TRAIL, or both proteins. B. HeLa cells treated for 48 h with ProSTAT, doxorubicin (DOX), or both compounds.

Fig. 12 shows the diagram (A) and properties (B, C) of the fusion protein ODD-ProSTAT (ProSTAT protein enriched with the cassette that increases its selectivity, namely the targeting cassette ODD which ensures specificity of the protein towards cells under hypoxia conditions). Cells of the PC3 line cultured under normoxia or hypoxia conditions were treated for 48 h with the fusion protein ODD-ProSTAT (30 μg/ml), the TRAIL protein (100 ng/ml) or both proteins. The level of the fusion protein ODD-ProSTAT was determined in cell extracts after electrophoretic separation of the proteins using anti-HA antibodies. Viability of the cells was measured by an MTT assay. A. Cassette organization of ODD-ProSTAT molecule. B. The level of ODD-ProSTAT protein in cellular extracts from the cells cultured under normoxia and hypoxia conditions. C. Viability of the cells treated with ODD-ProSTAT, TRAIL, or both proteins; cultured for 48 h under normoxia (dark bars) or hypoxia (light bars).

DESCRIPTION OF EMBODIMENTS

The following examples are presented merely to illustrate the invention and to clarify its various aspects, but are not intended to be limitative, and should not be equated with all its scope, which is defined in the appended claims.

EXAMPLES

In the following examples, unless otherwise indicated, standard materials and methods described in: Sambrook et al., Molecular cloning: A laboratory manual. 2nd edition. 1989. Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory Press were used, or the manufacturers' instructions for specific materials and methods were followed.

Example 1 :

Construction of the recombinant protein ProSTAT

DNA fragment encoding human BID (BID(L), isoform 1 , 194 aa in length; according to UniProtKB/Swiss-Prot P55957), without the START and STOP codons (SEQ ID NO: 1), was amplified by PCR using Pfx polymerase (Invitrogen) and primers BIDIong.EcoRI.For (CGGAATTCGACTGTGAGGTCAACAACGG) (SEQ ID NO: 9) and BIDIong.Hindlll.Rev (CCCAAGCTTGGTCCATCCCATTTCTGGCTAAGC) (SEQ ID NO: 10), and using plasmid IRATp 970C1135D (imaGenes), containing full length cDNA BID clone (BC036364, from 467 to 1054 nt, and a nucleotide sequence encoding BID(L) present between 629 to 1054 nt), as a template. Thus obtained DNA fragment was cloned into the expression vector pET28a (Novagen) between nucleotide sequences recognized by restriction enzymes EcoRI i Hindi 11 , which was previously modified as follows:

- a nucleotide sequence: TACCCATACGATGTTCCTGACTATGCGGGCTATCCCTATGACGTC CCCGACTATGCAGGATCCTATCCATATGACGTTCCAGATTACGCT (SEQ ID NO: 11), encoding three repeats of the HA epitope (fragment of the hemagglutinin protein, from an influenza virus), separated by one or two amino acid residues, with a final amino acid sequence of YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA (SEQ ID NO: 12) and recognized by anti-HA antibodies, was added between the sites recognized by restriction enzymes Notl and Xhol.

- a nucleotide sequence recognized by restriction enzyme BamHI was partly removed from between nucleotide sequences recognized by restriction enzymes Ndel and EcoRI (only GGA remained from the original sequence GGATCC), and DNA fragment of a nucleotide sequence set forth in SEQ ID NO: 3, coding TAT domain (amino acid residues from 47 to 57 in Tat protein of HIV-1 virus) was inserted at this site.

An amino acid sequence encoded by the nucleotide sequence SEQ ID NO: 1 is an active cassette of the fusion protein ProSTAT (SEQ ID NO: 2). An amino acid sequence coded by the nucleotide sequence SEQ ID NO: 3 is a transporting cassette of the fusion protein ProSTAT (SEQ ID NO: 4). The final nucleotide sequence encoding the fusion protein ProSTAT, inserted to expression vector pET28a (Novagen), is set forth in SEQ ID NO: 5. An amino acid sequence of the fusion protein ProSTAT is set forth in SEQ ID NO: 6.

