JP6972022B2 - グルタミンシンセターゼ遺伝子内相補ベクターを用いた高レベルのヘテロマータンパク質発現細胞の直接選択 - Google Patents
グルタミンシンセターゼ遺伝子内相補ベクターを用いた高レベルのヘテロマータンパク質発現細胞の直接選択 Download PDFInfo
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Description
本出願は、2016年5月11日に提出された米国仮出願第62/334,966号の利益を主張し、その全体が本明細書に参考として組み込まれる。
本出願は、ASCIIフォーマットで電子提出した配列表を含有し、参照により全体が本明細書に組み込まれる。2017年5月8日に作成された配列表のコンピューター可読フォーマットコピーは、A−2011−WO−PCT_SEQ_ST25.txtと名付けられ、サイズが79.1キロバイトである。
a.第一のポリペプチドをコードする第一の核酸と、
b.第二のポリペプチドをコードする第二の核酸であって、該第二のポリペプチドが選択マーカーの最初の変異サブユニットである第二の核酸とを含み、
前記第一の核酸の転写物が前記第二の核酸の転写物に作動可能に連結され、さらに
c.第三のポリペプチドをコードする第三の核酸であって、該第三のポリペプチドが前記第一のポリペプチドと会合してヘテロマー複合体を形成することができる第三の核酸と、
d.第四のポリペプチドをコードする第四の核酸であって、該第四のポリペプチドが前記選択マーカーの相補的変異サブユニットである第四の核酸とを含むベクターであって、
前記第三の核酸の転写物が前記第四の核酸の転写物に作動可能に連結され、
前記選択マーカーの最初の変異サブユニット及び相補的変異サブユニットが相互作用して選択活性を付与し、さらに、前記ベクターが哺乳類細胞にトランスフェクトされることができ、トランスフェクトされた細胞の選択を向上させることができる、ベクターを提供する。
e.第一のポリペプチドをコードする第一の核酸と、
f.第二のポリペプチドをコードする第二の核酸であって、該第二のポリペプチドが選択マーカーの最初のフラグメントである第二の核酸とを含み、
前記第一の核酸の転写物が前記第二の核酸の転写物に作動可能に連結され、さらに
g.第三のポリペプチドをコードする第三の核酸であって、該第三のポリペプチドが前記第一のポリペプチドと会合してヘテロマー複合体を形成することができる第三の核酸と、
h.第四のポリペプチドをコードする第四の核酸であって、該第四のポリペプチドが前記選択マーカーの相補的フラグメントである第四の核酸とを含むベクターであって、
前記第三の核酸の転写物が前記第四の核酸の転写物に作動可能に連結され、
前記選択マーカーの最初の変異サブユニット及び相補的変異サブユニットが相互作用して選択活性を付与し、さらに、前記ベクターが哺乳類細胞にトランスフェクトされるころができ、トランスフェクトされた細胞の選択を向上させることができる、ベクターを提供する。
a)選択マーカーの最初の変異サブユニットである第二のポリペプチドをコードする第二の核酸に作動可能に連結される第一のポリペプチドをコードする第一の核酸を含むバイシストロニック転写物をコードする第一のベクターと、
b)第四のポリペプチドをコードする第四の核酸に作動可能に連結される第三のポリペプチドをコードする第三の核酸を含むバイシストロニック転写物をコードする第二のベクターとを含む発現システムであって、該第四のポリペプチドが前記選択マーカーの相補的変異サブユニットであり、前記選択マーカーの最初の変異サブユニットと会合して選択活性を付与することができ、
さらに、前記第三のポリペプチドが前記第一のポリペプチドと会合してヘテロマー複合体を形成することができ、
さらに、前記発現システムが哺乳類細胞にトランスフェクトされることができ、前記細胞の選択を向上させることができる、発現システムを提供する。
c)選択マーカーの最初のフラグメントである第二のポリペプチドをコードする第二の核酸に作動可能に連結される第一のポリペプチドをコードする第一の核酸を含むバイシストロニック転写物をコードする第一のベクターと、
d)第四のポリペプチドをコードする第四の核酸に作動可能に連結される第三のポリペプチドをコードする第三の核酸を含むバイシストロニック転写物をコードする第二のベクターと含む発現システムであって、該第四のポリペプチドが前記選択マーカーの相補的フラグメントであり、前記選択マーカーの最初のフラグメントと会合して選択活性を付与することができ、
