JP2020103280A - Method for producing antibody having improved antibody-dependent cytotoxic activity - Google Patents
Method for producing antibody having improved antibody-dependent cytotoxic activity Download PDFInfo
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- JP2020103280A JP2020103280A JP2019224246A JP2019224246A JP2020103280A JP 2020103280 A JP2020103280 A JP 2020103280A JP 2019224246 A JP2019224246 A JP 2019224246A JP 2019224246 A JP2019224246 A JP 2019224246A JP 2020103280 A JP2020103280 A JP 2020103280A
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Abstract
Description
本発明は、高い抗体依存性細胞傷害（ＡＤＣＣ）活性を有した抗体を製造する方法に関する。より詳しくは、抗体を発現可能な宿主を培養する工程を最適化することで、当該宿主が発現する抗体が有するＡＤＣＣ作用を向上させる方法に関する。 The present invention relates to a method for producing an antibody having high antibody-dependent cellular cytotoxicity (ADCC) activity. More specifically, it relates to a method for improving the ADCC action of an antibody expressed by the host by optimizing the step of culturing a host capable of expressing the antibody.
現在、組換えタンパク質は幅広い分野で使用されている。近年の抗体医薬品に代表されるバイオ医薬品の成長によりその重要性はさらに高まっている。組換えタンパク質は主に大腸菌、酵母、昆虫細胞、哺乳細胞を宿主として製造されているが、発現させた組換えタンパク質の立体構造や、糖鎖付加といった翻訳後修飾等の理由から哺乳動物細胞を宿主として用いた組換えタンパク質発現系の重要性が増している。特にチャイニーズハムスター卵巣細胞（以下、ＣＨＯ細胞）は多くの組換えタンパク質発現の宿主として用いられる。また組換えＣＨＯ細胞由来の組換えタンパク質は、医薬品として使用できる安全性が確認されていることから、抗体医薬品を製造する際の宿主として最もよく使用される哺乳動物細胞である。 Currently, recombinant proteins are used in a wide range of fields. With the recent growth of biopharmaceuticals represented by antibody pharmaceuticals, their importance is increasing. Recombinant proteins are mainly produced in Escherichia coli, yeast, insect cells, and mammalian cells as hosts. However, because of the three-dimensional structure of the expressed recombinant protein and post-translational modification such as glycosylation, The importance of recombinant protein expression systems used as hosts is increasing. In particular, Chinese hamster ovary cells (hereinafter referred to as CHO cells) are used as a host for expressing many recombinant proteins. In addition, since the recombinant protein derived from recombinant CHO cells has been confirmed to be safe to use as a drug, it is a mammalian cell most often used as a host when producing an antibody drug.
抗体医薬品はモノクローナル抗体を主成分とした医薬品であるが、その治療効果には信号伝達阻害、細胞死（Ａｐｏｐｔｏｓｉｓ）の誘導、抗体依存性細胞傷害（ＡＤＣＣ）作用や補体依存性細胞傷害（ＣＤＣ）作用などがある。このうちＡＤＣＣ活性やＣＤＣ活性はエフェクター機能と呼ばれており免疫メカニズムを誘導することで癌等の目的細胞を傷害するため重要である。特にＡＤＣＣ活性は抗体医薬品の細胞傷害作用には重要な活性である。 The antibody drug is a drug whose main component is a monoclonal antibody, and its therapeutic effects include signal transduction inhibition, induction of cell death (Apoptosis), antibody-dependent cellular cytotoxicity (ADCC) action and complement-dependent cellular cytotoxicity (CDC). ) There is an action. Among these, ADCC activity and CDC activity are called effector functions, and are important because they induce an immune mechanism to damage target cells such as cancer. In particular, ADCC activity is an important activity for the cytotoxic action of antibody drugs.
抗体が有するＡＤＣＣ活性を向上させる方法として、抗体のＦｃ領域にある特定のアミノ酸残基を他のアミノ酸残基に置換する方法と、抗体のＦｃ領域に付加する糖鎖を改変する方法に大別される。抗体のＦｃ領域に付加する糖鎖には、特定のアミノ酸配列（例えば、Ａｓｎ−Ｘ−Ｓｅｒ／Ｔｈｒ）（Ｘは任意のアミノ酸残基を示す）のアスパラギン（Ａｓｎ）基の側鎖に付加するＮ型糖鎖と、セリン（Ｓｅｒ）またはスレオニン（Ｔｈｒ）残基の側鎖に結合するＯ型糖鎖がある。このうちＮ型糖鎖のコアフコースを欠損させることでＡＤＣＣ活性が向上する報告がされている（非特許文献１）。 Methods for improving the ADCC activity of an antibody are roughly classified into a method of substituting a specific amino acid residue in the Fc region of the antibody with another amino acid residue and a method of modifying the sugar chain added to the Fc region of the antibody. To be done. The sugar chain added to the Fc region of the antibody is added to the side chain of the asparagine (Asn) group of a specific amino acid sequence (for example, Asn-X-Ser/Thr) (X represents an arbitrary amino acid residue). There are N-type sugar chains and O-type sugar chains that bind to the side chains of serine (Ser) or threonine (Thr) residues. Among them, it has been reported that the ADCC activity is improved by deleting the core fucose of the N-type sugar chain (Non-Patent Document 1).
抗体を発現可能な哺乳動物細胞を培養し、得られた培養物中に含まれる前記哺乳動物細胞が発現した抗体を回収することで、前記抗体を製造する際、前記哺乳動物細胞の培養条件を変化させると、前記抗体のＦｃ領域に付加するＮ型糖鎖の構造が変化することが知られている（特許文献１）。しかしながら、実際に抗体のＦｃ領域へ付加する糖鎖は不均一であり、前記糖鎖の構造とＡＤＣＣ活性との関係を完全に結び付けることは難しい。従って、糖鎖構造を制御してＡＤＣＣ活性の高い抗体を製造する条件、特に前記抗体を発現可能な哺乳動物細胞を培養する条件の構築は困難であった。 Culturing mammalian cells capable of expressing the antibody, by recovering the antibody expressed by the mammalian cells contained in the obtained culture, when producing the antibody, the culture conditions of the mammalian cells It is known that the structure of the N-type sugar chain added to the Fc region of the antibody is changed by changing it (Patent Document 1). However, the sugar chains actually added to the Fc region of the antibody are heterogeneous, and it is difficult to completely link the relationship between the sugar chain structure and ADCC activity. Therefore, it was difficult to construct conditions for producing an antibody with high ADCC activity by controlling the sugar chain structure, particularly for culturing mammalian cells capable of expressing the antibody.
本発明の課題は、抗体を発現可能な哺乳動物細胞を培養する工程と、得られた培養物中に含まれる前記哺乳動物細胞が発現した抗体を回収する工程とを含む、抗体の製造方法において、高い抗体依存性細胞傷害活性を有する抗体を製造する方法を提供することにある。 The subject of the present invention comprises a step of culturing a mammalian cell capable of expressing an antibody, and a step of recovering the antibody expressed by the mammalian cell contained in the obtained culture, in a method for producing an antibody Another object of the present invention is to provide a method for producing an antibody having high antibody-dependent cellular cytotoxicity.
本発明者らは上記の課題を解決すべく鋭意検討した結果、抗体を発現可能な哺乳動物細胞を培養する工程において、前記哺乳動物細胞を一定量のマンガンイオンを添加した培地で培養することで、得られた培養物中に含まれる前記哺乳動物細胞が発現した抗体の抗体依存性細胞傷害が向上することを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have found that in the step of culturing a mammalian cell capable of expressing an antibody, the mammalian cell is cultured in a medium containing a fixed amount of manganese ion. The inventors have found that the antibody-dependent cellular cytotoxicity of the antibody expressed by the mammalian cells contained in the obtained culture is improved, and completed the present invention.
すなわち本発明は、以下に記載の態様を包含する。 That is, the present invention includes the embodiments described below.
（１）抗体を発現可能な哺乳動物細胞を培養する工程と得られた培養物中に含まれる前記哺乳動物細胞が発現した抗体を回収する工程とを含む抗体の製造方法において、前記培養工程を０．０１μＭから５０μＭのマンガンイオンを添加した培地で行なうことで、抗体依存性細胞傷害活性が向上した抗体を製造する方法。 (1) In a method for producing an antibody, which comprises a step of culturing a mammalian cell capable of expressing an antibody and a step of recovering an antibody expressed by the mammalian cell contained in the obtained culture, the culturing step comprising: A method for producing an antibody having improved antibody-dependent cellular cytotoxicity, which is carried out in a medium supplemented with 0.01 μM to 50 μM manganese ion.
（２）抗体がヒトＦｃ領域を含む抗体である、（１）に記載の製造方法。 (2) The production method according to (1), wherein the antibody comprises a human Fc region.
（３）（２）に記載の製造方法で得られた抗体とヒトＦｃγＲＩＩＩａとの親和性を評価することで、（２）に記載の製造方法における培養工程をモニタリングする方法。 (3) A method of monitoring the culture step in the production method according to (2) by evaluating the affinity between the antibody obtained by the production method according to (2) and human FcγRIIIa.
（４）（２）に記載の製造方法で得られた抗体とヒトＦｃγＲＩＩＩａとの親和性を評価することで、（２）に記載の製造方法における培地成分を評価する方法。 (4) A method for evaluating the medium components in the production method according to (2) by evaluating the affinity between the antibody obtained by the production method according to (2) and human FcγRIIIa.
（５）（２）に記載の製造方法で得られた抗体とヒトＦｃγＲＩＩＩａとの親和性評価を、（２）に記載の製造方法で得られた抗体とヒトＦｃγＲＩＩＩａ固定化分離剤との結合力に基づき行なう、（３）または（４）に記載の方法。 (5) The affinity evaluation between the antibody obtained by the production method described in (2) and human FcγRIIIa is evaluated by the binding force between the antibody obtained by the production method described in (2) and the human FcγRIIIa-immobilized separating agent. The method according to (3) or (4), which is performed based on
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明の製造方法は、抗体を発現可能な哺乳動物細胞を培養する際、０．０１μＭから５０μＭのマンガンイオンを培地に添加して培養することを特徴としている。培地中のマンガンイオンが前述した濃度範囲であれば、細胞の増殖性や抗体の生産性が著しく阻害されることはない。マンガンイオンの培地への添加は、水溶液中でマンガンイオンとして存在可能なマンガン化合物を添加すればよい。前記マンガン化合物の例として、塩化マンガン（ＭｎＣｌ２）や硫酸マンガン（ＭｎＳＯ４）が例示できる。マンガン化合物の添加量は、培地中のマンガンイオンが前述した濃度範囲となるよう添加すればよく、０．０３μＭから３０μＭとなるよう添加すると好ましく、０．１μＭから１０μＭとなるよう添加するとより好ましく、１μＭから１０μＭとなるよう添加するとさらにより好ましい。 The production method of the present invention is characterized by adding 0.01 to 50 μM manganese ion to a medium when culturing a mammalian cell capable of expressing an antibody. When the manganese ion concentration in the medium is within the above-mentioned concentration range, cell proliferation and antibody productivity are not significantly impaired. The manganese ion may be added to the medium by adding a manganese compound that can exist as manganese ion in the aqueous solution. Examples of the manganese compound include manganese chloride (MnCl 2 ) and manganese sulfate (MnSO 4 ). The amount of the manganese compound added may be such that the manganese ion in the medium is in the concentration range described above, preferably 0.03 μM to 30 μM, and more preferably 0.1 μM to 10 μM. It is even more preferable to add 1 μM to 10 μM.
本発明の製造方法で使用する哺乳動物細胞は、製造対象抗体を発現可能な細胞であれば特に制限はない。一例を示すと、ＣＨＯ細胞（ＣＨＯ−Ｋ１、ＣＨＯ−Ｓ、ＣＨＯ−ＤＧ４４およびＣＨＯ−ＤＸＢ１１）、マウス骨髄腫由来細胞（ＳＰ２／０、ＮＳ０）、ヒト胎児腎臓由来細胞（ＨＥＫ細胞）、ヒト白血病細胞由来細胞（ＨＬ−６０細胞）、ヒト子宮頸癌由来細胞（ＨｅＬａ細胞）およびアフリカミドリザルの腎細胞由来細胞（ＣＯＳ細胞）があげられる。中でも組換え抗体製造に汎用されるＣＨＯ細胞の使用が好ましい。 The mammalian cells used in the production method of the present invention are not particularly limited as long as they can express the antibody to be produced. As an example, CHO cells (CHO-K1, CHO-S, CHO-DG44 and CHO-DXB11), mouse myeloma-derived cells (SP2/0, NS0), human fetal kidney-derived cells (HEK cells), human leukemia Examples thereof include cell-derived cells (HL-60 cells), human cervical cancer-derived cells (HeLa cells) and African green monkey kidney cell-derived cells (COS cells). Above all, it is preferable to use CHO cells which are generally used for producing recombinant antibodies.
