JPWO2020033927A5 - - Google Patents

Description

からなる群より選択される、本発明1089の方法。
[本発明1091]
主剤および副剤の一方または両方が、抗体またはその抗原結合断片を含む、本発明1046～1063、1065～1076および1085～1090のいずれかの方法。
[本発明1092]
1つまたは複数の前記剤が、一価の抗体断片であるか、またはそれを含む、本発明1091の方法。
[本発明1093]
主剤および副剤の一方または両方が、Fabであるか、またはそれを含む、本発明1091または1092の方法。
[本発明1094]
オリゴマー粒子試薬が、
60nmよりも大きい、70nmよりも大きい、80nmよりも大きい、もしくは90nmよりも大きい半径、
50nm～150nm、75nm～125nm、80nm～115nm、もしくは90nm～110nmの半径（両端の値を含む）；または
90nm±15nmもしくは95nm±20～25nmの半径
を含む、本発明1001、1046～1063、1065～1076および1085～1093のいずれかの方法。
[本発明1095]
オリゴマー粒子試薬が、
少なくとも5×10 ⁷ g/molもしくは少なくとも1×10 ⁸ g/mol；および/または
5×10 ⁷ g/mol～5×10 ⁸ g/mol、1×10 ⁸ g/mol～5×10 ⁸ g/mol、もしくは1×10 ⁸ g/mol～2×10 ⁸ g/mol
の分子量を含む、本発明1001、1046～1063、1065～1076および1085～1094のいずれかの方法。
[本発明1096]
オリゴマー粒子試薬が、
少なくとも500のストレプトアビジンもしくはストレプトアビジンムテイン四量体、少なくとも1,000のストレプトアビジンもしくはストレプトアビジンムテイン四量体、少なくとも1,500のストレプトアビジンもしくはストレプトアビジンムテイン四量体、または少なくとも2,000のストレプトアビジンもしくはストレプトアビジンムテイン四量体；および/または
1,000～20,000のストレプトアビジンもしくはストレプトアビジンムテイン四量体、1,000～10,000のストレプトアビジンもしくはストレプトアビジンムテイン四量体、または2,000～5,000のストレプトアビジンもしくはストレプトアビジンムテイン四量体
を含む、本発明1001、1046～1063、1065～1076および1085～1088のいずれかの方法。
[本発明1097]
インキュベーションの少なくとも一部分の後または間のいずれかに、物質を細胞に加える工程をさらに含み、
該物質が、刺激試薬によるT細胞の刺激を終わらせるかまたは減らす、
本発明1001、1046～1063、1065～1076および1085～1096のいずれかの方法。
[本発明1098]
インキュベーションの少なくとも一部分の後または間のいずれかに、物質を細胞に加える工程をさらに含み、
該物質が、主剤および副剤とオリゴマー粒子試薬との間の結合を解消することができる、
本発明1046～1063、1065～1076および1085～1097のいずれかの方法。
[本発明1099]
前記物質が、フリーの結合パートナーであり、かつ/または競合試薬である、本発明1098の方法。
[本発明1100]
前記物質の存在が、T細胞中で主剤および副剤によって誘導または変調されたシグナルを終結または減少させる、本発明1098または1099の方法。
[本発明1101]
前記物質が、ストレプトアビジン結合ペプチド、ビオチンもしくは生物学的に活性な断片、またはビオチン類似体もしくは生物学的に活性な断片であるか、またはそれを含む、本発明1097～1100のいずれかの方法。
[本発明1102]
前記物質が、ストレプトアビジン結合ペプチドであるか、またはそれを含み、該ストレプトアビジン結合ペプチドが、

からなる群より選択される、本発明1101の方法。
[本発明1103]
前記物質が、ビオチンもしくは生物学的に活性な断片またはビオチン類似体もしくは生物学的に活性な断片であるか、またはそれを含む、本発明1101の方法。
[本発明1104]
前記物質が、オリゴマー粒子刺激試薬への曝露が開始された後42時間～120時間または約42時間～120時間（両端の値を含む）で加えられる、本発明1097～1103のいずれかの方法。
[本発明1105]
前記物質が、オリゴマー粒子試薬への曝露が開始された後72時間～96時間または約72時間～96時間で加えられる、本発明1097～1104のいずれかの方法。
[本発明1106]
前記物質が、オリゴマー粒子試薬への曝露が開始された後48時間または約48時間で加えられる、本発明1097～1105のいずれかの方法。
[本発明1107]
前記物質が、オリゴマー粒子試薬への曝露が開始された後72時間または約72時間で加えられる、本発明1097～1106のいずれかの方法。
[本発明1108]
前記物質が、オリゴマー粒子試薬への曝露が開始された後96時間または約96時間で加えられる、本発明1097～1107のいずれかの方法。
[本発明1109]
前記物質が、インキュベーションの後かつ採取の前に加えられる、本発明1097～1108のいずれかの方法。
[本発明1110]
前記物質が、インキュベーションの少なくとも一部分の間に加えられ、該インキュベーションが、該物質が加えられた後も継続する、本発明1097～1109のいずれかの方法。
[本発明1111]
操作されたT細胞の組成物を製造する方法であって、
（a）初代T細胞を含むインプット組成物を、複数のストレプトアビジン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粒子試薬を含む、細胞10 ⁶ 個あたり0.2μg～8μgまたは約0.2μg～8μgの刺激試薬に曝露する工程であって、該オリゴマー粒子試薬に、（i）CD3に特異的に結合する主剤と、（ii）CD28に特異的に結合する副剤とが付着しており、曝露が、組換えIL-2、IL-7、およびIL-15を有する無血清培地の存在下で実施され、それにより、刺激された集団を生成する、工程；
（b）該刺激された集団のT細胞に、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導入し、それにより、形質転換細胞の集団を生成する工程であって、導入が、組換えIL-2、IL-7、およびIL-15を有する無血清培地の存在下で実施される、工程；
（c）該形質転換細胞の集団を、静的条件下、24時間～96時間インキュベートする工程であって、任意で、インキュベーションが、約37℃の温度で、かつ該インキュベーションの少なくとも一部分の間、ビオチンもしくはその生物学的に活性な断片、任意でD-ビオチンまたはビオチン類似体もしくはその生物学的に活性な断片を含む物質を添加しながら実施される、工程；および
（d）該インキュベーションの後、形質転換集団のT細胞を採取し、それにより、操作された細胞の組成物を製造する工程であって、採取が、該刺激試薬への曝露が開始された後72～96時間の時点（両端の値を含む）で実施される、工程
を含み、
それにより、操作された細胞の組成物を製造する、方法。
[本発明1112]
インプット組成物が少なくとも300×10 ⁶ 個の生存初代T細胞を含む、本発明1001～1111のいずれかの方法。
[本発明1113]
インプット組成物が600×10 ⁶ 個または約600×10 ⁶ 個の生存初代T細胞を含む、本発明1001～1112のいずれかの方法。
[本発明1114]
インプット組成物中の細胞の少なくとももしくは少なくとも約80％、少なくとももしくは少なくとも約85％、少なくとももしくは少なくとも約90％、少なくとももしくは少なくとも約95％、少なくとももしくは少なくとも約96％、少なくとももしくは少なくとも約97％、少なくとももしくは少なくとも約98％、少なくとももしくは少なくとも約99％、約100％または100％がCD3+T細胞である、本発明1001～1113のいずれかの方法。
[本発明1115]
前記組成物中の細胞の少なくとももしくは少なくとも約80％、少なくとももしくは少なくとも約85％、少なくとももしくは少なくとも約90％、少なくとももしくは少なくとも約95％、少なくとももしくは少なくとも約96％、少なくとももしくは少なくとも約97％、少なくとももしくは少なくとも約98％、少なくとももしくは少なくとも約99％、約100％または100％がCD4+T細胞およびCD8+T細胞である、本発明1001～1114のいずれかの方法。
[本発明1116]
インプット組成物が、1.5：1～2.0：1のCD4+細胞とCD8+細胞の比を含む、本発明1001～1115のいずれかの方法。
[本発明1117]
インプット組成物が、1.2：1～0.8：1のCD4+細胞とCD8+細胞の比、任意で1：1または約1：1のCD4+T細胞とCD8+T細胞の比を含む、本発明1001～1116のいずれかの方法。
[本発明1118]
操作されたT細胞の組成物を製造する方法であって、
（a）初代T細胞を含むインプット組成物を、複数のストレプトアビジン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粒子試薬を含む、細胞10 ⁶ 個あたり0.4μg～8μgまたは約0.4μg～8μgの刺激試薬に曝露する工程であって、該オリゴマー粒子試薬に、（i）CD3に特異的に結合する主剤と、（ii）CD28に特異的に結合する副剤とが付着しており、
該インプット組成物が、2：1～1：2のCD4+T細胞とCD8+T細胞の比を含み、少なくとも300×10 ⁶ 個のCD4+およびCD8+T細胞を含み、曝露が、組換えIL-2、IL-7、およびIL-15を含む無血清培地の存在下で実施され、それにより、刺激された集団を生成する、工程；
（b）該刺激された集団のT細胞に、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導入し、それにより、形質転換細胞の集団を生成する工程であって、導入が、組換えIL-2、IL-7、およびIL-15を有する無血清培地の存在下で実施される、工程；
（c）形質転換細胞の集団を、静的条件下、24時間～72時間また約24時間～72時間インキュベートする工程であって、任意で、インキュベーションが、約37℃の温度で、かつ、該インキュベーションの少なくとも一部分の間、ビオチンもしくはその生物学的に活性な断片、任意でD-ビオチンまたはビオチン類似体もしくはその生物学的に活性な断片を含む物質を添加しながら実施され、添加が、該刺激試薬への曝露が開始された後48～72時間の時点（両端の値を含む）で実施される、工程；および
（d）該インキュベーションの後、形質転換集団のT細胞を採取し、それにより、操作された細胞の組成物を製造する工程であって、採取が、該刺激試薬への曝露が開始された後72～96時間の時点（両端の値を含む）で実施される、工程
を含み、
それにより、操作された細胞の組成物を製造する、方法。
[本発明1119]
前記物質が、刺激試薬への曝露が開始された後48時間または約48時間で加えられる、本発明1111～1118のいずれかの方法。
[本発明1120]
前記物質が、刺激試薬への曝露が開始された後72時間または約72時間で加えられる、本発明1111～1118のいずれかの方法。
[本発明1121]
前記物質が、刺激試薬への曝露が開始された後96時間または約96時間で加えられる、本発明1111～1118のいずれかの方法。
[本発明1122]
操作されたT細胞の組成物を製造する方法であって、
（a）初代T細胞を含むインプット組成物を、常磁性ビーズを含む刺激試薬に、2：1～0.5：1または約2：1～0.5：1のビーズと細胞の比で曝露する工程であって、該ビーズが、ポリスチレンコーティングおよび4.5μmまたは約4.5μmの直径を含む不活性常磁性ビーズであり、該ビーズに、（i）抗CD3抗体またはその抗原結合断片および抗CD28抗体またはその抗原結合断片が付着しており、該インプット組成物が、2：1～1：2のCD4+T細胞とCD8+T細胞の比を含み、少なくとも300×10 ⁶ 個のCD4+およびCD8+T細胞を含み、曝露が、組換えIL-2、IL-7、およびIL-15を含む無血清培地の存在下で実施され、それにより、刺激された集団を生成する、工程；
（b）該刺激された集団のT細胞に、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導入し、それにより、形質転換細胞の集団を生成する工程であって、導入が、組換えIL-2、IL-7、およびIL-15を有する無血清培地の存在下で実施される、工程；
（c）該形質転換細胞の集団を、静的条件下、48～72時間（両端の値を含む）インキュベートする工程であって、任意で、インキュベーションが約37℃の温度で実施される、工程；
（d）該インキュベーションの後、該細胞を磁場に曝露して該刺激試薬を該細胞から除去し、次いで該T細胞を採取し、それにより、操作された細胞の組成物を製造する工程であって、採取が、該刺激試薬への曝露が開始された後72～96時間の時点（両端の値を含む）で実施される、工程
を含み、
それにより、操作された細胞の組成物を製造する、方法。
[本発明1123]
インキュベーションの少なくとも一部分が、組換えIL-2、IL-7、およびIL-15を含む無血清培地の存在下で実行される、本発明1111～1122のいずれかの方法。
[本発明1124]
インキュベーションが、組換えサイトカインを欠く基礎培地中で実施される、本発明1111～1123のいずれかの方法。
[本発明1125]
インプット組成物が、450×10 ⁶ 個または約450×10 ⁶ 個の生存初代T細胞、500×10 ⁶ 個もしくは約500×10 ⁶ 個の生存初代T細胞、550×10 ⁶ 個もしくは約550×10 ⁶ 個の生存初代T細胞、600×10 ⁶ 個もしくは約600×10 ⁶ 個の生存初代T細胞、700×10 ⁶ 個もしくは約700×10 ⁶ 個の生存初代T細胞、800×10 ⁶ 個もしくは約800×10 ⁶ 個の生存初代T細胞、900×10 ⁶ 個もしくは約900×10 ⁶ 個の生存初代T細胞、または1,000×10 ⁶ 個もしくは約1,000×10 ⁶ 個の生存初代T細胞、または前記のいずれかの間の任意の値を含む、本発明1001～1124のいずれかの方法。
[本発明1126]
インプット組成物が少なくとも600×10 ⁶ 個または少なくとも約600×10 ⁶ 個の生存初代T細胞を含む、本発明1001～1125のいずれかの方法。
[本発明1127]
インプット組成物が900×10 ⁶ 個または最大900×10 ⁶ 個の生存初代T細胞を含む、本発明1001～1126のいずれかの方法。
[本発明1128]
インプット組成物が900×10 ⁶ 個または約900×10 ⁶ 個の生存初代T細胞を含む、本発明1001～1125のいずれかの方法。
[本発明1129]
インキュベーションが、導入の後24時間または約24時間実施される、本発明1044～1059、1061～1069、1081～1113、1115および1116のいずれかの方法。
[本発明1130]
インキュベーションが、導入の後48時間または約48時間実施される、本発明1001および1015～1129のいずれかの方法。
[本発明1131]
インキュベーションが、導入の後72時間または約72時間実施される、本発明1001および1015～1130のいずれかの方法。
[本発明1132]
刺激試薬への曝露の前に、生体試料からのCD3+T細胞またはCD4+T細胞および/またはCD8+T細胞を濃縮してインプット組成物を製造する工程を含む、本発明1001～1131のいずれかの方法。
[本発明1133]
刺激試薬への曝露の前に、生体試料からのCD3+T細胞を濃縮してインプット組成物を製造する工程を含む、本発明1001～1132のいずれかの方法。
[本発明1134]
濃縮が免疫親和性に基づく選択による、本発明1132または1133の方法。
[本発明1135]
免疫親和性に基づく選択が、アフィニティークロマトグラフィーマトリックス上に固定されたまたはそれに付着した抗体と、細胞を接触させることによって実施され、該抗体が、細胞表面マーカーに特異的に結合して、CD3+細胞の陽性選択を実施することができる、本発明1134の方法。
[本発明1136]
刺激試薬への曝露の前に、生体試料からのCD4+またはCD8+T細胞を濃縮してインプット組成物を製造する工程を含む、本発明1001～1132のいずれかの方法。
[本発明1137]
濃縮が、
（a）閉鎖系中で第一の選択を実行することであって、該第一の選択が、初代ヒトT細胞を含む試料からの（i）CD4+細胞および（ii）CD8+細胞の一方の濃縮を含み、それにより、該濃縮が第一の選択集団および非選択集団を生成する、前記実行すること；および
（b）閉鎖系中で第二の選択を実行することであって、該第二の選択が、該非選択集団からの（i）CD4+細胞および（ii）CD8+細胞の他方の濃縮を含み、それにより、該濃縮が第二の選択集団を生成し、該濃縮が、CD4+T細胞およびCD8+T細胞の別々の濃縮集団を製造する、前記実行すること
を含み、
該別々の濃縮集団がそれぞれ、該第一の選択集団または該第二の選択集団の一方からの細胞を含み、
インプット組成物が、CD4+T細胞の濃縮集団およびCD8+T細胞の濃縮集団の1つまたは複数からの細胞を含む、
本発明1136の方法。
[本発明1138]
CD4+T細胞およびCD8+T細胞の別々の濃縮集団が混合され、それにより、インプット組成物が製造される、本発明1136または1137の方法。
[本発明1139]
混合が閉鎖系中で実行され、任意で、該閉鎖系が自動化されている、本発明1138の方法。
[本発明1140]
第一および/または第二の選択における細胞の濃縮が、細胞表面マーカーの発現に基づいて陽性選択または陰性選択を実行することを含む、本発明1136～1139のいずれかの方法。
[本発明1141]
第一または第二の選択における細胞の濃縮が、細胞表面マーカーまたはCD4+もしくはCD8+細胞を濃縮するためのマーカーの発現に基づいて複数の陽性または陰性選択工程を実行することを含む、本発明1136～1140のいずれかの方法。
[本発明1142]
第一および/または第二の選択における細胞の濃縮が、免疫親和性に基づく選択を含む、本発明1136～1141のいずれかの方法。
[本発明1143]
第一および第二の選択における細胞の濃縮が免疫親和性に基づく選択を含む、本発明1142の方法。
[本発明1144]
免疫親和性に基づく選択が、アフィニティークロマトグラフィーマトリックス上に固定されたまたはそれに付着した抗体と、細胞を接触させることによって実施され、該抗体が、細胞表面マーカーに特異的に結合して、CD4+またはCD8+細胞の陽性または陰性選択を実施することができる、本発明1142または1143の方法。
[本発明1145]
抗体が、マトリックス上に固定された結合試薬と可逆性結合を形成することができる1つまたは複数の結合パートナーをさらに含み、該抗体が、接触の間にクロマトグラフィーマトリックスに可逆的に結合し；
該マトリックス上で該抗体が特異的に結合する細胞表面マーカーを発現する細胞を、該結合試薬と該結合パートナーとの間の該可逆性結合の切断によって該マトリックスから回収することができる、
本発明1135または1144の方法。
[本発明1146]
結合パートナーが、ビオチン、ビオチン類似体、および結合試薬に結合することができるペプチドの中から選択され；かつ
該結合試薬が、ストレプトアビジン、ストレプトアビジン類似体またはムテイン、アビジン、およびアビジン類似体またはムテインの中から選択される、
本発明1145の方法。
[本発明1147]
結合パートナーが、SEQ ID NO:64に記載されるアミノ酸の配列を含む；かつ/または
結合試薬が、SEQ ID NO: 75、80、76または63に記載されるアミノ酸の配列を含むストレプトアビジンムテインである、
本発明1145または1146の方法。
[本発明1148]
試料中の細胞をアフィニティークロマトグラフィーマトリックスに接触させた後、競合試薬を加えて結合パートナーと結合試薬との間の結合を切断し、それにより、選択された細胞を該マトリックスから回収する工程をさらに含む、本発明1145～1147のいずれかの方法。
[本発明1149]
競合試薬がビオチンまたはビオチン類似体である、本発明1148の方法。
[本発明1150]
1つまたは複数の選択における1つまたは複数の抗体が、3×10 ^-5 sec ^-1 よりも大きいまたは約3×10 ^-5 sec ^-1 よりも大きい、結合および細胞表面マーカーについての解離速度定数（k _off ）を有する、本発明1135および1144～1149のいずれかの方法。
[本発明1151]
1つまたは複数の選択における1つまたは複数の抗体が、約10 ^-3 ～10 ^-7 の範囲または約10 ^-7 ～約10 ^-10 の範囲の解離定数（K _d ）である、細胞表面マーカーに対する親和性を有する、本発明1135および1144～1150のいずれかの方法。
[本発明1152]
クロマトグラフィーマトリックスが、カラムである分離用容器に詰められている、本発明1135および1144～1151のいずれかの方法。
[本発明1153]
生体試料からのCD4+またはCD8+T細胞の濃縮が、
（a）CD4+細胞の濃縮が、CD4の表面発現に基づく陽性選択を含み；
（b）CD8+細胞の濃縮が、CD8の表面発現に基づく陽性選択を含み；または
（a）と（b）の両方
を含む、本発明1136～1152のいずれかの方法。
[本発明1154]
刺激試薬への曝露の前に、生体試料からのCD4+またはCD8+T細胞を濃縮する工程であって、濃縮が、試料の細胞を、インキュベーション組成物中でCD4に特異的に結合する第一の免疫親和性試薬およびインキュベーション組成物中でCD8に特異的に結合する第二の免疫親和性試薬と、該免疫親和性試薬が該試料中の細胞の表面上のCD4およびCD8分子にそれぞれ特異的に結合する条件下で接触させることを含む、工程；および
該第一および/または第二の免疫親和性試薬に結合した細胞を回収し、それにより、CD4+細胞およびCD8+細胞を含む濃縮組成物を生成する工程
を含み、
インプット組成物が、CD4+細胞およびCD8+細胞を含む濃縮組成物の細胞を含む、
本発明1001～1153のいずれかの方法。
[本発明1155]
免疫親和性試薬それぞれが抗体またはその抗原結合断片を含む、本発明1154の方法。
[本発明1156]
免疫親和性試薬がビーズの外側表面に固定されている、本発明1154または1155の方法。
[本発明1157]
ビーズが磁気ビーズである、本発明1156の方法。
[本発明1158]
