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WO2000020442A1 - Peptides antigenes specifiques aux tumeurs - Google Patents

Peptides antigenes specifiques aux tumeurs Download PDF

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Publication number
WO2000020442A1
WO2000020442A1 PCT/JP1999/005426 JP9905426W WO0020442A1 WO 2000020442 A1 WO2000020442 A1 WO 2000020442A1 JP 9905426 W JP9905426 W JP 9905426W WO 0020442 A1 WO0020442 A1 WO 0020442A1
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WIPO (PCT)
Prior art keywords
cancer
tumor
antibody
peptide
cells
Prior art date
Application number
PCT/JP1999/005426
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English (en)
Japanese (ja)
Inventor
Nobuo Satoh
Original Assignee
Suzuki, Nobutaka
Yamaguchi, Masaaki
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suzuki, Nobutaka, Yamaguchi, Masaaki filed Critical Suzuki, Nobutaka
Priority to AU60014/99A priority Critical patent/AU6001499A/en
Publication of WO2000020442A1 publication Critical patent/WO2000020442A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens

Definitions

  • the present invention relates to a tumor-specific antigen peptide, an antibody against the peptide, and a pharmaceutical composition comprising the peptide or the antibody.
  • a tumor-specific antigen peptide an antibody against the peptide
  • a pharmaceutical composition comprising the peptide or the antibody.
  • cancer cell membranes had a different structure from normal membranes.
  • This immunologically means that the antigen recognized by the immune cells is present on the cancer cell membrane.
  • One of the antigens is the so-called tumor rejection antigen that is recognized by T cells and expressed in a form linked to its own MHC.
  • the other is a tumor antigen on the cancer cell membrane recognized by NK cells and macrophages, which is thought to be expressed without binding to its own MHC.
  • the gene encoding the tumor rejection antigen has been isolated from human melanoma, and many more antigens have been found.
  • Such human tumor rejection antigens include, for example, the epitope sequence “EADPTGHSY” (referred to as MAGE-1), the epitope sequence “AARAVFLAL” (referred to as BAGE), the epitope sequence “YRPRPRRY” (referred to as GAGE-1), and the epitope sequence.
  • MAGE-1 epitope sequence
  • AARAVFLAL epitope sequence
  • GAGE-1 epitope sequence
  • antigens such as “VLPDVFIRC” (referred to as ⁇ - ⁇ )
  • MAGE-1 is not expressed in normal tissues except testis, suggesting high tumor specificity.
  • the expression of NA17-A is found in about 50% of melanomas, and it is slightly expressed in brain tumors and sarcomas, but slightly expressed in normal tissues.
  • melanocyte-specific tissue differentiation antigens or tumor-specific antigens derived from genetic changes associated with canceration have been identified (Robbins PF. And Kawakami Y. Human tumor antigens recognized by Teel Is . Curr. Opin. Immunol. 8: 628- 636, 1996. / Hideyuki Ikeda; CTL epitope of human melanoma. Annual Review Immunity 1996: 176-183, 1996. / Kyogo Ito, Shigeki Nanagata; Human tumor antigens and specific immunotherapy. History of Medicine 179: 117-121, 1996.).
  • An object of the present invention is to provide a tumor-specific antigen peptide, an antibody against the peptide, and a pharmaceutical composition containing the peptide or the antibody.
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, extracted antigens from cell membrane components of a cell line established from human thyroid cancer, and obtained a tumor-specific antigen map from the antigens. Successful isolation of the tide led to the completion of the present invention.
  • the present invention provides the following tumor-specific antigen peptide (A) or (B).
  • Tumor-specific antigen peptide consisting of the amino acid sequence represented by Phe Ser Ala Thr Asp Pro Lys
  • a tumor-specific antigen peptide which contains at least the amino acid sequence represented by Phe Ser Ala Thr Asp Pro Lys and is specifically expressed in a tumor.
  • the present invention is a tumor-specific antigen peptide that contains at least the 33rd to 39th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2, and is specifically expressed in a tumor.
  • the present invention provides the following ( ⁇ ′) or ( ⁇ ′) tumor-specific antigen peptide.
  • ( ⁇ ') a tumor-specific antigen peptide consisting of any of the amino acid sequences represented by SEQ ID NOs: 3 to 6
  • ( ⁇ ′) a tumor-specific antigen that includes an amino acid sequence in which one or several amino acids have been deleted, substituted or added in any of the amino acid sequences represented by SEQ ID NOs: 3 to 6, and is expressed in a tumor-specific manner.
  • the present invention is a DNA or RNA encoding a protein containing the peptide.
  • DNA include those described in (a) or (b) below
  • RNA examples include those described in (c) or (d) below.
  • RNA consisting of the nucleotide sequence represented by SEQ ID NO: 7
  • RNA that hybridizes with RNA consisting of the nucleotide sequence of SEQ ID NO: 7 under stringent conditions and encodes a protein containing a tumor-specifically expressed peptide.
  • the present invention is a recombinant vector containing the DNA or RNA.
  • the present invention is a transformant containing the recombinant vector.
  • the present invention provides a method for culturing the transformant, wherein the culture obtained is tumor-specific.
  • a method for producing the protein comprising collecting a protein containing dipeptide which is expressed in the protein.
  • the present invention relates to an antibody against the peptide.
  • the antibody include a monoclonal antibody or a polyclonal antibody or a fragment thereof (eg, Fab or F (ab ') 2 ).
  • the antibody of the present invention also includes a monoclonal antibody produced by a hybridoma having an accession number of FERM BP-6899.
  • the present invention is a leukocyte which reacts with the peptide or a leukocyte which binds with the antibody.
  • the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising the peptide or the antibody as an active ingredient.
  • the present invention is a diagnostic, prophylactic or therapeutic agent for tumors containing the peptide, antibody or leukocyte as an active ingredient.
  • tumors include thyroid cancer, breast cancer, stomach cancer, esophageal cancer, oral cancer, colorectal cancer, knee cancer, lung cancer, kidney cancer, bladder cancer, ovarian cancer, uterine cancer, vulvar cancer, skin cancer, melanoma, central nervous system Tumor, peripheral nerve tumor, gingival cancer, pharyngeal cancer, jaw cancer, mediastinal tumor, liver cancer, bile duct cancer, gallbladder cancer, renal pelvic tumor, ureteral cancer, testicular tumor, prostate cancer, choriocarcinoma, fallopian tube cancer, vaginal cancer Sarcoma, leukemia, erythroleukemia, multiple myeloma, malignant lymphoma, and carcinosarcoma.
  • the present invention is a reagent for measuring an antibody against a tumor-specific antigen, comprising the
  • the present invention is a method for detecting an antibody against a tumor-specific antigen, which comprises reacting the antibody with a body fluid or a tumor cell.
  • the present invention is a reagent for detecting a tumor-specific antigen, comprising the antibody.
  • the present invention is a method for detecting a tumor-specific antigen, which comprises reacting the antibody with a body fluid or a tumor cell.
  • the present invention basically relates to an antigen peptide having at least 7 amino acid sequences and an antibody against the peptide.
  • antibody means both monoclonal antibodies and polyclonal antibodies produced by mammals including humans, for example, and fragments of these antibodies (for example, Fab fragments, F (ab ′) 2 Fragments).
