Muc-1 protein cytoplasmic domain peptides as cancer inhibitors

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to MUC1 cytoplasmic domain peptides, and can be used in the anticancer therapy. A method for inhibiting MUC1-positive cancer cell in an individual involves administering into an individual the MUC1-peptide of the length of at least 6 sequential MUC1 residues and no more than 20 sequential MUC1residues and containing the sequence CQCRRK, wherein the amino terminal cysteine from CQCRRK is closed at its NH2 terminal by at least one amino acid residue, which shall not conform with the native transmembrane sequence MUC-1. Alternatively, there can be used MUC-1 peptide of the length of at least sequential MUC1 residues and no more than 20 sequential MUC1 residues, which contains the sequence CQCRRK with all amino acid residues of the above peptide being D-amino acids.

EFFECT: invention enables inhibiting MUC1oligomerisation effectively and inducing the tumour cell apoptosis and the tumour tissue necrosis in vivo.

80 cl, 16 dwg, 1 tbl, 3 ex

 

Background of the invention

This application claims priority under provisional application U.S. No. 61/106380, filed October 17, 2008, and provisional application U.S. No. 61/177109, filed may 11, 2009, the complete contents of which are incorporated into this description by reference.

1. The technical field to which the invention relates.

The invention relates to the regulation of cell growth and, more specifically, to regulate the growth of cancer cells. In particular, it was shown that MUC1 peptides derived from the sequence of a specific region of the cytoplasmic domain of MUC1 protein, inhibit oligomerization and nuclear translocation of MUC1, causing stunting and even the death of tumor cells expressing MUC1.

2. The level of technology

The mucines are intensively O-glycosylated proteins that are expressed predominantly by epithelial cells. Secreted and membrane-bound mucines form a physical barrier that protects the apical border of epithelial cells from damage caused by toxins, pathogens and other stress factors that occur at the boundary with the external environment. Transmembrane mucin 1 (MUC1) can also transmit the signal into the cell via its cytoplasmic domain. MUC1 has no homology by follow the Telenesti with other membrane-bound by mucines, with the exception of domain protein of the sea urchin sperm/enterokinase/agrin (SEA-domain) (Duraisamyet al., 2006). Due to the presence of this domain, MUC1 is translated as a single polypeptide, and then undergoes authorssvetlana on SEA domain (Macao, 2006).

N-terminal subunit of MUC1 (MUC1-N) contains a different number of tandem repeats with a high content of residues serine and threonine, modified by O-glycosylation (Siddiqui, 1988). MUC1-N is responsible for the glycocalyx of the cell and connected to the surface via non-covalent binding to the transmembrane C-terminal subunit MUC1 (MUC1-C) (Merlo, 1989). MUC1-C consists of a 58-amino acid extracellular domain, a 28-amino acid transmembrane domain and a 72 amino acid cytoplasmic domain, which interacts with various signaling molecules (Kufe, 2008). Dropping MUC1-N in a protective physical barrier leaves MUC1-C on the cell surface as a potential receptor for the transmission of intracellular signals that lead to the growth and survival of cells (Ramasamy Thursday,et al., 2007; Ahmadet al., 2007).

Currently available data indicate that carcinoma of the person using the function of MUC1 to enhance the oncogenic properties. In this case, transformation and loss of polarity of the high level of expression of MUC1 was observed throughout the cell surface EP is Telia mammary glands and other organs (Kufe, 1984). In another paper it was shown that overexpression of MUC1 causes independent of the attachment of growth and oncogenic properties (Liet al.2003a; Rainaet al., 2004; Renet al., 2004; Weiet al., 2005) at least partially due to the stabilization of β-catenin (Huanget al., 2005). Moreover, in accordance with the function of ensuring the survival of normal epithelial cells, overexpression of MUC1 gives stability to the carcinoma cells to induced stress apoptosis (Renet al., 2004; Yin and Kufe, 2003; Yinet al., 2004;Yinet al., 2007).

The loss of restrictions on the apical membrane enables the formation of complexes with growth factor receptor epidermal (EGFR) and coactively mediated EGFR signaling pathways (Liet al., 2001; Ramasamy Thursday,et al., 2007). Overexpression of MUC1 cell carcinoma is also associated with accumulation of MUC1-C in the cytosol and translocation of this subunit in the nucleus of the cell (Liet al., 2003b; Liet al., 2003c) and the mitochondrion (Renet al., 2004; Renet al., 2006). It is important to note that the oligomerization of MUC1-C is required for its transfer to the nucleus and interaction with different effectors (Lenget al., 2007). For example, the cytoplasmic domain of MUC1-C (MUC1-CD) functions as a substrate for c-Src (Liet al., 2001), c-Abl (Rainaet al., 2006), protein kinase Cδ (Renet al., 2002) and glycogen synthase kinase 3β (Liet al., 1998) and interacts directly with the effect the torus Wnt-signaling pathway, β-catenin (Yamamotoet al., 1997; Huanget al.2005) and the tumor suppressor, p53 (Weiet al., 2005). Thus, although oligomerization, apparently, is important, there is no direct evidence that the disruption of the formation of oligomers MUC1 would have a negative effect on tumor cells, less is known about how to perform this task.

Summary of the invention

Thus, the present invention relates to a method of inhibiting tumor cells expressing MUC1 (MUC1-positive tumor cells), the subject, including the introduction of a specified subject MUC1-peptide of length at least 4 consecutive rest of MUC1 and no more than 20 consecutive MUC1 residues and containing the sequence CQC, in which aminobenzoic cysteine of CQC is closed at its NH2-end of at least one amino acid residue that need not correspond to the native transmembrane sequence of MUC-1. The peptide may contain at least 5 consecutive MUC1 residues, at least 6 consecutive MUC1 residues, at least 7 consecutive MUC1 residues, at least 8 consecutive MUC1 residues and, more specifically, the sequence may include CQCR (SEQ ID NO:54), CQCRR (SEQ ID NO:50), CQCRRR (SEQ ID NO:51), CQCRRRR (SEQ ID NO:52), CQCRRK (SEQ ID NO:4) or CQCRRKN (SEQ ID NO:53). Peptide outstanging not more than 10 consecutive residues, 11 consecutive residues, 12 consecutive residues, 13 consecutive residues, 14 consecutive residues, 15 consecutive residues, 16 consecutive residues, 17 consecutive residues, 18 consecutive residues or 19 consecutive MUC1 residues. The peptide can be fused with domain transfer in cells, such as poly-D-R, poly-D-P or poly-D-K. the Peptide can contain all L-amino acids, all D-amino acids or a mixture of L - and D-amino acids.

MUC1-positive tumor cells may be cell carcinoma, the cell leukemia or cell myeloma, such as cell carcinoma of the prostate or breast cancer. The introduction may include intravenous, intraarterial, intratumoral, subcutaneous, superficial, or intraperitoneal administration, or local, regional, system, or continuous administration. Inhibition may include stunting specified tumor cells, apoptosis specified tumor cells and/or necrosis of tumor tissue, including the indicated tumor cell. The subject may be human.

The method may further include holding for the specified subject a second anti-cancer therapy. The second anti-cancer therapy can be surgery, chemotherapy, radiation therapy, hormonal therapy, therapy toxins, immunotherapy and cryotherapy who I am. The second anti-cancer therapy can be done prior to the introduction of this peptide after administration of the indicated peptide or simultaneously with the introduction of this peptide. The method may additionally include the stage of evaluation of the expression of MUC1 in cancer cells specified subject before the introduction of this peptide, and/or the method may optionally include the stage of evaluation of the effect of the indicated peptide on the expression of MUC1 in tumor specified entity.

The peptide can be introduced in an amount of 0.1-500 mg/kg/day or 10-100 mg/kg/day. The peptide can be administered daily, for example, within 7 days, 2 weeks, 3 weeks, 4 weeks, one month, 6 weeks, 8 weeks, two months, 12 weeks or 3 months. The peptide can be administered weekly, for example, in 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks or 12 weeks.

In another embodiment, the invention relates to pharmaceutical compositions containing: (a) MUC1-peptide of length at least 4 consecutive rest of MUC1 and no more than 20 consecutive MUC1 residues and containing the sequence CQC, in which aminobenzoic cysteine of CQC is closed at its NH2-end of at least one amino acid residue that need not correspond to the native transmembrane sequence of MUC-1; and (b) a pharmaceutically acceptable carrier, buffer or diluent. Length is and the peptide may comprise at least 5, 6, 7 or 8 consecutive MUC1 residues. The length of the peptide may be no more than 10 consecutive residues, 11 consecutive residues, 12 consecutive residues, 13 consecutive residues, 14 consecutive residues, 15 consecutive residues, 16 consecutive residues, 17 consecutive residues, 18 consecutive residues or 19 consecutive MUC1 residues. The peptide may be fused with a domain for transfer into the cell, such as poly-D-R, poly-D-P or poly-D-K, or domain for transduction of cells, such as domain for transduction of cells of the HIV protein tat. The length of the peptide may comprise at least 8 amino acid residues, and at least two that are not adjacent to each other, balance, form a bridge through their side chains. The bridge may include a linker chemically modified side chains or hydrocarbon stitching. Linkers may contain modifications that stabilize the alpha-helical structure of the indicated peptide. The buffer can contain β-mercaptoethanol, glutathione or ascorbic acid or other reducing agent, which supports the peptide in Monomeric state.

In yet another embodiment, the invention relates to a method of inhibiting oligomerization and nuclear transport MUC1 in a cell, comprising contacting cells expressing MUC1, MUC1-Pat the house of length at least 4 consecutive rest of MUC1 and no more than 20 consecutive MUC1 residues, and containing the sequence CQC, in which aminobenzoic cysteine of CQC is closed at its NH2-end of at least one amino acid residue that need not correspond to the native transmembrane sequence of MUC-1. The peptide may contain at least 5 consecutive MUC1 residues, at least 6 consecutive MUC1 residues, at least 7 consecutive MUC1 residues, at least 8 consecutive MUC1 residues and, more specifically, a sequence can contain CQCR, CQCRR, CQCRRR, CQCRRRR, CQCRRK or CQCRRKN. The peptide may contain not more than 10 consecutive residues, 11 consecutive residues, 12 consecutive residues, 13 consecutive residues, 14 consecutive residues, 15 consecutive residues, 16 consecutive residues, 17 consecutive residues, 18 consecutive residues or 19 consecutive MUC1 residues. The peptide can be fused with domain transfer in cells, such as poly-D-R, poly-D-P or poly-D-K. the Peptide can contain all L-amino acids, all D-amino acids or a mixture of L - and D-amino acids.

Cells expressing MUC1, may be a tumor cell, such as cell carcinoma, the cell leukemia or cell myeloma, such as cell carcinoma of the prostate or breast cancer. The tumor cell may be located in a living subject. The subject may be a human who ovec.

In yet another embodiment, the invention relates to peptidomimetic, which mimic the structure and ability to bind MUC1 MUC1-peptide of length at least 4 consecutive rest of MUC1 and no more than 20 consecutive MUC1 residues and containing the sequence CQC, in which aminobenzoic cysteine of CQC is closed at its NH2-end of at least one amino acid residue that need not correspond to the native transmembrane sequence of MUC-1. An additional variant embodiment of the invention relates to a MUC1-peptide with a length of at least 3 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues and containing the sequence CQC, all amino acid residues of the specified peptide are D-amino acids. The peptide may further comprise a sequence KRRCQC (SEQ ID NO:49).

A cancer cell can be, for example, the cell breast cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, stomach cancer, liver cancer, bone cancer, hematological cancer, cancer, neuronal tissue, melanoma, ovarian cancer, testicular cancer, prostate cancer, cervical cancer, vaginal cancer, or bladder cancer.

The invention encompasses methods of destruction of cancer cells. The methods may include receiving one subject is about or more additional treatments before, after or concurrent with the methods described above. Methods of treatment may represent, for example, one or more forms of ionizing radiation and/or one or more chemotherapeutic agents. One or more chemotherapeutic agents can be, for example, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosamine, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide, feranil, podophyllotoxin, tamoxifen, Taxol, transplatin, 5-fluorouracil, vincristine, vinblastine, methotrexate or an analog of any of the above compounds. Also, as combined methods of treatment provided by hormonal therapy, immunotherapy, treatment with toxins, cryotherapy and surgery.

Provides that any method or composition described in the present description can be applied to any other method or composition described in the present description.

The use of the singular in connection with the term "containing" ("including") in the claims and/or the specification may mean "one", but also corresponds to the value "one or more", "at least one" and "one or more". The word "PR is about" means plus or minus 5% from the specified number.

Other aims, characteristics and advantages of the present invention will be apparent from the following the following detailed description. However, it should be understood that the detailed description and specific examples, which show certain embodiments of the invention, given for illustrative purposes only, because of the detailed descriptions for specialists in this field will be apparent various changes and modifications are included in the nature and scope of the invention.

Brief description of figures

The following figures form part of the present description and are included to further demonstrate certain aspects of the present invention. The invention can be better understood with the help of one or more of these figures, together with a detailed description.

Fig.1A-D. MUC1/CQC-peptide blocks the MUC1 oligomerization. (Fig.1A) Schematic representation of the subunit of MUC1-C and shows the 72-amino acid sequence of MUC1-CD. N-terminal peptide MUC1/CQC (shaded sequence) and mutated N-terminal peptide MUC1/AQA length of 15 amino acids were synthesized with the transduction domain for poly-dArg. (Fig.1B) Peptides His-MUC1-CD (1.4 mg/ml) was immobilized on the sensor chip for the BIAcore system. Peptide MUC1/CQC was passed through the chip at a concentration of 10 μm. Source data binding was analyzed using the software on the especiany BIAevaluation software version 3.0 and led to model binding of Longwire 1:1. (Fig.1C) Purified peptide His-MUC1-CD (1.5 mg/ml) were incubated with PBS, MUC1/CQC, 200 μm, or MUC1/AQA, 200 μm, for 1 hour at room temperature. Proteins were separated by electrophoresis LTO-polyacrylamide gel non conditions and analyzed by Western blot turns with antibodies to MUC1-C. (Fig.1D) Cells 293 transient was transfusional for the expression of empty vector or GFP-MUC1-CD and Flag-MUC1-CD. 48 hours after transfection cells were treated MUC1/CQC or MUC1/AQA, 5 μm, for 3 days. Then the cells were collected for Western blot turns with antibodies to MUC1-C (left panel). Also spent immunoassay total cell lysates with anti-Flag antibodies and performed Western blot turns precipitation received with the indicated antibodies (right panel).

Fig.2A-C.Peptide MUC1/CQC blocks nuclear localization of MUC1-C. (Fig.2A) Cells were incubated with FITC labeled peptide MUC1/CQC (5 μm) for the indicated time periods and then analyzed using flow cytometry. In each of the panels included, the average fluorescence intensity (MFI). (Fig.2B-C) Cells ZR-75-1 (Fig.2B) and MCF-7 (Fig.2C) were incubated in the presence of 5 μm peptide MUC1/CQC or MUC1/AQA within 3 days. Performed the Western blot turns total cell lysates (OCP) (left panel) and nuclear lysates (right panel) with indicated antibodies.

