JP7397821B2 - モジュールポリペプチドライブラリならびにその作製方法及びその使用 - Google Patents
モジュールポリペプチドライブラリならびにその作製方法及びその使用 Download PDFInfo
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Description
本出願は、2015年9月1日に出願された米国特許仮出願第62/212,999号の利益を主張するものであり、その全体は本明細書に参照として組み込まれる。
本発明は、国立衛生研究所による認可番号EY016546、P50 GM081879、R01 CA196277、F32 GM006499、及び、R01 GM055040のもとで、政府による支援を受けて成立した。政府は、本発明において一定の権利を有する。
配列表は、本明細書にて、2016年8月29日に作成され、145KBのサイズを有するテキストファイル、「UCSF-518WO SeqList_ST25.txt」として提供される。テキストファイルの内容は、その全体が参照により本明細書に組み込まれる。
多くの真核細胞タンパク質は、タンパク質全体としての入力及び出力機能を制御または促進するモジュールドメインまたはモチーフを介して機能する。真核細胞タンパク質のモジュールドメインの再構成及び組換えにより、入力/出力関係を変化させて完全に新規の総合的な機能を有する新規のタンパク質が既に提供され始めている。入力ドメイン及び出力ドメインの簡便な組換えを用いて個々のモジュールタンパク質の改変を成功させることにより、新規タンパク質開発へのこのモジュール法の原理証明を提供してきた。
用語「ポリヌクレオチド」及び「核酸」は、本明細書において同じ意味で用いられ、任意の長さを有するヌクレオチドのポリマー形態(リボヌクレオチドまたはデオキシリボヌクレオチドのいずれか)のことを意味する。それゆえ、この用語は、一本鎖、二本鎖、もしくは多本鎖のDNAもしくはRNA、ゲノムDNA、cDNA、DNA-RNAハイブリッド、または、プリン塩基及びピリミジン塩基、もしくは、その他の天然ヌクレオチド塩基、化学的に修飾したヌクレオチド塩基もしくは生化学的に修飾したヌクレオチド塩基、非天然ヌクレオチド塩基、もしくは誘導体化されたヌクレオチド塩基を含むポリマー、を含むがこれらに限定されない。
本開示は、合成モジュールポリペプチドライブラリ、及び、このような合成モジュールポリペプチドライブラリをコードする核酸を提供する。合成モジュールポリペプチドライブラリの作製方法、及び、合成モジュールポリペプチドライブラリをコードする核酸の製造方法もまた提供する。合成モジュールポリペプチドライブラリをスクリーニングして、合成モジュールポリペプチドライブラリのメンバーに関連する選択した表現型を同定する方法もまた提供するが、このような方法は、インビトロアッセイ及びインビボアッセイの両方において有用である。
本開示の態様は、複数の合成モジュールポリペプチドをコードする核酸のバーコードライブラリに関する。本開示の態様は更に、それぞれの細胞が、個々のバーコード化核酸を発現する細胞ライブラリを含み、当該個々のバーコード化核酸は、固有合成モジュールポリペプチドまたは複数のこのような個々のバーコード化核酸をコードする。以下でより詳細に記載するように、本開示の態様は更に、このようなライブラリの作製方法、及び、このようなライブラリをスクリーニングして特定の表現型を検出するための方法を含む。
合成モジュールポリペプチドのライブラリ、及び、合成モジュールポリペプチドをコードする核酸を提供する。「モジュールポリペプチド」とは、モジュールが物理的に連結して単一ポリペプチド分子として共に機能するように、機能的に連結した2つ以上のモジュール構成要素を有する機能性タンパク質のことを意味する。モジュールポリペプチドのモジュールは、機能または活性と関連していてもしていなくてもよい。多くの実施形態では、合成モジュールポリペプチドのモジュール構成要素の少なくとも2つは、個々のタンパク質から誘導される。「個々のタンパク質」から誘導したモジュールは、機能的に関連しないまたは機能的に関連する異なるタンパク質から誘導してもよく、または、異種生物、同種生物、異なるオルソロガスタンパク質、異なるパラロガスタンパク質などから誘導してもよい。