  • a cell line is established from human thyroid cancer, an antigen peptide is extracted from the cell membrane component, and a polyclonal antibody or monoclonal antibody against the antigen peptide is prepared. Then, a monoclonal antibody that specifically reacts with thyroid cancer cells is selected. Next, the site where the tumor antigen recognized by the polyclonal antibody or the monoclonal antibody is present in the tumor cell is confirmed, and its antigenic determinant (epitope) is determined. The cell-killing effect of the polyclonal or monoclonal antibodies on tumors is confirmed, and peptides of epitope are chemically synthesized and used as cancer peptide peptides (tumor diagnostic, prophylactic or therapeutic agents).
  • leukocytes that specifically react with the above peptide or leukocytes that bind to an antibody (particularly, Fc portion) to the above peptide can also be used in the present invention.
  • a cell membrane component to be used as an immunogen (antigen) is prepared.
  • Preparation of the cell membrane component can be obtained as follows.
  • various cancer tissues or cancer cells or established cell lines can be used as a raw material.
  • a cancer tissue or cancer cell for example, a cancer cell or cancer cell extracted from a cancer patient such as thyroid cancer is crushed to obtain a cell suspension.
  • the established cell line is used as a raw material, a cell line established in advance is cultured in a large amount, and this is suspended in an appropriate buffer to form a cell suspension.
  • established cell lines include thyroid cancer cells such as TPC-1, TPC-2, and TPC-3.
  • these cell suspensions are subjected to an extraction treatment according to a known extraction method (Kurata Y and Okada S .: Immunol ogical studies of inso lub le l ipoprote ins. Int. Arch. Allergy, 29 : 495-509, 1966.).
  • a known extraction method Korean Y and Okada S .: Immunol ogical studies of inso lub le l ipoprote ins. Int. Arch. Allergy, 29 : 495-509, 1966.
  • the insoluble fraction is suspended in an appropriate buffer and centrifuged.
  • the centrifugation operation is repeated several times, and the obtained precipitate is suspended in sodium deoxyxycholate and stirred. Add cold acetone to the supernatant obtained by centrifugation and leave at -20 ° C overnight.
  • the precipitate obtained is centrifuged and dissolved in a small amount of buffer (10 mM PBS, etc.). Mix After removing the albumin, the mixture is concentrated and air-dried to prepare the ingredients. Next, the obtained membrane component is suspended in a suitable buffer (for example, containing 1M KC1, ImM monoacetic acid, 34 mM sodium citrate), and centrifuged to prepare an antigen peptide.
  • a suitable buffer for example, containing 1M KC1, ImM monoacetic acid, 34 mM sodium citrate
  • mice are immunized with the antigen prepared as described above.
  • the dose of the antigen per animal for mice is 0.1 to 10 mg when no adjuvant is used, and 1 to 100 zg when an adjuvant is used.
  • the adjuvant include carriers such as Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant.
  • Immunization is performed by administering to mammals (eg, rats, mice, egrets, humans, etc.).
  • the site of administration is intravenous, subcutaneous or intraperitoneal.
  • the interval of immunization is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 3 times at intervals of several days to several weeks, preferably at intervals of 2 to 3 weeks.
  • the antibody titer is measured 6 to 60 days after the last immunization, and blood is collected on the day when the maximum antibody titer is obtained to obtain an antiserum.
  • the antibody titer can be measured by an enzyme immunoassay (EUSA; enzyme-linked immunosorbent assay), a radioimmunoassay (RIA; radio immunoassay), or the like.
  • antibody purification from antiserum use known methods such as ammonium sulfate precipitation, ion exchange chromatography, gel filtration, and affinity chromatography, or use a combination of these methods. Can be purified.
  • an animal is immunized with the antigen peptide prepared as described above.
  • an adjuvant commercially available complete Freund's adjuvant, incomplete Freund's adjuvant, etc. may be mixed as described above for effective immunization.
  • Immunization is performed by administering to mammals (eg, rats, mice, and egrets).
  • mammals eg, rats, mice, and egrets.
  • the single dose of antigen is lmg per mouse.
  • the administration site is mainly intravenous, subcutaneous, or intraperitoneal.
  • the interval of immunity is particularly limited No, but at least 2-3 times at intervals of a few days to weeks, preferably 2-3 weeks.
  • the antibody-producing cells are collected.
  • Antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, and the like, with spleen cells being preferred.
  • a hybridoma for example, cell fusion between antibody-producing cells of a mammal such as a human and myeoma cells is performed.
  • myeloma cells to be fused with the antibody-producing cells generally available cell lines derived from animals such as human and mouse can be used.
  • a cell line to be used it has drug selectivity, cannot survive in HAT selection medium (including hypoxanthine, aminopterin and thymidine) in the unfused state, and can survive only in the state fused with antibody-producing cells.
  • HAT selection medium including hypoxanthine, aminopterin and thymidine
  • myeloid cell lines include mouse myeloid cell lines such as P3X63-Ag. 8. Ul (P3U1), P3 / NSI / l-Ag4-1, Sp2 / 0-Agl4.
  • the myeoma cells and the antibody-producing cells are subjected to cell fusion.
  • cell fusion antibody-producing cells and myeloma cells are mixed at a predetermined ratio (for example, 5: 1) in animal cell culture medium such as serum-free DMEM or RPMI-1640 medium, and then mixed with polyethylene glycol, etc.
  • the refusion reaction is carried out in the presence of a cell fusion promoter or by electric pulse treatment (for example, electroboration).
  • the target hybridoma is selected from the cells after the cell fusion treatment. For example, cells that are cultured in a medium containing hypoxanthine ( ⁇ ), aminobuterin (0.4 ⁇ ) and thymidine (16 ⁇ ) can be obtained, and the growing cells can be obtained as hybridomas. Next, it is screened whether the target antibody is present in the culture supernatant of the grown hybridoma. Screening of hybridomas may be performed according to a conventional method, and is not particularly limited. For example, a part of the culture supernatant contained in a well grown as a hybridoma is collected and subjected to enzyme immunoassay (ELISA; enzyme-linked immunosorbent assay), RIA (radioirarauno assay), etc. Can be cleaned.
  • ELISA enzyme immunoassay
  • RIA radio-linked immunosorbent assay
  • Cloning of the fused cells is performed by the limiting dilution method, etc. Establish a hybridoma, a somatic cell.
  • an ordinary cell culture method or the like can be employed as a method for collecting a monoclonal antibody from the established hybridoma.
  • Hybri-doma KTC-3 (name: hybri doma KTC-3), which produces mouse monoclonal antibodies, has been sent to FERM BP by the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture). -6899 as an international deposit under the Budapest Treaty (Original date of deposit: September 30, 1998).
  • the tumor-specific antigen peptide of the present invention has an amino acid sequence of Phe Ser Ala Thr Asp Pro Lys, It corresponds to amino acids 33-39 of the sequence (SEQ ID NO: 2) (referred to as “M1”).
  • Such amino acid sequences include, for example, those containing the 32nd to 40th amino acids of the amino acid sequence represented by SEQ ID NO: 2 (referred to as “Ala9”) and the 31st to 40th amino acids in addition to Ml described above.
  • Gly20 which includes the 30th to 49th amino acids (referred to as "Gly20").
