JP2015502162A - 弱毒化サルモネラ株を高収量で生産する方法 - Google Patents
弱毒化サルモネラ株を高収量で生産する方法 Download PDFInfo
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Abstract
Description
(1)野生型サルモネラ・タイフィ(Salmonella typhi)及び弱毒化サルモネラ・タイフィ(Salmonella typhi)Ty21aを含むサルモネラ・タイフィ(Salmonella typhi)
血管内皮増殖因子VEGF(Kd75−760pM)は、脈管系、特に血管及びリンパ管の様々な構成要素の増殖及び分化を制御する、6つの構造的に関連するタンパク質(VEGF−A [VEGFとしても知られる]、−B、−C、−D、−E及びPLGF [胎盤増殖因子、PGF又はPIGF−2としても知られる])のファミリーのメンバーである。血管新生におけるVEGFの役割は、VEGFR−2とこのタンパク質との相互作用を通じて媒介される。キナーゼ挿入ドメイン含有受容体(KDR)としても知られるVEGFR−2は、アミノ酸長1356、分子量200〜230kDaで、VEGF、VEGF−C及びVEGF−Dに対する高親和性の受容体である。内皮細胞のcDNAのスクリーニングを通じてヒトにおいて同定されたチロシンキナーゼ受容体の内、VEGF−R2は、既に発見されていた、胎児肝臓キナーゼ1(Flk−1)としても知られるマウスVEGFR−2と85%の配列同一性を有する。VEGFR−2は、内皮及び造血前駆体、そして内皮細胞、発生期の造血幹細胞、及び臍帯ストローマにおいて、通常発現している。しかしながら、休止期の成体の血管系において、VEGFR−2mRNAは下方制御されているように見える。
本発明に従い実施されるサルモネラ・タイフィ(Salmonella typhi)の弱毒変異株の製造プロセスは、アミノ酸及びペプチドの供給源としてペプトンを含有する培地中でサルモネラ・タイフィ(Salmonella typhi)の弱毒変異株を培養することを含む。本発明の方法に適した培地は、限定されないが、標準的なTSB培地や非動物起源のTSB培地を含む。標準的なTSB培地及び非動物起源のTSB培地のいずれも、2.5g/lのグルコースを含有する。通常、TSB又はTSB様培地中の当初グルコース量は、弱毒化サルモネラ・タイフィ(Salmonella typhi)株の培養の3〜5時間後に完全に消費され、培地中の一定のグルコースレベルを維持するために、3〜5時間ごとに新鮮なグルコースで置換されなければならない。異種抗原をコードする組換えDNA分子を任意で保有するサルモネラ・タイフィ(Salmonella typhi)の弱毒変異株の培養の過程でのグルコースの追加を省略することが、グルコースを追加する培養と比較して細胞増殖の増大をもたらすという知見は大変に驚異的なものであり、細菌細胞における特定の代謝経路がグルコースの非存在がきっかけとなることを示唆する。そのような効果は、TSB又はTSB様培地が培養プロセスの開始時点でグルコースを全く含有しない場合にも観察される。従って、本発明の方法は、より高い細胞収量及び最終的なDNAワクチンの収量と別に、発酵過程でのグルコース供給が不要となることにより、コストが安くなり、プロセスが単純になるという更なる利点が存在する。
空の、及び操作されたサルモネラ・タイフィ(Salmonella typhi)Ty21aの細胞の増殖を、TSB又はTSB様培地中、0〜30時間、25〜35℃、好ましくは30℃、pH6.5〜7.5、好ましくは7.0での培養により試験した。増殖は、OD又はCFU/ml(コロニー形成単位)で測定された。
RSLの調製の第一の段階は、Typhoral L(登録商標)カプセルから弱毒化サルモネラ・タイフィ(Salmonella typhi)Ty21a株を単離し、続いて当該弱毒化細菌を、プラスミドDNA (pVAX10.VR2−1)で形質転換することである。弱毒化サルモネラ・エンテリカ・セロヴァー・タイフィ(Salmonella enterica serovar typhi)Ty21a株を含有する市販のTyphoral L(登録商標)カプセルを、下記の組換え実験に使用されるS.typhiのストックの調製に使用した。当該プロセスは、前記カプセルの内容物を液体培養培地に播種し、更に当該液体培地を寒天培地にプレーティングして、単一の細菌コロニーを単離することからなる。単一のコロニーは単離され、液体培養培地中で増殖させられる。VAX.Ty21−1及びVAX.Ty21−2の2つの培養物がグリセロールで製剤化され、分注され(1mL)、そして−75±5℃で保存された。2つの培養物のそれぞれの同一性が、更に確認された。
プラスミド構築の原則は、以下の工程を有する二重鎖インビトロ遺伝子合成に基づく。
−7.58kBの全長pVAX10−VR2.1プラスミドを、最大1・5kBの5つの断片に分割した(ソフトウエア解析による)。各断片を、それぞれが両鎖のオリゴヌクレオチドの間で重複する領域を有する40〜50bpのオリゴヌクレオチドに分割した。