Fig.3A-D.Peptide MUC1/CQC causes inhibition of S-phase and necrosis. Cells ZR-75-1 (Fig.2A-B and MCF-7 (Fig.2C-D) were treated with peptide MUC1/CQC or MUC1/AQA (5 μm) for 3 and 4 days. The cells were fixed and analyzed the distribution of the phases of the cell cycle using flow cytometry (Fig.2A and 2C). The percentage of cells in G1-, S - and G2/M-phases included in the panel. Cells were also stained with propitiation and analyzed using flow cytometry to necrosis (Fig.2B and 2D). The percentage of necrotic cells included in the panel.

Fig.4A-E.The selectivity of the peptide MUC1/COC regarding breast cancer cells expressing MUC1. (Fig.4A) Cells ZR-75-1 stably infected "empty" lentiviruses (vector) or lentiviruses expressing kirk to MUC1. The Western blot turns lysates of infected cells was performed with the indicated antibodies. (Fig.4B) Cells ZR-75-1 carrying the vector, were left untreated (diamonds), and cells ZR-75-1 carrying the vector (squares) cells and ZR-75-1 bearing kirk to MUC1 (triangles) were treated with peptide MUC1/CQC during the specified time periods. The number of living cells was determined by the exclusion of the dye Trypanosoma blue. (Fig.4C) 293 Cells were left untreated (diamonds) and treated with 5 μm of peptide MUC1/CQC (squares) or MUC1/AQA (triangles) during the specified periods of time. The number of living cells was determined by the exclusion of the dye Trypanosoma blue. (Fig.4D) Cells MCF-10A were left untreated (left panel) and treated with 5 μm of peptide MUC1/CQC (middle panel) or MUC1/AQA (right panel). After 3 d the I analyzed the distribution of cell cycle phases. (Fig.4E) Cells MCF-10A were left untreated (diamonds) and treated with 5 μm of peptide MUC1/CQC (squares) or MUC1/AQA (triangles) during the specified periods of time. The number of living cells was determined by the exclusion of the dye Trypanosoma blue.

Fig.5A-C.Peptide MUC1/CQC inhibits the growth of xenografted tumors of the breast, ZR-75-1. (Fig.5A) 4-6-week old female mice Balb-c nu/nu implanted capsules with 17-β-estradiol. After 24 hours in the iliac region was injected subcutaneously breast cancer cells, ZR-75-1 enclosed in Matrigel. When tumor volume reached ~150 mm3mice were distributed matching pairs in groups and were injected with intraperitoneal injection of PBS (control media; filled squares), 50 mg/kg of peptide MUC1/AQA (control peptide; unfilled squares) or 10 mg/kg MUC1/CQC-peptide (filled triangles) daily for 21 days. Another group received 50 mg/kg of peptide MUC1/CQC daily for 6 days (unfilled triangles). Mice were weighed twice weekly, and the dimension of the tumor was performed every 4 days. (Fig.5B and 5C). On the 24th day (arrow) tumors collected from the control group and group treated with 50 mg/kg/day for 6 days, were stained with H&E (hematoxylin/eosin) (Fig.5B) and antibodies to MUC1 (Fig.5C).

Fig.6A-CenturyProlonged effects of MUC1/CQC-peptide on tumor ZR-75-1. (Fig.6A) Mice enter and by injection of the cells ZR-75-1, as described in the legend to Fig.5A. When tumor volume reached ~275 mm3mice were distributed matching pairs in groups and were injected with intraperitoneal injection of PBS (control media; solid squares), 50 mg/kg of peptide MUC1/AQA (control peptide; nesokrascheniya squares) or 30 mg/kg of peptide MUC1/CQC (filled squares) daily for 21 days. Control mice were scored on a 32-day, when tumor volume reached ~1200 mm3. For receiving the impact of the mice were monitored up to 52 days, when the tumors were collected for staining H&E (Fig.6B).

Fig.7.7-membered peptide MUC1 inhibits carcinoma of the prostate. Cells DU145 prostate cancer was treated with 5 µm short CQC-peptide (7-membered) or 5 µm long CQC-peptide (15-membered) for 4 days. Cell growth was measured using the MTT-assay. Data are presented as the percentage of growth inhibition relative to untreated cells (control).

Fig.8.Sequences of the United hydrocarbon blended MUC1-CD peptides.

Fig.9A.The effects of the United hydrocarbon blended MUC1-CD peptides on the growth of cells in non-small cell lung carcinoma H1650. To assess the sensitivity to inhibition of the function of MUC1 cells non-small cell lung carcinoma, H1650, were treated with 1 and 5 μm United hydrocarbon blended MUC1/CQC-peptides (GO-200-1B) for 7days. Treatment of cells N 5 μm GO-200-1B was accompanied by significant inhibition of growth and then decline in the number of cells.

Fig.9B. The effect of GO-200-2B on cell proliferation. Cell line H-1975 non-small cell lung carcinoma were grown in DMEM medium containing 10% temperature-inactivated fetal calf serum, 100 u/ml penicillin, 100 μg/ml streptomycin and 2 mmol/l L-glutamine. Cells were perseval through one day after treatment. Cells were treated with 5 μm GO-200-2B for 3 days, and cell viability was determined by exclusion Trypanosoma blue.

Fig.10.The effect of different MUC1-CD peptides (containing CQC-region) on the growth of hormone-dependent cell carcinoma of the breast. To determine the effect of exposure to different containing CQC-region MUC1-CD peptides, cells, breast carcinoma ZR-75-1 was treated with different peptides at a concentration of 5 μm for 4 days and was in control of cell proliferation. Undoubtedly there was a significant inhibition of cell growth compared to untreated cells.

Fig.11.The effect of different MUC1-CD peptides (containing CQC-region) on the growth of cells in non-small cell lung carcinoma. Cells non-small cell lung carcinoma A was treated with 5 ám GO-203, GO-203-2 or GO-203cyc within 7 days. The number of living cells was determined on day 7 the exception trypanosomatidae, and the percentage of growth inhibition was calculated by comparing the cell growth with untreated cells.

Fig.12.The effect of different MUC1-CD peptides (containing CQC-region) on the growth of cells in non-small cell lung carcinoma N. Cells non-small cell lung carcinoma N was treated with 5 μm containing various CQC-region MUC1-CD peptides for 6 days. The number of living cells was determined on day 6 the exception Trypanosoma blue. The results demonstrate that treatment of cells N 5 μm of various peptides was accompanied by significant inhibition of growth.

Fig.13.The effect of different MUC1-CD peptides (containing CQC-region) on the cell growth "triple negative" breast carcinoma. Cells "triple negative" breast carcinoma MDA-MB-231 were treated with 5 μm containing various CQC-region MUC1-CD peptides for 6 days. The number of living cells was determined on day 6 the exception Trypanosoma blue. The results demonstrate that treatment of cells MDA-MB-231 different peptides was accompanied by significant inhibition of growth.

Fig.14.The effect of shorter peptides GO-203 on cell proliferation of breast carcinoma ZR-75-1. The breast cancer cells of human ZR-75-1 were grown in RPMI1640 medium with 10% temperature-inactivated fetal calf serum, 100 u/ml penicillin, 100 µg/ml strepto is of CIN. Cells were treated with different peptides with a concentration of 5 μm daily for 4 days, and cell viability was determined by exclusion Trypanosoma blue. Unlike GO-210 processing cell carcinoma breast cancer ZR-75-1 5 µm peptides GO-203 (SEQ ID NO:53), GO-207 (SEQ ID NO:4), GO-208 (SEQ ID NO:50) and GO-209 (SEQ ID NO:54) daily for 4 days was accompanied by a significant inhibition of growth.

Fig.15A-D.Effects of GO-203 in combination with various anticancer drugs. Cells ZR-75-1 was treated with the indicated concentrations of cisplatin (Fig.15A), doxorubicin (Fig.15V), rh-TNF-α (Fig.15C) and Taxol (Fig.15D) separately and in combination with GO-203. For cisplatin, doxorubicin and Taxol treatment was consistent when the cells were treated with these agents for 72 hours, followed by exposure to 5 μm GO-203 within 72 hours. In the case of research with rh-TNF on cells was affected by different concentrations of rh-TNF alone and in combination with 5 μm GO-203 within 72 hours. To determine the survival rate of cells used MTS-analysis.

Fig.16.GO-203 has an additive or synergistic effect relative to anticancer agents. In the graphs showing Raman indexes on the various exposure levels (fraction of infected cells). The level of exposure received, the processing cells specified combination and anti-cancer drugs and GO-203.

Description of illustrative embodiments of the invention

I. the Present invention

The role of MUC1 protein in cancer intensively investigated by the authors of the invention and other scientists. As discussed above, protein MUC1 person is a heterodimeric glycoprotein, translated as a single polypeptide and cleaved at the N - and C-terminal subunits in the endoplasmic reticulum (Ligtenberget al., 1992; Macaoet al., 2006; Levitinet al., 2005). Broken overexpression of MUC1 that is found in most human carcinomas (Kufeet al., 1984), although not dependent attachment growth and carcinogenesis (Liet al., 2003a; Huanget al., 2003; Schroederet al., 2004; Huanget al., 2005). In other studies it was shown that overexpression of MUC1 confers resistance to apoptosis caused by oxidative stress and genotoxic anticancer agents (Yin and Kufe, 2003; Renet al., 2004; Rainaet al., 2004; Yinet al., 2004; Rainaet al., 2006; Yinet al., 2007).

The family of associated and secreted mucines functions, providing a protective barrier surface epithelial cells. If the damage of the epithelial layer, a tight connection between adjacent cells are destroyed, and loss of polarity, as the cells begin-induced heregulin recovery program (Vermeeret al., 2003). MUC1-N is reset with on top of the hair cells (Abe and Kufe, 1989), leaving MUC1-C to function as a transmitter of stress signals from the environment into the cell. In this case, the MUC1-C forms complexes on cell surfaces with members of the ErbB family of receptors, and MUC1-C translocases in the nucleus of the cell in response to stimulation heregulin (Liet al., 2001; Liet al., 2003c). MUC1-C also functions, bringing together signaling pathway ErbB-receptor and Wnt in the result of direct interaction between the cytoplasmic domain of MUC1 (MUC1-CD) and members of the catenin family (Huanget al., 2005; Liet al., 2003c; Yamamotoet al., 1997; Liet al., 1998; Liet al., 2001; Li and Kufe, 2001). In other studies it was demonstrated that MUC1-CD fosfauriliruetsa glycogen synthase kinase 3β, c-Src, protein kinase Cδ and c-Abl (Rainaet al., 2006; Liet al., 1998; Liet al., 2001; Renet al., 2002).

The mechanisms responsible for nuclear localization of MUC1-C is not clear. Containing a classical nuclear localization signal (NLS) proteins are imported into the cell nucleus, first contacting importing α, and then, in turn, importing β (Weis, 2003). The transport complex of importing α/β is attached to the nuclear pore by binding to nucleoporins, and moved through it by a mechanism dependent on the GTPase Ran. Classical NLS comprise either one of the signal area with a cluster of 4-5 basic amino acids or of two parcels with two main clusters s is nakilat, separated by a linker of 10-12 amino acids. MUC1-CD contains RRK-motif, which does not match the prototype NLS with one signal plot (Hodelet al., 2002). However, some proteins containing non-classical NLS, are transported through the nuclear pores, directly contacting importing β (Kauet al., 2004). Importin β is associated with several nucleoporins (Ryan and Wente, 2000), including Nup62, which is published as cytoplasmic and nucleoplasmic side of complexes of nuclear pores (Percipalleet al., 1997). In other studies it was shown that the import of β-catenin in the nucleus of the cell is carried out by importin and nucleoporin-independent mechanism (Suh and Gumbiner, 2003).

In 2006, the inventors reported that MUC1 is imported into the cell nucleus by a mechanism involving the binding of Nup62. The authors also demonstrated that MUC1 forms oligomers through CQC-motif in the cytoplasmic domain of MUC1, and that MUC1 oligomerization is required for nuclear import. In this study, the authors continued this work to further understand the role of CQC-motive plays in the formation of oligomers. The inventors have demonstrated that short peptides corresponding to this region can disrupt the formation of oligomers MUC1, inhibiting its transport into the nucleus of tumor cells. These peptides are able ingabire is the substance of the growth of tumor cells, and also to induce apoptosis in these cells and even necrosis in tumor tissue. These and other aspects of the invention are described in detail below.

II. MUC1

A. Structure

MUC1 is a glycoprotein mucinosa type, which is expressed on the apical borders of the normal secretory epithelial cells (Kufeet al., 1984). MUC1 forms heterodimer after synthesis in the form of a single polypeptide and cleavage of the precursor into two subunits in the endoplasmic reticulum (Ligtenberget al., 1992). Splitting can oposredovanie autocatalytic process (Levitanet al., 2005). N-terminal subunit of MUC1 with a molecular weight in excess of 250 kDa (MUC1 N-ter, MUC1-N), contains a different number 20-amino acid imperfect tandem repeats with highly conservative variations and modified by O-linked glycanase (Gendleret al., 1988; Siddiquiet al., 1988). MUC1-N is attached to the cell surface as a result of dimerization with C-terminal subunit mass of approximately 23 kDa (MUC1 C-ter, MUC1-C), which contains a 58-amino acid extracellular region, a 28-amino acid transmembrane domain and a 72 amino acid cytoplasmic domain (CD; SEQ ID NO:1) (Merloet al., 1989). The sequence of the MUC1 person is shown below:

Identifies the sequence of the cytoplasmic domain, and p is durkota part of the sequence corresponds to the peptide, the inhibitory oligomerization (SEQ ID NO:3) described in the examples.

During the transformation of normal epithelium to carcinoma abnormal MUC1 is expressed in the cytosol and across the cell membrane (Kufeet al., 1984; Pereyet al., 1992). Associated with the cellular membrane MUC1 is sent to endosome by clathrin-mediated endocytosis (Kinloughet al., 2004). In addition, MUC1, but not MUC1-N is transported to the cell nucleus (Balduset al., 2004; Huanget al., 2003; Liet al., 2003a; Liet al., 2003b; Liet al., 2003c; Weiet al., 2005; Wenet al.2003) and mitochondria (Renet al., 2004).

Century

MUC1 interacts with members of the ErbB family of receptors (Liet al.2001b; Liet al., 2003c; Schroederet al.2001) and with an effector of Wnt-signaling pathway, β-catenin (Yamamotoet al., 1997). The growth factor receptor epidermal and c-Src to phosphorylate the cytoplasmic domain of MUC1 (MUC1-CD) Y-46 and, thereby, enhance the binding of MUC1 and β-catenin (Liet al.2001a; Liet al., 2001b). The binding of MUC1 and β-catenin is also regulated by glycogen synthase kinase 3β and protein kinase Cδ (Liet al., 1998; Renet al., 2002). MUC1 colocalizes with β-catenin in the nucleus (Balduset al., 2004; Liet al., 2003a; Liet al., 2003c; Wenet al.2003) and together with it activates the transcription of target genes of Wnt-signaling pathway (Huanget al., 2003). In other studies it has been shown that MUC1 also communicates directly with p53 and regulates the TRANS is reply of target genes of p53 (Wei et al., 2005). It should be noted that overexpression of MUC1 is sufficient for the induction independent of the attachment of growth and oncogenic potential (Huanget al., 2003; Liet al.2003b; Renet al., 2002; Schroederet al., 2004).