本明細書に記載のライブラリは、それらタンパク質をコードする核酸配列が発現する、検出可能なシグナル産生タンパク質を含む。本明細書に記載のライブラリシステムに用いる特定の検出可能なシグナル産生タンパク質は様々であり、ある程度は選択した産生シグナル検出法に依存する。例えば、シグナルが光学的に検出される場合、例えば、蛍光顕微鏡またはフローサイトメトリー(蛍光活性化細胞分離法(FACS)を含む)を用いる場合、蛍光レポーターを用いる。
個々のライブラリメンバー内において、単一ポリペプチドをコードするモジュール構成要素間の連結部は通常、「インフレーム」に保持されるが、それは、マルチモジュールコード配列のコドン読み取り枠が1つのモジュールから次のモジュールへと維持されることを意味している。このような連結部は、本明細書においては、インフレーム連結部及び/またはインフレーム接合部と呼ばれることもある。マルチモジュールポリペプチドにおけるモジュール構成要素間のリンカーは通常、フレキシブルであり、及び/または、モジュールドメインの機能と干渉するアミノ酸残基を含まない。
核酸バーコードは、例えば、ハイブリダイゼーションをベースとした同定(すなわち、in situハイブリダイゼーション)、増幅をベースとした同定(すなわち、PCRベースの同定)、及び核酸シークエンシングが挙げられるがこれらに限定されない、任意の簡便な核酸配列同定法を用いて同定することが可能な、特定の固有核酸配列である。本開示のバーコードはモジュール特異的なバーコードであってもよく、それは、特定のバーコードの同定が結合したモジュールコード配列の明確な同定に相当するように、マルチモジュールライブラリにおけるそれぞれの固有モジュールが特定の固有バーコードと相関していることを意味する。
「ベクター特異的要素」とは、そのベクターの構築の前、その間、またはその後において、ベクターを作製、構築、増殖、維持、及び/または、アッセイするのに用いる要素のことを意味する。このようなベクター特異的要素としては、例えば、ベクターの使用中にベクターの増殖、クローニング及び選択に必要なベクター要素が挙げられるがこれらに限定されず、また例えば、複製起点、マルチクローニング部位、原核細胞プロモーター、ファージプロモーター、選択マーカー(例えば、抗生物質抵抗性遺伝子、コードされた酵素タンパク質、コードされた蛍光または発色タンパク質など)などを挙げることができるが、これらに限定されない。任意の好都合なベクター特異的要素は、必要に応じ、本明細書に記載のベクターとして有用であってもよい。
本開示は、合成モジュールポリペプチドをコードする核酸のライブラリを作製するための方法を提供する。ライブラリにおけるそれぞれの核酸は、モジュールポリペプチドの可変モジュール(複数可)を同定するマルチユニットバーコードを含み、複数の可変モジュールが存在し、それらの方向は互いに相対的である。多数の実施形態において、本開示は、合成モジュールポリペプチドをコードする得られた核酸のそれぞれが、インフレームモジュールのコード領域及びマルチユニットバーコードを含むことになるように(バーコードユニットの配列はインフレームモジュールの配列に対応する)、バーコード化モジュールコード核酸から、合成モジュールポリペプチドを段階的に組み合わせてアセンブリするための方法を提供する。一般的に、複数の可変モジュールがライブラリのそれぞれのメンバー内に含まれる場合、コアセンブリしたマルチユニットバーコードは、それぞれのライブラリメンバーにおける可変モジュールのアセンブリ記録を提供する。
全体として、本開示は、本明細書に記載のとおり、バーコード連結ポリペプチドモジュールコード核酸の入れ子アセンブリを用いたライブラリの作製方法を提供する。例えば、図12に示すように、モジュール特異的バーコード(102)と連結したポリペプチドモジュールをコードする配列(101)を含む核酸ベクター(100)を、ポリペプチドモジュールをコードする配列とモジュール特異的バーコードとの間の連結を開裂させることにより直鎖化(103)する。第1のモジュールコード配列及び第1のモジュール特異的バーコードを含むベクターの直鎖化に続き、第2のモジュールコード配列(104)及び第2のモジュールに特異的な第2のバーコード(105)を含む核酸を、第1のモジュールコード配列と第1のバーコードとの間に挿入(すなわち、入れ子)する(106)。アセンブリした核酸は、合成モジュールポリペプチドをコードするコード領域、及び、マルチユニットバーコード(すなわち、「バーコード」(BC))を含むバーコード領域を含む。一部の例においては、モジュールコード配列は、本明細書に記載のとおり、所望のリンカーをコードする配列によりそれらが連結するようにアセンブリされる。特定の例においては、モジュールコード配列は、リンカー配列を用いることなく、例えば、1つまたは複数のリンカーアミノ酸をコードするリンカー配列を用いることなく、第1のモジュールコード配列と第2のモジュールコード配列との間に非コードヌクレオチドを何ら介在させずに、などで連結されるようにアセンブリされる。それゆえ、「所望のリンカー配列」または「リンカー」という表記は、とりわけ図面内で用いる場合、任意のリンカーまたはリンカー配列が全く存在しないこと、及び、ポリペプチドモジュールとモジュールコード配列とが直接連結していることを包含する。
