WO2012023631A1 - Pharmaceutical composition for inhibiting proliferation of cancer cells which comprises heat shock protein (hsp) and eci301 polypeptide, and cancer treatment method using same - Google Patents

Pharmaceutical composition for inhibiting proliferation of cancer cells which comprises heat shock protein (hsp) and eci301 polypeptide, and cancer treatment method using same Download PDF

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WO2012023631A1
WO2012023631A1 PCT/JP2011/069107 JP2011069107W WO2012023631A1 WO 2012023631 A1 WO2012023631 A1 WO 2012023631A1 JP 2011069107 W JP2011069107 W JP 2011069107W WO 2012023631 A1 WO2012023631 A1 WO 2012023631A1
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eci301
cancer
hsp70
hsp
heat shock
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PCT/JP2011/069107
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French (fr)
Japanese (ja)
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祥司 横地
義郎 石渡
麻里子 石原
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株式会社Eci
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]

Definitions

  • the present invention relates to a pharmaceutical composition for treating cancer comprising heat shock protein (hereinafter referred to as “HSP”) and ECI301 polypeptide, and a method for treating cancer using the same.
  • HSP heat shock protein
  • ECI301 polypeptide ECI301 polypeptide
  • Cancer is defined as a malignant neoplasm anywhere in the human or animal body. Cancer destroys adjacent tissues by its uncontrolled growth and ultimately causes body death by physical blockage of vessels and organs (Non-Patent Document 1).
  • cancer treatment methods include surgery, radiation therapy, hyperthermia, chemotherapy, gene therapy, immunotherapy, and the like. However, these treatment methods are not effective in treating cancers that are distant from the treated site (such as metastatic cancer), and there are cancer cells that are less sensitive to treatment with severe side effects.
  • each method has various drawbacks such as being effective only for a specific limited patient, and a new method for cancer treatment is still required. In order to solve the above problems, a method of using HSP for cancer treatment has been developed in recent years.
  • HSP also called stress protein
  • HSP refers to a family of proteins that are universally expressed in almost all types of organisms from bacteria to humans. HSPs are classified into five families based on molecular weight, including HSP100, HSP90, HSP70, HSP60 and small HSP. Many members of these families are associated with heat shock and physiological stress (eg, environmental stimuli and developmental effects such as thermal, anoxia, alcohol, glucose starvation (glucose regulatory proteins that are also subgroups of HSP (ie GRP )), Expression is induced in response to tissue damage, nutritional deficiency, metabolic destruction, oxygen radicals, infection by intracellular pathogens, etc. (Non-patent Documents 2-6).
  • HSP is normally expressed at low to moderate levels in cells, but is expressed and accumulated at very high levels in cells when subjected to heat shock or physiological stress.
  • mammalian HSP70 is expressed at a low level that is difficult to detect in cells not subjected to heat shock, but is the protein expressed at the highest level in cells subjected to heat shock.
  • Non-Patent Document 7 Also, most (but not all) mammalian HSP90 and HSP60 are expressed at high levels in cells that have not undergone heat shock, but further expression is induced in cells that have undergone heat shock.
  • HSP is expressed and accumulated in cells in adverse conditions that have been subjected to heat shock and other physiological stresses, and has a function of protecting the cells (Non-Patent Document 10). Furthermore, HSP has been shown to be involved in biochemical and immunological processes even in cells that are not subjected to heat shock or other physiological stresses. That is, HSP has various types of chaperone functions. For example, members of the HSP70 family are present in the cytoplasm, nucleus, mitochondria, or endoplasmic reticulum (Non-patent Document 11), and protein translocation, folding and assembly. , As well as being involved in antigen presentation to cells of the immune system (Non-patent Document 12).
  • HSP inhibitor HSP is expressed and accumulated in cells in a disadvantageous state subjected to heat shock and other physiological stress, and has a function of protecting the cells. Therefore, HSP also has a cytoprotective action in cancer cells. It is thought that is fulfilled. In fact, it has been clarified that inhibition of the function of HSP90 results in selective degradation of signaling oncoproteins involved in cell proliferation, cell cycle control and apoptosis in cancer cells (Non-patent Document 13). . However, at present, no promising effect has been found in cancer treatment, and benzoquinone ansamycin and the like have been found as HSP90 inhibitors that can be used.
  • HSP-peptide complex Although HSP itself is not immunogenic, it forms a complex by noncovalently binding to an antigen peptide such as a cancer antigen, and the complex is an immunity specific for the antigen peptide.
  • Non-patent Documents 14-16; Patent Documents 1-3 The HSP-peptide complex is intended to activate specific immunity and remove cancer cells by taking specific or non-specific tumor antigens together with HSP into dendritic cells and dramatically improving antigen presentation. .
  • cancers vary greatly in antigen, and it is considered difficult to simply purify “immunogenic antigens” that can be used as antigens for all cancers.
  • the cancer antigen utilized for the complex is limited to a specific one, and the effect of the complex is also limited to a specific cancer type.
  • a non-specific antigen such as binding a tumor lysate directly to HSP without isolating and purifying the antigen
  • CMC Chemistry / Manufacture / Control
  • the present invention provides a therapeutic agent and a therapeutic method that are effective against various types of cancer regardless of the type of cancer (ie, different antigenicity).
  • the present invention was completed by finding that the product can significantly inhibit the growth of local and distant metastatic cancers, and both, regardless of the type of cancer. That is, the present invention has the following features.
  • a pharmaceutical composition for treating cancer comprising heat shock protein (HSP) 70 and a polypeptide represented by SEQ ID NO: 1 (ECI301).
  • [5] A combined medicine of heat shock protein (HSP) 70 and the polypeptide represented by SEQ ID NO: 1 (ECI301) for treating cancer.
  • a method for treating cancer comprising administering heat shock protein (HSP) 70 and the polypeptide represented by SEQ ID NO: 1 (ECI301) separately or simultaneously to a cancer patient.
  • the method of [8], wherein the cancer is a solid cancer.
  • the method of [9], wherein the solid cancer is lung cancer, colon cancer, or fibrosarcoma.
  • Heat shock protein (HSP) 70 and polypeptide represented by SEQ ID NO: 1 (ECI301) are administered to cancer patients at a ratio of 1: 0.4 to 16 by weight ratio [8] to [10] Either way.
  • Heat shock protein (HSP) 70 by weight ratio and polypeptide (ECI301) represented by SEQ ID NO: 1 are administered to cancer patients at a ratio of 1: 0.4 to 16, and used [12]
  • This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2010-183651 which is the basis of the priority of the present application.
  • FIG. 1 shows the amino acid sequence of ECI301, a derivative of human MIP-1 ⁇ .
  • FIG. 2 is a graph showing changes in volume of tumor tissue in mice over time by administration of HSP70 or HSP70 / ECI301 into the tail vein (iv).
  • FIG. 3 is a graph showing the volume of tumor tissue in each administration group in mice administered with HSP70 or HSP70 / ECI301 intravenously (iv) in the tail vein.
  • FIG. 4 shows tail vein (iv) administration of a formulation containing HSP70 at various doses (I: 0.125 ⁇ g; II: 0.5 ⁇ g; III: 5.0 ⁇ g) against ECI301 (2.0 ⁇ g) It is a graph which shows the volume of the tumor tissue in the done mouse.
  • FIG. 1 shows the amino acid sequence of ECI301, a derivative of human MIP-1 ⁇ .
  • FIG. 2 is a graph showing changes in volume of tumor tissue in mice over time by administration of HSP70 or HSP70 / ECI301 into the
  • HSP70 and ECI301 are each included in the following doses: i: 0.5 ⁇ g: 0; (ii: 0.1 ⁇ g: 0.4 ⁇ g; iii: 0.5 ⁇ g: 2.0 ⁇ g; iv: 2.5 ⁇ g: 10.0 ⁇ g) are administered in the tail vein (iv) and are graphs showing the volume of tumor tissue in mice. is there.
  • HSP70 and ECI301 are each included in the following doses: (I): 0.5 ⁇ g: 0; (II): 0.1 ⁇ g: 0.4 ⁇ g; (III): 0.5 ⁇ g: 2.0 ⁇ g) or a combination preparation containing HSP70 and human MIP-1 ⁇ (HSP70 and human MIP-1 ⁇ are included in the following doses, respectively) (IV): 0.1 ⁇ g: 0.4 ⁇ g; (V) : 0.5 ⁇ g: 2.0 ⁇ g) is a graph showing the volume of tumor tissue in mice administered intravenously (iv) in the tail vein.
  • FIG. 7 shows (A) a formulation containing HSP70 and ECI301 (containing HSP70 and ECI301 at doses of 0.5 ⁇ g and 2.0 ⁇ g, respectively) or (B) HSP70 and then ECI301 alone, respectively, in the tail vein ( iv) A graph showing the volume of tumor tissue in administered mice.
  • HSP70 and ECI301 are included at doses of 0.5 ⁇ g and 2.0 ⁇ g, respectively (i)) or a combination containing HSP60 and ECI301 (HSP60 and ECI301 respectively below)
  • Ii 0.5 ⁇ g: 0;
  • FIG. 9 is a graph showing changes over time in the volume of tumor tissue in mice by single administration of HSP70 or HSP90 or co-administration (iv) with ECI301.
  • FIG. 10 is a graph showing the volume of tumor tissue in mice by single administration of HSP70 or HSP90 or co-administration (iv) with ECI301.
  • FIG. 11 is a graph showing temporal changes in the volume of Colon 26 tumor tissue in mice after HSP70 or HSP70 / ECI301 administration into the tail vein (iv).
  • FIG. 12 is a graph showing changes over time in the volume of Lewis lung cancer tissue in mice after HSP70 or HSP70 / ECI301 administration into the tail vein (iv).
  • FIG. 13 is a graph showing changes over time in the volume of MethA tumor tissue in mice by HSP70 or HSP70 / ECI301 administration into the tail vein (iv).
  • FIG. 14 shows the results of an immunoprecipitation experiment performed using anti-MIP-1 ⁇ antibody-binding beads and ECI301 or anti-HSP70 antibody-binding beads and HSP70. The following samples are shown for each lane.
  • Lane 1-3 Anti-MIP-1 ⁇ antibody-binding beads; Lane 1: ECI301; Lane 2: ECI301 + HSP70; Lane 3: ECI301 + ⁇ MIP-1 ⁇ -biotin; Lane 4-6: Anti-HSP70 antibody-binding beads; Lane 4: HSP70; Lane 5 : HSP70 + ECI301; Lane 6: HSP70 + MIP-1 ⁇ ; Lane 7: molecular weight marker.
  • Primary antibody rabbit anti-MIP-1 ⁇ polyclonal-biotin label; secondary antibody: streptavidin-HRP label.
  • FIG. 15 shows the results of an immunoprecipitation experiment using anti-MIP-1 ⁇ antibody-binding beads and ECI301 or anti-HSP70 antibody-binding beads and HSP70.
  • Lane 1-3 Anti-MIP-1 ⁇ antibody-binding beads
  • Lane 1 ECI301
  • Lane 2 ECI301 + HSP70
  • Lane 3 ECI301 + ⁇ MIP-1 ⁇ -biotin
  • Lane 4-6 Anti-HSP70 antibody-binding beads
  • Lane 4 HSP70
  • Lane 5 HSP70 + ECI301
  • Lane 6 HSP70 + MIP-1 ⁇
  • Lane 7 molecular weight marker.
  • Primary antibody mouse anti-HSP70
  • secondary antibody anti-mouse IgG-HRP label.
  • the present invention provides a pharmaceutical composition for treating cancer, comprising heat shock protein (HSP) and the polypeptide represented by SEQ ID NO: 1 (ECI301).
  • HSP heat shock protein
  • the heat shock protein that can be used in the present invention includes all heat shock proteins except HSP90 and HSP60, and is preferably HSP70 protein.
  • the HSP70 protein is not particularly limited, but is preferably derived from human. By using a human-derived HSP70 protein, it is possible to minimize the immune rejection that occurs when administered to a human patient and increase the therapeutic effect.
  • the HSP70 protein is known, isolated from various biological species, and sequence information is disclosed.
  • the sequence information of human-derived HSP70 protein is registered in GenBank as accession numbers NP_002145, AAI26125, etc.
  • the sequence information of mouse-derived HSP70 protein is registered in GenBank as accession numbers NM_024172, etc., and these can be used.
  • the HSP70 protein having the amino acid sequence represented by SEQ ID NO: 2 can be used.
  • the “HSP70 protein” includes one to several amino acid deletions, substitutions, additions or insertions in the amino acid sequence represented by SEQ ID NO: 2, and the amino acid represented by SEQ ID NO: 2. Also included are proteins having the same activity as the HSP70 protein having the sequence.
  • “Same activity as HSP70 protein” means an activity that inhibits the growth of cancer cells when administered with an ECI301 polypeptide as detailed below.
  • the range of “1 to several” is not particularly limited, but for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, Particularly preferably, it is 1 to 3, or 1 or 2.
  • the “HSP70 protein” includes the amino acid sequence represented by SEQ ID NO: 2 and BLAST (Basic Local Alignment Search at the National Center for Biological Information) (basic local alignment search tool of the National Center for Biological Information). ) Etc.
  • the “HSP70 protein” includes an ortholog of the HSP70 protein having the amino acid sequence shown by SEQ ID NO: 2.
  • “HSP70 protein” itself may or may not have the activity of inhibiting the growth of cancer cells by administration alone.
  • the polypeptide represented by SEQ ID NO: 1 is a known polypeptide also referred to as “ECI301” (ECI Corporation).
  • ECI301 is a derivative of macrophage inflammatory protein-1 ⁇ (hereinafter referred to as “MIP-1 ⁇ ”), which is one of the chemotactic proteins that exist in the human body and has an immunostimulatory action, and the amino acid sequence of MIP-1 ⁇
  • MIP-1 ⁇ macrophage inflammatory protein-1 ⁇
  • FIG. 1 the aspartic acid at position 26 is a polypeptide consisting of 69 amino acids substituted with alanine (FIG. 1).
  • ECI301 can be used in combination with radiotherapy to achieve a remarkable tumor reduction effect and life extension effect including complete remission, and a reduction effect on tumors at non-irradiated sites not found in other anticancer agents (Shiraishi). K. et al., Clin Cancer Res. 2008 Feb 15; 14 (4): 1159-66).
  • ECI301 also includes a polypeptide having the deletion, substitution, addition or insertion of one to several amino acids in the amino acid sequence represented by SEQ ID NO: 1 and having the same activity as ECI301. included.
  • the same activity as ECI301 means an activity that inhibits the growth of cancer cells when administered together with the HSP70 protein.
  • ECI301 contains 90% or more, more preferably 95%, when calculated using the amino acid sequence shown in SEQ ID NO: 1, BLAST, etc. (for example, default or default parameters). As described above, a polypeptide having a sequence identity of 99% or more and having the same activity as ECI301 is also included. In the present invention, ECI301 itself may or may not have the activity of inhibiting the growth of cancer cells when administered alone. HSP70 protein and ECI301 can be produced and purified using genetic engineering techniques, as is well known to those skilled in the art. That is, DNA encoding the protein can be incorporated into an appropriate vector, the vector can be introduced into an appropriate host cell, and the protein can be expressed.
  • host cells well-known bacterial cells such as E. coli, insect cells such as yeast, SF9, and SF21, animal and plant cells such as COS1, COS7, CHO, HEK293, and the like can be used.
  • the expressed protein is obtained from the culture supernatant of the host cell by known methods used for protein purification, such as ammonium sulfate salting out, precipitation separation with an organic solvent (ethanol, methanol, acetone, etc.), ion exchange chromatography, isoelectricity.
  • HSP70 protein and / or ECI301 can be modified as appropriate to impart desired properties. For example, degradation in vivo can be suppressed or delayed by PEGylating HSP70 protein and / or ECI301.
  • the pharmaceutical composition of the present invention comprises, as an active ingredient, DNA encoding HSP70 protein and DNA encoding ECI301, or an expression vector containing DNA encoding HSP70 protein and DNA encoding ECI301. Containing expression vectors.
  • the DNA contained in the pharmaceutical composition is transcribed and translated in the introduced target cell, and can express and secrete HSP70 protein and / or ECI301.
  • a DNA encoding the HSP70 protein a DNA having a known nucleotide sequence registered in GenBank or the like can be used.
  • DNA (SEQ ID NO: 3) encoding HSP70 protein having the amino acid sequence shown by SEQ ID NO: 2 can be used.
  • the DNA encoding ECI301 is prepared by a known method such as specific mutagenesis by PCR, in which the base sequence encoding alanine is inserted into the base sequence site encoding aspartic acid at position 26 in the base sequence of human MIP-1 ⁇ . Although it can be obtained by introduction, DNA consisting of the base sequence represented by SEQ ID NO: 4 can be preferably used.
  • “DNA encoding HSP70 protein” and “DNA encoding ECI301” include a DNA encoding a secretory signal peptide at its 5 ′ end, 3 ′ end or any site, as necessary. Can be connected in a functional way.
  • any known signal can be used without particular limitation as long as the HSP70 protein and ECI301 transcribed and translated in the target cell can be secreted outside the cell.
  • “Link in a functional manner” means that the DNA encoding the HSP70 protein or ECI301 and the DNA encoding the secretory signal peptide so that the secretory signal peptide is accurately expressed in the target cell into which the DNA has been introduced. Is connected.
  • “linkage” may be directly linked or indirectly linked through a spacer having an appropriate length and sequence.
  • DNA encoding HSP70 protein and “DNA encoding ECI301” include deletion, substitution and addition of one to several nucleotides in the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4.
  • a DNA encoding a protein having the same activity as the HSP70 protein having an insertion and having the amino acid sequence shown by SEQ ID NO: 2 and a DNA encoding a polypeptide having the same activity as ECI301 are also included.
  • “Same activity as HSP70 protein” and “Same activity as ECI301” are as defined above.
  • DNA encoding HSP70 protein and “DNA encoding ECI301” hybridize under stringent conditions with DNA comprising a sequence complementary to the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4. And a DNA encoding a protein having the same activity as the HSP70 protein having the amino acid sequence shown in SEQ ID NO: 2 and a DNA encoding a polypeptide having the same activity as the ECI301.
  • Stringent conditions refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed.
  • SSC composition of 1 ⁇ SSC: 0.15M NaCl, 0 .15M sodium citrate, pH 7.0
  • 0.1-0.5% SDS in a solution containing 42-55 ° C.
  • 0.1-0.2 ⁇ SSC and 0.1 It refers to conditions for washing at 55 to 65 ° C. in a solution containing 0.5% SDS.
  • the “DNA encoding HSP70 protein” and the “DNA encoding ECI301” use the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 and the above BLAST and the like (for example, default or initial setting parameters).
  • DNA encoding HSP70 protein includes DNA encoding the ortholog of HSP70 protein having the amino acid sequence shown by SEQ ID NO: 2.
  • An expression vector containing DNA encoding HSP70 protein or DNA encoding ECI301 is obtained by using a genetic engineering technique well known to those skilled in the art, to an appropriate vector with DNA encoding HSP70 protein or DNA encoding ECI301. It can be produced by linking with a promoter and / or other regulatory sequences in a functional manner.
  • “to be ligated and inserted in a functional manner” means that HSP70 protein or ECI301 is expressed under the control of a promoter and / or other regulatory sequences in a cell into which the expression vector has been introduced. , Promoter and / or other regulatory sequences are linked and incorporated into the vector.
