Therapeutic preparations of gamma-delta T cells and natural killer cells, as well as methods for their manufacture and use.

JP7906202B2Active Publication Date: 2026-08-18オスペダーレ ペディアトリコ バンビーノ ジェズ
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Patent Information

Application Number
JP2024170336
Authority / Receiving Office
JP · JP
Patent Type
Patents
Current Assignee / Owner
Priority Date
2018-07-26
Filing Date
2024-09-30
Publication Date
2026-08-18
Estimated Expiration
2039-07-25

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【0060】 図面は、本技術のある特定の実施形態を例証するものであって、制限するものではない。例証を明快かつ容易にするために、図面は縮尺通りに記載されておらず、一部の例では様々な態様は、特定の実施形態の理解を容易にするために、誇張または拡大して示され得る。 特定の実施形態では、例えば以下の項目が提供される。 (項目1) NK細胞およびガンマ.デルタT細胞に関して富化された細胞の集団を含む組成物を製造する方法であって、 1または複数の対象から細胞を含有する試料を得ること; NK細胞およびガンマ.デルタT細胞を含む枯渇細胞集団を生成する条件下で前記試料からアルファ.ベータT細胞を枯渇させること;ならびに 前記枯渇細胞集団を(a)細胞接着ポリペプチドに免疫特異的に結合する少なくとも1種の外因性ポリペプチド、および(b)前記細胞接着ポリペプチドとは異なるポリペプチドに免疫特異的に結合し、前記試料集団の1種または複数種の細胞の表面上に発現される少なくとも1種の外因性ポリペプチドに接触させることを含む活性化条件に前記枯渇細胞集団を曝露すること;ならびに 前記枯渇細胞集団を少なくとも1種の補助的ポリペプチドに接触させることを含む拡大条件に前記枯渇細胞集団を曝露し、それによってNK細胞およびガンマ.デルタT細胞に関して富化された細胞の集団を含む組成物を生成すること を含む方法。 (項目2) NK細胞およびガンマ.デルタT細胞に関して富化された細胞の集団を含む組成物を製造する方法であって、 1または複数の対象から細胞を含有する試料を得ること; 前記試料を(a)細胞接着ポリペプチドに免疫特異的に結合する少なくとも1種の外因性ポリペプチド、および(b)前記細胞接着ポリペプチドとは異なるポリペプチドに免疫特異的に結合し、前記試料集団の1種または複数種の細胞の表面上に発現される少なくとも1種の外因性ポリペプチドに接触させることを含む活性化条件であって、(a)もしくは(b)が可溶性であるか、または(a)および(b)が可溶性である活性化条件に前記試料を曝露すること;ならびに 前記試料を少なくとも1種の補助的ポリペプチドに接触させることを含む拡大条件に前記試料を曝露し、それによってNK細胞およびガンマ.デルタT細胞に関して富化された細胞の集団を含む組成物を生成すること を含む方法。 (項目3) 前記少なくとも1種の補助的ポリペプチドが、前記集団におけるガンマ.デルタT細胞の量と比較したNK細胞の量が前記少なくとも1種の補助的ポリペプチドの量および/またはタイプに依存的であるように選択される、項目1または項目2に記載の方法。 (項目4) 前記少なくとも1種の補助的ポリペプチドが、前記枯渇細胞集団を前記少なくとも1種の補助的ポリペプチドに接触させた後に、前記細胞の集団におけるガンマ.デルタT細胞と比較してNK細胞の量を増加または減少させる、項目1~3のいずれかに記載の方法。(項目5) 前記活性化条件が、非ヒト動物由来の血清を含まない、項目1~4のいずれかに記載の方法。 (項目6) 前記拡大条件が、非ヒト動物由来の血清を含まない、項目1~5のいずれかに記載の方法。 (項目7) 前記活性化条件がフィーダー細胞を含まない、項目1~6のいずれかに記載の方法。 (項目8) 前記拡大条件がフィーダー細胞を含まない、項目1~7のいずれかに記載の方法。 (項目9) 前記試料が、末梢血、肝組織、骨髄、上皮組織、および臍帯血から選択される、項目1~8のいずれかに記載の方法。 (項目10) 前記試料が末梢血である、項目9に記載の方法。 (項目11) 前記末梢血試料が処理された試料である、項目10に記載の方法。 (項目12) 前記試料が臍帯血である、項目9に記載の方法。 (項目13) 前記臍帯血試料が処理された試料である、項目12に記載の方法。 (項目14) (b)における前記少なくとも1種の外因性ポリペプチドが、NK細胞活性化受容体、ガンマ.デルタT細胞活性化受容体、または両方に免疫特異的に結合する、項目1~13のいずれかに記載の方法。 (項目15) 前記受容体が、NKp30、NKp44、またはNKp46である、項目14に記載の方法。 (項目16) 前記受容体がNKp46である、項目15に記載の方法。 (項目17) (a)における前記外因性ポリペプチドが、CD2に免疫特異的に結合する、項目1~16のいずれかに記載の方法。 (項目18) (a)もしくは(b)、または(a)および(b)における前記外因性ポリペプチドが、抗体またはその抗原結合性断片である、項目1~17のいずれかに記載の方法。 (項目19) (a)または(b)のうちの少なくとも1つが可溶性である、項目1~18のいずれかに記載の方法。 (項目20) (a)および(b)における前記外因性ポリペプチドの両方が可溶性である、項目1~19のいずれかに記載の方法。 (項目21) (a)における前記外因性ポリペプチドまたは(b)における前記外因性ポリペプチドが基質に結合する、項目1~19のいずれかに記載の方法。 (項目22) 前記活性化条件が、前記試料または枯渇細胞集団を少なくとも2種の外因性ポリペプチドに接触させることを含む、項目1~21のいずれかに記載の方法。 (項目23) 第1の外因性ポリペプチドがCD2に免疫特異的に結合し、第2の外因性ポリペプチドがNKp46に免疫特異的に結合する、項目22に記載の方法。 (項目24) 前記第1の外因性ポリペプチドおよび/または前記第2の外因性ポリペプチドが、抗体またはその抗原結合性断片である、項目22または項目23に記載の方法。 (項目25) 前記活性化条件のポリペプチド構成要素が、 (a)前記細胞接着ポリペプチドCD2に免疫特異的に結合する外因性ポリペプチド;および (b)(a)における前記外因性ポリペプチドとは異なり、NKp46に免疫特異的に結合する外因性ポリペプチド から本質的になるかまたはそれからなる、項目1~24のいずれかに記載の方法。 (項目26) 前記少なくとも1種の補助的ポリペプチドが、サイトカインおよび/またはガンマ.デルタT細胞上の受容体に免疫特異的に結合するポリペプチドである、項目1~25のいずれかに記載の方法。 (項目27) 前記拡大条件が、前記試料または枯渇細胞集団をサイトカインである少なくとも1種の補助的ポリペプチド、および必要に応じてガンマ.デルタT細胞上の受容体に免疫特異的に結合する補助的ポリペプチドに接触させることを含む、項目26に記載の方法。 (項目28) 前記サイトカインがインターロイキン(IL)である、項目27に記載の方法。 (項目29) 前記少なくとも1種の補助的ポリペプチドが、IL-2、IL-4、IL-15、またはその任意の組合せを含む、項目26~28のいずれかに記載の方法。 (項目30) 前記拡大条件が、前記試料または枯渇細胞集団を (a)IL-2ポリペプチド、および必要に応じてガンマ.デルタT細胞上の受容体に免疫特異的に結合するポリペプチド; (b)IL-15ポリペプチド;または (c)IL-2ポリペプチドおよびIL-15ポリペプチド、ならびに必要に応じてガンマ.デルタT細胞上の受容体に免疫特異的に結合するポリペプチド に接触させることを含む、項目26~29のいずれかに記載の方法。 (項目31) 前記ガンマ.デルタT細胞上の前記受容体がCD3である、項目26~30のいずれかに記載の方法。 (項目32) 前記ガンマ.デルタT細胞上の受容体に免疫特異的に結合する前記ポリペプチドが、抗体またはその抗原結合性断片である、項目26~31のいずれかに記載の方法。 (項目33) 前記拡大条件が、前記試料を (a)IL-2ポリペプチド; (b)IL-15ポリペプチド; (c)IL-2ポリペプチドおよびIL-15ポリペプチド; (d)IL-2ポリペプチドおよびCD3に免疫特異的に結合する抗体;または (e)IL-2ポリペプチド、IL-15ポリペプチドおよびCD3に免疫特異的に結合する抗体 に接触させることを含む、項目32に記載の方法。 (項目34) CD3に免疫特異的に結合する前記抗体がOKT3である、項目33に記載の方法。 (項目35) 前記活性化および拡大条件が同時にまたは任意の順序で逐次的に実施される、項目1~34のいずれかに記載の方法。 (項目36) 前記少なくとも1種の外因性ポリペプチドが補助的ポリペプチドとしても機能することができる;または 前記少なくとも1種の補助的ポリペプチドが外因性ポリペプチドとしても機能することができる;または 前記少なくとも1種の外因性ポリペプチドが補助的ポリペプチドとしても機能することができ、かつ前記少なくとも1種の補助的ポリペプチドが外因性ポリペプチドとしても機能することができる、項目1~35のいずれかに記載の方法。 (項目37) (i)前記拡大条件が、前記試料または枯渇細胞集団をIL-2ポリペプチドに接触させることを含み; (ii)得られた、NK細胞およびガンマ.デルタT細胞に関して富化された前記細胞の集団が、約25~30%のNK細胞および約70~75%のガンマ.デルタT細胞を含む、項目33~36のいずれかに記載の方法。 (項目38) (i)前記拡大条件が、前記試料または枯渇細胞集団をIL-15ポリペプチドに接触させることを含み; (ii)得られた、NK細胞およびガンマ.デルタT細胞に関して富化された前記細胞の集団が、約80~99%のNK細胞および約1~20%のガンマ.デルタT細胞を含む、項目33~36のいずれかに記載の方法。 (項目39) (i)前記拡大条件が、前記試料または枯渇細胞集団をIL-2ポリペプチドおよびCD3に免疫特異的に結合する抗体に接触させることを含み; (ii)得られた、NK細胞およびガンマ.デルタT細胞に関して富化された前記細胞の集団が、約40~45%のNK細胞および約55~60%のガンマ.デルタT細胞を含む、項目33~36のいずれかに記載の方法。 (項目40) 前記拡大条件が、 前記試料または枯渇細胞集団を、1種または複数種の補助的ポリペプチドを含む第1の条件セットに接触させ、NK細胞のガンマ.デルタT細胞に対する第1の比を含む第1の細胞集団をもたらすこと;および 前記第1の細胞集団を、1種または複数種の補助的ポリペプチドを含む第2の条件セットに接触させ、NK細胞のガンマ.デルタT細胞に対する所望の最終的な比を含む第2の細胞集団をもたらすことを含み、前記第1の条件セットが前記第2の条件セットとは異なる、項目1~39のいずれかに記載の方法。 (項目41) 前記第1の細胞集団が前記第2の条件セットとの接触前に洗浄される、項目40に記載の方法。 (項目42) 前記第1の条件セットがIL-2を含み、前記第2の条件セットがIL-15を含む; 前記第1の条件セットがIL-15を含み、前記第2の条件セットがIL-2を含む; 前記第1の条件セットがIL-2およびCD3に免疫特異的に結合する抗体を含み、前記第2の条件セットがIL-15を含む;または 前記第1の条件セットがIL-15およびCD3に免疫特異的に結合する抗体を含み、前記第2の条件セットがIL-2およびCD3に免疫特異的に結合する抗体を含む、項目40または項目41に記載の方法。 (項目43) CD3に免疫特異的に結合する前記抗体がOKT3である、項目42に記載の方法。 (項目44) 前記試料を前記活性化および拡大条件に曝露する前に、前記試料からアルファ.ベータT細胞を枯渇させ、それによって枯渇細胞集団を生成すること;ならびに 前記枯渇細胞集団を前記活性化および拡大条件に供すること をさらに含み、 それによってNK細胞およびガンマ.デルタT細胞に関して富化された細胞の集団を含む組成物が得られる、項目2~43のいずれかに記載の方法。 (項目45) 活性化および拡大の前に、前記試料または前記枯渇細胞集団が、NK細胞またはガンマ.デルタT細胞について選択する条件にも、前記アルファ-ベータT細胞以外の細胞を枯渇させる条件にも曝露されない、項目1~44のいずれかに記載の方法。 (項目46) 活性化および拡大の前に、前記試料または前記枯渇細胞集団が、前記試料または前記枯渇細胞集団の全てのCD3+細胞を枯渇させる条件に曝露されない、項目1~45のいずれかに記載の方法。 (項目47) 前記試料または枯渇細胞集団の細胞が、活性化および拡大の前、間、または後に外因性核酸を含まない、項目1~46のいずれかに記載の方法。 (項目48) 前記試料または枯渇細胞集団の細胞が、活性化および拡大の前、間、または後に腫瘍壊死因子受容体も、キメラ抗原受容体(CAR)も、骨髄分化一次応答タンパク質も、自然免疫シグナル伝達アダプターをコードする外因性核酸も含まない、項目1~46のいずれかに記載の方法。 (項目49) 前記試料または枯渇細胞集団の細胞が、活性化および拡大の前、間、または後に遺伝子改変されない、項目1~48のいずれかに記載の方法。 (項目50) NK細胞およびガンマ.デルタT細胞に関して富化された前記細胞の集団を、それによって前記ガンマ.デルタ細胞が枯渇される処置に供することをさらに含み、得られた集団がNK細胞から本質的になるかまたはそれからなる、項目1~49のいずれかに記載の方法。 (項目51) NK細胞およびガンマ.デルタT細胞に関して富化された前記細胞の集団を、それによって前記NK細胞が枯渇される処置に供することをさらに含み、得られた集団がガンマ.デルタT細胞から本質的になるかまたはそれからなる、項目1~49のいずれかに記載の方法。 (項目52) NK細胞およびガンマ.デルタT細胞に関して富化された前記細胞の集団を、NK細胞についての正の選択に供することをさらに含み、それによってNK細胞から本質的になるかまたはそれからなる細胞集団が得られる、項目1~50のいずれかに記載の方法。 (項目53) NK細胞およびガンマ.デルタT細胞に関して富化された前記細胞の集団を、ガンマ.デルタ細胞についての正の選択に供することをさらに含み、それによってガンマ.デルタ細胞から本質的になるかまたはそれからなる細胞集団が得られる、項目1~49および51のいずれかに記載の方法。 (項目54) 前記拡大条件が、前記試料または枯渇細胞集団のフィーダー細胞フリー培地中での約1週間から約10週間のインキュベーションを含み、それによってNK細胞およびガンマ.デルタT細胞に関して富化された拡大した細胞の集団を含む組成物が得られる、項目1~53のいずれかに記載の方法。 (項目55) 前記培養条件が、前記試料または枯渇細胞集団のフィーダー細胞フリー培地中での約7日間、8日間、9日間、10日間、11日間、12日間、13日間、14日間、15日間、16日間、17日間、18日間、19日間、20日間、21日間、25日間、30日間、35日間、40日間、45日間、50日間、55日間、もしくは60日間もしくはそれより多くの日数、または約2週間、3週間、4週間、5週間、6週間、7週間、8週間、9週間、もしくは10週間のインキュベーションを含む、項目54に記載の方法。 (項目56) 前記NK細胞およびガンマ.デルタT細胞に関して富化された細胞の集団が、前記拡大条件下の30日間で約2logより大きく拡大される、項目1~55のいずれかに記載の方法。 (項目57) 前記細胞の集団が、前記拡大条件下の30日間で約3logより大きく拡大される、項目56に記載の方法。 (項目58) NK細胞およびガンマ.デルタT細胞に関して富化された前記拡大した細胞の集団が、疲弊した細胞を含まない、項目1~57のいずれかに記載の方法。 (項目59) NK細胞およびガンマ.デルタT細胞に関して富化された前記拡大した細胞の集団が、前記拡大条件の60日後に疲弊した細胞を含まない、項目1~58のいずれかに記載の方法。 (項目60) NK細胞およびガンマ.デルタT細胞に関して富化された前記拡大した細胞の集団におけるNK細胞の5%未満、4%未満、3%未満、または2%未満がPD-1マーカーを含み、および/または前記拡大した集団における総細胞もしくは前記拡大した集団における前記ガンマ.デルタT細胞の約20%、15%、10%、またはそれ未満が前記PD-1マーカーを含む、項目1~59のいずれかに記載の方法。 (項目61) NK細胞およびガンマ.デルタT細胞に関して富化された前記細胞の集団が、前記集団における総細胞数のパーセンテージとして以下の活性化マーカー: (a)90%またはそれより多くのKIR5; (b)10%またはそれより多くのSIGLEC-7; (c)60%またはそれより多くのKIR3D51; (d)10%またはそれより多くのKIR2DL1; (e)25%またはそれより多くのNKp30、NKp44、またはNKp46; (f)35%またはそれより多くのNKG2D; (g)90%またはそれより多くのDNAM1; (h)85%またはそれより多くのNTBA; (i)95%またはそれより多くのCD2;および (j)55%またはそれより多くのKIR3DS1 のうちの1つまたは複数を含む、項目1~60のいずれかに記載の方法。 (項目62) NK細胞およびガンマ.デルタT細胞に関して富化された前記細胞の集団が、80%またはそれより多くの自然免疫細胞を含む、項目1~61のいずれかに記載の方法。 (項目63) 前記細胞の約70%から約100%の間、または少なくとも約70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、もしくは100%がCD56+である、項目62に記載の方法。 (項目64) 前記細胞の約10%から約40%の間、または少なくとも約10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%、25%、26%、27%、28%、29%、30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、もしくは40%がCD16+である、項目62または項目63に記載の方法。 (項目65) 前記細胞の5%未満、4%未満、3%未満、または2%未満がCD57+である、項目62~64のいずれかに記載の方法。 (項目66) 前記試料または枯渇細胞集団が、活性化および拡大の間または後にCD4+CD8+細胞を含まない、項目1~65のいずれかに記載の方法。 (項目67) 前記活性化条件および前記拡大条件がビスホスホネートを含まない、項目1~66のいずれかに記載の方法。 (項目68) 前記ビスホスホネートがパミドロネートまたはゾレドロネートである、項目67に記載の方法。 (項目69) 前記ガンマ.デルタT細胞が、V.デルタ.1およびV.デルタ.2発現に関してポリクローナルである、項目1~68のいずれかに記載の方法。 (項目70) 前記ポリクローナルガンマ.デルタT細胞が、V.デルタ.1+およびV.デルタ.1-から選択される少なくとも1つの亜集団、ならびにV.デルタ.2+およびV.デルタ.2-から選択される少なくとも1つの亜集団を含む、項目69に記載の方法。 (項目71) 項目1~70のいずれかに記載の方法によって得ることができるまたは得られた組成物。 (項目72) 改変された末梢血細胞の集団を含む組成物であって、前記集団が 複数のNK細胞および複数のガンマ.デルタT細胞を含み; アルファ.ベータT細胞が枯渇され; フィーダー細胞を含まない、組成物。 (項目73) 前記細胞の約25%から約45%の間がNK細胞であり、前記細胞の約55%から約75%の間がガンマ.デルタT細胞である; 前記細胞の約25%から約30%の間がNK細胞であり、前記細胞の約70%から約75%の間がガンマ.デルタT細胞である; 前記細胞の約80%から約99%の間がNK細胞であり、前記細胞の約1%から約20%の間がガンマ.デルタT細胞である;または 前記細胞の約40%から約45%の間がNK細胞であり、前記細胞の約55%から約60%の間がガンマ.デルタT細胞である、項目72に記載の組成物。 (項目74) 前記細胞の30%またはそれより多くが活性化される、項目72または項目73に記載の組成物。 (項目75) 前記改変された細胞の集団が、前記集団における総細胞数のパーセンテージとして以下の活性化マーカー: (a)90%またはそれより多くのKIR5; (b)10%またはそれより多くのSIGLEC-7; (c)60%またはそれより多くのKIR3D51; (d)10%またはそれより多くのKIR2DL1; (e)25%またはそれより多くのNKp30、NKp44、またはNKp46; (f)35%またはそれより多くのNKG2D; (g)90%またはそれより多くのDNAM1; (h)85%またはそれより多くのNTBA; (i)95%またはそれより多くのCD2;および (j)55%またはそれより多くのKIR3DS1 のうちの1つまたは複数を含む、項目72~74のいずれかに記載の組成物。 (項目76) 前記改変された集団が、80%またはそれより多くの自然免疫細胞を含む、項目72~75のいずれかに記載の組成物。 (項目77) 前記改変された集団が、CD56+である活性化細胞傷害性細胞に関して富化されている、項目72~76のいずれかに記載の組成物。 (項目78) 前記改変細胞が、CD57-である活性化細胞傷害性細胞に関して富化されている、項目72~77のいずれかに記載の組成物。 (項目79) 前記集団が、CD56+CD57-である活性化細胞傷害性細胞に関して富化されている、項目72~78のいずれかに記載の組成物。 (項目80) 前記細胞の約80%から約100%の間、または少なくとも約80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、もしくは100%がCD56+である、項目77~79のいずれかに記載の組成物。 (項目81) 前記細胞の約10%から約40%の間、または少なくとも約10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%、25%、26%、27%、28%、29%、30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、もしくは40%がCD16+である、項目77~80のいずれかに記載の組成物。 (項目82) 前記細胞の5%未満、4%未満、3%未満、または2%未満がCD57+である、項目77~81のいずれかに記載の組成物。 (項目83) NK細胞およびガンマ.デルタT細胞以外の細胞を実質的に含まない、項目72~82のいずれかに記載の組成物。 (項目84) 5パーセント未満のNKT細胞を含む、項目72~83のいずれかに記載の組成物。 (項目85) 1パーセント未満のNKT細胞を含む、項目72~84のいずれかに記載の組成物。 (項目86) 0.1パーセント未満のNKT細胞を含む、項目72~85のいずれかに記載の組成物。 (項目87) 2パーセント未満のアルファ.ベータT細胞を含む、項目72~86のいずれかに記載の組成物。 (項目88) 1パーセント未満のアルファ.ベータT細胞を含む、項目72~87のいずれかに記載の組成物。 (項目89) 0.1パーセント未満のアルファ.ベータT細胞を含む、項目72~88のいずれかに記載の組成物。 (項目90) 前記集団におけるNK細胞のサブセットがCD16+細胞である、項目72~89のいずれかに記載の組成物。 (項目91) 大多数のガンマ.デルタT細胞がCD57-細胞である、項目72~90のいずれかに記載の組成物。 (項目92) 大多数のNK細胞がCD57-細胞である、項目72~91のいずれかに記載の組成物。 (項目93) 前記ガンマ.デルタT細胞が、V.デルタ.1およびV.デルタ.2発現に関してポリクローナルである、項目72~92のいずれかに記載の組成物。 (項目94) 前記ポリクローナルガンマ.デルタT細胞が、V.デルタ.1+およびV.デルタ.1-から選択される少なくとも1つの亜集団、ならびにV.デルタ.2+およびV.デルタ.2-から選択される少なくとも1つの亜集団を含む、項目93に記載の組成物。 (項目95) 大多数のガンマ.デルタT細胞がV.デルタ.1を発現し、少数のガンマ.デルタT細胞がV.デルタ.2を発現する;または 少数のガンマ.デルタT細胞がV.デルタ.1を発現し、大多数のガンマ.デルタT細胞がV.デルタ.2発現を発現する、項目72~94のいずれかに記載の組成物。 (項目96) 前記集団における少数の細胞がCD3陽性細胞であり、前記集団における大多数の細胞がCD3陰性細胞である;または 前記集団における大多数の細胞がCD3陽性細胞であり、前記集団における少数の細胞がCD3陰性細胞である、項目72~95のいずれかに記載の組成物。 (項目97) NK細胞のガンマ.デルタT細胞に対する比が1より大きい、項目72~96のいずれかに記載の組成物。 (項目98) NK細胞のガンマ.デルタT細胞に対する比が1未満である、項目72~96のいずれかに記載の組成物。 (項目99) 前記改変された細胞の集団が、約98~99%のNK細胞および約1~2%のガンマ.デルタT細胞を含む、項目97に記載の組成物。 (項目100) 前記改変された細胞の集団が、約25%から約45%の間のNK細胞および約55%から約75%の間のガンマ.デルタT細胞を含む、項目98に記載の組成物。 (項目101) 前記改変された細胞の集団が、約25~30%のNK細胞および約70~75%のガンマ.デルタT細胞を含む、項目98に記載の組成物。 (項目102) 前記改変された細胞の集団が、約40~45%のNK細胞および約55~60%のガンマ.デルタT細胞を含む、項目98に記載の組成物。 (項目103) 前記NK細胞および/または前記ガンマ.デルタT細胞の約50%から約99%もしくはそれより多くの間、または約50%より多くのもしくはそれに等しい、51%より多くのもしくはそれに等しい、52%より多くのもしくはそれに等しい、53%より多くのもしくはそれに等しい、54%より多くのもしくはそれに等しい、55%より多くのもしくはそれに等しい、56%より多くのもしくはそれに等しい、57%より多くのもしくはそれに等しい、58%より多くのもしくはそれに等しい、59%より多くのもしくはそれに等しい、60%より多くのもしくはそれに等しい、6%より多くのもしくはそれに等しい、62%より多くのもしくはそれに等しい、63%より多くのもしくはそれに等しい、64%より多くのもしくはそれに等しい、65%より多くのもしくはそれに等しい、66%より多くのもしくはそれに等しい、67%より多くのもしくはそれに等しい、68%より多くのもしくはそれに等しい、69%より多くのもしくはそれに等しい、70%より多くのもしくはそれに等しい、71%より多くのもしくはそれに等しい、72%より多くのもしくはそれに等しい、73%より多くのもしくはそれに等しい、74%より多くのもしくはそれに等しい、75%より多くのもしくはそれに等しい、76%より多くのもしくはそれに等しい、77%より多くのもしくはそれに等しい、78%より多くのもしくはそれに等しい、79%より多くのもしくはそれに等しい、80%より多くのもしくはそれに等しい、81%より多くのもしくはそれに等しい、82%より多くのもしくはそれに等しい、83%より多くのもしくはそれに等しい、84%より多くのもしくはそれに等しい、85%より多くのもしくはそれに等しい、86%より多くのもしくはそれに等しい、87%より多くのもしくはそれに等しい、88%より多くのもしくはそれに等しい、89%より多くのもしくはそれに等しい、90%より多くのもしくはそれに等しい、91%より多くのもしくはそれに等しい、92%より多くのもしくはそれに等しい、93%より多くのもしくはそれに等しい、94%より多くのもしくはそれに等しい、95%より多くのもしくはそれに等しい、96%より多くのもしくはそれに等しい、97%より多くのもしくはそれに等しい、98%より多くのもしくはそれに等しい、99%より多くのもしくはそれに等しい、80%より多くのもしくはそれに等しい、81%より多くのもしくはそれに等しい、82%より多くのもしくはそれに等しい、83%より多くのもしくはそれに等しい、84%より多くのもしくはそれに等しい、85%より多くのもしくはそれに等しい、86%より多くのもしくはそれに等しい、87%より多くのもしくはそれに等しい、88%より多くのもしくはそれに等しい、89%より多くのもしくはそれに等しい、90%より多くのもしくはそれに等しい、91%より多くのもしくはそれに等しい、92%より多くのもしくはそれに等しい、93%より多くのもしくはそれに等しい、94%より多くのもしくはそれに等しい、95%より多くのもしくはそれに等しい、96%より多くのもしくはそれに等しい、97%より多くのもしくはそれに等しい、98%より多くのもしくはそれに等しい、99%より多くのもしくはそれに等しい、またはその端数、最大100%がCD8+である、項目72~102のいずれかに記載の組成物。 (項目104) 前記NK細胞および/または前記ガンマ.デルタT細胞の2%未満がCD4+である、項目103に記載の組成物。 (項目105) 前記NK細胞および/または前記ガンマ.