本開示は、バーコード化合成モジュールポリペプチドのプールライブラリを作製するための方法を提供する。プールライブラリとは、ライブラリメンバーが共通の容器内及び/または共通の溶液中に存在し、かつ個々のライブラリメンバーを空間をあけて物理的に分離させる必要がないことを意味し、例えば、個々のライブラリメンバーは、ライブラリの構築中に(例えば、「ワンポットアセンブリ」におけるような)、または、個々のライブラリメンバーの構築後にプールされてもよい。例えば、組み合わせ入れ子アセンブリで構築したプール合成モジュールポリペプチドライブラリの場合、ライブラリメンバーのアセンブリ中に、ライブラリメンバーのアセンブリ完了前に、及び/または、ライブラリメンバーのアセンブリ完了後などに、ライブラリの構成要素をプールしてもよい。本発明で使用する場合、プールライブラリは、合成モジュールポリペプチドのみのライブラリに限定されず、合成モジュールポリペプチドを発現する細胞のプールライブラリ、合成モジュールポリペプチドをコードする核酸のプールライブラリなどもまた含む。
一部の例においては、コード領域及びバーコード領域を含む個々の核酸メンバーを区画化してもよい。プールライブラリ及び区画化構成要素を含むライブラリは、相互排他的である必要はなく、例えば、一部の例においては、ライブラリは、異なる回数プールしたライブラリ、及び異なる回数区画化した構成要素を含むライブラリの両方であってもよく、例えば、ライブラリを構築してもよく、例えば、ライブラリメンバーをプールで(例えば、ワンポットアセンブリで)アセンブリしてもよく、それから、例えば、核酸ライブラリメンバーを個々の細胞または非細胞区画へとトランスフェクションすることにより、続けて区画化してもよい。その他の例では、ライブラリメンバーをそれらのアセンブリの途中で区画化してもよく、その後、更なる処理(例えば、更なるアセンブリまたはスクリーニングが挙げられるがこれらに限定されない)のために続けてプールしてもよい。一般的に、本明細書に記載の入れ子アセンブリ及びクローニング手法に従いアセンブリした、バーコード化合成ポリペプチドコード核酸は、プールでアセンブリされ、その後区画化されてもされなくてもよい。
上記で少し詳細に説明したとおり、本発明で使用する場合、ライブラリは細胞ライブラリを含み、ライブラリの細胞は合成モジュールポリペプチドを発現する。例えば、ウイルストランスフェクション、エレクトロポレーション、リポフェクション、ボンバードメント、化学的形質転換、導入可能なキャリア(例えば、導入可能なキャリアタンパク質)の使用などが挙げられるがこれらに限定されない、任意の簡便な方法を用いて、合成モジュールポリペプチドをコードする核酸の形質転換を行ってもよい。一部の例においては、合成モジュールポリペプチドコード核酸を形質導入する細胞のことを、本明細書では、宿主細胞と呼ぶ。
ライブラリを作製する状況、及び/またはライブラリの意図する最終用途に応じて、本開示のライブラリを正規化してもしなくてもよい。本発明において記載するライブラリに関して使用する場合、「正規化」とは、それぞれのライブラリメンバーの相対量を、それぞれのライブラリメンバーの相対量を調節する前よりも少なくとも均等に近づくように調節することを意味する。一部の例においては、ライブラリを正規化することにより、最も多く発現するライブラリメンバーの量と最も少なく発現するライブラリメンバーとの間がより狭い範囲となるライブラリがもたらされる。一部の例においては、正規化により、最も少なく発現するライブラリメンバー(複数可)の量が上昇することになる。一部の例においては、正規化により、最も多く発現するライブラリメンバー(複数可)の量が低下することになる。
