JP2008539773A - 哺乳類宿主細胞における組換え抗体の高レベル発現 - Google Patents
哺乳類宿主細胞における組換え抗体の高レベル発現 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Abstract
【選択図】なし
Description
a)少なくとも第1および第2のポリペプチド鎖をコードする本発明による発現ベクターで哺乳類宿主細胞を形質移入する;
b)細胞の増殖を可能とし、細胞内で2つのポリペプチド鎖の発現およびアッセンブリーを行い複数のコピー数の第1および第2のポリペプチド鎖のそれぞれを含む分子を形成させ、および、形成された分子の分泌を可能とする、適切な条件下で宿主細胞を培養する;および
c)形成された分子を回収する。
使用する細胞
CHO細胞株CHOK1SV:細胞株CHO−K1の変種であり、懸濁性でタンパク質を含まない培地での増殖に適応化されている。
CHOK1SV細胞は、6mM L−グルタミンを添加したCD−CHO培地(Invitrogen)にて、懸濁振盪フラスコ(suspension shaker flasks)を用いて日常的に増殖させた。播種濃度は2x105細胞/mlとし、4日おきに細胞を分割した。フラスコに5%CO2を供給し、140rpmの円軌道の撹拌により36.5℃(35.5℃から37.0℃の間)でインキュベートした。
一過的形質転換を、懸濁増殖細胞を用いて行った。細胞を計測し、10%血清および6mM L−グルタミンを添加したDMEMベースの培地に1ウェル当り2.5x105生細胞となるように、24ウェルプレートのウェルに分配し、36.5℃で一晩インキュベートした。次の日、条件培地(conditioned medium)を、1mMの新鮮な培地(上記)と交換し、細胞を37℃で3時間インキュベートした。
形質移入に使用する細胞は、詳細を上記したとおり、細胞懸濁培養にて増殖させた。増殖している培養液からの細胞を遠心分離し、無血清培地で1度洗浄し、1.43x107細胞/mLの濃度で再懸濁した。0.7mLの細胞懸濁液および40μgのプラスミドDNAを電気穿孔法用キュベットに入れた。キュベットを、次に、電気穿孔法用装置に入れ、250Vおよび400μFのシングルパルスを送達した。形質移入の後、10%dFCSを添加した非選択性DMEMベースの培地を用いて、約2,500宿主細胞/ウェル(5x104/mL)で、96ウェルプレートに分配した。プレートを、10%CO2中で36.5℃(35.5℃から37.0℃)でインキュベートした。
96ウェル形質移入プレートを約3週間インキュベートし、コロニーを形成させた。できたコロニーを顕微鏡で観察し、コロニーがアッセイに適したサイズ(ウェルの底面の60%超を覆っている)であること、および各々のウェルに1つのコロニーのみが存在することを確認した。
サンプルの抗体濃度は、アッセンブルされたヒトIgGを測定するサンドウィッチELISAを用いて決定した。これは、抗ヒトFc抗体でコートした96ウェルプレート上への、サンプルおよび基準物質の捕獲を含む。結合した抗体は、ホースラディッシュ・ペルオキシダーゼがつながった抗ヒト軽鎖および色素生産性基質TMBで検出した。基準物質と比較すると、発色は、サンプル中に存在する抗体の濃度に比例した。
IgGの測定のためのプロテインA親和性クロマトグラフィー法を、Aglient1100HPLCで行った。IgG産物は、PorosプロテインA免疫検出カラムに選択的に結合する。非結合物質をカラムから洗浄除去し、残った結合抗体を溶媒のpHを下げることで放出させた。溶出物を280nmの吸光度でモニターし、一般的抗体基準物質によって産物を定量し(Chemstationソフトウェアを使用)、吸光係数の違いについて補正を行った。
a)重鎖cDNA
マニュアルに従ってLipofectamine−2000(Invitrogen)を用いてpcB72.3発現ベクター(図1に図示)をCHOK1SV細胞に一過的に形質移入することで、pcB72.3のHC cDNAバージョンを作製した。翌日、形質移入した細胞をcDNA合成のテンプレートとして使用して、その細胞から全RNAを抽出した。cB72.3 HC配列を、特異的なプライマーを用いてcDNAから増幅した。この配列はmRNA由来であるため、イントロン配列を欠いている。次に、この配列を、ベクターpEE6.4にクローン化し、ベクターpConK+VLと組み合わせて、pcB72.3 HC cDNAベクター(図2に図示)を作製した。