第一または第二の免疫親和性試薬に結合した細胞の回収が、細胞を磁場に曝露することを含む、本発明1157の方法。
[本発明1159]
生体試料が、対象、任意でヒト対象から得られた初代T細胞を含む、本発明1132～1158のいずれかの方法。
[本発明1160]
生体試料が、全血試料、バフィーコート試料、末梢血単核細胞（PBMC）試料、未分画T細胞試料、リンパ球試料、白血球試料、アフェレーシス産物、もしくは白血球アフェレーシス産物であるか、またはそれを含む、本発明1132～1159のいずれかの方法。
[本発明1161]
生体試料が、濃縮の前に事前にクライオ凍結されたアフェレーシス産物または白血球アフェレーシス産物である、本発明1132～1160のいずれかの方法。
[本発明1162]
採取が、刺激試薬への曝露が開始された後96時間または約96時間で実施される、本発明1001～1048および1052～1161のいずれかの方法。
[本発明1163]
採取が、刺激試薬への曝露が開始された後2日もしくは約2日または3日もしくは約3日で実施される、本発明1001～1048および1052～1161のいずれかの方法。
[本発明1164]
採取が、刺激試薬への曝露が開始された後72時間または約72時間で実施される、本発明1001～1048および1052～1161のいずれかの方法。
[本発明1165]
採取が、刺激試薬への曝露が開始された後48時間または約48時間で実施される、本発明1001～1048および1052～1161のいずれかの方法。
[本発明1166]
採取が、組み込まれたベクターがゲノム中で検出された時点で、ただし、二倍体ゲノムあたりの安定なiVCNを達成する前に実施される、本発明1001および1012～1165のいずれかの方法。
[本発明1167]
採取が、形質転換細胞の集団中の総VCNに対する組み込まれたベクターコピー数（iVCN）の分率が平均で0.8未満である時点で実施される、本発明1001および1012～1166のいずれかの方法。
[本発明1168]
採取が、形質転換細胞の集団のiVCNが平均で二倍体ゲノムあたり2.0コピー未満または約2.0コピー未満である時点で実施される、本発明1001および1012～1167のいずれかの方法。
[本発明1169]
細胞の採取が、細胞をすすぐまたは洗うことによって細胞デブリを除去することを含む、本発明1001～1168のいずれかの方法。
[本発明1170]
ナイーブ様細胞のパーセンテージが、集団中の全T細胞、該集団中の全CD4+T細胞、または該集団中の全CD8+T細胞もしくはその組換えタンパク質発現細胞の中で60％よりも高いまたは約60％よりも高い時点で、細胞が採取される、本発明1001および1030～1169のいずれかの方法。
[本発明1171]
ナイーブ様細胞および/またはセントラルメモリーT細胞のパーセンテージが、集団中の全T細胞、該集団中の全CD4+T細胞、または該集団中の全CD8+T細胞もしくはその組換えタンパク質発現細胞の中で60％よりも高いまたは約60％よりも高い時点で、細胞が採取される、本発明1001および1030～1169のいずれかの方法。
[本発明1172]
採取時、
ナイーブ様T細胞のパーセンテージが、集団中の全組換えタンパク質発現細胞の中で60％よりも高いまたは約60％よりも高い、任意で、該集団中の全組換えタンパク質発現T細胞の中で65％、70％、80％、90％、もしくは95％よりも高い、または約65％、70％、80％、90％、もしくは95％よりも高い；
ナイーブ様T細胞のパーセンテージが、該集団中の全組換えタンパク質発現CD4+T細胞の中で60％よりも高いまたは約60％よりも高い、任意で、該集団中の全組換えタンパク質発現CD4+細胞の中で65％、70％、80％、90％、もしくは95％よりも高い、または約65％、70％、80％、90％もしくは95％よりも高い；または
ナイーブ様T細胞のパーセンテージが、該集団中の全組換えタンパク質発現CD8+T細胞の中で60％よりも高いまたは約60％よりも高い、任意で、該集団中の全組換えタンパク質発現CD8+細胞の中で65％、70％、80％、90％、もしくは95％よりも高い、または約65％、70％、80％、90％、もしくは95％よりも高い、
本発明1170または1171の方法。
[本発明1173]
採取時、
ナイーブ様T細胞および/またはセントラルメモリーT細胞のパーセンテージが、集団中の全組換えタンパク質発現細胞の中で60％よりも高いまたは約60％よりも高い、任意で、該集団中の全組換えタンパク質発現T細胞の中で65％、70％、80％、90％、もしくは95％よりも高い、または約65％、70％、80％、90％、もしくは95％よりも高い；
ナイーブ様T細胞および/またはセントラルメモリーT細胞のパーセンテージが、該集団中の全組換えタンパク質発現CD4+T細胞の中で60％よりも高いまたは約60％よりも高い、任意で、該集団中の全組換えタンパク質発現CD4+細胞の中で65％、70％、80％、90％、もしくは95％よりも高い、または約65％、70％、80％、90％、もしくは95％よりも高い；または
ナイーブ様T細胞および/またはセントラルメモリーT細胞のパーセンテージが、該集団中の全組換えタンパク質発現CD8+T細胞の中で60％よりも高いまたは約60％よりも高い、任意で、該集団中の全組換えタンパク質発現CD8+細胞の中で65％、70％、80％、90％、もしくは95％よりも高い、または約65％、70％、80％、90％、もしくは95％よりも高い、
本発明1171の方法。
[本発明1174]
ナイーブ様T細胞、またはナイーブ様T細胞上に発現するマーカーに関して表面陽性であるT細胞が、CCR7+CD45RA+、CD27+CCR7+、またはCD62L-CCR7+である、本発明1170～1173のいずれかの方法。
[本発明1175]
ナイーブ様T細胞がCD27+CCR7+T細胞を含む、本発明1170～1174のいずれかの方法。
[本発明1176]
ナイーブ様T細胞がCCR7+CD45RA+T細胞を含む、本発明1170～1175のいずれかの方法。
[本発明1177]
操作された細胞の組成物の細胞を、凍結保存および/または対象への投与のために、任意で薬学的に許容される賦形剤の存在下で製剤化する工程をさらに含む、本発明1001～1176のいずれかの方法。
[本発明1178]
操作された細胞の組成物の細胞が凍結保護物質の存在下で製剤化される、本発明1177の方法。
[本発明1179]
凍結保護物質がDMSOを含む、本発明1178の方法。
[本発明1180]
操作された細胞の組成物の細胞が、容器、任意でバイアルまたはバッグの中に製剤化される、本発明1177～1179のいずれかの方法。
[本発明1181]
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がT細胞に導入される、本発明1001～1180のいずれかの方法。
[本発明1182]
ウイルスベクターがレトロウイルスベクターである、本発明1181の方法。
[本発明1183]
ウイルスベクターがレンチウイルスベクターまたはガンマレトロウイルスベクターである、本発明1180または1181の方法。
[本発明1184]
導入が形質導入アジュバントの存在下で実施される、本発明1001～1183のいずれかの方法。
[本発明1185]
形質導入アジュバントが、硫酸プロタミン、任意で1μg/ml～50μg/mlまたは約1μg/ml～50μg/mlの硫酸プロタミン、フィブロネクチン由来形質導入アジュバントまたはRetroNectinである、本発明1184の方法。
[本発明1186]
組換えタンパク質が、
疾患、障害、または病気の細胞または組織と関連する、それに特異的である、かつ/またはその上に発現する、標的抗原に結合することができる、組換え受容体
である、本発明1001～1185のいずれかの方法。
[本発明1187]
疾患、障害、または病気が、感染性疾患もしくは障害、自己免疫疾患、炎症性疾患、または腫瘍もしくはがんである、本発明1186の方法。
[本発明1188]
標的抗原が腫瘍抗原である、本発明1187の方法。
[本発明1189]
標的抗原が、αvβ6インテグリン（avb6インテグリン）、B細胞成熟抗原（BCMA）、B7-H3、B7-H6、炭酸脱水酵素9（CA9、別名CAIXまたはG250）、がん精巣抗原、がん精巣抗原1B（CTAG、別名NY-ESO-1およびLAGE-2）、がん胎児性抗原（CEA）、サイクリン、サイクリンA2、C-Cモチーフケモカインリガンド1（CCL-1）、CD19、CD20、CD22、CD23、CD24、CD30、CD33、CD38、CD44、CD44v6、CD44v7/8、CD123、CD133、CD138、CD171、コンドロイチン硫酸プロテオグリカン4（CSPG4）、上皮成長因子タンパク質（EGFR）、タイプIII上皮成長因子受容体変異（EGFR vIII）、上皮糖タンパク質2（EPG-2）、上皮糖タンパク質40（EPG-40）、エフリンB2、エフリン受容体A2（EPHa2）、エストロゲン受容体、Fc受容体様5（FCRL5；別名Fc受容体ホモログ5またはFCRH5）、胎児アセチルコリン受容体（胎児AchR）、葉酸結合タンパク質（FBP）、葉酸受容体アルファ、ガングリオシドGD2、O-アセチル化GD2（OGD2）、ガングリオシドGD3、糖タンパク質100（gp10O）、グリピカン-3（GPC3）、Gタンパク質共役型受容体クラスCグループ5メンバーD（GPRC5D）、Her2/neu（受容体チロシンキナーゼerb-B2）、Her3（erb-B3）、Her4（erb-B4）、erbB二量体、ヒト高分子量黒色腫関連抗原（HMW-MAA）、B型肝炎表面抗原、ヒト白血球抗原A1（HLA-A1）、ヒト白血球抗原A2（HLA-A2）、IL-22受容体アルファ（IL-22Rα）、IL-13受容体アルファ2（IL-13Rα2）、キナーゼインサートドメイン受容体（kdr）、カッパ軽鎖、L1細胞接着分子（L1-CAM）、L1-CAMのCE7エピトープ、ロイシンリッチリピート含有8ファミリーメンバーA（LRRC8A）、ルイスY、黒色腫関連抗原（MAGE）-A1、MAGE-A3、MAGE-A6、MAGE-A10、メソセリン（MSLN）、c-Met、マウスサイトメガロウイルス（CMV）、ムチン1（MUC1）、MUC16、ナチュラルキラーグループ2メンバーD（NKG2D）リガンド、メランA（MART-1）、神経細胞接着分子（NCAM）、がん胎児性抗原、メラノーマ優先発現抗原（PRAME）、プロゲステロン受容体、前立腺特異抗原、前立腺幹細胞抗原（PSCA）、前立腺特異膜抗原（PSMA）、受容体チロシンキナーゼ様オーファン受容体1（ROR1）、サバイビン、トロホブラスト糖タンパク質（TPBG、別名5T4）、腫瘍関連糖タンパク質72（TAG72）、チロシナーゼ関連タンパク質1（TRP1、別名TYRP1またはgp75）、チロシナーゼ関連タンパク質2（TRP2、別名ドーパクロムトートメラーゼ、ドーパクロムデルタイソメラーゼまたはDCT）、血管内皮細胞増殖因子受容体（VEGFR）、血管内皮細胞増殖因子受容体2（VEGFR2）、ウィルムス腫瘍1（WT-1）、病原体特異もしくは病原体発現抗原、またはユニバーサルタグと関連する抗原、および/またはビオチン化分子、および/またはHIV、HCV、HBV、もしくは他の病原体によって発現される分子の中から選択される、本発明1186～1188のいずれかの方法。
[本発明1190]
組換えタンパク質が、機能的非TCR抗原受容体またはTCRもしくはその抗原結合断片であるか、またはそれを含む、本発明1001～1048または1052～1189のいずれかの方法。
[本発明1191]
組換えタンパク質がキメラ抗原受容体（CAR）である、本発明1001～1048または1052～1190のいずれかの方法。
[本発明1192]
無血清培地が、
基礎培地中0.5mM～5mMのL-グルタミンのジペプチド形態；
0.5mM～5mMのL-グルタミン；および
少なくとも1つのタンパク質
を含み、血清を含まない、本発明1034～1042または1074～1191のいずれかの方法。
[本発明1193]
1つまたは複数の組換えサイトカインを欠く基礎培地が、L-グルタミンを、任意で約0.5mM～約5mMの濃度で、任意で約2mMの濃度で含む、本発明1001および1026～1192のいずれかの方法。
[本発明1194]
1つまたは複数の組換えサイトカインを欠く基礎培地が血清を含まない、本発明1001および1026～1191および1193のいずれかの方法。
[本発明1195]
基礎培地が、アルブミン、インスリン、またはトランスフェリンの1つまたは複数、任意でヒトまたは組換えアルブミン、インスリン、またはトランスフェリンの1つまたは複数から選択される少なくとも1つのタンパク質を含む、本発明1001、1026～1191および1193～1194のいずれかの方法。
[本発明1196]
本発明1001～1195のいずれかの方法によって製造された治療用T細胞組成物。
[本発明1197]
前記方法によって製造された複数の組成物における平均で、組成物中のT細胞の総数または該組成物中の組換えタンパク質を発現するT細胞の総数の少なくとも50％、60％、70％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、もしくは100％、または少なくとも約50％、60％、70％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、もしくは100％、または50％、60％、70％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、もしくは100％、または約50％、60％、70％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、もしくは100％が、ナイーブ様T細胞であるか、またはナイーブ様細胞上に発現するマーカーに関して表面陽性であるT細胞である、本発明1196の治療用T細胞組成物。
[本発明1198]
前記方法によって製造された複数の組成物における平均で、組成物中のT細胞の総数または該組成物中の組換えタンパク質を発現するT細胞の総数の少なくとも50％または少なくとも約50％または50％または約50％が、セントラルメモリーT細胞であるか、またはセントラルメモリーT細胞上に発現するマーカーに関して表面陽性であるT細胞である、本発明1196の治療用T細胞組成物。
[本発明1199]
平均で、組成物中のT細胞の総数または該組成物中の組換えタンパク質を発現するT細胞の総数の少なくとも50％または少なくとも約50％または50％または約50％が、ナイーブ様T細胞もしくはセントラルメモリーT細胞であるか、またはナイーブ様T細胞もしくはセントラルメモリーT細胞上に発現するマーカーに関して表面陽性であるT細胞である、本発明1196の治療用T細胞組成物。
[本発明1200]
ナイーブ様T細胞上に発現するマーカーが、CD45RA、CD27、CD28、およびCCR7からなる群より選択される、本発明1197または1199の治療用T細胞組成物。
[本発明1201]
ナイーブ様T細胞、またはナイーブ様T細胞上に発現するマーカーに関して表面陽性であるT細胞が、CCR7+CD45RA+、CD27+CCR7+、またはCD62L-CCR7+である、本発明1197、1199および1200のいずれかの治療用T細胞組成物。
[本発明1202]
ナイーブ様T細胞、またはナイーブ様T細胞上に発現するマーカーに関して表面陽性であるT細胞が、CD27+CCR7+T細胞を含み、組成物中の全受容体 ⁺ /CD8 ⁺ 細胞の少なくとも70％、80％、85％、または90％がCD27+CCR7+である、本発明1197および1199～1201のいずれかの治療用T細胞組成物。
[本発明1203]
ナイーブ様T細胞、またはナイーブ様T細胞上に発現するマーカーに関して表面陽性であるT細胞が、CD27+CCR7+T細胞を含み、組成物中の全受容体 ⁺ /CD4 ⁺ 細胞の少なくとも50％、60％、70％、80％、85％、または90％がCD27+CCR7+である、本発明1197および1199～1202のいずれかの治療用T細胞組成物。
[本発明1204]
ナイーブ様T細胞、またはナイーブ様T細胞上に発現するマーカーに関して表面陽性であるT細胞がCD27+CCR7+T細胞を含み、組成物中の全受容体 ⁺ /CD8 ⁺ 細胞の少なくとも50％、60％、70％、80％、85％、または90％がCD27+CCR7+であり、該組成物中の全受容体 ⁺ /CD4 ⁺ 細胞の少なくとも50％、60％、70％、80％、85％、または90％がCD27+CCR7+である、本発明1197および1199～1203のいずれかの治療用T細胞組成物。
[本発明1205]
組換え受容体を発現するCD4+T細胞および組換え受容体を発現するCD8+T細胞を含む、治療用T細胞組成物であって、
該組成物中の全受容体 ⁺ /CD8 ⁺ 細胞の少なくとも50％、60％、70％、80％、または90％がCD27+CCR7+であり、該組成物中の全受容体 ⁺ /CD4 ⁺ 細胞の少なくとも50％、60％、70％、80％、または90％がCD27+CCR7+である、
治療用T細胞組成物。
[本発明1206]
前記組成物中の細胞の少なくとももしくは少なくとも約80％、少なくとももしくは少なくとも約85％、少なくとももしくは少なくとも約90％、少なくとももしくは少なくとも約95％、少なくとももしくは少なくとも約96％、少なくとももしくは少なくとも約97％、少なくとももしくは少なくとも約98％、少なくとももしくは少なくとも約99％、約100％または100％がCD3+T細胞である、本発明1196～1205のいずれかの治療用T細胞組成物。
[本発明1207]
前記組成物中の細胞の少なくとももしくは少なくとも約80％、少なくとももしくは少なくとも約85％、少なくとももしくは少なくとも約90％、少なくとももしくは少なくとも約95％、少なくとももしくは少なくとも約96％、少なくとももしくは少なくとも約97％、少なくとももしくは少なくとも約98％、少なくとももしくは少なくとも約99％、約100％または100％がCD4+T細胞およびCD8+T細胞である、本発明1196～1206のいずれかの治療用T細胞組成物。
[本発明1208]
前記組成物中の細胞の少なくとももしくは少なくとも約90％がCD4+T細胞およびCD8+T細胞であり、該組成物中の全受容体 ⁺ /CD8 ⁺ 細胞の少なくとももしくは少なくとも約60％がCD27+CCR7+であり、該組成物中の全受容体 ⁺ /CD4 ⁺ 細胞の少なくとももしくは少なくとも約60％がCD27+CCR7+である、本発明1205～1207のいずれかの治療用T細胞組成物。
[本発明1209]
前記組成物中のT細胞の総数または該組成物中の組換え受容体を発現するT細胞の総数の少なくとも50％、60％、70％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、もしくは100％、または少なくとも約50％、60％、70％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、もしくは100％、または50％、60％、70％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、もしくは100％、または約50％、60％、70％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、もしくは100％が、CD27+CCR7+である、本発明1205～1208のいずれかの治療用T細胞組成物。
[本発明1210]
前記組成物中の受容体+/CD4+T細胞と受容体+/CD8+T細胞の比が、約1：3～約3：1である、本発明1197～1209のいずれかの治療用T細胞組成物。
[本発明1211]
前記組成物中の受容体+/CD4+T細胞と受容体+/CD8+T細胞の比が、約1：2～約2：1である、本発明1196～1210のいずれかの治療用T細胞組成物。
[本発明1212]
前記組成物中の受容体+/CD4+T細胞と受容体+/CD8+T細胞の比が、1：1または約1：1である、本発明1196～1211のいずれかの治療用T細胞組成物。
[本発明1213]
組換えタンパク質が、
疾患、障害、または病気の細胞または組織と関連する、それに特異的である、かつ/またはその上に発現する、標的タンパク質に結合することができる、組換え受容体
であるか、またはそれを含む、本発明1196～1212のいずれかの治療用組成物。
[本発明1214]
組換えタンパク質がキメラ抗原受容体（CAR）である、本発明1196～1213のいずれかの治療用組成物。
[本発明1215]
前記組成物中の生存T細胞の数が、25×10 ⁶ 個もしくは約25×10 ⁶ 個から、200×10 ⁶ 個もしくは約200×10 ⁶ 個であり、任意で、該組成物中の生存T細胞の数が、25×10 ⁶ 個もしくは約25×10 ⁶ 個から、100×10 ⁶ 個もしくは約100×10 ⁶ 個、25×10 ⁶ 個もしくは約25×10 ⁶ 個から、70×10 ⁶ 個もしくは約70×10 ⁶ 個、25×10 ⁶ 個もしくは約25×10 ⁶ 個から、50×10 ⁶ 個もしくは約50×10 ⁶ 個、50×10 ⁶ 個もしくは約50×10 ⁶ 個から、200×10 ⁶ 個もしくは約200×10 ⁶ 個、50×10 ⁶ 個もしくは約50×10 ⁶ 個から、100×10 ⁶ 個もしくは約100×10 ⁶ 個、50×10 ⁶ 個もしくは約50×10 ⁶ 個から、70×10 ⁶ 個もしくは約70×10 ⁶ 個、70×10 ⁶ 個もしくは約70×10 ⁶ 個から、200×10 ⁶ 個もしくは約200×10 ⁶ 個、70×10 ⁶ 個もしくは約70×10 ⁶ 個から、100×10 ⁶ 個もしくは約100×10 ⁶ 個、または100×10 ⁶ 個もしくは約100×10 ⁶ 個から、200×10 ⁶ 個もしくは約200×10 ⁶ 個（それぞれ両端の値を含む）である、本発明1196～1214のいずれかの治療用組成物。
[本発明1216]
前記組成物の量が、1.0mL～10mL（両端の値を含む）、任意で2mLもしくは約2mL、3mLもしくは約3mL、4mLもしくは約4mL、5mLもしくは約5mL、6mLもしくは約6mL、7mLもしくは約7mL、8mLもしくは約8mL、9mLもしくは約9mL、または10mLもしくは約10mL、または前記のいずれかの間の任意の値である、本発明1196～1215のいずれかの治療用組成物。
[本発明1217]
本発明1196～1216のいずれかの組成物と、該組成物を対象に投与するための指示書とを含む、製造物品。
[本発明1218]
