WO2024096038A1 - Method for producing active hgf - Google Patents

Method for producing active hgf Download PDF

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WO2024096038A1
WO2024096038A1 PCT/JP2023/039364 JP2023039364W WO2024096038A1 WO 2024096038 A1 WO2024096038 A1 WO 2024096038A1 JP 2023039364 W JP2023039364 W JP 2023039364W WO 2024096038 A1 WO2024096038 A1 WO 2024096038A1
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hgf
chain
cells
amino acid
acid sequence
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PCT/JP2023/039364
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French (fr)
Japanese (ja)
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善行 茅野
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クリングルファーマ株式会社
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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
    • C07K14/475—Growth factors; Growth regulators
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • This disclosure relates to a method for producing active HGF.
  • Hepatocyte growth factor is synthesized in vivo as an inactive single-chain HGF, and then activated by proteases to become an active double-chain HGF. Since single-chain HGF has no biological activity, conversion to double-chain HGF is essential for the expression of biological activity.
  • Proteases that activate HGF include HGF activator (HGFA), uPA (urokinase-type plasminogen activator), tPA (tissue-type plasminogen activator), matriptase, hepsin, and the like, which are contained in blood.
  • HGF activator HGFA
  • uPA urokinase-type plasminogen activator
  • tPA tissue-type plasminogen activator
  • matriptase hepsin, and the like
  • Fetal bovine serum has been added to culture of animal cells, but not using fetal bovine serum can reduce production costs and can avoid the possibility of contamination of protein preparations with viruses or abnormal prions derived from fetal bovine serum. Therefore, methods for producing active HGF, such as those used as pharmaceutical preparations, under serum-free conditions have been developed (Patent Documents 1 to 4). However, all of these methods require the production of single-chain HGF or its modified form and conversion to double-chain HGF by enzyme or chemical reaction.
  • the objective of this disclosure is to provide a method for producing active HGF by expressing both the HGF ⁇ chain and the HGF ⁇ chain in the same host cell.
  • active HGF can be recovered from the culture supernatant of host cells by inserting DNA encoding an amino acid sequence containing the HGF ⁇ chain and DNA encoding an amino acid sequence containing the HGF ⁇ chain into a vector for expressing a protein and then co-introducing the resulting vectors into host cells, and have continued their research based on this discovery.
  • a method for producing an active HGF comprising the steps of: Step 1: Step 2: A step of preparing DNA encoding an amino acid sequence containing an HGF ⁇ chain and a step of preparing DNA encoding an amino acid sequence containing an HGF ⁇ chain Step 3: Insert each DNA prepared in step 1 into a vector for expressing a protein. Step 4: Co-introducing the vectors obtained in step 2 into host cells and culturing the host cells to express HGF ⁇ -chain and HGF ⁇ -chain in the host cells. [2] A process for recovering active HGF from the culture supernatant after step 3.
  • each of the amino acid sequence containing the HGF ⁇ chain and the amino acid sequence containing the HGF ⁇ chain contains a signal sequence.
  • the signal sequence is an HGF signal sequence.
  • the HGF signal sequence is a human HGF signal sequence.
  • [6] The method according to any one of [1] to [5] above, characterized in that the DNA encoding an amino acid sequence including an HGF ⁇ chain and/or the DNA encoding an amino acid sequence including an HGF ⁇ chain is a DNA modified to contain codons suitable for the host cell.
  • the HGF ⁇ chain is a human HGF ⁇ chain.
  • the human HGF ⁇ chain consists of the amino acid sequence shown in SEQ ID NO: 13 or 19.
  • [9] The method according to any one of [1] to [8] above, wherein the HGF ⁇ chain is a human HGF ⁇ chain.
  • the human HGF ⁇ chain consists of the amino acid sequence shown in SEQ ID NO:16.
  • the host cell is selected from the group consisting of CHO-K1 cells, CHO-DG44 (DHFR-) cells, CHO-S cells, CHO-MK cells, CHO-GS cells, HEK293 cells, BHK-21 cells, PER.C6 cells, NS0 cells, SP2/0 cells, Sf9 cells, Sf21 cells, High Five cells, Saccharomyces cerevisiae, and Pichia pastoris.
  • the present disclosure provides a method for producing active HGF by expressing the HGF ⁇ chain and the HGF ⁇ chain in the same host cell.
  • active HGF can be produced without using serum and/or enzymes, reducing production costs and being highly advantageous industrially.
  • contamination with viruses and the like derived from serum can be avoided, making it possible to produce safe active HGF.
  • FIG. 1 shows the results of Western blotting in an example.
  • FIG. 2 shows the results of an evaluation test of cell proliferation-promoting activity in an example.
  • FIG. 3 shows the results of an evaluation test of cell proliferation-promoting activity in an example.
  • the method for producing activated HGF disclosed herein (also referred to as the method of the present disclosure in this specification) is characterized by including the following steps 1 to 4.
  • the method disclosed herein includes the following steps 1 to 4, and may include other steps as desired, or may consist of the following steps 1 to 4.
  • the method disclosed herein is preferably a method that does not use serum and/or enzymes.
  • Step 1 A step of preparing a DNA encoding an amino acid sequence containing an HGF ⁇ chain and a DNA encoding an amino acid sequence containing an HGF ⁇ chain.
  • Step 2 A step of inserting each of the DNAs prepared in step 1 into a vector for expressing a protein.
  • Step 3 A step of co-introducing the vectors obtained in step 2 into host cells and culturing the host cells to express the HGF ⁇ chain and the HGF ⁇ chain in the host cells.
  • Step 4 A step of recovering active HGF from the culture supernatant after step 3.
  • the method of the present disclosure comprises, as step 1, the step of preparing a DNA encoding an amino acid sequence containing an HGF ⁇ chain and a DNA encoding an amino acid sequence containing an HGF ⁇ chain.
  • HGF ⁇ chain and HGF ⁇ chain are proteins that constitute active HGF.
  • each of the ⁇ chain and ⁇ chain may be, for example, derived from humans or from animals other than humans (e.g., dogs, cats, rats, mice, rabbits, cows, chimpanzees, horses, pigs, sheep, etc.), but is preferably derived from humans (human HGF ⁇ chain, human HGF ⁇ chain).
  • humans e.g., dogs, cats, rats, mice, rabbits, cows, chimpanzees, horses, pigs, sheep, etc.
  • human HGF ⁇ chain human HGF ⁇ chain
  • a preferred example is one in which the ⁇ chain is human HGF ⁇ chain and the ⁇ chain is human HGF ⁇ chain.
  • Examples of the human HGF ⁇ chain include the human full-length HGF ⁇ chain consisting of the amino acid sequence shown in SEQ ID NO: 19, and the human 5-amino acid deleted HGF ⁇ chain consisting of the amino acid sequence shown in SEQ ID NO: 13.
  • Examples of the human HGF ⁇ chain include the human full-length HGF ⁇ chain consisting of the amino acid sequence shown in SEQ ID NO: 16.
  • Amino acid sequence information for HGF, HGF ⁇ chain, HGF ⁇ chain, etc. derived from animals other than humans can be obtained from publicly known databases (GenBank/EMBL/DDBJ, etc.).
  • the ⁇ -chain and ⁇ -chain of the present disclosure may each be a mutant having one to several amino acids added, deleted or substituted in the amino acid sequence (e.g., 2 to 150, more preferably 2 to 80, more preferably 2 to 70, more preferably 2 to 60, more preferably 2 to 50, more preferably 2 to 40, more preferably 2 to 30, more preferably 2 to 20, more preferably 2 to 10, or 2 to 5, or more preferably 1 to 5; the same applies below).
  • 2 to 150 more preferably 2 to 80, more preferably 2 to 70, more preferably 2 to 60, more preferably 2 to 50, more preferably 2 to 40, more preferably 2 to 30, more preferably 2 to 20, more preferably 2 to 10, or 2 to 5, or more preferably 1 to 5; the same applies below.
  • the ⁇ chain of the present disclosure includes a polypeptide having amino acids that show at least 80%, preferably 85%, and more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of the ⁇ chain.
  • the ⁇ chain of the present disclosure includes a polypeptide having amino acids that show at least 80%, preferably 85%, and more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of the ⁇ chain.
  • amino acid sequence containing HGF ⁇ chain and amino acid sequence containing HGF ⁇ chain may contain an amino acid sequence other than the amino acid sequence of the HGF ⁇ chain.
  • the amino acid sequence containing the HGF ⁇ chain may contain an amino acid sequence other than the amino acid sequence of the HGF ⁇ chain.
  • Examples of amino acid sequences other than the HGF ⁇ chain and the HGF ⁇ chain include signal sequences, affinity tags, and the like. All of these are within the technical scope of the present disclosure.
  • the amino acid sequence containing the HGF ⁇ chain and/or the amino acid sequence containing the HGF ⁇ chain preferably contains a signal sequence, and it is more preferable that each of the amino acid sequence containing the HGF ⁇ chain and the amino acid sequence containing the HGF ⁇ chain contains a signal sequence.
  • the amino acid sequence containing the HGF ⁇ chain and/or the amino acid sequence containing the HGF ⁇ chain contains a signal sequence
  • the ⁇ chain and the ⁇ chain expressed in the host cell can be actively secreted outside the host cell in step 3 described below.
  • the signal sequence include an HGF signal sequence, an insulin signal sequence, and an ⁇ -interferon signal sequence.
  • the human HGF signal sequence (an HGF signal sequence derived from a human; for example, the amino acid sequence shown in SEQ ID NO: 21) is preferably used as the signal sequence.
  • the signal sequence can be added using a known method, and for example, the method described in J. Biol. Chem., 264, 17619 (1989), Proc. Natl. Acad. Sci. , USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990), JP-A-5-336963, WO94/23021, and the like.
  • the DNA encoding an amino acid sequence containing the HGF ⁇ chain is not particularly limited as long as it is a DNA encoding an amino acid sequence containing the HGF ⁇ chain described above, but it is preferable that the sequence contains a base sequence of a signal sequence at the 5' end and/or a stop codon at the 3' end of the sequence.
  • An example of the DNA encoding the full-length human HGF ⁇ chain shown in SEQ ID NO: 19 is the base sequence shown in SEQ ID NO: 20.
  • An example of the DNA encoding the five amino acid deleted human HGF ⁇ chain shown in SEQ ID NO: 13 is the base sequence shown in SEQ ID NO: 14 or 15.
  • DNA encoding an amino acid sequence containing the HGF ⁇ chain suitable examples include DNA containing the base sequence shown in SEQ ID NO: 14, 15 or 20, such as DNA consisting of SEQ ID NO: 2, 3 or 8.
  • the DNA encoding an amino acid sequence containing the HGF ⁇ chain is not particularly limited as long as it is a gene encoding an amino acid sequence containing the HGF ⁇ chain described above, but it is preferable that the sequence contains a base sequence of a signal sequence at the 5' end and/or a stop codon at the 3' end of the sequence.
  • An example of a DNA encoding an amino acid sequence containing the human HGF ⁇ chain shown in SEQ ID NO:16 is the base sequence shown in SEQ ID NO:18.
  • a suitable example is a DNA containing the base sequence shown in SEQ ID NO: 17 or 18, such as a DNA consisting of SEQ ID NO: 5 or 6.
  • the base sequence information of genes for HGF, HGF ⁇ chain, HGF ⁇ chain, etc. derived from animals other than humans can be obtained from publicly known databases (GenBank/EMBL/DDBJ, etc.).
  • the DNA encoding an amino acid sequence containing the HGF ⁇ chain and/or the DNA encoding an amino acid sequence containing the HGF ⁇ chain is preferably DNA that has been modified to have codons suitable for the host cell according to a method known per se.
  • Hosts into which the above vectors are introduced include, for example, animal cells, insect cells, yeast cells, and the like.
  • Animal cells include, for example, Chinese hamster cells (sometimes abbreviated as CHO cells in this specification), monkey cells COS-7, Vero, dhfr gene-deficient CHO cells (hereinafter abbreviated as CHO (DHFR-) cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, human fetal kidney cells (HEK293 cells), Syrian hamster kidney (BHK-21 cells), fetal retina cells (PER.C6 cells), mouse myeloma cells (NS0 cells), mouse myeloma cells (SP2/0 cells), etc.
  • CHO Chinese hamster cells
  • Vero Vero, dhfr gene-deficient CHO cells
  • mouse L cells mouse AtT-20
  • mouse myeloma cells rat GH3, human FL cells
  • human fetal kidney cells HEK293 cells
  • BHK-21 cells human fetal retina cells
  • PER.C6 cells mouse myeloma cells
  • CHO cells include, for example, CHO-K1 cells, CHO-S cells, CHO-MK cells, CHO-GS cells, etc.
  • CHO (DHFR-) cells include, for example, CHO-DG44 (DHFR-) cells, etc.
  • yeast examples include Saccharomyces cerevisiae, Pichia pastoris, AH22R-, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, etc.
  • Saccharomyces cerevisiae examples include Saccharomyces cerevisiae AH22, etc.
  • insect cells examples include Sf9 cells (a clone isolate of Spodoptera frugiperda SF21 cells), Sf21 cells (Spodoptera frugiperda SF21 cells), and High Five cells (a BTI-TN-5B1-4 clone of the moth larva Trichoplusia ni).
  • the host cell is preferably selected from the group consisting of CHO-K1 cells, CHO-DG44 (DHFR-) cells, CHO-S cells, CHO-MK cells, CHO-GS cells, HEK293 cells, BHK-21 cells, PER. C6 cells, NS0 cells, SP2/0 cells, Saccharomyces cerevisiae and Pichia pastoris.
  • degeneracy of the genetic code refers, for example, to an oligonucleotide consisting of three nucleotides that codes for a given amino acid. Due to the degeneracy of the genetic code, most amino acids are coded for by multiple codons. It is known that the relative frequency of use of these different codons that code for the same amino acid varies in individual host cells. Therefore, to design a DNA sequence that codes for an amino acid sequence, one or more codons corresponding to each amino acid can be used.
  • a base sequence that codes for a single amino acid sequence possessed by a certain peptide may have multiple variations.
  • appropriate codons can be selected according to the codon usage of the host cell for expression into which a polynucleotide containing the DNA sequence or a vector containing the same is introduced, or the frequency or ratio of use of multiple codons can be appropriately adjusted.
  • the base sequence may be designed using codons that are frequently used in CHO cells. In accordance with the frequency of codon usage in the host cell, it is preferable to select codons that have high translation efficiency in the host cell species, and it is more preferable to select codons that have the highest translation efficiency.
  • a method of modifying codons to be suitable for the host cell can be carried out by referring to, for example, Gustafsson, C. et al. (Trends in biotechnology 22.7 (2004): 346-353). Information on codons used by various organisms is available from the Codon Usage Database (www.kazusa.or.jp/codon/) and the like.
  • the percentage of codons that are substituted to become codons suitable for the host cell may be 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, or 90-100%.
  • An example of a base sequence in which the base sequence shown in SEQ ID NO:15 (a base sequence encoding an amino acid sequence containing a human 5-amino acid deleted HGF ⁇ chain shown in SEQ ID NO:13) has been modified to have codons suitable for CHO cells is the base sequence shown in SEQ ID NO:14.
  • An example of a base sequence in which the base sequence shown in SEQ ID NO:3 (a base sequence encoding an amino acid sequence containing a human 5-amino acid deleted HGF ⁇ chain with a human HGF signal sequence bound to the N-terminus shown in SEQ ID NO:1) has been modified to have codons suitable for CHO cells is the base sequence shown in SEQ ID NO:2.
  • An example of a base sequence in which the base sequence shown in SEQ ID NO:18 (a base sequence encoding an amino acid sequence containing a human HGF ⁇ chain, as shown in SEQ ID NO:16) has been modified to have codons suitable for CHO cells is the base sequence shown in SEQ ID NO:17.
  • An example of a base sequence in which the base sequence shown in SEQ ID NO:6 (a base sequence encoding an amino acid sequence containing a human HGF ⁇ chain to which a human HGF signal sequence is attached at the N-terminus, as shown in SEQ ID NO:4) has been modified to have codons suitable for CHO cells is the base sequence shown in SEQ ID NO:5.
  • the base sequence shown in SEQ ID NO:24 (the base sequence encoding the signal sequence of human HGF shown in SEQ ID NO:21) is modified to have codons suitable for CHO cells, and examples of such sequences include the base sequences shown in SEQ ID NO:22 and SEQ ID NO:23.