In addition to the fusion protein ProSTAT of the amino acid sequence set forth in SEQ ID NO: 6, in experiments described in Example 9, concerning the properties of the protein, two other variants of the protein were also used: the mutant ProSTAT^{T58A S75A}, not phosphorylated by the CK2 kinase, and having threonine at site 58 and serine at site 75 with respect to the amino acid sequence set forth in SEQ ID NO: 2 substituted with alanine, and the mutant ProSTAT^D59E, not cleaved by caspase 8, and having aspartic acid at site 59 with respect to the amino acid sequence set forth in SEQ ID NO: 2 substituted with glutamic acid. All mutations were introduced using a standard method of site-directed mutagenesis and were verified by sequencing of the obtained DNA. Diagrams of the fusion protein ProSTAT and of its two variants, which illustrate the organization of transporting and active cassettes, and also cassettes 6xHis and HA used for identification of the protein, are shown on Fig. 1.

Example 2:

Expression and purification of the recombinant protein ProSTAT

Fusion protein ProSTAT was expressed in Escherichia coli BL21 (DE3) cells transformed with the plasmid containing the nucleotide sequence encoding the fusion protein ProSTAT (SEQ ID NO: 5) described in Example 1. The protein was purified using affinity chromatography on Ni- NTA agarose (Sigma Aldrich) and gel filtration on Applixchange-G25M (AppliChem Panreac). Fig. 2 shows the purity of the ProSTAT protein preparations isolated this way: the fusion protein ProSTAT and its mutants: ProSTAT^{T58A S75A} and ProSTAT^D59E, and also TRAIL protein used to induce apoptosis.

Example 3:

Evaluation of time- and dose-dependent uptake of the recombinant fusion protein ProSTAT into cells

The PC3, LNCaP, HeLa and A549 cells obtained from the European Collection of Cell Cultures (ECACC) were cultured in RPMI-1640 (in the case of cells of the PC3 and LNCaP line), F12K (in the case of cells of the A549 line) or DMEM medium (in the case of cells of the HeLa line), on dishes under standard conditions at 37°C in 5% C0₂. Fusion protein ProSTAT was directly added to the medium in the presence of trypsin inhibitor from soybean (final concentration 0.005%). To estimate the dose-dependent uptake of the fusion protein ProSTAT, different amounts of the protein (final concentration 0 - 40 μg/ml) were added to the culture of PC3 cells for 120 min. To estimate the time-dependent uptake of the fusion protein ProSTAT the protein was added to cultures of different cell lines to the final concentration 30 μg/ml (in the case of PC3 and LNCaP cells) or 40 Mg/ml (in the case of A549 and HeLa cells) for 0, 30, 60 or 120 min. Cells were then washed, trypsinized, washed once more, counted, suspended in Laemmli buffer to the concentration 2x10⁴ cells/μΙ and boiled for 20 min at 100°C. Protein concentration was measured using Bradford assay (Compton & Jones, 1985), and the samples were loaded on gel in amount corresponding to at least 30 ig protein/well. Proteins were separated in polyacrylamide gel under denaturing conditions and then transferred onto a PVDF membrane. Proteins were identified by Western blot method using anti-BID (1 : 1000, Santa Cruz) or anti- GAPDH (1 : 100 000, Sigma Aldrich) antibodies. The results were quantified by densitometry using ChemiDocXRS (BioRad). On the graphs shown on Fig. 3 the amount of the fusion protein ProSTAT in the cells is presented relative to endogenous BID (the level of endogenous BID taken as 100%).

As is shown on Fig. 3A, the fusion protein ProSTAT enters PC3 cells in a dose-dependent manner. The protein also enters the cells in an administration time-dependent manner (Fig. 3B- E). The kinetics of the uptake depends on the cell line: ProSTAT is most rapidly taken up by LNCaP (Fig. 3C), slower by PC3 and A549 cells (Fig. 3B and D), and the slowest by HeLa cells (Fig. 3E). Taken the above into consideration, it may be concluded that for a particular cell line and, eventually, for a particular cancer type, it is possible to maintain a stable intracellular level of ProSTAT by controlling both parameters, i.e. administration time and dose.

