Sparc-derivative antigenic peptides causing tumour rejection and medications containing such peptides

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to biotechnology. Described is a peptide capable to induce cytotoxic (killer)T-cells. Presented are medications containing the described peptide that are intended for induction of immunity against malignant tumours, for tumours treatment and/or prevention, for induction of antigen-presenting cells highly capable to induce tumour-reactive T-cells and for induction of tumour-reactive T-cells. Presented is an antibody specifically bonded to the described peptide. Described are an extracted tumour-reactive killer T-cell that is induced in vitro by way of stimulation by the described peptide, a tumour-reactive helper T-cell induced in vitro by way of stimulation by the described peptide, an antigen-presenting cell representing a complex of an HLA molecule and the described peptide.

EFFECT: invention allows to intensify immunity with a patient suffering from metastasis cancer or resistant cancer when surgery treatment, chemotherapy and radiation therapy are practically inapplicable.

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The technical FIELD

The present invention relates to new peptides that are effective as vaccines for malignant tumors highly expressing SPARC, such as gastric cancer, pancreatic cancer or malignant melanoma (melanoma), and medicines containing the above-mentioned peptides used for the treatment and/or prevention of tumors.

PRIOR art

Compared with Western countries, the incidence of gastric cancer is higher in Asian countries such as Japan and China. In the distribution of medical examinations, widespread use of endoscopes for the study of the digestive tract and the development of these procedures, it was possible to detect stomach cancer at an early stage, and thus, the number of patients suffering from this cancer, decreased. However, gastric cancer remains the second cause of death due to malignant neoplasms in Japan. Thus, gastric cancer remains the leading cause of death. Among several types of cancer of the stomach, diffuse (sirrodney) stomach cancer is more common in young people compared to any other type of stomach cancer (adenocarcinoma). Such diffuse (sirrodney) stomach cancer has a tendency to rapid progression, and often there are remote METAS the basics or peritoneal metastases, that gives a poor prognosis. In many cases skorosnogo stomach cancer has become impossible to carry out surgical removal of the tumor at the time of diagnosis. Even if is still possible to remove the tumor after treatment of cancer often recurs. Because of this, it is necessary to create a new treatment method.

The number of deaths from pancreatic cancer in Japan has tended to increase. In 2003, due to pancreatic cancer died 21148 people. Currently, pancreatic cancer is 6.8% of malignant tumors, which are the cause of death in Japan. That is, pancreatic cancer is the fifth leading cause of death after lung cancer, stomach cancer, colon cancer and liver cancer. Analyzed demographic data around the world, and was calculated age-specific mortality rates used to compare mortality in a heterogeneous age populations. As a result, in 2000 per 100,000 men, 8.6 people died due to pancreatic cancer in Japan, while 7,3 person died in the United States and 6.3-7.0 people died in the United Kingdom due to cancer of the same type. Per 100,000 women 4,9 person died due to pancreatic cancer in Japan, while 5.3 people died in the United States and 4.8-5.1 people died in the United States is the Kingdom because of cancer of the same type. Thus, mortality due to pancreatic cancer in Japan was the same as the level in Western countries.

Taking into account his or her age in 2000 (100,000 world population)for men, 8.6 people died due to pancreatic cancer in Japan, while 7,3 person died in the United States and 6.3-7.0 people died in the United Kingdom due to cancer of the same type. In addition, against women, 4.9 persons died due to pancreatic cancer in Japan, while 5.3 people died in the United States and 4.8-5.1 people died in the United Kingdom due to cancer of the same type. Thus, mortality due to pancreatic cancer in Japan was the same as the level in Western countries. Despite the development of diagnostic imaging, currently, approximately 40% of all Japanese patients with pancreatic cancer, suffers from progressing to cancer of the pancreas, leading to distant metastases, and, in the future, there are many cases in which the cancer is found after reaching locally common stage of cancer in which the tumor cannot be removed. The relative 5-year survival of all patients with pancreatic cancer is 43% in diagnosed cases in 1996. Although this level is usually higher than CNY survival rates (2-3%), it still remains low. In relation to factors in the development of pancreatic cancer it has been suggested that various factors, including lifestyle, such as Smoking, obesity, food, alcoholic drinks and coffee, as well as chronic pancreatitis, diabetes, genetic factors, etc. lead to cancer of the pancreas.

Pancreatic cancer has no specific symptoms, and therefore, in many cases, when certain symptoms appear, the cancer has already progressed. As a result, 5-year survival of all patients is 5% or less, and the prognosis after diagnosis is extremely negative. Because of the difficulties in the diagnosis of pancreatic cancer, the percentage of this type of cancer as the cause of death from cancer is gradually increasing in developed countries. Currently, multidisciplinary treatment, including surgical resection as the primary treatment, radiation therapy and chemotherapy. However, there were significant improvement of therapeutic effects, and, therefore, it is necessary to develop a new therapeutic strategy.

Melanoma is a type of skin cancer, which is often called malignant melanoma. Among several types of skin cancer, melanoma is a high probability of infiltration and metastasis and unee the highest degree of malignancy, and, therefore, the melanoma is a serious concern. Among the cells that make up the skin, some cells produce the pigment melanin. These cells are called melanocytes. When these melanocytes become malignant, melanoma occurs. In addition, the incidence of melanoma has increased, particularly among persons of the white race, as a result of increased exposure to ultraviolet rays due to the decrease of the ozone layer in the atmosphere, caused by the sharp deterioration of the environment.

