WO2001040311A1 - Chemokine slc-il2 fused protein and gene thereof - Google Patents

Chemokine slc-il2 fused protein and gene thereof Download PDF

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Publication number
WO2001040311A1
WO2001040311A1 PCT/JP2000/008325 JP0008325W WO0140311A1 WO 2001040311 A1 WO2001040311 A1 WO 2001040311A1 JP 0008325 W JP0008325 W JP 0008325W WO 0140311 A1 WO0140311 A1 WO 0140311A1
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Prior art keywords
amino acid
seq
acid sequence
fusion protein
cells
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PCT/JP2000/008325
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French (fr)
Japanese (ja)
Inventor
Koichiro Nakahara
Tsuneaki Sakata
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Shionogi & Co., Ltd.
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Priority to AU15518/01A priority Critical patent/AU1551801A/en
Priority to JP2001541065A priority patent/JP4553290B2/en
Publication of WO2001040311A1 publication Critical patent/WO2001040311A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to a fusion protein comprising a chemokine SLC and IL-2, a method for producing the fusion protein, a gene encoding the fusion protein, an expression vector for gene therapy containing the gene encoding the fusion protein, and the fusion protein.
  • the present invention relates to a pharmaceutical composition for treating cancer containing protein. Background art
  • Interleukin-2 is one of the cytokines produced mainly by activated T cells. Its physiological actions include proliferation and activation of T cells, proliferation of B cells and enhancement of antibody production, proliferation and activation of NK cells, activation of monocytes and macrophages (Smith, K., Annu Rev Immunol). 2: 319-333, 1984).
  • Human IL-1 (hIL-2) is made up of a precursor consisting of 15 amino acids, and the 20 N-terminal signal peptide is processed to produce 133 amino acids. It becomes a mature form of IL-2. Antitumor effects of direct systemic administration of hIL-2 have been reported in some tumors.
  • Chemokines are a general term for heparin-binding polypeptides having an endogenous leukocyte migration / activation activity. Chemokines are either conserved by the N-terminal two cysteine residues separated by a single amino acid (CXC) or adjacent (CC), with the exception of only one of the four conserved cysteine residues. Or separated by three amino acids (CX3C), there are four subfamilies.
  • CC-type chemokine SLC secondary lymphoid-tissue chemokine (named 6-C-kine, TCA-4, Exodus-2) is one of the CC-type chemokines.
  • TCA-4 TCA-4
  • Exodus-2 is one of the CC-type chemokines.
  • human chemokine SLC a precursor consisting of 134 amino acids is first produced, and the N-terminal 23 signal peptides are processed to form a mature SLC consisting of 11 amino acids. Become. It is known that the specific receptor for chemokine SLC is CCR7 (Yoshida, R., et al., J Biol Chem.
  • IL-2 does not impair its biological activity even when fused with another protein at its N-terminus.
  • chemokine does not lose its activity even if another protein is fused to its C-terminal. Therefore, the present inventors have proposed that by connecting the nucleotide sequence encoding the amino acid sequence of CC-type chemokine SLC that does not contain a stop codon and the nucleotide sequence encoding the mature protein of cytokine II-2.
  • a single gene and construct an artificial fusion gene encoding an artificial fusion protein having the migration activity of CC.
  • the produced fusion protein was a fusion protein having excellent properties showing the bioactivity of both chemokines SLC and IL-2 in a single molecule, and thus completed the present invention. That is, the present invention (1) a fusion protein having an amino acid sequence constituting chemokine SLC on the N-terminal side and containing an amino acid sequence constituting IL-2;
  • the amino acid sequence constituting chemokine SLC is the amino acid sequence from Ser at position 24 to Pro at position 134 of SEQ ID NO: 2, and the amino acid sequence constituting IL-2 is SEQ ID NO: 4
  • the fusion protein according to (5) wherein the fusion protein comprises the amino acid sequence described from Ser at position 24 to Thr at position 269 in SEQ ID NO: 6;
  • the amino acid sequence constituting the chemokine SLC is the amino acid sequence described from Ser at position 24 to Gly at position 133 of SEQ ID NO: 8, and the amino acid sequence constituting IL-2 is SEQ ID NO:
  • SEQ ID NO: 8 amino acid sequence described in Gly from position 1 to position 13 of Met, linker and SEQ ID NO: SEQ ID NO: described in Gin at position 16 from Ala in position 21
  • the fusion protein according to (15) above, wherein the fusion protein comprises an amino acid sequence described from Met at position 1 to Gin at position 284 of SEQ ID NO: 12;
  • amino acid sequence according to any one of (12) to (17) one or several amino acids include a substituted, deleted, inserted or added amino acid sequence, and the chemokine SLC activity And (19) a DNA encoding the fusion protein according to any one of (1) to (18) above;
  • SEQ ID NO: The DNA according to (19), comprising the nucleotide sequence from a at position 70 to t at position 808 described in 5; (21) the DNA of the above (20), which comprises the nucleotide sequence from a at position 1 to t at position 808 of SEQ ID NO: 5;
  • the fusion protein of the present invention is a fusion protein having chemokine SLC on its N-terminal side and containing IL-2.
  • chemokine SLC is not particularly limited, but is preferably a protein derived from human or mouse.
  • Mature human chemokine SLC is a protein having an amino acid sequence described from Ser at position 24 to Pro at position 134 of SEQ ID NO: 2.
  • Human chemokine SLC containing a signal sequence is a protein having an amino acid sequence described from Met at position 1 to Pro at position 134 in SEQ ID NO: 2.
  • Mature mouse chemokine SLC is a protein having an amino acid sequence described in Ser from SEQ ID NO: 24 to Gly in position 133 in SEQ ID NO: 8.
  • the murine chemokine SLC containing the signal sequence is a protein having an amino acid sequence described from Met at position 1 to Gly at position 133 in SEQ ID NO: 8.
  • IL-2 is also not particularly limited, but is preferably human or mouse protein.
  • Mature human IL-2 is a protein having the amino acid sequence of Ala at position 21 of SEQ ID NO: 4 to Thr at position 153 of SEQ ID NO: 4.
  • Human IL-2 containing a signal sequence is a protein having an amino acid sequence described from Met at position 1 to Thr at position 153 in SEQ ID NO: 4.
  • Mature mouse IL-2 is a protein having the amino acid sequence described in Ala at position 21 of SEQ ID NO: 10 to Gin at position 169 in SEQ ID NO: 10.
  • Mouse IL-2 containing a signal sequence is a protein having an amino acid sequence described from Met at position 1 of SEQ ID NO: 10 to Gin at position 169 of SEQ ID NO: 10.
  • the fusion protein of the present invention preferably has a chemokine SLC on the N-terminal side, is a fusion protein containing linker and IL-2, and may contain a signal sequence on the N-terminal side.
  • a fusion consisting of the amino acid sequence described in Ser from position 24 to ThT in position 269 of SEQ ID NO: 6 is preferred.
  • Even protein or SEQ ID NO: 26 Ser of position 24 in 6 A fusion protein consisting of the amino acid sequence of Thr from position 269 to position 269.
  • a fusion protein comprising the amino acid sequence described in Thr at position 269 from Met 1 at position 1 of SEQ ID NO: 6 or the amino acid sequence described in Thr at position 269 from Met at position 1 in SEQ ID NO: 26 Fusion protein.
  • a fusion protein consisting of the amino acid sequence of SEQ ID NO: 12 from Ser at position 24 to Gin at position 284 It is protein. More preferably, it is a fusion protein consisting of the amino acid sequence described from Met at position 1 of SEQ ID NO: 12 to Gin at position 284 of SEQ ID NO: 12.
  • linker is not particularly limited as long as it is at least one amino acid residue that does not cause steric hindrance to the proteins before and after it.
  • the amino acid residue has no more than 20 amino acids. Group. More preferably, it is an amino acid residue consisting of glycine and serine.
  • the fusion protein of the present invention includes “one or several amino acids in the amino acid sequence described from Ser at position 24 to Thr at position 269 in SEQ ID NO: 6; substitution, deletion, or insertion. Or a fusion protein containing an added amino acid sequence and having chemokine SLC activity and IL-2 activity ”;“ SEQ ID NO: 26 to Ser at position 24 to Thr at position 26 9 "A fusion protein containing one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence and having chemokine SLC activity and IL-2 activity", "SEQ ID NO: 12" In the amino acid sequence described in Ser from position 24 to Gin in position 284, one or several amino acids include a substituted, deleted, inserted or added amino acid sequence, and the chemokine SLC activity and IL -2 fusion tamper Quality "is also included.
  • the degree of “substitution, deletion, insertion or addition of amino acids” and their positions and the like are determined in the same manner as in the case where the modified protein is a chemocha in the same manner as the protein consisting of the amino acid sequence represented by SEQ ID NO: 6, 26 or 12.
  • the protein is not particularly limited as long as it has a protein having SLC activity and IL-2 activity. These amino acid sequence mutations occur naturally, for example, after mutation or translation. Although it may be caused by modification of DNA, it can be artificially modified based on the DNA of the present invention.
  • the protein of the present invention includes proteins encoded by all modified DNAs having the above-mentioned properties, regardless of the cause and means of such modification and mutation.
  • chemokine SLC activity refers to “migration ability of cells expressing CCR7 (Mark Birkenbach et al., J. Virol., 67: 2209-2220, 1993) that is a chemokine SLC-specific receptor”.
  • Means "Migration ability” means that inflammatory cells such as neutrophils, granulocytes, lymphocytes, or macula phage, and cells responsible for immunocompetence adhere to vascular endothelial cells, migrate out of blood vessels, and are damaged This means accumulation in tissues or tissues where antigens are present.
  • IL-2 activity means “proliferation ability of an IL-2-dependent cell line”.
  • the DNA of the present invention refers to “DNA encoding the protein of the present invention”.
  • the DNA of the present invention is preferably a DNA encoding a fusion protein comprising the amino acid sequence of SEQ ID NO: 6, 26 or 12, and more preferably the DNA of SEQ ID NO: 5.
  • a DNA containing the nucleotide sequence from a at position 0 to t at position 808, and a DNA containing the nucleotide sequence from a at position 70 to t at position 808 described in SEQ ID NO: 25 Is done.
  • the above DNA containing the base sequence from a at position 1 to a at position 852 in SEQ ID NO: 11 and the DNA from position a to position 852 at position 1 described in SEQ ID NO: 11 The above DNA containing the nucleotide sequence up to a is also included in the DNA of the present invention.
  • the DNA of the present invention also includes a DNA that hybridizes with the DNA of the present invention under stringent conditions, and encodes a protein that is tight and has chemokine SLC activity and IL-2 activity. “DNA that hybridizes to DNA under stringent conditions” can be obtained by using DNA in the coding region as a probe.
  • Hybridize under stringent conditions This means, for example, that in a solution of 6 ⁇ SSC, 0.5% SDS and 50% formamide, the solution is heated at 42 ° C., and then washed in a 0.1 ⁇ SSC, 0.5% SDS solution at 68 ° C. This indicates that a positive hybridization signal is still observed.
  • Production of a recombinant protein using the DNA of the present invention can be carried out, for example, based on many textbooks and documents such as the aforementioned Molecular Cloning. Specifically, a translation start codon is added upstream of the DNA to be expressed, and a translation stop codon is added downstream.
  • control transcription e.g., trp, lac, T7, SV40 early promoter
  • control gene such as a suitable base click evening one (e.g., pBR322, etc. P UC19, pSV ⁇ SPORT1)
  • a suitable base click evening one e.g., pBR322, etc. P UC19, pSV ⁇ SPORT1
  • a plasmid incorporating the DNA of the present invention is also included in the present invention.
  • the vector of the DNA of the present invention include a retrovirus vector, an adenovirus vector, and an adeno-associated virus vector into which the DNA of the present invention has been incorporated.
  • the DNA of the present invention is introduced into human somatic cells, which produce the fusion protein of the present invention.
  • the fusion protein is useful as an anticancer agent because it has the T cell migration activity of the chemokine SLC together with the immunostimulatory effect of IL-2. Therefore, the vector of the present invention can be used for gene therapy for treating these diseases.
  • the transformant of the present invention can be obtained by introducing the vector of the present invention into an appropriate host.
  • examples of the host include prokaryotic cells such as Escherichia coli, unicellular eukaryotic cells such as yeast, and cells of multicellular organisms such as insects and mammals.
  • prokaryotic cells such as Escherichia coli
  • unicellular eukaryotic cells such as yeast
  • cells of multicellular organisms such as insects and mammals.
  • a human somatic cell is used as a host.
  • Human somatic cells include patient-derived bone marrow cells, hepatocytes, fibroblasts, epidermal cells, muscle cells And the like.
  • the fusion protein prepared according to the present invention can be administered to humans for therapeutic purposes.
  • compositions can be prepared by mixing the fusion protein with buffers, stabilizers, bacteriostats and conventional excipients and additives used in parenteral pharmaceutical dosage forms. Therefore, the present invention also provides a pharmaceutical composition containing the fusion protein of the present invention.
  • FIG. 1 is a diagram showing a primer set for mouse SLC (mSLC) or mouse IL-2 (mIL-2) fragment / width.
  • FIG. 2 is a diagram showing a method for producing a retrovirus vector plasmid for transfection of an mSLC-mIL-2 fusion gene.
  • FIG. 3 is a diagram showing a method for producing a retrovirus vector pLX-IRES-EGFP from a retrovirus vector pLHDCX.
  • FIG. 4 is a diagram showing the number of migrated cells.
  • FIG. 5 is a diagram showing the additive number of CD4-positive T cells.
  • FIG. 6 is a diagram showing the additive number of CD8-positive T cells.
  • FIG. 7 is a diagram showing the tumor formation inhibitory effect of mSLC.
  • FIG. 8 is a diagram showing the tumor formation inhibitory effect of mIL2 and mSLC-IL2.
  • FIG. 9 is a diagram illustrating a method for preparing a retroviral vector for transfection of a human SLC (hSLC) -human IL-2 (hIL-2) fusion gene.
  • FIG. 10 is a diagram showing the tumor formation inhibitory effect of mSLC, mIL-2 and mSLC-mIL-2 alone or in combination with mSLC and mIL-2.
  • BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates mainly to novel fusion proteins.
  • the preparation of the DNA of the present invention, the preparation of the fusion protein of the present invention, the method for measuring the activity, the vector for gene therapy, and the pharmaceutical composition are described below.
  • a gene recombination technique known in the art a technique for producing a recombinant protein in animal cells, insect cells, yeast and Escherichia coli, a method for separating and purifying the expressed protein, and an analysis method Methods and immunological techniques are employed.
  • the gene having the nucleotide sequence encoding human CC-type chemokine SLC and human cytokine IL-2 used in the present invention is based on the sequence information (SEQ ID NO: 1 and SEQ ID NO: 2) taught by the present invention. It can be easily manufactured and obtained by general genetic engineering techniques (see, for example, Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989)). Specifically, a cDNA library is prepared from a suitable source in which the chemokines SLC and IL-2 are expressed according to a conventional method, and a desired clone is prepared from the library using an appropriate probe or antibody specific to the DNA of the present invention. (Proc. Natl. Acad.
  • cDNA libraries are commercially available, and in the present invention, those cDNA libraries, for example, various cDNA libraries available from Clontech can also be used.
  • An example of the cDNA library is a cDNA library derived from a human spleen node.
  • genes having nucleotide sequences encoding IL-2 and chemokine SLC can be obtained by conventional chemical methods such as the phosphoric acid triester method (Narang et al., Meth. Enzymol., 68, 90-108 (1979)). ) Or the phosphate diester method (Brown et al., Meth. Enzymol., 68, 109-151 (1979)).
  • the above-described nucleotide sequence encoding the chemokine SLC and the nucleotide sequence encoding the mature IL2 protein are connected by a conventional method to form a single gene.
  • an artificial fusion protein gene having, in the same molecule, the migratory activity against the CCR7-expressing cell line possessed by the CC-type chemokine SLC and the proliferation / activation ability of the T cell possessed by the cytotoxic IL2 is constructed.
  • the gene may have a gene encoding a linker between the gene encoding SLC and the gene encoding IL-2.
  • the protein of the present invention can be obtained by a genetic engineering technique (Science, 224, 1431 (1984); Biochem. Biophys. Res. Comm., 130, 692 (1985); Proc. Natl. Acad. Sci., USA., 80, 5990 (1983)). More specifically, a gene encoding a desired protein is inserted into an appropriate vector. This vector is introduced into a host cell to prepare a transformant. By culturing the transformant, a recombinant protein can be obtained.
  • the eukaryotic cells include cells such as vertebrates and yeasts.
  • the vertebrate cells include COS cells (Cell, 23, 175 (1981)) which are monkey cells and Chinese vertebrate cells. Hamster ovary cells are often used.
  • a vector having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like, which is usually located upstream of a gene to be expressed can be used. May be.
  • An example of such an expression vector is pSV2dhfr (Mol. Cell. Biol., 1, 854 (1981)), which has an initial promoter of SV40.
  • yeasts are generally used, and Saccharomyces yeasts can be used.
  • pAM82 Proc. Natl. Acad.
  • Escherichia coli and Bacillus subtilis are commonly used as prokaryotic hosts.
  • a plasmid vector capable of replication in the host bacterium is used, and a promoter sequence and an SD sequence are arranged upstream of the gene so that the desired gene can be expressed in the vector.
  • an expression plasmid to which a start codon necessary for initiation of protein synthesis has been added. Furthermore, when expressed in E.
  • the recognition sequence of Asp Asp Asp Asp Asp Lys (SEQ ID NO: 21) is inserted between the initiation codon and the coding region of the mature sequence of the gene immediately before the mature sequence.
  • the resulting recombinant protein is digested with enteroforce rice (manufactured by Invitrogen) to obtain the fusion protein in which the sequence of the N-terminal sequence is retained. Can be obtained.
  • E. coli K12 strain or the like is used.
  • pBR322 and its improved vector are often used as vectors, but not limited thereto, and various known strains and vectors can also be used.
  • the promoter for example, trp promoter, lpp promoter, lac promoter, PL / PR promoter and the like can be used.
  • various general methods can be adopted.
  • the obtained transformant can be cultured according to a conventional method, and the desired protein is produced by the culture.
  • the medium used for the culture various types commonly used depending on the host cell can be appropriately selected and used, and the culture can be carried out under conditions suitable for the host cell.
  • a vector containing the gene of the fusion protein of the present invention downstream of the pSVL SV40 late promoter is transferred to a monkey-derived cell.
  • Proteins are separated by various separation procedures using their physical and chemical properties (Biochemistry, 25 (25), 8274 (1986); Eur. J. Biochem., 163, 313 (1987), etc.). Can be separated and purified.
  • Examples of the method include salting out, centrifugation, osmotic shock, sonication, ultrafiltration, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, and high performance liquid chromatography.
  • Examples include various types of liquid chromatography such as chromatography, dialysis, and combinations thereof.
  • the amino acid sequence is replaced by deleting any amino acid sequence and introducing a desired amino acid or amino acid sequence.
  • a method known as protein engineering can be widely used for the substitution treatment of the amino acid sequence.
  • a site-diredted deletion method (Nucl. Acids Res., 11, 1645, 1983)
  • Site-specific mutagenesis Site-specific mutation
  • PCR mutation There are a production method, a method using restriction enzyme treatment, and a method using a synthetic gene.
  • site-directed mutagenesis for example, site-directed mutagenesis described in Molecuar Cloning: A Laboratory Manual, 2nd edition, Vol. 1-3, Sambrook, J. et al., Cold Spring Harber Laboratory Press, New York, 1989
  • a mutation is introduced into the DNA sequence of the present invention by using a method such as PCR or PCR.
  • the DNA sequence into which the mutation has been introduced by these methods can be prepared, for example, by using an appropriate vector and host system, for example, Molecuar Cloning: A Laboratory Manual, 2nd edition, Vol. It may be expressed genetically by the method described in Sambrook, J. et al., Cold Spring Harber Laboratory Press, New York, 1989.
  • kits such as Mutan TM -SuperExpress Km, Mutan TM _K (manufactured by Takara Shuzo) and Quik Change Site-Directed Mutagenesis Kit (manufactured by Stratagene) can be used.
  • the site-directed mutagenesis can be performed by first obtaining a single-stranded vector containing a DNA sequence encoding a protein in the sequence.
  • Oligonucleotide primers having the desired mutated sequence are generally synthesized, for example, by Clair et al. (Crea, R. et al., Proc. Natl. Acsd. Sci. USA, 75, 5765, 1978).
  • the primer is annealed with a single-stranded vector containing the DNA sequence, and a DNA polymerase such as Escherichia coli polymerase I Klenow fragment is allowed to act on the primer to complete the synthesis of the mutation-containing strand. .
  • a DNA polymerase such as Escherichia coli polymerase I Klenow fragment is allowed to act on the primer to complete the synthesis of the mutation-containing strand.
  • the first strand encodes the original non-mutated sequence and the second strand forms a heteroduplex with the desired mutation.
  • This double-stranded vector is then used to transform appropriate bacteria or cells, and clones are selected via hybridization to a radioactive probe consisting of a 32 P-labeled mutagenic primer. (Wallace, RB, Nucleic Acids Res., 9, 3647, 1981).
  • the selected clone contains a recombinant vector with the mutated sequence. After selecting such clones, the mutated protein region can be placed into an expression vector of the type used for transformation.
  • Chemokine SLC has chemotactic activity on T cells and B cells. Therefore, these cells are suspended in a migration assay buffer, and the By measuring the number of chemotactic cells, the chemokine SLC activity can be measured. Specifically, the gene product is added to a buffer for migration assay. After cell count
  • T cells or B cells preferably T cells or B cells expressing the chemokine SLC receptor (CCR7), are suspended in a buffer.
  • CCR7 chemokine SLC receptor
  • 1L-2 activity is usually performed in a bioassay using T cells that proliferate in an IL-2-dependent manner.
  • a system for measuring the biological activity of human IL-2 has been established (Basic and clinical Tatsu Sasaki et al. Vol. 22, No. 17, 29-42 1988). Therefore, the IL-2 biological activity of the gene product can be measured by the system. Preparation of the vector for gene therapy of the present invention
  • Vectors for using the fusion protein of the present invention for gene therapy include retrovirus vector (Mu MLV skeleton, HIV skeleton), adenovirus vector, and adeno-associated virus (AAV). Vectors and the like are conceivable. Gene therapy can also be performed using a plasmid incorporating the DNA of the present invention by using a gene gun or in vivo electroporation.
  • the promoter for expression in animal cells is not particularly limited as long as it is a promoter used in a normal animal cell culture expression system.
  • MuMLV LTR, etc. can be raised.
  • the CMV promoter is For example, it can be prepared from pRC / CMV (manufactured by Invitrogen) by ordinary genetic manipulation.
  • the above-described plasmid containing the promoter for expression in animal cells and the above-mentioned artificial fusion protein gene can be constructed using a conventional gene recombination method.
  • the gene encoding the fusion protein of the present invention is constructed by inserting it into the Hindlll site of pRC / CMV (manufactured by Invitrogen), which contains the CMV promoter, the poly (A) signal of pacific growth hormone, and the neomycin resistance gene. And other methods.
  • Gene therapy using the gene therapy vector of the present invention can be performed by allowing human somatic cells to acquire the DNA of the present invention and returning the recombinant cells to the patient, or by directly transferring the vector for gene therapy of the present invention to the affected part of the patient Can be administered.
  • the vector is introduced into somatic cells by a gene gun, microinjection, transfection or transduction. Can be introduced.
  • the vector is a virus
  • the DNA of the present invention can be introduced by infecting somatic cells with a virus incorporating the gene of the present invention.
  • somatic cells examples include bone marrow cells, hepatocytes, fibroblasts, epidermal cells, and muscle cells derived from patients.
  • Pharmaceutical composition of the present invention include bone marrow cells, hepatocytes, fibroblasts, epidermal cells, and muscle cells derived from patients.
  • the fusion protein of the present invention is useful as an anticancer agent because it has a T cell migration activity of chemokine SLC together with an immunostimulatory effect of IL-2.
  • the proteins also include pharmaceutically acceptable salts.
  • Such salts may be prepared by known methods, for example, non-toxic alkali metal salts such as sodium, potassium, lithium, calcium, magnesium, norium, and ammonium, alkaline earth metal salts, and ammonium salts. Is included.
  • a pharmaceutical preparation is prepared using a pharmaceutically effective amount of the protein as an active ingredient.
  • various forms can be selected according to the purpose of treatment, and typical examples are individual forms such as tablets, pills, powders, powders, granules, capsules and the like. It includes dosage forms and liquid dosage forms such as solutions, suspensions, emulsions, syrups, and elixirs.