Most mitochondrial proteins are encoded by nuclear DNA and imported into mitochondria with the complex vectors in the outer and inner mitochondrial membranes. Some mitochondrial proteins contain N-terminal mitochondrial signal sequence and interact with Tom20 in the outer mitochondrial membrane (Truscottet al., 2003). Other mitochondrial proteins contain internal signal sequence and interact with Tom70 receptor (Truscottet al., 2003). In recent studies it was shown that mitochondrial proteins without internal signal sequences are delivered to Tom70 using complex consisting of HSP70 and HSP90 (Younget al., 2003).

III. MUC1 peptides

A. Structure

The present invention provides for the development, production and use of different MUC1-peptide. The structural characteristics of these peptides are as follows. First, the peptides have no more than 20 consecutive amino acid residues of MUC1. Therefore, the term "peptide having no more than 20 consecutive residues, even including the term "containing" cannot interpret what I as including a greater number of consecutive amino acid residues MUC1. Secondly, the peptides must contain CQC-motif, and may also include CQCR-motive, CQCRR motive and CQCRRK-motive. Therefore, the peptide will have at least these three successive remainder of the MUC1-C domain. Thirdly, the peptide will have at least one amino acid residue attached to the NH2-the end of the first cysteine residue in CQC-motive, so the first cysteine residue will be "closed" at least one amino acid attached to it. This residue may belong to MUC1 (for example, to enter the transmembrane domain), can be a randomly chosen amino acid (any of the 20 natural amino acids or their analogues) or may be part of another peptide sequence (for example, the sequence used for cleaning, stabilizing sequence or domain, delivering into the cell).

Typically, the length of peptide is 50 amino acid residues or less, in turn, contains no more than 20 consecutive MUC1 residues from the. The total length can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 residues. Provided the size ranges of peptides from 4-50 residues, 5-50 residues, 6-50 residues, 7-50 residues, residues -25, 4-20 residues 5-20 residues, residues 6-20, 7-20 7-15 residues and residues. The number of consecutive amino acid residues of MUC1 may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Provided by the ranges of consecutive amino acid residues from 4-20 residues 5-20 residues, residues 6-20, 7-20 residues and residues 4-15, 5-15 residues 6-15 7-15 residues or residues.

In the present invention can be used in the L-configuration amino acids, D-configuration amino acids or their mixture. Although L-amino acids represent the vast majority of amino acids in proteins, D-amino acids are found in some proteins produced rare marine organisms, such as snails-cones. They are also common components of the composition of the cell wall of bacteria. D-serine may act as a neurotransmitter in the brain. L and D refer to the configuration of the amino acids are not optical activity of amino acids, and the optical activity of the isomer of glyceraldehyde from which this amino acid is theoretically possible to synthesize (D-glyceraldehyde is Pervouralsky; L-glyceraldehyde is levogyrate).

One form of the peptide, consisting only of D-amino acids, is retroinfection peptide. Retroinfection modification of natural polypeptides includes si the Tethyan the Assembly of amino acids with the stereochemistry of the α-carbon, the opposite stereochemistry of the corresponding L-amino acids, i.e., D-amino acids in the reverse order with respect to the natural peptide sequence. Retroinfection similar, therefore, will have the inverted ends and inverted (opposite) direction of peptide bonds (NH-CO, and not CO-NH), while approximately preserving the topology of the side chains, as in the natural peptide sequence. Cm. U.S. patent 6261569 included in the present description by reference.

As indicated above, the present invention involves the fusion or conjugation domain for delivery to the cell (also called a vector for gene delivery, or the domain for transduction of cells, CDD/CTD). These domains are well known in this field and are usually short amphipatic or cationic peptides and peptide derivatives, often containing multiple residues lysine and arginine (Fischer, 2007). Of particular interest are sequences of poly-D-Arg and poly-D-Lys (for example, programada residues, eight amino acid residues in length), while others are shown in table 1, below.

Table 1

Also, as mentioned above, is provided by the peptides, modified to usein vivoby adding at the amino - and/what do carboxy-end blocking agent to increase the life time of peptide in vivo. This can be useful in situations where degradation of the peptide ends under the action of proteases occurs before the injection of the peptide into the cell. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be joined to amino and/or carboxy-terminal residues of the injected peptide. These agents can be added either by chemical means in the course of peptide synthesis, or by using recombinant DNA technology methods known in this field. Alternative blocking agents, such as Pyroglutamate acid or other molecules known in this field can be joined to amino and/or carboxy-terminal residues.

B. Synthesis

It is preferable to obtain peptides using methods of solid-phase synthesis (Merrifield, 1963). Specialists in this field other well known methods of peptide synthesis (Bodanszkyet al., 1976; Peptide Synthesis, 1985; Solid Phase Peptide Synthelia, 1984). Appropriate protective groups used in such methods can be found in the above literature, as well as in the book Protective Groups in Organic Chemistry, 1973. Such synthesis methods include the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide C is PI. Usually, either the amino or carboxyl group of the first amino acid residue protects the corresponding selectively removable protective group. Other selectively removable protective group used for amino acids containing reactive side groups, such as lysine.

When using solid-phase synthesis as an example of protected amino acids or their derivatives attached to an inert solid substrate through their unprotected carboxyl or amino group. Then selectively removing the protective group of the amino or carboxyl group, and add the next amino acid in the sequence having a correspondingly protected complementary group (amino or carboxyl), which interacts with the rest, already attached to the solid substrate. Then remove the protective group of the amino or carboxyl group of the newly added amino acid residue, and then add the next amino acid (suitably protected), etc., After all of the planned amino acids were connected to each other in the correct order, any remaining protective groups are end and side groups (and the solid substrate) are removed sequentially or simultaneously, getting the final peptide. Preferably, the peptides according to the invention were free from benzylurea the data or methylbenzylamine amino acids. Such protective groups can be used during synthesis, but they should be removed before using the peptide. It may be necessary to conduct additional reactions described in the literature for the formation of intramolecular bonds that hold the conformation of the peptide.

In addition to the 20 standard amino acids that can be used, there is a wide range of "non-standard" amino acids. Two of them are encoded by the genetic code, but the squirrels are fairly rare. Selenocysteine is included in some proteins at UGA-codon, which normally represents a stop codon. Pyrrolidin used by some by methane-forming archaea in the enzymes that they use for the production of methane. It is encoded by the codon UAG. Examples of unconventional amino acids that do not occur in proteins include lanthionine, 2-aminoethanol acid, dehydroalanine and neurotransmitter gamma-aminobutyric acid. Non-standard amino acids are often found as intermediates in metabolicheskikh ways for the standard amino acids, such as ornithine or citrulline occur in the urea cycle, part of the catabolism of amino acids. Non-standard amino acids are usually formed as a result of modifications of the standard amino acids. For example, homocysteine is formed by transsylvania or desmetilirovaniya through an intermediate metabolite S-adenosylmethionine, whereas hydroxyproline occurs as a result of posttranslational modification of Proline.

C. Linkers

Linkers or agents for cross-linking can be used to merge MUC1-peptide with other protein sequences. Bifunctional reagents for cross-linking are used extensively for various purposes, including receipt of affinity media, modification and stabilization of various structures, identification of binding sites of ligands and receptors and structural studies. Homobifunctional reagents that carry two identical functional groups, was highly effective for the formation of cross-links between the same and different macromolecules or subunits of macromolecules, and for fusion polypeptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. Taking advantage of the different reactivity of the two different functional groups, it is possible to control both selectivity and consistency of cross-linking. Bifunctional reagents for cross-linking can be divided according to the specificity of their functional groups such as amino, sulfhydryl-, guanidino-, indole -, or carboxyl-specific groups. The most widely used what has been created are the reagents, aimed at free amino groups, due to their commercial availability, ease of synthesis and mild reaction conditions under which they can be used. Most heterobifunctional reagents for cross-linkage containing group, interact with primary amines, and the group, interact with thiols.

In another example, heterobifunctional reagents for cross-linking and methods of using the reagents for cross-linking are described in U.S. patent 5889155, the full content of which is incorporated into this description by reference. Reagents for cross-stitching combine nucleophilic hydrazide residue with electrophilic maleimide balance, allowing the combination in one example of aldehydes with free thiols. Reagent for cross-linking can be modified to cross-stitching the various functional groups and, thus, is suitable for cross-linking of polypeptides. When specific peptide does not contain a residue suitable for this reagent to cross-stitching in their original sequence, you can use conservative amino acid substitutions in the primary sequence, introduced genetic or synthetic methods.

Another use of the l is Kerov in the context of peptides as therapeutic agents is the so-called technology "peptides, United hydrocarbon blended" from the company Aileron Therapeutics. The fundamental approach for connecting peptide stitching is an approach in which two key residue in the peptide is modified by attaching linkers through the side chains of amino acids. After the synthesis of the linkers connecting through the catalyst, thus creating a bridge that physically hold the peptide in its natural α-helical form. In addition to supporting the natural patterns needed to interact with the molecule-target, this conformation also provides stability against peptidases, and for penetration into the cell. U.S. patents 7192713 and 7183059 that describe this technology is incorporated into this description by reference. Cm. article Schafmeisteret al., Journal of the American Chemical Society, 2000, 122(24): p. 5891-5892.

D. Structure, variants and analogs

The present invention is directed to peptides containing the sequence CQC. Defining this key structure in the formation of oligomers MUC1, the inventors also envisage the possibility of using variants of the sequence CQC. For example, some non-natural amino acids that satisfy the structural constraints of the sequence CQC, can be used as substituents without loss, and possibly with increased biological functions. The AOC is e, the authors present invention also provides for the use of structural analogs to simulate key sections of the peptides or polypeptides of the present invention. Such compounds are called peptidomimetics, you can use similar to the peptides of the present invention and, therefore, they are also their functional equivalents.

Some mimetics that mimic elements of secondary and tertiary structure of proteins, are described in Johnsonet al.(1993). The main argument in favor of the use of peptide mimetics is that the peptide skeleton proteins exists chiefly to maintain the orientation of amino acid side chains that facilitate molecular interactions, such as, for example, the interaction of antibodies and/or antigen. Thus, peptide mimetics are designed to provide molecular interactions similar to the natural interaction of molecules.

How to create specific structures described in this field. For example, the α-helix mimetics are disclosed in U.S. patents 5446128; 5710245; 5840833 and 5859184. How to create conformational restricted β-turns and β-bulges are described, for example, in U.S. patents 5440013; 5618914 and 5670155. Other types of mimetics turns include reversible and γ-turns. Mimetics reverse turns are disclosed in patents With Whom And 5475085 and 5929237, and mimetics γ-turns described in U.S. patent 5672681 and 5674976.

Used in the present description, the term "molecular modeling" means a quantitative and/or qualitative analysis of the structure and function of protein-protein physical interaction based on the information about the three-dimensional structure and models of protein-protein interactions. This term includes conventional numerical models of molecular dynamics and minimization of energy, interactive computer graphics model, the modified model of molecular mechanics, metric geometry and other restrictive model based on the structure. Molecular modeling is usually done with the use of computers, and can additionally be optimized using known methods. For the development of such compounds are particularly suitable computer program, using the data of x-ray analysis. To create three-dimensional models can be used, for example, programs such as RasMol. Computer programs such as INSIGHT (Accelrys, Burlington, MA), GRASP (Anthony Nicholls, Columbia University), Dock (Molecular Design Institute, University of California at San Francisco) and Auto-Dock (Accelrys), provide the possibility of additional manipulation and able to introduce new structures. The methods may include the additional step of removing the external device model the 3-dimensional structure of a connection is possible. In addition, you can compare 3-dimensional data potential connections with a computer database, for example, 3-dimensional structures.

Compounds according to the invention also can be developed interactively on the basis of structural information about the compounds described in the present description, using other methods of development/modeling compounds based on the structure (see, e.g., Jackson, 1997; Joneset al., 1996). Potential connection then you can test in the standard analysis methods known to experts in this field. Examples of the methods of analysis described in the present description.

3-dimensional structure of biological macromolecules (e.g. proteins, nucleic acids, carbohydrates and lipids) can be set based on the data obtained using several techniques. These techniques are most often used to assess the 3-dimensional structure of proteins include: (a) x-ray crystallography; (b) spectroscopy nuclear magnetic resonance (NMR); (c) analysis of the limitations of physical distance formed between specific sites on a macromolecule, for example, intramolecular chemical cross-links between amino acid residues on the protein (e.g., PCT/US00/14667, the description of which is fully incorporated into the present description by reference); and (d) methods of molecular simulation is, based on knowledge of the primary structure of the target protein, for example, simulation methods homologues, "reach out" algorithms orab initiostructural modeling using computer programs such as MONSSTER (modeling of new structures on the basis of secondary and tertiary constraints) (see, for example, international application number PCT/US99/11913, the description of which is fully incorporated into the present description by reference). Other methods of molecular modeling can also be used according to the present invention (e.g., Cohenet al., 1990; Naviaet al.in 1992, the description of which is fully incorporated into the present description by reference). All of these methods provide data that are amenable to computer analysis. Other spectroscopic methods that also may be suitable for the method of the present invention, but which do not provide structural information on biomolecules at the atomic level, include circular dichroism and fluorescence and spectroscopy in the UV/visible light. The preferred method of analysis is x-ray crystallography. Description of this technique and NMR spectroscopy below.

X-ray crystallography

X-ray crystallography is based on diffraction of x-rays with wavelength characteristics of the definition range of electronic clouds surrounding the nuclei of atoms in the crystal of interest of the molecule or molecular complex. In the method using the purified crystals of biological macromolecules or molecular complexes (but they often include solvents, cofactors, substrates or other ligands) to determine the resolution close to the atomic, atoms that constitute a particular biological macromolecule. A necessary condition for the resolution of the 3-dimensional structure by x-ray crystallography is a highly ordered crystal that will strongly scatter x-rays. In the way the x-ray beam is directed to correct the repeating lattice of many identical molecules, so that x-rays are scattered on the grill, giving the picture, which identifies the structure of the individual molecules. Highly ordered crystals, for example, molecules of globular proteins represent a large, spherical or ellipsoidal objects with inhomogeneous surface. The crystals contain large channels between individual molecules. These channels, which normally occupy more than half of the volume of the crystal, filled with disordered solvent molecules and protein molecules are in contact with each other only in a few small areas. This is the one reason why the structure of proteins in crystals are usually identical to the structures of proteins in solution.

Methods of obtaining crystals of the proteins of interest are described below. The formation of the crystals depends on various parameters, including pH, temperature, concentration of biological macromolecules, nature of solvent and precipitant, as well as from the presence of the added ions or ligands of the protein. For the selection of all parameters that will make a combination that gives a crystal suitable for x-ray analysis may be necessary to conduct many routine experiments on crystallization. Using automated devices for crystallization can automate and accelerate the reproducible production of a large number of experiments on crystallization (see, for example, U.S. patent 5790421, the description of which is fully incorporated into the present description by reference).