In Japan, cases of melanoma varies from 1.5 to 2 people per 100,000 in the General population. Therefore, estimated to be approximately 1500-2000 people a year developing melanoma. On the other hand, in Western countries, more than a dozen people developing melanoma per 100,000 in the General population. In particular, in Australia, twenty or more people developing melanoma per 100,000 in the General population, and, therefore, it is known that the incidence of melanoma in Australia is the highest in the world. In these conditions, people living in Europe, the United States and in Australia pay special attention to the symptoms of melanoma. Moreover, it has been unexpectedly observed that the occurrence of melanoma tends to increase from year to year in Japan, as well as in foreign countries. In accordance with the data of the last and the research, the annual number of deaths from melanoma is approximately 450 in Japan. Melanoma develops regardless of age. However, the incidence of this disease is increased in people older than 40, and is highest in people 60 and 70 years. The occurrence of this disease in childhood is extremely rare, but this does not mean that the disease never develops in childhood. Recently, the occurrence of melanoma has a tendency to increase in younger patients ages 20 and 30 years. Melanoma develops regardless of gender, and this disease affects both men and women. In the case of patients-Japanese place in which the most likely to develop melanoma, is the sole of the foot, and has 30% of all cases of melanoma. As the characteristics of patients-Japanese, melanoma develops on the foot and nail phalanges. In addition, in the case of patients in Western countries, melanoma develops in any skin areas such as the torso, hand, foot, face and head.

Currently, the methods that can be used to treat melanoma include surgery, chemotherapy and radiation therapy. However, as a therapy to relieve symptoms of metastatic cancer or drug-resistant cancer, in which the above-mentioned methods of treatment may not apply,the focus became immunotherapy to improve the immunity of patients suffering from cancer, a malignant tumor, in order to suppress the growth of malignant tumors. This immunotherapy is actually effective for some patients.

On the other hand, with the development of molecular biology and immunology of tumors in recent years, it has been shown that cytotoxic (killer) T-cells and helper T cells recognize peptides formed after the destruction of proteins, is highly and specifically expressed on malignant cells, which are presented on the surfaces of malignant cells or antigen-presenting cells via HLA molecules, and that they show immunoreactivity for the destruction of such malignant cells. Moreover, it was identified a huge number of tumor antigenic proteins and peptides derived from them, which stimulate an immune response to destroy malignant tumors, and it was suggested that the clinical application of antigen-specific immunotherapy of tumors.

The HLA class I molecules expressed on the surfaces of all nucleated cells of the body. Proteins are formed in the cytoplasm and nucleus, provide peptides formed as a result of destruction in the cells, and they are expressed on the surfaces of these cells. On the surface of healthy cells peptides derived from normal AutoLog is cnyh proteins, associated with HLA class I molecules and T cells of the immune system recognize and destroy these peptides associated with HLA class I molecules. On the other hand, in the process in which malignant cells are transformed into malignant tumor, such malignant cells can Express a huge number of proteins which are not expressed or expressed only in small quantities in healthy cells. If the peptide formed by the destruction in the cytoplasm of this protein, which is highly specifically expressed in malignant cell, binds to the HLA class I molecule and is expressed on the surface of the malignant cells, T-cell killer recognizes the peptide and only destroys malignant cells. In addition, by immunization of a patient such antigen specific to a malignant tumor, or peptide can destroy cancer cells and suppress the growth of malignant tumors without harming healthy cells. This is called immunotherapy of malignant tumors using antigen specific for malignant tumors. Moreover, the molecules of the HLA class II predominantly expressed on the surface of antigen-presenting cells. Such molecules HLA class II associated peptides, occurring them of the antigen, specific to a malignant tumor formed by the inclusion of an antigen specific to a malignant tumor, on the outside of the cell and destroying it in the cage, and it is expressed on the cell surface. T-cell helper cells that recognize peptides associated with HLA molecule class II is activated for the production of various cytokines that activate other immune cells in order to induce or enhance an immune response against the tumor.

Thus, if it can be developed immunotherapy directed to the antigen specifically expressed at a high level in such a malignant tumor, it can provide a therapeutic way to effectively correct only malignant tumors without harming healthy autologous organs. Moreover, it is expected that such immunotherapy may provide a therapeutic method that can be used in patients suffering from terminal cancer, in whom other methods of treatment cannot be done. In addition, if the antigen specific to a malignant tumor, and the peptide to enter it in the form of a vaccine to people with high risk of developing this type of cancer, then you will get the opportunity to prevent the occurrence of malignant tumors.

First, the authors present invention conducted Ana is from the gene expression of the entire genome. The inventors have analyzed 23040 types of genes in tissues of gastric cancer and healthy tissues, using micrometric cDNA. As a result, in 11 of 20 patients with diffuse infiltrating gastric cancer, the inventors have identified the secretory acidic and rich in cysteine protein (SPARC), which represents the product of the gene vysokodispersnogo in tissues of gastric cancer, and the expression level of which is 5 times or more higher than the expression level in healthy tissue (average of 130000 times higher on average) (Figure 1). Gene SPARC is expressed at low levels in normal adipose tissue, mammary gland, ovary, spinal cord, testicles, uterus, placenta, etc. However, the level of expression of the SPARC gene in any of the above bodies below the expression level in healthy mucous membrane of the stomach 5 times or less (Figure 2).

