WO2002102402A1 - Agents anticancereux - Google Patents

Agents anticancereux Download PDF

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Publication number
WO2002102402A1
WO2002102402A1 PCT/JP2002/005916 JP0205916W WO02102402A1 WO 2002102402 A1 WO2002102402 A1 WO 2002102402A1 JP 0205916 W JP0205916 W JP 0205916W WO 02102402 A1 WO02102402 A1 WO 02102402A1
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WIPO (PCT)
Prior art keywords
antigen
salt
substance
partial peptide
fusion protein
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PCT/JP2002/005916
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English (en)
Japanese (ja)
Inventor
Tsukasa Seya
Misako Matsumoto
Kenichiro Naito
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Takeda Chemical Industries, Ltd.
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Publication of WO2002102402A1 publication Critical patent/WO2002102402A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/30—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
    • 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
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or 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

Definitions

  • the present invention relates to a CTL inducer containing the Ml61 antigen or a partial peptide thereof or a salt thereof, and a method for preventing and treating cancer using the Ml61 antigen or a partial peptide thereof or a salt thereof.
  • the immune system is a biological defense system that has developed and evolved to distinguish between self and non-self and protect itself from external attacks. It consists of a phylogenetically more primitiv'e innate immunity and an acquired immunity centered on diverse lymphocytes.
  • the basic immune system is a system that recognizes bran and lipids that do not exist in the self, and includes the complement system, macrophages, dendritic cells, and NK cells.
  • the acquired immune system is a system that recognizes peptides, and lymphocytes are effector cells. The two do not work independently, but rather the activation of lymphocytes is induced by signals from the basic immune system, ie, cytokine production, costimulatory molecule expression, and antigen presentation. It has recently been suggested that the primary response by the basic immune system is very important for the induction of a selective secondary response by lymphocytes. It has come to be bathed (Science 27, 52, 50, 1996).
  • Activation of the basic immune system is important not only for protection against pathogenic microorganisms and virus infection, but also for antitumor immunity, and identification and functional analysis of various factors involved in activation have been performed.
  • the Ml61 antigen is a membrane protein having no sugar chain with a molecular weight of about 43 kDa.
  • cDNA analysis suggests that the Ml61 antigen is a soluble protein composed of 428 amino acids, including a signal peptide of 24 amino acids.
  • (Nature Medicine) 3, 11 volumes, 1 2 6 6, 1 9 9 7)
  • Southern hybridization and gene cloning using cDNA as a probe revealed that the gene product of Mycoplasma fermentans was a lipoprotein in which the N-terminal cysteine residue was modified with palmitic acid (Journal ⁇ Bob 'Bio-Mouth' Chemistry (J. Biol. Chera.) 273, 1 240 7, 1 9 98).
  • the Ml61 antigen gene contains a prokaryotic-specific promoter region and a Shine-Dalgarno sequence at the ribosome binding site, and the signal sequence contains a prokaryotic lipoprotein-specific signal sequence (AVS C). It is clear that there is.
  • the Ml61 antigen gene shows a 99% identity in nucleotide sequence with the M. fermentans gene monocyte differentiation I activation iactor P48; (fcfe (621 bp). Despite the fact that the terminal 114 amino acids show 97% concordance, the C-terminal amino acids are quite different, and both are different molecules in primary structure.
  • Ml61 antigen has the ability to activate complement C3 and also has the ability to induce cytokines, and THP-1 cells, THP-1 cells treated with retinoic acid to form macrophages, human Induces the production of IL-1 JS, TNF-j3, TNF-H, IL-16 and IL-8 from monocytes. In addition, production of IL-11 and IL-11, cytokines that affect ThlZTh2 differentiation from native T cells is also induced in human monocytes.
  • MA LP-2 and P48 have been reported as bioactive molecules derived from M. fermentans.
  • MALP-2 is a 14-amino acid lipotide in which 2 moles of palmitic acid is linked to the N-terminal cysteine residue, and acts on mouse macrophages to induce NO- production (Journal's Expertial 'Medison (J. Exp. Med.) 1 85, 195, 199 7).
  • the 14 amino acids are identical to the N-terminal amino acid of the Ml61 antigen.
  • MALP-404 was identified and found to be identical to the Ml61 antigen.
  • syrup 48 is a protein with a molecular weight of 48 kDa consisting of 16 1 amino acids, and 90 amino acids at the N-terminus are homologous to the Ml 61 antigen, and acts on human monocytes and myelomonocytic cell lines to cause inflammation. Induces the production of sexual cytokines (IL-1 ⁇ , TNF-HI, IL-16).
  • Dendritic cells are It is said to be a professional antigen-presenting cell and is the most important cell for lymphocyte activation. From progenitor cells present in the bone marrow, they differentiate into immature dendritic cells (iDCs) by the stimulation of cytodynamics and distribute to each organ. After eating the antigens, they become mature dendritic cells (mDCs), migrate to lymph nodes, and present antigens to lymphocytes. iDC has high antigen phagocytic ability, does not express costimulator, and does not activate lymphocytes.
  • mDC has no phagocytic ability and expresses costimulators such as CD80, CD86, and CD83, and activates lymphocytes.
  • the transition from iDC to mDC requires stimulation with in vivo molecules such as IL-11j3, TNF- and CD40L, or microbial components such as LPSBCG-CWS.
  • in vivo molecules such as IL-11j3, TNF- and CD40L, or microbial components such as LPSBCG-CWS.
  • Ml61 antigen differentiated from peripheral blood monocytes with GM-CSF and IL-14 are stimulated with purified Ml61 antigen, CD83 and CD86, markers of maturation, and IL-12P40 production are stimulated. Induction revealed that Ml61 antigen could induce dendritic cell maturation.
  • Mycoplasma is a microorganism that belongs between viruses and bacteria, and is characterized by having no cell wall. Expression of the Ml61 antigen is not found in all mycoplasma species and is specific for M. fermentans. M. fermentans infects humans intracellularly, but has not reported its own pathogenicity. Recently, it has been reported that it may be a cofactor that promotes the development of AIDS, and DNA is detected in peripheral blood of patients, but this point is not clear. By homology search, a molecule with high homology is found in a specific amino acid region of the Ml61 antigen.
  • lipoproteins such as Borreria, Treponema, and Listeria, all of which have a lipid bound to the N-terminal cysteine residue and activate the immune system (Matsumoto M. and Seya T., International Research Institute). 'Journal' of 'Molecular' Medicine (Int. J. Mol. Med.) 3: 291 -295, 1 999).
  • TLR4 Human Toll homologue
  • TLR Toll like receptor
  • An object of the present invention is to provide an excellent agent for preventing or treating cancer and the like. Disclosure of the invention
  • Ml61 antigen has unexpectedly excellent CTL inducing action
  • Ml61 antigen and cancer antigen It was unexpectedly found that cancer can be efficiently prevented and treated by unexpectedly using.
  • the present inventors have further studied based on these findings, and as a result, have completed the present invention.
  • a CTL inducer comprising the Ml 61 antigen or a partial peptide thereof or a salt thereof
  • a CTL induction method which comprises administering to a mammal an effective amount of the Ml61 antigen or a partial peptide thereof or a salt thereof,
  • Thl cell inducer comprising the Ml 61 antigen or a partial peptide thereof or a salt thereof
  • a Thl cell induction method which comprises administering to a mammal an effective amount of the Ml 61 antigen or a partial peptide thereof or a salt thereof,
  • M161 antigen or its partial peptide or a salt thereof for producing a Thl cell inducer
  • a medicine comprising a combination of the Ml 61 antigen or its partial peptide or a salt thereof and a cancer antigen
  • a method for preventing and treating cancer which comprises administering to a mammal a combination of an effective amount of the Ml61 antigen or a partial peptide thereof or a salt thereof and an effective amount of a cancer antigen,
  • a method for inducing cytoforce which comprises administering to a mammal an effective amount of the Ml61 antigen or a partial peptide thereof or a salt thereof and an effective amount of a cancer antigen in combination;
  • a method for inducing maturation of immature dendritic cells which comprises administering to a mammal a combination of an effective amount of the Ml 61 antigen or a partial peptide thereof or a salt thereof and an effective amount of a cancer antigen,
  • a method for promoting poor diet which comprises administering to a mammal a combination of an effective amount of the Ml61 antigen or a partial peptide thereof or a salt thereof and an effective amount of a cancer antigen,
  • a CTL induction method which comprises administering to a mammal a combination of an effective amount of an Ml 61 antigen or a partial peptide thereof or a salt thereof and an effective amount of a cancer antigen,
  • a method for inducing Thl cells comprising 'administering to a mammal a combination of an effective amount of the Ml61 antigen or a partial peptide thereof or a salt thereof and an effective amount of a cancer antigen'.
  • (36) a method for preventing or treating cancer, which comprises administering to a mammal an effective amount of the fusion protein or a salt thereof according to (26);
  • a method for inducing Thl cells which comprises administering to a mammal an effective amount of the fusion protein or a salt thereof according to (26),
  • (51) a method for screening a substance that enhances the activity of the Ml61 antigen or its partial peptide or a salt thereof, which comprises using the Ml61 antigen or its partial peptide or a salt thereof and a cancer antigen;
  • the Ml61 antigen or the Ml61 antigen obtained by the screening method according to the above (51). is a drug comprising a substance that enhances the activity of the partial peptide or a salt thereof,
  • a method for screening a substance that promotes induction of cytokines which comprises using the M161 antigen or a partial peptide thereof or a salt thereof, and a cancer antigen;
  • (60) a method for screening a substance that promotes the induction of Thl cells, characterized by using the Ml 61 antigen or a partial peptide thereof or a salt thereof cancer antigen;
  • (64) a method for screening a substance that induces maturation of immature dendritic cells, which comprises using Ml 61 antigen or a partial peptide thereof or a salt thereof, a cancer antigen and immature dendritic cells,
  • a medicament comprising a substance that promotes Thl cell induction, obtained by the screening method according to (79); (82) A method for screening a substance that induces maturation of immature dendritic cells, characterized by using the fusion protein or the salt thereof according to (26) and immature dendritic cells,
  • a medicament comprising a substance that induces maturation of immature dendritic cells, obtained by the screening method according to (82),
  • a method for screening a substance that promotes phagocytosis which comprises using the fusion protein according to (26) or a salt thereof and immature dendritic cells,
  • M161 antigen or its partial peptide or a salt thereof and a cancer antigen are used, and (1) Ml61 antigen or its partial peptide or a salt thereof and (2) TLR 2 and / or] 32 integrin Screening method for a substance that changes the binding to
  • (96) A substance obtained by the screening method according to (94), which comprises (1) a substance that alters the binding property between the fusion protein according to (26) or a salt thereof and (2) TLR2 and Z or 32 integrin. Medicine,
  • (98) a method for preventing or treating cancer, which comprises administering to a mammal an effective amount of a ligand or agonist for TLR2 and an effective amount of a ligand or agonist for Z or; 32 integrin;
  • a medicament comprising a fusion protein of Ml 61 antigen or its partial peptide and a cancer antigen or a salt thereof in combination with another anticancer agent
  • FIG. 1 shows the amino acid sequence of the Ml 61 antigen.
  • FIG. 2 shows the nucleotide sequence of DNA encoding the Ml61 antigen.
  • Ml61 antigen those described in Nature, Medicin (Nature Medicine) 3, 11, Vol. 126, p.