Crystallization of the polypeptide occurs in solutions in which the concentration of the polypeptide is greater than its maximum solubility (i.e., the solution of the polypeptide is saturated). Such solutions can be supported in equilibrium, reducing the concentration of the polypeptide, preferably by deposition of crystals of the polypeptide. You can often cause the formation of crystals of polypeptides from supersaturated what's solutions, adding agents that change the surface charge of the polypeptide, or disrupt the interaction between the polypeptide and the surrounding water, increasing interaction, leading to crystallization.

Crystallization is usually carried out at temperatures from 4°C to 20°C. is Often used substance known as "precipitators", to reduce the solubility of the polypeptide in the concentrated solution by the formation of energetically unfavorable precipitating the depletion layer around the molecules of the polypeptide (Weber, 1991). In addition to the precipitating in the solution for crystallization of the polypeptide sometimes add other materials. They include buffers to bring the solution pH and salt to reduce the solubility of the polypeptide. In this area there are various precipitators, including the following compounds: ethanol, 3-ethyl-2-4-pentanediol and many polyglycols, such as polyethylene glycol (PEG). Solutions for the deposition may include, for example, 13-24% PEG 4000, 5-41% of ammonium sulfate and 1.0-1.5 M sodium chloride, with a pH in the range of 5.0 to 7.5. Other additives may include 0.1 M Hepes, 2-4% of butanol, 20 to 100 mm sodium acetate, 50-70 mm citric acid, 120-130 mm sodium phosphate, 1 mm ethylenediaminetetraacetic acid (EDTA) and 1 mm dithiothreitol (DTT). These agents are produced in the buffers and added dropwise in various combinations to the crystallization buffer. Kristallizuetsya proteins can be the modified for example, phosphorylation or by using a simulator phosphate (e.g., tungstate, cacodylate or sulfate).

Commonly used methods of crystallization polypeptides include the following techniques: a volume in hanging drop, using seed and dialysis. In each of these ways, it is important to maintain a continuous crystallization process after the formation of the germ crystals by maintaining a supersaturated solution. During crystallization in the amount of the polypeptide is mixed with a precipitating to achieve a supersaturated solution, capacity, sealed and leave before the appearance of crystals. In the method using a dialysis polypeptide leave within a closed dialysis membrane, which is placed in a solution containing the precipitant. Equilibrium diffusion through the membrane increases the concentration of the polypeptide and precipitator, thereby causing saturation of the polypeptide.

In the preferred method of "hanging drops" (McPherson, 1976) original mix polypeptide create, adding a precipitant to a concentrated solution of the polypeptide. The concentration of the polypeptide and the precipitators are such that this initial solution, the polypeptide does not crystallize. A small drop of this mixture is placed on a glass plate, which is turned over and placed above the tank with the second solution. the ATEM system are tightly closed. Usually, the second solution contains a higher concentration of precipitator or other dehydrating agent. The difference in concentrations of the precipitant is the reason that the protein solution has a higher vapor pressure relative to the second solution. Since the system with two solutions hermetically closed, it establishes the equilibrium, and the water of the polypeptide mixture is transferred to the second solution. This balance increases the concentration of the polypeptide and precipitant solution of the polypeptide. When the critical concentration of the polypeptide and precipitator can be formed crystal of the polypeptide.

In another method of crystallization in concentrated solution of the polypeptide is injected seed. Typically, prepare a concentrated solution of the polypeptide, and this solution is injected embryo crystal of the polypeptide. If the concentration of the polypeptide and any of the precipitators are correct, then the germ of the crystal will provide the seed around which is formed larger crystal.

Another method of crystallization is electrocrystallization, which uses the dipole moments of the protein molecules, which themselves are placed in the Helmholtz layer at the electrode surface (see, for example, U.S. patent 5597457, the description of which is fully incorporated into the present description by reference)./p>

Some proteins may not be amenable to crystallization. However, the specialist there are several techniques by which you can cause crystallization. For example, the destruction of flexible fragments of the polypeptide at the amino - or carboxyl end of the protein can facilitate obtaining protein crystals. These fragments can be removed using methods of molecular biology or protein processing by proteases, such as trypsin, chymotrypsin or subtilisin.

In diffraction experiments or narrow parallel beam of x-rays is obtained from the x-ray source and is directed to the crystal for receiving the reflected beam. The incident primary beam causes damage to both the macromolecule and the solvent molecules. Therefore, to increase the lifetime of the crystal is cooled (for example, to a temperature in the range from -220°C to -50°C). To obtain all possible diffraction spots of the primary beam should hit the crystal in different directions, so that during the experiment the crystal rotate relative to the direction of the beam. Diffraction spots are recorded on film or using an electronic detector. Overexposed film digitize and quantify the scanning device, while the electronic detectors transmit signals that they detect, directly on to mputer. Electronic two-dimensional detectors significantly reduce the time required to collect and changes in the diffraction data. Each diffraction beam, which is detected in the form of spots on the film or detector plate has three characteristics: amplitude, which is measured by the intensity of the spots; the wavelength, which is determined by the x-ray source; and a phase that is lost in the experiments of x-ray analysis. All these three characteristics necessary for all reflected beams to determine the position of the atoms, causing the reflected beams. One way of determining the phases is called multiple isomorphous replacement (MIR), which requires the introduction of exogenous scatterer of x-rays (for example, heavy atoms, such as metal atoms) in the cell of the crystal. A more detailed description of MIR described in U.S. patent 6093573 (column 15), the disclosure of which is fully incorporated into the present description by reference.

The coordinates of the atoms represent the Cartesian coordinates (position in x, y and z), obtained by mathematical processing including Fourier synthesis, the data received from the pattern produced by diffraction of a monochromatic beam of x-rays by the atoms (scattering centers) of interest biological macromolecules in crystalline form. difraktsionnye data used to calculate electron density maps recurring elementary units of the crystal (cells). Maps of the electron density is used to measure the position coordinates of the atoms) of the individual atoms in the unit cell of the crystal. The absolute values of the coordinates of the atoms convey information about the spatial interactions between atoms, because the absolute values attributed to atomic coordinates, you can change the rotational and/or translational motion in x, y and/or z-axes, together or separately, while maintaining the same relative spatial interaction between atoms. Thus, we can assume that a biological macromolecule (e.g. protein), whose set of absolute values of atomic coordinates by using rotational or translational motions be customized so that it coincides with the set of pre-defined values from the analysis of another sample has the same atomic coordinates as the coordinates obtained for the other pattern.

For more detailed description of x-ray analysis can be obtained from simultaneously pending patent application U.S. No. 2005/0015232, U.S. patent 6093573 and international patent applications PCT/US99/18441, PCT/US99/11913 and PCT/US00/03745. A full description of all of these patent documents is hereby incorporated into this description by reference.

NMR spectroscopy

While for x-ray crystallography tre is described single crystals of the target macromolecule, NMR studies in solution under conditions close to physiological. However, obtained using the NMR structures are not as detailed as the patterns obtained on the basis of crystals.

Although the application of NMR spectroscopy until relatively recently, was limited to the establishment of the 3-dimensional structure of relatively small molecules (e.g. proteins, consisting of 100-150 amino acid residues), recent discoveries, including isotope tagging of interest molecules, and spectroscopy with optimized cross-relaxation (TROSY), allowed to extend the methodology for the analysis of much larger molecules, such as proteins with a molecular mass of 110 kDa (Wider, 2000).

In NMR radiofrequency radiation to determine the environment of atomic nuclei with non-zero magnetic moment in a homogeneous magnet field, pulsating with a specific radio frequency. Pulses violate the nuclear magnetization of these atoms with nuclei with nonzero spin. When you return the system to its equilibrium state is detected range of damped signals. Fourier-transformation of the decaying signal in the frequency range gives the one-dimensional NMR spectrum. The peaks in this spectrum are the chemical shifts of various active nuclei. The chemical shift for an atom is determined by its local who Elektronnyi environment. Two-dimensional NMR studies can provide information about the proximity of the various atoms in the structure and in three-dimensional space. The structure of proteins can be determined through a series of two (and sometimes 3 or 4)dimensional NMR studies and using the obtained information as constraints in a number of methods of spatial modeling protein folding.

More information on NMR spectroscopy, including a detailed description of how the baseline data obtained from the NMR experiment can be used to determine the 3-dimensional structure of macromolecules can be found in: Protein NMR Spectroscopy, Principles and Practice, (1996); Gronenbornet al.(1990); and Wider (2000),supra., full details are included in the present description by reference.

Also of interest are compounds of peptidomimetics, which are designed based on the amino acid sequences of the compounds of the present invention, representing peptides. Peptidomimetics are synthetic compounds having a three-dimensional conformational "motif", which essentially corresponds to the three-dimensional conformation of a selected peptide. The peptide motif is a combination of peptidomimetic, which is capable of inhibiting MUC1 oligomerization. Peptidomimetics can have additional characteristics that enhance their suitability for usein vivothat is what the best penetration into the cell and a longer half-life. Peptidomimetics typically have a frame that is partially or completely is not a peptide, but with side groups that are identical to the side groups of amino acid residues present in the peptide, on the basis of which developed peptidomimetic. In this area there are several types of chemical bonds, for example, ester, thioester, tiamina, retromedia restored carbonyl, demeterova and geometrinae communication that are typically used for replacement of peptide bonds during construction are resistant to proteases of peptidomimetics.

IV. Methods of treatment

A. Pharmaceutical compositions and routes of administration

If it is intended clinical application, it will be necessary to obtain pharmaceutical compositions in a form suitable for the intended application. In General, this process will include the preparation of compositions essentially free from pyrogenic compounds, and other impurities that are harmful to humans or animals.

In General, it is desirable to use appropriate salts and buffers to ensure the stability of the vectors used for delivery and for their introduction into target cells. For administration to the patient of recombinant cells also use buffers. Aqueous compositions of the present invention contain an effective amount of the vector relative activities is but cells dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions are also referred to as the inoculum. The phrase "pharmaceutically or pharmacologically acceptable refers to molecular compounds or compositions that do not give negative, allergic or other adverse reactions caused by the introduction of an animal or person. Used in the present description, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and agents that slow down the absorption, etc. In this area is well known for the ways in which such media and agents for pharmaceutically active substances. Except when any conventional medium or agent is incompatible with the vectors or cells according to the present invention, provided their use in therapeutic compositions. Also in the composition may include an auxiliary active ingredients.

Active composition of the present invention may include classic pharmaceutical preparations. The introduction of the compositions of the present invention can be held in any way, subject to the availability of the target tissue in this introduction. These routes include oral, nasal, buccal the th, rectal, vaginal or local road maintenance. Alternatively, the introduction can be performed using an orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions are typically administered in the form of pharmaceutically acceptable compositions described above. Of particular interest are the direct introduction into the tumor, the tumor perfusion or introduction to the place or region of the tumor, for example, in local vascular or lymphatic system, or in the bed of the removed tumor.

The active compounds can also enter parenterally or intraperitoneally. Solutions of the active compounds as free bases or pharmacologically acceptable salts can be prepared in water, respectively, mixed with a surface-active substance, such as hydroxypropylcellulose. It is also possible to prepare dispersions in glycerol, liquid polyethylene glycols and their mixtures, and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for extemporanea preparation of sterile solutions or dispersions for injection. In all cases the form must be sterile and liquid enough for l is Gogo injection through a syringe. It must be stable under the manufacture and storage and must be protected from contamination by microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyhydric alcohol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and so forth), suitable mixtures and vegetable oil. Proper fluidity can be maintained, for example, using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc., In many cases it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption introduced by injection compositions can be achieved by using in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared, including active compounds in the required amount in an appropriate solvent, if necessary, together with a number of other ingredients enumerated above, followed by sterilization by filtration is. Generally, dispersions are prepared, including a variety of sterile active ingredients into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, giving the powder active ingredient plus any additional desired ingredient from their solution, sterilized by filtration.

Used in the present description, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and agents that delay absorption, etc. In this area is well known for the ways in which such media and agents for pharmaceutically active substances. Except when any conventional medium or agent is incompatible with the active ingredient, they are to be used in therapeutic compositions. Also in the composition may include an auxiliary active ingredients.

For oral administration, the polypeptides of the present invention can be included in the composition with excipients and used in the form of a mouthwash for mouth and means for cleaning ubov, not intended for ingestion. The mouthrinse can be prepared, including the active ingredient in the required amount in an appropriate solvent, such as a solution of sodium borate (solution Dobell). Alternatively, the active ingredient can be included in an antiseptic mouthwash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient can also be atomized in means for cleaning the teeth, including gels, pastes, powders and suspensions. The active ingredient can be added in a therapeutically effective amount of a paste for brushing your teeth, which may include water, binders, abrasives, perfumes, blowing agents and moisturizers.

Compositions of the present invention can be in neutral form or as a salt. Pharmaceutically acceptable salts include the acid additive salts (formed with free amino groups of proteins), which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, almond, etc., Salts formed with the free carboxyl groups can also be obtained with inorganic bases, such as, for example, hydroxides of sodium, potassium, ammonium, calcium or iron (III), and such organic bases as isopr is pelamin, trimethylamine, histidine, procaine, etc.,

After composing solutions imposed in a way compatible with the dosage form, and quantity that is therapeutically effective. The compositions are easy to enter in a number of dosage forms such as solutions for injection, capsules for release of drugs, etc. For parenteral administration in an aqueous solution, for example, the solution, if necessary, must contain a buffer, and the liquid solvent is first necessary to give isotonicity with a sufficient amount of salt or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, specialists in this area known sterile aqueous medium, which can be used for the present invention. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml hypodermoclysis fluid, either enter by injection in the proposed location of the infusion (see, for example, "Remington''s Pharmaceutical Sciences, 15th edition, pages 1035-1038 and 1570-1580). Some changes dosage will be inevitable depending on the condition of the subject being treated. The man in charge of the drug is in any case to determine the appropriate dose for a particular subject is. Moreover, for introducing people, the drugs must meet the standards of sterility, progenote, General safety and purity according to the standards of the FDA (US quality supervision of food and drugs) for biological substances.

B. Types of cancer and subjects

Cancer cells, to which you can apply the methods of the present invention include in General any cell that expresses MUC1 and, more specifically, sverkhekspressiya MUC1. A suitable cancer cell may be a cancer cell, breast cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, stomach cancer, liver cancer, bone cancer, hematological cancer (e.g., leukemia or lymphoma), cancer of the nervous tissue, melanoma, ovarian cancer, testicular cancer, prostate cancer, cervical cancer, cancer of the vagina or bladder cancer. In addition, the methods of the present invention can be used in a wide range of species, for example, in humans, primates (excluding humans) (e.g., macaques, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, Guinea pigs, gerbils, hamsters, rats and mice.

