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WO2025141786A1 - Method for producing antibody - Google Patents

Method for producing antibody Download PDF

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Publication number
WO2025141786A1
WO2025141786A1 PCT/JP2023/046988 JP2023046988W WO2025141786A1 WO 2025141786 A1 WO2025141786 A1 WO 2025141786A1 JP 2023046988 W JP2023046988 W JP 2023046988W WO 2025141786 A1 WO2025141786 A1 WO 2025141786A1
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WO
WIPO (PCT)
Prior art keywords
antibody
fragment
copies
chain
cell
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PCT/JP2023/046988
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French (fr)
Japanese (ja)
Inventor
優也 石川
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Chugai Pharmaceutical Co Ltd
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Chugai Pharmaceutical Co Ltd
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Priority to PCT/JP2023/046988 priority Critical patent/WO2025141786A1/en
Priority to PCT/JP2024/046411 priority patent/WO2025143225A1/en
Priority to TW113151121A priority patent/TW202536182A/en
Publication of WO2025141786A1 publication Critical patent/WO2025141786A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a method for producing an antibody.
  • animal cells When using recombinant gene technology to produce recombinant antibodies useful as medicines, animal cells allow for complex post-translational modifications and folding that cannot be performed by prokaryotic cells, and therefore animal cells have come to be widely used as host cells for the production of recombinant antibodies.
  • a method for producing antibodies has been disclosed in which transient expression or stable expression is carried out using CHO (Chinese Hamster Ovary) cells that contain more copies of foreign DNA encoding the antibody's light chain than the DNA encoding the heavy chain (Non-Patent Document 4).
  • CHO Choinese Hamster Ovary
  • the foreign DNA encoding the antibody's light chain and the foreign DNA encoding the antibody's heavy chain are each introduced into the cells using separate vectors.
  • Reff ME Carner K, Chambers KS, Chinn PC, Leonard JE, Raab R et al. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood. 1994 Jan 15; 83(2):435-45. Presta LG, Chen H, O'Connor SJ, Chisholm V, Meng YG, Krummen L, et al. Humanization of an anti-vascular endothelial growt h factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res. 1997 Oct 15; 57(20):4593-9. Young-Kwang Lee, Joseph W. Brewer, Rachel Hellman, and Linda M. Hendershot.
  • the objective of the present invention is to provide a method for high antibody production.
  • the inventors produced CHO cells into which an expression vector containing four copies of DNA encoding an antibody light chain and two copies of DNA encoding the heavy chain was introduced, and found that a cell line with higher antibody production ability could be obtained than: 1) cells into which a vector containing two copies of the light chain and one copy of the heavy chain was introduced; 2) cells into which a vector containing six copies of the light chain and three copies of the heavy chain was introduced; and 3) cells into which a vector containing eight copies of the light chain and four copies of the heavy chain was introduced, thereby completing the present invention.
  • the gist of the present invention is as follows. (1) A method for producing an antibody or a fragment thereof using a cell into which a vector containing four copies of DNA encoding an antibody light chain or a fragment thereof and two copies of DNA encoding the antibody heavy chain or a fragment thereof has been introduced. (2) The method according to claim 1, comprising preparing a recombinant vector containing four copies of DNA encoding an antibody light chain or a fragment thereof and two copies of DNA encoding the antibody heavy chain or a fragment thereof, introducing the vector into a cell, and culturing the transformed cell into which the vector has been introduced to produce the antibody or a fragment thereof, thereby producing the antibody or a fragment thereof. (3) The method according to (1) or (2), wherein the cell is an animal cell.
  • the antibody is selected from the group consisting of an anti-CD137 antibody, an anti-latent TGF- ⁇ 1 antibody, an anti-latent myostatin antibody, an anti-complement (C1s) antibody, an anti-IL-8 antibody, an anti-IL-6 receptor antibody, an anti-IL-6 antibody, an anti-glypican-3 antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-GPIIb/IIIa antibody, an anti-TNF antibody, an anti-CD25 antibody, an anti-EGFR antibody, an anti-Her2/neu antibody, an anti-RSV antibody, an anti-CD33 antibody, an anti-CD52 antibody, an anti-IgE antibody, an anti-CD11a antibody, an anti-VEGF antibody, and an anti-VLA4 antibody, and is preferably an anti-CD137 agonist switch antibody STA551, an anti-latent TGF- ⁇ 1
  • the antibody is monoclonal antibody SOF10/RG6440, anti-latent TGF- ⁇ 1
  • the antibody is selected from the group consisting of an anti-CD137 antibody, an anti-latent TGF- ⁇ 1 antibody, an anti-latent myostatin antibody, an anti-complement (C1s) antibody, an anti-IL-8 antibody, an anti-IL-6 receptor antibody, an anti-IL-6 antibody, an anti-glypican-3 antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-GPIIb/IIIa antibody, an anti-TNF antibody, an anti-CD25 antibody, an anti-EGFR antibody, an anti-Her2/neu antibody, an anti-RSV antibody, an anti-CD33 antibody, an anti-CD52 antibody, an anti-IgE antibody, an anti-CD11a antibody, an anti-VEGF antibody, and an anti-VLA4 antibody, and is preferably an anti-CD137 agonist switch antibody STA551, an anti-latent TGF- ⁇ 1
  • the present invention by setting the copy numbers of the antibody L-chain and H-chain cDNA contained in the expression vector to a specific value (4 copies for L-chain and 2 copies for H-chain), it is possible to obtain transformed cells that produce a high amount of the desired recombinant antibody.
  • the present invention makes it possible to provide a novel method for producing recombinant antibodies with high production efficiency.
  • the H2L4 plasmid is composed of two copies of the heavy chain and four copies of the light chain of humanized anti-CD137 antibody genes.
  • the H3L6 plasmid is composed of three copies of the heavy chain and six copies of the light chain of humanized anti-CD137 antibody genes.
  • the H4L8 plasmid is composed of 4 copies of the heavy chain and 8 copies of the light chain of humanized anti-CD137 antibody genes.
  • 1 is a graph showing a comparison of titers among H2L4 plasmid-introduced clone cell lines (average value), H3L6 plasmid-introduced clone cell lines (average value), and H4L8 plasmid-introduced clone cell lines (average value).
  • the present invention provides a method for producing an antibody or a fragment thereof, which comprises producing an antibody or a fragment thereof using a cell into which a vector containing four copies of foreign DNA encoding an antibody L chain or a fragment thereof and two copies of foreign DNA encoding the antibody H chain or a fragment thereof has been introduced.
  • the four copies of DNA encoding the L chain or a fragment thereof and the two copies of DNA encoding the H chain or a fragment thereof are all contained in the same vector.
  • the present invention also provides a recombinant vector containing four copies of foreign DNA encoding an antibody light chain or a fragment thereof, and two copies of foreign DNA encoding the antibody heavy chain or a fragment thereof.
  • the present invention also provides a cell into which a vector containing four copies of foreign DNA encoding an antibody light chain or a fragment thereof and two copies of foreign DNA encoding the antibody heavy chain or a fragment thereof has been introduced.
  • the present invention also provides a cultured cell into which a vector containing four copies of foreign DNA encoding an antibody light chain or a fragment thereof and two copies of foreign DNA encoding the antibody heavy chain or a fragment thereof has been introduced.
  • the above-mentioned cells or cultured cells are preferably transformed cells into which a vector containing four copies of DNA encoding the L chain or a fragment thereof of a desired recombinant antibody and two copies of DNA encoding the H chain or a fragment thereof of the antibody has been introduced, and which produce the recombinant antibody or a fragment thereof.
  • the present inventors have demonstrated that, by using a transformed cell into which a vector containing four copies of DNA encoding the L chain and two copies of DNA encoding the H chain of a recombinant antibody has been introduced, 1) A transformed cell carrying two copies of the light chain and one copy of the heavy chain. 2) A transformed cell line carrying six copies of the light chain and three copies of the heavy chain. 3) We found that the production of the desired recombinant antibody was significantly increased compared to transformed cells containing eight copies of the light chain and four copies of the heavy chain.
  • the manufacturing method of the present invention is therefore characterized by using a transformed cell into which a vector containing four copies of DNA encoding the L chain or a fragment thereof and two copies of DNA encoding the H chain or a fragment thereof of a recombinant antibody is introduced.
  • a recombinant vector containing four copies of DNA encoding the L chain or a fragment thereof and two copies of DNA encoding the H chain or a fragment thereof of the desired antibody is prepared, the vector is introduced into cells, and the transformed cells into which the vector has been introduced are cultured to produce the desired antibody or a fragment thereof, thereby manufacturing the antibody or a fragment thereof.
  • the construction of the recombinant vector, the introduction of the vector into the host cell, and the selection of the transformed cells into which the vector has been introduced may be performed using general means well known to those skilled in the art. Those skilled in the art can also appropriately culture the transformed cells to produce the desired polypeptide.
  • culture equipment for animal cell culture include, for example, fermenter-type tank culture equipment, air lift-type culture equipment, culture flask-type culture equipment, spinner flask-type culture equipment, microcarrier-type culture equipment, fluidized bed-type culture equipment, hollow fiber-type culture equipment, roller bottle-type culture equipment, and packed tank-type culture equipment.
  • Cultivation may be performed using any method, such as batch culture, fed-batch culture, or continuous culture, but fed-batch culture or continuous culture is preferred, with fed-batch culture being more preferred.
  • chromatography besides affinity chromatography include, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These types of chromatography can be performed using liquid phase chromatography such as HPLC and FPLC.
  • the polypeptide can be treated with an appropriate polypeptide-modifying enzyme to optionally modify it or partially remove peptides.
  • polypeptide-modifying enzymes examples include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, and glucosidase.
  • the obtained antibody can be chemically modified to bind with various molecules such as polyethylene glycol (PEG) to produce a modified antibody.
  • PEG polyethylene glycol
  • the polypeptide can be mixed with a pharma- ceutical carrier or additive to produce a formulation, thereby producing a pharmaceutical product.
  • Examples of pharma- ceutically acceptable carriers and additives include water, pharma-ceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethylstarch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, and the like.
  • pharma-ceutically acceptable organic solvents include water, pharma-ceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose,
  • the purified polypeptide when used as an injectable formulation, can be dissolved in a solvent, such as physiological saline, buffer solution, glucose solution, etc., to which an adsorption inhibitor, such as Tween 80, Tween 20, gelatin, human serum albumin, etc., can be added.
  • a solvent such as physiological saline, buffer solution, glucose solution, etc.
  • an adsorption inhibitor such as Tween 80, Tween 20, gelatin, human serum albumin, etc.
  • the polypeptide may be lyophilized to form a dosage form that can be dissolved and reconstituted before use, and sugar alcohols and sugars, such as mannitol and glucose, can be used as excipients for lyophilization.
  • the effective dose of the antibody or a fragment thereof is appropriately selected depending on the type of antibody or fragment thereof, the type of disease to be treated or prevented, the age of the patient, the severity of the disease, etc.
  • the effective dose of the anti-glypican-3 antibody is selected in the range of 0.001 mg to 1000 mg per kg of body weight per administration.
  • a dose of 0.01 to 100,000 mg/body per patient can be selected. However, it is not limited to these doses.

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Abstract

The present application provides a method with which it is possible to efficiently produce a recombinant antibody at low cost. Specifically, the present invention provides a method for producing an antibody or a fragment thereof, the method being characterized by using a cell into which a vector containing four copies of a DNA coding for an L chain of an antibody or a fragment thereof and two copies of a DNA coding for an H chain of the antibody or a fragment thereof is introduced. Also provided are a recombinant vector containing four copies of a DNA coding for an L chain of an antibody or a fragment thereof and two copies of a DNA coding for an H chain of the antibody or a fragment thereof, a transformed cell into which such a vector is introduced, and the like.

Description

抗体の製造方法Methods for producing antibodies

　本発明は、抗体の製造方法に関する。 The present invention relates to a method for producing an antibody.

　遺伝子組換え技術を用いて、医薬として有用な組換え抗体を生産する際に、動物細胞を用いると、原核細胞が行い得ないような複雑な翻訳後修飾やフォールディングが可能となるため、動物細胞は組換え抗体生産のための宿主細胞として多用されてきている。 When using recombinant gene technology to produce recombinant antibodies useful as medicines, animal cells allow for complex post-translational modifications and folding that cannot be performed by prokaryotic cells, and therefore animal cells have come to be widely used as host cells for the production of recombinant antibodies.

　近年、抗体や生理活性タンパク質などの多くのバイオ医薬品が輩出されている。特に、抗体医薬の場合は通常1回の投与量がｍｇオーダーとなり、かなりの量の活性成分としての抗体が必要となる。組換え抗体を効率よく動物細胞に生産させる技術は、抗体医薬品の低コスト化につながり、患者への安定な供給を約束するものである。 In recent years, many biopharmaceuticals, including antibodies and biologically active proteins, have been produced. In particular, the dosage of antibody drugs is usually on the order of milligrams, so a considerable amount of antibody as the active ingredient is required. Technology to efficiently produce recombinant antibodies in animal cells will lead to lower costs for antibody drugs and promise a stable supply to patients.

　従って、より生産効率の高い組換え抗体の製造方法が望まれている。
　例えば、動物細胞で組換え抗体を効率よく生産するための発現系の開発が重要である。 Therefore, there is a need for a method for producing recombinant antibodies with higher production efficiency.
For example, it is important to develop an expression system for efficiently producing recombinant antibodies in animal cells.