  • Ml Phe Ser Ala Thr Asp Pro Lys (SEQ ID NO: 3)
  • Gly20 Ala Glu Pro Phe Ser Ala Thr Asp Pro Lys Ala He Gly His Arg Asn
  • RNA is prepared from tumor cells to obtain a protein partially containing these peptides (also referred to as “tumor antigen protein”) and DNA encoding the protein. Do.
  • Preparation of tumor antigen protein mRNA can be performed by a commonly used technique. For example, thyroid cancer cells and the like are treated with a guanidine reagent, a phenol reagent, and the like to obtain total RNA, and then the affinity column method using poly-U-sepharose using oligo dT-cellulose / sepharose 2B as a carrier, Alternatively, poly (A +) RNA (mRNA) is obtained by a batch method. Using the obtained mRNA as a gun, a single-stranded cDNA was synthesized using oligo dT primers and reverse transcriptase, and then double-stranded from the single-stranded cDNA. Synthesize cDNA. A cDNA library can be obtained by incorporating the thus obtained double-stranded cDNA into an appropriate expression vector (for example, gUl) to prepare a recombinant vector.
  • an appropriate expression vector for example, gUl
  • a method using a monoclonal antibody can be mentioned.
  • RNA encoding a tumor antigen protein can be obtained by incorporating the desired DNA into a plasmid vector, purifying the plasmid, and then using an in vitro transcription system.
  • a mutation can be introduced into at least a part (excluding the 33rd to 39th positions) of the amino acid sequence (SEQ ID NO: 2) of the tumor antigen protein. Therefore, the mutant is also included in the protein of the present invention as long as it exhibits tumor-specific antigenicity.
  • a method of introducing a mutation into the nucleotide sequence of a gene encoding the amino acid is employed.
  • Mutations can be introduced into a gene by a known method such as the Kunkel method or the Gapped duplex method.
  • a mutation is introduced based on a site-directed mutagenesis method using a mutant oligonucleotide as a primer.
  • Mutagenesis kits for example, Mutant-K (TAKARA), Mutant-G (TAKARA) ), And TAKARA's LA PCR in vitro Mutagenes is series kit).
  • the nucleotide sequence of the gene obtained as described in (1) or (2) above is determined.
  • the determination of the base sequence can be performed by a known method such as the Maxam-Gilbert chemical modification method or the dideoxynucleotide chain termination method using DNA polymerase.
  • SEQ ID NO: 1 exemplifies the nucleotide sequence of the DNA encoding the tumor antigen protein of the present invention
  • SEQ ID NO: 2 exemplifies the amino acid sequence of the protein
  • SEQ ID NO: 7 shows the nucleotide sequence of the RNA encoding the tumor antigen protein of the present invention
  • An example of an array is shown.
  • a peptide having the 33rd to 39th sequence of a protein consisting of this amino acid sequence is As long as it is expressed, one or more amino acids in the amino acid sequence may be mutated such as deletion, substitution or addition.
  • At least one, preferably about 1 to 10, and more preferably 1 to 5 amino acids of the amino acid sequence represented by SEQ ID NO: 2 may be deleted, and the amino acid sequence represented by SEQ ID NO: 2 At least one, preferably about 1 to 10, more preferably 1 to 5 amino acids may be added to the amino acid sequence, or at least one, preferably 1 amino acid sequence represented by SEQ ID NO: 2 Up to about 10, more preferably 1 to 5 amino acids may be replaced by other amino acids.
  • DNA or RNA encoding a protein containing a peptide that hybridizes with the above DNA (SEQ ID NO: 1) or RNA (SEQ ID NO: 7) under stringent conditions and is specifically expressed in a tumor is also included in the present invention. Included in DNA or RNA.
  • Stringent conditions are those where the sodium concentration is 0.1 X SSC and the temperature is 50 ° C.
  • the DNA of the present invention was introduced into Escherichia coli K12 (name: pRI T2T-625), and the FERM BP-6900 was sent to the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki Prefecture). (Deposit date: September 30, 1998).
  • the recombinant vector of the present invention can be obtained by ligating (inserting) a gene (DNA or RNA) encoding the tumor antigen protein of the present invention into an appropriate vector.
  • the vector for inserting the gene of the present invention is not particularly limited as long as it can be replicated in a host, and examples thereof include plasmid DNA and phage DNA.
  • Plasmid DNA includes Escherichia coli-derived plasmids (for example, pBR322, pBR325, pUC118, pUC119, pBluescript II SK +/-, pGEM4, pSP64, pSP65, etc.), and Bacillus subtilis-derived plasmid.
  • yeast-derived plasmid eg, YEpl3, YEp24, YCp50
  • phage DNA include fly phage (LgtlO, ⁇ gtll, ⁇ 13 ⁇ 18, M13mpl9, etc.).
  • animal viruses such as retrovirus, adenovirus or vaccinia virus, or insect virus vectors such as baculovirus can be used.
  • a fusion plasmid in which GST, GFP, His-tag, Myc-tag, etc. are linked can also be used.
  • the purified DNA is cleaved with an appropriate restriction enzyme, inserted into an appropriate vector DNA at a restriction enzyme site or a multicloning site, and ligated to a vector. Is adopted.
  • the gene of the present invention needs to be incorporated into a vector so that the function of the gene is exhibited. Therefore, in addition to the promoter and the gene of the present invention, a vector containing a cis element such as a re-enhancer, a splicing signal, a poly-A addition signal, a selection marker, a ribosome binding sequence, and the like are linked to the vector of the present invention. can do.
  • a selectable marker include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, and the like.
  • the transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the target gene can be expressed.
  • the host is not particularly limited as long as it can express the DNA of the present invention.
  • the genus Escherichia such as Escherichia coli
  • the genus Bacillus such as Bacillus subtilis
  • the strain Pseudomonas putida the strain Pseudomonas putida
  • Pseudoraonas putida and other bacteria belonging to the genus Pseudomonas, such as Saccharomyces cerevisiaej (Saccharomyces cerevisiaej, Schizosaccharomyces bomb).
  • the recombinant vector of the present invention is capable of autonomous replication in the bacterium and, at the same time, contains a promoter, a ribosome binding sequence, and a gene of the present invention. It is preferably composed of a transcription termination sequence. In addition, a gene that controls the promoter may be included.
  • Escherichia coli examples include Escherichia coli K12 and DH1
  • Bacillus subtilis examples include Bacillus subtilis MI114 and 207-21.
  • Any promoter can be used as long as it can be expressed in a host such as Escherichia coli.
  • promoters derived from Escherichia coli or phage such as trp promoter, lac promoter, P L promoter, and P R promoter, are used.
  • An artificially designed and modified promoter such as a tac promoter may be used.
  • the method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria.
  • a method using calcium ions Cohen, SN et al .: Pro Natl. Acad. Sci., USA, 69: 2110-2114 (1972)
  • an electroporation method Becker, DM et al .: Methods Enzymol., 194: 182-187 (1990)).
  • yeast When yeast is used as a host, for example, Saccharomyces cerevisiae and Schizosaccharomyces pombe are used.
  • the promoter is not particularly limited as long as it can be expressed in yeast.For example, gall promoter, gallO promoter, heat shock protein promoter, MFal promoter, PH05 promoter, PGK mouth motor, GAP Promoter, ADH promoter, A0X1 promoter and the like.
  • the method for introducing the recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast.