例えば、インビトロスクリーニング法及びインビボスクリーニング法が挙げられるがこれらに限定されない、合成モジュールポリペプチドライブラリのスクリーニング方法を提供する。「インビボスクリーニング」とは通常、生体の生物学的環境内でアッセイされる、複数の固有合成モジュールポリペプチドを含むライブラリのことを意味する。本明細書に記載の方法に従いインビボでアッセイ可能な生体としては、単細胞生物及び多細胞生物が挙げられる。
スクリーニング法(インビボまたはインビトロのどちらか)は通常、表現型を検出すること、及び、表現型に関連する1つまたは複数のライブラリメンバーを同定することを含む。本発明で使用する場合、用語「表現型」とは通常、特定のアッセイで検出された、分子、細胞、組織、器官または生体における特徴のことを意味し、例えば、分子表現型、細胞表現型、生物表現型、組織表現型、器官表現型、生物表現型などを挙げることができるがこれらに限定されない。特定のアッセイで検出された表現型は、所定の表現型、例えば、既知もしくは予想される表現型(例えば、既知または予想されるレベルの特定の特徴、既知または予想されるレベルの特徴の有無を含む)であってもよく、または、アッセイ時に同定されてもよく、例えば、新たに検出されたもしくは以前には同定されていなかった表現型(例えば、新たに検出されたまたは以前には同定されていなかったレベルの特定の特徴、新たに検出されたまたは以前には同定されていなかった特徴の有無などを含む)である。本明細書に記載の合成モジュールポリペプチドライブラリに関連する表現型を検出するための任意の簡便なアッセイは、このようなライブラリをスクリーニングするのに有用であってもよい。
(配列中、ITAMモチーフは、太字とし下線を引いている)。
または、
(配列中、ITAMモチーフは、太字とし下線を引いている)。
(配列中、ITAMモチーフは、太字とし下線を引いている)。
(配列中、ITAMモチーフは、太字とし下線を引いている)。
のアミノ酸配列である。一部の実施形態では、好適な細胞内シグナル伝達ドメインは、アミノ酸配列
の完全長に対して、少なくとも約85%の、少なくとも約90%の、少なくとも約95%の、少なくとも約98%の、または、少なくとも約99%のアミノ酸配列同一性を有するアミノ酸配列を含む。
のアミノ酸配列である。一部の実施形態では、好適な細胞内シグナル伝達ドメインは、アミノ酸配列
の完全長に対して、少なくとも約85%の、少なくとも約90%の、少なくとも約95%の、少なくとも約98%の、または、少なくとも約99%のアミノ酸配列同一性を有するアミノ酸配列を含む。
MNGDETKKVESEYIKKHHRHELVESQCSSTLVKHIKAPLHLVWSIVRRFDEPQKYKPFISRCVVQGKKLEVGSVREVDLKSGLPATKSTEVLEILDDNEHILGIRIVGGDHRLKNYSSTISLHSETIDGKTGTLAIESFVVDVPEGNTKEETCFFVEALIQCNLNSLADVTERLQAESMEKKI(配列番号:85)。
METSQKYHTCGSTLVQTIDAPLSLVWSILRRFDNPQAYKQFVKTCNLSSGDGGEGSVREVTVVSGLPAEFSRERLDELDDESHVMMISIIGGDHRLVNYRSKTMAFVAADTEEKTVVVESYVVDVPEGNSEEETTSFADTIVGFNLKSLAKLSERVAHLKL(配列番号:86)。
MKTSQEQHVCGSTVVQTINAPLPLVWSILRRFDNPKTFKHFVKTCKLRSGDGGEGSVREVTVVSDLPASFSLERLDELDDESHVMVISIIGGDHRLVNYQSKTTVFVAAEEEKTVVVESYVVDVPEGNTEEETTLFADTIVGCNLRSLAKLSEKMMELT(配列番号:87)。
MESSKQKRCRSSVVETIEAPLPLVWSILRSFDKPQAYQRFVKSCTMRSGGGGGKGGEGKGSVRDVTLVSGFPADFSTERLEELDDESHVMVVSIIGGNHRLVNYKSKTKVVASPEDMAKKTVVVESYVVDVPEGTSEEDTIFFVDNIIRYNLTSLAKLTKKMMK(配列番号:88)。
MANSESSSSPVNEEENSQRISTLHHQTMPSDLTQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMNRNNNNNNSSQVR(配列番号:89)。
MSSSPAVKGLTDEEQKTLEPVIKTYHQFEPDPTTCTSLITQRIHAPASVVWPLIRRFDNPERYKHFVKRCRLISGDGDVGSVREVTVISGLPASTSTERLEFVDDDHRVLSFRVVGGEHRLKNYKSVTSVNEFLNQDSGKVYTVVLESYTVDIPEGNTEEDTKMFVDTVVKLNLQKLGVAATSAPMHDDE(配列番号:90)。
