JP6444968B2 - 組換えポリペプチドの製造方法 - Google Patents
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- JP6444968B2 JP6444968B2 JP2016247585A JP2016247585A JP6444968B2 JP 6444968 B2 JP6444968 B2 JP 6444968B2 JP 2016247585 A JP2016247585 A JP 2016247585A JP 2016247585 A JP2016247585 A JP 2016247585A JP 6444968 B2 JP6444968 B2 JP 6444968B2
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- HPGGPRDJHPYFRM-UHFFFAOYSA-J tin(iv) chloride Chemical compound Cl[Sn](Cl)(Cl)Cl HPGGPRDJHPYFRM-UHFFFAOYSA-J 0.000 description 1
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
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Description
NfkBia(IKBα、nuclear factor κB inhibitor α)は、nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alphaの略称であり、細胞内のシグナル伝達に係わる転写因子であるNF-kappa Bの活性化に関与する。NfkBiaはNF-kappa Bの不活性化因子の一つである。新たに生合成されるNfkBiaは核内で誘導発現されることによって、NF-kappa BのDNA結合と転写活性を負に調節している(非特許文献1)。また、NfkBiaのように細胞増殖を阻害する一部の遺伝子発現は、ほとんどすべてのマウスやヒト腫瘍細胞で抑制されている(非特許文献2)。NF-kappa Bは、通常はNfkBiaのような不活性化因子と結合して存在しているが、種々の刺激により不活性化因子から遊離して活性化され、核内へ移行し、サイトカイン、成長因子、接着分子、細胞死制御因子その他の様々な標的遺伝子のプロモーター/エンハンサー領域にある特異的DNA配列(5’-GGGACTTTCC-3’、NFκB結合配列、kBモチーフ、NFkBレスポンスエレメント等と呼ばれる(配列番号35))に結合し、転写活性の調節に関与する(非特許文献3)。
(1)APESを発現し、且つ所望のポリペプチドをコードするDNAを導入した細胞を培養し、所望のポリペプチドを産生させることを含む、ポリペプチドの製造方法。
(1−1)細胞がAPESを強発現した細胞である、(1)の方法。
(2)APESが、ヒト、マウス、ラットまたはハムスター由来のNfkBiaの遺伝子のDNAまたはmRNAに塩基対形成により結合し、NfkBiaの遺伝子の発現を抑制し得る塩基配列を含む核酸分子である、(1)の方法。
(3)APESが、NfkBiaのmRNAの一部に塩基対形成により結合し得る19〜25塩基長の配列を含む30塩基長までの低分子RNAである、(2)の方法。
(4-1)APESが、NfkBiaのmRNAの一部に塩基対形成により結合し得る19〜25塩基長の配列を含む561塩基長までのmRNA型ノンコーディングRNAである、(2)の方法。
(4)APESが、NfkBiaのmRNAの一部に塩基対形成により結合し得る19〜25塩基長の配列を含む500塩基長までのmRNA型ノンコーディングRNAである、(2)の方法。
(5-1)APESが、NfkBiaのmRNAの一部に塩基対形成により結合し得る19〜25塩基長の配列を含む561〜1579塩基長のmRNA型ノンコーディングRNAである、(2)の方法。
(5)APESが、NfkBiaのmRNAの一部に塩基対形成により結合し得る19〜25塩基長の配列を含む500〜1000塩基長のmRNA型ノンコーディングRNAである、(2)の方法。
(6)19〜25塩基長の連続する配列が、配列番号2で示される塩基配列中の任意の部分配列である、(3)から(5)のいずれかの方法。
(7-1)APESが以下の塩基配列を含む核酸分子から選択される(6)の方法:
(a)配列番号1〜16及び29のいずれかの塩基配列からなるDNA;
