Cdh3-peptide and drug preparation containing it

FIELD: medicine.

SUBSTANCE: there are presented versions of the peptide (A) or (B) with the amino acid sequence of SEQ ID NO: 1 or 2 respectively presented in this description. The peptide has activity to induce a cytotoxic T-cell when an antigen-presenting cell carrying HLA-A2 (A*0201) presents it. There are described the versions of the peptide antibodies prepared by immunisation by the proper peptide. There are presented: an agent, methods for inducing: a cytotoxic (killer) T-cell, an antigen-presenting cell, CDH3 expressing cancer immunity; as well as a method of treating CDH3 expressing cancer on the basis of the peptide. What is presented is an isolated cytotoxic T-cell induced by said method on the basis of the peptide. There are described: the antigen-presenting cell and exosome presenting the complex containing the peptide and HLA-A2 (A*0201).

EFFECT: higher effectiveness of the use of the invention in treating CDH3 expressing cancer.

15 cl, 5 dwg, 2 tbl, 5 ex

 

The technical field

The present invention relates to new peptides suitable as vaccines against cancer, which is highly expressed P-cadherin (CDH3), such as pancreatic cancer, cholangiocellular cancer, gastrointestinal tract cancer, colon cancer and lung cancer, and the present invention relates to pharmaceutical agents, including peptide for the treatment and prevention of cancer.

The level of technology

The share of pancreatic cancer accounts for approximately 2-3% of all malignant tumors. Every year about 200,000 people around the world die from cancer of the pancreas, and the number of dead pancreatic cancer ranks fifth in importance among malignant tumors. In Japan annually kills about 20,000 people. Risk factors for developing pancreatic cancer include diabetes, chronic pancreatitis, Smoking and the like, and also has been reported that family history is also one of the risk factors. Performed various attempts for early diagnosis of the disease, including improving diagnostic imaging; however, in most patients the disease was diagnosed at late stages, when they showed resistance to chemotherapy. Thus, their five surviving the industry is approximately 9.7 per cent, and even in cases surgical removal of the tumor only about 13%. As a result in cancer of the pancreas is given the worst prognosis among cancer of the digestive system. Due to this difficulty diagnosing the gradual increase of cases of pancreatic cancer as cause of death from cancer, especially in developing countries. Although conducted multidisciplinary treatment of primary surgical removal, and other types of treatment, such as radiotherapy and chemotherapy, they have not dramatically improves therapeutic effects, and there is urgent need to develop new therapeutic strategies.

The share cholangiocellular cancer accounts for about 10% of primary liver cancer, and it represents the second most common cancer after liver cell carcinoma. This disease shows a weak clinical characteristics, and in many cases the cancer is detected in later stages, accompanied by metastasis in the lymph nodes, intrahepatic metastasis, and the like. Five-year survival is approximately 20%, and 35% in cases of surgical removal, but very weak, only 7.4% in the absence of surgical removal. Although surgical removal which is the only therapy which, as can be expected, leads to long-term survival, many patients at the time of detection are already in inoperable condition (percent of cases, surgical intervention: 66%incurable remover: 20%). Both parameters, the sensitivity to anticancer drugs and radiosensitivity of patients are very low and therefore it is desirable that a therapy for unresectable cases, including cases of incurable remove.

Compared with Western countries, the incidence of cancer of the gastrointestinal tract is the highest among Asian countries, such as Japan and China. Early detection of cancer of the gastrointestinal tract has been possible thanks to the proliferation of diagnostic tests and the development of tools endoscopy of the digestive system and methods of control, resulting in the number of patients has decreased. However, cancer of the gastrointestinal tract is still the second leading cause of death from malignant neoplasms among the Japanese, and the percentage of deaths is still high. Colon cancer is the second most common cancer in Western countries and is the third most common cause of death from malignant newly formed the th in Japan. Cancer of the gastrointestinal tract and colon cancer treated primarily with surgical resection and chemotherapy, radiation therapy and the like. Immunotherapy, which inhibits the growth of cancer by enhancing the immune system with cancer patients, attracts attention as a new therapy for metastatic and refractory cancer, against which it is impossible to use the previously mentioned therapies.

The incidence of lung cancer in recent years is constantly increasing in the world, and currently about one million people a year die from lung cancer. Mortality from lung cancer is also constantly increasing in Japan and, say, in 2015 will reach 123000 people. He is the leading cause of death from malignant neoplasms in Japan. The number of patients, it is believed, increases with the age of the population. Early detection and early treatment are important in the treatment of lung cancer. Recently, however, it was noted that a normal chest x-ray and sputum tests, conducted at medical examination, have little effect on the early detection of lung cancer and does not lead to a decrease in mortality from cancer. As the number of deaths from lung cancer, as it is constantly increasing, the development of a new terapeuticas the second strategy seems to be an urgent task.

On the one hand, the development of molecular biology and immunology of tumors revealed that cytotoxic T-cells (T-cells) and cells T-helper cells recognize peptides generated during the degradation of proteins that are specific to a high level expressed in cancer cells and presented on the surface of cancer cells or antigen-presenting cells via HLA molecules and T cells cause immune reaction that destroys the cancer cells. In addition, it was identified many proteins of tumor antigens and peptides derived from them, which stimulate such an immune reaction, which affects cancer, and is currently in the development stage of clinical application of antigen-specific immunotherapy tumors.

Molecule HLA class I is expressed on the surface containing all the core cells. It is expressed on the cell surface by binding to peptides generated by intracellular degradation of proteins produced in the cytoplasm or in the nucleus. On the surface of normal cells peptides derived from normal proteins bind to HLA molecules of class I, and T cells of the immune system does not identify them for the destruction of the cells. On the other hand, during the development of cancer cancer cells sometimes expressionvalue number of proteins, which hardly or very weakly expressed in normal cells. When molecules HLA class I associated with peptides generated by intracellular degradation of proteins that are specific, or at a high level expressed in cancer cells and then expressed on the surface of cancer cells, the cells are T-cells will recognize them and will destroy only the cancer cells. In addition, with the introduction of the individual such ofwholesale antigens or peptides, the immune response that destroys cancer cells and suppresses the development of cancer, can coil without harm to normal cells. This is called immunotherapy of cancer using ofwholesale of antanov. Molecules HLA class II molecules, mainly expressed on the surface of antigen-presenting cells. Molecules HLA class II molecules bind peptides derived from ofwholesale antigens, which are generated using intracellular degradation ofwholesale antigens incorporated into antigen-presenting cells from the outside of cells, and then expressed on the cell surface. Then, after their recognition, activated cells T-helper cells and induce or enhance the immune response against tumors by producing various cytokines that activate other immune CL the weave.

Accordingly, if you develop an immunotherapy that targets which are antigens that are specific to a high level expressed in these cancer cells, this treatment can effectively eliminate only the cancer cells, without causing any dangerous effect on your own normal organs. It is also expected that therapy may be used for patients with any end-stage cancer, for which you do not want to apply a different treatment. In addition, by pre-injection opukholespetsificheskaya antigen peptide as a vaccine to individuals with a high risk of developing such cancers, cancer can be prevented.

Although there are different therapies for the treatment of pancreatic cancer, the prognosis of cancer is very unfavorable in comparison with other types of cancer. This is because pancreatic cancer is difficult to detect at an early stage, it progresses rapidly and, thus, are often detected only very late stages. Although at present, surgical removal is the most promising radical treatment of resectable cases constitute only about 20% of the total. Surgery of the pancreas is also highly invasive, and demonstrate a very late stage blagopriyatny prognosis even after surgical removal. Do not remove the cases subjected to treatment with chemotherapy, which is mainly used gemcitabine, and with radiation therapy. However, many cases show resistance to treatment and have a weak cytoreductive effects, which is one of the reasons why pancreatic cancer is difficult to cure. Accordingly, if you develop immunotherapy, the target of which is an antigen that is specific and at a high level is expressed in cancer cells with cancer of the pancreas, such therapy can effectively eliminate only the cancer cells, without causing any dangerous effect on your own normal organs. It is also expected that it will become a therapy that can be applied to any patient with end-stage cancer. In addition, because pancreatic cancer often recurs at an early stage after removal, it is also expected that therapy will be applied as related postoperative therapy.

The applicants of the present invention previously conducted shirokopolosnyi analysis of gene expression 27648 human genes by analysis using cDNA-microarray to determine their expression profiles in 16 cases of cancer of the pancreas, fetal organs and in various normal organs of an adult man. As a result, they found that P-cadherin (CDH3) is highly expressed in cancer cells in most cases of cancer of the pancreas, while he barely expressed in normal organs of the adult. In addition, CDH3, as was observed, also has a high level of expression in cancer cells in most cases cholangiocellular cancer, cancer of the gastrointestinal tract, colon cancer, non-small cell lung cancer, testicular cancer, cervical cancer, osteosarcoma, soft tissue sarcoma and the like. This fact suggests that CDH3 can be opukholespetsificheskaya antigen of many cancers.

HLA-A2 is often observed in the human population, regardless of race, and about 30% of Japanese people are carriers of HLA-A2. Thus, if it can be identified peptide presented to cells of the T-killer cells using HLA-A2, it can be widely applied not only among the Japanese people, but also among Western Europeans and the like. Accordingly, the identification of tumor antigen peptides presented to T cells-the cells using HLA-A2, is an important task. Can be highly useful application of such peptides of tumor antigens for immunotherapy of lung cancer, whose prevalence and mortality rates are high around the world.

Information about what ocument prior art, relevant to the invention, opened in the present application is shown below.

[Non-patent document 1] Nakamura, T., et al., Oncogene 23: 2385-2400 (2004)

[Non-patent document 2] Obama, K., et al., Hepatology 41: 1339-1348 (2005)

[Non-patent document 3] Taniuchi, K., et al., Cancer Res 65: 3092-3099 (2005)

[Non-patent document 4] Soler, A. P., et al., Cancer 86: 1263-1272 (1999)

[Non-patent document 5] Paredes, J., et al., Clin Cancer Res 11: 5869-5877 (2005)

[Non-patent document 6] Ingunn, M., et al., J Clin Oncol 22: 1242-1252 (2004)

[Non-patent document 7] Glenn, L., et al., J Cell Biol 139: 1025-1032 (1997)

[Non-patent document 8] Bauer, R., et al., Exp. Mol. Pathol. 81: 224-230 (2006)

[Non-patent document 9] Muzon-Guerra, M.F., et al. Cancer 103: 960-969 (2005)

[Non-patent document 10] Marck, V.V., et al., Cancer Res. 65: 8774-8783 (2005)

Description of the invention

[Tasks solved by means of the invention]

The goal that should be achieved with the present invention represents a development of the methods for the implementation of immunotherapy, which inhibits the growth of cancer by enhancing the immune system with cancer patients, as a new therapy for metastatic and refractory cancers that can hardly be treated through surgical treatment, chemotherapy and radiation therapy used to treat cancer of the pancreas, cholangiocellular cancer, cancer of the gastrointestinal tract, cancer of the colon kick is, non-small cell lung cancer and the like. The present invention features identified peptides that are derived from proteins that are specific to a high level expressed in cancer cells and presented to T cells-the cells using HLA-A2. This allows immunotherapy, which can be used for approximately 30% of Japanese patients with different cancers, the cancer cells which Express a high level of CDH3.

[Means of solving problems]

The applicants of the present invention by analyzing the tissues of pancreatic cancer using cDNA microchip identified CDH3 (Registration No. GenBank NM_001793) as a gene that is highly expressed in cancer cells of the pancreas. To determine induced or no antitumor immunity using CDH3-specific T cell-killers, used transgenic for HLA-A2 mice expressing HLA-A2, carriers which are about 30% of the Japanese. Specifically, transgenic for HLA-A2 mice were immunized using bone-marrow-derived dendritic cells stimulated with human CDH3-peptide containing the motif binding to HLA-A2, in order to determine whether the induction of HLA-A2-restrictively patinopecten cells T-killers. The ELISPOT method used is ovali for detection of γ-interferon (IFN-γ), produced by cells of the T-killer cells, which are activated by recognition of peptide presented by HLA-A2, and thus determined, induced or not in the spleen cells of immunized mice cells T-cells specific to CDH3-peptide. In the applicants of the present invention have identified two new CDH3-peptide applicable for immunotherapy for HLA-A2-positive with cancer patients. In addition, it was found that CDH3-responsive cells CTL (CTL)induced by these peptides possess cytotoxicity specific to cancer cells expressing endogenous molecules CDH3 and HLA-A2, and the cells CTLs recognize target cells using restriction by HLA class I. in Addition, it was also found that the growth of tumors transplanted into mice NOD/SCID, was significantly suppressed by using intravenous injection of CD8-positive cells induced by peptides (method TLCs-adoptive immunity).