対象が疾患または病気を有し、任意で、組換え受容体が、該疾患または病気の細胞と関連するかまたはその上に発現もしくは存在する抗原を、特異的に認識するかまたはそれに特異的に結合する、本発明1217の製造物品。
[本発明1219]
疾患または病気を有するかまたは有することが疑われる対象を治療する方法であって、該対象に対し、本発明1196～1216のいずれかの組成物からのT細胞のある用量を投与する工程を含む、方法。
[本発明1220]
T細胞の前記用量が、1×10 ⁴ 個～50×10 ⁶ 個または約1×10 ⁴ 個～50×10 ⁶ 個（両端の値を含む）のT細胞を含む、本発明1219の方法。
[本発明1221]
T細胞の前記用量のT細胞が、全T細胞、全生存T細胞、全生存組換え受容体発現T細胞、全生存組換え受容体発現CD4+T細胞、または全生存組換え受容体発現CD8+T細胞である、本発明1219または1220の方法。
[本発明1222]
疾患または病気ががんである、本発明1219～1221のいずれかの方法。
[本発明1223]
疾患または病気を有する対象を治療する方法における使用のための組成物であって、任意で該疾患または病気ががんである、本発明1196～1216のいずれかの組成物。
[本発明1224]
疾患または病気を有する対象を治療するための医薬の製造のための使用であって、任意で該疾患または病気ががんである、本発明1196～1216のいずれかの組成物の使用。
[本発明1225]
前記組成物の細胞が、疾患または病気の細胞と関連するかまたはその上に発現もしくは存在する抗原を特異的に認識するかまたはそれに特異的に結合する組換え受容体を発現し、任意で、該組換え受容体がキメラ抗原受容体である、本発明1223の使用のための組成物または1224の使用。
In some embodiments, the T cells of the dose of T cells are total T cells, total viable T cells, total viable recombinant receptor-expressing T cells, total viable recombinant receptor-expressing CD4+ T cells or total viable recombinant Receptor-expressing CD8+ T cells. In certain embodiments, the disease or condition is cancer. In particular embodiments, the disease or condition is myeloma, leukemia or lymphoma. In some embodiments, the disease or condition is a B-cell malignancy and/or acute lymphocytic leukemia (ALL), adult ALL, chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL) and diffuse large cell-type B-cell lymphoma (DLBCL).
[Invention 1001]
A method of producing a composition of engineered T cells comprising:
(a) exposing an input composition comprising primary T cells to a stimulating reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules under conditions for stimulating the T cells, thereby stimulating wherein the stimulatory reagent comprises one or more intracellular signaling domains of one or more components of the TCR complex and one or more co-stimulatory molecules of one or capable of activating multiple intracellular signaling domains;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells;
(c) incubating the population of transformed cells for up to 96 hours, optionally the incubation being performed at a temperature of about 37° C., the incubation being in basal medium lacking one or more recombinant cytokines; a step performed in; and
(c) harvesting the T cells of the transformed population after said incubation, thereby producing a composition of engineered cells, said harvesting being initiated after exposure to said stimulating agent; Process performed at 48 to 120 hours (inclusive)
including
A method thereby producing a composition of engineered cells.
[Invention 1002]
A method of producing a composition of engineered T cells comprising:
(a) exposing an input composition comprising primary T cells to a stimulating reagent under conditions for stimulating T cells, thereby generating a stimulated population;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and
(c) harvesting T cells of the transformed population, wherein harvesting comprises
(i) at 24 to 120 hours after exposure to the stimulating agent is initiated, inclusive;
(ii) once integrated vector is detected in the genome, but before achieving a stable integrated vector copy number per diploid genome (iVCN);
(iii) before the total number of viable cells at harvest is 3-fold, 2-fold, or about 3-fold, about 2-fold, or the same or about the same as the number of total viable cells in the stimulated population; and/or
(iv) the percentage of CD27+CCR7+ total T cells in said population, total CD3+ T cells in said population, total CD4+ T cells in said population, or total CD8+ T cells in said population or at greater than or about 60% of the recombinant protein-expressing cells
process to be carried out
including
A method thereby producing a composition of engineered cells.
[Invention 1003]
Input composition is at least 300 x 10 ⁶ The method of the invention 1002, comprising viable primary T cells.
[Invention 1004]
A stimulatory reagent activates one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more co-stimulatory molecules The method of the invention 1002 or 1003 can.
[Invention 1005]
A method of producing a composition of engineered T cells comprising:
(a) at least 300 x 10 ⁶ exposing an input composition comprising viable primary T cells to a stimulating reagent under conditions for stimulating the T cells, thereby generating a stimulated population, wherein the stimulating reagent is a TCR capable of activating one or more intracellular signaling domains of one or more components of the complex and one or more intracellular signaling domains of one or more costimulatory molecules; process;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and
(c) harvesting T cells of the transformed population, wherein harvesting comprises
(i) at 48 to 120 hours after exposure to the stimulating agent is initiated, inclusive;
(ii) once integrated vector is detected in the genome of said transformed population, but before achieving a stable integrated vector copy number per diploid genome (iVCN);
(iii) before the total number of viable cells at harvest is 3-fold, 2-fold or about 3-fold, about 2-fold or the same or about the same as the number of total viable cells in the stimulated population; and /or
(iv) the percentage of naive-like T cells is total T cells in said population, total CD3+ cells in said population, total CD4+ T cells in said population, or total CD8+ T cells in said population or a combination thereof at greater than or about 60% of the recombinant protein-expressing cells
process to be carried out
including
A method thereby producing a composition of engineered cells.
[Invention 1006]
1005. The method of any of the inventions 1001-1005, wherein harvesting is performed when the integrated vector is detected in the genome, but before achieving a stable iVCN per diploid genome.
[Invention 1007]
A stable iVCN per diploid genome is achieved when the iVCN peaks and remains unchanged for longer than 24 hours or changes only within an acceptable margin of error. The method of invention 1006.
[Invention 1008]
per diploid genome when the fraction of iVCN to total vector copy number (VCN) in the diploid genome of a population of transformed cells is on average 1.0 or about 1.0, or within an acceptable error thereof The method of the invention 1006, wherein a stable iVCN of is achieved.
[Invention 1009]
1008. The method of any of claims 1001-1008, wherein harvesting is performed when the fraction of integrated vector copy number (iVCN) to total VCN of the population of transformed cells is on average less than 0.8.
[Invention 1010]
A method of producing a composition of engineered T cells comprising:
(a) exposing an input composition comprising primary T cells to a stimulating reagent under conditions for stimulating the T cells, thereby generating a stimulated population, wherein the stimulating reagent is a TCR capable of activating one or more intracellular signaling domains of one or more components of the complex and one or more intracellular signaling domains of one or more costimulatory molecules; process;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and
(c) harvesting the T cells of the transformed population, wherein harvesting is performed 48 to 120 hours (inclusive) after exposure to said stimulating agent is initiated, and at harvest, said The integrated vector copy number (iVCN) of the population of transformed cells averages from 0.4 copies to 2.0 copies per diploid genome, or from about 0.4 copies to 2.0 copies, inclusive.
including
A method thereby producing a composition of engineered cells.
[Invention 1011]
1011. The method of any of inventions 1001-1010, wherein harvesting is performed when the population of transformed cells has an average of less than or about 2.0 copies of iVCN per diploid genome.
[Invention 1012]
A method of producing a composition of engineered T cells comprising:
(a) exposing an input composition comprising primary T cells to a stimulating reagent under conditions for stimulating the T cells, thereby generating a stimulated population, wherein the stimulating reagent is a TCR capable of activating one or more intracellular signaling domains of one or more components of the complex and one or more intracellular signaling domains of one or more costimulatory molecules; process;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and
(c) harvesting T cells of the transformed population, wherein harvesting is performed at 48 to 120 hours (inclusive) after exposure to said stimulating agent is initiated, and , the percentage of naive-like cells and/or central memory T cells is equal to total T cells in said population, total CD4+ T cells in said population, or total CD8+ T cells in said population or cells expressing recombinant protein thereof of the process at or above 60% or about 60%
including
A method thereby producing a composition of engineered cells.
[Invention 1013]
at the time of collection,
the percentage of naive-like T cells is greater than or greater than about 60% among all recombinant protein-expressing cells in the population, optionally among all recombinant protein-expressing T cells in said population greater than 65%, 70%, 80%, 90%, or 95%, or greater than about 65%, 70%, 80%, 90%, or 95%;
the percentage of naive-like T cells is greater than or greater than about 40% of all recombinant protein-expressing CD4+ T cells in said population, optionally all recombinant protein-expressing CD4+ T cells in said population greater than 50%, 60%, 70%, 80%, 90%, or 95% of cells, or greater than about 50%, 60%, 70%, 80%, 90%, or 95%; or
the percentage of naive-like T cells is greater than or greater than about 40% of all recombinant protein-expressing CD8+ T cells in said population, optionally all recombinant protein-expressing CD8+ T cells in said population greater than 50%, 60%, 70%, 80%, 90%, or 95% of cells, or greater than about 50%, 60%, 70%, 80%, 90%, or 95%;
The method of any of the inventions 1001-1012.