(b)上記(a)の配列を含み、NfkBia遺伝子の 3’側 非翻訳領域の部分配列であるDNA;
(c)上記(a)または(b)の配列と1または数塩基を除き同一の塩基配列からなるDNA;
(d)上記(a)、(b)または(c)の転写産物であるRNA;
(e)上記(a)の配列に塩基対形成により結合しうる配列からなるDNAまたはRNA。
(7)APESが以下の塩基配列を含む核酸分子から選択される(6)の方法:
(a)配列番号4〜16の塩基配列からなるDNA
(b)上記(a)の転写産物であるRNA
(c)上記(a)の配列と1塩基を除き同一の塩基配列からなるDNA
(d)上記(c)の転写産物であるRNA
(e)上記(a)の配列に塩基対形成により結合しうる配列からなるDNAまたはRNA。
(8)細胞に、所望のポリペプチドをコードする外来のDNAが導入され、且つAPESが人為的に導入されている、(1)の方法。
(9)APESが人為的に導入された細胞が、APESがトランスフェクトされた細胞である、(8)の方法。
(10)APESが人為的に導入された細胞が、内因性APESの転写が活性化された細胞である、(8)の方法。
(11)細胞に、さらにタウリントランスポーターをコードするDNAが導入されている、(8)の方法。
(12)細胞に、さらにシステインスルフィン酸デカルボキシラーゼをコードするDNAが導入されている、(8)の方法。
(13)細胞に、さらにアラニントランスフェラーゼをコードするDNAが導入されている、(8)の方法。
(14)ポリペプチドが抗体である、(1)の方法。
(15)細胞がチャイニーズハムスター卵巣細胞である、(1)の方法。
(16)上記のいずれかに記載の方法で製造されたポリペプチドを含有する医薬品を製造する方法。
(17)以下の塩基配列を含み、APES活性を有する核酸分子(APES又はPPES)(但し配列番号1の核酸分子を除く):
(a)配列番号2〜16及び29のいずれかの塩基配列からなるDNA;
(b)上記(a)の配列を含み、NfkBia遺伝子の 3’側 非翻訳領域の部分配列であるDNA;
(c)配列番号1〜16及び29または(b)の配列と1または数塩基を除き同一の塩基配列からなるDNA;
(d)上記(a)、(b)または(c)の転写産物であるRNA;
(e)上記(a)の配列に塩基対形成により結合しうる配列からなるDNAまたはRNA。
(18)上記(17)記載の核酸分子を含むベクター。
(19)上記(17)記載の核酸分子又は(18)記載のベクターが人為的に導入された細胞。
(1)APES(Antibody Production Enhancing Sequence)
本発明は、APESを発現し、且つ所望のポリペプチドをコードするDNAを導入した細胞を培養し、所望のポリペプチドを産生させることを含む、ポリペプチドの製造方法を提供する。
さらに、本発明者らは、AI462015由来の一部の配列を有する核酸分子を宿主細胞に導入して発現させることにより、所望のポリペプチドの生産量を増加させることができることを見出した。
一態様において、APESは、NfkBiaの発現抑制または組換えポリペプチドの産生増大機能を有する核酸分子であり、ヒト、マウス、ラットまたはハムスター由来のNfkBiaの遺伝子のDNAまたはmRNAに塩基対形成により結合し得るRNAまたはDNAである。そのような核酸分子は、NfkBiaをコードするmRNAと相同または相補的な配列を含み、NfkBia遺伝子またはmRNAに結合してその発現を阻害することができると思われる。
(2)APESの発現
本発明においては、APESが発現した細胞、好ましくはAPESが強発現した細胞を用いることにより、当該細胞によるポリペプチドの産生量を増加させることを見出した。
APESが人為的に導入された細胞は当業者に公知の方法により作製することが可能であり、例えば、APESをコードするDNA配列をベクターに組込み、該ベクターを細胞に形質転換することにより作製することが可能である。