More specifically, the present invention features:

(1) a peptide corresponding to (A) or (B):

(A) a peptide comprising amino acid sequence SEQ ID NO: 1 or 2;

(B) a peptide comprising amino acid sequence SEQ ID NO: 1 or 2, where one, two or more amino acids replaced, deleterow, inserted and/or added, and where the peptide has an activity is induction of cytotoxic T-killer) T-cells;

(2) the peptide under item 1, where the second amino acid from the N-Terminus is a leucine or methionine;

(3) the peptide under item 1, where the C-terminal amino acid is a valine or leucine;

(4) an agent for inducing immunity against cancer comprising as active ingredient one or more peptides in p. 1;

(5) the agent for treating and/or preventing cancer, comprising as active ingredient one or more peptides in p. 1;

(6) an agent for inducing antigen-presenting cells having the activity of inducing cytotoxic T-cells (T-killer), comprising as active ingredient one or more peptides in p. 1;

(7) an agent for inducing antigen-presenting cells having the activity of inducing cytotoxic T-cells (T-killer), comprising as active ingredient one or more polynucleotides encoding the peptide under item 1;

(8) an agent for inducing cytotoxic T-cells (T-killer), comprising as active ingredient one or more peptides in p. 1;

(9) the antibody to the peptide under item 1;

(10) cell T-helper cells, cytotoxic T-cell (T-killer) or a group of immune cells, including those cells that are induced by the application of the peptide under item 1;

(11) the antigen-presenting cell, which is complex, involving the th peptide in p. 1 and HLA-antigen;

(12) the antigen-presenting cell according to p. 11, which is induced by the agent under item 6 or 7;

(13) actsoma, which is a complex comprising the peptide under item 1 and HLA-antigen;

(14) actsoma on p. 13, where HLA-antigen is an HLA-A2 (HLA-A2*0201);

(15) a method of inducing antigen-presenting cells having the activity of inducing cytotoxic T-cells (T-killer), including the stage of contacting the antigen presenting cells with the peptide under item 1;

(16) a method of inducing antigen-presenting cells having the activity of inducing cytotoxic T-cells (T-killer), including the state transfection of polynucleotide encoding the peptide under item 1, in the antigen-presenting cell;

(17) a method of inducing cytotoxic T-cells (T-killer), including the stage of contacting T cells with the peptide under item 1;

(18) a method of inducing immunity against cancer, including the stage of introduction of the subject peptide under item 1;

(19) a method of treating and/or preventing cancer, including the stage of introduction of the subject peptide under item 1;

(20) use of the peptide under item 1 for agent intended for inducing immunity against cancer;

(21) use of the peptide under item 1 for a medicinal product intended for the treatment and/or prevention of cancer.

To the capacity description drawings

In Fig. 1 illustrates an authentication scheme CDH3-peptide recognized by HLA-A2-restrictively cells of T-killers. (The day that were isolated spleen cells from immunized mice presented as “Day 0”).

In Fig. 2 depicts a graph showing the result of analysis of ELISPOT for 18 CDH3-peptides. The ELISPOT analysis was used to determine whether cells T-cells obtained from immunized mice that are specific to react with cells stimulated with CDH3-peptides, and to produce IFN-γ. Cells T-killer cells, induced with CDH3-4-peptide or CDH3-7-peptide-specific recognize cells KM-DC (BM-DC), stimulated with CDH3-peptides, and produced IFN-γ; however, T cells, killer induced by other peptides did not show CDH3-specific immune response. Thus, peptides CDH3-4 and CDH3-7, as confirmed, are epitope peptides that can induce CDH3-specific HLA-A2-restrictively cells T-killers. Non CDH3-peptides shown in Figure 2, correspond to the peptide numbers shown in the column “position” in Table 2, and not numbers of sequences SEQ ID NO described in this application.

In Fig. 3 shows photographs showing the results of ELISPOT analysis, detecting IFN-γ produced the CSOs cells T-killers, activated by specific recognition CDH3-peptides. CD4-negative cells of the spleen showed 283,7±40 spots/well in response to cell KM-DC stimulated with peptide CDH3-4655-663(to the left in A and above in B), while they showed 48,7±11.9 spots/well in response to cell KM-DK in the absence of peptide stimulus (to the right in A and the lower row (B) (P<0,05). Similarly, CD4-negative cells of the spleen showed 79,3±3.2 spots/well in response to cell KM-DC stimulated with peptide CDH3-7757-765(top row in C), while they showed 42,7±2.5 spots/well in response to cell KM-DK in the absence of peptide stimulus (bottom row in (C) (P<0,05).

The analysis was carried out twice and got the same results.

In Fig. 4 depicts line graphs showing the induction CDH3-specific human cells CTLs from cells of peripheral blood mononuclear MCPC (PBMC) of HLA-A2-positive healthy donors and patients with cancer. A: cells CTLs reactive to CDH3-peptide induced cell MCPC HLA-A2-positive healthy donors. After stimulation three times using autologous derived from monocytes dendritic cells DC (DC)stimulated with peptide CDH3-4655-663(above) or CDH3-7757-765(below)evaluated the cytotoxicity against CL is the current T2 (HLA-A2-positive, TAP-deficient), stimulated or not stimulated each peptide using standard analysis release51Cr. Cells CTLs showed cytotoxicity to the cells T2-stimulated peptide CDH3-4655-663(above) or CDH3-7757-765(below), but did not show cytotoxicity to the cells T2, not stimulated with peptide. B: Cells CTLs showed cytotoxicity to CDH3+HLA-A2+cells cell line HCT116 human, and squamous cell oral cancer cell lines HSC3, and PANC1 cells/CDH3, which are CDH3 - HLA-A2+cells cell line PANC1 pancreatic cancer man, transformed gene CDH3. However, cells CTLs do not exhibit cytotoxicity to CDH3 - HLA-A2+cells cell line SKHep1 liver cancer man, PANC1 cells, and to CDH3+HLA-A2 cells cell line PK8 pancreatic cancer man. C: CDH3-reactive cells CTLs induced from cells MCPC HLA-A2-positive patients with pancreatic cancer (PC), and patients with cancer of the gastrointestinal tract (GC)showed cytotoxicity to the cells HCT116 and PANC1/CDH3, but did not show cytotoxicity to cells PANC1 and PK8. D: Demonstrated inhibition of cytotoxicity using monoclonal antibodies to HLA class I. After the Incubus is the formation of target cells, SKHep1/CDH3 and HSC3, with a monoclonal antibody to HLA class I (W6/32, IgG2a) or with a monoclonal antibody to HLA-DR (H-DR-1, IgG2awithin hours, was added to cells CTLs induced from cells MCPC healthy donors stimulated with peptide CDH3-4655-663(left, middle) or CDH3-7757-765(on the right). The production of IFN-γ (left and right, IFN-γ-ELISPOT-analysis) and cytotoxicity (mid, analysis release51Cr) markedly inhibited using W6/32, but is not inhibited by using H-DR-1.

In Fig. 5 shows in vivo antitumor activity of cells CTLs induced with CDH3, against cancerous human cells transplanted mice NOD/SCID. A: Inhibition of cell growth cell line HCT116 (CDH3+HLA-A2+) cancer of the colon and rectum, vaccinated mice NOD/SCID, after transplantation of cells CTLs. When the tumor size reaches 25 mm2on the 7th day after the subcutaneous implantation of the tumors were inoculable intravenous human cells CTLs reactive to the peptide CDH3-4655-663(□) and peptide CDH3-7757-765(■). On day 14 the cells CTLs were inoculable again in the same way. Control CD8+T cells stimulated with HLA-A2-restrukturovaneho HIV-peptide did not show cytotoxicity (◊). Demonstrated the size of tumors in mice NOD/SCID who received two cycles CDH3-reactive to etoc CTLs (n=7), control CD8+T-cells (n=7), or individually PBS (○, n=7), 7 and on day 14. The tumor size is expressed in square millimeters. B: the tumor Size in each group is shown with a standard deviation ± SD (n=7).

The method of carrying out the invention

The terms “a”, “an” and “the” when used in this application denotes “at least one” unless and until determined otherwise.

Up until not specified differently, all technical and scientific terms used in this application have the same meaning, what is generally accepted among experts in this field belongs to the present invention.

The peptide according to the present invention is an epitope restrictively by HLA-A2, which represents the allele HLA typically found in populations of the Japanese and Europeans. Specifically, candidates of HLA-A2-binding peptides derived from CDH3, were selected using as an indicator of their affinity of binding to HLA-A2. The selected peptides was assessed by testing whether to the induction of T cells-killers in the body transgenic for HLA-A2 mice with dendritic cells derived from bone marrow cells (KM-DK) transgenic for HLA-A2 mice, stimulated with selected peptide. Cells T-cells were induced with p the power CDH3-4 (FILPVLGAV (SEQ ID NO: 1)) and CDH3-7 (FIIENLKAA (SEQ ID NO: 2)), in the body transgenic for HLA-A2 mice. Cells T-cells induced by these peptides showed an immune response to cells KM-DK, which was added to these peptides. However, these cells are T-cells did not show an immune response to cells KM-DK, to which the peptide was not added. These results demonstrate that peptides derived from CDH3, suitable for use as a peptide to induce an immune reaction against CDH3-presenting cells, and that the peptides derived from CDH3, are HLA-A2-restrictively epitope peptides. CDH3 has a high level of expressi in cancer cells in most cases of cancer, such as pancreatic cancer, cholangiocellular cancer, gastrointestinal tract cancer, colon cancer, non-small cell lung cancer, testicular cancer, cervical cancer, osteosarcoma and soft tissue tumors. This reveals that CDH3 suitable for use as a target for immunotherapy in many cancers.

(1) the Peptides according to the present invention, and agents for inducing immunity against cancer containing these peptides

The peptide according to the present invention is any of the following peptides:

(A) a peptide comprising amino acid sequence SEQ ID NO: 1 or 2;

(B) a peptide comprising the amino acid is tnou sequence of SEQ ID NO: 1 or 2, where one, two or more amino acids replaced, deleterow, inserted and/or added, and where the peptide has an activity of inducing T cell-killers;

(C) the peptide of (B), in which the second amino acid from the N-Terminus is a leucine or methionine; and

(D) peptide (B)in which the C-terminal amino acid is a valine or leucine.

The peptide according to the present invention is an epitope peptide containing less than 40 amino acids, preferably less than 20 amino acids, more preferably, less than 15 amino acids, which comprises amino acid sequence SEQ ID NO: 1 or 2, and has an activity of inducing T cell-killers. Alternatively, epitope peptide can include a peptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, where one, two or more amino acids replaced, deleterow, inserted and/or added while maintaining the activity of inducing T cell-killers. The number of replaced, deletirovannykh, inserted and/or added residues is usually 5 amino acids or less, preferably 4 amino acids or less, more preferably 3 amino acids or less, and more preferably 1 amino acid or 2 amino acids.

Variant-specific peptides (i.e. peptides comprising amino acid after which outermost, obtained by modifying the original amino acid sequence with substitution, deletion, insertion and/or addition of one, two or several amino acid residues)are known to retain the original biological activity (Mark DF, et al., (1984) Proc Natl Acad Sci USA 81: 5662-6; Zoller MJ and Smith M, (1982) Nucleic Acids Res 10: 6487-500; Dalbadie-G McFarland et al., (1982) Proc Natl Acad Sci USA 79: 6409-13). Amino acid changes are preferably retain the properties of the original amino acid side chains. Examples of properties of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or aggregate characteristics: aliphatic side chains (G, A, V, L, I, P); side chains containing hydroxyl group (S, T, Y); the side chains containing a sulfur atom (C, M); side chains containing carboxylic acids and amides (D, N, E, Q); the side chains containing base (R, K, H); and side chains containing aromatic component of a number of (H, F, Y, W), where the letters in brackets refer to the letter codes of amino acids.

