JP2004147649A - Head and neck cancer antigens - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antigen, an antibody, and a cytotoxic T cell required for antigen-specific immunotherapy using SEREX method in cancer such as head and neck cancer.
SOLUTION: Any of the following proteins (A) or (B) and antibodies having these as epitopes, killer T cells, cancer vaccines, cancer diagnostic probes, cancer diagnostic agents, and cancer preventive and therapeutic agents. (A) a protein having a specific amino acid sequence. (B) a protein having an amino acid sequence containing substitution, deletion, insertion or addition of one or several amino acids in a specific amino acid sequence, and having immunity-inducing activity;
[Selection diagram] None

Description

  The present invention relates to a protein specific to human cancer, a partial peptide thereof, a DNA encoding the same, an antibody having the above protein as an epitope, a killer T cell, a cancer vaccine, a probe for cancer diagnosis, a cancer diagnostic agent, and cancer prevention. Regarding therapeutic drugs.

  Human head and neck cancer is a disease that accounts for slightly less than 10% of all cancer patients, and the current treatment is a combination of radiation therapy and chemotherapy mainly of surgical therapy. Despite the recent development of new surgical methods and the discovery of new anti-cancer drugs, human head and neck cancers have a malignant tumor that has not seen much improvement in treatment results up to now, with a 5-year survival rate that has not changed much from 30 years ago. Known as one.

  In recent years, advances in molecular biology and cancer immunology have identified many genes encoding cancer antigens and proteins that can be antigens recognized by killer T cells that respond to cancer. Is growing. In immunotherapy, in order to reduce side effects, it is necessary that a peptide or protein recognized as an antigen is not present in normal cells but is specifically present only in cancer cells.

  On the other hand, SEREX (serological identification of antigens by recombinant expression cloning), a method to identify tumor antigens recognized by antibodies produced by cancer patients in response to their own cancer by incorporating gene expression cloning, was described in 1995 by Pfreundschuh. Established by them. The SEREX method has accelerated the identification of tumor antigens, and a database of about 1,700 tumor antigen candidates identified by the SEREX method has already been created. In addition, clinical trials of antitumor immunotherapy have been started targeting some of them. The present inventors have also used the SEREX method to identify antigens specific to some cancers and genes encoding them.

  The present invention has been accomplished as a result of further research, and provides an antigen, an antibody, and a cytotoxic T cell necessary for antigen-specific immunotherapy using the SEREX method in cancer such as head and neck cancer. The purpose is. More specifically, the present invention relates to a protein or peptide that exists specifically in cancer cells but not in normal cells and that can be recognized by cytotoxic T cells as an antigen, an antibody that recognizes these as an antigen, and a cell. It is intended to provide cytotoxic T cells. Another object of the present invention is to provide an effective antigen-specific immunotherapy using the above antigen or cytotoxic T cell.

That is, according to the present invention, any one of the following proteins (A) and (B) is provided.
(A) a protein having the amino acid sequence of SEQ ID NO: 1;
(B) a protein having an amino acid sequence including substitution, deletion, insertion and / or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 1 and having immunity-inducing activity;

According to another aspect of the present invention, there is provided a peptide comprising a part of the above-described protein of the present invention and having immunity-inducing activity.
The peptide of the present invention is preferably a peptide capable of activating cytotoxic T cells recognizing a cancer antigen protein.

According to another aspect of the present invention, there is provided a peptide having an amino acid sequence represented by any of the following formulas (1) to (8).
Val-Phe-Gly-Thr-Arg-Ile-Glu-Lys-Asp-Leu (1)
Lys-Phe-Glu-Asn-Ser-Ile-Ser-Arg-Leu (2)
Glu-Tyr-Leu-Ser-Leu-Ser-Asp-Lys-Ile (3)
Tyr-Phe-Lys-Val-Lys-Asp-Arg-Thr-Leu (4)
Asn-Tyr-Asn-Asn-Phe-Tyr-Arg-Phe-Leu (5)
Ile-Tyr-Lys-Asn-Ser-Leu-Ser-Glu-Ile (6)
Thr-Tyr-Ile-Ser-Arg-Arg-Glu-Lys-Phe (7)
Glu-Tyr-Ser-Lys-Glu-Cys-Leu-Lys-Glu-Phe (8)
According to still another aspect of the present invention, there is provided a DNA encoding the above-described protein of the present invention.

According to still another aspect of the present invention, there is provided any one of the following DNAs (a), (b) and (c).
(A) a DNA having the nucleotide sequence of SEQ ID NO: 2;
(B) a DNA that hybridizes with a DNA having the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having immunity-inducing activity.
(C) a DNA having a partial sequence of the DNA of the above (a) or (b) and encoding a protein having immunity-inducing activity;

According to still another aspect of the present invention, there is provided an antibody against the above-described protein or peptide of the present invention.
According to still another aspect of the present invention, there is provided a cytotoxic T cell induced by in vitro stimulation using the above-described protein or peptide of the present invention.

