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Description
本明細書において提供される態様のいずれか1つの方法によって生産される形質導入細胞の集団を含む組成物も、本明細書において提供される。任意のそのような態様のいくつかにおいて、組成物は冷凍保存物質（cyropreservant）をさらに含む。
[本発明1001]
初代T細胞の形質導入頻度を増加させるための方法であって、
（a）初代T細胞の集団を含む生物学的試料から、CCR7の表面発現が陽性である初代T細胞を選択する工程であって、それによって、CCR7＋初代T細胞が富化されているインプット集団を生成させる、工程、
（b）インプット集団を刺激性条件下でインキュベートする工程であって、それによって、刺激された組成物を生成させ、刺激条件が、TCR複合体の1つもしくは複数の構成要素の1つもしくは複数の細胞内シグナリングドメインおよび/または1つもしくは複数の共刺激分子の1つもしくは複数の細胞内シグナリングドメインを活性化することができる刺激性試薬の存在を含む、工程、および
（c）組換えタンパク質をコードする異種ポリヌクレオチドを含むウイルスベクター粒子を、刺激された組成物のT細胞と共にインキュベートする工程であって、それによって、形質導入細胞の集団を生成させる、工程
を含む、方法。
[本発明1002]
初代T細胞の形質導入頻度を増加させるための方法であって、
（a）初代T細胞の集団を含む生物学的試料から、CCR7の表面発現が陽性である初代T細胞を選択する工程であって、それによって、CCR7＋初代T細胞が富化されているインプット集団を生成させる、工程、および
（b）組換えタンパク質をコードする異種ポリヌクレオチドを含むウイルスベクター粒子をインプット細胞集団のT細胞と共にインキュベートする工程であって、それによって、形質導入細胞の集団を生成させる、工程
を含む、方法。
[本発明1003]
初代T細胞の形質導入頻度を増加させるための方法であって、
（a）CCR7＋T細胞が富化されているインプット初代T細胞集団を刺激性条件下でインキュベートする工程であって、それによって、刺激された組成物を生成させ、刺激条件が、TCR複合体の1つもしくは複数の構成要素の1つもしくは複数の細胞内シグナリングドメインおよび/または1つもしくは複数の共刺激分子の1つもしくは複数の細胞内シグナリングドメインを活性化することができる刺激性試薬の存在を含む、工程、および
（b）組換えタンパク質をコードする異種ポリヌクレオチドを含むウイルスベクター粒子を、刺激された組成物のT細胞と共にインキュベートする工程であって、それによって、形質導入細胞の集団を生成させる、工程
を含む、方法。
[本発明1004]
初代T細胞の形質導入頻度を増加させるための方法であって、
組換えタンパク質をコードする異種ポリヌクレオチドを含むウイルスベクター粒子を、CCR7＋T細胞が富化されているインプット初代T細胞集団のT細胞と共にインキュベートする工程であって、それによって、形質導入細胞の集団を生成させる、工程
を含む、方法。
[本発明1005]
インプット集団の少なくとも60％、少なくとも65％、少なくとも70％、少なくとも75％、少なくとも80％、少なくとも85％、少なくとも90％、少なくとも91％、少なくとも92％、少なくとも93％、少なくとも94％、少なくとも95％、少なくとも96％、少なくとも97％、少なくとも98％、少なくとも99％、または100％が、CCR7＋初代T細胞である、本発明1001～1004のいずれかの方法。
[本発明1006]
インプット集団の少なくとも90％、少なくとも91％、少なくとも92％、少なくとも93％、少なくとも94％、少なくとも95％、少なくとも96％、少なくとも97％、少なくとも98％、少なくとも99％、または100％が、CCR7＋初代T細胞である、本発明1001～1005のいずれかの方法。
[本発明1007]
インプット集団の少なくとも95％、少なくとも96％、少なくとも97％、少なくとも98％、少なくとも99％、または100％が、CCR7＋初代T細胞である、本発明1001～1006のいずれかの方法。
[本発明1008]
生物学的試料が血液試料である、本発明1001、1002および1005～1007のいずれかの方法。
[本発明1009]
生物学的試料が白血球アフェレーシス試料である、本発明1001、1002および1005～1007のいずれかの方法。
[本発明1010]
T細胞が、未分画T細胞であるか、富化もしくは単離されたCD3＋T細胞であるか、富化もしくは単離されたCD4＋T細胞であるか、または富化もしくは単離されたCD8＋T細胞である、本発明1001～1009のいずれかの方法。
[本発明1011]
インプット集団が、CD4＋T細胞またはCD8＋T細胞である細胞を、少なくとも80％、少なくとも85％、少なくとも90％、または少なくとも95％含む、本発明1001～1010のいずれかの方法。
[本発明1012]
インプット集団が、CD4＋T細胞である細胞を、少なくとも80％、少なくとも85％、少なくとも90％、または少なくとも95％含む、本発明1001～1011のいずれかの方法。
[本発明1013]
インプット集団が、CD8＋T細胞である細胞を、少なくとも80％、少なくとも85％、少なくとも90％、または少なくとも95％含む、本発明1001～1011のいずれかの方法。
[本発明1014]
インプット集団が、CD4＋T細胞およびCD8＋T細胞である細胞を、少なくとも80％、少なくとも85％、少なくとも90％、または少なくとも95％含む、本発明1001～1011のいずれかの方法。
[本発明1015]
CD4＋T細胞のCD8＋T細胞に対する比が、1：1、1：2、2：1、1：3、もしくは3：1であるか、または約1：1、1：2、2：1、1：3、もしくは3：1である、本発明1014の方法。
[本発明1016]
インプット集団が、CD3＋T細胞である細胞を、少なくとも80％、少なくとも85％、少なくとも90％、または少なくとも95％含む、本発明1001～1010のいずれかの方法。
[本発明1017]
インプット集団が、100×10
6
～500×10
6
個の総T細胞を含む、本発明1001～1016のいずれかの方法。
[本発明1018]
インプット集団が、200×10
6
～400×10
6
個の総T細胞、任意で300×10
6
または約300×10
6
個の総T細胞を含む、本発明1001～1017のいずれかの方法。
[本発明1019]
総T細胞が生存T細胞である、本発明1017または本発明1018の方法。
[本発明1020]
刺激された組成物の細胞のうちの少なくとも10％、少なくとも20％、少なくとも30％、少なくとも40％、少なくとも50％、または少なくとも60％が、
（i）HLA-DR、CD25、CD69、CD71、CD40L、および4-1BBからなる群より選択される表面マーカーを発現し、
（ii）IL-2、IFN-ガンマ、およびTNF-アルファからなる群より選択されるサイトカインの細胞内発現を含み、
（iii）細胞周期のG1期以降にあり、および/または
（iv）増殖能を有する、
本発明1001、1003、および1519のいずれかの方法。
[本発明1021]
刺激性試薬が、
TCR複合体のメンバーに特異的に結合する、任意でCD3に特異的に結合する、一次作用物質
を含む、本発明1001、1003、および1005～1020のいずれかの方法。
[本発明1022]
刺激性試薬が、T細胞共刺激分子に特異的に結合する二次作用物質をさらに含み、任意で、共刺激分子が、CD28、CD137（4-1-BB）、OX40、またはICOSから選択される、本発明1021の方法。
[本発明1023]
一次作用物質および/または二次作用物質が抗体を含み、任意で、刺激性試薬が、抗CD3抗体および抗CD28抗体またはそれらの抗原結合フラグメントとのインキュベーションを含む、本発明1021または本発明1022の方法。