In a preferred embodiment of the invention, the peptides of the present invention (immunogenic peptides) are nonapeptide (9 measures) or Decapeptide (10-steps).

In this application the peptide with what aktivnosti induce T cell-killers, refers to a peptide having the activity of inducing cell T-killer cells, which stimulates the cells of T-killer cells (cytotoxic T-cells/CTLs).

Order to obtain peptides with high affinity binding and activity of inducing T cell-killers amino acid sequence of a partial peptide of natural CDH3 can be changed by replacement, deletion or addition of one, two, or several amino acids. In this application, the term “several” means 5 or less, preferably 3 or less, more preferably 2 or less. Furthermore, since the known pattern of peptide sequences with high affinity to HLA antigens (Kubo RT, et al., (1994) J. Immunol., 152: 3913-24; Rammensee HG, et al., (1995) Immunogenetics. 41: 178-228; Kondo A et al. (1995) J.Immunol. 155: 4307-12), the peptides of the present invention (epitope peptides) can be modified to improve their affinity to HLA antigens on the basis of known patterns. For example, peptides with high affinity binding to HLA-2, can be obtained by replacing the second amino acid from N-Terminus to leucine or methionine. Similarly, peptides with high affinity binding to HLA-2, can also be obtained by replacing the C-terminal amino acids valine or leucine.

When the sequence of the epitope peptide is the same as part of the amino is islotes sequence of the endogenous or exogenous protein, with the excellent function, then this can be caused by side effects such as autoimmune disorders or allergic symptoms against specific substances. To avoid such side effects, the modified epitope peptide should not be identical to the amino acid sequences of known proteins. For this purpose it is necessary to homology search using databases to ensure that there is no endogenous or exogenous protein with excellent function, which showed 100%homology with the modified epitope peptide. This way you can avoid the risks caused by the above change in amino acid sequence to increase the affinity of binding to HLA antigen and/or for increasing the activity of inducing T cell-killers.

Although the above peptides having affinity binding to HLA-antigens, as expected, are highly effective as a vaccine against cancer, candidate peptides selected using as an indicator the high affinity, should be investigated to determine whether they have an activity of inducing T cell-killers. The activity of inducing T cell-killers can be confirmed using the: induction of antigen-presenting cells with the human the ical MHC-antigen (for example, B-lymphocytes, macrophages and dendritic cells), or, more specifically, by inducing dendritic cells derived from mononuclear leukocytes in human peripheral blood; stimulating them with the peptide of interest; then mix them with CD8-positive cells; measuring the cytotoxic activity against target cells. As the reaction system can be used transgenic animals that Express human HLA-antigen (as described, for example, BenMohamed L, et al., (2000) Hum. Immunol. 61 (8): 764-79, Related articles, Books, and library Linkout). For example, target cells can be radioactively labeled with51Cr or similar, and cytotoxic activity can be calculated from the radioactivity released from the target cells. Alternatively, target cells can be investigated using: measuring IFN-γ produced and released from cells by T-killer cells in the presence of antigen-presenting cells with immobilized peptide; and visualization of the area of production of IFN-γ in the culture medium using monoclonal antibodies to IFN-γ.

As demonstrated in the Examples, the result of the study peptide activity, the induction of T cell-killers demonstrates that peptides with high is some affinity binding to HLA-antigen, not necessarily possess high activity of inducing T cell-killers. However, peptides containing the amino acid sequence of CDH3-4 (FILPVLGAV (SEQ ID NO: 1)or CDH3-7 (FIIENLKAA (SEQ ID NO: 2)), show particularly high activity of inducing T cell-killers.

As described above, the present invention provides peptides having the activity of inducing T cell-killers, more specifically, peptides comprising amino acid sequence SEQ ID NO: 1 or 2, or their variants (i.e., amino acid sequence in which one, two or more amino acids replaced, deleterow, inserted and/or added). Amino acid sequences of peptides containing nine amino acids of SEQ ID NO: 1 or 2, or their variants preferably are not identical to the sequences of other endogenous proteins. Especially peptides with high binding affinity of HLA-A2 can be obtained by replacing the second amino acid from the N-terminal to leucine or methionine, and/or by replacing the C-terminal amino acids valine or leucine.

The peptides of the present invention may include modifications such as glycosylation, oxidation of the side chain and phosphorylation, unless the peptides do not lose their activity, induction of T cell-killers. Other modifications include, for example, D-amino acids or other and alogi amino acids, which can be used to increase the half-life of peptides in serum.

Methods of obtaining and production of the peptides of the present invention are not particularly limited. The peptides can be chemically synthesized or recombinant obtained using gene recombinant technology.

Chemically synthesized peptides of the present invention can be synthesized according to the methods of chemical synthesis, such as Fmoc method (a method that uses fluorenylmethoxycarbonyl) and t-Boc-method (method using tert-butyloxycarbonyl). The peptides of the present invention can also be synthesized using various commercially available peptide synthesizers.

The peptides of the present invention can be produced as recombinant proteins by obtaining DNA molecules containing nucleotide sequences encoding the peptides, or variants or homologues, and by introducing them into appropriate expressing system.

Used expressing vectors preferably can be any vector that can autonomously replicate in the cells of the host, or can be incorporated into the chromosome of the host cell, and contain a promoter in a suitable locus, enabling expression of the gene encoding the peptide. Transformants with the Yong, encoding the peptide of the present invention, can be obtained by introducing the above-mentioned expressing vector in the body-master. The body of the host can be bacterial, yeast, animal cells or insect cells, and the introduction of expressing the vector in the body-the owner may be carried out using known methods depending on the host body.

In the present invention the recombinant peptide of the present invention can be isolated by culturing transformants, obtained as described above, the generation and accumulation of the peptide in the culture, and collecting the polypeptide from the culture.

When transformants is prokaryotic, such as E. coli, or eukaryotic, such as yeast, the culture medium for culturing these microorganisms may be either natural or synthetic medium provided that it contains a carbon source, nitrogen source, minerals and the like, that can be used by microorganisms and allows efficient cultivation of transformant. The cultivation conditions may be conventional conditions used for culturing microorganisms. After cultivation, the peptide of the present invention can be isolated and purified from the culture of transformant using standard methods isolation of ochistki peptides.

Peptides comprising the amino acid sequence in which one, two, or several amino acids are substituted or added in the amino acid sequence of SEQ ID NO: 1 or 2, can be appropriately produced or obtained by the person skilled in the art based on the information of nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 1 or 2. Specifically, the gene encoding the peptide that contains the amino acid sequence in which one, two or more amino acids replaced, deleterow, inserted and/or added in the amino acid sequence of SEQ ID NO: 1 or 2, and which has an activity of inducing T cell-killers, can also be obtained using any method known to the person skilled in the art, a method such as chemical synthesis, methods of genetic engineering or mutagenesis. For example, using the method of site-directed mutagenesis, which is one of the techniques of genetic engineering, as it can help you to enter a specific mutation in a specific position. It can be carried out according to the methods described in the book, Molecular Cloning: A laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989 (hereinafter for brevity specified as Molecular Cloning 2nd Ed.), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) (hereinafter referred to for brevity as specified Current Protocls in Molecular Biology), and the like.

The above peptides according to the present invention can induce immunity against cancer, which is also shown below in the Examples. Thus, according to the present invention also provides agents for inducing immunity against cancer, containing the peptides of the present invention. Agents for inducing immunity according to the present invention can also be obtained in the form of a mixed composition by combining two or more of the epitope peptides. Agents for inducing immunity, presents the composition by combining many types of peptides, can be a cocktail or can be interconnected using standard methods. Epitope peptides, which should be combined, can be a peptide containing the amino acid sequence derived from the same gene, or may be a peptide containing the amino acid sequences derived from different genes. When the peptides of the present invention is administered to a subject, the peptides compactly presented on HLA-antigens and, therefore, induced cells T-cells that react with specific complexes formed between the introduced peptide and HLA-antigens. Alternatively, it may be derived antigen-predstavlyayushie the cells, which represent the peptides of the present invention on their cell surface, by contacting dendritic cells collected from the subject, with peptides of the present invention (or by stimulating dendritic cells collected from the subject, the peptides of the present invention). By introducing these antigen-presenting cells back each subject in the body of the subject induced cells T-killers, and as a result may increase the immune response to target cells presenting the peptides of the present invention.

When using in vitro or in vivo, preferably in vitro, the agents for inducing immunity against cancer according to the present invention can induce cells T-helper cells T-killers or group of immune cells, including the cells, whereby given immunity against cancer.

(2) Pharmaceutical agents for the treatment and/or prevention of cancer according to the present invention (cancer vaccine)

In the Examples it was demonstrated that the peptides of the present invention can induce in vivo cells T-cells specific to cancer cells. In addition, it was demonstrated in the previous invention that CDH3 has a high level of expression in cancer cells in most cases of cancer, such as pancreatic cancer W is Lesa cholangiocellular cancer, gastrointestinal tract cancer, colon cancer, non-small cell lung cancer, testicular cancer, cervical cancer, osteosarcoma, sarcoma of soft tissue, and the like. Accordingly, agents for the induction of immunity, including the peptides of the present invention, as expected, are effective agents for the treatment and/or prevention of cancer. That is, by injection into the body of the peptides of the present invention together with a suitable adjuvant or after stimulation with peptides of antigen-presenting cells such as dendritic cells, are induced or activated cells T-killers, attacking the tumor, and the result is expected to be a manifestation of antitumor effects. In addition, genes encoding the peptides of the present invention, can be incorporated into suitable vectors. Human antigen-presenting cells (dendritic cells, and so on) and bacteria such as BCG Mycobacterium tuberculosis that are transformed with recombinant DNA, or viruses, such as vaccinia virus, which contains integrated into the genome DNA encoding the peptide of the present invention, can effectively be used as live vaccines for the treatment and/or prevention of cancer in humans. Dosages and routes of administration for vaccines against cancer are the same, it is to smallpox vaccine or BCG vaccine.

With regard to the present invention, the term “vaccine” (also called immunogenic composition) refers to a substance that induces antitumor immunity or inhibits various cancers when insulinopenia animal. According to the present invention suggested that the peptide comprising amino acid sequence SEQ ID NO: 1 or 2, is an HLA-A2-restrictively epitope peptide, which induces a strong and specific immune response against CDH3-presenting cells. Accordingly, the present invention also includes methods of inducing antitumor immunity through the use of peptides comprising amino acid sequence SEQ ID NO: 1 or 2, or their variants that include substitutions, deletions or additions of one, two or more amino acids. As a rule, protivoopujolevy immunity includes the following immune response:

(1) the induction of T cell-killer cells against tumors containing cells expressing CDH3;

(2) the induction of antibodies that recognize tumors containing cells expressing CDH3; and

(3) inducing the production of anti-cancer cytokines.

When specific peptide induces one of these immune responses through insulinopenia animal, such a peptide is defined as oblad the Mering effect of inducing antitumor immunity. The induction of antitumor immunity using peptide can be detected by monitoring in vivo or in vitro responses of the immune system of the host body on the peptide.

For example, detection methods of inducing T cell-killers are well known. Alien substance which penetrates into a living organism, it seems T-cells and b-cells by functioning antigen-presenting cells (APC). T cells that respond to antigens presented by antigen-presenting cells, are differentiated antigen-specific manner in the cells of T-killer cells (also called cytotoxic T lymphocytes or CTLs) by stimulating antigens and then proliferate. In this application, this process is called “activation” T-cells. Inducing cells of T-killer using specific peptide can be assessed via submission of the peptide to the T cells using peptide-stimulated antigen-presenting cells, and then detected the induction of T cell-killers. In addition, antigen-presenting cells have the effect of activation of CD4+T-cells, CD8+T-cells, macrophages, eosinophils and NK cells. As CD4+T-cells are important in antitumor immunity, the effect of inducing antitumor immunity peptide can the t assessed with the effects of activation of these cells as indicators.

Methods of evaluating the effects of inducing cells of T-killer cells, where the cells of T-killer cells are induced with the use of dendritic cells (DC) as antigen-presenting cells, are well known in the prior art. Among antigen-presenting cells DK have the strongest effect of inducing T cell-killers. This method involves first contacting the test peptide with cells DC and then the contacting of the cells of DC with T cells. T cells with cytotoxic effects on target cells, are detected from T cells that came into contact with the cells DC. If T-cells exhibit cytotoxic activity against target cells, it means that the tested peptide has an activity of inducing cytotoxic T-cells. The activity of the cells of T-killer cells against target cells such as tumor cells, can be detected, for example, using lysis51Cr-labeled tumor cells as an indicator. Alternatively, the degree of destruction of tumor cells can be measured using an activity absorption3H-thymidine or using as an indicator the release of lactatedehydrogenase (LDH).