本発明の製造方法で使用する培地は、０．０１μＭから５０μＭのマンガンイオンを含み、かつ宿主である哺乳動物細胞が生育し抗体を発現可能な培地であれば、特に限定はない。一例を示すと、動物由来の血清が必要な培地（ＲＰＭＩ１６４０、Ｄ−ＭＥＭ等）や化学的に成分が決定されている培地（ＢａｌａｎＣＤ ＣＨＯ Ｇｒｏｗｔｈ Ａ［Ｉｒｖｉｎｅ Ｓｃｉｅｎｔｉｆｉｃ社］、ＦｒｅｅＳｔｙｌｅ ＣＨＯ Ｅｘｐｒｅｓｓｉｏｎ ＭｅｄｉｕｍＣＤ［Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ社］、ＯｐｔｉＣＨＯ［Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ社］、ＥＸ−ＣＥＬＬ ＣＤ ＣＨＯ Ｆｕｓｉｏｎ／ＥＸ−ＣＥＬＬ Ａｄｖａｎｃｅｄ ＣＨＯ Ｆｅｄ−ｂａｔｃｈ Ｍｅｄｉｕｍ［Ｍｅｒｃｋ社］およびＣＨＯｇｒｏ［Ｍｉｒｕｓ社］）に前述した濃度のマンガンイオンを添加した培地があげられる。さらに前述した培地に、栄養素、ホルモン、成長因子、特定イオン（ナトリウム、カリウム、カルシウム、マグネシウム等）、ビタミン、ヌクレオシド、ヌクレオチド、グルタミンのようなアミノ酸、無機塩（銅、亜鉛、コバルト、ニッケル等）、脂質、グルコースをはじめとする培地構成成分が含まれていてもよい。またＧ４１８、ピューロマイシン、ブラストサイジン、ゼオシン、ハイグロマイシン、フレオマイシン、カナマイシン、アンピシリンなどの抗生物質をさらに添加してもよい。 The medium used in the production method of the present invention is not particularly limited as long as it contains 0.01 μM to 50 μM manganese ion and can grow mammalian cells as a host and express antibodies. As an example, a medium that requires animal-derived serum (RPMI1640, D-MEM, etc.) or a medium whose components have been chemically determined (BalanCD CHO Growth A [Irvine Scientific], FreeStyle CHO Expression MediumCD[ThermoFertherFermo ], OptiCHO [Thermo Fisher], EX-CELL CD CHO Fusion/EX-CELL Advanced CHO Fed-batch Medium [Merck], and CHOgro [Mirus] with the above-mentioned concentration of the manganese ion added medium. To be Furthermore, nutrients, hormones, growth factors, specific ions (sodium, potassium, calcium, magnesium, etc.), vitamins, nucleosides, nucleotides, amino acids such as glutamine, inorganic salts (copper, zinc, cobalt, nickel, etc.) can be added to the aforementioned medium. , Mediums such as lipids and glucose may be contained. Further, antibiotics such as G418, puromycin, blasticidin, zeocin, hygromycin, phleomycin, kanamycin and ampicillin may be further added.
本発明の製造方法における培養工程は、宿主として用いる哺乳動物細胞や前記細胞で発現させる抗体に応じて適宜行なえばよい。一例として、
前述した濃度のマンガンイオンを添加した培地を入れたフラスコに、抗体を発現可能な哺乳動物細胞を接種後、当該フラスコを振盪させて培養してもよく、
前述した濃度のマンガンイオンを添加した培地を入れたバイオリアクターに、抗体を発現可能な哺乳動物細胞を接種後、回分培養、半回分培養（流加培養ともいう）、潅流培養またはそれらの組合せにより培養してもよい。
哺乳動物細胞がＣＨＯ細胞の場合、５％から８％のＣＯ２存在下、温度３０℃から３７℃
、ｐＨ６．８から７．４で培養することが好ましい。
The culturing step in the production method of the present invention may be appropriately performed depending on the mammalian cell used as a host and the antibody expressed by the cell. As an example,
A flask containing a medium containing the above-mentioned concentration of manganese ion is inoculated with mammalian cells capable of expressing the antibody, and the flask may be shaken to be cultured.
After inoculating mammalian cells capable of expressing antibody into a bioreactor containing a medium to which manganese ions are added at the above-mentioned concentration, batch culture, semi-batch culture (also referred to as fed-batch culture), perfusion culture, or a combination thereof is performed. You may culture.
When the mammalian cells are CHO cells, the temperature is 30°C to 37°C in the presence of 5% to 8% CO 2.
It is preferable to culture at pH 6.8 to 7.4.
本発明の方法で製造する抗体の一例として、ヒトＦｃ領域を含む抗体があげられる。具体的には、ヒト抗体、ヒト化抗体、ヒトと他の動物（マウスなど）とのキメラ抗体、ヒトＦｃ融合タンパク質などがあげられる。ヒトＦｃ領域を含む抗体がイムノグロブリンＧ（ＩｇＧ）の場合、４つのサブクラス（ＩｇＧ１、ＩｇＧ２、ＩｇＧ３、ＩｇＧ４）が知られているが、このうちＩｇＧ１とＩｇＧ３は抗体依存性細胞傷害（ＡＤＣＣ）活性が高い点で、本発明の方法で製造する抗体の好ましい態様といえる。さらにＩｇＧ１はＡＤＣＣ活性が特に高く、本発明の方法で製造する抗体の特に好ましい態様といえる。 An example of the antibody produced by the method of the present invention is an antibody containing a human Fc region. Specific examples thereof include human antibodies, humanized antibodies, chimeric antibodies of humans and other animals (mouse, etc.), human Fc fusion proteins and the like. When the antibody containing the human Fc region is immunoglobulin G (IgG), four subclasses (IgG1, IgG2, IgG3, IgG4) are known. Of these, IgG1 and IgG3 have antibody-dependent cellular cytotoxicity (ADCC) activity. It can be said that it is a preferred embodiment of the antibody produced by the method of the present invention because of its high value. Furthermore, IgG1 has a particularly high ADCC activity, which can be said to be a particularly preferred embodiment of the antibody produced by the method of the present invention.
本発明の製造方法で使用する、抗体を発現可能な哺乳動物細胞は、前記抗体をコードするポリヌクレオチドを含む発現ベクターで、前記哺乳動物細胞を形質転換し、作製すればよい。前記発現ベクターには、プロモーターおよび前記抗体をコードするポリヌクレオチドの他に、ポリＡや、組換え抗体の分泌発現に必要な分泌シグナルや、遺伝子増幅マーカー遺伝子や、宿主選択に用いる抗生物質耐性遺伝子や、遺伝子組換えのために用いる哺乳動物細胞以外の宿主での複製開始点等をさらに含んでもよい。 The mammalian cell capable of expressing an antibody used in the production method of the present invention may be produced by transforming the mammalian cell with an expression vector containing a polynucleotide encoding the antibody. In the expression vector, in addition to the promoter and the polynucleotide encoding the antibody, polyA, a secretory signal required for secretory expression of a recombinant antibody, a gene amplification marker gene, and an antibiotic resistance gene used for host selection Alternatively, a replication origin in a host other than mammalian cells used for gene recombination may be further included.
前記ポリＡはターミネーションシグナルを含んでいれば特に制限はなく、一例として、発現させる抗体由来のポリＡ、ＳＶ４０ウイルスゲノム由来のポリＡ、ヘルペスウイルスチミジンキナーゼのポリＡ、ウシ成長ホルモン由来のポリＡ、ウサギのβーグロビン遺伝子由来のポリＡがあげられる。 The poly A is not particularly limited as long as it contains a termination signal, and examples thereof include poly A derived from an antibody to be expressed, poly A derived from the SV40 virus genome, poly A of herpesvirus thymidine kinase, and poly A derived from bovine growth hormone. , Poly A derived from the rabbit β-globin gene.
前記分泌シグナルは発現抗体を分泌すれば特に制限はなく、その一例としては、発現させる組換え抗体由来の分泌シグナル、ヒトインターロイキン２（ＩＬー２）の分泌シグナル、アズロシジン前駆体の分泌シグナル、ヒト血清アルブミンの分泌シグナルがあげられる。 The secretory signal is not particularly limited as long as it secretes the expressed antibody, and examples thereof include a secretory signal derived from a recombinant antibody to be expressed, a secretory signal of human interleukin 2 (IL-2), a secretory signal of an azulocidin precursor, An example is a human serum albumin secretion signal.
前記遺伝子増幅マーカー遺伝子は、遺伝子増幅させる方法に適した遺伝子を用いればよい。例えばジヒドロ葉酸レダクターゼ（ｄｈｆｒ）／メトトレキサート（ＭＴＸ）法を用いる場合はｄｈｆｒ遺伝子を、グルタミン合成酵素（ＧＳ）／メチオニンスルホキシミン（ＭＳＸ）法を用いる場合はＧＳ遺伝子を、それぞれ用いればよい。 As the gene amplification marker gene, a gene suitable for the method of gene amplification may be used. For example, the dhfr gene may be used when the dihydrofolate reductase (dhfr)/methotrexate (MTX) method is used, and the GS gene may be used when the glutamine synthetase (GS)/methionine sulfoximine (MSX) method is used.
前記抗生物質耐性遺伝子は、宿主選択に用いる抗生物質に対応した耐性遺伝子を選択すればよく、一例として、Ｇ４１８耐性遺伝子、ピューロマイシン耐性遺伝子、ブラストサイジン耐性遺伝子、ゼオシン耐性遺伝子、ハイグロマイシン耐性遺伝子、フレオマイシン耐性遺伝子があげられる。 As the antibiotic resistance gene, a resistance gene corresponding to an antibiotic used for host selection may be selected, and as an example, G418 resistance gene, puromycin resistance gene, blasticidin resistance gene, zeocin resistance gene, hygromycin resistance gene. , And the phleomycin resistance gene.
前記複製開始点は、哺乳動物細胞以外の宿主が大腸菌である場合、大腸菌内でのコピー数が高くプラスミドＤＮＡの収量が多い、ＣｏｌＥ１が例示できる。 The replication origin can be exemplified by ColE1 which has a high copy number in E. coli and a high yield of plasmid DNA when the host other than mammalian cells is E. coli.
さらに前記発現ベクターには、プロモーターの働きを強めるためのエンハンサーをさらに含んでもよい。使用するエンハンサーに特に制限はなく、発現させる抗体や哺乳動物細胞を考慮し、適宜選択すればよい。一例としてサイトメガロウイルス（ＣＭＶ）由来のエンハンサーがあげられる。 Furthermore, the expression vector may further include an enhancer for enhancing the function of the promoter. The enhancer used is not particularly limited and may be appropriately selected in consideration of the antibody to be expressed and mammalian cells. An example is a cytomegalovirus (CMV)-derived enhancer.
また哺乳動物に導入した遺伝子（抗体をコードするポリヌクレオチド）が発現しやすくするために、前記発現ベクターにＬｏｘＰ遺伝子をさらに含ませてもよい。ゲノム中にＬｏｘＰ遺伝子を含んだ宿主細胞へ発現ベクターを導入する際に、Ｃｒｅリコンビナーゼによる相同組換えを行なうことで宿主細胞のゲノムへ部位特異的に組換えタンパク質をコードするポリヌクレオチドを導入することができる。また、宿主細胞のゲノムへ部位特異的に組換えタンパク質をコードするポリヌクレオチドを導入する方法としてＣＲＩＳＰＲ／Ｃａｓ９などを用いることもできる。 The expression vector may further contain a LoxP gene to facilitate expression of a gene (polynucleotide encoding an antibody) introduced into a mammal. When introducing an expression vector into a host cell containing the LoxP gene in its genome, homologous recombination with Cre recombinase is carried out to introduce a polynucleotide encoding a recombinant protein site-specifically into the genome of the host cell. You can In addition, CRISPR/Cas9 or the like can be used as a method for site-specifically introducing a polynucleotide encoding a recombinant protein into the genome of a host cell.
前記発現ベクターで哺乳動物細胞を形質転換するには、エレクトロポレーションやカチオニックリポソームを用いたリポフェクションなど、当業者が通常用いる形質転換法の中から、宿主として使用する哺乳動物細胞に合わせて適宜選択すればよい。 To transform a mammalian cell with the expression vector, electroporation, lipofection using a cationic liposome, or other transformation method commonly used by those skilled in the art is selected according to the mammalian cell used as the host. Just select it.
前述した方法で抗体を発現可能な哺乳動物細胞を培養後、得られた培養物中に含まれる前記哺乳動物細胞が発現した抗体を回収することで、ＡＤＣＣ活性が向上した抗体を製造する。抗体の回収方法の一例として、前記得られた培養物から、アフィニティークロマトグラフィー、イオン交換クロマトグラフィー、疎水クロマトグラフィー、ゲル濾過クロマトグラフィーなどのクロマトグラフィーによる精製操作を単独または組み合わせて抗体を回収する方法があげられる。前記方法は抗体を高効率かつ高純度に回収できる点で好ましい。 After culturing the mammalian cells capable of expressing the antibody by the method described above, the antibody expressed by the mammalian cells contained in the obtained culture is recovered to produce an antibody with improved ADCC activity. As an example of a method for recovering an antibody, a method for recovering an antibody from the obtained culture by a single or combination of purification operations by chromatography such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography and gel filtration chromatography. Can be given. The above method is preferable because the antibody can be recovered with high efficiency and high purity.