  • oral preparations are further classified into oral preparations, parenteral preparations, nasal preparations, vaginal preparations, suppositories, sublingual preparations, ointments, etc. according to the route of administration, and are prepared, formulated or prepared according to the usual methods. be able to.
  • the administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, age, sex and other conditions of the patient, degree of disease, and the like.
  • solid dosage forms such as tablets, pills, granules, capsules and the like, liquids, suspensions, and emulsions are orally administered.
  • the injection is administered intravenously, alone or mixed with a normal replenisher such as glucose or amino acids.
  • C The injection is administered intramuscularly, intradermally, subcutaneously, or intraperitoneally as needed.
  • the amount of the active ingredient of the compound of the present invention to be contained in the above pharmaceutical preparation and the dose thereof are not particularly limited, and are appropriately determined according to the desired therapeutic effect, administration method, treatment period, patient age, sex, and other conditions. Selected. In general, the dose is preferably about l to 10 mg per 1 kg of body weight per day, and the preparation can be administered once or several times a day.
  • mouse SLC gene fragment that does not include the termination codon of the mouse SLC (mSLC) gene (SEQ ID NO: ⁇ ) and has an XbaI site at the 3 ′ end
  • plasmid pT7-T3-D-Pac- mSLC (EST: clone number W67046, purchased from Genome Svstems)
  • EST clone number W67046, purchased from Genome Svstems
  • the obtained fragment was simultaneously digested with Sal I and Xba I and inserted into the Sal I and Xba I sites of Blue Script (+) (Stratagene) to construct plasmid pBS-mSLC (S / X). did.
  • the nucleotide sequence of the subcloned product was determined, and it was confirmed that there was no mutation due to the PCR method.
  • mIL-2 mouse IL-2
  • SEQ ID NO: 10 a plasmid containing the mIL-2 cDNA
  • Okayama- Using the primers shown in Fig. 1 (2) (mIL2-XbalF (SEQ ID NO: 15) and mIL2-Notl-R (SEQ ID NO: 16)) with Berg-MuIL2 as type I, an approximately 0.45 kb fragment was It was amplified by PCR.
  • the obtained fragment was co-digested with XbaI and NotI, subcloned into the XbaI and NotI sites of the commercially available vector BlueScript (+), and constructed plasmid pBS-mIL2 (X / N). .
  • the nucleotide sequence of the clone was determined, and it was confirmed that there was no mutation by PCR.
  • pBS-mSLC (S / X) is simultaneously digested with Sal I and Xba I, and the resulting 0.4 kb fragment is inserted into the Sal I and Xba I sites of pBS-mIL2 (X / N), resulting in a mouse SLC- the mouse IL2 fusion evening protein gene having between Sal I and Not I sites Bok, plus Mi de P BS-mSLC-IL2 were obtained ( Figure 2).
  • the artificial fusion protein gene (mSLC-IL-2) consists of 855 base pairs, and its structure is 399 bases encoding mouse SLC from the 5 'end, 6 bases encoding the linker, 6 bases of mouse IL-2 It consists of 445 bases encoding the 149 amino acid residues from the 21st N-terminal to the C-terminal and a termination codon (SEQ ID NO: 11).
  • Plasmid pBS-mSLC-IL2 was co-digested with restriction enzymes Sal I and Not I to obtain a Sail-Not I fragment of the mouse SLC-mouse IL-2 fusion protein gene.
  • This fragment retro viral vectors one P LX- IRES- EGFP of Sal I-Not mice an object than be incorporated into I site SLC- murine IL-2 fusion gene transfer retro virus base click evening one plasmid pLX — IRES—EGFP—mSLC-IL2 was obtained (FIG. 2).
  • PSPORT 1 (GIBCO / BRL) multicloning site EcoRl-Hind III is located at the EcoRl-Hind III site of the retrovirus vector pLX-IRES-EGFP in pLHDCX (Genbank accession No. M64754). Fragments were ligated, and the internal ribosome recognition site of encephalomyocarditis virus IRES fragment (Novagen) at the Notl-BamHl site was incorporated with an enhanced green fluorescent protein gene EGFP fragment (Clontech) (Fig. 3).
  • the transient viral transfection of retroviral vector-plasmid DNA results in an ecotropic virus.
  • a Bosc23 cell (ATCC CRL11554) capable of producing E. coli was used. 24 hours prior to Trang Sufuekushiyon plated suspended IX 1 0 6 pieces of normal medium (DMEM / 1 0% FCS) 2ml in a petri dish for culturing Bosc23 cells (diameter 35mm), 37 ° C, 5 % C 0 2 were cultured under.
  • the transfection was similarly performed for pLX-IRES-EGFP, pLX-IRES-EGFP-mSLC, and pLX-IRES-EGFP-mIL-2 for use in control experiments.
  • Balb / C mouse-derived fibroblasts CL.7 cells (ATCC TIB80) to be infected 24 hours before virus infection are transferred to a culture dish (35 mm in diameter) at 1 x 105 cells in normal medium (DMEM / 10% FCS). plated were suspended in 2 ml, were cultured in C0 2 under 37 ° C, 5%.
  • IL-2 activity is usually performed in a bioassay using T cells that proliferate in an IL-2-dependent manner.
  • a system for measuring the biological activity of human IL-2 has been established (Literature; Basic and Clinical Katsu Sasaki et al., Vol. 22 No. 17 Dec. 1988), and the above-mentioned transfected cultured cells CL.7-mSLCJL -2 produced an artificial fusion protein mSLC-IL-2 (SEQ ID NO: 12) produced in the medium.
  • the measurement was also performed on the culture supernatant of CL.7-mIL-2.
  • the IL-2 concentration is measured by mIL-2 ELISA (R & D), and the titer is determined based on the IL-2 concentration to obtain an appropriate concentration (approximately 2-3 ng / ml).
  • the medium was previously diluted with RPMI 16401 with 10% FCS.
  • a 96-well flat-bottom microplate manufactured by Sumitomo Bei-Client
  • 50 ⁇ 1 of RPMI 1640 with 10% FCS was added to the second and subsequent rows.
  • a titration medium containing human IL-2 200 JRU / ml, 50-1 as a maximum OD value control (ODmax) was dispensed.
  • ODmin minimum OD value control
  • RHu IL-2 (manufactured by Shionogi Pharmaceutical Co., Ltd .; trade name: Imnesis) is used as a standard in the top holes of the third and fourth rows so that both the standard sample and the measurement sample have the same dilution ratio.
  • Dissolve 50 JRU Japanese Reference Units
  • 50 JU1 of each diluted culture supernatant to the top hole of each of the second and subsequent rows in 50 JUL.
  • Each was added.
  • use a multi-channel pipette to mix well by repeating pipetting from the top row of the third row and thereafter, transfer to the wells that have been settled 50 1 at a time, and perform 2-fold serial dilution at the bottom. I went up to 8 levels.
  • the NK-7 cell solution cultured in the growth medium was centrifuged, and the supernatant was removed.
  • the cells were resuspended at 20,000 cells / 50 ⁇ 1 in the titration medium, and 50 ⁇ 1 of this cell suspension was inoculated into all the wells of the microplate.
  • MTT reagent PBS (-) (day Water Pharmaceutical) dissolve 0.3% of MTT (3-(4,5-Dimethyl-2-thiazolyl) -2 ) 5-diphenil-2Htetrazo] ium bromide, SIGMA) and mix with Milliporefil Yuichi (0.45m) Dispense filtered sterilization was intended) 25 ⁇ 1 to all the wells min, 37 ° (, a 5% C0 2 for 4 hours at below. broth microphone port in the plate, with Maruchichanerupipe' bets I moved to an empty microplate.
  • the dilution series of each sample consists of two columns, calculate the average value of each, plot the concentration dependence curve by plotting the dilution ratio of the sample on the horizontal axis and the OD value on the vertical axis. .
  • the dilution factor corresponding to the median absorbance of the maximum OD value control (ODmax) and the minimum OD value control (ODmin) in the plate was read from the graph.
  • the measurement coefficient (display force value / actual force value) of the standard product was calculated, and this coefficient was multiplied by the effective titer of each sample to determine the converted force value of each sample.
  • the IL-2 titer of the culture supernatant of CL.7-mSLC-IL2 measured this time was 242 JRU / ml. Since the IL-2 concentration of the culture supernatant of CL.7-mSLC-IL2 by ELISA was 10 ng / ml, the IL-2 titer per ng was 24.2 JRU. Since the IL-2 titer per 1 ng of the culture supernatant of CL.7-rnIL2 measured in the same manner was 18.4 JRU, the mouse SLC-mouse IL-2 fusion protein (mSLC-IL-2) It was found to have an IL-2 titer equal to or higher than that of IL-2 protein.
  • mSLC-IL-2 mouse SLC-mouse IL-2 fusion protein
  • 3 ⁇ 10 5 of the transfected cells obtained in Example 3 were added to a 3 ml culture solution.
  • DMEM / 10% FCS inoculated into a 35 mm diameter culture dish, and cultured at 37 ° C. under 5% CO 2 for 48 hours.
  • the culture supernatant containing the obtained various gene products was collected and filtered through Millipore Filter (0.45 Um). This was mixed with a buffer for migration assay (RPI1640, 10 mM HEPES, pH 7.4, containing 1% BSA) at a volume ratio of 2 times (containing 50% of culture supernatant) and 10 times (containing 10% of culture supernatant). And used for the following migration assy.
  • RPI1640 10 mM HEPES, pH 7.4, containing 1% BSA
  • a precursor B cell line B300-19 cell (B300-19-mCCR7), which stably expresses mouse CCR7, a specific receptor for mouse SLC, was used. This is obtained by introducing the mouse CCR7 expression plasmid pCAGGSneo-mCCR7 into which the mouse CCR7 gene has been introduced into the expression plasmid pCAGGSneo into the precursor B cell line B300-19 cells by the electroporation method, and selecting the drug with the drug G418. Mouse CCR7 expressing cells.
  • TCA precipitation was performed as follows to concentrate the protein in the medium.
  • One ml of the culture supernatant was mixed with 100/1 of 100% TCA, left on ice for 1 hour, and centrifuged at 12K rpm for 5 minutes. Wash the pellet with ice-cold acetone, dissolve in 25 ⁇ 1 3X SDS sample buffer, add 2.51 2-mercaptoethanol and 2.51 lM Tris-HCl, pH 8.0. And denatured by applying 10 CTC heat for 5 minutes.
  • Each 101 lysates were electrophoresed on a 15% -25% gradient SDS-polyacrylamide gel. At this time, Prestain Protein Maker and Broad Range (NEW ENGLAND BioLabs inc.) Were run in the adjacent lane.
  • the protein on the gel after electrophoresis was transferred to a ditrocellulose membrane (imobilonP, Milipore).
  • Nitrocellulose membrane after transfer is 5% (weight / volume) skim milk solution (dissolved skim milk made by Dii'co in T-PBS (PBS solution with 0.05% Tween20)) for 30 minutes at room temperature
  • Non-specific protein adsorption was inhibited by soaking in.
  • the ditrocellulose membrane was reacted with a goat anti-mouse IL-2 antibody (diluted 1000-fold with T-PBS for use) for about 2 hours.
  • Example 3 the mSLC-IL2 transgenic cell CL.7-mSLC-IL2 obtained in Example 3 was used as its parent strain. Fibroblasts CL.7 were intradermally administered to mouse Balb / C strain, and it was examined whether T cells infiltrated there. Obtained in Example 3 MSLC-IL2 transgenic cell (CL.7-mSLC-IL2), Vector as a control, MSLC, mIL2 transgenic cell it HANKS about it so that lm 1 x 10 8 cell number per ⁇ The cells were suspended in a buffer (manufactured by Gibco).
  • a female Balb / C mouse, 7 weeks old (purchased from Nippon Chillers-Liva) was inoculated intradermally with the above cell suspension (50 uI, 5 ⁇ 10 6 cells) on the back (each 2 in the cell group). Five days later, the transplantation site had a bulge of about 5 mm.Then, the mouse was euthanized, removed, immersed in an OTC compound (manufactured by Miles Laboratory), and frozen on dry ice. .
  • the plate was reacted with a egret anti-rat IgG antibody (Vector) for 30 minutes at room temperature, and washed twice with PBS (-) solution. After the purification, it was reacted with a 1% hydrogen peroxide solution (a mixture of 30% hydrogen peroxide solution and methanol at a volume ratio of 29 with a volume ratio of 29) for 30 minutes. After washing three times with a PBS (-) solution, a peroxidase label was developed using an EliteABC kit (Vector) and a DAB substrate kit. Counterstaining was also performed with methylene blue. For the stained sections, magnified images obtained with an optical microscope (OPTIPHOT) from Nippon Kogaku Co., Ltd.
  • OPTIPHOT optical microscope
  • Example 3 the CL.7-mSLC-IL2 transfected mSLC-IL-2 gene obtained in Example 3 was used.
  • Mouse Balb / C strain which is the animal derived from its parental fibroblast CL.7, was mixed with mouse Balb / C-derived colon cancer cell line Colon26, and then intradermally injected to evaluate the degree of tumor formation.
  • Vector and mIL2 transfected cells were examined as a control.
  • mice Female cell of a Balb / C mouse, 7 weeks old (purchased from Nippon Chill's Riva Co., Ltd.) on the back of the above cell suspension 100 1 (number of cells: 5 x 10 5 transgenic fibroblasts, Colcm26 and is intradermally inoculated with 1 X 10 6 cells) (each 5 animals in experiment 1, each cell group, experiment 2, Kakugunma 8 mice mouse). Thereafter, they were kept under normal conditions.
  • the major axis of the tumor and the length (minor axis) in the direction perpendicular to the tumor were measured with a vernier caliper, and the approximate value (major axis X minor axis X minor axis ⁇ 2) was taken as the tumor volume.
  • Figures 7 and 8 graphically represent the mean tumor volume (standard deviation of soil) of each group in each experiment.
  • Experiment 1 the results were 28 days after tumor inoculation.
  • the average tumor volume was 105% compared to the Vector group, and no effect of suppressing tumor formation was observed.
  • Experiment 2 shows the results on the 23rd day after tumor inoculation.
  • the average tumor volume was 77% compared to the vector group, indicating that the increase in tumor volume was delayed to some extent.
  • the tumor volume was even smaller in the mSLC-IL2 group, with an average tumor volume of 45% compared to the vector group and 59% compared to the mIL2 group.
  • a human SLC gene fragment that does not contain the termination codon of the human SLC (hSLC) gene (SEQ ID NO: 1) and has an XbaI site at the 3 'end is used as a vector for expression of a protein fused to an alfa phosphatase pDREF -SLC-AP (Nagira, M. et al., J. Biol. Chem., 272, 31, 19518-19524, 1997) is digested simultaneously with Sal I and Xba I. did.
  • a gene fragment encoding the mature sequence of human IL-2 (hIL-2) (SEQ ID NO: 4) was obtained.
  • the plasmid pIL2-50A having the cDNA of human IL2 (SEQ ID NO: 3) was designated as type I, and the primers (5'-hIL2-Spe I (SEQ ID NO: 22) and 3) were used.
  • '-hIL2-NotI (SEQ ID NO: 23)
  • the obtained fragment was co-digested with Spe I and Not I, subcloned into the Spe I and Not I sites of the commercially available vector Blue Script (+), and plasmid pBS-ML-2 (Spe I / N) was obtained. It was constructed. Determine the nucleotide sequence of the cloned product and confirm that there is no mutation by PCR.
  • the restriction enzymes Spe I and Xba I have the same sequence of the cohesive end after cleavage, and can bind.
  • a 0.4 kb fragment obtained by simultaneously digesting pDREF-SLC-AP with Sal I and Xba I was inserted into the Spe I and Not I sites of pBS-hIL-2 (Spe I / N), resulting in human Plasmid pBS-hSLC-IL2, which has the SLC-human IL-2 fusion protein gene between the Sal I and Not I sites, was obtained (FIG. 9).
  • the artificial fusion protein gene (hSLC-IL-2) consists of 810 base pairs and has a structure of 5, 402 bases encoding the human SLC gene from the end, 6 bases encoding the linker, and human IL-2. It consists of 399 bases coding for the 133 amino acid residues from the N-terminal 21st to the C-terminal and a stop codon (SEQ ID NO: 25).
  • Plasmid pBS-hSLC-IL2 was co-digested with restriction enzymes Sal I and Not I to obtain a Sal I—Not I fragment of the human SLC-human IL-2 fusion protein gene.
  • restriction enzymes Sal I and Not I restriction enzymes Sal I and Not I to obtain a Sal I—Not I fragment of the human SLC-human IL-2 fusion protein gene.
  • the desired retrovirus vector plasmid for transfecting the human SLC-human IL-2 fusion gene can be obtained.
  • pLX—IRES—EGFP—hSLC-IL2 was obtained (FIG. 9).
  • Retrovirus vector for human IL2 gene transfer for use in comparative experiments Plasmid pLX—IRES—EGFP—hIL-2 was inserted into the retroviral vector pLX—IRES-EGFP multicloning site at the human IL-2 gene fragment (pIL2-50A was type III, and )) (A fragment obtained by amplification with the PCR method using the primers 5′-WL2-Sal I (SEQ ID NO: 24) and 3′-WL2-Not I (SEQ ID NO: 23)). It was made more.
  • a retrovirus was produced in the same manner as in Example 3, and the gene was transfected into CL.7. -2 transgenic cells (CL.7-hIL2) and hSLC-IL2 transgenic cells (CL.7-hSLC-IL2) were obtained.
  • the obtained hSLC-IL2 gene-introduced cultured cells (CL.7-hSLC-IL2) and hIL2 gene-introduced cultured cells (CL.7-hIL2) were each converted into 3 x 105 cells in a 3 ml culture solution (DMEM / 10% was suspended in FCS), were seeded in 35mm diameter culture shear les, and cultured for 48 hours at 37 ° C, 5% C0 2 below. Culture supernatants containing the various gene products obtained were collected and filtered through a Millipore filter (0.45 m). The IL2 content of the culture supernatant was measured using a human IL2 ELISA (Human IL2 AN, ALYZA Immunoassay kit.
  • the IL2 concentration of the culture supernatant of CL.7-hSLC-IL2 was 25 ng / In ml
  • the concentration of IL2 in the culture supernatant of CL.7-hIL2 was 50 ng / ml
  • the expression of the transgene product was confirmed in each case.
  • Example 13 the biological activity of IL-2 was measured using the culture supernatant of the various transfected cells (CL.7-hSLC-IL2, CL.7-hIL2) obtained in Example 11. did.
  • the IL.2 titer of the culture supernatant of CL.7-hSLC-IL2 was 378 JRU / ml
  • the IL.2 titer of the culture supernatant of CL.7-hIL2 was 901 JRU / ml. Since the titer of IL2 per IL2 was 15.1 JRU for hSLC-IL2 and 18 JRU for hIL2, the human SLC-human IL-2 fusion protein was comparable to human IL-2 ⁇ IL. It was found to have a titer.
  • Example 13 the biological activity of IL-2 was measured using the culture supernatant of the various transfected cells (CL.7-hSLC-IL2, CL.7-hIL2) obtained in Example 11. did.
  • the culture of various gene-introduced cells (CL.7-hSLC-IL2, CL.7-hIL2) obtained in Example 11 and the vector-introduced cell (CL.7-Vector) described in Example 3 as a control
  • a buffer for migration assay (RPMI 1640, 10 mM HEPES, pH 7.4, containing 1% BSA) at a volume ratio of 2 times (containing 50% of culture supernatant), 10 times (10% of culture supernatant). % Containing) and used for the following migration assays.
  • a precursor B cell line L1.2 cell that stably expresses human CCR7, a specific receptor for human SLC. This was achieved by introducing the human CCR7 expression plasmid pCAGGSneo-CCR7 into which the human CCR7 gene was inserted into the expression plasmid pCAGGSneo by electroporation into the precursor B cell line L1.2 cells, and using the drug G418 Human CCR7 expressing cells obtained by selection o (Yoshida, R., et al., J Biol Chem. 273: 7118-7122, 1998)
  • fibroblast CL.7 cells from mouse Balb / C strain
  • colon cancer cell line Colon26 from mouse Balb / C
  • the antitumor effect was examined by comparing the degree of tumor formation.
  • the transgenic CL.7 from (1) to (7) was mixed with 1 ⁇ 10 5 Colon26 cells and transplanted into the back skin of mice for comparison.
  • HANKS was used so that each transfected fibroblast was 5 x 10 6 (in case of 5, a total of IX 10?), And Colon26 was a cell solution in which 106 IX were mixed in lml. The cells were suspended in a buffer (manufactured by Gibco).
  • FIG. 10 is a graph showing the average tumor volume (soil standard deviation) of each group 24 days after transplantation.
  • the average tumor volume was 102% compared to the Vector group, and no effect was observed in suppressing tumor formation.
  • the average tumor volume in the mIL2 group was 60% compared to the Vector group, indicating that the increase in tumor volume was delayed to some extent.
  • the tumor volume was even smaller in the mSLC-IL2 group, with an average tumor volume of 26% compared to the mIL2 group, showing a significantly stronger antitumor effect than the mIL2 group.
  • the fusion protein of the present invention has a T cell chemotactic effect as chemokine SLC in addition to an immune activating effect as IL-2, so that it can be administered in a therapy requiring immunostimulation of IL-2.
  • a T cell chemotactic effect as chemokine SLC in addition to an immune activating effect as IL-2, so that it can be administered in a therapy requiring immunostimulation of IL-2.

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Abstract

A fused protein which has the amino acid sequence of from Ser at the 24-position to Pro at the 134-position in SEQ ID NO:2 at the N-end and involves the amino acid sequence of from Ala at the 21-position to Thr at the 153-position in SEQ ID NO:4.