　EP2439269（特許文献１）には、抗体の重鎖及び／又は軽鎖ポリペプチドの遺伝子を高レベルで発現する形質転換細胞を樹立するために、ｍＲＮＡ不安定化配列を導入した薬剤選択マーカー遺伝子発現カセット、及び遺伝子発現安定化エレメントを含む発現ベクターを利用することが開示されている。 EP2439269 (Patent Document 1) discloses the use of an expression vector containing a drug selection marker gene expression cassette incorporating an mRNA destabilizing sequence and a gene expression stabilizing element to establish transformed cells that express antibody heavy and/or light chain polypeptide genes at high levels.

　組換え抗体を産生するための宿主細胞を作製する際には、通常その抗体のH鎖をコードするDNAを1コピーとL鎖をコードするDNAを1コピーずつ宿主細胞に導入する（非特許文献１及び２）。 When preparing host cells for producing recombinant antibodies, one copy each of the DNA encoding the antibody's H chain and L chain is usually introduced into the host cells (Non-Patent Documents 1 and 2).

　ところが、WO 2009/051108（特許文献２）には、抗体のL鎖をコードするDNAを抗体のH鎖をコードするDNAよりも多いコピー数で含む形質転換細胞を用いて抗体を高産生する方法が開示されている。WO 2009/051108では、所望の組換え抗体のH鎖をコードするDNAを1コピーとL鎖をコードするDNAを2コピー含むプラスミドが細胞に導入されている。 However, WO 2009/051108 (Patent Document 2) discloses a method for high antibody production using transformed cells that contain more copies of DNA encoding the antibody light chain than of the DNA encoding the antibody heavy chain. In WO 2009/051108, a plasmid containing one copy of DNA encoding the heavy chain and two copies of DNA encoding the light chain of the desired recombinant antibody is introduced into the cells.

　抗体分子を構成するH鎖ポリペプチドとL鎖ポリペプチドはBiP (Immunoglobulin heavy chain binding protein)のサポートでアセンブルし、その後foldingすることにより完全な抗体構造を完成する。このアセンブル過程はL鎖ポリペプチド依存的である(非特許文献３)。従ってL鎖遺伝子数比を高めL鎖ポリペプチドの比率を高める事によりH鎖ポリペプチドとL鎖ポリペプチドのアセンブルが促進し、産生量が増加するものと考えられる。 The heavy and light chain polypeptides that make up an antibody molecule assemble with the support of BiP (Immunoglobulin heavy chain binding protein), and then fold to complete the complete antibody structure. This assembly process is dependent on the light chain polypeptide (Non-Patent Document 3). Therefore, it is believed that by increasing the ratio of the number of light chain genes and the proportion of light chain polypeptides, the assembly of heavy and light chain polypeptides is promoted, and the amount produced is increased.

　抗体のL鎖をコードする外来DNAを、H鎖をコードするDNAよりも多いコピー数で含むCHO（チャイニーズハムスター卵巣）細胞を用いて、一過性発現（Transient Expression）又は恒常発現（Stable Expression）を行うことで、抗体を製造する方法が開示されている（非特許文献４）。この抗体製造方法では、抗体のL鎖をコードする外来DNAと、抗体のH鎖をコードする外来DNAをそれぞれ、別のベクターで細胞に導入している。 A method for producing antibodies has been disclosed in which transient expression or stable expression is carried out using CHO (Chinese Hamster Ovary) cells that contain more copies of foreign DNA encoding the antibody's light chain than the DNA encoding the heavy chain (Non-Patent Document 4). In this antibody production method, the foreign DNA encoding the antibody's light chain and the foreign DNA encoding the antibody's heavy chain are each introduced into the cells using separate vectors.

　しかし、特に、恒常発現系で組換え抗体を発現している恒常発現形質転換細胞（Stable Transformant）の場合、一過性発現系のものよりH鎖の発現量が低下するので、H鎖の発現量がより重要になることが示唆されている（非特許文献４）。よって、恒常発現系の場合、抗体の産生量を増加させるためにH鎖とL鎖の発現量の比をどのように制御するかは、不明である。 However, it has been suggested that the amount of H chain expression is more important in the case of stable transformants, which express recombinant antibodies in a constitutive expression system, since the amount of H chain expression is lower than in transient expression systems (Non-Patent Document 4). Therefore, in the case of a constitutive expression system, it is unclear how to control the ratio of H chain and L chain expression levels to increase antibody production.

EP2439269EP2439269 WO 2009/051108WO 2009/051108

Reff ME, Carner K, Chambers KS, Chinn PC, Leonard JE, Raab R et al. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood. 1994 Jan 15; 83(2):435-45.Reff ME, Carner K, Chambers KS, Chinn PC, Leonard JE, Raab R et al. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood. 1994 Jan 15; 83(2):435-45. Presta LG, Chen H, O'Connor SJ, Chisholm V, Meng YG, Krummen L, et al. Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res. 1997 Oct 15; 57(20):4593-9.Presta LG, Chen H, O'Connor SJ, Chisholm V, Meng YG, Krummen L, et al. Humanization of an anti-vascular endothelial growt h factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res. 1997 Oct 15; 57(20):4593-9. Young-Kwang Lee, Joseph W. Brewer, Rachel Hellman, and Linda M. Hendershot. BiP and immunoglobulin light chain cooperate to control the folding of heavy chain and ensure the fidelity of immunoglobulin assembly. Molecular Biology of the Cell, 1999, 10, 2209Young-Kwang Lee, Joseph W. Brewer, Rachel Hellman, and Linda M. Hendershot. BiP and immunoglobulin light chain cooperate to cont rol the folding of heavy chain and ensure the fidelity of immunoglobulin assembly. Molecular Biology of the Cell, 1999, 10, 2209 Stefan Schlatter, et al. On the Optimal Ratio of Heavy to Light Chain Genes for Efficient Recombinant Antibody Production by CHO Cells. Biotechnol. Prog., 2005, 21, 122Stefan Schlatter, et al. On the Optimal Ratio of Heavy to Light Chain Genes for Efficient Recombinant Antibody Production by CHO Cells. Biotechnol. Prog., 2005, 21, 122

　本発明は、抗体を高産生する方法を提供することを目的とする。 The objective of the present invention is to provide a method for high antibody production.

　本発明者らは、抗体のL鎖をコードするDNAを4コピー、H鎖をコードするDNAを2コピー含む発現用ベクターを導入したCHO細胞を作製したところ、1) L鎖2コピーとH鎖1コピーを含むベクターを導入した細胞、2) L鎖6コピーとH鎖3コピーを含むベクターを導入した細胞、3) L鎖8コピーとH鎖4コピーを含むベクターを導入した細胞よりも、抗体産生能の高い細胞株を取得できることを見出し、本発明を完成させるに至った。 The inventors produced CHO cells into which an expression vector containing four copies of DNA encoding an antibody light chain and two copies of DNA encoding the heavy chain was introduced, and found that a cell line with higher antibody production ability could be obtained than: 1) cells into which a vector containing two copies of the light chain and one copy of the heavy chain was introduced; 2) cells into which a vector containing six copies of the light chain and three copies of the heavy chain was introduced; and 3) cells into which a vector containing eight copies of the light chain and four copies of the heavy chain was introduced, thereby completing the present invention.

　本発明の要旨は以下の通りである。
（１）抗体のL鎖又はその断片をコードするDNAを4コピー、及び当該抗体のH鎖又はその断片をコードするDNAを2コピー含むベクターを導入した細胞を用いた、抗体又はその断片の製造方法。
（２）抗体のL鎖又はその断片をコードするDNAを4コピー、及び当該抗体のH鎖又はその断片をコードするDNAを2コピー含む組換えベクターを作製し、該ベクターを細胞に導入し、該ベクターが導入された形質転換細胞を培養して抗体又はその断片を産生させることにより、抗体又はその断片を製造する、請求項１に記載の方法。
（３）細胞が動物細胞である（１）または（２）に記載の方法。
（４）動物細胞がチャイニーズハムスター卵巣細胞である（１）～（３）のいずれかに記載の方法。
（５）抗体が、キメラ抗体、ヒト化抗体又はヒト抗体である（１）～（４）のいずれかに記載の方法。
（６）抗体が、スイッチ抗体、リサイクリング抗体、又はスイーピング抗体である（１）～（５）のいずれかに記載の方法。
（７）抗体が、抗CD137抗体、抗潜在型TGF-β1抗体、抗潜在型ミオスタチン抗体、抗補体(C1s)抗体、抗IL-8抗体、抗IL-6レセプター抗体、抗IL-6抗体、抗グリピカン-3抗体、抗CD3抗体、抗CD20抗体、抗GPIIb/IIIa抗体、抗TNF抗体、抗CD25抗体、抗EGFR抗体、抗Her2/neu抗体、抗RSV抗体、抗CD33抗体、抗CD52抗体、抗IgE抗体、抗CD11a抗体、抗VEGF抗体及び抗VLA4抗体からなる群より選択され、好ましくは、抗CD137アゴニストスイッチ抗体 STA551、抗潜在型 TGF-β1 モノクローナル抗体SOF10/RG6440、抗潜在型ミオスタチンスイーピング抗体GYM329/RG6237、抗補体(C1s)抗体RAY121、抗体S12preまたは抗IL-8リサイクリング抗体 AMY109である、（１）～（６）のいずれかに記載の方法。
（８）細胞が、抗体又はその断片を恒常的発現(stable expression)している、（１）～（７）のいずれかに記載の方法。
（９）抗体のL鎖又はその断片をコードするDNAを4コピー、当該抗体のH鎖又はその断片をコードするDNAを2コピー含む組換えベクター。
（１０）前記（９）記載のベクターが導入されている細胞。
（１１）抗体のL鎖又はその断片をコードする外来DNAを4コピーと当該抗体のH鎖又はその断片をコードする外来DNAを2コピーとを同一のベクター内に含む、当該ベクターを導入した培養細胞。
（１２）抗体又はその断片を恒常的発現している、（１０）又は（１１）に記載の細胞。
（１３）抗体が、抗CD137抗体、抗潜在型TGF-β1抗体、抗潜在型ミオスタチン抗体、抗補体(C1s)抗体、抗IL-8抗体、抗IL-6レセプター抗体、抗IL-6抗体、抗グリピカン-3抗体、抗CD3抗体、抗CD20抗体、抗GPIIb/IIIa抗体、抗TNF抗体、抗CD25抗体、抗EGFR抗体、抗Her2/neu抗体、抗RSV抗体、抗CD33抗体、抗CD52抗体、抗IgE抗体、抗CD11a抗体、抗VEGF抗体及び抗VLA4抗体からなる群より選択され、好ましくは、抗CD137アゴニストスイッチ抗体 STA551、抗潜在型 TGF-β1 モノクローナル抗体SOF10/RG6440、抗潜在型ミオスタチンスイーピング抗体GYM329/RG6237、抗補体(C1s)抗体RAY121、抗体S12preまたは抗IL-8リサイクリング抗体 AMY109である、（８）に記載のベクター、又は（９）～（１２）のいずれかに記載の細胞。
（１４）抗体が、ヒト化抗CD137抗体（例えばSTA551）である、（１）～（８）のいずれかに記載の方法、（９）に記載のベクター、又は（１０）～（１３）のいずれかに記載の細胞。
（１５）抗体又はその断片を含有する医薬品を製造する方法であって、（１）～（８）のいずれかに記載の方法で抗体又はその断片を製造し、得られた抗体を医薬的に許容される担体又は添加剤と混合して製剤化することにより、医薬品を製造する工程を含む、方法。 The gist of the present invention is as follows.
(1) A method for producing an antibody or a fragment thereof using a cell into which a vector containing four copies of DNA encoding an antibody light chain or a fragment thereof and two copies of DNA encoding the antibody heavy chain or a fragment thereof has been introduced.
(2) The method according to claim 1, comprising preparing a recombinant vector containing four copies of DNA encoding an antibody light chain or a fragment thereof and two copies of DNA encoding the antibody heavy chain or a fragment thereof, introducing the vector into a cell, and culturing the transformed cell into which the vector has been introduced to produce the antibody or a fragment thereof, thereby producing the antibody or a fragment thereof.
(3) The method according to (1) or (2), wherein the cell is an animal cell.
(4) The method according to any one of (1) to (3), wherein the animal cell is a Chinese hamster ovary cell.
(5) The method according to any one of (1) to (4), wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.
(6) The method according to any one of (1) to (5), wherein the antibody is a switching antibody, a recycling antibody, or a sweeping antibody.
(7) The antibody is selected from the group consisting of an anti-CD137 antibody, an anti-latent TGF-β1 antibody, an anti-latent myostatin antibody, an anti-complement (C1s) antibody, an anti-IL-8 antibody, an anti-IL-6 receptor antibody, an anti-IL-6 antibody, an anti-glypican-3 antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-GPIIb/IIIa antibody, an anti-TNF antibody, an anti-CD25 antibody, an anti-EGFR antibody, an anti-Her2/neu antibody, an anti-RSV antibody, an anti-CD33 antibody, an anti-CD52 antibody, an anti-IgE antibody, an anti-CD11a antibody, an anti-VEGF antibody, and an anti-VLA4 antibody, and is preferably an anti-CD137 agonist switch antibody STA551, an anti-latent TGF-β1 The method according to any one of (1) to (6), wherein the antibody is monoclonal antibody SOF10/RG6440, anti-latent myostatin sweeping antibody GYM329/RG6237, anti-complement (C1s) antibody RAY121, antibody S12pre, or anti-IL-8 recycling antibody AMY109.
(8) The method according to any one of (1) to (7), wherein the cell stably expresses the antibody or a fragment thereof.
(9) A recombinant vector comprising four copies of DNA encoding an antibody light chain or a fragment thereof and two copies of DNA encoding the antibody heavy chain or a fragment thereof.
(10) A cell into which the vector described in (9) above has been introduced.
(11) A cultured cell into which a vector has been introduced, the vector containing four copies of foreign DNA encoding an antibody light chain or a fragment thereof and two copies of foreign DNA encoding the antibody heavy chain or a fragment thereof.
(12) The cell according to (10) or (11), which constitutively expresses an antibody or a fragment thereof.
(13) The antibody is selected from the group consisting of an anti-CD137 antibody, an anti-latent TGF-β1 antibody, an anti-latent myostatin antibody, an anti-complement (C1s) antibody, an anti-IL-8 antibody, an anti-IL-6 receptor antibody, an anti-IL-6 antibody, an anti-glypican-3 antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-GPIIb/IIIa antibody, an anti-TNF antibody, an anti-CD25 antibody, an anti-EGFR antibody, an anti-Her2/neu antibody, an anti-RSV antibody, an anti-CD33 antibody, an anti-CD52 antibody, an anti-IgE antibody, an anti-CD11a antibody, an anti-VEGF antibody, and an anti-VLA4 antibody, and is preferably an anti-CD137 agonist switch antibody STA551, an anti-latent TGF-β1 The vector described in (8) or the cell described in any of (9) to (12), which is monoclonal antibody SOF10/RG6440, anti-latent myostatin sweeping antibody GYM329/RG6237, anti-complement (C1s) antibody RAY121, antibody S12pre, or anti-IL-8 recycling antibody AMY109.
(14) The method according to any one of (1) to (8), the vector according to (9), or the cell according to any one of (10) to (13), wherein the antibody is a humanized anti-CD137 antibody (e.g., STA551).
(15) A method for producing a pharmaceutical containing an antibody or a fragment thereof, comprising the steps of producing an antibody or a fragment thereof by a method according to any one of (1) to (8) and mixing the obtained antibody with a pharma- ceutical acceptable carrier or additive to form a formulation, thereby producing the pharmaceutical.