  According to still another aspect of the present invention, there is provided a cancer vaccine comprising the above-described protein or peptide of the present invention. According to still another aspect of the present invention, there is provided a cancer vaccine comprising the above-described DNA of the present invention, or a recombinant virus or bacterium containing the DNA. The cancer vaccine of the present invention preferably further comprises an adjuvant.

According to still another aspect of the present invention, there is provided a cancer diagnostic probe comprising the above-described DNA of the present invention, the protein of the present invention, or a partial peptide thereof.
According to still another aspect of the present invention, there is provided a cancer diagnostic agent comprising the above-described probe for cancer diagnosis of the present invention and / or the antibody of the present invention and / or the protein of the present invention or a partial peptide thereof. .
According to still another aspect of the present invention, there is provided a prophylactic / therapeutic agent for cancer comprising the above-described protein, peptide, antibody, and / or cytotoxic T cell of the present invention.
According to still another aspect of the present invention, there is provided a method of using the protein of the present invention or a partial peptide thereof as a cancer marker.

  The antigen of the present invention specifically appears only on cancer cells and testis cells. Therefore, the antigen peptide of the present invention or the DNA encoding the protein or peptide of the present invention can be used as an excellent anti-cancer vaccine with few side effects such as self-damage. Killer T cells stimulated and activated by the antigen of the present invention can be used as anticancer agents.

Hereinafter, embodiments of the present invention will be described in detail.
(1) Protein and Peptide of the Present Invention The protein related to head and neck cancer of the present invention is any one of the following (A) or (B).
(A) a protein having the amino acid sequence of SEQ ID NO: 1 (hereinafter also referred to as “KM-HN-1”);
(B) a protein having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, and / or additions in the amino acid sequence of SEQ ID NO: 1 and having immunity-inducing activity;

  As used herein, the term "protein having immunity-inducing activity" refers to a protein having an activity of inducing an immune response such as antibody production and cellular immunity. Among them, cytotoxic T cells (killer T cells / CTL) A protein having a T cell inducing activity that stimulates is particularly preferred.

  The range of “one or several” in the above “amino acid sequence including substitution, deletion, insertion and / or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 1” is not particularly limited, For example, it means about 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, and particularly preferably about 1 to 3.

  The method for obtaining and producing the protein of the present invention is not particularly limited, and may be any of a naturally-derived protein, a chemically synthesized protein, and a recombinant protein produced by a gene recombination technique. Recombinant proteins are preferred in that they can be produced in large quantities with relatively easy operations.

  When a naturally-occurring protein is obtained, it can be isolated from cells or tissues expressing the protein by appropriately combining methods for isolating and purifying the protein. When a chemically synthesized protein is obtained, for example, the protein of the present invention can be synthesized according to a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). . The protein of the present invention can also be synthesized using various commercially available peptide synthesizers.

  In order to produce the protein of the present invention as a recombinant protein, a DNA having a nucleotide sequence encoding the protein (for example, the nucleotide sequence of SEQ ID NO: 2) or a mutant or homologue thereof is obtained, and this is preferably used. The protein of the present invention can be produced by introducing it into an expression system.

  The expression vector is preferably any vector that can replicate autonomously in the host cell or can be integrated into the chromosome of the host cell, and contains a promoter at a position where the gene of the present invention can be expressed. Is used. In addition, a transformant having a gene encoding the protein of the present invention can be prepared by introducing the above expression vector into a host. The host may be any of bacteria, yeast, animal cells, and insect cells, and the expression vector may be introduced into the host by a known method appropriate for each host.

  In the present invention, the transformant having the gene of the present invention prepared as described above is cultured, the protein of the present invention is produced and accumulated in the culture, and the protein of the present invention is collected from the culture. Thus, the recombinant protein can be isolated.

  When the transformant of the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the microorganism. Any of a natural medium and a synthetic medium may be used as long as the medium can efficiently culture the transformant. The culture conditions may be the same as those usually used for culturing the microorganism. After culturing, the protein of the present invention can be isolated and purified from the culture of the transformant by a conventional protein isolation and purification method.

  In the amino acid sequence of SEQ ID NO: 1, a protein having an amino acid sequence containing substitution, deletion, insertion and / or addition of one or several amino acids is a DNA encoding the amino acid sequence of SEQ ID NO: 1. Those skilled in the art can appropriately produce or obtain the nucleotide sequence based on the information on the nucleotide sequence described in SEQ ID NO: 2, which shows an example of the sequence.

  That is, in the amino acid sequence of SEQ ID NO: 1, a gene (mutant gene) having a base sequence encoding a protein having an amino acid sequence containing substitution, deletion, insertion and / or addition of one or several amino acids is It can also be made by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques or mutagenesis. Specifically, a mutated DNA can be obtained by using a DNA having the base sequence of SEQ ID NO: 2 and introducing a mutation into these DNAs.

