WO2010010848A1 - Procédé de production d'anticorps dans une cellule de mammifère en culture en utilisant un chaperon moléculaire - Google Patents

Procédé de production d'anticorps dans une cellule de mammifère en culture en utilisant un chaperon moléculaire Download PDF

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WO2010010848A1
WO2010010848A1 PCT/JP2009/062952 JP2009062952W WO2010010848A1 WO 2010010848 A1 WO2010010848 A1 WO 2010010848A1 JP 2009062952 W JP2009062952 W JP 2009062952W WO 2010010848 A1 WO2010010848 A1 WO 2010010848A1
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protein
seq
gene
sequence listing
chop
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大祐 西宮
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第一三共株式会社
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    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/07—Heat shock proteins
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to a mammalian transformed cell whose foreign protein secretion ability is enhanced by a molecular chaperone and a transcription factor, and a method for producing the foreign protein using the same.
  • antibody drugs such as antibody drugs and therapeutic proteins are rapidly expanding the market.
  • antibody drugs do not cause harmful immune reactions even when administered to the human body, and are being actively developed due to their high specificity.
  • Post-translational modifications such as protein folding and sugar chain modification are essential for the physiological activity and antigenicity of protein drugs.
  • a secretory protein serving as a proteinaceous drug is transcribed and translated in the nucleus, and when a signal sequence is recognized by SRP (signal recognition particle), it passes through the translocon and enters the endoplasmic reticulum.
  • Secreted proteins are folded in the endoplasmic reticulum by unraveling the higher-order structure when passing through the translocon. Although protein folding is possible spontaneously, various molecular chaperones assist in folding.
  • the formation of the native three-dimensional structure formed in the endoplasmic reticulum is important for secretion, and proteins that are not correctly folded cannot enter the secretion pathway.
  • proteins with higher-order structure accumulate in the endoplasmic reticulum due to various environmental factors in the endoplasmic reticulum, inhibition of synthesis of membrane proteins and secreted proteins, etc., modification disorders such as addition of sugar chains and disulfide bonds, ER stress occurs.
  • Eukaryotic cells exhibit an endoplasmic reticulum stress response called Unfolded Protein Response (UPR) to avoid this critical situation.
  • URP Unfolded Protein Response
  • This stress response system is widely preserved in eukaryotic cells from yeast to mammalian cells, and is an important system that supports protein maturation and plays an essential role in protecting itself from cell death in an emergency. Yes.
  • UPR not only the mechanism that induces and regulates the transcription of molecular chaperones that repair accumulated abnormal proteins, but also ERAD (ER-associated degradation) that degrades and eliminates abnormal proteins to maintain endoplasmic reticulum homeostasis.
  • ERAD ER-associated degradation
  • examples of host cells for producing proteinaceous drugs include microorganisms, yeasts, insects, animal and plant cells, and transgenic animals and plants.
  • Post-translational modifications such as folding and sugar chain modification are indispensable for the physiological activity and antigenicity of protein drugs, so microorganisms that cannot perform complex post-translational modifications and plants with different sugar chain structures are not suitable as bioreactors.
  • Mammalian cells such as CHO cells, which have a sugar chain structure similar to that of humans, can be post-translationally modified, and are in close contact with humans, are now mainstream.
  • ERO1 or PDI1 when producing human serum albumin rich in SS bond, the production amount is improved 15 times. lactis et al. (Applied and Environmental Microbiology 2005 71 (8): 4359-4363). However, even if ERO1 and PDI1 are added at the same time, there is no further improvement, and the production period is only accelerated. In addition, IL-1 ⁇ having no SS bond has no effect.
  • Xbp-1 and ATF4 which play an important role as transcription factors in the secretory pathway and UPR.
  • EPO Epithropoietin
  • ATF4 Activating transcription factor 4
  • AT-III human antithrombin III
  • molecular chaperones are known not only for helping protein folding, but also for loosening aggregated proteins to fold.
  • HSP104 can perform a reaction impossible with other chaperones, such as solubilizing proteins from aggregates in cooperation with HSP70 (Cell 1998 94: 73-82).
  • An antibody molecule forms a disulfide bond between an antibody heavy chain and a light chain or an antibody heavy chain, and forms an intramolecular disulfide bond between the antibody heavy chain and the antibody light chain.
  • light chain tetramer molecules It is difficult to say that sufficient knowledge has been obtained about the types of molecular chaperones applicable to the production system of antibody molecules having such a complicated structure.
  • An object of the present invention is to provide means for producing highly secreted proteins, particularly proteins having a complex structure such as antibodies, in mammalian host cells.
  • the present inventors have co-expressed one or more specific molecular chaperone genes and transcription factors thereof with mammalian proteins to be expressed in mammalian cells.
  • the inventors have found that the secretion productivity of the foreign protein can be improved by making it possible to complete the present invention.
  • the present invention includes the following inventions.
  • a polynucleotide encoding a protein having promoting activity (C) Poly encoding a protein consisting of a base sequence having 90% or more homology to the base sequence shown in SEQ ID NO: 1, 3, 5, 65 or 69 in the sequence listing and having a foreign protein secretion promoting activity nucleotide.
  • the polynucleotide is one or more polynucleotides selected from the group consisting of the base sequences set forth in SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, and 23 in the sequence listing.
  • the transformed cell according to (3) The transformed cell according to (4), wherein the polynucleotide is one or two or more polynucleotides selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 13, 21, and 23 in the Sequence Listing. .
  • a protein having an amino acid sequence and having a foreign protein secretion promoting activity is one or more proteins selected from the group consisting of amino acid sequences set forth in SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 22 and 24 in the sequence listing.
  • the transformed cell according to 6 The transformed cell according to (7), wherein the protein is one or two or more proteins selected from the group consisting of the amino acid sequences described in SEQ ID NOs: 14, 22, and 24 in the Sequence Listing.