[本発明1024]
一次作用物質および/または二次作用物質が、固形支持体の表面に存在する、本発明1021～1023のいずれかの方法。
[本発明1025]
固形支持体がビーズであるかまたはビーズを含む、本発明1024の方法。
[本発明1026]
一次作用物質および二次作用物質が、複数のストレプトアビジン分子またはストレプトアビジンムテイン分子を含むオリゴマー型粒子試薬の表面に可逆的に結合している、本発明1021～1023のいずれかの方法。
[本発明1027]
複数のストレプトアビジン分子またはストレプトアビジンムテイン分子のそれぞれが、SEQ ID NO：34に示されるアミノ酸の配列におけるストレプトアビジン中の位置に関して位置44～47に対応する配列位置に、Val
44
-Thr
45
-Ala
46
-Arg
47
またはIle
44
-Gly
45
-Ala
46
-Arg
47
のアミノ酸配列を含む、本発明1026の方法。
[本発明1028]
複数のストレプトアビジン分子またはストレプトアビジンムテイン分子のそれぞれが、ストレプトアビジンムテイン分子であり、複数のストレプトアビジンムテイン分子のそれぞれが、
（a）SEQ ID NO：36、41、48～50、または53～55のいずれか1つに示されるアミノ酸の配列、
（b）SEQ ID NO：36、41、48～50、または53～55のいずれか1つに対して、少なくとも85％、86％、87％、88％、89％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、もしくは99％、またはそれ以上の配列同一性を呈し、かつVal44-Thr45-Ala46-Arg47またはIle44-Gly45-Ala46-Arg47に対応するアミノ酸配列を含有し、かつ/またはビオチン、ビオチン類似体、もしくはストレプトアビジン結合ペプチドに可逆的に結合する、アミノ酸の配列、または
（c）ビオチン、ビオチン類似体もしくはストレプトアビジン結合ペプチドに可逆的に結合する（a）もしくは（b）の機能的フラグメント
であるか、またはそれを含み、
任意で、複数のストレプトアビジンムテイン分子のそれぞれが、SEQ ID NO：36またはSEQ ID NO：41に示されるアミノ酸配列であるか、またはそれを含む、
本発明1026の方法。
[本発明1029]
形質導入細胞の集団が、組換えタンパク質を発現する細胞を、少なくとも20％、少なくとも25％、少なくとも30％、少なくとも35％、少なくとも40％、少なくとも45％、少なくとも50％、少なくとも55％、少なくとも60％、少なくとも65％、少なくとも70％、少なくとも75％、少なくとも80％、少なくとも85％、少なくとも90％、または少なくとも95％含む、本発明1001～1028のいずれかの方法。
[本発明1030]
形質導入細胞の集団が、組換えタンパク質を発現する細胞を、少なくとも55％、少なくとも60％、少なくとも65％、少なくとも70％、少なくとも75％、少なくとも80％、少なくとも85％、少なくとも90％、または少なくとも95％含む、本発明1001～1029のいずれかの方法。
[本発明1031]
形質導入細胞の集団における、組換えタンパク質を発現する細胞のパーセンテージが、選択工程によってCCR7＋初代T細胞が富化されなかった細胞組成物と比較して、少なくとも0.5倍、少なくとも1倍、少なくとも1.5倍、または少なくとも2倍大きい、本発明1001～1030のいずれかの方法。
[本発明1032]
ウイルスベクター粒子をインキュベートする工程が、ウイルスベクター粒子を、インプット集団または刺激された組成物と共にスピノキュレーションに付すことを含む、本発明1001～1031のいずれかの方法。
[本発明1033]
スピノキュレーションに付すことが、遠心分離チャンバの内側キャビティにおいて、ウイルスベクター粒子およびインプット集団または刺激された組成物を回転させることを含み、
回転が、
それぞれ両端の値を含め、500g～2500g、500g～2000g、500g～1600g、500g～1000g、600g～1600g、600g～1000g、1000g～2000g、もしくは1000g～1600g、または約500g～2500g、500g～2000g、500g～1600g、500g～1000g、600g～1600g、600g～1000g、1000g～2000g、もしくは1000g～1600g、または
少なくとも600g、800g、1000g、1200g、1600g、もしくは2000g、または少なくとも約600g、800g、1000g、1200g、1600g、もしくは2000g
である、キャビティ側壁の内面における相対遠心力で行われる、
本発明1032の方法。
[本発明1034]
スピノキュレーションに付すことが、
5分超もしくは約5分、10分超もしくは約10分、15分超もしくは約15分、20分超もしくは約20分、30分超もしくは約30分、45分超もしくは約45分、60分超もしくは約60分、90分超もしくは約90分、または120分超もしくは約120分、または
それぞれ両端の値を含め、5分～60分、10分～60分、15分～60分、15分～45分、30分～60分、もしくは45分～60分、または約5分～60分、10分～60分、15分～60分、15分～45分、30分～60分、もしくは45分～60分
である時間にわたって行われる、本発明1032または本発明1033の方法。
[本発明1035]
インキュベートする工程の少なくとも一部の間に、刺激された組成物および/またはウイルスベクター粒子を形質導入アジュバントと接触させる工程を、さらに含む、本発明1001、1003および1005～1034のいずれかの方法。
[本発明1036]
インキュベートする工程の少なくとも一部の間に、インプット集団および/またはウイルスベクター粒子を形質導入アジュバントと接触させる工程を、さらに含む、本発明1002、1004～1034のいずれかの方法。
[本発明1037]
接触させる工程が、ウイルスベクター粒子をインプット集団または刺激された組成物と共にスピノキュレーションに付す前に、またはそれと同時に、またはその後に、実行される、本発明1035または本発明1036の方法。
[本発明1038]
ウイルスベクター粒子のインキュベーションの少なくとも一部が、37℃±2℃または約37℃±2℃で実行される、本発明1001～1037のいずれかの方法。
[本発明1039]
ウイルスベクター粒子のインキュベーションの少なくとも一部が、スピノキュレーション後に実行される、本発明1001～1038のいずれかの方法。
[本発明1040]
ウイルスベクター粒子のインキュベーションの少なくとも一部が、2時間、4時間、12時間、18時間、24時間、30時間、36時間、48時間、60時間、もしくは72時間以下、または約2時間、4時間、12時間、18時間、24時間、30時間、36時間、48時間、60時間、もしくは72時間以下にわたって実行される、本発明1038または本発明1039の方法。
[本発明1041]
ウイルスベクター粒子のインキュベーションの少なくとも一部が、24時間または約24時間にわたって実行される、本発明1038～1040のいずれかの方法。
[本発明1042]
ウイルスベクター粒子のインキュベーションの総継続時間が、12時間、24時間、36時間、48時間、または72時間以下である、本発明1001～1041のいずれかの方法。
[本発明1043]
ウイルスベクター粒子がレンチウイルスベクター粒子である、本発明1001～1042のいずれかの方法。
[本発明1044]
レンチウイルスベクター粒子が複製欠損性である、本発明1043の方法。
[本発明1045]
ウイルスベクター粒子が、ウイルスエンベロープ糖タンパク質でシュードタイプ化されている、本発明1001～1044のいずれかの方法。
[本発明1046]