For example, the method can be carried out using a method of bringing a DNA having the base sequence of SEQ ID NO: 2 into contact with a drug as a mutagen, a method of irradiating ultraviolet rays, a genetic engineering technique, or the like. Site-directed mutagenesis method which is one of genetic engineering technique is useful because it is a method capable of introducing a specific mutation into a specific position, Molecular Cloning:. A laboratory Mannual , 2 nd Ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989 (hereinafter abbreviated as molecular cloning second edition), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) (hereinafter, Current Protocols. In molecular biology) and the like.

The present invention further relates to a peptide comprising a part of the above-described protein of the present invention and having immunity-inducing activity. The peptide of the present invention is preferably one that can activate cytotoxic T cells that recognize a cancer antigen protein. Specific examples of such peptides include those having any one of the following amino acid sequences, and HLA-A24-restricted cancer cytotoxic activity was observed in these eight peptides.
Val-Phe-Gly-Thr-Arg-Ile-Glu-Lys-Asp-Leu (1)
Lys-Phe-Glu-Asn-Ser-Ile-Ser-Arg-Leu (2)
Glu-Tyr-Leu-Ser-Leu-Ser-Asp-Lys-Ile (3)
Tyr-Phe-Lys-Val-Lys-Asp-Arg-Thr-Leu (4)
Asn-Tyr-Asn-Asn-Phe-Tyr-Arg-Phe-Leu (5)
Ile-Tyr-Lys-Asn-Ser-Leu-Ser-Glu-Ile (6)
Thr-Tyr-Ile-Ser-Arg-Arg-Glu-Lys-Phe (7)
Glu-Tyr-Ser-Lys-Glu-Cys-Leu-Lys-Glu-Phe (8)

    The peptide of the present invention can be synthesized according to a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). The peptide of the present invention can also be synthesized using various commercially available peptide synthesizers.

(2) DNA of the present invention
The DNA of the present invention is a DNA encoding the protein of the present invention described in (1) above, and is preferably any one of the following DNAs (a), (b) and (c).
(A) a DNA having the nucleotide sequence of SEQ ID NO: 2;
(B) a DNA that hybridizes with a DNA having the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having immunity-inducing activity.
(C) a DNA having a partial sequence of the DNA of the above (a) or (b) and encoding a protein having immunity-inducing activity;

  The above "hybridize under stringent conditions" refers to the base of DNA obtained by using colony hybridization, plaque hybridization, or Southern blot hybridization using DNA as a probe. Means a sequence, for example, after hybridization at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from a colony or plaque or a fragment of the DNA is immobilized, DNA that can be identified by washing the filter at 65 ° C. using a 1 to 2 × SSC solution (1 × SSC solution is 150 mM sodium chloride, 15 mM sodium citrate) can be mentioned. Hybridization can be performed according to the method described in Molecular Cloning, 2nd edition and the like.

  Examples of the DNA that hybridizes under stringent conditions include DNAs having a certain degree of homology with the base sequence of the DNA used as a probe, for example, 70% or more, preferably 80% or more, more preferably 90% or more. Above, more preferably 93% or more, particularly preferably 95% or more, most preferably 98% or more homologous DNA.

  The method for obtaining the DNA of the present invention is not particularly limited. Suitable probes and primers are prepared based on the information on the amino acid sequence and base sequence described in SEQ ID NO: 1 and SEQ ID NO: 2 in the Sequence Listing in this specification, and a human or other cDNA library is screened using them. Thus, the DNA of the present invention can be isolated. The cDNA library is preferably prepared from cells, organs or tissues expressing the DNA of the present invention.

  DNA having the base sequence described in SEQ ID NO: 2 can also be obtained by the PCR method. Using a human chromosomal DNA or cDNA library as a template, PCR is performed using a pair of primers designed to amplify the base sequence described in SEQ ID NO: 2. The PCR reaction conditions can be set as appropriate. For example, a reaction step consisting of 94 ° C. for 30 seconds (denaturation), 55 ° C. for 30 seconds to 1 minute (annealing), and 72 ° C. for 2 minutes (extension) is one cycle. Examples of such conditions include, for example, conditions in which the reaction is performed at 72 ° C. for 7 minutes after 30 cycles. Next, the amplified DNA fragment can be cloned into an appropriate vector that can be amplified in a host such as E. coli.

  Operations such as the preparation of the above-described probe or primer, construction of a cDNA library, screening of a cDNA library, and cloning of a target gene are known to those skilled in the art. For example, Molecular Cloning, 2nd edition, Current Protocols, Inc. -It can be performed according to the method described in Molecular Biology and the like.

(3) Antibody and cytotoxic cell of the present invention The present invention provides an antibody that recognizes a part or all of the above-described protein or peptide of the present invention as an epitope (antigen), and an in vitro stimulation using the protein or peptide. It also relates to induced cytotoxic (killer) T cells (CTL). In general, CTLs show stronger anti-tumor activity than antibodies.

  The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, and can be produced by a conventional method.