  • the DNA encoding an amino acid sequence containing the HGF ⁇ chain and the DNA encoding an amino acid sequence containing the HGF ⁇ chain may each be genomic DNA, a genomic DNA library, cDNA derived from cells or tissues, a cDNA library derived from cells or tissues, synthetic DNA, etc., but is preferably synthetic DNA.
  • the DNA encoding the amino acid sequence containing the HGF ⁇ chain and the DNA encoding the amino acid sequence containing the HGF ⁇ chain may each contain a restriction enzyme (e.g., HindIII, XhoI, etc.) recognition sequence, a linker, a start codon (also called a translation start codon, examples of which include ATG, etc.), a stop codon (also called a translation stop codon, examples of which include TAA, TGA, or TAG, etc.) etc.
  • a restriction enzyme e.g., HindIII, XhoI, etc.
  • the DNA encoding the amino acid sequence containing the HGF ⁇ chain and/or the DNA encoding the amino acid sequence containing the HGF ⁇ chain preferably contains a stop codon at the 3' end, and it is more preferable that the DNA encoding the amino acid sequence containing the HGF ⁇ chain and the DNA encoding the amino acid sequence containing the HGF ⁇ chain each contain a stop codon.
  • Step 1 a DNA encoding an amino acid sequence containing an HGF ⁇ -chain and a DNA encoding an amino acid sequence containing an HGF ⁇ -chain are prepared.
  • Methods for preparing each of the above DNAs include a method of isolating and extracting each DNA fragment from human or non-human animal cells and cloning it, a method using an amplification means such as PCR, and a method of synthesizing using a DNA synthesizer.
  • Step 2 The method of the present disclosure includes, as step 2, inserting each of the DNAs prepared in step 1 into a vector for expressing a protein.
  • each DNA prepared in step 1 can be inserted into a vector for expressing a protein.
  • the vector contains a promoter, a ribosome binding site, a terminator, a drug resistance gene (selection marker), etc., as necessary.
  • a vector for expressing the HGF ⁇ chain may be constructed in accordance with a conventional method, for example, so as to contain, in the order of downstream transcription, (1) a promoter, (2) a ribosome binding site, (3) a DNA encoding an amino acid sequence including the HGF ⁇ chain, and (4) a terminator.
  • a vector for expressing the HGF ⁇ chain may be constructed in accordance with a conventional method, for example, so as to contain, in the order of downstream transcription, (1) a promoter, (2) a ribosome binding site, (3) a DNA encoding an amino acid sequence including the HGF ⁇ chain, and (4) a terminator.
  • Vectors that can be used in the present disclosure include, when an animal cell is used as a host, pcDNA3.1, pCAGGS [Niwa, H., Yamamura, K. and Miyazaki, J., Gene, Vol. 108, pp.
  • pBK-CMV pZeoSV
  • pCAGGS pCXN2
  • pcDL-SR ⁇ Takebe, Y. et al., Mol. Cell. Biol, Vol. 8, pp. 466-472 (1988)] and the like.
  • yeast When yeast is used as a host, examples of the plasmid include pYES2 (Invitrogen) and pRB15 (ATCC37062).
  • the vector that can be used in the present disclosure is not particularly limited as long as it can be replicated or amplified in the host.
  • Insertion of DNA into a vector for protein expression The DNA prepared in step 1 is inserted into a vector.
  • the method of insertion is not particularly limited, and can be performed by a known method, for example.
  • the vector for example, the above-mentioned vector, preferably a plasmid, can be inserted.
  • a DNA encoding an amino acid sequence including an HGF ⁇ chain may be inserted into a certain vector, and a DNA encoding an amino acid sequence including an HGF ⁇ chain may be inserted into another vector.
  • each of the above DNAs may be inserted into one vector.
  • it can be confirmed by a known method, for example, sequence analysis by the Sanger method, restriction enzyme digestion, etc.
  • Step 3 The method of the present disclosure includes, as step 3, co-introducing the vectors obtained in step 2 into host cells and culturing the host cells to express HGF ⁇ chain and HGF ⁇ chain in the host cells.
  • Animal cells can be transformed, for example, according to the method described in "Cell Engineering Special Issue 8: New Cell Engineering Experimental Protocols, pp. 263-267 (1993) (published by Gakken Medical Shujunsha)" or "Virology, vol. 52, p. 456 (1973)".
  • Yeast can be transformed, for example, according to the method described in "Methods in Enzymology, vol. 194, p. 182-187 (1991)” or "Proc. Natl. Acad. Sci. USA, 75, p. 1929 (1978)".
  • the vectors obtained in step 2 can be co-introduced into host cells by known methods such as a physical method of applying a high voltage to cells for introduction, and a chemical method of imparting receptivity to cells by calcium treatment for introduction.
  • chemical methods include a method in which a vector for expressing HGF ⁇ and a vector for expressing HGF ⁇ are mixed with a transfection reagent such as Lipofectamine (registered trademark) and a known appropriate medium, incubated at room temperature appropriately (for example, about 10 minutes), and then the vector is added to host cells and incubated at about 37°C under about 5% CO2 conditions for about 4 hours.
  • the medium is preferably a serum-free medium such as Opti-MEM (registered trademark) I Reduced Serum Medium or P3000 reagent.
  • co-introduction means, for example, introduction so that HGF ⁇ and HGF ⁇ can be expressed in one cell. That is, when a DNA encoding an amino acid sequence including an HGF ⁇ -chain is inserted into one vector and a DNA encoding an amino acid sequence including an HGF ⁇ -chain is inserted into another vector, these vectors are introduced into the same host cell (introduction 1).When each of the DNAs is inserted into the same vector, the vector is introduced into a host cell (introduction 2).
  • the terms "to the extent” and “about” are used with the intention of including, for example, small deviations. Such ranges also include those within the experimental error (e.g., ⁇ 2, ⁇ 1, etc.) that is inherent in the standard method used to measure and/or quantify a given value or range. Thus, it can be said that “to the extent” and “about” are not necessarily ambiguous.
  • cells expressing both HGF ⁇ -chain and HGF ⁇ -chain may be selected.
  • Cells expressing both HGF ⁇ -chain and HGF ⁇ -chain can be selected using a selection marker such as a drug resistance gene carried by a vector. Alternatively, they can be selected by cloning.
  • the cells may be cells that transiently express HGF ⁇ -chain and HGF ⁇ -chain, or cells that stably express HGF ⁇ -chain and HGF ⁇ -chain, preferably stably expressing cells.
  • the host cells thus obtained are cultured by conventional means known to those skilled in the art.
  • the medium is preferably a serum-free medium such as Opti-MEM (registered trademark) I Reduced Serum Media, P3000 reagent, etc.
  • suitable medium include MEM medium containing about 5 to 20% fetal bovine serum (Science, 122, p501 (1952)), DMEM medium (Virology, 8, p396 (1959)), Ham's F-12 (containing L-glutamine and phenol red) medium, RPMI 1640 medium (The Journal of the American Medical Association, 199, p519 (1967)), 199 medium (Proceedings of the Society for the Biological Medicine, 73, p1 (1950)) or the like.
  • the pH is preferably about 6 to 8.
  • the culture is usually carried out at about 30°C to 40°C for about 15 to 60 hours, with aeration and stirring as necessary.
  • the culture is preferably carried out under 5% CO2 conditions.
  • examples of the medium include Sf-900III (trade name) (Thermo Fisher Scientific), EX-CELL405 (trade name) (Merck), and Express Five SFM (trade name) (Thermo Fisher Scientific).
  • the pH of the medium is preferably adjusted to about 5 to 7. Cultivation is usually carried out at about 20°C to 30°C for about 24 to 72 hours, with aeration and stirring as necessary.
  • examples of media include Burkholder's minimal medium (Bostian, K. L. et al., Proc. Natl. Acad. Sci. USA, 77, p. 4505 (1980)) and SD medium containing 0.5% casamino acids (Bitter, G. A. et al., Proc. Natl. Acad. Sci. USA, 81, p. 5330 (1984)).
  • the pH of the medium is preferably adjusted to about 5 to 8. Cultivation is usually carried out at about 20°C to 35°C for about 24 to 72 hours, with aeration and stirring as necessary.
  • DNA encoding an amino acid sequence including an HGF alpha chain and DNA encoding an amino acid sequence including an HGF beta chain are each prepared in step 1, and therefore, in step 3, the HGF alpha chain and the HGF beta chain are each expressed in a host cell.
  • Step 4 The method of the present disclosure includes, as step 4, after step 3, a step of recovering active HGF from the culture supernatant.
  • active HGF can be recovered or obtained by known methods, such as a method of collecting the culture medium, centrifuging it, and recovering the culture supernatant.
  • the active HGF may be one to which an affinity tag has been added in order to facilitate the purification.
  • the tag examples include a FLAG tag, a histidine tag, a c-Myc tag, an HA tag, an AU1 tag, a GST tag, an MBP tag, a fluorescent protein tag (e.g., GFP, YFP, RFP, CFP, BFP, etc.), an immunoglobulin Fc tag, etc.
  • the position to which the tag sequence is added may be the N-terminus or the C-terminus of the active HGF.
  • the active HGF to which an affinity tag has been added may be used as it is, or may be used after the affinity tag has been cleaved off.
  • the host cell is selected from the group consisting of CHO-K1 cells, CHO-DG44 (DHFR-) cells, CHO-S cells, CHO-MK cells and CHO-GS cells, and that the DNA encoding the human HGF ⁇ chain that has been modified to have codons suitable for the host cell comprises the base sequence shown in SEQ ID NO: 2, and/or the DNA encoding the human HGF ⁇ chain that has been modified to have codons suitable for the host cell comprises the base sequence shown in SEQ ID NO: 5.
  • the HGF can be confirmed to be an active HGF.
  • “equivalent” may mean that the activity is 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more compared to the activity of the active HGF positive control.
  • HGF activity can be confirmed by evaluating the proliferation and migration of cells to which HGF has been added, or the phosphorylation of Met, an HGF receptor, etc. Methods for confirming HGF activity are described in J. Immunol. Methods. 2001; 258: 1-11, Nat. Commun. 2015; 6: 6373, etc.
  • active HGF Due to its activity (pharmacological action), active HGF is useful as a wound healing agent such as a spinal cord injury therapeutic agent, a preventive or healing agent for skin ulcers, an angiogenesis promoter, a granulation proliferation promoter, a preventive or therapeutic agent for asthma, an agent for improving intellectual disability, a therapeutic agent for polyglutamine disease or an agent for suppressing the onset of the disease, a liver cirrhosis therapeutic agent, a kidney disease therapeutic agent, an epithelial cell proliferation promoter, an anticancer agent, an agent for preventing side effects in cancer therapy, a lung injury therapeutic agent, a gastric/duodenal injury therapeutic agent, a cranial nerve disorder therapeutic agent, an agent for preventing immunosuppressive side effects (for example, an agent for suppressing fibrosis of transplanted organs associated with the administration of immunosuppressants), a collagen decomposition promoter, a cartilage injury therapeutic agent, an arterial disease therapeutic agent, a pulmonary fibrosis therapeutic
  • DNA encoding an amino acid sequence containing an HGF ⁇ chain and DNA encoding an amino acid sequence containing an HGF ⁇ chain are each expressed in step 3, surprisingly, active HGF is recovered from the culture supernatant in step 4. It is presumed that thiol (SH) groups present in the HGF ⁇ -chain and ⁇ -chain expressed in step 3 are coupled in the cells to form disulfide bonds in step 3 or 4.
  • SH thiol
  • Step 1 DNA of SEQ ID NO: 2 (a base sequence in which the base sequence of a human 5 amino acid deletion HGF ⁇ chain in which the base sequence of a human HGF signal sequence is linked to the 5'-end side is modified to become a codon suitable for CHO, and a stop codon TGA is added to the 3'-end side) and DNA of SEQ ID NO: 5 (a base sequence in which the base sequence of a human HGF ⁇ chain in which the base sequence of a human HGF signal sequence is linked to the 5'-end side is modified to become a codon suitable for CHO, and a stop codon TGA is added to the 3'-end side) were added with restriction enzyme HindIII to the 5'-end and with XhoI recognition sequence to the 3'-end (two types of DNA; DNA of HGF ⁇ chain and DNA of HGF ⁇ chain). Specifically, DNA of a base sequence optimized for CHO codons
  • Step 2 The DNA of the HGF ⁇ chain obtained in step 1 and the plasmid pcDNA3.1(+) were mixed in a tube. Separately, the DNA of the HGF ⁇ chain obtained in step 1 and the plasmid pcDNA3.1(+) were mixed in a tube. The mixtures in each tube thus obtained were treated with HindIII and XhoI, and ligated with DNA ligase to prepare HGF ⁇ expression plasmid and HGF ⁇ expression plasmid in separate tubes. The prepared expression plasmids were confirmed to be correctly prepared by sequence analysis by the Sanger method and digestion with restriction enzymes.
  • CHO-K1 cells (RIKEN BioResource Research Center, RCB0285) were seeded at 5 x 10 5 cells/mL (2 mL volume) per well in a 6-well plate (Corning, 3516).
  • Ham's F-12 (containing L-glutamine and phenol red) medium (Fujifilm Wako Pure Chemical Industries, 087-08335) containing 10% fetal bovine serum (Nichirei Biosciences, 174012-500 mL) was used as the medium.
  • Step 4 The culture medium was collected 48 hours after gene transfer and centrifuged at 10,000 rpm for 1 minute, and the supernatant was recovered to prepare a sample for the example.
  • pHGF ⁇ pHGF ⁇ was prepared in the same manner as in the manufacturing method of the sample of the above Example, except that the plasmids (1.25 ⁇ g of HGF ⁇ expression plasmid and 1.25 ⁇ g of HGF ⁇ expression plasmid to be introduced into cells) were replaced with "1.25 ⁇ g of HGF ⁇ expression plasmid.”
  • pHGF ⁇ pHGF ⁇ was prepared in the same manner as in the manufacturing method of the sample of the above Example, except that the plasmids (1.25 ⁇ g of HGF ⁇ expression plasmid and 1.25 ⁇ g of HGF ⁇ expression plasmid to be introduced into cells) were replaced with "1.25 ⁇ g of HGF ⁇ expression plasmid.”
  • the filter paper, PVDF membrane, gel, and filter paper were placed in this order on the lower electrode plate of a blotting device (Bio-Rad, Trans-Blot SD Cell) using a Q-blot kit (ATTO, WSE-4055), and air bubbles were removed before sandwiching the upper electrode plate. Blotting was performed by applying a constant voltage of 12 V for 30 minutes using a power supply (Bio-Rad, Power Pack HC).
  • the PVDF membrane was incubated in blocking buffer (ATTO, AE-1475) at room temperature for 30 minutes, and then incubated overnight in a cold place with anti-HGF antibody (R&D systems, Anti-Human HGF Affinity Purified Polyclonal Ab, AF294-SP) diluted 2000-fold with PBST buffer containing 0.5% bovine serum albumin. After washing with PBST, the membrane was incubated at room temperature for 2 hours with rabbit anti-goat IgG-HRP (DAKO, P0449) diluted 1000-fold with PBST buffer containing 0.5% bovine serum albumin. After washing with PBST, the plate was reacted with a detection substrate (ATTO, WSE-7120S) and the luminescence was detected with a luminometer image analyzer (FUJIFILM, LAS-1000).
  • a detection substrate ATTO, WSE-7120S
  • FUJIFILM luminometer image analyzer
  • CM means CHO cell culture supernatant.
  • the method of the present disclosure can produce HGF in which an HGF ⁇ chain protein with a secretion signal cleaved and an HGF ⁇ chain protein with a secretion signal cleaved are heterodimerized by a disulfide bond.
  • Cell proliferation promoting activity evaluation test (Test method) 1. Preparation of cells Mv.1.Lu cells (RIKEN BioResource Research Center, RCB0996) were thawed and cultured in 5 mL of MEM medium (containing Earle's salts and L-glutamine) (Nacalai Tesque, Cat. No. 21442-25) containing 10% fetal bovine serum (Nichirei Biosciences, 174012-500 mL) and 1% MEM non-essential amino acid solution (x100) (Thermo Fisher, Cat. No. 11140-050) in a T-25 flask (Corning, 430639) at 37°C in the presence of 5% CO2 .