デルタT細胞の2%未満がCD8+CD4+である、項目103または項目104に記載の組成物。 (項目106) 前記NK細胞の約15%から約30%の間および/または前記ガンマ.デルタT細胞の約55%から85%の間の端数がCD8-CD4-である、項目103~105のいずれかに記載の組成物。 (項目107) 前記集団における前記細胞の約30%から約99%もしくはそれより多くの間、または少なくとも約30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、40%、41%、42%、43%、44%、45%、46%、47%、48%、49%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、91%、92%、93%、94%、95%、96%、97%、98%、99%、もしくはその端数、最大100%が、外因性ポリヌクレオチド、変異ポリヌクレオチド、欠失ポリヌクレオチド、またはその組合せを含む遺伝子改変をさらに含む、項目72~106のいずれかに記載の組成物。 (項目108) 前記集団における前記細胞の少なくとも約95%、96%、97%、98%、99%が前記遺伝子改変を含むか、または前記集団における前記細胞の約100%もしくは100%が前記遺伝子改変を含む、項目107に記載の組成物。 (項目109) 前記遺伝子改変が外因性ポリヌクレオチドを含む、項目107または項目108に記載の組成物。 (項目110) 前記外因性ポリヌクレオチドがレトロウイルスベクターまたはレンチウイルスベクター中にある、項目109に記載の組成物。 (項目111) 前記外因性ポリヌクレオチドが、前記改変された細胞集団の1種または複数種の細胞のゲノムに組み込まれる、項目109に記載の組成物。 (項目112) 前記集団における前記細胞がキメラ抗原受容体(CAR)を含む、項目107~111のいずれかに記載の組成物。 (項目113) 前記キメラ抗原受容体がCD19、GD2、HER3、B7H3、CD123、またはCD30のうちの1つまたは複数に免疫特異的に結合する結合分子部分を含む、項目112に記載の組成物。 (項目114) 前記複数のNK細胞および前記複数のガンマ.デルタT細胞を含む前記集団が、1より多くの対象の前記末梢血細胞に由来する、項目72~113のいずれかに記載の組成物。(項目115) 実施形態72~114のいずれかに記載の組成物および薬学的に許容される担体を含む医薬組成物。 (項目116) 遺伝子改変免疫細胞を作製する方法であって、 (a)外因性ポリヌクレオチドを、項目71~106のいずれかに記載の組成物に添加すること; (b)項目71~106のいずれかに記載の組成物の1種または複数種の細胞においてポリヌクレオチドを変異させること;または (c)項目71~106のいずれかに記載の組成物の1種または複数種の細胞においてポリヌクレオチドを欠失させること のうちの1つまたは複数を含む方法。 (項目117) 前記遺伝子改変が、レトロウイルス形質導入、レンチウイルス形質導入、電気穿孔、トランスフェクション、CRISPR/cas9、またはTALENSによる、項目116に記載の方法。 (項目118) 前記遺伝子改変が、(a)でのように外因性ポリヌクレオチドを添加することからなるか、またはそれから本質的になる、項目116または項目117に記載の方法。 (項目119) 前記遺伝子改変が、(a)でのように外因性ポリヌクレオチドを添加すること、および/または(b)でのようにポリヌクレオチドを変異させることを含み、前記外因性ポリヌクレオチドおよび/または前記変異ポリヌクレオチドが前記免疫細胞のゲノムに組み込まれる、項目116~118のいずれかに記載の方法。 (項目120) 前記組み込みが、電気穿孔、トランスフェクション、CRISPR/cas9、またはTALENSによる、項目119に記載の方法。 (項目121) 前記外因性ポリヌクレオチドがキメラ抗原受容体(CAR)をコードする、項目116~121のいずれかに記載の方法。 (項目122) 前記キメラ抗原受容体が、CD19、GD2、HER3、B7H3、CD123、またはCD30のうちの1つまたは複数に免疫特異的に結合する結合分子部分を含む、項目121の方法。 (項目123) 前記集団における前記細胞の約30%から約99%もしくはそれより多くの間、または少なくとも約30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、40%、41%、42%、43%、44%、45%、46%、47%、48%、49%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、91%、92%、93%、94%、95%、96%、97%、98%、99%、もしくはその端数、最大100%が前記遺伝子改変を含む、項目116~122のいずれかに記載の方法。 (項目124) 前記遺伝子改変が外因性ポリヌクレオチドを含む、項目123に記載の方法。 (項目125) 前記集団における前記細胞の約100%または100%が前記外因性ポリヌクレオチドを含む、項目124に記載の方法。 (項目126) 項目71~114のいずれかに記載の組成物、または項目115に記載の医薬組成物、必要に応じて使用のための指示、および必要に応じてサイトカインを含むキット。 (項目127) 前記組成物または前記医薬組成物が、セ氏マイナス4度またはそれ未満にある、項目126に記載のキット。 (項目128) 前記組成物または前記医薬組成物が、セ氏約マイナス75度からセ氏約マイナス80度にある、項目127に記載のキット。 (項目129) 細胞約1×105個から細胞約1×1012個を含む、項目126~128のいずれかに記載のキット。 (項目130) 前記サイトカインがインターロイキンポリペプチドである、項目126~129のいずれかに記載のキット。 (項目131) 前記インターロイキンペプチドがIL-2、IL-4、またはIL-15である、項目130に記載のキット。 (項目132) 非ヒト血清を含まないおよび/またはウシ血清を含まない、項目126~131のいずれかに記載のキット。 (項目133) 異種のものを含まない、項目126~132のいずれかに記載のキット。 (項目134) 外因性フィーダー細胞を含まない、項目126~133のいずれかに記載のキット。 (項目135) 単位剤形の、項目126~134のいずれかに記載のキット。 (項目136) 前記単位剤形が、細胞約1×106個から細胞約1×1012個である、実施形態E10に記載のキット。 (項目137) 各々が1または複数のドナー対象に由来する細胞を含む複数の容器を含む、異なるドナー対象に由来する細胞のコレクションであって、各容器が、項目71~114のいずれか一項に記載の組成物、項目115に記載の医薬組成物、または項目126~136のいずれかに記載のキットを含む、細胞のコレクション。 (項目138) がんまたは感染症を処置する方法であって、それを必要とする対象に、前記がんまたは感染症を処置するために有効な量の項目71~114のいずれか一項に記載の組成物、項目115に記載の医薬組成物、または項目126~136のいずれかに記載のキットを投与することを含み、前記組成物、前記医薬組成物、またはキットにおける細胞が前記対象に対して同種異系である方法。 (項目139) がんまたは感染症を処置する方法であって、それを必要とする対象に、前記がんまたは感染症を処置するために有効な量の項目71~114のいずれか一項に記載の組成物、項目115に記載の医薬組成物、または項目126~136のいずれかに記載のキットを投与することを含み、前記組成物、前記医薬組成物、またはキットにおける細胞が前記対象に対して自己である方法。 (項目140) 前記組成物を前記対象に2日またはそれより多くの別々の日に投与することを含む、項目138または項目139に記載の方法。 (項目141) 前記細胞のドナーが前記処置のレシピエントである、項目139または項目140に記載の方法。 (項目142) 前記細胞のドナーが前記処置のレシピエントではない、項目138または項目140に記載の方法。 (項目143) 前記処置のレシピエントが、前記ドナー由来のアルファ.ベータT細胞によって処置した場合にGvHDに罹りやすい、項目142に記載の方法。 (項目144) 前記処置が、約2週間から約4週間の間の間隔で約1単位投薬量から約36またはそれより多くの単位投薬量の間で投与される、項目138~143のいずれかに記載の方法。(項目145) 前記処置が、単一単位投薬量として毎日1回、2回、3回、4回、もしくは最大5回、または数日間、数週間もしくは数ヶ月間の間に1回、2回、3回、4回、5回、6回、7回、8回、9回、もしくは10回もしくはそれより多く、または2日毎、または週に1回、2回、3回、4回、5回、もしくは6回投与される、項目138~143のいずれかに記載の方法。 (項目146) 前記処置が静脈内(IV)、髄腔内または筋肉内(IM)、腹腔内(IP)、胸膜内、関節腔内に投与されるか、または前記がんもしくは感染症の部位もしくはその近傍に注射もしくは埋め込まれる、項目138~145のいずれかに記載の方法。 (項目147) 前記単位投薬量が、前記対象の体重1キログラムあたり細胞約104個から細胞約1010個の間、または対象あたり細胞約106個から細胞約1012個の間を含む、項目144~146のいずれかに記載の方法。 (項目148) 前記単位投薬量が、対象あたり細胞約1010個、または前記対象の体重1キログラムあたり細胞約108個である、項目147に記載の方法。 (項目149) 前記処置ががんのためである、項目138~148のいずれかに記載の方法。 (項目150) 前記がんが、肺がん、黒色腫、乳がん、前立腺がん、結腸がん、腎細胞癌、卵巣がん、神経芽腫、横紋筋肉腫、白血病もしくはリンパ腫、ホジキンリンパ腫もしくは小児急性リンパ芽球性白血病、非ホジキンリンパ腫、肥満細胞腫もしくは肥満細胞腫瘍、卵巣がんもしくは癌、膵臓がん、非小細胞肺がん、小細胞肺がん、肝細胞癌、網膜芽腫、乳腺腫瘍、結腸直腸癌、白血病、リンパ腫、急性リンパ芽球性白血病(ALL)もしくは急性リンパ性白血病、急性骨髄性白血病(AML)、組織球肉腫、脳腫瘍、星状細胞腫、膠芽腫、神経腫、結腸癌、子宮頸癌、肉腫、膀胱腫瘍、細網内皮組織の腫瘍、ウィルムス腫瘍、骨がん、骨肉腫、腎臓がん、または頭頸部がん、口腔がん、喉頭がん、転移疾患、または口腔咽頭がんから選択される、項目149に記載の方法。 (項目151) 第2の薬剤が、前記組成物、医薬組成物、またはキットと共投与される、項目149または項目150に記載の方法。 (項目152) 前記第2の薬剤が、がん関連抗原に免疫特異的に結合する抗体である、項目151に記載の方法。 (項目153) 前記がん関連抗原が、α-フェトプロテイン(AFP)、α-アクチニン-4、A3、A33抗体に特異的な抗原、ART-4、B7、B7-H3、Ba733、BAGE、BrE3抗原、CA125、CAMEL、CAP-1、炭酸脱水酵素IX、CASP-8/m、CCL19、CCL21、CD1、CD1a、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD22、CD23、CD25、CD29、CD30、CD32b、CD33、CD37、CD38、CD40、CD40L、CD44、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD66a-e、CD67、CD70、CD70L、CD74、CD79a、CD79b、CD80、CD83、CD95、CD123、CD126、CD132、CD133、CD138、CD147、CD154、CDC27、CDK-4/m、CDKN2A、CTLA4、CXCR4、CXCR7、CXCL12、HIF-1α、結腸特異抗原-p(CSAp)、CEA(CEACAM-5)、CEACAM-6、c-Met、DAM、EGFR、EGFRvIII、EGP-1(TROP-2)、EGP-2、ELF2-M、Ep-CAM、線維芽細胞成長因子(FGF)、Flt-1、Flt-3、葉酸受容体、G250抗原、GAGE、gp100、GRO-β、HLA-DR、HM1.24、ヒト絨毛ゴナドトロピン(HCG)およびそのサブユニット、HER2/neu、HMGB-1、低酸素症誘導因子(HIF-1)、HSP70-2M、HST-2、Ia、IGF-1R、IFN-γ、IFN-α、IFN-β、IFN-λ、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-2、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-23、IL-25、インスリン様成長因子-1(IGF-1)、KC4抗原、KS-1抗原、KS1-4、Le-Y、LDR/FUT、マクロファージ遊走阻止因子(MIF)、GD2、MAGE、MAGE-3、MART-1、MART-2、NY-ESO-1、TRAG-3、mCRP、MCP-1、MIP-1A、MIP-1B、MIF、MUC1、MUC2、MUC3、MUC4、MUC5ac、MUC13、MUC16、MUM-1/2、MUM-3、NCA66、NCA95、NCA90、膵臓がんムチン、PD1受容体、胎盤成長因子、p53、PLAGL2、前立腺酸性ホスファターゼ、PSA、PRAME、PSMA、PlGF、ILGF、ILGF-R、L-6、IL-25、RS5、RANTES、T101、SAGE、S100、サバイビン、サバイビン-2B、TAC、TAG-72、テネイシン、TRAIL受容体、TNF-α、Tn抗原、トムソン-フリードライヒ抗原、腫瘍壊死抗原、VEGFR、ED-Bフィブロネクチン、WT-1、17-1A抗原、補体因子C3、C3a、C3b、C5a、C5、血管新生マーカー、bcl-2、bcl-6、およびKrasからなる群から選択される、項目152に記載の方法。 (項目154) 前記抗体が、hR1(抗IGF-1R)、hPAM4(抗ムチン)、KC4(抗ムチン)、hA20(抗CD20)、hA19(抗CD19)、hIMMU31(抗AFP)、hLL1(抗CD74)、hLL2(抗CD22)、抗CD19/CD22二特異性抗体、RFB4(抗CD22)、hMu-9(抗CSAp)、hL243(抗HLA-DR)、hMN-14(抗CEACAM-5)、hMN-15(抗CEACAM-6)、hRS7(抗TROP-2)、hMN-3(抗CEACAM-6)、CC49(抗TAG-72)、J591(抗PSMA)、D2/B(抗PSMA)、G250(抗炭酸脱水酵素IX)、ジヌツキシマブ(抗GD2)、インフリキシマブ(抗TNF-α)、セルトリズマブペゴル(抗TNF-α)、アダリムマブ(抗TNF-α)、アレムツズマブ(抗CD52)、ベバシズマブ(抗VEGF)、セツキシマブ(抗EGFR)、ゲムツズマブ(抗CD33)、イブリツモマブチウキセタン(抗CD20)、パニツムマブ(抗EGFR)、リツキシマブ(抗CD20)、トシツモマブ(抗CD20)、GA101(抗CD20)、トラスツズマブ(抗HER2/neu)、トシリズマブ(抗IL-6受容体)、バシリキシマブ(抗CD25)、ダクリズマブ(抗CD25)、エファリズマブ(抗CD11a)、ムロモナブ-CD3(抗CD3受容体)、ナタリズマブ(抗α4インテグリン)、BWA-3(抗ヒストンH2A/H4)、LG2-1(抗ヒストンH3)、MRA12(抗ヒストンH1)、PR1-1(抗ヒストンH2B)、LG11-2(抗ヒストンH2B)、およびLG2-2(抗ヒストンH2B)から選択される、項目152または項目153に記載の方法。 (項目155) 前記処置が感染症のためである、項目138~148のいずれかに記載の方法。 (項目156) 前記感染症が、細菌病原体、真菌病原体、ウイルス病原体、または原虫病原体の存在によって特徴付けられる、項目155に記載の方法。 (項目157) 前記感染症が、ヘルペス、エボラ、ウエストナイルウイルス、ワクシニアウイルス、エプスタインバーウイルス、A型肝炎ウイルス(HAV);B型肝炎ウイルス(HBV);C型肝炎ウイルス(HCV);ヘルペスウイルス(例えば、HSV-1、HSV-2、HHV-6、CMV)、ヒト免疫不全ウイルス(HIV)、水疱性口内炎ウイルス(VSV)、Bacilli、Citrobacter、Cholera、Diphtheria、Enterobacter、Gonococci、Helicobacter pylori、Klebsiella、Legionella、Meningococci、mycobacteria、Pseudomonas、Pneumonococci、リケッチア細菌、Salmonella、Serratia、Staphylococci、Streptococci、Tetanus、Aspergillus(A.fumigatus、A.niger等)、Blastomyces dermatitidis、Candida(C.albicans、C.krusei、C.glabrata、C.tropicalis等)、Cryptococcus neoformans、Genus Mucorales(mucor、absidia、rhizopus)、Sporothrix schenkii、Paracoccidioides brasiliensis、Coccidioides immitis、Histoplasma capsulatum、Leptospirosis、Borrelia burgdorferi、蠕虫寄生生物(鉤虫、条虫、吸虫、扁形動物(例えば、Schistosomia)、Giardia lambia、trichinella、Dientamoeba Fragilis、Trypanosoma brucei、Trypanosoma cruzi、またはLeishmania donovaniからなる群から選択される、項目156に記載の方法。 (項目158) 複数のガンマデルタT細胞(γδ);複数のナチュラルキラー細胞(NK);またはγδ細胞およびNK細胞の組合せを含む細胞の治療的組成物。 (項目159) 前記γδおよび/またはNK細胞が組換えによって操作されるか、または遺伝子改変され、 必要に応じて前記γδおよび/またはNK細胞が、外因性または異種タンパク質を細胞外に発現するように組換えによって操作されるかまたは遺伝子改変され、 必要に応じて前記外因性、異種、またはキメラタンパク質がキメラ抗原受容体(CAR)または外因性もしくは異種T細胞受容体(TCR)であり、 必要に応じて前記外因性、異種、もしくはキメラタンパク質もしくはCARが、がん細胞もしくは腫瘍マーカーもしくは感染細胞に対して特異的である(特異的に結合することができる)か、または前記外因性、異種、もしくはキメラタンパク質もしくはCARが、がん細胞もしくは腫瘍マーカーもしくは感染細胞もしくは任意の疾患関連抗原を特異的に標的としてこれに結合することができる抗体に対して特異的である(または特異的に結合することができる)、項目158に記載の細胞の治療的組成物。 (項目160) 前記γδおよび/またはNK細胞がヒト細胞または動物細胞である、項目158または項目159に記載の細胞の治療的組成物。 (項目161) 前記治療的組成物が、静脈内(IV)、髄腔内、もしくは筋肉内(IM)投与のために製剤化されるか、または単位剤形で製剤化され、必要に応じて単位投薬量が細胞約102個から細胞1012個の間を含む、項目158~160のいずれかに記載の細胞の治療的組成物。 (項目162) 前記γδおよび/またはNK細胞がin vivo供給源から単離される、項目158~161のいずれかに記載の細胞の治療的組成物。 (項目163) 前記γδおよび/またはNK細胞が培養において拡大されるか、またはin vivo供給源から単離され、培養において拡大される、項目158~162のいずれかに記載の細胞の治療的組成物。 (項目164) 前記γδおよび/またはNK細胞が、in vivo供給源から単離され、いかなるフィーダー細胞も使用することなくまたはフィーダー細胞層を使用することなく培養において拡大され、それによってフィーダー細胞を欠如する拡大したγδおよび/またはNK細胞の集団を生成する、項目163に記載の細胞の治療的組成物。 (項目165) 前記γδおよび/またはNK細胞がin vivo供給源から単離され、フィーダー細胞またはフィーダー細胞層を使用して培養において拡大され、必要に応じて前記フィーダー細胞が実質的に除去され、および/または殺滅されて、フィーダー細胞を実質的に欠如する拡大したγδおよび/またはNK細胞の集団を生成する、項目163に記載の細胞の治療的組成物。 (項目166) 前記γδおよび/またはNK細胞のin vivo供給源が自己供給源に由来する(必要に応じて、前記γδおよび/またはNK細胞のレシピエントとなる個体に由来する)、または外因性、異種、もしくは同種異系供給源に由来する、項目158~165のいずれかに記載の細胞の治療的組成物。 (項目167) がん、腫瘍、機能障害細胞または感染細胞を処置する方法であって、 (a)それを必要とする個体に、先行項目のいずれかに記載の細胞の治療的組成物の治療有効量を投与すること、または (b)(i)先行項目のいずれかに記載の細胞の治療的組成物を提供するまたは提供したこと;および (ii)それを必要とする個体に、前記細胞の治療的組成物の治療有効量を投与することまたは投与したこと を含む方法。 (項目168) それを必要とする前記個体がヒトまたは動物である、項目167に記載の方法。 (項目169) 前記γδおよび/またはNK細胞がin vivo供給源から単離され、必要に応じて前記γδおよび/またはNK細胞のin vivo供給源が、同系もしくは自己供給源に由来する(必要に応じて、前記γδおよび/またはNK細胞のレシピエントとなる個体に由来する)、または外因性、異種、もしくは同種異系供給源、またはその組合せに由来する、項目167または項目168に記載の方法。 (項目170) 前記細胞の治療的組成物が、静脈内(IV)、髄腔内、もしくは筋肉内(IM)投与され、または前記がん、腫瘍、機能障害、もしくは感染細胞にもしくはその近傍に(近くに)注射もしくは埋め込まれ、必要に応じて前記細胞の治療的組成物が埋込物またはゲルとして送達され、必要に応じて前記ゲルがハイドロゲルである、項目167~169のいずれかに記載の方法。 (項目171) 前記細胞の治療的組成物が、単位剤形で投与され、必要に応じて単位投薬量が、細胞約102個から細胞1012個の間、または細胞104個から細胞1010個の間を含み;または1日投与量が細胞約102個から細胞1012個の間、または細胞104個から細胞1010個の間を含む、項目167~170のいずれかに記載の方法。 (項目172) 前記細胞の治療的組成物または単位剤形が、それを必要とする前記個体に数回(複数回)、または数日間、数週間もしくは数ヶ月間の間に2回、3回、4回、5回、6回、7回、8回、9回、もしくは10回またはそれより多く投与され、必要に応じて前記複数の単位剤形の各々が、毎日、2日毎、週に2回、3回、4回、5回、もしくは6回、または週に1回投与される、項目167~171のいずれかに記載の方法。 (項目173) 前記がんまたは腫瘍が、肺がん、黒色腫、乳がん、前立腺がん、結腸がん、腎細胞癌、卵巣がん、神経芽腫、横紋筋肉腫、白血病もしくはリンパ腫、ホジキンリンパ腫もしくは小児急性リンパ芽球性白血病、肥満細胞腫もしくは肥満細胞腫瘍、卵巣がんもしくは癌、膵臓がん、非小細胞肺がん、小細胞肺がん、肝細胞癌、網膜芽腫、乳腺腫瘍、結腸直腸癌、白血病、リンパ腫、急性リンパ芽球性白血病(ALL)もしくは急性リンパ性白血病、急性骨髄性白血病(AML)、組織球肉腫、脳腫瘍、星状細胞腫、膠芽腫、神経腫、結腸癌、子宮頸癌、肉腫、膀胱腫瘍、細網内皮組織の腫瘍、ウィルムス腫瘍、骨がん、骨肉腫、腎臓がん、または頭頸部がん、口腔がん、喉頭がん、または口腔咽頭がんである、項目167~172のいずれかに記載の方法。 (項目174) それを必要とする前記個体が、最初に、がん関連または腫瘍関連、感染症関連または疾患関連抗原に対する非コンジュゲート抗体を、それを必要とする前記個体に予め投与し、その後先行項目のいずれかに記載の細胞の治療的組成物を投与することによって、がん、感染症、または疾患に対する免疫応答を開始するように誘導され、前記細胞の治療的組成物の少なくとも一部が前記非コンジュゲート抗体に特異的に結合するポリペプチド、必要に応じてCARをその細胞表面上に発現する、項目167~173のいずれかに記載の方法。 (項目175) それを必要とする前記個体が、がん関連または腫瘍関連、感染症関連または疾患関連抗原に特異的に結合することが可能な抗体を投与され、その後先行項目のいずれかに記載の細胞の治療的組成物を投与され、前記細胞の治療的組成物の少なくとも一部が前記抗体に特異的に結合するポリペプチド、必要に応じてCARをその細胞表面上に発現し、 必要に応じて前記抗体が、前記細胞の治療的組成物の投与の前に、それと共に、またはその後に投与される、項目167~174のいずれかに記載の方法。 (項目176) 前記がん関連または腫瘍関連抗原が、α-フェトプロテイン(AFP)、α-アクチニン-4、A3、A33抗体に特異的な抗原、ART-4、B7、B7-H3、Ba733、BAGE、BrE3抗原、CA125、CAMEL、CAP-1、炭酸脱水酵素IX、CASP-8/m、CCL19、CCL21、CD1、CD1a、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD22、CD23、CD25、CD29、CD30、CD32b、CD33、CD37、CD38、CD40、CD40L、CD44、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD66a-e、CD67、CD70、CD70L、CD74、CD79a、CD79b、CD80、CD83、CD95、CD123、CD126、CD132、CD133、CD138、CD147、CD154、CDC27、CDK-4/m、CDKN2A、CTLA4、CXCR4、CXCR7、CXCL12、HIF-1α、結腸特異抗原-p(CSAp)、CEA(CEACAM-5)、CEACAM-6、c-Met、DAM、EGFR、EGFRvIII、EGP-1(TROP-2)、EGP-2、ELF2-M、Ep-CAM、線維芽細胞成長因子(FGF)、Flt-1、Flt-3、葉酸受容体、G250抗原、GAGE、gp100、GRO-β、HLA-DR、HM1.24、ヒト絨毛ゴナドトロピン(HCG)およびそのサブユニット、HER2/neu、HMGB-1、低酸素症誘導因子(HIF-1)、HSP70-2M、HST-2、Ia、IGF-1R、IFN-γ、IFN-α、IFN-β、IFN-λ、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-2、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-23、IL-25、インスリン様成長因子-1(IGF-1)、KC4抗原、KS-1抗原、KS1-4、Le-Y、LDR/FUT、マクロファージ遊走阻止因子(MIF)、GD2(神経外胚葉起源の腫瘍に発現されるジシアロガングリオシド)、MAGE、MAGE-3、MART-1、MART-2、NY-ESO-1、TRAG-3、mCRP、MCP-1、MIP-1A、MIP-1B、MIF、MUC1、MUC2、MUC3、MUC4、MUC5ac、MUC13、MUC16、MUM-1/2、MUM-3、NCA66、NCA95、NCA90、膵臓がんムチン、PD1受容体、胎盤成長因子、p53、PLAGL2、前立腺酸性ホスファターゼ、PSA、PRAME、PSMA、PlGF、ILGF、ILGF-R、L-6、IL-25、RS5、RANTES、T101、SAGE、S100、サバイビン、サバイビン-2B、TAC、TAG-72、テネイシン、TRAIL受容体、TNF-α、Tn抗原、トムソン-フリードライヒ抗原、腫瘍壊死抗原、VEGFR、ED-Bフィブロネクチン、WT-1、17-1A抗原、補体因子C3、C3a、C3b、C5a、C5、血管新生マーカー、bcl-2、bcl-6、およびKrasからなる群から選択される抗原である、項目167~175のいずれかに記載の方法。 (項目177) 前記抗体または前記非コンジュゲート抗体が、hR1(抗IGF-1R)、hPAM4(抗ムチン)、KC4(抗ムチン)、hA20(抗CD20)、hA19(抗CD19)、hIMMU31(抗AFP)、hLL1(抗CD74)、hLL2(抗CD22)、RFB4(抗CD22)、hMu-9(抗CSAp)、hL243(抗HLA-DR)、hMN-14(抗CEACAM-5)、hMN-15(抗CEACAM-6)、hRS7(抗TROP-2)、hMN-3(抗CEACAM-6)、CC49(抗TAG-72)、J591(抗PSMA)、D2/B(抗PSMA)、G250(抗炭酸脱水酵素IX)、インフリキシマブ(抗TNF-α)、セルトリズマブペゴル(抗TNF-α)、アダリムマブ(抗TNF-α)、アレムツズマブ(抗CD52)、ベバシズマブ(抗VEGF)、セツキシマブ(抗EGFR)、ゲムツズマブ(抗CD33)、イブリツモマブチウキセタン(抗CD20)、パニツムマブ(抗EGFR)、リツキシマブ(抗CD20)、トシツモマブ(抗CD20)、GA101(抗CD20)、トラスツズマブ(抗HER2/neu)、トシリズマブ(抗IL-6受容体)、バシリキシマブ(抗CD25)、ダクリズマブ(抗CD25)、エファリズマブ(抗CD11a)、ムロモナブ-CD3(抗CD3受容体)、ナタリズマブ(抗α4インテグリン)、BWA-3(抗ヒストンH2A/H4)、LG2-1(抗ヒストンH3)、MRA12(抗ヒストンH1)、PR1-1(抗ヒストンH2B)、LG11-2(抗ヒストンH2B)、およびLG2-2(抗ヒストンH2B)からなる群から選択される、項目174または175に記載の方法。