The tested peptides, which confirmed using these methods, they have activity of inducing T cell-killers, before whom represent peptides, with the effects of cell activation DK and, therefore, activity of inducing T cell-killers. Thus, peptides that induce cell T-killer cells against tumor cells will be suitable for use as vaccines against cancer cells, representing CDH3. In addition, antigen-presenting cells that have acquired the ability to induce cell T-killer cells against cancer cells by interacting with the peptide will be suitable for use as vaccines against cancer. In addition, the cells of T-killers who purchased cytotoxicity by presenting peptides of antigen-presenting cells, can also be used as vaccines against cancer cells, representing CDH3. A method of treating cancer using tumor immunity by antigen-presenting cells and T cell-killers called sitoemulatoreaz.

Typically, when using peptides for cytomander, the efficiency of induction of T cell-killers can be enhanced by combining a variety of peptides with different patterns. Thus, when stimulating cells DK using protein fragments, it is preferable to use a mixture of more than one type of peptide fragments.

The induction of antitumor immunity is using peptides can also be assessed using observations of inducing production of antibodies against tumors. For example, when antibodies against peptides induced in laboratory animals immunized with peptides, and when the growth, proliferation and/or metastasis of tumor cells is inhibited by these antibodies, we determined that peptides induce antitumor immunity.

Antitumor immunity can induction by administration of the vaccine of the present invention, and the induction of anti-tumor immunity enables treatment and prevention of cancer. Effects of cancer treatment or prevention of cancer may include the inhibition of cancerous cell growth, regression of cancer cells and suppress the development of cancer cells. The decrease in mortality of individuals with cancer, reducing levels of tumor markers in the blood and a reduction in detectable symptoms accompanying cancer, is also included in the effects of treatment or prevention of cancer. Such therapeutic or preventive effects of vaccines against cancer are preferably statistically significant compared to the effects of controls without the introduction of vaccines. For example, the effects observed at a significance level of 5% or less. Statistical methods that can be used to determine statistical significance, are, for example, t-test t-test, U-test Mann-Whitney or ANOVA.

In the present invention the subject is preferably a mammal. Examples include people of all primates except humans, mice, rats, dogs, cats, horses or cattle, but not limited to.

The peptides of the present invention can be administered to a subject in vivo or ex vivo. In addition, to obtain immunogenic compositions for the treatment or prevention of cancer can be used in immunogenic peptide of the present invention, i.e. the amino acid sequence of SEQ ID NO: 1 or 2, or nonapeptide selected from the variant-specific peptides.

More specifically, the present invention offers pharmaceutical agents for the treatment of a tumor or to prevent the growth and metastasis of tumors, and the like, comprising as active ingredients one or more of the peptides of the present invention. The peptides of the present invention is specifically suitable for use in the treatment of cancer of the pancreas, cholangiocellular cancer, cancer of the gastrointestinal tract, colon cancer, non-small cell lung cancer, testicular cancer, cervical cancer and tumors, such as osteosarcoma and sarcoma of soft tissue.

The peptides of the present invention can be administered directly to a subject as a pharmaceutical agent in the pharmaceutical compositions of POM is using standard methods of pharmaceutical compositions. Such pharmaceutical composition, if necessary, may include in addition to the peptides according to the present invention, pharmaceutically acceptable carriers, excipients and the like. The pharmaceutical agents of the present invention can be used for the treatment and prevention of various tumors.

In addition, for the effective creation of cellular immunity adjuvants can be mixed with pharmaceutical agents for the treatment and/or prevention of tumors, comprising as active ingredients one or more of the peptides of the present invention. Alternatively, the composition may be administered in conjunction with other active ingredients such as anti-tumor agents. Suitable pharmaceutical compositions also include granules. Suitable adjuvants are described in publications (Johnson AG., (1994) Clin. Environ. Rev., 7: 277-89). Examples of adjuvants include incomplete adjuvant's adjuvant, BCG, dimycolate trehalose (TDM), lipopolysaccharide (LPS), alum adjuvant, adjuvant based on silicon dioxide, aluminum phosphate, aluminum hydroxide, and potassium aluminum alum, but not limited to. In addition, can be used in a standard way liposomal pharmaceutical compositions, granular pharmaceutical composition in which the drug is attached to the granules having a diameter, amounting to several microns, and pharmaceutical compositions, in which the lipids associated with the above peptides. Methods of introduction can be a: oral administration, intradermal injection, subcutaneous injection, intravenous injection and the like, and may include systemic injection or local injection near the tumor target.

The dose of the peptides of the present invention can be adjusted appropriately, respectively, the disease being treated, the age and body weight of the patient, the route of administration and the like. The usual dose is 0.001 mg to 1000 mg, preferably 0.01 mg to 100 mg, and more preferably 0.1 mg to 10 mg, Preferably introduced from once every few days to once in a few months, but the person skilled in the art can easily select the appropriate dose and route of administration, and the selection and optimization of these parameters are entirely within the standard method. The form of pharmaceutical compositions is also not specifically limited, and it may be liofilizovannyh or granulated with the addition of auxiliary substances, such as sugar.

To increase the activity of inducing tumor-responsive T-cells to the pharmaceutical agents of the present invention can be added adjuvants, which include bacteria is further components of the BCG bacteria and the like, including muramyl dipeptide (MDP), ISCOM, cited in Nature, vol. 344, p.873 (1990), QS-21 number of saponin as described in J. Immunol. vol. 148, p.1438 (1992), liposome and aluminum hydroxide. In addition, can also be used as adjuvants Immunostimulants, such as lentinan, sizofiran, picibanil. Can also be used as adjuvants cytokines such substances that enhance the proliferation and differentiation of T cells, such as IL-2, IL-4, IL-12, IL-1, IL-6 and TNF, as well as CpG and lipopolysaccharide (LPS), which can activate your own immune system by binding to Toll-like receptors, and α-galactosylceramide.

Vaccine compositions of the present invention includes a component that stimulates cells T-killers. Lipids have been identified as substances that stimulate cells against viral antigens in vivo. For example, palmitic acid residues can join the ε-amino group and the α-amino group of lysine residue and then linked to immunogenic peptide of the present invention. Libidinously peptide can then be entered directly using any method of introduction into the micelle or particle being encapsulated in a liposome or emulsified in Freund. Another possible example of lipid stimulation is the stimulation with lipoprotein of Escherichia coli (E. coli), t is anyone as dipalmitoyl-S-glacierization-seryl-serine (P3CSS), is formed when a covalent bond with a suitable peptide (Deres K., et al., (1989) Nature 342: 561-4).

Immunogenic peptides of the present invention can also be expressed by viral vectors or bacterial vectors. Examples of suitable expressing vectors include viral hosts attenuated, such as vaccinia virus or variola virus in birds. For example, a vaccinia virus can be used as a vector for expression of the nucleotide sequence that encodes the peptide. Immunogenic peptides expressed by introducing a host cell, and this causes an immune response. Method of immunization using vaccinia virus vectors are described, for example, in U.S. patent No. 4722848. Bacille de Calmette et Guerin (BCG) may also be used. BCG vectors described in the publication Stover CK, et al., (1991) Nature 31: 456-60. The prior art known to a wide variety of other vectors suitable for use for therapeutic injection or immunization, including adeno - and adeno-associated viral vectors, retroviral vectors, vector bruchnotifosny Bacillus (Salmonella typhi), vectors neutralized toxin anthrax. See, for example, publish, Shata MT, et al., (2000) Mol. Med. Today 6: 66-71; Shedlock DJ and Weiner DB, et al., (2000) J. Leukoc. Biol. 68: 793-806; and Hipp JD, et al., (2000) In Vivo 14: 571-85.

In addition, for effective induction is one of T-killer cells in the body of the patient, in vitro add antigenic peptide for presentation of antigen to cells collected from the patient, or the cells of another individual that share part of the HLA allele (ALLO), and the cells are then administered to the patient intravascular or locally to the tumor. Alternatively, after induction of the cells of T-killer cells in vivo by adding the peptide to the peripheral blood lymphocytes of the patient and culturing in vivo, the cells can be administered to the patient intravascular or locally to the tumor. Such processing using cell transplantation have been carried out as a therapy against cancer, and it is well known to specialists in this field.

Types of cancer in the present invention are not specifically limited, and specific examples include esophageal cancer, breast cancer, thyroid cancer, colon cancer, pancreatic cancer, malignant melanoma, malignant lymphoma, osteosarcoma, pheochromocytoma, head and neck cancer, uterine cancer, ovarian cancer, brain tumor, chronic myeloid leukemia, acute myeloid leukemia, kidney cancer, prostate cancer, lung cancer, cancer of the gastrointestinal tract, liver cancer, gallbladder cancer, testicular cancer, thyroid cancer, bladder cancer, and sarcoma. Examples of cancers for which is a suitable application for the present image is ateneu, preferably represent pancreatic cancer, cholangiocellular cancer, gastrointestinal tract cancer, colon cancer, or lung cancer.

(3) the Antibody according to the present invention

The present invention relates to antibodies that recognize a portion of the peptide or whole peptide of the present invention, as mentioned above, the epitope (antigen), and also relates to cells of the T-killer cells, which are induced by using in vitro-stimulation using proteins or peptides. In General, the cells of T-killers demonstrate a stronger antitumor activity than antibodies.

In addition, like the peptides of the present invention, the antibodies of the present invention suitable for use as agents for preventing and/or treating cancers in which the cancer cells Express CDH3, provided that the agents can inhibit the activity of antigen CDH3 cancer cells. In one particular application of the peptides or antibodies of the present invention can be administered by themselves or together with a pharmaceutically acceptable carrier and/or diluent, if necessary, adjuvant, by injection or by spraying using transdermal absorption through the mucous membrane or similar method. More specifically, human serum albumin may b the th is given as an example of media referred to in this application as well as PBS, distilled water, and the like can be given as examples of the diluent.

Antibodies of the present invention can be a polyclonal antibody or monoclonal antibodies can be obtained by methods known in the art from the prior art.

For example, polyclonal antibodies can be obtained by immunization of mammals or birds using as antigen peptide of the present invention, using blood from mammals or birds, and by separating and purification of antibodies from the collected blood. For example, can be immunized mammals, such as mouse, hamster, Guinea pig, chicken, rat, rabbit, dog, goat, sheep and cattle or birds. Methods immunization known to specialists in this field, and the antigen may be, for example, two or three times at intervals components of 7-30 days. The dose may be, for example, about 0.05 mg to 2 mg of antigen in one introduction. Can be selected, suitable route of administration selected from subcutaneous, intradermal, intraperitoneal, intravenous, intramuscular and the like, but not limited to any of these ways. Furthermore, the antigen may be used after dissolving in a suitable b is fere, for example in a buffer containing a standard adjuvant, such as complete adjuvant's adjuvant or aluminum hydroxide.

Immunized mammals or birds fed within a certain period of time and when you increase the titer of antibodies them additionally subjected to immunization with antigen in amounts of, for example, 100 μg - 1000 μg. The blood sampling of the immunized mammal or bird species are carried out through one or two months after the last injection, and blood cells can be separated and cleaned using standard methods, such as centrifugation, precipitation using ammonium sulfate or polyethylene glycol, and chromatography such as gel filtration chromatography, ion exchange chromatography and affinity chromatography with obtaining polyclonal antibodies that recognize peptides of the present invention as polyclonal antisera.

Monoclonal antibodies can be obtained by making a hybrid. For example, hybridoma can be obtained by merging the cells that produce antibodies with cells of the myeloma cell lines. Hybridoma producing monoclonal antibodies of the present invention, can be obtained using methods merge cells, such as the methods described below.