　本発明では、抗体のL鎖とH鎖のcDNAのコピー数について、発現ベクターに含まれるコピー数を特定の数値（L鎖 4コピー及びH鎖 2コピー）とすることにより、所望の組換え抗体の産生量が高い形質転換細胞を取得することができる。本発明により、生産効率の高い組換え抗体の新規な製造方法を提供することが可能である。さらに H鎖 3 copy及びL鎖 6 copyで構成されるH3L6プラスミド及び H鎖 4 copy及びL鎖 8 copyで構成されるH4L8プラスミドよりもプラスミドサイズとして小さいことから、プラスミド構築が容易であること、細胞への導入効率が高いこと、宿主細胞ゲノム上に導入された後に安定的に保持されること等が期待される。 In the present invention, by setting the copy numbers of the antibody L-chain and H-chain cDNA contained in the expression vector to a specific value (4 copies for L-chain and 2 copies for H-chain), it is possible to obtain transformed cells that produce a high amount of the desired recombinant antibody. The present invention makes it possible to provide a novel method for producing recombinant antibodies with high production efficiency. Furthermore, since the plasmid size is smaller than the H3L6 plasmid, which is composed of 3 copies of the H-chain and 6 copies of the L-chain, and the H4L8 plasmid, which is composed of 4 copies of the H-chain and 8 copies of the L-chain, it is expected that the plasmid will be easily constructed, will be introduced efficiently into cells, and will be stably maintained after being introduced into the host cell genome.

ヒト化抗CD137抗体遺伝子のH鎖 2 copy及びL鎖 4 copyで構成されるH2L4プラスミドを示す。The H2L4 plasmid is composed of two copies of the heavy chain and four copies of the light chain of humanized anti-CD137 antibody genes. ヒト化抗CD137抗体遺伝子のH鎖 3 copy及びL鎖 6 copyで構成されるH3L6プラスミドを示す。The H3L6 plasmid is composed of three copies of the heavy chain and six copies of the light chain of humanized anti-CD137 antibody genes. ヒト化抗CD137抗体遺伝子のH鎖 4 copy及びL鎖 8 copyで構成されるH4L8プラスミドを示す。The H4L8 plasmid is composed of 4 copies of the heavy chain and 8 copies of the light chain of humanized anti-CD137 antibody genes. H2L4プラスミド導入クローン細胞株（平均値）、H3L6プラスミド導入クローン細胞株（平均値）、H4L8導入クローン細胞株（平均値）のタイター比較を示すグラフである。1 is a graph showing a comparison of titers among H2L4 plasmid-introduced clone cell lines (average value), H3L6 plasmid-introduced clone cell lines (average value), and H4L8 plasmid-introduced clone cell lines (average value).

　以下、本発明の実施の形態についてより詳細に説明する。
　本発明は、抗体のL鎖又はその断片をコードする外来DNAを4コピー、及び当該抗体のH鎖又はその断片をコードする外来DNAを2コピー含むベクターを導入した細胞を用いて抗体又はその断片を産生させることを含む、抗体又はその断片の製造方法を提供する。本発明の製造方法において、L鎖又はその断片をコードするDNA 4コピーとH鎖又はその断片をコードするDNA 2コピーは、全て、同一のベクター内に含まれる。 Hereinafter, the embodiment of the present invention will be described in more detail.
The present invention provides a method for producing an antibody or a fragment thereof, which comprises producing an antibody or a fragment thereof using a cell into which a vector containing four copies of foreign DNA encoding an antibody L chain or a fragment thereof and two copies of foreign DNA encoding the antibody H chain or a fragment thereof has been introduced. In the production method of the present invention, the four copies of DNA encoding the L chain or a fragment thereof and the two copies of DNA encoding the H chain or a fragment thereof are all contained in the same vector.

　本発明は、また、抗体のL鎖又はその断片をコードする外来DNAを4コピー、及び当該抗体のH鎖又はその断片をコードする外来DNAを2コピー含む組換えベクターを提供する。 The present invention also provides a recombinant vector containing four copies of foreign DNA encoding an antibody light chain or a fragment thereof, and two copies of foreign DNA encoding the antibody heavy chain or a fragment thereof.

　本発明は、また、抗体のL鎖又はその断片をコードする外来DNAを4コピー、及び当該抗体のH鎖又はその断片をコードする外来DNAを2コピー含むベクターを導入した細胞を提供する。 The present invention also provides a cell into which a vector containing four copies of foreign DNA encoding an antibody light chain or a fragment thereof and two copies of foreign DNA encoding the antibody heavy chain or a fragment thereof has been introduced.

　本発明は、また、抗体のL鎖又はその断片をコードする外来DNAを4コピー、及び当該抗体のH鎖又はその断片をコードする外来DNAを2コピー含むベクターを導入した培養細胞を提供する。 The present invention also provides a cultured cell into which a vector containing four copies of foreign DNA encoding an antibody light chain or a fragment thereof and two copies of foreign DNA encoding the antibody heavy chain or a fragment thereof has been introduced.

　本発明において、上述の細胞又は培養細胞は、所望の組換え抗体のL鎖又はその断片をコードするDNAを4コピー、及び当該抗体のH鎖又はその断片をコードするDNAを2コピー含むベクターを導入した形質転換細胞で、且つ当該組換え抗体又はその断片を産生する細胞であるとよい。 In the present invention, the above-mentioned cells or cultured cells are preferably transformed cells into which a vector containing four copies of DNA encoding the L chain or a fragment thereof of a desired recombinant antibody and two copies of DNA encoding the H chain or a fragment thereof of the antibody has been introduced, and which produce the recombinant antibody or a fragment thereof.

　本発明者らは、組換え抗体のL鎖をコードするDNAを4コピー、H鎖をコードするDNAを2コピー含むベクターを導入した形質転換細胞を用いることにより、
1) L鎖を2コピーとH鎖を1コピー導入した形質転換細胞、
2) L鎖を6コピーとH鎖を3コピー導入した形質転換細胞、
3) L鎖を8コピーとH鎖を4コピー含む導入した形質転換細胞
に比べ、明らかに優位に所望の組換え抗体の産生量が増加することを見出した。 The present inventors have demonstrated that, by using a transformed cell into which a vector containing four copies of DNA encoding the L chain and two copies of DNA encoding the H chain of a recombinant antibody has been introduced,
1) A transformed cell carrying two copies of the light chain and one copy of the heavy chain.
2) A transformed cell line carrying six copies of the light chain and three copies of the heavy chain.
3) We found that the production of the desired recombinant antibody was significantly increased compared to transformed cells containing eight copies of the light chain and four copies of the heavy chain.

　したがって、本発明の製造方法は、組換え抗体のL鎖又はその断片をコードするDNA 4コピーとH鎖又はその断片をコードするDNA 2コピーを同一のベクター内に含む、当該ベクターが導入された形質転換細胞を用いることを特徴とする。 The manufacturing method of the present invention is therefore characterized by using a transformed cell into which a vector containing four copies of DNA encoding the L chain or a fragment thereof and two copies of DNA encoding the H chain or a fragment thereof of a recombinant antibody is introduced.

　本発明の製造方法では、目的とする抗体のL鎖又はその断片をコードするDNAを4コピー、及びH鎖又はその断片をコードするDNAを2コピー含む組換えベクターを作製し、該ベクターを細胞に導入し、該ベクターが導入された形質転換細胞を培養して目的とする抗体又はその断片を産生させることにより、抗体又はその断片を製造する。組換えベクターの作製、宿主細胞へのベクターの導入及びベクターが導入された形質転換細胞の選抜は、当業者に周知の一般的な手段を用いて行えばよい。形質転換細胞を培養して目的のポリペプチドを産生させることも、当業者であれば適宜実施可能である。 In the manufacturing method of the present invention, a recombinant vector containing four copies of DNA encoding the L chain or a fragment thereof and two copies of DNA encoding the H chain or a fragment thereof of the desired antibody is prepared, the vector is introduced into cells, and the transformed cells into which the vector has been introduced are cultured to produce the desired antibody or a fragment thereof, thereby manufacturing the antibody or a fragment thereof. The construction of the recombinant vector, the introduction of the vector into the host cell, and the selection of the transformed cells into which the vector has been introduced may be performed using general means well known to those skilled in the art. Those skilled in the art can also appropriately culture the transformed cells to produce the desired polypeptide.

　本発明の製造方法は、上述のベクターが導入された形質転換細胞について、所望の組換え抗体又はその断片の産生量が高いクローン細胞株（複数種）を得るために当該抗体又はその断片の産生能による選抜を行うこと、そして、得られた各クローン細胞株を培養して抗体又はその断片を産生させることを含んでもよい。ここで、クローン細胞株とは、選抜された単一細胞にそれぞれ由来するものである。生産物を抗体医薬として用いる場合、クローン細胞株は、生産物の品質や細胞安定性に基づきさらに選抜され、生産培養に適した種培養株とされる。選抜されたクローン細胞株、すなわち種細胞株を拡大培養の後に通常数百本のバイアルに分注後に凍結保存したものをセルバンクとして抗体原薬の製造に利用する。 The manufacturing method of the present invention may include selecting transformed cells into which the above-mentioned vector has been introduced based on the ability to produce the desired recombinant antibody or a fragment thereof, in order to obtain clone cell lines (multiple types) that produce the antibody or a fragment thereof at a high level, and culturing each of the obtained clone cell lines to produce the antibody or a fragment thereof. Here, the clone cell lines are derived from the selected single cells. When the product is to be used as an antibody drug, the clone cell lines are further selected based on the quality of the product and the cell stability, and are used as seed culture lines suitable for production culture. The selected clone cell lines, i.e., the seed cell lines, are expanded and then usually dispensed into several hundred vials and frozen and stored, and are used as a cell bank for the manufacture of antibody drug substances.

　本発明の製造方法が適用可能な抗体は、如何なる抗原に対する抗体でもよい。例えば、抗CD137抗体、抗潜在型TGF-β1抗体、抗潜在型ミオスタチン抗体、抗補体(C1s)抗体、抗IL-8抗体、抗IL-6レセプター抗体、抗IL-6抗体、抗グリピカン-3抗体、抗CD3抗体、抗CD20抗体、抗GPIIb/IIIa抗体、抗TNF抗体、抗CD25抗体、抗EGFR抗体、抗Her2/neu抗体、抗RSV抗体、抗CD33抗体、抗CD52抗体、抗IgE抗体、抗CD11a抗体、抗VEGF抗体及び抗VLA4抗体などの組換え抗体が挙げられるが、これらに限定されない。 Antibodies to which the manufacturing method of the present invention can be applied may be antibodies against any antigen. Examples include, but are not limited to, recombinant antibodies such as anti-CD137 antibody, anti-latent TGF-β1 antibody, anti-latent myostatin antibody, anti-complement (C1s) antibody, anti-IL-8 antibody, anti-IL-6 receptor antibody, anti-IL-6 antibody, anti-glypican-3 antibody, anti-CD3 antibody, anti-CD20 antibody, anti-GPIIb/IIIa antibody, anti-TNF antibody, anti-CD25 antibody, anti-EGFR antibody, anti-Her2/neu antibody, anti-RSV antibody, anti-CD33 antibody, anti-CD52 antibody, anti-IgE antibody, anti-CD11a antibody, anti-VEGF antibody, and anti-VLA4 antibody.

　本発明の製造方法で生産される抗体には、ヒト、マウス、ラット、ハムスター、ウサギ、ヒツジ、ラクダ、サル等の動物由来のモノクローナル抗体だけでなく、キメラ抗体、ヒト化抗体、ヒト抗体、低分子化抗体など、人為的に改変した遺伝子組み換え型抗体も含まれる。 The antibodies produced by the manufacturing method of the present invention include not only monoclonal antibodies derived from animals such as humans, mice, rats, hamsters, rabbits, sheep, camels, and monkeys, but also artificially modified genetically engineered antibodies such as chimeric antibodies, humanized antibodies, human antibodies, and low molecular weight antibodies.