Other researchers reported that SPARC is expressed not only in the diffuse infiltrating cancer of the stomach, but is also expressed in cancer of the pancreas and melanoma. Moreover, the authors of the present invention found that SPARC is secreted in the serum of patients suffering from melanoma, and that SPARC may be appropriate tumor marker, especially for the early detection of melanoma (Japanese patent application No. 2004-303688; and Clinical Cancer Research 11: 8079-8088, 2005).

SPARC, not only is em an acidic secretory protein size 43-kDa, consisting of 286 amino acids. This protein is rich in cysteine and moves to the nucleus during the phase of cell division. In addition, SPARC controls the interaction between proteins of the extracellular matrix and the cell, so it can also be associated with control of cell growth. Because SPARC is expressed in osteoblasts, platelets and wound areas, it is believed that this protein is associated with the repair and regeneration of tissues. Moreover, it was also reported that SPARC is highly expressed in malignant tumors, such as melanoma or sarcoma, and in interacialsex the tumour cells, and that expression of SPARC correlates with prognosis, infiltration or metastasis of tumors.

[Non-patent document 1] Icuta Y et al, Clinical Cancer Research 11: 8079-8088, 2005.

[Patent document 1] Japanese patent application No. 2004-303688

Description of the INVENTION

Problems solved by the present invention

The aim of the present invention is to develop a method of enhancing immunity of a patient suffering from metastatic cancer or drug-resistant cancer, whose surgical treatment, chemotherapy and radiation therapy used as a treatment for tumors, vysokoagressivnyh SPARC, such as diffuse infiltrating gastric cancer, pancreatic cancer or melanoma, is not practically applicable, to relieve symptoms t the fir malignant tumors and for holding immunotherapy, the overwhelming growth of these malignant tumors. In other words, the present invention is the identification of a peptide derived from the protein SPARC, do overexpress in malignant tissue, which is able to induce a strong immune response to the aforementioned malignant tumor is not causing harmful effects in patients with malignant disease, and the use of identified peptides for immunotherapy of tumors. That is, the present invention is the identification of a peptide derived SPARC protein capable of inducing T-cells killer cells and T-cells helper person, reactive against tumors, and obtaining funds for immunotherapy of tumors in patients with various types of cancer, wescoexpress SPARC.

Solutions to these problems

The authors of the present invention have identified a gene SPARC, which is highly expressed in diffuse infiltrating cancer of the stomach, by conducting analysis using cDNA chips with the above-mentioned stomach cancer and various healthy tissues. The expression of SPARC has also been observed in some types of healthy tissue. However, the level of expression of SPARC in normal tissues is significantly lower expression level in malignant tissues. In order to verify the presence or absence of indukti the antitumor immunity SPARC - specific T cells by killer cells, were used mouse BALB/c mice, which Express murine molecule Kd, characteristics of the amino acid sequence of the bound peptide are identical to those of HLA-A24, which is the most frequent allele HLA class I in the population of Japan. Homology of amino acid sequence SPARC human and mouse is 95%. Thus, we synthesized peptides comprising amino acid sequences that are common to humans and mice having binding motif common to HLA-A24 human and Kdmouse. Then, BALB/c mice (Kd-expressing) were immunized dendritic cells derived from bone marrow, which was loaded with the mixture of peptides, the inventors investigated whether induced or not T-cells, killer cells, reactive against malignant cells expressing SPARC. Moreover, in respect of mice that previously had been subjected to the same method of immunization, as described above, the authors of the present invention investigated suppressed if the growth of transplanted malignant cells expressing murine SPARC, and extended or not the survival time of mice. Besides, it is also checked whether or not the negative effects together with the emergence of autoimmune phenomenon in mice immunized with the peptide. The result was the detection is but the peptide with any amino acid sequence of SEQ ID NO: 1-3 able to induce T-cell killers that destroy malignant cells expressing SPARC. Moreover, the growth of transplanted murine malignant cells expressing SPARC suppressed in mice immunized with the above peptide, and, consequently, the survival time of mice lengthened. The present invention was made on the basis of the obtained results.

The present invention relates to the following:

(1) the Peptide is chosen from:

(A) peptide, which consists of any amino acid sequence of SEQ ID NO: 1-3; or

(B) a peptide which consists of amino acid sequence containing substitution, or addition of one or several amino acids in relation to peptide comprising any amino acid sequence of SEQ ID NO: 1-3, and which has the ability to induce cytotoxic (killer) T cells.

(2) the Means of inducing immunity against malignant tumors, which contains at least one type of peptide according to item (1).

(3) a Medicinal product for the treatment and/or prevention of tumors, which contains at least one type of peptide according to item (1).

(4) Means for inducing antigen-presenting cells having high capacity induce the SQL T-cells, reactive against tumor that contains at least one type of peptide according to item (1).

(5) Means for the induction of T-cells reactive against tumors, which contains at least one type of peptide according to item (1).

(6) Means for inducing antigen-presenting cells having high capacity to induce T-cells reactive against tumor, which contains the gene encoding a peptide selected from:

(A) a peptide comprising any amino acid sequence of SEQ ID NO: 1-3; or

(B) a peptide consisting of the amino acid sequence containing substitution, or addition of one or several amino acids in relation to peptide comprising any amino acid sequence of SEQ ID NO: 1-3, and which has the ability to induce T-cell killers.

(7) the Antibody against the peptide according to item (1).

(8) T-cell killer, helper T-cell, or population of immune cells, containing such cells, which is induced by the peptide according to item (1).