  • the mammalian-derived cultured cell is any one cell selected from the group consisting of COS-1 cells, 293 cells, and CHO cells (CHO-K1, CHO dhfr-, CHO-S, etc.)
  • (11) The transformed cell according to any one of (1) to (10), into which a polynucleotide encoding a foreign protein is further introduced.
  • (12) The transformed cell according to (11), wherein the foreign protein is a multimeric protein.
  • the transformed cell according to (12), wherein the foreign protein is a heteromultimeric protein.
  • the transformed cell according to (13), wherein the foreign protein is an antibody or a functional fragment thereof.
  • a method for producing a protein wherein the transformed cell according to any one of (11) to (14) is cultured, and a foreign protein is obtained from the culture.
  • a polynucleotide encoding a human mutant CHOP protein comprising the base sequence set forth in SEQ ID NO: 65 in the Sequence Listing.
  • a human mutant CHOP protein comprising the amino acid sequence set forth in SEQ ID NO: 66 in the Sequence Listing.
  • a polynucleotide encoding a Chinese hamster mutant CHOP protein comprising the nucleotide sequence set forth in SEQ ID NO: 69 in the Sequence Listing.
  • a Chinese hamster mutant CHOP protein comprising the amino acid sequence set forth in SEQ ID NO: 70 in the Sequence Listing.
  • a chaperone gene and its transcription factor into a mammalian host cell, it is possible to produce a highly secreted protein having a complex structure such as an antibody in a correctly folded form.
  • a synergistic effect can be obtained with respect to protein high secretion production.
  • the produced protein can be used as a medicine, diagnostic agent or research reagent depending on its activity.
  • variant type CHOP protein The figure which compared the antibody expression enhancement effect which a human mutant CHOP and a Chinese hamster mutant CHOP show, and showed that there was no big difference in an effect by the species (ELISA method). The figure which compared the function of human mutant type and wild type CHOP, and confirmed that mutant type CHOP showed the stronger antibody expression enhancement effect.
  • the term “gene” includes not only DNA but also mRNA, cDNA and RNA thereof.
  • polynucleotide is used in the same meaning as nucleic acid, and includes DNA, RNA, probe, oligonucleotide, and primer.
  • polypeptide and “protein” are used without distinction.
  • the “functional fragment of an antibody” means a partial fragment of an antibody having an activity of binding to an antigen, and includes Fab, F (ab ′) 2, etc. As long as it has binding ability, it is not limited to these molecules.
  • Genes Used in Protein High Secretion Production System include genes encoding a series of molecular chaperones (hereinafter referred to as “chaperones”) that act for protein high secretion production, protein folding, denatured protein degradation and aggregation prevention in the endoplasmic reticulum. And a transcription factor that controls the chaperone gene.
  • one of the genes used for high protein secretion production includes a CHOP gene (CCAAT / enhancer binding protein homologous protein).
  • the CHOP gene is also called Gadd153 (growth arrest and DNA damage inducible gene 153) or DNA Damage-inductive Transscript 3 (DDIT3), and it is induced by genetic stress, cell proliferation E, and / or protein growth arrest P, A / C. / Enhancer binding protein) is also known as a transcription factor.
  • Gadd153 growth arrest and DNA damage inducible gene 153 or DNA Damage-inductive Transscript 3 (DDIT3)
  • DDIT3 DNA Damage-inductive Transscript 3
  • CHOP plays a part in the final signal when ER stress occurs, and when UPR is activated in the PERK pathway, it is said that when CHOP is induced, it activates an apoptotic signal and is involved in UPR. It is suggested that Hereinafter, the transcription factor CHOP gene related to UPR will be described.
  • CHOP is composed of an N-terminal transcription activation domain and a C-terminal bZip DNA binding domain.
  • the origin of the CHOP gene used in the present invention is not particularly limited as long as it is a gene encoding a CHOP protein.
  • a gene encoding human CHOP (GenBank registration number: P35638) (same registration number: NM_004083) is exemplified. be able to.
  • the base sequence of the gene encoding the human CHOP protein is described in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 2 in the sequence listing.
  • CHOP gene used in the present invention a gene (same registration number: NM_007837) encoding mouse CHOP (GenBank registration number: AAH13718) can also be mentioned.
  • the base sequence of the gene encoding mouse CHOP protein is described in SEQ ID NO: 3 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 4 in the sequence listing.
  • Examples of the CHOP gene used in the present invention include a gene encoding the Chinese hamster CHOP (GenBank registration number: P14607) (same registration number: M29238).
  • the base sequence of the gene encoding the Chinese hamster CHOP protein is described in SEQ ID NO: 5 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 6 in the sequence listing.
  • a Chinese hamster mutant CHOP gene newly cloned according to the present invention can also be used in the present invention.
  • the gene encoding the Chinese hamster mutant CHOP protein is described in SEQ ID NO: 69 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 70 in the sequence listing.
  • the CHOP used in the present invention may be derived from other biological species.
  • species-derived CHOP genes mammal-derived genes corresponding to the above-mentioned genes derived from humans, mice or Chinese hamsters are preferable.
  • GRP78 is also called immunoglobulin heavy chain-binding protein (BiP) or HSPA5, and is an endoplasmic reticulum (ER) endogenous HSP70 family molecular chaperone. It is a molecule involved in protein folding and assembly in the ER, and is also known as an endoplasmic reticulum stress sensor.
  • the origin of the GRP78 gene used in the present invention is not particularly limited as long as it is a gene encoding GRP78 protein.
  • a gene encoding human GRP78 (GenBank registration number: NP_005338) (same registration number: NM_005347) is mentioned. be able to.
  • the base sequence of the gene encoding the human GRP78 protein is described in SEQ ID NO: 7 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 8 in the sequence listing.
  • GRP94 is a molecular chaperone that is localized in the endoplasmic reticulum (ER) and is also known as gp96, a homolog of HSP90.
  • the origin of the GRP94 gene used in the present invention is not particularly limited as long as it is a gene encoding GRP94 protein.