  • MEM medium containing Earle's salts and L-glutamine
  • MEM medium containing Earle's salts and L-glutamine
  • x100 MEM non-essential amino acid solution
  • the medium in the flask was discarded. After washing with 2 mL of PBS (-) (Thermo Fisher, Cat. No. 20012-027), 2 mL of cell dissociation enzyme (Thermo Fisher, Cat. No. 12563011) was added and spread over the bottom of the flask. The cell dissociation enzyme was removed and the flask was left at 37°C for 3 minutes. 15 mL of RPMI1640 medium (Thermo Fisher, Cat. No. 11875-093) containing 2% fetal bovine serum (Nichirei Biosciences, 174012-500 mL) was added, and the cells were collected by pipetting.

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Abstract

The present invention addresses the problem of providing a method for producing active HGF in an industrially advantageous manner, by expressing HGF α chain and HGF β chain in the same host cell.　The present disclosure provides a method for producing active HGF, the method comprising the following steps 1-4. Step 1: A step for preparing DNA encoding an amino acid sequence including HGF α chain and DNA encoding an amino acid sequence including HGF β chain. Step 2: A step for inserting respective strands of DNA prepared in step 1 into vectors for expressing protein. Step 3: A step for expressing HGF α chain and HGF β chain both in a host cell by co-introducing the vectors obtained in step 2 into the host cell, and culturing the host cell. Step 4: A step for recovering active HGF from a culture supernatant after step 3.

Description

活性型ＨＧＦの製造方法Method for producing active HGF
　本開示は、活性型ＨＧＦの製造方法に関する。 This disclosure relates to a method for producing active HGF.
　肝細胞増殖因子（ＨＧＦ）は、生体内で不活性型の一本鎖ＨＧＦとして合成された後、プロテアーゼによって活性化され、活性型の二本鎖ＨＧＦになる。一本鎖ＨＧＦは生物活性を有さないことから、二本鎖ＨＧＦへの変換は生物活性の発現には必須である。ＨＧＦを活性化させるプロテアーゼとしては血液中に含まれる、ＨＧＦアクチベーター（ＨＧＦＡ）、ｕＰＡ(ウロキナーゼ型プラスミノーゲンアクチベーター）、ｔＰＡ（組織型プラスミノーゲンアクチベーター）、マトリプターゼ、ヘプシン等が知られている。
　従来、ＨＧＦの製造方法として、チャイニーズハムスター卵巣（ＣＨＯ）細胞等の動物細胞を用いる方法が知られている。動物細胞の培養にはウシ胎児血清が添加されてきたが、ウシ胎児血清を使用しないことにより生産コストの削減が可能なことに加え、タンパク質製剤にウシ胎児血清に由来するウイルスや異常プリオンが混入する可能性を回避することができるため、医薬製剤として用いられるような活性型ＨＧＦを無血清条件で製造するための方法が開発されている（特許文献１～４）。しかしこれらの方法は、いずれも、一本鎖ＨＧＦ又はその改変体を製造し、酵素又は化学反応により二本鎖ＨＧＦへ変換する必要があった。 Hepatocyte growth factor (HGF) is synthesized in vivo as an inactive single-chain HGF, and then activated by proteases to become an active double-chain HGF. Since single-chain HGF has no biological activity, conversion to double-chain HGF is essential for the expression of biological activity. Proteases that activate HGF include HGF activator (HGFA), uPA (urokinase-type plasminogen activator), tPA (tissue-type plasminogen activator), matriptase, hepsin, and the like, which are contained in blood.
Conventionally, methods using animal cells such as Chinese hamster ovary (CHO) cells have been known as methods for producing HGF. Fetal bovine serum has been added to culture of animal cells, but not using fetal bovine serum can reduce production costs and can avoid the possibility of contamination of protein preparations with viruses or abnormal prions derived from fetal bovine serum. Therefore, methods for producing active HGF, such as those used as pharmaceutical preparations, under serum-free conditions have been developed (Patent Documents 1 to 4). However, all of these methods require the production of single-chain HGF or its modified form and conversion to double-chain HGF by enzyme or chemical reaction.
特許第３２１３９８５号Patent No. 3213985 特許第３２３２７１４号Patent No. 3232714 特許第５０９３７８３号Patent No. 5093783 特許第６８６１２０１号Patent No. 6861201
　本開示の課題は、ＨＧＦα鎖とＨＧＦβ鎖を同一の宿主細胞内で各々発現させて、活性型ＨＧＦを製造する方法を提供することである。 The objective of this disclosure is to provide a method for producing active HGF by expressing both the HGF α chain and the HGF β chain in the same host cell.
　本発明者は、鋭意研究を重ねた結果、タンパク質を発現させるためのベクターに、ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ、及びＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡのそれぞれを挿入し、得られたベクターを宿主細胞に共導入することによって、宿主細胞の培養上清から活性型ＨＧＦを回収できるとの驚くべき新知見を得て、この新知見に基づいてさらに研究を進めた。 As a result of extensive research, the inventors have made the surprising discovery that active HGF can be recovered from the culture supernatant of host cells by inserting DNA encoding an amino acid sequence containing the HGF α chain and DNA encoding an amino acid sequence containing the HGF β chain into a vector for expressing a protein and then co-introducing the resulting vectors into host cells, and have continued their research based on this discovery.
　すなわち、本開示は、例えば以下の実施形態に関する。
〔１〕下記工程１～４の工程を含む、活性型ＨＧＦを製造する方法。
工程１：
　ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ及びＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡをそれぞれ作製する工程
工程２：
　工程１で作製されたそれぞれのＤＮＡを、タンパク質を発現させるためのベクターに挿入する工程
工程３：
　工程２で得られたベクターを宿主細胞に共導入し、宿主細胞を培養することにより、宿主細胞においてＨＧＦα鎖、及びＨＧＦβ鎖をそれぞれ発現させる工程
工程４：
　工程３の後に、培養上清から活性型ＨＧＦを回収する工程
〔２〕ＨＧＦα鎖を含むアミノ酸配列、及びＨＧＦβ鎖を含むアミノ酸配列のそれぞれが、シグナル配列を含むことを特徴とする前記〔１〕記載の方法。
〔３〕ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ、及びＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡのそれぞれが、終止コドンを含むことを特徴とする、前記〔１〕又は〔２〕記載の方法。
〔４〕シグナル配列が、ＨＧＦシグナル配列であることを特徴とする、前記〔２〕又は〔３〕記載の方法。
〔５〕ＨＧＦシグナル配列が、ヒトＨＧＦシグナル配列であることを特徴とする、前記〔４〕記載の方法。
〔６〕ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ、及び／又は、ＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡが、宿主細胞に適したコドンとなるように改変されているＤＮＡであることを特徴とする、前記〔１〕～〔５〕のいずれかに記載の方法。
〔７〕ＨＧＦα鎖が、ヒトＨＧＦα鎖であることを特徴とする、前記〔１〕～〔６〕のいずれかに記載の方法。
〔８〕ヒトＨＧＦα鎖が、配列番号１３又は１９で示されるアミノ酸配列からなることを特徴とする、前記〔７〕記載の方法。
〔９〕ＨＧＦβ鎖が、ヒトＨＧＦβ鎖であることを特徴とする、前記〔１〕～〔８〕のいずれかに記載の方法。
〔１０〕ヒトＨＧＦβ鎖が、配列番号１６で示されるアミノ酸配列からなることを特徴とする、前記〔９〕記載の方法。
〔１１〕宿主細胞が、ＣＨＯ－Ｋ１細胞、ＣＨＯ－ＤＧ４４（ＤＨＦＲ－）細胞、ＣＨＯ－Ｓ細胞、ＣＨＯ－ＭＫ細胞、ＣＨＯ－ＧＳ細胞、ＨＥＫ２９３細胞、ＢＨＫ－２１細胞、ＰＥＲ．Ｃ６細胞、ＮＳ０細胞、ＳＰ２／０細胞、Ｓｆ９細胞、Ｓｆ２１細胞、Ｈｉｇｈ　Ｆｉｖｅ細胞、Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ及びＰｉｃｈｉａ　ｐａｓｔｏｒｉｓからなる群から選択されることを特徴とする、前記〔１〕～〔１０〕のいずれかに記載の方法。
〔１２〕宿主細胞が、ＣＨＯ－Ｋ１細胞、ＣＨＯ－ＤＧ４４（ＤＨＦＲ－）細胞、ＣＨＯ－Ｓ細胞、ＣＨＯ－ＭＫ細胞及びＣＨＯ－ＧＳ細胞からなる群から選択されており、前記宿主細胞に適したコドンとなるように改変されているヒトＨＧＦα鎖をコードするＤＮＡが、配列番号２で示される塩基配列を含み、かつ／又は、前記宿主細胞に適したコドンとなるように改変されている、ヒトＨＧＦβ鎖をコードするＤＮＡが、配列番号５で示される塩基配列を含むことを特徴とする、前記〔６〕、〔７〕、〔９〕又は〔１１〕記載の方法。 That is, the present disclosure relates to the following embodiments, for example.
[1] A method for producing an active HGF, comprising the steps of:
Step 1:
Step 2: A step of preparing DNA encoding an amino acid sequence containing an HGF α chain and a step of preparing DNA encoding an amino acid sequence containing an HGF β chain
Step 3: Insert each DNA prepared in step 1 into a vector for expressing a protein.
Step 4: Co-introducing the vectors obtained in step 2 into host cells and culturing the host cells to express HGF α-chain and HGF β-chain in the host cells.
[2] A process for recovering active HGF from the culture supernatant after step 3. The method according to [1], wherein each of the amino acid sequence containing the HGF α chain and the amino acid sequence containing the HGF β chain contains a signal sequence.
[3] The method according to [1] or [2], characterized in that each of the DNA encoding an amino acid sequence including an HGF alpha chain and the DNA encoding an amino acid sequence including an HGF beta chain contains a stop codon.
[4] The method according to [2] or [3] above, wherein the signal sequence is an HGF signal sequence.
[5] The method according to [4] above, wherein the HGF signal sequence is a human HGF signal sequence.
[6] The method according to any one of [1] to [5] above, characterized in that the DNA encoding an amino acid sequence including an HGF α chain and/or the DNA encoding an amino acid sequence including an HGF β chain is a DNA modified to contain codons suitable for the host cell.
[7] The method according to any one of [1] to [6] above, wherein the HGF α chain is a human HGF α chain.
[8] The method according to [7] above, wherein the human HGF α chain consists of the amino acid sequence shown in SEQ ID NO: 13 or 19.
[9] The method according to any one of [1] to [8] above, wherein the HGF β chain is a human HGF β chain.
[10] The method according to [9] above, wherein the human HGF β chain consists of the amino acid sequence shown in SEQ ID NO:16.
[11] The method according to any one of [1] to [10] above, wherein the host cell is selected from the group consisting of CHO-K1 cells, CHO-DG44 (DHFR-) cells, CHO-S cells, CHO-MK cells, CHO-GS cells, HEK293 cells, BHK-21 cells, PER.C6 cells, NS0 cells, SP2/0 cells, Sf9 cells, Sf21 cells, High Five cells, Saccharomyces cerevisiae, and Pichia pastoris.
[12] The method according to [6], [7], [9] or [11], wherein the host cell is selected from the group consisting of CHO-K1 cells, CHO-DG44 (DHFR-) cells, CHO-S cells, CHO-MK cells and CHO-GS cells, and the DNA encoding the human HGF α chain modified to have codons suitable for the host cell comprises the base sequence shown in SEQ ID NO: 2, and/or the DNA encoding the human HGF β chain modified to have codons suitable for the host cell comprises the base sequence shown in SEQ ID NO: 5.
　本開示により、ＨＧＦα鎖とＨＧＦβ鎖を同一の宿主細胞内で各々発現させて、活性型ＨＧＦを製造する方法を提供することができる。本開示における製造方法を用いることにより、血清及び／又は酵素を用いずに活性型ＨＧＦを製造することができ、生産コストが削減され、工業的に非常に有利である。また、血清に由来するウイルス等の混入を回避でき、安全な活性型ＨＧＦを製造し得る。 The present disclosure provides a method for producing active HGF by expressing the HGF α chain and the HGF β chain in the same host cell. By using the production method disclosed herein, active HGF can be produced without using serum and/or enzymes, reducing production costs and being highly advantageous industrially. In addition, contamination with viruses and the like derived from serum can be avoided, making it possible to produce safe active HGF.
図１は、実施例におけるウエスタンブロットの結果を示す。FIG. 1 shows the results of Western blotting in an example. 図２は、実施例における細胞増殖促進活性の評価試験の結果を示す。FIG. 2 shows the results of an evaluation test of cell proliferation-promoting activity in an example. 図３は、実施例における細胞増殖促進活性の評価試験の結果を示す。FIG. 3 shows the results of an evaluation test of cell proliferation-promoting activity in an example.
　本開示の活性型ＨＧＦを製造する方法（本明細書において、本開示の方法ともいう。）は、下記工程１～４の工程を含むことを特徴とする。本開示の方法は、下記工程１～４の工程を含み、所望により、他の工程を含んでいてもよく、下記工程１～４の工程からなるものでもよい。本開示の方法は、血清及び／又は酵素を用いない方法であることが好ましい。 The method for producing activated HGF disclosed herein (also referred to as the method of the present disclosure in this specification) is characterized by including the following steps 1 to 4. The method disclosed herein includes the following steps 1 to 4, and may include other steps as desired, or may consist of the following steps 1 to 4. The method disclosed herein is preferably a method that does not use serum and/or enzymes.
工程１：
　ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ、及びＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡをそれぞれ作製する工程 Step 1:
A step of preparing a DNA encoding an amino acid sequence containing an HGF α chain and a DNA encoding an amino acid sequence containing an HGF β chain.
工程２：
　工程１で作製されたそれぞれのＤＮＡを、タンパク質を発現させるためのベクターに挿入する工程 Step 2:
A step of inserting each of the DNAs prepared in step 1 into a vector for expressing a protein.
工程３：
　工程２で得られたベクターを宿主細胞に共導入し、宿主細胞を培養することにより、宿主細胞においてＨＧＦα鎖及びＨＧＦβ鎖をそれぞれ発現させる工程 Step 3:
A step of co-introducing the vectors obtained in step 2 into host cells and culturing the host cells to express the HGF α chain and the HGF β chain in the host cells.
工程４：
　工程３の後に、培養上清から活性型ＨＧＦを回収する工程 Step 4:
A step of recovering active HGF from the culture supernatant after step 3.
〔工程１〕
　本開示の方法は、工程１として、ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ及びＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡをそれぞれ作製する工程を含む。 [Step 1]
The method of the present disclosure comprises, as step 1, the step of preparing a DNA encoding an amino acid sequence containing an HGF α chain and a DNA encoding an amino acid sequence containing an HGF β chain.
〔ＨＧＦα鎖及びＨＧＦβ鎖〕
　ＨＧＦα鎖（本開示において、単にα鎖又はＨＧＦαと表記することもある。）及びＨＧＦβ鎖（本開示において、単にβ鎖又はＨＧＦβと表記することもある。）は、活性型ＨＧＦを構成するタンパク質である。 [HGF α chain and HGF β chain]
The HGF α chain (in the present disclosure, sometimes simply referred to as α chain or HGFα) and the HGF β chain (in the present disclosure, sometimes simply referred to as β chain or HGFβ) are proteins that constitute active HGF.
　α鎖及びβ鎖のそれぞれの由来は、特に限定されないが、α鎖及びβ鎖のそれぞれは、例えば、ヒト由来であってもよく、ヒト以外の動物（例えば、イヌ、ネコ、ラット、マウス、ウサギ、ウシ、チンパンジー、ウマ、ブタ、ヒツジ等）等に由来するものであってもよいが、ヒト由来（ヒトＨＧＦα鎖、ヒトＨＧＦβ鎖）であることが好ましい。α鎖がヒトＨＧＦα鎖であり、かつ、β鎖がヒトＨＧＦβ鎖であることが好ましい例として挙げられる。 The origin of each of the α chain and β chain is not particularly limited, but each of the α chain and β chain may be, for example, derived from humans or from animals other than humans (e.g., dogs, cats, rats, mice, rabbits, cows, chimpanzees, horses, pigs, sheep, etc.), but is preferably derived from humans (human HGF α chain, human HGF β chain). A preferred example is one in which the α chain is human HGF α chain and the β chain is human HGF β chain.