  • vectors that can be used known vectors such as adenoviruses, adeno-associated viruses, retroviruses and other viral vectors, plasmid vectors, liposome vectors, and the like can be used (separate volume experimental medicine, gene).
  • adenoviruses adeno-associated viruses
  • retroviruses and other viral vectors plasmid vectors, liposome vectors, and the like
  • plasmid vectors liposome vectors, and the like
  • cancer refers to a malignant tumor that metastasizes and grows immortally. Accordingly, in this specification, the terms “cancer” and “tumor” are used interchangeably.
  • Solid cancer means tumor cells that grow as multicellular mass supported by blood vessels, such as oral cancer, colon cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, melanoma, renal cell cancer, stomach cancer, Pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, esophageal cancer, liver cancer, squamous cell carcinoma of the head and neck, skin cancer, bladder cancer, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, today Meningiomas, male embryos, endometrial hyperplasia, endometriosis, embryonal, fibrosarcoma, Kaposi's sarcoma, hemangiomas, cavernous hemangioma, hemangioblastoma, retinoblastoma, astrocytoma,
  • Subjects to be treated with the pharmaceutical composition of the present invention include humans and non-human mammals (domestic animals (cow, horses, goats, sheep and pigs) and pets (cats and dogs), etc.) suffering from the above cancer. Preferably, it is a human.
  • the pharmaceutical composition of the present invention is administered orally or parenterally (for example, intravenous administration, intraarterial administration, local administration by injection, administration to the abdominal cavity or thoracic cavity, subcutaneous administration, intramuscular administration, sublingual administration, transdermal For example, by absorption or rectal administration).
  • the pharmaceutical composition of this invention can be made into a suitable dosage form according to an administration route.
  • injections, suspensions, emulsifiers, ointments, creams, tablets, capsules, granules, powders, pills, fine granules, troches, rectal administration, oily suppositories, water-soluble It can be prepared in various pharmaceutical forms such as suppositories. These various preparations are commonly used excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, preservatives. , And can be produced by conventional methods using colorants, flavoring agents, and stabilizers.
  • Excipients include, for example, lactose, fructose, glucose, corn starch, sorbit and crystalline cellulose, sterile water, ethanol, glycerol, physiological saline, buffer, etc.
  • disintegrants include, for example, starch, sodium alginate, gelatin
  • binders include calcium carbonate, calcium citrate, dextrin, magnesium carbonate, and synthetic magnesium silicate.
  • methyl cellulose or a salt thereof, ethyl cellulose, gum arabic, gelatin, hydroxypropyl cellulose, and polyvinylpyrrolidone are lubricants.
  • stabilizers include amino acids such as arginine, histidine, lysine and methionine, human serum albumin , Gelatin, dextran 40, methylcellulose, sodium sulfite, sodium metasulfite, etc.
  • Other additives include syrup, petrolatum, glycerin, ethanol, propylene glycol, citric acid, sodium chloride, sodium nitrite and sodium phosphate Each is listed.
  • a pharmaceutical composition containing HSP70 protein and ECI301 will be described.
  • DNA encoding HSP70 protein and DNA encoding ECI301 are described below.
  • the pharmaceutical composition of the present invention may be formulated with either HSP70 protein or ECI301 so that even if it is formulated in one dosage form as a combination drug containing both HSP70 protein and ECI301, it can be used separately at the same time or at intervals. It may be formulated as a single agent containing. That is, the present invention may be a combined medicine of a single agent containing HSP70 protein and a single agent containing ECI301.
  • HSP70 protein and ECI301 are each suitably in the range of preferably 0.01 to 300000 ⁇ g / 10 mL, more preferably 0.1 to 30000 ⁇ g / 10 mL, and further preferably 10 to 3000 ⁇ g / 10 mL.
  • HSP70 protein and ECI301 are included in a weight ratio of 1: 0.4 to 16, preferably 1: 4.
  • the HSP70 protein and ECI301 may be bound by a covalent bond or a non-covalent bond.
  • the dose of the pharmaceutical composition of the present invention may vary depending on factors such as the patient's age, body weight, severity of disease, etc., but each of HSP70 protein and ECI301 can be administered at a dose of 0.00001 mg / kg body weight per administration.
  • An amount appropriately selected from the range of 100 mg, preferably 0.0001 mg to 10 mg, more preferably 0.050 mg to 1.0 mg can be administered.
  • a typical dose is in the range of 3.5 mg to 35 mg per administration of HSP70 protein and ECI301, respectively, for a human weighing 70 kg.
  • HSP70 protein and ECI301 are administered at a weight ratio of 1: 0.4 to 16, preferably 1: 4.
  • the pharmaceutical composition of the present invention is formulated as a single agent each containing HSP70 protein and ECI301, it is 0 to 60 minutes, preferably 0 to 15 minutes after administration of the first single agent. Within, the second single agent is administered.
  • the order of administration is not particularly limited.
  • the dosing schedule may also vary depending on factors such as the patient's age, weight, severity of the disease, etc., but the pharmaceutical composition of the invention may be administered to the patient once or multiple times (eg, 3 or more times), administered daily or continuously for 2-5 days per week for 1-8 weeks.
  • the pharmaceutical composition of the present invention contains DNA encoding the above HSP70 protein and DNA encoding ECI301 or a vector containing the DNA as an active ingredient
  • the DNA or vector can be used in various ways used in the field of gene therapy.
  • the method can be delivered to a subject.
  • DNA or a vector may be directly administered to a subject (in vivo method), or introduced into a cell collected from the subject, and a transformed cell expressing the target HSP70 protein and ECI301 is selected.
  • DNA or vectors may be administered to a subject (ex vivo method).
  • Gene delivery mechanisms that can be used to administer DNA or vectors to the tissue or cell of interest include colloidal dispersion systems, liposome-derived systems, artificial virus envelopes, and the like.
  • delivery systems can utilize macromolecular complexes, nanocapsules, microspheres, beads, oil-in-water emulsions, micelles, mixed micelles, liposomes, and the like.
  • DNA or vector cell introduction transformation
  • a general gene introduction method such as a calcium phosphate method, a DEAE dextran method, an electroporation method, or a lipofection method.
  • the optimal dosage and administration schedule of the pharmaceutical composition of the present invention can be appropriately determined by those skilled in the art by model experiments using experimental animals and tumor cells.
  • the pharmaceutical composition of the present invention can be used in combination with another cancer treatment method, an anticancer agent, and / or a cytokine.
  • Such treatment methods include surgery, radiation therapy, chemotherapy, immunotherapy, hyperthermia, and the like.
  • the anticancer agent is not particularly limited, and examples thereof include known ones (for example, cisplatin, carboplatin, interferon, asparaginase, tamoxifen, leuprolide, flutamide, megestrol, mitomycin, bleomycin, doxorubicin, irinotecan, taxol, etc.).
  • cytokine what has the effect
  • IL-1 ⁇ interleukin-1 ⁇
  • IL-1 ⁇ interleukin-1 ⁇
  • IL-2 interleukin-2
  • Interleukin-3 Interleukin-3
  • interleukin-4 interleukin-4
  • interleukin-5 IL-5
  • interleukin-6 IL-6
  • interleukin-7 IL-7)
  • Interleukin-8 interleukin-8
  • interleukin-9 interleukin-9
  • interleukin-10 IL-10
  • interleukin-11 IL-11
  • interleukin-12 IL-12
  • Interferon ⁇ IFN ⁇
  • IFN ⁇ interferon ⁇
  • IFN ⁇ tumor necrosis factor ⁇
  • TGF- ⁇ tumor necrosis factor ⁇
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte / macrophage colony stimulating factor
  • TGF- ⁇ transforming growth factor ⁇
  • the pharmaceutical composition of the present invention can be used in combination with a stimulus that increases the expression of HSP.
  • Stimulations that increase HSP expression include, but are not limited to, heat, anoxia, radiation, alcohol, specific inhibitors of energy metabolism, glutamine, HSP transcription factors, infections, and the like.
  • the content and dosage of HSP70 protein in the pharmaceutical composition of the present invention can be decreased by increasing the expression of HSP (particularly HSP70) in cancer cells.
  • tumor growth can be suppressed by approximately 20%, 30%, 40%, 50%, 60%, 70% or more.
  • the pharmaceutical composition of the present invention is greatly different from the composition using the conventional HSP-peptide complex in its action mechanism.
  • a conventional HSP-peptide complex is composed of HSP and a cancer antigen peptide, and activates immunity to the cancer antigen peptide to remove cancer cells. Therefore, the effect of the complex is limited only to cancer types having a specific cancer antigen peptide.
  • the pharmaceutical composition of the present invention does not contain a specific cancer antigen peptide as a peptide, but contains ECI301 which is a derivative of human MIP-1 ⁇ . Therefore, it acts on cancer cells by a mechanism completely different from the mechanism of action of the conventional HSP-peptide complex, and the effect is not limited to only cancer types having a specific cancer antigen peptide.
  • the present invention further includes a method for treating cancer using the pharmaceutical composition of the present invention. Cancers that can be treated by the method include solid cancers as defined above. EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
  • mice Six-week-old BALB / c male mice were purchased from Japan SLC and used after acclimation breeding.
  • HSP70 / ECI301 co-administration group a combination of HSP70 drug (500 ng HSP70 / 0.2 ml PBS) and ECI301 drug (2 ⁇ g ECI301 / 2 ⁇ l PBS) was administered into the tail vein (iv) on the 11th day of transplantation. (Once a day), administration was repeated in the same manner one day thereafter.
  • HSP70 drug 500 ng HSP70 / 0.2 ml PBS
  • the control group with the same amount of PBS as the administration group
  • the HSP70 / ECI301 co-administration group Administered.
  • the major axis and minor axis of the solid tumor were measured every 3 to 4 days in each group, and the tumor volume was calculated by the following formula.
  • Tumor volume (mm 3 ) major axis (mm) ⁇ minor axis (mm) ⁇ minor axis (mm) ⁇ 0.5236
  • FIG. 2 and FIG. FIG. 3 shows the results on the 16th day after the start of administration. As is clear from the results of FIGS. 2 and 3, tumor growth could be strongly suppressed by co-administration of HSP70 and ECI301.
  • ECI301 and human MIP-1 ⁇ were purchased from Japan SLC and used after acclimation breeding.
  • HSP70 / ECI301 co-administration and sequential administration experiment system Colon26 / BALB / c
  • Colon 26 cells were implanted subcutaneously into the right flank of BALB / c mice (4 ⁇ 10 5 cells per mouse).
  • Formulations containing 0.5 ⁇ g and 2.0 ⁇ g of HSP70 and ECI301, respectively, were prepared in the same manner as in Example 1 above, and in the tail vein once a day in the coadministration group (FIG. 7A) on the 9th day of transplantation (Iv) Administration, followed by administration in the same manner for 2 consecutive days.
  • HSP70 (0.5 ⁇ g) was administered into the tail vein (iv) on the 9th day after transplantation, and separated from the administration of HSP70 within 10 minutes, ECI301 (2.0 ⁇ g) was administered intravenously (iv) in the tail vein. Thereafter, it was administered in the same manner for 2 consecutive days.
  • the control group received only the same amount of PBS as the administration group.
  • the major axis and minor axis of the solid tumor were measured every 3 to 4 days in each group, and the tumor volume was calculated in the same manner as in Example 1 above.
  • FIG. FIG. 7 shows the results on the 11th day after the start of administration. It has been clarified that HSP70 and ECI301 exhibit a tumor growth inhibitory effect even when administered separately (FIG. 7B) without being co-administered as a combination drug.
  • each combination of HSP70 / ECI301 and HSP90 / ECI301 contains HSP70 or HSP90 (0.5 ⁇ g / 0.2 ml PBS) and ECI301 (2 ⁇ g / 2 ⁇ l PBS), respectively, and is co-administered
  • HSP70 or HSP90 0.5 ⁇ g / 0.2 ml PBS
  • ECI301 2 ⁇ g / 2 ⁇ l PBS
  • HSP70 single administration group HSP70 or HSP90 (0.5 ⁇ g / 0.2 ml PBS) mixed with 2 ⁇ l PBS, and in the control group, the same amount of PBS as in the administration group was administered in the same manner as in the co-administration group. did.
  • FIG. 9 and FIG. FIG. 10 shows the results on the 15th day after the start of administration.
  • the combination of HSP70 and ECI301 showed a tumor growth inhibitory effect with good reproducibility, but the combination of HSP90 and ECI301 showed no effect at all. From the above results, it has been clarified that HSP70 and HSP60 or HSP90 have completely different actions in the compounding agent with ECI301.
  • mice BALB / c 6-week-old male mice and C57BL / 6-line 6-week-old female mice were purchased from Japan SLC and used after acclimation breeding.
  • ECI301 ECI301 1mg / ml prepared in PBS aqueous solution HSP70 Mouse Recombinant Hsp70-A1 (Low Endotoxin) (ESP-502, ENZO BIOCHEM (former Assay Designs)) Experimental system prepared in HSP70 10 ⁇ g / 1 ml PBS Of the following tumor cells, Colon 26 cells and Meth A cells were transplanted subcutaneously into BALB / c mice, and Lewis lung cancer cells were subcutaneously implanted into C57BL / 6 mice on the right flank.
  • the combination of HSP70 / ECI301 contains HSP70 (0.5 ⁇ g / 0.2 ml PBS) and ECI301 (2 ⁇ g / 2 ⁇ l PBS), and the combination is administered to the co-administration group once a day for each group on the ninth day of transplantation.
  • Caudal vein (iv) was administered, and then administered in the same manner for 2 consecutive days.
  • HSP70 In the HSP70 or ECI301 single administration group, HSP70 (0.5 ⁇ g / 0.2 ml PBS) or ECI301 (2 ⁇ g / 2 ⁇ l PBS) mixed with 2 ⁇ l or 0.2 ml of PBS, respectively, the control group had the same amount as the administration group PBS was administered in the same manner as in the HSP70 / ECI301 co-administration group.
  • the major axis and minor axis of the solid tumor were measured every 3 to 4 days in each group, and the tumor volume was calculated in the same manner as in Example 1 above.
  • the results of each tumor cell are shown in FIGS.
  • the combination of HSP70 / ECI301 showed a tumor growth inhibitory effect with good reproducibility for all tumor cells. This result suggests that the combination drug is effective against any tumor cells of different strains.
  • SDS electrophoresis SDS-PAGE
  • Material Gel Nu-PAGE 10%: Invitrogen
  • Running buffer Invitrogen
  • Sample buffer Invitrogen Reduction buffer: Invitrogen Method
  • a sample was mixed with a sample buffer and a reducing buffer, treated at 70 ° C. for 10 minutes to prepare a sample, and then electrophoresed at 200 V constant pressure current for about 35 minutes according to the instruction manual attached to the Nu-PAGE product.
  • Staining was performed by blocking the transferred PVDF membrane with ChemiBlocker: 20 mM TBS-T (1%) 1: 1 solution for 30 minutes, and then adding a primary antibody: rabbit anti-MIP-1 ⁇ polyclonal-biotin label at room temperature. After incubation and washing for 60 minutes, secondary antibody: streptavidin-HRP label was added and incubated at R room temperature for 60 minutes. First, light was emitted using an ECL reagent according to the instruction manual attached to the product, and a color image was captured with an image analyzer LAS4000 Mini (Fuji Film) to obtain ImmunoBlot data of ECI301 and MIP-1 ⁇ (FIG. 14).
  • HSP70 co-precipitated during the immunoprecipitation using anti-MIP-1 ⁇ antibody-bound beads and ECI301 (lane 2). From these results, it was revealed that ECI301 and HSP70 have a strong affinity, that is, a specific adsorption action.
  • the growth of cancer cells in solid cancer can be inhibited.

Abstract

Provided is a cancer therapeutic agent which can treat solid cancer and various diseases or disorders associated with solid cancer without inducing any adverse side effect. Provided is a pharmaceutical composition for treating cancer, which comprises a heat shock protein (HSP) and a polypeptide represented by SEQ ID NO:1 (ECI301) as active ingredients.

Description

ヒートショックプロテイン（ＨＳＰ）とＥＣＩ３０１ポリペプチドを含む癌細胞の増殖を抑制するための医薬組成物ならびにそれを用いた癌の治療方法Pharmaceutical composition for inhibiting proliferation of cancer cells comprising heat shock protein (HSP) and ECI301 polypeptide
　本発明は、ヒートショックプロテイン（以下、「ＨＳＰ」と記載する）とＥＣＩ３０１ポリペプチドを含む癌を治療するための医薬組成物ならびにそれを用いた癌の治療方法に関するものである。 The present invention relates to a pharmaceutical composition for treating cancer comprising heat shock protein (hereinafter referred to as “HSP”) and ECI301 polypeptide, and a method for treating cancer using the same.