  • the elect-portion method and the spheroplast method Hinnen, A. et al .: Pro Natl. Acad. Sci., USA, 75: 1929-1933 (1978)
  • a lithium acetate method Itoh, H .: J. Bacteriol., 153: 163-168 (1983)
  • COS-7 When animal cells are used as hosts, COS-7, Vero, Chinese hamster ovary cells (CH0 cells), mouse myeloma cells, etc. are used.
  • the promoter used is SRa promoter, SV40 promoter, LTR promoter, etc.
  • An early gene promoter of the cytomegalovirus may be used.
  • Examples of a method for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.
  • a method of introducing the recombinant vector into insect cells for example, a calcium phosphate method, a lipofection method, an electoporation method, or the like is used.
  • the protein (tumor antigen protein) containing the peptide of the present invention can be produced by culturing the transformant prepared in the above 4 in vivo or in vitro and collecting from the culture. .
  • culture means any of a culture supernatant, a cultured cell, a cultured cell, or a cell or cell fragment.
  • the method for culturing the transformant of the present invention is performed according to a usual method used for culturing a host.
  • the medium for culturing the transformants obtained using microorganisms such as Escherichia coli and yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like, which can be used by the microorganisms, so that the cultivation of the transformants is efficient.
  • a natural medium or a synthetic medium may be used as long as the medium can be performed in a controlled manner.
  • Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose, and starch; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol.
  • nitrogen source examples include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, and other nitrogen-containing compounds, peptone, meat extract, and corn steep liquor. No.
  • the inorganic substances include potassium phosphate monobasic, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. Cultivation is usually performed at 37 ° C for 6 to 24 hours under aerobic conditions such as shaking culture or aeration and stirring culture. During the culture period, what is the PH? Keep at ⁇ 7.5. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like.
  • an antibiotic such as ampicillin-tetracycline may be added to the medium as needed.
  • an Indian user may be added to the medium as necessary.
  • an Indian user may be added to the medium as necessary.
  • isopropyl-9-D-thiogalactovyranoside (IPTG) or the like was transformed with an expression vector using a trp mouth motor.
  • IPTG isopropyl-9-D-thiogalactovyranoside
  • IAA indole acrylic acid
  • Examples of a medium for culturing a transformant obtained using animal cells as a host include commonly used RPMI-1640 medium, DMEM medium, or a medium obtained by adding fetal calf serum or the like to such a medium.
  • the cultivation is carried out 5% C0 2 presence, 1-30 days at 37 ° C.
  • antibiotics such as kanamycin and benicillin may be added to the medium as needed.
  • the cells are extracted by disrupting the cells or cells.
  • the culture solution is used as it is, or the bacterial cells or cells are removed by centrifugation or the like. Thereafter, common biochemical methods used for the isolation and purification of proteins, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., can be used alone or in appropriate combination.
  • the tumor antigen protein can be isolated and purified from the culture.
  • the peptide of the present invention has the sequence described in 2. (1) above, and can be obtained by ordinary peptide chemical synthesis.
  • amino acid sequence SEQ ID NOs: 3 to 6
  • one or several preferably Even if a mutation such as deletion, substitution or addition occurs, it is included in the scope of the present invention as long as the peptide having the mutation is cancer cell-specific.
  • the peptide of the present invention also includes its salts.
  • the peptide can be synthesized by a conventional method of synthesizing the peptide.
  • a conventional method of synthesizing the peptide For example, an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC method, an active ester method, a carboimidazole method, an oxidation-reduction method and the like can be mentioned.
  • both the solid phase synthesis method and the liquid phase synthesis method can be applied.
  • the amino acid capable of constituting the peptide of the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to synthesize the desired peptide.
  • the condensation method and the elimination of the protecting group may be performed by any known method [for example, Bodanszky, M and MA Ondett i, Pept i de Synthes is, Interscience Pub lishers, New York ( 1966), Schroeder and Luebke, The Peptide, Academic Press, New York (1965), Nobuo Izumiya et al., Basics and Experiments on Peptide Synthesis, Maruzen (1975), etc.].
  • the peptide of the present invention can be purified by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization.
  • the salt of the peptide of the present invention is preferably a physiologically acceptable acid addition salt or basic salt.
  • the acid addition salts include salts with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, and sulfuric acid, or acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, and citric acid.
  • salts with organic acids such as malic acid, oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid.
  • the basic salt include salts with inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide and magnesium hydroxide, and salts with organic bases such as caffeine, piperidine, trimethylamine and pyridine. Salts.
  • Salts can be prepared using a suitable acid such as hydrochloric acid, or a suitable base such as sodium hydroxide.
  • a suitable acid such as hydrochloric acid
  • a suitable base such as sodium hydroxide.
  • the standard protocol It can be prepared by treating with a col.
  • the treatment temperature is from 0 to 100 ° C, preferably room temperature.
  • the peptides of the present invention C-terminal, usually carboxyl (- C00H) is a group or carboxy Kishireto (-C00-), C-terminal amino-de (- C0NH 2) or an ester (- C00R) Derconnection also Good.
  • R in the ester includes an alkyl group having 1 to 12 carbon atoms, a cycloalkyl group having 3 to 10 carbon atoms, an aryl group having 6 to 12 carbon atoms, and an aralkyl group having 7 to 12 carbon atoms.
  • the peptide of the present invention includes those in which the N-terminal alanine residue of the alanine residue is protected with a protecting group, and complex peptides such as glycopeptides to which sugar chains are bound.
  • biochemical and physicochemical properties of the peptide of the present invention can be analyzed by mass spectrometry, nuclear magnetic resonance, electrophoresis, high performance liquid chromatography and the like.
  • leukocyte means a blood cell component collected from a host or a tumor-bearing host inoculated with the peptide as a vaccine, and includes lymphocytes (T lymphocytes, B lymphocytes), dendritic cells It consists of neutrophils, eosinophils, basophils, and monocytes.
  • the leukocytes react with the peptide of the present invention (referred to as peptide-reactive leukocytes) and can bind to an antibody (preferably the Fc portion) against the peptide of the present invention (antibody reaction).
  • Leukocytes or antibody-binding leukocytes Further, those belonging to these subgroups of leukocytes are also included in the leukocytes of the present invention. In the present invention, among these cell components, lymphocytes, monocytes, granulocytes and dendritic cells are preferred.
  • Peptide-reactive leukocyte refers to a cell that presents all the amino acids or a part of the antigenic phenotype (epitope) of the peptide to the major histocompatibility complex (MHC) on the cell surface.
  • MHC major histocompatibility complex
  • leukocyte is meant a leukocyte capable of binding to an antibody via a receptor for the antibody (located on the surface of the leukocyte).
  • the leukocyte of the present invention can be obtained by the following treatment.
  • lymphocytes blood, spleen, lymph nodes, etc. of humans or animals (mouse, rat, guinea pig, hamster, mouse, cat, dog, pig, monkey, etc.) It can be the source. It is prepared from peripheral blood lymphocytes (PBL), lymph node cells, tumor infiltrating lymphocytes (TIL), tumor local lymph node cells, etc. contained in these tissues or organs. That is, blood collected from a human or animal, or spleen or lymph node collected by laparotomy of an animal is suspended in an appropriate cell culture medium.