[Invention 1014]
at the time of collection,
the percentage of naive-like T cells and/or central memory T cells is greater than or greater than about 60% of all recombinant protein-expressing cells in the population, optionally all recombinants in the population greater than 65%, 70%, 80%, 90%, or 95%, or greater than about 65%, 70%, 80%, 90%, or 95% of protein-expressing T cells;
the percentage of naive-like T cells and/or central memory T cells is greater than or greater than about 40% of all recombinant protein-expressing CD4+ T cells in said population, optionally in said population greater than or about 50%, 60%, 70%, 80%, 90%, or 95% of all recombinant protein-expressing CD4+ cells of or greater than 95%; or
the percentage of naive-like T cells and/or central memory T cells is greater than or greater than about 40% among all recombinant protein-expressing CD8+ T cells in said population, optionally in said population greater than or about 50%, 60%, 70%, 80%, 90%, or 95% of all recombinant protein-expressing CD8+ cells of or higher than 95%,
The method of the invention 1012 or 1013.
[Invention 1015]
further comprising incubating the population of transformed cells for up to 96 hours after introduction and prior to harvesting;
optionally, the incubation is performed at a temperature of about 37°C,
The method of any of the inventions 1002-1014.
[Invention 1016]
The method of the invention 1001 or 1015, wherein incubation is carried out for up to 72 hours after introduction.
[Invention 1017]
The method of the invention 1001 or 1015, wherein incubation is carried out for up to 48 hours after introduction.
[Invention 1018]
The method of the invention 1001 or 1015, wherein incubation is carried out for up to 24 hours after introduction.
[Invention 1019]
The method of any of invention 1001 and 1015-1018, wherein the incubation is performed for at least 18 hours.
[Invention 1020]
The method of any of inventions 1001-1019, wherein the harvesting is performed within 96 hours after exposure to the stimulus is initiated.
[Invention 1021]
The method of any of inventions 1001-1019, wherein the harvesting is performed within 72 hours after exposure to the stimulant is initiated.
[Invention 1022]
The method of any of inventions 1001-1019, wherein harvesting is performed within 48 hours after exposure to the stimulus is initiated.
[Invention 1023]
The method of any of inventions 1001-1022, wherein one or both of exposing in (a) and introducing in (b) are performed in the presence of one or more recombinant cytokines.
[Invention 1024]
The method of any of the inventions 1001-1022, wherein the exposing in (a) and the introducing in (b) are performed in the presence of one or more recombinant cytokines.
[Invention 1025]
The method of any of inventions 1015-1024, wherein incubation is performed in the presence of one or more recombinant cytokines.
[Invention 1026]
The method of any of inventions 1015-1024, wherein incubation is performed in basal medium lacking one or more recombinant cytokines.
[Invention 1027]
Any of the invention 1005-1009 and 1012-1026, wherein the naive-like T cells or T cells that are surface positive for markers expressed on naive-like T cells are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+ method.
[Invention 1028]
The method of any of the invention 1005-1009 and 1012-1027, wherein the naive-like T cells comprise CD27+CCR7+ cells.
[Invention 1029]
The method of any of invention 1005-1009 and 1012-1028, wherein the naive-like T cells comprise CCR7+CD45RA+ T cells.
[Invention 1030]
A method of producing a composition of engineered T cells comprising:
(a) exposing an input composition comprising primary T cells to a stimulating reagent under conditions for stimulating T cells comprising the presence of one or more recombinant cytokines, thereby generating a stimulated population; generating, wherein the stimulatory reagent is one or more intracellular signaling domains of one or more components of the TCR complex and one or more of the co-stimulatory molecules of one or more cells capable of activating the intra-signal transduction domain;
(b) introducing a heterologous polynucleotide encoding a recombinant protein into the T cells of the stimulated population, thereby generating a population of transformed cells, wherein the introducing comprises one or more sets of a step performed in the presence of a replacement cytokine;
(c) incubating the population of transformed cells for up to 96 hours, optionally wherein the incubation is performed at a temperature of about 37° C. and the incubation is on a basis lacking one or more recombinant cytokines; a step performed in a culture medium; and
(c) harvesting the T cells of the transformed population after said incubation, thereby producing a composition of engineered cells, said harvesting being initiated after exposure to said stimulating agent; Process performed at 48 to 120 hours (inclusive)
including
A method thereby producing a composition of engineered cells.
[Invention 1031]
The method of any of inventions 1001 and 1023-1030, wherein the one or more recombinant cytokines is human.
[Invention 1032]
the one or more recombinant cytokines is recombinant IL-2, recombinant IL-7, and recombinant IL-15, optionally 10-200 IU/mL recombinant IL-2, 100 IU/mL-1,000 IU The method of any of the inventions 1001 and 1023-1031, wherein the method is selected from 10-200 IU/mL recombinant IL-7, and/or 10-200 IU/mL recombinant IL-15.
[Invention 1033]
the one or more recombinant cytokines is recombinant IL-2, recombinant IL-7, and recombinant IL-15, optionally 10-200 IU/mL recombinant IL-2, 100 IU/mL-1,000 IU The method of any of the inventions 1001 and 1023-1032 which is or comprises 10-200 IU/mL of recombinant IL-7/mL and 10-200 IU/mL of recombinant IL-15.
[Invention 1034]
The method of any of inventions 1001-1033, wherein one or both of exposing in (a) and introducing in (b) are performed in serum-free medium.
[Invention 1035]
The method of any of inventions 1001-1034, wherein the exposing in (a) and the introducing in (b) are performed in serum-free medium.
[Invention 1036]
the stimulating reagent
(i) a principal agent that specifically binds to a member of the TCR complex and optionally specifically binds to CD3, and
(ii) a secondary agent that specifically binds to a T cell co-stimulatory molecule
including
optionally, the co-stimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS;
The method of any of 1001, 1006-1009, 1011, 1013, 1014, 1016-1024 and 1032-1035 of the invention.
[Invention 1037]
the stimulating reagent
(i) a principal agent that specifically binds to a member of the TCR complex and optionally specifically binds to CD3, and
(ii) a secondary agent that specifically binds to a T cell co-stimulatory molecule
including
optionally, the co-stimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS;
The method of any of the inventions 1002-1035.
[Invention 1038]
The method of invention 1036 or 1037, wherein one or both of the primary and secondary agents comprises an antibody or antigen-binding fragment thereof.
[Invention 1039]
The method of any of inventions 1036-1038, wherein the primary and secondary agents comprise antibodies and, optionally, the stimulating reagents comprise anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof.
[Invention 1040]
The method of any of Inventions 1037-1039, wherein the primary and secondary agents are present on or attached to the surface of a solid support.
[Invention 1041]
The method of the invention 1040, wherein the solid support is or comprises beads.
[Invention 1042]
The method of invention 1041, wherein the ratio of beads to cells is less than 3:1.
[Invention 1043]
A method of producing a composition of engineered T cells comprising:
(a) an input composition comprising primary T cells, (i) a principal agent that specifically binds to a member of the TCR complex and optionally to CD3; exposing to a stimulating reagent comprising beads to which are bound a secondary agent, wherein the bead to cell ratio is less than 3:1 and the exposure is under conditions for stimulating T cells. thereby generating a stimulated population;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and
(c) harvesting T cells of the transformed population, wherein harvesting is performed at 48 to 120 hours, inclusive, after exposure to said stimulating agent has begun.
including
A method thereby producing a composition of engineered cells.
[Invention 1044]
The method of inventions 1041-1043, wherein the ratio of beads to cells is 1:1 or about 1:1.
[Invention 1045]
The method of any of Inventions 1041-1044, wherein the beads have attached thereto anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof.
[Invention 1046]
The method of invention 1036, 1038 or 1039, wherein the primary and secondary agents are reversibly bound to the surface of oligomeric particle reagents comprising a plurality of streptavidin or streptavidin mutein molecules.
[Invention 1047]
A method of producing a composition of engineered T cells comprising:
(a) exposing an input composition comprising primary T cells to a stimulating reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, wherein the oligomeric particle reagent comprises (i) a TCR-conjugated A primary agent that specifically binds to a member of the body, optionally specifically to CD3, and (ii) a secondary agent that specifically binds to a T-cell costimulatory molecule are reversibly bound such that exposure is , performed under conditions for stimulating T cells, thereby generating a stimulated population;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and
(c) harvesting T cells of said transformed population, wherein harvesting is performed at 48 to 120 hours, inclusive, after exposure to said stimulating agent has begun; process
including
A method thereby producing a composition of engineered cells.
[Invention 1048]
The exposure in (a) is 10 cells in the input composition ⁶ The method of the invention 1001, 1046 or 1047 practiced with an amount of stimulating reagent that is between or about 0.1 μg and 20 μg per individual, inclusive.
[Invention 1049]
An input composition comprising T cells was added to 10 cells in the input composition. ⁶ exposing to a stimulation reagent in an amount of 0.1 μg to 20 μg or about 0.1 μg to 20 μg per individual, inclusive, wherein the stimulation reagent comprises a plurality of streptavidin or streptavidin mutein molecules. A step comprising a reagent, wherein the exposure is performed under conditions to stimulate T cells, thereby generating a stimulated population.
A method of stimulating T cells, comprising:
[Invention 1050]
An input composition comprising T cells was added to 10 cells in the input composition. ⁶ exposing to a stimulating reagent in an amount of 0.1 μg to 2 μg or about 0.1 μg to 2 μg per individual, inclusive, wherein the stimulating reagent (i) specifically binds to a member of the TCR complex; and optionally a primary agent that specifically binds to CD3 and (ii) a secondary agent that specifically binds to a T cell co-stimulatory molecule, comprising a plurality of streptavidin or streptavidin mutein molecules. wherein the exposure is performed under conditions to stimulate T cells, thereby generating a stimulated population
A method of stimulating T cells, comprising:
[Invention 1051]
The method of invention 1049 or 1050, wherein the input composition comprises primary T cells.
[Invention 1052]
The amount of stimulating reagent is 10 cells per input composition. ⁶ The methods of inventions 1048, 1049 and 1051 that are 0.1 μg to 2 μg per individual, or about 0.1 μg to 2 μg, inclusive.
[Invention 1053]
further comprising incubating the population of transformed cells for up to 96 hours after introduction and prior to harvesting;
optionally, the incubation is performed at a temperature of about 37°C,
The method of any of the inventions 1043-1048 and 1052.
[Invention 1054]
A method of the invention 1053, wherein incubation is carried out for up to 72 hours after introduction.
[Invention 1055]
The method of invention 1053, wherein incubation is carried out for up to 48 hours after introduction.
[Invention 1056]
The method of invention 1053, wherein incubation is carried out for up to 24 hours after introduction.
[Invention 1057]
The method of any of the inventions 1043-1048 and 1052-1056, wherein harvesting is performed within 96 hours after exposure of the input composition to the stimulant is initiated.
[Invention 1058]
The method of any of the inventions 1043-1048 and 1052-1057, wherein the harvesting is performed within 72 hours after exposure of the input composition to the stimulant is initiated.
[Invention 1059]
The method of any of the inventions 1043-1048 and 1052-1058, wherein the harvesting is performed within 48 hours after exposure of the input composition to the stimulant is initiated.
[Invention 1060]
The method of any of the inventions 1043-1048 and 1052-1059, wherein one or both of exposing in (a) and introducing in (b) are performed in the presence of one or more recombinant cytokines.
[Invention 1061]
The method of any of the inventions 1043-1048 and 1052-1059, wherein the exposing in (a) and the introducing in (b) are performed in the presence of one or more recombinant cytokines.
[Invention 1062]
The method of any of the inventions 1043-1048 and 1052-1061, wherein incubation is performed in the presence of one or more recombinant cytokines.
[Invention 1063]
The method of any of the invention 1043-1048 and 1052-1061, wherein incubation is performed in basal medium lacking one or more recombinant cytokines.
[Invention 1064]
A method of producing a composition of engineered T cells comprising:
(a) an input composition comprising primary T cells, (i) a principal agent that specifically binds to a member of the TCR complex and optionally to CD3; exposing to a stimulating reagent comprising beads to which are selectively bound secondary agents, wherein the ratio of beads to cells is less than 3:1, and the exposure is to one or more recombinant cytokines. performed under conditions for stimulating T cells, including the presence thereof, thereby generating a stimulated population;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells, wherein the introduction comprises one or more sets of a step performed in the presence of a replacement cytokine;
(c) incubating the population of transformed cells for up to 96 hours, optionally the incubation being performed at a temperature of about 37° C., the incubation being in basal medium lacking one or more recombinant cytokines; a step performed in; and
(d) harvesting the T cells of the transformed population after said incubation, thereby producing a composition of engineered cells, said harvesting being initiated after exposure to said stimulating agent; Process performed at 48 to 120 hours (inclusive)
including
A method thereby producing a composition of engineered cells.
[Invention 1065]
A method of producing a composition of engineered T cells comprising:
(a) exposing an input composition comprising primary T cells to a stimulating reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, wherein the oligomeric particle reagent comprises (i) a TCR-conjugated a primary agent that specifically binds to a member of the body and optionally CD3, and (ii) a secondary agent that specifically binds to a T cell co-stimulatory molecule, wherein the exposure is one or performed under conditions for stimulating T cells, including the presence of a plurality of recombinant cytokines, thereby generating a stimulated population;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells, wherein the introduction comprises one or more sets of a step performed in the presence of a replacement cytokine;
(c) incubating the population of transformed cells for up to 96 hours, optionally the incubation being performed at a temperature of about 37° C., the incubation being in basal medium lacking one or more recombinant cytokines; a step performed in; and
(d) harvesting the T cells of the transformed population after said incubation, thereby producing a composition of engineered cells, said harvesting being initiated after exposure to said stimulating agent; Process performed at 48 to 120 hours (inclusive)
including
A method thereby producing a composition of engineered cells.
[Invention 1066]
The amount of stimulating reagent is 10 cells per input composition. ⁶ The method of any of the Inventions 1048-1063 and 1065 which is 0.4 μg to 8 μg or about 0.4 μg to 8 μg per individual, inclusive.
[Invention 1067]
The amount of stimulating reagent is 10 cells per input composition. ⁶ The method of any of the Inventions 1048-1063, 1065 and 1066 which is 0.8 μg to 4 μg or about 0.8 μg to 4 μg per individual, inclusive.
[Invention 1068]
The amount of stimulating reagent is 10 cells per input composition. ⁶ The method of any of inventions 1048-1063 and 1065-1067 that is at or about 0.8 μg per individual.
[Invention 1069]
The amount of stimulating reagent is 10 ⁶ The method of any of inventions 1048-1063 and 1065-1068 that is at or about 1.2 μg per individual.
[Invention 1070]
The amount of stimulating reagent is 10 ⁶ The method of any of inventions 1048-1063 and 1065-1068 that is at or about 1.8 μg per individual.
[Invention 1071]
The method of any of the inventions 1060-1070, wherein the one or more recombinant cytokines is human.
[Invention 1072]
the one or more recombinant cytokines is recombinant IL-2, recombinant IL-7, and/or recombinant IL-15, optionally 10-200 IU/mL recombinant IL-2, 100 IU/mL- The method of any of the inventions 1060-1071 selected from 1,000 IU/mL recombinant IL-7 and/or 10-200 IU/mL recombinant IL-15.
[Invention 1073]
the one or more recombinant cytokines is recombinant IL-2, recombinant IL-7, and recombinant IL-15, optionally 10-200 IU/mL recombinant IL-2, 100 IU/mL-1,000 IU The method of any of the inventions 1060-1072, which is or comprises 10-200 IU/mL of recombinant IL-7/mL and 10-200 IU/mL of recombinant IL-15.
[Invention 1074]
The method of any of the inventions 1001-1048 and 1052-1073, wherein one or both of the exposing in (a) and the introducing in (b) are performed in serum-free medium.
[Invention 1075]
The method of any of the inventions 1001-1048 and 1052-1074, wherein the exposing in (a) and the introducing in (b) are performed in serum-free medium.
[Invention 1076]
The method of any of the invention 1001, 1015-1048 and 1052-1075, wherein the incubation is performed in serum-free medium.
[Invention 1077]
The method of any of the invention 1041-1045, 1053-1064 and 1071-1076, wherein the ratio of beads to cells is 2:1 to 0.5:1 or about 2:1 to 0.5:1.
[Invention 1078]
The method of any of the inventions 1041-1045, 1053-1064 and 1071-1077, wherein the ratio of beads to cells is 1:1 or about 1:1.
[Invention 1079]
The present invention 1041-1045, 1053-, wherein the beads comprise a diameter greater than 3.5 μm or greater than about 3.5 μm but less than or equal to about 9 μm or less than or equal to about 8 μm or less than or equal to about 7 μm or less than or equal to about 6 μm or less than or equal to about 5 μm Any method from 1064 and 1071-1078.
[Invention 1080]
The method of any of the inventions 1041-1045, 1053-1064 and 1071-1079, wherein the beads comprise a diameter of 4.5 μm or about 4.5 μm.
[Invention 1081]
The method of any of the inventions 1041-1045, 1053-1064 and 1071-1080, wherein the beads are inert.
[Invention 1082]
The method of any of the inventions 1041-1045, 1053-1064 and 1071-1081, wherein the bead is or comprises a polystyrene surface.