　癌は、ヒトまたは動物の身体の任意の場所の悪性新生物として規定される。癌はその制御されない増殖によって隣接組織を破壊し、最終的に脈管および器官の物理的遮断によって身体の死を引き起こす（非特許文献１）。
　現在、癌の治療方法としては、外科的手術、放射線療法、温熱療法、化学療法、遺伝子治療、免疫療法などが挙げられる。しかしながら、これらの処置方法は、処置される部位から離れた癌（転移性癌など）を処置するのに有効ではない、重篤な副作用が伴う、処置に対して感受性の低い癌細胞が存在する、特定の限定された患者にのみに有効であること、等それぞれ様々な欠点を有しており、依然として、癌治療のための新たな方法が求められている。
　上記課題を解決すべく、近年、ＨＳＰを癌治療に利用する方法が開発されている。
　ＨＳＰは、ストレスタンパク質とも呼ばれており細菌からヒトまでのほとんど全ての種類の生物において普遍的に発現されるタンパク質のファミリーをいう。ＨＳＰは分子量に基づいて５つのファミリーに分類されており、ＨＳＰ１００、ＨＳＰ９０、ＨＳＰ７０、ＨＳＰ６０およびｓｍａｌｌ　ＨＳＰがある。これらのファミリーの多くのメンバーは、熱ショックや生理的ストレス（例えば、環境刺激および発達上の影響、例えば、温熱、無酸素、アルコール、グルコース飢餓（ＨＳＰのサブグループでもあるグルコース調節タンパク質（すなわちＧＲＰ）について）、組織損傷、栄養欠乏、代謝破壊、酸素ラジカル、細胞内病原体による感染など）に応答して、発現が誘導される（非特許文献２−６）。
　ＨＳＰは通常、細胞中において低~中程度のレベルで発現されているが、熱ショックや生理的ストレスを受けると細胞中に非常に高レベルで発現し蓄積する。例えば、哺乳動物のＨＳＰ７０は、熱ショックを受けていない細胞においては検出が困難である程の低レベルで発現しているが、熱ショックを受けた細胞においては最も高レベルに発現されるタンパク質の１つとなる（非特許文献７）。また、ほとんど（しかし全てではない）の哺乳動物のＨＳＰ９０およびＨＳＰ６０は、熱ショックを受けていない細胞においても高レベルで発現されているが、熱ショックを受けた細胞においてはさらに発現が誘導される（非特許文献８、９）。
　熱ショックや他の生理的ストレスに対する細胞性応答の研究から、ＨＳＰは熱ショックや他の生理的ストレスを受けた不利な状態にある細胞中に発現・蓄積され、当該細胞を保護する機能を有することが明らかにされた（非特許文献１０）。
　さらに、ＨＳＰは熱ショックや他の生理的ストレスを受けていない細胞においても生化学的および免疫学的プロセスに関与していることが明らかにされた。すなわち、ＨＳＰはさまざまな種類のシャペロン機能を有しており、例えば、ＨＳＰ７０ファミリーのメンバーは、細胞質、核、ミトコンドリア、または小胞体に存在し（非特許文献１１）、タンパク質の転移、折りたたみおよび組み立て、ならびに免疫系の細胞への抗原提示に関与していることが明らかにされた（非特許文献１２）。
　これらの知見に基づいて、ＨＳＰを標的とする、またはＨＳＰを利用する以下の癌治療剤の開発が進められている。
　ＨＳＰ阻害剤：ＨＳＰは熱ショックや他の生理的ストレスを受けた不利な状態にある細胞中に発現・蓄積され、当該細胞を保護する機能を有することから、癌細胞においてもＨＳＰは細胞保護作用を果たしていると考えられる。実際、ＨＳＰ９０の機能を阻害することによって、癌細胞における細胞増殖、細胞周期の制御およびアポトーシスに関与するシグナル伝達腫瘍性タンパク質の選択的分解が生じることが明らかにされている（非特許文献１３）。しかし、現在のところ癌治療において、有望な効果が見出されておらず、また利用し得るＨＳＰ９０阻害剤としては、ベンゾキノンアンサマイシンなどが見出されているが、これらＨＳＰ９０阻害剤のベンゾキノンアンサマイシン種のいくつかは、ＨＳＰ９０ＡＴＰアーゼ活性の阻害のため、例えば肝毒性を含めた有害な副作用を示す。
　ＨＳＰ−ペプチド複合体：ＨＳＰはそれ自体は免疫原性を有さないが、癌抗原などの抗原ペプチドと非共有結合して複合体を形成し、その複合体が当該抗原ペプチドに特異的な免疫を誘発できることが示された（非特許文献１４−１６；特許文献１−３）。ＨＳＰ−ペプチド複合体は、特定あるいは非特定の腫瘍抗原をＨＳＰと共に、樹状細胞に取り込ませ、抗原提示の飛躍的効率化によって、免疫を活性化させ、癌細胞を除去しようとするものである。しかしながら、癌は抗原的に非常に様々であり、あらゆる癌の抗原として利用できる「免疫原性抗原」を単利精製することは困難であると考えられる。したがって、複合体に利用する癌抗原は特定のものに限定され、当該複合体の効果も特定の癌種のみに限定される。一方、抗原を単離精製せずに、腫瘍ライセートをそのままＨＳＰに結合させるといった、非特定抗原を用いる場合、医薬品開発申請で求められる医薬品の品質に関する、Ｃｈｅｍｉｓｔｒｙ／Ｍａｎｕｆａｃｔｕｒｅ／Ｃｏｎｔｒｏｌ（ＣＭＣ）の要件を具備することができず、実質上、医薬品として利用し得ないものである。
　すなわち、現在までにＨＳＰを利用した有効な癌治療剤および治療方法は報告されていない。 Cancer is defined as a malignant neoplasm anywhere in the human or animal body. Cancer destroys adjacent tissues by its uncontrolled growth and ultimately causes body death by physical blockage of vessels and organs (Non-Patent Document 1).
Currently, cancer treatment methods include surgery, radiation therapy, hyperthermia, chemotherapy, gene therapy, immunotherapy, and the like. However, these treatment methods are not effective in treating cancers that are distant from the treated site (such as metastatic cancer), and there are cancer cells that are less sensitive to treatment with severe side effects. However, each method has various drawbacks such as being effective only for a specific limited patient, and a new method for cancer treatment is still required.
In order to solve the above problems, a method of using HSP for cancer treatment has been developed in recent years.
HSP, also called stress protein, refers to a family of proteins that are universally expressed in almost all types of organisms from bacteria to humans. HSPs are classified into five families based on molecular weight, including HSP100, HSP90, HSP70, HSP60 and small HSP. Many members of these families are associated with heat shock and physiological stress (eg, environmental stimuli and developmental effects such as thermal, anoxia, alcohol, glucose starvation (glucose regulatory proteins that are also subgroups of HSP (ie GRP )), Expression is induced in response to tissue damage, nutritional deficiency, metabolic destruction, oxygen radicals, infection by intracellular pathogens, etc. (Non-patent Documents 2-6).
HSP is normally expressed at low to moderate levels in cells, but is expressed and accumulated at very high levels in cells when subjected to heat shock or physiological stress. For example, mammalian HSP70 is expressed at a low level that is difficult to detect in cells not subjected to heat shock, but is the protein expressed at the highest level in cells subjected to heat shock. One (Non-Patent Document 7). Also, most (but not all) mammalian HSP90 and HSP60 are expressed at high levels in cells that have not undergone heat shock, but further expression is induced in cells that have undergone heat shock. (Non-Patent Documents 8 and 9).
From the study of cellular responses to heat shock and other physiological stresses, HSP is expressed and accumulated in cells in adverse conditions that have been subjected to heat shock and other physiological stresses, and has a function of protecting the cells (Non-Patent Document 10).
Furthermore, HSP has been shown to be involved in biochemical and immunological processes even in cells that are not subjected to heat shock or other physiological stresses. That is, HSP has various types of chaperone functions. For example, members of the HSP70 family are present in the cytoplasm, nucleus, mitochondria, or endoplasmic reticulum (Non-patent Document 11), and protein translocation, folding and assembly. , As well as being involved in antigen presentation to cells of the immune system (Non-patent Document 12).
Based on these findings, development of the following cancer therapeutic agents targeting HSP or using HSP is in progress.
HSP inhibitor: HSP is expressed and accumulated in cells in a disadvantageous state subjected to heat shock and other physiological stress, and has a function of protecting the cells. Therefore, HSP also has a cytoprotective action in cancer cells. It is thought that is fulfilled. In fact, it has been clarified that inhibition of the function of HSP90 results in selective degradation of signaling oncoproteins involved in cell proliferation, cell cycle control and apoptosis in cancer cells (Non-patent Document 13). . However, at present, no promising effect has been found in cancer treatment, and benzoquinone ansamycin and the like have been found as HSP90 inhibitors that can be used. Some of the species show adverse side effects including inhibition of HSP90ATPase activity, including for example hepatotoxicity.
HSP-peptide complex: Although HSP itself is not immunogenic, it forms a complex by noncovalently binding to an antigen peptide such as a cancer antigen, and the complex is an immunity specific for the antigen peptide. (Non-patent Documents 14-16; Patent Documents 1-3). The HSP-peptide complex is intended to activate specific immunity and remove cancer cells by taking specific or non-specific tumor antigens together with HSP into dendritic cells and dramatically improving antigen presentation. . However, cancers vary greatly in antigen, and it is considered difficult to simply purify “immunogenic antigens” that can be used as antigens for all cancers. Therefore, the cancer antigen utilized for the complex is limited to a specific one, and the effect of the complex is also limited to a specific cancer type. On the other hand, when using a non-specific antigen, such as binding a tumor lysate directly to HSP without isolating and purifying the antigen, the requirements of Chemistry / Manufacture / Control (CMC) regarding the quality of the drug required in the drug development application are met. It cannot be provided and cannot be used as a pharmaceutical product.
That is, no effective cancer therapeutic agent and treatment method using HSP has been reported so far.
ＷＯ　９６／１０４１１WO 96/10411 ＷＯ　９７／１０００１WO 97/10001 米国特許第５，９８５，２７０号US Pat. No. 5,985,270
　本発明は、癌の種類（すなわち、異なる抗原性）に左右されず様々な系統の癌に対して有効である、治療剤および治療方法を提供する。
　本発明者らは、上記課題を解決すべく、鋭意検討した結果、ＨＳＰ６０、ＨＳＰ９０以外のある種のＨＳＰ、特にＨＳＰ７０と、ある特定のアミノ酸配列を有するポリペプチド（ＥＣＩ３０１ポリペプチド）を含む混合組成物が、癌の種類に左右されることなく、局所的および遠位転移癌ならびにその両方の増殖を有意に阻害できることを見出し、本発明を完成させた。
　すなわち、本発明は以下の特徴を有する。
［１］　ヒートショックプロテイン（ＨＳＰ）７０と配列番号１で表されるポリペプチド（ＥＣＩ３０１）を含む、癌を治療するための医薬組成物。
［２］　癌が固形癌である、［１］の医薬組成物。
［３］　固形癌が肺癌、大腸癌、または線維肉腫である、［２］の医薬組成物。
［４］　重量比率でヒートショックプロテイン（ＨＳＰ）７０と配列番号１で表されるポリペプチド（ＥＣＩ３０１）を１：０．４~１６の比率で含む、［１］~［３］のいずれかの医薬組成物。
［５］　癌を治療するための、ヒートショックプロテイン（ＨＳＰ）７０と配列番号１で表されるポリペプチド（ＥＣＩ３０１）の組み合わせ医薬。
［６］　癌が固形癌である、［５］の組み合わせ医薬。
［７］　固形癌が肺癌、大腸癌、または線維肉腫である、［６］の組み合わせ医薬。
［８］　癌を治療する方法であって、ヒートショックプロテイン（ＨＳＰ）７０及び配列番号１で表されるポリペプチド（ＥＣＩ３０１）を別々にまたは同時に癌患者に投与することを含む、上記方法。
［９］　癌が固形癌である、［８］の方法。
［１０］　固形癌が肺癌、大腸癌、または線維肉腫である、［９］の方法。
［１１］　重量比率でヒートショックプロテイン（ＨＳＰ）７０と配列番号１で表されるポリペプチド（ＥＣＩ３０１）を１：０．４~１６の比率で癌患者に投与する、［８］~［１０］のいずれかの方法。
［１２］　癌患者における癌の治療に使用するための、ヒートショックプロテイン（ＨＳＰ）７０及び配列番号１で表されるポリペプチド（ＥＣＩ３０１）。
［１３］　癌が固形癌である、［１２］のヒートショックプロテイン（ＨＳＰ）７０及び配列番号１で表されるポリペプチド（ＥＣＩ３０１）。
［１４］　固形癌が肺癌、大腸癌、または線維肉腫である、［１３］のヒートショックプロテイン（ＨＳＰ）７０及び配列番号１で表されるポリペプチド（ＥＣＩ３０１）。
［１５］　重量比率でヒートショックプロテイン（ＨＳＰ）７０と配列番号１で表されるポリペプチド（ＥＣＩ３０１）を１：０．４~１６の比率で癌患者に投与して使用する、［１２］~［１４］のいずれかのヒートショックプロテイン（ＨＳＰ）７０及び配列番号１で表されるポリペプチド（ＥＣＩ３０１）。
　本明細書は本願の優先権の基礎である日本国特許出願２０１０−１８３６５１号の明細書および／または図面に記載される内容を包含する。 The present invention provides a therapeutic agent and a therapeutic method that are effective against various types of cancer regardless of the type of cancer (ie, different antigenicity).
As a result of intensive studies to solve the above problems, the present inventors have found that a mixed composition containing a certain HSP other than HSP60 and HSP90, particularly HSP70, and a polypeptide having a specific amino acid sequence (ECI301 polypeptide). The present invention was completed by finding that the product can significantly inhibit the growth of local and distant metastatic cancers, and both, regardless of the type of cancer.
That is, the present invention has the following features.
[1] A pharmaceutical composition for treating cancer, comprising heat shock protein (HSP) 70 and a polypeptide represented by SEQ ID NO: 1 (ECI301).
[2] The pharmaceutical composition of [1], wherein the cancer is a solid cancer.
[3] The pharmaceutical composition according to [2], wherein the solid cancer is lung cancer, colon cancer, or fibrosarcoma.
[4] The heat shock protein (HSP) 70 and the polypeptide represented by SEQ ID NO: 1 (ECI301) at a weight ratio of 1: 0.4 to 16 at a ratio of any one of [1] to [3] Pharmaceutical composition.
[5] A combined medicine of heat shock protein (HSP) 70 and the polypeptide represented by SEQ ID NO: 1 (ECI301) for treating cancer.
[6] The combined medicine of [5], wherein the cancer is a solid cancer.
[7] The combined medicine of [6], wherein the solid cancer is lung cancer, colon cancer, or fibrosarcoma.
[8] A method for treating cancer, comprising administering heat shock protein (HSP) 70 and the polypeptide represented by SEQ ID NO: 1 (ECI301) separately or simultaneously to a cancer patient.
[9] The method of [8], wherein the cancer is a solid cancer.
[10] The method of [9], wherein the solid cancer is lung cancer, colon cancer, or fibrosarcoma.
[11] Heat shock protein (HSP) 70 and polypeptide represented by SEQ ID NO: 1 (ECI301) are administered to cancer patients at a ratio of 1: 0.4 to 16 by weight ratio [8] to [10] Either way.
[12] A heat shock protein (HSP) 70 and a polypeptide represented by SEQ ID NO: 1 (ECI301) for use in the treatment of cancer in cancer patients.
[13] The heat shock protein (HSP) 70 of [12] and the polypeptide represented by SEQ ID NO: 1 (ECI301), wherein the cancer is a solid cancer.
[14] The heat shock protein (HSP) 70 according to [13] and the polypeptide represented by SEQ ID NO: 1 (ECI301), wherein the solid cancer is lung cancer, colon cancer, or fibrosarcoma.
[15] Heat shock protein (HSP) 70 by weight ratio and polypeptide (ECI301) represented by SEQ ID NO: 1 are administered to cancer patients at a ratio of 1: 0.4 to 16, and used [12] [14] The heat shock protein (HSP) 70 of any one and the polypeptide represented by SEQ ID NO: 1 (ECI301).
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2010-183651 which is the basis of the priority of the present application.
　図１は、ヒトＭＩＰ−１αの誘導体であるＥＣＩ３０１のアミノ酸配列を示す。
　図２は、ＨＳＰ７０またはＨＳＰ７０／ＥＣＩ３０１の尾静脈内（ｉｖ）投与による、マウスにおける腫瘍組織の体積変化を経時的に示すグラフ図である。
　図３は、ＨＳＰ７０またはＨＳＰ７０／ＥＣＩ３０１を尾静脈内（ｉｖ）投与されたマウスにおける、各投与群の腫瘍組織の体積を示すグラフ図である。
　図４は、ＥＣＩ３０１（２．０μｇ）に対してＨＳＰ７０を様々な用量（Ｉ：０．１２５μｇ；ＩＩ：０．５μｇ；ＩＩＩ：５．０μｇ）で含有する配合剤を尾静脈内（ｉｖ）投与したマウスにおける腫瘍組織の体積を示すグラフ図である。
　図５は、ＨＳＰ７０およびＥＣＩ３０１を重量比率にして１：４で含む、様々な用量のＨＳＰ７０およびＥＣＩ３０１を含有する配合剤（ＨＳＰ７０およびＥＣＩ３０１をそれぞれ以下の用量で含む。ｉ：０．５μｇ：０；ｉｉ：０．１μｇ：０．４μｇ；ｉｉｉ：０．５μｇ：２．０μｇ；ｉｖ：２．５μｇ：１０．０μｇ）を尾静脈内（ｉｖ）投与したマウスにおける腫瘍組織の体積を示すグラフ図である。
　図６は、ＨＳＰ７０およびＥＣＩ３０１を含有する配合剤（ＨＳＰ７０およびＥＣＩ３０１をそれぞれ以下の用量で含む。（Ｉ）：０．５μｇ：０；（ＩＩ）：０．１μｇ：０．４μｇ；（ＩＩＩ）：０．５μｇ：２．０μｇ）またはＨＳＰ７０およびヒトＭＩＰ−１αを含有する配合剤（ＨＳＰ７０およびヒトＭＩＰ−１αをそれぞれ以下の用量で含む。（ＩＶ）：０．１μｇ：０．４μｇ；（Ｖ）：０．５μｇ：２．０μｇ）を尾静脈内（ｉｖ）投与したマウスにおける腫瘍組織の体積を示すグラフ図である。
　図７は、（Ａ）ＨＳＰ７０およびＥＣＩ３０１を含有する配合剤（ＨＳＰ７０およびＥＣＩ３０１をそれぞれ、０．５μｇおよび２．０μｇの用量で含む）または（Ｂ）ＨＳＰ７０次いでＥＣＩ３０１をそれぞれ単独で、尾静脈内（ｉｖ）投与したマウスにおける腫瘍組織の体積を示すグラフ図である。
　図８は、ＨＳＰ７０およびＥＣＩ３０１を含有する配合剤（ＨＳＰ７０およびＥＣＩ３０１をそれぞれ、０．５μｇおよび２．０μｇの用量で含む（ｉ））またはＨＳＰ６０およびＥＣＩ３０１を含有する配合剤（ＨＳＰ６０およびＥＣＩ３０１をそれぞれ以下の用量で含む。（ｉｉ）：０．５μｇ：０；（ｉｉｉ）：０．１μｇ：２．０μｇ；（ｉｖ）：０．５μｇ：２．０μｇ；（ｖ）：２．５μｇ：２．０μｇ）を尾静脈内（ｉｖ）投与したマウスにおける腫瘍組織の体積を示すグラフ図である。
　図９は、ＨＳＰ７０またはＨＳＰ９０の単独投与あるいはＥＣＩ３０１との共投与（ｉｖ）によるマウスにおける腫瘍組織の体積変化を経時的に示すグラフ図である。
　図１０は、ＨＳＰ７０またはＨＳＰ９０の単独投与あるいはＥＣＩ３０１との共投与（ｉｖ）によるマウスにおける腫瘍組織の体積を示すグラフ図である。
　図１１は、ＨＳＰ７０またはＨＳＰ７０／ＥＣＩ３０１の尾静脈内（ｉｖ）投与によるマウスにおけるＣｏｌｏｎ２６腫瘍組織の体積変化を経時的に示すグラフ図である。
　図１２は、ＨＳＰ７０またはＨＳＰ７０／ＥＣＩ３０１の尾静脈内（ｉｖ）投与によるマウスにおけるルイス肺癌組織の体積変化を経時的に示すグラフ図である。
　図１３は、ＨＳＰ７０またはＨＳＰ７０／ＥＣＩ３０１の尾静脈内（ｉｖ）投与によるマウスにおけるＭｅｔｈＡ腫瘍組織の体積変化を経時的に示すグラフ図である。
　図１４は、抗ＭＩＰ−１α抗体結合ビーズとＥＣＩ３０１または抗ＨＳＰ７０抗体結合ビーズとＨＳＰ７０を用い行った免疫沈降実験の結果を示す。各レーンそれぞれ以下のサンプルを示す。レーン１−３：抗ＭＩＰ−１α抗体結合ビーズ；レーン１：ＥＣＩ３０１；レーン２：ＥＣＩ３０１＋ＨＳＰ７０；レーン３：ＥＣＩ３０１＋αＭＩＰ−１α−ビオチン；レーン４−６：抗ＨＳＰ７０抗体結合ビーズ；レーン４：ＨＳＰ７０；レーン５：ＨＳＰ７０＋ＥＣＩ３０１；レーン６：ＨＳＰ７０＋ＭＩＰ−１α；レーン７：分子量マーカー。一次抗体：ウサギ抗ＭＩＰ−１αポリクローナル−ビオチン標識；二次抗体：ストレプトアビジン−ＨＲＰ標識。
　図１５は、抗ＭＩＰ−１α抗体結合ビーズとＥＣＩ３０１または抗ＨＳＰ７０抗体結合ビーズとＨＳＰ７０を用い行った免疫沈降実験の結果を示す。各レーンそれぞれ以下のサンプルを示す。レーン１−３：抗ＭＩＰ−１α抗体結合ビーズ；レーン１：ＥＣＩ３０１；レーン２：ＥＣＩ３０１＋ＨＳＰ７０；レーン３：ＥＣＩ３０１＋αＭＩＰ−１α−ビオチン；レーン４−６：抗ＨＳＰ７０抗体結合ビーズ；レーン４：ＨＳＰ７０；レーン５：ＨＳＰ７０＋ＥＣＩ３０１；レーン６：ＨＳＰ７０＋ＭＩＰ−１α；レーン７：分子量マーカー。一次抗体：マウス抗ＨＳＰ７０；二次抗体：抗マウスＩｇＧ−ＨＲＰ標識。 FIG. 1 shows the amino acid sequence of ECI301, a derivative of human MIP-1α.