Example 4: Evaluation of cytotoxicity of the fusion protein ProSTAT

PC3 cells were cultured in 96-wells plates. The Fusion protein ProSTAT was directly added to the medium to the final concentration 0 - 40 μςΛηΙ, in the presence of trypsin inhibitor from soybean (final concentration 0.005%), and the cells were cultured for 24 h. After removal of the culture medium, MTT mixture (Twentyman & Luscombe, 1987) was added to the concentration 0.5 mg/ml and the cells were then cultured for additional 2 h. The formazan crystals were diluted in DMSO-isopropanol mixture (1 :1) and the absorbance was measured at 570 nm, using VICTOR Multilabel Plate Reader (PerkinElmer).

As can be seen on Fig. 4, the fusion protein ProSTAT present in the culture medium lowers cell viability only marginally (less than 10% decrease in viability) up to 40 μg/ml.

Example 5:

Evaluation of the sensitization of different cell lines by the fusion protein ProSTAT to TRAIL

Four cancer cell lines were compared in terms of their ability to be sensitized by ProSTAT: two lines derived from prostate cancer (PC3 and LNCaP), one line derived from non-small cell lung cancer (A549) and one line derived from cervix carcinoma (HeLa). Cells were cultured and their viability was measured as described in Example 4, except that MTT mixture was added to the concentration of 2.5 mg/ml in the case of HeLa cells. Fusion protein ProSTAT was added to the culture to the concentration of 30 μg/ml for cell lines PC3 and LNCaP or 40 μg/ml for cell lines A549 and HeLa, and TRAIL to the concentration of 100 ng/ml and the cells were grown for 24 hours. As it can be seen on Fig. 5, all cell lines except for LNCaP are sensitized by ProSTAT to TRAIL with the following efficiency: PC3>A549>HeLa. Taking into account different kinetics of ProSTAT entering into different cell lines described in Example 3, experiments using higher doses of the fusion protein ProSTAT were carried out. Increase of a dose of ProSTAT up to 50 μg/ml does not change the efficiency of sensitization of cell lines to TRAIL. Lack of sensitization of cell line LNCaP is most probably caused by insensitivity of these cells to TRAIL, resulting from constitutive expression of c-FLIP protein (Zhang et al., 2004).

Example 6:

Evaluation of sensitization of different cell lines by fusion protein ProSTAT to camptothecin (CPT)

The evaluation was performed as described in Example 5 except that CPT was added to the culture medium at the concentration 1 μΜ (0.2 μΜ for HeLa cells) instead of TRAIL. As it can be seen on Fig. 6, ProSTAT sensitizes to CPT only the PC3 cells in a statistically significant manner; the remaining cell lines are not sensitized in a statistically significant manner. Example 7:

Evaluation of sensitization of different cell lines by the fusion protein ProSTAT to doxorubicin (DOX)

The evaluation was performed as described in Example 5 except that doxorubicin was added to the culture medium at the concentration 1 μΜ instead of TRAIL, and that cells were treated with DOX for 48 h. As it can be seen on Fig. 7, ProSTAT sensitizes to DOX all cell lines in a statistically significant manner, except for LNCaP. LNCaP cell line is especially sensitive to DOX administrated without ProSTAT. Under the conditions presented on Fig. 7, treatment of LNCaP cells with DOX alone results in decrease of cell viability by about 85%. Lowering of the DOX dose to 0.5 μΜ, combined with shortening of administration time to 24 h reduces the decrease of cell viability caused by DOX alone to about 40%; however, even under the latter conditions sensitization of LNCaP cells by ProSTAT to DOX is not observed.