[Invention 1083]
The method of any of the inventions 1041-1045, 1053-1064 and 1071-1082, wherein the beads are paramagnetic or superparamagnetic.
[Invention 1084]
exposing the cells to a magnetic field either after or during incubation, thereby removing the stimulating reagent from the cells
prior to harvesting.
[Invention 1085]
Streptavidin or streptavidin mutein molecules bind or are capable of binding biotin, avidin, biotin analogs or muteins, avidin analogs or muteins, and/or biologically active fragments thereof. The method of any of Inventions 1001, 1046-1063 and 1065-1076.
[Invention 1086]
Each of the plurality of streptavidin mutein molecules has the amino acid sequence Va1 at sequence positions corresponding to positions 44-47 relative to positions in streptavidin in the sequence of amino acids set forth in SEQ ID NO: 61. ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ or Ile ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ The method of any of the inventions 1001, 1046-1063, 1065-1076 and 1085, comprising:
[Invention 1087]
Each of the plurality of streptavidin mutein molecules
(a) a sequence of amino acids set forth in any of SEQ ID NO: 62, 63, 68, 75-77, or 80-83;
(b) SEQ ID NO: 62, 63, 68, 75-77, or 80-83 with at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity and Va1 ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ or Ile ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ and/or reversibly binds to biotin, a biotin analogue, or a streptavidin-binding peptide; or
(c) a functional fragment of (a) or (b) that reversibly binds to biotin, a biotin analogue, or a streptavidin-binding peptide;
The method of any of the inventions 1001, 1046-1063, 1065-1076, 1085 and 1086, comprising:
[Invention 1088]
The method of any of the inventions 1001, 1046-1063, 1065-1076 and 1085-1087, wherein the plurality of streptavidin mutein molecules comprises the sequence of amino acids set forth in SEQ ID NO: 63 or 68.
[Invention 1089]
The method of any of the inventions 1046-1063, 1065-1076 and 1085-1088, wherein the primary and secondary agents each comprise a streptavidin-binding peptide.
[Invention 1090]
A streptavidin-binding peptide

A method of the present invention 1089 selected from the group consisting of:
[Invention 1091]
The method of any of the inventions 1046-1063, 1065-1076 and 1085-1090, wherein one or both of the main and secondary agents comprise an antibody or antigen-binding fragment thereof.
[Invention 1092]
The method of invention 1091, wherein one or more of said agents is or comprises a monovalent antibody fragment.
[Invention 1093]
The method of invention 1091 or 1092, wherein one or both of the primary and secondary agents is or comprises Fab.
[Invention 1094]
The oligomeric particle reagent is
a radius greater than 60 nm, greater than 70 nm, greater than 80 nm, or greater than 90 nm;
A radius of 50 nm to 150 nm, 75 nm to 125 nm, 80 nm to 115 nm, or 90 nm to 110 nm, inclusive; or
Radius of 90nm±15nm or 95nm±20~25nm
The method of any of the inventions 1001, 1046-1063, 1065-1076 and 1085-1093, comprising:
[Invention 1095]
The oligomeric particle reagent is
at least 5×10 ⁷ g/mol or at least 1 x 10 ⁸ g/mol; and/or
5×10 ⁷ g/mol to 5×10 ⁸ g/mol, 1×10 ⁸ g/mol to 5×10 ⁸ g/mol, or 1 x 10 ⁸ g/mol to 2×10 ⁸ g/mol
The method of any of the present invention 1001, 1046-1063, 1065-1076 and 1085-1094 comprising a molecular weight of
[Invention 1096]
The oligomeric particle reagent is
at least 500 streptavidin or streptavidin mutein tetramers, at least 1,000 streptavidin or streptavidin mutein tetramers, at least 1,500 streptavidin or streptavidin mutein tetramers, or at least 2,000 streptavidin or streptavidin mutein tetramers monomer; and/or
1,000-20,000 streptavidin or streptavidin mutein tetramers, 1,000-10,000 streptavidin or streptavidin mutein tetramers, or 2,000-5,000 streptavidin or streptavidin mutein tetramers
The method of any of the inventions 1001, 1046-1063, 1065-1076 and 1085-1088, comprising:
[Invention 1097]
further comprising adding a substance to the cells either after or during at least a portion of the incubation;
the substance terminates or reduces stimulation of the T cell by the stimulating agent;
The method of any of the inventions 1001, 1046-1063, 1065-1076 and 1085-1096.
[Invention 1098]
further comprising adding a substance to the cells either after or during at least a portion of the incubation;
the substance is capable of breaking the bond between the primary and secondary agents and the oligomeric particle reagent;
The method of any of the inventions 1046-1063, 1065-1076 and 1085-1097.
[Invention 1099]
The method of invention 1098, wherein said substance is a free binding partner and/or a competing reagent.
[Invention 1100]
The method of invention 1098 or 1099, wherein the presence of said substance terminates or reduces signals induced or modulated by the primary and secondary agents in T cells.
[Invention 1101]
The method of any of inventions 1097-1100, wherein said substance is or comprises a streptavidin binding peptide, biotin or biologically active fragment, or biotin analogue or biologically active fragment. .
[Invention 1102]
The substance is or comprises a streptavidin-binding peptide, wherein the streptavidin-binding peptide is

The method of the present invention 1101 selected from the group consisting of:
[Invention 1103]
The method of invention 1101, wherein said substance is or comprises biotin or a biologically active fragment or a biotin analogue or biologically active fragment.
[Invention 1104]
The method of any of Inventions 1097-1103, wherein the agent is added 42 hours to 120 hours, or about 42 hours to 120 hours, inclusive, after exposure to the oligomeric particle stimulating reagent is initiated.
[Invention 1105]
The method of any of Inventions 1097-1104, wherein the substance is added at or about 72 hours to 96 hours after exposure to the oligomeric particle reagent is initiated.
[Invention 1106]
The method of any of Inventions 1097-1105, wherein the substance is added at or about 48 hours after exposure to the oligomeric particle reagent is initiated.
[Invention 1107]
The method of any of Inventions 1097-1106, wherein the agent is added at or about 72 hours after exposure to the oligomeric particle reagent is initiated.
[Invention 1108]
The method of any of Inventions 1097-1107, wherein the agent is added at or about 96 hours after exposure to the oligomeric particle reagent is initiated.
[Invention 1109]
The method of any of Inventions 1097-1108, wherein said substance is added after incubation and before harvesting.
[Invention 1110]
1109. The method of any of inventions 1097-1109, wherein the substance is added during at least a portion of the incubation, and the incubation continues after the substance is added.
[Invention 1111]
A method of producing a composition of engineered T cells comprising:
(a) an input composition comprising primary T cells comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules; ⁶ exposing the oligomeric particle reagent to 0.2 μg to 8 μg or about 0.2 μg to 8 μg of stimulating reagent, wherein the oligomeric particle reagent comprises (i) a principal agent that specifically binds CD3; A binding adjuvant is attached and the exposure is performed in the presence of serum-free medium with recombinant IL-2, IL-7, and IL-15, thereby generating a stimulated population. , process;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells, wherein the introducing comprises recombinant IL-2 , IL-7, and IL-15, performed in the presence of serum-free medium;
(c) incubating the population of transformed cells under static conditions for 24 hours to 96 hours, optionally the incubation at a temperature of about 37° C. and during at least a portion of the incubation; a step carried out while adding a substance comprising biotin or a biologically active fragment thereof, optionally D-biotin or a biotin analogue or a biologically active fragment thereof; and
(d) harvesting the T cells of the transformed population after said incubation, thereby producing a composition of engineered cells, said harvesting being initiated after exposure to said stimulating agent; Process performed at 72 to 96 hours (inclusive)
including
A method thereby producing a composition of engineered cells.
[Invention 1112]
Input composition is at least 300 x 10 ⁶ The method of any of inventions 1001-1111, comprising viable primary T cells.
[Invention 1113]
Input composition is 600×10 ⁶ pcs or about 600 x 10 ⁶ The method of any of inventions 1001-1112, comprising viable primary T cells.
[Invention 1114]
at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least Or the method of any of inventions 1001-1113, wherein at least about 98%, at least or at least about 99%, about 100% or 100% are CD3+ T cells.
[Invention 1115]
at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least Or the method of any of inventions 1001-1114, wherein at least about 98%, at least or at least about 99%, about 100% or 100% are CD4+ T cells and CD8+ T cells.
[Invention 1116]
1115. The method of any of inventions 1001-1115, wherein the input composition comprises a ratio of CD4+ cells to CD8+ cells of 1.5:1 to 2.0:1.
[Invention 1117]
1001- of the invention, wherein the input composition comprises a ratio of CD4+ cells to CD8+ cells of 1.2:1 to 0.8:1, optionally a ratio of CD4+ T cells to CD8+ T cells of 1:1 or about 1:1. 1116 either way.
[Invention 1118]
A method of producing a composition of engineered T cells comprising:
(a) an input composition comprising primary T cells comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules; ⁶ 0.4 μg to 8 μg per individual, or about 0.4 μg to 8 μg, of a stimulating reagent comprising: (i) a principal agent that specifically binds CD3; Adheres to the secondary agent that binds,
The input composition comprises a ratio of CD4+ T cells to CD8+ T cells of 2:1 to 1:2 and contains at least 300×10 ⁶ CD4+ and CD8+ T cells, and the exposure is performed in the presence of serum-free medium containing recombinant IL-2, IL-7, and IL-15, thereby generating a stimulated population. , process;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells, wherein the introducing comprises recombinant IL-2 , IL-7, and IL-15, performed in the presence of serum-free medium;
(c) incubating the population of transformed cells under static conditions for 24 hours to 72 hours, or about 24 hours to 72 hours, optionally incubation is at a temperature of about 37°C and During at least a portion of the incubation is performed while adding a substance comprising biotin or a biologically active fragment thereof, optionally D-biotin or a biotin analogue or a biologically active fragment thereof, the addition being performed at 48-72 hours (inclusive) after exposure to the stimulating reagent is initiated; and
(d) harvesting the T cells of the transformed population after said incubation, thereby producing a composition of engineered cells, said harvesting being initiated after exposure to said stimulating agent; Process performed at 72 to 96 hours (inclusive)
including
A method thereby producing a composition of engineered cells.
[Invention 1119]
The method of any of Inventions 1111-1118, wherein the substance is added at or about 48 hours after exposure to the stimulating reagent is initiated.
[Invention 1120]
The method of any of Inventions 1111-1118, wherein the substance is added at or about 72 hours after exposure to the stimulating reagent is initiated.
[Invention 1121]
The method of any of Inventions 1111-1118, wherein the substance is added at or about 96 hours after exposure to the stimulating reagent is initiated.
[Invention 1122]
A method of producing a composition of engineered T cells comprising:
(a) exposing an input composition comprising primary T cells to a stimulating reagent comprising paramagnetic beads at a bead to cell ratio of 2:1 to 0.5:1 or about 2:1 to 0.5:1; wherein said beads are inert paramagnetic beads comprising a polystyrene coating and a diameter of 4.5 μm or about 4.5 μm, and are coated with (i) an anti-CD3 antibody or antigen-binding fragment thereof and an anti-CD28 antibody or antigen-binding thereof; fragments are attached and the input composition comprises a ratio of CD4+ T cells to CD8+ T cells of 2:1 to 1:2, ⁶ CD4+ and CD8+ T cells, and the exposure is performed in the presence of serum-free medium containing recombinant IL-2, IL-7, and IL-15, thereby generating a stimulated population. , process;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells, wherein the introducing comprises recombinant IL-2 , IL-7, and IL-15, performed in the presence of serum-free medium;
(c) incubating the population of transformed cells under static conditions for 48-72 hours inclusive, optionally wherein the incubation is performed at a temperature of about 37°C; ;
(d) after said incubation, exposing said cells to a magnetic field to remove said stimulating reagent from said cells and then harvesting said T cells, thereby producing a composition of engineered cells; and the collection is performed at 72 to 96 hours (inclusive) after exposure to the stimulating reagent is initiated.
including
A method thereby producing a composition of engineered cells.
[Invention 1123]
The method of any of inventions 1111-1122, wherein at least a portion of the incubation is performed in the presence of serum-free medium containing recombinant IL-2, IL-7 and IL-15.
[Invention 1124]
The method of any of Inventions 1111-1123, wherein incubation is performed in basal medium lacking recombinant cytokines.
[Invention 1125]
The input composition is 450 x 10 ⁶ pcs or about 450 x 10 ⁶ viable primary T cells, 500 x 10 ⁶ pcs or about 500 x 10 ⁶ viable primary T cells, 550 x 10 ⁶ pcs or about 550 x 10 ⁶ viable primary T cells, 600 x 10 ⁶ pcs or about 600 x 10 ⁶ viable primary T cells, 700 x 10 ⁶ pcs or about 700 x 10 ⁶ viable primary T cells, 800 x 10 ⁶ pcs or about 800 x 10 ⁶ viable primary T cells, 900 x 10 ⁶ pcs or about 900 x 10 ⁶ viable primary T cells, or 1,000 x 10 ⁶ pcs or about 1,000 x 10 ⁶ The method of any of the inventions 1001-1124, comprising viable primary T cells, or any value between any of the foregoing.
[Invention 1126]
Input composition is at least 600 x 10 ⁶ or at least about 600 x 10 ⁶ The method of any of the inventions 1001-1125, comprising viable primary T cells.
[Invention 1127]
Input composition is 900×10 ⁶ pcs or max 900×10 ⁶ The method of any of the inventions 1001-1126, comprising viable primary T cells.
[Invention 1128]
Input composition is 900×10 ⁶ pcs or about 900 x 10 ⁶ The method of any of the inventions 1001-1125, comprising viable primary T cells.
[Invention 1129]
The method of any of the inventions 1044-1059, 1061-1069, 1081-1113, 1115 and 1116, wherein the incubation is performed 24 hours or about 24 hours after introduction.
[Invention 1130]
The method of any of the inventions 1001 and 1015-1129, wherein incubation is performed for 48 hours or about 48 hours after introduction.
[Invention 1131]
The method of any of the inventions 1001 and 1015-1130, wherein incubation is performed for 72 hours or about 72 hours after introduction.
[Invention 1132]
Any of inventions 1001-1131, comprising enriching CD3+ T cells or CD4+ T cells and/or CD8+ T cells from the biological sample to produce an input composition prior to exposure to the stimulating reagent. method.
[Invention 1133]
1133. The method of any of inventions 1001-1132, comprising enriching CD3+ T cells from the biological sample to produce an input composition prior to exposure to the stimulating reagent.
[Invention 1134]
The method of invention 1132 or 1133, wherein the enrichment is by immunoaffinity-based selection.
[Invention 1135]
Immunoaffinity-based selection is performed by contacting the cells with an antibody immobilized on or attached to an affinity chromatography matrix, which antibody specifically binds to a cell surface marker to identify CD3+ cells. The method of the invention 1134, wherein the positive selection of
[Invention 1136]
1133. The method of any of inventions 1001-1132, comprising enriching CD4+ or CD8+ T cells from the biological sample to produce an input composition prior to exposure to the stimulating reagent.
[Invention 1137]
concentration, but
(a) performing a first selection in a closed system, said first selection enriching one of (i) CD4+ cells and (ii) CD8+ cells from a sample comprising primary human T cells; wherein said enrichment produces a first selected population and an unselected population; and
(b) performing a second selection in a closed system, said second selection comprising enrichment of the other of (i) CD4+ cells and (ii) CD8+ cells from said unselected population; wherein said enrichment produces a second selected population, said enrichment producing separate enriched populations of CD4+ T cells and CD8+ T cells.
including
each of said separate enriched populations comprises cells from one of said first selected population or said second selected population;
the input composition comprises cells from one or more of an enriched population of CD4+ T cells and an enriched population of CD8+ T cells;
The method of the invention 1136.
[Invention 1138]
The method of invention 1136 or 1137, wherein separate enriched populations of CD4+ T cells and CD8+ T cells are mixed thereby producing an input composition.
[Invention 1139]
1138. The method of invention 1138, wherein the mixing is performed in a closed system, optionally wherein said closed system is automated.
[Invention 1140]
The method of any of inventions 1136-1139, wherein enriching cells in the first and/or second selection comprises performing positive or negative selection based on expression of a cell surface marker.
[Invention 1141]
1136- of the invention, wherein the enrichment of cells in the first or second selection comprises performing multiple positive or negative selection steps based on expression of a cell surface marker or a marker to enrich for CD4+ or CD8+ cells. 1140 either way.
[Invention 1142]
The method of any of inventions 1136-1141, wherein enriching cells in the first and/or second selection comprises immunoaffinity-based selection.
[Invention 1143]
The method of invention 1142, wherein the enrichment of cells in the first and second selections comprises immunoaffinity-based selections.
[Invention 1144]
Immunoaffinity-based selection is performed by contacting the cells with antibodies immobilized on or attached to an affinity chromatography matrix, which antibodies specifically bind to cell surface markers, CD4+ or The method of invention 1142 or 1143, wherein positive or negative selection of CD8+ cells can be performed.
[Invention 1145]
the antibody further comprising one or more binding partners capable of forming a reversible bond with a binding reagent immobilized on a matrix, wherein the antibody reversibly binds to the chromatography matrix during contact;
Cells expressing a cell surface marker on the matrix to which the antibody specifically binds can be recovered from the matrix by cleavage of the reversible bond between the binding reagent and the binding partner.