本発明において、APESの強発現、及び/または、ポリペプチドを生産する目的においてベクターを使用する場合には、特に、発現ベクターが有用である。本発明において使用可能な発現ベクターとしては、例えば、哺乳動物由来の発現ベクター(例えば、pcDNA3 (Invitrogen社製)や、pEGF-BOS (Nucleic Acids. Res.1990, 18(17),p5322)、pEF 、pCDM8 )、昆虫細胞由来の発現ベクター(例えば「Bac-to-BAC baculovairus expression system」(GIBCO BRL社製)、pBacPAK8)、植物由来の発現ベクター(例えばpMH1、pMH2)、動物ウィルス由来の発現ベクター(例えば、pHSV、pMV、pAdexLcw )、レトロウィルス由来の発現ベクター(例えば、pZIpneo)、酵母由来の発現ベクター(例えば、「Pichia Expression Kit」( Invitrogen社製)、pNV11 、SP-Q01)、枯草菌由来の発現ベクター(例えば、pPL608、pKTH50)などが挙げられる。
本発明において用いる細胞は、所望のポリペプチドを産生できる天然の細胞であっても、所望のポリペプチドをコードするDNAを導入した細胞であってもよいが、所望のポリペプチドをコードするDNAを導入した形質転換細胞が好ましい。
本発明の宿主細胞は、例えば、所望のポリペプチドの製造や発現のための産生系として使用することができる。APESを強発現している宿主細胞に所望のポリペプチドをコードするDNAを導入すれば、所望のポリペプチドを高生産できる。本発明の宿主細胞には、タウリントランスポーター(TauT)あるいはアニオンエクスチェンジャー(AE1)をコードするDNA(ベクターに組み込まれていてもよい)がさらに導入されてもよい。本発明の宿主細胞には、さらにシステインスルフィン酸デカルボキシラーゼ(Cystein Sulfinic Acid Decarboxylase: CSAD)あるいはアラニントランスフェラーゼ(Alanine Transferase : ALT1)をコードするDNAが導入されていてもよい。詳細は、WO2007/119774、WO2008/114673、WO2009/020144並びにWO2009/054433を参照されたい。
本発明の方法で産生されるポリペプチドは特に限定されず、抗体(例えば、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、抗VLA4抗体など)や生理活性タンパク質(顆粒球コロニー刺激因子(G-CSF)、顆粒球マクロファージコロニー刺激因子(GM-CSF)、エリスロポエチン、インターフェロン、IL-1やIL-6等のインターロイキン、t-PA、ウロキナーゼ、血清アルブミン、血液凝固因子、PTHなど)など如何なるポリペプチドでもよいが、特に抗体が好ましい。抗体は、天然抗体、Fab、scFv、sc(Fv)2などの低分子化抗体、キメラ抗体、ヒト化抗体などの如何なる抗体であってもよい。
上述の宿主細胞を培養し、所望のポリペプチドを産生させて、そのポリペプチドを収集することにより、ポリペプチドを得ることができる。
具体的には、例えば、L-アラニン、L-アルギニン、L-アスパラギン、L-アスパラギン酸、L-システイン、L-シスチン、L-グルタミン、L-グルタミン酸、グリシン、L-ヒスチジン、L-イソロイシン、L-ロイシン、L-リジン、L-メチオニン、L-オルニチン、L-フェニルアラニン、L-プロリン、L-セリン、L-スレオニン、L-トリプトファン、L-チロシン、L-バリン等、好ましくはL-アラニン、L-アルギニン、L-アスパラギン、L-アスパラギン酸、L-シスチン、L-グルタミン、L-グルタミン酸、グリシン、L-ヒスチジン、L-イソロイシン、L-ロイシン、L-リジン、L-メチオニン、L-フェニルアラニン、L-プロリン、L-セリン、L-スレオニン、L-トリプトファン、L-チロシン、L-バリン等のアミノ酸類;i−イノシトール、ビオチン、葉酸、リポ酸、ニコチンアミド、ニコチン酸、p-アミノ安息香酸、パントテン酸カルシウム、塩酸ピリドキサール、塩酸ピリドキシン、リボフラビン、塩酸チアミン、ビタミンB12、アスコルビン酸等、好ましくはビオチン、葉酸、リポ酸、ニコチン酸アミド、パントテン酸カルシウム、塩酸ピリドキサール、リボフラビン、塩酸チアミン、ビタミンB12、アスコルビン酸等のビタミン類;塩化コリン、酒石酸コリン、リノール酸、オレイン酸、コレステロール等、好ましくは塩化コリン等の脂質因子;グルコース、ガラクトース、マンノース、フルクトース等、好ましくはグルコース等のエネルギー源;塩化ナトリウム、塩化カリウム、硝酸カリウム等、好ましくは塩化ナトリウム等の浸透圧調節剤;EDTA鉄、クエン酸鉄、塩化第一鉄、塩化第二鉄、硫酸第一鉄、硫酸第二鉄、硝酸第二鉄等、好ましくは塩化第二鉄、EDTA鉄、クエン酸鉄等の鉄源類;炭酸水素ナトリウム、塩化カルシウム、リン酸二水素ナトリウム、HEPES、MOPS等、好ましくは炭酸水素ナトリウム等のpH緩衝剤を含む培地を例示できる。