MNLAPIHDPSSSSTTTTSSSTPYGLTKDEFSTLDSIIRTHHTFPRSPNTCTSLIAHRVDAPAHAIWRFVRDFANPNKYKHFIKSCTIRVNGNGIKEIKVGTIREVSVVSGLPASTSVEILEVLDEEKRILSFRVLGGEHRLNNYRSVTSVNEFVVLEKDKKKRVYSVVLESYIVDIPQGNTEEDTRMFVDTVVKSNLQNLAVISTASPT(配列番号:91)。
MLAVHRPSSAVSDGDSVQIPMMIASFQKRFPSLSRDSTAARFHTHEVGPNQCCSAVIQEISAPISTVWSVVRRFDNPQAYKHFLKSCSVIGGDGDNVGSLRQVHVVSGLPAASSTERLDILDDERHVISFSVVGGDHRLSNYRSVTTLHPSPISGTVVVESYVVDVPPGNTKEETCDFVDVIVRCNLQSLAKIAENTAAESKKKMSL(配列番号:92)。
MRSPVQLQHGSDATNGFHTLQPHDQTDGPIKRVCLTRGMHVPEHVAMHHTHDVGPDQCCSSVVQMIHAPPESVWALVRRFDNPKVYKNFIRQCRIVQGDGLHVGDLREVMVVSGLPAVSSTERLEILDEERHVISFSVVGGDHRLKNYRSVTTLHASDDEGTVVVESYIVDVPPGNTEEETLSFVDTIVRCNLQSLARSTNRQ(配列番号:93)。
MPTSIQFQRSSTAAEAANATVRNYPHHHQKQVQKVSLTRGMADVPEHVELSHTHVVGPSQCFSVVVQDVEAPVSTVWSILSRFEHPQAYKHFVKSCHVVIGDGREVGSVREVRVVSGLPAAFSLERLEIMDDDRHVISFSVVGGDHRLMNYKSVTTVHESEEDSDGKKRTRVVESYVVDVPAGNDKEETCSFADTIVRCNLQSLAKLAENTSKFS(配列番号:94)。
MEMIGGDDTDTEMYGALVTAQSLRLRHLHHCRENQCTSVLVKYIQAPVHLVWSLVRRFDQPQKYKPFISRCTVNGDPEIGCLREVNVKSGLPATTSTERLEQLDDEEHILGINIIGGDHRLKNYSSILTVHPEMIDGRSGTMVMESFVVDVPQGNTKDDTCYFVESLIKCNLKSLACVSERLAAQDITNSIATFCNASNGYREKNHTETNL(配列番号:95)。
MEANGIENLTNPNQEREFIRRHHKHELVDNQCSSTLVKHINAPVHIVWSLVRRFDQPQKYKPFISRCVVKGNMEIGTVREVDVKSGLPATRSTERLELLDDNEHILSIRIVGGDHRLKNYSSIISLHPETIEGRIGTLVIESFVVDVPEGNTKDETCYFVEALIKCNLKSLADISERLAVQDTTESRV(配列番号:96)。
MMDGVEGGTAMYGGLETVQYVRTHHQHLCRENQCTSALVKHIKAPLHLVWSLVRRFDQPQKYKPFVSRCTVIGDPEIGSLREVNVKSGLPATTSTERLELLDDEEHILGIKIIGGDHRLKNYSSILTVHPEIIEGRAGTMVIESFVVDVPQGNTKDETCYFVEALIRCNLKSLADVSERLASQDITQ(配列番号:97)。
MPSELTPEERSELKNSIAEFHTYQLDPGSCSSLHAQRIHAPPELVWSIVRRFDKPQTYKHFIKSCSVEQNFEMRVGCTRDVIVISGLPANTSTERLDILDDERRVTGFSIIGGEHRLTNYKSVTTVHRFEKENRIWTVVLESYVVDMPEGNSEDDTRMFADTVVKLNLQKLATVAEAMARNSGDGSGSQVT(配列番号:98)。
MEEVSPAIAGPFRPFSETQMDFTGIRLGKGYCNNQYSNQDSENGDLMVSLPETSSCSVSGSHGSESRKVLISRINSPNLNMKESAAADIVVVDISAGDEINGSDITSEKKMISRTESRSLFEFKSVPLYGFTSICGRRPEMEDAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLKPRRKLKSKPLN(配列番号:99)。