C. Methods of treatment

The peptides or analogues, which inhibit the formation of oligomers MUC1, usually suitable as against the cancer therapeutic or prophylactic agents. You can enter a mammal (for example, patients with breast cancer) alone or in combination with other drugs and/or radiotherapy. Connections can also enter the subjects that are genetically and/or due to environmental effects (due, for example, physiological factors and/or environmental factors) are susceptible to cancer, for example, subjects who have a family history of cancer (e.g. breast cancer), subjects with chronic inflammation or exposed to chronic stress, or subjects who were exposed to natural or unnatural carcinogenic environmental conditions (for example, excessive exposure to sun radiation, industrial carcinogens or smoke).

When applying the methods of the present invention to subjects with cancer before the introduction of the optional connection to test cancer on the expression of MUC1 (the expression of MUC1 protein or MUC1 mRNA) by methods known in this field. In this case, subjects can be classified as having cancer with expression or overexpression of MUC1. Such methods can spendin vitroin cancer cells obtained from the subject. Alternatively, you can apply m is using the technique of visualization in vivousing, for example, radioactivedecay antibodies specific for MUC1. In addition, subjects with cancer, you can conduct testing of body fluids (e.g. blood or urine) to increase the level of protein MUC1 or MUC1 protein fragments.

The required dosage depends on the choice of route of administration; the nature of the composition; the nature of the disease of the patient; size, weight, and size of the workpiece, age and sex; other injected drugs; and the decision of the attending physician. Suitable dosages are in the range of from 0.0001 mg/kg to 100 mg/kg Expected to be significant variation in the required dosages due to the diversity of available connections and different effectiveness of different routes of administration. For example, it is expected that by oral administration will be required higher doses than in the case of intravenous injection. As is well known in this field, changes in the level of dosages can be selected using the standard empirical methods for optimization. The introduction may be single or multiple (e.g., 2-, 3-, 4-, 5-, 6-, 8-, 10-, 20-, 50-, 100-, 150- fold or more). The effectiveness of the delivery of the peptide can improve the conclusion of the polypeptide in a suitable carrier (e.g., polymeric microparticles or implantable devices), in particular, in the case of oral in which edenia.

V. Combined treatments

Tumor cells resistant to agents that damage DNA, pose a major problem in clinical Oncology. One goal of modern researches in the field of cancer is finding ways to enhance the effectiveness of chemo - and radiotherapy. One way is combining these traditional methods of treatment with gene therapy. In the context of the present invention, it is assumed that therapy MUC1-peptide can be used similarly in conjunction with chemotherapy, radiotherapy or immunotherapy intervention.

To kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or other treatment or reduce the malignant phenotype of tumor cells, using the methods and compositions according to the present invention, generally, is contact the target cell with a MUC1-peptide and at least one method of treatment. Such treatments will be performed with equal efficiency for the destruction of cells or inhibiting its proliferation. This process may include the simultaneous contact of the cells with agents/treatments. The process can be done by contact of the cell with a single composition or pharmacological composition, sod is Rasim both agents, or simultaneous contact of the cell with two distinct compositions or formulations, one composition comprises a MUC1-peptide, and the other includes the agent.

Alternatively, the effects of MUC1 may precede another action or follow him at intervals from one minute to weeks. In the variants of implementation, in which the cell is separately subjected to different impact and influence of MUC-1, in General, it should be noted that the period of time between each exposure was not too large, so that treatment methods were able to provide a preferred combined effect on the cell. In these cases it is assumed that the implementation of the contact of cells with two types of exposure in the range of about 12 to 24 hours between them, in the range of approximately 6-12 hours between them, or with an interval of only about 12 hours. However, in some situations, it may be desirable to significantly increase the time period of treatment; every few days(2, 3, 4, 5, 6 or 7) to several weeks(1, 2, 3, 4, 5, 6, 7 or 8) between the respective introductions.

Is also possible that will be desirable more than one introduction or MUC1-peptide or other treatment. Can be used in different combinations in which the MUC1-peptide is "A", and another method of treatment is "In", some of which are listed below:

Other combinations are also provided. On the other hand, for a cell with equal efficiency for the destruction of cells.

Agents or factors suitable for use in combination therapy include any chemical compound or treatment method that cause DNA damage when exposed to a cell. Such agents and factors include radiation and waves that induce DNA damage such as γ-irradiation, x-irradiation, UV-irradiation, microwaves, electron emission, etc., it is Assumed that the number of chemical compounds, also described as "chemotherapy" or "genotoxic agents will be suitable in the combined treatment methods disclosed in the present description. In the treatment of cancer according to the invention shall contact the tumor cells with an agent in addition to expression design. This can be accomplished by irradiating the localized tumor, such as x-rays, UV light, γ-rays or even microwaves. Alternatively, the tumor cell may be subjected to contact with the agent by introducing to the subject a therapeutically effective amount of the pharmaceutical composition.

In combination with the peptides of the present invention assumes the use of different classes of chemotherapeutic agents. For example the EP, selective antagonists of estrogen receptors ("SERM"), such as tamoxifen, 4-hydroxy-tamoxifen (apiexpo), palstics, raloxifene, bazedoxifene, clomiphene, femoral, dazoxiben, ormeloxifene and toremifene.

It is assumed that suitable chemotherapeutic agents include, for example, camptothecin, actinomycin-D, mitomycin C. the Invention also encompasses the use of a combination of one or more agents that damage DNA or radiation, or chemical compounds, such as the use of x-ray radiation with cisplatin or cisplatin with etoposide. The agent can be prepared and used in a combined therapeutic composition, or kit, by combining it with MUC1-peptide, as described above.

Heat shock protein 90 is a regulatory protein found in many eukaryotic cells. It has been shown that HSP90 inhibitors suitable for treating cancer. Such inhibitors include geldanamycin, 17-(allylamino)-17-demethoxygeldanamycin, PU-H71 and rifabutin.

Also provided by the agents, which are directly bound to DNA or form adducts with DNA. You can use agents, such as cisplatin and other DNA-alkylating agents. Cisplatin is widely used in the treatment of cancer with effective doses used in clinical application, the pillar is concerned 20 mg/m 2for 5 days every three weeks, only three of the course. Cisplatin is not absorbed orally and must be entered using intravenous, subcutaneous, intratumoral or intraperitoneal injection.

Agents that damage DNA, also include compounds that disrupt the DNA replication, mitosis and division of chromosomes. Such chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, etc. are Widely used in the clinic for the treatment of neoplasm such compounds injected bolus injections intravenously at doses ranging ranging from 25-75 mg/m221-day intervals for doxorubicin, up to 35-50 mg/m2for etoposide intravenously or doubling intravenous dose in the case of oral administration. Also provided by the inhibitors of the formation of microtubules, such as taxanes. These molecules are diterpenes produced by the plants of the genusTaxusand include paclitaxel and docetaxel.

Inhibitors of growth factor epidermis, such as iressa, mTOR, target of rapamycin in mammals, also known as FK506-binding protein 12-rapamycin associated protein 1 (FRAP1), is a serine/threonine a protein kinase that regulates cell growth, cell proliferation, cell movement, cleocin the e survival, protein synthesis and transcription. Therefore, rapamycin and its analogs (realogy") is intended for use in combination anticancer therapy according to the present invention.

Another possible material for combination therapy with the stated in the present description the peptides is TNF-α (tumor necrosis factor alpha), a cytokine involved in systemic inflammation and is a member of a group of cytokines that stimulate the acute phase reaction. The primary role of TNF is the regulation of immune cells. TNF can also induce apoptotic cell death, induce inflammation, and to inhibit tumorigenesis and viral replication.

Agents that disrupt the synthesis and accuracy predecessors and subunits of nucleic acids also cause DNA damage. Accordingly, there has been developed a number of precursors of nucleic acids. Particularly suitable agents that have been extensively tested and readily available. Accordingly, agents such as 5-fluorouracil (5-FU), are preferably used neoplastic tissue, making this agent is particularly suitable for infiltration of neoplastic cells. Despite the significant toxicity of 5-FU is used with a wide range of carriers, including carriers for the local administration, however, is typically used with intravenous doses varier is more fresh in the range from 3 to 15 mg/kg/day.

Other factors that cause DNA damage and are intensively used, include the well-known gamma-irradiation, x-irradiation and/or the directed delivery of radioisotopes to tumor cells. Also provides other forms of damaging DNA factors, such as microwaves and UV radiation. It is most likely that all these factors cause a variety of DNA damage, damage to the precursors of DNA, disruption of replication and DNA repair, and violation of the Assembly and maintenance of chromosomes. The ranges of doses for x-ray irradiation range from daily doses of 50-200 x-rays for extended periods of time (3-4 weeks) to single doses of 2000 to 6000 x-ray. The range of dosages for radioisotopes vary widely, and depends on the time half-life of the isotope, the strength and type of emitted radiation and absorption of neoplastic cells.

The person skilled in the art can refer to "Remington''s Pharmaceutical Sciences, 15th edition, Chapter 33, in particular pages 624-652. There will always be some changes to the dosage depending on the condition of the subject being treated. In any case, the person responsible for the introduction, will determine the appropriate dose for a specific subject. Moreover, for introducing people, the drugs must meet the standards of article is realnosti, progenote, General safety and purity required by the FDA standards (US quality supervision of food and drugs) for biological substances.

The inventors suggest that local or introduction MUC-1 peptides patients with cancer will be a very effective method for the treatment of clinically apparent disease. Similarly, chemotherapy or radiotherapy may be directed to the particular affected area of the body of the subject. Alternatively, in some circumstances, may be appropriate regional or systemic delivery of gene-expression constructs and/or agent, such as intensive metastasis.

In addition to combination therapies using MUC1 with chemo - and radiotherapy, also combination with immunotherapy, hormonal therapy, therapy with toxins and surgical intervention. In particular, you can use a targeted therapy, such as therapy with Avastin, Erbitux, Gleevec, Herceptin and Rituxan.

It should also be noted that any of the above therapies may prove useful by itself in the treatment of cancer.

VI. Examples

The following examples are included to demonstrate particular embodiments of the invention. Specialists in this area SL is blowing to consider, the techniques described in the following examples represent techniques that, as it was discovered by the inventors that work well in the application of the invention in practice, and, therefore, they can be regarded as the practical applications of the invention in practice. However, experts in this field should take into account that in specific embodiments of the present invention can make various changes, getting the same result without departing from the essence and scope of the invention.

Example 1 Materials and methods

Cultivation of cells

Cell lines breast cancer man, ZR-75-1, ZR-75-1/vector and ZR-75-1/MUC1siRNA (Renet al., 2004) were grown in RPMI1640 medium with 10% V / V heat inactivated fetal calf serum (HI-FBS), 100 u/ml penicillin and 100 µg/ml streptomycin (Invitrogen) in an incubator with humidity in 5% CO2at 37°C. Cells cancer human breast MCF-7 and 293 cells were grown on modified Dulbecco environment Needle (DMEM) with 10% HI-FBS, antibiotics and 2 mm L-glutamine. Epithelial cells human breast MCF-10A were grown in medium for growth of epithelial cells of the mammary glands (MEGM; Lonza). Cells were treated with peptides MUC1/CQC or MUC1/AQA synthesized in MIT Biopolymer Laboratory, Cambridge, MA. Cell viability was determined by exclusion of the trip the new blue.

Immunoassay and Western blot turns

Lysates of whole cells and nuclei were obtained as described (Lenget al., 2007). Immunoassay soluble proteins was performed with anti-Flag antibodies (Sigma, St. Louis, MO). Received immunoassay and soluble proteins were analyzed by Western blot turns with anti-His (Cell Signaling Technology, Danvers, MA), anti-GFP (Millipore, Danvers, MA), anti-Flag, anti-MUC1-C (Ab1; NeoMarkers, Fremont, CA), anti-Lamin B (EMD, La Jolla, CA) or anti-β-actin (Sigma) antibodies. Interactions were detected using conjugated with horseradish peroxidase secondary antibody and chemiluminescent reactions.

Transfection of cells

The 293 cells were transfusional vectors expressing GFP, GFP-MUC1-CD or Flag-MUC1-CD in the presence of Lipofectamine, as described (Lenget al., 2007).

The uptake of the peptide by cells

Cells were incubated with FITC labeled peptide MUC1/CQC (MIT Biopolymer Laboratory), washed with cold PBS, fixed in 1% wage paraformaldehyde in PBS and perform the analysis of the level of fluorescence using flow cytometry.

Analysis of the distribution of the phases of the cell cycle, apoptosis and necrosis

The cells were collected, washed with PBS, fixed with 80% ethanol, and incubated in PBS containing 40 μg/ml RNase and 40 μg/ml of propitiated for 30 min at 37°C. cell cycle Analysis was performed using flow cytometry. The DNA content in the fraction of sub-G1 assessed by staining fixed the haunted ethanol and permeabilizando citrate buffer cells propitiation and analysed using flow cytometry, as described (Yinet al., 2007). To assess necrosis cells were incubated with 1 μg/ml of propitiated in PBS for 45 min at room temperature and then analyzed using flow cytometry as described (Yinet al., 2007).

Xenograft model of breast cancer man

4-6 week old female mice Balb-c nu/nu (Charles River Laboratories, Wilmington, MA) weighing 18-22 grams implanted subcutaneously capsules with 17-β-estradiol (0.72 mg; Innovative Research, Sarasota, FL) using a trocar. After 24 hours, 1×107cells ZR-75-1, prisoners Christi Matri-Gel (BD Biosciences) were injected subcutaneously in the iliac region. When tumors became detectivesyme ~150 mm3(group 1) or 275 mm3(group 2) mice were distributed matching pairs in groups "influence" and "control". Each group contained 5 mice, each of which had a tag on his ear and was observed throughout the study. The first dose was administered on the day of distribution groups (day 1). Buffered phosphate saline solution (media), peptide MUC1/CQC and peptide MUC1/AQA were injected with intraperitoneal injections daily. Mice were weighed twice a week, and measurement of tumors with calipers were performed every 4 days. Tumor volume (V) was calculated using the formula V=W2×L/2, where W and L represent the smaller and larger diameters, respectively. Before killing mice perfusion the Li salt solution and then in phosphate buffered formalin through the heart. Tumors were excised, fixed by immersion way for 4 hours, dehydrational in a series of ethanol solutions of increasing concentration and placed in paraffin. Analysis of the tumors was performed using staining H&E (hematoxylin/eosin) and immunoperoxidase staining with antibodies to MUC1, as described (Kufe, 1984).

Drugs and cytokines

Cisdiamminedichloroplatinum (II), doxorubicin (adriamycin), Taxol (paclitaxel) were purchased from Sigma (St. Louis, MO). rh-TNF-alpha was acquired by the company Promega (Madison, WI). Peptide GO-203 was synthesized by Anaspec Inc.

In vitroanalysis of cytotoxicity and combined action of compounds

Cells were planted in 96-well microtiter plate with flat bottom holes (Fisher) in quantities of 1000 cells per well for 6-day experiment or 3000 cells per well for 3-day experiment. Then cells were cultured for 24 hours. Anticancer drugs and GO-203 was diluted to the indicated concentrations was added to the cells. GO-203 (5 µmol/l) was added every 24 hours for 72 hours. Cell survival was determined by adding to the cells MTS reagent and measuring the absorbance at 490 nm on microplate reader.