培地は、市販の動物細胞培養用培地、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)、 RPMI1640、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)などの培地を用いることも可能である。
宿主細胞が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。
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。
本発明の方法により製造されたポリペプチド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。
本発明によれば、所望のポリペプチドをコードするDNAを導入した動物細胞を培養して当該ポリペプチドを製造する方法において、宿主細胞中でnuclear factor κB inhibitor α(NfkBia)の発現量を低下させることを通じて、当該所望のポリペプチドの産生量を増加させることができる。NfkBia遺伝子は必須遺伝子であり、完全に発現を抑えると細胞死にいたる。従って、当該NfkBia遺伝子の発現を適度に抑制することが、本発明の方法において重要と考えられる。
NfkBia発現量を測定するに当たっては、対象となる細胞で発現しているNfkBia mRNAのTaqMan法で定量可能な配列を決定しなければならない。例えば、本検討で用いたNfkBia部分配列(配列番号19、28)とTaqManプローブセット(配列番号20-22)は、図12で示すことができ、このTaqManプローブの設計は、Primer Express(登録商標)Software(Applied Biosystems)などで行うことができる。上記のNfkBia部分配列(配列番号28)は、CHO K1 細胞でもNF-kappa-B inhibitor alpha-like配列として確認され、我々のPCRクローニング配列と一致した。このうち終始コドンTGA(907-909)の64塩基上流から132塩基上流までの領域の発現定量が可能である。
典型的な測定機器としては Applied Biosystems (ABI)社製の7900HI Sequence Detection Systemなどがあり、全てのキットおよび試薬が購入可能であるのでABI社推奨のプロトコールに従って定量することができる。
〔実施例1〕各種遺伝子導入CHO細胞のGeneChip解析実験
GeneChip実験は、AFFYMETRIX社のオリゴヌクレオチドアレイ(Affymetrix MOUSE430_2)を用いて通常の手順にしたがった。ただし、Hamster Arrayは商品化されていないためMouse Genome 430 2.0 Arrayを用いた。ハイブリダイゼーション条件の最適化によって、Test 3 array 上のMouse Gene16種のプローブ中、8種のプローブでPresent Callが得られるようになり、Mouseとの塩基配列相同性が約90%以上の場合は、Hamster転写産物の発現定量が可能になった。
実施例1で、MAb1(抗IL-6R抗体;tocilizumab、商品名 アクテムラ)高産生DG44細胞で転写産物AI462015の発現量が昂進されたが(図2)、異なる宿主細胞(CHO-DXB11s)に異なる抗体(MAb2:抗グリピカン3抗体;GC33(WO2006/006693参照))を高産生させた場合も同様にAI462015転写産物の発現昂進がみられた(図3)。
また、1Lジャー培養3日目の生産培養条件下においてもAI462015配列の異常な発現昂進がみられた。図4に示したように1Lジャー流加培養の10日目に約1200−1400mg/LのMAb2を産生する2種の抗体高産生細胞は5,000以上の高いGeneChipシグナル値を示した。培養条件の違いから、1Lジャー流加培養3日目のシグナル値はシェーカー培養の50%程度であったが、培養後期13日目にAI462015配列の発現強度はシェーカー継代培養と同程度にまで昂進され、異常に高いシグナル値を示した(図5)。一方、抗体産生量の低いMAb1強発現DXB11s細胞(加水分解物無添加のシェーカー培養7日目で300mg/L以下、加水分解物添加でも500mg/L以下)は、高産生化に寄与する加水分解物(Hy-Fish、Procine Lysate)を添加した条件でも、1Lジャー培養3日目の AI462015配列の発現昂進はみられなかった(図6)。