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Abstract

To provide a method for preparing natural immune cell compositions comprising gamma-delta (γδ) T cells and / or natural killer (NK) cells.SOLUTION: Provided is a method for producing a composition comprising 70% or more γδ T cells derived from a sample comprising cells derived from one or more subjects, the method comprising: a) depleting αβ T cells from the under a condition that generates a depleted cell population comprising NK cells and γδ T cells; b) exposing the depleted cell population to an activation condition comprising contacting the depleted cell population with: (a) an antibody or antigen binding fragment thereof that immunospecifically binds to cell adhesion molecule polypeptide CD2; and (b) an antibody or antigen binding fragment thereof that immunospecifically binds to NKp46; and c) exposing the depleted cell population to an expansion condition comprising contacting the depleted cell population with IL-2, thereby generating the composition comprising 70-75% γδ T cells.SELECTED DRAWING: None
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Description

[Technical Field]

[0001] Related patent applications This patent application claims the benefits of U.S. Provisional Patent Application No. 62 / 703,654, filed on 26 July 2018, titled "Therapeutic Preparations of Gamma-Delta T Cells and Natural Killer Cells and Methods for Making and Using Them," in the possession of Concetta Quintarelli et al., indicated by attorney reference number 6474-131400. The entire contents of the aforementioned application, including all text, tables, and drawings, are incorporated herein by reference for all purposes.

[0002] field This technology relates, in part, to innate immune cell compositions for therapeutic purposes, as well as to methods for preparing and using such compositions. [Background technology]

[0003] The immune response to antigen-presenting pathogens and other foreign antigen-presenting entities includes innate and adaptive defenses. The innate immune response is the first line of defense that is active and persistently functional in the host. Innate immune cells, such as NK cells and gamma-delta (γδ) T cells, do not recognize classical HLA antigens. The adaptive immune response is a response that specifically adapts to antigen-presenting substances, such as foreign bodies, cells, or microorganisms, and often takes several days to mature. Immunotherapy uses innate or modified (e.g., CAR-modified) adaptive immune cells (e.g., alpha-beta (αβ) T cells, dendritic cells, macrophages). For example, αβ T cells expand when they bind to ligands on antigen-presenting substances in vivo, which can take several days or even weeks. Similarly, αβ T cell binding occurs through one or more T cell surface molecules or complexes of molecules including CD4, CD8, and the T cell receptor (TCR), which recognize MHC class I and class II ligands encoded by the HLA gene complex (Miceli et al., Semin.Immunol., 3(3):133-141). (1991). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Miceli et al., Semin. Immunol., 3(3):133-141 (1991) [Overview of the project] [Means for solving the problem]

[0005] overview This specification provides compositions relating to immunology and medicine, methods for preparing such compositions, and methods for treatments using such compositions. In certain embodiments, compositions and methods, including products and kits, are provided for various cell therapies, comprising gamma delta T cells (γδ), natural killer cells (NK), or a combination of the two. Similarly, methods for preparing gamma delta T cells (γδ) and natural killer cells (NK) that can be genetically modified for use in these therapies are also provided. In alternative embodiments, genetically modified gamma delta T cells (γδ) and natural killer cells (NK) can be modified to express chimeric antigen receptors (CARs) or exogenous or heterologous T cell receptors (TCRs), which can then be used to directly or indirectly target any cell surface molecules, such as markers on cancer cells or infected cells.

[0006] In a particular embodiment, a method is provided for producing a composition containing a population of cells enriched with respect to NK cells and gamma delta T cells. In a particular embodiment of the method provided herein, a sample obtained from a donor (e.g., a tissue, organ, or blood sample from a subject that is a healthy subject or a patient treated by a population of cells) is exposed to an expansion condition which includes (a) an activation condition which includes at least one exogenous polypeptide that is immunospecifically bound to a cell adhesion polypeptide, and (b) an exogenous polypeptide that is immunospecifically bound to a polypeptide different from the cell adhesion polypeptide and expressed on the surface of one or more types of cells in the sample, thereby producing a composition containing a population of cells enriched with respect to NK cells and gamma delta T cells. The sample is sometimes exposed to the expansion condition sequentially after the activation condition. The methods provided herein involve about 30% to about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more of the cells in a population, or at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61% This can result in high levels of activation of up to 100%, including %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0007] The exogenous polypeptides and auxiliary polypeptides used in the methods provided herein can, in part, be readily identified, isolated, synthesized, or otherwise obtained, including from commercially available sources, based on available nucleic acids and amino acid sequences, as well as other knowledge regarding cell adhesion molecules and other immune cell molecules and receptors. In certain embodiments, the exogenous polypeptide is human. In some embodiments, the exogenous polypeptide is isolated. In certain embodiments, the auxiliary polypeptide is human. In some embodiments, the auxiliary polypeptide is isolated.

[0008] In certain embodiments of the methods provided herein, a sample is depleted of alpha-beta T cells and then exposed to activation and expansion conditions, and the resulting depleted cell population is then occasionally exposed to activation and expansion conditions. In certain embodiments, the exogenous polypeptide of (a) above, the exogenous polypeptide of (b) above, or both the exogenous polypeptide of (a) and the exogenous polypeptide of (b) above are soluble. In some embodiments, the exogenous polypeptide of (a) or the exogenous polypeptide of (b) is bound to a solid-phase substrate.

[0009] In some embodiments of the methods provided herein, at least one auxiliary polypeptide is selected such that the amount of NK cells relative to the amount of gamma-delta T cells in a population is dependent on the amount and / or type of at least one auxiliary polypeptide. In certain embodiments, the auxiliary polypeptide increases or decreases the amount of NK cells relative to gamma-delta T cells in a population of cells after contacting a depleted cell population with at least one auxiliary polypeptide. In certain embodiments, the duration of exposure of a sample or alpha-beta T cell depleted sample to expansion conditions is selected such that a desired ratio of NK cells to gamma-delta T cells is obtained.

[0010] In certain embodiments of the methods provided herein, the activation conditions do not include serum derived from non-human animals. In some embodiments of the methods provided herein, the expansion conditions do not include serum derived from non-human animals.

[0011] In the methods provided herein, a high percentage of cells are activated, often without the use of feeder cells. At times, at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 7 2%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, up to 100%, are activated without the use of feeder cells. Thus, in certain embodiments of the methods provided herein, the activation conditions do not include feeder cells. In some embodiments of the methods provided herein, the expansion conditions do not include feeder cells, and sometimes both the activation and expansion conditions do not include feeder cells.

[0012] Immune cells from any source can be used as a sample in the methods provided herein. In certain embodiments, the sample is selected from bone marrow, peripheral blood, liver tissue, epithelial tissue, and umbilical cord blood. In some embodiments of the methods provided herein, the sample does not originate from an embryonic source. In certain embodiments of the methods provided herein, the sample is peripheral blood, and in some embodiments, the peripheral blood sample is a processed sample that is processed before being subjected to alpha-beta T cell depletion in the methods provided herein. For example, a peripheral blood sample can be processed by density gradient centrifugation to separate and / or isolate a buffy coat containing leukocytes, platelets, granulocytes, etc., which can then be subjected to alpha-beta T cell depletion according to the methods provided herein. In certain embodiments, the buffy coat can be further subjected to Ficoll density gradient separation to obtain mononuclear cells (PBMCs), which can then be subjected to alpha-beta T cell depletion in the methods provided herein. In some embodiments, a peripheral blood sample may be apheresis to separate the plasma from the cells, and the cells may then be subjected to alpha-beta T cell depletion in the method provided herein. In certain embodiments of the method provided herein, the sample is umbilical cord blood, and sometimes the umbilical cord blood is treated umbilical cord blood that is processed before being subjected to alpha-beta T cell depletion in the method provided herein.

[0013] In certain embodiments of the methods provided herein, the exogenous polypeptide (b) binds immunospecifically to NK cell activating receptors, gamma-delta T cell activating receptors, or both. Such receptors include, but are not limited to, CD2, CD3, CD56, NKp30, NKp44, NKp46, NKG2A, NKG2C, NKG2D, KAR receptor, KIR receptor, SIGLEC-7, KIR3DS1, KIR3D51, KIR2DL1 (antibody: 11PB6), DNAM1, NTBA, HLA-DR, etc. In some embodiments, the receptor is NKp46. In certain embodiments of the methods provided herein, the exogenous polypeptide (a) binds immunospecifically to CD2. In some embodiments, the exogenous polypeptide (a) or (b), or (a) and (b), is an antibody or its antigen-binding fragment. For the purposes of this specification, the enumeration of “antibodies” includes full-length antibodies and some thereof, including antibody fragments. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotype (anti-Id) antibodies, or any of the antigen-binding fragments described above. Antibodies also include synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, and intrabodies. Antibodies may include any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or members of subclasses (e.g., IgG2a and IgG2b).

[0014] In certain embodiments of the methods provided herein, the activation condition comprises contacting a sample or depleted cell population with at least two exogenous polypeptides. In some embodiments, the first exogenous polypeptide is immunospecifically bound to CD2, and the second exogenous polypeptide is immunospecifically bound to NKp46. In some embodiments, the activation condition comprises, or is essentially, the first exogenous polypeptide immunospecifically bound to CD2 and the second exogenous polypeptide immunospecifically bound to NKp46. In certain embodiments, the exogenous polypeptide immunospecifically bound to CD2, the exogenous polypeptide immunospecifically bound to NKp46, or both the exogenous polypeptide immunospecifically bound to CD2 and the exogenous polypeptide immunospecifically bound to NKp46 are soluble. In certain embodiments, the first exogenous polypeptide and / or the second exogenous polypeptide are antibodies or antigen-binding fragments thereof.

[0015] In certain embodiments of the methods provided herein, the extension condition comprises, or is essentially derived from, at least one accessory polypeptide that is a cytokine, and / or a polypeptide that binds immunospecifically to a receptor on a gamma-delta T cell, and / or a portion thereof that binds immunospecifically to a receptor on a gamma-delta T cell. In certain embodiments, the cytokine is an interleukin, e.g., IL-1 (see, e.g., GenBank accession number BC008678.1), IL-2 (see, e.g., GenBank accession number S77834.1), IL-4 (see, e.g., GenBank accession number BC070123.1), IL-7 (see, e.g., GenBank accession number BC047698.1), IL-9 (see, e.g., GenBank accession number BC066285.1), IL-15 (see, e.g., GenBank accession numbers BC100962.1; 100963.1; 100961.1), IL-21 (see, e.g., GenBank accession number LC133256.1), or any combination thereof. In some embodiments of the methods provided herein, the cytokine is IL-2, IL-15, or a combination thereof. In certain embodiments, the extended conditions This comprises, or is essentially comprised of, polypeptides that immunospecifically bind to receptors on gamma-delta T cells, IL-2, IL-15, and gamma-delta T cells. In certain embodiments, the receptor on gamma-delta T cells is CD3. In some embodiments, the polypeptide that immunospecifically binds to the CD3 receptor on gamma-delta T cells is an antibody or its antigen-binding fragment, and in certain embodiments, the antibody is OKT3. In certain embodiments, the expansion condition comprises contacting a sample or depleted cell population with (a) IL-2 polypeptide; (b) IL-15 polypeptide; (c) IL-2 polypeptide and IL-15 polypeptide; (d) IL-2 polypeptide and an antibody that immunospecifically binds to CD3; or (e) IL-2 polypeptide, IL-15 polypeptide, and an antibody that immunospecifically binds to CD3. In some embodiments, the antibody that immunospecifically binds to CD3 is OKT3.

[0016] In certain embodiments, the expansion condition involves sequentially exposing a sample (e.g., a source sample or a sample cell population depleted of alpha and beta T cells) to one or more condition sets. In some embodiments, the sample is exposed to two expansion condition sets. In several embodiments, a population of cells exposed to a first expansion condition set is washed and then exposed to a second expansion condition set. In certain embodiments, (a) the first condition set includes IL-2 and the second condition set includes IL-15; (b) the first condition set includes IL-15 and the second condition set includes IL-2; (c) the first condition set includes antibodies that immunospecifically bind to IL-2 and CD3 and the second condition set includes IL-15; or (d) the first condition set includes antibodies that immunospecifically bind to IL-15 and CD3 and the second condition set includes antibodies that immunospecifically bind to IL-2 and CD3.

[0017] In certain embodiments of the methods provided herein, a first expansion set of conditions, comprising one or more auxiliary polypeptides, often results in a first cell population comprising a first ratio of NK cells to gamma-delta T cells, which can then be fine-tuned using a second expansion set of conditions to obtain a desired final ratio of NK cells to gamma-delta T cells, wherein the first set of conditions is different from the second set of conditions.

[0018] The methods provided herein allow a cell population to be exposed to activation and expansion conditions simultaneously or sequentially in any order. Furthermore, exogenous polypeptides may function as auxiliary polypeptides, and / or vice versa.

[0019] The immune cell compositions obtained by the methods provided herein are enriched with respect to innate immune cells, namely NK cells and gamma-delta T cells, compared to immune cell compositions found in nature (e.g., biological fluids or tissues). In nature, adaptive immune cells, namely alpha-beta T cells, are found in considerably larger quantities than NK cells and gamma-delta T cells. In contrast, in the compositions provided herein, alpha-beta T cells are absent or present in negligible to small amounts, with NK cells and gamma-delta T cells being the main immune cell components.

[0020] Depending on the expansion conditions, the innate immune cell-enriched composition obtained by the method provided herein may contain different amounts of NK cells compared to gamma-delta T cells. In certain non-limiting examples, (i) when the coordinating polypeptide is IL-2, the resulting cell population enriched with respect to NK cells and gamma-delta T cells often contains about 25-30% NK cells and about 70-75% gamma-delta T cells; (ii) when the coordinating polypeptide is IL-15, the resulting cell population enriched with respect to NK cells and gamma-delta T cells often contains about 80-99% NK cells and about 1-20% gamma-delta T cells; (iii) when the coordinating polypeptide is an antibody that immune-specifically binds to IL-2 and CD3, e.g., OKT3, the resulting cell population enriched with respect to NK cells and gamma-delta T cells often contains about 40- (iv) When the co-auxiliary polypeptide is IL-2 until day 20 of the extended condition and then switched to IL-15 until day 30, the percentage of gamma-delta T cells often increases from approximately 50% to approximately 70% compared to treatment with IL-2 alone, and often the percentage of NK cells decreases in a corresponding manner; and (v) when the co-auxiliary polypeptide is IL-15 until day 20 of the extended condition and then switched to IL-2 until day 30, the percentage of NK cells often increases from approximately 80% to approximately 90% compared to treatment with IL-15 alone, and often the percentage of gamma-delta T cells decreases in a corresponding manner.

[0021] In certain embodiments of the methods provided herein, the sample or depleted cell population is not exposed to the conditions selected for NK cells or gamma-delta T cells. In some embodiments, the sample or depleted cell population is not exposed to the conditions that deplete cells other than alpha-beta T cells.