MDEVSPAVAVPFRPFTDPHAGLRGYCNGESRVTLPESSCSGDGAMKDSSFEINTRQDSLTSSSSAMAGVDISAGDEINGSDEFDPRSMNQSEKKVLSRTESRSLFEFKCVPLYGVTSICGRRPEMEDSVSTIPRFLQVSSSSLLDGRVTNGFNPHLSAHFFGVYDGHGGSQVANYCRERMHLALTEEIVKEKPEFCDGDTWQEKWKKALFNSFMRVDSEIETVAHAPETVGSTSVVAVVFPTHIFVANCGDSRAVLCRGKTPLALSVDHKPDRDDEAARIEAAGGKVIRWNGARVFGVLAMSRSIGDRYLKPSVIPDPEVTSVRRVKEDDCLILASDGLWDVMTNEEVCDLARKRILLWHKKNAMAGEALLPAEKRGEGKDPAAMSAAEYLSKMALQKGSKDNISVVVVDLKGIRKFKSKSLN(配列番号:100)。
MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLYDPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLNEFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGEESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLVDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQYISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAAPDEIVADSFEALGANTIKEPGLCPSVSSNDQQVPSAVRYNGSKRVKPEEEEERDMKKSRGFDERELFSTAESSSSSSVFFVSQSCSLASEGKNLEGIQDSSDQITTSLGKNGCK(配列番号:101)。
MAASDEVNLIESRTVVPLNTWVLISNFKVAYNILRRPDGTFNRHLAEYLDRKVTANANPVDGVFSFDVLIDRRINLLSRVYRPAYADQEQPPSILDLEKPVDGDIVPVILFFHGGSFAHSSANSAIYDTLCRRLVGLCKCVVVSVNYRRAPENPYPCAYDDGWIALNWVNSRSWLKSKKDSKVHIFLAGDSSGGNIAHNVALRAGESGIDVLGNILLNPMFGGNERTESEKSLDGKYFVTVRDRDWYWKAFLPEGEDREHPACNPFSPRGKSLEGVSFPKSLVVVAGLDLIRDWQLAYAEGLKKAGQEVKLMHLEKATVGFYLLPNNNHFHNVMDEISAFVNAEC(配列番号:104)。
MAGGNEVNLNECKRIVPLNTWVLISNFKLAYKVLRRPDGSFNRDLAEFLDRKVPANSFPLDGVFSFDHVDSTTNLLTRIYQPASLLHQTRHGTLELTKPLSTTEIVPVLIFFHGGSFTHSSANSAIYDTFCRRLVTICGVVVVSVDYRRSPEHRYPCAYDDGWNALNWVKSRVWLQSGKDSNVYVYLAGDSSGGNIAHNVAVRATNEGVKVLGNILLHPMFGGQERTQSEKTLDGKYFVTIQDRDWYWRAYLPEGEDRDHPACNPFGPRGQSLKGVNFPKSLVVVAGLDLVQDWQLAYVDGLKKTGLEVNLLYLKQATIGFYFLPNNDHFHCLMEELNKFVHSIEDSQSKSSPVLLTP(配列番号:105)。
MAGSEEVNLIESKTVVPLNTWVLISNFKLAYNLLRRPDGTFNRHLAEFLDRKVPANANPVNGVFSFDVIIDRQTNLLSRVYRPADAGTSPSITDLQNPVDGEIVPVIVFFHGGSFAHSSANSAIYDTLCRRLVGLCGAVVVSVNYRRAPENRYPCAYDDGWAVLKWVNSSSWLRSKKDSKVRIFLAGDSSGGNIVHNVAVRAVESRIDVLGNILLNPMFGGTERTESEKRLDGKYFVTVRDRDWYWRAFLPEGEDREHPACSPFGPRSKSLEGLSFPKSLVVVAGLDLIQDWQLKYAEGLKKAGQEVKLLYLEQATIGFYLLPNNNHFHTVMDEIAAFVNAECQ(配列番号:106)。