　また、抗体の免疫グロブリンクラスも特に限定されるものではなく、ＩｇＧ１、ＩｇＧ２、ＩｇＧ３、ＩｇＧ４などのＩｇＧ、ＩｇＡ、ＩｇＤ、ＩｇＥ、ＩｇＭなどいずれのクラスでもよいが、医薬として用いる場合はＩｇＧ及びＩｇＭが好ましい。 The immunoglobulin class of the antibody is also not particularly limited, and may be any class such as IgG, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM, but IgG and IgM are preferred for pharmaceutical use.

　さらに、本発明において、所望の組換え抗体は、スイッチ抗体、リサイクリング抗体、スイーピング抗体などの、抗体エンジニアリング技術を用いて改変された抗体であってもよい。 Furthermore, in the present invention, the desired recombinant antibody may be an antibody modified using antibody engineering techniques, such as a switch antibody, recycling antibody, or sweeping antibody.

　抗体のL鎖をコードするDNA及びH鎖をコードするDNAは、一般的に以下のようにして調製することができる。抗体を発現する遺伝子を持つハイブリドーマ、細胞、ファージ、リボソームなどからmRNAを抽出する。このmRNAより逆転写酵素を用いる逆転写反応によりcDNAを作製する。L鎖遺伝子又はH鎖遺伝子と相補塩基配列を持つプライマーとcDNAを用いるPCRによりL鎖遺伝子又はH鎖遺伝子を増幅し、クローニング用プラスミドと結合することにより各遺伝子を取得する。 　DNA encoding the L chain and DNA encoding the H chain of an antibody can generally be prepared as follows: Extract mRNA from hybridomas, cells, phages, ribosomes, etc. that contain genes that express antibodies. Create cDNA from this mRNA by reverse transcription using reverse transcriptase. Amplify the L chain gene or H chain gene by PCR using cDNA and primers that have complementary base sequences to the L chain gene or H chain gene, and obtain each gene by combining with a cloning plasmid.

　本発明における所望の組換え抗体としてはwholeの抗体だけでなく、Fab、F(ab')2、Fvなどの抗体断片なども含まれる。抗体のL鎖の断片をコードするDNA及びH鎖の断片をコードするDNAである各遺伝子断片も上述と同様の方法でクローニングすることができる。 The desired recombinant antibody in the present invention includes not only whole antibodies but also antibody fragments such as Fab, F(ab')2, and Fv. Gene fragments that are DNA encoding an antibody L-chain fragment and DNA encoding an antibody H-chain fragment can also be cloned in the same manner as described above.

　生産物を抗体医薬として用いる場合、上述の方法で得られた、または別の方法で入手した遺伝子に基づき抗体鎖のアミノ酸配列を割り出してから、有効性、機能、物性などを向上させ得る改変を加えてもよい。そのような改変には、本明細書でも言及される抗体エンジニアリング技術を用いた改変が含まれる。 When the product is used as an antibody drug, the amino acid sequence of the antibody chain may be determined based on the gene obtained by the above method or obtained by another method, and then modifications may be made to improve efficacy, function, physical properties, etc. Such modifications include modifications using antibody engineering techniques, which are also mentioned in this specification.

　ベクターは、宿主細胞内において組換え抗体又はその断片をコードするDNAを保持したり、組換え抗体又はその断片を発現させたりするために有用である。本発明において、ベクターが導入される宿主細胞は特に制限はなく、例えば、大腸菌や種々の動物細胞などを用いることが可能である。 　Vectors are useful for maintaining DNA encoding a recombinant antibody or a fragment thereof in a host cell, and for expressing the recombinant antibody or a fragment thereof. In the present invention, there are no particular limitations on the host cell into which the vector is introduced, and for example, E. coli and various animal cells can be used.

　ベクターは、例えば、大腸菌（例えば、JM109、DH5α、HB101、XL1Blue）を宿主とする場合には、ベクターを増幅させ大量調製するために、大腸菌で増幅されるための「ori」をもち、さらに形質転換された大腸菌の選抜遺伝子（例えば、なんらかの薬剤（アンピシリンやテトラサイクリン、カナマイシン、クロラムフェニコール）により判別できるような薬剤耐性遺伝子）を有することが好ましい。公知のベクターの例としては、M13系ベクター、pUC系ベクター、pBR322、pBluescript、pCR-Scriptなどが挙げられる。また、cDNAのサブクローニング、切り出しを目的とした場合、上記ベクターの他に、例えば、pGEM-T、pDIRECT、pT7などが挙げられる。これらから派生した多くのベクターが流通しており、本発明でも使用可能である。 When E. coli (e.g., JM109, DH5α, HB101, XL1Blue) is used as the host, the vector preferably has an "ori" for amplification in E. coli in order to amplify the vector and prepare it in large quantities, and further has a selection gene for the transformed E. coli (e.g., a drug resistance gene that can be detected by a drug (ampicillin, tetracycline, kanamycin, chloramphenicol)). Examples of well-known vectors include M13 vectors, pUC vectors, pBR322, pBluescript, and pCR-Script. In addition to the above vectors, examples of vectors for the purpose of subcloning and excision of cDNA include pGEM-T, pDIRECT, and pT7. Many vectors derived from these are commercially available and can be used in the present invention.

　本発明の一態様において、ベクターは、動物細胞での抗体又はその断片の発現を目的とした発現ベクターである。発現ベクターとして、プラスミドベクター、或いはウイルスベクター、コスミドベクター、細菌人工染色体（ＢＡＣ）、酵母人工染色体（ＹＡＣ）等の非プラスミドベクターが使用できる。好ましい態様において、本発明の発現ベクターは、動物細胞での発現を目的としたプラスミドベクターである。本発明において、発現ベクターは、目的とする抗体のL鎖又はその断片をコードするDNAを4コピー、及びH鎖又はその断片をコードするDNAを2コピー含むように構築され、目的とする抗体分子または抗体断片の産生能が高い細胞株を樹立するために用いられる。 In one embodiment of the present invention, the vector is an expression vector intended for the expression of an antibody or a fragment thereof in an animal cell. As the expression vector, a plasmid vector or a non-plasmid vector such as a virus vector, a cosmid vector, a bacterial artificial chromosome (BAC), or a yeast artificial chromosome (YAC) can be used. In a preferred embodiment, the expression vector of the present invention is a plasmid vector intended for the expression in an animal cell. In the present invention, the expression vector is constructed so as to contain four copies of DNA encoding the L chain or a fragment thereof of the target antibody, and two copies of DNA encoding the H chain or a fragment thereof, and is used to establish a cell line having a high ability to produce the target antibody molecule or antibody fragment.

　哺乳動物細胞で機能する様々な遺伝子発現用ベクターが流通しており、目的に応じて必要なものを選ぶことができる。或いは、公知の遺伝子発現用ベクターの構成を適宜改変して用いることもできる。目的とする抗体分子が宿主細胞で効率よく発現するように設計した自作のベクターを用いてもよい。発現ベクターの基本的な構成要素として、下流の遺伝子を発現させるためのプロモーター／エンハンサー、目的遺伝子のタンパク質コード領域（ORF）、上流のORFの転写終結のためのポリA付加シグナル、薬剤選択や視覚化などのためのマーカー遺伝子、複製起点（大腸菌での複製起点 pUC ori、哺乳動物細胞での複製起点 SV40 oriなど）などが挙げられる。さらに、翻訳効率を向上させるため、ORF内開始コドンの前にKozak配列を配置してもよい。 Various gene expression vectors that function in mammalian cells are available, and the necessary one can be selected according to the purpose. Alternatively, the structure of a known gene expression vector can be modified as appropriate. A homemade vector designed to efficiently express the target antibody molecule in host cells may also be used. The basic components of an expression vector include a promoter/enhancer for expressing a downstream gene, the protein coding region (ORF) of the target gene, a polyA addition signal for terminating transcription of the upstream ORF, a marker gene for drug selection or visualization, and a replication origin (e.g., pUC ori, the replication origin in E. coli, and SV40 ori, the replication origin in mammalian cells). In addition, a Kozak sequence may be placed before the start codon in the ORF to improve translation efficiency.

　遺伝子発現ベクターの構築では、例えば、安定高発現細胞株を樹立するために、対象細胞内で効率よく機能する強力なプロモーター/エンハンサーを使用することが重要である。プロモーター/エンハンサーの下流に発現させたい遺伝子を挿入した発現ベクターを構築して使用する。代表的なものとして、例えばSV40プロモーター、MMLV-LTRプロモーター、EF-1αプロモーター、CMVプロモーター、マウスβグロビンプロモーター(mBGP)、SRαプロモーターなどが挙げられる。 When constructing a gene expression vector, for example to establish a stable, highly expressing cell line, it is important to use a strong promoter/enhancer that functions efficiently in the target cells. An expression vector is constructed and used in which the gene to be expressed is inserted downstream of the promoter/enhancer. Representative examples include the SV40 promoter, MMLV-LTR promoter, EF-1α promoter, CMV promoter, mouse β-globin promoter (mBGP), and SRα promoter.

　CAGプロモーターは、ニワトリ由来の改変β－アクチンプロモーターにCMVエンハンサーを結合したハイブリッドプロモーターとして構成される（Niwaら、Gene. (1991) 108, 193）。CAGプロモーターは細胞種に依存しない強力なプロモーターであり、CAGプロモーターによって目的遺伝子を転写する発現ベクターは、CMVプロモーターを基盤とする発現ベクターに比べて、目的タンパク質の高発現が期待できる。CAGプロモーターを改良したプロモーターも公知である（特許第5670330号（WO2010/015079））。別の改変CAGプロモーターであるeCAPEプロモーター（配列番号１３）は、抗体ポリペプチド鎖の発現のために本発明で利用可能な好ましいプロモーターの一例である。 The CAG promoter is constructed as a hybrid promoter by combining a CMV enhancer with a modified chicken-derived β-actin promoter (Niwa et al., Gene. (1991) 108, 193). The CAG promoter is a strong promoter independent of cell type, and an expression vector that transcribes a target gene using the CAG promoter is expected to express a higher target protein than an expression vector based on the CMV promoter. Promoters that are improved versions of the CAG promoter are also known (Patent No. 5670330 (WO2010/015079)). Another modified CAG promoter, the eCAPE promoter (SEQ ID NO: 13), is an example of a preferred promoter that can be used in the present invention for expressing antibody polypeptide chains.

　発現ベクターは、細胞への形質転換を選抜するための遺伝子を有することが好ましい。そのような遺伝子として、例えば、薬剤（ネオマイシン、G418、カナマイシン、ハイグロマイシン、アンピシリン、ピューロマイシンなど）により判別できるような薬剤耐性遺伝子や、セルソーティングにより選別できるような蛍光マーカー遺伝子等が挙げられる。 The expression vector preferably has a gene for selecting cells transformed with it. Examples of such genes include drug resistance genes that can be distinguished by drugs (neomycin, G418, kanamycin, hygromycin, ampicillin, puromycin, etc.) and fluorescent marker genes that can be selected by cell sorting.

　宿主細胞中で遺伝子コピー数を増幅させる、遺伝子増幅過程を経て高発現細胞を取得する場合、発現ベクターは選択マーカーとして、アミノグリコシドトランスフェラーゼ（APH）遺伝子、チミジンキナーゼ（TK）遺伝子、大腸菌キサンチングアニンホスホリボシルトランスフェラーゼ（Ecogpt）遺伝子、ジヒドロ葉酸還元酵素（dhfr）遺伝子等を含むことができる。 When obtaining highly expressing cells through a gene amplification process that amplifies the gene copy number in the host cell, the expression vector can contain the aminoglycoside transferase (APH) gene, the thymidine kinase (TK) gene, the Escherichia coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, the dihydrofolate reductase (dhfr) gene, etc. as a selection marker.

　polyAを持つmRNAは細胞内で安定する事が知られており、発現ベクターは、polyAを遺伝子に付加させるために必要なポリAシグナル、例えばマウスβグロビンpolyAシグナル、ウシ成長ホルモンpolyAシグナル(bGHpA)、SV40 polyAシグナルなどを有することが好ましい。 　It is known that mRNA with polyA is stable within cells, and it is preferable that the expression vector has a polyA signal required to add polyA to a gene, such as the mouse β-globin polyA signal, the bovine growth hormone polyA signal (bGHpA), or the SV40 polyA signal.

　本発明において、所望の抗体又はその断片を産生するために使用する発現ベクターの例として、L鎖発現ユニットを4コピーとH鎖発現ユニットを2コピー保持するプラスミドベクターが挙げられる。発現ユニットとは、プロモーターから遺伝子コード配列、ターミネータ配列（ポリAシグナル）までの遺伝子発現の単位である。典型的には、L鎖発現ユニットは、抗体L鎖遺伝子の上流にプロモーターを結合し、下流にpolyAシグナル配列を結合することにより作製される。同様に、H鎖発現ユニットは、抗体H鎖遺伝子の上流にプロモーターを結合し、下流にpolyAシグナル配列を結合することにより作製される。 In the present invention, an example of an expression vector used to produce a desired antibody or a fragment thereof is a plasmid vector carrying four copies of an L-chain expression unit and two copies of an H-chain expression unit. An expression unit is a unit of gene expression from a promoter to a gene coding sequence and a terminator sequence (polyA signal). Typically, an L-chain expression unit is produced by attaching a promoter upstream of an antibody L-chain gene and a polyA signal sequence downstream. Similarly, an H-chain expression unit is produced by attaching a promoter upstream of an antibody H-chain gene and a polyA signal sequence downstream.