  • amino acid sequence A the amino acid sequence described in Nature Medicine, Vol. 3, 11, Vol. 1, pp. 127, pp. 127 (hereinafter referred to as amino acid sequence A) Peptides having an amino acid sequence substantially the same as that used in the present invention may be used. These peptides may be used in warm-blooded animals (eg, guinea pigs, rats, mice, nits, puppies, pigs, higgies, puppies, monkeys, humans, etc.).
  • warm-blooded animals eg, guinea pigs, rats, mice, nits, puppies, pigs, higgies, puppies, monkeys, humans, etc.
  • Mammals and birds cells [eg, hepatocytes, spleen cells, nerve cells, glial cells, spleen] 3 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts Cell, fiber cell, muscle cell, adipocyte, immune cell (for example, macrophage, T cell, B cell, natural killer cell, mast cell, neutrophil, basophil, eosinophil, monocyte, etc.), megakaryocyte ball , Synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, or stromal cells, or precursors, stem cells, or cancerous cells of these cells] Any tissue [eg brain, parts of the brain (eg olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum, etc.)
  • a protein having an amino acid sequence substantially identical to the amino acid sequence A is defined as at least about 70%, preferably at least about 80%, more preferably at least about 90%, most preferably at least about the amino acid sequence A. And proteins having an amino acid sequence having about 95% or more homology.
  • Examples of a protein having an amino acid sequence substantially the same as the amino acid sequence A include, for example, an activity having substantially the same amino acid sequence as the amino acid sequence A and substantially the same activity as the protein having the amino acid sequence A. Are preferred.
  • Substantially the same quality of activity includes, for example, a signal transduction activity, a cytodynamic force-inducing activity, a maturation-inducing activity of immature dendritic cells, a phagocytosis-promoting activity, and a complement-activating activity.
  • Substantially equivalent indicates that their activities are qualitatively identical (eg, physiochemically or pharmacologically).
  • Ml61 antigen examples include, for example, one or two or more in the amino acid sequence A
  • amino acid sequence A has one or two or more amino acids ( Preferably, about 1 to 7, more preferably about 1 to 5, and still more preferably 1 to 3) amino acids with or without an amino acid sequence, amino acid sequence A or 1 in amino acid sequence A
  • amino acid sequence A An amino acid sequence in which two or more (preferably about 1 to 7, more preferably about 1 to 5, and still more preferably 1 to 3) amino acids are substituted with another amino acid, or a combination thereof A protein containing an amino acid sequence is also used.
  • the partial peptide of the Ml 61 antigen is the fragment peptide of the Ml 61 antigen described above, and is at least 10 or more, preferably about 10 to 50, and particularly preferably 10 to 15
  • a peptide which has about the same amount of amino acid residues and has substantially the same activity as the above-mentioned M161 antigen is used.
  • This partial peptide can be obtained from cells of warm-blooded animals (eg, mammals and birds such as guinea pigs, rats, mice, chicks, egrets, pigs, sheep, birds, monkeys, and humans).
  • warm-blooded animals eg, mammals and birds such as guinea pigs, rats, mice, chicks, egrets, pigs, sheep, birds, monkeys, and humans.
  • Substantially the same quality of activity includes, for example, signal transduction activity, cytokinin-inducing activity, immature dendritic cell maturation-inducing activity, phagocytosis-promoting activity, complement-activating activity, or selected from these.
  • One or more activities include signal transduction activity, cytokinin-inducing activity, immature dendritic cell maturation-inducing activity, phagocytosis-promoting activity, complement-activating activity, or selected from these.
  • One or more activities includes, for example, signal transduction activity, cytokinin-inducing activity, immature dendritic cell maturation-inducing activity, phagocytosis-promoting activity, complement-activating activity, or selected from these.
  • One or more activities includes, for example, signal transduction activity, cytokinin-inducing activity, immature dendritic cell maturation-inducing activity, phagocytosis-promoting activity, complement-activating activity, or selected from these.
  • Substantially equivalent indicates that their activities are qualitatively identical (eg, physiochemically or pharmacologically).
  • partial peptide of the MI161 antigen for example, a peptide containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is used.
  • a peptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 refers to a peptide having an amino acid sequence represented by SEQ ID NO: 1 that is about 70% or more, preferably about 80% or more. % Or more, more preferably about 90% or more, and most preferably about 95% or more.
  • Examples of the partial peptide of the Ml61 antigen include, for example, one or more (preferably about 1 to 7, more preferably 1 to 5) in the amino acid sequence represented by SEQ ID NO: 1. Amino acid sequence in which about 1 to 3 amino acids have been deleted, more preferably 1 or 2 (preferably) amino acid sequences represented by SEQ ID NO: 1.
  • amino acids to which an amino acid is added or inserted the amino acid represented by SEQ ID NO: 1
  • a peptide containing an amino acid sequence obtained by combining them, or the like may be used.
  • the Ml61 antigen or its partial peptide has the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide notation.
  • Ml61 antigen having an amino acid sequence represented by SEQ ID NO: 2 Ml61 antigen including a partial peptide of Ml61 antigen having an amino acid sequence represented by SEQ ID NO: 1, or a portion thereof
  • the peptide may have a carboxyl group (_COOH), a carboxylate (one COO—), an amide (_C ⁇ NH 2 ), or an ester (one COOR) at the C-terminus.
  • R in the ester e.g., methyl, Echiru, n- propyl, C, such as isopropyl or n- butyl - e alkyl group, for example, Shikuropen chill, C 3, such as cyclohexyl - 8 cycloalkyl group, for example, , phenyl, a - C 6, such as naphthyl - 12 Ariru group, e.g., benzyl, phenethyl, other C 6 _ 2 ⁇ Li one Roux C i-2 alkyl group such as ⁇ - naphthyl Rumechiru, general purpose as the oral ester Bivaloyloxymethyl ester and the like are used. '
  • the carboxyl group may be amidated or esterified and the Ml61 antigen or its partial peptide include.
  • ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • OH, C ⁇ OH, NH 2 , SH or the like on the side chain of the amino acid in the molecule has a suitable protecting group (eg, formyl group, acetyl) And complex peptides such as so-called glycopeptides to which sugar chains are bonded.
  • the synthesized Ml61 antigen or its partial peptide preferably has a lipid bound in the molecule.
  • the lipid is not particularly limited as long as it is a lipid capable of binding to the peptide protein, and examples thereof include fatty acids, acylglycerols, phospholipids, sphingolipids, glycolipids, glycerin ether, terpenoids, and sterols.
  • fatty acids acylglycerols
  • phospholipids phospholipids
  • sphingolipids glycolipids
  • glycerin ether glycerin ether
  • terpenoids terpenoids
  • Fatty acids include saturated fatty acids (such as acetic acid, propionic acid, butyric acid, cabronic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, and lignoceric acid).
  • saturated fatty acids such as acetic acid, propionic acid, butyric acid, cabronic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, and lignoceric acid).
  • Unsaturated fatty acids unsaturated fatty acids (oleic acid, batacenic acid, linoleic acid, ⁇ -linolenic acid, V-linolenic acid, arachidonic acid and other unsaturated fatty acids having 15 to 24 carbon atoms), ⁇ -eleo Stearic acid, tallic acid, isocyanic acid, lacrobacilic acid, vernolic acid, prostaglandin and the like are used, and among them, saturated fatty acids having 2 to 24 carbon atoms such as palmitic acid and stearic acid are preferable.
  • acyl glycerols also called neutral lipids
  • acyl glycerols refers to a monoester, diester or triester of glycerol and the above-mentioned fatty acid, and among them, triacylglycerol which is a triester of glycerol and a fatty acid is preferable.
  • a lipid that may contain glycerol, a fatty acid or (and) a nitrogen base in addition to phosphoric acid is used.
  • 3-s ⁇ -phosphatidylcholine, 3-s ⁇ -phosphatidinolethananol Noreamine, 3 s ⁇ -phosphatidinoleserine, 3-s ⁇ -phosphatidinoleethanolamine, 1-alkoxyphospholipid, 3-s ⁇ -phosphatidylinositol, 3-s ⁇ -phosphatidino reglycerol are used.
  • sphingolipid celeb mouth side, psychosine, sphingomyelin and the like are used.
  • glycolipid a lipid containing carbohydrate and diacylglycerol as main components and not containing phosphoric acid is used.
  • 3_s ⁇ -monogalactosyldiasyl glycerose, 3-s ⁇ -di Galactosinoresia sinore glycerone, 3-sn- (6-snore-ho-6-doxy-l-D-glycosyl) diasylglycerol, etc. are used.
  • dariserin ethers examples include 1-alkyl-1,2,3-diacyl-sn-glycerol, 1-alkene-2,2,3-diasinole-1 sn-glyceronole, For example, 1-alkyl-12-acetinolate sn-glyceronole-13-phosphorinolecoline is used.
  • an isoprenoid polymer As the terpenoid, an isoprenoid polymer, -rotin and the like are used.
  • sterol for example, cholesterol or an ester thereof is used.
  • fatty acids are preferred as the lipid, and saturated fatty acids having 2 to 24 carbon atoms, such as palmitic acid and stearic acid, are particularly preferred.
  • the binding position between the lipid and the M161 antigen or a partial peptide thereof is not particularly limited, but the N-terminus of the above-described M161 antigen or a partial peptide thereof is preferable.
  • the mode of binding between the lipid and the Ml61 antigen or a partial thereof: 7 ° -tide is not particularly limited, but may be on the side chain of the constituent amino acid of the Ml61 antigen or a partial peptide thereof.
  • they may be bonded via a hydroxy group, a carboxyl group, an amino group, a thiol group, or the like.
  • the lipid may be bound via the thiol group of the N-terminal cysteine residue. Good.
  • a partial peptide of the Ml61 antigen two carbon atoms of 2 to 2 are linked via the N-terminal cysteine residue of a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
  • Peptides to which four saturated fatty acids (particularly, palmitic acid) are bound are preferred.
  • MALP-2 palmitic acid
  • Binding of the Ml61 antigen or a partial peptide thereof to a lipid can be performed using a method known per se.
  • MA LP-2 is published in Journal Eta Sparimental 'Medicine (J. Exp. Med.), Vol. 185, Number 11, June 2, 1997, 1951-1958. Can be produced according to the method described in
  • a physiologically acceptable acid addition salt is particularly preferable.
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid
  • salts with inorganic bases eg, alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, aluminum and ammonium
  • organic bases eg, trimethylamine, triethylamine, pyridine
  • Picoline 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, hexahexylamine, dihexylhexanolamine, N, N, dibenzylethylenediamine, etc.
  • prodrugs of Ml61 antigen or its partial peptide include enzymes and stomach acids under physiological conditions in vivo.
  • a compound which is converted into the peptide or the like of the present invention by the reaction of the present invention that is, a compound which is enzymatically oxidized, reduced, hydrolyzed, etc. to be converted into the peptide of the present invention, etc.
  • a compound that changes to a peptide or the like is used.
  • a compound in which the amino group of the peptide or the like of the present invention is acylated, alkylated or phosphorylated for example, the amino group of the peptide or the like of the present invention has an amino group of eicosanoyl
  • a compound in which a hydroxyl group of a peptide or the like of the present invention is acylated, alkylated, phosphorylated, or borated for example, a hydroxyl group of a peptide or the like of the present invention is Acetylation, palmitoylation, propanoylation, pivaloylation, succ
  • the prodrug such as the peptide of the present invention can be prepared under physiological conditions as described in Hirokawa Shoten, 1990, “Development of Drugs,” Vol. 7, Molecular Design, pp. 163-198. It may be changed to the peptide or the like of the present invention.