ウイルスエンベロープ糖タンパク質がVSV-Gである、本発明1045の方法。
[本発明1047]
ウイルスベクター粒子が、20.0未満もしくは約20.0未満または10.0未満もしくは約10.0未満の感染多重度でインキュベートされる、本発明1001～1046のいずれかの方法。
[本発明1048]
ウイルスベクター粒子が、1.0 IU/細胞～10 IU/細胞もしくは2.0U/細胞～5.0 IU/細胞、または約1.0 IU/細胞～10 IU/細胞もしくは2.0U/細胞～5.0 IU/細胞の感染多重度でインキュベートされるか、または
ウイルスベクター粒子が、少なくとも1.6 IU/細胞、1.8 IU/細胞、2.0 IU/細胞、2.4 IU/細胞、2.8 IU/細胞、3.2 IU/細胞、3.6 IU/細胞、4.0 IU/細胞、5.0 IU/細胞、6.0 IU/細胞、7.0 IU/細胞、8.0 IU/細胞、9.0 IU/細胞、もしくは10.0 IU/細胞、または少なくとも約1.6 IU/細胞、1.8 IU/細胞、2.0 IU/細胞、2.4 IU/細胞、2.8 IU/細胞、3.2 IU/細胞、3.6 IU/細胞、4.0 IU/細胞、5.0 IU/細胞、6.0 IU/細胞、7.0 IU/細胞、8.0 IU/細胞、9.0 IU/細胞、もしくは10.0 IU/細胞の感染多重度でインキュベートされる、
本発明1001～1047のいずれかの方法。
[本発明1049]
刺激された組成物が、少なくとも50×10
6
細胞、100×10
6
細胞もしくは200×10
6
細胞、またはおよそ少なくとも50×10
6
細胞、100×10
6
細胞もしくは200×10
6
細胞、または約50×10
6
細胞、100×10
6
細胞もしくは200×10
6
細胞を含む、本発明1001、1003および1005～1048のいずれかの方法。
[本発明1050]
刺激された組成物が、両端の値を含め50×10
6
または約50×10
6
細胞～300×10
6
または約300×10
6
細胞、任意で両端の値を含め100×10
6
または約100×10
6
細胞～200×10
6
または約200×10
6
細胞を含む、本発明1001、1003および1005～1049のいずれかの方法。
[本発明1051]
インプット集団が、少なくとも50×10
6
細胞、100×10
6
細胞もしくは200×10
6
細胞、またはおよそ少なくとも50×10
6
細胞、100×10
6
細胞もしくは200×10
6
細胞、または約50×10
6
細胞、100×10
6
細胞もしくは200×10
6
細胞を含む、本発明1002および1004～1048のいずれかの方法。
[本発明1052]
インプット集団が、両端の値を含め50×10
6
または約50×10
6
細胞～300×10
6
または約300×10
6
細胞、任意で両端の値を含め100×10
6
または約100×10
6
細胞～200×10
6
または約200×10
6
細胞を含む、本発明1002および1004～1049のいずれかの方法。
[本発明1053]
ウイルス粒子と共にインキュベートされるT細胞が、少なくとも50×10
6
細胞、100×10
6
細胞もしくは200×10
6
細胞、またはおよそ少なくとも50×10
6
細胞、100×10
6
細胞もしくは200×10
6
細胞、または約50×10
6
細胞、100×10
6
細胞もしくは200×10
6
細胞を含む、本発明1001～1052のいずれかの方法。
[本発明1054]
ウイルス粒子と共にインキュベートされるT細胞が、両端の値を含め50×10
6
または約50×10
6
細胞～300×10
6
または約300×10
6
細胞、任意で両端の値を含め100×10
6
または約100×10
6
細胞～200×10
6
または約200×10
6
細胞を含む、本発明1001～1053のいずれかの方法。
[本発明1055]
組換えタンパク質が抗原受容体である、本発明1001～1054のいずれかの方法。
[本発明1056]
抗原受容体がトランスジェニックT細胞受容体（TCR）である、本発明1055の方法。
[本発明1057]
抗原受容体がキメラ抗原受容体（CAR）である、本発明1055の方法。
[本発明1058]
CARが、標的抗原に特異的に結合する細胞外抗原認識ドメイン、ITAMを含む細胞内シグナリングドメイン、および細胞外ドメインと細胞内シグナリングドメインとを連結する膜貫通ドメインを含む、本発明1057の方法。
[本発明1059]
細胞内シグナリングドメインがCD3ゼータ（CD3ζ）鎖の細胞内ドメインを含む、本発明1058の方法。
[本発明1060]
膜貫通ドメインがCD28の膜貫通部分を含む、本発明1058または本発明1059の方法。
[本発明1061]
細胞内シグナリングドメインがT細胞共刺激分子の細胞内シグナリングドメインをさらに含む、本発明1058～1060のいずれかの方法。
[本発明1062]
T細胞共刺激分子がCD28および41BBからなる群より選択される、本発明1061の方法。
[本発明1063]
抗原受容体が、疾患または状態と関連する抗原に特異的に結合するか、またはユニバーサルタグに特異的に結合する、本発明1055～1062のいずれかの方法。
[本発明1064]
疾患または状態が、がん、自己免疫疾患もしくは自己免疫障害、または感染性疾患である、本発明1063の方法。
[本発明1065]
形質導入細胞の集団が、異種ポリヌクレオチドが形質導入されているT細胞を含む、本発明1001～1064のいずれかの方法。
[本発明1066]
形質導入細胞の集団におけるT細胞の少なくとも20％、少なくとも25％、少なくとも30％、少なくとも35％、少なくとも40％、少なくとも45％、少なくとも50％、少なくとも55％、少なくとも60％、少なくとも65％、少なくとも70％、少なくとも75％、少なくとも80％、または少なくとも85％に、異種ポリヌクレオチドが形質導入されている、本発明1065の方法。
[本発明1067]
形質導入細胞の集団におけるT細胞の少なくとも55％、少なくとも60％、少なくとも65％、少なくとも70％、少なくとも75％、少なくとも80％、または少なくとも85％に、異種ポリヌクレオチドが形質導入されている、本発明1065または本発明1066の方法。
[本発明1068]
異種ポリヌクレオチドが形質導入されているT細胞の少なくとも95％、少なくとも96％、少なくとも97％、少なくとも98％、または少なくとも99％が、CCR7＋である、本発明1065～1067のいずれかの方法。
[本発明1069]
形質導入細胞の集団から、本方法によって生産された形質導入T細胞を回収または単離する工程を、さらに含む、本発明1065または本発明1066の方法。
[本発明1070]
形質導入細胞の複数の集団の間での、形質導入細胞の集団における、異種ポリヌクレオチドが形質導入されているT細胞のパーセンテージのばらつきが、30％以下、25％以下、20％以下、15％以下、または10％以下である、本発明1001～1069のいずれかの方法。
[本発明1071]
インビトロまたはエクスビボで実行される、本発明1001～1070のいずれかの方法。
[本発明1072]
本発明1001～1071のいずれかの方法によって生産された形質導入細胞の集団を含む、組成物。
[本発明1073]
冷凍保存物質をさらに含む、本発明1072の組成物。
Also provided herein are compositions comprising a population of transduced cells produced by the method of any one of the embodiments provided herein. In some of any such embodiments, the composition further comprises a cyropreservant.