  For example, a polyclonal antibody can be obtained by immunizing a mammal with the protein of the present invention as an antigen, collecting blood from the mammal, and separating and purifying the antibody from the collected blood. For example, mammals such as mice, hamsters, guinea pigs, chickens, rats, rabbits, dogs, goats, sheep, and cows can be immunized. The method of immunization is known to those skilled in the art, and for example, the antigen may be administered two to three times, for example, every 7 to 30 days. The dose can be, for example, about 0.05 to 2 mg of the antigen at one time. The administration route is also not particularly limited, and subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration and the like can be appropriately selected. The antigen can be used by dissolving it in an appropriate buffer, for example, an appropriate buffer containing a commonly used adjuvant such as complete Freund's adjuvant or aluminum hydroxide.

  After raising the immunized mammal for a certain period and the antibody titer increases, booster immunization can be performed using, for example, 100 μg to 1000 μg of the antigen. One to two months after the last administration, blood is collected from the immunized mammal, and the blood is subjected to, for example, centrifugation, precipitation using ammonium sulfate or polyethylene glycol, gel filtration chromatography, ion exchange chromatography, affinity. A polyclonal antibody recognizing the protein of the present invention can be obtained as a polyclonal antiserum by separation and purification by a conventional method such as chromatography such as chromatography.

  On the other hand, a monoclonal antibody can be obtained by preparing a hybridoma. For example, a hybridoma can be obtained by cell fusion between an antibody-producing cell and a myeloma cell line. The hybridoma producing the monoclonal antibody of the present invention can be obtained by the following cell fusion method.

  As antibody-producing cells, spleen cells, lymph node cells, B lymphocytes and the like from immunized animals are used. As the antigen, the protein of the present invention or its partial peptide is used. Mice, rats and the like can be used as immunized animals, and administration of the antigen to these animals is performed by a conventional method. For example, a suspension or emulsion of an adjuvant such as complete Freund's adjuvant or incomplete Freund's adjuvant and the protein of the present invention, which is an antigen, is administered to the animal several times by intravenous, subcutaneous, intradermal, intraperitoneal or the like. Immunize. Obtaining, for example, spleen cells as antibody-producing cells from an immunized animal, and fusing these with myeloma cells by a known method (G. Kohler et al., Nature, 256 495 (1975)) to produce a hybridoma. Can be.

  Examples of myeloma cell lines used for cell fusion include P3X63Ag8, P3U1 and Sp2 / 0 strains in mice. When performing cell fusion, a fusion promoter such as polyethylene glycol or Sendai virus is used, and for selection of hybridomas after cell fusion, hypoxanthine / aminopterin / thymidine (HAT) medium is used according to a conventional method. The hybridoma obtained by cell fusion is cloned by a limiting dilution method or the like. Further, if necessary, a cell line that produces a monoclonal antibody that specifically recognizes the protein of the present invention can be obtained by performing screening by an enzyme immunoassay using the protein of the present invention.

  To produce a monoclonal antibody of interest from the hybridoma thus obtained, the hybridoma may be cultured by a usual cell culture method or ascites formation method, and the monoclonal antibody may be purified from the culture supernatant or ascites. . Purification of the monoclonal antibody from the culture supernatant or ascites can be performed by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography and the like can be used in appropriate combination.

  Also, fragments of the above-mentioned antibodies are within the scope of the present invention. Antibody fragments include F (ab ') 2 fragments, Fab' fragments and the like.

Furthermore, labeled antibodies of the above-mentioned antibodies are also within the scope of the present invention. That is, the antibody of the present invention prepared as described above can be labeled and used. The types and methods of labeling antibodies are known to those skilled in the art. For example, an enzyme label such as horseradish peroxidase or alkaline phosphatase, a fluorescent label such as FITC (fluorescein isothiocyanate) or TRITC (tetramethylrhodamine B isothiocyanate), a label with a coloring substance such as colloidal metal and colored latex, biotin, etc. An affinity label or an isotope label such as ¹²⁵ I can be given. Analysis or measurement of the protein of the present invention (that is, cancer antigen) using the labeled antibody of the present invention can be performed by those skilled in the art such as enzyme antibody method, immunohistochemical staining method, immunoblot method, direct fluorescent antibody method and indirect fluorescent antibody method. Can be carried out according to a method known in the art.

  The present invention also relates to activated T cells induced by in vitro stimulation with a protein or peptide of the present invention. For example, when peripheral blood lymphocytes or tumor-infiltrating lymphocytes are stimulated in vitro with the protein or peptide of the present invention, tumor-reactive activated T cells are induced, and the activated T cells can be used effectively for adoptive immunotherapy. it can. In addition, the protein or peptide of the present invention can be expressed in vivo or in vitro in dendritic cells that are powerful antigen-presenting cells, and immunity can be induced by administering the antigen-expressing dendritic cells.