Cells of the spleen, the cells simpatiche the one node, B-lymphocytes and other cells of the immunized animals are used as cells that produce antibodies. The peptides of the present invention are used as antigen. Animals such as mouse and rat, can be used as the immunized animals, and the introduction of these animals antigens carried out by standard methods. For example, animals are subjected to immunization with intravenous, subcutaneous, intradermal, intraperitoneal, and so forth, the introduction of several times of a suspension or emulsion of the peptide of the present invention, which is an antigen, and an adjuvant, such as complete adjuvant's adjuvant or incomplete adjuvant's adjuvant. The cells producing the antibody, such as spleen cells, obtained from the immunized animals, and these cells can be fused with myeloma cells using known methods (G. Kohler et al., Nature, 256: 495 (1975)) to produce a hybrid.

Murine cell line P3X63Ag8, P3U1, Sp2/0 and so on can be cited as an example of myeloma cell lines used to merge cells. To merge cells used agent, stimulating the merger, such as polyethylene glycol or Sendai virus, and after merging cells for breeding hybrid standard method used environment gipoksantin/aminopterin/thymidine (HAT). Hybridoma, the floor is built using the merge cells clone using a method such as a method of serial dilutions. If necessary, cell lines producing monoclonal antibodies that recognize specific peptides of the present invention, can be obtained by using the screening method enzyme immunoassay using the peptides of the present invention.

In addition to the above methods immunized cells may be obtained by the stimulation of human lymphocytes, such as lymphocytes infected with EB virus, in vitro through the use of the peptides of the present invention, cells expressing the peptides, or their lysates. Human antibodies that bind to the peptides of the present invention, can also be obtained by merging these immunized lymphocytes obtained with human bone marrow cells such as U266 (Japanese Patent Application Kokai Publication No. (JP-A-S63-17688 (unexamined, published Japanese patent application)).

With the aim of producing targeted monoclonal antibody of the thus obtained hybridomas, hybridoma may be cultured using standard cultivation techniques or using methods with the formation of ascites, and monoclonal antibodies can be purified from supernatant cultures or from ascites monoclonal antibodies Purification of supernatant cultures or from ascites can be carried out using standard methods. For example, if necessary, can be used in combination fractionation using ammonium sulfate, gel filtration, ion exchange chromatography, affinity chromatography, and so forth.

In addition, transgenic animals having a group of genes of human antibodies can be immunized with the use of the peptides of the present invention, cells expressing the peptides, or their lysates. Cells producing antibodies can be collected from the immunized transgenic animals with obtaining hybridomas by fusion with the above cells, myeloma cell lines. Targeted monoclonal antibodies can then be produced by hybridomas (WO92-03918; WO94-02602; WO94-25585; WO94-33735; WO96-34096).

Alternatively, the immune cells that produce antibodies, such as the immunized lymphocytes, can also be immortality using oncogenes to obtain monoclonal antibodies.

Obtained in this way antibodies can also be modified using the techniques of genes (Borrbaeck and Larrick, (1990) Therapeutic Monoclonal Antibodies). For example, can be obtained recombinant antibodies by cloning DNA encoding the antibody is isolated from cells that produce antibodies, such as hybridoma and immunized lymphocytes, inserting the DNA into a suitable vector and by transformation the functions obtained structures in cells are the host.

Antibodies of the present invention can also be antibody fragments or modified antibodies, provided that they are associated with peptides of the present invention. Antibody fragments can be a Fab, F(ab')2, Fv or single-chain Fv fragment (scFv)in which Fv fragments derived from the H and L chains, linked together by using an appropriate linker (Huston et al., (1998) Proc Natl Acad Sci USA 85: 5879-83). More specifically, antibody fragments can be obtained by treating the antibody with an enzyme such as papain and pepsin (Co et al., (1994) J Immunol 152: 2968-76; Better and Horwitz, (1989) Methods Enzymol 178: 476-96; Pluckthun and Skerra, (1989) Methods Emzymol 178: 497-515; Lamoyi (1986) Methods Enzymol 121: 652-63; Rousseaux et al., (1986) Methods Enzymol 121: 663-9; Bird and Walker (1991) Trends Biotech 9: 132-7).

Antibodies of the present invention include modified antibodies, which are obtained by the blending of various molecules such as polyethylene glycol (PEG). Antibodies can be modified using standard methods of chemical modifications are known from the technical field.

Antibodies of the present invention include chimeric antibodies comprising variable plot derived from non-human antibody, and a constant plot derived from human antibodies, humanized antibodies, including hypervariable segment (CDR)derived from non-human is about antibodies, frame plot (FR)derived from a human antibody, and a constant plot derived from human antibodies. Such antibodies may be obtained using standard methods known from the technical field. Humanized antibodies are obtained by replacement of section CDR sequences of a human antibody CDR-plot rodents with target binding activity (Verhoeyen et al., (1988) Science 239: 1534-6). Accordingly, compared to chimeric antibodies, humanized antibodies are antibodies in which a shorter area of the human antibody is replaced by a corresponding portion of the origin of species other than human.

Can be obtained a complete human antibody containing human variable plot in addition to the human frame and constant area. For example, in in vitro method can be screened using recombinant library of bacteriophages containing fragments of human antibodies (Hoogenboom and Winter (1992) J Mol Biol 227: 381-8). Similarly, human antibodies can be obtained by introducing human immunoglobulin loci transgenic animals whose endogenous immunoglobulin genes partially or completely inactivated (US6150584, US5545807, US5545806, US5569825, US5625126, US5633425, US5661016).

Antibodies floor is Chennai, as stated above, can be purified to a homogeneous state using standard methods known from the technical field. For example, can be used conventional methods of separation and purification of proteins. Antibodies can be separated and purified using a combination of column chromatography such as affinity chromatography, filtration, ultrafiltration, vysalivaniya, dialysis, electrophoresis in SDS-polyacrylamide gel electrophoresis with isoelectric focusing and so on; but the methods of separation and purification are not limited to these methods (Antibodies: A Laboratory Manual, Ed Harlow and David Lane (1988) Cold Spring Harbor Laboratory). Column with protein A and column with protein G can be used as affinity columns. An example of a column with protein A can be HyperD, POROS and Separate F.F (Pharmacia).

The example is different from the affinity chromatography may be ion-exchange chromatography, hydrophobic chromatography, gel filtration, chromatography with reversed phase, adsorption chromatography, and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. et al.). Liquid chromatography, such as HPLC and FPLC, can also be used as chromatography.

The antigen-binding affinity of the antibodies of the present invention can be measured using, for example, determine the absorption, using terdapat the CSOs enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), immunofluorescent analysis; however, the methods are not restricted to these methods. For analysis by ELISA, antibodies of the present invention immobilized on the Cup, add the peptides of the present invention, and then add the sample containing supernatant culture of cells producing antibodies, or purified antibodies. In the next stage add secondary antibody containing detektiruya tag and recognizing antibody whose antigen-binding activity must be measured. After washing cups, add reagents for detection of the label on the secondary antibody, and determine the absorption or the like of the measured value. For example, the label for the secondary antibodies can be used enzymes such as alkaline phosphatase, and as reagents for detection can be used with substrates of enzymes, such as p-nitrophenylphosphate. BIAcore (Pharmacia) can also be used to evaluate the activity of antibodies.

Antibodies of the present invention can detect the peptides of the present invention contained in the samples. And it can be confirmed by the presence of the peptides of the present invention in cancer tissues, for example, by exposing biopsy samples of cancer tissues with antibodies for this is in the invention.

Before the stage of treatment and/or prevention of cancer with the use of the peptides of the present invention, before treatment can be done forecasts of subjects, effectively treated with the help confirm the expression of the peptides of the present invention in cancer cells, which should be treated using antibodies of the present invention.

In addition, as the antibodies of the present invention recognize peptide fragments CDH3, whose expression is increased in various cancer cells, it is expected that they are applicable not only for diagnosis but also for treatment.

(4) Cells T-helper cells T-killers or enclosing the group of immune cells

The present invention also relates to cells T-helper cells and T-killer cells or by enclosing the group of immune cells, induced with the help of in vitro stimulation with the use of the peptides of the present invention. For example, opuholeobraznye activated T-cells are induced when the peripheral blood lymphocytes stimulated in vitro with the use of the peptides of the present invention, and these activated T cells can be effectively used for adaptive immunotherapy. Also, dendritic cells, which are effective antigen-presenting cells, can be stimuleren is by using the peptides of the present invention or can be genetically transformed to the expression of these peptides, these cells can then be used to stimulate T cells in vivo or in vitro to induce antitumor immune response.

Preferably the cells are T-helper cells T-killers or enclosing the group of immune cells can be induced using in vitro stimulation with the use of the peptides of the present invention and Immunostimulants. The term adjuvant in the present description includes a cell growth factors or cytokines.

Tumors can be suppressed, and cancer can be prevented and/or be subjected to treatment with transfusions in the body cells T-helper cells, T cell-killers or involve groups of immune cells, as described above.

Cells T-helper cells T-killers or enclosing the group of immune cells that can suppress tumor, as described above, can also be obtained using the peptides of the present invention. Thus, the present invention provides cell culture environment containing the peptides of the present invention. Cells T-helper cells T-killers or enclosing the group of immune cells that can suppress tumor, as described above, can be obtained using such media cell cultures. In addition, the present invention features a kit of reagents for cell culture containing the ed cell culture, described above, and the vial cell culture to obtain cells T-helper cells, T cells killer or involve groups of immune cells.

(5) the Antigen-presenting actsoma

The present invention additionally offers endocytotic vesicles, called “ectosomal”, which are on the surface complex formed between the peptide of the present invention and an HLA antigen. Actsoma can be obtained, for example, using methods described in detail in Japanese translation, Japanese patent application Kohyo Publication No. (JP-A-H11-510507 (unexamined Japanese patent application in the national phase publication corresponding to the Japanese international publication) and JP-A (Kohyo) 2000-512161. Preferably, actsoma can be obtained using antigen-presenting cells derived from the target subject for the treatment and/or prevention of diseases. Alsosome of the present invention can inetservices as a cancer vaccine in a manner similar to the peptides of the present invention.

Antigen HLA-type, used in the present invention should be suitable antigen HLA-type of the subject in need of treatment t/or prevention of a disease. An example is HLA-A2, preferably HLA-A2 (HLA-A*0201). “HLA-A2” refers to a protein, while “HLA-A*0201” refers to a gene that is relevant to the ith segment of the protein (this term is used since there is no currently available terms designating segments of the protein).

(6) Methods of inducing antigen-presenting cells and T cell-killers

The present invention provides methods of inducing antigen-presenting cells using one or more of the peptides of the present invention. Antigen-presenting cells can be induced by stimulating dendritic cells induced from peripheral blood monocytes using one or more of the peptides of the present invention to stimulate cells. When the peptides of the present invention is administered to a subject, the antigen-presenting cells presenting on its surface the peptides of the present invention, can coil in the body of the subject. Alternatively, after contacting the antigen-presenting cells with the peptides of the present invention (or after stimulation of antigen-presenting cells by the peptides of the present invention), the cells can be administered to the subject as a vaccine using the method of ex vivo. For example, ex vivo-introduction may include stages:

(1) collecting the antigen-presenting cells from the subject; and

(2) contacting the antigen-presenting cell stage (1) with the peptides of the present invention (or the stimulation of antigen-ol is dostavlyaemykh cell stage (1) the peptides of the present invention).

Antigen-presenting cells, obtained in stage (2)can be administered to a subject as a vaccine.

The present invention also provides methods of inducing antigen-presenting cells with a high level of activity of inducing T cell-killers. The methods include in vitro-phase transfection in antigen-presenting cells of the gene, including polynucleotide encoding one or more peptides of the present invention. The gene, which should be transliterate, can be represented in the form of DNA or RNA. As a transfection method can be used appropriately different methods that are commonly used in engineering, such as lipofection, electroporation and calcium phosphate method, but not limited to. More specifically, the transfection can be carried out, as described in publications Reeves ME, et al., (1996) Cancer Res., 56: 5672-7; Butterfield LH, et al., (1998) J. Immunol., 161: 5607-13; Boczkowski D, et al., (1996) J Exp. Med., 184:465-72; and in published Japanese translation WO2000-509281. When genes transliterowany in antigen-presenting cells, they are transcribed and translated in these cells. Obtained in this way proteins are processed sequentially along the path of MHC class I or class II and presented on the surface of antigen-presenting cells in the form of a partial peptides by antigen-not only the existing path.