  • Ml61 antigen or a partial peptide thereof or a salt thereof can also be produced from the above-mentioned mammalian cells or tissues by a method known per se.
  • (2) can be produced according to the peptide synthesis method, or (3) can be produced by culturing a transformant containing DNA encoding the Ml61 antigen. .
  • the human or mammalian tissues or cells are homogenized and then extracted with an acid or the like, and the extract is subjected to reverse phase chromatography, ion exchange chromatography, etc.
  • the Ml61 antigen can be isolated and purified by combining the above chromatography.
  • the Ml61 antigen or its amide can be produced according to a peptide synthesis method known per se, or by cleaving the Ml61 antigen with an appropriate peptidase.
  • any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target Ml61 antigen is produced by condensing a partial peptide or amino acid capable of constituting the partial peptide with the remaining portion and, if the product has a protecting group, removing the protecting group. be able to. Examples of the known condensation method and elimination of the protecting group include the methods described in the following 1 to 5. ⁇ . Bodanszky and MA On detti, Peptide Synthesis, Interscience Pub 1 ishers, New York (1966)
  • a commercially available resin for peptide synthesis can be used for the synthesis of Ml61 antigen or its amide.
  • resins include, for example, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydrogen b carboxymethyl phenylalanine ⁇ Seto Ami Domechi Le resin, polyacrylamide resin, 4- (2,, 4 'Jime Tokishifue two Ruhido port Kishimechiru) phenoxy ⁇ , 4 one (2, 4, one dimethyl Tokishifue two Lou Fm oc-amino Ethyl) phenoxy resin and the like.
  • an amino acid having an amino group and a side chain functional group appropriately protected is condensed on a resin according to the sequence of the desired partial peptide according to various known condensation methods. .
  • the partial peptide is cleaved from the resin and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target Ml 61 antigen or its amide. .
  • carbodiimides are particularly preferable.
  • carposimids include DCC, N, N'-diisopropylcarbodiimide, N-ethyl-N, 1- (3-dimethylaminopropyl) carpoimide.
  • These activations involve the ability to add protected amino acids directly to the resin along with racemization inhibitors (eg, HOBt, HOOBt) or as symmetrical acid anhydrides or HOBt esters or HOOBt esters.
  • racemization inhibitors eg, HOBt, HOOBt
  • Pre-protected amino acid activity Can be added to the resin after the conversion.
  • the solvent used for activating the protected amino acid or condensing with the resin may be appropriately selected from solvents known to be usable for the peptide condensation reaction.
  • acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride, chloroform, trifluoroethanol Alcohols such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propio-tolyl; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof. Is used.
  • the reaction temperature is appropriately selected from a range known to be usable for the peptide bond formation reaction, and is usually appropriately selected from a range of about 120 ° C to 50 ° C.
  • the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
  • Examples of the protecting group for the starting amino group include Z, Boc, tert-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, C1-Z, and Br-Z Adamantyloxycarbonyl, trifluoroacetyl, phthaloynole, honolemil, 2-nitrophenylenosulfenyl, diphenylphosphinochioil, Fmoc and the like.
  • the carboxyl group can be, for example, alkyl esterified (for example, methyl, ethyl, propynole, butyl, tert-butyl, pentyl, cyclohexanol, cycloheptyl, cyclooctyl, cycloadatyl, 2-adamantyl, etc.).
  • alkyl esterified for example, methyl, ethyl, propynole, butyl, tert-butyl, pentyl, cyclohexanol, cycloheptyl, cyclooctyl, cycloadatyl, 2-adamantyl, etc.
  • Branched or cyclic alkyl esterification Branched or cyclic alkyl esterification
  • aralkyl esterification for example, benzyl ester, 4-12 trobenzinoleestenol, 4-methoxybenzinoleestenol, 4-chlorobenzinoleestenol, benzhydrin) Esterification
  • phenacyl esterification benzinoleoxyl hydronyl hydrazide, tert-butoxycarbonyl hydrazide, tritinolehydrazide and the like.
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • a lower alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group and an ethoxycarbonyl group, and the like are used.
  • a group suitable for ethereal dani include a benzyl group, a tetrahydrobiral group, a tert-butyl group, and the like.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, B z 1, C 1 2 - B zl, 2- nitrobenzyl, B r- Z, such as tert- Buchinore is used.
  • Examples of the protecting group for histidine imidazole include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, 'benzyloxymethyl, Bum, Boc, Trt, and Fmoc. Used.
  • activated carboxyl groups of the raw materials include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, esters with N-hydroxysuccinimide, HOB t), etc.
  • active esters eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, esters with N-hydroxysuccinimide, HOB t
  • the activated amino group in the raw material include, for example, Phosphate amide is used.
  • Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia Also used.
  • the elimination reaction by the above-mentioned acid treatment is generally performed at a temperature of about ⁇ 20 ° C. to 40 ° C.
  • the protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
  • the carboxyl-terminal amino acid is protected by amidating the strong oxyloxyl group, and then a peptide chain is added to the amino group side. After extending to a desired chain length, a partial peptide in which only the protecting group of the ⁇ -amino group at the ⁇ terminal of the peptide chain is removed and a partial peptide in which only the protecting group of the carboxyl group at the C terminal is removed are produced. Then, both partial peptides are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above.
  • the crude 161 antigen is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired 161 antigen.
  • ester form of Ml 61 antigen for example, after condensing the ⁇ -carboxyl group of the carboxyl terminal amino acid with the desired alcohol to form an amino acid ester, thus, an ester of the desired M61 antigen can be obtained.
  • the objective partial peptide can be isolated and purified by a combination of ordinary purification methods, for example, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like.
  • the Ml61 antigen obtained by the above method is a free form, it can be converted into an appropriate salt by a known method, and conversely, when it is obtained by a salt, the free form can be converted by a known method.
  • the DNA encoding the Ml61 antigen may be any DNA containing a base sequence encoding the Ml61 antigen.
  • Genomic DNA Any of a genomic DNA library, the above-described cDNA derived from cells and tissues, a cDNA isolated from the above-described cDNA library derived from cells and tissues by a method known per se, and synthetic DNA may be used. Specifically, c. Described in, for example, Nature, Medicine (Nature Medicine) 3, 1266, 1997; Journal of Biol. Biologica. Chem. (273), 12407, 1998. DNA can be used.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like.
  • the reverse transcription is performed directly by reverse transcription (hereinafter simply referred to as RT-PCR). It can also be released.
  • RT-PCR reverse transcription
  • the DNA encoding the Ml61 antigen of the present invention is, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 3, or represented by SEQ ID NO: 3.
  • nucleotide sequence that hybridizes with the nucleotide sequence under high stringency conditions and has substantially the same activity as the Ml61 antigen of the present invention (eg, signal transduction activity, cytokine-inducing activity, maturation of immature dendritic cells) Any DNA may be used as long as it encodes a peptide having a function of inducing phagocytosis, promoting phagocytosis, and activating a trap.
  • Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 3 include, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, Preferably, DNA containing a nucleotide sequence having about 95% or more homology or the like is used.
  • Hybridization is performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can be done. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed according to high stringent conditions.
  • the high stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, a temperature of about 50 to 70 ° C, and preferably about 60 to 65 ° C. The condition of C is shown. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
  • DNA encoding the Ml 61 antigen containing the amino acid sequence represented by SEQ ID NO: 2 a DNA containing the base sequence represented by SEQ ID NO: 3 (FIG. 2) or the like is used. .
  • Examples of the DNA encoding the partial peptide of the Ml 61 antigen include (1) DNA having the partial nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 3, or (2) DNA represented by SEQ ID NO: 3. It has a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence, and has substantially the same activity as the Ml61 antigen of the present invention (eg, signal transduction activity, cytokinin-inducing activity, immature dendritic cell).
  • a DNA having a partial nucleotide sequence of a DNA encoding a peptide having a maturation-inducing action, a phagocytosis-promoting action, a complement-activating action, etc. may be used.
  • DNA amplified in the PCR method using a synthetic DNA primer having a base sequence encoding a partial sequence of the Ml61 antigen or DNA incorporated in an appropriate vector is used.
  • the Ml 61 antigen was isolated from a genomic DNA, a genomic DNA library, the above-mentioned cell-tissue-derived cDNA, and the above-mentioned cell-tissue-derived cDNA library by a method known per se. Can be isolated by hybridization with a DNA fragment encoding a part or all of the DNA or a DNA fragment labeled with a synthetic DNA. Hybridization methods are described, for example, in Molecular Cloning 2nd (J. Sam rooke t. Al., Old Spring Spring Harbor Lab. Press, 1989). It can be performed according to a method or the like. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
  • Conversion of the DNA sequence of DNA is carried out by using a known kit, for example, Mutant TM -super Epress Km (Takara Shuzo Co., Ltd.), Mutant TM -K (Takara Shuzo Co., Ltd.), and the like.
  • Method, Ga pped du plex method, Kun It can be carried out according to a method known per se such as the ke 1 method or a method analogous thereto.
  • the cloned DNA encoding the Ml61 antigen can be used as it is depending on the purpose, or can be used after digesting with a restriction enzyme or adding a linker if desired.
  • the DNA may have ATG as a translation initiation codon at its 5 'end, and may have TAA, TGA or TAG as a translation termination codon at its 3' end. These translation initiation codons and translation termination codons are added using an appropriate synthetic DNA adapter; ] You can talk.
  • An expression vector for the Ml61 antigen can be prepared, for example, by (i) cutting out a DNA fragment of interest from DNA encoding the Ml61 antigen, and (mouth) placing the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by connecting.
  • Plasmids derived from Escherichia coli eg, pBR322, pBR325, pUC12, pUC13
  • plasmids derived from Bacillus subtilis eg, pUB110, pTP5, pC194
  • yeast-derived plasmids eg, pSH19, pSH15
  • bacteriophages such as phage, animal viruses such as retrovirus, vaccinia virus, and baculovirus, pA1-11, pXT1, pc / CMV , pRc / RSV, pcDNAI / Neo, and the like.
  • any promoter can be used as long as it is an appropriate promoter corresponding to the host used for gene expression.
  • SV40-derived promoter retrowinores promoter, metallothionein promoter, heat shock promoter, cytomegaloinoreles promoter, SR ⁇ promoter and the like can be mentioned.
  • the host is Escherichia, trp promoter, la. Promoter, rec A promoter,; IP L promoter, 1 pp promoter.
  • the host is Bacillus genus bacteria, SP 01 promoter, SP 02. promoter, pen P promoter.
  • the host is yeast, pHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable.
  • the host is an insect cell, a polyhedrin promoter, a P10 promoter, etc. Which is preferred.
  • the expression vector may further contain, if desired, an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like, if desired.
  • a selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin phosphorus resistant gene (hereinafter sometimes abbreviated as Amp r), neomycin resistant Gene (hereinafter sometimes abbreviated as Neo, G418 resistance) and the like.
  • dhfr dihydrofolate reductase
  • MTX metalhotrexate
  • Amp r ampicillin phosphorus resistant gene
  • Neo neomycin resistant Gene
  • a signal sequence suitable for the host can be added to the N-terminal side of the Ml61 antigen.
  • the host is a genus Escherichia, an alkaline phosphatase signal sequence, an OmpA signal sequence, etc.
  • the host is a Bacillus genus, an ⁇ -amylase ′ signal sequence, a subtilisin signal sequence, etc.
  • the host is a yeast, a mating factor signal sequence, an invertase. Signal sequence, etc.
  • an insulin signal sequence, a-interferon signal sequence, an antibody Molecules and signal sequences can be used.