　本発明で抗体又はその断片の製造や発現に用いる細胞は、動物細胞が好ましい。動物細胞としては、哺乳類細胞、例えば、ＣＨＯ細胞（J. Exp. Med. (1995) 108, 945）、ＣＯＳ細胞、３Ｔ３細胞、ミエローマ細胞、ＢＨＫ（baby hamster kidney）細胞、ＨｅＬａ細胞、Ｖｅｒｏ細胞、両生類細胞、例えばアフリカツメガエル卵母細胞（Valle, et al., Nature (1981) 291, 358-340）、あるいは昆虫細胞、例えば、Sf9 、Sf21、Tn5が知られている。 The cells used in the present invention for producing or expressing an antibody or a fragment thereof are preferably animal cells. Known animal cells include mammalian cells such as CHO cells (J. Exp. Med. (1995) 108, 945), COS cells, 3T3 cells, myeloma cells, BHK (baby hamster kidney) cells, HeLa cells, Vero cells, amphibian cells such as Xenopus oocytes (Valle, et al., Nature (1981) 291, 358-340), and insect cells such as Sf9, Sf21, and Tn5.

　動物細胞において、大量発現を目的とする場合には特にＣＨＯ細胞が好ましい。ＣＨＯ細胞としては、特に、ジヒドロ葉酸還元（DHFR）遺伝子を欠損したＣＨＯ細胞であるdhfr-CHO（Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220）やCHO K-1 （Proc. Natl. Acad. Sci. USA (1968) 60, 1275）を好適に使用することができる。細胞株であるdhfr欠失ＣＨＯ細胞として、DXB11株とDG44株の二種類が広く流通している。 In animal cells, CHO cells are particularly preferred when the aim is mass expression. As CHO cells, dhfr-CHO (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) and CHO K-1 (Proc. Natl. Acad. Sci. USA (1968) 60, 1275), which are CHO cells lacking the dihydrofolate reductase (DHFR) gene, are particularly suitable. Two types of dhfr-deficient CHO cell lines, the DXB11 and DG44 lines, are widely available.

　宿主細胞へのベクターの導入は、例えば、リン酸カルシウム法、DEAEデキストラン法、カチオニックリボソームDOTAP（ベーリンガーマンハイム社製）を用いた方法、エレクトロポレーション法、リポフェクションなどの周知の方法で行うことが可能である。　　　　　　 　Vectors can be introduced into host cells by well-known methods such as the calcium phosphate method, the DEAE dextran method, the cationic ribosome DOTAP (Boehringer Mannheim) method, electroporation, and lipofection.

　本発明の細胞は、一過性発現系(Transient Expression)で抗体又はその断片が発現されていてもよいし、恒常的発現系(Stable Expression)で発現されていてもよいが、恒常的発現系で発現されているものが好ましい。 The cells of the present invention may express an antibody or a fragment thereof in a transient expression system (Transient Expression) or in a stable expression system (Stable Expression), with the latter being preferred.

　一過性発現系とは、環状プラスミドをリン酸カルシウム法、エレクトロポレーション法、リポフェクション法などにより細胞内に取り込ませ発現させる方法である。環状プラスミドは染色体に挿入される効率が低く、目的遺伝子が染色体外に存在する事が多い。このため環状プラスミドからの目的遺伝子の発現は長期間維持することが難しい。 Transient expression systems are a method in which a circular plasmid is introduced into cells and expressed using techniques such as the calcium phosphate method, electroporation, or lipofection. Circular plasmids are not efficiently inserted into chromosomes, and the target gene often exists outside the chromosome. For this reason, it is difficult to maintain the expression of the target gene from a circular plasmid for a long period of time.

　恒常的発現系とは、制限酵素処理などにより作成した直鎖上プラスミドをリン酸カルシウム法、エレクトロポレーション法、リポフェクション法などにより細胞内に取り込ませ、染色体へプラスミドが挿入されることにより遺伝子を発現させる方法である。このため目的遺伝子の発現を長期間維持することが可能である。直鎖状プラスミドは環状プラスミドより染色体に挿入される効率が高く、目的遺伝子が染色体上に維持される効率も高くなる。またプラスミドへの薬剤耐性遺伝子の導入を行えば薬剤選抜が可能となり、目的遺伝子が染色体上に維持された細胞を効率的に選択することが出来るようになる。恒常的発現系で用いる動物細胞としては、CHO細胞、NS0細胞、SP2/0細胞などが挙げられ、CHO細胞が好ましい。 The constitutive expression system is a method in which a linear plasmid created by restriction enzyme treatment is introduced into cells using the calcium phosphate method, electroporation, lipofection, etc., and the plasmid is inserted into the chromosome, thereby expressing the gene. This makes it possible to maintain the expression of the target gene for a long period of time. Linear plasmids are more efficiently inserted into chromosomes than circular plasmids, and the efficiency of maintaining the target gene on the chromosome is also higher. Furthermore, drug selection becomes possible if a drug resistance gene is introduced into the plasmid, and cells in which the target gene is maintained on the chromosome can be efficiently selected. Examples of animal cells used in the constitutive expression system include CHO cells, NS0 cells, and SP2/0 cells, with CHO cells being preferred.

　一態様において、本発明は特定の抗体の製造を目的とする。目的の抗体として、以下の抗体が例示できる。
　抗CD137抗体は、例えば、WO2020/032230に記載される抗ヒトCD137抗体であり、さらに具体的には「STA551」と呼ばれる抗CD137アゴニスト抗体であり得る。STA551は、アデノシン 3 リン酸（ATP）依存的にCD137に結合してアゴニスト活性を発揮するスイッチ抗体である。 In one aspect, the present invention aims to produce a specific antibody. Examples of the antibody of interest include the following antibodies:
The anti-CD137 antibody may be, for example, an anti-human CD137 antibody described in WO2020/032230, and more specifically, an anti-CD137 agonist antibody called "STA551." STA551 is a switch antibody that binds to CD137 in an adenosine triphosphate (ATP)-dependent manner and exerts agonist activity.

　抗潜在型TGF-β1抗体は、例えば、WO2021/039945に記載される種交差性抗潜在型TGF-β1抗体であり、さらに具体的には「SOF10」又は「RG6440」と呼ばれるモノクローナル抗体であり得る。 The anti-latent TGF-β1 antibody may be, for example, a species-cross-reactive anti-latent TGF-β1 antibody described in WO2021/039945, and more specifically, a monoclonal antibody called "SOF10" or "RG6440."

　抗潜在型ミオスタチン抗体は、スイーピング抗体、例えば、「GYM329」または「RG6237」と呼ばれる抗体であり得る。
　抗IL-8抗体は、例えば、「AMY109」と呼ばれるリサイクリング抗体であり得る。 The anti-latent myostatin antibody may be a sweeping antibody, such as the antibody called "GYM329" or "RG6237."
The anti-IL-8 antibody can be, for example, the recycling antibody called "AMY109."

　好ましい一態様において、本発明では、抗体STA551の発現を目的として構築されたベクターを使用することができる。ベクターの具体例は、pSTA551-eCAPE-bGHpA-LLLLHH（図１）であり、抗体産生のための宿主細胞であるdhfr遺伝子欠失ＣＨＯ細胞に導入される。 In a preferred embodiment, the present invention can use a vector constructed for the purpose of expressing the antibody STA551. A specific example of the vector is pSTA551-eCAPE-bGHpA-LLLLHH (Figure 1), which is introduced into dhfr gene-deficient CHO cells, which are host cells for antibody production.

　pSTA551-eCAPE-bGHpA-LLLLHH は、抗体STA551のL鎖発現ユニットを4コピーとH鎖発現ユニットを2コピー含む。各発現ユニットにおいてはSTA551 H鎖（配列番号１１）またはL鎖（配列番号１２）の上流にeCAPEプロモーターが配置され、STA551 H鎖またはL鎖の下流にはポリAシグナルとしてbGHpAが配置されている。また本プラスミドにはDHFR遺伝子とそれに続くSV40 polyA (SV40pA)が含まれる。DHRFは本プラスミドが細胞の染色体上に導入された際に染色体内のプロモーターにより発現するように設計されており、形質転換体の選抜に寄与する。また、本プラスミドにはSV40プロモーターが含まれる。このプロモーターは細胞の染色体上に導入された際に事前に染色体上に設置されたハイグロマイシンB耐性遺伝子を発現させ、形質転換体の選抜に寄与する。また本プラスミドにはpUC oriとカナマイシン耐性遺伝子（kan R）が含まれる。pUC oriはプラスミドの調製時の大腸菌での複製開始点として機能する。カナマイシン耐性遺伝子はプラスミドを保持する大腸菌の選抜に寄与する。 pSTA551-eCAPE-bGHpA-LLLLHH contains four copies of the STA551 antibody light chain expression unit and two copies of the heavy chain expression unit. In each expression unit, the eCAPE promoter is located upstream of the STA551 heavy chain (sequence number 11) or light chain (sequence number 12), and bGHpA is located downstream of the STA551 heavy chain or light chain as a polyA signal. This plasmid also contains the DHFR gene followed by SV40 polyA (SV40pA). DHRF is designed to be expressed by a promoter in the chromosome when this plasmid is introduced into the chromosome of a cell, and contributes to the selection of transformants. This plasmid also contains the SV40 promoter. When this promoter is introduced into the chromosome of a cell, it expresses the hygromycin B resistance gene previously placed on the chromosome, and contributes to the selection of transformants. This plasmid also contains pUC ori and kanamycin resistance gene (kan R). pUC ori functions as the origin of replication in E. coli when the plasmid is prepared. The kanamycin resistance gene contributes to the selection of E. coli that harbor the plasmid.

　一態様において、本発明は、抗体のL鎖発現ユニットを4コピーとH鎖発現ユニットを2コピー含むベクターを導入したCHO細胞であり、各発現ユニットにおいてH鎖またはL鎖の上流にCAGプロモーターまたはeCAPEプロモーターが配置されており、抗体はSTA551、SOF10、RAY121、AMY109、GYM329、S12preのいずれかであることが好適であり、STA551、SOF10、またはRAY121であることも好適であり、STA551であることがさらに好適である。 In one aspect, the present invention relates to a CHO cell into which a vector containing four copies of an antibody L chain expression unit and two copies of an antibody H chain expression unit has a CAG promoter or an eCAPE promoter located upstream of the H chain or L chain in each expression unit, and the antibody is preferably any one of STA551, SOF10, RAY121, AMY109, GYM329, and S12pre, and is also preferably STA551, SOF10, or RAY121, and more preferably STA551.

　細胞を、目的とする遺伝子により形質転換し、形質転換された細胞を培養することにより、目的とする遺伝子がコードするポリペプチドが得られる。本発明では、抗体のL鎖又はその断片をコードするDNAを4コピー、及び当該抗体のH鎖又はその断片をコードするDNAを2コピー含むベクターを導入した細胞をin vitroで培養することにより、当該抗体又はその断片を産生させる。 　A polypeptide encoded by a gene of interest can be obtained by transforming a cell with the gene of interest and culturing the transformed cell. In the present invention, the antibody or a fragment thereof is produced by culturing in vitro cells into which a vector containing four copies of DNA encoding an antibody light chain or a fragment thereof and two copies of DNA encoding the antibody heavy chain or a fragment thereof has been introduced.

　本発明において、培養は、公知の方法に従い行うことができる。細胞の培養には、通常の細胞（好ましくは、動物細胞）培養で使用されている培地を用いることができ、動物細胞培養用培地であれば特に限定されない。これらには通常、アミノ酸、ビタミン類、脂質因子、エネルギー源、浸透圧調節剤、鉄源、pH緩衝剤を含む。さらに、例えば、微量金属元素、界面活性剤、増殖補助因子、ヌクレオシドなどを添加しても良い。 In the present invention, the culture can be carried out according to a known method. For cell culture, a medium used in normal cell (preferably animal cell) culture can be used, and there is no particular limitation as long as it is a medium for animal cell culture. These usually contain amino acids, vitamins, lipid factors, energy sources, osmotic regulators, iron sources, and pH buffers. In addition, for example, trace metal elements, surfactants, growth cofactors, nucleosides, etc. may be added.

　例えば、D-MEM (Dulbecco's Modified Eagle Medium)、 D-MEM/F-12 1:1 Mixture (Dulbecco's Modified Eagle Medium : Nutrient Mixture F-12)、 MEM、RPMI1640、IMDM、CHO-S-SFM II(Invitrogen社)、 CHO-SF (Sigma-Aldrich社)、 EX-CELL 301 (JRH biosciences社)、CD-CHO  (Invitrogen社)、 IS CHO-V (Irvine Scientific社)、 PF-ACF-CHO (Sigma-Aldrich社)、Basal10（味の素社）などの市販の細胞培養用培地を用いることも可能である。その際、牛胎児血清（FCS）等の血清補液を併用することもできる。あるいは、培地は無血清培地であってもよい。タンパク質の精製を考慮する場合、細胞株を無血清培地で培養するのが望ましい。 For example, commercially available cell culture media such as D-MEM (Dulbecco's Modified Eagle Medium), D-MEM/F-12 1:1 Mixture (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), MEM, RPMI1640, IMDM, CHO-S-SFM II (Invitrogen), CHO-SF (Sigma-Aldrich), EX-CELL 301 (JRH biosciences), CD-CHO (Invitrogen), IS CHO-V (Irvine Scientific), PF-ACF-CHO (Sigma-Aldrich), and Basal10 (Ajinomoto) can be used. In this case, serum supplements such as fetal calf serum (FCS) can also be used in combination. Alternatively, the medium may be serum-free. When considering protein purification, it is preferable to culture the cell line in serum-free medium.