  • a gene encoding human GRP94 (GenBank registration number: P14625) (same registration number: NM_003299) is mentioned. be able to.
  • the base sequence of the gene encoding human GRP94 protein is described in SEQ ID NO: 9 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 10 in the sequence listing.
  • PDIA4 is an enzyme localized in the endoplasmic reticulum (ER) and responsible for reorganization of disulfide (SS) bonds, and is also known as ERp72.
  • the origin of the PDIA4 gene used in the present invention is not particularly limited as long as it is a gene encoding PDIA4 protein.
  • a gene encoding human PDIA4 (GenBank registration number: P13667) (same registration number: NM_004911) is exemplified. be able to.
  • the base sequence of the gene encoding the human PDIA4 protein is described in SEQ ID NO: 11 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 12 in the sequence listing.
  • TPM3 is called Tropomyosin-3, Tropomyosin alpha-3 chain, Tropomyosin gamma, and is a dimer of coiled-coil protein. It is stable to filaments by polymerizing end-to-end along a large groove in actin filaments. And plays a role in regulating the access of other actin-binding proteins.
  • the origin of TPM3 gene used in the present invention is not particularly limited as long as it is a gene encoding TPM3 protein.
  • a gene encoding human TPM3 (GenBank registration number: Q63610) (same registration number: NM_153649) is mentioned. be able to.
  • the base sequence of the gene encoding the human TPM3 protein is described in SEQ ID NO: 13 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 14 in the sequence listing.
  • HSP90AB1 is a molecular chaperone called HSP90 ⁇ or HSP84 and localized in the cytoplasm.
  • the origin of the HSP90 ⁇ gene used in the present invention is not particularly limited as long as it is a gene encoding HSP90 ⁇ protein.
  • a gene encoding human HSP90AB1 (GENBANK registration number: P08238) (same registration number: NM_007355) is mentioned. be able to.
  • the base sequence of the gene encoding human HSP90AB1 protein is described in SEQ ID NO: 15 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 16 in the sequence listing.
  • PHB is a highly conserved gene from yeast to human, is localized in the inner mitochondrial membrane, and is thought to be a negative regulator of cell growth. Yeast is thought to have a chaperone-like function that binds to and stabilizes newly synthesized proteins in mitochondria.
  • the PHB gene used in the present invention is not particularly limited as long as it is a gene encoding a PHB protein.
  • a gene encoding human PHB (GenBank registration number: P35232) (same registration number: AK31649) is mentioned. be able to.
  • the base sequence of the gene encoding the human PHB protein is described in SEQ ID NO: 17 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 18 in the sequence listing.
  • ATP2A2 also known as Sarco / Endoplasmic Reticulum Calcium (SERCA) -ATPase
  • SERCA Sarco / Endoplasmic Reticulum Calcium
  • the ATP2A2 gene used in the present invention is not particularly limited as long as it is a gene encoding an ATP2A2 protein.
  • a gene encoding human ATP2A2 (GenBank registration number: P166615) (same registration number: NM_170665) is exemplified. be able to.
  • the base sequence of the gene encoding human ATPA2 protein is described in SEQ ID NO: 19 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 20 in the sequence listing.
  • MCM7 is called DNA replication licensing factor MCM7 or CDC47 homolog, and is one of the highly conserved mini-chromosome maintenance proteins (MCMs) important for the initiation of eukaryotic genome replication.
  • MCMs mini-chromosome maintenance proteins
  • the origin of the MCM7 gene used in the present invention is not particularly limited as long as it is a gene encoding MCM7 protein.
  • a gene encoding human MCM7 (GenBank accession number: P33993) (same registration number: NM_005916) is mentioned. be able to.
  • the base sequence of the gene encoding the human MCM7 protein is described in SEQ ID NO: 21 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 22 in the sequence listing.
  • EIF4A is called Eukaryotic initiation factor 4A-I, and has an ATP-dependent RNA helicase activity that dissolves RNA secondary structure in order for ribosomal small subunits to bind efficiently to mRNA.
  • the origin of the eIF4A gene used in the present invention is not particularly limited as long as it is a gene encoding the eIF4A protein.
  • a gene encoding human eIF4A (GenBank accession number: P60842) (same registration number: NM_001416) is mentioned. be able to.
  • the base sequence of the gene encoding the human eIF4A protein is described in SEQ ID NO: 23 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 24 in the sequence listing.
  • P5CS is also called Delta-1-pyroline-5-carboxylate synthetase (P5CS) and Gamma-glutamyl phosphate reductase (GPR), and belongs to the aldehyde dehydrogenase family, ⁇ -glutamyl phosphatase activity and ⁇ -glutamyl phosphate reductase Directs an ATP- and NADPH-dependent bifunctional mitochondrial enzyme with both activities.
  • the origin of the P5CS gene used in the present invention is not particularly limited as long as it is a gene encoding a P5CS protein.
  • a gene encoding human P5CS (GenBank accession number: P54886) (same registration number: NM_002860) is mentioned. be able to.
  • the base sequence of the gene encoding the human P5CS protein is described in SEQ ID NO: 25 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 26 in the sequence listing.
  • ILF3 is also called Interleukin enhancer-binding factor 3 (Nucleor factor of activated T-cells 90 kDa (NF-AT-90), and performs transcriptional regulation at the stage of mRNA elongation.
  • the ILF3 gene used in the present invention The origin of the gene is not particularly limited as long as it is a gene encoding ILF3 protein, but includes, for example, a gene encoding human ILF3 (GenBank registration number: Q12906) (same registration number: NM_0121818) Gene encoding human ILF3 protein
  • the nucleotide sequence of is described in SEQ ID NO: 27 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 28 in the sequence listing.
  • BAP37 also called BAP or Prohibitin-2 (PHB2)
  • PHB2 Prohibitin-2
  • the origin of BAP37 gene used in the present invention is not particularly limited as long as it is a gene encoding BAP37 protein.