The method of the invention 1135 or 1144.
[Invention 1146]
the binding partner is selected from among biotin, biotin analogues, and peptides capable of binding to the binding reagent; and
said binding reagent is selected from among streptavidin, streptavidin analogues or muteins, avidin and avidin analogues or muteins;
The method of the invention 1145.
[Invention 1147]
the binding partner comprises the sequence of amino acids set forth in SEQ ID NO:64; and/or
the binding reagent is a streptavidin mutein comprising a sequence of amino acids set forth in SEQ ID NO: 75, 80, 76 or 63;
The method of the invention 1145 or 1146.
[Invention 1148]
After contacting the cells in the sample with the affinity chromatography matrix, adding a competing reagent to cleave the bond between the binding partner and the binding reagent, thereby recovering the selected cells from the matrix. The method of any of the inventions 1145-1147, comprising:
[Invention 1149]
The method of invention 1148, wherein the competing reagent is biotin or a biotin analogue.
[Invention 1150]
One or more antibodies in one or more selections, 3 x 10 ^-Five sec ^-1 greater than or about 3 x 10 ^-Five sec ^-1 dissociation rate constants (k _off ) of any of the present invention 1135 and 1144-1149.
[Invention 1151]
One or more antibodies in one or more selections are about 10 ^-3 ～10 ^-7 range or about 10 ^-7 ~ about 10 ^-Ten dissociation constant (K _d ).
[Invention 1152]
The method of any of Inventions 1135 and 1144-1151, wherein the chromatographic matrix is packed in a separation vessel that is a column.
[Invention 1153]
enrichment of CD4+ or CD8+ T cells from a biological sample
(a) enrichment of CD4+ cells involves positive selection based on surface expression of CD4;
(b) enrichment of CD8+ cells comprises positive selection based on surface expression of CD8; or
both (a) and (b)
The method of any of the inventions 1136-1152, comprising:
[Invention 1154]
a step of enriching the CD4+ or CD8+ T cells from the biological sample prior to exposure to the stimulating reagent, wherein the enrichment renders the cells of the sample specifically binding CD4 in the incubation composition; an immunoaffinity reagent and a second immunoaffinity reagent that specifically binds to CD8 in the incubation composition, and said immunoaffinity reagent specifically for CD4 and CD8 molecules, respectively, on the surface of cells in said sample; contacting under binding conditions; and
collecting cells bound to said first and/or second immunoaffinity reagent, thereby producing an enriched composition comprising CD4+ and CD8+ cells;
including
the input composition comprises cells of an enriched composition comprising CD4+ cells and CD8+ cells;
The method of any one of Inventions 1001-1153.
[Invention 1155]
The method of invention 1154, wherein each immunoaffinity reagent comprises an antibody or antigen-binding fragment thereof.
[Invention 1156]
The method of invention 1154 or 1155, wherein the immunoaffinity reagent is immobilized on the outer surface of the bead.
[Invention 1157]
The method of invention 1156, wherein the beads are magnetic beads.
[Invention 1158]
1157. The method of invention 1157, wherein recovering cells bound to the first or second immunoaffinity reagent comprises exposing the cells to a magnetic field.
[Invention 1159]
The method of any of the inventions 1132-1158, wherein the biological sample comprises primary T cells obtained from a subject, optionally a human subject.
[Invention 1160]
the biological sample is or is a whole blood sample, buffy coat sample, peripheral blood mononuclear cell (PBMC) sample, unfractionated T cell sample, lymphocyte sample, white blood cell sample, apheresis product, or leukoapheresis product The method of any of the inventions 1132-1159, comprising:
[Invention 1161]
The method of any of the inventions 1132-1160, wherein the biological sample is a pre-cryofrozen apheresis or leukapheresis product prior to concentration.
[Invention 1162]
The method of any of the inventions 1001-1048 and 1052-1161, wherein harvesting is performed at or about 96 hours after exposure to the stimulating agent is initiated.
[Invention 1163]
The method of any of the inventions 1001-1048 and 1052-1161, wherein harvesting is performed 2 days or about 2 days or 3 days or about 3 days after exposure to the stimulating agent is initiated.
[Invention 1164]
The method of any of the inventions 1001-1048 and 1052-1161, wherein harvesting is performed at or about 72 hours after exposure to the stimulating agent is initiated.
[Invention 1165]
The method of any of the inventions 1001-1048 and 1052-1161, wherein harvesting is performed at or about 48 hours after exposure to the stimulating agent is initiated.
[Invention 1166]
The method of any of the inventions 1001 and 1012-1165, wherein harvesting is performed when the integrated vector is detected in the genome, but before achieving a stable iVCN per diploid genome.
[Invention 1167]
The method of any of Inventions 1001 and 1012-1166, wherein harvesting is performed when the fraction of integrated vector copy number (iVCN) to total VCN in the population of transformed cells is on average less than 0.8. .
[Invention 1168]
The method of any of the inventions 1001 and 1012-1167, wherein harvesting is performed when the population of transformed cells has an average of less than or about 2.0 copies of iVCN per diploid genome.
[Invention 1169]
The method of any of inventions 1001-1168, wherein harvesting the cells comprises removing cell debris by rinsing or washing the cells.
[Invention 1170]
the percentage of naive-like cells is greater than 60% among all T cells in the population, all CD4+ T cells in said population, or all CD8+ T cells in said population or recombinant protein-expressing cells thereof, or The method of any of inventions 1001 and 1030-1169, wherein the cells are harvested at greater than about 60%.
[Invention 1171]
The percentage of naive-like cells and/or central memory T cells is among all T cells in the population, all CD4+ T cells in said population, or all CD8+ T cells in said population or cells expressing recombinant protein thereof 1001 and 1030-1169 of any of the inventions 1001 and 1030-1169, wherein the cells are harvested at greater than 60% or greater than about 60% at .
[Invention 1172]
at the time of collection,
the percentage of naive-like T cells is greater than or greater than about 60% among all recombinant protein-expressing cells in the population, optionally among all recombinant protein-expressing T cells in said population greater than 65%, 70%, 80%, 90%, or 95%, or greater than about 65%, 70%, 80%, 90%, or 95%;
the percentage of naive-like T cells is greater than or greater than about 60% of all recombinant protein-expressing CD4+ T cells in said population, optionally all recombinant protein-expressing CD4+ T cells in said population greater than 65%, 70%, 80%, 90%, or 95% of cells, or greater than about 65%, 70%, 80%, 90%, or 95%; or
the percentage of naive-like T cells is greater than or greater than about 60% of all recombinant protein-expressing CD8+ T cells in said population, optionally all recombinant protein-expressing CD8+ T cells in said population greater than 65%, 70%, 80%, 90%, or 95% of cells, or greater than about 65%, 70%, 80%, 90%, or 95%;
The method of the invention 1170 or 1171.
[Invention 1173]
at the time of collection,
the percentage of naive-like T cells and/or central memory T cells is greater than or greater than about 60% of all recombinant protein-expressing cells in the population, optionally all recombinants in the population greater than 65%, 70%, 80%, 90%, or 95%, or greater than about 65%, 70%, 80%, 90%, or 95% of protein-expressing T cells;
the percentage of naive-like T cells and/or central memory T cells is greater than or greater than about 60% of all recombinant protein-expressing CD4+ T cells in said population, optionally in said population greater than 65%, 70%, 80%, 90%, or 95% of all recombinant protein-expressing CD4+ cells, or greater than about 65%, 70%, 80%, 90%, or 95% ;or
the percentage of naive-like T cells and/or central memory T cells is greater than or greater than about 60% among all recombinant protein-expressing CD8+ T cells in said population, optionally in said population of all recombinant protein-expressing CD8+ cells, or greater than about 65%, 70%, 80%, 90%, or 95% ,
The method of the invention 1171.
[Invention 1174]
The method of any of the inventions 1170-1173, wherein the naive-like T cells or T cells that are surface positive for markers expressed on naive-like T cells are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+.
[Invention 1175]
The method of any of inventions 1170-1174, wherein the naive-like T cells comprise CD27+CCR7+ T cells.
[Invention 1176]
The method of any of inventions 1170-1175, wherein the naive-like T cells comprise CCR7+CD45RA+ T cells.
[Invention 1177]
The present invention 1001 further comprising formulating the cells of the composition of engineered cells for cryopreservation and/or administration to a subject, optionally in the presence of a pharmaceutically acceptable excipient. Any method from ~1176.
[Invention 1178]
The method of invention 1177, wherein the cells of the composition of engineered cells are formulated in the presence of a cryoprotectant.
[Invention 1179]
The method of invention 1178, wherein the cryoprotectant comprises DMSO.
[Invention 1180]
The method of any of inventions 1177-1179, wherein the cells of the composition of engineered cells are formulated in a container, optionally a vial or bag.
[Invention 1181]
The method of any of inventions 1001-1180, wherein a viral vector containing a heterologous polynucleotide encoding a recombinant protein is introduced into the T cell.
[Invention 1182]
The method of Invention 1181, wherein the viral vector is a retroviral vector.
[Invention 1183]
The method of invention 1180 or 1181, wherein the viral vector is a lentiviral or gammaretroviral vector.
[Invention 1184]
The method of any of inventions 1001-1183, wherein the introduction is performed in the presence of a transduction adjuvant.
[Invention 1185]
1184. The method of the invention 1184, wherein the transduction adjuvant is protamine sulfate, optionally 1 μg/ml to 50 μg/ml or about 1 μg/ml to 50 μg/ml protamine sulfate, fibronectin-derived transduction adjuvant or RetroNectin.
[Invention 1186]
the recombinant protein
A recombinant receptor capable of binding a target antigen that is associated with, specific for, and/or expressed on a disease, disorder, or diseased cell or tissue
The method of any one of Inventions 1001-1185, wherein
[Invention 1187]
The method of invention 1186, wherein the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer.
[Invention 1188]
The method of invention 1187, wherein the target antigen is a tumor antigen.
[Invention 1189]
Target antigen is αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer testis antigen, cancer testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII) , epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp10O), glypican-3 (GPC3), G protein-coupled receptor class C group 5 member D (GPRC5D), Her2/neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer body, human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22 receptor alpha) 22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, containing leucine-rich repeats 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, mouse cytomegalovirus (CMV), Mucin 1 (MUC1), MUC16, Natural Killer Group 2 member D (NKG2D) ligand, Melan A (MART -1), nerve cell adhesion molecule (NCAM), carcinoembryonic antigen, melanoma preferentially expressed antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor somatic tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein (TPBG, aka 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1, aka TYRP1 or gp75), tyrosinase-related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor 1 (WT-1), The present invention 1186 selected among pathogen-specific or pathogen-expressed antigens, or antigens associated with universal tags, and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV, or other pathogens Any method from ~1188.
[Invention 1190]
The method of any of the inventions 1001-1048 or 1052-1189, wherein the recombinant protein is or comprises a functional non-TCR antigen receptor or TCR or antigen-binding fragment thereof.
[Invention 1191]
The method of any of inventions 1001-1048 or 1052-1190, wherein the recombinant protein is a chimeric antigen receptor (CAR).
[Invention 1192]
serum-free medium
dipeptide form of L-glutamine at 0.5 mM to 5 mM in basal medium;
0.5 mM to 5 mM L-glutamine; and
at least one protein
and is serum-free.
[Invention 1193]
Any of the inventions 1001 and 1026-1192, wherein the basal medium lacking one or more recombinant cytokines comprises L-glutamine, optionally at a concentration of about 0.5mM to about 5mM, optionally at a concentration of about 2mM. the method of.
[Invention 1194]
The method of any of the inventions 1001 and 1026-1191 and 1193, wherein the basal medium lacking one or more recombinant cytokines is serum-free.
[Invention 1195]
The invention 1001, 1026- wherein the basal medium comprises at least one protein selected from one or more of albumin, insulin or transferrin, optionally human or recombinant albumin, insulin or transferrin Any method of 1191 and 1193-1194.
[Invention 1196]
A therapeutic T cell composition produced by the method of any of the inventions 1001-1195.
[Invention 1197]
At least 50%, 60%, 70%, 80% of the total number of T cells in a composition or the total number of T cells expressing a recombinant protein in said composition, on average in a plurality of compositions produced by said method %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least about 50%, 60%, 70% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or 50%, 60%, 70% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or about 50%, 60%, 70% %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are naive-like T cells , or T cells that are surface positive for markers expressed on naive-like cells.
[Invention 1198]
at least 50% or at least about 50% or 50% of the total number of T cells in a composition or the total number of T cells expressing a recombinant protein in said composition, on average in a plurality of compositions produced by said method Or the therapeutic T cell composition of invention 1196, wherein about 50% are central memory T cells or T cells that are surface positive for markers expressed on central memory T cells.
[Invention 1199]
On average, at least 50% or at least about 50% or 50% or about 50% of the total number of T cells in the composition or the total number of T cells expressing the recombinant protein in said composition are naive-like T cells or The therapeutic T cell composition of invention 1196, which is a central memory T cell or a naive-like T cell or a T cell that is surface positive for markers expressed on central memory T cells.
[Invention 1200]
The therapeutic T cell composition of invention 1197 or 1199, wherein the marker expressed on naive-like T cells is selected from the group consisting of CD45RA, CD27, CD28 and CCR7.
[Invention 1201]
any of inventions 1197, 1199 and 1200, wherein the naive-like T cells or T cells that are surface positive for markers expressed on naive-like T cells are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+ A therapeutic T cell composition.
[Invention 1202]
Naive-like T cells, or T cells that are surface positive for markers expressed on naive-like T cells, comprise CD27+CCR7+ T cells, and all receptors in the composition ⁺ /CD8 ⁺ The therapeutic T cell composition of any of invention 1197 and 1199-1201, wherein at least 70%, 80%, 85%, or 90% of the cells are CD27+CCR7+.
[Invention 1203]
Naive-like T cells, or T cells that are surface positive for markers expressed on naive-like T cells, comprise CD27+CCR7+ T cells, and all receptors in the composition ⁺ /CD4 ⁺ The therapeutic T cell composition of any of inventions 1197 and 1199-1202, wherein at least 50%, 60%, 70%, 80%, 85%, or 90% of the cells are CD27+CCR7+.
[Invention 1204]
Naive-like T cells, or T cells that are surface positive for markers expressed on naive-like T cells, comprise CD27+CCR7+ T cells, and all receptors in the composition ⁺ /CD8 ⁺ at least 50%, 60%, 70%, 80%, 85%, or 90% of the cells are CD27+CCR7+ and all receptors in the composition ⁺ /CD4 ⁺ The therapeutic T cell composition of any of inventions 1197 and 1199-1203, wherein at least 50%, 60%, 70%, 80%, 85%, or 90% of the cells are CD27+CCR7+.
[Invention 1205]
A therapeutic T cell composition comprising CD4+ T cells expressing a recombinant receptor and CD8+ T cells expressing a recombinant receptor,
all receptors in the composition ⁺ /CD8 ⁺ at least 50%, 60%, 70%, 80%, or 90% of the cells are CD27+CCR7+ and all receptors in the composition ⁺ /CD4 ⁺ at least 50%, 60%, 70%, 80%, or 90% of the cells are CD27+CCR7+;
A therapeutic T cell composition.
[Invention 1206]
at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least Or the therapeutic T cell composition of any of inventions 1196-1205, wherein at least about 98%, at least or at least about 99%, about 100% or 100% are CD3+ T cells.
[Invention 1207]
at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least Or the therapeutic T cell composition of any of inventions 1196-1206, wherein at least about 98%, at least or at least about 99%, about 100% or 100% are CD4+ T cells and CD8+ T cells.
[Invention 1208]
At least or at least about 90% of the cells in said composition are CD4+ T cells and CD8+ T cells, and all receptors in said composition ⁺ /CD8 ⁺ at least or at least about 60% of the cells are CD27+CCR7+ and all receptors in the composition ⁺ /CD4 ⁺ The therapeutic T cell composition of any of inventions 1205-1207, wherein at least or at least about 60% of the cells are CD27+CCR7+.
[Invention 1209]
at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92 of the total number of T cells in said composition or the total number of T cells expressing a recombinant receptor in said composition %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least about 50%, 60%, 70%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or 50%, 60%, 70%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or about 50%, 60%, 70%, 80%, 85%, 90%, 91% %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the therapeutic T cells of any of invention 1205-1208 are CD27+CCR7+ Composition.
[Invention 1210]
The therapeutic T of any of inventions 1197-1209, wherein the ratio of receptor+/CD4+ T cells to receptor+/CD8+ T cells in said composition is from about 1:3 to about 3:1. cell composition.
[Invention 1211]
The therapeutic T of any of inventions 1196-1210, wherein the ratio of receptor+/CD4+ T cells to receptor+/CD8+ T cells in said composition is from about 1:2 to about 2:1. cell composition.
[Invention 1212]
The therapeutic T cell of any of inventions 1196-1211, wherein the ratio of receptor +/CD4+ T cells to receptor +/CD8+ T cells in said composition is 1:1 or about 1:1. Composition.
[Invention 1213]
the recombinant protein
A recombinant receptor capable of binding to a target protein associated with, specific for and/or expressed on a disease, disorder, or diseased cell or tissue
The therapeutic composition of any of the inventions 1196-1212 which is or comprises the same.