　ヒトＨＧＦα鎖としては、例えば配列番号１９で示されるアミノ酸配列からなるヒト全長型ＨＧＦα鎖、配列番号１３で示されるアミノ酸配列からなるヒト５アミノ酸欠失型ＨＧＦα鎖等が挙げられる。ヒトＨＧＦβ鎖としては、例えば、配列番号１６で示されるアミノ酸配列からなるヒト全長型ＨＧＦのβ鎖等が挙げられる。 Examples of the human HGF α chain include the human full-length HGF α chain consisting of the amino acid sequence shown in SEQ ID NO: 19, and the human 5-amino acid deleted HGF α chain consisting of the amino acid sequence shown in SEQ ID NO: 13. Examples of the human HGF β chain include the human full-length HGF β chain consisting of the amino acid sequence shown in SEQ ID NO: 16.
　ヒト以外の動物由来のＨＧＦ、ＨＧＦα鎖、ＨＧＦβ鎖等の、アミノ酸配列情報は、公知のデータベース（ＧｅｎＢａｎｋ/ＥＭＢＬ/ＤＤＢＪ等）から取得することができる。 Amino acid sequence information for HGF, HGF α chain, HGF β chain, etc. derived from animals other than humans can be obtained from publicly known databases (GenBank/EMBL/DDBJ, etc.).
　本開示の方法により製造されるＨＧＦが、実質的に活性型ＨＧＦと同等のＨＧＦ活性を有する限り、本開示におけるα鎖及びβ鎖は、それぞれ、そのアミノ酸配列の１～数個（例えば、２～１５０個、より好ましくは２～８０個、より好ましくは２～７０個、より好ましくは２～６０個、より好ましくは２～５０個、より好ましくは２～４０個、より好ましくは２～３０個、より好ましくは２～２０個、より好ましくは２～１０個、若しくは２～５個、又はより好ましくは１～５個である；以下において同じ。）のアミノ酸が付加、欠失又は置換を有する、変異体であってもよい。 As long as the HGF produced by the method of the present disclosure has HGF activity substantially equivalent to that of active HGF, the α-chain and β-chain of the present disclosure may each be a mutant having one to several amino acids added, deleted or substituted in the amino acid sequence (e.g., 2 to 150, more preferably 2 to 80, more preferably 2 to 70, more preferably 2 to 60, more preferably 2 to 50, more preferably 2 to 40, more preferably 2 to 30, more preferably 2 to 20, more preferably 2 to 10, or 2 to 5, or more preferably 1 to 5; the same applies below).
　また、本開示の方法により製造されるＨＧＦが、実質的に活性型ＨＧＦと同等のＨＧＦ活性を有する限り、本開示におけるα鎖には、α鎖のアミノ酸配列と、少なくとも８０％、好ましくは、８５％、より好ましくは少なくとも９０％、９１％、９２％、９３％、９４％、９５％、９６％、９７％、９８％又は９９％の配列同一性を示すアミノ酸を有するポリペプチドが含まれる。 Furthermore, so long as the HGF produced by the method of the present disclosure has HGF activity substantially equivalent to that of active HGF, the α chain of the present disclosure includes a polypeptide having amino acids that show at least 80%, preferably 85%, and more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of the α chain.
　また、本開示の方法により製造されるＨＧＦが、実質的に活性型ＨＧＦと同等のＨＧＦ活性を有する限り、本開示におけるβ鎖には、β鎖のアミノ酸配列と、少なくとも８０％、好ましくは、８５％、より好ましくは少なくとも９０％、９１％、９２％、９３％、９４％、９５％、９６％、９７％、９８％又は９９％の配列同一性を示すアミノ酸を有するポリペプチドが含まれる。 Furthermore, so long as the HGF produced by the method of the present disclosure has HGF activity substantially equivalent to that of active HGF, the β chain of the present disclosure includes a polypeptide having amino acids that show at least 80%, preferably 85%, and more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of the β chain.
〔ＨＧＦα鎖を含むアミノ酸配列及びＨＧＦβ鎖を含むアミノ酸配列〕
　ＨＧＦα鎖を含むアミノ酸配列は、ＨＧＦα鎖のアミノ酸配列以外のアミノ酸配列を含んでいてもよい。ＨＧＦβ鎖を含むアミノ酸配列は、ＨＧＦβ鎖のアミノ酸配列以外のアミノ酸配列を含んでいてもよい。ＨＧＦα鎖及びＨＧＦβ鎖以外のアミノ酸配列としては、例えば、シグナル配列、アフィニティータグ等が挙げられる。いずれも本開示の技術的範囲に含まれる。 [Amino acid sequence containing HGF α chain and amino acid sequence containing HGF β chain]
The amino acid sequence containing the HGF α chain may contain an amino acid sequence other than the amino acid sequence of the HGF α chain. The amino acid sequence containing the HGF β chain may contain an amino acid sequence other than the amino acid sequence of the HGF β chain. Examples of amino acid sequences other than the HGF α chain and the HGF β chain include signal sequences, affinity tags, and the like. All of these are within the technical scope of the present disclosure.
〔シグナル配列〕
　本開示におけるＨＧＦα鎖を含むアミノ酸配列及び／又はＨＧＦβ鎖を含むアミノ酸配列は、シグナル配列を含むことが好ましく、ＨＧＦα鎖を含むアミノ酸配列、及びＨＧＦβ鎖を含むアミノ酸配列のそれぞれが、シグナル配列を含むことがより好ましい。ＨＧＦα鎖を含むアミノ酸配列及び／又はＨＧＦβ鎖を含むアミノ酸配列が、シグナル配列を含むことにより、後述する工程３で、宿主細胞内で発現したα鎖及びβ鎖が、宿主細胞外に積極的に分泌され得る。シグナル配列としては、宿主細胞が動物細胞である場合には、ＨＧＦシグナル配列、インシュリン・シグナル配列、α－インターフェロン・シグナル配列等が例示され、本開示においては、シグナル配列として、ヒトＨＧＦシグナル配列（ヒト由来のＨＧＦシグナル配列；例えば、配列番号２１で示されるアミノ酸配列）が好ましく使用される。シグナル配列を付加する方法としては、公知の方法を用いて行うことができ、例えば、Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２６４，１７６１９（１９８９）、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．，ＵＳＡ，８６，８２２７（１９８９）、Ｇｅｎｅｓ Ｄｅｖｅｌｏｐ．，４，１２８８（１９９０）、特開平５－３３６９６３、ＷＯ９４／２３０２１等に記載の方法が挙げられる。 [Signal sequence]
In the present disclosure, the amino acid sequence containing the HGF α chain and/or the amino acid sequence containing the HGF β chain preferably contains a signal sequence, and it is more preferable that each of the amino acid sequence containing the HGF α chain and the amino acid sequence containing the HGF β chain contains a signal sequence. When the amino acid sequence containing the HGF α chain and/or the amino acid sequence containing the HGF β chain contains a signal sequence, the α chain and the β chain expressed in the host cell can be actively secreted outside the host cell in step 3 described below. When the host cell is an animal cell, examples of the signal sequence include an HGF signal sequence, an insulin signal sequence, and an α-interferon signal sequence. In the present disclosure, the human HGF signal sequence (an HGF signal sequence derived from a human; for example, the amino acid sequence shown in SEQ ID NO: 21) is preferably used as the signal sequence. The signal sequence can be added using a known method, and for example, the method described in J. Biol. Chem., 264, 17619 (1989), Proc. Natl. Acad. Sci. , USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990), JP-A-5-336963, WO94/23021, and the like.
〔ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ及びＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡ〕 [DNA encoding an amino acid sequence containing the HGF α chain and DNA encoding an amino acid sequence containing the HGF β chain]
　ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡとしては、前述してきたＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡであれば特に限定されないが、前記配列の５’末端にシグナル配列の塩基配列を含むこと、及び／又は、前記配列の３’末端に終止コドンを含むことが好ましい。 The DNA encoding an amino acid sequence containing the HGF α chain is not particularly limited as long as it is a DNA encoding an amino acid sequence containing the HGF α chain described above, but it is preferable that the sequence contains a base sequence of a signal sequence at the 5' end and/or a stop codon at the 3' end of the sequence.
　配列番号１９で示されるヒト全長型ＨＧＦα鎖をコードするＤＮＡとしては、例えば配列番号２０で示される塩基配列が挙げられる。配列番号１３で示されるヒト５アミノ酸欠失型ＨＧＦα鎖をコードするＤＮＡとしては、例えば配列番号１４又は１５で示される塩基配列が挙げられる。 An example of the DNA encoding the full-length human HGF α chain shown in SEQ ID NO: 19 is the base sequence shown in SEQ ID NO: 20. An example of the DNA encoding the five amino acid deleted human HGF α chain shown in SEQ ID NO: 13 is the base sequence shown in SEQ ID NO: 14 or 15.
　ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡとして、配列番号１４、１５又は２０で示される塩基配列を含む、例えば配列番号２、３又は８からなるＤＮＡ等が好適な例として挙げられる。 As DNA encoding an amino acid sequence containing the HGF α chain, suitable examples include DNA containing the base sequence shown in SEQ ID NO: 14, 15 or 20, such as DNA consisting of SEQ ID NO: 2, 3 or 8.
　ＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡとしては、前述してきたＨＧＦβ鎖を含むアミノ酸配列をコードする遺伝子であれば特に限定されないが、前記配列の５’末端にシグナル配列の塩基配列を含むこと、及び／又は、前記配列の３’末端に終止コドンを含むことが好ましい。 The DNA encoding an amino acid sequence containing the HGF β chain is not particularly limited as long as it is a gene encoding an amino acid sequence containing the HGF β chain described above, but it is preferable that the sequence contains a base sequence of a signal sequence at the 5' end and/or a stop codon at the 3' end of the sequence.
　配列番号１６で示されるヒトＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡとしては、例えば配列番号１８で示される塩基配列が挙げられる。 An example of a DNA encoding an amino acid sequence containing the human HGF β chain shown in SEQ ID NO:16 is the base sequence shown in SEQ ID NO:18.
　ＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡとして、配列番号１７又は１８で示される塩基配列を含む、例えば配列番号５又は６からなるＤＮＡ等が好適な例として挙げられる。 As a DNA encoding an amino acid sequence containing the HGF β chain, a suitable example is a DNA containing the base sequence shown in SEQ ID NO: 17 or 18, such as a DNA consisting of SEQ ID NO: 5 or 6.
　ヒト以外の動物由来のＨＧＦ、ＨＧＦα鎖、ＨＧＦβ鎖等の遺伝子の塩基配列情報は、公知のデータベース（ＧｅｎＢａｎｋ/ＥＭＢＬ/ＤＤＢＪ等）から取得することができる。 The base sequence information of genes for HGF, HGF α chain, HGF β chain, etc. derived from animals other than humans can be obtained from publicly known databases (GenBank/EMBL/DDBJ, etc.).
　ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ、及び／又は、ＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡは、自体公知の方法に従って、宿主細胞に適したコドンとなるように改変されているＤＮＡであることが好ましい。 The DNA encoding an amino acid sequence containing the HGF α chain and/or the DNA encoding an amino acid sequence containing the HGF β chain is preferably DNA that has been modified to have codons suitable for the host cell according to a method known per se.
〔宿主細胞〕
　上記ベクターを導入する宿主としては、例えば、動物細胞、昆虫細胞、酵母等が用いられる。 [Host cells]
Hosts into which the above vectors are introduced include, for example, animal cells, insect cells, yeast cells, and the like.
　動物細胞としては、例えば、チャイニーズハムスター細胞（本明細書において、ＣＨＯ細胞と略記することもある。）、サル細胞ＣＯＳ－７、Ｖｅｒｏ、ｄｈｆｒ遺伝子欠損ＣＨＯ細胞（以下、ＣＨＯ（ＤＨＦＲ－）細胞と略記）、マウスＬ細胞、マウスＡｔＴ－２０、マウスミエローマ細胞、ラットＧＨ３、ヒトＦＬ細胞、ヒト胎児腎細胞（ＨＥＫ２９３細胞）、シリアンハムスター腎（ＢＨＫ－２１細胞）、胎児の網膜細胞（ＰＥＲ．Ｃ６細胞）、マウス骨髄腫細胞（ＮＳ０細胞）、マウス骨髄腫細胞（ＳＰ２／０細胞）等が挙げられる。ＣＨＯ細胞としては、例えば、ＣＨＯ－Ｋ１細胞、ＣＨＯ－Ｓ細胞、ＣＨＯ－ＭＫ細胞、ＣＨＯ－ＧＳ細胞等が挙げられる。ＣＨＯ（ＤＨＦＲ－）細胞としては、例えば、ＣＨＯ－ＤＧ４４（ＤＨＦＲ－）細胞等が挙げられる。 Animal cells include, for example, Chinese hamster cells (sometimes abbreviated as CHO cells in this specification), monkey cells COS-7, Vero, dhfr gene-deficient CHO cells (hereinafter abbreviated as CHO (DHFR-) cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, human fetal kidney cells (HEK293 cells), Syrian hamster kidney (BHK-21 cells), fetal retina cells (PER.C6 cells), mouse myeloma cells (NS0 cells), mouse myeloma cells (SP2/0 cells), etc. Examples of CHO cells include, for example, CHO-K1 cells, CHO-S cells, CHO-MK cells, CHO-GS cells, etc. Examples of CHO (DHFR-) cells include, for example, CHO-DG44 (DHFR-) cells, etc.
　酵母としては、例えばＳａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ、Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ、ＡＨ２２Ｒ－、ＮＡ８７－１１Ａ、ＤＫＤ－５Ｄ、２０Ｂ－１２、Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ ｐｏｍｂｅ ＮＣＹＣ１９１３、ＮＣＹＣ２０３６等が挙げられる。Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅとして、例えば、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ ＡＨ２２等が挙げられる。 Examples of yeast include Saccharomyces cerevisiae, Pichia pastoris, AH22R-, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, etc. Examples of Saccharomyces cerevisiae include Saccharomyces cerevisiae AH22, etc.
　昆虫細胞としては、例えばＳｆ９細胞（Ｓｐｏｄｏｐｔｅｒｅ　ｆｒｕｇｉｐｅｒｄａＳＦ２１細胞のクローン分離株）、Ｓｆ２１細胞（Ｓｐｏｄｏｐｔｅｒｅ　ｆｒｕｇｉｐｅｒｄａＳＦ２１細胞）、Ｈｉｇｈ　Ｆｉｖｅ細胞（ガの幼虫Ｔｒｉｃｈｏｐｌｕｓｉａ　ｎｉのＢＴＩ－ＴＮ－５Ｂ１－４クローン）等が挙げられる。 Examples of insect cells include Sf9 cells (a clone isolate of Spodoptera frugiperda SF21 cells), Sf21 cells (Spodoptera frugiperda SF21 cells), and High Five cells (a BTI-TN-5B1-4 clone of the moth larva Trichoplusia ni).
　宿主細胞は、好ましくは、ＣＨＯ－Ｋ１細胞、ＣＨＯ－ＤＧ４４（ＤＨＦＲ－）細胞、ＣＨＯ－Ｓ細胞、ＣＨＯ－ＭＫ細胞、ＣＨＯ－ＧＳ細胞、ＨＥＫ２９３細胞、ＢＨＫ－２１細胞、ＰＥＲ．Ｃ６細胞、ＮＳ０細胞、ＳＰ２／０細胞、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ及びＰｉｃｈｉａ ｐａｓｔｏｒｉｓからなる群から選択される。 The host cell is preferably selected from the group consisting of CHO-K1 cells, CHO-DG44 (DHFR-) cells, CHO-S cells, CHO-MK cells, CHO-GS cells, HEK293 cells, BHK-21 cells, PER. C6 cells, NS0 cells, SP2/0 cells, Saccharomyces cerevisiae and Pichia pastoris.