  • a gene encoding human BAP37 (GenBank accession number: Q99623) (same registration number: NM_007273) is mentioned. be able to.
  • the base sequence of the gene encoding human BAP37 protein is described in SEQ ID NO: 29 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 30 in the sequence listing.
  • CALR is a multifunctional protein that functions as a major calcium (Ca2 +) binding protein in the endoplasmic reticulum (ER) lumen. It is also found in the nucleus, indicating that it functions in transcriptional regulation. CALR binds to the synthetic peptide KLGFFFKR, which is nearly identical to the amino acid sequence in the DNA binding region of the nuclear receptor superfamily.
  • the origin of the CALR gene used in the present invention is not particularly limited as long as it encodes a CALR protein.
  • a gene encoding human CALR (GenBank registration number: P27797) (same registration number: NM_004343) is mentioned. be able to.
  • the base sequence of the gene encoding the human CALR protein is described in SEQ ID NO: 31 in the sequence listing, and the amino acid sequence encoded thereby is described in SEQ ID NO: 32 in the sequence listing.
  • the chaperone and transcription factor used in the present invention may be chaperones and transcription factors derived from other species.
  • genes for chaperones and transcription factors derived from other species genes derived from mammals such as mice, rats, and Chinese hamsters corresponding to the above-mentioned genes derived from humans are preferable. Further, chaperones and transcription factors other than the above-mentioned chaperones and transcription factors may be used.
  • the above chaperone genes can be used alone or in combination of two or more.
  • two or more chaperone genes may be derived from the same or different species.
  • genes used in the present invention include human GRP78 gene, human CHOP gene, human GRP94 gene, human PDIA4 gene, human TPM3 gene, human HSP90 ⁇ gene, human PHB gene, human ATP2A2 gene, human MCM7 gene, human eIF4A
  • examples thereof include any one gene selected from the group consisting of a gene, a human P5CS gene, and a human ILF3 gene.
  • genes used in the present invention include human CHOP gene and human GRP78 gene combination, human CHOP gene and human GRP94 gene combination, human CHOP gene and human PDIA4 gene combination, human CHOP gene and human TPM3 gene Combination, combination of human CHOP gene and human HSP90 ⁇ gene, combination of human CHOP gene and human PHB gene, combination of human CHOP gene and human ATP2A2 gene, combination of human CHOP gene and human MCM7 gene, combination of human CHOP gene and human eIF4A gene Selected from the group consisting of a combination, a combination of a human CHOP gene and a human P5CS gene, and a combination of a human CHOP gene and a human ILF3 gene
  • One of the combination or Re can be mentioned.
  • Preferred examples of the combination of genes used in the present invention include a combination of a human CHOP gene and a human GRP94 gene, a combination of a human CHOP gene and a human PDIA4 gene, a combination of a human CHOP gene and a human TPM3 gene, a human CHOP gene and a human Selected from the group consisting of a combination of HSP90 ⁇ gene, a combination of human CHOP gene and human PHB gene, a combination of human CHOP gene and human ATP2A2 gene, a combination of human CHOP gene and human MCM7 gene, and a combination of human CHOP gene and human eIF4A gene Any one of the combinations may be mentioned.
  • More preferable examples of the combination of genes used in the present invention include a combination of a human CHOP gene and a human TPM3 gene, a combination of a human CHOP gene and a human MCM7 gene, and a combination of a human CHOP gene and a human eIF4A gene. Any one selected combination can be mentioned.
  • SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 And a gene encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in any one amino acid sequence selected from the group consisting of the amino acid sequences shown in FIG. Also good.
  • the number of amino acids that may be deleted, substituted and / or added is preferably 1 to several.
  • the number of “several” is not particularly limited, but means, for example, 20 or less, preferably 10 or less, more preferably 7 or less, and even more preferably about 5 or less.
  • the “mutation” here means a mutation artificially introduced mainly by a known mutant protein production method, but may be a similar naturally occurring mutation.
  • the gene used in the present invention consists of the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32. It may be a gene encoding a protein consisting of an amino acid sequence having 80% or more homology with any one amino acid sequence selected from the group and having a foreign protein secretion promoting activity.
  • the 80% or higher homology is preferably 85% or higher, more preferably 90% or higher, and most preferably 95% or higher.
  • Protein homology search can be performed, for example, using a program such as FASTA or BLAST for the DNA Database of Japan (DNA Database of JAPAN (DDBJ)).
  • “foreign protein secretion-promoting activity” is based on the protein folding action (disulfide bond formation, etc.) of the molecular chaperone in the endoplasmic reticulum, the refolding action of the denatured protein to the normal protein, and the action of inhibiting the aggregation of the denatured protein. This refers to the activity of highly secreting a correctly folded foreign protein in the host cell.
  • “having foreign protein secretion promoting activity” means that the above activity is represented by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30. And the activity of a protein having any one amino acid sequence selected from the group consisting of the amino acid sequences described in (32) and (32).
  • the chaperone gene used in the present invention consists of the nucleotide sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31. It may be a gene that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to any one polynucleotide selected from the group and encodes a protein having a foreign protein secretion promoting activity .
  • stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed.
  • nucleic acids with high homology that is, base sequences described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31.
  • a nucleic acid complementary strand consisting of a base sequence having a homology of 80% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more is hybridized with any one base sequence selected from An example is a condition in which a complementary strand of a nucleic acid composed of a soybean and a base sequence having a lower homology is not hybridized.
  • the sodium salt concentration is 15 to 750 mM, preferably 50 to 750 mM, more preferably 300 to 750 mM
  • the temperature is 25 to 70 ° C., preferably 50 to 70 ° C., more preferably 55 to 65 ° C.
  • formamide The condition is that the concentration is 0 to 50%, preferably 20 to 50%, more preferably 35 to 45%.