(4) Cancer vaccine of the present invention Since the DNA, protein and peptide of the present invention can induce cancer cell-specific cytotoxic T cells, they can be expected as therapeutic or preventive agents for cancer. For example, BCG bacteria transformed with the DNA of the present invention into an appropriate vector and transformed with the recombinant DNA, or a virus such as vaccinia virus with the DNA of the present invention integrated in the genome, can be used for treatment of human cancer. It can be used effectively as a live vaccine for prevention. The dose and administration method of the cancer vaccine are the same as those for ordinary vaccination and BCG vaccine.

  That is, the DNA of the present invention (as it is or in the form of a plasmid DNA incorporated into an expression vector), a recombinant virus or recombinant bacterium containing the DNA as it is or dispersed in an adjuvant can be used as a cancer vaccine in mammals including humans. Can be administered. Similarly, the peptide of the present invention can be administered as a cancer vaccine in a state of being dispersed with an adjuvant.

  Examples of the adjuvant that can be used in the present invention include incomplete Freund's adjuvant, BCG, trehalose dimycolate (TDM), lipopolysaccharide (LPS), alum adjuvant, silica adjuvant, and the like. For this reason, it is preferable to use Freund's incomplete adjuvant (FIA).

(5) Probe for Cancer Diagnosis, Cancer Diagnosis Agent, Cancer Prevention / Therapeutic Agent of the Present Invention The DNA of the present invention can be used as a diagnostic probe by extracting DNAs of various human cancers and examining their homology. . The probe and the antibody can be used as a cancer diagnostic agent. In addition, the protein or its partial peptide of the present invention can be used as a cancer diagnostic by examining the antibody titer in the serum of a cancer patient using the protein or its partial peptide. As the partial peptide of the protein of the present invention, any peptide can be used as long as it can detect an IgG antibody specific to KM-HN-1 which is the protein of the present invention. Examples include, but are not limited to, a peptide having the amino acid sequence of 37-524 of the amino acid sequence (KM-HN-1) described in No. 1.

  That is, the present invention relates to a cancer diagnostic probe comprising all or a part of the antisense strand of DNA or RNA encoding the protein of the present invention. The present invention further relates to a cancer diagnostic agent comprising the above-mentioned probe for cancer diagnosis or an antibody against the protein of the present invention. The cancer diagnostic probe of the present invention is all or part of the antisense strand of DNA (cDNA) or RNA (cRNA) encoding the protein of the present invention, and has a length (at least 20 bases) that can be established as a probe. Are preferred. For example, cancer can be diagnosed by detecting mRNA of the protein (cancer antigen) of the present invention obtained from a specimen using the above antisense strand. Specimens used for detection include, but are not limited to, genomic DNA and RNA or cDNA obtained from biopsies of cells, for example, blood, urine, saliva, tissue, etc. of a subject. . When such a sample is used, a sample amplified by PCR or the like may be used. Alternatively, cancer can be diagnosed by synthesizing the protein of the present invention or a partial peptide thereof and examining the antibody titer in the serum of the cancer patient by ELISA using the protein or its partial peptide.

  To date, a number of tumor-associated antigens have been identified, which have been classified into several. One of them is an antigen classified as a cancer / testis antigen. This antigen is expressed only on the testis in normal cells. The antigen of the present invention is one of the cancer / testis antigens.

  The type of cancer referred to in this specification is not particularly limited, and specific examples include head and neck cancer, brain tumor, chronic myelogenous leukemia, acute myeloid leukemia, malignant melanoma, malignant lymphoma, lung cancer, esophageal cancer, gastric cancer, and colon. Cancer, rectal cancer, colon cancer, kidney cancer, ureteral tumor, gallbladder cancer, bile duct cancer, biliary tract cancer, breast cancer, liver cancer, pancreatic cancer, testicular tumor, testicular cancer, maxillary cancer, tongue cancer, lip cancer, oral cancer, Pharyngeal, laryngeal, ovarian, uterine, prostate, thyroid, Kaposi's sarcoma, hemangiomas, leukemia, polycythemia vera, neuroblastoma, retinoblastoma, myeloma, cystoma, sarcoma, osteosarcoma, Examples include, but are not limited to, myoma, skin cancer, basal cell carcinoma, skin appendage cancer, skin metastatic cancer, cutaneous melanoma, and the like. Particularly preferred examples of cancers of interest are head and neck, esophagus, colon, hepatocellular, bile duct, breast, tongue, thyroid, stomach, lung, and pancreatic cancers.

  Since the protein or peptide of the present invention can induce cancer cell-specific cytotoxic T cells as T cell epitopes, it is useful as an agent for preventing or treating human cancer. The antibody of the present invention is also useful as a prophylactic / therapeutic agent for human cancer as long as it can inhibit the activity of the protein of the present invention, which is a cancer antigen. As an actual usage, the protein, peptide or antibody of the present invention is administered as an injection, as it is, or together with a pharmaceutically acceptable carrier and / or diluent and, if necessary, the following auxiliaries. Alternatively, it may be administered by percutaneous absorption through mucous membranes by spraying or the like. The carrier mentioned here is, for example, human serum albumin, and the diluent includes, for example, PBS and distilled water.