Data analysis

The value of the IC50for all cancer drugs were determined by nonlinear regression analysis of the m, using the program Graphpad Prism (GraphPad Software, San Diego, CA). To calculate the combination index used the CombiTool (version 2.001, 1MB Jena Biocomputing Group) for a range of doses in the presence of 5 mmol/l GO-203.

Example 2 - Results

The effects of MUC1/CQC-peptide to form oligomers MUC1

The cytoplasmic domain of MUC1 (MUC1-CD) contains CQC-motif, which is required for the formation of its oligomers and nuclear translocation (Lenget al., 2007). To determine whether inhibition of oligomerization of low molecular weight compounds, the inventors synthesized a peptide based on the N-terminal region of MUC1-CD, which contains CQC-motive (MUC1/CQC-peptide; Fig.1A). During the synthesis the peptide was included transducible poly-D-arginine domain, promote entry of the peptide into cells (Fischer, 2007) (Fig.1A). As control was synthesized similar peptide in which CQC-motif was replaced by AQA (MUC1/AQA-peptide; Fig.1A). To assess the binding of peptides with MUC1-CD inventors was immobilized His-tagged MUC1-CD on the sensor chip for the BIAcore system. Peptide MUC1/CQC contacted His-MUC1-CD with the dissociation constant (Kd) of 30 nm (Fig.1B), similar to the dissociation constant obtained for oligomers MUC1-CD (Lenget al., 2007). In contrast, this was not visible binding peptide MUC1/AQA (data not shown). Purified His-tagged MUC1-CD formed oligomers, detective is haunted by polyacrylamide gel electrophoresis (Fig.1C). Incubation of His-MUC1-CD peptide MUC1/CQC significantly reduced the formation of oligomers and increased the content of monomers (Fig.1C). Moreover, incubation with peptide MUC1/AQA gave minimal effect or did not give any effect (Fig.1C). To assess the MUC1 oligomerizationin vivothe 293 cells were transfusional vectors expressing GFP-MUC1-CD and Flag-MUC1-CD (Fig.1D, left). Complexes GFP-MUC1-CD and Flag-MUC1-CD was detected by co-deposition from lysates of cells treated with peptide (Fig.1D, right). Similar to the results obtainedin vitrothe incubation of transfected 293 cells with peptide MUC1/CQC was associated with the destruction of the interaction between Flag-MUC1-CD and GFP-MUC1-CD (Fig.1D, right). In addition, the peptide MUC1/AQA had no pronounced effect (Fig.1D, right). These results indicate that peptide MUC1/CQC binds to MUC1-CD and blocks the formation of oligomers MUC1-CDin vitroand in the cells.

Peptide MUC1/CQC blocks translocation of MUC1-C in the cell nucleus

The breast cancer cells of human ZR-75-1 and MCF-7 sverkhekspressiya endogenous MUC1 and, therefore, represent a potential model to assess the effects of peptide MUC1/CQC (Ramasamy Thursday,et al., 2007). To assess the absorption of peptide cells ZR-75-1 were incubated with 5 μm of peptide FITC-MUC1/CQC (Fig.2A). After 2 hours the analysis of cells using flow cytometry showed a significant increase in fluorescence intensity with rednam value (MFI) 145 (Fig.2A). Additional increase in MFI detected after 6 and 24 hours (Fig.2A). Oligomerization of MUC1-C is required for its nuclear import (Lenget al., 2007). Treatment of cells ZR-75-1 peptides MUC1/CQC or MUC1/AQA did not give any effect on cellular levels of MUC1-C (Fig.2B). However, in accordance with the effect of oligomerization processing peptide MUC1/CQC, but not MU1/AQA, was associated with a decrease in the level of MUC1-C in the nucleus (Fig.2B). Similar results were obtained for MCF-7 cells with reduced levels of nuclear MUC1-C in response to the processing of peptide MUC1/CQC (Fig.2C). These data indicate that peptide MUC1/CQC blocks oligomerization MUC1-C and, thus, blocks the transfer of MUC1-C in the nucleus of the cell.

Peptide MUC1/CQC inhibits the growth and induces cell necrosis

To determine the effect of peptide MUC1/CQC on cell growth of cells ZR-75-1 was treated with 5 mm MUC1/CQC within 72 hours and analyzed the distribution of cell cycle phases. Undoubtedly observed marked inhibition of the cell cycle in S-phase compared to untreated cells or cells treated with peptide MUC1/AQA (Fig.3A). To 96 hours, the number of stocks that are in the S phase was decreased, possibly due to dropout due to death of cells (Fig.3A). Were not observed or were observed very low accumulation of cells with DNA content in the sub-G1 fraction to confirm the induction of the of popdose (Fig.3A). However, treatment of cells ZR-75-1 peptide MUC1/CQC, but not MUC1/AQA, was associated with the induction of necrosis, which is detected after 72 hours and was more pronounced after 96 hours (Fig.3B). Cells MCF-7 were answered in a similar way to the peptide MUC1/CQC with growth inhibition in S-phase (Fig.3C) and the induction of necrosis (Fig.3D). These data indicate that peptide MUC1/CQC inhibits growth and causes cell necrosis breast cancer person.

The specificity of the peptide MUC1/CQC to expressing MUC1 cell carcinoma

To determine the selective activity of the peptide MUC1/CQC in the cells of breast carcinoma, which sverkhekspressiya endogenous MUC1, the inventors have conducted experiments with cells ZR-75-1, in which the expression of MUC1 was stably suppressed by using short interfering RNA to MUC1, MUC1siRNA (Fig.4A). In contrast to inhibition of growth and death control cells ZR-75-1/vector peptide MUC1/CQC had significantly less effect on the cells ZR-75-1/MUC1siRNA (Fig.4B). In addition, the peptide MUC1/CQC had no obvious effect on growth of MUC1-negative 293 cells (Fig.4C). Also conducted research on the untransformed cell line of epithelial breast cancer, MCF-10A (Muthuswamy, 2001; Soule, 1990), in which MUC1 is expressed, but at a level below the level found in cells ZR-75-1 and MCF-7 (Ahmadet al., 2007). It is noteworthy that in contrast to cells ZR-75-1 and MCF-7 pepti the MUC1/CQC did not influence the cell cycle (Fig.4D) and growth (Fig.4E) cells MCF-10A. These data indicate that peptide MUC1/CQC has a selective activity against cell carcinoma of the breast, which sverkhekspressiya endogenous MUC1.

Peptide MUC1/CQC inhibits oncogenesisin vivo

To determine the correlation between the introduction of peptide MUC1/CQC and influence on body weight five female Nude (nu/nu) mice were injected intraperitoneally (IP) once daily dose of 50 mg/kg injection of peptide for 11 days had no visible effect on the weight of individual mice. Moreover, there was no significant effect on body weight during the following 28 days after discontinuation of the introduction of the MUC1/CQC (data not shown). To assess the antitumor activity of cells ZR-75-1 (1×107) was injected subcutaneously in the iliac region of Nude mice. 12 days to mice with tumors of approximately 150 mm3introduced peptide MUC1/CQC at doses of 10 and 50 mg/kg/day. As a control, mice were injected only media or media with peptide MUC1/AQA. Introduction peptide MUC1/CQC at a concentration of 10 mg/kg/day for 21 days slowed tumor growth compared with the results obtained with peptide MUC1/AQA introduced at a concentration of 50 mg/kg/day (Fig.5A). In addition, the introduction of peptide MUC1/CQC at a concentration of 50 mg/kg/day blocked the tumor growth for the first 7 days of treatment (Fig.5A). After exposure ceased, and observed and re-growth of tumors in mice. Undoubtedly, there were no detected tumor growth over the next 17 days (Fig.5A). To assess, in particular, causes activity conducted histopathological study of tumors collected from control and exposed treatment of mice. Tumors of mice treated with MUC1/CQC (10 and 50 mg/kg), were markedly necrotic compared with tumors of mice treated with media or peptide MUC1/AQA (Fig.5B and data not shown). However, it should be noted that tumor cells also detected around the area of necrosis (Fig.5B). Sections of tumors were also stained with antibodies to MUC1. Treatment with peptide MUC1/AQA was associated with a significant decrease in the expression of MUC1 compared to control tumors and tumors treated with peptide MUC1/AQA (Fig.5C and data not shown).

Also conducted research on tumors of larger size (~275 mm3) (Fig.6A). Introduction peptide MUC1/CQC in the intermediate dose of 30 mg/kg/d for 21 days was associated with inhibition of tumor growth (Fig.6A). Moreover, there were no visible re-growth over the next 31 days after exposure (Fig.6A), which further indicated that the peptide MUC1/CQC effective in inhibition of tumor growth. Tumors collected at the 52nd day had extensive necrotic areas (Fig.6B) and reduced the expression of MUC1. These data indicate that peptide MUC1/CQC reduces the expression of MUC1 and is associated with induction of necrosis and prolonged inhibition of tumor growth.

MUC1-C-terminal CQC-peptides, United hydrocarbon crosslinking

Intracellular protein-protein interactions that regulate many biological pathways, often mediated α-helical structure of proteins. Helical peptides can disrupt or stabilize protein-protein interactions. Natural helical peptides have a large number of disadvantages as therapeutic agents due to low activity, instability and inefficient penetration into the cell. In recent studies it was shown that these problems can be overcome by chemical modification of α-helical peptides, called a "connection with hydrocarbon crosslinking".

The inventors used the sequence of the endogenous C-terminal peptide MUC1 (AIVYLIALAVCQCRRKNYG) and created two α-helical peptide, GO-200-1B, and GO-200-2B, using the connection hydrocarbon links:

To determine the effect of exposure to GO-200-1B on the growth of cells in non-small cell carcinoma of the lung, H-1650, cells were treated with 1 and 5 μm GO-200-1B within 7 days and controlled cell growth. The results showed that treatment of cells with 5 μm GO-200-1B was associated with a significant inhibition of growth (Fig.9A). In addition, another cell line non-small cell carcinoma of the lung, H-1975, about relatively 5 μm GO-200-2B for 3 days and was in control of cell growth, as well as cell death. The results demonstrate that treatment of cells with H-1975 peptide GO-200-2B for 3 days was associated with more than 80% inhibition of cell proliferation. Moreover, the impact of the GO-200-2B was also associated with a significant induction of cell death (Fig.9B). These data indicate that the peptides of the MUC1-C, United hydrocarbon crosslinking, effectively causing growth inhibition and death of MUC1-positive cells of human cancers.

Analogues of GO-203

In recent studies of the inventors have shown that the C-terminal peptide MUC1 (CQCRRKNYGQLDIFP) inhibits the growth of many carcinoma cell lines. The authors also demonstrated that shorter MUC1-C-terminal peptide, CQCRRKN, also has activity against the destruction of tumor cells. However, these MUC1-C-terminal peptides composed of L-amino acids. It is important to note that the peptides of L-amino acids susceptible to degradation by proteolytic enzymes, while it has been shown that peptides containing D-amino acids are more stable. Accordingly, the inventors have developed a fully programalso form the above-described C-terminal peptide MUC1, in which L-amino acids are replaced with D-amino acids (GO-203). In addition, to determine the minimum sequence of amino acid residues OFS-terminal region of MUC1, which is required to maintain the cytotoxic activity, the inventors have created a number of different variants of GO-203, described in Fig.8.

Set of tumor cell lines (hormone-dependent carcinoma of the mammary glands ZR-75-1; "triple negative" breast carcinoma MDA-MB-231; non-small cell lung carcinoma A; non-small cell lung carcinoma H-1975) was grown in medium RPMI-1640 with 10% V / V heat inactivated fetal calf serum, 100 u/ml penicillin, 100 μg/ml streptomycin and 2 mmol/l L-glutamine. Cells were processed separately by different analogues of GO-203 with a concentration of 5 μm (Fig.8) within 3-7 days, and viability was determined by exclusion Trypanosoma blue. The proliferation of different cell lines were compared with cells treated only with the media. The results demonstrate that the processing of the set of tumor cell lines of various analogues of 5 μm GO-203 associated with a significant inhibition of growth (Fig.10-14).

Combination therapy

The value of combination index (CI) obtained using CombiTool, inflicted on the chart relative to the fraction of infected cells as a result of a combination of drugs. The CI values were close to 1 for cisplatin and GO-203, indicating an additive effect. The CI values obtained for doxorubicin (is 25.50, 100, 200 nm), Taxol (25, 50, 100 nm) and TNF (10,20,40 ng/ml) with GO-203 were less than 1, indicating a strong additive effect or synergistic (Fig.15A-D and 16).

Example 3 - Discussion

Peptide MUC1/CQC blocking MUC1 oligomerization

Overexpression of MUC1 is sufficient for the induction independent of the attachment of growth and oncogenic potential (Liet al., 2003a; Huanget al., 2003; Huanget al., 2005). However, it is noteworthy that the transforming function of MUC1 is blocked by mutation of CQC-motif in the cytoplasmic domain for AQA (Lenget al., 2007). MUC1 forms oligomers, and CQC-motif required for its oligomerization (Lenget al., 2007). Moreover, the formation of oligomers is necessary for translocation of the a subunit MUC1-C in the cell nucleus (Lenget al., 2007). Other features subunit MUC1-C, such as activation of Wnt/β-catenin and IKKβ->NF-κB signaling pathways, also depend on the formation of oligomers MUC1-C (unpublished data). Based on these data, the inventors have concluded that a violation of MUC1 oligomerization of low molecular weight compound, may be to block the transforming function of MUC1. To do this, they synthesized a peptide based on the sequence of MUC1, which includes CQC-motif and poly-Arg-domain for delivery of the peptide into cells. Initial studies of peptide MUC1/CQC-peptide showed that it inhibits the oligomerization of MUC1-CDin vitro. As shown earlier is by using BIAcore analysis MUC1-CD forms a dimer with a dissociation constant (Kd) 33 nm (Lenget al., 2007). MUC1/CQC-peptide similar links MUCl-CD with a Kd of 30 nm. In addition, evidence that the peptide MUC1/AQA almost no effect on MUC1 oligomerization, support the hypothesis based oligomerization from CQC-motive. MUC1/CQC, but not MUC1/AQA, was also effective in inhibiting the oligomerization MUC1-CD cells. Therefore, these data indicate that peptide MUC1/CQC can be used for violations of MUC1 oligomerization and may, thus, functions of MUC1 in carcinoma cells human mammary gland.

The selectivity of the MUC1/CQC-peptide in relation to sverkhekspressiya MUC1 cell carcinoma

Domains for delivery into cells, such as poly-D-Arg, and their conjugates enter cells at least partially, by endocytosis, and then they need to get into the target compartment cells (Fischer, 2007). The occurrence of the MUC1/CQC-peptide in breast cancer cells, ZR-75-1, easily detected, and persisted for at least 24 hours. To a great extent and in accordance with the dependence of the nuclear translocation MUC1 from its oligomerization (Lenget al., 2007), the absorption by the cells of the MUC1/CQC-peptide was associated with a reduction in the level of MUC1-C in the core. Similar results were obtained on breast cancer cells, MCF-7, indicating that this response to peptide MUC1/CQC the e was cell-specific. In addition, it is important to note that the effects on these cells MUC1/CQC, but not MUC1/AQA, was associated with growth inhibition and induction of necrosis. It was important to establish, does MUC1/CQC cell death by a mechanism dependent on the expression of its target, or is non-specific cytotoxin. Inhibition of MUC1 expression in cells ZR-75-1 abolished the cytotoxic effects of MUC1/CQC. In contrast, the effect of peptide MUC1/CQC on non-malignant epithelial breast cancer cells, MCF-10A, produced minimal effect. These data indicate that sensitivity to the MUC1/CQC-peptide is dependent on overexpression of MUC1 and MUC1 function associated with malignant phenotype. Therefore, MUC1/CQC-peptide, apparently, has dominant-negative activity of election in respect of carcinoma cells, sverkhekspressiya MUC1.