さらに、前記第三のポリペプチドが前記第一のポリペプチドと会合してヘテロマー複合体を形成することができ、
さらに、前記発現システムが哺乳類細胞にトランスフェクトされることができ、前記細胞の選択を向上させることができる、発現システムを提供する。
本実施例では、遺伝子内相補性において有用である可能性が高いと同定されたマウスグルタミンシンセターゼ(GS)変異体の選択及び調製について記載する。マウスGSの4つのN末端残基(W60、N61、D63、及びS66)及び11のC末端残基(E134、E136、E196、E203、N248、H253、N255、R319、R324、E338及びR340)を、アラニン変異スキャンにおける評価のために選択した。個々の及び組み合わせの変異構築物を、pcDNA3.3ベクター(ネオマイシン耐性、Invitrogen Grand Island、NY)で作製し、これにより合計9個のN末端変異構築物及び16個のC末端変異構築物を得た。
この実施例では、組換えヘテロ二量体タンパク質、すなわちモノクローナル抗体(mAb)の発現におけるGSの変異体の影響について記載する。実施例1に記載の選択されたN末端及びC末端変異体を、mAb発現ベクターにクローニングし、複数の組み合わせの2ベクターシステムを構築した。ここで軽鎖(LC)の発現はIRESを介してmutGS−CTに連結され、重鎖(HC)はmutGS−NTに連結される。製造元の説明書に従って、CHO−K1 GSノックアウト細胞株をCompoZr(登録商標)ノックアウトZFNキット−CHO GS(Sigma−Aldrich St.Louis、MO)を用いて構築した。CHO−K1グルタミンシンセターゼノックアウト(CHO−K1 GS−KO)細胞株を前述のように培養した。
本実施例は、IRES GS結合の効率を低下させることによって、選択のストリンジェンシーを増加させることにより、トランスフェクトされたプールにおける抗体力価を増加させる方法について記載する。初期の構築物における配列は、GSの開始コドンに融合されたATG−12までの野生型EMCV配列である(Davies and Kaufman、J Virol 66,1924;1992)。効率的な選択及び増幅を可能にするために、以前に示された配列を用いることにより、IRESの効率が低下した(Aldrich et al., Biotechnol Prog 19, 1433;2003)。より効率的な結合における結合部のDNA配列(ED3)を、より効率の低い結合部のDNA配列(317)と以下比較する。ED3配列の下の数字は、WT EMCVで見出されるATGを示す。最後のATGはGSの開始コドンを示す。
本実施例では、これらの培養の選択性を高める別の戦略について記載する。トランスフェクションを実施例2に記載のように実施したが、選択の間、より高レベルのGS発現を選択するために、GSの阻害剤、MSXを培養に添加した。より効率的な(すなわち、非変異の)IRESを持つ3つのCT2−NT4培養のうち1つは回復したが、CT1−NT4または変異IRESを持つトランスフェクタントのいずれも回復しなかった。回復した単一の培養では、10日間の流加バッチ培養により0.29グラム/Lを得、比生産性は9.18p/c/dであった(上記表2参照)。
本実施例では、多数のmut IRES CT1−NT4トランスフェクションの結果について記載する。これらの構築物は、配列番号23に示される、より効率の低いIRESを有する。mut IRES CT1−NT4を含むベクターを用いて、合計88のCHO−K1 GSノックアウトトランスフェクションを実施した。88のトランスフェクションのうち合計10は選択から生存し、培養の約25日後に強く増殖するトランスフェクションプールが得られ、実施例2のより少ない数のトランスフェクションと同様の挙動を示した。モックトランスフェクションプールのGSプールは、グルタミン不含培地で17日後に死滅した。
本実施例では、酵素グルタミンシンセターゼの分子相補性アプローチについて記載する。該グルタミンシンセターゼ酵素は、2つの独立したフラグメントとして発現し、酵素活性は、2つのフラグメントの遺伝的相補性によって完全に再構成された。種々の残基でグルタミンシンセターゼ酵素を分割する一連の構築物を作製した。これらのポイントは、酵素の分子構造からの情報をベースとした。これらの構築物を、グルタミンシンセターゼ活性が欠損している細胞株を救済する能力について、細胞ベースのアッセイで試験した。この試験から、グルタミンシンセターゼ欠損細胞を完全に救済することができるいくつかのフラグメントが同定されたことから、これらのフラグメントが会合し、完全に機能的なグルタミンシンセターゼ酵素を作製できることを実証した。