〔宿主細胞に適したコドンとなるように改変されているＤＮＡ〕
　一般に、同じアミノ酸が異なるコドンによってコードされていることは、例えば、「遺伝コードの縮退」として公知である。「コドン」という用語は、例えば、所定のアミノ酸をコードしている３つのヌクレオチドからなるオリゴヌクレオチドを意味する。遺伝コードの縮退があるため、ほとんどのアミノ酸は複数のコドンによってコードされている。同じアミノ酸をコードするこれらの異なるコドンの相対的使用頻度は、個々の宿主細胞において異なることが知られている。そのため、アミノ酸配列をコードするＤＮＡ配列を設計するには、各アミノ酸に対応するコドンを１種又は２種以上使用することができる。すなわち、あるペプチドが有する単一のアミノ酸配列をコードする塩基配列は、複数のバリエーションを有し得る。かかるコドンの選択に際しては、該ＤＮＡ配列を含むポリヌクレオチド又はそれを含むベクターが導入される、発現用の宿主細胞のコドン使用（ｃｏｄｏｎ ｕｓａｇｅ）に応じて適宜コドンを選択したり、複数のコドンの使用の頻度又は割合を適宜調節したりすることができる。例えば、ＣＨО細胞を宿主細胞として用いる場合は、ＣＨО細胞において使用頻度が高いコドンを使用して塩基配列を設計してもよい。宿主細胞でのコドン使用頻度に対応して、その宿主細胞の生物種で、翻訳効率が高いようにコドンが選択されることが好ましく、翻訳効率が最も高いようにコドンが選択されることがより好ましい。宿主細胞に適したコドンとなるように改変する方法は、例えば、Ｇｕｓｔａｆｓｓｏｎ， Ｃ．ら（Ｔｒｅｎｄｓｉｎ ｂｉｏｔｅｃｈｎｏｌｏｇｙ２２．７ （２００４）： ３４６－３５３）等を参照して実行することができる。各種生物が使用するコドンの情報は、Ｃｏｄｏｎ Ｕｓａｇｅ Ｄａｔａｂａｓｅ（www.kazusa.or.jp/codon/）等から入手可能である。 [DNA modified to have codons suitable for the host cell]
In general, the fact that the same amino acid is coded for by different codons is known, for example, as "degeneracy of the genetic code." The term "codon" refers, for example, to an oligonucleotide consisting of three nucleotides that codes for a given amino acid. Due to the degeneracy of the genetic code, most amino acids are coded for by multiple codons. It is known that the relative frequency of use of these different codons that code for the same amino acid varies in individual host cells. Therefore, to design a DNA sequence that codes for an amino acid sequence, one or more codons corresponding to each amino acid can be used. That is, a base sequence that codes for a single amino acid sequence possessed by a certain peptide may have multiple variations. When selecting such codons, appropriate codons can be selected according to the codon usage of the host cell for expression into which a polynucleotide containing the DNA sequence or a vector containing the same is introduced, or the frequency or ratio of use of multiple codons can be appropriately adjusted. For example, when CHO cells are used as host cells, the base sequence may be designed using codons that are frequently used in CHO cells. In accordance with the frequency of codon usage in the host cell, it is preferable to select codons that have high translation efficiency in the host cell species, and it is more preferable to select codons that have the highest translation efficiency. A method of modifying codons to be suitable for the host cell can be carried out by referring to, for example, Gustafsson, C. et al. (Trends in biotechnology 22.7 (2004): 346-353). Information on codons used by various organisms is available from the Codon Usage Database (www.kazusa.or.jp/codon/) and the like.
　ヒト又はヒト以外の特定の動物由来のＨＧＦα鎖又はＨＧＦβ鎖を含むアミノ酸配列をコードする塩基配列において、宿主細胞に適したコドンとなるように置換するコドンの割合は、１０～１００％であってもよく、２０～１００％であってもよく、３０～１００％であってもよく、４０～１００％であってもよく、５０～１００％であってもよく、６０～１００％であってもよく、７０～１００％であってもよく、８０～１００％であってもよく、９０～１００％であってもよい。 In a base sequence that encodes an amino acid sequence containing an HGF α chain or HGF β chain derived from a human or a specific animal other than a human, the percentage of codons that are substituted to become codons suitable for the host cell may be 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, or 90-100%.
　配列番号１５で示される塩基配列（配列番号１３で示される、ヒト５アミノ酸欠失型ＨＧＦα鎖を含むアミノ酸配列をコードする塩基配列）が、ＣＨＯ細胞に適したコドンに改変されている塩基配列の例として、配列番号１４の塩基配列が挙げられる。配列番号３で示される塩基配列（配列番号１で示される、ヒトＨＧＦシグナル配列がＮ末端側に結合しているヒト５アミノ酸欠失ＨＧＦα鎖を含むアミノ酸配列をコードする塩基配列）が、ＣＨＯ細胞に適したコドンに改変されている塩基配列の例として、配列番号２の塩基配列が挙げられる。 An example of a base sequence in which the base sequence shown in SEQ ID NO:15 (a base sequence encoding an amino acid sequence containing a human 5-amino acid deleted HGF α chain shown in SEQ ID NO:13) has been modified to have codons suitable for CHO cells is the base sequence shown in SEQ ID NO:14. An example of a base sequence in which the base sequence shown in SEQ ID NO:3 (a base sequence encoding an amino acid sequence containing a human 5-amino acid deleted HGF α chain with a human HGF signal sequence bound to the N-terminus shown in SEQ ID NO:1) has been modified to have codons suitable for CHO cells is the base sequence shown in SEQ ID NO:2.
　配列番号１８で示される塩基配列（配列番号１６で示される、ヒトＨＧＦβ鎖を含むアミノ酸配列をコードする塩基配列）が、ＣＨＯ細胞に適したコドンに改変されている塩基配列の例として、配列番号１７の塩基配列が挙げられる。配列番号６で示される塩基配列（配列番号４で示される、ヒトＨＧＦシグナル配列がＮ末端側に結合しているヒトＨＧＦβ鎖を含むアミノ酸配列をコードする塩基配列）が、ＣＨＯ細胞に適したコドンに改変されている塩基配列の例として、配列番号５の塩基配列が挙げられる。 An example of a base sequence in which the base sequence shown in SEQ ID NO:18 (a base sequence encoding an amino acid sequence containing a human HGF β chain, as shown in SEQ ID NO:16) has been modified to have codons suitable for CHO cells is the base sequence shown in SEQ ID NO:17. An example of a base sequence in which the base sequence shown in SEQ ID NO:6 (a base sequence encoding an amino acid sequence containing a human HGF β chain to which a human HGF signal sequence is attached at the N-terminus, as shown in SEQ ID NO:4) has been modified to have codons suitable for CHO cells is the base sequence shown in SEQ ID NO:5.
　配列番号２４で示される塩基配列（配列番号２１で示されるヒトＨＧＦのシグナル配列をコードする塩基配列）が、ＣＨＯ細胞に適したコドンに改変されている塩基配列の例として、配列番号２２又は２３の塩基配列が挙げられる。 The base sequence shown in SEQ ID NO:24 (the base sequence encoding the signal sequence of human HGF shown in SEQ ID NO:21) is modified to have codons suitable for CHO cells, and examples of such sequences include the base sequences shown in SEQ ID NO:22 and SEQ ID NO:23.
　ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ、及び、ＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡは、それぞれ、ゲノムＤＮＡ、ゲノムＤＮＡライブラリー、細胞又は組織由来のｃＤＮＡ、細胞又は組織由来のｃＤＮＡライブラリー、合成ＤＮＡ等でもよいが、合成ＤＮＡであることが好ましい。 The DNA encoding an amino acid sequence containing the HGF α chain and the DNA encoding an amino acid sequence containing the HGF β chain may each be genomic DNA, a genomic DNA library, cDNA derived from cells or tissues, a cDNA library derived from cells or tissues, synthetic DNA, etc., but is preferably synthetic DNA.
　ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ、及び、ＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡのそれぞれにおいて、制限酵素（例えばＨｉｎｄＩＩＩ、ＸｈｏＩ等）認識配列、リンカー、開始コドン（翻訳開始コドンともいう。例としてＡＴＧ等が挙げられる。）、終止コドン（翻訳終止コドンともいう。例としてＴＡＡ、ＴＧＡ又はＴＡＧ等が挙げられる。）等を含んでいてもよい。本開示におけるＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ及び／又はＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡは、３’末端において終止コドンを含むことが好ましく、ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ、及びＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡそれぞれが、終止コドンを含むことがより好ましい。 The DNA encoding the amino acid sequence containing the HGF α chain and the DNA encoding the amino acid sequence containing the HGF β chain may each contain a restriction enzyme (e.g., HindIII, XhoI, etc.) recognition sequence, a linker, a start codon (also called a translation start codon, examples of which include ATG, etc.), a stop codon (also called a translation stop codon, examples of which include TAA, TGA, or TAG, etc.) etc. In the present disclosure, the DNA encoding the amino acid sequence containing the HGF α chain and/or the DNA encoding the amino acid sequence containing the HGF β chain preferably contains a stop codon at the 3' end, and it is more preferable that the DNA encoding the amino acid sequence containing the HGF α chain and the DNA encoding the amino acid sequence containing the HGF β chain each contain a stop codon.
〔各ＤＮＡをそれぞれ作製する工程〕
　工程１において、ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ、及び、ＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡをそれぞれ作製する。上記各ＤＮＡをそれぞれ作製する方法として、ヒト又はヒト以外の動物の細胞から、各ＤＮＡ断片を単離及び抽出しそれをクローニングする方法、ＰＣＲ等の増幅手段を用いる方法、ＤＮＡ合成機によって合成する方法等が挙げられる。 [Steps for preparing each DNA]
In step 1, a DNA encoding an amino acid sequence containing an HGF α-chain and a DNA encoding an amino acid sequence containing an HGF β-chain are prepared. Methods for preparing each of the above DNAs include a method of isolating and extracting each DNA fragment from human or non-human animal cells and cloning it, a method using an amplification means such as PCR, and a method of synthesizing using a DNA synthesizer.
〔工程２〕
　本開示の方法は、工程２として、工程１で作製されたそれぞれのＤＮＡを、タンパク質を発現させるためのベクターに挿入する工程を含む。 [Step 2]
The method of the present disclosure includes, as step 2, inserting each of the DNAs prepared in step 1 into a vector for expressing a protein.
〔タンパク質を発現させるためのベクター〕
　例えば、公知の制限酵素（例えばＨｉｎｄＩＩＩ、ＸｈｏＩ等）とＤＮＡリガーゼを用いることにより、工程１で作製されたそれぞれのＤＮＡを、タンパク質を発現させるためのベクターに挿入することが出来る。ベクターは、必要により、プロモーター、リボソーム結合部位、ターミネーター、薬剤耐性遺伝子（選択マーカー）等を含む。ＨＧＦα鎖を発現させるためのベクターは、常法に従い、例えば転写の下流方向に順番に、(１)プロモーター、（２）リボソーム結合部位、（３）ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ及び（４）ターミネーターを含むように構築されてもよい。ＨＧＦβ鎖を発現させるためのベクターは、常法に従い、例えば転写の下流方向に順番に、(１)プロモーター、（２）リボソーム結合部位、（３）ＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡ及び（４）ターミネーターを含むように構築されてもよい。本開示で用いることが出来るベクターとしては、動物細胞を宿主とする場合は、ｐｃＤＮＡ３．１、ｐＣＡＧＧＳ［Ｎｉｗａ，Ｈ．，Ｙａｍａｍｕｒａ，Ｋ．ａｎｄ Ｍｉｙａｚａｋｉ，Ｊ．，Ｇｅｎｅ，第１０８巻，ｐ．１９３－２００（１９９１）]、ｐＢＫ－ＣＭＶ、ｐＺｅｏＳＶ（インビトロゲン社、ストラジーン社）、ｐＣＡＧＧＳ及びｐＣＸＮ２［Ｎｉｗａ，Ｈ．，Ｙａｍａｍｕｒａ，Ｋ．ａｎｄ Ｍｉｙａｚａｋｉ，Ｊ．，Ｇｅｎｅ，第１０８巻，ｐ．１９３－２００（１９９１）、特開平０３‐１６８０８７］、ｐｃＤＬ-ＳＲα［Ｔａｋｅｂｅ，Ｙ．ら、Ｍｏｌ．Ｃｅｌｌ．Ｂｉｏｌ、第８巻，p．４６６－４７２（１９８８）］等が挙げられる。酵母を宿主とする場合は、ｐＹＥＳ２（Ｉｎｖｉｔｒｏｇｅｎ社製）又はｐＲＢ１５（ＡＴＣＣ３７０６２）等のプラスミドを挙げることができる。本開示で用いることが出来るベクターとしては、宿主内で複製又は増幅可能なベクターであれば特に限定はされない。 Vectors for expressing proteins
For example, by using known restriction enzymes (e.g., HindIII, XhoI, etc.) and DNA ligase, each DNA prepared in step 1 can be inserted into a vector for expressing a protein. The vector contains a promoter, a ribosome binding site, a terminator, a drug resistance gene (selection marker), etc., as necessary. A vector for expressing the HGF α chain may be constructed in accordance with a conventional method, for example, so as to contain, in the order of downstream transcription, (1) a promoter, (2) a ribosome binding site, (3) a DNA encoding an amino acid sequence including the HGF α chain, and (4) a terminator. A vector for expressing the HGF β chain may be constructed in accordance with a conventional method, for example, so as to contain, in the order of downstream transcription, (1) a promoter, (2) a ribosome binding site, (3) a DNA encoding an amino acid sequence including the HGF β chain, and (4) a terminator. Vectors that can be used in the present disclosure include, when an animal cell is used as a host, pcDNA3.1, pCAGGS [Niwa, H., Yamamura, K. and Miyazaki, J., Gene, Vol. 108, pp. 193-200 (1991)], pBK-CMV, pZeoSV (Invitrogen, Stragene), pCAGGS and pCXN2 [Niwa, H., Yamamura, K. and Miyazaki, J., Gene, Vol. 108, pp. 193-200 (1991), JP-A-03-168087], pcDL-SRα [Takebe, Y. et al., Mol. Cell. Biol, Vol. 8, pp. 466-472 (1988)] and the like. When yeast is used as a host, examples of the plasmid include pYES2 (Invitrogen) and pRB15 (ATCC37062). The vector that can be used in the present disclosure is not particularly limited as long as it can be replicated or amplified in the host.
〔タンパク質を発現させるためのベクターへのＤＮＡの挿入〕
　工程１で作製されたＤＮＡは、ベクターに挿入される。挿入方法は、特に限定されないが、例えば、公知の方法によって行うことができる。ベクターとしては、例えば、上記のベクター、好ましくはプラスミドに挿入することができる。あるベクターに、ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡを挿入し、別のベクターに、ＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡを挿入してもよい。また、前記各ＤＮＡをそれぞれ１つのベクターに挿入してもよい。作製した発現ベクターが正しく作製されているかを確認するには、公知の方法、例えば、サンガー法によるシーケンス解析、制限酵素消化等により、確認することができる。 Insertion of DNA into a vector for protein expression
The DNA prepared in step 1 is inserted into a vector. The method of insertion is not particularly limited, and can be performed by a known method, for example. As the vector, for example, the above-mentioned vector, preferably a plasmid, can be inserted. A DNA encoding an amino acid sequence including an HGF α chain may be inserted into a certain vector, and a DNA encoding an amino acid sequence including an HGF β chain may be inserted into another vector. Also, each of the above DNAs may be inserted into one vector. To confirm whether the expression vector prepared is correctly prepared, it can be confirmed by a known method, for example, sequence analysis by the Sanger method, restriction enzyme digestion, etc.
〔工程３〕
　本開示の方法は、工程３として、工程２で得られたベクターを宿主細胞に共導入し、宿主細胞を培養することにより、宿主細胞においてＨＧＦα鎖及びＨＧＦβ鎖をそれぞれ発現させる工程を含む。 [Step 3]
The method of the present disclosure includes, as step 3, co-introducing the vectors obtained in step 2 into host cells and culturing the host cells to express HGF α chain and HGF β chain in the host cells.
　動物細胞を形質転換するには、例えば「細胞工学別冊８新細胞工学実験プロトコール，ｐ２６３－２６７（１９９３）（学研メディカル秀潤社発行）」、「Ｖｉｒｏｌｏｇｙ，５２巻，ｐ４５６（１９７３）」等に記載の方法に従って行うことができる。酵母を形質転換するには、例えば「Ｍｅｔｈｏｄｓ ｉｎ Ｅｎｚｙｍｏｌｏｇｙ，１９４巻，ｐ１８２－１８７ （１９９１）」、「Ｐｒｏｃ. Ｎａｔｌ. Ａｃａｄ. Ｓｃｉ. ＵＳＡ，７５，ｐ１９２９ （１９７８）」等に記載の方法に従って行うことができる。 Animal cells can be transformed, for example, according to the method described in "Cell Engineering Special Issue 8: New Cell Engineering Experimental Protocols, pp. 263-267 (1993) (published by Gakken Medical Shujunsha)" or "Virology, vol. 52, p. 456 (1973)". Yeast can be transformed, for example, according to the method described in "Methods in Enzymology, vol. 194, p. 182-187 (1991)" or "Proc. Natl. Acad. Sci. USA, 75, p. 1929 (1978)".