Antitumor activity of MUC1/CQC-peptide

Domains for delivery into cells are used to introduce into cells for therapeutic compounds (Fischer, 2007). However, as for any of these agents, an important issue is the possibility of delivery of MUC1/CQC-peptidein vivowith effective therapeutic index, that is, antitumor activity and acceptable toxicity profile. When examining this question, the inventors have found that the introduction of the MUC1/CQC-peptide at 10 and 30 mg/kg/day during the course the e 21 was well tolerated by the animals without obvious acute toxic effects. The authors also found that the effects at these doses was effective for tumor growth. These results were opposite to the introduction of peptide MUC1/AQA in the amount of 50 mg/kg/day for 21 days, which did not possess antitumor activity. In addition, and quite unexpectedly, there was evidence of re-growth of the tumor after administration of peptide at a dose of 30 mg/kg/day for 21 days. The introduction of the MUC1/CQC-peptide in the amount of 50 mg/kg/day for 7 days also showed that tumor growth was stopped for long periods of time after exposure. These results may be explained, at least in part, on evidence that the impact of the MUC1/CQC-peptide is associated with induction of tumor necrosis.

In vitroactivity 7-dimensional peptide MUC1/CQC was similar to the activity of 15-dimensional peptide MUC1/CQC. Based on this information, it can be expected that the 7-dimensional peptide MUC1/CQC will be as active as an antitumor agent in the tumor modelsin vivo.

All compositions and/or methods disclosed and claimed in the present description, it is possible to carry out and perform without undue experimentation based on the present description. Although the compositions and methods of the present invention have been described in the preferred embodiments of the invention, for professionals in the data is the second region will be obvious, that you can change the composition and/or methods and in the steps or sequence of steps of a method described in the present description, without departing from the concept, nature and scope of the invention. More specifically, it will be obvious that the agents described in the present description, can be replaced by some agents, similar both chemically and physiologically, receiving the same or similar results. It is implied that all such similar substitutes and modifications apparent to experts in this field, enter the nature, scope and concept of the invention defined in the appended below the claims.

VII. Links

The following links, provided that they provided examples of experiments or other details supplementary to the description given in the present description, in connection with this included in the present description by reference:

U.S. patent 5440013

U.S. patent 5446128

U.S. patent 5475085

U.S. patent 5597457

U.S. patent 5618914

U.S. patent 5670155

U.S. patent 5672681

U.S. patent 5674976

U.S. patent 5710245

U.S. patent 5790421

U.S. patent 5840833

U.S. patent 5859184

U.S. patent 5889155

U.S. patent 5929237

U.S. patent 6093573

U.S. patent 6261569

The patent application U.S. 2005/0015232

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1. Method of inhibiting MUC1-positive tumor cells in an individual, comprising the introduction of a specified individual (a) MUC1-peptide with a length of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues and terasawa sequence CQCRRK (SEQ ID NO:4), in which aminobenzoic cysteine from CQCRRK closed at its NH2-end of at least one amino acid residue that need not correspond to the native transmembrane sequence of MUC-1, or (b) MUC1-peptide with a length of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues and containing a sequence CQCRRK, all amino acid residues of the specified peptide are D-amino acids.

2. The method according to p. 1, where the specified peptide contains at least 7 consecutive MUC1 residues.

3. The method according to p. 1, where the specified peptide contains no more than 15 consecutive MUC1 residues.

4. The method according to p. 1, where MUC1-positive tumor cell is cell carcinoma, the cell leukemia or cell myeloma.

5. The method according to p. 4, where cell carcinoma is cell carcinoma of the prostate or breast cancer.

6. Method of inhibiting MUC1-positive tumor cells in an individual, comprising the introduction of a specified individual (a) MUC1-peptide with a length of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues and containing a sequence CQCRRK (SEQ ID NO:4), in which aminobenzoic cysteine from CQCRRK closed at its NH2-end of at least one amino acid residue that need not correspond to the native transmembrane last the sequences of MUC-1, or (b) MUC1-peptide with a length of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues and containing a sequence CQCRRK, all amino acid residues of the specified peptide are D-amino acids
where the specified peptide fused with a domain for delivery into the cell.

7. The method according to p. 6, where the specified domain for delivery to the cell is poly-D-R, poly-D-P or poly-D-K.

8. The method according to p. 1 or 6, where the introduction includes intravenous, intraarterial, intratumoral, subcutaneous, local or intraperitoneal administration.

9. The method according to p. 1 or 6, where the introduction includes local, regional, system, or continuous administration.

10. The method according to p. 1 or 6, where the inhibition involves the induction of inhibition of growth of the indicated tumor cells, apoptosis specified tumor cells and/or necrosis of tumor tissue, containing the indicated tumor cell.

11. The method according to p. 1 or 6, further comprising holding for a specified individual second anti-cancer therapy.

12. The method according to p. 11, where the specified anti-cancer therapy is surgery, chemotherapy, radiotherapy, hormonal therapy, therapy, toxins, immune therapy and cryotherapy.

13. The method according to p. 11, where the specified second anti-cancer therapy prior to the introduction of the indicated peptide.

14. The method according to p. 11, the specified de anticancer therapy after the introduction of the indicated peptide.

15. The method according to p. 11, where the specified anti-cancer therapy is carried out simultaneously with the introduction of this peptide.

16. The method according to p. 1 or 6, where the specified individual is a person.

17. The method according to p. 1 or 6, where the specified peptide is administered in an amount of 0.1-500 mg/kg/day.

18. The method according to p. 1 or 6, where the specified peptide is administered in the amount of 10-100 mg/kg/day.

19. The method according to p. 1 or 6, where the specified peptide administered daily.

20. The method according to p. 19, where the specified peptide is administered daily for 7 days, 2 weeks, 3 weeks, 4 weeks, one month, 6 weeks, 8 weeks, two months, 12 weeks or 3 months.

21. The method according to p. 1 or 6, where the specified peptide is administered weekly.

22. The method according to p. 21, where the specified peptide is administered weekly for 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks or 12 weeks.

23. The method according to p. 1 or 6, where the specified peptide (a) contains all L-amino acids.

24. The method according to p. 1 or 6, where the specified peptide (b) contains all D-amino acids.

25. The method according to p. 1 or 6, further comprising the stage of evaluation of the expression of MUC1 in cancer cells specified individual prior to the introduction of the indicated peptide.

26. The method according to p. 1 or 6, further comprising the stage of evaluation of the effect of the indicated peptide on the expression of MUC1 in cancer cells of a specified individual.

27. The method according to p. 1 or 6, where the specified peptide represents p is ptid GO-201, having the amino acid sequence specified in SEQ ID NO:3.

28. Pharmaceutical composition for inhibiting MUC1-positive tumor cells in an individual, containing (a) an effective amount of MUC1-peptide with a length of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues and containing a sequence CQCRRK in which aminobenzoic cysteine from CQCRRK closed at its NH2-end of at least one amino acid residue that need not correspond to the native transmembrane sequence of MUC-1; and (b) a pharmaceutically acceptable carrier, buffer or diluent.

29. The composition according to p. 28, in which the specified peptide contains at least 7 consecutive MUC1 residues.

30. The composition according to p. 28, in which the specified peptide contains no more than 15 consecutive MUC1 residues.

31. Pharmaceutical composition for inhibiting MUC1-positive tumor cells in an individual, containing (a) an effective amount of MUC1-peptide with a length of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues and containing a sequence CQCRRK in which aminobenzoic cysteine from CQCRRK closed at its NH2-end of at least one amino acid residue that need not correspond to the native transmembrane placentas the activity MUC-1; and (b) a pharmaceutically acceptable carrier, buffer or diluent in which the specified MUC1 peptide fused with a domain for delivery to a cell or domain for transduction of cells.

32. The composition according to p. 31 in which the specified domain for delivery to the cell is poly-D-R, poly-D-P or poly-D-K.

33. The composition according to p. 28, where the specified peptide is a peptide of GO-201 having the amino acid sequence specified in SEQ ID NO:3.

34. Method of inhibiting oligomerization and nuclear transport of MUC1 in cancer cells, comprising contacting cells expressing MUC1, MUC1-peptide with a length of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues and containing a sequence CQCRRK in which aminobenzoic cysteine from CQCRRK closed at its NH2-end of at least one amino acid residue that need not correspond to the native transmembrane sequence of MUC-1.

35. The method according to p. 34, where the specified peptide contains at least 7 consecutive MUC1 residues.

36. The method according to p. 34, where the specified peptide contains no more than 15 consecutive MUC1 residues.

37. The method according to p. 34, where expressing MUC1 cell is a tumor cell.

38. The method according to p. 34, where MUC1-positive tumor cell is cell carcinoma, the cell leukemia or cell myeloma.

p> 39. The method according to p. 38, where cell carcinoma is cell carcinoma of the prostate or breast cancer.

40. The method according to p. 37, where the tumor cell is in a living individual.

41. The method according to p. 40, where the specified living individual is the man.

42. The method according to p. 34, where the specified peptide is a peptide of GO-201 having the amino acid sequence specified in SEQ ID NO:3.

43. MUC1-peptide with a length of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues and containing a sequence CQCRRK, all amino acid residues of the specified peptide are D-amino acids, where the specified peptide inhibits MUC1-positive tumor cells in an individual.

44. The peptide according to p. 43, where the specified peptide is a peptide of GO-201 having the amino acid sequence specified in SEQ ID NO:3.

45. The use of MUC1-peptide with a length of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues and containing a sequence CQCRRK (SEQ ID NO:4), in which aminobenzoic cysteine from CQCRRK closed at its NH2-end of at least one amino acid residue that need not correspond to the native transmembrane sequence of MUC-1, inhibition of MUC1-positive tumor cells in an individual, by introducing a specified individual is mu specified peptide.

46. The application of p. 45, where the specified peptide contains at least 7 consecutive MUC1 residues.

47. The application of p. 45, where the specified peptide contains no more than 15 consecutive MUC1 residues.

48. The application of p. 45, where MUC1-positive tumor cell is cell carcinoma, the cell leukemia or cell myeloma.

49. The application of p. 48, where cell carcinoma is cell carcinoma of the prostate or breast cancer.

50. The use of MUC1-peptide with a length of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues and containing a sequence CQCRRK (SEQ ID NO:4), in which aminobenzoic cysteine from CQCRRK closed at its NH2-end of at least one amino acid residue that need not correspond to the native transmembrane sequence of MUC-1, inhibition of MUC1-positive tumor cells in an individual, by introducing a specified individual specified peptide
where the specified peptide fused with a domain for delivery into the cell.

51. The application of p. 50, where the specified domain for delivery to the cell is poly-D-R, poly-D-P or poly-D-K.

52. The application of p. 45 or 50, where the introduction includes intravenous, intraarterial, intratumoral, subcutaneous, local or intraperitoneal administration.

53. The application of p. 45 or 50, where the introduction includes months is Noah, the zonal system or continuous introduction.

54. The application of p. 45 or 50, where inhibition involves the induction of inhibition of growth of the indicated tumor cells, apoptosis specified tumor cells and/or necrosis of tumor tissue, containing the indicated tumor cell.

55. The use of MUC1-peptide with a length of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues and containing a sequence CQCRRK (SEQ ID NO:4), in which aminobenzoic cysteine from CQCRRK closed at its NH2-end of at least one amino acid residue that need not correspond to the native transmembrane sequence of MUC-1, inhibition of MUC1-positive tumor cells in an individual, by introducing a specified individual specified peptide, optionally including for the specified individual second anti-cancer therapy.

56. The application of p. 55, where the specified anti-cancer therapy is surgery, chemotherapy, radiotherapy, hormonal therapy, therapy, toxins, immune therapy and cryotherapy.

57. The application of p. 55, where the specified second anti-cancer therapy prior to the introduction of the indicated peptide.

58. The application of p. 55, where the specified anti-cancer therapy is carried out after the introduction of the indicated peptide.

59. Primeneniia p. 55, where specified anti-cancer therapy is carried out simultaneously with the introduction of this peptide.

60. The application of p. 45 or 50, where the specified individual is a person.

61. The application of p. 45 or 50, where the specified peptide is administered in an amount of 0.1-500 mg/kg/day.

62. The application of p. 45 or 50, where the specified peptide is administered in the amount of 10-100 mg/kg/day.

63. The application of p. 45 or 50, where the specified peptide administered daily.

64. The application of p. 63, where the specified peptide is administered daily for 7 days, 2 weeks, 3 weeks, 4 weeks, one month, 6 weeks, 8 weeks, two months, 12 weeks or 3 months.

65. The application of p. 45 or 50, where the specified peptide is administered weekly.

66. The application of p. 65, where the specified peptide is administered weekly for 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks or 12 weeks.

67. The application of p. 45 or 50, where the specified peptide contains all L-amino acids.

68. The application of p. 45 or 50, where the specified peptide contains all D-amino acids.

69. The application of p. 45 or 50, where the specified peptide contains a mixture of L - and D-amino acids.

70. The use of MUC1-peptide with a length of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues and containing a sequence CQCRRK (SEQ ID NO:4), in which aminobenzoic cysteine from CQCRRK closed at its NH2-end of at least one AMI kislotnym balance, which should not correspond to the native transmembrane sequence of MUC-1, inhibition of MUC1-positive tumor cells in an individual, by introducing a specified individual specified peptide
additionally includes the stage of evaluation of the expression of MUC1 in cancer cells specified individual prior to the introduction of the indicated peptide, or stage estimates of the effect of the indicated peptide on the expression of MUC1 in cancer cells of a specified individual.

71. The application of p. 45 or 50, where the specified peptide is a peptide of GO-201 having the amino acid sequence specified in SEQ ID NO:3.

72. The use of MUC1-peptide with a length of at least 6 consecutive MUC1 residues and no more than 20 consecutive MUC1 residues and containing a sequence CQCRRK in which aminobenzoic cysteine from CQCRRK closed at its NH2-end of at least one amino acid residue that need not correspond to the native transmembrane sequence of MUC-1 for inhibition of oligomerization and nuclear transport of MUC1 in cancer cells by contacting cells expressing MUC1, with the indicated peptide.