　宿主細胞がdhfr遺伝子を欠失したＣＨＯ細胞である場合、培地中にヒポキサンチンとチミジンを添加する。宿主細胞がDXB11又はDG44株由来のプロリン栄養要求性ＣＨＯ細胞株である場合、培地中にプロリンを添加した方がよい可能性がある。 If the host cells are CHO cells lacking the dhfr gene, add hypoxanthine and thymidine to the medium. If the host cells are proline auxotrophic CHO cell lines derived from DXB11 or DG44 strains, it may be advisable to add proline to the medium.

　細胞がCHO細胞である場合、CHO細胞の培養は当業者に公知の方法を用いて行うことができる。例えば、通常、気相のCO2濃度が0－40%、好ましくは、2－10%の雰囲気下、30－39℃、好ましくは37℃程度で、培養することが可能である。 When the cells are CHO cells, the CHO cells can be cultured using methods known to those skilled in the art. For example, they can usually be cultured in an atmosphere with a CO2 concentration in the gas phase of 0-40%, preferably 2-10%, at 30-39°C, preferably around 37°C.

　所望の組換え抗体又はその断片を産生するために適当な細胞の培養期間は、通常１日～３ヶ月であり、好ましくは1日～2ヶ月、さらに好ましくは1日～1ヶ月である。 The culture period for cells suitable for producing the desired recombinant antibody or fragment thereof is usually 1 day to 3 months, preferably 1 day to 2 months, and more preferably 1 day to 1 month.

　また、動物細胞培養用の各種の培養装置としては、例えば発酵槽型タンク培養装置、エアーリフト型培養装置、カルチャーフラスコ型培養装置、スピンナーフラスコ型培養装置、マイクロキャリアー型培養装置、流動層型培養装置、ホロファイバー型培養装置、ローラーボトル型培養装置、充填槽型培養装置等を用いて培養することができる。 Various types of culture equipment for animal cell culture include, for example, fermenter-type tank culture equipment, air lift-type culture equipment, culture flask-type culture equipment, spinner flask-type culture equipment, microcarrier-type culture equipment, fluidized bed-type culture equipment, hollow fiber-type culture equipment, roller bottle-type culture equipment, and packed tank-type culture equipment.

　培養は、バッチ培養(batch culture)、流加培養(fed-batch culture)、連続培養(continuous culture)などのいずれの方法を用いてもよいが、流加培養又は連続培養が好ましく、流加培養がより好ましい。 Cultivation may be performed using any method, such as batch culture, fed-batch culture, or continuous culture, but fed-batch culture or continuous culture is preferred, with fed-batch culture being more preferred.

　組換え抗体又はその断片を産生するための細胞培養工程により得られた培養物から、抗体又はその断片を含む組成物を回収することができる。ここでいう「回収する」とは、細胞培養工程により得られた培養物ないし培養液から、抗体またはその断片を含む上清液（培養上清液）を回収することまたはフィルターを使用し抗体又はその断片を含む濾液を回収することをいう。このようにして回収した溶液は、後述のアフィニティカラムクロマトグラフィー等による精製、および濃縮の工程を経て、適当な濃度の抗体またはその断片を含む組成物に調整することができる。 A composition containing an antibody or a fragment thereof can be recovered from the culture obtained by the cell culture process for producing a recombinant antibody or a fragment thereof. "Recovering" here means recovering the supernatant (culture supernatant) containing the antibody or a fragment thereof from the culture or culture solution obtained by the cell culture process, or recovering the filtrate containing the antibody or a fragment thereof using a filter. The solution recovered in this way can be purified by affinity column chromatography or the like, as described below, and subjected to a concentration process to prepare a composition containing an antibody or a fragment thereof at an appropriate concentration.

　本発明の製造方法により得られた抗体又はその断片を、均一にまで精製することができる。抗体又はその断片の分離、精製は通常のポリペプチドで使用されている分離、精製方法を使用すればよい。例えば、アフィニティークロマトグラフィー等のクロマトグラフィーカラム、フィルター、限外濾過、塩析、透析、SDSポリアクリルアミドゲル電気泳動、等電点電気泳動等を適宜選択、組み合わせれば、抗体を分離、精製することができる(Antibodies : A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988) が、これらに限定されるものではない。上記で得られた抗体の濃度測定は吸光度の測定又は酵素結合免疫吸着検定法(Enzyme-linked immunosorbent assay；ELISA）等により行うことができる。 The antibodies or fragments thereof obtained by the production method of the present invention can be purified to homogeneity. Separation and purification of antibodies or fragments thereof can be performed using the separation and purification methods used for ordinary polypeptides. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but are not limited to these. The concentration of the antibodies obtained as above can be measured by measuring absorbance or enzyme-linked immunosorbent assay (ELISA), etc.

　アフィニティークロマトグラフィーに用いるカラムとしては、プロテインＡカラム、プロテインＧカラムが挙げられる。例えば、プロテインＡカラムを用いたカラムとして、Hyper D, POROS, Sepharose F. F. (Pharmacia) 等が挙げられる。 Columns used for affinity chromatography include Protein A columns and Protein G columns. For example, columns using Protein A columns include Hyper D, POROS, and Sepharose F. F. (Pharmacia).

　アフィニティークロマトグラフィー以外のクロマトグラフィーとしては、例えば、イオン交換クロマトグラフィー、疎水性クロマトグラフィー、ゲル濾過、逆相クロマトグラフィー、吸着クロマトグラフィー等が挙げられる(Strategies for Protein Purification and Characterization : A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996)。これらのクロマトグラフィーはHPLC、FPLC等の液相クロマトグラフィーを用いて行うことができる。 Other types of chromatography besides affinity chromatography include, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These types of chromatography can be performed using liquid phase chromatography such as HPLC and FPLC.

　なお、ポリペプチドを精製前又は精製後に適当なポリペプチド修飾酵素を作用させることにより、任意に修飾を加えたり、部分的にペプチドを除去したりすることもできる。ポリペプチド修飾酵素としては、例えば、トリプシン、キモトリプシン、リシルエンドペプチダーゼ、プロテインキナーゼ、グルコシダーゼなどが用いられる。また、得られた抗体に化学的な修飾を施すことによって、例えば、ポリエチレングリコール（PEG）等の各種分子と結合させて、抗体修飾物とすることもできる。 In addition, before or after purification, the polypeptide can be treated with an appropriate polypeptide-modifying enzyme to optionally modify it or partially remove peptides. Examples of polypeptide-modifying enzymes that can be used include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, and glucosidase. In addition, the obtained antibody can be chemically modified to bind with various molecules such as polyethylene glycol (PEG) to produce a modified antibody.

　本発明の方法により製造された抗体又はその断片が医薬として利用可能な生物学的活性を有する場合には、このポリペプチドを医薬的に許容される担体又は添加剤と混合して製剤化することにより、医薬品を製造することができる。 If the antibody or fragment thereof produced by the method of the present invention has biological activity that can be used as a medicine, the polypeptide can be mixed with a pharma- ceutical carrier or additive to produce a formulation, thereby producing a pharmaceutical product.

　医薬的に許容される担体及び添加剤の例として、水、医薬的に許容される有機溶剤、コラーゲン、ポリビニルアルコール、ポリビニルピロリドン、カルボキシビニルポリマー、カルボキシメチルセルロースナトリウム、ポリアクリル酸ナトリウム、アルギン酸ナトリウム、水溶性デキストラン、カルボキシメチルスターチナトリウム、ペクチン、メチルセルロース、エチルセルロース、キサンタンガム、アラビアゴム、カゼイン、寒天、ポリエチレングリコール、ジグリセリン、グリセリン、プロピレングリコール、ワセリン、パラフィン、ステアリルアルコール、ステアリン酸、ヒト血清アルブミン（ＨＳＡ）、マンニトール、ソルビトール、ラクトース、医薬添加物として許容される界面活性剤等が挙げられる。 Examples of pharma- ceutically acceptable carriers and additives include water, pharma-ceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethylstarch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, and the like.

　実際の添加物は、本発明治療剤の剤型に応じて上記の中から単独で又は適宜組み合わせて選ばれるが、もちろんこれらに限定するものではない。例えば、注射用製剤として使用する場合、精製されたポリペプチドを溶剤、例えば生理食塩水、緩衝液、ブドウ糖溶液等に溶解し、これに吸着防止剤、例えばＴｗｅｅｎ８０、Ｔｗｅｅｎ２０、ゼラチン、ヒト血清アルブミン等を加えたものを使用することができる。あるいは、使用前に溶解再構成する剤形とするために凍結乾燥したものであってもよく、凍結乾燥のための賦形剤としては、例えば、マンニトール、ブドウ糖等の糖アルコールや糖類を使用することができる。 Actual additives are selected from the above, either alone or in appropriate combination, depending on the dosage form of the therapeutic agent of the present invention, but are of course not limited to these. For example, when used as an injectable formulation, the purified polypeptide can be dissolved in a solvent, such as physiological saline, buffer solution, glucose solution, etc., to which an adsorption inhibitor, such as Tween 80, Tween 20, gelatin, human serum albumin, etc., can be added. Alternatively, the polypeptide may be lyophilized to form a dosage form that can be dissolved and reconstituted before use, and sugar alcohols and sugars, such as mannitol and glucose, can be used as excipients for lyophilization.

　抗体又はその断片の有効投与量は、抗体又はその断片の種類、治療や予防の対象とする疾患の種類、患者の年齢、疾患の重篤度などにより適宜選択される。例えば、抗体が抗グリピカン-3抗体であり、これを抗癌剤として使用する場合、抗グリピカン-3抗体の有効投与量は、一回につき体重１ｋｇあたり０．００１ｍｇから１０００ｍｇの範囲で選ばれる。あるいは、患者あたり０．０１～１０００００ｍｇ／ｂｏｄｙの投与量を選ぶことができる。しかしながら、これらの投与量に制限されるものではない。 The effective dose of the antibody or a fragment thereof is appropriately selected depending on the type of antibody or fragment thereof, the type of disease to be treated or prevented, the age of the patient, the severity of the disease, etc. For example, when the antibody is an anti-glypican-3 antibody and is used as an anti-cancer drug, the effective dose of the anti-glypican-3 antibody is selected in the range of 0.001 mg to 1000 mg per kg of body weight per administration. Alternatively, a dose of 0.01 to 100,000 mg/body per patient can be selected. However, it is not limited to these doses.

　抗体又はその断片の投与方法は、経口、非経口投与のいずれでも可能であるが、好ましくは非経口投与であり、具体的には、注射（例えば、静脈内注射、筋肉内注射、腹腔内注射、皮下注射などによる全身又は局所投与）、経鼻投与、経肺投与、経皮投与などが挙げられる。 The antibody or fragment thereof can be administered orally or parenterally, but is preferably administered parenterally. Specific examples include injection (for example, systemic or local administration by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.), intranasal administration, pulmonary administration, and transdermal administration.

　以下、本発明を実施例によって具体的に説明する。なお、これらの実施例は、本発明を説明するためのものであって、本発明の範囲を限定するものではない。
　実施例１および２では、ヒト化抗CD137抗体「STA551」のH鎖遺伝子（配列番号１１）及びL鎖遺伝子（配列番号１２）を用いてプラスミドを作製した。 The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of explaining the present invention and are not intended to limit the scope of the present invention.
In Examples 1 and 2, plasmids were prepared using the H chain gene (SEQ ID NO: 11) and L chain gene (SEQ ID NO: 12) of the humanized anti-CD137 antibody "STA551."

実施例1：H2L4プラスミド、H3L6プラスミド、H4L8プラスミドを用いたヒト化抗CD137抗体(STA551)の生産
　ヒト化抗CD137抗体H鎖遺伝子及びヒト化抗CD137抗体L鎖遺伝子のそれぞれ上流にeCAPEプロモーター（配列番号１３）を結合し、更に下流に bovine growth hormone (bgh) polyAシグナル配列を結合したH鎖発現ユニットとL鎖発現ユニットを作成した。ハイグロマイシン耐性遺伝子とDHFR遺伝子を組み込んだCHO細胞導入用ベクターにH鎖発現ユニットとL鎖発現ユニットを結合し、ヒト化抗CD137抗体遺伝子のH鎖 2 copy及びL鎖 4 copyで構成されるH2L4プラスミド（図１）、H鎖 3 copy及びL鎖 6 copyで構成されるH3L6プラスミド(図２)、H鎖 4 copy及びL鎖 8 copyで構成されるH4L8プラスミド(図３)をそれぞれ作成し、CHO細胞DXB11由来宿主細胞にエレクトロポレーションにより導入した。引き続き細胞を400 mg/mLのハイグロマイシンB存在下で培養することで、発現プラスミドが導入された細胞株を取得し、抗体産生量による選抜を行った。 Example 1: Production of humanized anti-CD137 antibody (STA551) using H2L4 plasmid, H3L6 plasmid, and H4L8 plasmid An H-chain expression unit and an L-chain expression unit were prepared by linking the eCAPE promoter (SEQ ID NO: 13) upstream of the humanized anti-CD137 antibody H-chain gene and the humanized anti-CD137 antibody L-chain gene, respectively, and further linking the bovine growth hormone (bgh) polyA signal sequence downstream. The H-chain expression unit and the L-chain expression unit were linked to a vector for CHO cell introduction incorporating a hygromycin resistance gene and a DHFR gene, and the H2L4 plasmid (Figure 1) consisting of 2 copies of the H-chain and 4 copies of the L-chain, the H3L6 plasmid (Figure 2) consisting of 3 copies of the H-chain and 6 copies of the L-chain, and the H4L8 plasmid (Figure 3) consisting of 4 copies of the H-chain and 8 copies of the L-chain were prepared, respectively, and introduced into CHO cell DXB11-derived host cells by electroporation. The cells were then cultured in the presence of 400 mg/mL hygromycin B to obtain cell lines into which the expression plasmid had been introduced, and selection was performed based on the amount of antibody production.