本発明で製造する抗体が、ヒトＦｃ領域を含む抗体である場合、前記抗体とヒトＦｃγＲＩＩＩａとの親和性を評価することで、抗体を発現可能な哺乳動物細胞の培養状態（培養工程）をモニタリングできる。ヒトＦｃ領域を含む抗体に付加するＮ型糖鎖を欠損させると前記抗体とヒトＦｃγＲＩＩＩａとの親和性が著しく低下する。またヒトＦｃ領域を含む抗体が有するＡＤＣＣ活性は、当該Ｆｃ領域と免疫細胞表面上のヒトＦｃγＲＩＩＩａとの親和性（結合性）と関連することが知られている（Ｎｏｒｄｓｔｒｏｍ．Ｊ，Ｌ， ｅｔ ａｌ．，Ｂｒｅａｓｔ Ｃａｎｃｅｒ Ｒｅｓ．，１３，６，（２０１１））。従って、ヒトＦｃ領域を含む抗体とヒトＦｃγＲＩＩＩａとの親和性を評価することで、製造する抗体が有するＡＤＣＣ活性をモニタリングでき、抗体を発現可能な哺乳細胞の培養工程をモニタリングできる。一例として、得られたヒトＦｃ領域を含む抗体とヒトＦｃγＲＩＩＩａとの親和性が低下した場合、当該抗体が有するＡＤＣＣ活性が低下しているため、当該抗体が有するＡＤＣＣ活性を向上すべく、マンガンイオン濃度などの培地成分や培養条件（二酸化炭素濃度、温度、ｐＨ、時間など）を適宜調整する。 When the antibody produced in the present invention is an antibody containing a human Fc region, the culture state (culture step) of mammalian cells capable of expressing the antibody is monitored by evaluating the affinity between the antibody and human FcγRIIIa. it can. When the N-type sugar chain added to the antibody containing the human Fc region is deleted, the affinity between the antibody and human FcγRIIIa is markedly reduced. It is known that the ADCC activity of an antibody containing a human Fc region is associated with the affinity (binding) between the Fc region and human FcγRIIIa on the surface of immune cells (Nordstrom. J, L, et al. , Breast Cancer Res., 13, 6, (2011)). Therefore, by evaluating the affinity between the antibody containing the human Fc region and human FcγRIIIa, the ADCC activity of the antibody to be produced can be monitored, and the process of culturing the mammalian cells capable of expressing the antibody can be monitored. As an example, when the affinity between the obtained antibody containing the human Fc region and human FcγRIIIa is decreased, the ADCC activity of the antibody is decreased, so that the ADCC activity of the antibody is improved, the manganese ion is increased. Medium components such as concentration and culture conditions (carbon dioxide concentration, temperature, pH, time, etc.) are adjusted appropriately.
ヒトＦｃ領域を含む抗体とヒトＦｃγＲＩＩＩａとの親和性評価の好ましい態様として、ヒトＦｃ領域を含む抗体とヒトＦｃγＲＩＩＩａ固定化分離剤との結合力に基づく評価があげられる。ヒトＦｃγＲＩＩＩａを担体に固定化して得られるヒトＦｃγＲＩＩＩａ固定化分離剤を充填したカラムに、ヒトＦｃ領域を含む抗体をアプライすると、前記抗体が付加した糖鎖構造の違いに基づき分離され（特開２０１５−０８６２１６号公報、ＷＯ２０１８／１５０９７３号）、かつ前記抗体が有するＡＤＣＣ活性の違いに基づき分離される（特開２０１６−０２３１５２号公報、ＷＯ２０１８／１５０９７３号）。従って、前記分離パターンの形状に基づき、本発明における、抗体を発現可能な哺乳動物細胞の培養状態（培養工程）のモニタリングができる。具体的には、ヒトＦｃγＲＩＩＩａ固定化分離剤を充填したカラムを用いてヒトＦｃ領域を含む抗体を分離すると、ＡＤＣＣ活性が高い抗体がＡＤＣＣ活性が低い抗体よりも遅れて溶出される（すなわちヒトＦｃγＲＩＩＩａ固定化分離剤との結合力が強い）。従って、前記分離により得られた溶出パターンのピーク面積またはピーク高さから、ＡＤＣＣ活性が高い抗体が溶出されるピーク（画分）の量および／または割合を算出し、当該量および／または割合が低下した場合、当該抗体が有するＡＤＣＣ活性が低下しているため、当該抗体が有するＡＤＣＣ活性を向上すべく、マンガンイオン濃度などの培地成分や培養条件（二酸化炭素濃度、溶存酸素濃度、温度、ｐＨ、時間など）を適宜調整する。 A preferred embodiment of the affinity evaluation between an antibody containing a human Fc region and human FcγRIIIa is an evaluation based on the binding force between an antibody containing a human Fc region and a human FcγRIIIa-immobilized separating agent. When an antibody containing a human Fc region is applied to a column filled with a human FcγRIIIa-immobilized separating agent obtained by immobilizing human FcγRIIIa on a carrier, the antibody is separated based on the difference in sugar chain structure added to the antibody (JP-A-2015). No. 086216, WO2018/150973) and based on the difference in ADCC activity of the antibody (Japanese Patent Laid-Open No. 2016-023152, WO2018/150973). Therefore, based on the shape of the separation pattern, the culture state (culture step) of the mammalian cells capable of expressing the antibody in the present invention can be monitored. Specifically, when a human Fc region-containing antibody is separated using a column packed with a human FcγRIIIa-immobilized separating agent, an antibody with high ADCC activity elutes later than an antibody with low ADCC activity (ie, human FcγRIIIa). Strong binding force with immobilized separating agent). Therefore, from the peak area or peak height of the elution pattern obtained by the separation, the amount and/or ratio of the peak (fraction) at which the antibody with high ADCC activity is eluted is calculated, and the amount and/or ratio is calculated. If it decreases, the ADCC activity of the antibody is decreased, so in order to improve the ADCC activity of the antibody, medium components such as manganese ion concentration and culture conditions (carbon dioxide concentration, dissolved oxygen concentration, temperature, pH) , Time, etc.) are adjusted appropriately.
また、培養条件や細胞株を同条件とし培地成分のみを変えて培養を行なうことで、よりＡＤＣＣ活性が高くなる培地成分の評価を行なうことができる。具体的には異なる培地で培養することで得られた抗体を前記ヒトＦｃγＲＩＩＩａ固定化分離剤により評価を行ない、得られた分離パターンの形状やピーク（画分）の量および／または割合に基づき得られた抗体のＡＤＣＣ活性を比較することで培地の抗体に対する評価を行なう。 In addition, by culturing under the same culture conditions and cell lines and changing only the medium components, it is possible to evaluate the medium components having a higher ADCC activity. Specifically, the antibody obtained by culturing in a different medium is evaluated by the above-mentioned human FcγRIIIa-immobilized separating agent, and the antibody is obtained based on the shape of the obtained separation pattern and the amount and/or ratio of peaks (fractions). The ADCC activity of the obtained antibodies is compared to evaluate the medium for the antibody.
なお本明細書においてヒトＦｃγＲＩＩＩａとは、
（Ａ）ヒトＦｃγＲＩＩＩａ（ＵｎｉＰｒｏｔ Ｎｏ．Ｐ０８６３７）のアミノ酸配列のうち１７番目のグリシンから１９２番目のグルタミンまでのアミノ酸残基を少なくとも含む、Ｆｃ結合性タンパク質、または
（Ｂ）ヒトＦｃγＲＩＩＩａ（ＵｎｉＰｒｏｔ Ｎｏ．Ｐ０８６３７）のアミノ酸配列のうち１７番目のグリシンから１９２番目のグルタミンまでのアミノ酸残基を少なくとも含み、ただし当該１７番目から１９２番目までのアミノ酸残基において、１以上のアミノ酸残基が欠失、他のアミノ酸残基に置換、または付加されたポリペプチドを含む、Ｆｃ結合性タンパク質、
のことを意味する。また前記（Ｂ）の好ましい態様として、
特開２０１５−０８６２１６号公報で開示のＦｃ結合性タンパク質、
特開２０１６−１６９１９７号公報で開示のＦｃ結合性タンパク質、
特開２０１７−１１８８７１号公報で開示のＦｃ結合性タンパク質、
ＷＯ２０１８／１５０９７３号で開示のＦｃ結合性タンパク質、
ＷＯ２０１９／０８３０４８号で開示のＦｃ結合性タンパク質、
があげられる。
In this specification, human FcγRIIIa means
(A) An Fc-binding protein containing at least an amino acid residue from glycine at position 17 to glutamine at position 192 in the amino acid sequence of human FcγRIIIa (UniProt No. P08637), or (B) human FcγRIIIa (UniProt No. P08637). ) At least the amino acid residues from the 17th glycine to the 192nd glutamine in the amino acid sequence, provided that one or more amino acid residues in the 17th to 192nd amino acid residues are deleted, An Fc-binding protein comprising a polypeptide substituted or added to an amino acid residue,
Means that. Further, as a preferable mode of the above (B),
Fc binding protein disclosed in JP-A-2005-086216,
Fc binding protein disclosed in JP-A-2016-169197,
Fc-binding protein disclosed in JP-A-2017-118871,
An Fc binding protein disclosed in WO2018/150973,
An Fc binding protein disclosed in WO2019/083048,
Can be given.
また本発明において、ＡＤＣＣ活性が向上した抗体とは、例えば、ヒトＦｃγＲＩＩＩａ固定化分離剤を充填したカラムを用いた分離により得られた結果（溶出パターン）のうち、ＡＤＣＣ活性の高い抗体が位置するピーク面積またはピーク高さの割合が、マンガンイオン無添加培地で培養したときの前記割合と比較し、２％以上、好ましくは５％以上、より好ましくは１０％以上、さらに好ましくは１５％以上、さらにより好ましくは２０％以上向上した抗体のことを意味する。 In the present invention, the antibody with improved ADCC activity refers to, for example, an antibody with high ADCC activity in the results (elution pattern) obtained by separation using a column packed with a human FcγRIIIa-immobilized separating agent. The ratio of the peak area or the peak height is 2% or more, preferably 5% or more, more preferably 10% or more, still more preferably 15% or more, as compared with the above ratio when cultured in a manganese ion-free medium. Even more preferably, it means an antibody with an improvement of 20% or more.
本発明は、抗体を発現可能な哺乳動物細胞を培養する工程と得られた培養物中に含まれる前記哺乳動物細胞が発現した抗体を回収する工程とを含む抗体の製造方法において、前記培養工程を０．０１μＭから５０μＭのマンガンイオンを添加した培地で行なうことを特徴としている。本発明により、抗癌剤など抗体依存性細胞障害活性を必要とする抗体を効率的に製造できる。 The present invention provides a method for producing an antibody, which comprises a step of culturing a mammalian cell capable of expressing an antibody and a step of recovering the antibody expressed by the mammalian cell contained in the obtained culture, wherein the culturing step is performed. Is carried out in a medium supplemented with 0.01 μM to 50 μM manganese ion. According to the present invention, an antibody that requires antibody-dependent cellular cytotoxicity such as an anticancer agent can be efficiently produced.
また本発明で製造する抗体がヒトＦｃ領域を含む抗体の場合、前記抗体とヒトＦｃγＲＩＩＩａとの親和性を評価することで、本発明の製造方法における培養工程のモニタリングや培地成分の評価が行なえる。 When the antibody produced in the present invention is an antibody containing a human Fc region, the affinity between the antibody and human FcγRIIIa can be evaluated to monitor the culture step and evaluate the medium components in the production method of the present invention. ..
以下、実施例および比較例を用いて、本発明をさらに詳細に説明するが、本発明はこれら例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples and comparative examples, but the present invention is not limited to these examples.
実施例１ 抗インターロイキン６レセプター（ＩＬ−６Ｒ）抗体発現細胞の構築
（１）以下の方法で抗ＩＬ−６Ｒ抗体を哺乳動物細胞で発現可能なベクターを構築した。
（１−１）配列番号１に記載のジヒドロ葉酸レダクターゼ（ｄｉｈｙｄｒｏｆｏｌａｔｅ ｒｅｄｕｃｔａｓｅ、ｄｈｆｒ）およびＳＶ４０のＰｏｌｙＡをコードする遺伝子に制限酵素ＳａｃＩＩ認識配列列（ＣＣＧＣＧＧ）を５’末端および３’末端の両方に付加した遺伝子を全合成し（Ｉｎｔｅｇｒａｔｅｄ ＤＮＡ Ｔｅｃｈｎｏｌｏｇｉｅｓ社に委託）プラスミドにクローニングした。
（１−２）（１−１）で作製したプラスミドで大腸菌ＪＭ１０９株を形質転換した。得られた形質転換体を培養し、プラスミドを抽出したのち、制限酵素ＳａｃＩＩで消化することで、ｄｈｆｒ−ＳＶ４０ＰｏｌｙＡをコードする遺伝子を調製しｄｈｆｒ−Ｐ１と命名した。
（１−３）ｐＩＲＥＳベクター（Ｃｌｏｎｔｅｃｈ社製）を鋳型として、配列番号２（５’−ＴＣＣ［ＣＣＧＣＧＧ］ＧＣＧＧＧＡＣＴＣＴＧＧＧＧＴＴＣＧＡＡＡＴＧＡＣＣＧ−３’）および配列番号３（５’−ＴＣＣ［ＣＣＧＣＧＧ］ＧＧＴＧＧＣＴＣＴＡＧＣＣＴＴＡＡＧＴＴＣＧＡＧＡＣＴＧ−３’）に記載の配列からなるオリゴヌクレオチドプライマー（配列番号２および３中の角かっこは制限酵素ＳａｃＩＩ認識配列を示している）を用いてＰＣＲを行なった。具体的には、表１に示す組成の反応液を調製し、当該反応液を９８℃で３０秒間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で５分間の第３ステップを１サイクルとする反応を２５サイクル繰り返ことで実施した。このＰＣＲにより、ｐＩＲＥＳベクターのうちネオマイシン耐性遺伝子を除いた領域を増幅した。
Example 1 Construction of cells expressing anti-interleukin 6 receptor (IL-6R) antibody (1) A vector capable of expressing anti-IL-6R antibody in mammalian cells was constructed by the following method.