Description

明細: ケモカイン SLC-IL2 融合夕ンパク質とその遺伝子 技術分野  Description: Chemokine SLC-IL2 fusion protein and its gene
本発明は、 ケモカイン SLC と IL-2からなる融合夕ンパク質およびその製造方 法、 該融合タンパク質をコードする遺伝子、 該融合タンパク質をコードする遺伝 子を含む遺伝子治療用発現べクタ一及び該融合夕ンパク質を含む癌治療用医薬組 成物に関する。 背景技術  The present invention relates to a fusion protein comprising a chemokine SLC and IL-2, a method for producing the fusion protein, a gene encoding the fusion protein, an expression vector for gene therapy containing the gene encoding the fusion protein, and the fusion protein. The present invention relates to a pharmaceutical composition for treating cancer containing protein. Background art
インタ一ロイキン— 2 ( IL-2) は、 主に活性化された T細胞より産生されるサ ィ トカインの一つである。 その生理作用は T 細胞の増殖 ·活性化、 B細胞の增殖 と抗体産生能の増強、 NK 細胞増殖と活性化、 単球 · マクロファージの活性化な どである （Smith, K.， Annu Rev Immunol. 2: 319-333， 1984) 。 ヒ トインタ一口 ィキン一 2 (hIL-2)は、 1 5 3個のアミノ酸ならなる前駆体がまず作られ、 N末端 の 2 0個のシグナルペプチドがプロセッシングを受けて、 1 3 3個のアミノ酸か らなる成熟型の IL-2 となる。 hIL-2の直接的全身投与による抗腫瘍効果がいく つかの腫瘍で報告されている。 また腫瘍細胞に hIL-2遺伝子を導入することによ る腫瘍原性の低下は報告されているが、 腫瘍免疫の誘導は弱いと考えられている ( Dranoff, G. et al., Pro Natl Acad Sci USA. 90, 3569-3543, 1993) 。 ケモカインは内因性の白血球遊走 · 活性化作用を有するへパリン結合性ポリぺ プチドの総称である。 ケモカインは保存されている 4つのシスティン残基のうち N 末端の 2つのシスティン残基が 1つのアミノ酸で隔てられているか（CXC)、 隣 り合っているか（CC)、 例外的に 1つのみであったり、 三つのアミノ酸で隔てられ ているか（CX3C)によって、 4つのサブファミ リ一に分類される。 CC型ケモカイ ン SLC secondary lymphoid-tissue chemokine、 称 6-C-kine、 TCA-4、 Exodus-2) は CC型ケモカインの 1つである。 ヒ トケモカイン SLCは、 1 3 4個 のアミノ酸からなる前駆体がまず作られ、 N末端の 2 3個のシグナルべプチドが プロセッシングを受けて、 1 1 1個のアミノ酸からなる成熟型の SLCとなる。 ケ モカイ ン SLC に対する特異的受容体は CCR7 である こ とが知られている (Yoshida, R., et al., J Biol Chem. 273: 7118-7122, 1998) o ケモカイン SLCは、 CCR7を発現するナイーブ T細胞、 メモリー Τ細胞、 Β細胞に対する遊走活性を 有している ( Gunn, M. D., et al., Proc Natl Acad Sci USA. 95: 258-263, 1998) 。 発明の開示 Interleukin-2 (IL-2) is one of the cytokines produced mainly by activated T cells. Its physiological actions include proliferation and activation of T cells, proliferation of B cells and enhancement of antibody production, proliferation and activation of NK cells, activation of monocytes and macrophages (Smith, K., Annu Rev Immunol). 2: 319-333, 1984). Human IL-1 (hIL-2) is made up of a precursor consisting of 15 amino acids, and the 20 N-terminal signal peptide is processed to produce 133 amino acids. It becomes a mature form of IL-2. Antitumor effects of direct systemic administration of hIL-2 have been reported in some tumors. Although the reduction of tumorigenicity by introducing the hIL-2 gene into tumor cells has been reported, the induction of tumor immunity is considered to be weak (Dranoff, G. et al., Pro Natl Acad Sci USA. 90, 3569-3543, 1993). Chemokines are a general term for heparin-binding polypeptides having an endogenous leukocyte migration / activation activity. Chemokines are either conserved by the N-terminal two cysteine residues separated by a single amino acid (CXC) or adjacent (CC), with the exception of only one of the four conserved cysteine residues. Or separated by three amino acids (CX3C), there are four subfamilies. CC-type chemokine SLC secondary lymphoid-tissue chemokine (named 6-C-kine, TCA-4, Exodus-2) is one of the CC-type chemokines. In human chemokine SLC, a precursor consisting of 134 amino acids is first produced, and the N-terminal 23 signal peptides are processed to form a mature SLC consisting of 11 amino acids. Become. It is known that the specific receptor for chemokine SLC is CCR7 (Yoshida, R., et al., J Biol Chem. 273: 7118-7122, 1998) o Chemokine SLC expresses CCR7 It has a chemotactic activity against naive T cells, memory Τ cells, and Β cells (Gunn, MD, et al., Proc Natl Acad Sci USA. 95: 258-263, 1998). Disclosure of the invention
IL-2 はその N 末端に別の夕ンパク質を融合してもその生理活性を損なわない 例が知られている。 一方、 ケモカインはその C末端に別のタンパク質を融合させ ても其の活性が失われない事例が知られている。 そこで、 本発明者ら.は、 停止コ ドンを含まない CC型ケモカイン SLC のアミノ酸配列をコードする塩基配列と サイ トカイン IL-2の成熟型蛋白をコ一ドする塩基配列とを接続することにより、 単一の遺伝子とし、 CC.型ケモカイン SLC としての遊走活性およびサイ トカイン IL-2 としての生理活性を同一分子内に有する人工融合タンパク質をコードする 人工融合遺伝子を構築し、 この遺伝子に係る真核細胞発現べクタ一、 さらに該発 現ベクター導入動物細胞株を樹立し、 その培養上清中に人工融合夕ンパク質を生 産することに成功した。  It is known that IL-2 does not impair its biological activity even when fused with another protein at its N-terminus. On the other hand, it is known that chemokine does not lose its activity even if another protein is fused to its C-terminal. Therefore, the present inventors have proposed that by connecting the nucleotide sequence encoding the amino acid sequence of CC-type chemokine SLC that does not contain a stop codon and the nucleotide sequence encoding the mature protein of cytokine II-2. A single gene, and construct an artificial fusion gene encoding an artificial fusion protein having the migration activity of CC. Type chemokine SLC and the physiological activity of cytokine IL-2 in the same molecule. We established a nuclear cell expression vector and an animal cell line transfected with the expression vector, and succeeded in producing an artificially fused protein in the culture supernatant.
生産された融合夕ンパク質は、 単一分子内にケモカイン SLC、 IL-2の両方の生 理活性を示すすぐれた性質を有する融合タンパク質であることを確認し、 本発明 を完成した。 すなわち、 本発明は、 ( 1 ) N末端側にケモカイン SLCを構成するアミノ酸配列を有し、 かつ IL-2を 構成するァミノ酸配列を含む融合夕ンパク質 ； It was confirmed that the produced fusion protein was a fusion protein having excellent properties showing the bioactivity of both chemokines SLC and IL-2 in a single molecule, and thus completed the present invention. That is, the present invention (1) a fusion protein having an amino acid sequence constituting chemokine SLC on the N-terminal side and containing an amino acid sequence constituting IL-2;
(2 ) ケモカイン SLCおよび IL-2がマウスまたはヒ ト由来である上記 （ 1 ) に 記載の融合夕ンパク質 ；  (2) the fusion protein according to (1), wherein the chemokines SLC and IL-2 are derived from mouse or human;
( 3 ) ケモカイン SLC を構成するアミノ酸配列が配列番号 ： 2の 24位の Ser から 1 34位の Proに記載のアミノ酸配列であり、かつ IL-2を構成するアミノ酸 配列が配列番号： 4の 2 1位の Alaから 1 5 3位の Thrに記載のァミノ酸配列で ある上記 （ 1 ) または （2 ) に記載の融合タンパク質 ；  (3) The amino acid sequence constituting chemokine SLC is the amino acid sequence from Ser at position 24 to Pro at position 134 of SEQ ID NO: 2, and the amino acid sequence constituting IL-2 is SEQ ID NO: 4 The fusion protein according to the above (1) or (2), which is an amino acid sequence according to Ala at position 1 to Thr at position 153;
( 4 ) 配列番号 ： 2の 24位の Serカゝら 1 34位の Proに記載のァミノ酸配列、 リンカ一及び配列番号： 4の 2 1位の Alaから 1 5 3位の Thrに記載のァミノ酸 配列を含む上記 （ 1 ) から （ 3 ) のいずれかに記載の融合タンパク質 ；  (4) SEQ ID NO: 2 amino acid sequence described in Ser at position 24 in position 134, Pro in position 134, linker and sequence described in Ala in position 21 from position 21 in Thr in position 153 A fusion protein according to any one of the above (1) to (3), which comprises an amino acid sequence;
( 5 ) 配列番号 ： 2の 24位の Serから 1 34位の Proに記載のァミノ酸配列、 リンカ一及び配列番号： 4の 2 1位の Alaから 1 5 3位の Thrに記載のァミノ酸 配列からなる上記 （4) に記載の融合タンパク質 ；  (5) SEQ ID NO: Amino acid sequence from Ser at position 24 to Pro at position 134, Linker and Amino acid described from Thr at position Ala at position 21 from SEQ ID NO: 4 The fusion protein according to the above (4), which comprises a sequence;
( 6 ) 配列番号 ： 2の 1位の Metから 1 3 4位の Proに記載のァミノ酸配列、 リ ンカー及び配列番号： 4の 2 1位の Alaから 1 5 3位の Thrに記載のァミノ酸配 列からなる上記 （4) に記載の融合タンパク質 ；  (6) SEQ ID NO: Amino acid sequence described in Met at position 1 from position 1 to Pro at position 134, linker, and amino acid described as Ala at position 21 from Ala to position Thr at position 15 The fusion protein according to the above (4), which comprises an acid sequence;
(7 ) 上記 （ 5 ) に記載の融合タンパク質が、 配列番号 ： 6の 24位の Serから 2 6 9位の Thrに記載のァミノ酸配列からなる融合夕ンパク質 ；  (7) the fusion protein according to (5), wherein the fusion protein comprises the amino acid sequence described from Ser at position 24 to Thr at position 269 in SEQ ID NO: 6;
( 8 ) 上記 （ 6 ) に記載の融合夕ンパク質が、 配列番号 ： 6の 1位の Metから 2 6 9位の Thrに記載のァミノ酸配列からなる融合夕ンパク質 ；  (8) the fusion protein according to (6) above, which comprises an amino acid sequence described from Met at position 1 of SEQ ID NO: 6 to Thr at position 269 in SEQ ID NO: 6;
( 9 ) 上記 （ 5 ) 記載の融合夕ンパク質が、 配列番号 ： 2 6の 24位の Serから 2 6 9位の Thrに記載のアミノ酸配列からなる融合夕ンパク質 ；  (9) the fusion protein according to (5) above, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 26 from Ser at position 24 to Thr at position 269;
( 1 0 ) 上記 （ 6 ) 記載の融合タンパク質が、 配列番号 ： 2 6の 1位の Metから 2 6 9位の Thrに記載のアミノ酸配列からなる融合夕ンパク質 ；  (10) The fusion protein according to (6) above, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 26 from Met at position 1 to Thr at position 269;
( 1 1 ) 上記 （ 3 ) から （ 1 0) のいずれかに記載のアミノ酸配列において 1 も しくは数個のアミノ酸が、 置換、 欠失、 挿入または付加されたアミノ酸配列を含 み、 かつケモカイン SLC活性および IL-2活性を有する融合夕ンパク質 ； (11) In the amino acid sequence according to any one of (3) to (10) above, Or a fusion protein in which several amino acids have a substituted, deleted, inserted or added amino acid sequence and have chemokine SLC activity and IL-2 activity;
( 1 2) ケモカイン SLCを構成するアミノ酸配列が配列番号： 8の 24位の Ser から 1 3 3位の Glyに記載のァミノ酸配列であり、かつ IL-2を構成するァミノ酸 配列が配列番号 ： 1 0の 2 1位の Alaから 1 6 9位の Ginに記載のァミノ酸配列 である上記 （ 1 ) または （ 2) に記載の融合タンパク質 ；  (12) The amino acid sequence constituting the chemokine SLC is the amino acid sequence described from Ser at position 24 to Gly at position 133 of SEQ ID NO: 8, and the amino acid sequence constituting IL-2 is SEQ ID NO: The fusion protein according to (1) or (2), which is the amino acid sequence described in Gin at position 21 from Ala to position 169 at 10:
( 1 3 ) 配列番号 ： 8の 24位の Serカゝら 1 3 3位の Glyに記載のァミノ酸配列、 リンカー及び配列番号： 1 ◦の 2 1位の Alaから 1 6 9位の Ginに記載のァミノ 酸配列を含む上記 （ 1 2 ) に記載の融合タンパク質 ；  (13) SEQ ID NO: 8 amino acid sequence described in Gly at position 13 of Sercapra at position 24, linker and sequence number: from Ala at position 21 to Gin at position 169 The fusion protein according to (12), which comprises the amino acid sequence described above;
( 1 4) 配列番号 ： 8の 24位の Serから 1 3 3位の Glyに記載のァミノ酸配列、 リンカ一及び配列番号： 1 0の 2 1位の Alaから 1 6 9位の Ginに記載のァミノ 酸配列からなる上記 （ 1 3 ) 記載の融合タンパク質 ；  (14) SEQ ID NO: Amino acid sequence described in Gly from position Ser 24 to position 13 3 in Linker 8, linker and SEQ ID NO: Described in Gin position 16 from Ala in position 21 A fusion protein according to the above (13), comprising the amino acid sequence of
( 1 5) 配列番号： 8の 1位の Metから 1 3 3位の Glyに記載のァミノ酸配列、 リンカー及び配列番号： 1 0の 2 1位の Alaから 1 6 9位の Ginに記載のアミノ 酸配列からなる上記 （ 1 3 ) 記載の融合夕ンパク質 ；  (15) SEQ ID NO: 8 amino acid sequence described in Gly from position 1 to position 13 of Met, linker and SEQ ID NO: SEQ ID NO: described in Gin at position 16 from Ala in position 21 The fusion protein according to the above (13), which comprises an amino acid sequence;
( 1 6 ) 上記 （ 1 4) 記載の融合夕ンパク質が、 配列番号 ： 1 2の 24位の Ser から 284位の Ginに記載のァミノ酸配列からなる融合夕ンパク質 ；  (16) the fusion protein according to (14) above, wherein the fusion protein comprises the amino acid sequence of Ser at position 24 to Gin at position 284 of SEQ ID NO: 12;
( 1 7) 上記 （ 1 5 ) 記載の融合夕ンパク質が、 配列番号 ： 1 2の 1位の Metか ら 284位の Ginに記載のアミノ酸配列からなる融合タンパク質 ；  (17) the fusion protein according to (15) above, wherein the fusion protein comprises an amino acid sequence described from Met at position 1 to Gin at position 284 of SEQ ID NO: 12;
( 1 8) 上記 （ 1 2 ) から （ 1 7 ) のいずれかに記載のアミノ酸配列において 1 もしくは数個のアミノ酸が、 置換、 欠失、 挿入または付加されたアミノ酸配列を 含み、 かつケモカイン SLC活性および IL-2活性を有する融合夕ンパク質 ； ( 1 9 ) 上記 （ 1 ) から （ 1 8) のいずれかに記載の融合夕ンパク質をコ一ドす る DNA；  (18) In the amino acid sequence according to any one of (12) to (17), one or several amino acids include a substituted, deleted, inserted or added amino acid sequence, and the chemokine SLC activity And (19) a DNA encoding the fusion protein according to any one of (1) to (18) above;
( 2 0 ) 配列番号 ： 5に記載の 7 0位の aから 8 0 8位の tまでの塩基配列を含 む上記 （ 1 9 ) 記載の DNA； ( 2 1 ) 配列番号 ： 5に記載の 1位の aから 80 8位の tまでの塩基配列を含む 上記 （2 0 ) 記載の DNA； (20) SEQ ID NO: The DNA according to (19), comprising the nucleotide sequence from a at position 70 to t at position 808 described in 5; (21) the DNA of the above (20), which comprises the nucleotide sequence from a at position 1 to t at position 808 of SEQ ID NO: 5;
(2 2 ) 配列番号 ： 2 5に記載の 7 0位の aから 8 0 8位の tまでの塩基配列を 含む上記 （ 1 9 ) 記載の DNA；  (22) SEQ ID NO: The DNA according to (19), comprising the nucleotide sequence from a at position 70 to t at position 808 described in 25;
(2 3 ) 配列番号 ： 2 5に記載の 1位の aから 8 08位の tまでの塩基配列を含 む上記 （ 2 2 ) 記載の DNA；  (23) SEQ ID NO: DNA according to (22) above, comprising the nucleotide sequence from a at position 1 to t at position 808 in 25;
(24) 配列番号 ： 1 1に記載の 1位の aから 85 2位の aまでの塩基配列を含 む上記 （ 1 9 ) 記載の DNA；  (24) SEQ ID NO: DNA according to (19) above, comprising the nucleotide sequence from a at position 1 to a at position 852 of 11
(2 5 ) 配列番号 ： 1 1に記載の 1位の aから 8 5 2位の aまでの塩基配列を含 む上記 （ 24) 記載の DNA；  (25) SEQ ID NO: DNA according to (24) above, comprising the nucleotide sequence from a at position 1 to a at position 852 in 11;
(2 6) 上記 （ 1 9) から （2 5) のいずれかに記載の DNA とス ト リンジェン トな条件でハイプリダイズし、 かつケモカイン SLC活性および IL-2活性を有す る融合夕ンパク質をコードする DNA；  (26) A fusion protein that hybridizes with the DNA according to any one of (19) to (25) above under stringent conditions and has chemokine SLC activity and IL-2 activity. DNA encoding
(2 7 ) 上記 （ 1 9 ) から （2 6 のいずれかに記載の DNAを含むプラスミ ド ； (2 8) 上記 （ 1 9 ) から （ 2 6) のいずれかに記載の DNA を含む発現べクタ  (27) a plasmid containing the DNA according to any one of (19) to (26); (28) an expression vector containing the DNA according to any of (19) to (26) above. Kuta
( 2 9 ) 遺伝子治療用である上記 （28) に記載の発現べクタ一 ； (29) The expression vector according to the above (28), which is used for gene therapy;
( 3 0) 上記 （ 28) または （ 2 9) に記載の発現べクタ一を宿主に導入して得 られる形質転換体 ；  (30) a transformant obtained by introducing the expression vector according to (28) or (29) into a host;
( 3 1 ) 上記 （ 3 0 ) に記載の形質転換体を培養する工程、 および産生された上 記 （ 1 ) から （ 1 8) のいずれかに記載の融合タンパク質を培養培地から回収す る工程を包含する、 該融合タンパク質の製造方法 ；  (31) a step of culturing the transformant according to (30), and a step of collecting the produced fusion protein according to any one of (1) to (18) from the culture medium A method for producing the fusion protein;
(3 2 ) 上記 （ 1 ) から （ 1 8) のいずれかに記載の融合タンパク質を含む医薬 組成物 ；  (32) a pharmaceutical composition comprising the fusion protein according to any one of (1) to (18);
( 3 3 ) 上記 （ 2 9 ) 記載のベクタ一を用いた癌の治療方法 ；  (33) a method for treating cancer using the vector according to (29);
( 34) 上記 （ 3 2 ) 記載の医薬組成物を用いた癌の治療方法 ； および ( 3 5 ) 癌治療薬を製造するための上記 （ 3 2 ) 記載の医薬組成物の使用、 に関する。 本発明の融合夕ンパク質は、その N末端側にケモカイ ン SLCを有し、かつ IL-2 を含む融合タンパク質である。 (34) a method for treating cancer using the pharmaceutical composition according to (32); (35) Use of the pharmaceutical composition according to (32) for producing a therapeutic agent for cancer. The fusion protein of the present invention is a fusion protein having chemokine SLC on its N-terminal side and containing IL-2.
「ケモカイン SLC」 の由来は特に限定されないが、 好ましくはヒ トまたはマウ ス由来のタンパク質である。 成熟ヒ トケモカイン SLCは、 配列番号： 2の 2 4位 の Serから 1 3 4位の Proに記載のアミノ酸配列を有する夕ンパク質である。 シ グナル配列を含むヒ トケモカイン SLCは、 配列番号 ： 2の 1位の Metから 1 3 4位の Proに記載のアミノ酸配列を有するタンパク質である。 成熟マウスケモカ ィン SLCは、 配列番号： 8の 2 4位の Serから 1 3 3位の Glyに記載のアミノ酸 配列を有する夕ンパク質である。シグナル配列を含むマウスケモカイン SLCは、 配列番号： 8の 1位の Metから 1 3 3位の Glyに記載のァミノ酸配列を有する夕 ンパク質である。 「IL-2」 も、 その由来は特に限定されないが、 好ましくはヒ ト またはマウス由来の夕ンパク質である。 成熟ヒ ト IL-2は、 配列番号： 4の 2 1位 の Alaから 1 5 3位の Thrに記載のアミノ酸配列を有するタンパク質である。 シ グナル配列を含むヒ ト IL-2は、 配列番号 ： 4の 1位の Metから 1 5 3位の Thr に記載のアミノ酸配列を有するタンパク質である。 成熟マウス IL-2 は、 配列番 号： 1 0の 2 1位の Alaから 1 6 9位の Ginに記載のァミノ酸配列を有する夕ン パク質である。 シグナル配列を含むマウス IL-2は、 配列番号： 1 0の 1位の Met から 1 6 9位の Ginに記載のアミノ酸配列を有するタンパク質である。  The origin of "chemokine SLC" is not particularly limited, but is preferably a protein derived from human or mouse. Mature human chemokine SLC is a protein having an amino acid sequence described from Ser at position 24 to Pro at position 134 of SEQ ID NO: 2. Human chemokine SLC containing a signal sequence is a protein having an amino acid sequence described from Met at position 1 to Pro at position 134 in SEQ ID NO: 2. Mature mouse chemokine SLC is a protein having an amino acid sequence described in Ser from SEQ ID NO: 24 to Gly in position 133 in SEQ ID NO: 8. The murine chemokine SLC containing the signal sequence is a protein having an amino acid sequence described from Met at position 1 to Gly at position 133 in SEQ ID NO: 8. The origin of “IL-2” is also not particularly limited, but is preferably human or mouse protein. Mature human IL-2 is a protein having the amino acid sequence of Ala at position 21 of SEQ ID NO: 4 to Thr at position 153 of SEQ ID NO: 4. Human IL-2 containing a signal sequence is a protein having an amino acid sequence described from Met at position 1 to Thr at position 153 in SEQ ID NO: 4. Mature mouse IL-2 is a protein having the amino acid sequence described in Ala at position 21 of SEQ ID NO: 10 to Gin at position 169 in SEQ ID NO: 10. Mouse IL-2 containing a signal sequence is a protein having an amino acid sequence described from Met at position 1 of SEQ ID NO: 10 to Gin at position 169 of SEQ ID NO: 10.
本発明の融合夕ンパク質は、 好ましくは N末端側にケモカイン SLC を有し、 リ ンカ一及び IL-2を含む融合夕ンパク質であり、 N末端側にシグナル配列を含ん でいてもよい。 本発明の融合夕ンパク質がヒ ト由来のケモカイン SLC と IL-2を 含む場合には、 好ましくは、 配列番号 ： 6の 24位の Serから 269位の ThTに記 載のアミノ酸配列からなる融合夕ンパク質または配列番号 ： 2 6の 24 位の Ser から 269位の Thrに記載のアミノ酸配列からなる融合夕ンパク質である。 さらに 好ましくは、 配列番号： 6の 1位の Metから 269位の Thrに記載のァミノ酸配列 からなる融合タンパク質または配列番号 ： 2 6の 1位の Met から 269位の Thr に記載のアミノ酸配列からなる融合タンパク質である。 本発明の融合夕ンパク質 がマウス由来のケモカイン SLC と IL-2を含む場合には、 配列番号 ： 1 2の 2 4 位の Serから 2 8 4位の Ginに記載のアミノ酸配列からなる融合夕ンパク質であ る。 更に好ましくは、 配列番号 ： 1 2の 1位の Metから 2 8 4位の Ginに記載の アミノ酸配列からなる融合タンパク質である。 The fusion protein of the present invention preferably has a chemokine SLC on the N-terminal side, is a fusion protein containing linker and IL-2, and may contain a signal sequence on the N-terminal side. When the fusion protein of the present invention contains human-derived chemokines SLC and IL-2, a fusion consisting of the amino acid sequence described in Ser from position 24 to ThT in position 269 of SEQ ID NO: 6 is preferred. Even protein or SEQ ID NO: 26 Ser of position 24 in 6 A fusion protein consisting of the amino acid sequence of Thr from position 269 to position 269. More preferably, a fusion protein comprising the amino acid sequence described in Thr at position 269 from Met 1 at position 1 of SEQ ID NO: 6 or the amino acid sequence described in Thr at position 269 from Met at position 1 in SEQ ID NO: 26 Fusion protein. When the fusion protein of the present invention contains mouse-derived chemokines SLC and IL-2, a fusion protein consisting of the amino acid sequence of SEQ ID NO: 12 from Ser at position 24 to Gin at position 284 It is protein. More preferably, it is a fusion protein consisting of the amino acid sequence described from Met at position 1 of SEQ ID NO: 12 to Gin at position 284 of SEQ ID NO: 12.
「リンカ一」とは、 前後の夕ンパク質に対して立体障害を起さない 1以上のアミ ノ酸残基であれば特に制限されないが、 好ましくはアミノ酸数が 2 0以下のアミ ノ酸残基である。 更に好ま しくは、 グリシン、 セリンからなるアミノ酸残基であ る。  The term “linker” is not particularly limited as long as it is at least one amino acid residue that does not cause steric hindrance to the proteins before and after it. Preferably, the amino acid residue has no more than 20 amino acids. Group. More preferably, it is an amino acid residue consisting of glycine and serine.