  • PBL peripheral blood lymphocytes
  • TIL tumor infiltrating lymphocytes
  • Cell culture media include basic media commonly used for culturing animal cells, such as minimal basic media (MEM), RPMI-1640 medium, Ham F12 medium, Dulbecco's modified Eagle's medium (DMEM), etc. .
  • the cell culture solution preferably contains fetal calf serum (FCS) and the like.
  • a lymphocyte fraction is obtained by a normal lymphocyte separation method, for example, Ficoll density gradient centrifugation, Percoll density gradient centrifugation, or the like.
  • a normal lymphocyte separation method for example, Ficoll density gradient centrifugation, Percoll density gradient centrifugation, or the like.
  • these tissues or organs are minced with scissors, and the spleen is removed as it is, and various organs and tissues are enzymes such as collagenase and DNase.
  • a chelating agent such as EDTA
  • each is placed on a metal mesh and gently pressed from above to allow the internal lymphocytes to migrate out of the tissue.
  • the cells that have passed through the metal mesh are gently pipetted, allowed to stand for a while, and then the supernatant is obtained.
  • Free lymphocytes are obtained by removing the tissue fragments and cell aggregates in the sediment.
  • the above-mentioned lymphocyte suspension is cultured in a Petri dish for 1 hour to obtain a cell group attached to the Petri dish wall. After removing the non-adherent cells, collect the adherent cells by adding a chelating agent.
  • the population of cells obtained in this way contains dendritic cells, monocytes (macrophages) and neutrophils in proportions of about 10%, about 40% and about 40%, respectively. These three types of cells coexist with magnetic particles to engulf monocytes and neutrophils. Magnetic particles Collect phagocytic cells in a magnetic field and remove them to obtain dendritic cells.
  • monocytes are obtained from the above phagocytic cell group using an anti-MHC class II antibody or an anti-CD11b antibody.
  • Neutrophils are obtained by the action of an anti-CD157 antibody.
  • the above-mentioned non-adherent cell group is obtained by reacting an anti-CD116 antibody or an anti-CDW125 antibody or an anti-CDW128 antibody.
  • the subgroup of leukocytes includes granulocytes, lymphocytes and monocytes. These subgroups are morphologically distinguished to obtain content. Also, to separate each group, It is prepared by utilizing the difference in adhesion and surface structure.
  • the confirmation that the leukocytes obtained as described above have a function of binding to the peptide of the present invention is performed by confirming the action of enhancing nucleic acid synthesis ability (antigenesis phenomenon) after antigen binding. Confirmation of the ability to bind to the antibody of the present invention (particularly the Fc portion) is performed by using an antibody-dependent biological activity test such as enhancement of phagocytosis in phagocytic cells (opsonin effect). .
  • the pharmaceutical composition containing the peptide, the antibody or the leukocyte as an active ingredient and the preventive, diagnostic or therapeutic agent for tumor of the present invention may contain a pharmaceutically acceptable carrier or additive.
  • a pharmaceutically acceptable carrier or additive examples include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, Pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, acceptable as a pharmaceutical additive And artificial cell structures such as ribosomes.
  • the additives to be used are appropriately or in combination selected from the above according to the dosage form of the present invention.
  • the target for use is not particularly limited.
  • diagnosis, treatment or prevention of at least one kind of tumor such as cancer, sarcoma, and benign tumor can be used as a specific purpose. Regardless of whether these diseases are used alone, in combination, or in combination with other diseases other than those listed above, use of the peptide, antibody or leukocyte of the present invention is a subject of the use. Can be.
  • these cancer types are not particularly limited, and include, for example, thyroid cancer, breast cancer, Gastric cancer, esophageal cancer, oral cancer, colon cancer, knee cancer, lung cancer, kidney cancer, bladder cancer, ovarian cancer, uterine cancer, vulvar cancer, skin cancer, melanoma, central or peripheral nerve tumor (meningioma, nerve Glial tumors, acoustic nerve tumors, including neurofibrosarcoma, brain and spinal cord 'peripheral nerve tumors', gingival cancer, pharyngeal cancer, jaw cancer (including maxillary cancer; squamous cell carcinoma), mediastinal tumor (including thymic cancer), Liver cancer, cholangiocarcinoma, gallbladder cancer, renal pelvic tumor, ureteral cancer, testicular tumor, prostate cancer, choriocarcinoma, fallopian tube cancer, vaginal
  • the diagnostic, prophylactic or therapeutic agent containing the peptide or antibody of the present invention can be administered orally or parenterally for a peptide and parenterally for an antibody.
  • a solid preparation such as tablets, granules, powders, and pills to be applied thereto, or a liquid preparation such as a liquid preparation or a syrup
  • granules and powders can be made into unit dosage forms as capsules, or in the case of liquid preparations, they can be dried products to be re-dissolved when used.
  • oral solid preparations usually contain additives, such as binders, excipients, lubricants, disintegrants, and wetting agents, which are generally used in pharmaceutical compositions.
  • Oral liquid preparations usually contain additives, such as stabilizers, buffers, flavoring agents, preservatives, fragrances, and coloring agents, which are generally used in preparations, in their compositions.
  • peptide, antibody or the peptide-reactive leukocyte of the present invention When the peptide, antibody or the peptide-reactive leukocyte of the present invention is administered parenterally, injections, suppositories and the like may be used.
  • injections For injections, they will usually be presented in unit dose ampoules or in multi-dose containers, which may be reconstituted with a suitable carrier for use, for example, sterile pyrogen-free water. These dosage forms usually contain additives, such as emulsifiers and suspending agents, which are generally used in pharmaceutical compositions.
  • Injection techniques include, for example, intravenous infusion, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and intradermal injection.
  • the dose varies depending on the age of the subject, the route of administration, and the number of administrations, and can be widely varied.
  • an effective amount of the peptide of the present invention and a suitable diluent and pharmacologically usable is 4 g to 20 ⁇ g / kg body weight at a time and is administered at intervals of one to four weeks.
  • the effective amount of the antibody is 100 g to 100 rng / kg body weight at a time, and it is administered every 1 to 4 weeks.
  • parenteral administration such as intravenous injection, intratumoral injection, subcutaneous injection, intraperitoneal administration and the like can be adopted as the administration method.
  • the dosage varies depending on the age, sex, administration route, administration frequency and the like of the administration subject, and can be varied over a wide range.
  • the effective amount (the number of effective leukocytes) to be administered as a combination of the effective amount of the leukocyte of the present invention and an appropriate diluent and a pharmacologically usable carrier is 1 ⁇ 10 3 to 1 ⁇ 10 9 / cell. kg body weight / day, given once to several times a day.
  • the leukocyte of the present invention When the leukocyte of the present invention is administered parenterally, additives such as a stabilizer, a buffer, a preservative, and an isotonicity agent can be contained, and the leukocyte is prepared as needed.
  • the patient after collecting blood from the patient and screening for the target leukocyte, the patient may be immediately returned to the patient.
  • the degree of leukocyte activation Is preferably increased.
  • CTL cytotoxic lymphocytes
  • the CTL ⁇ dendritic cells are separated and identified by a flow cytometer using a complex of the peptide and a fluorescent dye bound from the blood to identify the tumor. Can be determined.
  • coexistence of the peptide or peptide-presenting cells with leukocytes in the blood can be used to test for an increase in nucleic acid synthesis to indicate the presence of a tumor.
  • the peptide of the present invention can be administered orally, it can also be used as a functional food.