  • the conditions for washing the filter after hybridization are usually that the sodium salt concentration is 15 to 600 mM, preferably 50 to 600 mM, more preferably 300 to 600 mM, and the temperature is 50 to 70 ° C., preferably The temperature is 55 to 70 ° C, more preferably 60 to 65 ° C.
  • the amino acid mutation (deletion, substitution, and / or addition) can be introduced by a technique known in the art such as the Kunkel method or the Gapped duplex method, or a method analogous thereto, for example, site-specific.
  • Mutation introduction kits using the mutagenesis method for example, Mutant-K (manufactured by Takara Bio Inc.) or Mutant-G (manufactured by Takara Bio Inc.), LA PCR in vitro Mutagenesis series kit manufactured by Takara Bio Inc., etc.
  • Mutant-K manufactured by Takara Bio Inc.
  • Mutant-G manufactured by Takara Bio Inc.
  • the above-mentioned gene used for protein high-secretion production and the gene encoding a foreign protein that is the target of high-secretory expression described below are prepared by preparing mRNA and using reverse transcriptase. It can be obtained by a general method for synthesizing cDNA. As a general method, for example, a cDNA library derived from a cell or tissue expressing the target gene is isolated by screening using a DNA probe synthesized based on the gene fragment. can do. Preparation of mRNA can be performed by a method usually used in the art.
  • the above cells or tissues are treated with a guanidinin reagent, a phenol reagent or the like to obtain total RNA, and then, by an affinity column method using an oligo (dT) cellulose column or poly U-sepharose using Sepharose 2B as a carrier.
  • poly (A) + RNA (mRNA) is obtained by a batch method.
  • poly (A +) RNA may be further fractionated by sucrose density gradient centrifugation or the like.
  • single-stranded cDNA was synthesized using oligo dT primer and reverse transcriptase, and double-stranded using DNA synthase I, DNA ligase, RNase H, etc.
  • cDNA library can be prepared using a plasmid vector. Thereafter, a strain (positive clone) having the target DNA may be selected from the cDNA library.
  • Genomic DNA can be extracted by, for example, the method of Cryer et al. (Methods in Cell Biology, 12, 39-44 (1975)) and P.I. It can be carried out according to the method of Philippsen et al. (Methods Enzymol., 194, 169-182 (1991)).
  • a yeast protoplast is prepared, and from the protoplast, a generally known DNA extraction method, an alcohol precipitation method after removing cell residues under high salt concentration, a phenol or chloroform extraction A conventional method such as an alcohol precipitation method may be used.
  • the target gene can also be obtained, for example, by the PCR method (PCR Technology. Henry A. Errich, Attackton press (1989)).
  • PCR method PCR Technology. Henry A. Errich, Attackton press (1989)
  • 20 to 30-mer synthetic single-stranded DNA is used as a primer
  • genomic DNA is used as a template.
  • the amplified gene is used after confirming the base sequence.
  • a gene library is prepared by a conventional method, (b) a desired clone is selected from the prepared gene library, and the clone is amplified.
  • the gene library is a fragment obtained by partially digesting chromosomal DNA obtained from a cell line of an organism as a collection source by a conventional method with an appropriate restriction enzyme and ligating the obtained fragment to an appropriate vector.
  • a plasmid known as a vector for preparing a commonly known gene library can be used, and a phage vector or a cosmid can also be widely used.
  • a host for transformation or transduction a host corresponding to the type of the vector may be used.
  • Selection of clones retaining the target gene fragment is carried out by colony hybridization method, plaque hybridization method or the like using a labeled probe containing a sequence specific to the target gene from the above gene library.
  • the target gene can also be chemically synthesized.
  • a method of producing two pairs of complementary oligonucleotides and annealing them, a method of ligating several annealed DNAs with DNA ligase, or producing several partially complementary oligonucleotides by PCR Genes can be synthesized by, for example, filling a gap.
  • Determination of the DNA sequence of a gene can be performed by a conventional method, for example, the dideoxy method (Sanger et al., Proc. Natl. Acad. Sci., USA, 74, 5463-5467 (1977)). Furthermore, the determination of the DNA base sequence can be easily performed by using a commercially available sequence kit or the like.
  • the vector of the present invention is a vector containing one of the above chaperone genes, a vector containing one or more of the above chaperone genes, or a combination of two or more of the above chaperone genes.
  • a vector comprising is provided.
  • a chaperone gene in a host cell it may be transformed using a vector containing each gene alone, or may be transformed using a single vector containing a plurality of genes.
  • the expression vector may contain a gene encoding a foreign protein.
  • an expression vector containing a gene encoding a foreign protein may be prepared separately, and when prepared separately, each vector is cotransfected (co-introduced) into a host cell.
  • the gene encoding the foreign protein is not particularly limited, but various enzyme genes such as ⁇ -amylase gene and ⁇ -galactosidase gene, particularly pharmaceuticals such as erythropoietin (EPO) and granulocyte colony stimulating factor (G-CSF).
  • EPO erythropoietin
  • G-CSF granulocyte colony stimulating factor
  • Glycosyltransferase genes necessary for the production of the above useful glycoproteins various interferon genes such as interferon ⁇ and interferon ⁇ , which are physiologically active physiologically active proteins, various interleukin genes such as IL1 and IL2, erythropoietin (EPO) Examples include genes, various cytokine genes such as granulocyte colony-stimulating factor (G-CSF) gene, growth factor genes, antibody genes and the like. These genes may be obtained by any technique.
  • the present invention is particularly effective for proteins with high hydrophobicity and proteins that are difficult to produce secretively, such as forming a complex. Therefore, the foreign protein includes a multimeric protein such as an antibody or a functional product thereof. Heteromultimers that are fragments are included.
  • humanized anti-DR5 antibody (mouse anti-human DR5 antibody TRA-8 (Nature Med. 2001, 7 (8), 954-60) is a humanized antibody, (Hereinafter referred to as hTRA-8) was selected.
  • hTRA-8 human anti-human DR5 antibody
  • the foreign protein to be subjected to high secretion production using the present invention is not limited to hTRA-8.