FIG. 2 is a graph showing changes in volume of tumor tissue in mice over time by administration of HSP70 or HSP70 / ECI301 into the tail vein (iv).
FIG. 3 is a graph showing the volume of tumor tissue in each administration group in mice administered with HSP70 or HSP70 / ECI301 intravenously (iv) in the tail vein.
FIG. 4 shows tail vein (iv) administration of a formulation containing HSP70 at various doses (I: 0.125 μg; II: 0.5 μg; III: 5.0 μg) against ECI301 (2.0 μg) It is a graph which shows the volume of the tumor tissue in the done mouse.
FIG. 5 is a formulation containing various doses of HSP70 and ECI301, including HSP70 and ECI301 in a weight ratio of 1: 4 (HSP70 and ECI301 are each included in the following doses: i: 0.5 μg: 0; (ii: 0.1 μg: 0.4 μg; iii: 0.5 μg: 2.0 μg; iv: 2.5 μg: 10.0 μg) are administered in the tail vein (iv) and are graphs showing the volume of tumor tissue in mice. is there.
FIG. 6 shows a formulation containing HSP70 and ECI301 (HSP70 and ECI301 are each included in the following doses: (I): 0.5 μg: 0; (II): 0.1 μg: 0.4 μg; (III): 0.5 μg: 2.0 μg) or a combination preparation containing HSP70 and human MIP-1α (HSP70 and human MIP-1α are included in the following doses, respectively) (IV): 0.1 μg: 0.4 μg; (V) : 0.5 μg: 2.0 μg) is a graph showing the volume of tumor tissue in mice administered intravenously (iv) in the tail vein.
FIG. 7 shows (A) a formulation containing HSP70 and ECI301 (containing HSP70 and ECI301 at doses of 0.5 μg and 2.0 μg, respectively) or (B) HSP70 and then ECI301 alone, respectively, in the tail vein ( iv) A graph showing the volume of tumor tissue in administered mice.
FIG. 8 shows a combination containing HSP70 and ECI301 (HSP70 and ECI301 are included at doses of 0.5 μg and 2.0 μg, respectively (i)) or a combination containing HSP60 and ECI301 (HSP60 and ECI301 respectively below) (Ii): 0.5 μg: 0; (iii): 0.1 μg: 2.0 μg; (iv): 0.5 μg: 2.0 μg; (v): 2.5 μg: 2.0 μg Is a graph showing the volume of tumor tissue in mice administered intravenously (iv) in the tail vein.
FIG. 9 is a graph showing changes over time in the volume of tumor tissue in mice by single administration of HSP70 or HSP90 or co-administration (iv) with ECI301.
FIG. 10 is a graph showing the volume of tumor tissue in mice by single administration of HSP70 or HSP90 or co-administration (iv) with ECI301.
FIG. 11 is a graph showing temporal changes in the volume of Colon 26 tumor tissue in mice after HSP70 or HSP70 / ECI301 administration into the tail vein (iv).
FIG. 12 is a graph showing changes over time in the volume of Lewis lung cancer tissue in mice after HSP70 or HSP70 / ECI301 administration into the tail vein (iv).
FIG. 13 is a graph showing changes over time in the volume of MethA tumor tissue in mice by HSP70 or HSP70 / ECI301 administration into the tail vein (iv).
FIG. 14 shows the results of an immunoprecipitation experiment performed using anti-MIP-1α antibody-binding beads and ECI301 or anti-HSP70 antibody-binding beads and HSP70. The following samples are shown for each lane. Lane 1-3: Anti-MIP-1α antibody-binding beads; Lane 1: ECI301; Lane 2: ECI301 + HSP70; Lane 3: ECI301 + αMIP-1α-biotin; Lane 4-6: Anti-HSP70 antibody-binding beads; Lane 4: HSP70; Lane 5 : HSP70 + ECI301; Lane 6: HSP70 + MIP-1α; Lane 7: molecular weight marker. Primary antibody: rabbit anti-MIP-1α polyclonal-biotin label; secondary antibody: streptavidin-HRP label.
FIG. 15 shows the results of an immunoprecipitation experiment using anti-MIP-1α antibody-binding beads and ECI301 or anti-HSP70 antibody-binding beads and HSP70. The following samples are shown for each lane. Lane 1-3: Anti-MIP-1α antibody-binding beads; Lane 1: ECI301; Lane 2: ECI301 + HSP70; Lane 3: ECI301 + αMIP-1α-biotin; Lane 4-6: Anti-HSP70 antibody-binding beads; Lane 4: HSP70; Lane 5 : HSP70 + ECI301; Lane 6: HSP70 + MIP-1α; Lane 7: molecular weight marker. Primary antibody: mouse anti-HSP70; secondary antibody: anti-mouse IgG-HRP label.
　以下、本発明を詳細に説明する。
　本発明は、ヒートショックプロテイン（ＨＳＰ）と配列番号１で表されるポリペプチド（ＥＣＩ３０１）を含む、癌を治療するための医薬組成物を提供する。
　本発明に用い得るヒートショックプロテインには、ＨＳＰ９０およびＨＳＰ６０を除く全てのヒートショックプロテインが含まれるが、好ましくはＨＳＰ７０タンパク質である。
　本発明において、ＨＳＰ７０タンパク質は、特に限定されるものではないが、ヒト由来であることが好ましい。ヒト由来のＨＳＰ７０タンパク質を用いることによって、ヒト患者に投与された際に生じる免疫拒絶反応を最小に抑え、治療の効果を上げることができる。
　ＨＳＰ７０タンパク質は公知であり、様々な生物種から単離され、配列情報が公開されている。ヒト由来ＨＳＰ７０タンパク質の配列情報は、例えばアクセッション番号ＮＰ＿００２１４５，ＡＡＩ２６１２５等として、マウス由来ＨＳＰ７０タンパク質の配列情報は、例えばアクセッション番号ＮＭ＿０２４１７２等として、ＧｅｎＢａｎｋに登録されておりこれらを利用することができる。本発明においては、配列番号２で示されたアミノ酸配列を有するＨＳＰ７０タンパク質を用いることができる。
　また、本発明において「ＨＳＰ７０タンパク質」には、配列番号２で示されたアミノ酸配列に１~数個のアミノ酸の欠失、置換、付加または挿入を有し、かつ配列番号２で示されたアミノ酸配列を有するＨＳＰ７０タンパク質と同じ活性を有するタンパク質も含まれる。「ＨＳＰ７０タンパク質と同じ活性」とは、下記に詳述するＥＣＩ３０１ポリペプチドと共に投与された場合に、癌細胞の増殖を阻害する活性を意味する。本明細書において、「１から数個」の範囲は特には限定されないが、例えば、１から２０個、好ましくは１から１０個、より好ましくは１から７個、さらに好ましくは１から５個、特に好ましくは１から３個、あるいは１個または２個である。
　さらに、本発明において「ＨＳＰ７０タンパク質」には、配列番号２で示されたアミノ酸配列とＢＬＡＳＴ（Ｂａｓｉｃ　Ｌｏｃａｌ　Ａｌｉｇｎｍｅｎｔ　Ｓｅａｒｃｈ　Ｔｏｏｌ　ａｔ　ｔｈｅ　Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｌｏｇｉｃａｌ　Ｉｎｆｏｒｍａｔｉｏｎ（米国国立生物学情報センターの基本ローカルアラインメント検索ツール））等（例えば、デフォルトすなわち初期設定のパラメータ）を用いて計算したときに、８０％以上、８５％以上、９０％以上、より好ましくは９５％以上、最も好ましくは９９％以上の配列同一性を有し、かつ配列番号２で示されたアミノ酸配列を有するＨＳＰ７０タンパク質と同じ活性を有するタンパク質も含まれる。
　したがって、本発明において「ＨＳＰ７０タンパク質」には、配列番号２で示されたアミノ酸配列を有するＨＳＰ７０タンパク質のオルソログも含まれる。
　本発明において「ＨＳＰ７０タンパク質」自体は、単独の投与によって癌細胞の増殖を阻害する活性を有していても、有していなくても良い。
　本発明において、配列番号１で表されるポリペプチドは「ＥＣＩ３０１」（株式会社ＥＣＩ）とも称される公知のポリペプチドである。以下、本明細書において「配列番号１で表されるポリペプチド」を「ＥＣＩ３０１」と記載する。ＥＣＩ３０１はヒトの体内に存在し、免疫賦活作用を有する走化性タンパク質のひとつであるマクロファージ炎症性タンパク質−１α（以下、「ＭＩＰ−１α」と記載）の誘導体であり、ＭＩＰ−１αのアミノ酸配列において２６位のアスパラギン酸が、アラニンに置換された６９アミノ酸からなるポリペプチドである（図１）。ＥＣＩ３０１は放射線治療と併用することによって、完全寛解を含む顕著な腫瘍縮小効果と延命効果ならびに他の制癌剤に認められない非放射線照射部位の腫瘍に対する縮小効果が得られることが知られている（Ｓｈｉｒａｉｓｈｉ　Ｋ．ら、Ｃｌｉｎ　Ｃａｎｃｅｒ　Ｒｅｓ．２００８　Ｆｅｂ　１５；１４（４）：１１５９−６６）。
　また本発明において、「ＥＣＩ３０１」には、配列番号１で示されたアミノ酸配列に１~数個のアミノ酸の欠失、置換、付加または挿入を有し、かつＥＣＩ３０１と同じ活性を有するポリペプチドも含まれる。「ＥＣＩ３０１と同じ活性」とは、上記ＨＳＰ７０タンパク質と共に投与された場合に、癌細胞の増殖を阻害する活性を意味する。
　さらに、本発明において「ＥＣＩ３０１」には、配列番号１で示されたアミノ酸配列とＢＬＡＳＴ等（例えば、デフォルトすなわち初期設定のパラメータ）を用いて計算したときに、９０％以上、より好ましくは９５％以上、最も好ましくは９９％以上の配列同一性を有し、かつＥＣＩ３０１と同じ活性を有するポリペプチドも含まれる。
　本発明において、ＥＣＩ３０１自体は、単独の投与によって癌細胞の増殖を阻害する活性を有していても、有していなくても良い。
　ＨＳＰ７０タンパク質およびＥＣＩ３０１は、当業者にとって周知であるように、遺伝子工学的手法を用いて、製造・精製することができる。すなわち、当該タンパク質をコードするＤＮＡを適当なベクターに組込み、このベクターを適当な宿主細胞に導入し、当該タンパク質を発現させることが可能である。宿主細胞としては、周知の大腸菌などの細菌細胞、酵母、ＳＦ９、ＳＦ２１などの昆虫細胞、ＣＯＳ１、ＣＯＳ７、ＣＨＯ、ＨＥＫ２９３等、動物および植物細胞を用いることが可能である。発現されたタンパク質は、宿主細胞の培養上清より、タンパク質精製に用いられる公知の方法、例えば、硫安塩析、有機溶媒（エタノール、メタノール、アセトン等）による沈殿分離、イオン交換クロマトグラフィー、等電点クロマトグラフィー、ゲルろ過クロマトグラフィー、疎水性クロマトグラフィー、吸着カラムクロマトグラフィー、基質または抗体などを利用したアフィニティークロマトグラフィー、逆相カラムクロマトグラフィーなどのクロマトグラフィー、精密ろ過、限外ろ過、逆浸透ろ過等の濾過処理など、を１つまたは複数組み合わせて用いて精製することが可能である。
　本発明において、ＨＳＰ７０タンパク質および／またはＥＣＩ３０１は所望の性質を付与すべく適宜修飾することができる。例えば、ＨＳＰ７０タンパク質および／またはＥＣＩ３０１をＰＥＧ化することによって、生体内での分解を抑制または遅延することができる。
　また、別の実施形態として、本発明の医薬組成物は、有効成分としてＨＳＰ７０タンパク質をコードするＤＮＡおよびＥＣＩ３０１をコードするＤＮＡ、あるいはＨＳＰ７０タンパク質をコードするＤＮＡを含む発現ベクターおよびＥＣＩ３０１をコードするＤＮＡを含む発現ベクターを含む。
　当該医薬組成物に含まれるＤＮＡは、導入された標的細胞において転写・翻訳され、ＨＳＰ７０タンパク質および／またはＥＣＩ３０１を発現・分泌することが可能である。
　ＨＳＰ７０タンパク質をコードするＤＮＡとしては、ＧｅｎＢａｎｋなどに登録された公知のヌクレオチド配列を有するＤＮＡを用いることができる。本発明においては、上記配列番号２で示されたアミノ酸配列を有するＨＳＰ７０タンパク質をコードするＤＮＡ（配列番号３）を用いることができる。
　ＥＣＩ３０１をコードするＤＮＡは、ヒトＭＩＰ−１αの塩基配列において、２６位のアスパラギン酸をコードする塩基配列部位に、アラニンをコードする塩基配列を、ＰＣＲによる特異的変異導入法などの公知の手法によって導入することによって得ることができるが、好ましくは配列番号４で示される塩基配列からなるＤＮＡを利用することができる。
　本発明において、「ＨＳＰ７０タンパク質をコードするＤＮＡ」および「ＥＣＩ３０１をコードするＤＮＡ」には、必要に応じて、その５’末端、３’末端または任意の部位に、分泌シグナルペプチドをコードするＤＮＡを機能しうるかたちで連結することができる。分泌シグナルペプチドとしては、標的細胞において転写・翻訳されたＨＳＰ７０タンパク質およびＥＣＩ３０１が細胞外へと分泌され得る限り、特に限定されることなく公知のものを利用することができる。「機能しうるかたちで連結する」とは、当該ＤＮＡが導入された標的細胞において、分泌シグナルペプチドが正確に発現されるように、ＨＳＰ７０タンパク質またはＥＣＩ３０１をコードするＤＮＡと分泌シグナルペプチドをコードするＤＮＡとを連結することを意味する。ここで「連結」は直接連結されても良いし、適当な長さ及び配列のスペーサーを介して間接的に連結されても良い。
　本発明において、「ＨＳＰ７０タンパク質をコードするＤＮＡ」および「ＥＣＩ３０１をコードするＤＮＡ」には、配列番号３および配列番号４に示される塩基配列において、１から数個のヌクレオチドの欠失、置換、付加または挿入を有し、かつそれぞれ配列番号２で示されたアミノ酸配列を有するＨＳＰ７０タンパク質と同じ活性を有するタンパク質をコードするＤＮＡおよびＥＣＩ３０１と同じ活性を有するポリペプチドをコードするＤＮＡも含まれる。「ＨＳＰ７０タンパク質と同じ活性」および「ＥＣＩ３０１と同じ活性」とは、上に定義するとおりである。
　また、「ＨＳＰ７０タンパク質をコードするＤＮＡ」および「ＥＣＩ３０１をコードするＤＮＡ」には、配列番号３および配列番号４に示される塩基配列に相補的な配列からなるＤＮＡとストリンジェントな条件下でハイブリダイズし、かつそれぞれ配列番号２で示されたアミノ酸配列を有するＨＳＰ７０タンパク質と同じ活性を有するタンパク質をコードするＤＮＡおよびＥＣＩ３０１と同じ活性を有するポリペプチドをコードするＤＮＡも含まれる。「ストリンジェントな条件」とは、いわゆる特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件をいい、例えば、２~６×ＳＳＣ（１×ＳＳＣの組成：０．１５Ｍ　ＮａＣｌ，０．０１５Ｍ　クエン酸ナトリウム，ｐＨ　７．０）および０．１~０．５％ＳＤＳを含有する溶液中４２~５５℃にてハイブリダイズを行い、０．１~０．２×ＳＳＣおよび０．１~０．５％ＳＤＳを含有する溶液中５５~６５℃にて洗浄を行う条件をいう。
　さらに、「ＨＳＰ７０タンパク質をコードするＤＮＡ」および「ＥＣＩ３０１をコードするＤＮＡ」には配列番号３および配列番号４に示される塩基配列と、上記ＢＬＡＳＴ等（例えば、デフォルトすなわち初期設定のパラメータ）を用いて計算したときに、８０％以上、８５％以上、９０％以上、より好ましくは９５％以上、最も好ましくは９９％以上の同一性を有する塩基配列からなり、かつかつそれぞれ配列番号２で示されたアミノ酸配列を有するＨＳＰ７０タンパク質と同じ活性を有するタンパク質をコードするＤＮＡおよびＥＣＩ３０１と同じ活性を有するポリペプチドをコードするＤＮＡも含まれる。
　したがって、本発明において「ＨＳＰ７０タンパク質をコードするＤＮＡ」には、配列番号２で示されたアミノ酸配列を有するＨＳＰ７０タンパク質のオルソログをコードするＤＮＡも含まれる。
　ＨＳＰ７０タンパク質をコードするＤＮＡまたはＥＣＩ３０１をコードするＤＮＡを含む発現ベクターは、当業者に周知である遺伝子工学的手法を用いて、適当なベクターに、ＨＳＰ７０タンパク質をコードするＤＮＡまたはＥＣＩ３０１をコードするＤＮＡをプロモーターおよび／またはその他の制御配列と機能し得るかたちで連結して挿入することによって作製することができる。ここで「機能し得るかたちで連結して挿入する」とは、当該発現ベクターが導入された細胞において、プロモーターおよび／またはその他の制御配列の制御の下、ＨＳＰ７０タンパク質またはＥＣＩ３０１が発現されるように、プロモーターおよび／またはその他の制御配列を連結してベクターに組み込むことを意味する。
　用いることができるベクターとしては、アデノウイルス、アデノ随伴ウイルス、レトロウイルス等のウイルスベクター、プラスミドベクター、リポソームベクターなど（これらに限定されない）が公知のベクターを利用することができる（別冊実験医学、遺伝子治療の基礎技術、羊土社、１９９６；別冊実験医学、遺伝子導入＆発現解析実験法、羊土社、１９９７；日本遺伝子治療学会編、遺伝子治療開発研究ハンドブック、エヌ・ティー・エス、１９９９）。
　本明細書において、「癌」とは、不死的に転移および増殖する悪性腫瘍を指す。したがって、本明細書において、「癌」と「腫瘍」なる用語は、相互互換的に用いられる。
　本発明の医薬組成物を用いて治療し得る癌としては、固形癌が挙げられ、当該固形癌は原発癌および転移癌のいずれであっても良い。
　固形癌とは、血管により支持される多細胞塊として成長する腫瘍細胞を意味し、例えば、口腔内癌、大腸癌、結腸直腸癌、肺癌、乳癌、脳腫瘍、黒色腫、腎細胞癌、胃癌、膵臓癌、子宮頚癌、子宮内膜癌、卵巣癌、食道癌、肝臓癌、頭頚部扁平上皮癌、皮膚癌、膀胱癌、尿路癌、前立腺癌、絨毛癌、咽頭癌、喉頭癌、きょう膜腫、男性胚腫、子宮内膜過形成、子宮内膜症、胚芽腫、線維肉腫、カポジ肉腫、血管腫、海綿状血管腫、血管芽腫、網膜芽腫、星状細胞腫、神経線維腫、稀突起謬腫、髄芽腫、神経芽腫、神経膠腫、横紋筋肉腫、謬芽腫、骨原性肉腫、平滑筋肉腫、甲状肉腫およびウィルムス腫瘍などが挙げられるが、これらに限定されない。特に好ましくは、大腸癌、肺癌および線維肉腫である。
　本発明の医薬組成物を用いた治療対象には、上記癌を罹患するヒトおよび非ヒト哺乳類（家畜（ウシ、ウマ、ヤギ、ヒツジおよびブタ）およびペット（ネコおよびイヌ）など）が含まれるが、好ましくはヒトである。
　本発明の医薬組成物は、経口投与または非経口投与（例えば、静脈内投与、動脈内投与、注射による局所投与、腹腔または胸腔への投与、皮下投与、筋肉内投与、舌下投与、経皮吸収または直腸内投与など）によって投与することができる。
　また、本発明の医薬組成物は、投与経路に応じて適当な剤形とすることができる。具体的には注射剤、懸濁剤、乳化剤、軟膏剤、クリーム剤、錠剤、カプセル剤、顆粒剤、散剤、丸剤、細粒剤、トローチ錠、直腸投与剤、油脂性坐剤、水溶性坐剤等の各種製剤形態に調製することができる。
　これらの各種製剤は、通常用いられている賦形剤、増量剤、結合剤、浸潤剤、崩壊剤、表面活性剤、滑沢剤、分散剤、緩衝剤、保存剤、溶解補助剤、防腐剤、着色料、香味剤、および安定化剤などを用いて常法により製造することができる。
　賦形剤としては、例えば、乳糖、果糖、ブドウ糖、コーンスターチ、ソルビットおよび結晶セルロース、滅菌水、エタノール、グリセロール、生理食塩水、緩衝液などが、崩壊剤としては、例えば澱粉、アルギン酸ナトリウム、ゼラチン、炭酸カルシウム、クエン酸カルシウム、デキストリン、炭酸マグネシウムおよび合成ケイ酸マグネシウムなどが、結合剤としては、例えばメチルセルロースまたはその塩、エチルセルロース、アラビアゴム、ゼラチン、ヒドロキシプロピルセルロースおよびポリビニルピロリドンなどが、滑沢剤としては、タルク、ステアリン酸マグネシウム、ポリエチレングリコールおよび硬化植物油などが、安定化剤としては、例えばアルギニン、ヒスチジン、リジン、メチオニンなどのアミノ酸、ヒト血清アルブミン、ゼラチン、デキストラン４０、メチルセルロース、亜硫酸ナトリウム、メタ亜硫酸ナトリウムなどが、その他の添加剤としては、シロップ、ワセリン、グリセリン、エタノール、プロピレングリコール、クエン酸、塩化ナトリウム、亜硝酸ソーダおよびリン酸ナトリウムなどがそれぞれ挙げられる。
　以下、特に断りのない限り、ＨＳＰ７０タンパク質およびＥＣＩ３０１を含有する医薬組成物について記載するが、ＨＳＰ７０タンパク質をコードするＤＮＡおよびＥＣＩ３０１をコードするＤＮＡ、あるいはＨＳＰ７０タンパク質をコードするＤＮＡを含む発現ベクターおよびＥＣＩ３０１をコードするＤＮＡを含む発現ベクターを含む医薬組成物についても同様である。
　本発明の医薬組成物は、ＨＳＰ７０タンパク質およびＥＣＩ３０１を共に含有する配合剤として一の剤型に製剤化したものでも、同時にまたは間隔を空けて別々に使用できるように、ＨＳＰ７０タンパク質あるいはＥＣＩ３０１のいずれかを含有する単剤として製剤化したものであってもよい。すなわち、本発明は、ＨＳＰ７０タンパク質を含有する単剤およびＥＣＩ３０１を含有する単剤の組み合わせ医薬であってもよい。単剤として製剤化する場合は、当該製剤はそれぞれ異なる投与形態であっても同一の投与形態であってよい。あるいは、ＨＳＰ７０タンパク質およびＥＣＩ３０１のいずれかをそれぞれ含有する単剤を、使用直前に混合して用いても良い。
　本発明の医薬組成物には、ＨＳＰ７０タンパク質およびＥＣＩ３０１をそれぞれ、好ましくは０．０１~３０００００μｇ／１０ｍＬ、より好ましくは０．１~３００００μｇ／１０ｍＬ、さらに好ましくは１０~３０００μｇ／１０ｍＬ、の範囲で適宜含めることができるが、配合剤または単剤の混合に際しては、ＨＳＰ７０タンパク質およびＥＣＩ３０１を重量比にして、１：０．４~１６、好ましくは１：４の比率で含める。下記実施例にて詳述するように、当該比率でＨＳＰ７０タンパク質およびＥＣＩ３０１を用いた場合に、最も有効に癌細胞の増殖を阻害することができる。