[Invention 1214]
The therapeutic composition of any of Inventions 1196-1213, wherein the recombinant protein is a chimeric antigen receptor (CAR).
[Invention 1215]
The number of viable T cells in said composition is 25 x 10 ⁶ pcs or about 25 x 10 ⁶ from 200 x 10 ⁶ pcs or about 200 x 10 ⁶ and optionally the number of viable T cells in said composition is 25 x 10 ⁶ pcs or about 25 x 10 ⁶ from 100 x 10 ⁶ pcs or about 100 x 10 ⁶ pcs, 25×10 ⁶ pcs or about 25 x 10 ⁶ from 70 x 10 ⁶ pcs or about 70 x 10 ⁶ pcs, 25×10 ⁶ pcs or about 25 x 10 ⁶ from 50 x 10 ⁶ pcs or about 50 x 10 ⁶ pcs, 50×10 ⁶ pcs or about 50 x 10 ⁶ from 200 x 10 ⁶ pcs or about 200 x 10 ⁶ pcs, 50×10 ⁶ pcs or about 50 x 10 ⁶ from 100 x 10 ⁶ pcs or about 100 x 10 ⁶ pcs, 50×10 ⁶ pcs or about 50 x 10 ⁶ from 70 x 10 ⁶ pcs or about 70 x 10 ⁶ pcs, 70×10 ⁶ pcs or about 70 x 10 ⁶ from 200 x 10 ⁶ pcs or about 200 x 10 ⁶ pcs, 70×10 ⁶ pcs or about 70 x 10 ⁶ from 100 x 10 ⁶ pcs or about 100 x 10 ⁶ pcs, or 100 x 10 ⁶ pcs or about 100 x 10 ⁶ from 200 x 10 ⁶ pcs or about 200 x 10 ⁶ (each inclusive).
[Invention 1216]
The amount of said composition is between 1.0 mL and 10 mL inclusive, optionally 2 mL or about 2 mL, 3 mL or about 3 mL, 4 mL or about 4 mL, 5 mL or about 5 mL, 6 mL or about 6 mL, 7 mL or about 7 mL. , 8 mL or about 8 mL, 9 mL or about 9 mL, or 10 mL or about 10 mL, or any value between any of the foregoing.
[Invention 1217]
An article of manufacture comprising the composition of any of the inventions 1196-1216 and instructions for administering the composition to a subject.
[Invention 1218]
The subject has a disease or disorder and optionally the recombinant receptor specifically recognizes or is specific to an antigen associated with or expressed or present on cells of said disease or disorder An article of manufacture of the present invention 1217 that binds.
[Invention 1219]
A method of treating a subject having or suspected of having a disease or condition comprising administering to said subject a dose of T cells from the composition of any of the inventions 1196-1216 ,Method.
[Invention 1220]
The dose of T cells is 1 x 10 ^Four pcs ~ 50 x 10 ⁶ pcs or about 1 x 10 ^Four pcs ~ 50 x 10 ⁶ The method of the invention 1219, comprising 10 T cells, inclusive.
[Invention 1221]
Said dose of T cells T cells are total T cells, total viable T cells, total viable recombinant receptor-expressing T cells, total viable recombinant receptor-expressing CD4+ T cells, or total viable recombinant receptor-expressing CD8 The method of invention 1219 or 1220, which is a +T cell.
[Invention 1222]
The method of any of inventions 1219-1221, wherein the disease or condition is cancer.
[Invention 1223]
The composition of any of inventions 1196-1216 for use in a method of treating a subject having a disease or condition, optionally wherein said disease or condition is cancer.
[Invention 1224]
Use of any of the compositions of inventions 1196-1216 for the manufacture of a medicament for treating a subject with a disease or condition, optionally wherein said disease or condition is cancer.
[Invention 1225]
the cells of said composition express a recombinant receptor that specifically recognizes or specifically binds to an antigen associated with, expressed or present on, a diseased or diseased cell; The composition for use of 1223 or the use of 1224 of the invention, wherein said recombinant receptor is a chimeric antigen receptor.

Claims (58)

A method for producing a composition of engineered T cells, the method comprising:
(a) exposing an input composition comprising primary T cells to a stimulating reagent under conditions for stimulating T cells, thereby generating a stimulated population;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells;
(c) incubating the population of transformed cells, wherein incubation is performed in basal medium lacking any recombinant cytokines; and
(d) harvesting T cells of said transformed population, thereby producing a composition of engineered cells;
A method, including said stimulating reagent comprises an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules ; and one or more intracellular signaling domains of one or more components of a TCR complex; or can activate one or more intracellular signaling domains of a plurality of co-stimulatory molecules , and
said sampling is performed at 48 to 120 hours (inclusive) after exposure to said stimulating reagent is initiated;
The method of Claim 1. said exposing and introducing is in the presence of one or more recombinant cytokines , and
said incubation is carried out for up to 96 hours,
3. The method of claim 1 or 2. A method for producing a composition of engineered T cells, the method comprising:
(a) exposing an input composition comprising primary T cells to a stimulating reagent under conditions for stimulating T cells, thereby generating a stimulated population;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and (c) transforming the T cells of the transformed population into a step of collecting, wherein the collecting is
(i) at 24 to 120 hours after exposure to the stimulating agent is initiated, inclusive;
(ii) once integrated vector is detected in the genome, but before achieving a stable integrated vector copy number per diploid genome (iVCN);
(iii) before the total number of viable cells at harvest is more than three times, two times, or about three times, about two times the number of total viable cells in the stimulated population, or is the same or about the same; and/or (iv) the percentage of naive-like T cells exceeds total T cells in said population, total CD3+ T cells in said population, total CD4+ T cells in said population, total T cells in said population, and/or at greater than or about 60% of the recombinant protein-expressing cells of CD8+ T cells, or any of those cells in said population,
A method thereby producing a composition of engineered cells. A method for producing a composition of engineered T cells, the method comprising:
(a) exposing an input composition comprising at least 300×10 ⁶ viable primary T cells to a stimulating reagent under conditions for stimulating the T cells, thereby generating a stimulated population; wherein the stimulatory reagent comprises one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more co-stimulatory molecules; capable of activating the
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and (c) transforming the T cells of the transformed population into a step of collecting, wherein the collecting is
(i) at 48 to 120 hours after exposure to the stimulating agent is initiated, inclusive;
(ii) once integrated vector is detected in the genome of said transformed population, but before achieving a stable integrated vector copy number per diploid genome (iVCN);
(iii) a time point before the total number of viable cells at harvest is 3-fold, 2-fold or about 3-fold, more than about 2-fold, or equal or approximately equal to the number of total viable cells in the stimulated population; and/or (iv) the percentage of naive-like T cells is total T cells in said population, total CD3+ cells in said population, total CD4+ T cells in said population, total CD8+ T cells in said population at greater than or about 60% of the recombinant protein-expressing cells, or any of those cells in said population;
A method thereby producing a composition of engineered cells. collection is
(a) at the time integrated vector is detected in the genome, but before achieving a stable integrated vector copy number per diploid genome ( iVCN ) ; A stable iVCN per genome is
(i) is achieved when the iVCN reaches a peak and remains unchanged for a period of more than 24 hours or changes only within a tolerance ; or
(ii) achieved when the fraction of iVCN to total vector copy number (VCN) in the diploid genome of the population of transformed cells is on average 1.0 or about 1.0, or is within an acceptable error thereof; ;
(b) when the fraction of iVCN to total VCN in the population of transformed cells is on average less than 0.8 ; and/or
(c) when the iVCN of the population of transformed cells averages less than or less than about 2.0 copies per diploid genome;
A method according to any one of claims 1-5 . A method for producing a composition of engineered T cells, the method comprising:
(a) exposing an input composition comprising primary T cells to a stimulating reagent under conditions for stimulating the T cells, thereby generating a stimulated population, wherein the stimulating reagent is a TCR capable of activating one or more intracellular signaling domains of one or more components of the complex and one or more intracellular signaling domains of one or more costimulatory molecules; process;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and (c) transforming the T cells of the transformed population into a step of harvesting, wherein harvesting is performed 48 to 120 hours (inclusive) after exposure to the stimulating agent is initiated, and at the time of harvesting the population of transformed cells contains integrated vector wherein the copy number (iVCN) averages between 0.4 and 2.0 copies per diploid genome, or between about 0.4 and 2.0 copies, inclusive;
A method thereby producing a composition of engineered cells. A method for producing a composition of engineered T cells, the method comprising:
(a) exposing an input composition comprising primary T cells to a stimulating reagent under conditions for stimulating the T cells, thereby generating a stimulated population, wherein the stimulating reagent is a TCR capable of activating one or more intracellular signaling domains of one or more components of the complex and one or more intracellular signaling domains of one or more costimulatory molecules; process;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and (c) transforming the T cells of the transformed population into a step of harvesting, wherein harvesting is performed at 48 to 120 hours (inclusive) after exposure to the stimulating agent is initiated, and naive-like cells and/or central memory T The percentage of cells is 60% among all T cells in said population, all CD4+ T cells in said population, all CD8+ T cells in said population, or cells expressing said recombinant protein in said population higher than or about 60% higher than
A method thereby producing a composition of engineered cells. the stimulating reagent
(i) a primary agent that specifically binds to a member of the TCR complex and optionally specifically binds to CD3; and (ii) a secondary agent that specifically binds to a T cell co-stimulatory molecule,
optionally, the co-stimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS ;
optionally, one or both of the primary and secondary agents comprise antibodies or antigen-binding fragments thereof ; optionally, the stimulating reagent comprises anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof ;
A method according to any one of claims 1-8 . 10. The method of claim 9 , wherein the primary and secondary agents are present on or attached to the surface of a solid support , and optionally the solid support is or comprises beads. A method for producing a composition of engineered T cells, the method comprising:
(a) an input composition comprising primary T cells, (i) a principal agent that specifically binds to a member of the TCR complex and optionally to CD3; exposing to a stimulating reagent comprising beads to which are bound a secondary agent, wherein the bead to cell ratio is less than 3:1 and the exposure is under conditions for stimulating T cells. thereby generating a stimulated population;
(b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and (c) transforming the T cells of the transformed population into collecting, wherein the collecting is performed at 48 to 120 hours, inclusive, after exposure to the stimulating reagent is initiated;
A method thereby producing a composition of engineered cells. said stimulating agent is an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, a primary agent and a secondary agent are reversibly bound to the surface of said oligomeric particle reagent; 10. The method of claim 9 , wherein the method is carried out using an amount of stimulating reagent that is, inclusive, between 0.1 μg and 20 μg per 10 ⁶ cells in the input composition. A method for producing a composition of engineered T cells, the method comprising:
(a) exposing an input composition comprising primary T cells to a stimulating reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, wherein the oligomeric particle reagent comprises (i) a TCR-conjugated A primary agent that specifically binds to a member of the body, optionally specifically to CD3, and (ii) a secondary agent that specifically binds to a T-cell costimulatory molecule are reversibly bound such that exposure is , performed under conditions for stimulating T cells, thereby generating a stimulated population;
(b) introducing a heterologous polynucleotide encoding a recombinant protein into T cells of said stimulated population, thereby generating a population of transformed cells; and (c) T cells of said transformed population. wherein the harvesting is performed at 48 to 120 hours, inclusive, after exposure to the stimulating reagent is initiated;
A method thereby producing a composition of engineered cells. A method for stimulating T cells, comprising an input composition comprising T cells , optionally primary T cells , in an amount of 0.1 μg to 20 μg or about 0.1 μg to 20 μg per 10 ⁶ cells in said input composition. (inclusive), wherein the stimulating reagent comprises (i) a principal agent that specifically binds to a member of the TCR complex and (ii) a T cell stimulatory molecule that specifically binds to comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules reversibly bound to binding secondary agents , wherein exposure is performed under conditions for stimulating T cells, thereby stimulating generating a modified population. A method for stimulating T cells, comprising an input composition comprising T cells , optionally primary T cells, in an amount of 0.1 μg to 2 μg or about 0.1 μg to 2 μg per 10 ⁶ cells in the input composition. (inclusive), wherein the stimulating agent (i) a principal agent that specifically binds to a member of the TCR complex and optionally specifically binds to CD3 and ( ii) comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules reversibly bound to secondary agents that specifically bind T cell co-stimulatory molecules, such that exposure stimulates T cells; thereby generating a stimulated population. After said introducing and prior to said harvesting, said method further comprises incubating the population of transformed cells, optionally
incubation is performed at a temperature of about 37°C;
the cell is more than about 1, 2, 3, 4, or 4 days, or at least more than 1, 2, 3, 4, or 4 days after introduction of the heterologous polynucleotide encoding the recombinant protein and/or
the incubation is carried out for up to or up to about 120 hours, or up to or up to about 108 hours,
A method according to any one of claims 1-13. one or both of said exposing and said introducing is performed in the presence of one or more recombinant cytokines , optionally said exposing and said introducing are performed in the presence of one or more recombinant cytokines The method of any one of claims 1-13 and 16 , wherein 18. The method of any one of claims 6 , 9, 10, 12, 16, and 17 , wherein incubation is performed in basal medium lacking any recombinant cytokine. 19. Any one of claims 1-3, 6, 9, 10, 12 and 16-18, wherein said incubation is performed for up to 96 hours , optionally incubation is performed at a temperature of about 37°C. method . a basal medium lacking any recombinant cytokines , comprising L-glutamine, optionally at a concentration of about 0.5 mM to about 5 mM, optionally at a concentration of about 2 mM ;
the basal medium lacking any recombinant cytokine is serum-free and/or
the basal medium lacking one or more recombinant cytokines comprises at least one protein selected from albumin, insulin, and transferrin , optionally human or one or more of recombinant albumin, insulin, and transferrin;
20. The method of any one of claims 1-3, 6, 9, 10, 12, and 16-19 . the one or more recombinant cytokines is recombinant IL-2, recombinant IL-7, and/or recombinant IL-15, optionally 10-200 IU/mL recombinant IL-2, 100 IU/mL- 3, selected from 1,000 IU/mL recombinant IL-7, and/or 10-200 IU/mL recombinant IL-15 , and optionally one or more recombinant cytokines are human ; The method of any one of 6, 9, 10, 12, and 16-20 . 22. The method of any one of claims 1-21, wherein one or more of exposing , introducing , and incubating is performed in serum-free medium. (a) a streptavidin or streptavidin mutein molecule binds or can bind to biotin, avidin, a biotin analog or mutein, an avidin analog or mutein, and/or biologically active fragments thereof; can ;
(b) each of the plurality of streptavidin mutein molecules comprising:
(i) the amino acid sequence Va1 ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ at sequence positions corresponding to positions 44-47 relative to the position in streptavidin in the sequence of amino acids set forth in SEQ ID NO: 61; or ^Ile44 - ^Gly45 - ^Ala46 - ^Arg47 ;
(ii) a sequence of amino acids set forth in any of SEQ ID NOs: 62, 63, 68, 75-77, and 80-83;
(iii) any of SEQ ID NOs: 62, 63, 68, 75-77, and 80-83 with at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92; %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity and streptavidin in the sequence of amino acids set forth in SEQ ID NO: 61 A biotin, biotin analogue, or streptavidin binding peptide comprising an amino acid sequence corresponding to Va1 ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ or Ile ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ on a position basis and/or A sequence of amino acids that reversibly binds to; and/or
(iv) a functional fragment of (ii) or (iii) that reversibly binds to biotin, a biotin analogue, or a streptavidin-binding peptide;
The method of any one of claims 2, 3 , 6, 9, and 12-22 . Claims that the primary and secondary agents each comprise a streptavidin-binding peptide, and optionally the streptavidin-binding peptide is an amino acid sequence set forth in any of SEQ ID NOs: 64-67, 70, 73, and 74 The method of any one of paragraphs 9-13 and 16-23. One or both of the primary and secondary agents comprises an antibody or antigen-binding fragment thereof , optionally one or both of the primary and secondary agents is or comprises a monovalent antibody fragment, optionally a Fab , claims 9-13 , and 16-24. The oligomeric particle reagent is
a radius of 50 nm to 150 nm, 75 nm to 125 nm, 80 nm to 115 nm, or 90 nm to 110 nm, inclusive;
5 x ¹⁰⁷ g/mol to 5 x ¹⁰⁸ g/mol, 1 x ¹⁰⁸ g/mol to 5 x ¹⁰⁸ g/mol, or 1 x ¹⁰⁸ g/mol to 2 x ¹⁰⁸ g/mol ; and /or
1,000 to 20,000 streptavidin or streptavidin mutein tetramers, 1,000 to 10,000 streptavidin or streptavidin mutein tetramers, or 2,000 to 5,000 streptavidin or streptavidin mutein tetramers. , 6, 9, and 12-25 . further comprising adding a substance to the cells either after or during at least a portion of the incubation;
the substance is capable of terminating or reducing stimulation of T cells by the stimulating reagent and/or breaking the bond between the primary and secondary agents and the oligomeric particle reagent;
optionally, the substance is or comprises a streptavidin-binding peptide, biotin or a biologically active fragment, or a biotin analogue or biologically active fragment ;
Optionally, the substance is or comprises a streptavidin-binding peptide, wherein the streptavidin-binding peptide is

selected from the group consisting of
27. The method of any one of claims 1-3, 6, 9, 10, 12, and 16-26 . The substance is added 42 hours to 120 hours, or about 42 hours to 120 hours, inclusive, after exposure to the oligomeric particle stimulating reagent is initiated ; 28. The method of claim 27 , added at or about 48 hours after exposure to is initiated. 29. The method of any one of claims 1-3, 6, 9, 10, 12, and 16-28, wherein said incubation is performed under static conditions, optionally in an incubator. The exposure is 0.2 μg to 8 μg per 10 ⁶ cells or about 0.2 μg to 8 μg , 0.4 μg to 8 μg or about 0.4 μg to 8 μg per 10 6 cells, or ^0.8 μg to 4 μg or about 0.8 μg per 10 ⁶ cells. μg to 4 μg of stimulation reagent , optionally with an amount of stimulation reagent of 0.8 μg or about 0.8 μg per 10 ⁶ cells, 1.2 μg or about 1.2 μg per 10 6 ^{cells ,} or 30. The method of any one of claims 1-9 and 12-29, which is 1.8 μg or about 1.8 μg. at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, at least or at least about 99%, about 100% or 100% are CD3+ T cells , or CD4+ T cells and CD8+ T cells ; , a ratio of CD4+ cells to CD8+ cells of 1.5:1 to 2.0:1 or 1.2:1 to 0.8:1, optionally including a ratio of CD4+ T cells to CD8+ T cells of 1:1 or about 1:1, The method of any one of claims 1-30 . said exposure is at or about 0.4 μg to 8 μg of stimulating reagent per 10 ⁶ cells, said stimulating reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules ; (i) a main agent that specifically binds to CD3 and (ii) a secondary agent that specifically binds to CD28 are attached to the oligomer particle reagent, and the exposure is to recombinant IL-2, IL-7 , and in the presence of serum-free medium containing IL-15,
wherein said input composition comprises a ratio of CD4+ T cells to CD8+ T cells of 2:1 to 1:2 and comprises at least 300×10 ⁶ CD4+ and CD8+ T cells;
said introducing is performed in the presence of serum-free medium with recombinant IL-2, IL-7, and IL-15 ;
said incubation is carried out under static conditions for 24 hours to 72 hours or about 24 hours to 72 hours , optionally incubation is carried out at a temperature of about 37° C.;
said sampling is performed at 72 to 96 hours (inclusive) after exposure to said stimulating reagent is initiated ;
The method further comprises adding during at least a portion of said incubation a substance comprising biotin or a biologically active fragment thereof, optionally D-biotin or a biotin analogue or biologically active fragment thereof. wherein said addition is performed at a time point of 48-72 hours (inclusive) after exposure to said stimulating reagent is initiated;
32. The method of any one of claims 1-3, 6, 9, 12, and 16-31. The exposure is with a stimulating reagent comprising paramagnetic beads in a bead to cell ratio of 2:1 to 0.5:1 or about 2:1 to 0.5:1 , the beads comprising polystyrene inert paramagnetic beads comprising a coating and a diameter of 4.5 μm or about 4.5 μm, having attached thereto (i) an anti-CD3 antibody or antigen-binding fragment thereof and an anti-CD28 antibody or antigen-binding fragment thereof; the exposure is performed in the presence of serum-free medium containing recombinant IL-2, IL-7, and IL-15;
wherein said input composition comprises a ratio of CD4+ T cells to CD8+ T cells of 2:1 to 1:2 and comprises at least 300×10 ⁶ CD4+ and CD8+ T cells;
said introducing is performed in the presence of serum-free medium with recombinant IL-2, IL-7, and IL-15 ;
said incubation is performed under static conditions for 48-72 hours (inclusive) , optionally incubation is performed at a temperature of about 37°C ;
said sampling is performed at 72 to 96 hours (inclusive) after exposure to said stimulating reagent is initiated ;
The method further comprises, after incubation and prior to harvesting, exposing the cells to a magnetic field to remove the stimulating reagent from the cells.