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • Escherichia Escherichia coli K12, DH1 [Procedures of the National Academy of Sciences, Obesity, Science, Ob, the USA (Proc.) N at 1. Acad. S ci. USA), Vol. 60, 16 (1966)], JM103 (Nucleic A cids Research) , 9 turns, 3 09 (1 9 8 1), J ⁇ 2 2 1 [Journal of More Journalof Molecular Biology], 120 volumes, 5 17 (1 9 78)], HB 101 [Journal of molecular biology, 41 volumes, 459] (1969)], C600 [Genetics, Vol. 39, 440 (1954)] and the like are used.
  • Bacillus bacteria examples include, for example, Bacillus S. subtilis MI 114 (Gene, 24 vol., 255 (1 983)), 207-221 (Journal of Biochemistry) J ournalof Bioch em istry), 95, 8 7 (1 984)].
  • yeast examples include Saccharomyces cerevisiae AH22, AH22R—, NA87-11A, DKD-5D, 2OB—12, and Schizosaccharomyces bomb (Schizosacchar omy cesp).
  • omb e) NCYC 1913, NCYC 2036, Saccharomyces pichia nostris (S acchar omy cespicjia pastoris) and the like are used.
  • insect cells for example, when the virus is Ac NPV, a cell line derived from a larva of night rob moth (S podopterafrugiperdaee l 1; S f cell), an MG1 cell derived from the midgut of T richoplusiani, an egg of T richoplusiani High Five TM cells, cells derived from Mame strabrassicae or cells derived from Estigme naacrea are used.
  • the virus is BmNP V
  • a silkworm-derived cell line Boomby Xmorri N cell; BmN cell
  • Sf cells include, for example, Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, J.L., et al., In vivo, 13, 2 1 3—2 17 and 1977 7) are used.
  • insects for example, silkworm larvae and the like are used [Maeda et al., Nature, Vol. 3115, 592 (19985)].
  • animal cells examples include monkey cells COS-7, Veto, Chinese hamster cells CHO, DHFR gene-deficient Chinese hamster cells CHO (d hfr-CHO cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, and the like.
  • Transformation of Bacillus spp. Is performed, for example, according to the method described in Molecular & Generic Genetics, 168, 11 (1 79). be able to.
  • To transform yeast for example, the method described in Methodsin Enzymology, 194, 182—187 (1 991), Processing's Ob. "National” Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 75 volumes, 1992 (1978), etc. It can be done according to the method.
  • Transformation of insect cells or insects can be performed, for example, according to the method described in Bio Z Technology (Bio / ⁇ eChnolology), 6, 47-55 (1.988).
  • a liquid medium is suitable as a medium used for the cultivation, and a carbon source necessary for the growth of the transformant is contained therein.
  • carbon sources include glucose, dextrin, soluble starch, and sucrose.
  • nitrogen sources include for example, inorganic or organic substances such as ammonium salts, nitrates, corn chip liquor, peptone, potato zein, meat extract, soybean meal, potato extract, and inorganic substances such as calcium chloride, sodium dihydrogen phosphate, Magnesium chloride and the like.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5 to 8.
  • Examples of a medium for culturing Escherichia bacteria include, for example, an M9 medium containing glucose and casamino acid [mirror (Mi 11 er), a journal “Ob esperimentin”, “Molecular genetics”. 1 of E perimentsin Molecular Genetics), 431-433, Old Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3] 3-indolylacrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium of the genus Escherichia, cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.
  • the cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring may be added.
  • the culture medium When culturing a transformant in which the host is an insect cell or an insect, the culture medium is immobilized as Graces Insect Medium Grace, TCC, Nature, 195, 788 (1962)). 10% blood Those to which additives such as Qing are appropriately added are used.
  • the pH of the medium is adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.
  • the medium When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], DMEM medium (Viro 1 ogy, 8 vol. 396 (1959)), RPMI 1640 medium [Journa 1 of the American Medicine] (Journa 1 of the American Medicine) As sociation) 1 99 51 9 (1 967)], 199 medium [Proceding of Society for the Biological Medicine], 73, 1 (1 950)].
  • the pH is between about 6 and 8. Culture is usually performed at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and agitation are added as necessary.
  • the M161 antigen can be produced in a culture medium or a transformant. .
  • the Ml61 antigen can be separated and purified from the culture by, for example, the following method.
  • Ml61 antigen When extracting Ml61 antigen from cultured cells or cells, after culturing, cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to sonication, lysozyme and z or freeze-thawing. After the cells or cells are destroyed by the method described above, a method of obtaining a crude peptide extract by centrifugation or filtration is used as appropriate.
  • the buffer solution may contain a peptide converting agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (trade name).
  • Ml61 antigen is secreted into the culture solution, after completion of the culture, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected.
  • Purification of the Ml61 antigen contained in the culture supernatant or the extract thus obtained can be carried out by appropriately combining known separation and purification methods.
  • These known separation and purification methods mainly include methods using solubility such as salting-out> solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • a method using the difference in hydrophobicity, a method using the difference in isoelectric point such as isoelectric focusing, and the like are used.
  • the Ml61 antigen thus obtained is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained in a salt form, it is known per se Can be converted into a free form or another salt by the method of or a method analogous thereto.
  • the Ml61 antigen produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification.
  • an appropriate protein modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
  • the Ml61 antigen or a salt thereof thus produced can be detected by enzyme immunoassay using a specific antibody or the like.
  • the cancer antigens used in the present invention include, for example, cancer cells or extracts thereof, carcinogenic proteins or peptides, proteins or peptides selectively expressed on cancer cells, tumors, destructive tumors, apoptosis of cancer cells Small bodies are used. These cancer antigens may be isolated from the patient or may be present in the body of the cancer patient.
  • cancer cells it is preferable to use cancer cells to be treated as the cancer cells.
  • the lunar ulcer it is preferable to use a tumor to be treated.
  • the destroyed tumor for example, it is preferable to use a tumor in which a tumor to be treated is destroyed.
  • a tumor to be treated is destroyed.
  • the purpose is to treat melanoma
  • cancer antigens include HER2 protein, mutant p53 protein, Oncogenic proteins such as atypical ras protein, oncoproteins such as papillomavirus-derived E-7 protein, tumor marker proteins such as PSA (prostate specific antigen) and CEA (oncofetal antigen), and serological expression cloning ( SEREX) and the like.
  • a protein in which the above-described Ml61 antigen or a partial peptide thereof and the above-mentioned cancer antigen are linked at any position is used, and particularly, the Ml61 antigen
  • a protein in which a cancer antigen is linked to the C-terminus of a partial peptide thereof is preferable.
  • the fusion protein in which the above-mentioned cancer antigen, Ml61 antigen or a partial peptide thereof and the cancer antigen are linked can be produced according to the above-mentioned method for producing the Ml61 antigen.
  • a cancer antigen is synthesized in the same manner following the C-terminal amino acid of the Ml61 antigen, and a transformant containing DNA encoding the Ml61 antigen is used.
  • the DNA encoding the cancer antigen is inserted directly or immediately following the appropriate linker sequence upstream of the termination codon of the DNA encoding the Ml 61 antigen. It can be produced according to the production method by culturing a transformant containing DNA encoding the antigen.
  • Ml61 antigen or its partial peptide or its salt or its prodrug (hereinafter abbreviated as Ml61 antigen of the present invention) is activated by dendritic cells, followed by cross-priming (class I-class I switching) and then class I. Induces CTL by presenting antigen to CTL. That is, CTL can be induced without using Thl cells.
  • the M161 antigen induces cytokines such as IL-12, IL-18, INF ⁇ , and TNF- ⁇ , induces Thl cells, and produces the above cytokines in a chain reaction. Can also induce CTLs.
  • cytokines such as IL-12, IL-18, INF ⁇ , and TNF- ⁇
  • the Ml61 antigen, its partial peptide, or a salt thereof, or a prodrug thereof has an excellent CTL-inducing effect and / or Thl-cell-inducing effect. Therefore, the pharmaceutical composition containing the Ml61 antigen of the present invention Objects can be mammals (eg, mice, rats, hamsters, egrets, cats, dogs, dogs, higgies, sanoles, humans). CTL inducer and / or Thl cell inducer. CTL [Cytotoxic T Lymphocytes] are also called killer cells, which are cytotoxic cells and T lymphocytes that have the ability to break down target cells. Are cells that specifically attack cancer cells presenting anti-menorrhea and express anti-menorrhea.
  • Immature dendritic cells contain a signaling receptor (TLR 2) and an antigen uptake receptor (complement receptor CR3, or 2 integrins), and signal activation of immature dendritic cells
  • TLR 2 signaling receptor
  • antigen uptake receptor complement receptor CR3, or 2 integrins
  • Antigen uptake induces antigen presentation of dendritic cells.
  • the Ml61 antigen binds to these two types of receptors and is taken up by endosomes of dendritic cells.It can induce antigen presentation and induce cytokines and CTLs. Thus, an excellent cancer immune system can be established.
  • the medicament obtained by combining the Ml61 antigen of the present invention with a cancer antigen and the fusion protein of the present invention or a salt thereof can be used to (1) induce cytocytoin from THP-1 cells, macrophages, human mononuclear cells, etc. (Induced action of cytoforce-in production), (2) Induced maturation of immature dendritic cells, (3) Promoted phagocytosis, (4) Activated tumor immune mechanism, (5) Induced CTL, ( 6) Since it has a Thl cell-inducing action, it induces cytokines in mammals (for example, mice, rats, hamsters, rabbits, cats, dogs, dogs, sheep, monkeys, monkeys, humans, etc.).
  • mammals for example, mice, rats, hamsters, rabbits, cats, dogs, dogs, sheep, monkeys, monkeys, humans, etc.
  • an anticancer agent such as an inducer, an agent for inducing the maturation of immature dendritic cells, an agent for promoting phagocytosis, an activator of the tumor immune system, a CTL inducer, or a Thl cell inducer.
  • an inducer an agent for inducing the maturation of immature dendritic cells
  • an agent for promoting phagocytosis an activator of the tumor immune system
  • a CTL inducer a CTL inducer
  • Thl cell inducer a Thl cell inducer
  • cytokines examples include interferon, 1 ⁇ 8 or 1 ⁇ , interleukin-1, 2, 3, 6, 8, 10, 12, or 18, tumor necrosis factor ( ⁇ NF), lymphotoxin, colony stimulating factor (CSF), erythropoietin, epidermal growth factor (EGF), fibroblast growth factor (FGF) and the like.
  • ⁇ NF tumor necrosis factor
  • CSF colony stimulating factor
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • interferon-y, interleukin-12 or interleukin-18 are preferable.
  • a medicine comprising a combination of the Ml61 antigen and a cancer antigen and the fusion of the present invention.
  • Synthetic protein or a salt thereof can be used in mammals (eg, mice, rats, hamsters, egrets, cats, dogs, dogs, pigs, higgins, monkeys, humans, etc.) (eg, breast cancer, prostate cancer, spiroma, Stomach cancer, lung cancer, colon cancer (colon cancer, rectal cancer, anal cancer), esophageal cancer, duodenal cancer, head and neck cancer (tongue cancer, pharyngeal cancer), brain tumor, schwannoma, non-small cell lung cancer, small cell lung cancer, Liver cancer, kidney cancer, bile duct cancer, uterine cancer (child cancer, cervical cancer)., Ovarian cancer, bladder cancer, skin cancer, hemangiomas, malignant lymphoma, malignant melanoma, thyroid cancer, bone tumor, hemangiomas , Hemangiofibromas, retina
  • the Ml61 antigen, cancer antigen, fusion protein of the present invention or a salt thereof of the present invention has low toxicity and is safe.