〔工程２で得られたベクターの宿主細胞への共導入〕
　細胞に高電圧をかけて導入する物理的方法、カルシウム処理により細胞に受容能を付与して導入する化学的方法等の公知の方法により、工程２で得られたベクターを宿主細胞に共導入し得る。化学的方法として、例えば、ＨＧＦαを発現させるためのベクター及びＨＧＦβを発現させるためのベクターを、Ｌｉｐｏｆｅｃｔａｍｉｎｅ（登録商標）等のトランスフェクション試薬及び公知の適当な培地と混ぜ、室温で適宜（例えば、１０分程度）インキュベートした後、当該ベクターを宿主細胞に添加し、約３７℃で、５％程度ＣＯ２条件下で４時間程度インキュベートする方法等が挙げられる。培地としては、Ｏｐｔｉ－ＭＥＭ（登録商標） Ｉ Ｒｅｄｕｃｅｄ Ｓｅｒｕｍ Ｍｅｄｉｕｍ、Ｐ３０００ ｒｅａｇｅｎｔ等の、血清を含まない培地であることが好ましい。本開示において、共導入とは、例えば、１つの細胞においてＨＧＦα及びＨＧＦβを発現できるように導入することを意味する。すなわち、あるベクターに、ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡを挿入し、別のベクターに、ＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡを挿入した場合、これらのベクターを、同一の宿主細胞に導入する（導入１）。同一のベクターに前記各ＤＮＡを挿入した場合、当該ベクターを、宿主細胞に導入する（導入２）。 [Co-introduction of the vectors obtained in step 2 into host cells]
The vectors obtained in step 2 can be co-introduced into host cells by known methods such as a physical method of applying a high voltage to cells for introduction, and a chemical method of imparting receptivity to cells by calcium treatment for introduction. Examples of chemical methods include a method in which a vector for expressing HGFα and a vector for expressing HGFβ are mixed with a transfection reagent such as Lipofectamine (registered trademark) and a known appropriate medium, incubated at room temperature appropriately (for example, about 10 minutes), and then the vector is added to host cells and incubated at about 37°C under about 5% CO2 conditions for about 4 hours. The medium is preferably a serum-free medium such as Opti-MEM (registered trademark) I Reduced Serum Medium or P3000 reagent. In the present disclosure, co-introduction means, for example, introduction so that HGFα and HGFβ can be expressed in one cell. That is, when a DNA encoding an amino acid sequence including an HGF α-chain is inserted into one vector and a DNA encoding an amino acid sequence including an HGF β-chain is inserted into another vector, these vectors are introduced into the same host cell (introduction 1).When each of the DNAs is inserted into the same vector, the vector is introduced into a host cell (introduction 2).
　本明細書において、「程度」及び「約」とは、例えば少々逸脱した場合も含ませる意図で使用する用語である。このような範囲は、所与の値又は範囲の測定及び／又は定量に使用される標準の方法に特有である実験誤差内（例えば±２、±１等）の場合も含まれる。従って、「程度」、「約」は必ずしも不明瞭ではないといえる。 In this specification, the terms "to the extent" and "about" are used with the intention of including, for example, small deviations. Such ranges also include those within the experimental error (e.g., ±2, ±1, etc.) that is inherent in the standard method used to measure and/or quantify a given value or range. Thus, it can be said that "to the extent" and "about" are not necessarily ambiguous.
〔宿主細胞の培養〕
　培養開始前に、ＨＧＦα鎖及びＨＧＦβ鎖の両方を発現する細胞を選抜してもよい。ＨＧＦα鎖及びＨＧＦβ鎖の両方を発現する細胞は、ベクターが有する薬剤耐性遺伝子等の選択マーカーを用いて選抜することができる。また、クローニングにより選抜することができる。
　細胞は、ＨＧＦα鎖及びＨＧＦβ鎖を一過性に発現する細胞でもよく、ＨＧＦα鎖及びＨＧＦβ鎖を安定発現する細胞でもよい。好ましくは、安定発現細胞である。 Cultivation of host cells
Before starting the culture, cells expressing both HGF α-chain and HGF β-chain may be selected. Cells expressing both HGF α-chain and HGF β-chain can be selected using a selection marker such as a drug resistance gene carried by a vector. Alternatively, they can be selected by cloning.
The cells may be cells that transiently express HGF α-chain and HGF β-chain, or cells that stably express HGF α-chain and HGF β-chain, preferably stably expressing cells.
　このようにして得られた宿主細胞を常とう手段を用いる当業者に知られた方法によって培養する。宿主が動物細胞である場合、培地は、Ｏｐｔｉ－ＭＥＭ（登録商標） Ｉ Ｒｅｄｕｃｅｄ Ｓｅｒｕｍ Ｍｅｄｉａ、Ｐ３０００ ｒｅａｇｅｎｔ等の、血清を含まない培地であることが好ましいが、約５～２０％のウシ胎児血清を含むＭＥＭ培地（Ｓｃｉｅｎｃｅ，１２２，ｐ５０１（１９５２））、ＤＭＥＭ培地（Ｖｉｒｏｌｏｇｙ，８，ｐ３９６ （１９５９））、Ｈａｍ’ｓ Ｆ－１２（Ｌ－グルタミン、フェノールレッド含有）培地、ＲＰＭＩ １６４０培地（Ｔｈｅ Ｊｏｕｒｎａｌ ｏｆ ｔｈｅ Ａｍｅｒｉｃａｎ Ｍｅｄｉｃａｌ Ａｓｓｏｃｉａｔｉｏｎ， １９９，ｐ５１９（１９６７））、１９９培地（Ｐｒｏｃｅｅｄｉｎｇ ｏｆ ｔｈｅ Ｓｏｃｉｅｔｙ ｆｏｒ ｔｈｅ Ｂｉｏｌｏｇｉｃａｌ Ｍｅｄｉｃｉｎｅ，７３，ｐ１ （１９５０））等であってもよい。ｐＨは約６～８であるのが好ましい。培養は通常約３０℃～４０℃で約１５～６０時間行い、必要に応じて通気や撹拌を加える。培養は５％ＣО２条件下で行うことが好ましい。 The host cells thus obtained are cultured by conventional means known to those skilled in the art. When the host is an animal cell, the medium is preferably a serum-free medium such as Opti-MEM (registered trademark) I Reduced Serum Media, P3000 reagent, etc., but other suitable medium include MEM medium containing about 5 to 20% fetal bovine serum (Science, 122, p501 (1952)), DMEM medium (Virology, 8, p396 (1959)), Ham's F-12 (containing L-glutamine and phenol red) medium, RPMI 1640 medium (The Journal of the American Medical Association, 199, p519 (1967)), 199 medium (Proceedings of the Society for the Biological Medicine, 73, p1 (1950)) or the like. The pH is preferably about 6 to 8. The culture is usually carried out at about 30°C to 40°C for about 15 to 60 hours, with aeration and stirring as necessary. The culture is preferably carried out under 5% CO2 conditions.
　宿主が昆虫細胞である場合、培地としては、例えばＳｆ－９００ＩＩＩ（商標名）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）、ＥＸ－ＣＥＬＬ４０５（商標名）（Ｍｅｒｃｋ）、Ｅｘｐｒｅｓｓ　Ｆｉｖｅ　ＳＦＭ（商標名）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）等が挙げられる。培地のｐＨは約５～７に調整するのが好ましい。培養は通常約２０℃～３０℃で約２４～７２時間行い、必要に応じて通気や撹拌を加える。 When the host is an insect cell, examples of the medium include Sf-900III (trade name) (Thermo Fisher Scientific), EX-CELL405 (trade name) (Merck), and Express Five SFM (trade name) (Thermo Fisher Scientific). The pH of the medium is preferably adjusted to about 5 to 7. Cultivation is usually carried out at about 20°C to 30°C for about 24 to 72 hours, with aeration and stirring as necessary.
　宿主が酵母である場合、培地としては、例えばバークホールダー（Ｂｕｒｋｈｏｌｄｅｒ）最小培地（Ｂｏｓｔｉａｎ, Ｋ. Ｌ. ら、Ｐｒｏｃ. Ｎａｔｌ. Ａｃａｄ. Ｓｃｉ. ＵＳＡ，７７，ｐ４５０５ （１９８０)）や０.５％カザミノ酸を含有するＳＤ培地（Ｂｉｔｔｅｒ，Ｇ．Ａ．ら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，８１，ｐ５３３０（１９８４））等が挙げられる。培地のｐＨは約５～８に調整するのが好ましい。培養は通常約２０℃～３５℃で約２４～７２時間行い、必要に応じて通気や撹拌を加える。 When the host is yeast, examples of media include Burkholder's minimal medium (Bostian, K. L. et al., Proc. Natl. Acad. Sci. USA, 77, p. 4505 (1980)) and SD medium containing 0.5% casamino acids (Bitter, G. A. et al., Proc. Natl. Acad. Sci. USA, 81, p. 5330 (1984)). The pH of the medium is preferably adjusted to about 5 to 8. Cultivation is usually carried out at about 20°C to 35°C for about 24 to 72 hours, with aeration and stirring as necessary.
〔宿主細胞におけるＨＧＦα鎖及びＨＧＦβ鎖のそれぞれの発現〕
　本開示の方法では、工程１でＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ、及び、ＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡをそれぞれ作製しているので、工程３において、宿主細胞においてＨＧＦα鎖及びＨＧＦβ鎖は、それぞれ発現される。 [Expression of HGF α-chain and HGF β-chain in host cells]
In the method of the present disclosure, DNA encoding an amino acid sequence including an HGF alpha chain and DNA encoding an amino acid sequence including an HGF beta chain are each prepared in step 1, and therefore, in step 3, the HGF alpha chain and the HGF beta chain are each expressed in a host cell.
〔工程４〕
　本開示の方法は、工程４として、工程３の後に、培養上清から活性型ＨＧＦを回収する工程を含む。
　工程３の後に、公知の方法、例えば、培養液を採取し、遠心分離し、培養上清を回収する方法等により、活性型ＨＧＦを回収又は取得することができる。
　その後精製する場合は、その精製を容易にするために、活性型ＨＧＦは、アフィニティータグが付加されたものであってもよい。タグとしては、ＦＬＡＧタグ、ヒスチジンタグ、ｃ－Ｍｙｃタグ、ＨＡタグ、ＡＵ１タグ、ＧＳＴタグ、ＭＢＰタグ、蛍光タンパク質タグ（例えばＧＦＰ、ＹＦＰ、ＲＦＰ、ＣＦＰ、ＢＦＰ等）、イムノグロブリンＦｃタグ等が挙げられる。タグ配列が付加される位置は、活性型ＨＧＦのＮ末端であってもよく、Ｃ末端であってもよい。また、アフィニティータグを付加した活性型ＨＧＦは、そのまま使用されてもよく、アフィニティータグを切り離してから使用されてもよい。 [Step 4]
The method of the present disclosure includes, as step 4, after step 3, a step of recovering active HGF from the culture supernatant.
After step 3, active HGF can be recovered or obtained by known methods, such as a method of collecting the culture medium, centrifuging it, and recovering the culture supernatant.
In the case of subsequent purification, the active HGF may be one to which an affinity tag has been added in order to facilitate the purification. Examples of the tag include a FLAG tag, a histidine tag, a c-Myc tag, an HA tag, an AU1 tag, a GST tag, an MBP tag, a fluorescent protein tag (e.g., GFP, YFP, RFP, CFP, BFP, etc.), an immunoglobulin Fc tag, etc. The position to which the tag sequence is added may be the N-terminus or the C-terminus of the active HGF. In addition, the active HGF to which an affinity tag has been added may be used as it is, or may be used after the affinity tag has been cleaved off.
〔好ましい組み合わせ〕
　本開示の方法において、宿主細胞が、ＣＨＯ－Ｋ１細胞、ＣＨＯ－ＤＧ４４（ＤＨＦＲ－）細胞、ＣＨＯ－Ｓ細胞、ＣＨＯ－ＭＫ細胞及びＣＨＯ－ＧＳ細胞からなる群から選択されており、前記宿主細胞に適したコドンとなるように改変されているヒトＨＧＦα鎖をコードするＤＮＡが、配列番号２で示される塩基配列を含み、かつ／又は、前記宿主細胞に適したコドンとなるように改変されているヒトＨＧＦβ鎖をコードするＤＮＡが、配列番号５で示される塩基配列を含むことが好ましい。 [Preferred combination]
In the method of the present disclosure, it is preferred that the host cell is selected from the group consisting of CHO-K1 cells, CHO-DG44 (DHFR-) cells, CHO-S cells, CHO-MK cells and CHO-GS cells, and that the DNA encoding the human HGF α chain that has been modified to have codons suitable for the host cell comprises the base sequence shown in SEQ ID NO: 2, and/or the DNA encoding the human HGF β chain that has been modified to have codons suitable for the host cell comprises the base sequence shown in SEQ ID NO: 5.
〔活性型ＨＧＦ〕
　例えば、本開示の方法により製造されるＨＧＦが、ポジティブコントロールの活性型ＨＧＦの活性と比較して、実質的に同等のＨＧＦ活性を有していることを確認することで、そのＨＧＦは、活性型ＨＧＦであることを確認できる。この場合における同等とは、活性型ＨＧＦのポジティブコントロールの活性と比較して、活性が５０％以上であってもよく、６０％以上であってもよく、７０％以上であってもよく、８０％以上であってもよく、９０％以上であってもよく、１００％以上であってもよい。 [Active HGF]
For example, by confirming that the HGF produced by the method of the present disclosure has substantially the same HGF activity as that of a positive control active HGF, the HGF can be confirmed to be an active HGF. In this case, "equivalent" may mean that the activity is 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more compared to the activity of the active HGF positive control.
　具体的には、ＨＧＦを添加した細胞の増殖性、遊走あるいはＨＧＦの受容体であるＭｅｔのリン酸化等を評価することにより、ＨＧＦ活性を確認することができる。ＨＧＦ活性の確認方法は、Ｊ．Ｉｍｍｕｎｏｌ．Ｍｅｔｈｏｄｓ．２００１；２５８：１－１１，　Ｎａｔ．Ｃｏｍｍｍｕｎ．２０１５；６：６３７３等に記載されている。 Specifically, HGF activity can be confirmed by evaluating the proliferation and migration of cells to which HGF has been added, or the phosphorylation of Met, an HGF receptor, etc. Methods for confirming HGF activity are described in J. Immunol. Methods. 2001; 258: 1-11, Nat. Commun. 2015; 6: 6373, etc.
〔活性型ＨＧＦの活性〕
　活性型ＨＧＦはその活性（薬理作用）から、脊髄損傷治療剤等の創傷治療剤、皮膚潰瘍の予防又は治癒剤、血管新生促進剤、肉芽増生促進剤、喘息の予防又は治療剤、知的障害の改善剤、ポリグルタミン病の治療剤又は発病抑制剤、肝硬変治療剤、腎疾患治療剤、上皮細胞増殖促進剤、抗ガン剤、ガン療法用副作用防止剤、肺障害治療剤、胃・十二指腸損傷治療剤、脳神経障害治療剤、免疫抑制副作用防止剤（例えば、免疫抑制剤投与に伴う移植臓器の線維化抑制剤等）、コラーゲン分解促進剤、軟骨障害治療剤、動脈疾患治療剤、肺線維症治療剤、肝臓疾患治療剤、血液凝固異常治療剤、血漿低蛋白治療剤、神経障害改善剤、造血幹細胞増加剤、育毛促進剤、糸球体スリット蛋白の誘導剤又は維持剤等として有用である。これらの活性は、例えば、特許第５４１９０４５号、特許第４５３７９５６号、ＷＯ２００７／１２２９７６、ＷＯ２００５／０２３２８７、特開２０１１－１７３９００等に記載されている。 [Activity of active HGF]
Due to its activity (pharmacological action), active HGF is useful as a wound healing agent such as a spinal cord injury therapeutic agent, a preventive or healing agent for skin ulcers, an angiogenesis promoter, a granulation proliferation promoter, a preventive or therapeutic agent for asthma, an agent for improving intellectual disability, a therapeutic agent for polyglutamine disease or an agent for suppressing the onset of the disease, a liver cirrhosis therapeutic agent, a kidney disease therapeutic agent, an epithelial cell proliferation promoter, an anticancer agent, an agent for preventing side effects in cancer therapy, a lung injury therapeutic agent, a gastric/duodenal injury therapeutic agent, a cranial nerve disorder therapeutic agent, an agent for preventing immunosuppressive side effects (for example, an agent for suppressing fibrosis of transplanted organs associated with the administration of immunosuppressants), a collagen decomposition promoter, a cartilage injury therapeutic agent, an arterial disease therapeutic agent, a pulmonary fibrosis therapeutic agent, a liver disease therapeutic agent, a blood coagulation abnormality therapeutic agent, a plasma low protein therapeutic agent, an agent for improving nerve disorders, an agent for increasing hematopoietic stem cells, a hair growth promoter, an inducer or maintainer of glomerular slit protein, and the like. These activities are described, for example, in Japanese Patent No. 5419045, Japanese Patent No. 4537956, WO2007/122976, WO2005/023287, JP2011-173900A, etc.