　本発明の医薬組成物において、ＨＳＰ７０タンパク質とＥＣＩ３０１は、共有結合または非共有結合によって結合されていても良い。
　本発明の医薬組成物の投与量は、患者の年齢、体重、疾患の重篤度などの要因によって変化し得るが、ＨＳＰ７０タンパク質およびＥＣＩ３０１をそれぞれ、１回の投与につき体重１ｋｇあたり０．００００１ｍｇ~１００ｍｇ、好ましくは０．０００１ｍｇ~１０ｍｇ、さらに好ましくは０．０５０ｍｇ~１．０ｍｇの範囲から適宜選択される量を投与することができる。典型的な投与量としては７０ｋｇの体重のヒトに対して、ＨＳＰ７０タンパク質およびＥＣＩ３０１をそれぞれ、１回の投与につき３．５ｍｇ~３５ｍｇの範囲である。投与に際しては、ＨＳＰ７０タンパク質およびＥＣＩ３０１を重量比にして、１：０．４~１６、好ましくは１：４の比率で投与する。本発明の医薬組成物が、ＨＳＰ７０タンパク質およびＥＣＩ３０１をそれぞれ含有する単剤として製剤化されている場合には、第一の単剤を投与してから、０~６０分、好ましくは０~１５分以内に、第二の単剤を投与する。投与の順番は、特に限定されない。投与スケジュールも、患者の年齢、体重、疾患の重篤度などの要因によって変化し得るが、本発明の医薬組成物を患者に対して、１日に１回または複数回（例えば、２回、３回またはそれ以上）、毎日または週に２~５日の連続投与を１~８週間にわたって、投与する。
　本発明の医薬組成物が、有効成分として上記ＨＳＰ７０タンパク質をコードするＤＮＡおよびＥＣＩ３０１をコードするＤＮＡあるいは当該ＤＮＡを含むベクターを含む場合、当該ＤＮＡあるいはベクターは遺伝子治療の分野で用いられている種々の方法で被験体に送達することができる。例えば、ＤＮＡあるいはベクターを被験体に直接投与（ｉｎ　ｖｉｖｏ法）してもよいし、または被験体から採取した細胞に導入して、目的のＨＳＰ７０タンパク質およびＥＣＩ３０１を発現する形質転換細胞を選択してからその細胞を被験体に投与してもよい（ｅｘ　ｖｉｖｏ法）。目的の組織または細胞にＤＮＡあるいはベクターを投与するために使用し得る遺伝子送達機構には、コロイド分散系、リポソーム誘導系、人工ウイルスエンベロープなどが含まれる。例えば、送達系は巨大分子複合体、ナノカプセル、ミクロスフェア、ビーズ、水中油型乳剤、ミセル、混合ミセル、リポソーム等を利用することができる。またＤＮＡあるいはベクターの細胞導入（形質転換）は、例えば、リン酸カルシウム法、ＤＥＡＥデキストラン法、エレクトロポレーション法、またはリポフェクション法等の一般的な遺伝子導入法を用いて行うことができる。
　本発明の医薬組成物の最適な投与量および投与スケジュールは、実験動物や腫瘍細胞を用いたモデル実験によって、当業者であれば適宜決定することができる。
　本発明の医薬組成物は、別の癌治療方法や抗癌剤、および／またはサイトカインと併用することができる。このような治療方法としては、外科的手術、放射線療法、化学療法、免疫療法、温熱療法などが挙げられる。抗癌剤としては、特に限定されることなく、公知のもの（例えば、シスプラチン、カルボプラチン、インターフェロン、アスパラギナーゼ、タモキシフェン、ロイプロリド、フルタミド、メゲストロール、マイトマイシン、ブレオマイシン、ドキソルビシン、イリノテカン、タキソールなど）が挙げられる。また、サイトカインとしては、腫瘍に対する免疫応答を増強する作用を有するものが好ましく、例えば、インターロイキン−１α（ＩＬ−１α）、インターロイキン−１β（ＩＬ−１β）、インターロイキン−２（ＩＬ−２）、インターロイキン−３（ＩＬ−３）、インターロイキン−４（ＩＬ−４）、インターロイキン−５（ＩＬ−５）、インターロイキン−６（ＩＬ−６）、インターロイキン−７（ＩＬ−７）、インターロイキン−８（ＩＬ−８）、インターロイキン−９（ＩＬ−９）、インターロイキン−１０（ＩＬ−１０）、インターロイキン−１１（ＩＬ−１１）、インターロイキン−１２（ＩＬ−１２）、インターフェロンα（ＩＦＮα）、インターフェロンβ（ＩＦＮβ）、インターフェロンγ（ＩＦＮγ）、腫瘍壊死因子α（ＴＮＦα）、腫瘍壊死因子β（ＴＮＦβ）、顆粒球コロニー刺激因子（Ｇ−ＣＳＦ）、顆粒球／マクロファージコロニー刺激因子（ＧＭ−ＣＳＦ）、及びトランスフォーミング増殖因子β（ＴＧＦ−β）が挙げられるが、これらに限定されない。
　また、本発明の医薬組成物は、ＨＳＰの発現を上昇させる刺激と併用することができる。ＨＳＰの発現を上昇させる刺激としては、温熱、無酸素、放射線、アルコール、エネルギー代謝の特定のインヒビター、グルタミン、ＨＳＰ転写因子、感染などが挙げられるがこれらに限定されない。当該刺激によって、癌細胞において、ＨＳＰ（特に、ＨＳＰ７０）の発現を上昇させることによって、本発明の医薬組成物におけるＨＳＰ７０タンパク質の含有量や投与量を減少させることができる。
　本発明の医薬組成物を投与することによって、腫瘍の増殖をおよそ２０％、３０％、４０％、５０％、６０％、７０％またはそれ以上、抑制することができる。
　本発明の医薬組成物は、従来のＨＳＰ−ペプチド複合体を用いた組成物とその作用機構が大きく異なっている。従来のＨＳＰ−ペプチド複合体は、ＨＳＰと癌抗原ペプチドからなり、当該癌抗原ペプチドに対する免疫を活性化させ、癌細胞を除去しようとするものである。したがって、当該複合体の効果は、特定の癌抗原ペプチドを有する癌種のみに限定される。一方、本発明の医薬組成物は、ペプチドとして特定の癌抗原ペプチドを含有するものではなく、ヒトＭＩＰ−１αの誘導体であるＥＣＩ３０１を含有する。したがって、従来のＨＳＰ−ペプチド複合体の作用機構とは全く異なる機構によって癌細胞に作用するものであり、その効果は、特定の癌抗原ペプチドを有する癌種のみに限定されるものではない。また、有効成分として、腫瘍ライセートをそのままＨＳＰに結合させるといった、非特定抗原を用いることもないため、医薬品開発申請で求められる医薬品の品質に関する、Ｃｈｅｍｉｓｔｒｙ／Ｍａｎｕｆａｃｔｕｒｅ／Ｃｏｎｔｒｏｌ（ＣＭＣ）の要件を具備することができ、医薬品として実用性を有するものである。
　本発明は、さらに本発明の医薬組成物を用いた癌の治療法を包含する。該方法により治療し得る癌としては、上に定義したような固形癌が含まれる。
　以下に実施例を示して本発明を具体的に説明するが、本発明はこれらの実施例に限定されるものでない。 Hereinafter, the present invention will be described in detail.
The present invention provides a pharmaceutical composition for treating cancer, comprising heat shock protein (HSP) and the polypeptide represented by SEQ ID NO: 1 (ECI301).
The heat shock protein that can be used in the present invention includes all heat shock proteins except HSP90 and HSP60, and is preferably HSP70 protein.
In the present invention, the HSP70 protein is not particularly limited, but is preferably derived from human. By using a human-derived HSP70 protein, it is possible to minimize the immune rejection that occurs when administered to a human patient and increase the therapeutic effect.
The HSP70 protein is known, isolated from various biological species, and sequence information is disclosed. The sequence information of human-derived HSP70 protein is registered in GenBank as accession numbers NP_002145, AAI26125, etc., and the sequence information of mouse-derived HSP70 protein is registered in GenBank as accession numbers NM_024172, etc., and these can be used. In the present invention, the HSP70 protein having the amino acid sequence represented by SEQ ID NO: 2 can be used.
In the present invention, the “HSP70 protein” includes one to several amino acid deletions, substitutions, additions or insertions in the amino acid sequence represented by SEQ ID NO: 2, and the amino acid represented by SEQ ID NO: 2. Also included are proteins having the same activity as the HSP70 protein having the sequence. “Same activity as HSP70 protein” means an activity that inhibits the growth of cancer cells when administered with an ECI301 polypeptide as detailed below. In the present specification, the range of “1 to several” is not particularly limited, but for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, Particularly preferably, it is 1 to 3, or 1 or 2.
Furthermore, in the present invention, the “HSP70 protein” includes the amino acid sequence represented by SEQ ID NO: 2 and BLAST (Basic Local Alignment Search at the National Center for Biological Information) (basic local alignment search tool of the National Center for Biological Information). ) Etc. (eg, default or default parameters) when calculated using 80% or more, 85% or more, 90% or more, more preferably 95% or more, most preferably 99% or more. A protein having the same activity as the HSP70 protein having the amino acid sequence shown in SEQ ID NO: 2 is also included.
Therefore, in the present invention, the “HSP70 protein” includes an ortholog of the HSP70 protein having the amino acid sequence shown by SEQ ID NO: 2.
In the present invention, “HSP70 protein” itself may or may not have the activity of inhibiting the growth of cancer cells by administration alone.
In the present invention, the polypeptide represented by SEQ ID NO: 1 is a known polypeptide also referred to as “ECI301” (ECI Corporation). Hereinafter, the “polypeptide represented by SEQ ID NO: 1” is referred to as “ECI301” in the present specification. ECI301 is a derivative of macrophage inflammatory protein-1α (hereinafter referred to as “MIP-1α”), which is one of the chemotactic proteins that exist in the human body and has an immunostimulatory action, and the amino acid sequence of MIP-1α In FIG. 1, the aspartic acid at position 26 is a polypeptide consisting of 69 amino acids substituted with alanine (FIG. 1). It is known that ECI301 can be used in combination with radiotherapy to achieve a remarkable tumor reduction effect and life extension effect including complete remission, and a reduction effect on tumors at non-irradiated sites not found in other anticancer agents (Shiraishi). K. et al., Clin Cancer Res. 2008 Feb 15; 14 (4): 1159-66).
In the present invention, “ECI301” also includes a polypeptide having the deletion, substitution, addition or insertion of one to several amino acids in the amino acid sequence represented by SEQ ID NO: 1 and having the same activity as ECI301. included. “The same activity as ECI301” means an activity that inhibits the growth of cancer cells when administered together with the HSP70 protein.
Furthermore, in the present invention, “ECI301” contains 90% or more, more preferably 95%, when calculated using the amino acid sequence shown in SEQ ID NO: 1, BLAST, etc. (for example, default or default parameters). As described above, a polypeptide having a sequence identity of 99% or more and having the same activity as ECI301 is also included.
In the present invention, ECI301 itself may or may not have the activity of inhibiting the growth of cancer cells when administered alone.
HSP70 protein and ECI301 can be produced and purified using genetic engineering techniques, as is well known to those skilled in the art. That is, DNA encoding the protein can be incorporated into an appropriate vector, the vector can be introduced into an appropriate host cell, and the protein can be expressed. As host cells, well-known bacterial cells such as E. coli, insect cells such as yeast, SF9, and SF21, animal and plant cells such as COS1, COS7, CHO, HEK293, and the like can be used. The expressed protein is obtained from the culture supernatant of the host cell by known methods used for protein purification, such as ammonium sulfate salting out, precipitation separation with an organic solvent (ethanol, methanol, acetone, etc.), ion exchange chromatography, isoelectricity. Point chromatography, gel filtration chromatography, hydrophobic chromatography, adsorption column chromatography, affinity chromatography using substrates or antibodies, chromatography such as reverse phase column chromatography, microfiltration, ultrafiltration, reverse osmosis filtration It is possible to purify by using one or a combination of a plurality of filtration treatments.
In the present invention, the HSP70 protein and / or ECI301 can be modified as appropriate to impart desired properties. For example, degradation in vivo can be suppressed or delayed by PEGylating HSP70 protein and / or ECI301.
In another embodiment, the pharmaceutical composition of the present invention comprises, as an active ingredient, DNA encoding HSP70 protein and DNA encoding ECI301, or an expression vector containing DNA encoding HSP70 protein and DNA encoding ECI301. Containing expression vectors.
The DNA contained in the pharmaceutical composition is transcribed and translated in the introduced target cell, and can express and secrete HSP70 protein and / or ECI301.
As a DNA encoding the HSP70 protein, a DNA having a known nucleotide sequence registered in GenBank or the like can be used. In the present invention, DNA (SEQ ID NO: 3) encoding HSP70 protein having the amino acid sequence shown by SEQ ID NO: 2 can be used.
The DNA encoding ECI301 is prepared by a known method such as specific mutagenesis by PCR, in which the base sequence encoding alanine is inserted into the base sequence site encoding aspartic acid at position 26 in the base sequence of human MIP-1α. Although it can be obtained by introduction, DNA consisting of the base sequence represented by SEQ ID NO: 4 can be preferably used.
In the present invention, “DNA encoding HSP70 protein” and “DNA encoding ECI301” include a DNA encoding a secretory signal peptide at its 5 ′ end, 3 ′ end or any site, as necessary. Can be connected in a functional way. As the secretory signal peptide, any known signal can be used without particular limitation as long as the HSP70 protein and ECI301 transcribed and translated in the target cell can be secreted outside the cell. “Link in a functional manner” means that the DNA encoding the HSP70 protein or ECI301 and the DNA encoding the secretory signal peptide so that the secretory signal peptide is accurately expressed in the target cell into which the DNA has been introduced. Is connected. Here, “linkage” may be directly linked or indirectly linked through a spacer having an appropriate length and sequence.
In the present invention, “DNA encoding HSP70 protein” and “DNA encoding ECI301” include deletion, substitution and addition of one to several nucleotides in the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4. Alternatively, a DNA encoding a protein having the same activity as the HSP70 protein having an insertion and having the amino acid sequence shown by SEQ ID NO: 2 and a DNA encoding a polypeptide having the same activity as ECI301 are also included. “Same activity as HSP70 protein” and “Same activity as ECI301” are as defined above.