また、 本発明の融合タンパク質には、 「配列番号 ： 6の 2 4位の Serから 2 6 9位の Thrに記載のアミノ酸配列において 1 も しくは数個のアミノ酸が、 置換、 欠失、 挿入または付加されたアミノ酸配列を含み、 かつケモカイン SLC活性およ び IL-2活性を有する融合夕ンパク質」 、 「配列番号 ： 2 6の 2 4位の Serから 2 6 9位の Thrに記載のアミノ酸配列において 1 もしくは数個のアミノ酸力 置換、 欠失、 挿入または付加されたアミノ酸配列を含み、 かつケモカイン SLC活性およ び IL-2活性を有する融合夕ンパク質」 、 「配列番号 ： 1 2の 2 4位の Serから 2 8 4位の Ginに記載のアミノ酸配列において 1 もしくは数個のアミノ酸が、 置換、 欠失、 挿入または付加されたアミノ酸配列を含み、 かつケモカイン SLC活性およ び IL-2活性を有する融合タンパク質」 も含まれる。 「アミノ酸の置換、 欠失、 挿 入又は付加」の程度及びそれらの位置等は、改変されたタンパク質が、配列番号 ： 6、 2 6または 1 2で示されるアミノ酸配列からなるタンパク質と同様にケモカ イン SLC活性および IL-2活性を有するタンパク質であれば特に制限されない。 なお、 これらアミノ酸配列の変異等は、 天然において、 例えば突然変異や翻訳後 の修飾等により生じる場合もあるが、 本発明 DNA に基づいて人為的に改変する こともできる。 本発明のタンパク質は、 このような改変 · 変異の原因 · 手段を等 を問わず、 上記特性を有する全ての改変 DNA によりコ一ドされるタンパク質を 含む。 In addition, the fusion protein of the present invention includes “one or several amino acids in the amino acid sequence described from Ser at position 24 to Thr at position 269 in SEQ ID NO: 6; substitution, deletion, or insertion. Or a fusion protein containing an added amino acid sequence and having chemokine SLC activity and IL-2 activity ”;“ SEQ ID NO: 26 to Ser at position 24 to Thr at position 26 9 "A fusion protein containing one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence and having chemokine SLC activity and IL-2 activity", "SEQ ID NO: 12" In the amino acid sequence described in Ser from position 24 to Gin in position 284, one or several amino acids include a substituted, deleted, inserted or added amino acid sequence, and the chemokine SLC activity and IL -2 fusion tamper Quality "is also included. The degree of “substitution, deletion, insertion or addition of amino acids” and their positions and the like are determined in the same manner as in the case where the modified protein is a chemocha in the same manner as the protein consisting of the amino acid sequence represented by SEQ ID NO: 6, 26 or 12. The protein is not particularly limited as long as it has a protein having SLC activity and IL-2 activity. These amino acid sequence mutations occur naturally, for example, after mutation or translation. Although it may be caused by modification of DNA, it can be artificially modified based on the DNA of the present invention. The protein of the present invention includes proteins encoded by all modified DNAs having the above-mentioned properties, regardless of the cause and means of such modification and mutation.
本発明において 「ケモカイン SLC活性」 とは、 「ケモカイン SLC特異的受容 体である CCR7 ( Mark Birkenbach et al., J. Virol., 67: 2209-2220, 1993 ) を発 現する細胞の遊走能」 を意味する。 「遊走能」 とは、 好中球、 顆粒球、 リンパ球 あるいはマク口ファ一ジなどの炎症性細胞や免疫担当細胞の血管内皮細胞への吸 着、 血管外への移動、 障害を受けた組織や抗原の存在する組織への集積を意味す る。 「IL-2活性」 とは、 「IL-2依存性細胞株の増殖能」 を意味する。 本発明の DNAとは、 「本発明の夕ンパク質をコードする DNA」 を指す。 本発 明の DNA として、 好ましくは、 配列番号 ： 6、 2 6または 1 2に記載のァミノ 酸配列からなる融合タンパク質をコードする DNA であり、 更に好ましくは、 配 列番号： 5に記載の Ί 0位の aから 8 0 8位の tまでの塩基配列を含む DNA、 酉己 列番号： 2 5に記載の 7 0位の aから 8 0 8位の tまでの塩基配列を含む DNAが 例示される。 配列番号 ： 5に記載の 1位の aから 8 0 8位の tまでの塩基配列を 含む DNA、 配列番号： 2 5に記載の 1位の aから 8 0 8位の tまでの塩基配列を 含む DNAも本発明の DNAに含まれる。 更に、 配列番号： 1 1に記載の 1位の a から 8 5 2位の aまでの塩基配列を含む上記の DNA および配列番号 ： 1 1 に記 載の 1位の aから 8 5 2位の aまでの塩基配列を含む上記の DNA も本発明の DNAに含まれる。 本発明の DNAとス ト リンジェン トな条件でハイブリダィズし、 かっかつケモカイ ン SLC活性および IL-2活性を有する夕ンパク質をコードする DNA も、 本発明の DNA に含まれる。 「DNA にス ト リ ンジェントな条件でハイ ブリダィズする DNA」 は、 コード領域の DNAをプローブとして用いることによ り得ることが出来る。 ここで、 「ス ト リンジヱントな条件でハイプリダイズする」 とは、 例えば、 6 X SSC、 0.5%SDSおよび 50%ホルムアミ ドの溶液中で 42°Cに て加温した後、 0.1xSSC、 0.5%SDS の溶液中で 68°Cにて洗浄する条件でも依然 として陽性のハイブリダイズのシグナルが観察されることを表す。 本発明の DNA を用いて、 組換えタンパク質を生産するには、 例えば、 前述の Molecular Cloning等の多くの教科書や文献に基づいて実施することができる。 具体的には、 発現させ'たい DNA の上流に翻訳閧始コ ドンを付加し、 下流には翻 訳終止コ ドンを付加する。 さらに、 転写を制御するプロモーター配列 （例えば、 trp、 lac、 T7、 SV40 初期プロモーター） 等の制御遺伝子を付加し、 適当なべク 夕一 （例えば、 pBR322、 PUC19、 pSV · SPORT1など） に組み込むことにより、 宿主細胞内で複製し、 機能する発現プラスミ ドを作製する。 本発明の DNA を組 み込んだプラスミ ドも本発明に含まれる。 本発明の DNAのべクタ一としては、本発明の DNAを組み込んだレ トロウィル スベクタ一、 アデノウイルスベクターやアデノ随伴ウィルスベクタ一なども含ま れる。 本発明のベクタ一をヒ トに投与することにより、 本発明の DNA がヒ ト体 細胞に導入され、 該体細胞は本発明の融合タンパク質を産生する。 融合タンパク 質は、 IL-2の免疫活性化作用と共にケモカイン SLCの T細胞遊走活性を有する ため、 抗癌剤として有用である。 従って、 本発明のベクタ一は、 これら疾患の治 療を目的とした遺伝子治療に用いることができる。 In the present invention, “chemokine SLC activity” refers to “migration ability of cells expressing CCR7 (Mark Birkenbach et al., J. Virol., 67: 2209-2220, 1993) that is a chemokine SLC-specific receptor”. Means "Migration ability" means that inflammatory cells such as neutrophils, granulocytes, lymphocytes, or macula phage, and cells responsible for immunocompetence adhere to vascular endothelial cells, migrate out of blood vessels, and are damaged This means accumulation in tissues or tissues where antigens are present. “IL-2 activity” means “proliferation ability of an IL-2-dependent cell line”. The DNA of the present invention refers to “DNA encoding the protein of the present invention”. The DNA of the present invention is preferably a DNA encoding a fusion protein comprising the amino acid sequence of SEQ ID NO: 6, 26 or 12, and more preferably the DNA of SEQ ID NO: 5. A DNA containing the nucleotide sequence from a at position 0 to t at position 808, and a DNA containing the nucleotide sequence from a at position 70 to t at position 808 described in SEQ ID NO: 25 Is done. SEQ ID NO: DNA containing the nucleotide sequence from a at position 1 to t at position 808 described in 5; SEQ ID NO: 25 DNA comprising the base sequence from a at position 1 to t at position 808 described in 25 Included DNA is also included in the DNA of the present invention. Further, the above DNA containing the base sequence from a at position 1 to a at position 852 in SEQ ID NO: 11 and the DNA from position a to position 852 at position 1 described in SEQ ID NO: 11 The above DNA containing the nucleotide sequence up to a is also included in the DNA of the present invention. The DNA of the present invention also includes a DNA that hybridizes with the DNA of the present invention under stringent conditions, and encodes a protein that is tight and has chemokine SLC activity and IL-2 activity. “DNA that hybridizes to DNA under stringent conditions” can be obtained by using DNA in the coding region as a probe. Here, "Hybridize under stringent conditions" This means, for example, that in a solution of 6 × SSC, 0.5% SDS and 50% formamide, the solution is heated at 42 ° C., and then washed in a 0.1 × SSC, 0.5% SDS solution at 68 ° C. This indicates that a positive hybridization signal is still observed. Production of a recombinant protein using the DNA of the present invention can be carried out, for example, based on many textbooks and documents such as the aforementioned Molecular Cloning. Specifically, a translation start codon is added upstream of the DNA to be expressed, and a translation stop codon is added downstream. Moreover, the incorporation of the promoter sequences that control transcription (e.g., trp, lac, T7, SV40 early promoter) was added to control gene, such as a suitable base click evening one (e.g., pBR322, etc. P UC19, pSV · SPORT1) This creates an expression plasmid that replicates and functions in the host cell. A plasmid incorporating the DNA of the present invention is also included in the present invention. Examples of the vector of the DNA of the present invention include a retrovirus vector, an adenovirus vector, and an adeno-associated virus vector into which the DNA of the present invention has been incorporated. By administering the vector of the present invention to a human, the DNA of the present invention is introduced into human somatic cells, which produce the fusion protein of the present invention. The fusion protein is useful as an anticancer agent because it has the T cell migration activity of the chemokine SLC together with the immunostimulatory effect of IL-2. Therefore, the vector of the present invention can be used for gene therapy for treating these diseases.
本発明の形質転換体は、 本発明のベクターを適当な宿主に導入することにより 得ることができる。 融合タンパク質の製造を目的として形質転換体を作製する場 合には、 宿主としては、 大腸菌などの原核細胞、 酵母のような単細胞真核細胞、 昆虫、 哺乳類などの多細胞生物の細胞などが挙げられる。 遺伝子治療を目的とし て形質転換体を作製する場合には、 宿主としては、 ヒ ト体細胞を用いる。 ヒ ト体 細胞としては、 患者由来の骨髄細胞、 肝細胞、 線維芽細胞、 表皮細胞、 筋肉細胞 などが挙げられる。 本発明により調製された融合夕ンパク質は、 治療目的のためにヒ トに投与しえ る。 融合タンパク質を緩衝剤、 安定剤、 静菌剤ならびに医薬の非経口投与形態に 使用される慣用的な賦形剤および添加剤に混合することにより、 医薬組成物と し て調製することができる。 従って、 本発明は、 本発明の融合タンパク質を含む医 薬組成物をも提供するものである。 図面の簡単な説明 The transformant of the present invention can be obtained by introducing the vector of the present invention into an appropriate host. When producing a transformant for the purpose of producing a fusion protein, examples of the host include prokaryotic cells such as Escherichia coli, unicellular eukaryotic cells such as yeast, and cells of multicellular organisms such as insects and mammals. Can be When producing a transformant for the purpose of gene therapy, a human somatic cell is used as a host. Human somatic cells include patient-derived bone marrow cells, hepatocytes, fibroblasts, epidermal cells, muscle cells And the like. The fusion protein prepared according to the present invention can be administered to humans for therapeutic purposes. Pharmaceutical compositions can be prepared by mixing the fusion protein with buffers, stabilizers, bacteriostats and conventional excipients and additives used in parenteral pharmaceutical dosage forms. Therefore, the present invention also provides a pharmaceutical composition containing the fusion protein of the present invention. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 マウス SLC ( mSLC) またはマウス IL-2 ( mIL-2) 断片增幅用プライ マ一セッ トを示す図である。  FIG. 1 is a diagram showing a primer set for mouse SLC (mSLC) or mouse IL-2 (mIL-2) fragment / width.
図 2は、 mSLC-mIL-2融合遺伝子導入用レ トロウィルスベクタ一プラスミ ドの 作製方法を示した図である。  FIG. 2 is a diagram showing a method for producing a retrovirus vector plasmid for transfection of an mSLC-mIL-2 fusion gene.
図 3は、 レ トロウィルスベクタ一 pLHDCXからレ トロウィルスベクタ一 pLX— IRES - EGFPの作製方法を示した図である。  FIG. 3 is a diagram showing a method for producing a retrovirus vector pLX-IRES-EGFP from a retrovirus vector pLHDCX.
図 4は、 遊走細胞数を示した図である。  FIG. 4 is a diagram showing the number of migrated cells.
図 5は、 CD4陽性 T細胞の增加数を示した図である。  FIG. 5 is a diagram showing the additive number of CD4-positive T cells.
図 6は、 CD8陽性 T細胞の增加数を示した図である。  FIG. 6 is a diagram showing the additive number of CD8-positive T cells.
図 7は、 mSLCによる腫瘍形成抑制効果を示した図である。  FIG. 7 is a diagram showing the tumor formation inhibitory effect of mSLC.
図 8は、 mIL2及び mSLC-IL2による腫瘍形成抑制効果を示した図である。 図 9は、 ヒ ト SLC (hSLC) —ヒ ト IL-2 ( hIL-2) 融合遺伝子導入用レ トロウイ ルスべクタ一プラスミ ドの作製方法を示した図である。  FIG. 8 is a diagram showing the tumor formation inhibitory effect of mIL2 and mSLC-IL2. FIG. 9 is a diagram illustrating a method for preparing a retroviral vector for transfection of a human SLC (hSLC) -human IL-2 (hIL-2) fusion gene.
図 1 0は、 mSLC、 mIL-2及び mSLC-mIL-2の単独、 または mSLC と mIL-2 の併用による腫瘍形成抑制効果を示した図である。 発明を実施するための最良の形態 本発明は、 おもに新規融合タンパク質に関する。 FIG. 10 is a diagram showing the tumor formation inhibitory effect of mSLC, mIL-2 and mSLC-mIL-2 alone or in combination with mSLC and mIL-2. BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates mainly to novel fusion proteins.
以下に本発明 DNA の調製、 本発明融合タンパク質の調製、 活性の測定方法、 遺 伝子治療用ベクター、 医薬組成物について説明する。 本明細書において、 特に指 示のない限り、 当該分野で公知である遺伝子組換え技術、 動物細胞、 昆虫細胞、 酵母および大腸菌での組換えタンパク質の生産技術、 発現したタンパク質の分離 精製法、 分析法および免疫学的手法が採用される。 本発明融合夕ンパク質をコードする DNA配列 The preparation of the DNA of the present invention, the preparation of the fusion protein of the present invention, the method for measuring the activity, the vector for gene therapy, and the pharmaceutical composition are described below. In the present specification, unless otherwise specified, unless otherwise indicated, a gene recombination technique known in the art, a technique for producing a recombinant protein in animal cells, insect cells, yeast and Escherichia coli, a method for separating and purifying the expressed protein, and an analysis method Methods and immunological techniques are employed. DNA sequence encoding the fusion protein of the present invention
本発明において用いられるヒ ト CC 型ケモカイン SLC 及びヒ トサイ トカイン IL-2をコードする塩基配列を有する遺伝子は、 本発明により教示された配列情報 (配列番号 ： 1及び配列番号 ： 2 ) に基づいて一般的遺伝子工学的手法により容 易に製造 ·取得することができる（Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989)等参照）。 具体的にはケモカイン SLCや IL-2が発現される適当 な起源より、 常法に従って cDNAライブラリ一を調製し、 該ライブラリ一から本 発明 DNA に特有の適当なプローブや抗体を用いて所望のクローンを選択するこ とにより実施できる (Proc. Natl. Acad. Sci.， USA., 78, 6613 (1981); Science, 22, 778 (1983)等参照）。 cDNAの起源としては、 本発明の DNAを発現する各種の細 胞、 組織やこれらに由来する培養細胞等が例示される。 これらからの全 RNA の 分離、 mRNAの分離 .精製、 cDNAの取得とそのクローニング等はいずれも常法 に従い実施できる。 また、 cDNA ライブラリ一は市販されており、 本発明におい てはそれら cDNAライブラ リー、 例えば Clontech社より市販の各種 cDNAラィ ブラリ一等を用いることもできる。 cDNA ライブラ リ一としては、 ヒ トひ臓節由 来の cDNAライブラリーが例示できる。  The gene having the nucleotide sequence encoding human CC-type chemokine SLC and human cytokine IL-2 used in the present invention is based on the sequence information (SEQ ID NO: 1 and SEQ ID NO: 2) taught by the present invention. It can be easily manufactured and obtained by general genetic engineering techniques (see, for example, Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989)). Specifically, a cDNA library is prepared from a suitable source in which the chemokines SLC and IL-2 are expressed according to a conventional method, and a desired clone is prepared from the library using an appropriate probe or antibody specific to the DNA of the present invention. (Proc. Natl. Acad. Sci., USA., 78, 6613 (1981); Science, 22, 778 (1983), etc.). Examples of the origin of cDNA include various cells and tissues expressing the DNA of the present invention, and cultured cells derived therefrom. Isolation of total RNA, isolation and purification of mRNA, acquisition of cDNA, and cloning of cDNA can be performed according to a conventional method. In addition, cDNA libraries are commercially available, and in the present invention, those cDNA libraries, for example, various cDNA libraries available from Clontech can also be used. An example of the cDNA library is a cDNA library derived from a human spleen node.
また、 IL-2 およびケモカイン SLC をコードする塩基配列を有する遺伝子は、 慣用の化学的方法、 例えばリ ン酸三エステル法 （Narang et al., Meth. Enzymol., 68, 90- 108 (1979))またはリン酸ニエステル法（ Brown et al., Meth. Enzymol., 68, 109- 151 (1979)) により合成され得る。 In addition, genes having nucleotide sequences encoding IL-2 and chemokine SLC can be obtained by conventional chemical methods such as the phosphoric acid triester method (Narang et al., Meth. Enzymol., 68, 90-108 (1979)). ) Or the phosphate diester method (Brown et al., Meth. Enzymol., 68, 109-151 (1979)).
上記のケモカイン SLC をコ一ドする塩基配列と IL2 の成熟型夕ンパク質をコ ードする塩基配列を通常の方法により接続し、 単一の遺伝子とする。 これにより、 CC型ケモカイン SLCの有する CCR7発現細胞株に対する遊走活性および サイ 卜力イン IL2の有する T 細胞の増殖 ·活性化能等を同一分子内に有する人工融合 タンパク質遺伝子を構築される。 該遺伝子は、 SLC をコードする遺伝子と IL-2 をコードする遺伝子との間にリ ンカ一をコードする遺伝子を有していてもよい。 本発明融合タンパク質の調製  The above-described nucleotide sequence encoding the chemokine SLC and the nucleotide sequence encoding the mature IL2 protein are connected by a conventional method to form a single gene. As a result, an artificial fusion protein gene having, in the same molecule, the migratory activity against the CCR7-expressing cell line possessed by the CC-type chemokine SLC and the proliferation / activation ability of the T cell possessed by the cytotoxic IL2 is constructed. The gene may have a gene encoding a linker between the gene encoding SLC and the gene encoding IL-2. Preparation of the fusion protein of the present invention
( 1 ) 融合タンパク質の発現  (1) Expression of fusion protein
本発明のタンパク質は、 本発明の DNA配列情報に従って、 遺伝子工学的手法 (Science, 224, 1431 (1984); Biochem. Biophys. Res. Comm., 130， 692 (1985); Proc. Natl. Acad. Sci., USA., 80， 5990 (1983)等）により得ることができる。 より 詳細には、 所望のタンパク質をコ一ドする遺伝子を適当なベクターに組み込む。 このべク夕一を宿主細胞に導入して形質転換体を作成する。 該形質転換体を培養 することにより組換えタンパク質を得ることができる。  The protein of the present invention can be obtained by a genetic engineering technique (Science, 224, 1431 (1984); Biochem. Biophys. Res. Comm., 130, 692 (1985); Proc. Natl. Acad. Sci., USA., 80, 5990 (1983)). More specifically, a gene encoding a desired protein is inserted into an appropriate vector. This vector is introduced into a host cell to prepare a transformant. By culturing the transformant, a recombinant protein can be obtained.
ここで宿主細胞としては、 真核生物及び原核生物のいずれも用いることができ る。 該真核生物の細胞には、 脊椎動物、 酵母等の細胞が含まれ、 脊椎動物細胞と しては、 例えばサルの細胞である COS細胞（Cell, 23, 175 (1981))やチヤィニー ズ · ハムスター卵巣細胞等がよく利用される。  Here, any of eukaryotes and prokaryotes can be used as host cells. The eukaryotic cells include cells such as vertebrates and yeasts. Examples of the vertebrate cells include COS cells (Cell, 23, 175 (1981)) which are monkey cells and Chinese vertebrate cells. Hamster ovary cells are often used.
発現ベクターとしては、 通常発現しょうとする遺伝子の上流に位置するプロモ 一ター、 RNAのスプライス部位、 ポリアデニル化部位及び転写終了配列等を保有 するものを使用でき、 これは更に必要により複製起点を有していても良い。 該発 現べクタ一の例と しては、 例えば、 SV40 の初期プロモータ一を保有する pSV2dhfr(Mol. Cell. Biol., 1， 854 (1981))等を例示できる。 また、 真核微生物と しては、 酵母が一般によく用いられ、 中でもサッカロミセス属酵母を利用できる。 該酵母の発現ベクターとしては、 例えば酸性ホスファターゼ遺伝子に対するプロ モータ一を有する pAM82(Proc. Natl. Acad. Sci., USA., 80, 1 (1983))等を利用で きる。 原核生物の宿主としては、 大腸菌や枯草菌が一般によく利用される。 これらを 宿主とする場合、 例えば該宿主菌中で複製が可能なプラスミ ドベクターを用い、 このべクタ一中に所望の遺伝子が発現できるように該遺伝子の上流にプロモ一夕 —及び SD配列、 更に蛋白合成開始に必要な閧始コ ドンを付与した発現プラスミ ドを利用するのが好ましい。 更に、 大腸菌等で発現される場合、 シグナル配列は 認識されないので、 N末端の配列に保持された成熟型配列を得る為の工夫が必要 である。 その例としては、 閧始コ ドンと該遺伝子の成熟型配列のコード領域との 間にェンテロ力イネ一スの認識配列 Asp Asp Asp Asp Lys (配列番号 ： 2 1 ) を、 成熟型配列の直前に Lysが続くように挿入し、 得られた組換え夕ンパク質をェン テロ力イネ一ス （Invitrogen社製） にて消化することによって N末端の配列の配 列の保持された該融合タンパク質を得ることができる。上記宿主としては、 E . coli K12株等が利用される。 ベクターとしては一般に pBR322及びその改良べクタ一 がよく利用されるが、 これらに限定されず公知の各種の菌株及びベクターも利用 できる。 プロモ一夕一としては、 例えば trpプロモータ一、 lppプロモーター、 lac プロモータ一、 PL/PRプロモーター等を使用できる。 所望の組換え DNA の宿主細胞への導入方法及びこれによる形質転換方法とし ては、 一般的な各種方法を採用できる。 また得られる形質転換体は、 常法に従い 培養でき、 該培養により所望のタンパク質が産生される。 該培養に用いられる培 地としては、 宿主細胞に応じて慣用される各種のものを適宜選択利用でき、 その 培養も宿主細胞に適した条件下で実施できる。 例えば、 pSVL SV40後期プロモ一 ターの下流に本発明融合タンパク質の遺伝子を含むベクタ一を、 サル由来細胞 COS-7に導入することによって形質転換体を作成し、 この形質転換体を 5% C02 存在下、 37°Cで 3日間培養することにより、 本発明の融合タンパク質が産生され 得る。 タ ンパク質は、 その物理的性質、 化学的性質等を利用 した各種の分離操作 (Biochemistry, 25(25), 8274 (1986); Eur. J. Biochem., 163, 313 (1987)等）により 分離 ·精製できる。 該方法としては、 塩析法、 遠心分離、 浸透圧ショック法、 超 音波破砕、 限外濾過、 ゲル濾過、 吸着クロマトグラフィー、 イオン交換クロマ ト グラフィ一、 ァフィ二ティ一クロマ トグラフィー、 高速液体クロマ トグラフィー 等の各種液体クロマトグラフィー、 透析法、 これらの組み合わせ等を例示できる。 As an expression vector, a vector having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like, which is usually located upstream of a gene to be expressed, can be used. May be. An example of such an expression vector is pSV2dhfr (Mol. Cell. Biol., 1, 854 (1981)), which has an initial promoter of SV40. As eukaryotic microorganisms, yeasts are generally used, and Saccharomyces yeasts can be used. As an expression vector for the yeast, for example, pAM82 (Proc. Natl. Acad. Sci., USA., 80, 1 (1983)) having a promoter for the acid phosphatase gene can be used. Escherichia coli and Bacillus subtilis are commonly used as prokaryotic hosts. When these are used as a host, for example, a plasmid vector capable of replication in the host bacterium is used, and a promoter sequence and an SD sequence are arranged upstream of the gene so that the desired gene can be expressed in the vector. Further, it is preferable to use an expression plasmid to which a start codon necessary for initiation of protein synthesis has been added. Furthermore, when expressed in E. coli or the like, since the signal sequence is not recognized, it is necessary to devise a method for obtaining a mature sequence retained at the N-terminal sequence. As an example, the recognition sequence of Asp Asp Asp Asp Lys (SEQ ID NO: 21) is inserted between the initiation codon and the coding region of the mature sequence of the gene immediately before the mature sequence. And the resulting recombinant protein is digested with enteroforce rice (manufactured by Invitrogen) to obtain the fusion protein in which the sequence of the N-terminal sequence is retained. Can be obtained. As the above host, E. coli K12 strain or the like is used. In general, pBR322 and its improved vector are often used as vectors, but not limited thereto, and various known strains and vectors can also be used. As the promoter, for example, trp promoter, lpp promoter, lac promoter, PL / PR promoter and the like can be used. As a method for introducing a desired recombinant DNA into a host cell and a transformation method using the same, various general methods can be adopted. The obtained transformant can be cultured according to a conventional method, and the desired protein is produced by the culture. As the medium used for the culture, various types commonly used depending on the host cell can be appropriately selected and used, and the culture can be carried out under conditions suitable for the host cell. For example, a vector containing the gene of the fusion protein of the present invention downstream of the pSVL SV40 late promoter is transferred to a monkey-derived cell. Create a transformant by introducing into COS-7, the transformants 5% C0 2 presence by culturing for 3 days at 37 ° C, fusion proteins of the present invention can be produced. Proteins are separated by various separation procedures using their physical and chemical properties (Biochemistry, 25 (25), 8274 (1986); Eur. J. Biochem., 163, 313 (1987), etc.). Can be separated and purified. Examples of the method include salting out, centrifugation, osmotic shock, sonication, ultrafiltration, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, and high performance liquid chromatography. Examples include various types of liquid chromatography such as chromatography, dialysis, and combinations thereof.