−インビトロ合成されたオリゴヌクレオチドは、T4ポリヌクレオチドキナーゼとインキュベーションしてリン酸化された。
−適切な条件下で重複したオリゴヌクレオチドをアニールさせた後、Taq DNAリガーゼ酵素が、並列したオリゴヌクレオチドを接続した。
−ライゲーション工程の終了後、ライゲーションしたプラスミド断片(〜1.5kB)の収量が増大するように、外側の位置にアニールするプライマーを使用して、PCRを実施した。
−アガロースゲル電気泳動を実施して、PCR産物を単離した。
−単離したPCR産物を、TOPOベクター(Invitrogen K#4575−40)にクローニングし、育種用のTOP10 E. coliに形質転換した。
−TOPOプラスミドの単離後、制限及び配列の評価を実施した。
−単離された並列した断片を、オーバーラップPCRを通じて組立てた。当該プロセスの後、pVAX10.VR2−1プラスミドを線形的に組立てた。
−XhoI制限消化(pVAX10.VR2−1プラスミド中に制限酵素部位が1つ存在する。図2.1.S.1.2.2−1)し、T4リガーゼを通じて共有結合させ、当該環状プラスミドを、育種用のE.coliに形質転換した。
−最終的なプラスミド配列の評価の後、pVAX10.VR2−1プラスミドを、S. typhi Ty21a細菌株に形質転換した。
下記表2は、発酵の過程でグルコースを供給する/しない製造プロセスをまとめたものである。
生細菌癌ワクチンの生産は、弱毒化サルモネラ・エンテリカ・セロヴァー・タイフィ(Salmonella enterica serovar typhi)Ty21a株(pVAX10.VR2−1プラスミド含有)の発酵を基礎とする。増殖後、細胞懸濁物は、クロスフロー濾過で濃縮され、透析濾過により洗浄された。洗浄された細胞懸濁物の5種類の希釈物を、2Rガラスバイアルに充填した。
−生産プロセスのタイムテーブルを計画するためのプレカルチャー1及び2の増殖速度の評価
−高い細胞密度(OD600値)を達成するためのグルコース補給の影響の調査
−VK1a:500ml TSB培地、3l Corningフラスコ+0.5ml MCB,30℃、120rpm
−VK1b:500ml TSB培地、3l Corningフラスコ+0.5ml MCB,30℃、120rpm
−VK2:1000ml TSB培地、3l Corningフラスコ+75ml VK1b(OD1.6),30℃、120rpm
−VK1ak:500ml TSB培地+25mg/l硫酸カナマイシン、2l Corningフラスコ+0.5ml MCB,30℃、120rpm
−VK1bk:500ml TSB培地+25mg/l硫酸カナマイシン、3l Corningフラスコ+0.5mlMCB,30℃、120rpm
−VK2ak:1000ml TSB培地+25mg/l硫酸カナマイシン、3l Corningフラスコ+75ml VK1ak(OD1,8),30℃、120rpm
−VK2bk:1000ml TSB培地+25mg/l硫酸カナマイシン、3l Corningフラスコ+75ml VK1ak(OD1,8),30℃、120rpm
3つのプレカルチャー(1、2、3)の増殖試験が、2l Erlenmeyerフラスコ中で、及び生産株と同様に2段階の培養で、実施された。
−VK1a:500ml TSB培地、2l Corningフラスコ+0.5ml RCB、30℃、120rpm
−VK1b:500ml TSB培地、2l Corningフラスコ+0.5ml RCB、30℃、120rpm+グルコース追加
−VK2a:500ml TSB培地、2l Corningフラスコ+38ml VK1a(OD5.4)、30℃、120rpm
−VK2b:500ml TSB培地、2l Corningフラスコ+38ml VK1a(OD5.4)、30℃、120rpm+グルコース追加
−VK3a:500ml TSB培地、2L Corningフラスコ+38ml VK1b(OD5.1;5時間後最初のグルコース追加)、30℃、120rpm
−VK3b:500ml TSB培地、2L Corningフラスコ+38ml VK1b(OD5.1;5時間後最初のグルコース追加)、30℃、120rpm+グルコース追加
実施例5に示す癌ワクチン生産株VXM01において実施されたものと同一の実験アプローチが使用された。実施例5との唯一の違いは、当該株がプラスミドpVAX10.VR2−1で形質転換されている点である。これらの調査は、生産株S. typhi Ty21a:pVAX10−VR2.1 (p)のグルコース代謝に関連する増殖特性が、プラスミドに影響されず空の株の特性に影響されるという仮説を検証するために行われた。両株の増殖及びグルコース追加を、図11及び12に記載する。
Claims (16)
- ガラクトースエピメラーゼ活性を欠如し、発現カセットを有する1コピー以上の組換えDNA分子を有するサルモネラ・タイフィ(Salmonella typhi)の弱毒変異株を増殖させる方法であって、当該株を、発酵スケールで、ほぼ中性の当初pH値の、ペプトンを含有する緩衝培地中で培養する工程を含み、当該発酵の過程での培地中のグルコースの量が、定常期に達する前に当該グルコースの量が0にまで減少するように調整される、当該方法。