  The dose can be, for example, 0.01 mg to 100 mg per administration of the protein, peptide or antibody of the present invention per adult, but is not limited to this range. The form of the preparation is not particularly limited either, and may be freeze-dried or granulated by adding an excipient such as sugar.

  Examples of adjuvants for enhancing cytotoxic T cell-inducing activity that can be added to the agent of the present invention include bacterial cell components such as BCG bacteria, ISCOM described in Nature, vol. 344, p873 (1990), J. Immunol. Vol. 148, p1438 (1992), such as saponin QS-21, liposome, and aluminum hydroxide. In addition, immunostimulants such as lentinan, schizophyllan, and picibanil can also be used as adjuvants. In addition, cytokines such as IL-2, IL-4, IL-12, IL-1, IL-6, and TNF that enhance the proliferation and differentiation of T cells can also be used as adjuvants.

  In addition, the antigen peptide is added to cells collected from a patient or cells having the same HLA paprotype in a test tube in order to present the antigen, and then administered to a patient's blood vessel. Can also be induced. In addition, by adding the peptide to peripheral blood lymphocytes of a patient and culturing the peptide in a test tube, cytotoxic T cells can be induced in the test tube and then returned to the blood vessel of the patient. Such treatment by cell transfer has already been performed as a cancer treatment method, and is a method well known to those skilled in the art.

A target antigen for specific anti-tumor immunotherapy requires that the antigen be a recognition antigen for cytotoxic T cells (killer T cells / CTL). The antigen of the present invention increased killer T cell inducing activity in vitro in HLA-A24, which is common in Japanese. Thus, injection of the antigen of the present invention into the body induces and activates CTL, and as a result, an antitumor effect can be expected. In addition, when stimulated with the antigen of the present invention, activated T cells are induced in the tumor, and the activated T cells can be effectively used for adoptive immunotherapy by injecting the activated T cells into the affected area.
Hereinafter, the antigen of the present invention, its production method, and effects will be described with reference to examples, but the present invention is not limited to these examples. KM-HN-1 which is the head and neck cancer antigen of the present invention can be detected by, for example, the SEREX method as described in the following examples.

Example 1
《Collecting cancer cells》
First, 5 kinds of head and neck squamous cell carcinoma cell lines and RPM11640 (containing 10% fetal bovine serum, 100 U / ml penicillin, 0.1 μg / ml streptomycin, 2 mM L-glutamine and 10 mM HEPES) were used. Cultivation was carried out under conditions of 5 ° C., 5% CO ₂ and 100% humidity until the concentration reached 1 × 10 ⁷ .

《Collection of serum》
Serum was collected from six patients with squamous cell carcinoma of the head and neck. The serum after blood collection was stored at -80 ° C. An antibody against E. coli and λ phage was removed from this serum sample using a column packed with a lysate of E. coli and phage and Sepharose 4B, and then diluted 100- to 800-fold before use.

<< Preparation of RNA / cDNA >>
Total RNA was extracted from the head and neck squamous cell carcinoma cell line by a modified acid-guanidium-phenol-chloroform method, and mRNA was converted to poly (A) using oligo (dT) Oligotex-dT30 super mRNA Purification Kit (Takara Shuzou, Kyoto, Japan). ) Purified by selection method. CDNA was synthesized from 5 μg of the purified poly (A) RNA by a known method using a reverse transcriptase.

<< Preparation of cDNA library / protein >>
It was inserted into a λZAP express vector using a ZAP-cDNA Synthesis Kit (Stratagene, La Jolla, CA) to prepare a phage cDNA library. After infecting Escherichia coli with the λ phage cDNA library prepared by the above method, the cells were cultured at 42 ° C. for 4 hours on an NZY plate medium to form plaques. The plate was covered with a nitrocellulose membrane impregnated with isopropyl β-D-thiogalactoside (IPTG) at 37 ° C. for 3 hours to produce a protein encoded by cDNA incorporated into λ phage in the plaque.

《Immuno screening》
The protein produced by the above method was transferred to a nitrocellulose membrane. The nitrocellulose after the blocking was washed, and reacted with the serum diluted 100 to 800 times at room temperature for 2 hours. After washing, Horseradish Oeroxidase (HRP) -labeled mouse anti-human IgG antibody was reacted with the membrane as a secondary antibody. After washing, chemiluminescence was detected on an X-ray film, checked against the photographic plate, and positive plaques were picked up with surrounding negative plaques. A plaque corresponding to the color reaction positive site was collected from a 15 cm NZY agarose plate and dissolved in SM buffer (100 mM NaCl, 10 mM MgSO ₄ , 50 mM Tris-HCl, 0.01% gelatin; pH 7.5). Was. The secondary and tertiary screenings were repeated in the same manner as described above until the chromogenic positive plaques were unified, and a single phage clone to which the IgG antibody in the serum reacted was obtained. By the above method, a total of 28 positive clones were isolated, 12 from cDNA derived from the head and neck squamous cell carcinoma cell line and 16 from normal testis-derived cDNA.