( 2 ) 変異体の作製 (2) Preparation of mutant
アミノ酸配列は、 任意のアミノ酸配列を欠失させ、 所望のアミノ酸、 ないしは ァミノ酸配列を導入することによって置換される。 ァミノ酸配列の置換処理には、 プロテインエンジニアリングとして知られる方法が広く利用できるが、 例えば、 Site-diredted deletion (部位指定削除） 法 ( Nucl. Acids Res., 11, 1645, 1983) Site-specific mutagenesis (部位特異的変異） 法 （Zoller, M. J. et al., Methods in Enzymol., 100， 468, 1983、 Kunkel. T.A. et al., Methods in Enzymol., 154, 367-382, 1987) 、 PCR 突然変異生成法、 制限酵素処理と合成遺伝子の利用によ る方法等がある。  The amino acid sequence is replaced by deleting any amino acid sequence and introducing a desired amino acid or amino acid sequence. A method known as protein engineering can be widely used for the substitution treatment of the amino acid sequence. For example, a site-diredted deletion method (Nucl. Acids Res., 11, 1645, 1983) Site-specific mutagenesis (Site-specific mutation) method (Zoller, MJ et al., Methods in Enzymol., 100, 468, 1983, Kunkel. TA et al., Methods in Enzymol., 154, 367-382, 1987), PCR mutation There are a production method, a method using restriction enzyme treatment, and a method using a synthetic gene.
部位特異的変異法であれば、 例えば Molecuar Cloning: A Laboratory Manual 第 2版第 1 - 3卷 Sambrook, J.ら著、 Cold Spring Harber Laboratory Press出 版 New York 1989年に記載の部位特異的変異誘発法や PCR法などの方法を用い、 本発明の DNA配列に変異を導入する。  For site-directed mutagenesis, for example, site-directed mutagenesis described in Molecuar Cloning: A Laboratory Manual, 2nd edition, Vol. 1-3, Sambrook, J. et al., Cold Spring Harber Laboratory Press, New York, 1989 A mutation is introduced into the DNA sequence of the present invention by using a method such as PCR or PCR.
これら方法により変異が導入された DNA配列は、 適当なベクターおよび宿主 系を用いて、 例えば Molecuar Cloning: A Laboratory Manual第 2版第 1 一 3卷 Sambrook, J.ら著、 Cold Spring Harber Laboratory Press出版 New York 1989 年に記載の方法により、 遺伝子工学的に発現させればよい。 例えば、 Mutan TM -SuperExpress Km, Mutan TM _K (宝酒造社製） 、 Quik Change Site- Directed Mutagenesis Kit ( Stratagene社製） といったキッ トが使用できる。 一般に、 部位特異的変異法は、 まず、 タンパク質をコードする DNA 配列をそ の配列中に含む一本鎖ベクターを得ることによって実施することができる。 所望 の突然変異した配列を持つォリゴヌクレオチドプライマ一を、 一般的には合成に よって、 例えばクレア等 （Crea，： R. et al., Proc. Natl. Acsd. Sci. U.S.A., 75, 5765, 1978) の方法によって製造する。 次に、 このプライマ一を一本鎖の本 DNA配列 含有ベクターとァニ一リングし、 大腸菌ポリメラーゼ I クレノウフラグメン トの ような DNA重合酵素を作用させて、 突然変異含有鎖の合成を完成する。 このよ うにして、 第一の鎖は元の非突然変異配列をコードしており、 第二の鎖は所望の 突然変異を有しているへテロ二本鎖が形成される。 次いで、 この二本鎖ベクター を用いて、 適当な細菌、 または細胞を形質転換し、 32 P—標識突然変異生成プライ マ一から成る放射性プローブへのハイブリダィゼ一シヨンを介してクロ一ンを選 択する （Wallace, R.B., Nucleic Acids Res., 9, 3647, 1981 ) 。 選択されたクロー ンには、 突然変異した配列を有する組換えべクタ一を含んでいる。 このようなク ローンを選択した後、 突然変異した本夕ンパク質の領域を形質転換に使用される 型の発現ベクターに入れることができる。 The DNA sequence into which the mutation has been introduced by these methods can be prepared, for example, by using an appropriate vector and host system, for example, Molecuar Cloning: A Laboratory Manual, 2nd edition, Vol. It may be expressed genetically by the method described in Sambrook, J. et al., Cold Spring Harber Laboratory Press, New York, 1989. For example, kits such as Mutan ™ -SuperExpress Km, Mutan ™ _K (manufactured by Takara Shuzo) and Quik Change Site-Directed Mutagenesis Kit (manufactured by Stratagene) can be used. In general, the site-directed mutagenesis can be performed by first obtaining a single-stranded vector containing a DNA sequence encoding a protein in the sequence. Oligonucleotide primers having the desired mutated sequence are generally synthesized, for example, by Clair et al. (Crea, R. et al., Proc. Natl. Acsd. Sci. USA, 75, 5765, 1978). Next, the primer is annealed with a single-stranded vector containing the DNA sequence, and a DNA polymerase such as Escherichia coli polymerase I Klenow fragment is allowed to act on the primer to complete the synthesis of the mutation-containing strand. . In this way, the first strand encodes the original non-mutated sequence and the second strand forms a heteroduplex with the desired mutation. This double-stranded vector is then used to transform appropriate bacteria or cells, and clones are selected via hybridization to a radioactive probe consisting of a 32 P-labeled mutagenic primer. (Wallace, RB, Nucleic Acids Res., 9, 3647, 1981). The selected clone contains a recombinant vector with the mutated sequence. After selecting such clones, the mutated protein region can be placed into an expression vector of the type used for transformation.
以降、 （ 1 ) で示した組換えタンパク質の調整方法に従い、 変異体を宿主細胞 に産生させることができる。 ケモカイン SLC活性の測定  Thereafter, a mutant can be produced in a host cell according to the method for preparing a recombinant protein described in (1). Chemokine SLC activity measurement
ケモカイン SLCは、 T細胞および B細胞に対して遊走活性を有する。 従って、 これら細胞を遊走アツセィ用緩衝液に懸濁し、 遺伝子産物の添加により生じる遊 走細胞の数を測定することにより、ケモカイン SLC活性を測定することができる。 具体的には、 遺伝子産物を遊走アツセィ用緩衝液に添加する。 細胞数測定後のChemokine SLC has chemotactic activity on T cells and B cells. Therefore, these cells are suspended in a migration assay buffer, and the By measuring the number of chemotactic cells, the chemokine SLC activity can be measured. Specifically, the gene product is added to a buffer for migration assay. After cell count
T細胞または B細胞、 このましくは、 ケモカイン SLC受容体 （CCR7) を発現す る T細胞または B細胞を緩衝液に懸濁する。 T cells or B cells, preferably T cells or B cells expressing the chemokine SLC receptor (CCR7), are suspended in a buffer.
トランスウエルチヤンバーの上部のゥヱルに細胞を含んだ緩衝液を接種し、 下 部のゥエルには、 細胞を含まない遊走アツセィ用緩衝液を加え、 37°C 5%C02 下で 4時間培養する。  Inoculate the buffer containing cells into the upper well of the transwell chamber, add the cell-free migration buffer to the lower well, and incubate at 37 ° C 5% C02 for 4 hours. .
下部のゥエルに遊走した細胞を回収し、 細胞数を測定することにより遺伝子産物 の遊走活性を測定することが可能である。 It is possible to measure the migration activity of the gene product by collecting the cells that have migrated to the lower well and counting the number of cells.
IL-2活性の測定 Measurement of IL-2 activity
1L-2活性は通常、 IL-2依存的に増殖する T細胞を用いるバイオアツセィでおこ なわれる。 ヒ ト IL-2の生物活性を測定するシステムは確立している （基礎と臨床 佐々木緊ら Vol . 22， No . 17, 29-42 1988) 。 従って、 該システムによって遺伝 子産物の IL-2生物活性の測定することができる。 本発明の遺伝子治療用ベクターの作成  1L-2 activity is usually performed in a bioassay using T cells that proliferate in an IL-2-dependent manner. A system for measuring the biological activity of human IL-2 has been established (Basic and clinical Tatsu Sasaki et al. Vol. 22, No. 17, 29-42 1988). Therefore, the IL-2 biological activity of the gene product can be measured by the system. Preparation of the vector for gene therapy of the present invention
本発明融合夕ンパク質を遺伝子治療に使用するためのベクターとしては、 レ ト ロウィルスべクタ一 （M u M L V骨格の物、 HIV骨格の物） 、 アデノウイルスべ クタ一、 アデノ随伴ウィルス （AAV) ベクタ一等が考えられる。 遺伝子銃やイン ビボ . エレク ト口ポレーション法を用いれば本発明 DNA を組み込んだプラスミ ドを用いて遺伝子治療を行うことも可能である。  Vectors for using the fusion protein of the present invention for gene therapy include retrovirus vector (Mu MLV skeleton, HIV skeleton), adenovirus vector, and adeno-associated virus (AAV). Vectors and the like are conceivable. Gene therapy can also be performed using a plasmid incorporating the DNA of the present invention by using a gene gun or in vivo electroporation.
動物細胞内で発現させるためのプロモ一夕一としては、 通常の動物培養細胞発 現系において用いられるプロモーターであれば特に制限されるものではないが、 たとえばサイ トメガロウィルス初期プロモータ一 （以下 CMV プロモーターと記 す。 ） 、 MuMLV LTR等を上げることが出来る。 なお、 CMVプロモータ一は、 例えば pRC/CMV(Invitrogen社製） から、 通常の遺伝子操作により調製すること ができる。 The promoter for expression in animal cells is not particularly limited as long as it is a promoter used in a normal animal cell culture expression system. For example, a cytomegalovirus early promoter (CMV) Promoter.), MuMLV LTR, etc. can be raised. The CMV promoter is For example, it can be prepared from pRC / CMV (manufactured by Invitrogen) by ordinary genetic manipulation.
上記の動物細胞内で発現させるためのプロモーターおよび前記の人工融合夕ン パク質遺伝子を含むプラスミ ドは通常の遺伝子組み換え方法を用いて構築するこ とが出来る。 例えば、 本発明の融合タンパク質をコードする遺伝子を CMV プロ モータ一とゥシ成長ホルモンのポリ Aシグナル、 ネオマイシン耐性遺伝子を保有 する pRC/CMV(Invitrogen社製）の Hindlll部位に揷入することにより構築する 方法等をあげることができる。  The above-described plasmid containing the promoter for expression in animal cells and the above-mentioned artificial fusion protein gene can be constructed using a conventional gene recombination method. For example, the gene encoding the fusion protein of the present invention is constructed by inserting it into the Hindlll site of pRC / CMV (manufactured by Invitrogen), which contains the CMV promoter, the poly (A) signal of pacific growth hormone, and the neomycin resistance gene. And other methods.
本発明遺伝子治療用ベクターを用いた遺伝子治療は、ヒ ト体細胞に本発明 DNA を獲得させ、 この組換え細胞を患者に戻すことにより、 あるいは本発明遺伝子治 療用ベクタ一を直接患者の患部に投与することができる。  Gene therapy using the gene therapy vector of the present invention can be performed by allowing human somatic cells to acquire the DNA of the present invention and returning the recombinant cells to the patient, or by directly transferring the vector for gene therapy of the present invention to the affected part of the patient Can be administered.
遺伝子治療用べクタ一をヒ ト体細胞に導入する方法としては、 ベクターがブラ スミ ドの場合には、 遺伝子銃、 マイクロインジェクション、 トランスフエクショ ンまたはトランスダクシヨンによりべクタ一を体細胞へ導入することができる。 ベクタ一がウィルスの場合には、 体細胞に本発明遺伝子を組み込んだウィルスを 感染させることで本発明 DNAを導入することができる。  As a method for introducing a vector for gene therapy into human somatic cells, when the vector is a plasmid, the vector is introduced into somatic cells by a gene gun, microinjection, transfection or transduction. Can be introduced. When the vector is a virus, the DNA of the present invention can be introduced by infecting somatic cells with a virus incorporating the gene of the present invention.
体細胞としては、 患者由来の骨髄細胞、 肝細胞、 線維芽細胞、 表皮細胞、 筋肉 細胞などが挙げられる。 本発明の医薬組成物  Examples of somatic cells include bone marrow cells, hepatocytes, fibroblasts, epidermal cells, and muscle cells derived from patients. Pharmaceutical composition of the present invention
本発明の融合夕ンパク質は、 IL-2の免疫活性化作用と共にケモカイン SLCの T 細胞遊走活性を有するため、 抗癌剤として有用である。  The fusion protein of the present invention is useful as an anticancer agent because it has a T cell migration activity of chemokine SLC together with an immunostimulatory effect of IL-2.
該タンパク質には、 医薬的に許容される塩もまた包含される。 かかる塩には、 周知の方法により調整される、 例えばナト リウム、 カリウム、 リチウム、 カルシ ゥム、 マグネシウム、 ノ リウム、 アンモニゥム等の無毒性アルカリ金属塩、 アル 力リ土類金属塩及びアンモニゥム塩等が包含される。 該タンパク質の薬学的有効量を活性成分として、 医薬製剤が調整される。 該医 薬製剤の投与単位形態としては、 各種の形態が治療目的に応じて選択でき、 その 代表的なものとしては、 錠剤、 丸剤、 散剤、 粉末剤、 顆粒剤、 カプセル剤等の個 体投与形態や、 液剤、 懸濁液剤、 乳剤、 シロップ、 エリキシル等の液剤投与形態 が含まれる。 これらは更に、 投与経路に応じて経口剤、 非経口剤、 経鼻剤、 経膣 剤、 坐剤、 舌下剤、 軟膏剤等に分類され、 それぞれ通常の方法に従い、 調合、 成 形もしくは調製することができる。 The proteins also include pharmaceutically acceptable salts. Such salts may be prepared by known methods, for example, non-toxic alkali metal salts such as sodium, potassium, lithium, calcium, magnesium, norium, and ammonium, alkaline earth metal salts, and ammonium salts. Is included. A pharmaceutical preparation is prepared using a pharmaceutically effective amount of the protein as an active ingredient. As the dosage unit form of the pharmaceutical preparation, various forms can be selected according to the purpose of treatment, and typical examples are individual forms such as tablets, pills, powders, powders, granules, capsules and the like. It includes dosage forms and liquid dosage forms such as solutions, suspensions, emulsions, syrups, and elixirs. These are further classified into oral preparations, parenteral preparations, nasal preparations, vaginal preparations, suppositories, sublingual preparations, ointments, etc. according to the route of administration, and are prepared, formulated or prepared according to the usual methods. be able to.
上記医薬製剤の投与方法は、 特に制限がなく、 各種製剤形態、 患者の年齢、 性 別その他の条件、 疾患の程度等に応じて決定される。 例えば、 腚剤、 丸剤、 顆粒 剤、 カプセル剤等の個体投与形態や、 液剤、 懸濁液剤、 乳剤は経口投与される。 注射剤は単独又はブドウ糖やアミノ酸等の通常の補液と混合して静脈投与される c 更に注射剤は、 必要に応じて単独で筋肉内、 皮内、 皮下もしくは腹腔内投与され る。 The administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, age, sex and other conditions of the patient, degree of disease, and the like. For example, solid dosage forms such as tablets, pills, granules, capsules and the like, liquids, suspensions, and emulsions are orally administered. The injection is administered intravenously, alone or mixed with a normal replenisher such as glucose or amino acids. C The injection is administered intramuscularly, intradermally, subcutaneously, or intraperitoneally as needed.
上記医薬製剤中に含有されるべき本発明化合物の有効成分量およびその投与量 は、 特に限定されず、 所望の治療効果、 投与方法、 治療期間、 患者の年齢、 性別 その他の条件に応じて適宜選択される。 一般的には、 該投与量は、 1 日当たり体 重 l k g当たり、 約 l〜 1 0 m g程度とするのがよく、 該製剤は 1 日に 1〜数回 に分けて投与することができる。 実施例  The amount of the active ingredient of the compound of the present invention to be contained in the above pharmaceutical preparation and the dose thereof are not particularly limited, and are appropriately determined according to the desired therapeutic effect, administration method, treatment period, patient age, sex, and other conditions. Selected. In general, the dose is preferably about l to 10 mg per 1 kg of body weight per day, and the preparation can be administered once or several times a day. Example
本発明を以下の実施例によりさらに説明する。  The present invention is further described by the following examples.
実施例 1 Example 1
マウス SLC-マウス IL-2融合夕ンパク質遺伝子の構築 Construction of mouse SLC-mouse IL-2 fusion protein gene
マウス SLC ( mSLC) 遺伝子 （配列番号 ： Ί ) の終止コ ドンを含まず 3'末端に Xba I 部位を有するようなマウス SLC 遺伝子断片を得るために、 プラス ミ ド pT7-T3-D-Pac-mSLC ( EST:クローン番号 W67046、 Genome Svstems社より購 入） を鍩型として、 図 1 ( 1 ) に示したプライマー （mSLC-Sall-F (配列番号 ： 13) 及び mSLC-Xbal-R (配列番号 ： 14) ) を用いて、 約 0.4 k bの断片を PCR 法により増幅した。 得られた断片を、 Sal I 及び Xba I で同時切断して、 Blue Script (+) (Stratagene 社）の Sal I、 Xba I 部位に挿入 し、 プラス ミ ド pBS- mSLC(S/X)を構築した。 サブクローン化したものの塩基配列を決定し、 PCR法に よる変異のないことを確認した。 In order to obtain a mouse SLC gene fragment that does not include the termination codon of the mouse SLC (mSLC) gene (SEQ ID NO: Ί) and has an XbaI site at the 3 ′ end, plasmid pT7-T3-D-Pac- mSLC (EST: clone number W67046, purchased from Genome Svstems) Using the primers shown in FIG. 1 (1) (mSLC-Sall-F (SEQ ID NO: 13) and mSLC-Xbal-R (SEQ ID NO: 14)) as a type I fragment, a fragment of about 0.4 kb was used. Was amplified by the PCR method. The obtained fragment was simultaneously digested with Sal I and Xba I and inserted into the Sal I and Xba I sites of Blue Script (+) (Stratagene) to construct plasmid pBS-mSLC (S / X). did. The nucleotide sequence of the subcloned product was determined, and it was confirmed that there was no mutation due to the PCR method.
マウス IL-2 ( mIL-2) の成熟型配列 （配列番号 ： 10) をコードする遺伝子断片 を得るために、 mIL-2 の cDNA (配列番号 ： 9 )を有 しているプラス ミ ド Okayama-Berg-MuIL2を鈸型として、図 1 (2)に示したプライマー（mIL2-Xba l-F (配列番号 ： 15) 及び mIL2-Notl-R (配列番号 16) ) を用いて、 約 0.45 k bの 断片を PCR法により增幅した。 得られた断片は Xba I及び Not Iで同時消化し、 市販のベクタ一 Blue Script(+)の Xba I、 Not I部位にサブクローン化し、 プラス ミ ド pBS-mIL2(X/N)を構築した。 クローン化したものの塩基配列を決定し、 PCR 法による変異のないことを確認した。  In order to obtain a gene fragment encoding the mature sequence of mouse IL-2 (mIL-2) (SEQ ID NO: 10), a plasmid containing the mIL-2 cDNA (SEQ ID NO: 9), Okayama- Using the primers shown in Fig. 1 (2) (mIL2-XbalF (SEQ ID NO: 15) and mIL2-Notl-R (SEQ ID NO: 16)) with Berg-MuIL2 as type I, an approximately 0.45 kb fragment was It was amplified by PCR. The obtained fragment was co-digested with XbaI and NotI, subcloned into the XbaI and NotI sites of the commercially available vector BlueScript (+), and constructed plasmid pBS-mIL2 (X / N). . The nucleotide sequence of the clone was determined, and it was confirmed that there was no mutation by PCR.
pBS-mSLC(S/X)を Sal I及び Xba Iで同時切断して、 得られる 0.4 k bの断片 を、 pBS-mIL2(X/N)の Sal I、 Xba I部位に挿入し、 結果としてマウス SLC-マウ ス IL-2融合夕ンパク質遺伝子を Sal I と Not I サイ 卜の間に持つ、 プラス ミ ド PBS-mSLC-IL2 が得られた （図 2) 。 人工融合タンパク質遺伝子 （mSLC-IL-2) は、 8 5 5塩基対からなり、 その構造は 5'末端からマウス SLCをコードする 399 塩基、 リンカ一をコードする配列 6塩基、 マウス IL-2の N末端 21番目から C末 端までの 1 4 9アミノ酸残基をコードする 4 4 7塩基と終止コ ドンからなる（配 列番号 ： 11)。 実施例 2 pBS-mSLC (S / X) is simultaneously digested with Sal I and Xba I, and the resulting 0.4 kb fragment is inserted into the Sal I and Xba I sites of pBS-mIL2 (X / N), resulting in a mouse SLC- the mouse IL2 fusion evening protein gene having between Sal I and Not I sites Bok, plus Mi de P BS-mSLC-IL2 were obtained (Figure 2). The artificial fusion protein gene (mSLC-IL-2) consists of 855 base pairs, and its structure is 399 bases encoding mouse SLC from the 5 'end, 6 bases encoding the linker, 6 bases of mouse IL-2 It consists of 445 bases encoding the 149 amino acid residues from the 21st N-terminal to the C-terminal and a termination codon (SEQ ID NO: 11). Example 2
レ ト口ウィルスべク夕一 pLX— IRES— EGFP— mSLC-IL2 (pLXIE - mSLC-IL2) の構築 プラスミ ド pBS-mSLC-IL2を制限酵素 Sal I、 Not Iで同時消化して、 マウス SLC-マウス IL-2融合タンパク質遺伝子の Sai l— Not I断片を得た。 この断片を、 レ トロウィルスベクタ一 PLX— IRES— EGFPの Sal I-Not I部位に組み込むこと より 目的とするマウス SLC-マウス IL-2融合遺伝子導入用レ トロウィルスべク 夕一プラスミ ド pLX— IRES— EGFP— mSLC-IL2を得た（図 2) 。 Construction of lettovirus virus Yuichi pLX—IRES—EGFP—mSLC-IL2 (pLXIE-mSLC-IL2) Plasmid pBS-mSLC-IL2 was co-digested with restriction enzymes Sal I and Not I to obtain a Sail-Not I fragment of the mouse SLC-mouse IL-2 fusion protein gene. This fragment retro viral vectors one P LX- IRES- EGFP of Sal I-Not mice an object than be incorporated into I site SLC- murine IL-2 fusion gene transfer retro virus base click evening one plasmid pLX — IRES—EGFP—mSLC-IL2 was obtained (FIG. 2).
レ ト ロウィルスベクタ一pLX— IRES— EGFP は、 レ ト ロウイルスベクタ一 pLHDCX (Genbank accession No. M64754) の EcoRl-Hind III部位に pSPORT 1 ( GIBCO/BRL社） マルチクローニングサイ ト EcoRl-Hind III断片を連結し、 その中の Notl-BamHl部位に脳心筋炎ウィルス （encephalomyocarditis virus) の内部リボソーム認識部位 IRES断片 （Novagen社） —増強型緑色蛍光タンパク 質遺伝子 EGFP断片 （クロンテック社） を組み込んだものである （図 3) 。  PSPORT 1 (GIBCO / BRL) multicloning site EcoRl-Hind III is located at the EcoRl-Hind III site of the retrovirus vector pLX-IRES-EGFP in pLHDCX (Genbank accession No. M64754). Fragments were ligated, and the internal ribosome recognition site of encephalomyocarditis virus IRES fragment (Novagen) at the Notl-BamHl site was incorporated with an enhanced green fluorescent protein gene EGFP fragment (Clontech) (Fig. 3).