[0022] Cells of compositions prepared by the methods provided herein can be further genetically modified to express exogenous polynucleotides such as tumor necrosis factor receptors, chimeric antigen receptors (CARs), myeloid differentiation primary response proteins, or innate immune signaling adapters. Cells can also be modified to mutate or deplete one or more polypeptides.

[0023] In certain embodiments of the methods provided herein, a composition containing gamma delta cells and NK cells can be subjected to a treatment to obtain a composition comprising or essentially consisting of NK cells or gamma delta cells. The treatment may be depletion, for example, obtaining NK cells by depleting all gamma delta (CD3+) cells using an anti-CD3 antibody, or positive selection, for example, selecting gamma delta T cells using an anti-CD3 antibody. In some embodiments, the anti-CD3 antibody, for example, the OKT3 antibody, can be conjugated to a solid phase.

[0024] In embodiments of the methods provided herein, the activation conditions, expansion conditions, or activation and expansion conditions include incubation of the sample or depleted cell population in feeder cell-free medium for approximately 1 hour, 2 hours, 5 hours, 10 hours, 12 hours, 15 hours, 20 hours, or several days, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, or 60 days, or more, or several weeks, for example, approximately 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In certain embodiments, the activation condition is a period ranging from approximately 1 hour, 5 hours, 10 hours, 12 hours, 15 hours, or 20 hours to approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week. In some embodiments, the activation condition is a period ranging from approximately 12 hours, 24 hours, 36 hours, or 2 days to approximately 3 days, 4 days, 5 days, 6 days, or 1 week, or from approximately 2 days to approximately 4 or 5 days, or from approximately 3 days to approximately 4 days. In certain embodiments, the expansion condition is a period ranging from 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week. In certain embodiments, the expansion condition is implemented in a continuous cycle, with each cycle independently running for approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 1 week, 2 weeks, 3 weeks, or longer. In some embodiments, the number of expansion cycles is greater than 1, for example, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 cycles. In certain embodiments, each expansion cycle is approximately 7 days long. In some embodiments, the number of expansion cycles is 3.

[0025] In some embodiments, the enlarged cell population enriched with enlarged NK cells and gamma-delta T cells does not contain exhausted cells. In certain embodiments, the enlarged cell population enriched with enlarged NK cells and gamma-delta T cells does not contain exhausted cells after 60 days of enlargement conditions.

[0026] In certain embodiments, a population of cells enriched with respect to NK cells and gamma delta T cells obtained by the method provided herein contains 80% or more innate immune cells. In some embodiments, between about 80% and about 100% of the cells, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, up to 100%, are innate immune cells.

[0027] In some embodiments of the methods provided herein, the activation condition, expansion condition, or both the activation condition and the expansion condition do not contain a bisphosphonate. Examples of exemplary bisphosphonates include, but are not limited to, clodronate, etidronate, alendronate, pamidronate, zoledronate (zoledronic acid), and neridronate.

[0028] In certain embodiments of the methods provided herein, the compositions obtained by the methods provided herein contain gamma delta T cells that are polyclonal with respect to V. delta.1 and V. delta.2 expression. The relative amounts of V. delta.1 and V. delta.2 cells can be adjusted by one or more of the expansion conditions, e.g., the absence of bisphosphonates, the selection of auxiliary polypeptides, and the duration for which the sample or depleted cell population is subjected to the expansion conditions. In certain non-limiting examples, if the expansion conditions include polypeptides that immunospecifically bind to IL-2 and CD3 (e.g., OKT3), then (i) V. delta.1 cells may be between about 75% and about 95% of gamma delta T cells, sometimes between about 80% and about 90% of gamma delta T cells, and (ii) V. delta.2 cells may be between about 10% and about 25% of gamma delta T cells, sometimes between about 10% and about 15%, or 20% of gamma delta T cells. In certain embodiments, if the expansion condition includes IL-2 (e.g., does not include IL-15 and does not include polypeptides that immune-specifically bind to CD3), then (i) V. delta.1 cells may be between approximately 30% and 60% of gamma delta T cells, sometimes between approximately 35% and 55% of gamma delta T cells, and (ii) V. delta.2 cells may be between approximately 35% and 60% of gamma delta T cells, sometimes between approximately 40% and 50%, or 55%, of gamma delta T cells. In certain embodiments, if the expansion condition includes IL-15 (e.g., does not include IL-2 and does not include polypeptides that immune-specifically bind to CD3), then (i) V. delta.1 cells may be between about 10% and about 30% of gamma delta T cells, sometimes between about 20% and about 25% of gamma delta T cells, and (ii) V. delta.2 cells may be between about 65% and about 80% of gamma delta T cells, sometimes between about 70% and about 75%, or even 80% of gamma delta T cells.

[0029] In certain embodiments, a composition is provided herein that contains a cell population comprising a plurality of NK cells and a plurality of gamma-delta T cells, and in which alpha-beta T cells are depleted. In some embodiments, the composition does not contain feeder cells. Similarly, in certain embodiments herein, a composition is provided herein that contains a cell population comprising a plurality of NK cells and a plurality of gamma-delta T cells, in which alpha-beta T cells are depleted, and in which feeder cells are not. In certain embodiments, the cell population is a population of modified peripheral blood cells. In some embodiments, the compositions provided herein contain (i) NK cells between about 25% and about 45% and gamma-delta T cells between about 55% and about 75%; (ii) NK cells between about 25% and about 30% and gamma-delta T cells between about 70% and about 75%; (iii) NK cells between about 80% and about 99% and gamma-delta T cells between about 1% and about 20%; or (iv) NK cells between about 40% and about 45% and gamma-delta T cells between about 55% and about 60%.

[0030] In certain embodiments, 30% or more of the cells in the compositions provided herein are activated. In some embodiments, between about 30% to about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more of the cells in a population, or at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 6% of the cells in a population are activated. 0%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, up to 100%.

[0031] In some embodiments, the compositions provided herein contain a population of cells comprising one or more of the following activation markers as a percentage of the total number of cells in the population: (a) 90% or more of the cells in the population express KIR5; (b) 10% or more of the cells in the population express SIGLEC-7; (c) 60% or more of the cells in the population express KIR3D51; (d) 10% or more of the cells in the population express KIR2DL1; (e) 25% or more of the cells in the population express NKp30, NKp44, and / or NKp46; (f) 35% or more of the cells in the population express NKG2D; (g) 90% or more of the cells in the population express DNAM1; (h) 85% or more of the cells in the population express NTBA; and (i) 95% or more of the cells in the population express CD2.

[0032] In certain embodiments, the compositions provided herein contain 80% or more innate immune cells. In some embodiments, the compositions are enriched with respect to activated cytotoxic cells that are CD56+; in certain embodiments, between about 80% and about 100% of the cells, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are CD56+. In some embodiments, the compositions provided herein are enriched with respect to activated cytotoxic cells that are CD57-. In certain embodiments, the compositions provided herein are enriched with respect to activated cytotoxic cells that are CD56+CD57-. In some embodiments, between about 10% and about 40% of the cells, or at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% of the cells are CD16+. In certain embodiments, less than 5%, less than 4%, less than 3%, or less than 2% of the cells in the composition provided herein are CD57+.

[0033] In certain embodiments, the compositions provided herein are substantially free of cells other than NK cells and gamma delta T cells. In some embodiments, the compositions provided herein contain about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.9% or less, about 0.8% or less, about 0.7% or less, about 0.6% or less, about 0.5% or less, about 0.4% or less, about 0.3% or less, about 0.2% or less, about 0.1% or less, or less NKT The composition contains cells and / or approximately 5% or less, approximately 4% or less, approximately 3% or less, approximately 2% or less, approximately 1% or less, approximately 0.9% or less, approximately 0.8% or less, approximately 0.7% or less, approximately 0.6% or less, approximately 0.5% or less, approximately 0.4% or less, approximately 0.3% or less, approximately 0.2% or less, approximately 0.1% or less, or less alpha-beta T cells. In certain embodiments, the subset of NK cells in the composition is CD16+ cells. In some embodiments, the majority of gamma-delta T cells, or the majority of NK cells, or the majority of both gamma-delta T cells and NK cells are CD57- cells.

[0034] In certain embodiments, the gamma delta T cells of the compositions provided herein are polyclonal with respect to V. delta 1 and V. delta 2 expression. In some embodiments, the gamma delta T cell population of the compositions provided herein is: (i) between about 75% and about 95% V. delta 1 cells, e.g., between about 80% and about 90% V. delta 1 cells; and between about 5% and about 25% V. delta 2 cells, e.g., between about 10% and about 15% or 20% V. delta 2 cells; or (ii) between about 30% and about 60% V. delta 1 cells, e.g., between about 35% and about 55% (iii) containing V. delta.1 cells and V. delta.2 cells between approximately 35% and 60%, for example between approximately 40% and 50% or 55%; or (iii) containing V. delta.1 cells between approximately 10% and 30%, for example between approximately 20% and 25%; and V. delta.2 cells between approximately 65% ​​and 80%, for example between approximately 70% and 75% or 80%.

[0035] In certain embodiments, the composition provided herein contains between approximately 50% and approximately 99% or more of NK cells and / or gamma delta T cells, or approximately 50% or more, approximately 51% or more, approximately 52% or more, approximately 53% or more, approximately 54% or more, approximately 55% or more, approximately 56% or more, approximately 57% or more, approximately 58% or more, approximately 59% or more, approximately 60% or more, approximately 61% or more, approximately 62% or more, approximately 63% or more, approximately 64% or more, approximately 65% ​​or more, approximately 66% or more, approximately 67% or more, approximately 68% or more, approximately 69% or more, approximately 70% or more, approximately 71% More than or equal to, approximately 72% more than or equal to, approximately 73% more than or equal to, approximately 74% more than or equal to, approximately 75% more than or equal to, approximately 76% more than or equal to, approximately 77% more than or equal to, approximately 78% more than or equal to, approximately 79% more than or equal to, approximately 80% more than or equal to, approximately 81% more than or equal to, approximately 82% more than or equal to, approximately 83% more than or equal to, approximately 84% more than or equal to, approximately 85% more than or equal to, approximately 86% more than or equal to, approximately 87% more than or equal to, approximately 88% more than or equal to, approximately 89% more than or equal to, approximately 90% more than or equal to, approximately 91% more than or equal to, approximately 92% more than or equal to, approximately 93% more than or equal to, approximately 94% more than or equal to, approximately 95% more than or equal toCD8+ is approximately 96% or more, 97% or more, 98% or more, 99% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or any fraction thereof, up to 100%. In some embodiments, less than 2% of NK cells and / or gamma-delta T cells are CD4+. In multiple embodiments, less than 2% of NK cells and / or gamma-delta T cells are CD8+CD4+. In certain embodiments, a fraction of approximately 15% to 30% of NK cells and / or approximately 55% to 85% of gamma-delta T cells are CD8-CD4-.

[0036] In certain embodiments of the compositions provided herein, between about 30% and about 99% or more of the cells in a population, or at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70% %, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or fractions thereof, up to 100%, further comprises gene modifications including exogenous polynucleotides, mutant polynucleotides, deletion polynucleotides, or combinations thereof. In some embodiments, the gene modification includes exogenous polynucleotides. Exogenous polynucleotides are sometimes found in retroviral vectors or lentiviral vectors, and sometimes they are incorporated into the genomes of one or more cell species in a modified cell population.

[0037] Exogenous polynucleotides may, in certain embodiments, encode exogenous or heterologous T cell receptors, tumor necrosis factor receptors, chimeric antigen receptors (CARs), myeloid differentiation primary response proteins, and innate immune signaling adapters, or other proteins or polypeptides of interest, and in some embodiments may include promoters or other regulators of gene expression. In some embodiments, the exogenous polynucleotide is a regulatory sequence, such as a promoter or enhancer.

[0038] In certain embodiments, the exogenous polynucleotide encodes a chimeric antigen receptor (CAR), and the cells in the composition contain the CAR. The CAR is a recombinant receptor that provides both antigen-binding and T-cell activation functions (e.g., Sadelain et al., Cancer Discov., See 3(4):388-398 (2013). When immune cells, such as T cells, are engineered (genetically modified) to express CARs, this provides the immune cells with a novel and / or improved ability to target a protein or antigen of interest. In certain embodiments, the target protein or antigen of interest may be a cancer antigen or an infectious disease antigen, some of which are known and / or identifiable in the art. In some embodiments, the CAR may be CD19 (see, e.g., GenBank accession number AH005421.2), GD2 (diciaroganglioside; see, e.g., Schulz et al., Cancer Res., 44(12):5914-5920 (1984)), HER3 (see, e.g., GenBank accession number AH005421.2). It contains a binding molecule moiety that immunospecifically binds to one or more of the following: (see GenBank accession number M34309.1), B7H3 (see, for example, GenBank accession number BC062581.1), CD123 (see, for example, GenBank accession number BC035407.1; BX296563.3), or CD30 (see, for example, GenBank accession number M83554.1; AY498860.1).

[0039] Any composition provided herein can be further treated to remove either gamma-delta T cells or NK cells, thereby producing a composition containing substantially all NK cells or substantially all gamma-delta T cells. For example, a composition provided herein can be treated with an anti-CD3 antibody to deplete the gamma-delta T cells from a mixture of NK cells and gamma-delta T cells, leaving a composition containing substantially all or all NK cells, or alternatively, an anti-CD3 antibody can be used to isolate a substantially pure gamma-delta T cell population from the mixture.

[0040] In certain embodiments, a therapeutic composition (or therapeutic combination) of cells is provided, comprising a plurality of gamma delta T cells (γδ); a plurality of natural killer cells (NK); or a combination of γδ and NK cells. In certain embodiments, the γδ and / or NK cells are recombinantly engineered or genetically modified, in which case the γδ and / or NK cells are recombinantly engineered or genetically modified to express an exogenous or heterologous protein extracellularly, in which case the exogenous, heterologous, or chimeric protein is a chimeric antigen receptor (CAR) or an exogenous or heterologous T cell receptor (TCR), in which case the exogenous, heterologous, or chimeric protein or CAR is specific to (can specifically bind to) cancer cells or tumor markers or infected cells, or the exogenous, heterologous, or chimeric protein or CAR is specific to (can specifically bind to) an antibody that can specifically target and bind to cancer cells or tumor markers or infected cells, or any disease-associated antigen.

[0041] In some embodiments, γδ and / or NK cells are human or animal cells. In certain embodiments, the therapeutic composition (or therapeutic combination) is formulated for intravenous (IV), intrathecal, intramuscular (IM), intraperitoneal (IP), or intratumoral (IT) administration, intraarticular, or injected or implanted at or near the site of cancer or infection, or as needed for about 10 2 from 10 12 Formulated in unit dosage forms containing cells between individuals,

[0042] Alternatively, it may be formulated for intramuscular (IM) administration, or approximately 10 times as needed. 2 from 10 12 It is formulated as a unit dosage form containing cells between individuals.

[0043] In some embodiments, γδ and / or NK cells are isolated from an in vivo source. In some embodiments, γδ and / or NK cells are expanded in culture, or isolated from an in vivo source and expanded in culture. In certain embodiments, γδ and / or NK cells are isolated from an in vivo source and expanded in culture without the use of any feeder cells or feeder cell layers, thereby generating an expanded population of γδ and / or NK cells lacking feeder cells. In some embodiments, γδ and / or NK cells are isolated from an in vivo source and expanded in culture using feeder cells or feeder cell layers, with feeder cells substantially removed and / or killed as necessary, thereby generating an expanded population of γδ and / or NK cells substantially lacking feeder cells.

[0044] In certain embodiments, the in vivo source of γδ and / or NK cells is either self-sourced (derived from the individual that will be the recipient of the γδ and / or NK cells, if applicable) or derived from exogenous, heterologous, or homogeneous sources.

[0045] Similarly, in certain embodiments herein, pharmaceutical compositions comprising any of the compositions provided herein and a pharmaceutically acceptable carrier are also provided.

[0046] In certain embodiments, methods for producing genetically modified immune cells are provided herein by adding exogenous polynucleotides to compositions provided herein, mutating polynucleotides in one or more cell types of compositions provided herein, or deleting polynucleotides in one or more cell types of compositions provided herein. In some embodiments, the genetic modification is an exogenous polynucleotide. Methods used to produce compositions provided herein often result in highly activated cells that facilitate the introduction of exogenous polynucleotides, for example, by retroviral or lentiviral transduction. In certain embodiments, the methods for producing genetically modified immune cells provided herein often involve a ratio of approximately 30% to approximately 99% or more of cells, or at least approximately 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%. , 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any fraction thereof, up to 100%, results in a composition containing genetically modified material.

[0047] In certain embodiments, provided herein is a kit comprising any one of the compositions or pharmaceutical compositions provided herein, instructions for use as needed, and cytokines as needed. The compositions, pharmaceutical compositions, or kits provided herein can be stored at refrigerated temperatures, such as 10 degrees Celsius or lower, such as 9 degrees Celsius, 8 degrees Celsius, 7 degrees Celsius, 6 degrees Celsius, 5 degrees Celsius, 4 degrees Celsius, 3 degrees Celsius, 2 degrees Celsius, 1 degree Celsius, minus 4 degrees Celsius or lower, or at freezing temperatures (e.g., minus 15 degrees Celsius, minus 20 degrees Celsius, minus 25 degrees Celsius, minus 30 degrees Celsius, minus 35 degrees Celsius, minus 40 degrees Celsius, minus 45 degrees Celsius, minus 50 degrees Celsius, minus 55 degrees Celsius, minus 60 degrees Celsius, minus 65 degrees Celsius, minus 70 degrees Celsius, minus 75 degrees Celsius, minus 80 degrees Celsius, minus 85 degrees Celsius or lower), as needed for storage and / or transportation. In certain embodiments, the kit comprises between about 1×10 5 cells and about 1×10 12 cells, such as about 1×10 6 cells, 1×10 7 cells, 1×10 8 cells, 1×10 9 cells, or 1×10 10It contains [number] units. In certain embodiments, the kits provided herein may contain cytokines. In some embodiments, the cytokines may include TNF (see, e.g., GenBank accession number KJ892290.1; AY214167.1), IFNγ (see, e.g., GenBank accession number J00219.1), interleukins IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10 (see, e.g., GenBank accession number U16720.1), and IL-12 (see, e.g., GenBank accession number AF404773.1 for IL-12A; e.g., IL For -12B, see GenBank accession number AF512686.1), IL-15, IL-18 (see, for example, GenBank accession numbers BC007461.1;BC007007.1), IL-21, CCL4 (see, for example, GenBank accession numbers CR542119.1;KJ901727.1;KJ901726.1), RANTES (see, for example, GenBank accession number GQ504011.1), and TGFβ (see, for example, National Center for Biotechnology Information (NCBI) accession number NM_000660.7). In certain embodiments, the kit contains the composition or pharmaceutical composition provided herein in unit dosage form.

[0048] In certain embodiments, products and kits are provided for practicing the methods provided herein, including therapeutic combinations of cells provided herein. In certain embodiments, the products and kits further include instructions for practicing the methods provided herein. In some embodiments, the products and kits further include antibodies capable of specifically binding to cancer-related, or tumor-related, infection-related, or disease-related antigens. The products provided herein may include implants containing therapeutic combinations of cells provided herein.

[0049] Similarly, in certain embodiments, a method for treating cancer or infection is provided herein, comprising administering to a subject in need of such treatment one of the compositions, pharmaceutical compositions, or kits provided herein in an amount effective for treating cancer or infection. The treatment may be administered in an autologous or allogeneic context. In some embodiments, the donor of the sample from which the composition, pharmaceutical composition, or kit is produced is the recipient of the treatment. In certain embodiments, the treatment may be administered on two or more separate days, and in certain embodiments, the treatment may be administered in multiple doses. In certain embodiments, the treatment may be administered at intervals of about two weeks to about four weeks, in doses ranging from about one unit to about 36 or more units. In some embodiments, the treatment is administered as a single unit dose once, twice, three, four, or up to five times daily, or once, two, three, four, five, six, seven, eight, nine, or ten times, or more times, over several days, weeks, or months, or every two days, or once, two, three, four, five, or six times per week. The treatment can be administered intravenously (IV), intrathecally, or intramuscularly (IM), intraperitoneally (IP), intrapleurally, or intraarticularly, or to the site of cancer or infection or its vicinity, with approximately 10 cells per kilogram of the subject's body weight. 4 From about 10 10 Between individuals, or approximately 10 cells per subject 6 From about 10 12 It is administered by injection or implantation in unit doses per individual. In certain embodiments, the unit dose is approximately 10 cells per subject. 10 Approximately 10 cells per kilogram of body weight of the subject. 8 It is an individual.

[0050] In certain embodiments of the treatment methods provided herein, the treatment is for cancer. In some embodiments, cancer is selected from lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia or lymphoma, Hodgkin lymphoma or pediatric acute lymphoblastic leukemia, non-Hodgkin lymphoma, mast cell tumor or mast cell tumor, ovarian cancer or cancer, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, retinoblastoma, mammary gland tumor, colorectal cancer, leukemia, lymphoma, acute lymphoblastic leukemia (ALL) or acute lymphoblastic leukemia, acute myeloid leukemia (AML), histiocytic sarcoma, brain tumor, astrocytoma, glioblastoma, neuroma, colon cancer, cervical cancer, sarcoma, bladder tumor, reticuloendothelial tumor, Wilms' tumor, bone cancer, osteosarcoma, kidney cancer, or head and neck cancer, oral cancer, laryngeal cancer, metastatic disease, or oropharyngeal cancer.

[0051] In certain embodiments of the treatment methods provided herein, the second agent is co-administered with a composition, pharmaceutical composition, or kit. In some embodiments, the second agent is an antibody that binds immunospecifically to a cancer-associated antigen. In certain embodiments, cancer-associated antigens include α-fetoprotein (AFP), α-actinin-4, antigens specific to A3, A33 antibodies, ART-4, B7, B7-H3, Ba733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, C D66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD123, CD126, CD132, CD133 , CD138, CD147, CD154, CDC27, CDK-4 / m, CDKN2A, CTLA4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen- p(CSAp), CEA(CEACAM-5), CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, EGP-1(TROP-2), EGP-2, ELF 2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-β, HLA-DR, HM1.24. Human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, HMGB-1, hypoxia-inducing factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL -15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor 1 (IGF-1), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR / FUT, macrophage migration Inhibiting factor (MIF), GD2, MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC 1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, PD1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-R, L-6, IL-25, RS5, RANTES, T101, SAGE The following are selected from the group consisting of S100, Survivin, Survivin-2B, TAC, TAG-72, Tenascin, TRAIL receptor, TNF-α, Tn antigen, Thomson-Friedreich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factors C3, C3a, C3b, C5a, C5, angiogenesis markers, bcl-2, bcl-6, and Kras.

[0052] In certain embodiments, cells of the compositions provided herein can be manipulated to express antibodies that bind to cancer-related antigens. In some embodiments, the antibodies are co-administered with a second agent. In certain embodiments, the cancer-related antigens include hR1 (anti-IGF-1R), hPAM4 (anti-mucin), KC4 (anti-mucin), hA20 (anti-CD20), hA19 (anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22), anti-CD19 / CD22 bispecific antibody, RFB4 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 ( Anti-CEACAM-5), hMN-15 (anti-CEACAM-6), hRS7 (anti-TROP-2), hMN-3 (anti-CEACAM-6), CC49 (anti-TAG-72), J591 (anti-PSMA), D2 / B (anti-PSMA), G250 (anti-carbonic anhydrase IX), dinutuximab (anti-GD2), infliximab (anti-TNF-α), certolizumab pegol (anti-TNF-α), adalimumab (anti-TNF-α), Alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab (anti-CD20), GA101 (anti-CD20), trastuzumab (anti-HER2 / neu), tocilizumab (anti-IL-6 receptor), basiliximab (anti-C) The following are selected: D25), daclizumab (anti-CD25), efalizumab (anti-CD11a), muromonab-CD3 (anti-CD3 receptor), natalizumab (anti-α4 integrin), BWA-3 (anti-histone H2A / H4), LG2-1 (anti-histone H3), MRA12 (anti-histone H1), PR1-1 (anti-histone H2B), LG11-2 (anti-histone H2B), and LG2-2 (anti-histone H2B).