The present invention additionally provides methods of inducing T cell-killer, using one or more of the peptides of the present invention. By administering to the subject one or more peptides of the present invention, the cells of T-killers can coil in the body of a subject, increasing in this way the immune system, which targets the cancer cells, representing CDH3, in tumor tissues. Alternatively, activated cells, T-killers can induction by contacting in vitro antigen-presenting cells obtained from the subject, and CD8-positive cells with one or more peptides of the present invention, and by way of additional contact mononuclear leukocytes of peripheral blood antigen-presenting cells in vitro to stimulate cells. In ex vivo methods of treatment, the immune system, which targets the cancer cells, representing CDH3, in the tumor tissues of the subject, can be strengthened with the return of activated T cells-killer subject. For example, the methods include the steps:

(1) collecting the antigen-presenting cells from a subject;

(2) contacting the antigen-presenting cell stage (1) with the peptides of the present invention (or the stimulation of antigen-presenting cells stage (1) peptides present Adamu invention);

(3) mixing and culturing the antigen-presenting cell stage (2) with CD8+T-cells to induce cytotoxic T-cells; and

(4) collection CD8+T-cells from a common culture stage (3).

CD8+T cells with cytotoxic activity obtained in stage (4), can be administered to a subject as a vaccine.

In the present invention are isolated cells T-killer cells, which are induced using one or more of the peptides of the present invention. Preferably, the cells of T-killer cells, induced using the method according to the present invention, receive from the subject, a disease which should be treated and/or prevented. They can be administered in combination with other agents, including antigen-presenting cells or actsoma representing one or more peptides of the present invention. The obtained cells T-cells are specific for target cells presenting the peptide, which is the same that is used for induction. Target cells are those cells that Express endogenous CDH3 or those transliterowany gene CDH3. Cells presenting on its surface the peptides of the present invention, stimulating peptides on nastasemarian, such as cancer cells, pancreatic cancer, cholangiocellular cancer, cancer of the gastrointestinal tract, colon cancer, non-small cell lung cancer, testicular cancer, cervical cancer, osteosarcoma and soft tissue sarcoma, can be a target for attack.

The present invention also provides antigen-presenting cells that present a complex formed between an HLA antigen and one or more peptides of the present invention. Antigen-presenting cells expressing one or more peptides of the present invention or a nucleotide encoding such peptides, preferably harvested from a subject, a disease which should be treated and/or prevented. The peptides of the present invention, antigen-presenting cells presenting the peptides, actsoma or activated cells, T-killers can be administered as a vaccine in combination with other agents.

The present invention additionally explained using the Examples described below. However, it is not limited to these Examples.

All references to the prior art cited in this specification are introduced in this application with references.

Examples

[Example 1]

Expression of CDH3 in malignant tumors

According to previous analyses the application of cDNA microarray was detected, the expression of CDH3 increased in various malignant tumors, including cancers of the gastrointestinal tract, colon cancer and so on, compared with expression in normal adjacent tissues (table 1) (Nakamura T, et al., Oncogene 2004; 23: 2385-2400; Kitahara O, Cancer Res 2001; 61: 3544-3549., Obama K, et al., Hepatology 2005; 41: 1339-1348.).

[Table 1]
NA positive ratio* (%)The relative expression (mean value)
Pancreatic cancer16/161001900000
Cancer of the testis10/10100396000
Tumor of soft tissue21/21100248000
Cholangiocellular cancer19/191003600
Non-small cell lung cancer35/379573000
Cancer of the colon and rectum31/349184000
Cervical cancer14/19741500
Cancer of the gastrointestinal tract20/287135000
Bladder cancer24/347130
Small cell lung cancer3/14217
Breast cancer5/8161
Prostate cancer2/5741500
Renal cell carcinoma0/2000
Cancer of the esophagus0/1902
*“Positive” means, when the ratio of the relative expression (cancerous tissue/normal tissue) is >5.

[Example 2]

The choice of the composition of CDH3-peptide having affinity to HLA-A2

Search human amino acid sequence CDH3 was performed using a system BIMAS, and was selected 18 peptides in descending order of the expected affinity of binding to HLA-A2 (table 2).

HLA-A2-restrictively epitopes T cell-killers identified in the present invention, shown with the use of underscores.

[Example 3]

First, dendritic cells (DC) induced from bone marrow cells transgenic for HLA-A2 mice using a previously described method (Komori H et al. Clinical Cancer Research 12: 2689-2697, 2006). Then thus obtained cells KM-DC stimulated with CDH3-peptides (10 μm) and then were injected intraperitoneally transgenic for HLA-A2 mice in the amount of 5×105cells/mouse. After immunization by injection twice weekly intervals cells in the spleen of the mouse was collected and used for detection of cells of T-killers. In order to detect the induction of T cell-killers derived from CD8+T-cells, used cells of the spleen, which were obtained by removing CD4+T-cells is the use of pellets MACS after removal of the spleen.

The Figure 1 presents the Protocol definition CDH3-peptide recognized by HLA-A2-restrictionenzyme cells T-cells in transgenic for HLA-A2 mice. Day, when spleen cells were collected from immunized mice presented as “Day 0”.

Day 21: (1) the Induction of bone-marrow-derived dendritic cells (hereinafter in the present description, called “KM-DK”) was initiated by adding GM-CSF to the bone marrow cells transgenic for HLA-A2 mice.

Day 14: (2) a Mixture of three types of CDH3-peptides were added to the induced cells KM-DK. After two hours, cells KM-DK was administered intraperitoneally in an amount of 5×105cells/mouse.

(1) and (2) was repeated twice with one week interval.

Day 0: spleen Cells were collected from the immunized transgenic for HLA-A2 mice and were cultured together with cells KM-DK, which again incubated with CDH3-peptide for two hours and were cultured for six days.

Day 6: To detect cells T-cells that recognize specific CDH3-peptides, T cells producing interferon gamma (IFN-γ), was evaluated quantitatively by using ELISPOT analysis after antigenic stimulation. Stimulated using CDH3-peptide cells KM-DK and not stimulated cells KM-DC were used as target cells.

Research activity CDH3-specific T cells to which lerow using ELISPOT analysis:

In order to confirm that the cells of T-killers-specific reactive CDH3 with the production of IFN-γ, really exist among these cells, we conducted the survey using ELISPOT analysis. IFN-γ were detected using a set of reagents Mouse IFN-γ ELISPOT Set (BD Biosciences). When cells T-killer cells (effector) react to stimulating cells (the target) and produce IFN-γ, IFN-γ is detected in the form of red spots. Cells KM-DK or stimulated CDH3-peptide cells KM-DC were used as target cells. First ELISPOT tablet (BD Biosciences) were coated murine antibody to IFN-γ for 18 hours and then blocked with 10% FCS/RPMI for two hours. Effector cells (100 μl/well) and target cells (100 μl/per well) were mixed and cultured for 22 hours at 37°C. the Experiment was performed with respect to the effector/target (E/T)of 10:1. The tablet is then washed with sterile water, treated with biotinylated mouse antibodies to IFN-γ for two hours and then treated with streptavidin-horseradish peroxidase for one hour. IFN-γ-positive spots were detected in the substrate solution. Software MINERVA TECH to research using automatic analyzer used for counting spots. As a result, observed immune response CD3-specific T cell natural killer cell, T-killers, induced with peptide CDH3-4 or CDH3-7, while not observed CDH3-specific immune response to T cell-killer induced by other peptides (Figures 2 and 3).

The result of the analysis of ELISPOT for cells T-cells induced with peptide CDH3-4 (SEQ ID NO: 1) and peptide CDH3-7 (SEQ ID NO: 2)shown in Figure 3.

T cells-killers showed 283,7±40.0 spots/well in response to cell KM-DC stimulated with peptide CDH3-4 (SEQ ID NO: 1), whereas they showed 48,7±11.9 spots/well in the presence of cells KM-DK without stimulating peptide (P<0,05). Similarly, the cells of T-killers showed 79,3±3.2 spots/well in response to cell KM-DC stimulated with peptide CDH3-7 (SEQ ID NO: 2), whereas they showed 42.7 spots/well in the presence of cells KM-DK without stimulation with peptide (P<0,05).

Statistical analysis:

Bilateral student test was used to assess statistical significance in the data, obtained by ELISPOT analysis, and data size of tumors between groups with different processing. The value of P<0.05 is considered as significant. Statistical analysis was performed using commercially available statistical package software (SPSS for Windows (TM), version 11.0, Chicago, IL, USA).

[Example 4]

Cell lines expressia HLA:

Cancer cells of the pancreas of a human cell line PANC1 cells, oral cancer cell lines HSC3, and cells deficient in TAP and HLA-A2 (A*0201)-positive cell line T2, used to assess the cytotoxic activity, were purchased in the Cell Bank, Riken (Tsukuba, Japan). Cells pancreatic cancer human cell lines PK8 were kindly provided by the Center for Cellular Resources for Biomedical Research, Institute of development, Aging and Cancer, Tohoku University. Cells colon cancer cell line HCT116 were kindly provided by Others. B Vogelstein (B. Vogelstein, Johns Hopkins University (Baltimore, MD). The cells of the liver cancer cell line SKHep1 were kindly provided by Professor Kyogo ito (Kyogo Ito), University of Qurum (Column, Japan). The expression of HLA-A2 was determined by flow cytometry using anti-HLA-A2 monoclonal antibody (mAb) BB7.2 (One Lambda, Inc., Canoga Park, CA, USA) for the selection of HLA-A2-positive blood donors and cell lines target for analysis of cytotoxicity. These cells were maintained in RPMI medium 1640 or DMEM with addition of 10% FCS in an atmosphere of 5% CO2at 37 degrees C.

Lentiviral transfer gene transfer:

Indirect lentivirusnye vectors gene transfer was performed as described previously (Tahara-Hanaoka S, et al. Exp Hematol 2002; 30: 11-17). Briefly, 17 µg seminariruumis, etc) the ditch CSII-CMV-RfA and CSIIEF-RfA (Miyoshi H, et al. J Virol 1998; 72: 8150-8157), bearing CDH3-cDNA, and 10 μg pCMV-VSV-G-RSV-Rev and pCAG-HIVgp was transfusional using Lipofectamine 2000 (Invitrogen Corporation, CA, USA) in 293T cells grown in 10-cm culture vial. After 60 hours the culture medium was removed, and viral particles were besieged by centrifugation (50000×g for two hours). Sediment suspended in 50 μl of medium RPMI 1640 was added 10 μl of viral suspension to PANC1 cells or SKHep1, which were scattered in flat-bottomed 96-well-plate with a density of 5×104cells per well. The expression transfitsirovannykh CDH3 was confirmed using Western blot analysis.

The induction of CDH3-reactive human cells CTLs:

Cells MCPC obtained from treated heparin blood of HLA-A2-positive patients with pancreatic cancer, patients with cancer of the gastrointestinal tract, patients with colorectal cancer, or healthy donors were isolated by centrifugation in a density gradient solution of Ficoll-Conray. Derived from mononuclear peripheral blood cells (monocytes) cells DC were obtained according to previously published method (Yoshitake Y, et al. Clin Cancer Res 2004; 10: 6437-6448, Komori H, et al. Clin Cancer Res 2006; 12: 2689-2697). Cells DC were stimulated with 20 μg/ml of the candidate peptide in the presence of 4 μg/ml β2-microglobulin (Sigma-Aldrich, St. Louis, MO, USA) for two hours at 37°C in medium AIM-V (Ivitrogen), containing 2%inactivated by heating autologous plasma. These cells DC are then irradiated (40 Gy) and incubated with CD8-positive cells. Incubation was performed in 24-hole tablets, which were prepared so that each well contained 2 ml of medium AIM-V with the addition of 2% autologous plasma, 1×105stimulated with peptide cells DK, 2×106CD8+T-cells and 5 ng/ml human recombinant IL-7 (Wako, Osaka, Japan). After two days in these cultures was added human recombinant IL-2 (PeproTec Inc.) to a final concentration of 20 IU/ml Carried out two additional incentives once a week using the same peptide-stimulated autologous DC using the same procedure on the 7th and 14th day. Six days after the last stimulation of antigen-specific response induced cells CTLs was evaluated by analyzing the release of51Cr and ELISPOT analysis for IFN-γ. Various cancer cells or stimulated with peptide T2 cells (5×103cells/per well), used as target cells, were cultured together with cells CTLs with a suitable ratio of the effector/target for analysis release51Cr using a known method (Komori H, et al., Clin Cancer Res 2006; 12: 2689-2697).