(1-1) Addition of a restriction enzyme SacII recognition sequence sequence (CCGCGG) to both the 5'end and the 3'end of the gene encoding dihydrofolate reductase (dihydrofolate reductase, dhfr) shown in SEQ ID NO: 1 and PolyA of SV40. The gene was totally synthesized (consigned to Integrated DNA Technologies) and cloned into a plasmid.
(1-2) Escherichia coli JM109 strain was transformed with the plasmid prepared in (1-1). The resulting transformant was cultured, the plasmid was extracted, and then the gene encoding dhfr-SV40PolyA was prepared by digesting with the restriction enzyme SacII, and named as dhfr-P1.
(1-3) Using the pIRES vector (manufactured by Clontech) as a template, SEQ ID NO: 2 (5′-TCC[CCGCGG]GCGGGACTCTGGGGTCTGAAATGACCG-3′) and SEQ ID NO:3 (5′-TCC[CCGCGG]GGTGGGCTCTAGCCTTAAGTTCGAGACTG-3′). PCR was carried out using an oligonucleotide primer having the described sequence (the brackets in SEQ ID NOS: 2 and 3 represent the restriction enzyme SacII recognition sequence). Specifically, a reaction solution having the composition shown in Table 1 was prepared, and the reaction solution was heat-treated at 98° C. for 30 seconds, followed by the first step at 98° C. for 10 seconds, the second step at 55° C. for 5 seconds, and the second step at 72° C. The reaction was carried out by repeating 25 cycles of the third step of 1 minute at 5° C. for 25 cycles. By this PCR, the region excluding the neomycin resistance gene in the pIRES vector was amplified.
（１−４）（１−３）で作製したＰＣＲ産物を精製後、制限酵素ＳａｃＩＩで消化し、（１−２）で調製したｄｈｆｒ−Ｐ１とライゲーションした。当該ライゲーション産物で大腸菌ＪＭ１０９株を形質転換し、培養した形質転換体からプラスミドを抽出することでｄｈｆｒ遺伝子を含んだ発現ベクターｐＩＲＥＳ−ｄｈｆｒを得た。 (1-4) After purifying the PCR product prepared in (1-3), it was digested with the restriction enzyme SacII and ligated with dhfr-P1 prepared in (1-2). Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain an expression vector pIRES-dhfr containing the dhfr gene.
（２）（１）で作製したｐＩＲＥＳ−ｄｈｆｒを鋳型として配列番号４（５’−ＴＴＴＡＡＡＴＣＡ［ＧＣＧＧＣＣＧＣ］ＧＣＡＧＣＡＣＣＡＴＧＧＣＣＴＧＡＡＡＴＡＡＣＣＴＣＴＧ−３’）および配列番号５（５’−ＧＣＡＡＧＴＡＡＡＡＣＣＴＣＴＡＣＡＡＡＴＧＴＧＧＴＡＡＡ［ＣＧＡＴＣＧ］ＣＴＣＣＧＧＴＧＣＣＣＧＴー３’）に記載の配列からなるオリゴヌクレオチドプライマー（配列番号４中の角かっこは制限酵素ＮｏｔＩ認識配列を、配列番号５中の角かっこは制限酵素ＰｖｕＩ認識配列を、それぞれ示している）を用いてＰＣＲを行なった。具体的には、表２に示す組成の反応液を調製し、当該反応液を９８℃で１分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル繰り返すことで実施した。このＰＣＲにより増幅したＰＣＲ産物（ＳＶ４０プロモーター、ｄｈｆｒ、ＳＶ４０のＰｏｌｙＡまでの領域を）をｄｈｆｒ−Ｐ２と命名した。 (2) Using the pIRES-dhfr prepared in (1) as a template, SEQ ID NO: 4 (5′-TTTAAATCA[GCGGCCGC]GCAGCACCATGGCCTGAAATAACCTCTTG-3′) and SEQ ID NO:5 (5′-GCAAGTAAAACCTCTACAAATCGTGTAAA]CGTGCTAGA[CGTGCTAGAG]CGTGCTAGAG] PCR was performed using an oligonucleotide primer having the sequence of SEQ ID NO: 4 (the brackets in SEQ ID NO: 4 represent the restriction enzyme NotI recognition sequence, and the brackets in SEQ ID NO: 5 represent the restriction enzyme PvuI recognition sequence). .. Specifically, a reaction solution having the composition shown in Table 2 was prepared, the reaction solution was heat-treated at 98° C. for 1 minute, and then the first step was performed at 98° C. for 10 seconds, the second step was performed at 55° C. for 5 seconds, 72 The reaction was carried out by repeating 30 cycles of the third step for 1 minute at 1° C. as one cycle. The PCR product (SV40 promoter, dhfr, the region up to PolyA of SV40) amplified by this PCR was designated as dhfr-P2.
（３）ヒト抗体の重鎖定常領域を含んだｐＦＵＳＥｓｓ−ＣＨＩｇ−ｈＧ１（ＩｎｖｉｖｏＧｅｎ社製）、ヒト抗体の軽鎖定常領域を含んだｐＦＵＳＥ２ｓｓ−ＣＬＩｇ−ｈｋ（ＩｎｖｉｖｏＧｅｎ社製）および（２）で作製したｄｈｆｒ−Ｐ２をそれぞれ制限酵素ＮｏｔＩおよびＰｖｕＩで消化した後、精製しライゲーションした。当該ライゲーション産物で大腸菌ＪＭ１０９株を形質転換し、培養した形質転換体からプラスミドを抽出することでＳＶ４０プロモーター、ｄｈｆｒ、ＳＶ４０のＰｏｌｙＡを含んだｐＦＵＳＥｓｓ−ＣＨＩｇ−ｈＧ１およびｐＦＵＳＥ２ｓｓ−ＣＬＩｇ−ｈｋを得た。ｐＦＵＳＥｓｓ−ＣＨＩｇ−ｈＧ１にＳＶ４０プロモーター、ｄｈｆｒおよびＳＶ４０のＰｏｌｙＡを組込んだプラスミドをｐＦＵ−ＣＨＩｇ−ｄｈｆｒと命名し、ｐＦＵＳＥ２ｓｓ−ＣＬＩｇ−ｈｋにＳＶ４０プロモーター、ｄｈｆｒおよびＳＶ４０のＰｏｌｙＡを組込んだプラスミドをｐＦＵ−ＣＬＩｇ−ｄｈｆｒと命名した。 (3) pFUSEss-CHIg-hG1 (manufactured by InvivoGen) containing a heavy chain constant region of a human antibody, pFUSE2ss-CLIg-hk (manufactured by InvivoGen) containing a human antibody light chain constant region, and (2) The digested dhfr-P2 was digested with restriction enzymes NotI and PvuI, respectively, and then purified and ligated. Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain pFUSEss-CHIg-hG1 and pFUSE2ss-CLIg-hk containing SV40 promoter, dhfr, and PolyA of SV40. A plasmid in which SV40 promoter, dhfr and PolyA of SV40 were incorporated into pFUSEss-CHIg-hG1 was named pFU-CHIg-dhfr, and a plasmid in which SV40 promoter, dhfr and PolyA of SV40 were incorporated into pFUSE2ss-CLIg-hk was pFU. It was named -CLIg-dhfr.
（４）配列番号６に記載のアミノ酸配列からなる抗インターロイキン６レセプター（以下、ＩＬ−６Ｒ）抗体の重鎖可変領域をコードする配列番号７に記載のポリヌクレオチドの５’末端に制限酵素ＥｃｏＲＩ認識配列（ＧＡＡＴＴＣ）とフレームシフト抑制のためグアニン（Ｇ）を付加し、３’末端に制限酵素ＮｈｅＩ認識配列（ＧＣＴＡＧＣ）を付加した遺伝子を全合成しプラスミドにクローニングした（ＦＡＳＭＡＣ社に委託）。作製したプラスミドをｐＵＣ−ＶＨ６Ｒと命名した。また、配列番号８に記載のアミノ酸配列からなる抗ＩＬ−６Ｒ抗体の軽鎖可変領域をコードする配列番号９に記載のポリヌクレオチドの５‘末端に制限酵素ＥｃｏＲＩ認識配列（ＧＡＡＴＴＣ）とフレームシフト抑制のためグアニン（Ｇ）を付加し、３’末端に制限酵素ＢｓｉＷＩ認識配列（ＣＧＴＡＣＧ）を付加した遺伝子を全合成しプラスミドにクローニングした（ＦＡＳＭＡＣ社に委託）。作製したプラスミドをｐＵＣ−ＶＬ６Ｒと命名した。 (4) Restriction enzyme EcoRI at the 5'end of the polynucleotide of SEQ ID NO: 7 encoding the heavy chain variable region of the anti-interleukin 6 receptor (hereinafter IL-6R) antibody consisting of the amino acid sequence of SEQ ID NO: 6 A gene having a recognition sequence (GAATTC) and guanine (G) added to suppress frame shift and a restriction enzyme NheI recognition sequence (GCTAGC) added to the 3'end was totally synthesized and cloned into a plasmid (outsourced to FASMAC). The prepared plasmid was named pUC-VH6R. In addition, a restriction enzyme EcoRI recognition sequence (GAATTC) and a frame shift inhibitor are added to the 5'end of the polynucleotide shown in SEQ ID NO: 9 encoding the light chain variable region of the anti-IL-6R antibody consisting of the amino acid sequence shown in SEQ ID NO: 8. Therefore, a gene in which guanine (G) was added and a restriction enzyme BsiWI recognition sequence (CGTACG) was added to the 3'end was totally synthesized and cloned into a plasmid (outsourced to FASMAC). The prepared plasmid was named pUC-VL6R.
（５）（４）で作製したｐＵＣ−ＶＨ６Ｒおよび（３）で作製したｐＦＵ−ＣＨＩｇ−ｄｈｆｒをそれぞれ制限酵素ＥｃｏＲＩ、ＮｈｅＩで消化後、精製し、ライゲーションした。当該ライゲーション産物で大腸菌ＪＭ１０９株を形質転換し、培養した形質転換体からプラスミドを抽出することで、抗ＩＬ−６Ｒ抗体の重鎖（Ｈ鎖）を発現するプラスミドｐＦＵ−６ＲＨ−ｄｈｆｒを得た。また（４）で作製したｐＵＣ−ＶＬ６Ｒおよび（３）で作製したｐＦＵ−ＣＬＩｇ−ｄｈｆｒをそれぞれ制限酵素ＥｃｏＲＩ、ＢｓｉＷＩで消化後、精製し、ライゲーションした。当該ライゲーション産物で大腸菌ＪＭ１０９株を形質転換し、培養した形質転換体からプラスミドを抽出することで、抗ＩＬ−６Ｒ抗体の軽鎖（Ｌ鎖）を発現するプラスミドｐＦＵ−６ＲＬ−ｄｈｆｒを得た。 (5) The pUC-VH6R prepared in (4) and the pFU-CHIg-dhfr prepared in (3) were digested with restriction enzymes EcoRI and NheI, respectively, purified, and ligated. Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain a plasmid pFU-6RH-dhfr expressing the heavy chain (H chain) of the anti-IL6R antibody. Further, pUC-VL6R prepared in (4) and pFU-CLIg-dhfr prepared in (3) were digested with restriction enzymes EcoRI and BsiWI, respectively, purified, and ligated. Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain a plasmid pFU-6RL-dhfr expressing the light chain (L chain) of the anti-IL-6R antibody.
実施例２ 抗ＩＬ−６Ｒ抗体高発現細胞の構築
（１）実施例１で作製したｐＦＵ−６ＲＨ−ｄｈｆｒおよびｐＦＵ−６ＲＬ−ｄｈｆｒを、ＣＨＯ細胞（ＤＧ４４株）にＮｅｏｎ Ｔｒａｎｓｆｅｃｔｉｏｎ Ｓｙｓｔｅｍ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）を用いて遺伝子導入した。その後、５０μｇ／ｍＬのカナマイシン、４０ｍＬ／ＬのＧｌｕｔａＭＡＸ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）を含んだＣＤ ＯｐｔｉＣＨＯ Ｍｅｄｉｕｍ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）で形質転換細胞を培養抗ＩＬ−６Ｒ抗体発現細胞を得た。その後、培地に５０ｎｇ／ｍＬのメトトレキサート
（ＭＴＸ）を添加することで遺伝子増幅を行なった。
Example 2 Construction of cells with high expression of anti-IL-6R antibody (1) pFU-6RH-dhfr and pFU-6RL-dhfr prepared in Example 1 were used as CHO cells (DG44 strain) in the Neoon Transfection System (Thermo Fisher Scientific). Gene transfer was carried out using Then, transformed cells were cultured with CD OptiCHO Medium (manufactured by Thermo Fisher Scientific) containing 50 μg/mL kanamycin and 40 mL/L GlutaMAX (manufactured by Thermo Fisher Scientific) to obtain anti-IL-6R antibody-expressing cells. Then, gene amplification was performed by adding 50 ng/mL methotrexate (MTX) to the medium.