[0053] In certain embodiments of the treatment methods provided herein, the treatment is for an infection. In some embodiments, the infection is characterized by the presence of a bacterial pathogen, fungal pathogen, viral pathogen, or protozoan pathogen. In embodiments of the treatment methods provided herein, the infection is herpes, Ebola, West Nile virus, vaccinia virus, Epstein-Barr virus, hepatitis A virus (HAV); hepatitis B virus (HBV); hepatitis C virus (HCV); herpesviruses (e.g., HSV-1, HSV-2, HHV-6, CMV), human immunodeficiency virus (HIV), varicella-stomatitis virus (VSV), Bacilli, Citrobacter, Cholera, Diphtheria, Enterobacter, Gonococci, Helicobacter pylori, Klebsiella, Legionella, Meningococci, mycobacteria, Pseudomonas, Pneumonococci, Rickettsia bacteria, Salmonella, Serratia, Staphylococci, Streptococci, Tetanus, Aspergillus (A.fumigatus, A.niger, etc.), Blastomyces dermatitidis, Candida (C.albicans, C.krusei, C.glabrata, C.tropicalis, etc.), Cryptococcus neoformans, Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Paracoccidioides brasiliensis, Coccidioides immitis, Histoplasma capsulatum, Leptospirosis, Borrelia The group is selected from burgdorferi, helminth parasites (hookworms, tapeworms, trematodes, flatworms (e.g., Schistosomia), Giardia lambia, trichinella, Dientamoeba Fragilis, Trypanosoma brucei, Trypanosoma cruzi, or Leishmania donovani).

[0054] In some embodiments, a method for treating cancer, tumor, dysfunctional cells, or infected cells as described herein, comprising (a) administering a therapeutically effective amount of a therapeutic composition of cells described herein to an individual in need thereof, or (b) (i) providing or having provided a therapeutic composition of cells described herein; and (ii) The present invention provides a method comprising administering or having administered a therapeutically effective amount of a therapeutic composition of cells to an individual in need of such a treatment.

[0055] In certain embodiments of the treatment methods provided herein, the individuals requiring them are human or animal. In some embodiments, γδ and / or NK cells are isolated from an in vivo source, and the in vivo source of γδ and / or NK cells may, if necessary, be a syngeneic or self-source (derived, if necessary, from the individual that will be the recipient of the γδ and / or NK cells), or from an exogenous, heterogeneous, or allogeneic source, or a combination thereof. In embodiments of the methods provided herein, the therapeutic composition or combination of cells is administered intravenously (IV), intrathecally, or intramuscularly (IM), or injected or implanted inside or near cancer, tumor, dysfunctional, or infected cells, and the therapeutic composition of cells may, if necessary, be delivered as an implant or gel, and the gel may, if necessary, be a hydrogel.

[0056] In some embodiments of the treatment methods provided herein, the therapeutic composition or combination of cells is administered in unit dosage form, and the unit dose, if necessary, is approximately 10 cells. 2 from 10 12 Between individuals, or cells 10 4 from 10 10 The dose includes between cells; or the daily dose is approximately 10 cells. 2 from 10 12 Between individuals, or cells 10 4 from 10 10This includes between individuals. In certain embodiments, a therapeutic composition or combination or unit dosage form of cells is administered several times (multiple times) or two, three, four, five, six, seven, eight, nine, or ten times or more times to an individual in need over several days, weeks, or months, and each of the multiple unit dosage forms is administered daily, every other day, twice, three, four, five, or six times a week, or once a week, as needed.

[0057] In certain embodiments of the treatment methods provided herein, an individual in need of treatment is first administered a non-conjugate antibody against a cancer-related or tumor-related, infection-related or disease-related antigen, and then administered a therapeutic cell composition described herein to induce an immune response to cancer, infection, or disease, wherein at least a portion of the therapeutic cell composition expresses a polypeptide, optionally CAR, that specifically binds to the non-conjugate antibody on its cell surface. In certain embodiments, an individual in need is administered an antibody capable of specifically binding to a cancer-related or tumor-related, infection-related or disease-related antigen, and then administered a therapeutic cell composition described herein, wherein at least a portion of the therapeutic cell composition expresses a polypeptide, optionally CAR, that specifically binds to the antibody on its cell surface, and optionally the antibody is administered before, together with, or after the administration of the therapeutic cell composition.

[0058] Details of a particular embodiment of this technology are described in the accompanying drawings and the following description. Other features, purposes, and advantages of this technology will become apparent from the description and drawings, as well as from the claims.

[0059] All publications, patents, and patent applications cited herein are expressly incorporated herein by reference for all purposes.