  • the pharmaceutical composition comprising the MI161 antigen or the fusion protein or a salt thereof of the present invention has low toxicity and can be prepared according to a method known per se generally used in a method for producing a pharmaceutical preparation.
  • the l61 antigen or the fusion protein of the present invention or a salt thereof is mixed as it is or with a pharmacologically acceptable carrier, for example, tablets (including sugar-coated tablets, film-coated tablets), powders, granules, capsules It can be safely administered orally or parenterally (eg, topically, rectally, intravenously, etc.) as a pharmaceutical preparation such as a drug, (including soft capsules), liquid, injection, suppository, sustained release, etc. it can.
  • these drugs can be separately or simultaneously prepared into a pharmaceutical preparation and administered orally or parenterally as a pharmaceutical composition. it can.
  • the drugs can be administered separately by mixing them with a diluent at the time of use.However, the separately formulated products can be administered simultaneously or with a time lag. May be separately administered to the same subject.
  • Kit products for administration of separately formulated products using a diluent or the like at the time of use for example, an ampoule containing a powdered individual drug and two or more drugs mixed at the time of use and dissolved Kits, which contain diluents for injection, etc.
  • kit products for administering the separately formulated individual preparations to the same subject simultaneously or separately with a time lag for example, individual Tablets containing the same drug in the same or separate bags and, if necessary, two or more drugs at the same time Or a kit for tablets for separate administration with a time lag
  • a time lag for example, individual Tablets containing the same drug in the same or separate bags and, if necessary, two or more drugs at the same time Or a kit for tablets for separate administration with a time lag
  • the present invention also provides an embodiment in which a preparation of Ml61 antigen alone is administered to a patient holding a cancer antigen, and the Ml61 antigen coexists in a cancer antigen-rich environment. Included in medicine.
  • Pharmaceutically acceptable carriers that may be used in the production of the pharmaceutical composition of the present invention include various organic or inorganic carrier materials commonly used as pharmaceutical materials, such as excipients in solid pharmaceuticals. , Lubricants, binders and disintegrants, or solvents, dissolution aids, suspending agents, isotonic agents, buffers and soothing agents in liquid preparations. Further, if necessary, usual additives such as preservatives, antioxidants, coloring agents, sweeteners, adsorbents, wetting agents and the like can be used in appropriate amounts.
  • shell morphs examples include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light caffeic anhydride and the like.
  • lubricant examples include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
  • Binders include, for example, crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropinoresenolylose, hydroxypropinolemethinoresenololose, polyvinylinolepyrrolidone, starch, sucrose, gelatin , Methylcellulose, carboxymethylcellulose sodium and the like.
  • Disintegrants include, for example, starch, carboxymethylcellulose, carboxymethyl / resenolerose kanoresidum, sodium canolepoxmethinolestarch, L-hydroxypropyl xypropylcellulose and the like.
  • solvent examples include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil and the like.
  • dissolution aids for example, polyethylene glycol, propylene glycol,
  • a suspending agent for example, stearyl triethanolamine, sodium lauryl sulfate Surfactants such as thorium, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate; and the like; for example, polyvinyl alcohol, polyvinylpyrrolidone, canoleboximetinolecellulose sodium, Hydrophilic macromolecules such as methinoresenorelose, hydroxymethinoresenorelose, hydroxyxetinoresenorelose, and hydroxypropylcellulose.
  • Surfactants such as thorium, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate; and the like; for example, polyvinyl alcohol, polyvinylpyrrolidone, canoleboximetinolecellulose sodium, Hydrophilic macromolecule
  • tonicity agent examples include glucose, D-sorbitol, sodium salt, glycerin, D-mannitol and the like.
  • buffers such as phosphate, acetate, carbonate, and citrate.
  • Examples of the soothing agent include benzyl alcohol and the like.
  • preservatives include parahydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
  • Antioxidants include, for example, sulfite, ascorbic acid, ⁇ -tocopherol and the like.
  • the content of the Ml61 antigen of the present invention or the fusion protein of the present invention or a salt thereof varies depending on the form of the preparation, but is usually about 0.1 relative to the whole preparation. 1100% by weight, preferably about 10-99.9% by weight, more preferably about 20-90% by weight. / 0 or so.
  • the content of the cancer antigen varies depending on the form of the preparation, but is usually about 10 to 99.9% by weight based on the whole preparation. Is about 20 to 90% by weight.
  • the content of components other than the Ml61 antigen of the present invention or the fusion protein of the present invention or a salt thereof varies depending on the form of the preparation. It is about 10 to 99.9% by weight, preferably about 20 to 90% by weight.
  • the dosage of the pharmaceutical composition of the present invention varies depending on the administration route, symptoms, age of the patient, and the like.For example, when the composition is orally administered for the purpose of treating or treating cancer, the body weight is 1 kg per day. Per the Ml61 antigen of the present invention or the fusion protein of the present invention or a salt thereof. About 0.05 to 50 mg, preferably about 0.05 to 10 mg, and more preferably about 0.2 to 4 mg can be administered in 1 to 3 divided doses.
  • the pharmaceutical composition of the present invention can be used in an appropriate amount in combination with or in combination with an appropriate amount of another drug, in addition to the Ml61 antigen of the present invention, the fusion protein of the present invention, or a salt thereof.
  • compositions for such combination or combined use include, for example, various anticancer agents that can be used for the treatment of cancer (in the present specification, the M161 antigen of the present invention or a partial peptide thereof) However, for the sake of convenience, it may be referred to as “another anticancer agent” for the sake of distinction from an anticancer agent containing the prodrug or a salt thereof).
  • drugs with low immunosuppressive effects such as endocrine therapy drugs (such as LH-RH agonists and antagonists, 'I live hormone antagonists, sex hormone synthesis inhibitors), and genes such as tyrosine kinases that are selective for cancer products (EGF receptor, HER 2 / erb - 2 s HER 3 / erb _ 3, HER 4 / erb- 4, PDGF receptor, VEGF receptor, etc.) pharmaceutical and targeting, and more cancer vaccine therapy agents No.
  • endocrine therapy drugs such as LH-RH agonists and antagonists, 'I live hormone antagonists, sex hormone synthesis inhibitors
  • genes such as tyrosine kinases that are selective for cancer products (EGF receptor, HER 2 / erb - 2 s HER 3 / erb _ 3, HER 4 / erb- 4, PDGF receptor, VEGF receptor, etc.
  • tumor antigens or similar tumor cell-derived proteins, partial peptides thereof or fusion proteins containing them (2) these proteins or peptides are encoded and can be expressed in vivo DNA fragments and ribosomes containing them, and (3) viruses or plasmids containing the DNA fragments.
  • tumor cell-derived proteins examples include melanoma-related antigens (eg, MAGE-1, MAGE-3, MART-11, gpl 00, tyrosine kinase, etc.), prostate-specific antigens (P SA), HER2 protein, MUC-1 mucin, hCG, gastrin, heat shock protein, human papilloma mouse E7 protein, carcinoembryonic antigen (CEA), mutant Ras protein and the like.
  • melanoma-related antigens eg, MAGE-1, MAGE-3, MART-11, gpl 00, tyrosine kinase, etc.
  • P SA prostate-specific antigens
  • HER2 protein MUC-1 mucin
  • hCG gastrin
  • heat shock protein human papilloma mouse E7 protein
  • CEA carcinoembryonic antigen
  • the pharmaceutical composition of the present invention has excellent anticancer activity even when used as a single agent. Further, the effect can be further enhanced by using it in combination with other anticancer agents (combination of multiple drugs). it can. Other benefits of the combination include the use of each other's drugs The amount can be reduced, which reduces side effects and quality of life for cancer patients: Quality of Life (eg, performance stasis and pain reduction, edema suppression, appetite enhancement, weight gain) Improvement).
  • Quality of Life eg, performance stasis and pain reduction, edema suppression, appetite enhancement, weight gain
  • the other anti-cancer agent when another anti-cancer agent is used in combination, the other anti-cancer agent induces apoptosis of cancer cells at the cancer site of the cancer patient to induce the release of the cancer antigen, and the cancer antigen of the present invention (eg, , Apoptotic bodies) and Ml 6
  • concomitant drugs that can be used in combination with the pharmaceutical composition of the present invention are specifically exemplified below.
  • hormone therapeutic agent examples include: phosphestrol, getylstinolebestroneol, chlorotrianiserin, medroxyprogesterone citrate, megestrol acetate, chlormadinone acetate, cyproterone acetate, danazol, aryl estrenolone, Gestrinone, mepanoretricin, raloxifene, honoremeloxifene, reponoremeloxifen, antiestrogen (eg, tamoxifen taenoate, toremifene taenoate, etc.), pill preparation, mepithiostan, test mouth ratataton, aminoglutetimid, LH-RHagonist (eg , Goserelin acetate, pserelin, ryuprorelin, etc.), droloxifene, epitiostano monole, ethurestradiol sunole
  • chemotherapeutic agent examples include an alkylating agent, an antimetabolite, an anticancer antibiotic, a plant-derived anticancer agent and the like.
  • alkylating agent examples include nitrogen mustard, nitrogen mustard hydrochloride-N-oxide, chloramptinole, cyclophosphamide, diphosphamide, zotepa, carbocon, improsulfon tosylate, busulfan, and hydrochloric acid.
  • antimetabolites include, for example, mercaptopurine, 6-mercaptopurine riboside, thioinosine, methotrexate, enositabine, cytarabine, citarabine otafosufate, ancitabine hydrochloride, 5-FU drugs (eg, fluoroperacil , Tegafur, UFT, Doxyfluridine, Carmofur, Garocitabine, Emitefur, etc.), Aminopterin, Leucovorin calcium, Tabloid, Butocin, Forineit calcium, Lepofluorinate calcium, Cladribine, Emitefir, Fludarabine, Gemcitabine, Gemcitabine , Pentostatin, pyrithrexime, idoxyduridine, mitguazone, thiazofurin, ambamustine and the like.
  • 5-FU drugs eg, fluoroperacil , Tegafur, UFT, Doxyfluridine, Carmofur, Garocitabine
  • anticancer antibiotics include actinomycin D, actinomycin C, mitomycin C, chromomycin A3, bleomycin hydrochloride, bleomycin sulfate, ⁇ promycin sulfate, daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride, and hydrochloride hydrochloride. Pyrarubicin, epilubicin hydrochloride, neocarzinostatin, mythramycin, sarcomycin, carcinophilin, mitotane, zolubicin hydrochloride, mitoxantrone hydrochloride, idarubicin hydrochloride and the like.
  • plant-derived anticancer agent examples include etoposide, etoposide phosphate, vinblastine sulfate, vincristine sulfate, vindesine sulfate, teniposide, paclitaxel, docetaxel, vinorelbine and the like.
  • BRM immunotherapeutic agent J
  • examples of the "immunotherapeutic agent (BRM) J include, for example, picibanil, krestin, schizophyllan, lentinan, ubenimetas, interferon, interleukin, macrophage colony stimulating factor, granulocyte colony stimulating factor, erythropoietin, lymphotoxin, BCG vaccine And corynebacterium parvum, levamisole, polysaccharide K, procodazole and the like.
  • the “cell growth factor” in the “drug that inhibits the action of the cell growth factor and its receptor” may be any substance that promotes cell growth, and usually has a molecular weight of 20, Factors that exert an effect at a low concentration by binding to the receptor with a peptide of 000 or less include (1) EGF (epidermal growth factor) or an activity substantially the same as it.