The attempt was made to induce CDH3-specific cells CTLs from kleeck HLA-A2-positive healthy donors and patients having cancer, stimulating peptides CDH3-4655-663and CDH3-7757-765. CD8+ T cells, sorted from cells MCPC, incubated with derived from autologous mononuclear cells (monocytes) cells DC stimulated each peptide. After three boosts with analysis release51Cr and using ELISPOT analysis for IFN-γ was evaluated the effect of killing cells against peptide-stimulated cells T2 (Figure 4A). Cells CTLs induced from cells MCPC healthy donors demonstrated the effect that kills cells against T2 cells stimulated with peptides CDH3-4655-663or CDH3-7757-765but not against T2 cells that are not stimulated with peptides. Similar responses were observed in relation to other donors. These results reveal that these cells CTLs have peptide-specific cytotoxicity.

Then tested the cytotoxic activity of these cells CTLs cells against human cancer cell lines expressing CDH3 and HLA-A2. As shown in Figure 4B, CDH3-reactive cells CTLs stimulated with peptide CDH3-4655-663demonstrated in healthy donors cytotoxicity to HCT116 (CDH3+, HLA-A2+), HSC3 (CDH3+, HLA-A2+), and PANC1/CDH3 (CDH3+, HLA-A2+), in which the gene CDH3 was transfirieran in PANC1 cells; however, they did not show the same effect in relation to PNC1 (CDH3-, HLA-A2+), SKHep1 (CDH3-, HLA-A2+), and PK8 (CDH3+, HLA-A2-). Similarly cells CTLs stimulated with peptide CDH3-7757-765demonstrated cytotoxicity against HSC3, but not to PANC1, PK8 and SKHep1. These cytotoxic activity was observed for cells CTLs obtained from different patients with cancer (Figure 4C).

In order to confirm whether such peptides to processionals from CDH3 protein in vivo, used PANC1/CDH3 and SKHep1/CDH3 (CDH3+, HLA-A2+), in which the gene CDH3 was transfirieran in SKHep1 cells as target cells. As shown in Figure 4C, the cells CTLs induced by stimulation with peptide CDH3-4655-663or CDH3-7757-765demonstrated cytotoxicity against HCT116, PANC1/CDH3, and SKHep1/CDH3, but not against PANC1, SKHep1 and PK8. The above results suggest that in natural conditions, these peptides are processed and presented on the surface of cancer cells using molecules HLA-A2. CDH3-reactive cells CTLs have the cytotoxicity specific to cancer cells, which Express endogenous molecules simultaneously CDH3 and HLA-A2.

Confirmation of restriction by HLA class I:

To confirm whether the induced cells CTLs to recognize target cells by way of restriction of HLA class I, the cancerous target cells were incubated with 10 μg/ml of monoclonal antibodies and the t-HLA class I mAb (W6/32) or with 10 μg/ml anti-HLA-DR mAb (H-DR-1) within one hour before co-cultivation of cells CTLs cells and cancer cell lines for analysis release 51Cr or ELISPOT analysis, and examined using known methods, the effects of monoclonal antibodies mAb on the cytotoxic activity of the cells CTLs or production in IFN-γ (Gomi S, et al., J Immunol 1999; 163: 4994-5004). As a result, the antibody anti-HLA-class I could inhibit the production of IFN-γ by a statistically significant amount in the ELISPOT analysis for cells CTLs generated by stimulation with peptide CDH3-4655-663against SKHep1/CDH3 (Figure 4D, left, P<0,01). It could also inhibit the cytotoxic activity against HCT116 cells in the analysis release51Cr (Figure 4D, middle). Similarly antibody, anti-class I could inhibit the production of IFN-γ by a statistically significant amount in the ELISPOT analysis for cells CTLs generated by stimulation with peptide CDH3-7757-765against HSC3 cells (Figure 4D, right, P<0,01). These results reveal that the induced cells CTLs recognize expressing CDH3 target cells by way of restriction by HLA-class.

[Example 5]

Adoptive immunotherapy

In vivo activity against cancer induced with human CDH3 cells CTLs were used for adoptive immunization of mice NOD/SCID:

In order to evaluate therapeutic effect of the introduction of CDH3-reactive cells CTLs mice, which transplanted CDH3-positive human cancer cells and, conducted an experimental adoptive immunotherapy, as described previously (Komori H, et al. Clin Cancer Res 2006; 12: 2689-2697). Briefly, HCT116 cells (4×106cells), positive for both HLA-A2 and endogenous CDH3, inoculable mice NOD/SCID via subcutaneous injection into the right side. When the tumor size reached 25 mm2on the 7th day after insulinopenia tumors of mice that were injected with an intravenous line cells CTLs specific for the peptide CDH3-4655-663or CDH3-7757-765as a negative control, line CD8+T-cells stimulated with HLA-A2-restrukturovaneho HIV-peptide (SLYNTYATL, SEQ ID NO: 19), obtained from five healthy donors and suspended in 100 μl (4×106). T-cells again were injected with intravenous on day 14. The sizes of tumors were measured twice a week and was evaluated by measuring two perpendicular to each other diameters using a compass. Bilateral student test was used to assess statistical significance of tumor size. The value of P<0.05 is considered as significant. Statistical analysis was performed using commercially available statistical package software (SPSS for Windows (TM), version 11.0, Chicago, IL, USA).

Control stimulated HIV-peptide CD8+ T cells did not show cytotoxicity in vitro against HCT116 cells. And estimated the size of the of whole seven individual mice in each group (Figure 5A) and mean tumor size ± standard deviation in each group (Figure 5B). Control T-cell line and one PBS showed no inhibitory effect on tumor growth. The tumor size of mice inoculated stimulated using CDH3 cells CTLs was significantly smaller than the size of tumors in mice inoculated with control induced with HIV-peptide CD8+ T cells one or PBS (P<0,001). These results reveal the effectiveness of adaptive transplantation therapy using CDH3-reactive human cells CTLs against CDH3+ human tumor in mice NOD/SCID.

Discussion:

In this study the authors present invention identified Cadherin 3 (CDH3)/P-cadherin as the new TAA through analysis of pancreatic cancer using cDNA-microarray chip. Based on the analysis using cDNA microchip found that CDH3 expressively at high levels in cancer cells pancreatic cancer and expressively at a low level in cancer cells of testicular cancer and breast cancer. The expression of CDH3 was hardly detected in the other critical organs. In addition, the data obtained with the use of the microchip and real-time PCR (RT-PCR)demonstrated that CDH3 expressively in cancer cells with cancer of the gastrointestinal tract and in colon cancer, and cancer of the pancreas, but barely expressively in analogion the x normal tissues. It was reported that CDH3 was sverkhekspressiya in most cancer tissues of the pancreas, whereas normal cells of the ducts and acinar cells in the pancreas almost did not show expression of CDH3 using immunohistochemical staining (Taniuchi K, et al. Cancer Res 2005; 65: 3092-3099). These results suggest that CDH3 may represent a new target for immunotherapy for the above-mentioned cancer, which is associated with a low risk of inducing an autoimmune response.

The cadherin family is classified into two different subfamilies, including Cadherin 1 (CDH1)/E-cadherin, Cadherin 2 (CDH2)/N-cadherin and Cadherin 3 (CDH3)/P-cadherin according to their tissue distribution. CDH1 is the predominant member of the family catherinew, which is expressed in all epithelial tissues. It is assumed that CDH1 functions as suppressing tumor factor that negatively regulates invasion and metastasis of cancer cells (Frixen U H, et al. J Cell Biol 1991; 113: 173-185, Berx G, et al. Genomics 1995; 26: 281-289, Oka H, et al. Cancer Res 1993; 53: 1696-1701). Expression of CDH2 is increased in cancer cells at invasive cancer, and CDH2 promotes invasive phenomenon through interaction with the receptor fibroblast growth factor (FGF) and through subsequent reaction cascade (Suyama K, et al. Cancer Cell 2002; 2: 301-314). Expression and role of CDH3 in R is the same diseases not clear. In earlier research Taniuchi et al. suggested that the increased expression of CDH3, probably is a factor that enhances the invasiveness of cancer cells pancreatic cancer through interaction with p120ctn and GTP-Asai family of Rho, Rac1 and CDC (Taniuchi K, et al. Cancer Res 2005; 65: 3092-3099). In other earlier studies suggested that CDH3 is also a factor in increased invasiveness and poor prognosis in breast cancer (Palacios J, et al. Am J Pathol 1995; 146: 605-612, Paredes J, et al. Clin Cancer Res 2005; 11: 5869-5877, Peralta Soler A, et al. Cancer 1999; 86: 1263-1272) and endometrial cancer (Stefansson I, M, et al. J Clin Oncol 2004; 22: 1242-1252.).

All taken together previously published messages, the target's response to vaccines against cancer in clinical trials is so low value as of 2.6% (Rosenberg S A, et al. Nat Med 2004; 10: 909-915). One option is that cancer cells avoid immune system due to deletion, mutation or down-regulation of tumor-associated antigens (TAA) in consequence of immuno-inducing therapy. Based on the view that tumor cells cannot lose antigens that are required for carcinogenesis, CDH3 will be suitable for use by the candidate TAA for immunotherapy against cancer.

In the present invention, the authors of the present invention identified among the 18 candidate peptides selected with the aid of the completion algorithm BIMAS, two HLA-A2-restrictively CDH3 epitope peptide, which confirmed that they induce HLA-A2-restrictively mouse cells in transgenic CTLs in HLA-A2.1 (HHD) mice. In addition, the authors of the present invention confirmed that CDH3-reactive cells CTLs were generated with the use of these peptides from cells MCPC obtained from healthy donors and from patients with cancer (Figure 4). These cells CTLs showed the effect of killing cells, not only with respect to T2 cells stimulated with the relevant peptide, but also in relation to the cells of cancer cell lines expressing CDH3 and HLA-A2. Based on the above suggested that these CDH3-peptides (CDH3-4655-663and CDH3-7757-765) are produced naturally when the processing of the CDH3 protein in cancer cells, are presented on the cell surface together with molecules of HLA-A2 and then recognized by cells CTLs.

Cytotoxicity CDH3-reactive cells CTLs of the present invention was confirmed not only in vitro by analyzing the release of51Cr, but also in vivo using TLCs-adoptive immunotherapy. As shown in Figure 5, intravenous injection of CD8+ cells induced with peptides of the present invention significantly inhibited the growth of tumors transplanted into mice NOD/SCID, CPA is in control of CD8+ cells and other controls.

HLA-A2 (A*0201) represents one of the most common HLA alleles in different ethnic groups, including Asians, Africans, African Americans, and Europeans (M. Browning et al. Immunol Today 1996; 17: 165-170). Thus, the peptides identified in the present invention, which are presented to cells of the T-killer cells by HLA-A2, have the potential clinical use around the world, if their safety and efficacy in cancer immunotherapy demonstrated in research medicine. In addition, the identification of peptides that are presented to cells and T-killer cells by HLA-A2, the media is often found not only in Japan but also among people around the world will likely lead to the development of drugs for immunotherapy against cancer, applicable for approximately 30% of patients with pancreatic cancer, worldwide.

Industrial applicability

HLA-A2 is the allele HLA class I carriers which are about 30% of the population of the Japanese. When transgenic mice expressing human HLA-A2, subjected to immunization with CDH3-peptides of the present invention, the peptides can induce cytotoxic T cells that recognize peptides associated with molecules HLA-A2, with the induction of the immune response. There is a high probability that also the people these peptides can induce human cytotoxic T-cells, which destroy cancer cells expressing the complexes of peptides and molecules HLA-A2. Thus, the peptides of the present invention can be used for immunotherapy against cancer of the pancreas, cholangiocellular cancer, cancer of the gastrointestinal tract, colon cancer and lung cancer in HLA-A2-positive patients. Thus, the peptides are expected to improve the quality of life of patients by suppressing proliferation and/or progression of such cancers.

1. Peptide to induce cytotoxic T-cells in its submission to the antigen-presenting cell bearing HLA-A2 (A*0201), selected from the following (A) or (B);
(A) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2;
(B) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2, in which the second amino acid from the N-Terminus is a leucine or methionine, and/or C-terminal amino acid is a valine or leucine, and where the peptide has an activity of inducing cytotoxic (killer) T cells.