　選抜されたH2L4プラスミド導入クローン細胞株6株、H3L6プラスミド導入クローン細胞株９株、H4L8プラスミド導入クローン細胞株６株について、細胞培養装置ambr15（Sartorius社）を用いたフェドバッチ培養により抗体産生量の比較を行った。培養には培地としてBasal 10（味の素社）を用い、初発培養液量10 mL、培養温度37℃、攪拌速度 800 rpmの条件で実施した。 The antibody production amounts of the six selected clone cell lines introduced with the H2L4 plasmid, the nine clone cell lines introduced with the H3L6 plasmid, and the six clone cell lines introduced with the H4L8 plasmid were compared by fed-batch culture using the ambr15 cell culture device (Sartorius). Culture was performed using Basal 10 (Ajinomoto) as the medium, with an initial culture volume of 10 mL, a culture temperature of 37°C, and an agitation speed of 800 rpm.

　培養開始後14日目に培養液中の抗体濃度を測定した結果、H2L4プラスミド導入クローン細胞株 6株は他のクローンよりも優位に抗体産生量が高かった(図４の左３つ)。 The antibody concentration in the culture medium was measured 14 days after the start of culture, and the six clone cell lines into which the H2L4 plasmid had been introduced showed significantly higher antibody production than the other clones (the three on the left in Figure 4).

実施例２：異なる培地を用いた場合
　H2L4プラスミド導入クローン細胞株13株、H3L6プラスミド導入クローン細胞株6株、H4L8プラスミド導入クローン細胞株5株に対し、実施例１と同様の培養評価を異なる培地STM2.1（Chugai proprietary chemically defined medium）を用いて実施した。その結果、異なる培地を用いてもH2L4プラスミド導入クローン細胞株は高い抗体産生量を示した(図４の右３つ)。 Example 2: When different media were used 13 clone cell lines introduced with H2L4 plasmid, 6 clone cell lines introduced with H3L6 plasmid, and 5 clone cell lines introduced with H4L8 plasmid were subjected to culture evaluation similar to that in Example 1 using a different medium, STM2.1 (Chugai proprietary chemically defined medium). As a result, the clone cell lines introduced with H2L4 plasmid showed high antibody production even when using different media (the three on the right in Figure 4).

　以上より、H鎖 2 copyに対してL鎖 4 copyを宿主細胞に導入することで、それ以外のコピー数よりも抗体産生量を高められることを確認した。 From the above, it was confirmed that introducing two copies of the heavy chain and four copies of the light chain into host cells can increase antibody production more than with other copy numbers.

　本明細書で引用した全ての刊行物、特許及び特許出願をそのまま参考として本明細書にとり入れるものとする。 All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

　配列について
　STA551は、抗体名「A551-SCF057aPh/B379-LamLib」としてWO2020/032230に記載されている。WO2020/032230中で、A551-SCF057aPh/B379-LamLibの構成とアミノ酸配列は以下のとおり記載されている（表５１、表５２等を参照）。ここで、便宜上、各配列はWO2020/032230に記載のSEQ IDで表示している。
[VH] A551 (VH: SEQ 51, HCDR1: SEQ 7, HCDR2: SEQ 15, HCDR3: SEQ 20)
[CH] SCF057aPh (SEQ: 82)
[VL] B379 (VL: SEQ 60, LCDR1: SEQ 25, LCDR2: SEQ 26, LCDR3: SEQ 27)
[CL] Lamlib (SEQ: 63) Regarding the sequence , STA551 is described in WO2020/032230 under the antibody name "A551-SCF057aPh/B379-LamLib". In WO2020/032230, the structure and amino acid sequence of A551-SCF057aPh/B379-LamLib are described as follows (see Tables 51, 52, etc.). Here, for convenience, each sequence is shown by the SEQ ID described in WO2020/032230.
[VH] A551 (VH: SEQ 51, HCDR1: SEQ 7, HCDR2: SEQ 15, HCDR3: SEQ 20)
[CH] SCF057aPh (SEQ: 82)
[VL] B379 (VL: SEQ 60, LCDR1: SEQ 25, LCDR2: SEQ 26, LCDR3: SEQ 27)
[CL] Lamlib (SEQ: 63)

　すなわち、H鎖（A551-SCF057aPh）は可変領域A551と定常領域SCF057aPhが組み合わされたものであり、L鎖（B379-LamLib）は可変領域B379と定常領域LamLibが組み合わされたものである。これらのアミノ酸配列に、本願の配列表ではそれぞれ配列番号１～１０を割り当てる。抗体STA551のH鎖遺伝子及びL鎖遺伝子のヌクレオチド配列に、本願の配列表では配列番号１１と１２を割り当てる。 In other words, the H chain (A551-SCF057aPh) is a combination of the variable region A551 and the constant region SCF057aPh, and the L chain (B379-LamLib) is a combination of the variable region B379 and the constant region LamLib. These amino acid sequences are assigned SEQ ID NOs: 1 to 10, respectively, in the sequence listing of the present application. The nucleotide sequences of the H chain gene and L chain gene of the antibody STA551 are assigned SEQ ID NOs: 11 and 12 in the sequence listing of the present application.

　抗体STA551 （WO2020/032230に記載の「A551-SCF057aPh/B379-LamLib」）の配列（配列番号１～１２）およびeCAPEプロモータの配列（配列番号１３）を以下に示す。
表１：H鎖可変領域のアミノ酸配列
[VH] A551 (VH: SEQ 51, HCDR1: SEQ 7, HCDR2: SEQ 15, HCDR3: SEQ 20 - WO2020/032230に記載の配列番号で表示)

Figure JPOXMLDOC01-appb-T000001
The sequences (SEQ ID NOs: 1 to 12) of the antibody STA551 ("A551-SCF057aPh/B379-LamLib" described in WO2020/032230) and the sequence of the eCAPE promoter (SEQ ID NO: 13) are shown below.
Table 1: Amino acid sequence of the heavy chain variable region
[VH] A551 (VH: SEQ 51, HCDR1: SEQ 7, HCDR2: SEQ 15, HCDR3: SEQ 20 - represented by sequence numbers described in WO2020/032230)
Figure JPOXMLDOC01-appb-T000001

表２：H鎖定常領域のアミノ酸配列
[CH] SCF057aPh (SEQ: 82 -WO2020/032230に記載の配列番号で表示)

Figure JPOXMLDOC01-appb-T000002
Table 2: Amino acid sequence of the H chain constant region
[CH] SCF057aPh (SEQ: 82 -represented by the sequence number given in WO2020/032230)
Figure JPOXMLDOC01-appb-T000002

表３：L鎖可変領域のアミノ酸配列
[VL] B379 (VL: SEQ 60, LCDR1: SEQ 25, LCDR2: SEQ 26, LCDR3: SEQ 27 - WO2020/032230に記載の配列番号で表示)

Figure JPOXMLDOC01-appb-T000003
Table 3: Amino acid sequence of the light chain variable region
[VL] B379 (VL: SEQ 60, LCDR1: SEQ 25, LCDR2: SEQ 26, LCDR3: SEQ 27 - represented by sequence numbers described in WO2020/032230)
Figure JPOXMLDOC01-appb-T000003

表４：L鎖定常領域のアミノ酸配列
[CL] Lamlib (SEQ: 63 -WO2020/032230に記載の配列番号で表示)

Figure JPOXMLDOC01-appb-T000004
Table 4: Amino acid sequence of the light chain constant region
[CL] Lamlib (SEQ: 63 -represented by the sequence number listed in WO2020/032230)
Figure JPOXMLDOC01-appb-T000004

「STA551」のH鎖遺伝子及びL鎖遺伝子のDNA配列
>STA551_H（配列番号１１）
ATGGAGctgGGGCTGcgcTGGGTTTTCCTCGTTGCTATAttagaaGGTGTCCAGTGTgaa
GTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTCAAACCTGGGGGGTCCCTGAGACTCTCC
TGTGCTGCCTCTGGATTCACCTTCTCTacctttACCATGAATTGGGTCCGCCAAGCTCCC
GGCAAGGGGCTGGAATGGGTGTCATCCATTTCatcaaaAgggTCtTATATTgagTATGCT
GAATCCttcaaagtcCGATTCACCATCTCAAGAGACAACGCCAAGAACTCTCTGTATCTG
CAAATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTATTACTGTGCCAGATATGGTatc
aaaaatgagctgaatTGGGTTTTTGACTACTGGGGCCAAGGCACCCTGGTGACAGTGTCC
TCTGCCTCCACAAAaGGCCCATCCGTGTTCCCCCTGGCTCCTTCCTCTAAGTCTACCTCC
GGCGGCACAGCCGCTCTGGGATGTCTGGTGAAGGATTATTTCCCTGAACCCGTGACCGTG
TCTTGGAACTCCGGCGCCCTGACCTCCGGCGTGCACACATTTCCCGCCGTGCTACAGTCC
TCTGGCCTGTACTCTCTGTCCTCTGTGGTGACAGTGCCCTCCTCTTCCCTGGGCACCCAG
ACATATATCTGTAATGTTAATCACAAGCCCTCCAATACAAAAGTGGACAAGAGAGTGGAG
CCTAAGTCTTGTGATAAGACCCATACATGCCCCCCTTGTCCTGCCCCTGAACTGtggaac
GGCCCTTCAGTGTTCCTGTTTCCACCCAAACCAAAGGACaccCTGATGATCTCAAGGACC
CCTGAGGTGACATGCGTGGTGGTGGACGTGTCTgacGAGgacCCCGAAGTGAAGTTTAAT
TGGTACGTGGATGGCGTGGAAGTGCATAATGCCAAGACAAAGCCCAGAGAGGAGCAGTAC
AACtcaACaTATAGGGTGGTGTCTGTGCTGaccGTGCTGCACCgGGACTGGCTGAACGGC
AAGGAGTATAAGTGCAAGGTGTCCAATaaaGCCTTGCCTaagCCCATCGAGaaaACCATC
TCTAAGGCTAAGGGACAGcggCGGGAGCCTCAAGTGTACACACTGCCTCCATCCAGAGAG
GAGATGACCAAGAACCAAGTGTCTCTGACATGTCTGGTCAAGGGCTTCTATCCATCTGAC
ATCGCTGTGGAGTGGGAGTCTAATGGCCAGCCCGAGAACAATTACAAGACCACACCCCCT
GTGCTGGACTCTGATGGCTCCTTCTTTCTGTATTCCAAACTGACCGTGGATAAGTCTAGG
TGGCAGCAGGGCAACGTGTTCTCCTGTTCTGTCATGCACGAGGCACTGCACAACCATTAC
ACCCAGaagTCACTGTCACTGTCACCTTGA
  DNA sequences of the H-chain and L-chain genes of "STA551"
>STA551_H (SEQ ID NO: 11)
ATGGAGctgGGGCTGcgcTGGGTTTTCCTCGTTGCTATAttagaaGGTGTCCAGTGTgaa
GTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTCAAACCTGGGGGGTCCCTGAGACTCTCC
TGTGCTGCCTCTGGATTCACCTTCTCTacctttACCATGAATTGGGTCCGCCAAGCTCCC
GGCAAGGGGCTGGAATGGGTGTCATCCATTTCatcaaaAgggTCtTATATTgagTATGCT
GAATCCttcaaagtcCGATTCACCATCTCAAGAGACAACGCCAAGAACTCTCTGTATCTG
CAAATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTATTACTGTGCCAGATATGGTatc
aaaaatgagctgaatTGGGTTTTTGACTACTGGGGCCAAGGCACCCTGGTGACAGTGTCC
TCTGCCTCCACAAAaGGCCCATCCGTGTTCCCCCTGGCTCCTTCCTCTAAGTCTACCTCC
GGCGGCACAGCCGCTCTGGGATGTCTGGTGAAGGATTATTTCCCTGAACCCGTGACCGTG
TCTTGGAACTCCGGCGCCTGACCTCCGGCGTGCACACATTTCCCGCCGTGCTACAGTCC
TCTGGCCTGTACTCTCTGTCCTCTGTGGTGACAGTGCCCTCCTCTTCCCTGGGCACCCAG
ACATATATCTGTAATGTTAATCACAAGCCCTCCAATACAAAGTGGACAAGAGAGTGGAG
CCTAAGTCTTGTGATAAGACCCATACATGCCCCCCTTGTCCTGCCCCTGAACTGtggaac
GGCCCTTCAGTGTTCCTGTTTCCACCCAAACCAAAGGACaccCTGATGATCTCAAGGACC
CCTGAGGTGACATGCGTGGTGGTGGACGTGTCTgacGAGgacCCCGAAGTGAAGTTTAAT
TGGTACGTGGATGGCGTGGAAGTGCATAATGCCAAGACAAAGCCCAGAGAGGAGCAGTAC
AACtcaACaTATAGGGTGGTGTCTGTGCTGaccGTGCTGCACCgGGACTGGCTGAACGGC
AAGGAGTATAAGTGCAAGGTGTCCAATaaaGCCTTGCCTaagCCCATCGAGaaaACCATC
TCTAAGGCTAAGGGACAGcggCGGGAGCCTCAAGTGTACACACTGCCTCCATCCAGAGAG
GAGATGACCAAGAACCAAGTGTCTCTGACATGTCTGGTCAAGGGCTTCTATCCATCTGAC
ATCGCTGTGGAGTGGGAGTCTAATGGCCAGCCCGAGAACAATTACAAGACCACACCCCCT
GTGCTGGACTCTGATGGCTCCTTCTTTCTGTATTCCAAACTGACCGTGGATAAGTCTAGG
TGGCAGCAGGGCAACGTGTTCTCCTGTTCTGTCATGCACGAGGCACTGCACAACCATTAC
ACCCAGaagTCACTGTCACTGTCACCTTGA
 