[0060] The drawings illustrate, but do not limit, certain embodiments of the present technology. For clarity and ease of illustration, the drawings are not drawn to scale, and in some examples, various aspects may be exaggerated or enlarged to facilitate understanding of the particular embodiment. In certain embodiments, for example, the following items are provided: (Item 1) A method for producing a composition comprising a population of cells enriched with respect to NK cells and gamma delta T cells, Obtain a sample containing cells from one or more subjects; Depleting alpha-beta T cells from the sample under conditions that generate a depleted cell population including NK cells and gamma-delta T cells; and Exposing the depleted cell population to activation conditions that include (a) contact with at least one exogenous polypeptide that immunospecifically binds to a cell adhesion polypeptide, and (b) at least one exogenous polypeptide that immunospecifically binds to a polypeptide different from the cell adhesion polypeptide and is expressed on the surface of one or more types of cells in the sample population; and Exposing the depleted cell population to expansion conditions that include contacting the depleted cell population with at least one auxiliary polypeptide, thereby generating a composition containing a population of cells enriched with respect to NK cells and gamma delta T cells. A method that includes this. (Item 2) A method for producing a composition comprising a population of cells enriched with respect to NK cells and gamma delta T cells, Obtain a sample containing cells from one or more subjects; Activation conditions comprising contacting the sample with (a) at least one exogenous polypeptide that immunospecifically binds to a cell adhesion polypeptide, and (b) at least one exogenous polypeptide that immunospecifically binds to a polypeptide different from the cell adhesion polypeptide and is expressed on the surface of one or more types of cells in the sample population, wherein (a) or (b) is soluble, or (a) and (b) are soluble; and Exposing the sample to expansion conditions that include contacting the sample with at least one auxiliary polypeptide, thereby generating a composition containing a population of cells enriched with respect to NK cells and gamma delta T cells. A method that includes this. (Item 3) The method according to item 1 or item 2, wherein the at least one auxiliary polypeptide is selected such that the amount of NK cells compared to the amount of gamma delta T cells in the population is dependent on the amount and / or type of the at least one auxiliary polypeptide. (Item 4) The method according to any one of items 1 to 3, wherein the at least one auxiliary polypeptide increases or decreases the amount of NK cells compared to gamma delta T cells in the cell population after contacting the depleted cell population with the at least one auxiliary polypeptide. (Item 5) The method according to any one of items 1 to 4, wherein the activation conditions described above do not include serum derived from non-human animals. (Item 6) The method according to any of items 1 to 5, wherein the aforementioned expansion condition does not include serum derived from non-human animals. (Item 7) The method according to any one of items 1 to 6, wherein the activation conditions described above do not include feeder cells. (Item 8) The method according to any one of items 1 to 7, wherein the aforementioned expansion conditions do not include feeder cells. (Item 9) The method according to any one of items 1 to 8, wherein the sample is selected from peripheral blood, liver tissue, bone marrow, epithelial tissue, and umbilical cord blood. (Item 10) The method according to item 9, wherein the sample is peripheral blood. (Item 11) The method according to item 10, wherein the peripheral blood sample is a processed sample. (Item 12) The method according to item 9, wherein the sample is umbilical cord blood. (Item 13) The method according to item 12, wherein the umbilical cord blood sample is a processed sample. (Item 14) The method according to any one of items 1 to 13, wherein the at least one exogenous polypeptide in (b) is immunospecifically bound to NK cell activating receptor, gamma delta T cell activating receptor, or both. (Item 15) The method according to item 14, wherein the receptor is NKp30, NKp44, or NKp46. (Item 16) The method according to item 15, wherein the receptor is NKp46. (Item 17) The method according to any one of items 1 to 16, wherein the exogenous polypeptide in (a) binds immunospecifically to CD2. (Item 18) The method according to any one of items 1 to 17, wherein the exogenous polypeptide in (a) or (b), or in (a) and (b), is an antibody or an antigen-binding fragment thereof. (Item 19) The method according to any one of items 1 to 18, wherein at least one of (a) or (b) is soluble. (Item 20) The method according to any one of items 1 to 19, wherein both of the exogenous polypeptides in (a) and (b) are soluble. (Item 21) The method according to any one of items 1 to 19, wherein the exogenous polypeptide in (a) or the exogenous polypeptide in (b) is bound to a substrate. (Item 22) The method according to any one of items 1 to 21, wherein the activation condition includes contacting the sample or depleted cell population with at least two exogenous polypeptides. (Item 23) The method according to item 22, wherein a first exogenous polypeptide is immune-specifically bound to CD2 and a second exogenous polypeptide is immune-specifically bound to NKp46. (Item 24) The method according to item 22 or item 23, wherein the first exogenous polypeptide and / or the second exogenous polypeptide is an antibody or an antigen-binding fragment thereof. (Item 25) The polypeptide components of the aforementioned activation conditions are (a) an exogenous polypeptide that binds immunospecifically to the cell adhesion polypeptide CD2; and (b) Unlike the exogenous polypeptide in (a), this exogenous polypeptide binds immunospecifically to NKp46. A method of any of items 1-24, which is essentially derived from or consists of. (Item 26) The method according to any one of items 1 to 25, wherein the at least one auxiliary polypeptide is a polypeptide that binds immunospecifically to cytokines and / or receptors on gamma delta T cells. (Item 27) The method according to item 26, wherein the expansion condition comprises contacting the sample or depleted cell population with at least one co-auxiliary polypeptide which is a cytokine, and optionally with a co-auxiliary polypeptide which binds immunospecifically to a receptor on gamma delta T cells. (Item 28) The method according to item 27, wherein the cytokine is interleukin (IL). (Item 29) The method according to any one of items 26 to 28, wherein the at least one auxiliary polypeptide comprises IL-2, IL-4, IL-15, or any combination thereof. (Item 30) The aforementioned expansion conditions apply to the sample or depleted cell population. (a) IL-2 polypeptide, and polypeptides that, if necessary, immunospecifically bind to receptors on gamma-delta T cells; (b) IL-15 polypeptide; or (c) IL-2 polypeptide and IL-15 polypeptide, and polypeptides that, if necessary, immunospecifically bind to receptors on gamma delta T cells. A method according to any of items 26-29, including bringing into contact with the object. (Item 31) The method according to any one of items 26 to 30, wherein the receptor on the gamma-delta T cell is CD3. (Item 32) The method according to any one of items 26 to 31, wherein the polypeptide that immune-specifically binds to the receptor on the gamma-delta T cell is an antibody or an antigen-binding fragment thereof. (Item 33) The aforementioned magnification conditions apply to the sample (a) IL-2 polypeptide; (b) IL-15 polypeptide; (c) IL-2 polypeptide and IL-15 polypeptide; (d) An antibody that binds immunospecifically to IL-2 polypeptide and CD3; or (e) Antibodies that bind immunospecifically to IL-2 polypeptide, IL-15 polypeptide, and CD3. The method described in item 32, which includes bringing into contact with the object. (Item 34) The method according to item 33, wherein the antibody that binds immunospecifically to CD3 is OKT3. (Item 35) The method according to any one of items 1 to 34, wherein the activation and expansion conditions are carried out simultaneously or sequentially in any order. (Item 36) The aforementioned at least one exogenous polypeptide can also function as an auxiliary polypeptide; or The aforementioned at least one auxiliary polypeptide can also function as an exogenous polypeptide; or The method according to any one of items 1 to 35, wherein the at least one exogenous polypeptide can also function as an auxiliary polypeptide, and the at least one auxiliary polypeptide can also function as an exogenous polypeptide. (Item 37) (i) The expansion condition includes contacting the sample or depleted cell population with IL-2 polypeptide; (ii) The method according to any of items 33 to 36, wherein the obtained population of cells enriched with respect to NK cells and gamma delta T cells contains approximately 25-30% NK cells and approximately 70-75% gamma delta T cells. (Item 38) (i) The expansion condition includes contacting the sample or depleted cell population with IL-15 polypeptide; (ii) The method according to any of items 33 to 36, wherein the obtained population of cells enriched with respect to NK cells and gamma delta T cells contains approximately 80–99% NK cells and approximately 1–20% gamma delta T cells. (Item 39) (i) The expansion condition includes contacting the sample or depleted cell population with an antibody that immunospecifically binds to IL-2 polypeptide and CD3; (ii) The method according to any of items 33 to 36, wherein the obtained population of cells enriched with respect to NK cells and gamma delta T cells contains approximately 40–45% NK cells and approximately 55–60% gamma delta T cells. (Item 40) The aforementioned expansion conditions are, The aforementioned sample or depleted cell population is exposed to a first set of conditions containing one or more auxiliary polypeptides to yield a first cell population containing a first ratio of NK cells to gamma delta T cells; and The method according to any one of items 1 to 39, comprising exposing the first cell population to a second set of conditions comprising one or more auxiliary polypeptides to yield a second cell population having a desired final ratio of NK cells to gamma delta T cells, wherein the first set of conditions is different from the second set of conditions. (Item 41) The method according to item 40, wherein the first cell population is washed before contact with the second set of conditions. (Item 42) The first set of conditions includes IL-2, and the second set of conditions includes IL-15; The first set of conditions includes IL-15, and the second set of conditions includes IL-2; The first set of conditions comprises antibodies that bind immunospecifically to IL-2 and CD3, and the second set of conditions comprises IL-15; or The method according to item 40 or item 41, wherein the first set of conditions comprises antibodies that immunospecifically bind to IL-15 and CD3, and the second set of conditions comprises antibodies that immunospecifically bind to IL-2 and CD3. (Item 43) The method according to item 42, wherein the antibody that binds immunospecifically to CD3 is OKT3. (Item 44) Before exposing the sample to the activation and expansion conditions, alpha and beta T cells are depleted from the sample, thereby generating a population of depleted cells; and The depleted cell population is subjected to the activation and expansion conditions. It further includes, The method according to any one of items 2 to 43, thereby obtaining a composition containing a population of cells enriched with respect to NK cells and gamma delta T cells. (Item 45) The method according to any one of items 1 to 44, wherein, prior to activation and expansion, the sample or the depleted cell population is not exposed to the conditions for selection of NK cells or gamma-delta T cells, nor to the conditions for depleting cells other than alpha-beta T cells. (Item 46) The method according to any one of items 1 to 45, wherein, prior to activation and expansion, the sample or the depleted cell population is not exposed to conditions that deplete all CD3+ cells in the sample or the depleted cell population. (Item 47) The method according to any one of items 1 to 46, wherein the cells of the sample or depleted cell population are free of exogenous nucleic acids before, during, or after activation and expansion. (Item 48) The method according to any of items 1 to 46, wherein the cells of the sample or depleted cell population do not contain tumor necrosis factor receptors, chimeric antigen receptors (CARs), primary myeloid differentiation response proteins, or exogenous nucleic acids encoding innate immune signaling adapters before, during, or after activation and expansion. (Item 49) The method according to any of items 1 to 48, wherein the cells of the sample or depleted cell population are not genetically modified before, during, or after activation and expansion. (Item 50) The method according to any one of items 1 to 49, further comprising subjecting a population of cells enriched with respect to NK cells and gamma delta T cells to a treatment which thereby depletes the gamma delta cells, wherein the resulting population is essentially composed of or consists of NK cells. (Item 51) The method according to any one of items 1 to 49, further comprising subjecting a population of cells enriched with respect to NK cells and gamma delta T cells to a treatment which thereby depletes the NK cells, wherein the resulting population is essentially composed of or consists of gamma delta T cells. (Item 52) The method according to any one of items 1 to 50, further comprising subjecting the population of cells enriched with respect to NK cells and gamma delta T cells to positive selection for NK cells, thereby obtaining a population of cells essentially derived from or consisting of NK cells. (Item 53) The method according to any one of items 1-49 and 51, further comprising subjecting the cell population enriched with respect to NK cells and gamma delta T cells to positive selection for gamma delta cells, thereby obtaining a cell population essentially consisting of or derived from gamma delta cells. (Item 54) The method according to any one of items 1 to 53, wherein the expansion conditions include incubation of the sample or depleted cell population in feeder cell-free medium for about 1 to 10 weeks, thereby obtaining a composition comprising an expanded cell population enriched with respect to NK cells and gamma delta T cells. (Item 55) The method according to item 54, wherein the culture conditions include incubation of the sample or depleted cell population in feeder cell-free medium for approximately 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 30, 35, 40, 45, 50, 55, or 60 days or more, or incubation for approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. (Item 56) The method according to any one of items 1 to 55, wherein the population of cells enriched with respect to NK cells and gamma delta T cells is expanded by more than approximately 2 log over 30 days under the expansion conditions. (Item 57) The method according to item 56, wherein the population of cells is expanded by more than 3 log over 30 days under the expansion conditions. (Item 58) The method according to any one of items 1 to 57, wherein the expanded cell population enriched with respect to NK cells and gamma delta T cells does not contain exhausted cells. (Item 59) The method according to any one of items 1 to 58, wherein the expanded cell population enriched with respect to NK cells and gamma delta T cells does not contain exhausted cells after 60 days of the expansion conditions. (Item 60) The method according to any one of items 1 to 59, wherein less than 5%, less than 4%, less than 3%, or less than 2% of NK cells in the expanded cell population enriched with respect to NK cells and gamma delta T cells contain the PD-1 marker, and / or about 20%, 15%, 10%, or less of the total cells in the expanded population or the gamma delta T cells in the expanded population contain the PD-1 marker. (Item 61) The aforementioned cell population enriched with respect to NK cells and gamma delta T cells is defined as the following activation markers as a percentage of the total cell number in the population: (a) 90% or more of KIR5; (b) 10% or more of SIGLEC-7; (c) 60% or more KIR3D51; (d) 10% or more of KIR2DL1; (e) 25% or more of NKp30, NKp44, or NKp46; (f) 35% or more NKG2D; (g) 90% or more DNAM1; (h) 85% or more NTBA; (i) 95% or more of CD2; and (j) 55% or more KIR3DS1 A method described in any of items 1 through 60, including one or more of the above. (Item 62) The method according to any one of items 1 to 61, wherein the cell population enriched with respect to NK cells and gamma delta T cells contains 80% or more innate immune cells. (Item 63) The method according to item 62, wherein between approximately 70% and approximately 100% of the cells, or at least approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are CD56+. (Item 64) The method according to item 62 or item 63, wherein between approximately 10% and approximately 40% of the cells, or at least approximately 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% are CD16+. (Item 65) The method according to any one of items 62-64, wherein less than 5%, less than 4%, less than 3%, or less than 2% of the cells are CD57+. (Item 66) The method according to any of items 1 to 65, wherein the sample or depleted cell population does not contain CD4+CD8+ cells during or after activation and expansion. (Item 67) The method according to any one of items 1 to 66, wherein the activation condition and the expansion condition do not include bisphosphonates. (Item 68) The method according to item 67, wherein the bisphosphonate is pamidronate or zoledronate. (Item 69) The method according to any one of items 1 to 68, wherein the gamma delta T cells are polyclonal with respect to V delta 1 and V delta 2 expression. (Item 70) The method according to item 69, wherein the polyclonal gamma delta T cells comprise at least one subpopulation selected from V. delta.1+ and V. delta.1-, and at least one subpopulation selected from V. delta.2+ and V. delta.2-. (Item 71) A composition that can be obtained or obtained by any method described in item 1 to 70. (Item 72) A composition comprising a population of modified peripheral blood cells, wherein the population is It contains multiple NK cells and multiple gamma-delta T cells; Alpha and beta T cells are depleted; A composition that does not contain feeder cells. (Item 73) Approximately 25% to 45% of the aforementioned cells are NK cells, and approximately 55% to 75% of the aforementioned cells are gamma-delta T cells; Approximately 25% to 30% of the aforementioned cells are NK cells, and approximately 70% to 75% of the aforementioned cells are gamma-delta T cells; Approximately 80% to 99% of the cells are NK cells, and approximately 1% to 20% of the cells are gamma delta T cells; or The composition according to item 72, wherein approximately 40% to approximately 45% of the cells are NK cells and approximately 55% to approximately 60% of the cells are gamma delta T cells. (Item 74) The composition according to item 72 or 73, which activates 30% or more of the aforementioned cells. (Item 75) The aforementioned modified cell population is identified by the following activation markers as a percentage of the total number of cells in the population: (a) 90% or more of KIR5; (b) 10% or more of SIGLEC-7; (c) 60% or more KIR3D51; (d) 10% or more of KIR2DL1; (e) 25% or more of NKp30, NKp44, or NKp46; (f) 35% or more NKG2D; (g) 90% or more DNAM1; (h) 85% or more NTBA; (i) 95% or more of CD2; and (j) 55% or more KIR3DS1 A composition according to any one of items 72 to 74, comprising one or more of the following. (Item 76) The composition according to any one of items 72 to 75, wherein the modified population contains 80% or more innate immune cells. (Item 77) The composition according to any one of items 72 to 76, wherein the modified population is enriched with respect to activated cytotoxic cells that are CD56+. (Item 78) The composition according to any one of items 72 to 77, wherein the modified cells are enriched with respect to activated cytotoxic cells that are CD57-. (Item 79) The composition according to any one of items 72 to 78, wherein the population is enriched with respect to activated cytotoxic cells that are CD56+CD57-. (Item 80) The composition according to any of items 77 to 79, wherein between approximately 80% and approximately 100% of the cells, or at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are CD56+. (Item 81) The composition according to any of items 77 to 80, wherein between about 10% and about 40% of the cells, or at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% are CD16+. (Item 82) The composition according to any one of items 77 to 81, wherein less than 5%, less than 4%, less than 3%, or less than 2% of the cells are CD57+. (Item 83) A composition according to any one of items 72 to 82, which substantially contains cells other than NK cells and gamma delta T cells. (Item 84) A composition according to any one of items 72 to 83, containing less than 5 percent NKT cells. (Item 85) A composition according to any one of items 72 to 84, containing less than 1 percent NKT cells. (Item 86) A composition according to any one of items 72 to 85, containing less than 0.1 percent NKT cells. (Item 87) A composition according to any of items 72 to 86, containing less than 2 percent alpha-beta T cells. (Item 88) A composition according to any one of items 72 to 87, containing less than 1 percent alpha-beta T cells. (Item 89) A composition according to any of items 72 to 88, containing less than 0.1 percent alpha-beta T cells. (Item 90) The composition according to any one of items 72 to 89, wherein a subset of NK cells in the population is CD16+ cells. (Item 91) A composition according to any of items 72-90, wherein the majority of gamma delta T cells are CD57- cells. (Item 92) A composition according to any of items 72-91, wherein the majority of NK cells are CD57- cells. (Item 93) The composition according to any one of items 72 to 92, wherein the gamma delta T cells are polyclonal with respect to V delta 1 and V delta 2 expression. (Item 94) The composition according to item 93, wherein the polyclonal gamma delta T cells comprise at least one subpopulation selected from V. delta.1+ and V. delta.1-, and at least one subpopulation selected from V. delta.2+ and V. delta.2-. (Item 95) The majority of gamma-delta T cells express V.delta.1, and a small number of gamma-delta T cells express V.delta.2; or A composition according to any one of items 72 to 94, wherein a small number of gamma delta T cells express V. delta.1 and the majority of gamma delta T cells express V. delta.2. (Item 96) A small number of cells in the population are CD3-positive cells, and the majority of cells in the population are CD3-negative cells; or The composition according to any one of items 72 to 95, wherein the majority of cells in the population are CD3-positive cells, and a small number of cells in the population are CD3-negative cells. (Item 97) A composition according to any one of items 72 to 96, wherein the ratio of NK cells to gamma delta T cells is greater than 1. (Item 98) A composition according to any one of items 72 to 96, wherein the ratio of NK cells to gamma delta T cells is less than 1. (Item 99) The composition according to item 97, wherein the population of modified cells comprises approximately 98-99% NK cells and approximately 1-2% gamma delta T cells. (Item 100) The composition according to item 98, wherein the population of modified cells comprises between approximately 25% and 45% NK cells and between approximately 55% and 75% gamma delta T cells. (Item 101) The composition according to item 98, wherein the population of modified cells comprises approximately 25-30% NK cells and approximately 70-75% gamma delta T cells. (Item 102) The composition according to item 98, wherein the population of modified cells comprises approximately 40-45% NK cells and approximately 55-60% gamma delta T cells. (Item 103) Between approximately 50% and approximately 99% or more of the NK cells and / or the gamma delta T cells, or between approximately 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 6% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73 More than or equal to %, more than or equal to 74%, more than or equal to 75%, more than or equal to 76%, more than or equal to 77%, more than or equal to 78%, more than or equal to 79%, more than or equal to 80%, more than or equal to 81%, more than or equal to 82%, more than or equal to 83%, more than or equal to 84%, more than or equal to 85%, more than or equal to 86%, more than or equal to 87%, more than or equal to 88%, more than or equal to 89%, more than or equal to 90%, more than or equal to 91%, more than or equal to 92%, more than or equal to 93%, more than or equal to 94%, more than or equal to 95%, more than or equal to 96%, more than or equal to 97%, more than or equal to 98%,Compositions described in any of items 72-102, where more than or equal to 99%, more than or equal to 80%, more than or equal to 81%, more than or equal to 82%, more than or equal to 83%, more than or equal to 84%, more than or equal to 85%, more than or equal to 86%, more than or equal to 87%, more than or equal to 88%, more than or equal to 89%, more than or equal to 90%, more than or equal to 91%, more than or equal to 92%, more than or equal to 93%, more than or equal to 94%, more than or equal to 95%, more than or equal to 96%, more than or equal to 97%, more than or equal to 98%, more than or equal to 99%, or any fraction thereof, up to 100% being CD8+. (Item 104) The composition according to item 103, wherein less than 2% of the NK cells and / or gamma-delta T cells are CD4+. (Item 105) The composition according to item 103 or item 104, wherein less than 2% of the NK cells and / or gamma delta T cells are CD8+CD4+. (Item 106) The composition according to any one of items 103 to 105, wherein between approximately 15% and 30% of the NK cells and / or between approximately 55% and 85% of the gamma delta T cells, the fractional part is CD8-CD4-. (Item 107) Between approximately 30% and approximately 99% or more of the cells in the population, or at least approximately 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75% A composition according to any one of items 72 to 106, wherein 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any fraction thereof, up to 100%, further comprises a genetic modification including an exogenous polynucleotide, a mutant polynucleotide, a deletion polynucleotide, or a combination thereof. (Item 108) The composition according to item 107, wherein at least about 95%, 96%, 97%, 98%, and 99% of the cells in the population contain the gene modification, or about 100% or 100% of the cells in the population contain the gene modification. (Item 109) The composition according to item 107 or item 108, wherein the gene modification comprises an exogenous polynucleotide. (Item 110) The composition according to item 109, wherein the exogenous polynucleotide is contained in a retroviral vector or a lentiviral vector. (Item 111) The composition according to item 109, wherein the exogenous polynucleotide is incorporated into the genome of one or more cells of the modified cell population. (Item 112) The composition according to any one of items 107 to 111, wherein the cells in the population contain a chimeric antigen receptor (CAR). (Item 113) The composition according to item 112, wherein the chimeric antigen receptor comprises a binding molecule that immunospecifically binds to one or more of CD19, GD2, HER3, B7H3, CD123, or CD30. (Item 114) The composition according to any one of items 72 to 113, wherein the population comprising the plurality of NK cells and the plurality of gamma delta T cells is derived from the peripheral blood cells of one or more subjects. (Item 115) A pharmaceutical composition comprising the composition described in any of embodiments 72 to 114 and a pharmaceutically acceptable carrier. (Item 116) A method for producing genetically modified immune cells, (a) Adding an exogenous polynucleotide to any of the compositions described in items 71 to 106; (b) Mutating polynucleotides in one or more cells of the composition described in any of items 71 to 106; or (c) Deleting polynucleotides in one or more cells of the composition described in any of items 71 to 106. A method that includes one or more of the following. (Item 117) The gene modification is performed by retroviral transduction, lentiviral transduction, electroporation, transfection, CRISPR / Cas9, or TALENS, as described in item 116. (Item 118) The method according to item 116 or item 117, wherein the gene modification consists of, or is essentially derived from, the addition of an exogenous polynucleotide as in (a). (Item 119) The method according to any one of items 116 to 118, wherein the gene modification comprises adding an exogenous polynucleotide as in (a) and / or mutating a polynucleotide as in (b), and the exogenous polynucleotide and / or the mutated polynucleotide are incorporated into the genome of the immune cell. (Item 120) The aforementioned integration is by electroporation, transfection, CRISPR / CAS9, or TALENS, as described in item 119. (Item 121) The method according to any one of items 116 to 121, wherein the exogenous polynucleotide encodes a chimeric antigen receptor (CAR). (Item 122) The method of item 121, wherein the chimeric antigen receptor includes a binding molecule that immunospecifically binds to one or more of CD19, GD2, HER3, B7H3, CD123, or CD30. (Item 123) Between approximately 30% and approximately 99% or more of the cells in the population, or at least approximately 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% The method according to any one of items 116 to 122, wherein 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any fraction thereof, up to 100%, contains the aforementioned gene modification. (Item 124) The method according to item 123, wherein the gene modification includes an exogenous polynucleotide. (Item 125) The method according to item 124, wherein approximately 100% or 100% of the cells in the population contain the exogenous polynucleotide. (Item 126) A kit comprising a composition described in any of items 71-114, or a pharmaceutical composition described in item 115, instructions for use as appropriate, and cytokines as appropriate. (Item 127) The kit according to item 126, wherein the composition or the pharmaceutical composition is at minus 4 degrees Celsius or below. (Item 128) The kit according to item 127, wherein the composition or the pharmaceutical composition is located at approximately minus 75 degrees Celsius to approximately minus 80 degrees Celsius. (Item 129) cells approximately 1 x 10 5 From a single cell, approximately 1 x 10⁻⁶ cells 12 A kit containing one of the items listed in any of items 126-128. (Item 130) The kit described in any of items 126-129, wherein the cytokine is an interleukin polypeptide. (Item 131) The kit according to item 130, wherein the interleukin peptide is IL-2, IL-4, or IL-15. (Item 132) A kit as described in any of items 126-131, which does not contain non-human serum and / or bovine serum. (Item 133) A kit that does not contain any different types of materials, as described in any of items 126-132. (Item 134) A kit that does not contain exogenous feeder cells, as described in any of items 126-133. (Item 135) A kit of any of the unit dosage forms listed in items 126-134. (Item 136) The aforementioned unit dosage form is for approximately 1 × 10 cells 6 From a single cell, approximately 1 x 10⁻⁶ cells 12 A kit according to embodiment E10, comprising one unit. (Item 137) A collection of cells derived from different donor subjects, comprising a plurality of containers, each containing cells derived from one or more donor subjects, wherein each container contains a composition described in any one of items 71 to 114, a pharmaceutical composition described in item 115, or a kit described in any one of items 126 to 136. (Item 138) A method for treating cancer or an infection, comprising administering to a subject in need thereof an amount effective for treating the cancer or infection of a composition according to any one of items 71 to 114, a pharmaceutical composition according to item 115, or a kit according to any one of items 126 to 136, wherein the cells in the composition, the pharmaceutical composition, or the kit are allogeneic to the subject. (Item 139) A method for treating cancer or an infection, comprising administering to a subject in need thereof an amount effective for treating the cancer or infection of a composition according to any one of items 71 to 114, a pharmaceutical composition according to item 115, or a kit according to any one of items 126 to 136, wherein the cells in the composition, the pharmaceutical composition, or the kit are self to the subject. (Item 140) The method according to item 138 or item 139, comprising administering the composition to the subject on two or more separate days. (Item 141) The method according to item 139 or item 140, wherein the cell donor is the recipient of the treatment. (Item 142) The method according to item 138 or item 140, wherein the cell donor is not the recipient of the treatment. (Item 143) The method according to item 142, wherein the recipient of the treatment is susceptible to GvHD when treated with alpha-beta T cells derived from the donor. (Item 144) The procedure described above is administered at intervals of approximately 2 to 4 weeks, ranging from approximately 1 unit dose to approximately 36 or more units doses, as described in any of items 138 to 143. (Item 145) The method according to any of items 138-143, wherein the treatment is administered as a single unit dose once, two, three, four, or up to five times daily, or once, two, three, four, five, six, seven, eight, nine, or ten times or more over several days, weeks, or months, or every two days, or once, two, three, four, five, or six times per week. (Item 146) The method according to any of items 138-145, wherein the procedure is administered intravenously (IV), intrathecally or intramuscularly (IM), intraperitoneally (IP), intrapleurally, or intraarticularly, or is injected or implanted at or near the site of the cancer or infection. (Item 147) The aforementioned unit dose is approximately 10 cells per kilogram of body weight of the subject. 4 From one cell to about 10 10 Between individuals, or approximately 10 cells per subject 6 From one cell to about 10 12 The method described in any of items 144-146, including between individuals. (Item 148) The aforementioned unit dose is approximately 10 cells per target. 10 Each cell, or approximately 10 cells per kilogram of body weight of the subject. 8 The method described in item 147, which is one of several items. (Item 149) The method described in any of items 138-148, wherein the treatment is for cancer. (Item 150) The method according to item 149, wherein the cancer is selected from lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia or lymphoma, Hodgkin lymphoma or pediatric acute lymphoblastic leukemia, non-Hodgkin lymphoma, mast cell tumor or mast cell tumor, ovarian cancer or cancer, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, retinoblastoma, mammary gland tumor, colorectal cancer, leukemia, lymphoma, acute lymphoblastic leukemia (ALL) or acute lymphoblastic leukemia, acute myeloid leukemia (AML), histiocytic sarcoma, brain tumor, astrocytoma, glioblastoma, neuroma, colon cancer, cervical cancer, sarcoma, bladder tumor, reticuloendothelial tumor, Wilms' tumor, bone cancer, osteosarcoma, kidney cancer, or head and neck cancer, oral cancer, laryngeal cancer, metastatic disease, or oropharyngeal cancer. (Item 151) The method according to item 149 or item 150, wherein a second drug is administered co-administered with the composition, pharmaceutical composition, or kit. (Item 152) The method according to item 151, wherein the second drug is an antibody that binds immunely to a cancer-associated antigen. (Item 153) The aforementioned cancer-related antigens include α-fetoprotein (AFP), α-actinin-4, antigens specific to A3, A33 antibodies, ART-4, B7, B7-H3, Ba733, BAGE, BrE3 antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e , CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD123, CD126, CD132, CD133, CD13 8, CD147, CD154, CDC27, CDK-4 / m, CDKN2A, CTLA4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen-p(C SAp), CEA (CEACAM-5), CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-β, HLA-DR, HM1.24. Human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, HMGB-1, hypoxia-inducing factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15 , IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR / FUT, macrophage migration inhibitory factor Child (MIF), GD2, MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC 2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, PD1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-R, L-6, IL-25, RS5, RANTES, T101, SAGE, S100, mackerel The method described in item 152, selected from the group consisting of Ivin, Survivin-2B, TAC, TAG-72, Tenascin, TRAIL receptor, TNF-α, Tn antigen, Thomson-Friedreich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factors C3, C3a, C3b, C5a, C5, angiogenesis markers, bcl-2, bcl-6, and Kras. (Item 154) The antibodies are hR1 (anti-IGF-1R), hPAM4 (anti-mucin), KC4 (anti-mucin), hA20 (anti-CD20), hA19 (anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74). , hLL2 (anti-CD22), anti-CD19 / CD22 bispecific antibody, RFB4 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEACAM-5), hMN -15 (anti-CEACAM-6), hRS7 (anti-TROP-2), hMN-3 (anti-CEACAM-6), CC49 (anti-TAG-72), J591 (anti-PSMA), D2 / B (anti-PSMA), G250 (anti-carbonic anhydrase IX), dinutuximab (anti-GD2), infliximab (anti-TNF-α), certolizumab pegol (anti-TNF-α), adalimumab (anti-TNF-α), alemtuzumab (anti-CD52), ve Bacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab (anti-CD20), GA101 (anti-CD20), trastuzumab (anti-HER2 / neu), tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25), daclizumab (anti-CD25) The method described in item 152 or item 153, selected from ephalizumab (anti-CD11a), muromonab-CD3 (anti-CD3 receptor), natalizumab (anti-α4 integrin), BWA-3 (anti-histone H2A / H4), LG2-1 (anti-histone H3), MRA12 (anti-histone H1), PR1-1 (anti-histone H2B), LG11-2 (anti-histone H2B), and LG2-2 (anti-histone H2B). (Item 155) The method described in any of items 138-148, wherein the treatment is due to an infection. (Item 156) The method according to item 155, wherein the infection is characterized by the presence of a bacterial pathogen, a fungal pathogen, a viral pathogen, or a protozoan pathogen. (Item 157) The aforementioned infections include herpes, Ebola, West Nile virus, vaccinia virus, Epstein-Barr virus, hepatitis A virus (HAV); hepatitis B virus (HBV); hepatitis C virus (HCV); herpesviruses (e.g., HSV-1, HSV-2, HHV-6, CMV), human immunodeficiency virus (HIV), vesicular stomatitis virus (VSV), Bacilli, Citrobacter, Cholera, Diphtheria, Enterobacter, Gonococci, Helicobacter pylori, Klebsiella, Legionella, Meningococci, mycobacteria, Pseudomonas, Pneumonococci, Rickettsia bacteria, Salmonella, Serratia, Staphylococci, Streptococci, Tetanus, Aspergillus (A. fumigatus, A. niger, etc.), and Blastomyces. dermatitidis, Candida (C.albicans, C.krusei, C.glabrata, C.tropicalis, etc.), Cryptococcus neoformans, Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Paracoccidioides brasiliensis, Coccidioides immitis, Histoplasma The method described in item 156, selected from the group consisting of capsulatum, Leptospirosis, Borrelia burgdorferi, helminth parasites (hookworms, tapeworms, trematodes, flatworms (e.g., Schistosomia), Giardia lambia, trichinella, Dientamoeba Fragilis, Trypanosoma brucei, Trypanosoma cruzi, or Leishmania donovani. (Item 158) A therapeutic cell composition comprising multiple gamma delta T cells (γδ); multiple natural killer cells (NK); or a combination of γδ cells and NK cells. (Item 159) The γδ and / or NK cells are manipulated by recombination or genetically modified, If necessary, the γδ and / or NK cells are recombinantly manipulated or genetically modified to express exogenous or heterologous proteins extracellularly. If necessary, the exogenous, heterologous, or chimeric protein may be a chimeric antigen receptor (CAR) or an exogenous or heterologous T cell receptor (TCR). A therapeutic cell composition according to item 158, wherein, if necessary, the exogenous, heterologous, or chimeric protein or CAR is specific to (can specifically bind to) cancer cells, tumor markers, or infected cells, or the exogenous, heterologous, or chimeric protein or CAR is specific to (or can specifically bind to) an antibody that can specifically target and bind to cancer cells, tumor markers, infected cells, or any disease-associated antigen. (Item 160) A therapeutic cell composition according to item 158 or item 159, wherein the γδ and / or NK cells are human cells or animal cells. (Item 161) The therapeutic composition may be formulated for intravenous (IV), intrathecal, or intramuscular (IM) administration, or formulated in unit dosage forms, with the unit dose being approximately 10 cells as needed. 2 From one cell to 10 12 A therapeutic composition of cells as described in any of items 158 to 160, including between cells. (Item 162) A therapeutic composition of cells according to any one of items 158-161, wherein the γδ and / or NK cells are isolated from an in vivo source. (Item 163) A therapeutic composition of cells according to any of items 158-162, wherein the γδ and / or NK cells are expanded in culture or isolated from an in vivo source and expanded in culture. (Item 164) A therapeutic cell composition according to item 163, wherein the γδ and / or NK cells are isolated from an in vivo source and expanded in culture without the use of any feeder cells or a feeder cell layer, thereby generating an expanded population of γδ and / or NK cells lacking feeder cells. (Item 165) A therapeutic cell composition according to item 163, wherein the γδ and / or NK cells are isolated from an in vivo source, expanded in culture using feeder cells or a layer of feeder cells, and the feeder cells are substantially removed and / or killed as necessary to produce an expanded population of γδ and / or NK cells substantially lacking feeder cells. (Item 166) A therapeutic cell composition according to any of items 158 to 165, wherein the in vivo source of the γδ and / or NK cells is derived from a self-source (or, if necessary, from the individual that will be the recipient of the γδ and / or NK cells), or from an exogenous, heterologous, or allogeneic source. (Item 167) A method for treating cancer, tumor, dysfunctional cells, or infected cells, (a) administering to an individual in need of it a therapeutically effective amount of the therapeutic composition of cells described in any of the preceding items, or (b)(i) providing or having provided a therapeutic composition of cells described in any of the preceding items; and (ii) administering to an individual in need of it an effective therapeutic amount of the therapeutic composition of the cells or having administered such an amount to an individual. A method that includes this. (Item 168) The method according to item 167, wherein the individual requiring it is a human or an animal. (Item 169) The method according to item 167 or 168, wherein the γδ and / or NK cells are isolated from an in vivo source, and the in vivo source of the γδ and / or NK cells is, if necessary, derived from a syngeneic or self-source (or, if necessary, from the individual that will be the recipient of the γδ and / or NK cells), or from an exogenous, heterogeneous, or homogeneous source, or a combination thereof. (Item 170) The method according to any one of items 167-169, wherein the therapeutic composition of the cells is administered intravenously (IV), intrathecally, or intramuscularly (IM), or injected or implanted in or near the cancer, tumor, dysfunction, or infected cells, and the therapeutic composition of the cells is delivered as an implant or gel, and the gel is a hydrogel, if necessary. (Item 171) The therapeutic composition of the cells is administered in unit dosage form, and if necessary, the unit dose is approximately 10 cells 2 From one cell to 10 12 Between individuals, or cells 10 4 From one cell to 10 10 Including between cells; or a daily dose of approximately 10 cells 2 From one cell to 10 12 Between individuals, or cells 10 4 From one cell to 10 10 The method described in any of items 167-170, including between individuals. (Item 172) The method according to any of items 167-171, wherein the therapeutic composition or unit dosage form of the cells is administered to the individual in need several times (multiple times), or two, three, four, five, six, seven, eight, nine, or ten times or more over several days, weeks or months, and each of the multiple unit dosage forms is administered daily, every other day, twice, three, four, five, or six times per week, or once per week, as needed. (Item 173) The method according to any one of items 167-172, wherein the cancer or tumor is lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia or lymphoma, Hodgkin lymphoma or pediatric acute lymphoblastic leukemia, mast cell tumor or mast cell tumor, ovarian cancer or cancer, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, retinoblastoma, mammary gland tumor, colorectal cancer, leukemia, lymphoma, acute lymphoblastic leukemia (ALL) or acute lymphoblastic leukemia, acute myeloid leukemia (AML), histiocytic sarcoma, brain tumor, astrocytoma, glioblastoma, neuroma, colon cancer, cervical cancer, sarcoma, bladder tumor, reticuloendothelial tumor, Wilms' tumor, bone cancer, osteosarcoma, kidney cancer, or head and neck cancer, oral cancer, laryngeal cancer, or oropharyngeal cancer. (Item 174) The method according to any one of items 167-173, wherein the individual requiring the treatment is first administered a non-conjugate antibody against a cancer-related or tumor-related, infection-related or disease-related antigen, and then administered a therapeutic composition of cells described in any of the preceding items to induce an immune response to cancer, infection, or disease, wherein at least a portion of the therapeutic composition of cells expresses a polypeptide, optionally a CAR, that specifically binds to the non-conjugate antibody on its cell surface. (Item 175) The individual requiring it is administered an antibody capable of specifically binding to cancer-related or tumor-related, infection-related or disease-related antigens, and subsequently administered a therapeutic composition of cells described in any of the preceding items, wherein at least a portion of the therapeutic composition of cells expresses a polypeptide that specifically binds to the antibody, and optionally expresses a CAR on its cell surface. The method according to any one of items 167 to 174, wherein the antibody is administered, if necessary, before, together with, or after, the therapeutic composition of the cells. (Item 176) The aforementioned cancer-related or tumor-related antigens include antigens specific to α-fetoprotein (AFP), α-actinin-4, A3, A33 antibodies, ART-4, B7, B7-H3, Ba733, BAGE, BrE3 antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66 a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD123, CD126, CD132, CD133, C D138, CD147, CD154, CDC27, CDK-4 / m, CDKN2A, CTLA4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen-p (CSAp), CEA(CEACAM-5), CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, EGP-1(TROP-2), EGP-2, ELF2 -M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-β, HLA-DR, HM1.24. Human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, HMGB-1, hypoxia-inducing factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-1 8, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR / FUT, macrophage migration inhibitory factor (MIF), GD2 (disiaroganglioside expressed in tumors of neuroectoderm origin), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, M UC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, PD1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-R, L-6, IL-25, RS5, RANTES, T101, SAGE, S100, Survivin The method described in any of items 167-175, wherein the antigen is selected from the group consisting of Survivin-2B, TAC, TAG-72, Tenascin, TRAIL receptor, TNF-α, Tn antigen, Thomson-Friedreich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factors C3, C3a, C3b, C5a, C5, angiogenesis markers, bcl-2, bcl-6, and Kras. (Item 177) The aforementioned antibody or the aforementioned non-conjugate antibody is hR1 (anti-IGF-1R), hPAM4 (anti-mucin), KC4 (anti-mucin), hA20 (anti-CD20), hA19 (anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22), RFB4 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEACAM-5), hM N-15 (anti-CEACAM-6), hRS7 (anti-TROP-2), hMN-3 (anti-CEACAM-6), CC49 (anti-TAG-72), J591 (anti-PSMA), D2 / B (anti-PSMA), G250 (anti-carbonic anhydrase IX), infliximab (anti-TNF-α), certolizumab pegol (anti-TNF-α), adalimumab (anti-TNF-α), alemtuzumab (anti-CD52), bevacizumab (anti-VEG F) Cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab (anti-CD20), GA101 (anti-CD20), trastuzumab (anti-HER2 / neu), tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), ephalizumab The method described in item 174 or 175, selected from the group consisting of B (anti-CD11a), muromonab-CD3 (anti-CD3 receptor), natalizumab (anti-α4 integrin), BWA-3 (anti-histone H2A / H4), LG2-1 (anti-histone H3), MRA12 (anti-histone H1), PR1-1 (anti-histone H2B), LG11-2 (anti-histone H2B), and LG2-2 (anti-histone H2B). [Brief explanation of the drawing]