  • EGF E.g., EGF, haledalin (HER2 ligand), etc.
  • Insulin or a substance having substantially the same activity as it e.g., insulin, IGF (insulin-like growth factor) -1, IGF-2, etc.
  • FGF fibroblast growth factor
  • acidic FGF basic FGF
  • KGF fibroblast growth factor
  • CSF colony stimulating factor
  • ⁇ ⁇ erythropoietin
  • IL-2 interleukin-2
  • NGF nerve growth factor
  • PDGF platelet-derived growth factor
  • TGF j3 transforming growth factor; 6
  • HF hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • the “cell growth factor receptor” may be any receptor as long as it has the ability to bind to the above-mentioned cell growth factor, and specifically, EGF receptor, Hallegulin receptor (HER 2), insulin receptor-11, insulin receptor-1, IGF receptor, F.GF receptor-11 or FGF receptor-12, and the like.
  • EGF receptor EGF receptor
  • Hallegulin receptor HER 2
  • insulin receptor-11 insulin receptor-1
  • IGF receptor F.GF receptor-11 or FGF receptor-12
  • Examples of the “drug that inhibits the action of cell growth factor” include Herceptin (HER 2 receptor antibody), Daribec (imatinib mesylate), Iressa (gefitinib, ZD1839), and other oxazole derivatives having a tyrosine kinase inhibitory activity.
  • the salt is preferably a pharmaceutically acceptable salt, for example, a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, and the like.
  • a salt with an inorganic base include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; aluminum salt; ammonium salt and the like.
  • Suitable examples of salts with organic bases include, for example, trimethylamine, triethylamine, pyridine, picolin, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N ,, '-di.
  • Salts with benzylethylenediamine and the like can be mentioned.
  • the salt with an inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like.
  • Preferred examples of salts with organic acids include, for example, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, ⁇ -Salts with toluenesulfonate and the like.
  • Preferable examples of the salt with a basic amino acid include salts with arginine, lysine, ortin and the like.
  • Preferred examples of the salt with an acidic amino acid include salts with aspartic acid, glutamic acid, and the like. Is mentioned.
  • topoisomerase 1I inhibitor eg, irinotecan, topotecan, etc.
  • topoisomerase Ze II inhibitors eg, sobuzoxane
  • differentiation inducers eg, retinoids, vitamins D, etc.
  • angiogenesis inhibitors eg, t
  • Ml 61 antigen of the present invention fusion protein of the present invention or a salt thereof (hereinafter referred to as M
  • the combination of the M161 antigen and the like of the present invention and the concomitant drug is not limited, and the Ml61 antigen and the like of the present invention
  • the concomitant drug may be administered to the subject at the same time or at different times.
  • the dose of the concomitant drug may be in accordance with the dose clinically used, and can be appropriately selected depending on the administration target, administration route, disease, combination and the like.
  • the administration form of the Ml61 antigen and the like of the present invention and the concomitant drug is not particularly limited, as long as the Ml61 antigen and the like of the present invention and the concomitant drug are combined at the time of administration.
  • Such administration forms include, for example, (1) administration of a single preparation obtained by simultaneously preparing the Ml 61 antigen or the like of the present invention and a concomitant drug, (2) Ml 61 of the present invention Simultaneous administration of the two preparations obtained by separately formulating the 61 antigen etc. and the concomitant drug via the same administration route, (3) separately formulating the Ml 61 antigen etc.
  • the concomitant drug (4) different preparations obtained by separately preparing the Ml61 antigen or the like of the present invention and a concomitant drug; Simultaneous administration by administration route, (5) Administration of two types of preparations obtained by separately preparing the Ml 61 antigen or the like of the present invention and a concomitant drug at different time points by different administration routes (for example, Ml 61 antigen etc. of the present invention ⁇ administration in the order of concomitant drugs or administration in the reverse order).
  • these administration forms are collectively abbreviated as the concomitant drug of the present invention.
  • an anticancer combination drug when it is intended for the prevention and treatment of cancer, it is also called an anticancer combination drug.
  • the concomitant drug of the present invention has low toxicity.
  • the Ml61 antigen of the present invention or (and) the above concomitant drug is mixed with a pharmacologically acceptable carrier according to a method known per se to obtain a medicament. It can be a composition.
  • the same carriers as those used in the aforementioned pharmaceutical composition of the present invention can be used.
  • the content of the Ml61 antigen and the like of the present invention in the concomitant drug of the present invention depends on the form of the preparation. Usually, about 0.1 to: 100% by weight of L, preferably about 10 to 99.9% by weight, more preferably about 20 to 90% by weight based on the whole preparation. %.
  • the content of the concomitant drug in the concomitant drug of the present invention varies depending on the form of the preparation. Usually, about 0.1 to 100% by weight, preferably about 10 to 99.9% by weight, based on the whole preparation. More preferably, it is about 20 to 90% by weight.
  • the content of the components other than the Ml61 antigen of the present invention and the concomitant drug may vary depending on the form of the preparation. Generally, about 10 to 99.9% by weight, preferably about 10 to 99.9% by weight based on the whole preparation. Is about 20 to 90% by weight.
  • the mixing ratio of the Ml61 antigen or the like of the present invention and the concomitant drug in the concomitant drug of the present invention can be appropriately selected depending on the administration subject, administration route, disease and the like.
  • the dose of the concomitant drug of the present invention varies depending on the type of the concomitant drug such as the Ml61 antigen of the present invention, the administration route, the symptoms, the age of the patient, etc., for example, for the purpose of treating cancer.
  • ⁇ 4 mg can be administered in 1-3 divided doses.
  • the same content may be used when the Ml61 antigen and the like and the concomitant drug of the present invention are separately formulated.
  • the Ml61 antigen or the like of the present invention and a pharmaceutical composition containing the concomitant drug are administered at the same time.
  • the pharmaceutical composition containing the concomitant drug may be administered, or the pharmaceutical composition containing the M161 antigen or the like of the present invention may be administered. May be administered first, followed by administration of a pharmaceutical composition containing the concomitant drug.
  • the time difference varies depending on the active ingredient, dosage form and administration method to be administered.
  • the pharmaceutical composition containing the concomitant drug is administered first, the pharmaceutical composition containing the concomitant drug is used.
  • the pharmaceutical composition containing the Ml 61 antigen or the like of the present invention is administered first, after administration of the pharmaceutical composition containing the Ml 61 antigen or the like of the present invention, it is preferably within 1 minute to 1 day, preferably 10 minutes or less.
  • the pharmaceutical composition or the concomitant drug of the present invention can be used, for example, for (1) surgery, (2) gene therapy, (3) vasopressor chemotherapy using angiotensin II, (4) hyperthermia, (5) freezing It can be used before or after therapy, (6) laser ablation, (7) radiation therapy, etc., or before or after treatment combining two or three of these.
  • treatment with the pharmaceutical composition or the concomitant drug of the present invention, and supportive care [: (i) antibiotics against complications of various infectious diseases (for example, pansporin etc.) 3-latatams, macrolides such as clarithromycin System)), (ii) administration of high-calorie infusion for improving nutritional disorders, administration of amino acid preparations and multivitamin preparations, (iii) morphine administration for pain relief, '(iv) nausea, vomiting, appetite To administer drugs that improve side effects such as anorexia, diarrhea, leukopenia, thrombocytopenia, decreased hemoglobin concentration, hair loss, liver damage, kidney damage, DIC, fever, and (V) to suppress multidrug resistance of cancer And the like).
  • antibiotics against complications of various infectious diseases for example, pansporin etc. 3-latatams, macrolides such as clarithromycin System
  • administration of high-calorie infusion for improving nutritional disorders administration of amino acid preparations and multivitamin preparations
  • morphine administration for pain relief '(iv) nausea
  • the pharmaceutical composition or the concomitant agent of the present invention is orally administered (including sustained release), intravenous administration (including bolus, infusion, clathrate), subcutaneous and It is preferably administered by intramuscular injection (including bolus, infusion, sustained release), transdermal, intratumoral and proximal administration.
  • the time when the pharmaceutical composition of the present invention or the concomitant drug is administered before the radiation therapy is, for example, about 10 minutes to about 24 hours before surgery or the like.
  • the time when the pharmaceutical composition or the concomitant drug of the present invention is administered after surgery or the like is, for example, about 10 minutes to about 24 hours after surgery or the like.
  • a medicament obtained by combining the Ml61 antigen of the present invention and a cancer antigen can be produced and used in the same manner as in the above-mentioned concomitant drug of the present invention, except that “the concomitant drug” is replaced with “cancer antigen”. Further, the above-mentioned concomitant drug can be used in combination with a medicine obtained by combining the Ml61 antigen and a cancer antigen of the present invention.
  • preferred embodiments of the medicament of the present invention will be described with reference to an example.
  • HER2 a receptor-type protein having tyrosine kinase activity
  • HER2 may be used as a cancer antigen and administered in combination with the Ml61 antigen.
  • HER2 used as a cancer antigen is preferably used as an apoptosis body.
  • Administration of appropriate amounts of M161 antigen and HER2 to HER2-positive patients activates dendritic cells, induces antigen presentation, induces cytokine production, and induces cell-mediated immunity such as CTL You.
  • a concomitant drug such as a tyrosine kinase inhibitor
  • a concomitant drug such as a tyrosine kinase inhibitor
  • an HER2 inhibitor for example, a tyrosine kinase inhibitor of HER2
  • the cancer antigen and the Ml 6.1 antigen or a salt thereof administered in an appropriate amount activate dendritic cells, induce antigen presentation and induce cytokine production, and induce cell-mediated immunity such as CTL.
  • Ml61 antigen of the present invention or a partial peptide thereof or a salt thereof (hereinafter abbreviated as Ml61 antigen of the present invention) and a cancer antigen, and if desired, dendritic cells (preferably immature dendritic cells), macrophages, etc.
  • activated antigen-presenting cells preferably, cells expressing TLR2 and Z or j32 integrin
  • X an anticancer effect
  • a substance that promotes the induction of CTL (substance X3), and / or
  • Substance promoting Thl cell induction (Substance X4)
  • Substances that promote phagocytosis (substance X6)
  • Etc. can be screened.
  • Ml61 antigen (i) cancer antigens and (iii) TLR2 and receptors expressing cells such as Z or 2 integrin (eg, immature dendritic cells, etc.)
  • Substance that alters (inhibits or promotes) the binding of Ml61 antigen or cancer antigen to TLR 2 and Z or 2 integrins (substance ⁇ 8)
  • Etc. can be screened.
  • TLR2 and Z or] 32 integrin Ml61 antigen and cancer antigen were brought into contact with cells expressing TLR2 and Z or] 32 integrin
  • Ml61 antigen and cancer antigen were brought into contact with cells expressing TLR2 and / or 32 integrin.
  • TLR 2 and / or 2 integrin-mediated cell stimulatory activity e.g., promoting cytokine production induction, immature dendritic cell maturation, antiphagocytosis, CTL Substance Xl ⁇ , characterized by measuring and comparing
  • a receptor such as TLR2 and / or j32 integran used in the screening method of the present invention
  • a known receptor can be used as a receptor.
  • the receptor can be extracted from tissues or organs of a warm-blooded animal using a method known per se.
  • receptors that have been expressed in large amounts using genetic recombination technology are suitable for screening.
  • human TLR2 for example, a DNA encoding human TLR2 (Rock, FL et al., Proceeding of the National. USA (Proc. Natl. Accad. Sci. USA), 95, 588-593, 1998; GenBabk AAC 341 33) in mammalian or insect cells. it can.