>STA551_L（配列番号１２）
ATGGCCTGGtcCcctCTGCTCCTCACCCTCCTCgctcactgcACAGGGTCCTGGGCCCAA
TCCGCACTGACTCAACCTCCATCCGCCTCTGGTTCCCCTGGACAGACAGTTACCATCTCC
TGTacTGGCaccTCAACTGATGTGGGTtAtTACgaATATGTATCCTGGTATCAACAGCAC
CCTGGAAAGGCTCCTAAACTGATGATTTATgagACTTCCAAAcgtctgTCTGGTGTCCCT
GATCGTTTTTCCGGCTCCAAATCCGGCAACACTGCCTCCCTGACTGTTTCCGGTTTGCAA
GCTGAGGACGAGGCAGACTATtacTGCTCATCcTATcgttatgaAcatcaggttTCcTTC
GGCGGAGGGACCAAACTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCTGTCACTCTG
TTCCCACCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTCGTATGTCTCATATCT
GACTTCTACCCTGGCGCCGTGACAGTGGCCTGGAAGGCAGATTCCTCACCCGTCAAGGCT
GGCGTGGAaACCACCACACCCTCCAAACAATCTAACAACAAATACGCTGCCTCATCCTAT
CTGTCCCTGACTCCTGAGCAGTGGAAGTCCCACAGATCCTACTCCTGCCAAGTCACACAT
GAAGGGTCCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCCTGA
  >STA551_L (SEQ ID NO: 12)
ATGGCCTGGtcCcctCTGCTCCTCACCCTCCTCgctcactgcACAGGGTCCTGGGCCCAA
TCCGCACTGACTCAACCTCCATCCGCCTCTGGTTCCCTGGACAGACAGTTACCATCTCC
TGTacTGGCaccTCAACTGATGTGGGTtAtTACgaATATGTATCCTGGTATCAAACAGCAC
CCTGGAAAGGCTCCTAAACTGATGATTTATgagACTTCCAAAcgtctgTCTGGTGTCCCT
GATCGTTTTTCCGGCTCCAAATCCGGCAACACTGCCTCCTGACTGTTTCCGGTTTGCAA
GCTGAGGACGAGGCAGACTATtacTGCTCATCcTATcgttatgaAcatcaggttTCcTTC
GGCGGAGGGACCAAACTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCTGTCACTCTG
TTCCCACCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTCGTATGTCTCATATCT
GACTTCTACCCTGGCGCCGTGACAGTGGCCTGGAAGGCAGATTCCTCACCCGTCAAGGCT
GGCGTGGAaACCACCACACCCTCCAAACAATCTAACAACAAATACGCTGCCTCATCCTAT
CTGTCCCTGACTCCTGAGCAGTGGAAGTCCCACAGATCCTACTCCTGCCAAGTCACACAT
GAAGGGTCCACCGTGGAGAAGACAGTGGCCCTACAGAATGTTCCTGA
 

eCAPEプロモータのDNA配列（配列番号１３）
GTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA
GCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGC
CCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAG
GGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTAC
ATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG
CCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACG
TATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCA
TCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATAGCCAATCAGAG
CGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAA
GCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGC
CGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGG
GCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGGGGGGACGGCTGCCTTCGGGGGGG
ACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAA
CCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCT
GTCTCATCATTTTGGCAAAccggt
  DNA sequence of the eCAPE promoter (SEQ ID NO: 13)
GTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA
GCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGC
CCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAG
GGACTTTCCATTGACGTCATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTAC
ATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG
CCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACG
TATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCA
TCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTATAATAGCCAATCAGAG
CGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAA
GCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGC
CGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGG
GCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGGGGGGACGGCTGCCTTCGGGGGGG
ACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAA
CCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCT
GTCTCATCATTTTGGCAAAccggt

　本発明は、抗体の生産に利用することができる。本発明は、あらゆる抗体産生細胞へ応用可能である。 The present invention can be used to produce antibodies. The present invention can be applied to any antibody-producing cell.

Claims (16)

　抗体のL鎖又はその断片をコードするDNAを4コピー、及び当該抗体のH鎖又はその断片をコードするDNAを2コピー含むベクターを導入した細胞を用いた抗体又はその断片の製造方法。 A method for producing an antibody or a fragment thereof using cells transfected with a vector containing four copies of DNA encoding an antibody light chain or a fragment thereof and two copies of DNA encoding the antibody heavy chain or a fragment thereof. 　細胞が動物細胞である、請求項１に記載の方法。 The method of claim 1, wherein the cells are animal cells. 　動物細胞がチャイニーズハムスター卵巣細胞である、請求項２に記載の方法。 The method according to claim 2, wherein the animal cell is a Chinese hamster ovary cell. 　抗体が、キメラ抗体、ヒト化抗体又はヒト抗体である、請求項１～３のいずれかに記載の方法。 The method according to any one of claims 1 to 3, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody. 　抗体が、スイッチ抗体、リサイクリング抗体、又はスイーピング抗体である、請求項１～３のいずれかに記載の方法。 The method according to any one of claims 1 to 3, wherein the antibody is a switching antibody, a recycling antibody, or a sweeping antibody. 　抗体が、抗CD137抗体、抗潜在型TGF-β1抗体、抗潜在型ミオスタチン抗体、抗補体(C1s)抗体、抗IL-8抗体、抗IL-6レセプター抗体、抗IL-6抗体、抗グリピカン-3抗体、抗CD3抗体、抗CD20抗体、抗GPIIb/IIIa抗体、抗TNF抗体、抗CD25抗体、抗EGFR抗体、抗Her2/neu抗体、抗RSV抗体、抗CD33抗体、抗CD52抗体、抗IgE抗体、抗CD11a抗体、抗VEGF抗体及び抗VLA4抗体からなる群より選択される、請求項１～３のいずれかに記載の方法。 The method according to any one of claims 1 to 3, wherein the antibody is selected from the group consisting of anti-CD137 antibody, anti-latent TGF-β1 antibody, anti-latent myostatin antibody, anti-complement (C1s) antibody, anti-IL-8 antibody, anti-IL-6 receptor antibody, anti-IL-6 antibody, anti-glypican-3 antibody, anti-CD3 antibody, anti-CD20 antibody, anti-GPIIb/IIIa antibody, anti-TNF antibody, anti-CD25 antibody, anti-EGFR antibody, anti-Her2/neu antibody, anti-RSV antibody, anti-CD33 antibody, anti-CD52 antibody, anti-IgE antibody, anti-CD11a antibody, anti-VEGF antibody and anti-VLA4 antibody. 　抗体が、抗CD137アゴニストスイッチ抗体 STA551、抗潜在型 TGF-β1 モノクローナル抗体SOF10/RG6440、抗潜在型ミオスタチンスイーピング抗体GYM329/RG6237、抗補体(C1s)抗体RAY121、抗体S12preまたは抗IL-8リサイクリング抗体 AMY109である、請求項６に記載の方法。 The method of claim 6, wherein the antibody is anti-CD137 agonist switch antibody STA551, anti-latent TGF-β1 monoclonal antibody SOF10/RG6440, anti-latent myostatin sweeping antibody GYM329/RG6237, anti-complement (C1s) antibody RAY121, antibody S12pre or anti-IL-8 recycling antibody AMY109. 　抗体が、抗CD137抗体 STA551 である、請求項６に記載の方法。 The method of claim 6, wherein the antibody is the anti-CD137 antibody STA551. 　細胞が、抗体又はその断片を恒常的発現(stable expression)している、請求項１～３のいずれかに記載の方法。 The method according to any one of claims 1 to 3, wherein the cells have stable expression of the antibody or a fragment thereof. 　抗体のL鎖又はその断片をコードするDNAを4コピー、及び当該抗体のH鎖又はその断片をコードするDNAを2コピー含む組換えベクター。 A recombinant vector containing four copies of DNA encoding an antibody light chain or a fragment thereof, and two copies of DNA encoding the antibody heavy chain or a fragment thereof. 　請求項１０に記載のベクターが導入されている細胞。 A cell into which the vector described in claim 10 has been introduced. 　抗体又はその断片を恒常的発現している、請求項１１に記載の細胞。 The cell according to claim 11, which constitutively expresses an antibody or a fragment thereof. 　抗体が、抗CD137抗体、抗潜在型TGF-β1抗体、抗潜在型ミオスタチン抗体、抗補体(C1s)抗体、抗IL-8抗体、抗IL-6レセプター抗体、抗IL-6抗体、抗グリピカン-3抗体、抗CD3抗体、抗CD20抗体、抗GPIIb/IIIa抗体、抗TNF抗体、抗CD25抗体、抗EGFR抗体、抗Her2/neu抗体、抗RSV抗体、抗CD33抗体、抗CD52抗体、抗IgE抗体、抗CD11a抗体、抗VEGF抗体及び抗VLA4抗体からなる群より選択される、請求項１０に記載のベクター、または請求項１１若しくは１２に記載の細胞。 The vector according to claim 10 or the cell according to claim 11 or 12, wherein the antibody is selected from the group consisting of anti-CD137 antibody, anti-latent TGF-β1 antibody, anti-latent myostatin antibody, anti-complement (C1s) antibody, anti-IL-8 antibody, anti-IL-6 receptor antibody, anti-IL-6 antibody, anti-glypican-3 antibody, anti-CD3 antibody, anti-CD20 antibody, anti-GPIIb/IIIa antibody, anti-TNF antibody, anti-CD25 antibody, anti-EGFR antibody, anti-Her2/neu antibody, anti-RSV antibody, anti-CD33 antibody, anti-CD52 antibody, anti-IgE antibody, anti-CD11a antibody, anti-VEGF antibody and anti-VLA4 antibody. 　抗体が、抗CD137アゴニストスイッチ抗体 STA551、抗潜在型 TGF-β1 モノクローナル抗体SOF10/RG6440、抗潜在型ミオスタチンスイーピング抗体GYM329/RG6237、抗補体(C1s)抗体RAY121、抗体S12preまたは抗IL-8リサイクリング抗体 AMY109である、請求項１３に記載のベクターまたは細胞。 The vector or cell of claim 13, wherein the antibody is anti-CD137 agonist switch antibody STA551, anti-latent TGF-β1 monoclonal antibody SOF10/RG6440, anti-latent myostatin sweeping antibody GYM329/RG6237, anti-complement (C1s) antibody RAY121, antibody S12pre or anti-IL-8 recycling antibody AMY109. 　抗体が、抗CD137抗体 STA551 である、請求項１３に記載のベクターまたは細胞。 The vector or cell of claim 13, wherein the antibody is the anti-CD137 antibody STA551. 　抗体又はその断片を含有する医薬品を製造する方法であって、請求項１～９のいずれかに記載の方法で抗体又はその断片を製造し、得られた抗体を医薬的に許容される担体又は添加剤と混合して製剤化することにより、医薬品を製造する工程を含む、方法。
  A method for producing a pharmaceutical containing an antibody or a fragment thereof, comprising the steps of producing an antibody or a fragment thereof by the method according to any one of claims 1 to 9, and mixing the obtained antibody with a pharma- ceutical carrier or additive to form a formulation, thereby producing the pharmaceutical.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014525247A (en) * 2011-08-19 2014-09-29 アルダーバイオ・ホールディングズ・エルエルシー Multi-copy method for producing multi-subunit proteins such as antibodies in high concentrations and purity in transformed microorganisms such as Pichia pastoris
WO2020032230A1 (en) * 2018-08-10 2020-02-13 中外製薬株式会社 Anti-cd137 antigen-binding molecule and utilization thereof
JP2021036864A (en) * 2019-08-28 2021-03-11 中外製薬株式会社 Species-crossing anti-latent TGF-β1 antibody and usage

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2701338C (en) * 2007-10-15 2018-09-18 Chugai Seiyaku Kabushiki Kaisha Method for production of antibody
JP4525863B1 (en) * 2009-06-05 2010-08-18 東洋紡績株式会社 Expression vector for establishment of high-productivity cells and high-productivity cells
IL275462B1 (en) * 2017-12-22 2025-09-01 Genentech Inc Targeted integration of nucleic acids

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014525247A (en) * 2011-08-19 2014-09-29 アルダーバイオ・ホールディングズ・エルエルシー Multi-copy method for producing multi-subunit proteins such as antibodies in high concentrations and purity in transformed microorganisms such as Pichia pastoris
WO2020032230A1 (en) * 2018-08-10 2020-02-13 中外製薬株式会社 Anti-cd137 antigen-binding molecule and utilization thereof
JP2021036864A (en) * 2019-08-28 2021-03-11 中外製薬株式会社 Species-crossing anti-latent TGF-β1 antibody and usage

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHUSAINOW, J. ET AL.: "A Study of Monoclonal Antibody-Producing CHO Cell Lines: What Makes a Stable High Producer?", BIOTECHNOLOGY AND BIOENGINEERING, vol. 102, no. 4, 8 October 2008 (2008-10-08), pages 1182 - 1196, XP002525682, DOI: 10.1002/BIT.22158 *
KAMATA-SAKURAI, M. ET AL.: "Antibody to CD 137 Activated by Extracellular Adenosine Triphosphate Is Tumor Selective and Broadly Effective In Vivo without Systemic Immune Activation", CANCER DISCOVERY, vol. 11, no. 1, 25 August 2020 (2020-08-25), pages 158 - 175, XP055964092, DOI: 10.1158/2159-8290.CD-20-0328 *

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