Claims (17)
- a)第一のポリペプチドをコードする第一の核酸と、
b)グルタミンシンセターゼのN末端変異体(mutGS−NT)をコードする第二の核酸と、ここで、前記mutGS−NTは、W60A N61A D63A D63R S66A及びD76Aからなる群から選択される2つ又はそれ以上の変異を含み、前記第一の核酸の転写物が前記第二の核酸の転写物に作動可能に連結される、
さらに、
c)第三のポリペプチドをコードする第三の核酸と、ここで、前記第三のポリペプチドは前記第一のポリペプチドと会合してヘテロマー複合体を形成することができる、
d)グルタミンシンセターゼのC末端変異体(mutGS−CT)をコードする第四の核酸と、ここで、前記mutGS−CTは、E134A E136A E196A E203A N248A H253A N255A R319A及びR324Aからなる群から選択される1つ以上の変異を含み、前記第三の核酸の転写物が前記第四の核酸の転写物に作動可能に連結される、
を含む、ベクターであって、
前記グルタミンシンセターゼのN末端変異体及び前記グルタミンシンセターゼのC末端変異体は相互作用して選択活性を付与し、さらに、前記ベクターは哺乳類細胞にトランスフェクトされることができ、トランスフェクトされた細胞の選択を向上させることができる、ベクター。 - a)第二のポリペプチドをコードする第二の核酸に作動可能に連結される第一のポリペプチドをコードする第一の核酸を含むバイシストロニック転写物をコードする第一のベクターと、ここで、前記第二のポリペプチドは、グルタミンシンセターゼのN末端変異体(mutGS−NT)であり、前記mutGS−NTは、W60A N61A D63A D63R S66A及びD76Aからなる群から選択される2つ又はそれ以上の変異を含む、b)第四のポリペプチドをコードする第四の核酸に作動可能に連結される第三のポリペプチドをコードする第三の核酸を含むバイシストロニック転写物をコードする第二のベクターと、ここで、前記第四のポリペプチドは、グルタミンシンセターゼのC末端変異体(mutGS−CT)であり、前記mutGS−CTは、前記グルタミンシンセターゼのN末端変異体に会合して選択活性を付与することができる、E134A E136A E196A E203A N248A H253A N255A R319A及びR324Aからなる群から選択される1つ以上の変異を含む、
を含む発現システムであって、
さらに、前記第三のポリペプチドが前記第一のポリペプチドと会合してヘテロマー複合体を形成することができ、
さらに、前記発現システムが哺乳類細胞にトランスフェクトされることができ、前記細胞の選択を向上させることができる、発現システム。 - 前記ヘテロマー複合体が免疫グロブリンである、請求項1又は2に記載のベクター又は発現システム。
- 前記第一の核酸が免疫グロブリン重鎖をコードし、前記第三の核酸が免疫グロブリン軽鎖をコードする、請求項1〜3のいずれか一項に記載のベクター又は発現システム。
- a)前記第一の核酸と前記第二の核酸との間の部位、
b)前記第三の核酸と前記第四の核酸との間の部位、及び
c)第一と第二の核酸との間、及び第三と第四の核酸との間、の両方の部位、
からなる群から選択される部位に、配列内リボソーム進入部位(IRES)が現れる、請求項1〜4のいずれか一項に記載のベクター又は発現システム。 - 前記配列内リボソーム進入部位が配列番号23を含む、請求項5に記載のベクター又は発現システム。
- 前記mutGS−NTが、W60A N61A D63A(mutGS−NT1;配列番号6)、W60A N61A D63A S66A(mutGS−NT2;配列番号10)、W60A N61A D63A S66A D76A(mutGS−NT3;配列番号25)及びW60A N61A D63R S66A(mutGS−NT4;配列番号19)からなる群から選択され、且つ、前記mutGS−CTが、E134A E136A E196A E203A(mutGS−CT1;配列番号21)及びN248A H253A N255A(mutGS−CT2;配列番号22)からなる群から選択される、請求項1〜6のいずれか一項に記載のベクター又は発現システム。
- 請求項1〜7のいずれか一項に記載の前記ベクター又は発現システムによりトランスフェクト、形質転換又は形質導入された、単離された宿主細胞。