  • the peptide of the present invention can be administered as it is or as it is contained in an enteric capsule.
  • dissolve in a liquid (preferably water) to an appropriate concentration and add it to food by mixing, dipping, coating, spraying, etc. obtain.
  • the present peptide can be used as a functional food such as meat, fish, and vegetables.
  • the amount can be 0.001 mg to 1 mg, preferably 0.01 to 0.5 mg.
  • a tumor cell-specific antigen by reacting a purified antibody with a measurement target (a body fluid such as blood, saliva, tears, or a tumor tissue) and use it for cancer diagnosis. it can.
  • a measurement target a body fluid such as blood, saliva, tears, or a tumor tissue
  • Detection of the tumor antigen contained in the measurement object can be performed by ELISA.
  • a cancer antigen in a measurement object is adsorbed to each well of a 96-well ELISA plate. After blocking with 1% BSA-PBS, the antibody of the present invention (polyclonal antibody or monoclonal antibody KTC-3) is reacted.
  • a biotinylated goat anti-mouse Ig is reacted. After washing with PBS, the complex of biotinylated peroxidase and avidin was reacted (ABC method), and diaminobenzidine (0.5 mg / ml in 0.05M Tris-HCl pH7.5) and 3.1% peroxide were added. hydrogen (H 2 0 2) was added, allowed to develop. Then, the absorbance is measured at 4 90 nm in ELISA Li one da scratch.
  • the detection of tumor antigens can also be carried out by using the ECL Electronosimetry (Electrochemi luminescence I band unoassay) method (Yusaburo Namba, Toshihito Kanashima; Electrochemical Imnoassay, clinical test: 293-300, 1998).
  • ECL Electronosimetry Electronosimetry (Electrochemi luminescence I band unoassay) method (Yusaburo Namba, Toshihito Kanashima; Electrochemical Imnoassay, clinical test: 293-300, 1998).
  • a tumor cell-specific antibody it is possible to detect a tumor cell-specific antibody by reacting a purified antigen peptide with a measurement target (a body fluid such as blood, saliva, tears or a tumor tissue), and use the antibody for diagnosis of cancer. it can.
  • a measurement target a body fluid such as blood, saliva, tears or a tumor tissue
  • Detection of the tumor-specific antibody contained in the measurement target can be performed by ELISA.
  • each well of an ELISA 96-well plate is immobilized with the peptide of the present invention (Ml, AlalO, Map 10, etc.), blocked with 1% BSA-PBS, and the measurement target is reacted. After washing with PBS, react with the biotinylated goat anti-mouse lg.
  • Piotin The complex of peroxidase hydrochloride and avidin is reacted (ABC method), and diaminobenzidine (0.5 mg / ml in 0.05 M Tris-HCl pH 7.5) and 3.1% hydrogen peroxide (H 2 0 2) was added, allowed to develop. Then, measure the absorbance at 490 mn with an ELISA reader.
  • the detection of a tumor-specific antibody can also be performed using the ECL immunoassay (Electrochemiluminescence Immunoassay) method.
  • an antitumor agent or a radioisotope is bound to a tumor-specific antibody, so that cancer can be treated by so-called missile therapy or cancer can be detected.
  • the antibody When an antibody to which a radioisotope is bound is administered to a human or animal, the antibody aggregates at the cancer lesion, and the location of the cancer lesion can be known by detecting the radioisotope. This detection can be performed by photographing with a scintillation counter or a scintillation camera.
  • an antitumor agent or a radioisotope capable of destroying cancer cells or cancer tissue is bound to the antibody and administered to the body.
  • radioisotope examples include i3i I and the like.
  • examples of the antitumor agent bound to the antibody of the present invention include alkylating agents such as cyclophosphamide, busulfan, and nitrogen mustard; antimetabolites such as methotrexate, 5-FU, and Ara-C; and actinomycin D. And antibiotics such as adriamycin and MMC; plant alkaloids such as vinblastine, vincristine and VP-16; hormonal drugs such as prednisolone, estrogen, androgen, and progesterone; and other cisplatin.
  • 131 1 is bound to an antibody, the antibody accumulates in cancer lesions and 13
  • FIG. 1 is a photograph showing fluorescent antibody staining of a normal thyroid cell culture (NTC-1) with the KTC-3 antibody.
  • FIG. 2 is a photograph showing fluorescent antibody staining of a human papillary thyroid cancer cell line (TPC-1) with the KTC-3 antibody.
  • FIG. 3 is a photograph showing immunohistochemical staining of human papillary thyroid carcinoma using the KTC-3 antibody.
  • FIG. 4 is a photograph showing immunohistochemical staining of undifferentiated human thyroid cancer using the KTC-3 antibody.
  • FIG. 5 is a photograph showing the localization of tumor antigens in human papillary thyroid cancer cell line (TPC-1).
  • FIG. 6 is a photograph showing the cell killing effect of the KTC-3 antibody on a human papillary thyroid cancer cell line (TPC-1).
  • FIG. 7 shows the results of separation of the KTC-3 antibody using a Protein A Sepharose column.
  • FIG. 8 is a photograph showing the cell killing effect of the Fab portion of the KTC-3 antibody on the human papillary thyroid cancer cell line (TPC-1) (after 24 hours of culture).
  • FIG. 9 is a photograph showing the cell killing effect (after 72 hours of culture) of the Fab portion of the KTC-3 antibody on the human papillary thyroid cancer cell line (TPC-1).
  • FIG. 10 shows the results of analysis of the amino acid sequence of the S-1 tumor antigen by the N14 profile method (hydrophycity plot).
  • FIG. 11 is a photograph showing the reactivity of the KTC-3 antibody with the peptide of the present invention.
  • FIG. 12 is a photograph showing the reactivity of the KTC_3 antibody with the peptide of the present invention.
  • FIG. 13 is a photograph showing the expression results of S-1 antigen in cancer tissues and normal tissues.
  • FIG. 14 is a photograph showing the expression result of S-1 antigen in cancer tissue and normal tissue.
  • FIG. 15 is a photograph showing the result of expressing the S-1 antigen in a cancer tissue.
  • FIG. 16 is a photograph showing a result of detecting a cancer by scintigram.
  • This cell line was cultivated in large quantities, and a modification of the method of Smith et al. (Smith JT, et al., Exp. Cell Res. 13: 96-102, 1957.) for the purpose of extracting the protein portion of the cell membrane.
  • antigens were prepared as follows. That is, 5 ⁇ 10 7 cells obtained by culturing at 37 ° C.
  • the resulting precipitate was centrifuged and dissolved in a small amount of 10 mM PBS (phosphate buffer). This was removed with Blue Sepharose CL-6B (Pharmacia) to remove mixed albumin. The obtained eluted fraction was concentrated and air-dried.
  • the preparation of the hybridoma was performed according to a known method. That is, the extract obtained from the above membrane components was suspended in complete adjuvant, and injected five times subcutaneously into Balb / c mice every two weeks. Two days after the final immunization, cells were separated from the spleen, and lymphocytes were removed by separating blood cells using a centrifuge.