　本開示の方法では、工程３でＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ、及び、ＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡをそれぞれ発現させているにもかかわらず、驚くべきことに、工程４において、培養上清から活性型ＨＧＦが回収される。
　工程３において発現されるＨＧＦα鎖及びβ鎖それぞれに有するチオール（ＳＨ）基が、細胞内においてカップリングし、工程３又は４において、ジスルフィド結合が形成されていると推測される。 In the method of the present disclosure, although DNA encoding an amino acid sequence containing an HGF α chain and DNA encoding an amino acid sequence containing an HGF β chain are each expressed in step 3, surprisingly, active HGF is recovered from the culture supernatant in step 4.
It is presumed that thiol (SH) groups present in the HGF α-chain and β-chain expressed in step 3 are coupled in the cells to form disulfide bonds in step 3 or 4.
　本開示における配列番号の説明を表１に示す。 The sequence numbers in this disclosure are explained in Table 1.
　本開示は、本開示の効果を奏する限り、本開示の技術的範囲内において、上記の構成を種々組み合わせた態様を含む。 This disclosure includes various combinations of the above configurations within the technical scope of this disclosure, so long as the effects of this disclosure are achieved.
　次に、実施例を挙げて本開示のいくつかの実施形態をさらに具体的に説明するが、本開示の実施形態はこれらの実施例により何ら限定されるものではなく、多くの変形が本開示の技術的思想内で当分野において通常の知識を有する者により可能である。 Next, some embodiments of the present disclosure will be described in more detail with reference to examples, but the embodiments of the present disclosure are in no way limited to these examples, and many modifications are possible within the technical ideas of the present disclosure by those with ordinary skill in the art.
（１）実施例のサンプル（ｐＨＧＦα＋ｐＨＧＦβ）の製造方法
〔工程１〕
　配列番号２のＤＮＡ（ヒトＨＧＦシグナル配列の塩基配列が５’末端側に結合しているヒト５アミノ酸欠失ＨＧＦα鎖の塩基配列が、ＣＨＯに適したコドンとなるように改変され、３’末端側に終止コドンＴＧＡが付加されている塩基配列）、及び、配列番号５のＤＮＡ（ヒトＨＧＦシグナル配列の塩基配列が５’末端側に結合しているヒトＨＧＦβ鎖の塩基配列が、ＣＨＯに適したコドンとなるように改変され、３’末端側に終止コドンＴＧＡが付加されている塩基配列）のそれぞれの５’末端に、制限酵素ＨｉｎｄＩＩＩが付加され、３’末端に、ＸｈｏＩ認識配列が付加されたＤＮＡ（２種類のＤＮＡ；ＨＧＦα鎖のＤＮＡ及びＨＧＦβ鎖のＤＮＡ）を合成した。具体的には、ジェンスクリプトジャパン株式会社に委託してＯｐｔｉｍｕｍＧｅｎｅアルゴリズムを用いてＣＨＯのコドンに最適化した塩基配列のＤＮＡを、ＤＮＡプリンティング技術を用いて合成した。 (1) Method for producing the sample of the embodiment (pHGFα+pHGFβ) [Step 1]
DNA of SEQ ID NO: 2 (a base sequence in which the base sequence of a human 5 amino acid deletion HGF α chain in which the base sequence of a human HGF signal sequence is linked to the 5'-end side is modified to become a codon suitable for CHO, and a stop codon TGA is added to the 3'-end side) and DNA of SEQ ID NO: 5 (a base sequence in which the base sequence of a human HGF β chain in which the base sequence of a human HGF signal sequence is linked to the 5'-end side is modified to become a codon suitable for CHO, and a stop codon TGA is added to the 3'-end side) were added with restriction enzyme HindIII to the 5'-end and with XhoI recognition sequence to the 3'-end (two types of DNA; DNA of HGF α chain and DNA of HGF β chain). Specifically, DNA of a base sequence optimized for CHO codons using the OptimumGene algorithm was commissioned to GenScript Japan Co., Ltd. and synthesized using DNA printing technology.
〔工程２〕
　工程１で得られたＨＧＦα鎖のＤＮＡ、及び、プラスミドｐｃＤＮＡ３．１（＋）をチューブ内で混合した。それとは別に、工程１で得られたＨＧＦβ鎖のＤＮＡ、及び、プラスミドｐｃＤＮＡ３．１（＋）をチューブ内で混合した。このようにして得られた各チューブ内の混合物を、ＨｉｎｄＩＩＩ及びＸｈｏＩで処理し、ＤＮＡリガーゼで連結させ、ＨＧＦα発現プラスミド及びＨＧＦβ発現プラスミドをそれぞれ別々のチューブにて作製した。作製した発現プラスミドはサンガー法によるシーケンス解析及び制限酵素消化により、正しく作製されたことを確認した。 [Step 2]
The DNA of the HGF α chain obtained in step 1 and the plasmid pcDNA3.1(+) were mixed in a tube. Separately, the DNA of the HGF β chain obtained in step 1 and the plasmid pcDNA3.1(+) were mixed in a tube. The mixtures in each tube thus obtained were treated with HindIII and XhoI, and ligated with DNA ligase to prepare HGF α expression plasmid and HGF β expression plasmid in separate tubes. The prepared expression plasmids were confirmed to be correctly prepared by sequence analysis by the Sanger method and digestion with restriction enzymes.
〔工程３〕
　遺伝子導入の前日にＣＨＯ－Ｋ１細胞（理化学研究所バイオリソース研究センター、ＲＣＢ０２８５）を６ウェルプレート（コーニング、３５１６）に１ウェルあたり５×１０５ｃｅｌｌｓ／ｍＬ（液量２ｍＬ）で播種した。培地として、１０％ウシ胎児血清（ニチレイバイオサイエンス, １７４０１２－５００ｍＬ）含有Ｈａｍ’ｓ Ｆ－１２（Ｌ－グルタミン、フェノールレッド含有）培地（富士フイルム和光純薬、０８７－０８３３５）を用いた。細胞に導入するＨＧＦα発現プラスミド１．２５μg及びＨＧＦβ発現プラスミド１．２５μgと、Ｌｉｐｏｆｅｃｔａｍｉｎｅ ３０００（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ, Ｌ３０００００１）７．５μＬ、Ｏｐｔｉ－ＭＥＭ（登録商標）Ｉ Ｒｅｄｕｃｅｄ Ｓｅｒｕｍ Ｍｅｄｉｕｍ (Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ、３１９８５０６２)２５０μＬ及びＰ３０００ ｒｅａｇｅｎｔ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ、Ｌ３０００００１)５μＬと混ぜ、室温で１０分間インキュベートしたのち、上記の細胞培養培地に添加した。４時間後に、各ウェルに、Ｏｐｔｉ－ＭＥＭ(登録商標) Ｉ Ｒｅｄｕｃｅｄ Ｓｅｒｕｍ Ｍｅｄｉｕｍ ５００μＬを加え、３７℃、５％ＣＯ２の条件で培養した。 [Step 3]
The day before gene transfer, CHO-K1 cells (RIKEN BioResource Research Center, RCB0285) were seeded at 5 x 10 5 cells/mL (2 mL volume) per well in a 6-well plate (Corning, 3516). Ham's F-12 (containing L-glutamine and phenol red) medium (Fujifilm Wako Pure Chemical Industries, 087-08335) containing 10% fetal bovine serum (Nichirei Biosciences, 174012-500 mL) was used as the medium. 1.25 μg of the HGFα expression plasmid and 1.25 μg of the HGFβ expression plasmid to be introduced into the cells were mixed with 7.5 μL of Lipofectamine 3000 (Thermo Fisher Scientific, L3000001), 250 μL of Opti-MEM (registered trademark) I Reduced Serum Medium (Thermo Fisher Scientific, 31985062), and 5 μL of P3000 reagent (Thermo Fisher Scientific, L3000001), incubated at room temperature for 10 minutes, and then added to the above cell culture medium. After 4 hours, 500 μL of Opti-MEM® I Reduced Serum Medium was added to each well, and the cells were cultured under conditions of 37° C. and 5% CO 2 .
〔工程４〕
　遺伝子導入から４８時間後に培養液を採取し、１０，０００ｒｐｍで１分間、遠心分離し、上清を回収し、実施例のサンプルを製造した。 [Step 4]
The culture medium was collected 48 hours after gene transfer and centrifuged at 10,000 rpm for 1 minute, and the supernatant was recovered to prepare a sample for the example.
（２）対照サンプルの製造方法
（２－１）一本鎖ＨＧＦ（ｐＳＲα－ＨＧＦ）
　上記実施例のサンプルの製造方法におけるプラスミド（細胞に導入するＨＧＦα発現プラスミド１．２５μg及びＨＧＦβ発現プラスミド１．２５μg）を「一本鎖のヒト５アミノ酸欠失ＨＧＦ（配列番号１４）発現プラスミド２．５μｇ」に置き換えた以外は、当該方法と同様にして一本鎖ＨＧＦ（ｐＳＲα－ＨＧＦ）を製造した。 (2) Method for producing control sample (2-1) Single-chain HGF (pSRα-HGF)
Single-stranded HGF (pSRα-HGF) was produced in the same manner as in the manufacturing method of the sample of the above Example, except that the plasmids (1.25 μg of HGF α expression plasmid and 1.25 μg of HGF β expression plasmid to be introduced into cells) were replaced with "2.5 μg of single-stranded human 5 amino acid deleted HGF (SEQ ID NO: 14) expression plasmid."
（２－２）ｐＨＧＦα
　上記実施例のサンプルの製造方法におけるプラスミド（細胞に導入するＨＧＦα発現プラスミド１．２５μg及びＨＧＦβ発現プラスミド１．２５μg）を「ＨＧＦα発現プラスミド１．２５μg」に置き換えた以外は、当該方法と同様にしてｐＨＧＦαを作製した。 (2-2) pHGFα
pHGFα was prepared in the same manner as in the manufacturing method of the sample of the above Example, except that the plasmids (1.25 μg of HGFα expression plasmid and 1.25 μg of HGFβ expression plasmid to be introduced into cells) were replaced with "1.25 μg of HGFα expression plasmid."
（２－３）ｐＨＧＦβ
　上記実施例のサンプルの製造方法におけるプラスミド（細胞に導入するＨＧＦα発現プラスミド１．２５μg及びＨＧＦβ発現プラスミド１．２５μg）を「ＨＧＦβ発現プラスミド１．２５μg」に置き換えた以外は、当該方法と同様にしてｐＨＧＦβを作製した。 (2-3) pHGFβ
pHGFβ was prepared in the same manner as in the manufacturing method of the sample of the above Example, except that the plasmids (1.25 μg of HGFα expression plasmid and 1.25 μg of HGFβ expression plasmid to be introduced into cells) were replaced with "1.25 μg of HGFβ expression plasmid."
（２－４）ポジティブコントロール（ＳＴＤ）
　活性型ＨＧＦのポジティブコントロールとして、組換えヒトＨＧＦタンパク質（ＫＰ－１００、クリングルファーマ株式会社製）を用いた。
（２－５）ネガティブコントロール（ｍｏｃｋ）
　ネガティブコントロールとして、ＣＨＯ細胞培養上清を用いた。 (2-4) Positive control (STD)
As a positive control for active HGF, recombinant human HGF protein (KP-100, Kringle Pharma Inc.) was used.
(2-5) Negative control (mock)
As a negative control, CHO cell culture supernatant was used.
ウエスタンブロット：
（実験方法）
　ＨＧＦ発現培養上清は還元剤であるジチオスレイトール（ＡＴＴＯ, ＡＥ－１４３０）の存在、又は非存在下でＳＤＳサンプルバッファー（ＡＴＴＯ, ＡＥ－１４３０）と混合し、５０℃で１０分間インキュベートしたのち、ＳＤＳ－ＰＡＧＥ用ゲル（コスモバイオ, ＭＵＬＴＩＧＥＬ（登録商標） ＩＩ ｍｉｎｉ ８/１６ （１３Ｗ）, ４１７２６９）にアプライし、２０ｍＡの定電流で通電した。サンプルバッファーの染色液がゲルの下端に達した時、通電を終了した。ゲルを洗浄液（ＡＴＴＯ, ＷＳＥ－４０５５）で洗浄後、Ｑブロットキット(ＡＴＴＯ, ＷＳＥ－４０５５)を用いて、ブロッティング装置（Ｂｉｏ－Ｒａｄ, Ｔｒａｎｓ－Ｂｌｏｔ ＳＤ Ｃｅｌｌ）の下部電極板上にろ紙、ＰＶＤＦメンブレン、ゲル、ろ紙の順にのせ、気泡を取り除いて上部電極板で挟んだ。パワーサプライ(Ｂｉｏ－Ｒａｄ, パワーパックＨＣ)を用いて１２Ｖの定電圧で３０分間通電し、ブロッティングした。ブロッティングが終了したＰＶＤＦメンブレンはブロッキンバッファー（ＡＴＴＯ, ＡＥ－１４７５）中で室温３０分間インキュベートし、０．５％ウシ血清アルブミン含有ＰＢＳＴバッファーで２０００倍に希釈した抗ＨＧＦ抗体(Ｒ＆Ｄ ｓｙｓｔｅｍｓ, Ａｎｔｉ－Ｈｕｍａｎ ＨＧＦ Ａｆｆｉｎｉｔｙ Ｐｕｒｉｆｉｅｄ Ｐｏｌｙｃｌｏｎａｌ Ａｂ, ＡＦ２９４－ＳＰ)で一晩、冷所でインキュベートした。ＰＢＳＴで洗浄した後、０．５％ウシ血清アルブミン含有ＰＢＳＴバッファーで１０００倍希釈したウサギ抗ヤギＩｇＧ－ＨＲＰ（ＤＡＫＯ, Ｐ０４４９）と室温で２時間インキュベートした。ＰＢＳＴで洗浄した後、検出基質(ＡＴＴＯ, ＷＳＥ－７１２０Ｓ)と反応させたのち発光をルミノイメージアナライザー（富士フイルム、ＬＡＳ－１０００）で検出した。 Western Blot:
(experimental method)
The HGF-expressing culture supernatant was mixed with SDS sample buffer (ATTO, AE-1430) in the presence or absence of dithiothreitol (ATTO, AE-1430) as a reducing agent, and incubated at 50°C for 10 minutes. The mixture was then applied to an SDS-PAGE gel (Cosmo Bio, MULTIGEL (registered trademark) II mini 8/16 (13W), 417269) and a constant current of 20 mA was applied. When the staining solution of the sample buffer reached the bottom of the gel, the application of the current was stopped. After washing the gel with a washing solution (ATTO, WSE-4055), the filter paper, PVDF membrane, gel, and filter paper were placed in this order on the lower electrode plate of a blotting device (Bio-Rad, Trans-Blot SD Cell) using a Q-blot kit (ATTO, WSE-4055), and air bubbles were removed before sandwiching the upper electrode plate. Blotting was performed by applying a constant voltage of 12 V for 30 minutes using a power supply (Bio-Rad, Power Pack HC). After blotting, the PVDF membrane was incubated in blocking buffer (ATTO, AE-1475) at room temperature for 30 minutes, and then incubated overnight in a cold place with anti-HGF antibody (R&D systems, Anti-Human HGF Affinity Purified Polyclonal Ab, AF294-SP) diluted 2000-fold with PBST buffer containing 0.5% bovine serum albumin. After washing with PBST, the membrane was incubated at room temperature for 2 hours with rabbit anti-goat IgG-HRP (DAKO, P0449) diluted 1000-fold with PBST buffer containing 0.5% bovine serum albumin. After washing with PBST, the plate was reacted with a detection substrate (ATTO, WSE-7120S) and the luminescence was detected with a luminometer image analyzer (FUJIFILM, LAS-1000).