《Homology search of isolated antigen gene》
The insert DNA was amplified by the PCR method and used for the subsequent analysis. The resulting PCR product was subjected to Big Dye DNA sequencing kit (PE Biosystems, CA) A sequencing to determine the nucleotide sequence. The nucleotide sequences of the 28 genes determined above were compared with gene information registered in the NCBI data bank using the homology search program BLAST (Basic Local Alignment Search Tool).

<< KM-HN-1 >>
As a result, it was confirmed that 28 positive clones were 19 types of genes. One of these genes was novel DNA. One of them was named KM-HN-1. This gene was located on chromosome 4, was a protein of 833 amino acids, and contained a coiled coil domain and a leucine zipper motif (FIG. 1). The amino acid sequence of KM-HN-1 is described in SEQ ID NO: 1, and the nucleotide sequence is described in SEQ ID NO: 2.

<< RT-PCR >>
The cDNA of the positive clone obtained by the above method was propagated by RT-PCR. In the RT-PCR reaction, the unification treatment was repeated at 94 ° C. for 5 minutes, the annealing temperature was 58 ° C., and 30 cycles were repeated. As a control, standardization was performed using β-actin mRNA.

<< Confirmation of KM-HN-1 expression >>
Examination of the presence or absence of KM-HN-1 in various cancer tissues and normal tissues for lung cancer, tongue cancer, pancreatic cancer, brain tumor, thyroid cancer, stomach cancer, laryngeal cancer, and submandibular gland cancer using RT-PCR method did. As a result, as shown in FIG. 2, it was found that KM-HN-1 appeared in tongue cancer, thyroid cancer, gastric cancer, lung cancer, and pancreatic cancer tissues, and was expressed only in testis in normal tissues.

Example 2
<< Peptide Constituting KM-HN-1 >>
Based on the amino acid structure identified by KM-HN-1, 13 kinds of peptides (SEQ ID NOS: 3 to 15) of Table 1 consisting of 9 or 10 amino acids were synthesized by the Fmoc / PyBOP method and used in the following tests. .

<< Preparation of HLA-A24 binding peptide based on HLA-A24 binding motif >>
Using the HLA-peptide bond prediction from the amino acid sequence of KM-HN-1, 13 types of peptides shown in Table 1 consisting of 9 or 10 amino acids which are considered to bind to HLA-A24 were synthesized by the Fmoc / PyBOP method. Were used for the following tests.

<< Measurement of CTL inducing activity >>
Blood of 30 CC was collected from five patients at Kumamoto University Hospital and four head and neck cancer patients and three healthy people at Saga Medical University Hospital. It was tested whether 13 kinds of peptides derived from KM-HN-1 obtained from 8 head-and-neck cancer patients positive for HLA-A24 and 3 healthy PBMC have the ability to induce cancer-reactive killer T cells. . After culturing for 4 weeks, killer T cell activity in PBMC was determined by measuring with a ⁵¹ Cr release assay. The results are shown in FIG.

Similarly, to determine the CTL epitope peptide of HLA-A24 in humans of KM-HN-1, peripheral blood lymphocytes of HLA-A24 healthy humans were stimulated three times with 13 kinds of peptides and subjected to ⁵¹ Cr release assay. , KM-HN-1 and HLA-A24-expressing human esophageal cancer cell line TE3 and lung cancer cell line PC-9 were examined for cytotoxic activity. The results are shown in Table 2 below.

The following peptides (1) to (8):
Val-Phe-Gly-Thr-Arg-Ile-Glu-Lys-Asp-Leu (1)
Lys-Phe-Glu-Asn-Ser-Ile-Ser-Arg-Leu (2)
Glu-Tyr-Leu-Ser-Leu-Ser-Asp-Lys-Ile (3)
Tyr-Phe-Lys-Val-Lys-Asp-Arg-Thr-Leu (4)
Asn-Tyr-Asn-Asn-Phe-Tyr-Arg-Phe-Leu (5)
Ile-Tyr-Lys-Asn-Ser-Leu-Ser-Glu-Ile (6)
Thr-Tyr-Ile-Ser-Arg-Arg-Glu-Lys-Phe (7)
Glu-Tyr-Ser-Lys-Glu-Cys-Leu-Lys-Glu-Phe (8)
Is a CTL line induced by HLA-A24 but does not express KM-HN-1 but does not damage HSC-2, but a head and neck cancer cell line that expresses KM-HN-1 on HLA-A24 It specifically damages HSC-3, esophageal cancer cell line TE3, and lung cancer cell line PC-9.

《Peptide vaccine》
The above results may provide evidence that peptides (1) to (8) can be used as anti-cancer vaccines.

<< CTL cancer therapeutics >>
In addition, killer T cells that recognize these KM-HN-1 and / or peptides (1) to (8) as antigens do not damage normal cells and have cytotoxic activity only on head and neck cancer, esophageal cancer or lung cancer. It can be used as a therapeutic agent for head and neck cancer, esophageal cancer or lung cancer with few side effects.