Example 8:

Evaluation of synergy between ProSTAT and different drugs for particular cell lines The cells were treated as follows: ProSTAT at 30 μg/ml for PC3 and LNCaP cells or 40 μg/ml for A549 and HeLa cells; TRAIL 100 ng/ml; camptothecin 0.2 μΜ for HeLa cells or 1 μΜ for the remaining cells; doxorubicin 1 μΜ. Treatment time 24 h in experiments with TRAIL or camptothecin and 48 h in experiments with doxorubicin. Cell viability was measured by an MTT assay as described in Example 5. The evaluation was done using a coefficient of drug interaction (CDI) which was calculated as follows: (viability of cells treated with ProSTAT+drug)/(viability of cells treated with ProSTAT)x(viability of cells treated with the drug). The following ways of drug interaction identified by CDI are distinguished: antagonism (CDI>1), additivity (CDI=1), weak synergy (CDI=0.8 - 0.9), moderate synergy (CDI=0.7 - 0.8), significant synergy (CDI<0.7). Table 1

Cell line TRAIL camptothecin doxorubicin

PC3 0.7663 0.8831 0.7165

A549 0.7929 0.9347 0.9360

HeLa 0.8653 0.9418 0.5129

LNCaP 0.9697 0.9572 0.9102 As can be seen in Table 1 , weak synergy is observed for ProSTAT+camptothecin acting on PC3 cells, and for ProSTAT+TRAIL acting on HeLa cells; moderate synergy for ProSTAT+TRAIL acting on PC3 or A549 cells, and also for ProSTAT+DOX acting on PC3 cells; and significant synergy for ProSTAT+DOX acting on HeLa cells. Example 9:

Evaluation of selectivity of the fusion protein ODD-ProSTAT (ProSTAT protein fused with the ODD targeting cassette)

A nucleotide sequence encoding the ODD cassette was added to the nucleotide sequence encoding the fusion protein ProSTAT, according to the following protocol. DNA fragment encoding the human ODD domain, 56 amino acid residues in length (a fragment of HIF1a protein, GenBank AAC50152.1 , from 548 to 603 amino acid residue) was amplified by PCR using Pfu Turbo polymerase (Agilent) and primers HIF1548For (CGGAATTCAACCCATTTTCTACTCAGGACACA) (SEQ ID NO: 13) and HIF1603Rev (CGGAATTCCTGGAATACTGTAACTGTGCTTTG) (SEQ ID NO: 14), and using plasmid pGEX- 4T-1-HIF1a (Chachami et al., 2005) as a template. The so obtained DNA fragment was cloned into the expression vector for the ProSTAT protein described in Example 1 at the site recognized by restriction enzyme EcoRI, between the sequences encoding the TAT domain and the BID protein. A final nucleotide sequence coding the ODD-ProSTAT protein is set forth in SEQ ID NO: 8. When compared to the nucleotide sequence encoding the ProSTAT protein, the inserted fragment encoding the ODD domain begins at position 106 and ends at position 273 of SEQ ID NO: 8. An amino acid sequence of the fusion protein ODD-ProSTAT is set forth in SEQ ID NO: 7. As compared to the amino acid sequence of the ProSTAT protein, the fragment comprising the ODD domain begins at position 36 and end at position 91 of SEQ ID NO: 7.

PC3 cells were cultured under either normal (21 %; normoxia) or decreased oxygen concentration (1 %; hypoxia), in the presence of the ODD-ProSTAT fusion protein (30 μg/ml), the TRAIL protein (100 ng/ml) or both proteins for 48 h. The level of ODD-ProSTAT was evaluated in cell extracts after electrophoretic separation of proteins using anti-HA antibodies (1 :2000, Sigma Aldrich). Cell viability was measured after 48 h of culture by an MTT assay, as described in Example 5. As can be seen on Fig. 12B, in the cells cultured under normoxia, the fusion protein ODD- ProSTAT is completely degraded after 48 h, whereas it is still present in the cells cultured under hypoxia conditions. Moreover, as can be seen on Fig. 12C, the ODD-ProSTAT protein sensitizes the cells cultured for 48 h under hypoxia to TRAIL, whereas the cells cultured for the same time under normoxia are not sensitized. The above results demonstrate that the protein ProSTAT can be given additional functions, advantageously selectivity towards cancer cells, if an additional cassette, advantageously a targeting cassette, more advantageously the ODD cassette, is added to it.

Cited literature included as references:

Chachami, G. et al. (2005) Biochim. Biophys. Res. Commun. 331 , 464-470.

Compton, S.J. & Jones, C.G. (1985) Anal. Biochem. 151 , 369-374.