（２）（１）でＭＴＸ処理をした細胞を限外希釈法により単クローン化し、下記に記載のＥＬＩＳＡ（Ｅｎｚｙｍｅ−Ｌｉｎｋｅｄ ＩｍｍｕｎｏＳｏｒｂｅｎｔ Ａｓｓａｙ）を用いて、抗ＩＬ−６Ｒ抗体を安定的に高生産可能な細胞を選択した。
（２−１）抗ヒトＦａｂ抗体（Ｂｅｔｈｙｌ社製）を、９６穴マイクロプレートのウェルに１μｇ／ｗｅｌｌで固定化した（４℃で一晩）。固定化終了後、２％（ｗ／ｖ）のＳＫＩＭ ＭＩＬＫ（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ社製）および１５０ｍＭ塩化ナトリウムを含んだ２０ｍＭのトリス塩酸緩衝液（ｐＨ７．４）によりブロッキングした。
（２−２）洗浄緩衝液（０．０５％［ｗ／ｖ］のＴｗｅｅｎ ２０（商品名）と１５０ｍＭのＮａＣｌとを含む２０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ８．０））で洗浄後、抗体を含んだ培養上清を添加し、抗体と固定化タンパク質とを反応させた（３０℃で１時間）。
（２−３）反応終了後、前記洗浄緩衝液で洗浄し、１００ｎｇ／ｍＬに希釈したペルオキシターゼで標識された抗ヒトＦｃ抗体（Ｂｅｔｈｙｌ社製）を１００μＬ／ｗｅｌｌで添加した。
（２−４）３０℃で１時間反応し、前記洗浄緩衝液で洗浄した後、ＴＭＢ Ｐｅｒｏｘｉｄａｓｅ Ｓｕｂｓｔｒａｔｅ（ＫＰＬ社製）を５０μＬ／ｗｅｌｌで添加した。その後、１Ｍのリン酸を５０μＬ／ｗｅｌｌで添加することで発色を止め、マイクロプレートリーダー（テカン社製）を用いて４５０ｎｍの吸光度を測定し、測定値の高い抗ＩＬ−６Ｒ抗体高生産細胞株を選択した。
(2) The cells treated with MTX in (1) can be monocloned by the limiting dilution method, and stable high production of anti-IL-6R antibody can be achieved by using the following ELISA (Enzyme-Linked Immunosorbent Assay). Cells were selected.
(2-1) An anti-human Fab antibody (manufactured by Bethyl) was immobilized in wells of a 96-well microplate at 1 μg/well (4° C. overnight). After completion of immobilization, blocking was performed with 20 mM Tris-HCl buffer (pH 7.4) containing 2% (w/v) SKIM MILK (manufactured by Becton Dickinson) and 150 mM sodium chloride.
(2-2) After washing with a washing buffer (20 mM Tris-HCl buffer (pH 8.0) containing 0.05% [w/v] Tween 20 (trade name) and 150 mM NaCl), the antibody was washed. The containing culture supernatant was added to react the antibody with the immobilized protein (30° C. for 1 hour).
(2-3) After the reaction was completed, the reaction mixture was washed with the washing buffer, and an anti-human Fc antibody labeled with peroxidase (manufactured by Bethyl) diluted to 100 ng/mL was added at 100 μL/well.
(2-4) After reacting at 30° C. for 1 hour and washing with the washing buffer, TMB Peroxidase Substrate (manufactured by KPL) was added at 50 μL/well. After that, color development was stopped by adding 1 M phosphoric acid at 50 μL/well, the absorbance at 450 nm was measured using a microplate reader (manufactured by Tecan), and a cell line with high production of anti-IL-6R antibody was measured. Was selected.
（３）ＭＴＸ濃度を段階的（５０ｎＭ、５００ｎＭ、１μＭ、２μＭ、４μＭ、８μＭ、１６μＭ、３２μＭ、６４μＭ）に上昇させながら、限外希釈を行ない（２）に記載のＥＬＩＳＡでクローン選択を行なうことを繰り返した。その結果、抗ＩＬ−６Ｒ抗体高生産細胞株を得た。 (3) Perform clonal selection by the ELISA described in (2) while increasing the MTX concentration stepwise (50 nM, 500 nM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM). Was repeated. As a result, a cell line with high production of anti-IL-6R antibody was obtained.
実施例３ 抗ヒトｇｐ１３０受容体（ｇｐ１３０Ｒ）抗体高発現細胞の構築
（１）配列番号１０に記載のアミノ酸配列からなる抗ヒトｇｐ１３０受容体（ｇｐ１３０Ｒ）抗体の重鎖可変領域をコードする配列番号１１に記載のポリヌクレオチドの５’末端に制限酵素ＥｃｏＲＩ認識配列（ＧＡＡＴＴＣ）とフレームシフト抑制のためグアニン（Ｇ）を付加し、３’末端に制限酵素ＮｈｅＩ認識配列（ＧＣＴＡＧＣ）を付加した遺伝子を全合成しプラスミドにクローニングした（ＦＡＳＭＡＣ社に委託）。作製したプラスミドをｐＵＣ−ＶＨｇｐ１３０と命名した。また、配列番号１２に記載のアミノ酸配列からなる抗ヒトｇｐ１３０Ｒ抗体の軽鎖可変領域をコードする配列番号１３に記載のポリヌクレオチドの５’末端に制限酵素ＥｃｏＲＩ認識配列（ＧＡＡＴＴＣ）とフレームシフト抑制のためグアニン（Ｇ）を付加し、３’末端に制限酵素ＢｓｉＷＩ認識配列（ＣＧＴＡＣＧ）を付加した遺伝子を全合成しプラスミドにクローニングした（ＦＡＳＭＡＣ社に委託）。作製したプラスミドをｐＵＣ−ＶＬｇｐ１３０と命名した。
Example 3 Construction of anti-human gp130 receptor (gp130R) antibody highly expressing cells (1) SEQ ID NO: 11 encoding heavy chain variable region of anti-human gp130 receptor (gp130R) antibody having the amino acid sequence set forth in SEQ ID NO: 10 The polynucleotide having the restriction enzyme EcoRI recognition sequence (GAATTC) and guanine (G) added for frameshift suppression at the 5'end and the 3'terminal addition of the restriction enzyme NheI recognition sequence (GCTAGC) at the 5'end Synthesized and cloned into a plasmid (outsourced to FASMAC). The prepared plasmid was designated as pUC-VHgp130. In addition, a restriction enzyme EcoRI recognition sequence (GAATTC) at the 5'end of the polynucleotide shown in SEQ ID NO: 13 encoding the light chain variable region of the anti-human gp130R antibody consisting of the amino acid sequence shown in SEQ ID NO: 12 and a frameshift inhibitor Therefore, a gene in which guanine (G) was added and a restriction enzyme BsiWI recognition sequence (CGTACG) was added to the 3'end was totally synthesized and cloned into a plasmid (outsourced to FASMAC). The prepared plasmid was named pUC-VLgp130.
（２）（１）で作製したｐＵＣ−ＶＨｇｐ１３０および実施例１（３）で作製したｐＦＵ−ＣＨＩｇ−ｄｈｆｒをそれぞれ制限酵素ＥｃｏＲＩ、ＮｈｅＩで消化後、精製し、ライゲーションした。当該ライゲーション産物で大腸菌ＪＭ１０９株を形質転換し、培養した形質転換体からプラスミドを抽出することで、抗ヒトｇｐ１３０Ｒ抗体の重鎖（Ｈ鎖）を発現するプラスミドｐＦＵ−ｇｐ１３０Ｈ−ｄｈｆｒを得た。また（１）で作製したｐＵＣ−ＶＬｇｐ１３０および実施例１（３）で作製したｐＦＵ−ＣＬＩｇ−ｄｈｆｒをそれぞれ制限酵素ＥｃｏＲＩ、ＢｓｉＷＩで消化後、精製し、ライゲーションした。当該ライゲーション産物で大腸菌ＪＭ１０９株を形質転換し、培養した形質転換体からプラスミドを抽出することで、抗ヒトｇｐ１３０Ｒ抗体の軽鎖（Ｌ鎖）を発現するプラスミドｐＦＵ−ｇｐ１３０Ｌ−ｄｈｆｒを得た。 (2) pUC-VHgp130 produced in (1) and pFU-CHIg-dhfr produced in Example 1(3) were digested with restriction enzymes EcoRI and NheI, respectively, purified, and ligated. Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain a plasmid pFU-gp130H-dhfr expressing the heavy chain (H chain) of the anti-human gp130R antibody. In addition, pUC-VLgp130 prepared in (1) and pFU-CLIg-dhfr prepared in Example 1 (3) were digested with restriction enzymes EcoRI and BsiWI, respectively, purified, and ligated. Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain a plasmid pFU-gp130L-dhfr expressing the light chain (L chain) of the anti-human gp130R antibody.
（３）（２）で得られたｐＦＵ−ｇｐ１３０Ｌ−ｄｈｆｒを鋳型として、配列番号１４（５’−ＧＣＣＴＣＴＴＣＣＣＧＧＧＣＣＧＡＧＣＴＧＧＴＧＣＴＧＡＣＴＣ−３’）および配列番号１５（５’−ＡＡＴ［ＧＣＧＧＣＣＧＣ］ＴＡＣＴＡＡＣＡＣＴＣＴＣＣＣＣＴＧＴＴＧＡＡＧＣ−３’）（配列番号１５中の角かっこは制限酵素ＮｏｔＩ認識配列を示している）に記載の配列からなるオリゴヌクレオチドプライマーを用いてＰＣＲを行なった。具体的には、表３に示す組成の反応液を調製し、当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１５分間の第３ステップを１サイクルとする反応を３０サイクル繰り返すことで実施した。このＰＣＲにより抗ヒトｇｐ１３０Ｒ抗体のＬ鎖全長をコードする遺伝子を増幅し、精製後に得られたＰＣＲ産物をｈｇｐ１３０−Ｌ１と命名した。 (3) Using the pFU-gp130L-dhfr obtained in (2) as a template, SEQ ID NO: 14 (5'-GCCTCTTCCCGGGCCGAGCTGGGTGCTGAACTC-3') and SEQ ID NO: 15 (5'-AAT[GCGGCCGC]TACTAACACTCTCCCCCTGTGAAGC-3'). PCR was carried out using an oligonucleotide primer having the sequence described in "(15) shows the restriction enzyme NotI recognition sequence". Specifically, a reaction solution having the composition shown in Table 3 was prepared, and the reaction solution was heat-treated at 98° C. for 5 minutes, then at 98° C. for 10 seconds in the first step, and at 55° C. for 5 seconds in the second step. The reaction was carried out by repeating 30 cycles of the third step for 1 minute at 15° C. for 30 cycles. A gene encoding the full-length L chain of the anti-human gp130R antibody was amplified by this PCR, and the PCR product obtained after purification was named hgp130-L1.
（４）（３）で得られたｈｇｐ１３０−Ｌ１を鋳型として、配列番号１６（５’−ＣＴＡ［ＧＡＡＴＴＣ］ＧＣＣＡＣＣＡＴＧＡＣＣＣＧＧＣＴＧＡＣＣ−３’）（配列番号１６中の角かっこは制限酵素ＥｃｏＲＩ認識配列を示している）および配列番号１５に記載の配列からなるオリゴヌクレオチドプライマーを用いて、配列番号１７に記載のアミノ酸配列からなるシグナル配列（ＭＴＲＬＴＶＬＡＬＬＡＧＬＬＡＳＳＲＡ）を付加するため、配列番号１７に記載のアミノ酸配列をコードする配列番号１８（５’−ＡＴＧＡＣＣＣＧＧＣＴＧＡＣＣＧＴＧＣＴＧＧＣＣＣＴＧＣＴＧＧＣＴＧＧＣＣＴＧＣＴＣＧＣＣＴＣＴＴＣＣＣＧＧＧＣＣ−３’）に記載のポリヌクレオチドを添加してＰＣＲを行なった。具体的には、表４に示す組成の反応液を調製し、当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１．５分間の第３ステップを１サイクルとする反応を３０サイクル繰り返すことで実施した。このＰＣＲにより、抗ヒトｇｐ１３０Ｒ抗体のＬ鎖全長をコードする遺伝子に配列番号１７に記載のシグナル配列をコードする遺伝子を付加した遺伝子を増幅し、精製後に得られたＰＣＲ産物をｈｇｐ１３０−Ｌ２と命名した。 (4) Using the hgp130-L1 obtained in (3) as a template, SEQ ID NO: 16 (5'-CTA [GAATTC] GCCACCCATGACCCGGCTGACC-3') (the brackets in SEQ ID NO: 16 indicate the restriction enzyme EcoRI recognition sequence). And a signal sequence (MTRLTVLLALLGLASSRA) consisting of the amino acid sequence of SEQ ID NO: 17 is added using an oligonucleotide primer consisting of the sequence of SEQ ID NO: 15 to encode the amino acid sequence of SEQ ID NO: 17. PCR was carried out by adding the polynucleotide described in SEQ ID NO: 18 (5′-ATGACCCCGGCTGACCGTGCTGGCCCCTGCTGCTGGCCTGTCCGCCCTCTCCCGGGCC-3′). Specifically, a reaction solution having the composition shown in Table 4 was prepared, and the reaction solution was heat-treated at 98° C. for 5 minutes, then at 98° C. for 10 seconds in the first step, and at 55° C. for 5 seconds in the second step. The reaction was carried out by repeating 30 cycles of the third step for 1 minute at 1.5° C. for 30 cycles. A gene obtained by adding the gene encoding the signal sequence of SEQ ID NO: 17 to the gene encoding the full-length L chain of the anti-human gp130R antibody was amplified by this PCR, and the PCR product obtained after purification was named hgp130-L2. did.