In addition, “DNA encoding HSP70 protein” and “DNA encoding ECI301” hybridize under stringent conditions with DNA comprising a sequence complementary to the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4. And a DNA encoding a protein having the same activity as the HSP70 protein having the amino acid sequence shown in SEQ ID NO: 2 and a DNA encoding a polypeptide having the same activity as the ECI301. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, 2 to 6 × SSC (composition of 1 × SSC: 0.15M NaCl, 0 .15M sodium citrate, pH 7.0) and 0.1-0.5% SDS in a solution containing 42-55 ° C., and 0.1-0.2 × SSC and 0.1 It refers to conditions for washing at 55 to 65 ° C. in a solution containing 0.5% SDS.
Further, the “DNA encoding HSP70 protein” and the “DNA encoding ECI301” use the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 and the above BLAST and the like (for example, default or initial setting parameters). An amino acid comprising a base sequence having an identity of 80% or more, 85% or more, 90% or more, more preferably 95% or more, and most preferably 99% or more when calculated, and represented by SEQ ID NO: 2, respectively Also included are DNA encoding a protein having the same activity as the HSP70 protein having a sequence and DNA encoding a polypeptide having the same activity as ECI301.
Therefore, in the present invention, “DNA encoding HSP70 protein” includes DNA encoding the ortholog of HSP70 protein having the amino acid sequence shown by SEQ ID NO: 2.
An expression vector containing DNA encoding HSP70 protein or DNA encoding ECI301 is obtained by using a genetic engineering technique well known to those skilled in the art, to an appropriate vector with DNA encoding HSP70 protein or DNA encoding ECI301. It can be produced by linking with a promoter and / or other regulatory sequences in a functional manner. Here, “to be ligated and inserted in a functional manner” means that HSP70 protein or ECI301 is expressed under the control of a promoter and / or other regulatory sequences in a cell into which the expression vector has been introduced. , Promoter and / or other regulatory sequences are linked and incorporated into the vector.
As vectors that can be used, known vectors such as adenoviruses, adeno-associated viruses, retroviruses and other viral vectors, plasmid vectors, liposome vectors, and the like can be used (separate volume experimental medicine, gene). Basic Technology of Treatment, Yodosha, 1996; Separate Volume Experimental Medicine, Gene Transfer & Expression Analysis Experiment Method, Yodosha, 1997; edited by Japanese Society of Gene Therapy, Gene Therapy Development Research Handbook, NTS, 1999).
As used herein, “cancer” refers to a malignant tumor that metastasizes and grows immortally. Accordingly, in this specification, the terms “cancer” and “tumor” are used interchangeably.
Examples of cancer that can be treated using the pharmaceutical composition of the present invention include solid cancer, and the solid cancer may be either primary cancer or metastatic cancer.
Solid cancer means tumor cells that grow as multicellular mass supported by blood vessels, such as oral cancer, colon cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, melanoma, renal cell cancer, stomach cancer, Pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, esophageal cancer, liver cancer, squamous cell carcinoma of the head and neck, skin cancer, bladder cancer, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, today Meningiomas, male embryos, endometrial hyperplasia, endometriosis, embryonal, fibrosarcoma, Kaposi's sarcoma, hemangiomas, cavernous hemangioma, hemangioblastoma, retinoblastoma, astrocytoma, nerve fiber Tumors, oligodendroma, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, glioblastoma, osteogenic sarcoma, leiomyosarcoma, thyroid sarcoma, and Wilms tumor. It is not limited. Particularly preferred are colon cancer, lung cancer and fibrosarcoma.
Subjects to be treated with the pharmaceutical composition of the present invention include humans and non-human mammals (domestic animals (cow, horses, goats, sheep and pigs) and pets (cats and dogs), etc.) suffering from the above cancer. Preferably, it is a human.
The pharmaceutical composition of the present invention is administered orally or parenterally (for example, intravenous administration, intraarterial administration, local administration by injection, administration to the abdominal cavity or thoracic cavity, subcutaneous administration, intramuscular administration, sublingual administration, transdermal For example, by absorption or rectal administration).
Moreover, the pharmaceutical composition of this invention can be made into a suitable dosage form according to an administration route. Specifically, injections, suspensions, emulsifiers, ointments, creams, tablets, capsules, granules, powders, pills, fine granules, troches, rectal administration, oily suppositories, water-soluble It can be prepared in various pharmaceutical forms such as suppositories.
These various preparations are commonly used excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, preservatives. , And can be produced by conventional methods using colorants, flavoring agents, and stabilizers.
Excipients include, for example, lactose, fructose, glucose, corn starch, sorbit and crystalline cellulose, sterile water, ethanol, glycerol, physiological saline, buffer, etc., and disintegrants include, for example, starch, sodium alginate, gelatin, Examples of binders include calcium carbonate, calcium citrate, dextrin, magnesium carbonate, and synthetic magnesium silicate. For example, methyl cellulose or a salt thereof, ethyl cellulose, gum arabic, gelatin, hydroxypropyl cellulose, and polyvinylpyrrolidone are lubricants. Are talc, magnesium stearate, polyethylene glycol and hydrogenated vegetable oil, and examples of stabilizers include amino acids such as arginine, histidine, lysine and methionine, human serum albumin , Gelatin, dextran 40, methylcellulose, sodium sulfite, sodium metasulfite, etc. Other additives include syrup, petrolatum, glycerin, ethanol, propylene glycol, citric acid, sodium chloride, sodium nitrite and sodium phosphate Each is listed.
Hereinafter, unless otherwise specified, a pharmaceutical composition containing HSP70 protein and ECI301 will be described. DNA encoding HSP70 protein and DNA encoding ECI301, or an expression vector containing DNA encoding HSP70 protein and ECI301 are described below. The same applies to a pharmaceutical composition containing an expression vector containing the encoding DNA.
The pharmaceutical composition of the present invention may be formulated with either HSP70 protein or ECI301 so that even if it is formulated in one dosage form as a combination drug containing both HSP70 protein and ECI301, it can be used separately at the same time or at intervals. It may be formulated as a single agent containing. That is, the present invention may be a combined medicine of a single agent containing HSP70 protein and a single agent containing ECI301. When formulated as a single agent, the formulations may be in different dosage forms or in the same dosage form. Alternatively, single agents each containing either HSP70 protein or ECI301 may be mixed and used immediately before use.
In the pharmaceutical composition of the present invention, HSP70 protein and ECI301 are each suitably in the range of preferably 0.01 to 300000 μg / 10 mL, more preferably 0.1 to 30000 μg / 10 mL, and further preferably 10 to 3000 μg / 10 mL. When mixing the compounding agent or single agent, HSP70 protein and ECI301 are included in a weight ratio of 1: 0.4 to 16, preferably 1: 4. As described in detail in the Examples below, when HSP70 protein and ECI301 are used in this ratio, the growth of cancer cells can be most effectively inhibited.
In the pharmaceutical composition of the present invention, the HSP70 protein and ECI301 may be bound by a covalent bond or a non-covalent bond.
The dose of the pharmaceutical composition of the present invention may vary depending on factors such as the patient's age, body weight, severity of disease, etc., but each of HSP70 protein and ECI301 can be administered at a dose of 0.00001 mg / kg body weight per administration. An amount appropriately selected from the range of 100 mg, preferably 0.0001 mg to 10 mg, more preferably 0.050 mg to 1.0 mg can be administered. A typical dose is in the range of 3.5 mg to 35 mg per administration of HSP70 protein and ECI301, respectively, for a human weighing 70 kg. In administration, HSP70 protein and ECI301 are administered at a weight ratio of 1: 0.4 to 16, preferably 1: 4. When the pharmaceutical composition of the present invention is formulated as a single agent each containing HSP70 protein and ECI301, it is 0 to 60 minutes, preferably 0 to 15 minutes after administration of the first single agent. Within, the second single agent is administered. The order of administration is not particularly limited. The dosing schedule may also vary depending on factors such as the patient's age, weight, severity of the disease, etc., but the pharmaceutical composition of the invention may be administered to the patient once or multiple times (eg, 3 or more times), administered daily or continuously for 2-5 days per week for 1-8 weeks.
When the pharmaceutical composition of the present invention contains DNA encoding the above HSP70 protein and DNA encoding ECI301 or a vector containing the DNA as an active ingredient, the DNA or vector can be used in various ways used in the field of gene therapy. The method can be delivered to a subject. For example, DNA or a vector may be directly administered to a subject (in vivo method), or introduced into a cell collected from the subject, and a transformed cell expressing the target HSP70 protein and ECI301 is selected. And the cells may be administered to a subject (ex vivo method). Gene delivery mechanisms that can be used to administer DNA or vectors to the tissue or cell of interest include colloidal dispersion systems, liposome-derived systems, artificial virus envelopes, and the like. For example, delivery systems can utilize macromolecular complexes, nanocapsules, microspheres, beads, oil-in-water emulsions, micelles, mixed micelles, liposomes, and the like. In addition, DNA or vector cell introduction (transformation) can be performed using a general gene introduction method such as a calcium phosphate method, a DEAE dextran method, an electroporation method, or a lipofection method.
The optimal dosage and administration schedule of the pharmaceutical composition of the present invention can be appropriately determined by those skilled in the art by model experiments using experimental animals and tumor cells.
The pharmaceutical composition of the present invention can be used in combination with another cancer treatment method, an anticancer agent, and / or a cytokine. Such treatment methods include surgery, radiation therapy, chemotherapy, immunotherapy, hyperthermia, and the like. The anticancer agent is not particularly limited, and examples thereof include known ones (for example, cisplatin, carboplatin, interferon, asparaginase, tamoxifen, leuprolide, flutamide, megestrol, mitomycin, bleomycin, doxorubicin, irinotecan, taxol, etc.). Moreover, as cytokine, what has the effect | action which enhances the immune response with respect to a tumor is preferable, for example, interleukin-1 (IL-1α), interleukin-1β (IL-1β), interleukin-2 (IL-2). ), Interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7) ), Interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12) ), Interferon α (IFNα), interferon β (IFNβ), interferon γ (IFNγ), tumor necrosis factor α (TNF) α), tumor necrosis factor β (TNFβ), granulocyte colony stimulating factor (G-CSF), granulocyte / macrophage colony stimulating factor (GM-CSF), and transforming growth factor β (TGF-β). However, it is not limited to these.
Moreover, the pharmaceutical composition of the present invention can be used in combination with a stimulus that increases the expression of HSP. Stimulations that increase HSP expression include, but are not limited to, heat, anoxia, radiation, alcohol, specific inhibitors of energy metabolism, glutamine, HSP transcription factors, infections, and the like. By this stimulation, the content and dosage of HSP70 protein in the pharmaceutical composition of the present invention can be decreased by increasing the expression of HSP (particularly HSP70) in cancer cells.
By administering the pharmaceutical composition of the present invention, tumor growth can be suppressed by approximately 20%, 30%, 40%, 50%, 60%, 70% or more.
The pharmaceutical composition of the present invention is greatly different from the composition using the conventional HSP-peptide complex in its action mechanism. A conventional HSP-peptide complex is composed of HSP and a cancer antigen peptide, and activates immunity to the cancer antigen peptide to remove cancer cells. Therefore, the effect of the complex is limited only to cancer types having a specific cancer antigen peptide. On the other hand, the pharmaceutical composition of the present invention does not contain a specific cancer antigen peptide as a peptide, but contains ECI301 which is a derivative of human MIP-1α. Therefore, it acts on cancer cells by a mechanism completely different from the mechanism of action of the conventional HSP-peptide complex, and the effect is not limited to only cancer types having a specific cancer antigen peptide. In addition, since non-specific antigens such as tumor lysate directly bound to HSP are not used as active ingredients, it has the requirements of Chemistry / Manufacture / Control (CMC) regarding the quality of drugs required in drug development applications. Can be used as a pharmaceutical product.
The present invention further includes a method for treating cancer using the pharmaceutical composition of the present invention. Cancers that can be treated by the method include solid cancers as defined above.
EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
腫瘍に対するＥＣＩ３０１および／またはＨＳＰ７０投与の効果
＜実験方法＞
マウス
　６週齢ＢＡＬＢ／ｃ系雄性マウスを日本ＳＬＣより購入し、馴化飼育の後に使用した。
薬物
ＥＣＩ３０１
ＥＣＩ３０１（株式会社ＥＣＩ）
　ＥＣＩ３０１　１ｍｇ／ｍｌ　ＰＢＳ水溶液に調製
ＨＳＰ７０
Ｍｏｕｓｅ　Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｓｐ７０−Ａ１　（Ｌｏｗ　Ｅｎｄｏｔｏｘｉｎ）（ＥＳＰ−５０２，ＥＮＺＯ　ＢＩＯＣＨＥＭ（旧Ａｓｓａｙ　Ｄｅｓｉｇｎｓ））
　ＨＳＰ７０　１０μｇ／１ｍｌＰＢＳに調製
実験系
　Ｃｏｌｏｎ２６　／ＢＡＬＢ／ｃ
　Ｃｏｌｏｎ２６細胞をＢＡＬＢ／ｃマウス右側腹部に皮下移植した（マウス１匹につき４×１０５細胞）。
　移植１１日目に、移植したＣｏｌｏｎ２６細胞の腫瘍径が約０．５~０．８ｃｍになった時点で３群（対照群、ＨＳＰ７０単独投与群およびＨＳＰ７０／ＥＣＩ３０１共投与群）に分けた（ｎ＝６）。
　ＨＳＰ７０／ＥＣＩ３０１共投与群には、ＨＳＰ７０薬物（５００ｎｇ　ＨＳＰ７０／０．２ｍｌ　ＰＢＳ）に、ＥＣＩ３０１薬物（２μｇ　ＥＣＩ３０１／２μｌＰＢＳ）を混合した配合剤を、移植１１日目に尾静脈内（ｉｖ）投与し（１日１回）、その後１日おいて再度、同様に投与した。
　ＨＳＰ７０単独投与群には、ＨＳＰ７０薬物（５００ｎｇ　ＨＳＰ７０／０．２ｍｌ　ＰＢＳ）に、２μｌＰＢＳを混合したものを、対照群には投与群と同量のＰＢＳを、上記ＨＳＰ７０／ＥＣＩ３０１共投与群と同様に投与した。
　群分け後、各群３~４日おきに固形腫瘍の長径、短径を測定し、下記の式で腫瘍体積を計算した。
腫瘍体積（ｍｍ３）＝長径（ｍｍ）×短径（ｍｍ）×短径（ｍｍ）×０．５２３６
　結果を図２および図３に示す。図３は投与開始後、１６日目の結果を示す。
　図２および図３の結果より明らかなように、ＨＳＰ７０およびＥＣＩ３０１の共投与によって、腫瘍の増殖を強く抑制することができた。 Effect of ECI301 and / or HSP70 administration on tumor <Experimental method>
Mice Six-week-old BALB / c male mice were purchased from Japan SLC and used after acclimation breeding.
Drug ECI301
ECI301 (ECI Corporation)
ECI301 1mg / ml prepared in PBS aqueous solution HSP70
Mouse Recombinant Hsp70-A1 (Low Endotoxin) (ESP-502, ENZO BIOCHEM (former Assay Designs))
HSP70 10 μg / 1 ml PBS prepared experimental system Colon26 / BALB / c
Colon 26 cells were implanted subcutaneously into the right flank of BALB / c mice (4 × 10 5 cells per mouse).
On the 11th day of transplantation, when the tumor diameter of the transplanted Colon 26 cells reached about 0.5 to 0.8 cm, the tumor was divided into 3 groups (control group, HSP70 single administration group and HSP70 / ECI301 co-administration group) (n = 6).
To the HSP70 / ECI301 co-administration group, a combination of HSP70 drug (500 ng HSP70 / 0.2 ml PBS) and ECI301 drug (2 μg ECI301 / 2 μl PBS) was administered into the tail vein (iv) on the 11th day of transplantation. (Once a day), administration was repeated in the same manner one day thereafter.
For the HSP70 single administration group, HSP70 drug (500 ng HSP70 / 0.2 ml PBS) mixed with 2 μl PBS, the control group with the same amount of PBS as the administration group, and the HSP70 / ECI301 co-administration group Administered.
After grouping, the major axis and minor axis of the solid tumor were measured every 3 to 4 days in each group, and the tumor volume was calculated by the following formula.
Tumor volume (mm 3 ) = major axis (mm) × minor axis (mm) × minor axis (mm) × 0.5236
The results are shown in FIG. 2 and FIG. FIG. 3 shows the results on the 16th day after the start of administration.
As is clear from the results of FIGS. 2 and 3, tumor growth could be strongly suppressed by co-administration of HSP70 and ECI301.
配合剤におけるＨＳＰ７０／ＥＣＩ３０１の用量の検討
＜実験方法＞
マウス
　６週齢ＢＡＬＢ／ｃ系雄性マウスを日本ＳＬＣより購入し、馴化飼育の後に使用した。
薬物
ＥＣＩ３０１
ＥＣＩ３０１（株式会社ＥＣＩ）
　ＥＣＩ３０１　１ｍｇ／ｍｌ　ＰＢＳ水溶液に調製
ＨＳＰ７０
Ｍｏｕｓｅ　Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｓｐ７０−Ａ１　（Ｌｏｗ　Ｅｎｄｏｔｏｘｉｎ）（ＥＳＰ−５０２，ＥＮＺＯ　ＢＩＯＣＨＥＭ（旧Ａｓｓａｙ　Ｄｅｓｉｇｎｓ））
　ＨＳＰ７０　１０μｇ／１ｍｌＰＢＳに調製
実験系
　Ｃｏｌｏｎ２６　／ＢＡＬＢ／ｃ
　Ｃｏｌｏｎ２６細胞をＢＡＬＢ／ｃマウス右側腹部に皮下移植した（マウス１匹につき４×１０５細胞）。移植７日目に、移植したＣｏｌｏｎ２６細胞の腫瘍径が約０．５~１．０ｃｍになった時点で群わけした（ｎ＝７）。
　ＨＳＰ７０およびＥＣＩ３０１を以下の用量で含む配合剤を調製し、移植７日目に各群に尾静脈内（ｉｖ）に１日１回投与し、その後２日間連続して同様に投与した。対照群には配合剤投与群と同量のＰＢＳのみを投与した。
Figure JPOXMLDOC01-appb-I000001
　群分け後、各群３~４日おきに固形腫瘍の長径、短径を測定し、上記実施例１と同様に、腫瘍体積を計算した。
　結果を図４に示す。図４は投与開始後、８日目の結果を示す。
　配合剤におけるＨＳＰ７０とＥＣＩ３０１の比率は、重量比率で、ＨＳＰ７０の１に対して０．４~１６の範囲内で十分に腫瘍増殖抑制を示すことが明らかとなった。特に、ＨＳＰ７０：ＥＣＩ３０１の重量比率が、１：４（ＩＩ群）であるとき、最も効果が高かった。
　そこで、配合剤における重量比率をＨＳＰ７０：ＥＣＩ３０１＝１：４に維持しながら、配合剤に含まれるＨＳＰ７０およびＥＣＩ３０１の用量を変えてその効果を検討した。
　ＨＳＰ７０およびＥＣＩ３０１を以下の用量で含む配合剤を調製し、上記の方法でそれぞれ投与した。対照群には配合剤投与群と同量のＰＢＳのみを投与した。
Figure JPOXMLDOC01-appb-I000002
　結果を図５に示す。図５は投与開始後、１１日目の結果を示す。
　ＨＳＰ７０とＥＣＩ３０１を上記の用量で用いた配合剤はいずれも十分に腫瘍増殖抑制を示すことが明らかとなった。すなわち、マウス一匹当たりＨＳＰ７０にして０．１~２．５（μｇ）、ＥＣＩ３０１にして０．４~１０（μｇ）の用量を含有する配合剤は、腫瘍増殖抑制作用を有することが明らかになった。この投与量は、マウスの体重が約２０~２５ｇであることから、ＨＳＰ７０にして４０~１２５μｇ／ｋｇ、ＥＣＩ３０１にして１６~５００μｇ／ｋｇに相当する。 Examination of dose of HSP70 / ECI301 in combination drug <Experimental method>
Mice Six-week-old BALB / c male mice were purchased from Japan SLC and used after acclimation breeding.