比較対照実験に使用するためにマウス SLC 遺伝子導入用レ トロウィルスべク 夕一プラスミ ド pLX— IRES— EGFP— mSLC、 及び、 マウス IL-2遺伝子導入用 レ トロウィルスベクタ一プラスミ ド pLX— IRES— EGFP— mIL2をレ トロウィル スベクタ—pLX— IRES— EGFP のマルチクローニングサイ トにマウス SLC遺伝 子断片 （pT7-T3-D-Pac-mSLC を鍩型にして、 図 1 の（3)のプライマ一（mSLC- EcoRl-F (配列番号 ： 17) 及び mSLC-Notl-R (配列番号 ： 18) ) で増幅して得 られた断片） 、 マウス IL- 2遺伝子断片 （pOkayama-Berg-MuIL2を鐯型にして、 図 1の（4)のプライマ一 （mIL2-EcoRl-F (配列番号 ： 19) 及び mIL2-Notl-R (配 列番号 ： 20) ) で増幅して得られた断片） をそれぞれ揷入することにより作製し た。 実施例 3 ― Retrovirus vector for mouse SLC gene transfer Yuichi Plasmid pLX—IRES—EGFP—mSLC and retrovirus vector for mouse IL-2 gene transfer pLX—IRES— EGFP- mIL2 Les the Torowiru Subekuta - p LX- IRES- by a multiple cloning site in the mouse SLC gene fragment (pT7-T3-D-Pac -mSLC of EGFP in鍩型, primer one (3) of FIG. 1 (mSLC- EcoRl-F (SEQ ID NO: 17) and mSLC-Notl-R (SEQ ID NO: 18)) fragment obtained by amplifying with)鐯mouse IL- 2 gene fragment (pOkayama-Ber g -MuIL2 Each of the primers (fragments obtained by amplification with mIL2-EcoRl-F (SEQ ID NO: 19) and mIL2-Notl-R (SEQ ID NO: 20)) in (4) of FIG. It was made by inserting. Example 3 ―
非増殖型レ ト口ウィルスの産生及び遺伝子導入線維芽細胞の樹立  Production of non-proliferative reticulovirus and establishment of transgenic fibroblasts
組換えレ トロウイルスの産生系と して、 レ トロウイルスベクタ一プラスミ ド DNA を一過性に トランスフエクシヨンする事によりェコ トロピックなウィルス が産生可能なパッケージング Bosc23細胞 （ATCC CRL11554) を用いた。 トラン スフエクシヨンの 24時間前に、 Bosc23細胞を培養用シャーレ （直径 35mm) に I X 1 0 6個を通常の培地 （DMEM/ 1 0 % FCS) 2mlに 懸濁して撒き、 37°C、 5 % C 0 2下で培養した。 トランスフエクシヨンは LipofectAMIN T M試薬（Life Technology, Inc) を用いて行い、 試薬の指示書に従ってレ トロウイルスベクタ一 プラス ミ ド pLX— IRES— EGFP— mSLC-IL2の DNA2 〃 gを LipofectAMIN 6 1と共に Bosc23細胞に導入した。 As a recombinant retrovirus production system, the transient viral transfection of retroviral vector-plasmid DNA results in an ecotropic virus. A Bosc23 cell (ATCC CRL11554) capable of producing E. coli was used. 24 hours prior to Trang Sufuekushiyon plated suspended IX 1 0 6 pieces of normal medium (DMEM / 1 0% FCS) 2ml in a petri dish for culturing Bosc23 cells (diameter 35mm), 37 ° C, 5 % C 0 2 were cultured under. Transfection was performed using LipofectAMIN ™ reagent (Life Technology, Inc), and according to the reagent instructions, retroviral vector plasmid pLX—IRES—EGFP—mSLC-IL2 2 g of Bosc23 together with LipofectAMIN61 was used. The cells were introduced.
対照実験に使用するために pLX— IRES— EGFP、 pLX— IRES— EGFP— mSLC、 pLX- IRES - EGFP - mIL-2についても同様にトランスフエクシヨンを行った。  The transfection was similarly performed for pLX-IRES-EGFP, pLX-IRES-EGFP-mSLC, and pLX-IRES-EGFP-mIL-2 for use in control experiments.
48時間後、 培養上清を回収し、 細胞片等を除去するために孔径 0.45 z mのフ ィルターにて、 ろ過した液体を感染ウィルス含有液として以下の感染実験に使用 した。 ウィルス感染の 24 時間前に感染させる Balb/C マウス由来の繊維芽細胞 CL.7細胞 （ATCC TIB80) を培養用シャーレ （直径 35mm) に 1 X 105個を通常 の培地 （DMEM/10% FCS) 2mlに 懸濁して撒き、 37°C、 5% C02下で培養した。 感染時、 培養シャーレより培養液を除去し、 代わりに上記の感染ウィルス含有液 742.5 〃 1にウィルス感染促進を目的としてポリプレン溶液 （10mg/ml) を 7.5 (1 1を混合したものを加えて 37° (：、 5% C02下で約 8時間感染を行った。 その後、 感染ウィルス含有液を取り除き新鮮な培地 （DMEM/10% FCS) を 2ml加えて、 さらに 37° ( 、 5% C〇2下で 48時間培養した。 After 48 hours, the culture supernatant was recovered, and the liquid filtered with a filter having a pore size of 0.45 zm was used as an infectious virus-containing solution in the following infection experiments in order to remove cell debris and the like. Balb / C mouse-derived fibroblasts CL.7 cells (ATCC TIB80) to be infected 24 hours before virus infection are transferred to a culture dish (35 mm in diameter) at 1 x 105 cells in normal medium (DMEM / 10% FCS). plated were suspended in 2 ml, were cultured in C0 2 under 37 ° C, 5%. At the time of infection, remove the culture solution from the culture dish, and add 7.5% (11) of a polyprene solution (10 mg / ml) to the above-mentioned solution containing infectious virus 742.5〃1 for the purpose of promoting virus infection. ° (:., under 5% C0 2 was about 8 hours infection then added 2ml fresh medium (DMEM / 10% FCS) was removed and infected virus-containing solution further 37 ° (, 5% C_〇 Cultured under 2 for 48 hours.
こう して得られた細胞集団ではレ トロウイルスベクタ一 LX— IRES— EGFP の 感染により感染細胞のみ EGFPの発現による緑色蛍光 （励起波長 488nm , 蛍光波 長 507nm) が見られるため FACSにより感染細胞の識別、 選択が可能である。 こ の事を利用して実際に上記の感染させた細胞集団よりの感染細胞のみ濃縮を行う ために、 FACStar Plus ( Becton社） を用いて GFP陽性の細胞の濃縮を行った。 各ウィルスに関して 90 %以上の GFP 陽性の細胞群 （CL.7-mSLC-IL2、 CL.7- Vector、 CL.7-mSLC、 CL.7- mIL2) が得られた。 実施例 4 In the cell population thus obtained, only infected cells are infected with the retrovirus vector LX-IRES-EGFP, and green fluorescence (excitation wavelength 488 nm, fluorescence wavelength 507 nm) due to EGFP expression is observed. Identification and selection are possible. To take advantage of this fact, GFP-positive cells were enriched using FACStar Plus (Becton) to enrich only infected cells from the infected cell population. A GFP-positive cell group (CL.7-mSLC-IL2, CL.7-Vector, CL.7-mSLC, CL.7-mIL2) of 90% or more for each virus was obtained. Example 4
融合タンパク質 mSLC-IL2の IL-2生物活性の測定  Measurement of IL-2 biological activity of fusion protein mSLC-IL2
IL-2活性は通常、 IL-2依存的に増殖する T細胞を用いるバイオアツセィでおこ なわれる。 ヒ ト IL-2の生物活性を測定するシステムは確立しており （文献；基礎 と臨床 佐々木緊ら Vol . 22 No . 17 Dec . 1988) 、 それに従って上記の遺伝子 導入培養細胞 CL.7-mSLCJL-2 が培地中に産生する人工融合タンパク質 mSLC- IL-2 (配列番号 ： 12) の IL2生物活性の測定した。  IL-2 activity is usually performed in a bioassay using T cells that proliferate in an IL-2-dependent manner. A system for measuring the biological activity of human IL-2 has been established (Literature; Basic and Clinical Katsu Sasaki et al., Vol. 22 No. 17 Dec. 1988), and the above-mentioned transfected cultured cells CL.7-mSLCJL -2 produced an artificial fusion protein mSLC-IL-2 (SEQ ID NO: 12) produced in the medium.
比較のため、 CL.7-mIL-2 の培養上清についても測定を行った。 各培養上清に ついては、 mIL-2 ELISA ( R& D社） により IL-2濃度を測定し、 それを基に測定 に適当な濃度 （約 2 ~ 3ng/ ml ) になるよう に力価測定培地 10 % FCS 加 RPMI 16401にてあらかじめ希釈した。 For comparison, the measurement was also performed on the culture supernatant of CL.7-mIL-2. For each culture supernatant, the IL-2 concentration is measured by mIL-2 ELISA (R & D), and the titer is determined based on the IL-2 concentration to obtain an appropriate concentration (approximately 2-3 ng / ml). The medium was previously diluted with RPMI 16401 with 10% FCS.
96穴平底マイクロプレート （住友べ一クライ ト製） の 2列め以降の穴に、 力価 測定培地 10% FCS加 RPMI 1640].を 50〃 1を分注した。 第 1列目の穴には、 最 大 OD値対照 （ODmax) としてヒ ト IL-2 200JRU/mlを加えた力価測定培地、 50〃 1を分注した。 第 2列目には IL-2を加えず、 最小 OD値対照 （ODmin) と した。 標準品、 測定試料とも各希釈率デュプリケイ トとなるように 3列目と 4列 目の一番上の穴には標準として rHu IL-2(シオノギ製薬製。 商品名 ： ィムネ一ス） を力価測定培地に 50 JRU (Japanese Reference Units)/mlに溶解したものを 50 JUL 1, 5列目以降は 2列ずつの一番上の穴にあらかじめ希釈済みの各培養上清を 50 JU 1 をそれぞれ加えた。次にマルチチャネルピぺッ トを用いて 3列目以降の一 番上のゥヱルからピペッティ ングを繰り返しよく混和した後、 50 1ずつすぐし たのゥエルに移し、 2倍段階希釈を一番下のゥエルまで 8段階行った。  In a 96-well flat-bottom microplate (manufactured by Sumitomo Bei-Client), 50〃1 of RPMI 1640 with 10% FCS was added to the second and subsequent rows. In the wells of the first column, a titration medium containing human IL-2 200 JRU / ml, 50-1 as a maximum OD value control (ODmax) was dispensed. In the second column, no IL-2 was added and served as a minimum OD value control (ODmin). RHu IL-2 (manufactured by Shionogi Pharmaceutical Co., Ltd .; trade name: Imnesis) is used as a standard in the top holes of the third and fourth rows so that both the standard sample and the measurement sample have the same dilution ratio. Dissolve 50 JRU (Japanese Reference Units) / ml in the titration medium, and add 50 JU1 of each diluted culture supernatant to the top hole of each of the second and subsequent rows in 50 JUL. Each was added. Next, use a multi-channel pipette to mix well by repeating pipetting from the top row of the third row and thereafter, transfer to the wells that have been settled 50 1 at a time, and perform 2-fold serial dilution at the bottom. I went up to 8 levels.
増殖培地中で培養した NK- 7細胞液を遠心し、 上澄みを除去した。 力価測定培 地に 20,000cells/50〃 1に再懸濁し、 この細胞浮遊液 50〃 1ずつをマイクロプレ ―トの全穴に接種した。 37° (：、 5% C02下で 16時間培養後、 MTT試薬（PBS (-) (日 水製薬） に、 MTT ( 3 - (4, 5-Dimethyl-2-thiazolyl)-2) 5-diphenil-2Htetrazo]ium bromide, SIGMA社） 、 0.3%を溶解しミ リポアフィル夕一 （0.45 m) でろ過滅 菌したもの） 25〃 1を全穴に分注し、 37° (、 5% C02下で 4時間培養した。 マイク 口プレート内の培養液を、 マルチチャネルピぺッ トを用いて空のマイクロプレー トに移しかえた。 The NK-7 cell solution cultured in the growth medium was centrifuged, and the supernatant was removed. The cells were resuspended at 20,000 cells / 50〃1 in the titration medium, and 50〃1 of this cell suspension was inoculated into all the wells of the microplate. 37 ° (:, after 16 hours at 5% C0 2 under, MTT reagent (PBS (-) (day Water Pharmaceutical), dissolve 0.3% of MTT (3-(4,5-Dimethyl-2-thiazolyl) -2 ) 5-diphenil-2Htetrazo] ium bromide, SIGMA) and mix with Milliporefil Yuichi (0.45m) Dispense filtered sterilization was intended) 25〃 1 to all the wells min, 37 ° (, a 5% C0 2 for 4 hours at below. broth microphone port in the plate, with Maruchichanerupipe' bets I moved to an empty microplate.
培養液の抜き取られた全穴に、 溶出液 （0.04N塩酸—ィソプロパノ一ル液） 100 〃 1を加えマイクロミキサー （三光純薬） で 5分間振とう し、 産生された MTTフ オルマザンを充分に溶出させた。 次に移しかえた各穴の培養液を、 マルチチヤネ ルビぺッ トで元の各穴に戻した後、 マイクロプレート用分光光度計マルチスキヤ ン MC ( Flow社） を使用し、 吸光度 （OD560nm) を測定した。 光度計に接続し たパーソナルコンピューターに測定値を入力し、 力価計算を行った。 各試料の希 釈シリーズは 2列からなっているので各々の平均値を求め、 横軸にサンプルの希 釈倍率、縦軸に OD値をとつたグラフ上にプロッ ト し、濃度依存曲線を描く。各々 の試料について、プレート内の最大 0 D値対照（ODmax)、最小 OD値対照（ODmin) の吸光度の中間値と一致する希釈倍率をグラフより読み取った。 次に標準品の測 定係数 （表示力値/実測力値） を算出し、 この係数を各試料の実行力価に乗じ、 各試料の換算力値を決定した。 今回測定した CL.7-mSLC-IL2の培養上清の IL-2 力価は 242JRU/ml であった。 CL.7-mSLC-IL2の培養上清の ELISAによる IL-2 濃度は 10ng/mlであったので、 lngあたりの IL-2力価は 24.2JRUである。 同様 にして測定した CL.7-rnIL2 の培養上清については lng あたりの IL-2 力価は 18.4JRUであったので、 マウス SLC-マウス IL-2融合タンパク質 （mSLC-IL-2) はマウス IL-2夕ンパク質と同等以上の IL-2力価を有することが判明した。 実施例 5  Add 100 〃 1 of eluate (0.04N hydrochloric acid-isopropanol solution) to all the holes from which the culture solution has been removed, shake with a micromixer (Sanko Junyaku) for 5 minutes, and sufficiently produce the MTT formazan. Eluted. Next, the transferred culture solution in each well is returned to the original well by multi-channel bit, and the absorbance (OD560nm) is measured using a microplate spectrophotometer Multiscan MC (Flow). did. The measured values were input to a personal computer connected to a photometer, and the titer was calculated. Since the dilution series of each sample consists of two columns, calculate the average value of each, plot the concentration dependence curve by plotting the dilution ratio of the sample on the horizontal axis and the OD value on the vertical axis. . For each sample, the dilution factor corresponding to the median absorbance of the maximum OD value control (ODmax) and the minimum OD value control (ODmin) in the plate was read from the graph. Next, the measurement coefficient (display force value / actual force value) of the standard product was calculated, and this coefficient was multiplied by the effective titer of each sample to determine the converted force value of each sample. The IL-2 titer of the culture supernatant of CL.7-mSLC-IL2 measured this time was 242 JRU / ml. Since the IL-2 concentration of the culture supernatant of CL.7-mSLC-IL2 by ELISA was 10 ng / ml, the IL-2 titer per ng was 24.2 JRU. Since the IL-2 titer per 1 ng of the culture supernatant of CL.7-rnIL2 measured in the same manner was 18.4 JRU, the mouse SLC-mouse IL-2 fusion protein (mSLC-IL-2) It was found to have an IL-2 titer equal to or higher than that of IL-2 protein. Example 5
融合タンパク質のケモカイン SLCとしての遊走活性の確認  Confirmation of migration activity of fusion protein as chemokine SLC
実施例 3によって得られた各種遺伝子導入細胞を 3 X 105個を 3ml の培養液 ( DMEM/10% FCS ) に懸濁して、 直径 35mm培養シャーレに接種し、 37°C、 5% C02下にて 48時間培養した。 得られた各種遺伝子産物を含む培養上清を回収し、 ミ リポアフ ィルタ一 （0.45 U m ) でろ過した。 これを遊走ァッセィ用緩衝液 ( RPMI1640, 10mM HEPES,pH7.4, 1%BSA含有） にて、 体積比で 2倍 （培 養上清 50%含有） 、 10倍 （培養上清 10%含有） に希釈し、 以下の遊走アツセィに 使用した。 ケモカイン SLC としての活性を測定するためにマウス SLCの特異的 受容体であるマウス CCR7を安定発現する前駆 B細胞株 B300— 19細胞 （B300 - 19 - mCCR7) を用いた。 これは発現ブラスミ ド pCAGGSneo にマウス CCR7 遺伝子を揷入したマウス CCR7発現プラスミ ド pCAGGSneo- mCCR7を前駆 B細 胞株 B300— 19細胞にエレク トロポレーシヨン法により導入し、 薬剤 G418にて 薬剤選択することにより得られたマウス CCR7発現細胞である。 3 × 10 5 of the transfected cells obtained in Example 3 were added to a 3 ml culture solution. (DMEM / 10% FCS), inoculated into a 35 mm diameter culture dish, and cultured at 37 ° C. under 5% CO 2 for 48 hours. The culture supernatant containing the obtained various gene products was collected and filtered through Millipore Filter (0.45 Um). This was mixed with a buffer for migration assay (RPI1640, 10 mM HEPES, pH 7.4, containing 1% BSA) at a volume ratio of 2 times (containing 50% of culture supernatant) and 10 times (containing 10% of culture supernatant). And used for the following migration assy. To measure the activity as a chemokine SLC, a precursor B cell line B300-19 cell (B300-19-mCCR7), which stably expresses mouse CCR7, a specific receptor for mouse SLC, was used. This is obtained by introducing the mouse CCR7 expression plasmid pCAGGSneo-mCCR7 into which the mouse CCR7 gene has been introduced into the expression plasmid pCAGGSneo into the precursor B cell line B300-19 cells by the electroporation method, and selecting the drug with the drug G418. Mouse CCR7 expressing cells.
B300- 19 - mCCR7を血球計算盤にて細胞数測定後、 遠心により細胞を回収し、 I X lO^Cells/mlとなるように遊走ァッセィ用緩衝液（RPMI 1640、 lOmM HEPES, pH7.4、 1%BSA含有） で再懸濁し、 100〃 1ずつ、 トランスウエルチヤンバ一 ( 3 Pi m pore size. Coaster社製） の上部のゥエルに接種した。 下部のゥエルには、 600 ju 1の希釈後の培養上清あるいは対照としての遊走ァッセィ用緩衝液を加え、 37°C 5%C〇2下で 4時間培養した。 上部のゥエルを取り外し、 下部のゥヱルに遊 走した細胞を回収し、 FACStar Plus ( Becton社） を用いて細胞数を測定した。 その結果を図 4に示す。  After measuring the cell count of B300-19-mCCR7 with a hemocytometer, collect the cells by centrifugation, and run a buffer for migration assay (RPMI 1640, lOmM HEPES, pH 7.4, 1) to IX lO ^ Cells / ml. % BSA) and inoculated in 100 μl aliquots into the upper wells of a transwell chamber (3 Pim pore size, manufactured by Coaster). The culture well after dilution of 600 ju 1 or a buffer for migration assay as a control was added to the lower well, and the cells were cultured at 37 ° C. under 5% C 2 for 4 hours. The upper well was removed, the cells that had migrated to the lower well were collected, and the number of cells was counted using FACStar Plus (Becton). Fig. 4 shows the results.
CL.7-mSLC-IL2の培養上清では 2倍希釈液で、 接種細胞の 16%、 10倍希釈液 で 3.5%の遊走細胞が見られた。 CL.7- mSLCの培養上清では、 2倍希釈液で接種 細胞の 8%の遊走細胞が見られた。 CL.7- mIL2、 CL.7-VRctorの培養上清ではど ちらの希釈倍率でも、 遊走細胞数は測定限界（0.166%)以下であった。マウス SLC- マウス IL-2融合夕ンパク質 （mSLC-IL-2) はマウス CCR7発現細胞 B300，19— mCCR7に対する遊走活性を有していることが判明した。 実施例 6 In the culture supernatant of CL.7-mSLC-IL2, 16% of the inoculated cells were found in the 2-fold dilution and 3.5% of the inoculated cells were found in the 10-fold dilution. In the culture supernatant of CL.7-mSLC, migrating cells of 8% of the inoculated cells were found at the 2-fold dilution. CL.7- mIL2, even CL.7-V R ctor flickering dilution ratio etc. supernatants culture of the number of migrated cells were determined limit (0.166%) or less. The mouse SLC-mouse IL-2 fusion protein (mSLC-IL-2) was found to have chemotactic activity against mouse CCR7 expressing cells B300, 19-mCCR7. Example 6
融合夕ンパク質のィムノブロッ トによる分子量の同定  Molecular weight identification of fusion proteins by immunoblotting
実施例 3によって得られた mSLC-IL2遺伝子導入細胞 CL.7-mSLC-IL2、 対照 として CL.7-Vectorをそれぞれについて 5 X 105個を 2mlの培養液 （DMEM/10% FCS ) に懸濁して、 直径 35mm 培養シャーレに接種し、 37° (：、 5% C02下にて 24 時間培養後培地を除き、 OPTI-MEM ( Gibco/BRL) 3ml に置き換えてさらに 37°C、 5% CO2下にて 24時間培養した。 5 x 105 cells of the mSLC-IL2 transfected cell CL.7-mSLC-IL2 obtained in Example 3 and CL.7-Vector as a control were suspended in 2 ml of a culture solution (DMEM / 10% FCS). Nigoshi and were inoculated into 35mm diameter culture dish, 37 ° (:, 5% C0 2 at the bottom the medium was removed after 24 hours incubation, further 37 ° C by replacing the OPTI-MEM (Gibco / BRL) 3ml, 5% The cells were cultured under CO2 for 24 hours.
得られた各種遺伝子産物を含む培養上清を回収し、 ミ リポアフィルタ一 （0.45 ju m) でろ過した。 培地内蛋白を濃縮するために以下のように TCA沈殿を行つ た。 1mlの培養上清を 100 / 1の 100%TCAを加えて混合し、 氷上に 1時間静置 し、 12Krpmで 5分間遠心した。 沈殿したペレッ トを氷冷ァセ トンにて、 洗浄し、 25〃 1の 3 X SDSサンプルバッファ一に溶解し、 2.5 1の 2-mercaptoethanol と 2.5 1の lM Tris-HCl,pH8.0を加えて 10CTCの熱を 5分間加えて変性させた。 各々 10 1のライセ一トを 15%— 25%グラジェン ト SDS—ポリアク リルアミ ドゲ ルによる電気泳動を行った。 その際、 隣接するレーンに Prestain Protein Maker、 Broad Range ( NEW ENGLAND BioLabs inc. ) を泳動した。  Culture supernatants containing the obtained various gene products were collected and filtered through a Millipore filter (0.45 jum). TCA precipitation was performed as follows to concentrate the protein in the medium. One ml of the culture supernatant was mixed with 100/1 of 100% TCA, left on ice for 1 hour, and centrifuged at 12K rpm for 5 minutes. Wash the pellet with ice-cold acetone, dissolve in 25〃1 3X SDS sample buffer, add 2.51 2-mercaptoethanol and 2.51 lM Tris-HCl, pH 8.0. And denatured by applying 10 CTC heat for 5 minutes. Each 101 lysates were electrophoresed on a 15% -25% gradient SDS-polyacrylamide gel. At this time, Prestain Protein Maker and Broad Range (NEW ENGLAND BioLabs inc.) Were run in the adjacent lane.