  • the foreign protein that can be used for testing the effect of the present invention is not limited to hTRA-8.
  • An expression vector may be constructed as a protein expression unit by appropriately adding an expression control region to a chaperone gene and a gene encoding a foreign protein.
  • the protein expression unit has at least a promoter region, the gene, and a transcription terminator region (poly A addition signal) in the direction of the transcription reading frame.
  • the promoter that can be used here may be a constitutive expression promoter or an inducible expression promoter.
  • constitutive expression promoters include various natural promoters such as SV40 early promoter, adenovirus E1A promoter, CMV (cytomegalovirus) promoter, EF-1 ⁇ (human elongation factor-1) promoter, HSP70 promoter, MT promoter, RSV promoter, UBC promoter, actin promoter, SR ⁇ promoter which is an artificial (fusion) promoter, CAG promoter and the like.
  • the poly A addition sequence may be any sequence that has an activity of causing transcription termination with respect to transcription from the promoter, and may be of the same or different gene as the promoter gene.
  • the protein to be produced is denatured and does not cause aggregation, or a promoter that can achieve an expression level that does not exceed the capacity of the secreted protein is appropriately selected, or adjustments such as weakening the activity are made.
  • a promoter that can achieve an expression level that does not exceed the capacity of the secreted protein is appropriately selected, or adjustments such as weakening the activity are made.
  • multimeric proteins molecules that form heteromultimers are susceptible to the above-mentioned effects.
  • molecules such as antibodies are heterotetramers in which two heavy chains and two light chains are associated with each other. The expression level is an important factor for achieving this.
  • the expression vector of the present invention can contain a selection marker for selecting a transformant.
  • a selection marker for selecting a transformant for example, by using drug resistance markers that confer resistance to drugs such as cerulenin, aureobasidin, zeocin, canavanine, cycloheximide, hygromycin, blasticidin, tetracycline, kanamycin, ampicillin, tetracycline, neomycin, etc. It is possible to select converters.
  • a transformant can be selected by using as a marker a gene that imparts solvent resistance to ethanol and the like, osmotic pressure resistance to glycerol and salts, resistance to metal ions such as copper, and the like.
  • the transformed cell of the present invention is the above-mentioned 1. Of the gene of 2.
  • the host cell to be transformed is a eukaryotic cell, preferably a mammalian cell, more preferably a human, mouse or rat, hamster, monkey or bovine cell.
  • the present invention aims to obtain a host cell reinforced with an endoplasmic reticulum (ER) essential for secretion, it can be applied to other mammalian cells or yeast.
  • ER endoplasmic reticulum
  • any method can be used for introducing an expression vector into a host cell as long as the transgene is stably present in the host and can be appropriately expressed.
  • calcium phosphate method Ito et al., (1984) Agric. Biol. Chem., 48, 341
  • electroporation method Becker, DM et al. (1990) Methods. Enzymol., 194, 182-187
  • spheroplast method Creggh et al., Mol. Cell. Biol., 5, 3376 (1985)
  • lithium acetate method Itoh, H. (1983) J. Bacteriol. 153, 163) -168
  • Lipo Ekushon method or the like can be mentioned.
  • a foreign protein is produced by transforming the transformed cell described in the above item “3.” with the gene encoding the foreign protein expressed in the expression "2.”
  • the transformed cells introduced using can be cultured by a known method, collected from the culture, and purified.
  • the “foreign protein” means that the transformed cell described in the above item “3.” is not produced at all or is produced in a small amount, and it is difficult to prepare a large amount from the transformed cell itself. It means protein.
  • the “culture” means not only the culture supernatant but also cultured cells or cell disruptions.
  • a foreign protein that can be produced using the transformed cell described in the item “3.” not only a monomer protein but also a multimeric protein can be selected. When producing a heteromultimeric protein composed of a plurality of different subunits, it is necessary to introduce a plurality of genes encoding these subunits into the transformed cells described in the item “3.” There is.
  • the method of culturing the transformed cell in the medium can be performed according to a usual method used for culturing the host cell.
  • the transformed cell When the transformed cell is a mammalian cell, it is cultured under conditions of 37 ° C., 5% or 8% CO 2 , and the culture time is about 24 to 1000 hours. It can be carried out by batch culture, fed-batch culture or continuous culture.
  • Confirmation of the expression product of the foreign protein gene from the above culture can be performed by SDS-PAGE, Western analysis, ELISA, or the like.
  • ordinary protein isolation and purification methods may be used.
  • the target protein is produced intracellularly after culturing, the target protein is collected by disrupting the cells with an ultrasonic disrupter, a French press, a Manton Gaurin homogenizer, a dynomill or the like.
  • the culture solution is used as it is, or the cells are removed by centrifugation or the like.
  • the target protein is collected by extraction with an organic solvent, etc., and if necessary, various chromatography (hydrophobic chromatography, reverse phase chromatography, affinity chromatography, ion exchange chromatography, etc.), gel filtration using a molecular sieve May be isolated and purified using a method such as electrophoresis, electrophoresis using polyacrylamide gel or the like alone or in combination.
  • various chromatography hydrophobic chromatography, reverse phase chromatography, affinity chromatography, ion exchange chromatography, etc.
  • gel filtration using a molecular sieve May be isolated and purified using a method such as electrophoresis, electrophoresis using polyacrylamide gel or the like alone or in combination.
  • the above culture method and purification method are examples, and are not limited to these.
  • the amino acid sequence of the purified gene product can be confirmed by known amino acid analysis, for example, automatic amino acid sequencing by Edman degradation.
  • the heteromultimeric protein produced using the production method described in the item “4.” examples include antibody proteins.
  • the antibody protein is a tetrameric protein composed of two molecules of a heavy chain polypeptide and two molecules of a light chain polypeptide. Therefore, in order to obtain an antibody protein in a form that maintains the antigen-binding ability, it is necessary to introduce both the heavy chain and light chain genes into the transformed cells described in the item “3.”. In this case, the heavy and light chain genes may be present on the same expression vector or may be present on different expression vectors.