(9) the Antigen-presenting cell, which is a complex molecule HLA and peptide according to item (1).

(10) the Antigen-presenting cell according to item (9), which is induced by means of paragraph (4) or (6).

The BEST OPTION of carrying out the INVENTION

(1) the Peptide according to the present izobreteniya the containing means, inducing antitumor immunity

The peptide of the present invention is described in the following:

(A) a peptide which consists of any amino acid sequence of SEQ ID NO: 1-3; or

(B) a peptide which consists of amino acid sequence containing substitution, or addition of one or several amino acids in relation to peptide comprising any amino acid sequence of SEQ ID NO: 1-3, and which has the ability to induce T-cell killers.

The term "peptide possessing the ability to induce cytotoxic T-cells" is used in this description to refer to a peptide having the activity to stimulate T-cells, killer cells, reactive against tumors.

Method of receipt/production of a peptide of the present invention is not limited in a particular way. Can be used either chemically synthesized peptide or recombinant peptide, obtained through genetic recombination.

When receiving a chemically synthesized peptide, the peptide of the present invention can be synthesized by means of chemical synthesis method such as Fmoc (forevermore.actually method or tBoc method (t-butyloxycarbonyl method), for example.

In addition, the peptide of the present invention can also be synthesized various types and commercially available peptide synthesizers.

In those cases, when the peptide of the present invention is produced in the form of a recombinant protein, get a DNA having a nucleotide sequence encoding the aforementioned protein, its mutated form or its homolog, and then it is injected in the preferred expression system, to obtain the peptide of the present invention.

As an expression vector, preferably, can be used vector capable of Autonomous replication in a cell-the owner of, or able to integrate into the chromosome of the host cell. Use the expression vector containing the promoter in position, is able to Express the gene encoding the peptide. Furthermore, the transformant having a gene encoding the peptide of the present invention, can be obtained by incorporating the above-mentioned expression vector into the host. As the owner may be used any of the bacterial, yeast cells, animal cells and insect. The expression vector may be introduced into the host in accordance with the known method, depending on the type of the host.

In the present invention, the transformant obtained above, cultivate, and the peptide of the present invention then is produced and accumulated in the culture. After this, the peptide of the present invention is collected from the culture to highlight the recombinant who CSOs peptide.

In those cases, when such a transformant is a prokaryotic cell, such asEscherichia colior eukaryotes, such as yeast, the medium used for the cultivation of such microorganisms can be any natural or synthetic environment, provided that it contains a carbon source, nitrogen source, inorganic salts and the like that can be assimilated by the above-mentioned microorganisms, able to effectively carry out the cultivation of transformant. Moreover, such cultivation can be performed under conditions, which are usually used for culturing the aforementioned microorganisms. After completion of cultivation, the peptide of the present invention can be isolated and purified from the culture of transformant in accordance with the generally accepted method of isolation and purification of peptides.

The term "one or several amino acids"used in the present description to refer mainly from 1 to 10 amino acids, preferably from 1 to 8 amino acids, more preferably 1 to 5 amino acids, and particularly preferably from 1 to 3 amino acids (e.g. 1, 2, or 3 amino acids).

A peptide consisting of an amino acid sequence that contains a substitution, or addition of one or several amino acids in respect to the peptide consisting of any amino acid consequently the particular SEQ ID NO: 1-3, can be properly received or acquired by experts in this field on the basis of information relating to any amino acid sequence of SEQ ID NO: 1-3. In other words, the peptide consists of an amino acid sequence that contains a substitution, or addition of one or several amino acids with respect to any amino acid sequence of SEQ ID NO: 1-3, and which has the ability to induce cytotoxic T-cells, can be obtained in any way specified, well-known experts in this field, for example, the above-mentioned chemical synthesis by methods of genetic engineering or by mutagenesis. For example, suitable site-directed mutagenesis, which is a genetic engineering method, since it is a way of introducing specific mutations in a specific position. Such site-directed mutagenesis can be performed using the method described in Molecular Cloning: A laboratory Manual, 2ndEd, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989 (hereinafter abbreviated as Molecular Cloning 2ndEd), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology), etc.

As described later in the examples mentioned above, the peptide of the present invention is able to induce an immune response against malignant tumors. Thus, the crust is ASEE the invention relates to a tool, inducing an immune response against malignant tumors, which contains the peptide of the present invention.

The means of inducing an immune response according to the present invention used against malignant tumors, is usedin vitro, ex vivoorin vivoand, preferably,ex vivoso that it could induce T cells killer cells, helper T cells or a population of immune cells, containing such cells, thereby providing anti-tumor immunity.

(2) the Antibody according to the present invention

The present invention also relates to an antibody that recognizes a portion of the above-mentioned peptide of the present invention or the peptide as a whole epitope (antigen), and T-cells, killer cells, induced byex vivoorin vitrostimulation using the above-mentioned peptide. Basically, it is known that such T cells killers antineoplastic activity, more activity of the antibody.

The antibody of the present invention may be either a polyclonal antibody or a monoclonal antibody. This antibody can be obtained by the conventional method.