AI462015配列発現量の高さが抗体産生ポテンシャルの高さと相関することを示すため、図6で抗体産生ポテンシャルの低かったMAb1強発現DXB11s細胞にAI462015配列の一部を発現するプラスミドを導入し、強発現させて抗体産生ポテンシャルを比較した。
実施例1で述べたように、AI462015配列はマウスゲノム12のNfkBia 遺伝子の3’側の非翻訳領域近傍(3’側78bp)の相補鎖上に存在すること、AI462015配列に含まれる22塩基(AAGTACCAAAATAATTACCAAC:配列番号10)はヒトNfkBia 遺伝子の3’側非翻訳領域(1492-1513)の相補鎖と同一配列であり、さらにラット、アカゲザル、イヌ、ウマなど種を超えて保存されていることからmicroRNAとしてRNA干渉してNfkBia mRNAを分解する可能性があること、あるいはAI462015発現を定量できるAFFYMETRIX社のオリゴヌクレオチドアレイ(Affymetrix MOUSE430_2)上の特異的プローブ配列領域(CATATACAACATTTACAAGAAGGCGACACAGACCTTAGTTGG:配列番号16)42bpの前半部分に相当する5’側71番目のCからの21塩基(CATATACAACATTTACAAGAA:配列番号15)がラットNfkBia mRNAの1478から1498塩基目の相補配列であることから、AI462015配列由来の核酸分子がNfkBia mRNA にRNA干渉し、発現を抑制することで抗体高産生CHO細胞のホメオスタシスを維持する可能性が考えられた(ノックアウトマウスのlethality はpostnatal)。(注記:後に、AI462015の転写産物はマウスNfkBia 遺伝子の3’側513塩基の非翻訳領域の相補鎖に相当することが判明した。実施例8参照。また、マウスGeneChipで定量されたAI462015の71番目から112番目の配列(配列番号16)はCHO細胞での転写産物として確認された。)
そこで、抗体産生ポテンシャルが高かったAI462015高発現細胞でのNfkBia mRNA 発現量を定量し、その発現が抑制されていることを確認することにした。
microRNAを解析するために、図15に示したようにMir-XTM miRNA First-Strand Sythesis Kit (Clontech)を用いて、継代培養中のMAb1(抗IL-6R抗体)高産生DXB11s細胞とMAb1(抗IL-6R抗体)高産生TAUT強発現DXB11s細胞、さらに抗体遺伝子導入前のDXB11s宿主細胞から調製したsmall RNAの3’側にpoly(A)タグを付加したのち、オリゴdTを3’側にPCRプライマー配列(mRQ 3’Primer)を5’側にもつアダプターをプライミングして一次鎖cDNAを合成した。得られたcDNAを鋳型にして、mRQ 3’ primer と 予想されたAPES配列由来のmicroRNA-specific Primer(APES 40-61 5’ primer, あるいは APES 71-91 5’ primer)、さらに ポジティブコントロールのU6 snRNA 5’primerを用いてqPCR反応(95℃ 5sec, 60℃ 20sec, 30cycles)をおこなった。PCR反応液は、精製後、3%アガロースゲルで電気泳動した。図16に示したように、APES 40-61 5’primerとU6 snRNA 5’ primerによるPCR反応で目的の大きさのバンドがみられた。レーン1,2,3で示したように、APES 40-61(AAGTACCAAAATAATTACCAAC:配列番号10)22塩基がMAb1(抗IL-6R抗体) 高産生細胞中で高発現していた。ポジティブコントロールのU6 snRNA(レーン4)の発現量はいずれの細胞においても同レベルであったこと、またAPES 71-91(CATATACAACATTTACAAGAA:配列番号15)の存在は確認されなかったことから(data not shown)、種を超えて配列が保存されているAPES 40-61(22塩基)がmicroRNAとして抗体高産生化に寄与すると考えられた。