  • the antibody gene used for antibody production has a specific nucleotide sequence as long as the combination of the heavy and light chain polypeptides transcribed and translated from the antibody gene retains the activity of binding to any antigen protein. It is not limited to an antibody gene having
  • antibody functional fragment means a partial fragment of an antibody having an activity of binding to an antigen, and includes Fab, F (ab ′) 2, etc., but has the ability to bind to an antigen. As long as it is not limited to these molecules. Genes encoding these functional fragments can be obtained by genetically modifying the gene encoding the full-length antibody protein molecule.
  • Plasmids, restriction enzymes, DNA modifying enzymes and the like used in the examples of the present invention are commercially available and can be used in accordance with conventional methods. Also well known to those skilled in the art are the procedures used for DNA cloning, nucleotide sequence determination, host cell transformation, transformation cell culture, enzyme collection from the resulting culture, purification, etc. It can be known from the literature.
  • pcDNA3.1 (-) (Invitrogen) was treated with restriction enzymes XbaI and HindIII, the target fragment was separated by 1% agarose electrophoresis, cut out from the gel, purified, and this DNA fragment was used as a vector along with the above-mentioned ligation. Reaction and transformation were performed. Ligation reaction was performed using In-fusion 2.0 Dry-Down PCR Cloning Kit (Clontech). In the transformation, first, the frozen competent cell JM109 was thawed, 10 ⁇ l of the solution after the above ligation reaction was added to the solution, and the mixture was allowed to stand on ice for 30 minutes. Then, heat shock was performed at 42 ° C. for 45 seconds and ice-cooled for 5 minutes.
  • the target plasmid was obtained from the colonies cultured on the LB plate by the alkali method.
  • the pcDNA3.1 ( ⁇ ) hTRA-8 light chain expression vector was constructed by determining the base sequence of the hTRA-8 light chain in the plasmid obtained by the alkaline method.
  • This DNA fragment and pcDNA3.1 (-) are treated with restriction enzymes XhoI and HindIII, followed by separation and purification of the target fragment by agarose gel electrophoresis, ligation reaction, transformation, and culture of the plate. Acquired.
  • the Ligation reaction was performed according to the protocol using a LigaFast TM Rapid DNA Ligation System (Promega).
  • the pcDNA3.1 ( ⁇ ) hTRA-8 heavy chain expression vector was constructed by determining the base sequence of the hTRA-8 heavy chain in the plasmid obtained by the alkaline method.
  • hTRA-8 is given the general name of tigatuzumab, and the amino acid sequences of the heavy chain and the light chain are known.
  • hTRA-8 by synthesizing the hTRA-8 gene according to conventional methods based on these known sequence information and cloning the obtained gene into an appropriate expression vector. is there. It is also possible to test the effect of the present invention using a synthesized hTRA-8 gene.
  • chaperone and transcription factor expression vector (2-1) Cloning of human-derived chaperone and transcription factor Reverse transcription reaction using RNA extracted from 293 cells using RNeasy Kit (Qiagen) as template and ReveTraAce (TOYOBO) To synthesize cDNA. Using the synthesized cDNA as a template, DNA encoding each protein was amplified with the primers shown in (2-2), and the target fragment was separated by agarose gel electrophoresis and the DNA fragment was purified. Each DNA fragment was cloned into pcDNA3.1 Directional TOPO. A clone containing the target gene was selected by determining the nucleotide sequence of the target gene in the cloned plasmid.
  • FIG. 3 shows a comparison of base sequences of human wild-type CHOP and human mutant CHOP
  • FIG. 4 shows a comparison of amino acid sequences.
  • Example 1-3 1-3
  • human constructed in Example 2 Each of the derived protein expression vectors was introduced into a cell, the antibody was transiently expressed, and an ELISA method was performed to measure the amount of the antibody secreted into the culture medium.
  • an empty vector in which no chaperone gene was inserted was added to an antibody expression vector, and the amount of DNA was adjusted to introduce the gene into the cell.
  • the host cell was COS-1, and Lipofectamine 2000 was used as the gene introduction reagent.
  • COS-1 was an ⁇ -MEM medium containing 10% FCS and cultured under conditions of 37 ° C. and 5% CO 2.
  • ELISA was performed by the following method. To a 96-well plate coated with 50 ng / well of anti-kappa Light Chain, 100 ⁇ l of the decellularized culture supernatant was added and allowed to stand at 37 ° C. for 1 hour. Next, after removing the sample (culture supernatant) and washing each well with 200 ⁇ l of PBS-Tween (0.05%), 100 ⁇ l of HRP-labeled anti-human IgG (Fc) was added to the well. It left still at 1 degreeC for 1 hour.
  • Host cells are COS-1, 293F cells, CHO-S, CHO-K1, and gene introduction reagents are Lipofectamine 2000 (Invitrogen) for COS-1 and CHO-K1, and MAX reagents (Invitrogen) for 293F cells and CHO-S. )
  • COS-1 is ⁇ -MEM medium containing 10% FCS
  • CHO-S is Free style CHO medium
  • CHO-K1 is F-12 medium containing 10% FCS
  • 293 cells at 37 ° C under 5% CO2.
  • was cultured in a free style 293 medium under conditions of 37 ° C. and 8% CO 2 and after 48 hours of transfection, the culture solution was collected and subjected to ELISA.
  • the ELISA operation was performed by the method described in Example 3.
  • Antibody expression enhancement effect by a combination of CHOP and chaperone or CHOP and transcription factor A gene is introduced into a cell in the same manner as in Example 3 by combining a mutant CHOP expression vector and another protein (chaperone or transcription factor) expression vector and culturing. The amount of antibody secreted into the liquid was measured by ELISA. The ELISA operation was performed by the method described in Example 3.