（５）（４）で得られたｈｇｐ１３０−Ｌ２および図１に記載の発現ベクターｐＥＦｄをそれぞれ制限酵素ＥｃｏＲＩおよびＮｏｔＩで消化し、精製後ライゲーションした。当該ライゲーション産物で大腸菌ＪＭ１０９株を形質転換し、培養した形質転換体からプラスミドを抽出することで哺乳動物細胞において抗ヒトｇｐ１３０Ｒ抗体のＬ鎖を発現可能なｐＥＦｄ−ｇｐ１３０Ｌを得た。なお、図１に記載のｐＥＦｄベクターは配列番号１９に記載のＥＦ１αプロモーターのうち６２１番目のグアニンから７９８番目のグアニンまでのヌクレオチドが欠損しているプロモーター（ＥＦｄ ｐｒｏ）を有しており、ＳＶ４０のｐｏｌｙＡおよびｄｈｆｒ遺伝子も有している（特開２０１９−１０６９７６号公報）。 (5) The hgp130-L2 obtained in (4) and the expression vector pEFd shown in FIG. 1 were digested with restriction enzymes EcoRI and NotI, respectively, and purified and ligated. Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain pEFd-gp130L capable of expressing the L chain of the anti-human gp130R antibody in mammalian cells. The pEFd vector shown in FIG. 1 has a promoter (EFd pro) in which the nucleotides from the 621st guanine to the 798th guanine of the EF1α promoter shown in SEQ ID NO: 19 are deleted, and the pEFd vector of SV40 It also has polyA and dhfr genes (JP-A-2019-106976).
（６）（２）で得られたｐＦＵ−ｇｐ１３０Ｈ−ｄｈｆｒを鋳型として、配列番号２０（５’−ＧＣＣＴＣＴＴＣＣＣＧＧＧＣＣＣＡＧＧＴＴＣＡＡＣＴＣＣＡＧ−３’）および配列番号２１（５’−ＡＡＴ［ＧＣＧＧＣＣＧＣ］ＴＡＴＣＡＴＴＴＡＣＣＣＧＧＡＧＡＣＡＧＧＧＡＧＡＧ−３’）（配列番号２１中の角かっこは制限酵素ＮｏｔＩ認識配列を示している）に記載の配列からなるオリゴヌクレオチドプライマーを用いてＰＣＲを行なった。具体的には、表３に示す組成の反応液を調製し、当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１．５分間の第３ステップを１サイクルとする反応を３０サイクル繰り返すことで実施した。このＰＣＲにより、抗ヒトｇｐ１３０Ｒ抗体のＨ鎖全長をコードする遺伝子を増幅し精製後に得られたＰＣＲ産物をｈｇｐ１３０−Ｈ１と命名した。 (6) SEQ ID NO: 20 (5′-GCCCTTCCCCGGGCCCAGGTTCAACTCCAG-3′) and SEQ ID NO: 21 (5′-AAT[GCGGCCGC]TATCATTTTACCCGGAGACAGGGAGAG-3′) using the pFU-gp130H-dhfr obtained in (2) as a template. PCR was carried out using an oligonucleotide primer having the sequence described in (21) indicates the restriction enzyme NotI recognition sequence. Specifically, a reaction solution having the composition shown in Table 3 was prepared, and the reaction solution was heat-treated at 98° C. for 5 minutes, then at 98° C. for 10 seconds in the first step, and at 55° C. for 5 seconds in the second step. The reaction was carried out by repeating 30 cycles of the third step for 1 minute at 1.5° C. for 30 cycles. The PCR product obtained by amplifying the gene encoding the full-length H chain of the anti-human gp130R antibody by this PCR and purifying was named hgp130-H1.
（７）（６）で得られたｈｇｐ１３０−Ｈ１を鋳型として、配列番号１６および配列番号２１に記載の配列からなるオリゴヌクレオチドプライマーを用いて、配列番号１７に記載のアミノ酸配列からなるシグナル配列を付加するため、配列番号１８に記載のポリヌクレオチドを添加してＰＣＲを行なった。具体的には、表４に示す組成の反応液を調製し、当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１．５分間の第３ステップを１サイクルとする反応を３０サイクル繰り返すことで実施した。このＰＣＲにより、抗ヒトｇｐ１３０Ｒ抗体のＨ鎖全長をコードする遺伝子に配列番号１７に記載のシグナル配列をコードする遺伝子を付加した遺伝子を増幅し、精製後に得られたＰＣＲ産物をｈｇｐ１３０−Ｈ２と命名した。 (7) Using the hgp130-H1 obtained in (6) as a template and an oligonucleotide primer consisting of the sequences set forth in SEQ ID NO: 16 and SEQ ID NO: 21, a signal sequence consisting of the amino acid sequence set forth in SEQ ID NO: 17 was obtained. To add, the polynucleotide of SEQ ID NO: 18 was added and PCR was performed. Specifically, a reaction solution having the composition shown in Table 4 was prepared, and the reaction solution was heat-treated at 98° C. for 5 minutes, then at 98° C. for 10 seconds in the first step, and at 55° C. for 5 seconds in the second step. The reaction was carried out by repeating 30 cycles of the third step for 1 minute at 1.5° C. for 30 cycles. A gene obtained by adding the gene encoding the signal sequence of SEQ ID NO: 17 to the gene encoding the full-length H chain of the anti-human gp130R antibody was amplified by this PCR, and the PCR product obtained after purification was named hgp130-H2. did.
（８）（７）で得られたｈｇｐ１３０−Ｈ２および図１に記載の発現ベクターｐＥＦｄをそれぞれ制限酵素ＥｃｏＲＩおよびＮｏｔＩで消化し、精製後ライゲーションした。当該ライゲーション産物で大腸菌ＪＭ１０９株を形質転換し、培養した形質転換体からプラスミドを抽出することで哺乳動物細胞において抗ヒトｇｐ１３０Ｒ抗体を発現可能なｐＥＦｄ−ｇｐ１３０Ｈを得た。 (8) The hgp130-H2 obtained in (7) and the expression vector pEFd shown in FIG. 1 were digested with restriction enzymes EcoRI and NotI, respectively, and purified and ligated. Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain pEFd-gp130H capable of expressing anti-human gp130R antibody in mammalian cells.
（９）（５）で得られたｐＥＦｄ−ｇｐ１３０Ｌ、（８）で得られたｐＥＦｄ−ｇｐ１３０Ｈを用いた以外は実施例２と同様の方法で高発現細胞の構築を行ない、ＭＴＸ濃度を５０ｎＭ、２５０ｎＭ、１μＭと段階的にあげることで抗ヒトｇｐ１３０Ｒ抗体高発現細胞を構築した。 (9) High expression cells were constructed in the same manner as in Example 2 except that pEFd-gp130L obtained in (5) and pEFd-gp130H obtained in (8) were used, and MTX concentration was 50 nM. Anti-human gp130R antibody high expressing cells were constructed by stepwise increasing to 250 nM and 1 μM.
実施例４ フラスコ培養におけるマンガンイオン添加の効果（その１）
（１）実施例２で得られた抗ＩＬ−６Ｒ抗体高発現細胞または実施例３で得られた抗ヒトｇｐ１３０Ｒ抗体高発現細胞を、５０μｇ／ｍＬのカナマイシン、４０ｍＬ／ＬのＧｌｕｔａＭＡＸ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）を含む２０ｍＬのＢａｌａｎＣＤ ＣＨＯ Ｇｒｏｗｔｈ Ａ ｍｅｄｉｕｍ（Ｉｒｖｉｎｅ Ｓｃｉｅｎｔｉｆｉｃ社製）を入れた１２５ｍＬの三角フラスコ（Ｃｏｒｎｉｎｇ社製）に接種し、１３０ｒｐｍ、３７℃、８％ＣＯ２の条件下で振盪培養した。
Example 4 Effect of Manganese Ion Addition on Flask Culture (Part 1)
(1) The anti-IL-6R antibody high-expressing cells obtained in Example 2 or the anti-human gp130R antibody high-expressing cells obtained in Example 3 were mixed with 50 μg/mL kanamycin and 40 mL/L GlutaMAX (Thermo Fisher Scientific). (Manufactured by the company) containing 20 mL of BalanCD CHO Growth A medium (manufactured by Irvine Scientific) was inoculated into a 125 mL Erlenmeyer flask (manufactured by Corning), and shake-cultured under conditions of 130 rpm, 37° C. and 8% CO 2 . ..
（２）細胞数が１×１０６ｃｅｌｌｓ／ｍＬ以上になったところで、５０μｇ／ｍＬのカナマイシン、４０ｍＬ／ＬのＧｌｕｔａＭＡＸを含んだＢａｌａｎＣＤ ＣＨＯ Ｇｒｏｗｔｈ Ａ ｍｅｄｉｕｍに０．２×１０６ｃｅｌｌｓ／ｍＬとなるように前記細胞を接種し、フィルター滅菌した５０ｍＭの硫酸マンガンを終濃度で０μＭ（添加せず）、０．０１μＭ、０．１μＭ、１μＭ、５μＭ、１０μＭもしくは５０μＭとなるように添加し、総量を２０ｍＬに調製して１２５ｍＬの三角フラスコで、１３０ｒｐｍ、３７℃、８％ＣＯ２の条件下で１０日間、振盪培養した。培養途中で培養液をサンプリングし、生細胞数をＣｏｕｎｔｅｓｓ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）を使用して測定し、抗体生産性はヒトＩｇＧ１（シグマ社製）を標準品とした検量線に基づき実施例２（２）に記載のＥＬＩＳＡ法にて測定した。 (2) When the cell number becomes 1×10 6 cells/mL or more, 0.2×10 6 cells/mL is obtained in BalanCD CHO Growth A medium containing 50 μg/mL kanamycin and 40 mL/L GlutaMAX. The above cells were inoculated as described above, and 50 mM manganese sulfate filter-sterilized was added to a final concentration of 0 μM (no addition), 0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM or 50 μM, and the total amount was added. It was adjusted to 20 mL and shake-cultured in a 125 mL Erlenmeyer flask for 10 days under the conditions of 130 rpm, 37° C. and 8% CO 2 . The culture solution was sampled during the culture, and the number of viable cells was measured using Countess (Thermo Fisher Scientific), and the antibody productivity was determined based on a calibration curve using human IgG1 (Sigma) as a standard example. It was measured by the ELISA method described in 2(2).
（３）培養終了後の培養液を遠心分離によって細胞および不純物を除去し、得られた上清を、１．０ｍＬのＭａｂＳｅｌｅｃｔ ＳｕＲｅ ＬＸ（ＧＥヘルスケア社製）をオープンカラムに詰めて作製した分離カラム（１５０ｍＭの塩化ナトリウムを含んだ２０ｍＭのＴｒｉｓ−ＨＣｌ（ｐＨ７．４）で平衡化済）にアプライした。 (3) Cells and impurities were removed from the culture solution after completion of the culture by centrifugation, and the obtained supernatant was prepared by packing 1.0 mL of MabSelect SuRe LX (manufactured by GE Healthcare) into an open column. It was applied to a column (equilibrated with 20 mM Tris-HCl (pH 7.4) containing 150 mM sodium chloride).
（４）前記平衡化に用いた緩衝液１０ｍＬで前記分離カラムを洗浄後、０．１Ｍのグリシン塩酸緩衝液（ｐＨ３．０）４ｍＬで前記分離カラムに吸着した抗体を溶出した。溶出液に１ｍＬの１Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）を加えることでｐＨを中性領域に戻し、限外ろ過膜で濃縮しながら５０ｍＭのクエン酸緩衝液（ｐＨ６．５）に緩衝液交換することで、培地に添加したマンガンイオン濃度が異なる、高純度な抗ＩＬ−６Ｒ抗体および抗ヒトｇｐ１３０Ｒ抗体を得た。 (4) After washing the separation column with 10 mL of the buffer used for the equilibration, the antibody adsorbed on the separation column was eluted with 4 mL of 0.1 M glycine-hydrochloric acid buffer (pH 3.0). The pH is returned to the neutral range by adding 1 mL of 1 M Tris-HCl (pH 8.0) to the eluate, and the buffer is exchanged with a 50 mM citrate buffer (pH 6.5) while concentrating with an ultrafiltration membrane. Thus, high-purity anti-IL-6R antibody and anti-human gp130R antibody having different manganese ion concentrations added to the medium were obtained.