[Invention 1001]
1. A method for increasing the transduction frequency of primary T cells, the method comprising:
(a) Selecting primary T cells positive for surface expression of CCR7 from a biological sample comprising a population of primary T cells, whereby an input population is enriched for CCR7+ primary T cells. A process of generating
(b) incubating the input population under stimulatory conditions, thereby producing a stimulated composition, wherein the stimulatory conditions affect one or more of the one or more components of the TCR complex; and/or the presence of a stimulatory reagent capable of activating the intracellular signaling domain of the one or more costimulatory molecules.
(c) incubating viral vector particles comprising a heterologous polynucleotide encoding a recombinant protein with T cells of the stimulated composition, thereby generating a population of transduced cells.
including methods.
[Invention 1002]
1. A method for increasing the transduction frequency of primary T cells, the method comprising:
(a) Selecting primary T cells positive for surface expression of CCR7 from a biological sample comprising a population of primary T cells, whereby the input population is enriched for CCR7+ primary T cells. producing a process, and
(b) incubating viral vector particles comprising a heterologous polynucleotide encoding a recombinant protein with T cells of the input cell population, thereby generating a population of transduced cells.
including methods.
[Present invention 1003]
1. A method for increasing the transduction frequency of primary T cells, the method comprising:
(a) incubating an input primary T cell population enriched in CCR7+ T cells under stimulatory conditions, thereby generating a stimulated composition, wherein the stimulatory conditions are the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components and/or one or more intracellular signaling domains of one or more costimulatory molecules; including, steps, and
(b) incubating viral vector particles comprising a heterologous polynucleotide encoding a recombinant protein with T cells of the stimulated composition, thereby generating a population of transduced cells.
including methods.
[Present invention 1004]
1. A method for increasing the transduction frequency of primary T cells, the method comprising:
incubating viral vector particles containing a heterologous polynucleotide encoding a recombinant protein with T cells of an input primary T cell population enriched for CCR7+ T cells, thereby producing a population of transduced cells. make, process
including methods.
[Present invention 1005]
at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of the input population , at least 96%, at least 97%, at least 98%, at least 99%, or 100% are CCR7+ primary T cells.
[Present invention 1006]
At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the input population is CCR7+ first generation The method of any one of the present invention 1001 to 1005, which is a T cell.
[Present invention 1007]
The method of any of the inventions 1001-1006, wherein at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the input population are CCR7+ primary T cells.
[Present invention 1008]
The method of any of the inventions 1001, 1002 and 1005-1007, wherein the biological sample is a blood sample.
[Present invention 1009]
The method of any of the inventions 1001, 1002 and 1005-1007, wherein the biological sample is a leukocyte apheresis sample.
[Present invention 1010]
The T cells are unfractionated T cells, enriched or isolated CD3+ T cells, enriched or isolated CD4+ T cells, or enriched or isolated CD8+ T cells. The method according to any one of the present inventions 1001 to 1009.
[Present invention 1011]
The method of any of the inventions 1001-1010, wherein the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD4+ T cells or CD8+ T cells.
[Invention 1012]
The method of any of the inventions 1001-1011, wherein the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD4+ T cells.
[Present invention 1013]
The method of any of the inventions 1001-1011, wherein the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD8+ T cells.
[Present invention 1014]
The method of any of the inventions 1001-1011, wherein the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD4+ T cells and CD8+ T cells.
[Present invention 1015]
The ratio of CD4+ T cells to CD8+ T cells is 1:1, 1:2, 2:1, 1:3, or 3:1 or about 1:1, 1:2, 2:1, 1:3 , or 3:1, the method of the invention 1014.
[Invention 1016]
The method of any of the inventions 1001-1010, wherein the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD3+ T cells.
[Invention 1017]
The method of any of the inventions 1001-1016, wherein the input population comprises 100×10 6 to 500×10 6 total T cells.
[Invention 1018]
The method of any of the inventions 1001-1017 , wherein the input population comprises 200×10 6 to 400×10 6 total T cells, optionally 300×10 6 or about 300×10 6 total T cells.
[Invention 1019]
The method of invention 1017 or invention 1018, wherein the total T cells are viable T cells.
[Invention 1020]
at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% of the cells of the stimulated composition,
(i) expressing a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L, and 4-1BB;
(ii) comprising intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-gamma, and TNF-alpha;
(iii) is in the G1 phase of the cell cycle or later, and/or
(iv) has the ability to proliferate;
The method of any of inventions 1001, 1003, and 1519.
[Present invention 1021]
The irritating reagent is
A primary agent that specifically binds to a member of the TCR complex, optionally to CD3
The method of any of the inventions 1001, 1003, and 1005-1020, comprising:
[Invention 1022]
The stimulatory reagent further comprises a second agent that specifically binds to a T cell costimulatory molecule, optionally the costimulatory molecule being selected from CD28, CD137(4-1-BB), OX40, or ICOS. The method of the present invention 1021.
[Invention 1023]
According to the invention 1021 or the invention 1022, the primary agent and/or the secondary agent comprises an antibody, and optionally the stimulatory reagent comprises incubation with anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof. Method.
[Invention 1024]
The method of any of the inventions 1021-1023, wherein the primary agent and/or the secondary agent is present on the surface of the solid support.
[Invention 1025]
1024. The method of the invention 1024, wherein the solid support is or comprises beads.
[Invention 1026]
The method of any of the inventions 1021-1023, wherein the primary agent and the secondary agent are reversibly bound to the surface of an oligomeric particle reagent comprising a plurality of streptavidin molecules or streptavidin mutein molecules.
[Invention 1027]
Each of the plurality of streptavidin molecules or streptavidin mutein molecules has a Val 44 -Thr 45 -Ala at a sequence position corresponding to positions 44-47 with respect to the positions in streptavidin in the sequence of amino acids shown in SEQ ID NO : 34 . The method of the invention 1026, comprising the amino acid sequence of 46 -Arg 47 or Ile 44 -Gly 45 -Ala 46 -Arg 47 .
[Invention 1028]
Each of the plurality of streptavidin molecules or streptavidin mutein molecules is a streptavidin mutein molecule, and each of the plurality of streptavidin mutein molecules comprises:
(a) Sequence of amino acids shown in any one of SEQ ID NO: 36, 41, 48-50, or 53-55,
(b) at least 85%, 86%, 87%, 88%, 89%, 89%, 90% for any one of SEQ ID NO: 36, 41, 48-50, or 53-55; exhibiting sequence identity of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, and Val44-Thr45-Ala46-Arg47 or Ile44-Gly45 - a sequence of amino acids that contains an amino acid sequence corresponding to Ala46-Arg47 and/or binds reversibly to biotin, a biotin analog, or a streptavidin-binding peptide, or
(c) a functional fragment of (a) or (b) that reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide;
is or contains
Optionally, each of the plurality of streptavidin mutein molecules is or comprises the amino acid sequence set forth in SEQ ID NO:36 or SEQ ID NO:41.