  • CHO-S was cultured in a free style CHO medium under conditions of 37 ° C. and 5% CO 2. Six days after gene introduction, the culture supernatant was collected, and the culture solution was purified using a protein A column. Apoptosis activity measurement and antigen binding activity measurement were performed using the purified protein solution.
  • Apoptosis activity was measured by the following method. Serial dilution (200 ng) of purified hTRA-8 and sample (hTRA-8 expressed and purified in CHO-S) with Goat Affinity Purified Antibodies to Human IgG Fc solution adjusted to 1 ⁇ g / ml using RPMI 1640 with 10% serum / Ml, 20 ng / ml, 2 ng / ml, 0.2 ng / ml) and added to the assay plate at 50 ⁇ l / well. This plate was seeded with 50 ⁇ l of Jurkat cells adjusted to 1 ⁇ 10 3 cells / ml and incubated at 37 ° C. under 5% CO 2 for 72 hours.
  • the amount of ATP in each well was measured with CellTiter-Glo TM Luminescent Cell Viability Assay (Promega) to produce cell viability.
  • Jurkat was cultured using RPMI 1640 containing 10% serum under conditions of 37 ° C. and 5% CO 2. The results are shown in FIG.
  • the apoptotic activity of the expressed antibody was consistent with or without CHOP.
  • the antibody obtained by co-introducing the antibody expression vector and the mutant CHOP expression vector had the same apoptotic activity as the antibody obtained separately by introducing only the antibody expression vector into the cell ( Data not shown).
  • the antigen binding activity measurement by competitive ELISA was performed by the following method.
  • HDR5-His (6 ⁇ His fused to human DR5 C-end) was diluted with a coating buffer (SDT) adjusted to a 1-fold concentration, added to the immuno plate at 50 ⁇ L / well, and left at 4 ° C. overnight or 37 ° C. And left for 2 hours.
  • the plate was washed with 200 ⁇ l of PBS-Tween (0.05%), 150 ⁇ l of PBS containing 1% BSA was added, and the plate was allowed to stand at 37 ° C. for 1 hour.
  • Biotin-labeled hTRA-8 was serially diluted with PBS-Tween (0.05%) containing 1% BSA (10-fold dilution in order from 200 ⁇ g / ml), and a sample (hTRA-8 expressed and purified with CHO-S) In the same manner, 25 ⁇ l / well was added to each of the plates from which 1% BSA-containing PBS was removed, and the plate was allowed to stand at 37 ° C. for 2 hours or at 4 ° C. overnight.
  • the antigen binding activities of the expressed antibodies were the same regardless of the presence or absence of CHOP, and the antigen binding activity measurement was the same.
  • the antibody obtained by co-introducing the antibody expression vector and the mutant CHOP expression vector had the same antigen-binding activity as the antibody obtained separately by introducing only the antibody expression vector into the cell. (Data not shown). This suggests that CHOP enhances only the expression level without affecting the function of the foreign gene (antibody).
  • Chinese hamster mutant CHOP and human mutant CHOP showed a high antibody expression enhancing effect.
  • Chinese hamster mutant CHOP and human mutant CHOP have 87% homology in the base sequence and 88% homology in the amino acid sequence. That is, it was confirmed that the CHOP gene having 85% or more homology with the human CHOP gene has an antibody expression enhancing effect regardless of the species.
  • DNA encoding human wild-type CHOP was amplified, the target fragment was separated by agarose gel electrophoresis, and the DNA fragment was purified. Furthermore, PCR (94 ° C. for 15 seconds, 55 ° C. for 30 seconds, 68 ° C. for 30 seconds ⁇ 30 cycles) was performed using the purified DNA as a template and the hCHOP amplification primer described in Example 2.
  • DNA encoding human wild-type CHOP was amplified, the target fragment was separated by agarose gel electrophoresis, and the DNA fragment was purified, and each DNA fragment was cloned into pcDNA3.1 Directional TOPO.
  • a clone containing the target gene was selected by determining the nucleotide sequence of the target gene in the cloned plasmid.
  • the human mutant CHOP expression vector and the human wild-type CHOP expression vector were each introduced into COS-1 cells together with the antibody expression vector, and the amount of antibody secreted into the culture medium was determined by ELISA. It was measured.
  • an empty vector into which the CHOP gene was not inserted was added to the antibody expression vector, and the amount of DNA was adjusted to introduce the gene into the cells.
  • the ELISA operation was performed by the method described in Example 3. The results are shown in FIG.
  • Human mutant-type CHOP and human wild-type CHOP showed an antibody expression enhancing effect, but mutant-type CHOP showed a higher effect.
  • Human mutant CHOP and human wild-type CHOP have 99% homology in the nucleotide sequence and 97% homology in the amino acid sequence, but the place where the mutation is located is in the transcriptional activation domain, and the mutation enhances the expression of the antibody. The possibility that it became high was suggested.
  • Mammalian host cells into which a polynucleotide encoding the molecular chaperone protein of the present invention has been introduced can be used as a high-secretory production system for medical proteins or antibodies.

Abstract

L'invention concerne un moyen de sécrétion et de production d'une protéine, en particulier une protéine dont la structure est complexe comme un anticorps, à un niveau de sécrétion élevé dans une cellule hôte de mammifère. L'invention concerne spécifiquement une cellule transformée portant au moins un gène de protéine chaperonne moléculaire et un procédé pour la sécrétion d'une protéine étrangère à un niveau de sécrétion élevé en utilisant la cellule transformée.
PCT/JP2009/062952 2008-07-22 2009-07-17 Procédé de production d'anticorps dans une cellule de mammifère en culture en utilisant un chaperon moléculaire WO2010010848A1 (fr)

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WO2022191253A1 (fr) * 2021-03-10 2022-09-15 住友ファーマ株式会社 Procédé de production d'une protéine de fusion

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WO2022191253A1 (fr) * 2021-03-10 2022-09-15 住友ファーマ株式会社 Procédé de production d'une protéine de fusion

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