（５）ヒトＦｃγＲＩＩＩａを固定化した担体（分離剤）を詰めたカラム（ＦｃＲ９＿Ｆカラム、ＷＯ２０１８／１５０９７３号の実施例５に記載）を用いて、下記の方法により（４）で得られた抗ＩＬ−６Ｒ抗体および抗ヒトｇｐ１３０Ｒ抗体を分析した。
（５−１）ＦｃＲ９＿Ｆカラムを高速液体クロマトグラフィー装置（島津製作所社製）に接続し、カラムオーブンで前記カラムを２５℃の恒温状態に維持し、５０ｍＭのクエン酸緩衝液（ｐＨ６．５）を流速１．０ｍＬ／ｍｉｎで１０分間流すことにより前記カラムを平衡化した。
（５−２）（４）で得た抗ＩＬ−６Ｒ抗体および抗ヒトｇｐ１３０Ｒ抗体を（５−１）で用いた緩衝液で１．０ｍｇ／ｍＬに希釈し、当該希釈抗体溶液を流速１．０ｍＬ／ｍｉｎ
にて１０μＬ添加した。
（５−３）（５−１）で用いた緩衝液を流速１．０ｍＬ／ｍｉｎで２分間流した後、５０
ｍＭのクエン酸緩衝液（ｐＨ４．５）によるｐＨグラジエント（１８分間で５０ｍＭのク
エン酸緩衝液（ｐＨ４．５）が１００％となるグラジエント）でＦｃＲ９＿Ｆカラムに吸
着した抗体を溶出した。
(5) Using a column (FcR9_F column, described in Example 5 of WO2018/150973) packed with a carrier (separating agent) on which human FcγRIIIa is immobilized, the anti-IL obtained in (4) by the following method The -6R antibody and anti-human gp130R antibody were analyzed.
(5-1) The FcR9_F column was connected to a high performance liquid chromatography apparatus (manufactured by Shimadzu Corporation), the column was kept at a constant temperature of 25° C. in a column oven, and a 50 mM citrate buffer solution (pH 6.5) was used. The column was equilibrated by flowing at a flow rate of 1.0 mL/min for 10 minutes.
(5-2) The anti-IL-6R antibody and the anti-human gp130R antibody obtained in (4) were diluted to 1.0 mg/mL with the buffer solution used in (5-1), and the diluted antibody solution was flowed at a flow rate of 1. 0 mL/min
Then, 10 μL was added.
(5-3) After flowing the buffer solution used in (5-1) at a flow rate of 1.0 mL/min for 2 minutes, 50
The antibody adsorbed on the FcR9_F column was eluted with a pH gradient of mM citrate buffer (pH 4.5) (gradient at which 50 mM citrate buffer (pH 4.5) was 100% in 18 minutes).
培地中に添加したマンガンイオン濃度の違いによる、抗ＩＬ−６Ｒ抗体の生産性を比較した結果を図２に、抗ＩＬ−６Ｒ抗体高発現細胞数（生細胞数）の推移を図３に、抗ＩＬ−６Ｒ抗体のＦｃＲ９＿Ｆカラムによる分析結果を図４に、抗ｇｐ１３０Ｒ抗体の生産性を比較した結果を図５に、抗ｇｐ１３０Ｒ抗体高発現細胞数（生細胞数）の推移を図６に、抗ＩＬ−６Ｒ抗体のＦｃＲ９＿Ｆカラムによる分析結果を図７に、それぞれ示す。また図４に記載の分析結果にある各ピークの面積割合を表５に、図７に記載の分析結果にある各ピークの面積割合を表６に、それぞれ示す。 The results of comparing the productivity of the anti-IL6R antibody due to the difference in the manganese ion concentration added to the medium are shown in FIG. 2, and the transition of the number of highly expressing anti-IL-6R antibody cells (the number of viable cells) is shown in FIG. The results of analysis of the anti-IL-6R antibody by the FcR9_F column are shown in FIG. 4, the results of comparison of the productivity of the anti-gp130R antibody are shown in FIG. 5, the transition of the number of high-expressing anti-gp130R antibody cells (viable cell number) is shown in FIG. The analysis results of the anti-IL-6R antibody by the FcR9_F column are shown in FIG. 7, respectively. The area ratio of each peak in the analysis result shown in FIG. 4 is shown in Table 5, and the area ratio of each peak in the analysis result shown in FIG. 7 is shown in Table 6.
表５および表６より、抗ＩＬ−６Ｒ抗体高発現細胞、抗ｇｐ１３０Ｒ抗体高発現細胞数とも、培地に０．０１μＭから５０μＭのマンガンイオンを添加して培養すると、マンガンイオン未添加時と比較し、ＦｃＲ９＿Ｆカラムとの結合力が強いピーク（ピーク３）の割合が増加していることがわかる。ＡＤＣＣ活性の高い抗体ほど、ＦｃγＲＩＩＩａ固定化分離剤との結合力が強まる（特開２０１６−０２３１５２号公報、ＷＯ２０１８／１５０９７３号）。すなわち図４および図７におけるピーク３の示す割合が増加する。このことから、抗体を発現可能な哺乳動物細胞を培養する際、０．０１μＭから５０μＭ（特に０．１μＭから５０μＭ）のマンガンイオンを添加した培地で培養することで、前記哺乳細胞が発現する抗体が有するＡＤＣＣ活性が向上することがわかる。 From Table 5 and Table 6, both anti-IL-6R antibody high-expressing cells and anti-gp130R antibody high-expressing cell numbers were compared with those when no manganese ion was added when the culture was performed by adding 0.01 μM to 50 μM manganese ion to the medium. It can be seen that the ratio of peaks (peak 3) having strong binding force with the FcR9_F column is increasing. The higher the ADCC activity of the antibody, the stronger the binding strength to the FcγRIIIa-immobilized separating agent (JP-A-2016-023152, WO2018/150973). That is, the ratio of peak 3 in FIGS. 4 and 7 increases. Therefore, when culturing mammalian cells capable of expressing antibodies, the antibodies expressed by the mammalian cells can be obtained by culturing in a medium containing manganese ions of 0.01 μM to 50 μM (particularly 0.1 μM to 50 μM). It can be seen that the ADCC activity possessed by is improved.
実施例５ フラスコ培養におけるマンガンイオン添加の効果（その２）
使用した培地をＣＤ Ｏｐｔｉ ＣＨＯ Ｍｅｄｉｕｍ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）とし、培養期間を１１日間とした他は、実施例４と同様の方法を用いて、実施例２で得られた抗ＩＬ−６Ｒ抗体高発現細胞、および実施例３で得られた抗ヒトｇｐ１３０Ｒ抗体高発現細胞を培養し、抗体生産性および生細胞数の測定、ならびに得られた培養物中に含まれる前記細胞が発現した抗体のＦｃＲ９＿Ｆカラムによる分析を行なった。
Example 5 Effect of Manganese Ion Addition on Flask Culture (Part 2)
The anti-IL-6R antibody obtained in Example 2 was used in the same manner as in Example 4 except that the medium used was CD Opti CHO Medium (manufactured by Thermo Fisher Scientific) and the culture period was 11 days. The high-expressing cells and the anti-human gp130R antibody-high-expressing cells obtained in Example 3 were cultivated, the antibody productivity and the number of viable cells were measured, and the antibody expressed by the cells contained in the obtained culture was expressed. Analysis by FcR9_F column was performed.
培地中に添加したマンガンイオン濃度の違いによる、抗ＩＬ−６Ｒ抗体の生産性を比較した結果を図８に、抗ＩＬ−６Ｒ抗体高発現細胞数（生細胞数）の推移を図９に、抗ＩＬ−６Ｒ抗体のＦｃＲ９＿Ｆカラムによる分析結果を図１０に、抗ｇｐ１３０Ｒ抗体の生産性を比較した結果を図１１に、抗ｇｐ１３０Ｒ抗体高発現細胞数（生細胞数）の推移を図１２に、抗ＩＬ−６Ｒ抗体のＦｃＲ９＿Ｆカラムによる分析結果を図１３に、それぞれ示す。また図１０に記載の分析結果にある各ピークの面積割合を表７に、図１３に記載の分析結果にある各ピークの面積割合を表８に、それぞれ示す。 The results of comparing the productivity of the anti-IL6R antibody due to the difference in the manganese ion concentration added to the medium are shown in FIG. 8, and the transition of the number of high-expressing anti-IL-6R antibody cells (the number of viable cells) is shown in FIG. FIG. 10 shows the results of analysis of the anti-IL-6R antibody by the FcR9_F column, FIG. 11 shows the results of comparing the productivity of the anti-gp130R antibody, and FIG. 12 shows the transition of the number of high-expressing anti-gp130R antibody cells (the number of viable cells). The results of analysis of the anti-IL-6R antibody by the FcR9_F column are shown in FIG. 13, respectively. The area ratio of each peak in the analysis result shown in FIG. 10 is shown in Table 7, and the area ratio of each peak in the analysis result shown in FIG. 13 is shown in Table 8.
表７および表８より、培地としてＢａｌａｎＣＤ ＣＨＯ Ｇｒｏｗｔｈ Ａ ｍｅｄｉｕｍを用いたとき（実施例４）と同様、抗ＩＬ−６Ｒ抗体高発現細胞、抗ｇｐ１３０Ｒ抗体高発現細胞数とも、培地に０．０１μＭから５０μＭのマンガンイオンを添加して培養すると、マンガンイオン未添加時と比較し、ＦｃＲ９＿Ｆカラムとの結合力が強いピーク（ピーク３）の割合が増加していることがわかる。なお培地に５０μＭのマンガンイオンを添加して培養したときは、ＦｃγＲＩＩＩａ固定化分離剤との結合力がさらに弱い（すなわちＡＤＣＣ活性が非常に弱い抗体に相当する）ピーク（ピーク０）が確認された（図１０および図１３）。 From Table 7 and Table 8, as in the case of using BalanCD CHO Growth A medium as the medium (Example 4), the number of anti-IL-6R antibody high-expressing cells and anti-gp130R antibody high-expressing cell numbers was 0.01 μM to the medium. When 50 μM manganese ion was added and cultured, the ratio of peaks (peak 3) having a strong binding force with the FcR9_F column was increased as compared with the case where manganese ion was not added. When 50 μM manganese ion was added to the medium and cultured, a peak (peak 0) having a weaker binding force with the FcγRIIIa-immobilized separating agent (that is, corresponding to an antibody having very weak ADCC activity) was confirmed. (FIGS. 10 and 13).
実施例６ バイオリアクターを用いたバッチ培養におけるマンガンイオン添加の効果（その１）
（１）５０μｇ／ｍＬのカナマイシン、４０ｍＬ／ＬのＧｌｕｔａＭＡＸ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）を含んだ１００ｍＬのＢａｌａｎＣＤ ＣＨＯ Ｇｒｏｗｔｈ Ａ ｍｅｄｉｕｍを入れた５００ｍＬの三角フラスコ（Ｃｏｒｎｉｎｇ社製）に、実施例２で作製した抗ＩＬ−６Ｒ抗体高発現細胞を接種し、１３０ｒｐｍ、３７℃、８％ＣＯ２の条件下で振盪培養した。
Example 6 Effect of Manganese Ion Addition on Batch Culture Using Bioreactor (Part 1)
(1) Prepared in Example 2 in a 500 mL Erlenmeyer flask (manufactured by Corning) containing 100 mL of BalanCD CHO Growth A medium containing 50 μg/mL kanamycin and 40 mL/L GlutaMAX (manufactured by Thermo Fisher Scientific). The anti-IL-6R antibody high-expressing cells described above were inoculated, and cultured by shaking under the conditions of 130 rpm, 37° C., and 8% CO 2 .
（２）校正したｐＨ計、溶存酸素（ＤＯ）計をセットした２Ｌの滅菌済ジャーファーメンター（バイオット社製）に、５０μｇ／ｍＬのカナマイシン、４０ｍＬ／ＬのＧｌｕｔａＭＡＸを含んだ９００ｍＬのＢａｌａｎＣＤ ＣＨＯ Ｇｒｏｗｔｈ Ａ ｍｅｄｉｕｍを入れ、（１）で培養した抗ＩＬ−６Ｒ抗体高発現細胞を０．２×１０６ｃｅｌｌｓ／ｍＬとなるよう接種後、全量を１Ｌとなるよう前述の培地を追加した。前記培地には終濃度で５μＭとなるよう硫酸マンガンを添加している。 (2) 900 mL of BalanCD CHO Growth containing 50 μg/mL kanamycin and 40 mL/L GlutaMAX in a 2 L sterilized jar fermenter (manufactured by Biot) in which a calibrated pH meter and a dissolved oxygen (DO) meter are set. A medium was added thereto, and the cells highly expressing the anti-IL-6R antibody cultured in (1) were inoculated to give 0.2×10 6 cells/mL, and the above-mentioned medium was added so that the total amount became 1 L. Manganese sulfate was added to the medium to a final concentration of 5 μM.