[0061] [Figure 1] Figure 1 shows the expansion of αβTCRneg mononuclear cells as a function of the number of days the cells were activated with antibodies and expanded under feeder-free culture conditions, as analyzed on day 10 of culture. [Figure 2] Figure 2 shows the analysis of the composition of the activated and expanded αβTCRneg mononuclear cell population (referred to herein as the INNATE-K or BINATE cell population). [Figure 3] Figure 3 shows the frequency and distribution of T cell lineage markers, CD4 (helper) and CD8 (cytotoxic), on γδ TCR+ cells in a 10-day sample of activated αβ TCRneg cells expanded in feeder-free culture, as analyzed by flow cytometry. [Figure 4] Figure 4 shows the analysis of the growth rate of CAR.19 INNATE-K cells over 10 days. [Figure 5] Figure 5 shows data from co-culture cytotoxicity assays in which INNATE-K cells and INNATE-CAR.19 cells were co-cultured with CD19+ leukemia (221) or CD19+ lymphoma (Daudi) cell lines. [Figure 6] Figure 6 shows the total number of expanded INNATE-NK cells cultured in vitro for a long period on a plate. [Figure 7] Figure 7 shows a comparison of the total number of cells in the plate and the INNATE-NK population expanded in the bioreactor. [Figure 8] Figure 8 shows the subset cell composition at different time points in in vitro INNATE-NK expansion. [Figure 9] Figure 9 shows the expression of chimeric antigen receptor (CAR) molecules in INNATE-NK CAR cells under long-term in vitro culture. [Figure 10] Figure 10 shows the expression of activation and cell-lysing molecules after expansion of the INNATE-NK and INNATE-NK CAR populations. [Figure 11] Figure 11 shows the absence of exhaustion in INNATE-NK cells and INNATE-NK CAR cells expanded in a feeder-free environment. [Figure 12]Figure 12 shows a cytotoxic co-culture assay with INNATE-NK or INNATE-NK-CAR.19 and four tumor cell lines. Column A: 221, CD19+ leukemia cell line; Column B: Daudi, CD19+ lymphoma cell line; Column C: BV173, CD19+ (diversely expressing) pre-B tumor cell line; and Column D: KARPAS, CD19- tumor cell line. [Figure 13] Figures 13 and 14 show the % specific lysis of primary tumor cells as a function of the ratio of effector (E) to target (T) for both INNATE-NK cell and INNATE-NK-CAR.19 cell populations in various test runs. [Figure 14] Figures 13 and 14 show the % specific lysis of primary tumor cells as a function of the ratio of effector (E) to target (T) for both INNATE-NK cell and INNATE-NK-CAR.19 cell populations in various test runs. [Figure 15] Figure 15 shows the percentage of residual primary CD19+ tumors after co-culture with effector cells (INNATE-NK cells and INNATE-NK-CAR.19 cell populations) compared to a control condition in which primary CD19+ leukemic blasts were seeded in the absence of effector cells. [Figure 16] Figure 16 shows the survival curves of animals administered with INNATE-NK and INNATE-NK-CAR.19 cells. [Figure 17] Figure 17 shows the total BINATE expansion expressed by the total cell number over time (i.e., expansion of activated αβTCRneg cells in BINATE medium supplemented with either IL-2 or IL-15 in a flask, and expansion of the activated αβTCRneg cell population with IL-15 in a bioreactor). [Figure 18]Figure 18 shows the total BINATE expansion expressed by the total cell number over time (i.e., expansion of activated αβTCRneg cells in BINATE medium supplemented with either IL-2, IL-15, IL-2 / OKT3, IL-2 / IL-15, or IL-2 / IL-15 / OKT3 in a flask). [Figure 19] Figure 19 shows the phenotypic analysis for each marker type (activation, cytotoxicity, and exhaustion / immaturity) related to the conditions for expansion by IL-15. [Figure 20] Figure 20 shows the phenotypic analysis for each marker type (activation, cytotoxicity, and exhaustion / immaturity) related to the conditions for expansion by IL-2. [Figure 21] Figure 21 shows the phenotypic analysis for each marker type (activation, cytotoxicity, and exhaustion / immaturity) related to IL-2 / OKT3 expansion conditions. [Figure 22] Figure 22 shows the cell counts recorded approximately every 7 days for the BINATE and BINATE.CARGD2 populations. [Figure 23] Figure 23 shows the in vivo maintenance of human BINATE cells in both mouse blood and mouse liver. [Figure 24] Figure 24 is a flowchart illustrating a modified form as an example of the process for isolating a pure NK cell population and / or a pure γδT(gd) cell population. [Modes for carrying out the invention]

[0062] In certain embodiments, this specification provides therapeutic immune cell compositions in which a significant number or majority of immune cells are innate immune cells. Such compositions can generate an innate immune response in a subject after administration. The innate immune system is active and constantly functional in the host, responds rapidly (within minutes to hours post-infection), and responds non-specifically, offering the potential for use as "off-the-shelf" immunotherapy for a broader patient population. Innate immune cells, e.g., NK cells and gamma-delta (γδ) T cells, do not recognize classical HLA antigens and can therefore be used in off-the-shelf (allogeneic) situations while mitigating graft-versus-host disease (GvHD). Innate immune cells can be augmented ex vivo, thereby avoiding cytokine release syndrome (CRS). By utilizing innate immune cell compositions in which the components (e.g., NK cells, gamma-delta (γδ) T cells) and their quantities can be adjusted, a series of synergistic attacks against a variety of cancers, including solid tumors and hematological malignancies, infectious diseases, etc., can be provided.

[0063] In contrast, certain immune cell compositions not produced by the methods described herein contain the majority of adaptive immune cells instead of innate immune cells. When utilizing immune cell compositions in which the majority of cells are adaptive immune cells, recognition occurs through HLA antigens, and there is a significant risk of GvHD, particularly when the adaptive immune cells used in immunotherapy originate from an allogeneic source. Therefore, the adaptive immune cells utilized in such compositions most often originate from the patient / subject (self) or from a designated "matched" donor, which limits the repertoire of immunotherapy, i.e., its ability to be used in an "off-the-shelf" manner in large patient populations. Most cell therapies utilizing such compositions originate from the self or from the least specific donor. This means that the dose for each patient must be individually adjusted to suit that patient. Thus, current T cell-based therapeutic compositions, such as cell therapies using chimeric antigen receptors (CARs) or exogenous T cell receptors (TCRs) (as αβ T cells), cannot be used "off the shelf". The manufacturing process can take six weeks or longer, during which time the patient's disease, such as cancer or infection, may progress. Similarly, some of these patient-specific manufacturing runs fail for a variety of reasons, including general manufacturing failures as well as specific failures due to depletion of the patient-donor immune system after chemotherapy or radiotherapy. In addition, the in vivo expansion of αβ T cells (or CAR-modified, "CAR-T" cells) sometimes causes a massive and rapid release of cytokines into the bloodstream, leading to CRS that can be severe or life-threatening.

[0064] Therefore, the therapeutic immune cell compositions described herein offer advantages over cell compositions containing the majority of adaptive immune cells. The therapeutic immune cell compositions described herein are also modified or altered compared to naturally occurring biological samples obtained from the subject. The therapeutic immune cell compositions, methods of preparation, and therapeutic uses are described in detail below.

[0065] Method for producing a natural immune cell composition This specification provides a method for producing innate immune cell compositions. Compositions produced by the method provided herein often contain a mixture of two activated innate immune cell populations: natural killer (NK) cells and gamma-delta T cells.

[0066] The immune cell compositions obtained by the methods provided herein are enriched with respect to innate immune cells, namely NK cells and gamma-delta T cells, compared to immune cell compositions in nature (e.g., biological fluids and tissues). In nature, adaptive immune cells, namely alpha-beta T cells, are found in considerably larger quantities than NK cells and gamma-delta T cells. In contrast, in the compositions provided herein, alpha-beta T cells are absent or present in negligible to small amounts, with NK cells and gamma-delta T cells being the main immune cell components.

[0067] For example, in human peripheral blood, in the peripheral blood of healthy human donors, (i) in individuals under 40 years of age, the median percentage of NK cells in PBMCs is 5%, and the median percentage of T cells (combined CD8+ and CD4+ populations; since gamma-delta T cells are typically CD8-CD4-, this represents alpha-beta T cells) is 53%; and (ii) in individuals older than 40 years of age, the median percentage of NK cells in PBMCs is 10%, and the median percentage of T cells (combined CD8+ and CD4+ populations) is 37% (Lepone et al., J. Circ. Biomark., 5(5):1-17 (2016)). Thus, peripheral In the blood, individuals older than 40 years of age have approximately four circulating alpha-beta T cells for every one circulating NK cell, while individuals under 40 years of age have approximately ten alpha-beta T cells for every one circulating NK cell. Therefore, the ratio of NK cells to alpha-beta T cells in circulating blood ranges from approximately 1:4 to approximately 1:10. On the other hand, in the compositions provided herein, NK cells constitute 20% or more of the composition, up to a maximum of 99%, while alpha-beta T cells are practically absent, constitute less than 2% of the composition, and generally constitute approximately 0%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of the composition. Thus, in the compositions provided herein, the ratio of NK cells to alpha-beta T cells is at least 10:1, and generally considerably higher than 10:1, for example, about 15:1 or higher, about 20:1 or higher, about 25:1 or higher, about 30:1 or higher, about 35:1 or higher, about 40:1 or higher, about 45:1 or higher, about 50:1 or higher, about 55:1 or higher, about 60:1 or higher, about 65:1 or higher, about 70:1 or higher, about 75:1 or higher, about 80:1 or higher, about 85:1 or higher, about 90:1 or higher, about 95 :1 or higher, approximately 100:1 or higher, approximately 150:1 or higher, approximately 200:1 or higher, approximately 250:1 or higher, approximately 300:1 or higher, approximately 350:1 or higher, approximately 400:1 or higher, approximately 450:1 or higher, approximately 500:1 or higher, approximately 550:1 or higher, approximately 600:1 or higher, approximately 650:1 or higher, approximately 700:1 or higher, approximately 750:1 or higher, approximately 800:1 or higher, approximately 850:1 or higher, approximately 900:1 or higher, approximately 950:1 or higher, or approximately 1000:1 or higher.Thus, although alpha and beta T cells are naturally present in much larger quantities than NK cells, the compositions provided herein show a significant increase in the NK cell population compared to the alpha and beta T cell population.

[0068] For example, regarding gamma delta T cells in peripheral blood, less than 10% of T cells, generally about 5%, are gamma delta T cells, while the remainder are alpha and beta T cells (Esin et al., Scand. J. Immunol., 43(5):593-596 (1996); Radestad et al., J. Immunol. Res., Article ID 578741 (2014)). Therefore, in circulating blood... The ratio of gamma-delta T cells to alpha-beta T cells is in the range of approximately 1:10 to approximately 1:20. On the other hand, in the compositions provided herein, gamma-delta T cells constitute 1% or more of the composition, generally between 2% or more and up to 70-75%, while alpha-beta T cells are not actually present and constitute less than 2% of the composition, generally around 0%, or less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%. Thus, even assuming a composition in which gamma-delta T cells are present at 1% and alpha-beta T cells are present at 2%, the ratio of gamma-delta T cells to alpha-beta T cells is at least 0.5:1. Generally, the ratio of gamma-delta T cells to alpha-beta T cells in the compositions provided herein is quite high, for example, about 5:1 or higher, about 10:1 or higher, about 15:1 or higher, about 20:1 or higher, about 25:1 or higher, about 30:1 or higher, about 35:1 or higher, about 40:1 or higher, about 45:1 or higher, about 50:1 or higher, about 55:1 or higher, about 60:1 or higher, about 65:1 or higher, about 70:1 or higher, about 75:1 or higher, about 80:1 or Higher than that, approximately 85:1 or higher, approximately 90:1 or higher, approximately 95:1 or higher, approximately 100:1 or higher, approximately 150:1 or higher, approximately 200:1 or higher, approximately 250:1 or higher, approximately 300:1 or higher, approximately 350:1 or higher, approximately 400:1 or higher, approximately 450:1 or higher, approximately 500:1 or higher, approximately 550:1 or higher, approximately 600:1 or higher, approximately 650:1 or higher, approximately 700:1 or higher, or approximately 750:1 or higher, or higher.

[0069] As used herein, the term “enriched” means that the following two ratios in the compositions provided herein: (i) the ratio of gamma-delta T cells to alpha-beta T cells, and (ii) the ratio of NK cells to alpha-beta T cells are higher than these ratios in nature, for example, in biological samples such as peripheral blood. When used herein in general, “enriched” means that the ratio of gamma delta T cells to alpha beta T cells in the compositions provided herein is increased by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 times, or more, compared to the ratio in a biological sample, e.g., tissue, umbilical cord blood, or peripheral blood. In the case of NK cells, as generally used herein, “enriched” means that the ratio of NK cells to alpha / beta T cells in the compositions provided herein is at least 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, or 1000-fold, or greater, compared to the ratio in a biological sample, e.g., tissue, umbilical cord blood, or peripheral blood. In certain embodiments, the term “enriched” means that the compositions provided herein have a ratio of NK cells to alpha-beta T cells greater than 1, and a ratio of gamma-delta T cells to alpha-beta T cells (this ratio is generally less than 1 in nature).

[0070] In peripheral blood, adaptive immune cells, namely B cells and alpha-beta T cells, constitute the majority of lymphocytes. On the other hand, the compositions produced by the methods provided herein are enriched with respect to innate immune cells, namely NK cells and gamma-delta T cells. The terms alpha-beta T cells and αβ T cells are used interchangeably herein and refer to adaptive immune T cells, while the terms gamma-delta T cells and γδ T cells are used interchangeably herein and refer to innate immune T cells.

[0071] Immunotherapy compositions composed of innate immune cells are advantageous compared to compositions containing adaptive immune cells for a variety of reasons. Innate immune responses are nonspecific and are initiated against the target of interest (e.g., cancer or infectious disease) within minutes to hours after infection. Innate immune cells can be expanded ex vivo. The expansion conditions provided herein can result in compositions of NK cells and gamma-delta T cells that remain unexhausted for up to 60 days after expansion, allowing for long-term storage (suitable for stockpiling and re-administration if needed) and cost-effective administration of multiple doses. Adaptive immune cells, on the other hand, initiate and expand an antigen-specific immune response upon binding to a target antigen in vivo, but this can take days or even weeks. Furthermore, unlike innate immunity, adaptive immune responses are HLA antigen-dependent, meaning that cells must be self-referential or "matched" to the recipient patient / subject to minimize or avoid GvHD (graft-versus-host disease). Compositions prepared by the methods provided herein that contain innate immune cells, such as NK cells and gamma-delta (γδ) T cells, do not require HLA antigen recognition to mediate killing and can therefore be used in a wider range of off-the-shelf (allogeneic) settings while avoiding GvHD. Adaptive immune cells can also produce large amounts of cytokines when expanded in vivo, potentially leading to cytokine release syndrome (CRS), whereas innate immune cells can be expanded ex vivo, thereby minimizing or avoiding CRS.

[0072] In the methods provided herein, a sample containing cells, such as peripheral blood or umbilical cord blood, is obtained from a subject. The subject is generally a healthy donor, but may also be a patient requiring treatment with an immunotherapy composition prepared by the methods provided herein. In certain embodiments, when the sample is peripheral blood, a universal donor bank of peripheral blood can be used as a source of the sample. The sample is sometimes subjected to activation conditions, which include contacting the sample with (a) at least one exogenous polypeptide that immunospecifically binds to a cell adhesion polypeptide, and (b) at least one exogenous polypeptide that immunospecifically binds to a polypeptide different from the cell adhesion polypeptide and is expressed on the surface of one or more types of cells in the sample population. Examples of cell adhesion polypeptides include, but are not limited to, CD2 (see, e.g., GenBank accession numbers KJ905161.1;KJ896558.1), LFA-1 (see, e.g., GenBank accession numbers BC005861.2), LFA-3 (see, e.g., GenBank accession number BC005930.1), CD8 (see, e.g., GenBank accession numbers AH003215.2;AY039664.1 for CD8A; see, e.g., GenBank accession numbers KJ896562.1;BC100911.1;BC100912.2;BC100913.1;BC100914.1 for CD8B), and CD4 (see, e.g., GenBank accession numbers M35160.1;DQ892052.2).Examples of polypeptides expressed on the surface of one or more cell types in a sample population, such as NK cells and / or gamma-delta cells, include CD2 (see, e.g., GenBank accession numbers KJ905161.1; KJ896558.1), CD3 (see, e.g., GenBank accession number AB583162.1 for CD3γ; see, e.g., GenBank accession number AB583139.1 for CD3ε; see, e.g., GenBank accession number AH002612.2 for CD3δ), CD 56 (see, for example, GenBank accession number U63041.1;BC047244.1;BC029119.1), NKp30 (see, for example, GenBank accession number AB055881.1), NKp44 (see, for example, GenBank accession number BC166647.1), NKp46 (see, for example, GenBank accession number BC064806.1;AY346373.1), NKG2A (see, for example, GenBank accession number AF461812.1;BC053840.1), PD-1 ( For example, see GenBank accession number L27440.1), NKG2C (for example, see GenBank accession numbers BC093644.1;BC112039.1), NKG2D (for example, see GenBank accession numbers AF461811.1;BC039836.1), KAR receptor, KIR receptor, SIGLEC-7 (for example, see GenBank accession numbers AF193441.1;AF170485.1), KIR3DS1 (for example, see GenBank accession number EU156175.1) Examples include, but are not limited to, KIR2DL1 (e.g., GenBank accession numbers LT984790.1;LT984791.1; antibody: 11PB6), DNAM1 (e.g., GenBank accession numbers BC074787.2;U56102.1), NTBA (e.g., GenBank accession numbers BC114495.1;BC113893.1), HLA-DR (e.g., GenBank accession number AH001506.2 for α; AH002824.2 for β), etc.Cell activation generally initiates an innate immune response in cells of the compositions provided herein, which can be used to target diseases in subjects requiring treatment of such diseases, such as cancer or infectious diseases.

[0073] A polypeptide immunospecifically binds to a region of another molecule (i.e., an epitope) if it reacts to or associates with that epitope more frequently, more rapidly, for a longer period, and / or with higher affinity compared to an alternative epitope. For example, a polypeptide that immunospecifically binds to a first epitope (e.g., an antibody or its antigen-binding fragment) is an antibody that binds to this first epitope with higher affinity, avidity, more readily, and / or for a longer period than it binds to a second epitope or other epitopes. A polypeptide that immunospecifically binds to a first target may or may not bind specifically or preferentially to a second target, and immunospecific binding is not necessarily exclusive binding.

[0074] In certain embodiments of the methods provided herein, the activation condition includes contacting a sample with at least two exogenous polypeptides. In some embodiments, the first exogenous polypeptide binds immunospecifically to CD2, and the second exogenous polypeptide binds immunospecifically to NKp46. In some embodiments, the activation condition consists of, or is essentially derived from, the first exogenous polypeptide that binds immunospecifically to CD2 and the second exogenous polypeptide that binds immunospecifically to NKp46. The phrase "essentially derived from," as used herein, means that the presence of components other than those listed does not substantially alter the activity of the listed components. Thus, for example, in the context of the activation condition described above, the activation condition may include one or more components other than the first exogenous polypeptide that binds immunospecifically to CD2 and the second exogenous polypeptide that binds immunospecifically to NKp46, which do not substantially alter the activity of the first and second exogenous polypeptides. In certain embodiments, the first exogenous polypeptide and / or the second exogenous polypeptide are antibodies or antigen-binding fragments thereof.

[0075] Furthermore, the sample is often subjected to expansion conditions, which include contacting the sample with at least one auxiliary polypeptide to generate a composition containing a population of cells enriched with respect to NK cells and gamma delta T cells. In certain embodiments of the methods provided herein, the expansion conditions include at least one auxiliary polypeptide that is a cytokine and / or a polypeptide that immune-specifically binds to a receptor on gamma delta T cells. In certain embodiments, the cytokine is an interleukin, e.g., IL-1, IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or any combination thereof. In some embodiments of the methods provided herein, the cytokine is IL-2, IL-15, or a combination thereof. In several embodiments, the expansion conditions consist of, or are essentially, a polypeptide that immune-specifically binds to a receptor on gamma delta T cells, including IL-2, IL-15, and a polypeptide. In certain embodiments, the receptor on gamma delta T cells is CD3. In some embodiments, the polypeptide that immunospecifically binds to the CD3 receptor on gamma delta T cells is an antibody or an antigen-binding fragment thereof, and in certain embodiments, the antibody is OKT3. In certain embodiments, the expansion condition comprises, or is essentially thereof, contacting a sample with (a) IL-2 polypeptide; (b) IL-15 polypeptide; (c) IL-2 polypeptide and IL-15 polypeptide; (d) IL-2 polypeptide and an antibody that immunospecifically binds to CD3; or (e) IL-2 polypeptide, IL-15 polypeptide, and an antibody that immunospecifically binds to CD3. In some embodiments, the antibody that immunospecifically binds to CD3 is OKT3.

[0076] In some embodiments, the activation conditions consist of, or consist essentially of, contacting the sample with a first exogenous polypeptide that immunospecifically binds to CD2 and a second exogenous polypeptide that immunospecifically binds to NKp46, and the expansion conditions consist of, or consist essentially of, contacting the sample with (a) an IL-2 polypeptide; (b) an IL-15 polypeptide; (c) an IL-2 polypeptide and an IL-15 polypeptide; (d) an IL-2 polypeptide and an antibody that immunospecifically binds to CD3; or (e) an IL-2 polypeptide, an IL-15 polypeptide, and an antibody that immunospecifically binds to CD3. In some embodiments, the antibody that immunospecifically binds to CD3 is OKT3.

[0077] In certain embodiments, the methods provided herein for manufacturing a composition include depleting alpha-beta T cells from the sample prior to activation and expansion, thereby generating a depleted cell population, and then subjecting them to activation and expansion. For example, alpha-beta T cells can be depleted from the sample using an antibody that immunospecifically binds to an alpha-beta T cell receptor, such as an alpha-beta TCR. As used herein, the term "depleted" means that substantially all of the component being depleted (e.g., alpha-beta T cells) has been removed from the sample, e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, up to about 100% of the component being depleted has been removed from the sample. Generally, the term "depleted cell population" as used herein refers to a population of cells derived from a sample after depletion of alpha-beta T cells from the sample. In certain embodiments, the sample is further depleted of B cells using, for example, a polypeptide that immunospecifically binds to a B cell receptor, such as CD19.

[0078] For example, if the sample is a peripheral blood sample, the sample can be treated by density gradient centrifugation to separate and / or isolate a buffy coat containing leukocytes, platelets, granulocytes, etc., which can then be subjected to alpha-beta T cell depletion according to the method provided herein. In certain embodiments, mononuclear cells (PBMCs) can be obtained by further Ficoll density gradient separation of the buffy coat, which can then be subjected to alpha-beta T cell depletion according to the method provided herein. In some embodiments, the peripheral blood sample can be apheresis to separate the plasma from the cells (e.g., using a Terumo Optia instrument), and in certain embodiments, the cells can then be subjected to alpha-beta T cell depletion according to the method provided herein. Alpha-beta T cell depletion can be carried out by methods known to those skilled in the art. In certain embodiments, alpha-beta T cells can be depleted using a Miltenyi LS column. In some embodiments, alpha-beta T cell depletion can be performed on the apheresis product using a Miltenyi Clinimacs separation device. The depleted cell populations can be cryopreserved (e.g., at -70°C, -75°C, -80°C, -85°C or lower) if necessary or desirable, and can be stored until activated and expanded. Non-limiting examples of culture conditions for activation and expansion include the activation and expansion conditions described in the Examples section of this specification for cell populations containing NK cells and gamma delta cells, or NK MACS® medium supplemented with 5% AB serum (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA)) or other media used in the art (e.g., R&D Systems, CellGenix).