Method of the invention 1026.
[Invention 1029]
The population of transduced cells has at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60 cells expressing the recombinant protein. %, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.
[Invention 1030]
The population of transduced cells comprises at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least The method of any of the inventions 1001-1029, comprising 95%.
[Present invention 1031]
The percentage of cells expressing the recombinant protein in the population of transduced cells is at least 0.5-fold, at least 1-fold, at least 1.5-fold compared to a cell composition in which CCR7+ primary T cells were not enriched by the selection step. , or at least twice as large.
[Invention 1032]
The method of any of the inventions 1001-1031, wherein the step of incubating the viral vector particles comprises subjecting the viral vector particles to spinoculation with the input population or stimulated composition.
[Present invention 1033]
Subjecting to spinoculation comprises spinning the viral vector particles and the input population or stimulated composition in an inner cavity of a centrifugation chamber;
The rotation is
Including the values at both ends, 500g to 2500g, 500g ~ 2000g, 500g ~ 1600g, 500g ~ 1000g, 600G ~ 1600g, 600G ~ 1000g, 1000G ~ 2000g, or about 500G to 2500g, 500g ~ 2000g, 500g ~ 2000g, 500g to 1600g, 500g to 1000g, 600g to 1600g, 600g to 1000g, 1000g to 2000g, or 1000g to 1600g, or
at least 600g, 800g, 1000g, 1200g, 1600g, or 2000g, or at least about 600g, 800g, 1000g, 1200g, 1600g, or 2000g
is carried out by the relative centrifugal force on the inner surface of the cavity side wall,
Method of the invention 1032.
[Present invention 1034]
To be attached to spinoculation,
More than 5 minutes or about 5 minutes, more than 10 minutes or about 10 minutes, more than 15 minutes or about 15 minutes, more than 20 minutes or about 20 minutes, more than 30 minutes or about 30 minutes, more than 45 minutes or about 45 minutes, 60 minutes more than or about 60 minutes, more than or about 90 minutes, or more than or about 120 minutes, or
Including the values at both ends, 5 minutes to 60 minutes, 10 minutes to 60 minutes, 15 minutes to 60 minutes, 15 minutes to 45 minutes, 30 minutes to 60 minutes, or 45 minutes to 60 minutes, or approximately 5 minutes to 60 minutes. minutes, 10 minutes to 60 minutes, 15 minutes to 60 minutes, 15 minutes to 45 minutes, 30 minutes to 60 minutes, or 45 minutes to 60 minutes
A method of the invention 1032 or the invention 1033 carried out over a period of time.
[Invention 1035]
The method of any of the inventions 1001, 1003 and 1005-1034, further comprising contacting the stimulated composition and/or viral vector particles with a transduction adjuvant during at least a portion of the incubating step.
[Invention 1036]
The method of any of the inventions 1002, 1004-1034, further comprising contacting the input population and/or viral vector particles with a transduction adjuvant during at least a portion of the incubating step.
[Present invention 1037]
The method of invention 1035 or invention 1036, wherein the contacting step is performed before, concurrently with, or after subjecting the viral vector particles to spinoculation with the input population or stimulated composition.
[Invention 1038]
The method of any of the inventions 1001-1037, wherein at least a portion of the incubation of the viral vector particles is performed at or about 37°C ± 2°C.
[Invention 1039]
The method of any of the inventions 1001-1038, wherein at least part of the incubation of the viral vector particles is performed after spinoculation.
[Invention 1040]
At least a portion of the incubation of the viral vector particles is less than or equal to 2 hours, 4 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours, 60 hours, or 72 hours, or about 2 hours, 4 hours. , 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours, 60 hours, or 72 hours or less.
[Present invention 1041]
The method of any of the inventions 1038-1040, wherein at least a portion of the incubation of the viral vector particles is carried out for 24 hours or about 24 hours.
[Present invention 1042]
The method of any of the inventions 1001-1041, wherein the total duration of incubation of the viral vector particles is 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours or less.
[Invention 1043]
The method according to any of the inventions 1001 to 1042, wherein the viral vector particle is a lentiviral vector particle.
[Present invention 1044]
1043. The method of the invention 1043, wherein the lentiviral vector particle is replication-defective.
[Invention 1045]
The method of any of the inventions 1001-1044, wherein the viral vector particle is pseudotyped with a viral envelope glycoprotein.
[Invention 1046]
1045. The method of the invention 1045, wherein the viral envelope glycoprotein is VSV-G.
[Invention 1047]
The method of any of the inventions 1001-1046, wherein the viral vector particles are incubated at a multiplicity of infection of less than or about 20.0 or less than or about 10.0.
[Invention 1048]
Viral vector particles have a multiplicity of infection of 1.0 IU/cell to 10 IU/cell or 2.0U/cell to 5.0 IU/cell, or about 1.0 IU/cell to 10 IU/cell or 2.0U/cell to 5.0 IU/cell. incubated with or
viral vector particles of at least 1.6 IU/cell, 1.8 IU/cell, 2.0 IU/cell, 2.4 IU/cell, 2.8 IU/cell, 3.2 IU/cell, 3.6 IU/cell, 4.0 IU/cell, 5.0 IU/cell , 6.0 IU/cell, 7.0 IU/cell, 8.0 IU/cell, 9.0 IU/cell, or 10.0 IU/cell, or at least about 1.6 IU/cell, 1.8 IU/cell, 2.0 IU/cell, 2.4 IU/cell, 2.8 IU/cell, 3.2 IU/cell, 3.6 IU/cell, 4.0 IU/cell, 5.0 IU/cell, 6.0 IU/cell, 7.0 IU/cell, 8.0 IU/cell, 9.0 IU/cell, or 10.0 IU/cell incubated at a multiplicity of infection of
The method according to any one of the inventions 1001 to 1047.
[Invention 1049]
The stimulated composition contains at least 50 x 10 6 cells, 100 x 10 6 cells or 200 x 10 6 cells, or about at least 50 x 10 6 cells, 100 x 10 6 cells or 200 x 10 6 cells, or about 50 The method of any of the inventions 1001, 1003 and 1005-1048, comprising ×10 6 cells, 100 × 10 6 cells or 200 × 10 6 cells .
[Invention 1050]
If the stimulated composition is between 50 x 10 6 or about 50 x 10 6 cells, inclusive to 300 x 10 6 or about 300 x 10 6 cells, optionally 100 x 10 6 or about 100 cells, inclusive The method of any of the inventions 1001, 1003 and 1005-1049, comprising from × 10 6 cells to 200 × 10 6 or about 200 × 10 6 cells.
[Present invention 1051]
The input population is at least 50 x 10 6 cells, 100 x 10 6 cells or 200 x 10 6 cells, or about at least 50 x 10 6 cells, 100 x 10 6 cells or 200 x 10 6 cells, or about 50 x 10 6 cells. The method of any of the invention 1002 and 1004-1048, comprising cells, 100×10 6 cells or 200×10 6 cells.