For example, a polyclonal antibody can be obtained by immunization of a mammal or bird peptide of the present invention used as an antigen, then get the Rove this mammal or bird, and then the separation and purification of the antibodies obtained from the blood. For example, can be immunized mammals or birds, for example, mice, hamsters, Guinea pigs, chickens, rats, rabbits, dog, goat, sheep, or horses were. This method of immunization known to specialists in this field. For example, the antigen can be administered 2 or 3 times at intervals of from 7 to 30 days. At a dose of from about 0.05 to 2 mg of antigen, for example, you can enter only once. Route of administration not specifically limited and, depending on the situation can be selected subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, etc. in Addition, the antigen can be dissolved in a suitable buffer containing adjuvant, for example, in a suitable buffer containing commonly used adjuvant, such as complete adjuvant's adjuvant or aluminum hydroxide, and can be used such antigen.

Immunized thus mammals or birds are grown within a certain period of time. After that, if the antibody titer is increased, it is possible to carry out repeated immunization with the use of from 100 to 1000 μg of antigen, for example. After one or two months after the last immunization, the immunized mammal or bird take blood. Thus obtained blood (polyclonal EN is vyvorotka) then separated and purified in the usual way, includes centrifugation, precipitation using ammonium sulfate or polyethylene glycol, chromatography such as gel filtration chromatography, ion exchange chromatography or affinity chromatography, etc. to obtain polyclonal antibodies, which recognize the peptide of the present invention.

On the other hand, a monoclonal antibody can be obtained using hybridoma. For example, this hybridoma can be obtained by cell fusion of cells producing antibodies, and myeloma cells. Hybridoma, which produces the monoclonal antibody of the present invention, can be obtained in the following way merge cells.

As antitelomerase cells using T cell spleen, cell lymph node, lymphocytes or the like, obtained from the immunized animal. As antigen, using the peptide of the present invention. As the immunized animal, you can use a mouse, rat or similar. The antigen is administered to this animal in accordance with a customary method. For example, suspension or emulsified liquid containing adjuvant, such as complete adjuvant's adjuvant or incomplete adjuvant's adjuvant, and the peptide of the present invention used as an antigen, is administered to the animal by NR is trivinho introduction, subcutaneous injection, intradermal, intraperitoneal administration, etc. several times to immunize the animal. After that antitelobrazuyuschih cell, such as cell spleen, receive from the immunized animal, and the thus obtained cell spleen then drain with a myeloma cell in accordance with the known method (G Kohler et al., Nature, 256 495 (1975)), and thus, hybridoma.

Examples of strains of myeloma cells used in cell fusion include strain P3X63Ag8, strain P3U1 and strain Sp2/0, in the case of mice. When performing this merge cells, use activator mergers, such as polyethylene glycol or Sendai virus. For breeding hybridoma after cell fusion, use the environment gipoksantin-aminopterin-thymidine (HAT) in accordance with a customary method. Hybridoma obtained as a result of merging cells, clone using the method of limiting dilutions. Further, if necessary, conduct a screening enzyme linked immunosorbent assay using the peptide of the present invention, obtaining strain of cells producing a monoclonal antibody specifically recognizing the peptide of the present invention.

To obtain monoclonal antibodies of interest, from the thus obtained hybridoma, this hybridoma can be grown, about cnym method of culturing cells or a way to obtain ascitic fluid, and monoclonal antibody of interest can be purified from the culture supernatant or ascitic fluid in accordance with a customary method. For example, fractionation using ammonium sulfate, gel filtration, ion exchange chromatography, affinity chromatography and other methods can be combined, depending on the situation, and used.

Moreover, fragments of the above antibodies are also included in the scope of the present invention. Examples of such fragments of antibodies include the fragment F(ab')2 and Fab fragment'.

(3) T-cells, killer cells, helper T cells or population of immune cells, containing such cells

The present invention also relates to T-cell killer, helper T-cell or population of immune cells, containing such cells, which are induced by stimulation of thein vitrousing the peptide of the present invention. For example, when the peripheral blood lymphocytes or lymphocytes infiltrating the tumor stimulatein vitrothe peptide of the present invention, induced by activated T-cells showing reactivity against the tumor. Thus, activated T cells can be effectively used for adoptive immunotherapy of cancer. Moreover, the peptide of the present invention has the ability to Express the example in dendritic cells, which are potent antigen-presenting cellsin vivoorin vitroand dendritic cells expressing the antigenic peptide is then injected to the induction of an immune response to the tumor.

Preferably, T-cells, killer cells, helper T cells or population of immune cells, containing such cells, can be induced by stimulation of theex vivoorin vitrousing the peptide of the present invention and immunostimulant. Examples of such immunostimulant used in the present invention include T-cell growth factor and a cytokine.

Thus obtained T-cells, killer cells, helper T cells or a population of immune cells, containing such cells, transferred into the body so that the tumor could be suppression and to prevent and/or treat a malignant tumor.

Next, using the peptide of the present invention, it is possible to get T-killer cells, helper T cells or a population of immune cells, containing such cells, which are ways to suppress tumor growth, as described above. In this regard, the present invention relates to a cell culture solution containing T-cells, realoneplayer against the tumor, and the peptide of the present invention. Using this cell culture solution can be obtained T-cells is Ellery, helper T-cells or population of immune cells, containing such cells, which are able to suppress tumor growth. Moreover, the present invention also relates to a cell culture kit to get T-killer cells, helper T cells or populations of immune cells, containing such cells, which contains the above-mentioned cell culture solution and the flask for cell culture.