2. The agent for inducing immunity against cancer expressing CDH3, comprising as active ingredient one or more peptides according to claim 1 and a pharmaceutically acceptable carrier or diluent, or adjuvant.

3. The agent according to claim 2 for the treatment of cancer, Express the dominant CDH3.

4. Antibody to the peptide consisting of the amino acid sequence of SEQ ID NO: 1, which is obtained by a process comprising immunizing peptide.

5. Antibody to the peptide consisting of the amino acid sequence of SEQ ID NO: 2, which is obtained by a process comprising immunizing peptide.

6. Isolated actsoma, which is a complex comprising the peptide according to claim 1 and HLA-A2 (A*0201).

7. Method of inducing antigen-presenting cells having the activity of inducing a cytotoxic (killer) T-cells, comprising a stage of bringing the antigen-presenting cells bearing HLA-A2 (a*0201), in contact with the peptide according to claim 1.

8. The method of inducing a cytotoxic (killer) T-cells, including the stage of culturing the antigen-presenting cells bearing HLA-A2 (a*0201), brought into contact with the peptide according to claim 1, CD8+T-cell.

9. Isolated cytotoxic (killer) T cell, which is induced by the method according to item 8.

10. An isolated antigen-presenting cell, which is a complex comprising the peptide according to claim 1 and HLA-A2 (a*0201).

11. Antigen-presenting cell of claim 10, which is induced by the method according to claim 7.

12. Method of inducing immunity against cancer expressing CDH3, including the stage of introduction of the subject peptide according to claim 1.

13. A method of treating cancer, expressio the future CDH3, including the stage of introduction of the subject peptide according to claim 1.

14. The use of the peptide according to claim 1 to obtain an agent for inducing immunity against cancer expressing CDH3.

15. The use of the peptide according to claim 1 for obtaining a medicinal product for the treatment of cancers expressing CDH3.



 

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SUBSTANCE: invention refers to biotechnology, more specifically to prepare cell lines used for monoclonal antibody technology. What is prepared is genetically and phenotypically stable D11 human multiple myeloma cell line preserving a histogenetic phenotype of human multiple myeloma suitable for growing in a medium containing 8-azaguanine.

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2 dwg, 1 tbl, 1 ex

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Antibodies // 2482131

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35 cl, 4 ex, 3 tbl

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3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to expression constructs, and may be used for immunoglobulin expression. An expression vector contains one open reading frame (sORF) insert which contains a first sequence of nucleic acid coding a first polypeptide; a first intermediate sequence of nucleic acid coding a first protein cleavage site containing an autoprocessing element with an intein segment providing proteolytic sORF polypeptide cleavage between the first polypeptide and the intein segment and the second polypeptide, but not ligation of said first polypeptide with said second polypeptide; and a second sequence of nucleic acid coding the second polypeptide. The expression vector is able to express a mammalian polypeptide coding sORF and cleaved in said first protein cleavage site in a host cell; consisting of the first polypeptide - an immunoglobulin heavy chain, and the second polypeptide - an immunoglobulin light chain able to be assembled into a multimer.

EFFECT: invention provides functional antibody production with 'correct' setup and assembly.

40 cl, 9 dwg, 57 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to expression constructs, and may be used for immunoglobulin expression. An expression vector contains one open reading frame (sORF) insert which contains a first sequence of nucleic acid coding a first polypeptide; a first intermediate sequence of nucleic acid coding a first protein cleavage site containing an autoprocessing element with an intein segment providing proteolytic sORF polypeptide cleavage between the first polypeptide and the intein segment and the second polypeptide, but not ligation of said first polypeptide with said second polypeptide; and a second sequence of nucleic acid coding the second polypeptide. The expression vector is able to express a mammalian polypeptide coding sORF and cleaved in said first protein cleavage site in a host cell; consisting of the first polypeptide - an immunoglobulin heavy chain, and the second polypeptide - an immunoglobulin light chain able to be assembled into a multimer.

EFFECT: invention provides functional antibody production with 'correct' setup and assembly.

40 cl, 9 dwg, 57 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used to obtain monoclonal antibodies against the Yersinia pestis V antigen. The strain of hybrid animal cells Mus musculus 2B8 is obtained by immunising BALB/c mice. The mice are immunised by four-time administration of a recombinant V antigen in a dose of 100 mcg/mouse. On the third day after the last immunication, splenocytes of immune mice (1×108 cells) are hybridised with mouse myeloma cells RZ-X63 Ag/8-653 (1×107 cells). The fusion agent used is polyethylene glycol (Sigma, USA). Hybridisation is followed by selection, screening, cloning and cryopreservation of the hybridoma. The strain is deposited in the state collection of pathogenic microorganisms and cell cultures (GKPM-Obolensk) under number N-20.

EFFECT: strain of hybrid cultured cells, which produces monoclonal antibodies which are specific to the Y pestis V antigen, is suitable for constructing test systems for detecting plague pathogens.

8 dwg, 3 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used to obtain monoclonal antibodies against the Yersinia pestis V antigen. The strain of hybrid animal cells Mus musculus 5G6 is obtained by immunising BALB/c mice. The mice are immunised by four-time administration of a recombinant V antigen in a dose of 100 mcg/mouse. On the third day after the last immunication, splenocytes of immune mice (1×108 cells) are hybridised with mouse myeloma cells RZ-X63 Ag/8-653 (1×107 cells). The fusion agent used is polyethylene glycol (Sigma, USA). Hybridisation is followed by selection, screening, cloning and cryopreservation of the hybridoma. The strain is deposited in the state collection of pathogenic microorganisms and cell cultures (GKPM-Obolensk) under number N-19.

EFFECT: strain of hybrid cultured cells, which produces monoclonal antibodies which are specific to the Y pestis V antigen, is suitable for constructing test systems for detecting plague pathogens.

8 dwg, 2 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented invention refers to medical microbiology, particularly to molecular-genetic typing of the strains Helicobacter pylori. What is presented is a method for differentiation of the strains H.pylori by multilocal VNTR-typing with the PCR involving oligonucleotide primers on VNTR-comprising loci of H.pylori - HpA, HpD, HpE and HpF; the strains H.pylori are differentiated by a number of repetitions in the amplified fragments in each VNTR-comprising locus of H.pylori - HpA, HpD, HpE and HpF that enables genetic typing of the studied strains.

EFFECT: what is presented is the method for differentiation of the strains Hpylori by multilocal VNTR-typing.

1 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and virology. Any two E2 encoding nucleic acid molecules have the identity percentage less than 75% in any part of 40 or more unbroken nucleotides. There are also described an infectious viral particle and a composition containing the mentioned expression vector.

EFFECT: what is disclosed is the expression vector containing the nucleic acid molecules coding at least two papillomavirus E2 polypeptides.

34 cl, 3 dwg, 1 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and virology. Any two E2 encoding nucleic acid molecules have the identity percentage less than 75% in any part of 40 or more unbroken nucleotides. There are also described an infectious viral particle and a composition containing the mentioned expression vector.

EFFECT: what is disclosed is the expression vector containing the nucleic acid molecules coding at least two papillomavirus E2 polypeptides.

34 cl, 3 dwg, 1 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention relates to biotechnology, and represents a method for preparing L-arginine with the use of the bacteria of to the genus Escherichia, which is modified in such a way that the astCADBE operon in the mentioned bacterium is inactivated. The bacterium is grown in a culture medium, after that L-arginine is recovered from a culture fluid.

EFFECT: method enables providing higher productivity of the bacteria producing L-arginine and producing high-yield L-arginine.

4 cl, 2 tbl, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to genetic engineering and may be used in biotechnology to produce proteins of practical interest, which are a product of silent genes or low expression genes. What is presented is a method for producing a recombinant protein of human renalase 2 involving a) preparing a complete cDNA sequence, and b) expressing the prepared sequence in E.coli, wherein the stage (a) is performed by PCR amplification of each exon using specific primer pairs and a genomic DNA as a matrix, followed by twinning requiring no pre-treatment and involving the same method for adjacent exons at first, and then being the product of their amplicon combining. In this case, the primers twice exceeding exons in number are selected so that the forward primer contain a terminal sequence of the preceding exon and an initial sequence of the exon to be copied; the downstream primers contain only a sequence complementary to the end of the amplified exons, while the forward and downstream primers flanking the complete coding sequence contain restriction sites necessary for integration into a plasmid vector.

EFFECT: preparing the proteins of practical interest which are the products of silent genes or low expression genes.

2 tbl, 7 dwg, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to genetic engineering and may be used in biotechnology to produce proteins of practical interest, which are a product of silent genes or low expression genes. What is presented is a method for producing a recombinant protein of human renalase 2 involving a) preparing a complete cDNA sequence, and b) expressing the prepared sequence in E.coli, wherein the stage (a) is performed by PCR amplification of each exon using specific primer pairs and a genomic DNA as a matrix, followed by twinning requiring no pre-treatment and involving the same method for adjacent exons at first, and then being the product of their amplicon combining. In this case, the primers twice exceeding exons in number are selected so that the forward primer contain a terminal sequence of the preceding exon and an initial sequence of the exon to be copied; the downstream primers contain only a sequence complementary to the end of the amplified exons, while the forward and downstream primers flanking the complete coding sequence contain restriction sites necessary for integration into a plasmid vector.

EFFECT: preparing the proteins of practical interest which are the products of silent genes or low expression genes.

2 tbl, 7 dwg, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. There are presented versions of a CD20 modified antibody or its antigen-binding fragment. Each version is characterised by the fact that it comprises a variable region of a light chain and a variable region of a heavy chain and induces a higher level of apoptosis as compared with B-Lyl murine antibody. There are presented versions of the compositions to enhance the effector functions. One of such compositions contains antibodies wherein at least 20% of the Fc oligosaccharides are bisectional and non-fucosylated, whereas the other one contains antibodies wherein at least 50% of the oligosaccharides are non-fucosylated. Also, there are described: versions of a host cell to produce the antibodies, an expression vector, as well as the versions of the coding polynucleotides, a method for producing the antibodies in a cell and using the antibodies for preparing a drug for treating disorders treated by B-cells depletion.

EFFECT: use of the inventions provides the antibodies with the improved therapeutic properties, including with higher Fc receptor binding and enhanced effector function that can be find application in treating tumors.

36 cl, 3 ex, 9 tbl, 26 dwg

Antibodies // 2482131

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. There are presented the antibodies that bind to the P-selectin glycoprotein ligand-1 (PSGL-1), as well as methods for stimulating the death of activated T-cells and simulating T-cell immune response in a patient by the use of the antibodies under the invention, and pharmaceutical compositions containing the antibodies under the invention. There are also disclosed nucleic acids, expression vectors and host cells for producing the antibodies under the invention.

EFFECT: invention may can find further application in therapy of the PSGL-1 associated diseases.

35 cl, 4 ex, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: synthetic oligonucleotide primers and probes are disclosed to detect genomes of 1st, 4th and 16th serotypes of the bluetongue disease virus. Also the method is described to detect genomes of 1st, 4th and 16th serotypes of the bluetongue disease virus using such synthetic oligonucleotide primers and probes.

EFFECT: method makes it possible to perform determination of a serotype of the bluetongue disease virus with the help of PCR with a stage of reverse transcription in real time mode, and also to increase specificity and sensitivity, to reduce time to perform work aimed at determination of a serotype of the bluetongue disease virus in samples of an investigated biological material.

4 cl, 4 tbl

FIELD: biotechnologies.

SUBSTANCE: inventions represent synthetic oligonucleotide primers: P1 - 5'-TGGTTACTATTCCATCACCATT-3' (annealing site 11-32 base pairs), P2 - 5'-CGAAACGTCACTTTCGCAAC-3' (annealing site 259-278 base pairs) and method for detection of DNA of infectious anaemia virus of chickens with their help. The method consists in the fact that primers flank the section of the virus genome, which includes CpG islands and VNTR repetitions in the polymerase chain reaction in real time mode. In case of positive reaction, a peak of the melting curve 92C° appears, and when the reaction is confirmed by means of electrophoresis a fragment is visualised in the gel that corresponds to the size of 268 base pairs.

EFFECT: method of diagnostics makes it possible to determine quantitative content of a virus in tissues and may be used to diagnose infectitious anaemia of chickens.

2 cl, 3 dwg, 4 tbl, 5 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

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