[Invention 1052]
The input population is between 50×10 6 or about 50×10 6 cells inclusive to 300×10 6 or about 300×10 6 cells, optionally 100×10 6 or about 100×10 6 inclusive. The method of any of the invention 1002 and 1004-1049, comprising ˜200×10 6 cells or about 200×10 6 cells.
[Present invention 1053]
the T cells incubated with the viral particles are at least 50×10 6 cells, 100×10 6 cells or 200×10 6 cells, or about at least 50×10 6 cells, 100×10 6 cells or 200×10 6 cells; or about 50×10 6 cells, 100×10 6 cells or 200×10 6 cells.
[Invention 1054]
T cells incubated with viral particles range from 50 x 10 6 or about 50 x 10 6 cells to 300 x 10 6 or about 300 x 10 6 cells, optionally 100 x 10 6 inclusive or about 100×10 6 cells to 200×10 6 or about 200×10 6 cells.
[Present invention 1055]
The method of any of the inventions 1001-1054, wherein the recombinant protein is an antigen receptor.
[Invention 1056]
1055. The method of the invention 1055, wherein the antigen receptor is a transgenic T cell receptor (TCR).
[Present invention 1057]
1055. The method of the invention 1055, wherein the antigen receptor is a chimeric antigen receptor (CAR).
[Invention 1058]
1057. The method of the invention 1057, wherein the CAR comprises an extracellular antigen recognition domain that specifically binds a target antigen, an intracellular signaling domain that includes an ITAM, and a transmembrane domain that connects the extracellular domain and the intracellular signaling domain.
[Invention 1059]
1058. The method of the invention, wherein the intracellular signaling domain comprises an intracellular domain of the CD3 zeta (CD3ζ) chain.
[Invention 1060]
The method of invention 1058 or invention 1059, wherein the transmembrane domain comprises a transmembrane portion of CD28.
[Present invention 1061]
The method of any of the inventions 1058-1060, wherein the intracellular signaling domain further comprises an intracellular signaling domain of a T cell costimulatory molecule.
[Invention 1062]
1061. The method of the invention 1061, wherein the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB.
[Invention 1063]
The method of any of the inventions 1055-1062, wherein the antigen receptor specifically binds an antigen associated with a disease or condition or specifically binds a universal tag.
[Present invention 1064]
1063. The method of the invention 1063, wherein the disease or condition is cancer, an autoimmune disease or disorder, or an infectious disease.
[Invention 1065]
The method of any of the inventions 1001-1064, wherein the population of transduced cells comprises T cells that have been transduced with a heterologous polynucleotide.
[Invention 1066]
at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least of the T cells in the population of transduced cells 1065. The method of the invention 1065, wherein the method is 70%, at least 75%, at least 80%, or at least 85% transduced with the heterologous polynucleotide.
[Invention 1067]
at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the T cells in the population of transduced cells are transduced with the heterologous polynucleotide; Invention 1065 or method of invention 1066.
[Invention 1068]
The method of any of the inventions 1065-1067, wherein at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the T cells transduced with the heterologous polynucleotide are CCR7+.
[Invention 1069]
The method of the invention 1065 or the invention 1066, further comprising the step of recovering or isolating the transduced T cells produced by the method from the population of transduced cells.
[Invention 1070]
The variation in the percentage of T cells transduced with the heterologous polynucleotide in the population of transduced cells between the populations of transduced cells is 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, the method of any one of Inventions 1001 to 1069.
[Present invention 1071]
The method of any of the inventions 1001-1070, performed in vitro or ex vivo.
[Invention 1072]
A composition comprising a population of transduced cells produced by the method of any of the inventions 1001-1071.
[Invention 1073]
The composition of the invention 1072 further comprising a cryopreservative substance.
Claims (39)
（a）初代T細胞の集団を含む生物学的試料から、CCR7の表面発現が陽性である初代T細胞を選択する工程であって、それによって、CCR7＋初代T細胞が富化されているインプット集団を生成させる、工程、
（b）インプット集団を刺激性条件下でインキュベートする工程であって、それによって、刺激された組成物を生成させ、刺激条件が、TCR複合体の1つもしくは複数の構成要素の1つもしくは複数の細胞内シグナリングドメインおよび/または1つもしくは複数の共刺激分子の1つもしくは複数の細胞内シグナリングドメインを活性化することができる刺激性試薬の存在を含む、工程、および
（c）組換えタンパク質をコードする異種ポリヌクレオチドを含むウイルスベクター粒子を、刺激された組成物のT細胞と共にインキュベートする工程であって、それによって、形質導入細胞の集団を生成させる、工程
を含む、方法。 1. A method for increasing the transduction frequency of primary T cells, the method comprising:
(a) Selecting primary T cells positive for surface expression of CCR7 from a biological sample comprising a population of primary T cells, whereby an input population is enriched for CCR7+ primary T cells. A process of generating
(b) incubating the input population under stimulatory conditions, thereby producing a stimulated composition, wherein the stimulatory conditions affect one or more of the one or more components of the TCR complex; (c) the recombinant protein A method comprising incubating a viral vector particle comprising a heterologous polynucleotide encoding a T cell with a stimulated composition of T cells, thereby generating a population of transduced cells.
（a）初代T細胞の集団を含む生物学的試料から、CCR7の表面発現が陽性である初代T細胞を選択する工程であって、それによって、CCR7＋初代T細胞が富化されているインプット集団を生成させる、工程、および
（b）組換えタンパク質をコードする異種ポリヌクレオチドを含むウイルスベクター粒子をインプット細胞集団のT細胞と共にインキュベートする工程であって、それによって、形質導入細胞の集団を生成させる、工程
を含む、方法。 1. A method for increasing the transduction frequency of primary T cells, the method comprising:
(a) Selecting primary T cells positive for surface expression of CCR7 from a biological sample comprising a population of primary T cells, whereby an input population is enriched for CCR7+ primary T cells. (b) incubating viral vector particles comprising a heterologous polynucleotide encoding the recombinant protein with T cells of the input cell population, thereby generating a population of transduced cells. , a method comprising a step.
（a）CCR7＋T細胞が富化されているインプット初代T細胞集団を刺激性条件下でインキュベートする工程であって、それによって、刺激された組成物を生成させ、刺激条件が、TCR複合体の1つもしくは複数の構成要素の1つもしくは複数の細胞内シグナリングドメインおよび/または1つもしくは複数の共刺激分子の1つもしくは複数の細胞内シグナリングドメインを活性化することができる刺激性試薬の存在を含む、工程、および
（b）組換えタンパク質をコードする異種ポリヌクレオチドを含むウイルスベクター粒子を、刺激された組成物のT細胞と共にインキュベートする工程であって、それによって、形質導入細胞の集団を生成させる、工程
を含む、方法。 1. A method for increasing the transduction frequency of primary T cells, the method comprising:
(a) incubating an input primary T cell population enriched in CCR7+ T cells under stimulatory conditions, thereby generating a stimulated composition, wherein the stimulatory conditions are the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components and/or one or more intracellular signaling domains of one or more costimulatory molecules; (b) incubating viral vector particles comprising a heterologous polynucleotide encoding a recombinant protein with T cells of the stimulated composition, thereby producing a population of transduced cells. A method including a step of causing.