(4) the Drug according to the present invention for the treatment and/or prevention of a tumor (cancer vaccine)

Since the peptide of the present invention is able to induce T-cell killers that are specific to malignant cells, it can be offered as a means for the treatment and/or prevention of malignant tumors. For example, bacteria such as BCG (Bacillus Calmette-GuErin), which have been transformed with recombinant DNA obtained by introducing the gene encoding the peptide of the present invention, in a suitable vector, or viruses such as vaccinia virus, the genome of which DNA encoding the peptide of the present invention, was included, can effectively be used as a live vaccine for the treatment and/or prevention of human malignancies. It should be noted that the dose and route of administration of anti-cancer vaccines are the same as dose and route of administration in the beam of ordinary smallpox vaccination or vaccination with BCG.

In other words, the DNA encoding the peptide of the present invention (which is used as it is or in the form of plasmid DNA integrated into the expression vector), or recombinant virus or recombinant bacterium containing the above DNA, you can enter as a cancer vaccine mammals, including humans, either directly or in dispergirovannom in Freund. Similarly, the peptide of the present invention can also be entered as a cancer vaccine in dispergirovannom in Freund.

Examples of the adjuvant used in the present invention include incomplete adjuvant's adjuvant, BCG, dimycolate trehalose (TDM), lipopolysaccharide (LPS), aluminum adjuvant and silicon adjuvant. From the point of view of the ability to induce antibodies, preferably used incomplete adjuvant's adjuvant (IFA).

The type of cancer is not an individual in the present description. Specific examples of malignant tumors include stomach cancer, colon cancer, esophageal cancer, pancreatic cancer, liver cancer, gallbladder cancer, cholangiocarcinoma, lung cancer, breast cancer, thyroid cancer, melanoma (malignant melanoma), skin cancer, osteosarcoma, a pheochromocytoma, a cancer of the tissues of the head and neck, brain tumor, chronic myelogenous leukemia, acute myelogenous leukemia, placecast the military lymphoma, kidney cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, ovarian cancer and carcinoma of the soft tissues. Of them are malignant tumors that highly Express SPARC, such as gastric cancer (particularly diffuse infiltrating gastric cancer), pancreatic cancer and melanoma (malignant melanoma), are typical examples.

The peptide of the present invention acts as a T-cell epitope for the induction of T-cells and killer cells that are specific to malignant cells, or helper T cells. Thus, the peptide of the present invention is used as a tool for the prevention and/or treatment of human malignancies. Furthermore, if the antibody of the present invention is able to inhibit the activity of SPARC, antigen malignant tumors, this antibody is also applicable as a means for the prevention and/or treatment of human malignancies. As the actual use of the peptide or antibody of the present invention can be introduced in the form of injectable product directly or together with a pharmaceutically acceptable carrier and/or diluent, and, if necessary, together with listed below excipients. Moreover, peptide or antibody of the present invention can also be entered in the ways the Ohm, as a spray, through a percutaneous absorption through the mucous. The term "carrier"used in the present description, means, for example, serum albumin human. In addition, as a diluent can be used PBS, distilled water or the like.

As the dose of the peptide or antibody of the present invention can be input in the range from 0.01 to 100 mg per adult one introduction. However, the dose is not limited to the above range. Also not specifically limited to the dosage form. May also be available lyophilized product or granules obtained by adding excipient, such as sugar.

Examples of excipients that can be added to the tool of the present invention, to enhance inducing activity of T-cells reactive against tumor include: muramyl-dipeptide (MDP); bacterial components, such as bacteria BCG; ISCOM, described in Nature, vol. 344, p. 873 (1990); saponin QS-21, described in J Immunol vol. 148, p. 1438 (1992); liposomes; and aluminum oxide. Additionally, Immunostimulants, such as lentinan, sizofiran or picibanil can also be used as auxiliary substances. Other examples of products used in the present invention as an auxiliary substances include cytokines to enhance ROS is a or differentiation of T cells, such as IL-2, IL-4, IL-12, IL-1, IL-6 or TNF; galactosylceramide to activate NKT cells; CpG, which binds to Toll-like receptor to activate the natural immune system; and lipopolysaccharide (LPS).

Moreover, the above-mentioned antigenic peptide typein vitrothe cells derived from HLA-A24-positive patients or allogeneic cells, isolated from somebody else who is HLA-A24-positive, with subsequent antigen presentation by these cells. After that, cells are introduced into a blood vessel of the patient, so that T cells could efficiently coil in the body of the patient. Further, the peptide of the present invention is added to the peripheral blood lymphocytes of the patient, and the resulting mixture was then cultivated byin vitro.By this T-cell killers can coilin vitroand then you can enter them into a blood vessel of the patient. Such treatment, which includes the transfer of cells, was performed as a method of treatment of malignant tumors, and, therefore, this method is well known to specialists in this field.

By introducing a peptide of the present invention in the body, induced T-cells reactive against tumor, and as a result, you can expect antitumor activity. Moreover, the stimulation of lymphocytes by the peptide of the present inventionex vivoorin vitro induced by activated T cells. Activated T-cells injected into the affected area. Thus, this technique can be effectively used for adoptive immunotherapy.

The present invention will be further described in the following examples. However, these examples are not intended to limit the scope of the present invention.