Drug ECI301
ECI301 (ECI Corporation)
ECI301 1mg / ml prepared in PBS aqueous solution HSP70
Mouse Recombinant Hsp70-A1 (Low Endotoxin) (ESP-502, ENZO BIOCHEM (former Assay Designs))
HSP70 10 μg / 1 ml PBS prepared experimental system Colon26 / BALB / c
Colon 26 cells were implanted subcutaneously into the right flank of BALB / c mice (4 × 10 5 cells per mouse). On the 7th day after transplantation, when the tumor diameter of the transplanted Colon 26 cells became about 0.5 to 1.0 cm, the cells were divided into groups (n = 7).
Formulations containing HSP70 and ECI301 at the following doses were prepared, and each group was administered once a day in the tail vein (iv) on the seventh day of transplantation, and thereafter in the same manner for two consecutive days. The control group received only the same amount of PBS as the combination drug administration group.
Figure JPOXMLDOC01-appb-I000001
After grouping, the major axis and minor axis of the solid tumor were measured every 3 to 4 days in each group, and the tumor volume was calculated in the same manner as in Example 1 above.
The results are shown in FIG. FIG. 4 shows the results on the 8th day after the start of administration.
It was revealed that the ratio of HSP70 and ECI301 in the combination was sufficiently tumor suppressive within the range of 0.4 to 16 with respect to 1 of HSP70 in terms of weight ratio. In particular, the effect was highest when the weight ratio of HSP70: ECI301 was 1: 4 (Group II).
Therefore, while maintaining the weight ratio in the formulation at HSP70: ECI301 = 1: 4, the effect was examined by changing the dose of HSP70 and ECI301 contained in the formulation.
Formulations containing HSP70 and ECI301 at the following doses were prepared and administered by the methods described above. The control group received only the same amount of PBS as the combination drug administration group.
Figure JPOXMLDOC01-appb-I000002
The results are shown in FIG. FIG. 5 shows the results on day 11 after the start of administration.
It has been clarified that the combination drugs using HSP70 and ECI301 at the above doses sufficiently suppress tumor growth. That is, it is clear that a combination drug containing a dose of 0.1 to 2.5 (μg) as HSP70 and 0.4 to 10 (μg) as ECI301 per mouse has a tumor growth inhibitory effect. became. This dose corresponds to 40 to 125 μg / kg for HSP70 and 16 to 500 μg / kg for ECI301 since the weight of the mouse is about 20 to 25 g.
ＥＣＩ３０１とヒトＭＩＰ−１αの比較
＜実験方法＞
マウス
　６週齢ＢＡＬＢ／ｃ系雄性マウスを日本ＳＬＣより購入し、馴化飼育の後に使用した。
薬物
ＥＣＩ３０１
ＥＣＩ３０１（株式会社ＥＣＩ）
　ＥＣＩ３０１　１ｍｇ／ｍｌ　ＰＢＳ水溶液に調製
ＨＳＰ７０
Ｍｏｕｓｅ　Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｓｐ７０−Ａ１　（Ｌｏｗ　Ｅｎｄｏｔｏｘｉｎ）（ＥＳＰ−５０２，ＥＮＺＯ　ＢＩＯＣＨＥＭ（旧Ａｓｓａｙ　Ｄｅｓｉｇｎｓ））
　ＨＳＰ７０　１０μｇ／１ｍｌＰＢＳに調製
ヒトＭＩＰ−１α
　ヒトＭＩＰ−１αをコードするＤＮＡを用いて形質転換したＢｒｅｖｉｂａｃｉｌｌｕｓ　ｃｈｏｓｈｉｎｅｎｓｉｓ（グラム陽性菌）を用いて生産し、精製したもの（Ｐｒｏｔｅｉｎ　Ｅｘｐｒｅｓｓ社に委託）
実験系
　Ｃｏｌｏｎ２６　／ＢＡＬＢ／ｃ
　Ｃｏｌｏｎ２６細胞をＢＡＬＢ／ｃマウス右側腹部に皮下移植した（マウス１匹につき４×１０５細胞）。
　移植９日目に、移植したＣｏｌｏｎ２６細胞の腫瘍径が約０．５~０．８ｃｍになった時点で群わけした（ｎ＝７）。
　ＨＳＰ７０およびＥＣＩ３０１、あるいはＨＳＰ７０およびヒトＭＩＰ−１αを以下の用量で含む配合剤を調製し、移植９日目に各群に尾静脈内（ｉｖ）に１日１回投与し、その後２日間連続して同様に投与した。対照群には配合剤投与群と同量のＰＢＳのみを投与した。
Figure JPOXMLDOC01-appb-I000003
　群分け後、各群３~４日おきに固形腫瘍の長径、短径を測定し、上記実施例１と同様に、腫瘍体積を計算した。
　結果を図６に示す。図６は投与後、１１日目の結果を示す。
　ＨＳＰ７０およびＥＣＩ３０１を含有する配合剤は用量依存的に腫瘍増殖抑制を示すが、ＨＳＰ７０およびヒトＭＩＰ−１αを含有する配合剤は、腫瘍増殖抑制効果を示さなかった。この結果より、ＥＣＩ３０１とヒトＭＩＰ−１αとは、ＨＳＰ７０との配合剤において全く異なる作用を有することが明らかとなった。 Comparison of ECI301 and human MIP-1α <Experimental method>
Mice Six-week-old BALB / c male mice were purchased from Japan SLC and used after acclimation breeding.
Drug ECI301
ECI301 (ECI Corporation)
ECI301 1mg / ml prepared in PBS aqueous solution HSP70
Mouse Recombinant Hsp70-A1 (Low Endotoxin) (ESP-502, ENZO BIOCHEM (former Assay Designs))
HSP70 10 μg / 1 ml prepared in PBS Human MIP-1α
Produced and purified using Brevibacillus choshinensis (Gram positive bacteria) transformed with DNA encoding human MIP-1α (consigned to Protein Express)
Experimental system Colon26 / BALB / c
Colon 26 cells were implanted subcutaneously into the right flank of BALB / c mice (4 × 10 5 cells per mouse).
On the 9th day after transplantation, when the tumor diameter of the transplanted Colon 26 cells became about 0.5 to 0.8 cm, the cells were divided into groups (n = 7).
Preparations containing HSP70 and ECI301 or HSP70 and human MIP-1α at the following doses were prepared, and each group was administered once daily in the tail vein (iv) on the 9th day after transplantation, and then for 2 consecutive days. Were administered in the same manner. The control group received only the same amount of PBS as the combination drug administration group.
Figure JPOXMLDOC01-appb-I000003
After grouping, the major axis and minor axis of the solid tumor were measured every 3 to 4 days in each group, and the tumor volume was calculated in the same manner as in Example 1 above.
The results are shown in FIG. FIG. 6 shows the results on day 11 after administration.
The combination containing HSP70 and ECI301 showed tumor growth suppression in a dose-dependent manner, but the combination containing HSP70 and human MIP-1α did not show a tumor growth suppression effect. From this result, it became clear that ECI301 and human MIP-1α have completely different actions in the combination with HSP70.
ＨＳＰ７０／ＥＣＩ３０１の共投与および逐次投与
実験系
　Ｃｏｌｏｎ２６　／ＢＡＬＢ／ｃ
　上記実施例３と同様に、Ｃｏｌｏｎ２６細胞をＢＡＬＢ／ｃマウス右側腹部に皮下移植した（マウス１匹につき４×１０５細胞）。
　移植９日目に、移植したＣｏｌｏｎ２６細胞の腫瘍径が約０．５~０．８ｃｍになった時点で群わけした（ｎ＝７）。
　ＨＳＰ７０およびＥＣＩ３０１をそれぞれ０．５μｇおよび２．０μｇ含有する配合剤を上記実施例１と同様に調製し、移植９日目に共投与群（図７（Ａ））に１日１回尾静脈内（ｉｖ）投与し、その後２日間連続して同様に投与した。一方、逐次投与群（図７（Ｂ））には、移植９日目にＨＳＰ７０（０．５μｇ）を尾静脈内（ｉｖ）投与した後、１０分間以内にＨＳＰ７０の投与とは分離して、ＥＣＩ３０１（２．０μｇ）を尾静脈内（ｉｖ）投与した。その後２日間連続して同様に投与した。対照群には投与群と同量のＰＢＳのみを投与した。
　群分け後、各群３~４日おきに固形腫瘍の長径、短径を測定し、上記実施例１と同様に、腫瘍体積を計算した。
　結果を図７に示す。図７は投与開始後、１１日目の結果を示す。
　ＨＳＰ７０とＥＣＩ３０１とは、配合剤として共投与せず、別個に投与した場合（図７（Ｂ））においても、腫瘍増殖抑制効果を示すことが明らかとなった。 HSP70 / ECI301 co-administration and sequential administration experiment system Colon26 / BALB / c
As in Example 3 above, Colon 26 cells were implanted subcutaneously into the right flank of BALB / c mice (4 × 10 5 cells per mouse).
On the 9th day after transplantation, when the tumor diameter of the transplanted Colon 26 cells became about 0.5 to 0.8 cm, the cells were divided into groups (n = 7).
Formulations containing 0.5 μg and 2.0 μg of HSP70 and ECI301, respectively, were prepared in the same manner as in Example 1 above, and in the tail vein once a day in the coadministration group (FIG. 7A) on the 9th day of transplantation (Iv) Administration, followed by administration in the same manner for 2 consecutive days. On the other hand, in the sequential administration group (FIG. 7B), HSP70 (0.5 μg) was administered into the tail vein (iv) on the 9th day after transplantation, and separated from the administration of HSP70 within 10 minutes, ECI301 (2.0 μg) was administered intravenously (iv) in the tail vein. Thereafter, it was administered in the same manner for 2 consecutive days. The control group received only the same amount of PBS as the administration group.
After grouping, the major axis and minor axis of the solid tumor were measured every 3 to 4 days in each group, and the tumor volume was calculated in the same manner as in Example 1 above.
The results are shown in FIG. FIG. 7 shows the results on the 11th day after the start of administration.
It has been clarified that HSP70 and ECI301 exhibit a tumor growth inhibitory effect even when administered separately (FIG. 7B) without being co-administered as a combination drug.
ＨＳＰ７０とその他のＨＳＰとの比較
＜実験方法＞
マウス
　６週齢ＢＡＬＢ／ｃ系雄性マウスを日本ＳＬＣより購入し、馴化飼育の後に使用した。
薬物
ＥＣＩ３０１
ＥＣＩ３０１（株式会社ＥＣＩ）
　ＥＣＩ３０１　１ｍｇ／ｍｌ　ＰＢＳ水溶液に調製
ＨＳＰ７０
Ｍｏｕｓｅ　Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｓｐ７０−Ａ１　（Ｌｏｗ　Ｅｎｄｏｔｏｘｉｎ）（ＥＳＰ−５０２，ＥＮＺＯ　ＢＩＯＣＨＥＭ（旧Ａｓｓａｙ　Ｄｅｓｉｇｎｓ））
　ＨＳＰ７０　１０μｇ／１ｍｌＰＢＳに調製
ＨＳＰ６０
Ｍｏｕｓｅ　Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｓｐ６０　（Ｌｏｗ　Ｅｎｄｏｔｏｘｉｎ）（ＥＮＺＯ　ＢＩＯＣＨＥＭ）
　ＨＳＰ６０　１０μｇ／１ｍｌＰＢＳに調製
ＨＳＰ９０
Ｍｏｕｓｅ　Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｓｐ９０　（Ｌｏｗ　Ｅｎｄｏｔｏｘｉｎ）（ＥＮＺＯ　ＢＩＯＣＨＥＭ）
　ＨＳＰ９０　１０μｇ／１ｍｌＰＢＳに調製
実験系
　Ｃｏｌｏｎ２６　／ＢＡＬＢ／ｃ
　Ｃｏｌｏｎ２６細胞をＢＡＬＢ／ｃマウス右側腹部に皮下移植した（マウス１匹につき４×１０５細胞）。
　移植９日目に、移植したＣｏｌｏｎ２６細胞の腫瘍径が約０．５~１ｃｍになった時点で群わけした（ｎ＝７）。
　ＨＳＰ７０またはＨＳＰ６０およびＥＣＩ３０１を以下の用量で含む配合剤を調製し、移植９日目に各群に１日１回尾静脈内（ｉｖ）投与し、その後２日間連続して同様に投与した。対照群には配合剤投与群と同量のＰＢＳのみを投与した。
Figure JPOXMLDOC01-appb-I000004
　群分け後、各群２~３日おきに固形腫瘍の長径、短径を測定し、上記実施例１と同様に、腫瘍体積を計算した。
　結果を図８に示す。図８は投与開始後、１１日目の結果を示す。
　ＨＳＰ７０とＥＣＩ３０１の配合剤は再現良く腫瘍増殖抑制効果を示したが（図８（ｉ））、ＨＳＰ６０とＥＣＩ３０１の配合剤は実質的に効果を示さなかった（図８（ｉｉ）−（ｖ））。
　次に、ＨＳＰ９０の効果について検討した。
　上記同様に群わけしたマウスに対して、ＨＳＰ７０／ＥＣＩ３０１およびＨＳＰ９０／ＥＣＩ３０１の各配合剤は、ＨＳＰ７０またはＨＳＰ９０（０．５μｇ／０．２ｍｌ　ＰＢＳ）とＥＣＩ３０１（２μｇ／２μｌＰＢＳ）をそれぞれ含み、共投与群には各配合剤を、移植９日目に１日１回尾静脈内（ｉｖ）投与し、その後２日間連続して同様に投与した。
　ＨＳＰ７０単独投与群には、ＨＳＰ７０またはＨＳＰ９０（０．５μｇ／０．２ｍｌ　ＰＢＳ）に、２μｌＰＢＳを混合したものを、対照群には投与群と同量のＰＢＳを、上記共投与群と同様に投与した。
　結果を図９および図１０に示す。図１０は投与開始後、１５日目の結果を示す。
　図９および図１０の結果より明らかなように、ＨＳＰ７０とＥＣＩ３０１の配合剤は再現良く腫瘍増殖抑制効果を示したが、ＨＳＰ９０とＥＣＩ３０１の配合剤は全く効果を示さなかった。
　以上の結果より、ＨＳＰ７０とＨＳＰ６０またはＨＳＰ９０とは、ＥＣＩ３０１との配合剤において全く異なる作用を有することが明らかとなった。 Comparison between HSP70 and other HSPs <Experimental method>
Mice Six-week-old BALB / c male mice were purchased from Japan SLC and used after acclimation breeding.
Drug ECI301
ECI301 (ECI Corporation)
ECI301 1mg / ml prepared in PBS aqueous solution HSP70
Mouse Recombinant Hsp70-A1 (Low Endotoxin) (ESP-502, ENZO BIOCHEM (former Assay Designs))
HSP70 prepared in 10 μg / 1 ml PBS HSP60
Mouse Recombinant Hsp60 (Low Endotoxin) (ENZO BIOCHEM)
HSP60 10μg / 1ml prepared in PBS HSP90
Mouse Recombinant Hsp90 (Low Endotoxin) (ENZO BIOCHEM)
HSP90 10μg / 1ml PBS prepared experimental system Colon26 / BALB / c
Colon 26 cells were implanted subcutaneously into the right flank of BALB / c mice (4 × 10 5 cells per mouse).
On the 9th day after transplantation, when the tumor diameter of the transplanted Colon 26 cells became about 0.5 to 1 cm, the cells were divided into groups (n = 7).
Formulations containing HSP70 or HSP60 and ECI301 at the following doses were prepared, and each group was administered once a day via the tail vein (iv) on the 9th day of transplantation, and thereafter in the same manner for 2 consecutive days. The control group received only the same amount of PBS as the combination drug administration group.
Figure JPOXMLDOC01-appb-I000004
After grouping, the major axis and minor axis of the solid tumor were measured every 2-3 days for each group, and the tumor volume was calculated in the same manner as in Example 1 above.
The results are shown in FIG. FIG. 8 shows the results on the 11th day after the start of administration.
The combination of HSP70 and ECI301 showed a tumor growth inhibitory effect with good reproducibility (FIG. 8 (i)), but the combination of HSP60 and ECI301 showed virtually no effect (FIG. 8 (ii)-(v)). ).
Next, the effect of HSP90 was examined.
For the mice grouped in the same manner, each combination of HSP70 / ECI301 and HSP90 / ECI301 contains HSP70 or HSP90 (0.5 μg / 0.2 ml PBS) and ECI301 (2 μg / 2 μl PBS), respectively, and is co-administered Each combination was administered to the group once a day via the caudal vein (iv) on the 9th day of transplantation, and thereafter in the same manner for 2 consecutive days.
In the HSP70 single administration group, HSP70 or HSP90 (0.5 μg / 0.2 ml PBS) mixed with 2 μl PBS, and in the control group, the same amount of PBS as in the administration group was administered in the same manner as in the co-administration group. did.
The results are shown in FIG. 9 and FIG. FIG. 10 shows the results on the 15th day after the start of administration.
As is clear from the results of FIGS. 9 and 10, the combination of HSP70 and ECI301 showed a tumor growth inhibitory effect with good reproducibility, but the combination of HSP90 and ECI301 showed no effect at all.
From the above results, it has been clarified that HSP70 and HSP60 or HSP90 have completely different actions in the compounding agent with ECI301.