32. The method of any one of claims 1-3, 6, 9, 10, 16-25, 29, and 31. The input composition comprises 450 x ¹⁰⁶ or about 450 x ¹⁰⁶ viable primary T cells, 500 x ¹⁰⁶ or about 500 x ¹⁰⁶ viable primary T cells, 550 x ¹⁰⁶ or about 550 x 10 ⁶ viable primary T cells, 600×10 ⁶ or about 600×10 ⁶ viable primary T cells, 700×10 ⁶ or about 700×10 ⁶ viable primary T cells, 800×10 ⁶ or about 800×10 ⁶ viable primary T cells, 900×10 ⁶ or about 900×10 ⁶ viable primary T cells, 1,000×10 ⁶ or about 1,000×10 ⁶ viable primary T cells , 1,100 x 10 ⁶ or about 1,100 x 10 ⁶ viable primary T cells, or 1,200 x 10 ⁶ or about 1,200 x 10 ⁶ viable primary T cells, or
at least 450×10 ⁶ or at least about 450×10 ⁶ viable primary T cells, at least 500×10 ⁶ or at least about 500×10 ⁶ viable primary T cells, at least 550×10 ⁶ or at least about 550 x106 viable primary T cells, at least 600 x ^{106 or} at least about 600 x 106 ^viable primary T cells, at least 700 x ¹⁰⁶ or at least about 700 x ¹⁰⁶ viable primary T cells, at least 800×10 ⁶ or at least about 800×10 ⁶ viable primary T cells, at least 900×10 ⁶ or at least about 900×10 ⁶ viable primary T cells, at least 1,000×10 ⁶ or at least about 1,000 × 10 ⁶ viable primary T cells, at least 1,100 × 10 ⁶ or at least about 1,100 × 10 ⁶ viable primary T cells, or at least 1,200 × 10 ⁶ or at least about 1,200 × 10 ⁶ viable primary T cells
34. The method of any one of claims 1-33, comprising Prior to exposure to the stimulating reagent,
enriching CD3+ T cells or CD4+ T cells and/or CD8+ T cells from the biological sample to produce an input composition ; and/or
enriching CD4+ or CD8+ T cells from a biological sample to produce an input composition , optionally said enrichment producing separate enriched populations of CD4+ T cells and CD8+ T cells; wherein said separate enriched populations are mixed to produce an input composition
35. The method of any one of claims 1-34, comprising concentration, but
(a) performing a first selection in a closed system, said first selection enriching one of (i) CD4+ cells and (ii) CD8+ cells from a sample comprising primary human T cells; and (b) performing a second selection in a closed system, wherein said enrichment produces a first selected population and an unselected population; and comprises enriching the other of (i) CD4+ cells and (ii) CD8+ cells from said unselected population, whereby said enrichment produces a second selected population, said enrichment produces a second selected population of CD4+ T cells and producing separate enriched populations of CD8+ T cells;
each of said separate enriched populations comprises cells from one of said first selected population or said second selected population;
the input composition comprises cells from one or more of an enriched population of CD4+ T cells and an enriched population of CD8+ T cells;
Optionally, the enrichment of cells in the first and/or second selection comprises immunoaffinity-based selection, optionally the immunoaffinity-based selection is immobilized on or attached to an affinity chromatography matrix carried out by contacting the cells with an antibody that specifically binds to a cell surface marker and is capable of performing positive or negative selection of CD4+ or CD8+ cells;
36. The method of claim 35 . a step of enriching the CD4+ or CD8+ T cells from the biological sample prior to exposure to the stimulating reagent, wherein the enrichment renders the cells of the sample specifically binding CD4 in the incubation composition; an immunoaffinity reagent and a second immunoaffinity reagent that specifically binds to CD8 in the incubation composition, and said immunoaffinity reagent specifically to CD4 and CD8 molecules, respectively, on the surface of cells in said sample; and recovering cells bound to said first and/or second immunoaffinity reagents, thereby producing an enriched composition comprising CD4+ and CD8+ cells. including the step of
the input composition comprises an enriched composition of cells comprising CD4+ cells and CD8+ cells;
optionally, each immunoaffinity reagent comprises an antibody or antigen-binding fragment thereof and/or the immunoaffinity reagent is immobilized on the outer surface of the bead;
37. The method of any one of claims 1-36. the biological sample comprises primary T cells obtained from a subject, optionally a human subject;
the biological sample is or is a whole blood sample, buffy coat sample, peripheral blood mononuclear cell (PBMC) sample, unfractionated T cell sample, lymphocyte sample, white blood cell sample, apheresis product, or leukoapheresis product including and/or
the biological sample is a pre-cryofrozen apheresis or leukoapheresis product prior to concentration;
The method of any one of claims 35-37 . 39. The method of any one of claims 1-13 and 16-38, wherein the harvesting is performed 36 hours to 120 hours or about 36 hours to about 120 hours after exposure to the stimulating agent is initiated. The percentage of naive-like T cells and/or central memory T cells is equal to total T cells in the population, total CD4+ T cells in the population, total CD8+ T cells in the population, or recombination thereof in the population at greater than or about 60% of the protein-expressing cells,
the percentage of naive-like T cells and/or central memory T cells is greater than or greater than about 60% of all recombinant protein-expressing cells in the population, optionally all recombinants in the population at greater than 65%, 70%, 80%, 90%, or 95%, or greater than about 65%, 70%, 80%, 90%, or 95% of protein-expressing T cells ,
the percentage of naive-like T cells and/or central memory T cells is greater than or greater than about 60% among all recombinant protein-expressing CD4+ T cells in said population, optionally in said population greater than 65%, 70%, 80%, 90%, or 95% of all recombinant protein-expressing CD4+ cells, or greater than about 65%, 70%, 80%, 90%, or 95% At the time
the percentage of naive-like T cells and/or central memory T cells is greater than or greater than about 60% of all recombinant protein-expressing CD8+ T cells in said population, optionally in said population greater than 65%, 70%, 80%, 90%, or 95% of all recombinant protein-expressing CD8+ cells, or greater than about 65%, 70%, 80%, 90%, or 95% at a time and/or
When naive-like T cells or T cells that are surface positive for markers expressed on naive-like T cells are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+ ,
40. The method of any one of claims 1-39 , wherein the cells are harvested. Sampling is the percentage of CD27+CCR7+ total T cells in the population, total CD3+ T cells in the population, total CD4+ T cells in the population, total CD8+ T cells in the population, or any combination thereof in the population optionally at greater than or about 60% of the recombinant protein-expressing cells, total T cells in the population, total CD3+ T cells in the population, total CD4+ T cells in the population, greater than 65%, 70%, 80%, 90%, or 95%, or about 65%, 70%, 80% of all CD8+ T cells in the population, or the recombinant protein-expressing cells in the population 41. The method of any one of claims 1-40, wherein the method is performed at a time point greater than %, 90%, or 95%. A composition of engineered cells is collected as an output composition in a container, and the output composition is about or at least about 10 x 10 cells in one or more containers. ⁶ , about or at least about 20×10 ⁶ , about or at least about 25×10 ⁶ , about or at least about 50×10 ⁶ , about or at least about 100×10 ⁶ , about or at least about 200×10 ⁶ , about or at least about 400×10 ⁶ , about or at least about 600×10 ⁶ , about or at least about 800×10 ⁶ , about or at least about 1000×10 ⁶ , about or at least about 1200×10 ⁶ , about or at least about 1400×10 ⁶ , about or at least about 1600×10 ⁶ , about or at least about 1800×10 ⁶ , about or at least about 2000×10 ⁶ , about or at least about 2500×10 ⁶ , about or at least about 3000×10 ⁶ , or about or at least about 4000×10 ⁶ 42. The method of any one of claims 1-41, comprising 1 total cells, optionally total viable cells. The cells of the composition of engineered cells are formulated for cryopreservation and/or administration to a subject, optionally in the presence of a pharmaceutically acceptable excipient or cryoprotectant, optionally DMSO . 43. The method of any one of claims 1-42, further comprising a step. 44. Any of claims 1-43, wherein a viral vector comprising a heterologous polynucleotide encoding a recombinant protein is introduced into the T cell , optionally the viral vector is a retroviral vector, a lentiviral vector, or a gammaretroviral vector . or the method described in item 1. transduction is performed in the presence of a transduction adjuvant , optionally protamine sulfate, fibronectin-derived transduction adjuvant, or RetroNectin , and/or
the introduction is performed in the presence of 1 μg/ml to 50 μg/ml or about 1 μg/ml to 50 μg/ml of protamine sulfate;
The method of any one of claims 1-13 and 16-44. The recombinant protein is a recombinant receptor capable of binding to a target antigen associated with, specific for, and/or expressed on a disease, disorder, or diseased cell or tissue . ,
optionally the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer ; and/or
the target antigen is a tumor antigen,
46. The method of any one of claims 1-45 . the recombinant protein is or comprises a functional non-TCR antigen receptor or TCR or antigen-binding fragment thereof ; and/or
the recombinant protein is a chimeric antigen receptor (CAR);
47. The method of any one of claims 1-13 or 16-46. serum-free medium
dipeptide form of L-glutamine at 0.5 mM to 5 mM in basal medium;
0.5 mM to 5 mM L-glutamine; and at least one protein, serum free ,
optionally, the basal medium comprises at least one protein selected from one or more of albumin, insulin, or transferrin, optionally human or recombinant albumin, insulin, or transferrin;
48. The method of any one of claims 22-47 . A therapeutic T cell composition produced by the method of any one of claims 1-48 . On average in a plurality of compositions produced by the method,
at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92% of the total number of T cells in the composition or the total number of T cells expressing the recombinant protein in said composition, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92% %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are naive-like T cells or surface positive for markers expressed on naive-like cells is a T cell and/or
at least 50% or at least about 50% or 50% or about 50% of the total number of T cells in the composition or the total number of T cells expressing the recombinant protein in said composition are naive-like T cells or central memory T cells or a T cell that is surface positive for markers expressed on naive-like T cells or central memory T cells ,
optionally, the marker expressed on naive-like T cells is selected from the group consisting of CD45RA, CD27, CD28 and CCR7 ; and/or
naive-like T cells or T cells that are surface positive for markers expressed on naive-like T cells are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+;
50. The therapeutic T cell composition of claim 49 . naive-like T cells, or T cells that are surface positive for markers expressed on naive-like T cells, comprise CD27+CCR7+ T cells, and at least 70% of all receptor ⁺ /CD8 ⁺ cells in the composition; 80%, 85%, or 90% are CD27+CCR7+
naive-like T cells, or T cells that are surface positive for markers expressed on naive-like T cells, comprise CD27+CCR7+ T cells, and at least 50% of all receptor ⁺ /CD4 ⁺ cells in the composition; 60%, 70%, 80%, 85%, or 90% are CD27+CCR7+ and/or
Naive-like T cells, or T cells that are surface positive for markers expressed on naive-like T cells, comprise CD27+CCR7+ T cells, and at least 50% of all receptor ⁺ /CD8 ⁺ cells in the composition, 60 %, 70%, 80%, 85%, or 90% are CD27+CCR7+ and at least 50%, 60%, 70%, 80%, 85% of all receptor ⁺ /CD4 ⁺ cells in the composition , or 90% CD27+CCR7+,
51. The therapeutic T cell composition of claim 50 . A therapeutic T cell composition comprising CD4+ T cells expressing a recombinant receptor and CD8+ T cells expressing a recombinant receptor,
at least 50%, 60%, 70%, 80%, or 90% of all receptor ⁺ /CD8 ⁺ cells in said composition are CD27 + CCR7 + and all receptor ⁺ /CD4 ⁺ cells in said composition are CD27+CCR7+ at least 50%, 60%, 70%, 80%, or 90% of
A therapeutic T cell composition. at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, at least or at least about 99%, about 100% or 100% are CD3+ T cells , and/or
at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, at least or at least about 99%, about 100% or 100% are CD4+ T cells and CD8+ T cells,
53. The therapeutic T cell composition of any one of claims 49-52. at least or at least about 90% of the cells in said composition are CD4+ T cells and CD8+ T cells, and at least or at least about 60% of all receptor ⁺ /CD8 ⁺ cells in said composition are CD27+CCR7+ and at least or at least about 60% of all receptor ⁺ /CD4 ⁺ cells in said composition are CD27+CCR7+ , and/or
at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92 of the total number of T cells in said composition or the total number of T cells expressing a recombinant receptor in said composition %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least about 50%, 60%, 70%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or 50%, 60%, 70%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or about 50%, 60%, 70%, 80%, 85%, 90%, 91% %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are CD27+CCR7+
54. The therapeutic T cell composition of any one of claims 49-53 . The ratio of receptor+/CD4+ T cells to receptor+/CD8+ T cells in said composition is from about 1:3 to about 3:1 or from about 1:2 to about 2:1, optionally 1:1. 55. The therapeutic T cell composition of any one of claims 49-54 , which is 1 or about 1:1 . Whether the recombinant protein is a recombinant receptor capable of binding to a target protein associated with, specific for, and/or expressed on a disease, disorder, or diseased cell or tissue , or contains it, and/or
the recombinant protein is a chimeric antigen receptor (CAR);
The therapeutic composition according to any one of claims 49-55 . 57. The composition of any one of claims 49-56 and instructions for administering the composition to a subject , optionally wherein the subject has a disease or condition; An article of manufacture wherein the receptor specifically recognizes or specifically binds to an antigen associated with or expressed or present on the disease or diseased cells. 57. The composition of any one of claims 49-56 for use in a method of treating a subject having a disease or condition, optionally wherein said disease or condition is cancer.