WO2001040311A1 - Proteine fusionnee de chimiokine slc-il2 - Google Patents

Proteine fusionnee de chimiokine slc-il2 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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English (en)
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/ja
Publication of WO2001040311A1 publication Critical patent/WO2001040311A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—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

L'invention concerne une protéine fusionnée comportant la séquence d'acides aminés comprise entre la sérine en position 24 et la proline en position 134 dans la séquence ID NO :2 à l'extrémité N, et impliquant la séquence d'acides aminés comprise entre l'alanine en position 21 et la thrombine en position 153 dans la séquence ID NO :4.
PCT/JP2000/008325 1999-11-30 2000-11-27 Proteine fusionnee de chimiokine slc-il2 WO2001040311A1 (fr)

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CN100342017C (zh) * 2004-05-10 2007-10-10 中国医学科学院肿瘤医院肿瘤研究所 基因重组趋化抗原疫苗
CN102028958A (zh) * 2010-12-24 2011-04-27 中国医学科学院肿瘤研究所 以细菌纳米磁小体为载体的复合肿瘤基因疫苗及其制备方法
JP2016503434A (ja) * 2012-12-05 2016-02-04 ナショナル チュン シン ユニバーシティ ケモカイン−サイトカイン融合タンパク質およびその利用

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WO2000038706A2 (fr) * 1998-12-31 2000-07-06 Chiron Corporation Methodes de traitement du cancer et de mediation de la chimiotaxie des cellules dendritiques

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EP0118617A2 (fr) * 1982-12-15 1984-09-19 Ajinomoto Co., Inc. Interleukine-2 polypeptides
EP0177357A1 (fr) * 1984-10-05 1986-04-09 Schering Biotech Corporation Clones de cADN codant pour polypeptides montrant une activité de murine-interleukine-2
EP0288809A1 (fr) * 1987-04-16 1988-11-02 Hoechst Aktiengesellschaft Protéines bifonctionnelles
WO1997023639A1 (fr) * 1995-12-22 1997-07-03 Toray Industries, Inc. Procede de production de proteines fusionnees biologiquement actives
WO1998014581A1 (fr) * 1996-10-02 1998-04-09 Schering Corporation Chemokines mammaliennes
WO2000038706A2 (fr) * 1998-12-31 2000-07-06 Chiron Corporation Methodes de traitement du cancer et de mediation de la chimiotaxie des cellules dendritiques

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100342017C (zh) * 2004-05-10 2007-10-10 中国医学科学院肿瘤医院肿瘤研究所 基因重组趋化抗原疫苗
CN102028958A (zh) * 2010-12-24 2011-04-27 中国医学科学院肿瘤研究所 以细菌纳米磁小体为载体的复合肿瘤基因疫苗及其制备方法
JP2016503434A (ja) * 2012-12-05 2016-02-04 ナショナル チュン シン ユニバーシティ ケモカイン−サイトカイン融合タンパク質およびその利用
EP2930189A4 (fr) * 2012-12-05 2016-06-08 Nat Univ Chung Hsing Protéine de fusion chimiokine-cytokine et son utilisation
AU2012396113B2 (en) * 2012-12-05 2017-03-30 National Chung Hsing University Chemokine-cytokine fusion protein and application thereof

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