本開示は、特定の検出表現型に関連するライブラリメンバーを同定するための方法を含む。理論に束縛されるものではないが、それぞれのマルチモジュール合成ポリペプチドにそれぞれの対応するマルチユニットバーコードを加えた整合アセンブリにより、アセンブリに続いて、それぞれの固有合成モジュールポリペプチドを同定することが可能となる。上記のとおり、それぞれの合成モジュールポリペプチドコード核酸のバーコード領域は、それぞれの合成モジュールポリペプチドを構成する個々のモジュールの同一性だけではなく、そのモジュールの特定の配置(本明細書では、構造と呼ぶ)もまた提供する。それゆえ、バーコード領域をシークエンシングすることにより、それぞれのライブラリメンバーの同一性及び構造を同定することができる。
ライブラリ構築に必要な要素を含むバーコード化モジュールコード核酸断片を作製するために、ポリペプチドモジュール(すなわち、共調節ドメイン(すなわち、共刺激または共抑制))をコードする核酸を、シークエンシング及びType IIS制限酵素消化に好適なクローニングベクターにサブクローニングした。サブクローニング後、ベクターは、モジュールコード配列、モジュールコード配列の前後に存在する最適なGly/Serリンカー配列、モジュール特異的バーコード配列、モジュール特異的バーコード配列の前後に存在する3´クローニングホモロジーアーム、モジュールコード配列とモジュール特異的バーコード配列との間のBamHI制限部位、ならびに、モジュールコード配列の5´末端側及びモジュール特異的バーコード配列の3´末端側の両方に存在するType IIS制限酵素部位を含んでいた(図1)。サブクローニングしたベクターインサートをシークエンシングし、挿入した共調節ドメインの同一性を確認した。この組み合わせライブラリに用いた共調節ドメイン及びそれらの対応するタンパク質配列を表1に提供する。
入れ子アセンブリにより、61種の可変CARモジュール(表2、図25に記載)を、2次元(2D)合成モジュールCARポリペプチドをコードするバーコード化核酸ライブラリへとアセンブリした。MiSeq system(Illumina Inc.,Hayward,CA)を利用したsequencing by synthesis(SBS)法を用いてライブラリをディープシークエンシングして、アセンブリしたそれぞれのライブラリメンバーのリードカウントを測定した(図26)。
ライブラリパーツの正規化を用いた、組み合わせライブラリ内のクローン分布を向上させる方法を開発した。
Claims (15)
- エクスビボおよび/またはインビトロで、細胞内の合成モジュールポリペプチドに関連する選択した表現型を同定する方法であって、
a)核酸のバーコードライブラリを宿主細胞へ導入することにより、遺伝子組換え宿主細胞の不均質な集団を生成することであって、このとき核酸の前記バーコードライブラリが複数のメンバーを含み、複数のメンバーのそれぞれが、異なる合成モジュールポリペプチドをコードするヌクレオチド配列を含み、前記ヌクレオチド配列が、2つ以上の可変モジュールをコードし、互いにインフレームにある2つ以上のコード配列を含むコード領域と、2つ以上の固有バーコードを含むバーコード領域とを含み、このとき前記コード領域内のそれぞれの可変モジュールが、前記バーコード領域内の特定の固有バーコードと相関している、生成することと、
b)刺激物に応答して選択した表現型を示す遺伝子組換え宿主細胞を前記不均質な集団内において同定することと、
c)(b)の同定した宿主細胞から、シークエンシングにより前記バーコード領域を同定および/または定量化することにより、前記選択した表現型を示す合成モジュールポリペプチドおよび/またはその可変モジュールを同定することと
を含み、前記宿主細胞が哺乳動物の初代細胞および不死化細胞株から選択され、前記合成モジュールポリペプチドがモジュール受容体である、方法。 - 核酸のバーコードライブラリを作製する方法であって、この核酸のバーコードライブラリが複数のメンバーを含み、当該複数のメンバーのそれぞれが固有合成モジュールポリペプチドをコードするヌクレオチド配列を含み、前記方法が、
第1のバーコード配列に連結した第1のモジュールコード配列を含む第1のポリヌクレオチドを、第2のバーコード配列に連結した第2のモジュールコード配列を含む第2のポリヌクレオチドと、前記第2のモジュールコード配列と前記第2のバーコード配列との間の連結部において、前記第1のポリヌクレオチドを前記第2のポリヌクレオチドへと挿入して、バーコード化バイモジュールポリヌクレオチドを生成するのに十分な条件下で、接触させることを含み、