Example 3
<< Protein Constituting KM-HN-1 >>
The amino acid sequence 37-524 of SEQ ID NO: 1 identified by KM-HN-1 was prepared as a synthetic protein using Escherichia coli strain DH5α and used in the following tests.

《Measurement of KM-HN-1 specific IgG antibody in serum of cancer patient》
Blood was collected from esophageal cancer, stomach cancer, lung cancer, hepatocellular carcinoma, colorectal cancer, head and neck cancer, pancreatic cancer, bile duct cancer, breast cancer and healthy subjects, and the serum was separated. KM-HN-1 specific IgG antibodies were measured by ELISA using KM-HN-1 protein, serum and anti-human IgG antibodies. Specifically, after a human KM-HN-1 synthetic protein prepared using Escherichia coli was stuck to a 96-well plate, 100-fold diluted serum from various cancer patients was added and reacted. Thereafter, HRP-conjugated mouse anti-human IgG antibody and O-phenylenediamine as a chromogenic substrate were added, and the absorbance was measured using an absorbance meter. FIG. 4 shows the results.

  As can be seen from the results of FIG. 4, when the average value of the healthy subjects + standard deviation is defined as the upper limit of normality, the antibody positive rate in healthy subjects is as low as 6.3%, whereas the antibody positive rate in various cancer patients is low. The positive rate was as high as 40 to 100%. Therefore, it was demonstrated that the protein of the present invention or a fragment thereof can be used as a diagnostic agent for cancer. That is, the KM-HN-1 specific IgG antibody titer was proved to be useful as a tumor marker.

FIG. 1 shows the amino acid sequence and base sequence of the protein KM-HN-1 which forms the antigen of the present invention. FIG. 2 is a diagram comparing the expression levels of the protein KM-HN-1 of the present invention in normal cells and cancer cells using the RT-PCR method. The amount of mRNA in each tissue is normalized with β-actin. FIG. 3 is a graph showing the cancer-inducing activity of peptide-specific killer T cells using ⁵¹ Cr release assay. FIG. 4 shows the results of measuring KM-HN-1 specific IgG antibodies in the serum of cancer patients.

Claims (16)

Any one of the following proteins (A) and (B):
(A) a protein having the amino acid sequence of SEQ ID NO: 1;
(B) a protein having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, and / or additions in the amino acid sequence of SEQ ID NO: 1 and having immunity-inducing activity; A peptide comprising a part of the protein according to claim 1 and having immunity-inducing activity. The peptide according to claim 2, which is capable of activating a cytotoxic T cell recognizing a cancer antigen protein. The peptide according to claim 2 or 3, which has an amino acid sequence according to any of the following formulas (1) to (8).
Val-Phe-Gly-Thr-Arg-Ile-Glu-Lys-Asp-Leu (1)
Lys-Phe-Glu-Asn-Ser-Ile-Ser-Arg-Leu (2)
Glu-Tyr-Leu-Ser-Leu-Ser-Asp-Lys-Ile (3)
Tyr-Phe-Lys-Val-Lys-Asp-Arg-Thr-Leu (4)
Asn-Tyr-Asn-Asn-Phe-Tyr-Arg-Phe-Leu (5)
Ile-Tyr-Lys-Asn-Ser-Leu-Ser-Glu-Ile (6)
Thr-Tyr-Ile-Ser-Arg-Arg-Glu-Lys-Phe (7)
Glu-Tyr-Ser-Lys-Glu-Cys-Leu-Lys-Glu-Phe (8) A DNA encoding the protein according to claim 1. DNA of any of the following (a), (b) or (c):
(A) a DNA having the nucleotide sequence of SEQ ID NO: 2;
(B) a DNA that hybridizes with a DNA having the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having immunity-inducing activity.
(C) a DNA having a partial sequence of the DNA of the above (a) or (b) and encoding a protein having immunity-inducing activity; An antibody against the protein according to claim 1 or the peptide according to any one of claims 2 to 4. A cytotoxic T cell induced by in vitro stimulation using the protein according to claim 1 or the peptide according to any one of claims 2 to 4. A cancer vaccine comprising the protein according to claim 1 or the peptide according to any one of claims 2 to 4. The cancer vaccine according to claim 9, further comprising an adjuvant. A cancer vaccine comprising the DNA according to claim 5 or a recombinant virus or a bacterium containing the DNA. The cancer vaccine according to claim 11, further comprising an adjuvant. A cancer diagnostic probe comprising the DNA according to claim 5 or 6, or the protein or a partial peptide thereof according to claim 1. A cancer diagnostic comprising the cancer diagnostic probe according to claim 13 and / or the antibody according to claim 7 and / or the protein or a partial peptide thereof according to claim 1. Prevention and prevention of cancer comprising the protein according to claim 1, the peptide according to any one of claims 2 to 4, the antibody according to claim 7, and / or the cytotoxic T cell according to claim 8. Therapeutic drugs. A method for using the protein according to claim 1 or a partial peptide thereof as a cancer marker.

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