- CHO、VERO、BHK、HeLa、Cos、MDCK、293、3T3、骨髄腫細胞株及びWI38細胞からなる群から選択される、請求項8に記載の宿主細胞。
- ヘテロマー複合体の産生方法であって、前記ヘテロマー複合体が請求項8又は9に記載の宿主細胞によって発現される条件下で前記宿主細胞を培養する工程を含む、前記ヘテロマー複合体の産生方法。
- 前記宿主細胞が、請求項1〜7のいずれか一項に記載のベクターにより形質転換又は形質導入された場合、前記ヘテロマー複合体が抗体である、請求項10に記載の方法。
- 前記ヘテロマー複合体を単離することをさらに含む、請求項10又は11に記載の方法。
- N末端変異グルタミンシンセターゼ(mutGS−NT)をコードする第二の核酸に作動可能に連結される抗体の重鎖をコードする第一の核酸を含む第一のベクターと、C末端変異グルタミンシンセターゼ(mutGS−CT)をコードする第四の核酸に作動可能に連結される抗体の軽鎖をコードする第三の核酸を含む第二のベクターとを含む発現システムであって、
前記mutGS−NTは、W60A N61A D63A D63R S66A及びD76Aからなる群から選択される2つ又はそれ以上の変異を含み、前記mutGS−CTが、E134A E136A E196A E203A N248A H253A N255A R319A及びR324Aからなる群から選択される1つ以上の変異を含み、
グルタミンシンセターゼの各変異サブユニットが単独発現時には選択活性を持たず、mutGS−NTとmutGS−CTとの共発現では、グルタミンシンセターゼ活性を付与し、前記発現システムが哺乳類細胞にトランスフェクトされることができ、トランスフェクトされた細胞の選択を向上させることができる、発現システム。 - 前記mutGS−NTが、W60A N61A D63A(mutGS−NT1;配列番号6)、W60A N61A D63A S66A(mutGS−NT2;配列番号10)、W60A N61A D63A S66A D76A(mutGS−NT3;配列番号25)及びW60A N61A D63R S66A(mutGS−NT4;配列番号19)からなる群から選択され、且つ、前記mutGS−CTが、E134A E136A E196A E203A(mutGS−CT1;配列番号21)及びN248A H253A N255A(mutGS−CT2;配列番号22)からなる群から選択される、請求項13に記載の発現システム。
- W60A N61A D63A D63R S66A及びD76A、並びにその組み合わせからなる群から選択される変異を含むN末端変異グルタミンシンセターゼ(mutGS−NT)をコードする第二の核酸に作動可能に連結される所望のポリペプチドをコードする第一の核酸を含むバイシストロニック転写物をコードする第一のベクターと、
E134A E136A E196A E203A N248A H253A N255A R319A及びR324A、並びにその組み合わせからなる群から選択される変異を含むC末端変異グルタミンシンセターゼ(mutGS−CT)をコードする第四の核酸に作動可能に連結される第三の核酸を含むバイシストロニック転写物をコードする第二のベクターと
を含む発現システムであって、前記N末端変異グルタミンシンセターゼ及び前記C末端変異グルタミンシンセターゼは会合して選択活性を付与し、前記発現システムが哺乳類細胞にトランスフェクトされることができ、前記トランスフェクトされた細胞の選択を向上させることができ、さらに、前記第一の核酸が抗体重鎖をコードし、前記第三の核酸が抗体軽鎖をコードする、発現システム。 - 前記mutGS−NTが、W60A N61A D63A(mutGS−NT1;配列番号6)、W60A N61A D63A S66A(mutGS−NT2;配列番号10)、W60A N61A D63A S66A D76A(mutGS−NT3;配列番号25)及びW60A N61A D63R S66A(mutGS−NT4;配列番号19)からなる群から選択され、且つ、前記mutGS−CTが、E134A E136A E196A E203A(mutGS−CT1;配列番号21)及びN248A H253A N255A(mutGS−CT2;配列番号22)からなる群から選択される、請求項15に記載の発現システム。
- 請求項13〜16のいずれか一項に記載の発現システムによりトランスフェクト、形質転換又は形質導入された、単離された宿主細胞。
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