（結果）
　結果を図１に示す。尚、図１において、ＣＭは、ＣＨＯ細胞培養上清を意味する。 (result)
The results are shown in Figure 1. In Figure 1, CM means CHO cell culture supernatant.
　図１から明らかなように、実施例のサンプル（ｐＨＧＦα＋ｐＨＧＦβ）（ｎ＝２）では、非還元条件で、一本鎖ＨＧＦと同等の位置にバンドが認められ、一方で、還元条件で、ｐＨＧＦα及びｐＨＧＦβと同等の位置にバンドが認められた。
　また、実施例のサンプルでは、還元条件及び非還元条件において、ＳＴＤ（ポジティブコントロール）と同等の位置にバンドが認められた。
　以上のことから、実施例のサンプル（ｐＨＧＦα＋ｐＨＧＦβ）には、ヘテロ二量化されているヒトＨＧＦが含まれると考えられる。すなわち、本開示の方法により、分泌シグナルが切断されたＨＧＦα鎖タンパク質と分泌シグナルが切断されたＨＧＦβ鎖タンパク質がジスルフィド結合でヘテロ二量化されているＨＧＦを作製できることが確認された。 As is clear from Figure 1, in the example sample (pHGFα + pHGFβ) (n = 2), under non-reducing conditions, a band was observed at a position equivalent to that of single-chain HGF, while under reducing conditions, bands were observed at positions equivalent to that of pHGFα and pHGFβ.
In addition, in the sample of the example, a band was observed at the same position as that of STD (positive control) under reducing and non-reducing conditions.
From the above, it is considered that the sample of the embodiment (pHGFα+pHGFβ) contains heterodimerized human HGF. That is, it was confirmed that the method of the present disclosure can produce HGF in which an HGF α chain protein with a secretion signal cleaved and an HGF β chain protein with a secretion signal cleaved are heterodimerized by a disulfide bond.
　細胞増殖促進活性の評価試験：
（試験方法）
１．細胞の準備
　Ｍｖ.１.Ｌｕ細胞（理化学研究所バイオリソース研究センター、 ＲＣＢ０９９６）を融解し、Ｔ－２５フラスコ（コーニング、４３０６３９）に入れた１０％ウシ胎児血清（ニチレイバイオサイエンス, １７４０１２－５００ｍＬ）及び１％ＭＥＭ非必須アミノ酸溶液（×１００）(Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ、Ｃａｔ. Ｎｏ.１１１４０－０５０)含有ＭＥＭ培地（アール塩，Ｌ－グルタミン含有）（ナカライテスク，Ｃａｔ. Ｎｏ. ２１４４２－２５）５ｍＬで３７℃，５％ＣＯ２存在下で培養した。３日後にフラスコ内の培地を廃棄した。ＰＢＳ（－）(Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ、Ｃａｔ. Ｎｏ. ２００１２－０２７)２ｍＬでの洗浄後、細胞解離酵素（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ，Ｃａｔ. Ｎｏ. １２５６３０１１）２ｍＬを加えて，フラスコ底面にゆきわたらせた。細胞解離酵素を除き、３７℃で３分間放置した。２％ウシ胎児血清（ニチレイバイオサイエンス、１７４０１２－５００ｍＬ）含有ＲＰＭＩ１６４０培地(Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ，Ｃａｔ. Ｎｏ. １１８７５－０９３)１５ｍＬを加えて、ピペッティングにより細胞を回収した。細胞浮遊液５０μＬに０．４％トリパンブルー染色液(富士フイルム和光純薬、Ｃａｔ. Ｎｏ. ２０７－１７０８１)５０μＬを加え、混合後、血球計算盤を用いて生細胞及び死細胞の数を計数した。計数値より生細胞数及び生存率を算出した。２％ウシ胎児血清含有ＲＰＭＩ１６４０培地を用いて、生細胞密度を０．５×１０５ｃｅｌｌｓ／ｍＬに調整した。細胞浮遊液に濃度が５００ｐｇ／ｍＬとなるようにＴＧＦ－β１( Ｒ＆Ｄ，Ｃａｔ. Ｎｏ. ２４０－Ｂ－００２)を加えた。 Cell proliferation promoting activity evaluation test:
(Test method)
1. Preparation of cells Mv.1.Lu cells (RIKEN BioResource Research Center, RCB0996) were thawed and cultured in 5 mL of MEM medium (containing Earle's salts and L-glutamine) (Nacalai Tesque, Cat. No. 21442-25) containing 10% fetal bovine serum (Nichirei Biosciences, 174012-500 mL) and 1% MEM non-essential amino acid solution (x100) (Thermo Fisher, Cat. No. 11140-050) in a T-25 flask (Corning, 430639) at 37°C in the presence of 5% CO2 . After 3 days, the medium in the flask was discarded. After washing with 2 mL of PBS (-) (Thermo Fisher, Cat. No. 20012-027), 2 mL of cell dissociation enzyme (Thermo Fisher, Cat. No. 12563011) was added and spread over the bottom of the flask. The cell dissociation enzyme was removed and the flask was left at 37°C for 3 minutes. 15 mL of RPMI1640 medium (Thermo Fisher, Cat. No. 11875-093) containing 2% fetal bovine serum (Nichirei Biosciences, 174012-500 mL) was added, and the cells were collected by pipetting. 50 μL of 0.4% trypan blue staining solution (FUJIFILM Wako Pure Chemical Industries, Cat. No. 207-17081) was added to 50 μL of the cell suspension, and after mixing, the number of live and dead cells was counted using a hemocytometer. The number of live cells and survival rate were calculated from the counted value. The live cell density was adjusted to 0.5 x 10 5 cells/mL using RPMI 1640 medium containing 2% fetal bovine serum. TGF-β1 (R&D, Cat. No. 240-B-002) was added to the cell suspension to a concentration of 500 pg/mL.
２．細胞増殖促進活性の確認
　この細胞浮遊液５０μＬを９６ウェルマイクロプレート各ウェルに添加し、３７℃、５％ＣＯ２存在下で２時間培養した。各サンプルを２％ウシ胎児血清含有ＲＰＭＩ１６４０培地で希釈し、５０μＬをウェルに添加した(ｎ＝３)。３７℃、５％ＣＯ２存在下で，７２時間培養した。培養終了後、ＭＴＳ試薬(プロメガ，Ｃｅｌｌ Ｔｉｔｅｒ ９６ Ａｑｕｅｏｕｓ Ｏｎｅ Ｓｏｌｕｔｉｏｎ Ｃｅｌｌ Ｐｒｏｌｉｆｅｒａｔｉｏｎ Ａｓｓａｙ，Ｃａｔ.Ｎｏ. Ｇ３５８０)２０μＬを各ウェルに添加した。３７℃，５％ ＣＯ２存在下で４時間インキュベートし、マイクロプレートリーダーで４９２ｎｍの吸光度を測定した。 2. Confirmation of cell proliferation promoting activity 50 μL of this cell suspension was added to each well of a 96-well microplate and cultured at 37°C and 5% CO2 for 2 hours. Each sample was diluted with RPMI1640 medium containing 2% fetal bovine serum, and 50 μL was added to the well (n=3). Culture was performed at 37°C and 5% CO2 for 72 hours. After the culture was completed, 20 μL of MTS reagent (Promega, Cell Titer 96 Aqueous One Solution Cell Proliferation Assay, Cat. No. G3580) was added to each well. The cells were incubated at 37°C and 5% CO2 for 4 hours, and the absorbance at 492 nm was measured using a microplate reader.
（結果）
　結果を図２及び３に示す。図２から明らかなように、実施例のサンプルは、一本鎖ＨＧＦが血清により活性化されたもの（図２において、一本鎖ＨＧＦと表記する）及びポジティブコントロールと同等の細胞増殖促進活性を示すことが確認された。また、図３から明らかなように、実施例のサンプルは、一本鎖ＨＧＦが血清により活性化されたもの（図３において、一本鎖ＨＧＦと表記する）と同等の細胞増殖促進活性を示すことが確認された。一方、図３から明らかなように、ネガティブコントロールの細胞増殖促進活性は確認されなかった。これらのことから、実施例のサンプルは、一本鎖ＨＧＦが血清により活性化されたもの又はポジティブコントロールと同様の細胞増殖促進活性を有する活性型ＨＧＦを含むことが確認された。 (result)
The results are shown in Figures 2 and 3. As is clear from Figure 2, it was confirmed that the sample of the Example exhibited cell proliferation promoting activity equivalent to that of the single-chain HGF activated by serum (referred to as single-chain HGF in Figure 2) and the positive control. Also, as is clear from Figure 3, it was confirmed that the sample of the Example exhibited cell proliferation promoting activity equivalent to that of the single-chain HGF activated by serum (referred to as single-chain HGF in Figure 3). On the other hand, as is clear from Figure 3, no cell proliferation promoting activity was confirmed for the negative control. From these findings, it was confirmed that the sample of the Example contains active HGF having cell proliferation promoting activity equivalent to that of the single-chain HGF activated by serum or the positive control.

Claims (12)

  1. 　下記工程１～４の工程を含む、活性型ＨＧＦを製造する方法。
    工程１：
    　ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ、及びＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡをそれぞれ作製する工程
    工程２：
    　工程１で作製されたそれぞれのＤＮＡを、タンパク質を発現させるためのベクターに挿入する工程
    工程３：
    　工程２で得られたベクターを宿主細胞に共導入し、宿主細胞を培養することにより、宿主細胞においてＨＧＦα鎖及びＨＧＦβ鎖をそれぞれ発現させる工程
    工程４：
    　工程３の後に、培養上清から活性型ＨＧＦを回収する工程     A method for producing active HGF, comprising the steps of:
    Step 1:
    Step 2: preparing a DNA encoding an amino acid sequence containing an HGF α chain and a DNA encoding an amino acid sequence containing an HGF β chain
    Step 3: Insert each DNA prepared in step 1 into a vector for expressing a protein.
    Step 4: Co-introducing the vectors obtained in step 2 into host cells and culturing the host cells to express HGF α-chain and HGF β-chain in the host cells.
    A step of recovering active HGF from the culture supernatant after step 3.
  2. 　ＨＧＦα鎖を含むアミノ酸配列、及びＨＧＦβ鎖を含むアミノ酸配列のそれぞれが、シグナル配列を含むことを特徴とする請求項１記載の方法。 The method according to claim 1, characterized in that the amino acid sequence containing the HGF α chain and the amino acid sequence containing the HGF β chain each contain a signal sequence.
  3. 　ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ、及びＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡのそれぞれが、終止コドンを含むことを特徴とする、請求項１又は２記載の方法。 The method according to claim 1 or 2, characterized in that the DNA encoding an amino acid sequence including the HGF α chain and the DNA encoding an amino acid sequence including the HGF β chain each contain a stop codon.
  4. 　シグナル配列が、ＨＧＦシグナル配列であることを特徴とする、請求項２記載の方法。 The method according to claim 2, characterized in that the signal sequence is an HGF signal sequence.
  5. 　ＨＧＦシグナル配列が、ヒトＨＧＦシグナル配列であることを特徴とする、請求項４記載の方法。 The method according to claim 4, characterized in that the HGF signal sequence is a human HGF signal sequence.
  6. 　ＨＧＦα鎖を含むアミノ酸配列をコードするＤＮＡ、及び／又は、ＨＧＦβ鎖を含むアミノ酸配列をコードするＤＮＡが、宿主細胞に適したコドンとなるように改変されているＤＮＡであることを特徴とする、請求項１記載の方法。 The method according to claim 1, characterized in that the DNA encoding an amino acid sequence containing the HGF α chain and/or the DNA encoding an amino acid sequence containing the HGF β chain is DNA that has been modified to have codons suitable for the host cell.
  7. 　ＨＧＦα鎖が、ヒトＨＧＦα鎖であることを特徴とする、請求項１記載の方法。 The method of claim 1, wherein the HGF α chain is a human HGF α chain.
  8. 　ヒトＨＧＦα鎖が、配列番号１３又は１９で示されるアミノ酸配列からなることを特徴とする、請求項７記載の方法。 The method according to claim 7, characterized in that the human HGF α chain consists of the amino acid sequence shown in SEQ ID NO: 13 or 19.
  9. 　ＨＧＦβ鎖が、ヒトＨＧＦβ鎖であることを特徴とする、請求項１記載の方法。 The method according to claim 1, characterized in that the HGF β chain is a human HGF β chain.
  10. 　ヒトＨＧＦβ鎖が、配列番号１６で示されるアミノ酸配列からなることを特徴とする、請求項９記載の方法。 The method according to claim 9, characterized in that the human HGF β chain consists of the amino acid sequence shown in SEQ ID NO: 16.
  11. 　宿主細胞が、ＣＨＯ－Ｋ１細胞、ＣＨＯ－ＤＧ４４（ＤＨＦＲ－）細胞、ＣＨＯ－Ｓ細胞、ＣＨＯ－ＭＫ細胞、ＣＨＯ－ＧＳ細胞、ＨＥＫ２９３細胞、ＢＨＫ－２１細胞、ＰＥＲ．Ｃ６細胞、ＮＳ０細胞、ＳＰ２／０細胞、Ｓｆ９細胞、Ｓｆ２１細胞、Ｈｉｇｈ　Ｆｉｖｅ細胞、Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ及びＰｉｃｈｉａ　ｐａｓｔｏｒｉｓからなる群から選択されることを特徴とする、請求項１記載の方法。 The method according to claim 1, characterized in that the host cell is selected from the group consisting of CHO-K1 cells, CHO-DG44 (DHFR-) cells, CHO-S cells, CHO-MK cells, CHO-GS cells, HEK293 cells, BHK-21 cells, PER. C6 cells, NS0 cells, SP2/0 cells, Sf9 cells, Sf21 cells, High Five cells, Saccharomyces cerevisiae and Pichia pastoris.
  12. 　宿主細胞が、ＣＨＯ－Ｋ１細胞、ＣＨＯ－ＤＧ４４（ＤＨＦＲ－）細胞、ＣＨＯ－Ｓ細胞、ＣＨＯ－ＭＫ細胞及びＣＨＯ－ＧＳ細胞からなる群から選択されており、前記宿主細胞に適したコドンとなるように改変されているヒトＨＧＦα鎖をコードするＤＮＡが、配列番号２で示される塩基配列を含み、かつ／又は、前記宿主細胞に適したコドンとなるように改変されているヒトＨＧＦβ鎖をコードするＤＮＡが、配列番号５で示される塩基配列を含むことを特徴とする、請求項６、７、９又は１１記載の方法。 The method according to claim 6, 7, 9 or 11, characterized in that the host cell is selected from the group consisting of CHO-K1 cells, CHO-DG44 (DHFR-) cells, CHO-S cells, CHO-MK cells and CHO-GS cells, and the DNA encoding the human HGF α chain modified to have codons suitable for the host cell contains the base sequence shown in SEQ ID NO: 2, and/or the DNA encoding the human HGF β chain modified to have codons suitable for the host cell contains the base sequence shown in SEQ ID NO: 5.
PCT/JP2023/039364 2022-11-01 2023-11-01 Method for producing active hgf WO2024096038A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1385533A (en) * 2001-12-05 2002-12-18 协和生物工程研究所有限公司 Process for preparing recombinant human liver cell grouth factor
WO2017159722A1 (en) * 2016-03-17 2017-09-21 エーザイ・アール・アンド・ディー・マネジメント株式会社 Method for producing activated hepatocyte growth factor (hgf)
CN110607304A (en) * 2019-07-25 2019-12-24 广州凌腾生物医药有限公司 Recombinant expression method of hepatocyte growth factor

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1385533A (en) * 2001-12-05 2002-12-18 协和生物工程研究所有限公司 Process for preparing recombinant human liver cell grouth factor
WO2017159722A1 (en) * 2016-03-17 2017-09-21 エーザイ・アール・アンド・ディー・マネジメント株式会社 Method for producing activated hepatocyte growth factor (hgf)
CN110607304A (en) * 2019-07-25 2019-12-24 广州凌腾生物医药有限公司 Recombinant expression method of hepatocyte growth factor

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