- 発酵の過程で培地中にグルコースが追加されず、開始時点でのグルコースの量が、定常期に達する前に枯渇する、請求項1に記載の方法。
- 前記サルモネラ・タイフィ(Salmonella typhi)の弱毒変異株が、サルモネラ・タイフィ(Salmonella typhi)Ty21aである、請求項1又は2のいずれか1項に記載の方法。
- 前記発現カセットが、真核細胞発現カセットであり、好ましくはVEGF受容体タンパク質をコードし、好ましくは、当該VEGF受容体タンパク質が、ヒトVEGFR−2及びそれと約80%以上の同一性を有する類似体からなる群から選択され、特に当該ヒトVEGFR−2は、配列番号1に示すアミノ酸配列を有する、請求項1〜3のいずれか1項に記載の方法。
- 前記緩衝培地が、非動物起源のペプトンを含有し、好ましくは、非動物起源のTryptic Soy Broth (TSB)である、請求項1〜4のいずれか1項に記載の方法。
- 前記培地の体積が約10l、好ましくは約10〜10,000l、より好ましくは約30〜1,000l、最も好ましくは約100〜500lである、請求項1〜5のいずれか1項に記載の方法。
- 前記当初グルコース濃度が、細菌の最少培地のグルコース濃度以下であり、好ましくは前記当初グルコース濃度が、約0〜4g/lである、請求項1〜6のいずれか1項に記載の方法。
- 前記当初pH値が約5〜9、好ましくは約6〜8、より好ましくは約6.5〜7.5である、請求項1〜7のいずれか1項に記載の方法。
- 前記pH値が、前記培養の過程で、約約6〜8、好ましくは約6.5〜7.5に調整される、請求項1〜8のいずれか1項に記載の方法。
- 増殖の進行が、(i)光学密度(OD)の測定、好ましくは(ia)培養物の光学密度のin−situでのモニタリングによる測定、又は(ib)試料の取得及び当該試料の光学密度の測定、又は(ii)細胞密度の測定、好ましくは(iia)顕微鏡下での細胞密度測定、又は(iib)電気抵抗による細胞密度の測定、又は(iic)フローサイトメトリーによる細胞密度の測定、又は(iii)試料の取得及び寒天培地への播種による、コロニー形成単位(CFU)の測定、により判定される、請求項1〜9のいずれか1項に記載の方法。
- 前記細胞が、光学密度が約6、好ましくは光学密度が約5〜6に達する前に回収される、請求項1〜10のいずれか1項に記載の方法。
- 前記サルモネラ・タイフィ(Salmonella typhi)の弱毒変異株が、サルモネラ・タイフィ(Salmonella typhi)Ty21aであり、前記組換えDNA分子が、カナマイシン耐性遺伝子、pMB1ori、及びCMVプロモーターの制御下にあるヒトVEGFR−2をコードする真核細胞発現カセットを有し、特に、ヒトVEGFR−2をコードする発現カセットの核酸配列が、配列番号2に記載されたものである、請求項1〜11のいずれか1項に記載の方法。
- ガラクトースエピメラーゼ活性を欠如し、発現カセットを有する1コピー以上の組換えDNA分子を有するサルモネラ・タイフィ(Salmonella typhi)の弱毒変異株であって、当該株を、発酵スケールで、ほぼ中性の当初pH値の、ペプトンを含有する緩衝培地中で培養する工程を含む増殖方法であって、当該発酵の過程での培地中のグルコースの量が、定常期に達する前に当該グルコースの量が0にまで減少するように調整され、好ましくは発酵の過程で培地中にグルコースが追加されず、開始時点でのグルコースの量が、定常期に達する前に枯渇する、当該増殖方法により取得が可能な、当該弱毒変異株。
- 前記発現カセットが、真核細胞発現カセットであり、好ましくはVEGF受容体タンパク質をコードし、好ましくは、当該VEGF受容体タンパク質が、ヒトVEGFR−2及びそれと約80%以上の同一性を有する類似体からなる群から選択され、特に当該ヒトVEGFR−2は、配列番号1に示すアミノ酸配列を有する、請求項13に記載の弱毒変異株。
- サルモネラ・タイフィ(Salmonella typhi)Ty21aであり、前記組換えDNA分子が、カナマイシン耐性遺伝子、pMB1ori、及びCMVプロモーターの制御下にあるヒトVEGFR−2をコードする真核細胞発現カセットを有し、特に、ヒトVEGFR−2をコードする発現カセットの核酸配列が、配列番号2に記載されたものである、請求項14に記載の弱毒変異株。
- サルモネラ・タイフィ(Salmonella typhi)Ty21aの弱毒変異株であって、VEGF受容体タンパク質をコードする真核細胞発現カセットを有する、1コピー以上の組換えDNA分子を有し、好ましくは、当該VEGF受容体タンパク質が、ヒトVEGFR−2及びそれと約80%以上の同一性を有する類似体からなる群から選択され、特に当該ヒトVEGFR−2は、配列番号1に示すアミノ酸配列を有する、ワクチンとして使用される、当該弱毒変異株。
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