  • normal thyroid cells NTC-1, human fetal kidney cells HEK-1 and thyroid cancer cells TPC-1 are spread on a vinyl plate at a rate of 5 to 5 ⁇ 10, and poly-L-lysin (MW: 520,000) is used. hand 4. Attach in 2 hours. The cells were then fixed using 0.25% dartaraldehyde for 15 minutes at room temperature. Blocking was performed at 37 ° C. for 2 hours in the presence of 1% BSA-PBS-0.25% NaN 3 . Next, the supernatant of each of the hybridomas was added and reacted at 37 ° C for 1 hour, and peroxidase-linked anti-mouse Ig was allowed to act at 37 ° C for 1 hour.
  • KTC-3 antibody a hybridoma producing the monoclonal antibody of the present invention was obtained and named KTC-3.
  • KTC-3 antibody the antibody produced by hybridoma KTC-3 is referred to as “KTC-3 antibody”.
  • Hybridoma KTC-3 (named hybridoma KTC-3), which produces KTC-3 antibody, was sent to FERM BP-I by the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki Prefecture). It has been deposited internationally under the Budapest Treaty as 6899 (Original deposit date: September 30, 1998).
  • TPC-2 Human papillary thyroid cancer +
  • TUC-1 Human anaplastic thyroid cancer
  • KTC-3 antibody was reacted with TPC-1 and observed by immunoelectron microscopy.
  • KTC-3 antibody as primary antibody, gold colloid-labeled anti-mouse antibody Globulin antibodies were used.
  • cultured TPC-1 cells were treated according to the following procedure.
  • Dispersion Disperse cells in TBS containing 0.5% BSA
  • Gold colloid Gold colloid-labeled anti-mouse goat noglin purine antibody, rinsed three times with TBS at room temperature for 30 minutes
  • the tumor antigen extracted from the TPC-1 cell membrane was subjected to various chemical pretreatments on an ELISA plate, and then reacted with the KTC-3 antibody, and the color development was measured (Table 2).
  • the color of tumor antigen was significantly reduced by treatment with proteolytic enzymes, trypsin and pronase E, compared to that of untreated (none). However, treatment with sugar was unaffected. This indicates that this antigen is a protein.
  • the effect of the KTC-3 antibody on the human thyroid cancer cell line TPC-1 was examined.
  • the KTC-3 antibody was added to the TPC-1 cell culture system, and TPC-1 cells (derived from human thyroid cancer) were observed 72 hours later (FIG. 6).
  • thyroid cancer cells TPC-1 and hybridoma KTC-3 were each cultured. RPMI1640-10% fetal bovine serum was used for both culture solutions. Twenty-four hours after the culture, the culture solution of TPC-1 was replaced with the culture supernatant of the hybridoma. Observation was started from this point. As a result, 72 hours later, all cancer cells had died with trypan blue staining.
  • the KTC-3 antibody was separated into a Fab portion and an Fc portion by papain treatment, and the Fab portion was recovered using a Protein A Sepharose column as follows (FIG. 7).
  • Hypridoma KTC-3 was collected and saturated ammonium sulfate was added to make it 45% saturated, and the protein was precipitated. This was collected in a dialysis tube and desulfurized with a large amount of water.
  • gel filtration was performed with Sepharose CL-6B to obtain the antibody KTC-3 at the first peak.
  • affinity chromatography using protein A went. That is, using IgG Sepharose 6 Fast Flow (Pharmacia), elute the Fab part with 50raM Tris buffer, pH7.6, 150mM NaCl, and then Fc part with 0.5M acetic acid pH3.4 (adjusted with ammonium acetate). Was eluted.
  • a culture solution (RPMI1640-10% fetal bovine serum) 9 was prepared for Fab portion 1 obtained by affinity chromatography. This was replaced with a culture of TPC-1 cells (RPMI1640-10% fetal bovine serum) that had been cultured in advance. After 72 hours, the presence or absence of trypan blue staining positive cells (dead cells) was observed.
  • the KTC-3 antibody reacted specifically with cells derived from thyroid, stomach, colon and kidney cancers.
  • the tumor antigen recognized by the KTC-3 antibody was present on the cancer cell membrane.
  • the tumor antigen recognized by the KTC-3 antibody was not sugar but protein.
  • KTC-3 antibody worked cytocidal against thyroid cancer. A similar effect was seen with the Fab portion alone.
  • a cDNA library of the thyroid cancer cell line TPC-1 was prepared.
  • a double-stranded cDNA was synthesized using Amersham's cDNA synthesis system Brass, using the poly (A) + RNA 5 ⁇ g as a type II.
  • a cDNA library was prepared by the Adabuter method, and GIGAPACK II of Stratagene was used. Packaged using Gold. The reaction conditions followed the protocols of Amersham and Stratagene.
  • thyroid cancer tumor antigen protein As a result, a partial fragment of a gene encoding a thyroid cancer tumor antigen protein was obtained, and its sequence was represented by SEQ ID NO: 1.
  • the amino acid sequence of a protein (tumor antigen protein) encoded by the base sequence represented by SEQ ID NO: 1 is shown in SEQ ID NO: 2.
  • the thyroid cancer tumor antigen protein was named S-1 tumor antigen.
  • S-1 tumor antigen a tumor antigen protein of thyroid cancer
  • ATP6F proteolipid subunit
  • vacuolar H + -ATPase proton pump
  • the amino acid residues 32 to 40 were synthesized, and the amino acid residues 17 to 26 (lie Tyr Gly He He Met Ala lie Val lie) were used as a control to examine the reactivity with the KTC-3 antibody. Reactivity was measured in the Example except that filter paper was used instead of ELISA plate. The test was performed in the same manner as in the isci warfare L. As a result, the antibody reacted only with amino- 4 ⁇ 4 u (Fig. 11). Further, peptides were sequentially removed one by one from the N-terminal side and the C-terminal side, and the reaction with the KTC-3 antibody was examined.
  • RT-PCR was performed using the RT-PCR high kit from T0Y0B0 according to the instructions.
  • a PCR was performed using cDNA obtained by reverse transcription of 25 ⁇ g of total RNA extracted from various tumor tissues as type III.
  • the PCR was performed 33 times with the conditions of 94 ° C for 30 seconds, 56 ° C for 30 seconds and 72 ° C for 1 minute as one cycle.
  • the following primers were used as primers for the S-1 antigen.
  • Forward primer (F1 or F2 primer)
  • F1 primer (forward primer 1): 5'-CAAGAACCTGGTCAGCATCATC-3, (SEQ ID NO: 10) (corresponding to the 9th to 30th nucleotides in the nucleotide sequence described in SEQ ID NO: 1)
  • F2 primer 5'-TGTGAGGCTGTGGCCATCTA-3 '(SEQ ID NO:
  • R primer reverse primer 5 '-CCCCACCCACACATATCATC-3' (SEQ ID NO:
  • the size of the DNA fragment amplified by these primers is 374 bp when using the F1 primer and 349 bp when using the F2 primer.
  • a primer for the housekeeping gene (daricelaldehyde triphosphate dehydrogenase (GAPDH)) (included in the kit) was used.
  • the size of the DNA fragment amplified by these primers is 450 bp. It was confirmed that no band appeared when the reverse transcription reaction was not performed (-RT) (Fig. 14, lanes 4, 8, 13, and 17).
  • the size marker is lOObp ladder (Pharmacia).