組換えタンパク質をコードする異種ポリヌクレオチドを含むウイルスベクター粒子を、CCR7＋T細胞が富化されているインプット初代T細胞集団のT細胞と共にインキュベートする工程であって、それによって、形質導入細胞の集団を生成させる、工程
を含む、方法。 1. A method for increasing the transduction frequency of primary T cells, the method comprising:
incubating viral vector particles containing a heterologous polynucleotide encoding a recombinant protein with T cells of an input primary T cell population enriched for CCR7+ T cells, thereby producing a population of transduced cells. A method including a step of causing.
インプット集団が、CD3＋T細胞である細胞を、少なくとも80％、少なくとも85％、少なくとも90％、または少なくとも95％含む、
請求項1～8のいずれか一項記載の方法。 the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD4+ T cells , CD8+ T cells, or CD4+ T cells and CD8+ T cells ; and/or
the input population comprises at least 80%, at least 85%, at least 90%, or at least 95% of cells that are CD3+ T cells ;
A method according to any one of claims 1 to 8 .
（i）HLA-DR、CD25、CD69、CD71、CD40L、および4-1BBからなる群より選択される表面マーカーを発現し、
（ii）IL-2、IFN-ガンマ、およびTNF-アルファからなる群より選択されるサイトカインの細胞内発現を含み、
（iii）細胞周期のG1期以降にあり、および/または
（iv）増殖能を有する、
請求項1、3、および5～12のいずれか一項記載の方法。 at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% of the cells of the stimulated composition,
(i) expressing a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L, and 4-1BB;
(ii) comprising intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-gamma, and TNF-alpha;
(iii) is in the G1 phase of the cell cycle or later, and/or (iv) has proliferative capacity;
13. The method according to any one of claims 1, 3, and 5-12 .
TCR複合体のメンバーに特異的に結合する、任意でCD3に特異的に結合する、一次作用物質を含み、
任意で、刺激性試薬が、T細胞共刺激分子に特異的に結合する二次作用物質をさらに含み、任意で、共刺激分子が、CD28、CD137（4-1-BB）、OX40、またはICOSから選択される、
請求項1、3、および5～13のいずれか一項記載の方法。 The irritating reagent is
comprising a primary agent that specifically binds to a member of the TCR complex, optionally that specifically binds to CD3;
Optionally, the stimulatory reagent further comprises a secondary agent that specifically binds to a T cell costimulatory molecule, optionally the costimulatory molecule is CD28, CD137(4-1-BB), OX40, or ICOS. selected from
14. The method according to any one of claims 1, 3, and 5-13 .
一次作用物質および/または二次作用物質が、固形支持体の表面に存在する、任意で、ここで、固形支持体がビーズであるかまたはビーズを含む、
請求項14記載の方法。 The primary agent and/or the secondary agent comprises an antibody, and optionally the stimulatory reagent comprises incubation with anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof ; and/or
The primary agent and/or the secondary agent are present on the surface of a solid support, optionally wherein the solid support is or comprises beads .
15. The method according to claim 14 .
複数のストレプトアビジン分子またはストレプトアビジンムテイン分子のそれぞれが、ストレプトアビジンムテイン分子であり、複数のストレプトアビジンムテイン分子のそれぞれが、
（a）SEQ ID NO：36、41、48～50、または53～55のいずれか1つに示されるアミノ酸の配列、
（b）SEQ ID NO：36、41、48～50、または53～55のいずれか1つに対して、少なくとも85％、86％、87％、88％、89％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、もしくは99％、またはそれ以上の配列同一性を呈し、かつVal44-Thr45-Ala46-Arg47またはIle44-Gly45-Ala46-Arg47に対応するアミノ酸配列を含有し、かつ/またはビオチン、ビオチン類似体、もしくはストレプトアビジン結合ペプチドに可逆的に結合する、アミノ酸の配列、または
（c）ビオチン、ビオチン類似体もしくはストレプトアビジン結合ペプチドに可逆的に結合する（a）もしくは（b）の機能的フラグメントであるか、またはそれを含み、任意で、複数のストレプトアビジンムテイン分子のそれぞれが、SEQ ID NO：36またはSEQ ID NO：41に示されるアミノ酸配列であるか、またはそれを含む、
請求項16記載の方法。 Each of the plurality of streptavidin molecules or streptavidin mutein molecules has a Val 44 -Thr 45 -Ala at a sequence position corresponding to positions 44-47 with respect to the positions in streptavidin in the sequence of amino acids shown in SEQ ID NO:34. 46 -Arg 47 or Ile 44 -Gly 45 -Ala 46 -Arg 47 ; or
Each of the plurality of streptavidin molecules or streptavidin mutein molecules is a streptavidin mutein molecule, and each of the plurality of streptavidin mutein molecules comprises:
(a) Sequence of amino acids shown in any one of SEQ ID NO: 36, 41, 48-50, or 53-55,
(b) at least 85%, 86%, 87%, 88%, 89%, 89%, 90% for any one of SEQ ID NO: 36, 41, 48-50, or 53-55; exhibiting sequence identity of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, and Val44-Thr45-Ala46-Arg47 or Ile44-Gly45 - a sequence of amino acids that contains an amino acid sequence corresponding to Ala46-Arg47 and/or binds reversibly to biotin, a biotin analog, or a streptavidin-binding peptide, or
(c) is or comprises a functional fragment of (a) or (b) that reversibly binds biotin, a biotin analog, or a streptavidin-binding peptide, optionally each of a plurality of streptavidin mutein molecules; is or contains the amino acid sequence shown in SEQ ID NO: 36 or SEQ ID NO: 41,
17. The method according to claim 16 .
形質導入細胞の集団における、組換えタンパク質を発現する細胞のパーセンテージが、選択工程によってCCR7＋初代T細胞が富化されなかった細胞組成物と比較して、少なくとも0.5倍、少なくとも1倍、少なくとも1.5倍、または少なくとも2倍大きい、
請求項1～17のいずれか一項記載の方法。 The population of transduced cells has at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60 cells expressing the recombinant protein. %, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or containing at least 95% ; and/or
The percentage of cells expressing the recombinant protein in the population of transduced cells is at least 0.5-fold, at least 1-fold, at least 1.5-fold compared to a cell composition in which CCR7+ primary T cells were not enriched by the selection step. , or at least twice as large ,
18. The method according to any one of claims 1 to 17 .
ウイルスベクター粒子のインキュベーションの少なくとも一部が、スピノキュレーション後に実行される； および/または
ウイルスベクター粒子のインキュベーションの少なくとも一部が、2時間、4時間、12時間、18時間、24時間、30時間、36時間、48時間、60時間、もしくは72時間以下、または約2時間、4時間、12時間、18時間、24時間、30時間、36時間、48時間、60時間、もしくは72時間以下にわたって実行される、
請求項1～21のいずれか一項記載の方法。 at least a portion of the incubation of the viral vector particles is performed at or about 37°C ± 2°C ;
at least a portion of the incubation of the viral vector particles is performed after spinoculation; and/or
At least a portion of the incubation of the viral vector particles is less than or equal to 2 hours, 4 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours, 60 hours, or 72 hours, or about 2 hours, 4 hours. , run for less than 12, 18, 24, 30, 36, 48, 60, or 72 hours ;
22. A method according to any one of claims 1 to 21 .
ウイルスベクター粒子が、ウイルスエンベロープ糖タンパク質でシュードタイプ化されている、任意で、ここで該ウイルスエンベロープ糖タンパク質がVSV-Gである、
請求項1～22のいずれか一項記載の方法。 the viral vector particle is a lentiviral vector particle, optionally wherein the lentiviral vector particle is replication-defective; and/or
Optionally, the viral vector particle is pseudotyped with a viral envelope glycoprotein, wherein the viral envelope glycoprotein is VSV-G .