EXAMPLES [Example 1]

(1) Analysis micrometrical cDNA

As for isecheno malignant tissues of a patient with diffuse infiltrating cancer of the stomach, dividing cancerous tissue and non-cancerous tissues was performed using exciting laser Microdissection, and dissected both malignant and non-malignant segments. Then from each tissue were extracted RNA. When cDNA was synthesized from this RNA using the reverse transcription reaction, the cDNA derived from malignant tissue, was fluorescently labeled Cy5, and cDNA obtained from non-cancerous tissue was fluorescently labeled Cy3. After that, two cDNA preparation was stirred to obtain target DNA. Hybridization was carried out on a glass slide, on which were ordered cDNA probes, and nonspecific binding was then removed by washing. After that, the fluorescence images obtained after hybridization, recorded using a CCD camera or a fluorescent scanner and then they were shown using pseudo color (Cy5: red; Cy3 green). At the same time, the expected ratio of the two types of fluorescence intensity (K/H), and indicated as the profile of gene expression. Moreover, expression 23040 types of genes in malignant and non-malignant segments 20 patients with diffuse infiltrating gastric cancer were subjected to a comparative study, and was selected 15 genes, the ratio of expression in malignant tissue/non-cancerous tissue was 5 or higher (figure 1).

Was then analyzed the expression profile of 23040 types of genes in healthy tissues 29 bodies (including 4 embryonic organs), and were selected genes, the expression level was lower in these healthy bodies. In the case of SPARC, which was selected as a tumor antigen in this experiment, the ratio of the expression in malignant/non-malignant segments was equal to 5 or higher (133359 times on average) in 11 of 20 patients with diffuse infiltrating gastric cancer. SPARC expressively in some healthy tissues, but the expression levels in these non-cancerous tissues were significantly lower levels of expression in malignant tissues (Figure 2).

[Example 2]

(2) Selecting the peptide repertoires SPARC HLA-A24 human and murine Kd-restrictively peptide repertoires SPARC

In SPARC human and mouse having a homology of 95%, the following clause shall tidy were selected from a fragment of SPARC, amino acid sequence of which were common to human and mouse. All amino acid sequences of the molecules SPARC investigated on the basis of known information in relation to amino acid sequences that are typically observed in peptides having strong affinity of binding with molecules HLA-A24 human and murine Kd. The repertoires of peptides, which presumably had a high affinity binding in respect of the molecules of two types, were selected using a computer program. As a result, the quality of the candidate peptides were selected 4 types of peptides (table 1).

Table 1
SPARC-derived peptides may have a binding affinity of both molecules HLA-A24 and mouse molecules Kd
PeptideSequenceThe starting positionIndex linking
A24Kd
SPARC - Kd-1DYIGPCKYI14375 400
SPARC - Kd-2HFFATKCTL123201382
SPARC - Kd-3EFPLRMRDWL16130960
SPARC - Kd-4MYIFPVHWQF225210120
Determined using http://bimas.dcrt.nih.qov/cqi-bin/molbio/kenparker combpform

SPARC Kd-1:SEQ ID NO: 1

SPARC Kd-3: SEQ ID NO: 2

SPARC Kd-4: SEQ ID NO: 3

(3) the Vaccine to dendritic cells (DC)

Dendritic cells bone marrow (BM-DC) were obtained from bone marrow cells derived from bone marrow BALBc mice, using GM-CSF as described previously by the inventors (Nakatsura, T. et al, Clin Cancer Res 10, 8630-8640, 2004). Thus obtained BM-DC were cultured with a mixture of 4 types of peptides, selected as described above, at a concentration of 10 μm for 2 hours, and sensitized with peptide BM-DC (5×105cells then were injected intraperitoneally to each mouse. After a break of 1 week the same peptide sensitized BM-DC were twice injected into mice so that the mouse was highly immunized. the donkey this, received cells from the spleen of the mouse and evaluated the induction of T-cell killer. In order to accurately analyze the induction of T-cells and killer cells derived from T cells CD8+after receiving spleen T cells CD4+T was removed from the obtained spleen cells using MACS beads, and the remaining cells are then used for analysis.

To obtain and characteristics of the SPARC-specific T cells killers used the following protocols (date of receipt of spleen cells from the immunized mouse was designated as Day 0).

Day -21 (1) GM-CSF was added to bone marrow cells of BALB/c mice to begin the induction of dendritic cells from bone marrow (hereinafter called BM-DC).

Day -14 (2) a Mixture of 4 types SPARC peptides were added to the induced BM-DC, and 2 hours later, 5×105cells were injected intraperitoneally to each mouse.

Stage (1) and (2) was repeated twice every two weeks.

Day 0. Were obtained from spleen cells of immunized BALB/c mice. These cells were co-cultured with BM-DC, pre-sensitised each SPARC peptide for 2 hours. After this, the resulting cells were co-cultured for 6 days.

Day 6. To identify T-cells and killer cells that specifically recognize peptides SPARC, conducted the analysis with the release of Cr. Cells T2Kd, RL male 1 cell line, cell line A and eth cell line BALB/3T3 were selected as tumor target cells for T-cell killer.

(4) analysis of the cytotoxic effects of SPARC-specific T cells killer cells by analyzing the release of Cr

Cytotoxic activity induced SPARC-specific T cells killer cells against tumor cells was analyzed as follows. Cells T2Kdrepresent cell line, obtained by introduction of a gene Kdin the murine cell line T2, which does not expresses a gene TAP. Only in the case when the peptide is added from the outside, binds to the molecule MHC class I, due to lack of TAP is stable expression of complex molecules MHC class I and peptide on the cell surface. Cell line RL male 1 is a leukemic T-cells of mice BALB-c and expresses SPARC. The above-mentioned two types of cells does not Express SPARC. On the other hand, cell line Meth A and cell line BALB/3T3 represent cell line fibrosarcoma and cell line of fibroblasts obtained from BALB/c mice, respectively. Both of these cell lines Express SPARC. The above cell lines were labeled with radioactive chromium (Cr), and then they were cultured with the above-mentioned T-killer cells for 4 hours. Then, the culture supernatant was extracted and estimated the amount of radioactive Cr released from dead cells, to determine the cytotoxic and the activity.