73. The application of p. 72, where the specified peptide contains at least 7 consecutive MUC1 residues.

74. The application of p. 72, where the specified peptide contains no more than 15 consecutive mod the s MUC1.

75. The application of p. 72, where expressing MUC1 cell is a tumor cell.

76. The application of p. 72, where MUC1-positive tumor cell is cell carcinoma, the cell leukemia or cell myeloma.

77. The application of p. 76, where cell carcinoma is cell carcinoma of the prostate or breast cancer.

78. The application of p. 75, where the tumor cell is in a living individual.

79. The application of p. 78, where the specified living individual is the man.

80. The application of p. 72, where the specified peptide is a peptide of GO-201 having the amino acid sequence specified in SEQ ID NO:3.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula (I) and (II) and a method for [18F]-fluorination of biomolecules biomolecule, particularly peptides, using a compound of formula (I).

EFFECT: obtained 18F-labelled compounds are useful as imaging agents, especially in positron emission tomography (PET).

10 cl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to a peptide of general formula A-Thr-Lys-Pro-B-C-D-X, where A is 0, Met, Met(0), Thr, Ala, His, Phe, Lys, Gly; B is 0, Gly, Asp, Trp, Gin, Asn, Tyr, Pro, Arg; C is 0, Arg, Phe, Tyr, Gly, His, Pro, Lys; D is 0, Val, Gly, Tyr, Trp, Phe, His; X is OH, OCH3, NH2, where 0 denotes the absence of an amino acid residue, under the condition that if A≠0, then B and/or C, and/or D≠0, if B≠0, then C and/or D≠0, except tetrapeptide peptides, as well as peptides Phe-Thr-Lys-Pro-Gly, Thr-Lys-Pro-Pro-Arg, Thr-Lys-Pro-Arg-Gly, having a genital or sexual function stimulating activity.

EFFECT: improved method.

7 dwg, 5 tbl, 9 ex

FIELD: biotechnologies.

SUBSTANCE: composition for improvement of brain function as active ingredient includes peptide X-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu-Y (where X is absent or represents He or Asn-Ile; and Y is absent or represents Val-Met), peptide X-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu-Y (where X is absent or represents Thr-Gln-Thr-Pro, Pro-Leu-Thr-Gln-Thr-Pro, Leu-Thr-Gln-Thr-Pro or Pro; and Y is absent or represents Val-Met) or their salts. Method for improvement of brain function involves introduction of the above peptide or its salt.

EFFECT: invention allows effective prevention of amnesia and strengthening of memory at peroral use of a low dose of the above peptides.

35 cl, 6 dwg, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to virology, and concerns the Newcastle disease virus strain. The characterised strain NDV/Mallard/Adigeya/8/2008 is recovered from a dead duck, and deposited in the Collection of Cultures of the State Research Centre for Virology and Biotechnology "Vector" under registration number No. V-512.

EFFECT: presented strain may be used to study the oncolytic properties and mechanisms, and to develop the anticancer candidate preparations based thereon.

9 dwg, 2 ex

FIELD: biotechnologies.

SUBSTANCE: peptide is presented, which is produced from protein WT1, which is able to bind with a molecule HLA-A*1101 and to induce CTL, having a sequence SEQ ID NO:6 or SEQ ID NO:7, presented in the description. Besides, a peptide dimer is described, which is used for the same purposes and comprises two peptide monomers selected from a group of peptides that includes SEQ ID NO:7, SEQ ID NO:3, SEQ ID NO:8 and SEQ ID NO:9, available in the description. A nucleic acid is presented, which codes the specified peptide and the expression vector, which contains the specified nucleic acid. A pharmaceutical composition is described for treatment or prevention of cancer of an individual, positive by HLA-A*1101, which contains the specified peptide, nucleic acid or vector. A WT1-specific CTL is described, which is induced by the specified peptide or dimer, and an antigen-presenting cell that presents the peptide. There is data on the method and set for induction of the WT1-specific CTL and for induction of the antigen-presenting cell. The method of in vitro diagnostics of cancer in an individual positive by HLA-A*1101 is presented, which includes incubation of the specified CTL or the antigen-presenting cell with a sample received from an individual positive by HLA-A*1101, and detection of the quantity of the specified CTL or antigen-presenting cell.

EFFECT: invention makes it possible to expand assortment of peptides that bind with HLA-A*1101 and are produced from the antigen WT1.

14 cl, 1 tbl, 14 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and specifically to an isolated decapeptide or nonapeptide, capable for inducing cytotoxic T cells, as well as said peptides in which 1 amino acid is substituted, a polynucleotide which codes said peptides, a pharmaceutical composition and a vaccine, which contain said peptides, a method of inducing antigen-presenting cells, a method of inducing cytotoxic T cells, an isolated cytoxic CD8+ T cell, a dendritic cell which induces CTL and a method of treating a disease associated with high expression of SEQ ID NO: 1, 3 and/or 5 genes. The isolated decapeptide or nonapeptide, which is capable of inducing cytotoxic T cells, contains amino acid sequences selected from a group consisting of SEQ ID NO: 7, 8, 12, 9, 10, 11, 192, 195, 197, 209, 225, 226, 228, 230, 240, 241, 243, 244, 253, 254 and 255. The pharmaceutical composition for treating a disease associated with high expression of SEQ ID NO: 1, 3 and/or 5 genes contains one or more said peptides and a pharmaceutically acceptable carrier. The method of treating a disease associated with high expression of SEQ ID NO: 1, 3 and/or 5 genes in an individual involves administering to the said individual a vaccine containing one or more said peptides.

EFFECT: invention enables to obtain peptides which are used to treat a disease associated with high expression of SEQ ID NO: 1, 3 and/or 5 genes.

22 cl, 26 dwg, 7 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to cosmetics, and concerns a method of application of a hair dye substance involving the stages a) preparation of the hair dye substance; b) preparation of a composition containing a hair binding peptide containing from approximately 7 to approximately 50 amino acids; and c) application of the hair dye substance and the composition containing a hair binding peptide on hair for a time sufficient for hair binding of the hair dye substance and the hair binding peptide. Also, the invention discloses the hair dye compositions and hair conditioning compositions containing the non-attached hair binding peptide and the hair dye substance or hair conditioner respectively.

EFFECT: invention provides more adequate after-shampooing colour retention relative to that if using the hair dye substance only.

20 cl, 8 tbl, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. Offered is an agent for viruses; encephalomyocarditis, influenza type A, herpes simplex type 2, representing peptides of general formula A-B-C-D-Gly-Pro-X, where A - 0, Met, Ala, Lys, Gly, Glu, Arg, His, Phe, Thr; B - 0; Arg, Asp, Gly, Glu, Phe, Pro, Arg, Tyr, His, Lys; C - 0, Arg, Asp, Gly, Glu, Tyr, Yal, Phe; D- 0, Pro, His, Arg, Lys, Tyr, Thr, X - 0, Arg, Gly, Glu, Asp, Leu, where 0 is the absence of an amino acid residue.

EFFECT: invention extends the range of products for antiviral control.

13 tbl, 16 ex

FIELD: medicine.

SUBSTANCE: invention relates to biologically active peptides, able to prevent sharp increase of permeability of vessel endothelium. Claimed is peptide of formula H-(N-Me)-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 and its pharmaceutically acceptable salts.

EFFECT: peptide can be applied as anti-edema medication in various fields of medicine.

1 dwg, 2 ex

FIELD: virology.

SUBSTANCE: invention proposes different compositions able to induce production of antibodies against Tat HIV-1 that can inhibit multiplication of HIV-1. Also, invention proposes a method for induction of antibodies raised against Tat HIV-1, in vitro method for assay of the presence of antibodies and their titer values, a method for reducing HIV-1 virus levels, sequence of synthetic nucleic acid and synthetic molecule. Proposed group of inventions can be used for inhibition of multiplication of HIV-1 in infected patients and for attenuation of HIV-1 multiplication after the primary infection in early infected persons.

EFFECT: valuable methods and compositions.

39 cl, 7 dwg, 9 tbl, 5 ex

FIELD: metallurgy.

SUBSTANCE: invention relates to casein succinylate of iron (III) wherein iron content varies from 4.5 wt % to 7 wt %, water solubility exceeds 92% while phosphorus-to-nitrogen ratio exceeds 5 wt %.

EFFECT: additionally, invention relates to production of iron (III) and to pharmaceutical composition containing casein succinylate of iron (III).

17 cl, 4 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented group of inventions refers to biotechnology, and concerns a DLK1-Fc fused protein and using it for the metastases inhibition, a polynucleotide coding such a protein, an expression vector containing the polynucleotide, a host cell producing the above fused protein, a method for producing the fused protein by culturing the above host cell, a composition containing the above fused protein, and a method for the metastases inhibition. The characterised fused protein contains a DLK1 extracellular soluble domain consisting of the amino acid sequence SEQ ID NO:4 and Fc domain of a human antibody.

EFFECT: group of inventions can be used for preparing a therapeutic agent for reduction of cancer cell migration and the metastases inhibition.

11 cl, 36 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to biochemistry. Application of a fused protein to obtain a composition for the body weight reduction is described. The fused protein contains a domain of transduction, a signal of mitochondrial localisation and a domain of a mitochondrial factor of transcription A, binding polynucleotide (TFAM), containing a group with high mobility. Methods of treating obesity by means of the said protein are described.

EFFECT: invention extends an arsenal of means for treating obesity.

9 cl, 5 dwg, 2 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of production of casein calcium chloride of technical casein by precipitation, and can be used in microbiological studies for production of components of storing media of cultures of microorganisms, and also production of calcium co-precipitates for food industry.

EFFECT: improvement of the method.

2 cl, 1 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, namely to leukolectins, and can be used in medicine. What is prepared is the polypeptide leukolectin characterised by SEQ ID NO:1-8. The recombinant preparation is ensured by using a nucleic acid coding it and integrated into an expression vector which is used to transform a host cell. Testing absence-presence or determining an amount of the polypeptide leukolectin are ensured by using an antibody or an antigen-binding fragment of a variable region of the above antibody which is specifically bound to the polypeptide leukolectin. The polypeptide leukolectin or the nucleic acid coding it are used as ingredients of a pharmaceutical composition in therapy of pathological disorders of skin and mucous membranes.

EFFECT: invention enables treating or preventing autoimmune disorders of skin, inflammatory diseases of skin or mucous membrane, or injured skin in an animal effectively.

16 cl, 19 dwg, 3 tbl, 12 ex

Antibody to epha2 // 2525133

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of immunology, medicine and biotechnology. Claimed are versions of anti-EPHA2 antibodies. Claimed antibodies are bound with polypeptide, consisting of amino acids 426-534 in SEQ ID NO:8. Also described are hybridomes, which produce such antibodies, and pharmaceutical compositions and methods of application of said antibodies and compositions.

EFFECT: invention can be used in medicine.

74 cl, 14 dwg, 14 ex, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, in particular to immunogens based on antigenic tau-peptide, and can be used in medicine. Obtained is immunogen, which contains antigenic tau-peptide, consisting of amino acid sequence, selected from SEQ ID NO:6, 8-19, 21-26, 105 and 108-112, covalently bound with immunogenic carrier by means of linker, represented by formula (G)nC, where n equals 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Linker can be located either on C-terminal (peptide -(G)nC), or on N-terminal (C(G)n-peptide) of peptide. Obtained immunogens are used as base for creation of pharmaceutical compositions for treatment of tau-associated neurological disorders.

EFFECT: invention makes it possible to induce immune response against tau autoantigen in efficient way.

12 cl, 10 dwg, 5 tbl, 16 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel non-branched carbamate derivatives of some peptides Wnt-5a, in particular to N-butyloxycarbonyl derivative, their pharmaceutical compositions and their application for treatment of gastric melanoma and cancer.

EFFECT: obtaining novel non-branched carbamate derivatives of some peptides Wnt-5a.

7 cl, 9 dwg, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology, namely to muteins of human tear lipocalin, and can be used in medicine. Mutein of human tear lipocalin (hTLc) has identifiable affinity of binding with human receptor Met (c-Met) receptor tyrosine kinase, or its domain, or fragment of human c-Met. Mutein contains from 6 to 18 amino acid substitutions relative to amino acid sequence of mature lipocalin of human tear liquid (SWISSPROT DATABANK ENTRY P31025; SEQ ID NO:36), selected from group, consisting of Arg 26→Thr, Val, Pro, Ser, Gly; Glu 27→Gln, Gly, Val, Ser; Phe 28→Met, Asp; Pro 29→Leu, Ile, Ala, Trp; Glu 30→Leu, Gly, Arg, Phe; Met 31→Ser; Asn 32→Leu, Arg, Val, Gln; Leu 33→Tyr, Val, Ile, Thr, Phe; Glu 34→Val, Arg, Ala; Leu 56→Asn; Ile 57→Gln; Ser 58→Ile, Val; Asp 80→Tyr; Lys 83→Ala; Glu 104→Asp; Leu 105→Thr; His 106→Trp and Lys 108→Gly. Mutein can also additionally contain the following substitutions: Cys 61→Ser; Cys 101→Ser; Cys 153→Ser; Arg 111→Pro; Lys 114→Trp; Thr 37→Ser; Met 39→Ile, Leu; Asn 48→Ser; Lys 52→Thr, Met; Met 55→Leu; Lys 65→Arg, Leu; Ala 79→Leu, Ser; Ala 86→Thr; Ile 89→Ser, Gln, Thr, His; Thr 40→Cys; Glu 73→Cys; Arg 90→Cys; Asp 95→Cys; Lys 121→Cys; Asn 123→Cys and Glu 131→Cys.

EFFECT: invention makes it possible to efficiently treat pathological disorders, which involve pathway HGF/c-Met, as well as to perform identification of human c-Met in sample.

40 cl, 16 dwg, 9 tbl, 25 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. What is disclosed is a vaccine representing four RNAs coding a prostate-specific antigen (PSA), a prostate-specific membrane antigen (PSMA), a prostate stem cell antigen (PSCA) and a six-transmembrane epithelial antigen of the prostate (STEAP). The vaccine is applicable for treating prostate carcinoma, preferentially neo-adjuvant and/or hormone resistant prostate carcinoma, as well as related diseases or disorders. Using the vaccine and a kit are also disclosed. The invention can be used in medicine.

EFFECT: preparing the vaccine for treating prostate carcinoma.

16 cl, 23 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biochemistry, in particular to single variable domain, aimed against IL-6R, to polypeptide and construction, directed against IL-6R, containing said single variable domain, as well as to methods of obtaining them. Disclosed are nucleic acids, coding said single variable domain, polypeptide and construction, as well as genetic constructions, containing said nucleic acids. Described are host cells and host organisms, containing said nucleic acids. Invention also deals with composition for blocking interaction of IL-6/IL-6R, containing effective quantity of described single variable domain, polypeptide, construction, nucleic acid or genetic construction. Also disclosed is method of prevention and/or treatment of at least one of diseases or disorders, associated with IL-6, IL-6R, complex IL-6/IL-6R and/or signal pathways, in which IL-6, IL-6R or complex IL-6/IL-6R is involved and/or biological functions and reactions, win which IL-6, IL-6R or complex IL-6/IL-6R takes part with application of described single variable domain, polypeptide, construction or composition.

EFFECT: invention makes it possible to block interaction of IL-6/IL-6R effectively with increased affinity and biological activity.

25 cl, 70 dwg, 56 tbl, 61 ex

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