23. A method according to any one of claims 1 to 22 .
ウイルスベクター粒子が、1.0 IU/細胞～10 IU/細胞もしくは2.0U/細胞～5.0 IU/細胞、または約1.0 IU/細胞～10 IU/細胞もしくは2.0U/細胞～5.0 IU/細胞の感染多重度でインキュベートされるか、または
ウイルスベクター粒子が、少なくとも1.6 IU/細胞、1.8 IU/細胞、2.0 IU/細胞、2.4 IU/細胞、2.8 IU/細胞、3.2 IU/細胞、3.6 IU/細胞、4.0 IU/細胞、5.0 IU/細胞、6.0 IU/細胞、7.0 IU/細胞、8.0 IU/細胞、9.0 IU/細胞、もしくは10.0 IU/細胞、または少なくとも約1.6 IU/細胞、1.8 IU/細胞、2.0 IU/細胞、2.4 IU/細胞、2.8 IU/細胞、3.2 IU/細胞、3.6 IU/細胞、4.0 IU/細胞、5.0 IU/細胞、6.0 IU/細胞、7.0 IU/細胞、8.0 IU/細胞、9.0 IU/細胞、もしくは10.0 IU/細胞の感染多重度でインキュベートされる、
請求項1～23のいずれか一項記載の方法。 the viral vector particles are incubated at a multiplicity of infection of less than or about 20.0 or less than or about 10.0;
Viral vector particles have a multiplicity of infection of 1.0 IU/cell to 10 IU/cell or 2.0U/cell to 5.0 IU/cell, or about 1.0 IU/cell to 10 IU/cell or 2.0U/cell to 5.0 IU/cell. or the viral vector particles are incubated at least 1.6 IU/cell, 1.8 IU/cell, 2.0 IU/cell, 2.4 IU/cell, 2.8 IU/cell, 3.2 IU/cell, 3.6 IU/cell, 4.0 IU /cell, 5.0 IU/cell, 6.0 IU/cell, 7.0 IU/cell, 8.0 IU/cell, 9.0 IU/cell, or 10.0 IU/cell, or at least about 1.6 IU/cell, 1.8 IU/cell, 2.0 IU/cell cells, 2.4 IU/cell, 2.8 IU/cell, 3.2 IU/cell, 3.6 IU/cell, 4.0 IU/cell, 5.0 IU/cell, 6.0 IU/cell, 7.0 IU/cell, 8.0 IU/cell, 9.0 IU/cell cells, or incubated at a multiplicity of infection of 10.0 IU/cell.
24. A method according to any one of claims 1 to 23 .
刺激された組成物が、両端の値を含め50×10 6 または約50×10 6 細胞～300×10 6 または約300×10 6 細胞、任意で両端の値を含め100×10 6 または約100×10 6 細胞～200×10 6 または約200×10 6 細胞を含む、
請求項1、3および5～24のいずれか一項記載の方法。 The stimulated composition contains at least 50 x 10 6 cells, 100 x 10 6 cells or 200 x 10 6 cells, or about at least 50 x 10 6 cells, 100 x 10 6 cells or 200 x 10 6 cells, or about 50 containing ×10 6 cells, 100 × 10 6 cells or 200 × 10 6 cells ; and/or
If the stimulated composition is between 50 x 10 6 or about 50 x 10 6 cells, inclusive to 300 x 10 6 or about 300 x 10 6 cells, optionally 100 x 10 6 or about 100 cells, inclusive containing ×10 6 cells to 200 × 10 6 or about 200 × 10 6 cells,
25. The method according to any one of claims 1, 3 and 5-24 .
インプット集団が、両端の値を含め50×10 6 または約50×10 6 細胞～300×10 6 または約300×10 6 細胞、任意で両端の値を含め100×10 6 または約100×10 6 細胞～200×10 6 または約200×10 6 細胞を含む、
請求項2および4～24のいずれか一項記載の方法。 The input population is at least 50 x 10 6 cells, 100 x 10 6 cells or 200 x 10 6 cells, or about at least 50 x 10 6 cells, 100 x 10 6 cells or 200 x 10 6 cells, or about 50 x 10 6 cells. cells, comprising 100×10 6 cells or 200×10 6 cells ; and/or
The input population is between 50×10 6 or about 50×10 6 cells inclusive to 300×10 6 or about 300×10 6 cells, optionally 100×10 6 or about 100×10 6 inclusive. comprising ~200×10 6 cells or about 200×10 6 cells;
25. A method according to any one of claims 2 and 4 to 24 .
ウイルス粒子と共にインキュベートされるT細胞が、両端の値を含め50×10 6 または約50×10 6 細胞～300×10 6 または約300×10 6 細胞、任意で両端の値を含め100×10 6 または約100×10 6 細胞～200×10 6 または約200×10 6 細胞を含む、
請求項1～26のいずれか一項記載の方法。 the T cells incubated with the viral particles are at least 50×10 6 cells, 100×10 6 cells or 200×10 6 cells, or about at least 50×10 6 cells, 100×10 6 cells or 200×10 6 cells; or comprising about 50×10 6 cells, 100×10 6 cells or 200×10 6 cells ; and/or
T cells incubated with viral particles range from 50 x 10 6 or about 50 x 10 6 cells to 300 x 10 6 or about 300 x 10 6 cells, optionally 100 x 10 6 inclusive or about 100×10 6 cells to 200×10 6 or about 200×10 6 cells ;
27. A method according to any one of claims 1 to 26 .
膜貫通ドメインがCD28の膜貫通部分を含む； および/または
細胞内シグナリングドメインがT細胞共刺激分子の細胞内シグナリングドメインをさらに含み、任意で、ここでT細胞共刺激分子がCD28および41BBからなる群より選択される、
請求項30記載の方法。 the intracellular signaling domain comprises the intracellular domain of the CD3 zeta (CD3ζ) chain ;
the transmembrane domain comprises a transmembrane portion of CD28; and/or
the intracellular signaling domain further comprises an intracellular signaling domain of a T cell costimulatory molecule, optionally wherein the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB;
31. The method of claim 30 .
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US202062967005P | 2020-01-28 | 2020-01-28 | |
| US62/967,005 | 2020-01-28 | ||
| PCT/US2021/015333 WO2021154887A1 (en) | 2020-01-28 | 2021-01-27 | Methods for t cell transduction |
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2021
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- 2021-01-27 CN CN202180023558.XA patent/CN115427550A/en active Pending
- 2021-01-27 US US17/796,224 patent/US20230090117A1/en active Pending
- 2021-01-27 EP EP21707507.6A patent/EP4097218A1/en active Pending
- 2021-01-27 KR KR1020227029607A patent/KR20220146480A/en unknown
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