  • Complementary DNA is usually used as the DNA fragment encoding the receptor of interest, but is not necessarily limited to this.
  • a gene fragment or synthetic DNA may be used.
  • the DNA fragment is required to be a baculovirus belonging to an insect host, such as nucleopolyhedropathy quinores, nuclearpol yh edrosisvirus; It is preferable to incorporate the gene into the downstream of the polyhedrin promoter of NPV), the promoter derived from SV40, the promoter of retro-inoles, the metamouth thionine promoter, the human heat shock promoter, the cytomegalovirus promoter and the SR ⁇ promoter.
  • the amount and quality of the expressed receptor can be detected by a method known per se. P. et al., The Journal of 'Biological' Chemistry (J. Bio 1.
  • the receptor may be a receptor purified according to a method known per se, or a cell containing the receptor or a membrane fraction thereof.
  • the cell when a cell containing a receptor is used, the cell may be fixed with daltaraldehyde, formalin, or the like.
  • the immobilization method can be performed according to a method known per se.
  • the cell containing the receptor refers to a host cell expressing the receptor, and the host cell includes Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like.
  • the membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a method known per se after cell disruption.
  • Cell crushing methods include crushing cells with a Potter-E1Vehj em-type homogenizer, crushing with a Perling Blender ⁇ polytron (Kine 11 matica), crushing with ultrasonic waves, Crushing by forcing cells out of a thin nozzle while applying pressure with a French press or the like can be mentioned.
  • centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used.
  • the cell lysate spin the cell lysate at low speed (500-3000 rpm) for a short period of time (typically about 1-10 minutes), and centrifuge the supernatant at higher speed (15000-30000 rpm) for 30 minutes to 2 hours.
  • the obtained precipitate is used as a membrane fraction.
  • the membrane fraction is rich in the expressed receptor and membrane components such as cell-derived phospholipids and membrane partial peptides.
  • the amount of the receptor cells or membrane fraction containing the receptor, 1 is preferably 10 8 molecules from cells per 10 2, from 10 5 is to 10 7 molecules are preferred.
  • the receptor fraction is preferably a natural receptor fraction or a recombinant receptor fraction having an activity equivalent thereto.
  • the equivalent activity is the Ml 61 antigen Shows the same binding activity and the like.
  • Ml 61 antigen for example, it may be utilized, such as [3 H], [125 I], [14 C], Ml 61 antigen labeled with like [135 S].
  • a cell containing the receptor or a membrane thereof is used.
  • Prepare a sample of the receptor by suspending the sample in a buffer suitable for screening.
  • the buffer may be any buffer such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a buffer which does not inhibit the binding between the M161 antigen and the receptor, such as a tris-monohydrochloride buffer. .
  • a surfactant such as CHAP S, Tween-80 (trade name) (manufactured by Kao-Ichi Atlas Co., Ltd.), digitonin, and deoxycholate can be added to the buffer.
  • a protease inhibitor such as PMSF, leptin, E-64 (manufactured by Peptide Research Institute), or pepstatin can be added for the purpose of suppressing the degradation of the receptor and Ml61 antigen by protease.
  • the Ml 61 antigen receptor solution from 0.
  • NS beta as 100% specific binding
  • a test compound having 50% or less can be selected as a candidate substance having competitive inhibition ability, while a test compound having a specific binding amount ( ⁇ -NSB) of 150% or more has binding promoting ability. It can be selected as a candidate compound.
  • the cell stimulating activity through the receptor for example, cytokine inducing action, immaturity
  • the receptor for example, cytokine inducing action, immaturity
  • the cell stimulating activity through the receptor for example, cytokine inducing action, immaturity
  • CTL induction for example, Thl cell induction, arachidonic acid release, acetylcholine release, intracellular Ca2 + release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production
  • cells containing the receptor are cultured in a multiwell plate or the like. Before performing screening, replace the cells with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., incubate for a certain period of time, and then extract cells or collect supernatant. Quantify the product produced according to each method.
  • a substance for example, arachidonic acid
  • an inhibitor for the degrading enzyme may be added to perform the assay.
  • activities such as cAMP production suppression can be detected as production suppression effects on cells whose basic production has been increased by forskolin or the like.
  • cells expressing an appropriate receptor are required.
  • a cell line having a natural receptor eg, immature dendritic cells
  • a cell line expressing the above-mentioned recombinant receptor, and the like are preferable.
  • Substances XI to X8 are classified into those that act on the M61 antigen (substance XI), those that act on the receptor (substances X7 to 8), or those that express the receptor or cells induced by it. 'It can be distinguished by acting on the sexual immune system (substances X 2-6).
  • test compounds for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. are used, and these compounds are novel compounds. Or a known compound.
  • the screening kit for substance X e.g., X1 to X8 contains Ml61 antigen, a cancer antigen, and a cell containing a receptor or an extract fraction of a cell containing a receptor. is there.
  • Examples of the screening kit of the present invention include the following.
  • Receptor sample '' CHO cells expressing the receptor were subcultured on a 12-well plate with 5 x 10 5 holes.
  • PMB [(B-NS B) / (B. One NSB)] X 100 PMB: Percent Maximum Binding
  • the substance obtained by using the screening method or the screening kit of the present invention is a substance (X) having an anticancer effect, specifically,
  • Substances that activate TLR 2 and Z or] 32 integrins (substance X7), ⁇ ⁇ Alter (especially promote) the binding of Ml61 antigen and cancer antigen to TLR 2 and Z or 2 integrins Substance (Substance X8)
  • Examples of the substance include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like. These compounds may be novel compounds or known compounds.
  • salt of the substance those similar to the above-mentioned salts of the fusion protein of the present invention are used.
  • a pharmaceutical composition containing the substance X (e.g., X 1 to X8) obtained by the screening method of the present invention is a safe anti-phagocytic agent, a cytokine induction promoter, a immature dendritic cell for mammals. It is useful as a maturation inducer, CTL inducer, Thl cell inducer, and the like.
  • cancer eg, breast, prostate, knee, stomach, lung, colon (colon, rectum, anal), esophagus, duodenum, head and neck (tongue, pharyngeal), brain tumor , Schwannomas, non-small cell lung cancer, small cell lung cancer, liver cancer, kidney cancer, bile duct cancer, uterine cancer (child cancer, cervical cancer), ovarian cancer, bladder cancer, skin cancer, hemangiomas, malignant lymphoma , Malignant black Tumors, thyroid cancer, bone tumors, hemangiomas, angiofibromas, retinal sarcomas, penile carcinomas, pediatric solid tumors, positron sarcomas, positron sarcomas due to AIDS, maxillary sinus tumors, fibrous histiocytomas, smooth It is useful as a preventive / therapeutic agent for malignant tumors such as sarcomas and rhabdomyosarcomas) and leukemia
  • a pharmaceutical composition containing these substances X can be produced and used in the same manner as the above-mentioned pharmaceutical composition containing the fusion protein of the present invention.
  • Substances that inhibit the induction of Thl cells (substance Y4)
  • Substances that inhibit phagocytosis (of dendritic cells) (substance Y6),
  • ⁇ A substance that inhibits the activity of TLR 2 and / or j32 integrin (substance Y 7), ⁇ A substance that changes (particularly inhibits) the binding of Ml61 antigen and cancer antigen to TLR 2 and Z or 32 integrin (Substance ⁇ 8)
  • Such a substance can be used in the same manner as the substances ⁇ and C1 described below.
  • the action of the fusion protein of the present invention by using the fusion protein of the present invention or a salt thereof (hereinafter abbreviated as the fusion protein of the present invention) and a receptor such as TLR2 and ⁇ or] 32 integrin, (1) the action of the fusion protein of the present invention Substance that enhances ():
  • Etc. can be screened.
  • TLR 2 and / or] 32 integrin-expressing cells eg, ⁇ -1 cells, macrophages
  • fusion protein of the present invention When the fusion protein of the present invention is contacted with cells expressing TLR2 and ⁇ or 02 integrin, and when the fusion protein and test compound of the present invention are contacted with cells expressing TLR2 and Z or 2 integrin Cell stimulatory activity via TLR 2 and ⁇ or J32 integrin (eg, induces cytokinin production, matures immature dendritic cells, promotes phagocytosis, induces CTL, induces Thl cells
  • the present invention provides a method for screening for substances A, B or C, characterized by measuring and comparing the effects.
  • a receptor such as TLR 2 and Z or 2 integrin used in the screening method of the present invention
  • a known receptor can be used as a receptor such as TLR 2 and Z or 2 integrin used in the screening method of the present invention.
  • the receptor can be extracted from tissues or organs of a warm-blooded animal using a method known per se. However, it is extremely difficult to obtain human-derived organs. Because of the difficulty, receptors that are expressed in large amounts using genetic recombination techniques are suitable for screening.
  • human TLR2 for example, the DNA encoding human TLR2 (Rock, FL et al., Proceeding of National Inc. USA (Proc. Natl. Acc. Sci. USA), Vol. 95, 588-593, 1998; GenBabk AAC 341 33) in mammalian or insect cells. it can. '
  • Complementary DNA is usually used as a DNA fragment encoding the receptor of interest, but is not necessarily limited thereto.
  • a gene fragment or a synthetic DNA may be used.
  • NP V nucleopolyhedrovirus and nuclearpol yh edrosisvirus.
  • NP V nucleopolyhedrovirus and nuclearpol yh edrosisvirus.
  • NP V nuclearpolyh edrosisvirus
  • the amount and quality of the expressed receptor can be detected by a method known per se. For example, it is described in the literature [Namb i. P. et al., The 'Journal of Biological' Chemistry (J. Biol. Chem.), 267, 19555-199559, 1992]. This can be done according to the method described above.
  • the receptor may be a receptor purified according to a method known per se, or a cell containing the receptor or a membrane fraction thereof may be used. .
  • the cell When a cell containing a receptor is used in the screening method of the present invention, the cell may be fixed with daltaraldehyde, formalin, or the like.
  • the immobilization method can be performed according to a method known per se.
  • the cells containing the receptor include host cells expressing the receptor.
  • Cells include E. coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like.
  • the membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a method known per se after cell disruption. Cells can be disrupted by crushing the cells with a Potter-E 1 Vehj em-type homogenizer, crushing with a Perling Plender ⁇ ⁇ Polytron (Kine 11 matica), sonication, Crushing by forcing cells out of a thin nozzle while applying pressure with a French press or the like can be mentioned.
  • centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used.
  • the cell lysate is centrifuged at low speed (500 to 3000 rpm) for a short time (typically about 1 to 10 minutes), and the supernatant is centrifuged at a higher speed (15000 to 30000 rpm) for 30 minutes to 2 hours.
  • the obtained precipitate is used as a membrane fraction.
  • the membrane fraction is rich in the expressed receptor and membrane components such as cell-derived phospholipids and membrane partial peptides.
  • the amount of the receptor cells or membrane fraction containing the receptor, 1 is preferably 10 8 molecules from cells per 10 2, from 10 5 is to 10 7 molecules are preferred.
  • an appropriate receptor fraction and a labeled fusion protein of the present invention are required.
  • the receptor fraction a natural receptor fraction or a recombinant receptor fraction having an activity equivalent thereto is desirable.
  • equivalent activity means equivalent binding activity to the fusion protein of the present invention.
  • the fusion protein labeled the present invention for example, [3 H], [125 1] (14
  • a cell containing the receptor or its membrane fraction is screened.
  • a receptor sample is prepared by suspending in a buffer suitable for screening.