抗体産生用宿主細胞DXB11/TAUTから、1Lジャー流加培養14日目に3.9g/LのMAb1(抗IL-6R抗体)を産生する抗体高産生細胞(DXB11/TAUT/MAb1)が得られ、TAUTの生存率維持能によって培養31日目に8.1g/Lを産生したが、実生産を考慮して培養14日目に高産生にするには、細胞最高到達密度(4.1 x10e6 cells/mL)を増加させる必要があった。APES強発現によるNfkbia mRNAの発現抑制(実施例4)がNfkbの活性化を促進するのであれば、増殖関連遺伝子の発現が亢進されるため、細胞最高到達密度は上がる可能性がある。APESと同様に抗体高産生化に寄与したALT1の共発現用プラスミドをそれぞれ(pPur-APES165, pPur-ALT1, 図17)、上記抗体高産生細胞DXB11/TAUT/MAb1(親株)に導入して高増殖な上位3株ずつを選抜し、シェーカー流加培養をおこなうと、APES165強発現細胞の細胞最高到達密度の平均値は(11.5±1.7)x10e6 cells/mLであり、ALT1強発現細胞の(8.9±1.8) x10e6 cells/mL以上に高増殖な細胞が得られた。さらにシェーカー流加培養14日目の抗体産生量の平均値は、APES強発現細胞:4.4±0.6 g/L, ALT1強発現細胞:4.0±0.6 g/Lと導入前のDXB11/TAUT/MAb1細胞:3.4g/L以上に高くなったことから、APES強発現効果はTAUT強発現効果に独立してポジティブに作用することが示された(図18)。APES強発現による正の効果は1L-Jar流加培養において顕著であり、それぞれシェーカー流加培養での高増殖細胞を比較すると、APES強発現株は最も高増殖で、培養12日目で5.3g/Lと親株の3.2g/L, ALT1強発現株4.4g/Lに対して短期間培養で高産生である長所が示された(図19)。以上の結果に基づき、抗体産生用宿主細胞DXB11/TAUTをより高増殖な宿主細胞に改変することにし、APES165強発現宿主DXB11/TAUT/APESを作成した。DXB11/TAUT宿主にpPur-APES165をエレクトロポレーション法で遺伝子導入し、薬剤選抜後に生存率、増殖ともに良好であった宿主候補の9株について、継代培養時のAPES snRNA (small non-coding RNA)発現量を定量した。APES発現量の高かったDXB11/TAUT/APES宿主候補株は培養時の生細胞密度が高く、相関(R2=0.70)が示された(図20)。
〔実施例7〕APES強発現による抗体産生細胞の高産生化例2
実施例3と同様に、MAb1強発現DXB11s細胞にAI462015転写産物の5’側の部分配列を発現するプラスミドを導入し、抗体産生ポテンシャルを比較した。
〔実施例8〕APESに関する遺伝子解析
実施例1においては、出願時の遺伝子情報に基づいて、「AI462015は437塩基のmRNA型ノンコーディングRNAであるが、その配列はマウスゲノム12のNfkBia 遺伝子の3’側の非翻訳領域近傍(56590831- 56590397)の相補鎖上に存在する」と記述したが、しかし、その後のGeneBankの情報更新によって、AI462015の転写産物である437塩基はマウスNfkBia 遺伝子の3’側 非翻訳領域(513塩基)の相補鎖に相当することが明らかになった(図23)。図24に示したように、出願後に公開されたCHO-K1細胞のゲノム配列上にAI462015の相同配列が存在すること(配列番号25:AI462015;配列番号26-27:CHO-K1ゲノム)、さらに、抗体高産生なCHO細胞においてNfkbia の発現抑制(実施例4)がみられたことから、CHO細胞ではAI462015の相同配列が高発現されて機能するものと考えられる。
本明細書で引用した全ての刊行物、特許およ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。
Claims (3)
- 以下の塩基配列からなる、APES(Antibody Production Enhancing Sequence)活性を有する核酸分子:
(a)配列番号2、4〜16及び29のいずれかの塩基配列からなるDNA;
(b)配列番号1〜16及び29のいずれかの塩基配列と1塩基を除き同一の塩基配列からなるDNA;または
(c)上記(a)または(b)の転写産物であるRNA
であって、APES活性は、宿主細胞内のNfkBiaの発現を抑制することにより、Nf-kappa Bを活性化し、それにより組換えポリペプチド産生能を向上させる作用である、核酸分子。 - 請求項1記載の核酸分子を含むベクター。
- 請求項1記載の核酸分子又は請求項2記載のベクターが人為的に導入された細胞。
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