泳動後のゲル上の夕ンパク質は二トロセルロース膜 （imobilonP, Milipore社） に移した。 トランスファ一後の二トロセルロース膜は 5% (重量/容積） スキムミ ルク容液 （Dii'co社製スキムミルクを T-PBS ( 0.05% Tween20入り PBS溶液） に て溶解したもの） に 30分間、 室温にて浸す事により非特異的な蛋白吸着を阻害し た。 二トロセルロース膜は、 やぎ抗マウス IL-2抗体 （T-PBSにて 1000倍に希釈 して使用。 ） に約 2時間反応させた。 その後、 T-PBSにて 3回洗浄し、 ペルォキ シダ一ゼ標識抗やぎ TgG抗体 （Cappel社製、 2000倍に希釈して使用） で約 30 分間反応し、 その後 T-PBSにて 3回洗浄したのちに、 ECL試薬 （アマシャム社） にて、 ペルォキシダ一ゼ標識を発光させ、 それを X線フィルムで感光させた。 そ の結果、 対照である CL.7-Vector のレーンにはバン ドは見られないが CL.7- mSLC-IL2のレーンには、 約 32kDの位置にのみ特異的なバン ドが見られた。 こ れは一次構造から予測される分子量とほぼ一致することが判明した。 実施例 7 The protein on the gel after electrophoresis was transferred to a ditrocellulose membrane (imobilonP, Milipore). Nitrocellulose membrane after transfer is 5% (weight / volume) skim milk solution (dissolved skim milk made by Dii'co in T-PBS (PBS solution with 0.05% Tween20)) for 30 minutes at room temperature Non-specific protein adsorption was inhibited by soaking in. The ditrocellulose membrane was reacted with a goat anti-mouse IL-2 antibody (diluted 1000-fold with T-PBS for use) for about 2 hours. Then, wash three times with T-PBS, react with peroxidase-labeled anti-goat TgG antibody (Cappel, diluted 2000 times) for about 30 minutes, and then wash three times with T-PBS After that, peroxidase label was emitted with ECL reagent (Amersham) and exposed to X-ray film. As a result, no band was observed in the lane of the control CL.7-Vector, but CL.7-Vector In the mSLC-IL2 lane, a specific band was observed only at a position of about 32 kD. This was found to be almost consistent with the molecular weight predicted from the primary structure. Example 7
T細胞の組織免疫染色による同定 Identification of T cells by tissue immunostaining
融合タンパク質 SLC-IL2 が実際に生体内で T細胞を遊走させる能力を有する のかを調べるために、 実施例 3で得られた mSLC-IL2 遺伝子導入細胞 CL.7- mSLC-IL2をその親株である線維芽細胞 CL.7の由来であるマウス Balb/Cス ト レ インに皮内投与し、 そこに T細胞が浸潤してくるかを調べた。 実施例 3で得られ た mSLC-IL2遺伝子導入細胞 （CL.7-mSLC-IL2) 、 対照として Vector、 mSLC、 mIL2 遺伝子導入細胞それそれについて lm〗 あたり 1 x 108細胞数となるように HANKS緩衝液 （Gibco社製） に懸濁した。 In order to investigate whether the fusion protein SLC-IL2 actually has the ability to migrate T cells in vivo, the mSLC-IL2 transgenic cell CL.7-mSLC-IL2 obtained in Example 3 was used as its parent strain. Fibroblasts CL.7 were intradermally administered to mouse Balb / C strain, and it was examined whether T cells infiltrated there. Obtained in Example 3 MSLC-IL2 transgenic cell (CL.7-mSLC-IL2), Vector as a control, MSLC, mIL2 transgenic cell it HANKS about it so that lm 1 x 10 8 cell number per〗 The cells were suspended in a buffer (manufactured by Gibco).
Balb/C マウスの雌、 7 週齢 （日本チヤ一ルス -リバ一社より購入） の背中に上 記の細胞懸濁液 50 u I (細胞数 5 X 106) を皮内接種した （各細胞群で 2匹ずつ） 。 5日後には移植部位は 5mm ぐらいの膨らみとなっているので、それをマウスを安 楽死させたあとに摘出し、 OTC compound (Miles Laboratory社製） に浸して ド ライアイス上にて凍結させた。 A female Balb / C mouse, 7 weeks old (purchased from Nippon Chillers-Liva) was inoculated intradermally with the above cell suspension (50 uI, 5 × 10 6 cells) on the back (each 2 in the cell group). Five days later, the transplantation site had a bulge of about 5 mm.Then, the mouse was euthanized, removed, immersed in an OTC compound (manufactured by Miles Laboratory), and frozen on dry ice. .
これを、 クライオスタツ トを用いて 8 mの厚さの切片を作製し、 スライ ドガ ラス上に乗せ風乾させた。 その後、 マイナス 2(TCに冷やしておいた固定液 （40% ァセ トン、 60%メ夕ノ一ル、 容積比） に 15分間浸して固定し、 PBS (—） 溶液に て 2回洗浄後、 ブロッキング液 （20%正常ゥサギ血清、 80% PBS ( - ) 溶液、 容 積比） に 30分間反応させ非特異的吸着を阻止した。 それから、 PBS (—） 溶液に て 2回洗浄後、 ラヅ ト抗マウス CD4単クローン抗体 （CEDARLANE社製） 及び ラッ ト抗マウス CD8単クローン抗体 （CEDARLANE社製） にて約 60分問、 室温 にて反応させた。 反応後、 PBS ( - ) 溶液にて 3回洗浄後、 ゥサギ抗ラッ 卜 IgG 抗体 （Vector社製） に 30分間、 室温で反応させた。 PBS ( - ) 溶液にて 2回洗 浄後、 1 %過酸化水素液 （30%過酸化水素水を容積比 1 に対してメタノールを容 積比 29の割合で混合したもの） に 30分間反応させた。 PBS (—） 溶液にて 3回 洗浄後、 ペルォキシダーゼ標識を EliteABCキッ ト （Vector社製） 、 DAB基質キ ッ トを使用して発色させた。 メチレンブルーにて対比染色もおこなった。 染色し た切片は日本光学社光学顕微鏡 OPTIPHOT で得られた拡大画像をフジフィルム 社デジタルカメラ HC— 2000でパーソナルコンピュ一夕一に取り込んで、 各サン プルについて任意の 3視野について染色陽性の細胞数の同定し、 各群 2匹ずつな ので計 6視野の平均値 （土標準偏差） を図 5及び図 6に表した。 Using a cryostat, 8 m-thick sections were prepared, placed on a slide glass, and air-dried. Then, fix by immersing in minus 2 (fixed solution (40% acetone, 60% methanol, volume ratio) cooled in TC) for 15 minutes, and wash twice with PBS (-) solution. After reacting with a blocking solution (20% normal normal heron serum, 80% PBS (-) solution, volume ratio) for 30 minutes to prevent nonspecific adsorption, and washing twice with PBS (-) solution,反 応 After reacting for about 60 minutes at room temperature with anti-mouse CD4 monoclonal antibody (CEDARLANE) and rat anti-mouse CD8 monoclonal antibody (CEDARLANE). After washing 3 times, the plate was reacted with a egret anti-rat IgG antibody (Vector) for 30 minutes at room temperature, and washed twice with PBS (-) solution. After the purification, it was reacted with a 1% hydrogen peroxide solution (a mixture of 30% hydrogen peroxide solution and methanol at a volume ratio of 29 with a volume ratio of 29) for 30 minutes. After washing three times with a PBS (-) solution, a peroxidase label was developed using an EliteABC kit (Vector) and a DAB substrate kit. Counterstaining was also performed with methylene blue. For the stained sections, magnified images obtained with an optical microscope (OPTIPHOT) from Nippon Kogaku Co., Ltd. were captured at a personal computer overnight using a Fujifilm Digital Camera HC-2000, and the number of cells stained positive in any three visual fields for each sample. Since two animals were identified in each group, the average value (soil standard deviation) of a total of six visual fields was shown in FIGS. 5 and 6.
Vector遺伝子導入細胞移植群は CD4、 CD8 に関しても浸潤細胞は僅かに見ら れた。 mSLC、 mIL2遺伝子導入細胞移植群共に CD4、 CD8陽性細胞の明らかな 增加が見られた。 さらに mSLC-IL2遺伝子導入細胞移植群では mSLC、 mIL2遺 伝子導入細胞移植群と比較してもよりさらに多くの浸潤細胞が見られた。 実施例 8  In the vector transfected cell transplantation group, infiltrating cells were slightly observed in CD4 and CD8. In both the mSLC and mIL2 gene transfected cell transplant groups, a clear increase in CD4 and CD8 positive cells was observed. Furthermore, in the mSLC-IL2 transgenic cell transplantation group, even more infiltrating cells were observed as compared with the mSLC and mIL2 gene transfected cell transplantation group. Example 8
生体内投与のモデル実験 Model experiment of in vivo administration
融合夕ンパク質 SLC-IL2 が実際に生体内で腫瘍形成を抑制する能力を有する のかを調べるために、 実施例 3で得られた mSLC-IL- 2遺伝子導入細胞 CL.7- mSLC-IL2をその親株である線維芽細胞 CL.7の由来動物であるマウス Balb/Cス ト レイ ンに、 マウス Balb/C 由来の大腸癌細胞株 Colon26 と混合した後に皮内投 与し、 腫瘍形成の程度をしてくるかを Vector、 mIL2遺伝子導入細胞を対照群と して調べた。  To investigate whether the fusion protein SLC-IL2 actually has the ability to suppress tumor formation in vivo, the CL.7-mSLC-IL2 transfected mSLC-IL-2 gene obtained in Example 3 was used. Mouse Balb / C strain, which is the animal derived from its parental fibroblast CL.7, was mixed with mouse Balb / C-derived colon cancer cell line Colon26, and then intradermally injected to evaluate the degree of tumor formation. Vector and mIL2 transfected cells were examined as a control.
2度の実験を行い実験 1では Vector、 mSLC遺伝子導入細胞間で比較を、 実験 2では Vector、 mIL2、 mSLC-IL2 遺伝子導入細胞間で比較を行った。 どちらの 実験も同じプロ トコールで行った。 実施例 3で得られたそれそれの遺伝子導入線 維芽細胞が I X 107細胞数と、 Colon26が 5 X 106細胞数が 1mlに混合した細胞液 となるように HANKS緩衝液 （ Gibco社製） に懸濁した。 Balb/C マウスの雌、 7 週齢 （日本チヤ一ルス 'リバ一社より購入） の背中に上 記の細胞懸濁液 100 1 (細胞数は遺伝子導入線維芽細胞 5 X 105個、 Colcm26は 1 X 106個） を皮内接種した （実験 1では各細胞群で 5匹ずつ、 実験 2では各群マ ウス 8匹） 。 その後、 通常の環境下で飼育を続けた。 腫瘍の測定は、 腫瘍の長径 と、 その直角方向の長さ （短径） をノギスにて測定し、 その値より近似値 （長径 X短径 X短径 ÷ 2 ) を腫瘍体積とした。 図 7及び図 8は各実験での各群の平均腫 癟体積 （土標準偏差） をグラフに表したものである。 Two experiments were performed. In Experiment 1, a comparison was made between the Vector and mSLC-transfected cells, and in Experiment 2, a comparison was made between the Vector, mIL2, and mSLC-IL2 transfected cells. Both experiments were performed using the same protocol. HANKS buffer solution (manufactured by Gibco) so that each of the gene transfer lines obtained in Example 3 was a cell solution obtained by mixing IX 10 7 cell numbers and Colon 26 with 5 × 10 6 cell numbers in 1 ml. ). Female cell of a Balb / C mouse, 7 weeks old (purchased from Nippon Chill's Riva Co., Ltd.) on the back of the above cell suspension 100 1 (number of cells: 5 x 10 5 transgenic fibroblasts, Colcm26 and is intradermally inoculated with 1 X 10 6 cells) (each 5 animals in experiment 1, each cell group, experiment 2, Kakugunma 8 mice mouse). Thereafter, they were kept under normal conditions. For the measurement of the tumor, the major axis of the tumor and the length (minor axis) in the direction perpendicular to the tumor were measured with a vernier caliper, and the approximate value (major axis X minor axis X minor axis ÷ 2) was taken as the tumor volume. Figures 7 and 8 graphically represent the mean tumor volume (standard deviation of soil) of each group in each experiment.
実験 1では腫瘍接種後 28日目の結果であるが、 mSLC群では Vector群と比べ て平均腫瘍体積は 105%であり、 腫瘍形成を抑制する効果が見られなかった。 実 験 2では腫瘍接種後 2 3 日目の結果であるが、 mIL2 群ではべクタ一群と比べて 平均腫瘍体積は 77%であり、 ある程度腫瘍の体積増加を遅らせることが分かる。 mSLC-IL2群ではさらに腫瘍体積は小さくなつていて、 ベクター群と比べて平均 腫瘍体積は 45%であり、 mIL2群と比べても平均腫瘍体積は 59%であった。  In Experiment 1, the results were 28 days after tumor inoculation. In the mSLC group, the average tumor volume was 105% compared to the Vector group, and no effect of suppressing tumor formation was observed. Experiment 2 shows the results on the 23rd day after tumor inoculation. In the mIL2 group, the average tumor volume was 77% compared to the vector group, indicating that the increase in tumor volume was delayed to some extent. The tumor volume was even smaller in the mSLC-IL2 group, with an average tumor volume of 45% compared to the vector group and 59% compared to the mIL2 group.
これらの結果から、この腫瘍形成モデルでは対照群である Vector遺伝子導入細 胞と同様に mSLC 遺伝子導入細胞は腫瘍抑制効果が見られないこと、 mIL2、 mSLC-IL2 遺伝子導入細胞ともに腫癟抑制効果は見られるがその程度は mSLC- IL2遺伝子導入細胞の方が有意に強いことが分かった。 実施例 9  These results indicate that, in this tumorigenic model, the mSLC-transfected cells did not show a tumor-suppressing effect as in the control group, Vector-transfected cells, and that the tumor-suppressing effects of both mIL2 and mSLC-IL2 It was found that the degree was significantly stronger in the mSLC-IL2 gene transfected cells. Example 9
ヒ ト SLC-ヒ ト IL-2融合蛋白質遺伝子の構築 Construction of human SLC-human IL-2 fusion protein gene
ヒ ト SLC ( hSLC) 遺伝子 （配列番号： 1) の終止コ ドンを含まず 3 ' 末端に Xba I部位を有するようなヒ ト SLC 遺伝子断片はアル力リ フォスファタ一ゼ融合夕 ンパク発現用ベクター pDREF-SLC-AP (Nagira, M. et al., J. Biol. Chem.， 272, 31, 19518- 19524, 1997) を Sal I及び Xba I で同時切断して切り出される約 0.4kbの断片を利用した。  A human SLC gene fragment that does not contain the termination codon of the human SLC (hSLC) gene (SEQ ID NO: 1) and has an XbaI site at the 3 'end is used as a vector for expression of a protein fused to an alfa phosphatase pDREF -SLC-AP (Nagira, M. et al., J. Biol. Chem., 272, 31, 19518-19524, 1997) is digested simultaneously with Sal I and Xba I. did.
ヒ ト IL-2 ( hIL-2) の成熟型配列 （配列番号 ： 4 )をコードする遺伝子断片を得 るために、 ヒ ト IL2の cDNA (配列番号 ： 3) を有しているプラスミ ド pIL2-50A を鍩型と して、 プライマ一 （5'-hIL2-Spe I (配列番号 ： 22) 及び 3'-hIL2-Not I (配列番号 ： 23) ) を用いて、 約 0.4 k bの断片を PCR法により増幅した。 得 られた断片は Spe I及び Not Iで同時消化し、 市販のベクタ一 Blue Script(+)の Spe I、 Not I部位にサブクローン化し、 プラスミ ド pBS-ML-2(Spe I /N)を構築 した。 クローン化したものの塩基配列を決定し、 PCR法により変異のないことをA gene fragment encoding the mature sequence of human IL-2 (hIL-2) (SEQ ID NO: 4) was obtained. For this purpose, the plasmid pIL2-50A having the cDNA of human IL2 (SEQ ID NO: 3) was designated as type I, and the primers (5'-hIL2-Spe I (SEQ ID NO: 22) and 3) were used. Using '-hIL2-NotI (SEQ ID NO: 23)), a fragment of about 0.4 kb was amplified by PCR. The obtained fragment was co-digested with Spe I and Not I, subcloned into the Spe I and Not I sites of the commercially available vector Blue Script (+), and plasmid pBS-ML-2 (Spe I / N) was obtained. It was constructed. Determine the nucleotide sequence of the cloned product and confirm that there is no mutation by PCR.
V^.口'。、した。 V ^ .Mouth '. ,did.
制限酵素 Spe I と Xba Iは切断後の付着端の配列が同じであり、 結合可能であ る。 pDREF-SLC-APを Sal I及び Xba Iで同時切断して得られる 0.4kbの断片 を pBS-hIL-2(Spe I /N)の Spe I、 Not I部位に揷入し、 結果としてヒ ト SLC- ヒ ト IL-2 融合蛋白質遺伝子を Sal I と Not Iサイ トの間に持つ、 プラスミ ド pBS-hSLC-IL2 が得られた （図 9 ) 。 人工融合蛋白質遺伝子 （hSLC-IL-2) は、 810塩基対からなり、 その構造は 5， 末端からヒ ト SLC遺伝子をコードする 402 塩基、 リンカ一をコードする配列 6塩基、 ヒ ト IL-2の N末端 21番目から C末端 までの 133 ァミノ酸残基をコ一ドする 399 塩基と終止コ ドンからなる（配列番 号 ： 25)。 実施例 10  The restriction enzymes Spe I and Xba I have the same sequence of the cohesive end after cleavage, and can bind. A 0.4 kb fragment obtained by simultaneously digesting pDREF-SLC-AP with Sal I and Xba I was inserted into the Spe I and Not I sites of pBS-hIL-2 (Spe I / N), resulting in human Plasmid pBS-hSLC-IL2, which has the SLC-human IL-2 fusion protein gene between the Sal I and Not I sites, was obtained (FIG. 9). The artificial fusion protein gene (hSLC-IL-2) consists of 810 base pairs and has a structure of 5, 402 bases encoding the human SLC gene from the end, 6 bases encoding the linker, and human IL-2. It consists of 399 bases coding for the 133 amino acid residues from the N-terminal 21st to the C-terminal and a stop codon (SEQ ID NO: 25). Example 10
レ ト ロウィルスベクタ一 pLX— IRES— EGFP— hSLC-IL2 ( pLXIE - hSLC-IL2) の構築 Construction of retrovirus vector pLX—IRES—EGFP—hSLC-IL2 (pLXIE-hSLC-IL2)
プラスミ ド pBS-hSLC-IL2を制限酵素 Sal I、Not Iで同時消化して、ヒ ト SLC- ヒ ト IL-2融合蛋白質遺伝子の Sal I— Not I断片を得た。 この断片を、 レ トロゥ ィルスべクタ一 pLX— IRES— EGFP の Sal I -Not I部位に組み込むことにより 目的とするヒ ト SLC-ヒ ト IL-2融合遺伝子導入用レ トロウイルスベクタ一プラス ミ ド pLX— IRES— EGFP— hSLC-IL2を得た（図 9 ) 。  Plasmid pBS-hSLC-IL2 was co-digested with restriction enzymes Sal I and Not I to obtain a Sal I—Not I fragment of the human SLC-human IL-2 fusion protein gene. By incorporating this fragment into the SalI-NotI site of retroviral vector pLX-IRES-EGFP, the desired retrovirus vector plasmid for transfecting the human SLC-human IL-2 fusion gene can be obtained. pLX—IRES—EGFP—hSLC-IL2 was obtained (FIG. 9).