  • the buffer contains a pH of 4 to 10 (preferably pH 6 to 8).
  • a buffer that does not inhibit the binding of the fusion protein of the present invention to a receptor such as a phosphate buffer or a Tris-HCl buffer! / It may be shifted.
  • a surfactant such as CHAPS, Tween-80 (trade name) (manufactured by Kao-Ichi Atlas Co., Ltd.), digitonin, and deoxycholate can be added to the buffer.
  • protease inhibitors such as PMSF, leptin, E-64 (manufactured by Peptide Research Laboratories) and pepstatin can be added for the purpose of suppressing the degradation of the receptor and the fusion protein of the present invention by the protease.
  • the Ml 61 antigen receptor solution from 0.
  • test compound with 50% or less can be selected as a candidate substance with competitive inhibition ability, while a test compound with a specific binding amount (B-NSB) of, for example, 150% or more has the ability to promote binding. It can be selected as a candidate compound.
  • a receptor-mediated cell stimulating activity for example, a cytokine-inducing action, an immature dendritic cell maturation-inducing action, Phagocytosis-promoting action, tumor immune mechanism activating action, CTL-inducing action, Thl cell-inducing action, arachidonic acid release, acetylcholine release, intracellular Ca2 + release, intracellular cAMP production, intracellular cGMP production, Activity to promote or suppress inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, decrease in pH, etc.) using known methods or commercially available measurement kits. Can be measured.
  • a receptor-mediated cell stimulating activity for example, a cytokine-inducing action, an immature dendritic cell maturation-inducing action, Phagocytosis-promoting action, tumor immune mechanism activating action, CTL-inducing action, Thl cell-inducing action, arachidonic acid
  • cells containing the receptor are cultured in a multiwell plate or the like. Poison fresh media or cells before screening After replacing the buffer with an appropriate buffer that does not show the property, add the test compound, etc., incubate for a certain period of time, extract the cells or collect the supernatant, and quantitate the generated product according to each method. I do.
  • a substance for example, arachidonic acid
  • an inhibitor for the degrading enzyme may be added to perform the assay.
  • activities such as inhibition of cAMP production can be detected as production inhibitory effects on cells whose basic production has been increased by forskolin or the like.
  • cells expressing an appropriate receptor are required.
  • a cell line having a natural receptor, a cell line expressing the above-mentioned recombinant receptor and the like are desirable.
  • the classification of substances A and B and substance C can be distinguished by directly acting on the fusion protein of the present invention or binding to the receptor. That is, among the substances obtained by the screening method [3], the substances not selected by the screening method [1] or [2] are the substances A or B, and are selected by the screening method [1] or [2].
  • the substance can be determined to be substance C.
  • test compounds for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. are used, and these compounds are novel compounds. Or a known compound.
  • the screening kit for the substance A, B or C contains the fusion protein of the present invention, a cell containing the receptor or an extract fraction of a cell containing the receptor. Examples of the screening kit of the present invention include the following.
  • It may be sterilized by filtration through a 0.45 ⁇ filter and stored at 4 ° C, or may be prepared at use.
  • CHO cells expressing the receptor and passaged 1 2 well plates at 5 X 1 0 5 or Z holes, 3 7. C, 5% C0 2, 9 with 5% air 2 days followed by culturing.
  • the fusion protein of the present invention is dissolved to a concentration of 1 mM in PBS containing 0.1% serum albumin (manufactured by Sigma) and stored at 120 ° C.
  • the substance obtained by using the screening method or the screening kit of the present invention is, as described above,
  • An antagonist which binds to a receptor, but has a cell-stimulating activity through the receptor (eg, promoting phagocytosis, activating the tumor immune system, inducing cytokines, inducing immature dendritic cell maturation, etc.)
  • Substances that do not show promoting or inhibiting activities (substance C 1))
  • 2Agonist A substance that binds to a receptor and exhibits a cell-stimulating activity through the receptor (Substance
  • Examples of the substance include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like. These compounds may be novel compounds or known compounds.
  • salt of the substance those similar to the above-mentioned salts of the fusion protein of the present invention are used.
  • a pharmaceutical composition containing the substance A can be used to safely promote poor food intake in mammals. It is useful as an agent, a cytokine induction promoter, an agent for inducing the maturation of immature dendritic cells, and the like.
  • cancer eg, breast, prostate, Teng, stomach, lung, colon, colon, rectum, anal
  • esophagus duodenum, head and neck (tongue, pharyngeal)
  • brain tumor eg., Schwannomas, non-small cell lung cancer, small cell lung cancer, liver cancer, kidney cancer, bile duct cancer, uterine cancer (uterine body cancer, cervical cancer), ovarian cancer, bladder cancer, skin cancer, hemangiomas, malignant Lymphoma, malignant melanoma, thyroid cancer, bone tumor, hemangiomas, hemangiofibromas, retinal sarcoma, penile cancer, pediatric solid carcinoma, positive sarcoma, positive sarcoma due to AIDS, maxillary sinus tumor, fibrous histiocytoma , Leiomyosarcoma, rhabdomyosarcoma, etc.), prevention of malignant tumors such as leukemia- Useful as a therapeutic.
  • a pharmaceutical composition containing the substance B can inhibit safe cytokine induction in mammals. It is useful as an agent (inhibitor of cytokine production), inhibitor of maturation of immature dendritic cells, inhibitor of phagocytosis, inhibition of CTL induction, etc. Furthermore, it is useful as a preventive and therapeutic agent for allergic diseases and autoimmune diseases. Since the substance C 1 (antagonist) obtained by the screening method of the present invention can suppress the action of the fusion protein of the present invention or a salt thereof, a pharmaceutical composition containing the substance C 1 can be used in mammals.
  • cytokine induction inhibitor site force-in production inhibitor
  • an inhibitor of immature dendritic cell maturation induction an antiphagocytic inhibitor
  • a CTL induction inhibitor a CTL induction inhibitor
  • it is useful as a prophylactic / therapeutic agent for allergic diseases and autoimmune diseases.
  • substance C 2 (agonist) obtained by the screening method of the present invention has the same action as the fusion protein of the present invention or a salt thereof, a pharmaceutical composition containing the substance C 2 Is useful as a safe cytokine inducer for mammals, an inducer of maturation of immature dendritic cells, an antiphagocytic agent, a CTL inducer, and the like.
  • cancer eg, breast, prostate, knee, stomach, lung, colon (colon, rectum, anal), esophagus, duodenum, head and neck (tongue, pharyngeal), brain tumor , Schwannomas, non-small cell lung cancer, small cell lung cancer, liver cancer, kidney cancer, bile duct cancer, uterine cancer (uterine body cancer, cervical cancer), ovarian cancer, bladder cancer, skin cancer, hemangiomas, malignant lymphoma , Malignant melanoma, thyroid cancer, bone tumor, hemangiomas, hemangiofibromas, retinal sarcoma, penile cancer, pediatric solid carcinoma, positron sarcoma, positron sarcoma due to AIDS, maxillary sinus tumor, fibrous histiosphere Tumors, leiomyosarcoma, rhabdomyosarcoma, etc.), leukemia and other malignant tumors.
  • an N-terminal partial peptide containing M161 antigen or a binding lipid thereof is used as the ligand or agonist for TLR2.
  • Ml61 antigen or a partial peptide thereof is used as the ligand or agonist for ⁇ 2 integrin.
  • Examples of the substance that activates TLR 2 and / or 2 integrin include Ml61 antigen, the fusion protein of the present invention, substance C2 or a salt thereof, and the like.
  • the pharmaceutical composition containing the fusion protein of the present invention or the substance C2 or a salt thereof and the dose to a mammal are the same as described above.
  • bases, amino acids, and the like are indicated by abbreviations, which are based on the abbreviations based on I UPAC-I UB Commionion Biochemical Nomenclature or the abbreviations commonly used in the art. See below.
  • amino acids can have optical isomers, the L-form is indicated unless otherwise specified.
  • Trt Trityl
  • HOB t 1—Hydroxybenztriazole
  • sequence numbers in the sequence listing in the present specification indicate the following sequences.
  • a mixture of 10.0 g of Ml 61 antigen, 60.0 g of lactose and 35.0 g of corn starch was granulated through a 1 ⁇ mesh sieve using 30 ml of a 10% by weight aqueous gelatin solution (3.0 g as gelatin). After drying, it was dried at 40 ° C and sieved again. The obtained granules were mixed with 2.0 g of magnesium stearate and compressed. The resulting central tablets were coated with a sucrose, titanium dioxide, talc and gum arabic suspension in water. The coated tablets were polished with beeswax to give 100 tablets.
  • Ml 61 Antigen 10 Og and magnesium stearate 3.0 g were granulated with 70 ml of an aqueous solution of soluble starch (7.0 g as soluble starch), dried, lactose 70.0 g and corn starch 50 0 g. The mixture was compressed to give 100 tablets.
  • Test Example 1 Antitumor activity of M161 antigen
  • Ml 6 1 antigen (2. 5 ⁇ g) destruction tumor (B 1 6 melanoma, 5 X 1 0 6) and four times with subcutaneous injection to the co, every 7 days Eight C 5 7 BL / 6 prei Thigh unization and then Tumors (B16 melanoma, 5 ⁇ 10 6 ) were inoculated. Four weeks later, the size of the tumor was measured from above the skin with a vernier caliper. Table 1 shows the results.
  • the Ml61 antigen or its partial peptide or a salt thereof has an excellent CTL inducing action.
  • cancer can be efficiently prevented or treated.

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Abstract

La présente invention concerne l'antigène M161, son fragment peptidique ou ses sels qui ont un excellent effet d'induction CTL, etc. L'utilisation combinée de l'antigène M161, de son fragment peptidique ou de ses sels, avec un antigène cancéreux rend possible une prévention/un traitement efficace du cancer.
PCT/JP2002/005916 2001-06-15 2002-06-13 Agents anticancereux WO2002102402A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005041982A1 (fr) * 2003-10-31 2005-05-12 Kurume University Therapie combinant un vaccin peptidique et un traitement a l'estramustine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000073432A2 (fr) * 1999-06-01 2000-12-07 Cornell Research Foundation, Inc. Activation de cellules dendritiques pour accroitre l'immunite

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000073432A2 (fr) * 1999-06-01 2000-12-07 Cornell Research Foundation, Inc. Activation de cellules dendritiques pour accroitre l'immunite

Non-Patent Citations (3)

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Title
KAUFMANN A. ET AL.: "Induction of cytokines and chemokines in human monocytes by mycoplasma fermentants-derived lipoprotein MALP-2", INFECT. IMMUN., vol. 67, no. 12, 1999, pages 6303 - 6308, XP002203724 *
NISHIGUCHI M.: "Mycoplasma fermentans lipoprotein M161Ag-induced cell activation is mediated by toll-like receptor 2: role of N-terminal hydrophobic portion in its multiple functions", J. IMMUNOL., vol. 166, no. 4, February 2001 (2001-02-01), pages 2610 - 2616, XP002951598 *
XIANG JIM ET AL.: "Efficient antitumor immunity derived from enhancedmaturation of dendritic cells which hadphagocytosed apoptotic tumor cells", PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL MEETING, vol. 42, March 2001 (2001-03-01), pages 25, XP002955897 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005041982A1 (fr) * 2003-10-31 2005-05-12 Kurume University Therapie combinant un vaccin peptidique et un traitement a l'estramustine
WO2005041981A1 (fr) * 2003-10-31 2005-05-12 Kurume University Traitement combine vaccin peptidique et estramustine

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