遺伝子最適化は、cB72.3のHCおよびLCをコードするcDNA配列において行った。最適化は、全ての可能性あるネガティブに作用する配列を除去するため、およびコドンの利用を最適化するために配列に対して行った。そして、遺伝子アッセンブリーアプローチを用いて、最適な配列を合成した。アッセンブリーの後、SEQ ID No.1および3に示されるHCおよびLCをコードする配列を、HindIIIおよびEcoRIサイトによってそれぞれベクターpEE6.4(HCのため)およびベクターpEE12.4(LCのため)にクローン化した。次に、NotIおよびPvuI制限酵素サイトをクローニングし、HCおよびLC発現カセットを組み合わせることで、DGVを作製した。このように作製したベクターpcB72.3 Geneart HCおよびpcB72.3 Geneart HC/LCは図3に図示される。
cDNAおよびGeneartベクターのために、DNAのバルクの調製物をQiagen Maxiprepキットを用いて作製した。全ての形質移入のために、ベクターDNAはPvuIによる消化によって直線化した。消化したプラスミドを、アガロースゲルで泳動し、完全に直線化していることを確認した。DNAをフェノール:クロロホルム抽出で精製し、40μgに分割した。それぞれの一定分量に含まれるDNAは、0.1倍量の3M酢酸ナトリウム(pH5.2)および2倍量の冷却100%エタノールを添加し、−20℃±5℃で必要な程度冷却することで沈殿させた。
標準的電気穿孔法を用いてベクター構築物をCHOK1SV細胞に形質移入した。細胞を96ウェルプレート中に播いた。次の日、選択培地を50μMで添加した。
ゲノムとcDNAとの間の重鎖配列の比較
pcB72.3のHCをコードする配列からイントロンを除去する効果を、安定的形質移入によって評価した。形質移入のそれぞれのセットの104コロニーを24ウェルプレートで過増殖させ、それぞれの細胞株由来の抗体濃度を決定した。その結果を図4および表1に要約した。データから、pcB72.3 HCコード配列におけるイントロンの存在は、抗体産生レベルに何も影響がないことが示された。プロモーター配列中のイントロンは、最大の遺伝子発現のために必要であり、それゆえこのイントロンだけは遺伝子発現への影響に寄与するらしいことがわかった。
DNA配列最適化の効果を、安定的形質移入において評価した。形質移入体のそれぞれのセットの104コロニーを24ウェルプレートで過増殖させ、それぞれの細胞株由来の抗体濃度を決定した。この場合、遺伝子最適化配列は、標準的なゲノムの型式ではなくcDNAの型式であるので、データはコントロールのcDNAバージョンと比較した。結果を図5および表2に要約した。
ベクターは、IgG1、IgG2およびIgG4の重鎖および軽鎖遺伝子をそれぞれ含むよう作製されており、重鎖および軽鎖遺伝子の定常領域は遺伝子最適化されており、一方で両遺伝子の可変領域は最適化されていない。従って、定常領域が最適化され可変領域が最適化されていないIgG1遺伝子を含むpCnG1GAベクター、定常領域が最適化され可変領域が最適化されていないIgG2遺伝子を含むpCnG2GAベクター、および定常領域が最適化され可変領域が最適化されていないIgG4遺伝子を含むpCnG4GAベクターを作製した。
Claims (20)
- 第1のポリペプチド鎖をコード化する第1の合成ヌクレオチド配列を含む第1の転写単位および第2のポリペプチド鎖をコード化する第2の合成ヌクレオチド配列を含む第2の転写単位を少なくとも含む哺乳類発現ベクターであって、ここにおいて、前記第1および第2の合成ヌクレオチド配列は、天然のヌクレオチド配列に基づいており、および、ここにおいて、前記第1のおよび第2のポリペプチド鎖は、少なくとも1コピーまたは複数コピーのそれぞれの前記第1および第2のポリペプチド鎖を含む分子を形成することが可能であり、前記第1および第2の両ヌクレオチド配列のコドン組成は、前記天然のヌクレオチド配列の由来となった宿主種とは異なる所定の哺乳類宿主種の遺伝子のコドンバイアスに適合していることが特徴付けられる哺乳類発現ベクター。