比較対照実験に使用するためにヒ ト IL2遺伝子導入用レ トロウイルスベクタ一 プラスミ ド pLX— IRES— EGFP— hIL-2 をレ トロウィルスベクタ一 pLX— IRES - EGFPのマルチクローニングサイ トにヒ ト IL-2遺伝子断片（ pIL2-50Aを錶型 にして、図 1の（3)のプライマ一（5'-WL2-Sal I (配列番号： 24)及び 3'-WL2-Not I (配列番号 ： 23) ) で PCR 法で増幅して得られた断片） を挿入することによ り作製した。 実施例 11 Retrovirus vector for human IL2 gene transfer for use in comparative experiments Plasmid pLX—IRES—EGFP—hIL-2 was inserted into the retroviral vector pLX—IRES-EGFP multicloning site at the human IL-2 gene fragment (pIL2-50A was type III, and )) (A fragment obtained by amplification with the PCR method using the primers 5′-WL2-Sal I (SEQ ID NO: 24) and 3′-WL2-Not I (SEQ ID NO: 23)). It was made more. Example 11
非増殖型レ トロウィルスの産生及び遺伝子導入線維芽細胞の樹立 Production of non-replicating retrovirus and establishment of transgenic fibroblasts
プラスミ ド pLX— IRES— EGFP— hIL-2及び pLX— IRES— EGFP— hSLC-IL2 を用いて、 実施例 3 と同様の方法によってレ トロウイルスを産生し、 CL.7への 遺伝子導入を行い hIL-2遺伝子導入細胞 （CL.7-hIL2) 及び hSLC-IL2遺伝子導 入細胞 （CL.7-hSLC-IL2) を得た。 得られた hSLC-IL2 遺伝子導入培養細胞 ( CL.7-hSLC-IL2) 及び hIL2遺伝子導入培養細胞 （CL.7-hIL2) をそれそれ ： 3 X 105個を 3mlの培養液 （DMEM/10% FCS)に懸濁して、 直径 35mm培養シヤー レに接種し、 37°C、 5% C02下にて 48時間培養した。 得られた各種遺伝子産物を 含む培養上清を回収し、 ミ リポアフィルタ一 （0.45 m) でろ過した。 この培養 上清中の IL2含量をヒ ト IL2 ELISA( Human IL2 AN、ALYZA Immunoassay kit. Genzyme TECHNE 社）を用いて測定したところ CL.7-hSLC-IL2 の培養上清の IL2濃度は 25ng/mlで、 CL.7-hIL2の培養上清の IL2濃度は 50ng/mlであり、 そ れそれ導入遺伝子産物の発現が確認された。 これらのサンプルを実施例 12、 13 の生物活性測定実験使用した。 実施例 12 Using the plasmids pLX-IRES-EGFP-hIL-2 and pLX-IRES-EGFP-hSLC-IL2, a retrovirus was produced in the same manner as in Example 3, and the gene was transfected into CL.7. -2 transgenic cells (CL.7-hIL2) and hSLC-IL2 transgenic cells (CL.7-hSLC-IL2) were obtained. The obtained hSLC-IL2 gene-introduced cultured cells (CL.7-hSLC-IL2) and hIL2 gene-introduced cultured cells (CL.7-hIL2) were each converted into 3 x 105 cells in a 3 ml culture solution (DMEM / 10% was suspended in FCS), were seeded in 35mm diameter culture shear les, and cultured for 48 hours at 37 ° C, 5% C0 2 below. Culture supernatants containing the various gene products obtained were collected and filtered through a Millipore filter (0.45 m). The IL2 content of the culture supernatant was measured using a human IL2 ELISA (Human IL2 AN, ALYZA Immunoassay kit. Genzyme TECHNE). The IL2 concentration of the culture supernatant of CL.7-hSLC-IL2 was 25 ng / In ml, the concentration of IL2 in the culture supernatant of CL.7-hIL2 was 50 ng / ml, and the expression of the transgene product was confirmed in each case. These samples were used for the biological activity measurement experiments in Examples 12 and 13. Example 12
融合蛋白質 hSLC-IL-2の IL-2生物活性の測定 Measurement of IL-2 biological activity of fusion protein hSLC-IL-2
実施例 11によって得られた各種遺伝子導入細胞（CL.7-hSLC-IL2、CL.7-hIL2) の培養上清を、 実施例 4 と同様の方法を用いて IL-2 の生物活性を測定した。 CL.7-hSLC-IL2の培養上清の IL2力価は 378JRU/mlで、 CL.7-hIL2の培養上清 の IL2力価は 901JRU/mlであった。 lngの IL2あたりの IL2力価は hSLC-IL2 で 15.1JRUであり、 hIL2で 18JRUであったので、 ヒ ト SLC-ヒ ト IL-2融合蛋 白質はヒ ト IL-2搔白質と遜色ない IL2力価を有することが判明した。 実施例 13 Using the same method as in Example 4, the biological activity of IL-2 was measured using the culture supernatant of the various transfected cells (CL.7-hSLC-IL2, CL.7-hIL2) obtained in Example 11. did. The IL.2 titer of the culture supernatant of CL.7-hSLC-IL2 was 378 JRU / ml, and the IL.2 titer of the culture supernatant of CL.7-hIL2 was 901 JRU / ml. Since the titer of IL2 per IL2 was 15.1 JRU for hSLC-IL2 and 18 JRU for hIL2, the human SLC-human IL-2 fusion protein was comparable to human IL-2 搔 IL. It was found to have a titer. Example 13
融合蛋白質のケモカイ ン SLCとしての遊走活性の確認 Confirmation of migration activity of fusion protein as chemokine SLC
実施例 11によって得られた各種遺伝子導入細胞（CL.7-hSLC-IL2、 CL.7-hIL2) および対照と して実施例 3に記載のベクター導入細胞 （CL.7-Vector) の培養上 清をそのまま、あるいは遊走ァヅセィ用緩衝液（RPMI 1640、10mM HEPES,pH7.4, 1%BSA含有） にて、 体積比で 2倍 （培養上清 50%含有） 、 10倍 （培養上清 10% 含有） に希釈し、 以下の遊走ァッセィに使用した。 ケモカイン SLCとしての活性 を測定するためにヒ ト SLC の特異的受容体であるヒ ト CCR7 を安定発現する前 駆 B 細胞株 L1.2 細胞 （L1.2-CCR7 ) を用いた。 これは発現プラス ミ ド pCAGGSneo に ヒ ト CCR7 遺伝子を挿入 した ヒ ト CCR7 発現プラス ミ ド pCAGGSneo-CCR7を前駆 B細胞株 L1.2細胞にエレク ト口ポレーション法により 導入し、 薬剤 G418にて薬剤選択することにより得られたヒ ト CCR7発現細胞で ある o (Yoshida, R., et al., J Biol Chem. 273: 7118-7122, 1998)  The culture of various gene-introduced cells (CL.7-hSLC-IL2, CL.7-hIL2) obtained in Example 11 and the vector-introduced cell (CL.7-Vector) described in Example 3 as a control Use the supernatant as it is or with a buffer for migration assay (RPMI 1640, 10 mM HEPES, pH 7.4, containing 1% BSA) at a volume ratio of 2 times (containing 50% of culture supernatant), 10 times (10% of culture supernatant). % Containing) and used for the following migration assays. To measure the activity as a chemokine SLC, a precursor B cell line L1.2 cell (L1.2-CCR7) that stably expresses human CCR7, a specific receptor for human SLC, was used. This was achieved by introducing the human CCR7 expression plasmid pCAGGSneo-CCR7 into which the human CCR7 gene was inserted into the expression plasmid pCAGGSneo by electroporation into the precursor B cell line L1.2 cells, and using the drug G418 Human CCR7 expressing cells obtained by selection o (Yoshida, R., et al., J Biol Chem. 273: 7118-7122, 1998)
L1.2-CCR7 を血球計算盤にて細胞数測定後、 遠心によ り細胞を回収し、 I X KFCells/mlとなるように遊走アツセィ用緩衝液 （RPMI1640、 10mM HEPES、 pH7.4、 1%BSA含有） で再懸濁し、 100〃 1ずつ、 トランスウエルチヤンバ一 ( 3 μ. m pore size, Coaster社製） の上部のゥエルに接種した。 下部のゥエルには、 600 / 1の希釈後の培養上清あるいは対照としての遊走アツセィ用緩衝液を加え、 37°C 5%C02下で 4時間培養した。 上部のゥエルを取り外し、 下部のゥエルに遊 走した細胞を回収し、 Flow cytometer EPICS XL ( Coulter社） を用いて細胞数 を測定した。 CL.7-hSLC-IL2の培養上清では原液で接種細胞の 1.6.6%、 2倍希釈液で 9%、 10倍希釈液で 1.1%の遊走細胞が見られた。 一方 CL.7- hIL2、 CL.7-Vectorの培 養上清ではどの希釈倍率でも、 遊走細胞数は 0.3%以下であった。 この結果より ヒ ト SLC-ヒ ト IL-2融合蛋白質はヒ ト CCR7発現細胞 L1.2-CCR7に対する遊走活 性すなわちケモカイン SLCとしての生物活性を保持していることが判明した。 実施例 14 After measuring the cell number of L1.2-CCR7 using a hemocytometer, collect the cells by centrifugation, and use a buffer for migration assay (RPMI1640, 10 mM HEPES, pH 7.4, 1% to IX KFCells / ml). The mixture was resuspended in BSA) and inoculated in 100 μl aliquots into the upper wells of a transwell chamber (3 μm pore size, manufactured by Coaster). At the bottom of Ueru, migration Atsusi buffer as culture supernatant or control after dilution of 600/1 was added and 4 hours at 5% C0 2 under 37 ° C. The upper well was removed, the cells that had migrated to the lower well were collected, and the cell number was measured using a Flow cytometer EPICS XL (Coulter). In the culture supernatant of CL.7-hSLC-IL2, 1.6.6% of the inoculated cells were found in the undiluted solution, 9% in the 2-fold diluted solution, and 1.1% of the migrated cells in the 10-fold diluted solution. On the other hand, in the culture supernatant of CL.7-hIL2 and CL.7-Vector, the number of migrated cells was 0.3% or less at any dilution ratio. From this result, it was revealed that the human SLC-human IL-2 fusion protein retains the migration activity against human CCR7-expressing cells L1.2-CCR7, that is, the biological activity as a chemokine SLC. Example 14
生体内投与のモデル実験 （併用との比較） Model experiment of in vivo administration (comparison with combination)
mSLC、 mIL2遺伝子導入細胞を同時に投与する場合と、 mSLC-IL2遺伝子導入 細胞を単独で投与する場合とでの抗腫瘍効果を比較するために以下の実験を行つ た。  The following experiment was performed to compare the antitumor effects of the case where mSLC and mIL2 gene transfected cells were simultaneously administered and the case where mSLC-IL2 gene transfected cells were administered alone.
実施例 8と同様に各遺伝子を導入した繊維芽細胞 CL.7細胞 （マウス Balb/Cス トレイン由来） と大腸癌細胞株 Colon26 (マウス Balb/C由来） とを混合した後に 皮内投与し、 腫瘍形成の程度を比較することにより抗腫瘍効果を調べた。  As in Example 8, fibroblast CL.7 cells (from mouse Balb / C strain) into which each gene was introduced were mixed with colon cancer cell line Colon26 (from mouse Balb / C), and then intradermally administered. The antitumor effect was examined by comparing the degree of tumor formation.
実験群としては、 下記 5種類の細胞群を用いた。 As the experimental group, the following five types of cell groups were used.
① Vector導入細胞 （CL.7-Vector) 5 X 105個  ① Vector transfected cells (CL.7-Vector) 5 x 105 cells
② mSLC導入細胞 （CL.7-mSLC) 5 X 105個  ② mSLC transfected cells (CL.7-mSLC) 5 x 105 cells
③ mlL2導入細胞 （CL.7-mIL2) 5 X 105個  ③ mlL2 transfected cells (CL.7-mIL2) 5 x 105 cells
④ mSLC-IL2遺伝子導入細胞 （ CL.7-mSLC-IL2) 5 X 105個  ④ mSLC-IL2 transfected cells (CL.7-mSLC-IL2) 5 x 105 cells
⑤ mSLC導入細胞 （CL.7-mSLC) 5 X 10 個と mIL2導入細胞 （CL.7-mIL2) 5 X 105個 ⑤ 5 x 10 cells transfected with mSLC (CL.7-mSLC) and 5 x 105 cells transfected with mIL2 (CL.7-mIL2)
①から⑤の遺伝子導入 CL . 7と Colon26細胞 1 x 105個とそれぞれを混合して マウスの背中皮内に移植して比較を行った。 The transgenic CL.7 from (1) to (7) was mixed with 1 × 10 5 Colon26 cells and transplanted into the back skin of mice for comparison.
具体的には、 それぞれの遺伝子導入線維芽細胞が 5 X 106個 （⑤の場合は計 I X 10?個） と、 Colon26が I X 106個が lmlに混合した細胞液となるように HANKS 緩衝液 （Gibco社製） に懸濁した。 Specifically, HANKS was used so that each transfected fibroblast was 5 x 10 6 (in case of ⑤, a total of IX 10?), And Colon26 was a cell solution in which 106 IX were mixed in lml. The cells were suspended in a buffer (manufactured by Gibco).
Balb/C マウスの雌、 7 週齢 （日本チヤ一ルス'リバ一社より購入） の背中に上 記の細胞懸濁液 100〃 1 (細胞数は遺伝子導入線維芽細胞 5 X 105個 （⑤の場合は 計 I X 106個） 、 Colon26は I X 105個） を皮内接種した （各群マウス 8匹） 。 そ の後、 通常の環境下で飼育を続けた。 腫瘍の測定は、 腫瘍の長径と、 その直角方 向の長さ （短径） をノギスにて測定し、 その値より近似値 （長径 X短径 X短径÷ 2 ) を腫瘍体積とした。 図 10は移植後 24日目の各群の平均腫瘍体積 （土標準偏 差） をグラフに表したものである。 Female cell of a Balb / C mouse, 7 weeks old (purchased from Nippon Chillers Riva Co., Ltd.) on the back of the above cell suspension 100〃1 (number of cells: 5 x 10 5 transgenic fibroblasts ( total IX 10 6 cells in the case of ⑤), Colon 26 is IX 10 5 cells) were inoculated intradermally (8 mice per group). Subsequently, they were kept under normal conditions. For the measurement of the tumor, the major axis of the tumor and the length (minor axis) in the direction perpendicular to the tumor were measured with a vernier caliper, and the approximate value (major axis X minor axis X minor axis ÷ 2) was used as the tumor volume. Figure 10 is a graph showing the average tumor volume (soil standard deviation) of each group 24 days after transplantation.
mSLC群では Vector群と比べて平均腫瘍体積は 102%であり、 腫瘍形成を抑制 する効果が見られなかった。 mIL2群では Vector群と比べて平均腫瘍体積は 60% であり、 ある程度腫瘍の体積増加を遅らせることが分かる。 mSLC-IL2群ではさ らに腫瘍体積は小さくなっていて、 mIL2群と比べても平均腫瘍体積は 26%であ り、 mIL2群よりも有意に強い抗腫瘍効果が見られた。 一方 mSLC、 mIL2遺伝 子導入細胞を同時に投与した場合、 mIL2群よりも高い抗腫瘍効果が得られたが、 mSLC-IL2群と比較すると抗腫瘍効果は低く、 ばらつきも大きく、 mSLC、 mIL2 遺伝子導入細胞を同時に投与するより、 mSLC-IL2遺伝子導入細胞を単独で投与 する方がより高い抗腫癟効果が得られることが分かった。 産業上の利用可能性  In the mSLC group, the average tumor volume was 102% compared to the Vector group, and no effect was observed in suppressing tumor formation. The average tumor volume in the mIL2 group was 60% compared to the Vector group, indicating that the increase in tumor volume was delayed to some extent. The tumor volume was even smaller in the mSLC-IL2 group, with an average tumor volume of 26% compared to the mIL2 group, showing a significantly stronger antitumor effect than the mIL2 group. On the other hand, when mSLC and mIL2 transfected cells were simultaneously administered, a higher antitumor effect was obtained than in the mIL2 group, but the antitumor effect was lower and more variable than in the mSLC-IL2 group, and the mSLC and mIL2 gene transfection was greater. It was found that administration of the mSLC-IL2 gene-transduced cells alone yielded a higher antitumor effect than administration of the cells simultaneously. Industrial applicability
本発明の融合タンパク質は、 IL-2としての免疫活性化作用に加えて、 ケモカイ ン SLC としての T細胞走化作用を具備することにより、 IL-2 を免疫賦活を必要 とする治療で投与された際に投与部位により多くの T細胞を呼び寄せることによ り、 より多くの T細胞に作用が可能であり、 その結果、 より高い治療効果が得ら れる。  The fusion protein of the present invention has a T cell chemotactic effect as chemokine SLC in addition to an immune activating effect as IL-2, so that it can be administered in a therapy requiring immunostimulation of IL-2. By attracting more T cells to the administration site at the time, more T cells can be acted on, and as a result, a higher therapeutic effect can be obtained.

Claims

請求の範囲 The scope of the claims
1 . N末端側にケモカイン SLCを構成するアミノ酸配列を有し、 かつ IL-2を構 成するアミノ酸配列を含む融合夕ンパク質。 1. A fused protein having an amino acid sequence constituting the chemokine SLC on the N-terminal side and containing an amino acid sequence constituting IL-2.
2 . ケモカイン SLCおよび IL-2がヒ トまたはマウス由来である請求項 1 に記載 の融合タンパク質。  2. The fusion protein according to claim 1, wherein the chemokines SLC and IL-2 are derived from human or mouse.
3 . ケモカイン SLCを構成するァミノ酸配列が配列番号： 2の 2 4位の Serから 1 3 4位の Proに記載のァミノ酸配列であり、かつ IL-2を構成するァミノ酸配列 が配列番号： 4の 2 1位の Alaから 1 5 3位の Thrに記載のァミノ酸配列である 請求項 1 または 2に記載の融合タンパク質。  3. The amino acid sequence that constitutes the chemokine SLC is the amino acid sequence from Ser at position 24 to Ser at position 13 of Pro in SEQ ID NO: 2, and the amino acid sequence that constitutes IL-2 is SEQ ID NO: The fusion protein according to claim 1 or 2, which is an amino acid sequence described in Ala at position 21 of 4 to Thr at position 153.
4 . 配列番号 ： 2の 2 4位の Serから 1 3 4位の Proに記載のアミノ酸配列、 リ ンカ一及び配列番号： 4の 2 1位の Alaから 1 5 3位の Thrに記載のァミノ酸配 列を含む請求項 1から 3のいずれかに記載の融合夕ンパク質。  4. SEQ ID NO: Amino acid sequence from Ser at position 2-4 to Pro at position 134, linker and amino acid sequence from Ala at position 21 to Thr 3 at position 15-3 4. The fusion protein according to claim 1, which contains an acid sequence.
5 . 配列番号 ： 2の 2 4位の Serから 1 3 4位の Proに記載のァミノ酸配列、 リ ンカー及び配列番号： 4の 2 1位の Alaから 1 5 3位の Thrに記載のァミノ酸配 列からなる請求項 4記載の融合夕ンパク質。  5. SEQ ID NO: Amino acid sequence described in Ser at position 134 of position 2 to Pro in position 134, linker and amino acid described in Thr from position Ala of position 21 in SEQ ID NO: 4 5. The fusion protein according to claim 4, comprising an acid sequence.
6 . 配列番号 ： 2の 1位の Metから 1 3 4位の Proに記載のァミノ酸配列、 リ ン 力一及び配列番号： 4の 2 1位の Alaから 1 5 3位の Thrに記載のアミノ酸配列 からなる請求項 4記載の融合夕ンパク質。  6. SEQ ID NO: Amino acid sequence described in Met at position 1 from position 1 to position 134 in Pro, amino acid sequence described in SEQ ID NO: 4 from Ala in position 21 to position 13 in Thr 5. The fusion protein according to claim 4, which comprises an amino acid sequence.
7 . 請求項 5記載の融合夕ンパク質が、 配列番号 ： 6の 2 4位の Serから 2 6 9 位の Thrに記載のァミノ酸配列からなる融合夕ンパク質。 7. The fusion protein according to claim 5, which comprises an amino acid sequence according to SEQ ID NO: 6 from Ser at position 24 to Thr at position 269.
8 . 請求項 6記載の融合夕ンパク質が、 配列番号 ： 6の 1位の Metから 2 6 9位 の Thrに記載のアミノ酸配列からなる融合夕ンパク質。  8. The fusion protein according to claim 6, comprising the amino acid sequence of Met at position 1 of SEQ ID NO: 6 to Thr at position 269 of SEQ ID NO: 6.
9 . 請求項 5記載の融合夕ンパク質が、 配列番号 ： 2 6の 2 4位の Serから 2 6 9位の Thrに記載のアミノ酸配列からなる融合タンパク質。  9. A fusion protein comprising the amino acid sequence of SEQ ID NO: 26 from Ser at position 24 to Thr at position 269, wherein the fusion protein according to claim 5 is SEQ ID NO: 26.
1 0 . 請求項 6記載の融合タンパク質が、 配列番号 ： 2 6の 1位の Metから 2 6 9位の Thrに記載のアミノ酸配列からなる融合タンパク質。 10. The fusion protein according to claim 6, wherein the Met at position 1 of SEQ ID NO: 26 is 26 A fusion protein comprising the amino acid sequence of Thr at position 9.
1 1 . 請求項 3から 1 0のいずれかに記載のアミノ酸配列において 1 も しくは数 個のアミノ酸が、 置換、 欠失、 挿入または付加されたアミノ酸配列を含み、 かつ ケモカイ ン SLC活性および IL-2活性を有する融合夕ンパク質。  11. The amino acid sequence according to any one of claims 3 to 10, wherein one or several amino acids include a substituted, deleted, inserted, or added amino acid sequence, and has chemokine SLC activity and IL. Fusion protein with -2 activity.
1 2 . ケモカイン SLC を構成するァミノ酸配列が配列番号 ： 8の 2 4位の Ser から 1 3 3位の Glyに記載のアミノ酸配列であり、かつ IL-2を構成するァミノ酸 配列が配列番号： 1 0の 2 1位の Alaから 1 6 9位の Ginに記載のアミノ酸配列 である請求項 1 または 2に記載の融合夕ンパク質。  12. The amino acid sequence that constitutes the chemokine SLC is the amino acid sequence from Ser at position 24 to Gly at position 13 of SEQ ID NO: 8, and the amino acid sequence that constitutes IL-2 is SEQ ID NO: The fusion protein according to claim 1 or 2, wherein the amino acid sequence is an amino acid sequence from Ala at position 21 to Gin at position 169.
1 3 . 配列番号 ： 8の 2 4位の Serから 1 3 3位の Glyに記載のアミノ酸配列、 リンカ一及び配列番号： 1 0の 2 1位の Alaから 1 6 9位の Ginに記載のァミノ 酸配列を含む請求項 1 2に記載の融合夕ンパク質。  13. SEQ ID NO: Amino acid sequence described in Gly at position 133 from Ser at position 24 of 8, linker and SEQ ID NO: Described as Gin at position Ala of position 21 from Ala at position 21 13. The fusion protein according to claim 12, comprising an amino acid sequence.
1 4 . 配列番号 ： 8の 2 4位の Serから 1 3 3位の Glyに記載のアミノ酸配列、 リンカ一及び配列番号： 1 0の 2 1位の Alaから 1 6 9位の Ginに記載のァミノ 酸配列からなる請求項 1 3記載の融合タンパク質。  14. SEQ ID NO: 8 Amino acid sequence described in Gly at positions 13 to 3 from Ser at position 24, Linker 1 and SEQ ID NO: Amino acid described in Gin at position 16 from Ala at position 21 14. The fusion protein according to claim 13, comprising an amino acid sequence.
1 5 . 配列番号： 8の 1位の Metから 1 3 3位の Glyに記載のアミノ酸配列、 リ ンカ一及び配列番号： 1 0の 2 1位の Alaから 1 6 9位の Ginに記載のアミノ酸 配列からなる請求項 1 3記載の融合夕ンパク質。  15. SEQ ID NO: 8 Amino acid sequence described in Gly from position 1 to position 133 of Met, linker and SEQ ID NO: SEQ ID NO: Description of amino acid sequence described in Ala in position 21 to Gin in position 169 14. The fusion protein according to claim 13, which comprises an amino acid sequence.
1 6 . 請求領 1 4記載の融合タンパク質が、 配列番号 ： 1 2の 2 4位の Serから 2 8 4位の Ginに記載のアミノ酸配列からなる融合タンパク質。  16. The fusion protein according to claim 14, wherein the fusion protein comprises the amino acid sequence of Ser at position 24 of SEQ ID NO: 12 to Gin at position 284 of SEQ ID NO: 12.
1 7 . 請求項 1 5記載の融合夕ンパク質が、 配列番号 ： 1 2の 1位の Metから 2 8 4位の Ghiに記載のアミノ酸配列からなる融合タンパク質。 17. A fusion protein according to claim 15, wherein the fusion protein comprises the amino acid sequence from Met at position 1 of SEQ ID NO: 12 to Ghi at position 284 of SEQ ID NO: 12.
1 8 . 請求項 1 2から 1 7のいずれかに記載のアミノ酸配列において 1 もしくは 数個のアミノ酸が、 置換、 欠失、 揷入または付加されたアミノ酸配列を含み、 か っケモカイ ン SLC活性および IL-2活性を有する融合夕ンパク質。 18. The amino acid sequence according to any one of claims 12 to 17, wherein one or several amino acids include a substituted, deleted, inserted or added amino acid sequence, and the chemokine SLC activity and Fusion protein having IL-2 activity.
1 9 . 請求項 1から 1 8のいずれかに記載の融合夕ンパク質をコードする DNA。 19. A DNA encoding the fusion protein according to any one of claims 1 to 18.
2 0 . 配列番号 ： 5に記載の 7 0位の aから 8 0 8位の tまでの塩基配列を含む 請求項 1 9記載の DNA。 20. SEQ ID NO: Includes the nucleotide sequence from a at position 70 to t at position 808 described in 5. A DNA according to claim 19.
2 1 . 配列番号 ： 5に記載の 1位の aから 8 0 8位の tまでの塩基配列を含む請 求項 2 0記載の DNA。  21. The DNA according to claim 20, which comprises the nucleotide sequence from a at position 1 to t at position 808 in SEQ ID NO: 5.
2 2 . 配列番号 ： 2 5に記載の 7 0位の aから 8 0 8位の tまでの塩基配列を含 む請求項 1 9記載の DNA。  22. The DNA according to claim 19, comprising the nucleotide sequence from a at position 70 to t at position 808 described in SEQ ID NO: 25.
2 3 . 配列番号 ： 2 5に記載の 1位の aから 8 0 8位の tまでの塩基配列を含む 請求項 2 2記載の DNA。  23. The DNA according to claim 22, comprising the nucleotide sequence from a at position 1 to t at position 808 in SEQ ID NO: 25.
2 4 . 配列番号 ： 1 1に記載の 1位の aから 8 5 2位の aまでの塩基配列を含む 請求項 1 9記載の DNA。  24. The DNA according to claim 19, comprising the base sequence from a at position 1 to a at position 852 in SEQ ID NO: 11.
2 5 . 配列番号 ： 1 1 に記載の 1位の aから 8 5 2位の aまでの塩基配列を含む 請求項 2 4記載の DNA。 25. The DNA according to claim 24, comprising the base sequence from a at position 1 to a at position 852 in SEQ ID NO: 11.
2 6 . 請求項 1 9から 2 5のいずれかに記載の DNA とス ト リ ンジェン トな条件 でハイブリダイズし、 かつケモカイン SLC活性および IL-2活性を有する融合夕 ンパク質をコードする DNA。  26. A DNA that hybridizes with the DNA according to any one of claims 19 to 25 under stringent conditions and encodes a fusion protein having chemokine SLC activity and IL-2 activity.
2 7 . 請求項 1 9から 2 6のいずれかに記載の DNAを含むプラスミ ド。 27. A plasmid comprising the DNA according to any one of claims 19 to 26.
2 8 . 請求項 1 9から 2 6のいずれかに記載の DNAを含む発現べクタ一。 28. An expression vector containing the DNA according to any one of claims 19 to 26.
2 9 . 遺伝子治療用である請求項 2 8に記載の発現べクタ一。  29. The expression vector according to claim 28, which is used for gene therapy.
3 0 . 請求項 2 8または 2 9に記載の発現べクタ一を宿主に導入して得られる形 質転換体。  30. A transformant obtained by introducing the expression vector according to claim 28 or 29 into a host.
3 1 . 請求項 3 0に記載の形質転換体を培養する工程、 および産生された請求項 1から 1 8のいずれかに記載の融合タンパク質を培養培地から回収する工程を包 含する、 該融合タンパク質の製造方法。 31. A method comprising the steps of: culturing the transformant according to claim 30; and recovering the produced fusion protein according to any one of claims 1 to 18 from a culture medium. A method for producing a protein.
3 2 . 請求項 1から 1 8のいずれかに記載の融合タンパク質を含む医薬組成物。  32. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 18.
3 3 . 請求項 2 9記載のベクタ一を用いた癌の治療方法。 33. A method for treating cancer using the vector according to claim 29.
3 4 . 請求項 3 2記載の医薬組成物を用いた癌の治療方法。 34. A method for treating cancer using the pharmaceutical composition according to claim 32.
3 5 . 癌治療薬を製造するための請求項 3 2記載の医薬組成物の使用。  35. Use of the pharmaceutical composition according to claim 32 for producing a therapeutic agent for cancer.
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