As a result, no cytotoxicity was observed in the cells T2Kdand cells RL male 1, which is not expressed SPARC. However, cytotoxicity was observed specific cells T2Kdthat expressed peptides SPARC in the case of molecules Kdon the surface by the load SPARC Kdpeptides 1, 3, and 4, as well as against Meth A and BALB/3T3, which spontaneously expressed SPARC (figure 3).

[Example 3]

(5) Induction of antitumor immunity in BALB/c mice by immunization with peptide SPARC

(Method)

BM-DC were cultured with a mixture of peptides SPARC Kd-1, 3, and 4 (10 μm each) for 2 hours. Then, 5×105cells were injected intraperitoneally to each mouse. After a break of 1 week, loaded with peptide BM-DC were injected into mice in the same way that the mouse was immunized in the amount of two times. After seven days, the cell line mouse fibrosarcoma, Meth A (1×106cells, which highly expresses murine SPARC, subcutaneously transplanted mice. Thus, the studied tumor growth and survival time of mice.

(The results)

The results are shown in figure 4. Prophylactic administration of BM-DC, sensitised SPARC peptide could induce inhibition of the growth of the subcutaneously transplanted Meth A, and to induce prolonged survival of mice.

INDUSTRIAL APPLICATIONS IS the NEED

HLA-A24 (A*2402) is the most frequent allele HLA class I, which has approximately 60% of the Japanese. Structural motifs of peptides associated with molecules of KdBALB/c mice, substantially similar structural motifs of peptides associated with molecules HLA-A24 human. In this regard, it is shown that the immunization of BALB/c mice specific peptide when the peptide binds with a molecule Kdand, thereby, induces T-cell killers, and if these T cells killer cells to destroy cancer cells that Express a complex of the peptide and the molecules of the Kdhigh probability that this peptide also binds to HLA-A24 and induces T-cell killers person that can destroy cancer cells. Therefore, the peptide of the present invention can be used for immunotherapy of patients with diffuse infiltrating gastric cancer, pancreatic cancer and melanoma, with HLA-A24. It is expected that the quality of life of patients can be improved by suppressing the growth or progression of such malignant tumors under the action of anti-cancer immunotherapy based on SPARC-peptides.

BRIEF DESCRIPTION of DRAWINGS

The figure 1 shows the level of expression of the most important 15 genes that expressibility in tissues of gastric cancer at a higher level, h is m in normal gastric mucosa, which were identified by analysis with microarrays cDNA in 20 patients with diffuse infiltrating gastric cancer. Taking into account the results obtained, as well as the results of the analysis with cDNA microarrays healthy tissue, as described below, of the above 15 genes, the secretory acidic and rich in cysteine protein (SPARC), is specifically expressed in malignant tissue, was selected as the most ideal tumor-specific antigen in diffuse infiltrating cancer of the stomach.

The figure 2 shows the results obtained by analyzing the expression of SPARC in 25 major types of human organs and 4 types of embryonic organs, using cDNA chips. The SPARC gene expression was observed even in some healthy tissues, but the expression level in these healthy tissues was significantly lower level in malignant tissue.

The figure 3 shows the cytotoxicity SPARC-positive tumor cells under the action of CTLs that were induced with the use of murine Kd-restrukturovaneho SPARC Kd-1, Kd-3 or Kd-4 epitope peptide in BALB/c mice.

The figure 4 shows the suppression of the growth of the subcutaneously transplanted malignant cells Meth and A prolonged survival of BALB/c mice by pre-injection of dendritic cells, the scientists from the bone marrow (BM-DC), loaded with a mixture of peptides SPARC Kd-1, Kd-3 and Kd-4.

1. A peptide which has the ability to induce cytotoxic (killer) T cells selected from (a) and (b):
(A) peptide, which consists of any amino acid sequence of SEQ ID NO:1-3; or
(B) a peptide which consists of amino acid sequence containing substitution, or addition of one or more amino acids relative to the peptide comprising any amino acid sequence of SEQ ID NO:1-3.

2. The means of inducing immunity directed against malignant tumors, which contains at least one type of peptide according to claim 1.

3. Medicinal product for the treatment and/or prevention of tumors, which contains at least one type of peptide according to claim 1.

4. Means for inducing antigen-presenting cells having high capacity to induce T-cells reactive against tumor that contains at least one type of peptide according to claim 1.

5. Tool for the induction of T-cells reactive against tumors, which contains at least one type of peptide according to claim 1.

6. The antibody that specifically binds to the peptide according to claim 1, obtained by using the peptide according to claim 1.

7. Selected T-cell killer, reactive in from the Oseni tumors, which is induced by in vitro stimulation with the peptide according to claim 1.

8. Dedicated helper T-cell reactive against tumors, which is induced by in vitro stimulation of peptides according to claim 1.

9. The population of immune cells that contains the selected T-cell killer according to claim 7.

10. Selected antigen presenting cell, which is a complex molecule HLA and peptide according to claim 1, which is induced by the tool according to claim 4.



 

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