- 前記第1および第2のヌクレオチド配列のコドン組成がCHO細胞の遺伝子のコドンバイアスに適合している、請求項1に記載の哺乳類発現ベクター。
- 第1のポリペプチド鎖をコード化する第1の合成ヌクレオチド配列を含む第1の転写単位および第2のポリペプチド鎖をコード化する第2の合成ヌクレオチド配列を含む第2の転写単位を少なくとも含む哺乳類発現ベクターであって、ここにおいて、前記第1および第2の合成ヌクレオチド配列は、天然のヌクレオチド配列に基づいており、および、ここにおいて、前記第1のおよび第2のポリペプチド鎖は、少なくとも1コピーまたは複数コピーのそれぞれの前記第1および第2のポリペプチド鎖を含む分子を形成することが可能であり、前記第1および第2の両ヌクレオチド配列のコドン組成は、それらのmRNAが高効率で転写されるように改変されていることが特徴付けられる哺乳類発現ベクター。
- 前記2つの合成ヌクレオチド配列が、対応する前記天然ヌクレオチド配列とは異なるGC含量および/またはGC分布を有する、請求項1から3の何れか1項に記載の哺乳類発現ベクター。
- 前記2つの合成ヌクレオチド配列が、対応する前記天然ヌクレオチド配列とは異なるAT含量および/またはAT分布を有する、請求項1から4の何れか1項に記載の哺乳類発現ベクター。
- 前記2つの合成ヌクレオチド配列が、対応する前記天然ヌクレオチド配列よりもシス作用性配列モチーフを少なく含む、請求項1から5の何れか1項に記載の哺乳類発現ベクター。
- 前記2つの合成ヌクレオチド配列が、内部TATAボックス、chiサイトおよび/またはリボソームエントリーサイトをより少なく含む、請求項6に記載の哺乳類発現ベクター。
- 前記2つの合成ヌクレオチド配列が、ARE、INSおよび/またはCRS配列因子をより少なく含む、請求項6または7に記載の哺乳類発現ベクター。
- 前記2つの合成ヌクレオチド配列が反復配列をより少なく含み、そのため、それらのRNAが前記天然のヌクレオチド配列よりも少なく二次構造を形成する、請求項6から8の何れか1項に記載の哺乳類発現ベクター。
- 前記2つの合成ヌクレオチド配列が、潜在性スプライスドナーおよびアクセプターサイトをより少なく含む、請求項6から9の何れか1項に記載の哺乳類発現ベクター。
- 前記2つの合成ヌクレオチド配列が、対応する前記天然のヌクレオチド配列よりも少なく選択的開始サイトを含む、請求項1から10の何れか1項に記載の哺乳類発現ベクター。
- 前記合成ヌクレオチド配列にコードされる前記第1および第2のポリペプチド鎖が、前記天然のヌクレオチド配列によってコードされる対応するポリペプチド鎖と同じアミノ酸配列を有する、請求項1から11の何れか1項に記載の哺乳類発現ベクター。
- 前記合成ヌクレオチド配列にコードされる前記第1および第2のポリペプチド鎖が、前記天然のヌクレオチド配列によってコードされる対応するポリペプチド鎖と異なるアミノ酸配列を有する、請求項1から11の何れか1項に記載の哺乳類発現ベクター。
- 前記合成ヌクレオチド配列にコードされる前記第1および第2のポリペプチド鎖が、前記天然のヌクレオチド配列によってコードされる対応するポリペプチドと比較して、グリコシル化サイトが少ないまたはグリコシル化サイトが修正された、請求項13に記載の哺乳類発現ベクター。
- 前記第1の合成ヌクレオチド配列が、抗体の軽鎖またはその断片をコードする、請求項1から14の何れか1項に記載の哺乳類発現ベクター。
- 前記第2の合成ヌクレオチド配列が、抗体の重鎖またはその断片をコードする、請求項1から14の何れか1項に記載の哺乳類発現ベクター。
- 前記第1および第2のポリペプチド鎖が抗体またはその断片を形成し得る、請求項1から16の何れか1項に記載の哺乳類発現ベクター。
- 前記第1および/または第2の合成ヌクレオチド配列がエフェクタータンパク質の遺伝子に融合している、請求項1から17の何れか1項に記載の哺乳類発現ベクター。
- 前記第1および第2のポリペプチド鎖が、エフェクタータンパク質が抗体またはその断片に結合した融合タンパク質を形成し得る、請求項18に記載の哺乳類発現ベクター。
- 選択マーカー、好ましくはグルタミン合成酵素(GS)マーカーをコードする第3の転写単位を含む、請求項1から19の何れか1項に記載の哺乳類発現ベクター。
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