Danhier, F. et al. (2012) J. Control. Release 161 , 505-522.

Dearnaley, D.P. et al. (1999) Lancet 353,267-272.

European age-standardized rates calculated by the Statistical Information Team at Cancer

Research UK (201 1) using data from GLOBOCAN 2008, v1.2 http://globocan.iarc.fr

Ferlay, J. et al. (2010) Int. J. Cancer 127, 2893-2917.

Fukazawa, T. et al. (2003) J. Biol. Chem. 278, 25428-25434.

Fukazawa, T. et al. (2009) Int. J. Cancer. 125, 1975-1984.

Global Health Observatory Data Repository. Cancer Mortality and Morbidity. Situation and

Trends. WHO.

Gizzo, S. et al. (2013) Oncol. Rep. 30, 2545-2554.

Griffith, T.S. & Kemp, T.J. (2003) Cancer Chemother. Pharmacol. 52, 175-184

Heidenreich, A. et al. (2008) Eur. Urol. 53, 68-80.

Huang et al. (1998) Proc. Natl. Acad. Sci. USA 95, 7987-7992.

Jassem, J. et al. (eds) (2013) Nowotwory uktadu moczowo-ptciowego. Via Medica, Gdahsk.

Kantari, C. & Walczak, H. (201 1) Biochim Biophys Acta 1813, 558-563.

Kazhdan, I. et al. (2006) Cancer Gene Ther.13, 141-149.

Kotoula, V. et al. (2002) Liver 22, 57-69.

Liu, Y. et al. (2011) Mol. Cell. Biol. 31 , 4298^1309.

Markman, M. (2014) Expert Rev. Clin. Pharmacol. 7, 219-223.

Melosky, B. (2014) Front. Oncol. 4:256.

Miao, J. et al. (2006) Int. J. Cancer 119, 1985-1993.

Orzechowska, E.J. et al. (2014) BMC Cancer 14:771.

Robinson, J.R. (ed) (1978) Sustained and Controlled Release Drug Delivery Systems., Marcel

Dekker Inc., New York.

Salomon-Perzyhska, M. et al. (2014) Ginekol. Pol. 85, 461-465.

Song, G. et al. (2008) Apoptosis 13, 693-701.

Tang, D.G. & Porter, AT. (1997) Prostate 32, 284-293.

The Cancer Market Outlook to 2016. Competitive landscape, market size, pipeline analysis, and growth opportunities (201 1) Reference Code: BI00042-009. Tsuno, T. et al. (2012) J. Immunother. 35, 23-31.

Twentyman, P.R. & Luscombe, M. (1987) Br. J. Cancer 56, 279-285.

Yi, X. et al. (2003) J. Biol. Chem. 278, 16992-16999.

van Beers, M.M.C & Bardor, M. (2012) Biotechnol. J. 7, 1-12.

Vaupel, P. & Kelleher, D.K. (2013) Adv. Exp. Med. Biol. 765, 299-305.

Wang, G. et al. (2006) J. Cell. Biochem. 99, 382-391.

Wirth, T. et al. (2003) Cancer Res. 63, 3181-3188.

Zhang, X. et al. (2004) Cancer Res. 64, 7086-7091. Sequence listing

SEQ ID NO: 1 - Nucleotide sequence encoding the active cassette;

SEQ ID NO: 2 - Amino acid sequence of the active cassette;

SEQ ID NO: 3 - Nucleotide sequence encoding the transporting cassette;

SEQ ID NO: 4 - Amino acid sequence of the transporting cassette;

SEQ ID NO: 5 - Nucleotide sequence encoding the advantageous fusion protein ProSTAT; SEQ ID NO: 6 - Amino acid sequence of the advantageous fusion protein ProSTAT;

SEQ ID NO: 7 - Amino acid sequence of the advantageous fusion protein ODD-ProSTAT; SEQ ID NO: 8 - Nucleotide sequence encoding the advantageous fusion protein ODD- ProSTAT;

SEQ ID NO: 9 - Nucleotide sequence of the primer BIDIong.EcoRI.For;

SEQ ID NO: 10 - Nucleotide sequence of the primer BIDIong.Hindlll.Rev;

SEQ ID NO: 11 - Nucleotide sequence encoding the tag 3xHA;

SEQ ID NO: 12 - Amino acid sequence of the tag 3xHA;

SEQ ID NO: 13 - Nucleotide sequence of the primer HIF1548For;

SEQ ID NO: 14 - Nucleotide sequence of the primer HIF1603Rev.