前記バーコード化バイモジュールポリヌクレオチドが、前記第2のバーコード配列に連結した前記第1のバーコード配列に連結した前記第1のモジュールコード配列にインフレームで連結した前記第2のモジュールコード配列を含み、
各固有合成モジュールポリペプチドがモジュール受容体である、方法。 - 前記モジュール受容体がキメラ抗原受容体(CAR)ポリペプチドである、請求項1又は2に記載の方法。
- 前記複数のメンバーが、抗原結合ドメイン、特異的結合ドメインまたは特異的結合パートナータンパク質、共刺激ドメイン、共抑制ドメイン、細胞内タンパク質、膜貫通ドメイン、およびこれらの組み合わせからなる群から選択されるモジュールドメインを含むモジュール受容体をコードするヌクレオチド配列を含む、請求項3に記載の方法。
- 前記CARが、1つまたは複数のT細胞共調節ドメインを含む、請求項3に記載の方法。
- 前記共調節ドメインが、配列番号19~42のアミノ酸配列に少なくとも95%の配列同一性を有する、請求項5に記載の方法。
- 前記CARが、抗BCMA、抗CD123、抗CD138、抗CD171、抗CD19、抗CD22、抗CD30、抗CD33、抗CD7、抗CD70、抗CEA、抗EGFRvIII、抗EPCAM、抗EphA2、抗ErbB、抗FAP、抗GD2、抗GPC3、抗HER2、抗IL1RAP、抗Kappa、抗LeY、抗Meso、抗MG7、抗MUC1、抗NKG2D、抗PSCA、および抗ROR1抗原結合ドメインから選択される抗原結合ドメインを含む、請求項3に記載の方法。
- 前記CARが、1つまたは複数の免疫受容体チロシンベース活性化モチーフ(ITAM)を含む細胞内シグナル伝達ドメインポリペプチドを含む、請求項3に記載の方法。
- 前記細胞内シグナル伝達ドメインポリペプチドが、配列番号107~132のアミノ酸配列に少なくとも95%の配列同一性を有する、請求項8に記載の方法。
- 前記CARが、それぞれの鎖に二量体化ドメインを含む条件付き活性化CARであり、その二量体結合対が、FK506結合タンパク質(FKBP)およびFKBP;FKBPおよびカルシニューリン触媒サブユニットA;FKBPおよびシクロフィリン;FKBPおよびFKBP-ラパマイシン結合タンパク質;ジャイレースB(GyrB)およびGyrB;ジヒドロ葉酸還元酵素(DHFR)およびDHFR;DmrBおよびDmrB;PylおよびABI;Cry2およびCIB1;GAIおよびGID1から選択される、請求項3に記載の方法。
- 前記宿主細胞が、T細胞およびNK細胞から選択される、請求項1に記載の方法。
- 核酸のバーコードライブラリであって、
それぞれが、固有合成モジュールポリペプチドをコードするヌクレオチド配列を含む、複数の固有ポリヌクレオチドを含み、それぞれの固有ポリヌクレオチドが、
i)第2のモジュールをコードする第2のコード配列にインフレームで連結した第1のモジュールをコードする第1のコード配列を含む、前記固有合成モジュールポリペプチドをコードするコード領域と、
ii)前記第2のコード配列に特異的な第2のバーコードに連結した前記第1のコード配列に特異的な第1のバーコードを含むマルチユニットバーコード領域と、
を含み、前記第1および第2のバーコードが、前記第1および第2のコード配列と比較して逆順の5´トゥ3´であり、
それぞれのマルチユニットバーコード領域をシークエンシングすることにより、それぞれの固有合成モジュールポリペプチドの同定が可能となり、
各固有合成モジュールポリペプチドがモジュール受容体である、バーコードライブラリ。 - 前記第1および第2のコード配列が、非コードヌクレオチドを何ら介在させずに直接連結している、請求項12に記載のライブラリ。
- インビトロでプールされ、プールライブラリが少なくとも96の固有ライブラリメンバーを含む、請求項12または13に記載のライブラリ。
- それぞれが、請求項12から14のいずれか一項に記載の核酸のバーコードライブラリの固有ポリヌクレオチドを含む、複数の細胞を含み、
それぞれのバーコード領域をシークエンシングすることにより、ライブラリのそれぞれの細胞のそれぞれの固有合成モジュールポリペプチドの同定が可能となり、前記細胞が哺乳動物の初代細胞および不死化細胞株から選択される、細胞ライブラリ。
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