様々な固形腫瘍に対するＨＳＰ７０／ＥＣＩ３０１配合剤の効果
＜実験方法＞
マウス
　ＢＡＬＢ／ｃ系６週齢雄性マウス、Ｃ５７ＢＬ／６系の６週齢雌性マウスをそれぞれ日本ＳＬＣより購入し、馴化飼育の後に使用した。
薬物
ＥＣＩ３０１
ＥＣＩ３０１（株式会社ＥＣＩ）
　ＥＣＩ３０１　１ｍｇ／ｍｌ　ＰＢＳ水溶液に調製
ＨＳＰ７０
Ｍｏｕｓｅ　Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｓｐ７０−Ａ１　（Ｌｏｗ　Ｅｎｄｏｔｏｘｉｎ）（ＥＳＰ−５０２，ＥＮＺＯ　ＢＩＯＣＨＥＭ（旧Ａｓｓａｙ　Ｄｅｓｉｇｎｓ））
　ＨＳＰ７０　１０μｇ／１ｍｌＰＢＳに調製
実験系
　以下の腫瘍細胞のうちＣｏｌｏｎ２６細胞、Ｍｅｔｈ　Ａ細胞はＢＡＬＢ／ｃマウスに、ルイス肺癌細胞はＣ５７ＢＬ／６マウスに、それぞれ右側腹部に皮下移植した。
Ｃｏｌｏｎ２６細胞　　　　　　４×１０５細胞
ルイス肺癌細胞　　　　　　　　４×１０５細胞
Ｍｅｔｈ　Ａ細胞　　　　　　　４×１０５細胞
（各細胞数はマウス一匹あたりの移植数を示す）
　移植１０日目に、移植した腫瘍細胞の腫瘍径が約０．５~０．８ｃｍになった時点で群わけした（ｎ＝７）。
　ＨＳＰ７０／ＥＣＩ３０１の配合剤は、ＨＳＰ７０（０．５μｇ／０．２ｍｌＰＢＳ）とＥＣＩ３０１（２μｇ／２μｌＰＢＳ）を含み、共投与群には当該配合剤を、移植９日目に各群に１日１回尾静脈内（ｉｖ）投与し、その後２日間連続して同様に投与した。
　ＨＳＰ７０またはＥＣＩ３０１単独投与群には、ＨＳＰ７０（０．５μｇ／０．２ｍｌ　ＰＢＳ）またはＥＣＩ３０１（２μｇ／２μｌＰＢＳ）にそれぞれＰＢＳを２μｌまたは０．２ｍｌ混合したものを、対照群には投与群と同量のＰＢＳを、上記ＨＳＰ７０／ＥＣＩ３０１共投与群と同様に投与した。
　群分け後、各群３~４日おきに固形腫瘍の長径、短径を測定し、上記実施例１と同様に、腫瘍体積を計算した。
　各腫瘍細胞の結果を図１１~１３に示す。
　ＨＳＰ７０／ＥＣＩ３０１の配合剤は、いずれの腫瘍細胞に対しても再現良く腫瘍増殖抑制効果を示した。この結果は、当該配合剤が系統の異なるいずれの腫瘍細胞に対しても有効であることを示唆する。 Effect of HSP70 / ECI301 combination on various solid tumors <Experimental method>
Mice BALB / c 6-week-old male mice and C57BL / 6-line 6-week-old female mice were purchased from Japan SLC and used after acclimation breeding.
Drug ECI301
ECI301 (ECI Corporation)
ECI301 1mg / ml prepared in PBS aqueous solution HSP70
Mouse Recombinant Hsp70-A1 (Low Endotoxin) (ESP-502, ENZO BIOCHEM (former Assay Designs))
Experimental system prepared in HSP70 10 μg / 1 ml PBS Of the following tumor cells, Colon 26 cells and Meth A cells were transplanted subcutaneously into BALB / c mice, and Lewis lung cancer cells were subcutaneously implanted into C57BL / 6 mice on the right flank.
Colon 26 cell 4 × 10 5 cell Lewis lung cancer cell 4 × 10 5 cell Meth A cell 4 × 10 5 cell (each cell number indicates the number of transplants per mouse)
On the 10th day after transplantation, when the tumor diameter of the transplanted tumor cells reached about 0.5 to 0.8 cm, the cells were divided into groups (n = 7).
The combination of HSP70 / ECI301 contains HSP70 (0.5 μg / 0.2 ml PBS) and ECI301 (2 μg / 2 μl PBS), and the combination is administered to the co-administration group once a day for each group on the ninth day of transplantation. Caudal vein (iv) was administered, and then administered in the same manner for 2 consecutive days.
In the HSP70 or ECI301 single administration group, HSP70 (0.5 μg / 0.2 ml PBS) or ECI301 (2 μg / 2 μl PBS) mixed with 2 μl or 0.2 ml of PBS, respectively, the control group had the same amount as the administration group PBS was administered in the same manner as in the HSP70 / ECI301 co-administration group.
After grouping, the major axis and minor axis of the solid tumor were measured every 3 to 4 days in each group, and the tumor volume was calculated in the same manner as in Example 1 above.
The results of each tumor cell are shown in FIGS.
The combination of HSP70 / ECI301 showed a tumor growth inhibitory effect with good reproducibility for all tumor cells. This result suggests that the combination drug is effective against any tumor cells of different strains.
ＨＳＰ７０とＥＣＩ３０１との相互作用の検討
実験方法
（１）抗体結合ビーズの作製
材料
抗ＭＩＰ−１α（マウス）：Ｒ＆Ｄ
抗ＨＳＰ７０（マウス）：ＳｔｒｅｓｓＭａｒｋ
Ｃｏ−ＩＰキット：Ｉｎｖｉｔｒｏｇｅｎ
方法
　製品添付の取扱説明書に従って、各抗体５０μｇをＣｏ−ＩＰキット中の１０ｍｇビーズに結合させ、１ｍＬのバッファーに懸濁した。
（２）電気泳動サンプルの作製：免疫沈降法Ｉｍｍｕｎｏｐｒｅｃｉｐｉｔａｔｉｏｎ
材料
抗ＭＩＰ−１α結合ビーズ
抗ＨＳＰ７０結合ビーズ
薬物
ＥＣＩ３０１　１ｍｇ／ｍｌ　ＰＢＳ水溶液
ＨＳＰ７０
Ｍｏｕｓｅ　Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｓｐ７０−Ａ１　（Ｌｏｗ　Ｅｎｄｏｔｏｘｉｎ）（ＥＳＰ−５０２，ＥＮＺＯ　ＢＩＯＣＨＥＭ（旧Ａｓｓａｙ　Ｄｅｓｉｇｎｓ）　）
ウサギαＭＩＰ−１α−ビオチン：Ｒ＆Ｄ
ＨＲＰ−ストレプトアビジン
ｍｕ　ａＨＳＰ７０
ＨＲＰ−α　マウス　ＩｇＧ
方法
　５μｇ抗体／１ｍｇ　ビーズ／１００μｌに、ＨＳＰ７０、ＥＣＩ３０１、ｈＭＩＰ−１α、をそれぞれ、あるいは混合して、添加し、４℃２４時間ローテーターでインキュベーションした。
非結合成分を十分に洗浄し除去した後、ビーズをＳＤＳ電気泳動に供した。
（３）ＳＤＳ電気泳動（ＳＤＳ−ＰＡＧＥ）
材料
Ｇｅｌ：Ｎｕ−ＰＡＧＥ　１０％：Ｉｎｖｉｔｒｏｇｅｎ
泳動バッファー：Ｉｎｖｉｔｒｏｇｅｎ
サンプルバッファー：Ｉｎｖｉｔｒｏｇｅｎ
還元用バッファー：Ｉｎｖｉｔｒｏｇｅｎ
方法
　サンプルをサンプルバッファー、還元用バッファーと混ぜて、７０℃，１０分間処理しサンプル調整した後、Ｎｕ−ＰＡＧＥ　製品添付の取扱説明書に従って２００Ｖ定圧電流で約３５分間電気泳動した。
（４）Ｉｍｍｕｎｏ　Ｂｌｏｔｉｎｇ
材料
ｉＢｌｏｔ　（Ｉｎｖｉｔｒｏｇｅｎ）
ＰＶＤＦ，Ｍｉｎｉ　（Ｉｎｖｉｔｒｏｇｅｎ）
ＣｈｅｍｉＢｌｏｃｋｅｒ：２０ｍＭＴＢＳ−Ｔ（１％）
ウサギ抗ＭＩＰ−１α　ポリクローナル−ビオチン標識（Ｒ＆Ｄ）
ストレプトアビジン−ＨＲＰ標識，　（Ｊａｃｓｏｎ）
ウサギ抗ＭＩＰ−１α　ポリクローナル−ビオチン標識　（Ｒ＆Ｄ）
ストレプトアビジン−ＨＲＰ標識
Ｎｏｖｅｘ　ＥＣＬ　ＨＲＰ　Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｔ　Ｓｕｂｓｔｒａｔｅ　Ｒｅａｇｅｎｔ　Ｋｉｔ：Ｃａｔ　Ｎｏ．ＷＰ２０００５　（Ｉｎｖｉｔｒｏｇｅｎ）
方法
　ＢｌｏｔｔｉｎｇはｉＢｌｏｔ（Ｉｎｖｉｔｒｏｇｅｎ）を使用して、製品添付の取扱説明書に従って行った。
　染色は、転写したＰＶＤＦメンブレンをＣｈｅｍｉＢｌｏｃｋｅｒ：２０ｍＭＴＢＳ−Ｔ（１％）　１：１溶液で３０分間、ブロッキングを行った後に、一次抗体：ウサギ抗ＭＩＰ−１αポリクローナル−ビオチン標識を添加し、室温にて６０分間、インキュベーションし、洗浄した後に、二次抗体：ストレプトアビジン−ＨＲＰ標識を添加し、Ｒ室温にて６０分間、インキュベーションした。
　まずここで、ＥＣＬ試薬を用いて製品添付の取扱説明書に従って発光させ、イメージアナライザーＬＡＳ４０００　Ｍｉｎｉ（富士フイルム）で発色画像を取り込みＥＣＩ３０１、ＭＩＰ−１αのＩｍｍｕｎｏＢｌｏｔデータを得た（図１４）。
　次にメンブレンを洗浄した後、一次抗体：マウス　抗ＨＳＰ７０を添加し、室温にて６０分間、インキュベーションし、二次抗体：抗マウスＩｇＧ−ＨＲＰ標識を添加し、室温にて６０分間、インキュベーションした。
　上記と同様にＥＣＬ試薬を用いて製品添付の取扱説明書に従って発色させ、イメージアナライザーで発色画像を取り込みＨＳＰ７０のＩｍｍｕｎｏＢｌｏｔデータを得た（図１５）。
　結果を図１４および図１５に示す。
　図１４に示すように、抗ＨＳＰ７０抗体結合ビーズとＨＳＰ７０を用いた免疫沈降に際して、ＥＣＩ３０１は共沈を起こした（レーン５）。その強度はｈＭＩＰ−１α（レーン６）よりも明らかに強かった。
　さらに、図１５に示すように、抗ＭＩＰ−１α抗体結合ビーズとＥＣＩ３０１を用いた免疫沈降に際して、ＨＳＰ７０は共沈を起こした（レーン２）。
　これらの結果からＥＣＩ３０１とＨＳＰ７０は強い親和性、すなわち、特異的吸着作用が存在することが明らかになった。 Experimental Method for Examination of Interaction between HSP70 and ECI301 (1) Preparation Material of Antibody-Binding Bead Anti-MIP-1α (mouse): R & D
Anti-HSP70 (mouse): StressMark
Co-IP kit: Invitrogen
Method According to the instruction manual attached to the product, 50 μg of each antibody was bound to 10 mg beads in the Co-IP kit and suspended in 1 mL of buffer.
(2) Preparation of electrophoresis sample: immunoprecipitation method Immunoprecipitation
Materials Anti-MIP-1α binding beads Anti-HSP70 binding beads Drug ECI301 1 mg / ml PBS aqueous solution HSP70
Mouse Recombinant Hsp70-A1 (Low Endotoxin) (ESP-502, ENZO BIOCHEM (former Assay Designs))
Rabbit αMIP-1α-Biotin: R & D
HRP-Streptavidin mu aHSP70
HRP-α mouse IgG
Method HSP70, ECI301, and hMIP-1α were added to 5 μg antibody / 1 mg beads / 100 μl, respectively, or mixed and incubated at 4 ° C. for 24 hours.
After thoroughly washing away unbound components, the beads were subjected to SDS electrophoresis.
(3) SDS electrophoresis (SDS-PAGE)
Material Gel: Nu-PAGE 10%: Invitrogen
Running buffer: Invitrogen
Sample buffer: Invitrogen
Reduction buffer: Invitrogen
Method A sample was mixed with a sample buffer and a reducing buffer, treated at 70 ° C. for 10 minutes to prepare a sample, and then electrophoresed at 200 V constant pressure current for about 35 minutes according to the instruction manual attached to the Nu-PAGE product.
(4) Immunoblotting
Materials iBlot (Invitrogen)
PVDF, Mini (Invitrogen)
ChemiBlocker: 20 mM TBS-T (1%)
Rabbit anti-MIP-1α polyclonal-biotin labeling (R & D)
Streptavidin-HRP labeling, (Jacson)
Rabbit anti-MIP-1α polyclonal-biotin label (R & D)
Streptavidin-HRP labeled Novex ECL HRP Chemiluminescent Substrate Reagent Kit: Cat No. WP20005 (Invitrogen)
Method Blotting was performed using iBlot (Invitrogen) according to the instruction manual attached to the product.
Staining was performed by blocking the transferred PVDF membrane with ChemiBlocker: 20 mM TBS-T (1%) 1: 1 solution for 30 minutes, and then adding a primary antibody: rabbit anti-MIP-1α polyclonal-biotin label at room temperature. After incubation and washing for 60 minutes, secondary antibody: streptavidin-HRP label was added and incubated at R room temperature for 60 minutes.
First, light was emitted using an ECL reagent according to the instruction manual attached to the product, and a color image was captured with an image analyzer LAS4000 Mini (Fuji Film) to obtain ImmunoBlot data of ECI301 and MIP-1α (FIG. 14).
Next, after washing the membrane, primary antibody: mouse anti-HSP70 was added and incubated at room temperature for 60 minutes, secondary antibody: anti-mouse IgG-HRP label was added, and incubated at room temperature for 60 minutes.
In the same manner as described above, the color was developed using the ECL reagent according to the instruction manual attached to the product, and the developed image was captured by an image analyzer to obtain ImmunoBlot data of HSP70 (FIG. 15).
The results are shown in FIG. 14 and FIG.
As shown in FIG. 14, ECI301 co-precipitated during the immunoprecipitation using anti-HSP70 antibody-binding beads and HSP70 (lane 5). The intensity was clearly stronger than hMIP-1α (lane 6).
Furthermore, as shown in FIG. 15, HSP70 co-precipitated during the immunoprecipitation using anti-MIP-1α antibody-bound beads and ECI301 (lane 2).
From these results, it was revealed that ECI301 and HSP70 have a strong affinity, that is, a specific adsorption action.
　本発明を用いることにより、固形癌における癌細胞の増殖を阻害することができる。従って、固形癌および関連する様々な疾患または障害を、治療することが可能となる。
　本発明により、ＨＳＰ７０およびＥＣＩ３０１を有効成分として含む、癌細胞の増殖を抑制可能な医薬組成物を提供することができ、癌および関連する様々な疾患または障害を治療するための新たな治療剤および治療方法として利用されることが大いに期待される。
　本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。
By using the present invention, the growth of cancer cells in solid cancer can be inhibited. Thus, it becomes possible to treat solid cancers and various related diseases or disorders.
According to the present invention, it is possible to provide a pharmaceutical composition containing HSP70 and ECI301 as active ingredients and capable of suppressing the growth of cancer cells, a new therapeutic agent for treating cancer and various diseases or disorders related thereto, and It is highly expected to be used as a treatment method.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (15)

  1. 　ヒートショックプロテイン（ＨＳＰ）７０と配列番号１で表されるポリペプチド（ＥＣＩ３０１）を含む、癌を治療するための医薬組成物。 A pharmaceutical composition for treating cancer, comprising heat shock protein (HSP) 70 and a polypeptide represented by SEQ ID NO: 1 (ECI301).
  2. 　癌が固形癌である、請求項１記載の医薬組成物。 The pharmaceutical composition according to claim 1, wherein the cancer is a solid cancer.
  3. 　固形癌が肺癌、大腸癌、または線維肉腫である、請求項２記載の医薬組成物。 The pharmaceutical composition according to claim 2, wherein the solid cancer is lung cancer, colon cancer, or fibrosarcoma.
  4. 　重量比率でヒートショックプロテイン（ＨＳＰ）７０と配列番号１で表されるポリペプチド（ＥＣＩ３０１）を１：０．４~１６の比率で含む、請求項１~３のいずれか１項記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 3, comprising heat shock protein (HSP) 70 and polypeptide represented by SEQ ID NO: 1 (ECI301) in a weight ratio of 1: 0.4 to 16. object.
  5. 　癌を治療するための、ヒートショックプロテイン（ＨＳＰ）７０と配列番号１で表されるポリペプチド（ＥＣＩ３０１）の組み合わせ医薬。 A combination medicine of heat shock protein (HSP) 70 and the polypeptide represented by SEQ ID NO: 1 (ECI301) for treating cancer.
  6. 　癌が固形癌である、請求項５記載の組み合わせ医薬。 The combined medicine according to claim 5, wherein the cancer is solid cancer.
  7. 　固形癌が肺癌、大腸癌、または線維肉腫である、請求項６記載の組み合わせ医薬。 The combination medicine according to claim 6, wherein the solid cancer is lung cancer, colon cancer, or fibrosarcoma.
  8. 　癌を治療する方法であって、ヒートショックプロテイン（ＨＳＰ）７０及び配列番号１で表されるポリペプチド（ＥＣＩ３０１）を別々にまたは同時に癌患者に投与することを含む、上記方法。 A method for treating cancer, which comprises administering heat shock protein (HSP) 70 and the polypeptide represented by SEQ ID NO: 1 (ECI301) separately or simultaneously to a cancer patient.
  9. 　癌が固形癌である、請求項８記載の方法。 The method according to claim 8, wherein the cancer is solid cancer.
  10. 　固形癌が肺癌、大腸癌、または線維肉腫である、請求項９記載の方法。 The method according to claim 9, wherein the solid cancer is lung cancer, colon cancer, or fibrosarcoma.
  11. 　重量比率でヒートショックプロテイン（ＨＳＰ）７０と配列番号１で表されるポリペプチド（ＥＣＩ３０１）を１：０．４~１６の比率で癌患者に投与する、請求項８~１０のいずれか１項記載の方法。 The heat shock protein (HSP) 70 and the polypeptide represented by SEQ ID NO: 1 (ECI301) are administered to cancer patients at a weight ratio of 1: 0.4 to 16, respectively. The method described.
  12. 　癌患者における癌の治療に使用するための、ヒートショックプロテイン（ＨＳＰ）７０及び配列番号１で表されるポリペプチド（ＥＣＩ３０１）。 A heat shock protein (HSP) 70 and a polypeptide represented by SEQ ID NO: 1 (ECI301) for use in the treatment of cancer in cancer patients.
  13. 　癌が固形癌である、請求項１２記載のヒートショックプロテイン（ＨＳＰ）７０及び配列番号１で表されるポリペプチド（ＥＣＩ３０１）。 The heat shock protein (HSP) 70 according to claim 12 and the polypeptide represented by SEQ ID NO: 1 (ECI301), wherein the cancer is solid cancer.
  14. 　固形癌が肺癌、大腸癌、または線維肉腫である、請求項１３記載のヒートショックプロテイン（ＨＳＰ）７０及び配列番号１で表されるポリペプチド（ＥＣＩ３０１）。 The heat shock protein (HSP) 70 according to claim 13 and the polypeptide represented by SEQ ID NO: 1 (ECI301), wherein the solid cancer is lung cancer, colon cancer or fibrosarcoma.
  15. 　重量比率でヒートショックプロテイン（ＨＳＰ）７０と配列番号１で表されるポリペプチド（ＥＣＩ３０１）を１：０．４~１６の比率で癌患者に投与して使用する、請求項１２~１４のいずれか１項記載のヒートショックプロテイン（ＨＳＰ）７０及び配列番号１で表されるポリペプチド（ＥＣＩ３０１）。 The heat shock protein (HSP) 70 and the polypeptide represented by SEQ ID NO: 1 (ECI301) in a weight ratio are administered to a cancer patient at a ratio of 1: 0.4 to 16, and used. A heat shock protein (HSP) 70 according to claim 1 and a polypeptide represented by SEQ ID NO: 1 (ECI301).
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