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Claims

Claims

1. A recombinant fusion protein ProSTAT, comprising a transporting cassette which enables the protein to enter into the cell, and an active cassette which selectively sensitizes cancer cells, preferably of prostate cancer, cervix carcinoma, non-small cell lung cancer, to anticancer drugs inducing apoptosis, wherein the active cassette comprises the amino acid sequence of the human BID protein or a functional variant thereof, and wherein the transporting cassette comprises a short peptide derived from the TAT protein.

2. The recombinant protein according to claim 1 , wherein the transporting cassette comprises the sequence of SEQ ID NO: 4.

3. The recombinant protein according to claims 1 or 2, wherein active cassette comprises the sequence of the human protein BID(L), isoform 1 , preferably the sequence of SEQ ID NO: 2.

4. The recombinant protein according to claims 1 -3, wherein it comprises the sequence of SEQ ID NO: 6.

5. The recombinant protein according to any of claims 1-4, wherein it further comprises targeting cassette which enhances selectivity, and wherein preferably the targeting cassette which enhances selectivity is ODD (oxygen destruction domain).

6. The recombinant protein according to claim 5, wherein it comprises the fusion protein ODD-ProSTAT having the sequence of SEQ ID NO: 7.

7. The recombinant protein according to any of claims 1-6, wherein it is a functional variant of the recombinant protein ProSTAT protected against phosphorylation by the CK2 kinase.

8. The recombinant protein according to claim 7, wherein mutations T58A and S75A have been introduced into the active cassette, preferably mutations T58A and S75A in SEQ ID NO: 2.

9. A pharmaceutical composition comprising the recombinant protein according to any of claims 1-8.

10. The pharmaceutical composition of claim 9, wherein the composition comprises pharmaceutically acceptable substances which lower the sensitivity to proteolytic degradation and/or reduce immunogenicity of the recombinant fusion protein ProSTAT of claims 1-8.

11. The pharmaceutical composition of claims 9-10, characterized in that it further comprises an anticancer compound which induces apoptosis, preferably doxorubicin, TRAIL protein, camptothecin or derivatives thereof.

12. The recombinant protein of claims 1-8, or the pharmaceutical composition of claims 9-11 , for use as a medicament.

13. The recombinant protein according to any of claims 1-8, or the pharmaceutical composition of claims 9-11 , for use in the treatment of cancer, preferably prostate cancer, cervix carcinoma, non-small cell lung cancer.

14. The recombinant protein of claims 1-8, or the pharmaceutical composition of claims 9-11 , for use according to claims 12-13, wherein the recombinant protein or the pharmaceutical composition are used in combination with at least one anticancer drug which induces apoptosis, preferably doxorubicin, TRAIL protein, camptothecin derivatives or other anticancer drugs.

15. An expression vector comprising the sequence of the recombinant protein according to any of claims 1-8, which enables production and purification of the recombinant fusion protein ProSTAT.

16. The expression vector according to claim 15, wherein said vector comprises the sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 8.

17. Use of the recombinant protein according to any of claims 1-8, or of the pharmaceutical composition of claims 9-11 , as a medicament in the treatment of cancer, preferably prostate cancer, cervix carcinoma, non-small cell lung cancer.

18. Use of the recombinant protein according to any of claims 1-8, or of the pharmaceutical composition of claims 9-11 , as an agent which selectively sensitizes cancer cells to anticancer drugs which induce apoptosis.

19. The use of claim 18, wherein cancer cells are cells of prostate cancer, cervix carcinoma, non-small lung cancer.

20. The use of claims 18-19, wherein recombinant protein or the pharmaceutical composition are used in combination with at least one anticancer drug which induces apoptosis, advantageously doxorubicin, TRAIL protein, camptothecin derivatives or other anticancer drugs.