  • thyroid cancer well differentiated adenocarcinoma, undifferentiated adenocarcinoma
  • breast cancer adenocarcinoma
  • stomach cancer adenocarcinoma
  • esophagus cancer adenocarcinoma, squamous cell carcinoma
  • oral cancer squamous cell carcinoma
  • Colorectal cancer adenocarcinoma
  • knee cancer adenocarcinoma
  • lung cancer adenocarcinoma, squamous cell carcinoma, small cell carcinoma
  • kidney cancer adenocarcinoma
  • bladder cancer transitional cell carcinoma
  • ovarian cancer Adenocarcinoma), endometrial cancer (adenocarcinoma), cervix cancer (adenocarcinoma, squamous cell carcinoma), vulvar cancer (squamous cell carcinoma), skin cancer (squamous cell carcinoma), melanoma, central or peripheral nerve tumor (Including meningioma, glio
  • SI antigen F2 / R normal ovarian tissue
  • SI antigen F2 / R ovarian cancer: adenocarcinoma
  • SI antigen F2 / R normal endometrial tissue
  • SI antigen F2 / R endometrial cancer: adenocarcinoma
  • SI antigen F2 / R Oral cancer (squamous cell carcinoma)
  • SI antigen F2 / R Esophageal cancer (squamous cell carcinoma)
  • SI antigen F2 / R Vulvar cancer (squamous cell carcinoma)
  • SI antigen F2 / R endometrial cancer (adenocarcinoma)
  • SI antigen F2 / R Lung cancer (adenocarcinoma)
  • SI antigen F2 / R Lung cancer (Squamous cell carcinoma)
  • SI antigen F2 / R Vulvar cancer (squamous cell carcinoma)
  • SI antigen F2 / R Breast cancer (adenocarcinoma) H. SI antigen F2 / R (-RT)
  • SI antigen F2 / R Ovarian cancer (adenocarcinoma)
  • SI antigen F2 / R Liver cancer (adenocarcinoma) (Case 1)
  • SI antigen F2 / R Liver cancer (adenocarcinoma) (Case 2)
  • SI antigen F2 / R Liver cancer (adenocarcinoma) (Case 3)
  • SI antigen F2 / R Liver cancer (adenocarcinoma) (Case 5)
  • a cDNA library of the thyroid cancer cell line TPC-1 was prepared, and a tumor antigen protein (S-1 tumor antigen) that reacts with the KTC-3 antibody was identified.
  • the amino acid sequence of the protein is as follows: Proteolipid Subunit (ATP6F) of vacuolar H + -ATPase (proton pump)
  • All S-1 tumor antigens that can be tested by the present inventors are thyroid cancer (highly differentiated adenocarcinoma, undifferentiated adenocarcinoma), breast cancer (adenocarcinoma), and gastric cancer (adenocarcinoma).
  • Esophageal cancer adenocarcinoma, squamous cell carcinoma
  • Oral cancer squamous cell carcinoma
  • Colorectal cancer adenocarcinoma
  • Tengler cancer adenocarcinoma
  • Adenocarcinoma squamous cell carcinoma, small cell carcinoma
  • kidney cancer adenocarcinoma
  • bladder cancer transitional cell carcinoma
  • ovarian cancer adenocarcinoma
  • endometrial cancer adenocarcinoma
  • cervical cancer gland
  • Cancer squamous cell carcinoma
  • vulvar carcinoma squamous cell carcinoma
  • skin cancer squamous cell carcinoma
  • melanoma central or peripheral nerve tumor (meningiomas, glioma, auditory nerve tumor, neurofibrosarcoma including brain and spinal cord) ⁇ Peripheral nerve tumor), gingival cancer (squamous cell carcinoma), pharyngeal cancer (squamous cell carcinoma), jaw cancer (including maxillary carcinoma; squamous cell carcinoma), mediastinal tumor (including thymic carcinoma), liver cancer (glandular) Cancer), bile duct cancer (adenocarcinoma), gallbladder cancer (adenocarcinoma),
  • S-1 tumor antigen was very minor or hardly detectable in normal tissues.
  • the present inventor synthesized various peptides containing the sequence of M-1 peptide by the Fastmoc method using a peptide synthesizer (Model 433A>) manufactured by Applied Biosystems Inc.
  • the synthesized peptides were AlalO (Glu Pro Phe Ser Ala Thr Asp Pro Lys Ala), MAP10 using MAP resin (AlalO was linked to MAP resin in eight branches), Gly20 (Ala Glu Pro Phe Ser Ala Thr Ala Thr). Asp Pro Lys Alylie Gly His Arg Asn Tyr His Ala Gly) and MAP20 (Gly20 is linked to MAP resin in four branches).
  • the present inventors have confirmed the antitumor activity and safety of these peptides from experiments of intradermal, subcutaneous, and oral (enteric microcapsules or enteric capsules) administration of the peptides of the present invention to mice.
  • MKN45 (1 ⁇ 10 7 ), a gastric cancer cell line, was transplanted into 4-week-old nude mice and bred for 1 week. In addition, four mice were used for each of the peptide administration group and the control group.
  • the peptide (AlalO) was subcutaneously administered to the peptide-administered group mice at a dose of 200 g / 100 g / week. This operation was performed three times at two-week intervals, and the survival status of the mice and the presence or absence of shrinkage of the tumor were observed.
  • the KTC-3 antibody of the present invention was labeled with 131 1 and administered to tumor-bearing mice, and the location of cancer was detected from the distribution of 131 1.
  • mice were administered 131 1 labeled KTC- 3 antibodies into mice. After transplantation, 131 1 was detected by a scintillation counter.
  • the antibodies of the present invention accumulated in cancer tissues (FIG. 16).
  • the upper small scintigram is for the thyroid gland, and the lower large scintigram is for the cancer tissue.
  • the present invention provides a tumor-specific antigen peptide, an antibody against the peptide, and a pharmaceutical composition containing the peptide or antibody. Since the peptides and antibodies of the present invention have an antitumor effect, they are useful for treating and preventing cancer, and are also useful for diagnosing cancer and the like.

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Abstract

L'invention concerne des peptides antigènes spécifiques aux tumeurs, tels que définis sous les points (A) et (B) ci-après. (A) Il s'agit de peptides antigènes spécifiques aux tumeurs comprenant la séquence d'acides aminés représentée par Phe Ser Ala Thr Asp Pro Lys. (B) Il s'agit de peptides antigènes spécifiques aux tumeurs comprenant au moins la séquence d'acides aminés représentée par Phe Ser Ala Thr Asp Pro Lys et assujettis à une expression spécifique aux tumeurs.
PCT/JP1999/005426 1998-10-05 1999-10-01 Peptides antigenes specifiques aux tumeurs WO2000020442A1 (fr)

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WO2024071008A1 (fr) * 2022-09-26 2024-04-04 愛知県 Procédé permettant d'isoler une molécule d'antigène

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WO1998021328A2 (fr) * 1996-11-13 1998-05-22 Sagami Chemical Research Center Proteines humaines possedant des domaines de transmembrane et adn codant ces proteines

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WO1998021328A2 (fr) * 1996-11-13 1998-05-22 Sagami Chemical Research Center Proteines humaines possedant des domaines de transmembrane et adn codant ces proteines

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NISHIGORI H. ET AL.: "Identification and Characterization of the Gene Encoding a Second Proteolipid Subunit of Human Vacuolar H+-ATPase (ATP6F)", GENOMICS, vol. 50, no. 2, June 1998 (1998-06-01), pages 222 - 228, XP002925799 *

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