（３）培地および細胞を入れたジャーファーメンターを制御装置（ＢＣＰ：バイオット社製）にセットし、８％ＣＯ２を１００ｍＬ／分で流しながら、３７℃、１００ｒｐｍで１０日間バッチ培養した。なお培養中、ｐＨはＣＯ２と０．５Ｍの炭酸水素ナトリウム水溶液を添加することで制御し、ＤＯは３７℃での飽和溶存酸素量の５０％量を保つよう制御した。培養途中で培養液を４０から８０ｍＬサンプリングし、生細胞数をＣｏｕｎｔｅｓｓ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）を使用して測定し、抗体生産性を実施例２（２）に記載のＥＬＩＳＡ法（検量線作成のための標準品としてヒトＩｇＧ１（シグマ社製）を使用）にて測定した。また前記サンプリングした培養液から、実施例４（３）から（４）に記載の精製方法を用いて抗体を精製し、実施例４（５）に記載の方法でＦｃＲ９＿Ｆカラムによる抗体分析を行なった。 (3) A jar fermenter containing a medium and cells was set in a controller (BCP: manufactured by Biot Co.), and batch culture was performed at 37° C. and 100 rpm for 10 days while flowing 8% CO 2 at 100 mL/min. During the culture, pH was controlled by adding CO 2 and 0.5 M aqueous sodium hydrogen carbonate solution, and DO was controlled so as to maintain 50% of the saturated dissolved oxygen amount at 37°C. During the culture, 40 to 80 mL of the culture solution was sampled, the number of viable cells was measured using Countess (manufactured by Thermo Fisher Scientific), and the antibody productivity was determined by the ELISA method described in Example 2 (2) (calibration curve preparation). Human IgG1 (manufactured by Sigma) was used as a standard for the measurement. The antibody was purified from the sampled culture medium using the purification method described in Example 4(3) to (4), and the antibody was analyzed by the FcR9_F column by the method described in Example 4(5). ..
比較例１
実施例６（２）で硫酸マンガンを添加しないこと、および実施例６（３）での培養期間が９日間であること以外は、実施例６と同様な方法で培養し、生細胞数および抗体生産性の測定ならびにＦｃＲ９＿Ｆカラムによる抗体分析を行なった。
Comparative Example 1
Culture was carried out in the same manner as in Example 6 except that manganese sulfate was not added in Example 6(2), and the culture period in Example 6(3) was 9 days, and the viable cell count and antibody were used. Productivity measurements and antibody analysis with FcR9_F column were performed.
実施例６および比較例１における抗体生産性の推移を図１４に、生細胞数の推移を図１５に、それぞれ示す。図１４および図１５の結果より、培地へのマンガンイオンの添加による、抗体生産性および細胞増殖への影響はないといえる。 The changes in antibody productivity in Example 6 and Comparative Example 1 are shown in FIG. 14, and the changes in the number of viable cells are shown in FIG. 15, respectively. From the results of FIGS. 14 and 15, it can be said that the addition of manganese ions to the medium has no effect on antibody productivity and cell growth.
比較例１で得られた培養９日目の抗ＩＬ−６Ｒ抗体のＦｃＲ９＿Ｆカラムによる分析結果および、実施例６で得られた培養１０日目の抗ＩＬ−６Ｒ抗体のＦｃＲ９＿Ｆカラムによる分析結果を図１６に示す。また図１６に記載の分析結果にある各ピークの面積割合を表９に示す。培地にマンガンイオンを添加して培養（実施例６）すると、マンガンイオン未添加時（比較例１）と比較し、ＦｃＲ９＿Ｆカラムとの結合力が強いピーク（ピーク３）の割合が増加していることがわかる（比較例１：２７．９％［培養９日後の培養液］、実施例６：３４．７％［培養１０日後の培養液］）。 The analysis result by the FcR9_F column of the anti-IL-6R antibody on the 9th day of culture obtained in Comparative Example 1 and the analysis result by the FcR9_F column of the anti-IL-6R antibody on the 10th day of culture obtained in Example 6 are shown. 16 shows. Table 9 shows the area ratio of each peak in the analysis result shown in FIG. When manganese ions were added to the medium and cultured (Example 6), the proportion of peaks (peak 3) having a strong binding force with the FcR9_F column was increased as compared with the case where manganese ions were not added (Comparative Example 1). (Comparative example 1: 27.9% [culture medium after 9 days of culture], Example 6: 34.7% [culture medium after 10 days of culture]).
以上の結果から、ジャーファーメンターでのバッチ培養においても、フラスコ培養のときと同様、培地にマンガンイオンを添加することで、抗体を発現可能な哺乳動物細胞が発現した抗体が有するＡＤＣＣ活性が向上することがわかる。 From the above results, even in batch culture with a jar fermenter, as in the case of flask culture, by adding manganese ion to the medium, the ADCC activity of the antibody expressed by the mammalian cells capable of expressing the antibody is improved. I understand that
実施例７ バイオリアクターを用いたバッチ培養におけるマンガンイオン添加の効果（その２）
（１）５０μｇ／ｍＬのカナマイシン、３０ｍＬ／ＬのＧｌｕｔａＭＡＸ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）を含んだ５０ｍＬのＥＸ−ＣＥＬＬ Ａｄｖａｎｃｅｄ ＣＨＯ Ｆｅｄ−ｂａｔｃｈ Ｍｅｄｉｕｍを加えた２５０ｍＬの三角フラスコ（Ｃｏｒｎｉｎｇ社製）に、実施例２で作製した抗ＩＬ−６Ｒ抗体高発現細胞を接種し、１３０ｒｐｍ、３７℃、８％ＣＯ２の条件下で振盪培養した。
Example 7 Effect of Manganese Ion Addition on Batch Culture Using Bioreactor (Part 2)
(1) A 250-mL Erlenmeyer flask (manufactured by Corning) to which 50 mL of EX-CELL Advanced CHO Fed-batch Medium containing 50 μg/mL of kanamycin and 30 mL/L of GlutaMAX (manufactured by Thermo Fisher Scientific) was added. The anti-IL-6R antibody high-expressing cells prepared in Example 2 were inoculated and shake-cultured under the conditions of 130 rpm, 37° C., and 8% CO 2 .
（２）校正したｐＨ計、溶存酸素（ＤＯ）計をセットした３器の２５０ｍＬのジャーファーメンター（バイオット社製）に、５０μｇ／ｍＬのカナマイシン、３０ｍＬ／ＬのＧｌｕｔａＭＡＸを含んだ１００ｍＬのＥＸ−ＣＥＬＬ Ａｄｖｅｎｃｅｄ ＣＨＯ Ｆｅｄ−ｂａｔｃｈ Ｍｅｄｉｕｍをそれぞれ加え、（１）で培養した抗ＩＬ−６Ｒ抗体高発現細胞を０．２×１０６ｃｅｌｌｓ／ｍＬとなるよう各リアクターに接種した後、全量を１１０ｍＬとなるよう前述の培地をそれぞれ追加した。前記培地には終濃度でそれぞれ１μＭ、５μＭ、１０μＭとなるよう硫酸マンガンを添加している。 (2) 100 mL EX-containing 50 μg/mL kanamycin and 30 mL/L GlutaMAX in three 250 mL jar fermenters (manufactured by Biot Co.) set with a calibrated pH meter and a dissolved oxygen (DO) meter. CELL Advanced CHO Fed-batch Medium was added to each, and the cells with high anti-IL-6R antibody-expressing cells cultivated in (1) were inoculated into each reactor at 0.2×10 6 cells/mL, and the total volume was 110 mL. As mentioned above, each of the above-mentioned media was added. Manganese sulfate was added to the medium so that the final concentrations were 1 μM, 5 μM, and 10 μM, respectively.
（３）培地および細胞を加えたジャーファーメンターを制御装置（Ｂｉｏ Ｊｒ．８：バイオット社製）にセットし、８％ＣＯ２を１００ｍＬ／分で流しながら、３７℃、１３０ｒｐｍで１０日間バッチ培養した。なお培養中、ｐＨはＣＯ２と０．５Ｍの炭酸水素ナトリウム水溶液を添加することでｐＨ７．１を保つよう制御し、ＤＯは３７℃での飽和溶存酸素量の５０％量を保つよう制御した。培養途中で培養液を１から２ｍＬサンプリングし、生細胞数をＶｉ−ＣＥＬＬ ＸＲ（ベックマン・コールター社製）を使用して測定し、抗体生産性をＣｅｄｅｘ Ｂｉｏ（ロシュ・ダイアグノスティックス社製）を使用して測定した。また培養終了後の培養液から、実施例４（３）から（４）に記載の精製方法を用いて抗体を精製し、実施例４（５）に記載の方法でＦｃＲ９＿Ｆカラムによる抗体分析を行なった。 (3) A jar fermenter containing a medium and cells was set in a controller (Bio Jr. 8: manufactured by Biot), and batch culture was performed at 37° C. and 130 rpm for 10 days while flowing 8% CO 2 at 100 mL/min. did. During the culture, the pH was controlled to maintain pH 7.1 by adding CO 2 and 0.5 M aqueous sodium hydrogen carbonate solution, and the DO was controlled to maintain 50% of the saturated dissolved oxygen amount at 37°C. .. During the culture, 1 to 2 mL of the culture solution was sampled, the number of viable cells was measured using Vi-CELL XR (manufactured by Beckman Coulter), and the antibody productivity was Cedex Bio (manufactured by Roche Diagnostics). Was measured using. In addition, the antibody is purified from the culture broth after the culturing using the purification method described in Example 4(3) to (4), and the antibody is analyzed by the FcR9_F column by the method described in Example 4(5). It was
比較例２
実施例７（２）で硫酸マンガンを添加しないこと以外は、実施例７と同様の方法で培養し、生細胞数および抗体生産性の測定ならびにＦｃＲ９＿Ｆカラムによる抗体分析を行なった。
Comparative example 2
Culture was performed in the same manner as in Example 7 except that manganese sulfate was not added in Example 7(2), and the number of viable cells and antibody productivity were measured and the antibody was analyzed by the FcR9_F column.
実施例７および比較例２における抗体生産性の推移を図１７に、生細胞数の推移を図１８に、それぞれ示す。図１７および図１８の結果より、培地へのマンガンイオンの添加による、抗体生産性および細胞増殖への影響はないといえる。 The changes in antibody productivity in Example 7 and Comparative Example 2 are shown in FIG. 17, and the changes in the number of viable cells are shown in FIG. 18, respectively. From the results of FIGS. 17 and 18, it can be said that the addition of manganese ion to the medium has no effect on antibody productivity and cell growth.
比較例２および実施例７で得られた培養１０日目の抗ＩＬ−６Ｒ抗体のＦｃＲ９＿Ｆカラムによる分析結果を図１９に示す。また図１９に記載の分析結果にある各ピークの面積割合を表１０に示す。培地にマンガンイオンを添加して培養（実施例７）すると、マンガンイオン未添加時（比較例２）と比較し、ＦｃＲ９＿Ｆカラムとの結合力が強いピーク（ピーク３）の割合が増加しており、さらにマンガンイオン濃度が高いほどピーク３の割合が増加していることがわかる（比較例２：３５．９％、実施例７：３６．５％（１μＭマンガンイオン添加）、３８．４％（５μＭマンガンイオン添加）、４１．０％（１０μＭマンガンイオン添加））。 FIG. 19 shows the results of analysis of the anti-IL-6R antibody on day 10 of culture obtained in Comparative Example 2 and Example 7 using the FcR9_F column. Table 10 shows the area ratio of each peak in the analysis result shown in FIG. When manganese ions were added to the medium and cultured (Example 7), the proportion of peaks (peak 3) having a strong binding force with the FcR9_F column increased as compared with the case where manganese ions were not added (Comparative Example 2). Further, it can be seen that the ratio of peak 3 increases as the manganese ion concentration increases (Comparative Example 2: 35.9%, Example 7: 36.5% (1 μM manganese ion added), 38.4% ( 5 μM manganese ion addition), 41.0% (10 μM manganese ion addition)).
以上の結果から、ジャーファーメンターでのバッチ培養において、培地に添加したマンガンイオンの濃度が高いほど、抗体を発現可能な哺乳動物細胞が発現した抗体が有するＡＤＣＣ活性が向上することがわかる。一方で、実施例４の結果（図３と図６）および実施例５の結果（図９と図１２）から、より高濃度のマンガンイオン（５０μＭ）を添加すると細胞増殖が阻害され、抗体生産量が低下することがわかる。このことから、細胞増殖を阻害しない程度の濃度（０．０３μＭから３０μＭ、好ましくは０．１μＭから１０μＭ、より好ましくは１μＭから１０μＭ）のマンガンイオンを添加すれば、抗体産生量を低下させずに高いＡＤＣＣ活性を有する抗体を発現できるといえる。 From the above results, it can be seen that in batch culture with a jar fermenter, the higher the concentration of manganese ion added to the medium, the more the ADCC activity possessed by the antibody expressed by the mammalian cells capable of expressing the antibody. On the other hand, from the results of Example 4 (FIGS. 3 and 6) and the results of Example 5 (FIGS. 9 and 12), addition of a higher concentration of manganese ion (50 μM) inhibits cell growth and antibody production. It can be seen that the amount decreases. From this, if manganese ion is added at a concentration that does not inhibit cell growth (0.03 μM to 30 μM, preferably 0.1 μM to 10 μM, more preferably 1 μM to 10 μM), the amount of antibody produced does not decrease. It can be said that an antibody having high ADCC activity can be expressed.
Claims (5)
前記培養工程を０．０１μＭから５０μＭのマンガンイオンを添加した培地で行なうことで、抗体依存性細胞傷害活性が向上した抗体を製造する方法。 In the method for producing an antibody, which comprises a step of culturing a mammalian cell capable of expressing an antibody, and a step of collecting the antibody expressed by the mammalian cell contained in the obtained culture,
A method for producing an antibody having improved antibody-dependent cellular cytotoxicity by performing the culturing step in a medium supplemented with 0.01 to 50 µM manganese ion.
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