[0079] In certain embodiments, the cell population is not exposed to conditions that are positively selective for NK cells or gamma delta T cells prior to the activation and expansion conditions described herein.

[0080] In certain embodiments of the methods provided herein, a sample or a depleted cell population is subjected to activation conditions under which the exogenous polypeptide of (a), the exogenous polypeptide of (b), or both the exogenous polypeptide of (a) and the exogenous polypeptide of (b) are soluble. In multiple embodiments, the soluble exogenous polypeptide of (a), the soluble exogenous polypeptide of (b), or the soluble exogenous polypeptides of both (a) and (b) are an antibody or an antigen-binding fragment thereof. As used herein, the term “soluble” in connection with a component, such as a polypeptide, means that the component is not bound to a solid phase or support and is in a homogeneous single-phase or emulsion state under culture conditions (e.g., activation conditions, expansion conditions). In some embodiments, the exogenous polypeptide of (a) or (b), such as an antibody, binds to a solid phase or support. Examples of solid phase supports include, but are not limited to, silica, glass (e.g., glass, controlled pore glass (CPG)), nylon, Wang resin, Merrifield resin, Sephadex, Sepharose, cellulose, magnetic beads, Dynabeads, metal surfaces (e.g., steel, gold, silver, aluminum, silicon, and copper), plastic materials (e.g., polyethylene, polypropylene, polyamide, polyester, polyvinylidene fluoride (PVDF)). The solid phase support can be in any desired form, including, but not limited to, beads, chips, capillaries, plates, membranes, wafers, combs, pins, substantially flat surfaces, arrays of pits or nanoliter wells, and other geometric patterns and shapes known to those of skill in the art. Using a soluble exogenous polypeptide rather than a polypeptide bound to a solid phase support can reduce steric hindrance while increasing the scalability of the method (e.g., for GMP manufacturing). In certain embodiments, the first exogenous polypeptide is a soluble anti-CD2 antibody and the second polypeptide is a soluble anti-NKp46 antibody. In certain embodiments, either the anti-CD2 antibody or the anti-NKp46 antibody is bound to a solid phase support.

[0081] Antibodies, such as polyclonal and monoclonal antibodies, can be prepared using standard methods (e.g., Kohler et al., Nature 256:495-497 (1975); Kohler et al., Eur. J. Immunol. 6:511-519 (1976); and WO02 / 464). (See 55). For example, to produce polyclonal antibodies, an immune response to the target antigen is induced in a host animal. Blood is then collected from the host animal, and the serum fraction containing the secreted antibodies is separated from the cell fraction using methods known to those skilled in the art. To produce monoclonal antibodies, the animals are immunized by standard methods to produce antibody-secreting somatic cells. These cells are then isolated from the immunized animals and fused with myeloma cells. Somatic cells capable of producing antibodies, particularly B cells, can be used to fused with myeloma cell lines. These somatic cells may be derived from the lymph nodes, spleen, and peripheral blood of the primed animals. Special myeloma cell lines for use in hybridoma-producing fusion techniques have been prepared from lymphoid tumors (Kohler and Milstein, Eur. J.). Immunol. 6:511-519 (1976); Shulman et al., Nature, 276:269-282 (1978); Volk et al., J. Virol., 42:220-227 (1982). These cell lines have three useful properties. They possess the following characteristics. First, they facilitate the selection of fusion hybridomas from non-fusion hybridomas that also endlessly self-reproduce by having enzyme deficiencies that prevent them from growing in selective media that support hybridoma growth. Second, they possess antibody-producing ability and are unable to produce endogenous light-chain or heavy-chain immunoglobulins. Third, they efficiently fuse with other cells. Other methods for producing hybridomas and monoclonal antibodies are well known to those skilled in the art. It is customary to produce antibodies against any polypeptide, e.g., antigenic markers on an immune cell population.

[0082] In certain embodiments of the methods provided herein, the activation conditions, expansion conditions, or both activation and expansion conditions are feeder cell-free. The term “free” as used herein (e.g., feeder cell-free or feeder cell-free, serum-free, serum-free from non-human animals, exhausted cells-free) means that the conditions are substantially free of components (i.e., feeder cells, serum, exhausted cells, or other components referred herein), i.e., at least 80%, 85%, 90%, 95%, generally 95% or more, e.g., 96%, 97%, 98%, 99%, or more, up to 100%. In certain embodiments of the methods provided herein, the sample, activation conditions, expansion conditions, or activation and expansion conditions, or all of the foregoing, are free of exogenous cells, exogenous feeder cells, irradiated cells, and / or irradiated feeder cells. Exogenous cells and exogenous feeder cells are generally cells derived from a different subject or a different part of a subject compared to the subject or part of a subject from which the sample cells are obtained and subjected to activation and / or expansion conditions. In non-restrictive examples, (i) the sample cells are derived from a subject of a first species and the exogenous cells are derived from a subject of a second species (e.g., the sample cells are derived from humans and the exogenous cells are derived from a non-human animal, e.g., a rodent or monkey), and (ii) the sample cells are derived from the peripheral blood of a human subject and the exogenous cells are derived from a different part of the same subject (e.g., from umbilical cord blood or organs of the same subject, or from a different human subject). A composition is generally considered "not containing" a particular component if the sample has not come into contact with that component during one or all of the following: pre-activation and / or expansion processing, activation, and expansion.

[0083] Reliance on feeder cells such as K562 cells can limit the location and method of cell culture and significantly increase the cost of cell culture. The use of feeder cells can also be problematic due to variability in cell culture caused by undefined biological factors originating from the feeder cells themselves. In addition, feeder cells can introduce unwanted agents (e.g., retroviruses, other pathogens, and immunogenic nonhuman sialic acids). There is a possibility of introducing Neu5Gc) into compositions prepared by the methods provided herein, which may be undesirable for certain applications, such as transplantation. While we do not wish to be constrained by theory, the methods provided herein allow exogenous polypeptides that immunospecifically bind to cell adhesion polypeptides to generate an "endogenous feeder cell-like layer," thereby generating a significantly activated and expanded cell population without the need for exogenous feeder cells.

[0084] In certain embodiments of the methods provided herein, the selection of auxiliary polypeptides and / or expansion time in expansion conditions can be adjusted to suit desired relative amounts of NK cells and gamma delta T cells. For example, compositions containing relatively more NK cells and relatively fewer gamma delta T cells generally have greater applicability to solid tumors, while compositions containing relatively more gamma delta T cells and relatively fewer NK cells generally have greater applicability to hematological malignancies. The auxiliary polypeptides and / or expansion reaction can be selected and carried out for a time that facilitates obtaining a composition having the desired ratio of NK cells and gamma delta T cells.The desired ratio of NK cells to gamma delta T cells can be obtained by selecting specific enrichment conditions, and non-restrictive examples of combinations are as follows: (i) when the coordinating polypeptide is IL-2, the resulting cell population enriched with respect to NK cells and gamma delta T cells often contains about 25-30% NK cells and about 70-75% gamma delta T cells; (ii) when the coordinating polypeptide is IL-15, the resulting cell population enriched with respect to NK cells and gamma delta T cells often contains about 80-99% NK cells and about 1-20% gamma delta T cells; (iii) when the coordinating polypeptide is an antibody that immunospecifically binds to IL-2 and CD3, e.g., OKT3, the resulting NK cells and gamma delta T cells With respect to delta T cells, the enriched cell population often contains approximately 40-45% NK cells and approximately 55-60% gamma delta T cells; (iv) when the co-auxiliary polypeptide is IL-2 until day 20 of the expansion condition and then switched to IL-15 until day 30, the percentage of gamma delta T cells often increases from approximately 50% to approximately 70% compared to treatment with IL-2 alone, and the percentage of NK cells decreases accordingly; and (v) when the co-auxiliary polypeptide is IL-15 until day 20 of the expansion condition and then switched to IL-2 until day 30, the percentage of NK cells often increases from approximately 80% to approximately 90% compared to treatment with IL-15 alone, and the percentage of gamma delta T cells decreases accordingly.

[0085] The duration of the expansion conditions (e.g., comparing 12 days and 25 days) can also control the relative amounts of NK cells and gamma delta T cells in the compositions prepared by the methods provided herein. For example, the length of incubation of a sample or depleted cell population in feeder cell-free medium under expansion conditions may be about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 30, 35, 40, 45, 50, 55, or 60 days or more, or about 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In certain embodiments of the methods provided herein, the composition is depleted of cells, and in some embodiments, the composition is depleted of cells at least 60 days after expansion conditions. Preparing cell compositions that are substantially free of exhausted cells will increase the ex vivo availability of expanded cytotoxic cell compositions, enabling immediate treatment and multiple doses of many current immunotherapies (e.g., CAR-T using alpha and beta T cells) at minimal cost.

[0086] In certain embodiments of the methods provided herein, activation, or expansion, or activation and expansion conditions, without the presence of bisphosphonates, result in polyclonal populations of V. delta.1 and V. delta.2 gamma delta T cells. While the use of bisphosphonates, such as zoledronate and pamidronate, may be more favorable to one clonal population of gamma delta T cells than to other populations, this may limit the repertoire of compositions for certain tumors and infections. In some embodiments, the absence of bisphosphonates in the methods provided herein generates compositions that are polyclonal with respect to gamma delta T cells, thereby increasing the range of cancers and infectious diseases for which they can be administered as immunotherapies.

[0087] A non-limiting example of the outline of steps for carrying out the method provided herein is as follows: (1) Thaw and culture a sample from the donor (e.g., peripheral blood directly from the donor or from a blood bank) as needed; (2) If desired, subject the culture to alpha-beta T cell depletion, and B cell depletion as needed; (3) Subject the sample or any remaining cells after depletion (depleted cell population) to activation conditions for 3-4 days to generate activated cells; (4) If desired, transduce the activated cells with exogenous polynucleotides using, for example, a retroviral vector or lentiviral vector; (5) After activation in (3) or transduction in (4), subject the cells to expansion conditions on a 7-day cycle, wash between cycles, and generally perform 2-3 cycles to generate expanded cells, which may sometimes be used immediately, or sometimes refrigerated, maintained on ice, or frozen for transport and / or storage until needed for immunotherapy. The ratio of NK cells to gamma delta T cells can be adjusted, if desired, by selecting the co-opposite polypeptide(s) used during expansion, including switching co-opposite polypeptides during the 7-day cycle, and by controlling the expansion time.

[0088] In a particular embodiment, the composition resulting from a sample cell population exposed to activation and expansion conditions has the following characteristics: (i) A ratio greater than 1 (for example, a ratio of approximately 1.5 or greater, 2 or greater, 3 or greater, 4 or greater, or 5 or greater; approximately 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 or greater ratio) ratio of NK cells to gamma delta T cells; or (ii) The ratio of NK cells to gamma-delta T cells less than 1 (for example, ratios of about 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.1 or less, 0.05 or less, 0.01 or less; ratios of about 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, 0.001 or less); It has the following features as needed: (iii) The ratio of NK cells to alpha / beta T cells greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or greater than that; (iv) The ratio of gamma-delta T cells to alpha-beta T cells greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or greater than that; (v) Percentage of NK cells of 20% or more of total cells; (vi) Percentage of gamma-delta T cells relative to total cells, 2%, 3%, 4%, 5%, or greater; (vii) Approximately 75% to 95% of gamma-delta T cells (e.g., approximately 80% to 90% of gamma-delta T cells (e.g., approximately 85%)) express V.delta.1; (viii) Approximately 10% to 25% (e.g., approximately 10% to 20% (e.g., approximately 15% of gamma delta T cells) express V delta 2; (ix) Approximately 30% to 60% (approximately 35% to 55%) of gamma-delta T cells express V.delta.1; (x) Approximately 35% to 60% (e.g., approximately 40% to 50% or 55%) of gamma-delta T cells express V.delta-2; (xi) Approximately 10% to 30% of gamma-delta T cells (for example, approximately 20% to 25% of gamma-delta T cells) express V.delta.1; (xii) Approximately 65% ​​to 80% (e.g., approximately 70% to 80% (e.g., approximately 75% of gamma delta T cells) express V. delta 2; (xiii) Approximately 80% or more of the total cells (e.g., approximately 90% or more of the cells) express KIR5; (xiv) Approximately 5% or more of the total cells (e.g., approximately 10% or more of the cells) express SIGLEC-7; (xv) Approximately 50% or more of the total cells (e.g., approximately 60% or more of the cells) express KIR3D51; (xvi) Approximately 5% or more of the total cells (e.g., approximately 10% or more of the cells) express KIR2DL1; (xvii) Approximately 20% or more of total cells (e.g., approximately 25% or more of cells) express NKp30, NKp44, or NKp46; (xviii) Approximately 25% or more of the total cells (e.g., 35% or more of the cells) express NKG2D; (xix) Approximately 80% or more of the total cells (e.g., approximately 90% or more of the cells) express DNAM1; (xx) Approximately 75% or more of the total cells (e.g., approximately 85% or more of the cells) express NTBA; (xxi) Approximately 85% or more of the total cells express CD2 (for example, approximately 95% or more of the cells); (xxii) Approximately 80% to 100% of total cells express CD56 (for example, at least approximately 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%); (xxiii) Approximately 51% to 100% of total cells (e.g., at least approximately 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) do not express CD57; (xxiv) Approximately 10% to 40% of total cells express CD16 (for example, at least approximately 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%); (xxv) Approximately 10% or less of total cells express CD57 (e.g., approximately 5% or less, 4% or less, 3% or less, 2% or less of cells); (xxvi) Approximately 50% or more of the total cells (e.g., approximately 55% or more of the cells) express KIR3DS1; (xxvii) Approximately 5% or less of the total cells (e.g., approximately 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less) are NKT cells; (xxviii) Approximately 5% or less of the total cells (e.g., approximately 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less) are alpha or beta T cells; (xxix) About 50% or more of NK cells and / or gamma delta cells (e.g., about 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 6%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%) %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any fraction thereof, up to 100%) expressing CD8; (xxx) Approximately 5% or less of NK cells and / or gamma delta T cells (e.g., approximately 4% or less, approximately 3% or less, approximately 2% or less, or approximately 1% or less of NK cells and / or gamma delta T cells) express CD4; (xxxi) less than or about 5% of NK cells and / or gamma-delta T cells (e.g., less than or about 4%, less than or about 3%, less than or about 2%, or less than or about 1% of NK cells and / or gamma-delta T cells) express CD8 and CD4; (xxxii) from about 15% to about 35% of NK cells (e.g., 16%, 17%, 18%, 19%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34% of NK cells) do not express CD8 and CD4; and (xxxiii) from about 55% to about 85% of gamma-delta T cells (e.g., about 56%, 58%, 60%, 62%, 63%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84% of gamma-delta T cells) do not express CD8 or CD4 having one or more of the above.

[0089] Innate immune cell composition A particular composition prepared by the manufacturing method described herein is referred to as a BINATE composition. The BINATE composition contains two natural cell types, namely NK cells and gamma-delta T cells, and the relative amounts of these two cell types can be adjusted to treat solid tumors, blood cancers, or infectious diseases by selecting suitable auxiliary polypeptide(s) or polypeptides including the order of their use and the period of expansion conditions.

[0090] In some embodiments, the BINATE composition does not contain feeder cells, and these compositions often do not contain feeder cells (e.g., they do not contain exogenous feeder cells and / or irradiated feeder cells). In certain embodiments, the activation and expansion conditions do not contain bisphosphonates, thereby allowing polyclonal gamma delta T cells to have a broader repertoire of activity against tumors and infectious diseases (i.e., polyclonal with respect to V. delta 1 and V. delta 2 gamma delta T cells). This innate immune cell platform, interchangeably referred to herein as BINATE cells, INNATE cells, or INNATE-K cells, can comprehensively bind to both tumor signals and receptors, providing readily available and versatile therapies for solid tumors, hematological malignancies, and infectious diseases. The improved safety of innate immunotherapy makes it possible to use it in community hospitals, reducing healthcare costs and providing effective treatment to more patients.

[0091] In certain embodiments, the BINATE cell compositions provided herein can be further treated using markers to actively select a target cell population or remove an undesirable cell population to produce compositions containing substantially all NK cells (hereinafter referred to as "INNATE-NK") or substantially all gamma delta T cells.

[0092] The BINATE compositions provided herein can be developed to target both solid tumors and hematological malignancies by a) non-genetically modified cells in post-transplant or other situations / indications; b) non-genetically modified cells administered in combination with other therapeutic agents, e.g., commercially available antibodies for cancer treatment; or c) genetically modified cells by, for example, mutation of endogenous polynucleotides, deletion of endogenous polynucleotides, addition of exogenous mutant polynucleotides (wild-type is present in non-modified cells), or addition of heterogeneous exogenous polynucleotides, e.g., CAR polynucleotides (BINATE.CAR). The CAR-modified INNATE-K (same as BINATE) or INNATE-NK population is indicated herein by any of the following terms:

[0093] Generally, in the case of immune cells modified by CAR, the cells are called INNATE-CAR, INNATE-K.CAR, INNATE-NK.CAR, and BINATE.CAR, with a period or hyphen followed by the suffix "CAR"; and

[0094] In the case of immune cells modified with a specific CAR, such as a CAR targeting CD19, the cells are interchangeably referred to as BINATE.CD19, BINATE.CAR19, or BINATE.CAR.CD19, with a period or hyphen followed by the suffix "CD19", "CAR19", or "CAR.CD19".

[0095] The manufacturing methods provided herein involve a ratio of approximately 30% to approximately 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more of cells in a population, or at least approximately 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62% of cells in a population. This results in BINATE cell populations with high levels of activation of up to 100%, including 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher. In comparison, inducing an innate immune response in vivo activates 10% or less of cells.

[0096] These activated cells are highly cytotoxic and have a significant proportion of CD56+CD16+ cells (for example, about 40% of the NK cell population in the compositions provided herein may be CD56+CD16+). Where used herein, a "+" symbol or term "plus" in relation to a description of a cell marker indicates that the marker is expressed in the cell (or on the cell surface), while a "-" symbol or term "minus" in relation to a description of a cell marker indicates that the marker is not present or undetectable.

[0097] The compositions provided herein exhibit high cytotoxicity in BINATE cells, but the majority of cells have low CD57 marker levels (high CD57 marker levels indicate cytotoxicity, but also indicate that the cells are closer to aging and therefore have a shorter lifespan (see Kared et al., Cancer Immunol. Immunotherap., 65(4):441-452 (2016))), indicating low maturity (i.e., they are further from aging). As used herein, the term “majority” means more than 50% of the cells in a population, generally 50.5% or more, e.g., 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%. , 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any fraction thereof, up to 100%. BINATE cell compositions prepared by the manufacturing methods provided herein also have low levels of exhaustion markers, such as PD-1 and TIM-3. Non-limiting examples of activation / cytotoxic / exhaustion marker phenotypes for BINATE compositions obtained under different expansion conditions according to the manufacturing methods provided herein are summarized below. [Table A]

[0098] In certain embodiments, cells of the BINATE compositions provided herein can be genetically modified. Certain non-limiting examples of genetic modification include: (i) adding an exogenous polynucleotide encoding a polypeptide having the desired activity; (ii) adding an exogenous regulatory polynucleotide (e.g., a primer or enhancer) that alters or adds an endogenous polynucleotide or modifies the expression of an endogenous polypeptide having the desired activity; (iii) altering and / or interfering with the endogenous polynucleotide encoding a polypeptide having the desired activity (e.g., insertion mutagenesis); (iv) partially or completely deleting a regulatory polynucleotide that modulates the expression of a polypeptide having the desired activity, thereby interfering with its regulation; and / or (v) partially or completely deleting a coding sequence encoding a polypeptide having the desired activity, thereby reducing or eliminating its activity (e.g., knockout mutagenesis).

[0099] In some embodiments, the BINATE composition is genetically modified by adding exogenous (regulatory or coding) polynucleotides. Highly activated cells of the BINATE composition are genetically modified with high efficiency, often in 80% or more of the cells in the population, and generally at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%. Transduction can occur at 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or fractions thereof, up to a maximum of 100%. This transduction rate is considerably higher than the rate often observed for adaptive immune cells (e.g., about 9% for alpha and beta T cells).

[0100] In certain embodiments, the exogenous polynucleotide encodes a chimeric antigen receptor (CAR), and the cells in the composition contain the CAR (referred to as "BINATE.CAR cells"). In some embodiments, the CAR contains a binding molecule moiety that immunospecifically binds to one or more of the following: CD19, GD2, HER3, B7H3, CD123, or CD30.

[0101] Using the BINATE and BINATE.CAR cells provided herein, a variety of cancers and infectious diseases can be targeted (e.g., GD2 / HER3 / B7H3: lung / bronchial, prostate, breast, colon, pancreatic, ovarian; CD123: leukemia; CD30: non-Hodgkin lymphoma; other cancers including liver and intrahepatic cholangiocarcinoma, esophageal cancer, bladder cancer, kidney and renal pelvis cancer, uterine cancer, brain / nervous system cancer). GD2, a disiaroganglioside, has also been found to be expressed on the surface of tumor cells of neuroectoderm origin. Tumors with GD2 expression have a high mortality rate (childhood tumors - neuroblastoma, retinoblastoma, sarcoma; adult tumors - melanoma, non-small cell lung cancer, breast cancer). Monoclonal antibodies currently being tested as treatments are limited by their toxicity to neuropathic pain. Initial testing with the BINATE.CAR.GD2 construct showed comparable high levels of transduction in both NK cells and gamma delta T cells (approximately 80% in NK cells and approximately 40-60% in gamma delta T cells); expression was stable and maintained throughout the BINATE culture period. In addition, in vitro killing of solid tumor cells was observed (Example 18).

[0102] Pharmaceutical compositions and kits Any of the compositions provided herein can be formulated as a pharmaceutical composition with a pharmaceutically acceptable carrier. Similar to the compositions, the pharmaceutical compositions provided herein can be used to treat cancer and infectious diseases. Similarly, kits containing the compositions or pharmaceutical compositions provided herein, and, if applicable, instructions for use, are also provided herein. In certain embodiments, the kits provided herein may contain cytokines. Innate immune cells control opportunistic invasions by a wide range of viral, fungal, bacterial, and parasitic pathogens by coordinating with other cells, in part, by releasing excessive amounts of cytokines and chemokines, thereby integrating the immune response.

[0103] A pharmaceutical composition or kit sometimes contains a specific dosage of therapeutic cells, and sometimes provides a unit dosage of therapeutic cells. In a particular embodiment, the unit dosage is approximately 10 cells per kilogram of body weight of the intended subject. 4 From about 10 10 Approximately 10 cells per individual or subject 6 From about 10 12 Between individuals (for example, about 10 cells per subject) 10 Approximately 10 cells per kilogram of body weight of an individual or intended subject 8 It is (one).

[0104] A pharmaceutical composition or kit may contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” means that it is approved by a federal or state regulatory authority for use in animals, more specifically in humans, or that it is listed in the United States Pharmacopeia or other generally recognized pharmacopoeia. The term “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic agent is administered. If the composition is administered by infusion, it may be dispensed into an infusion bottle containing sterile pharmaceutical-grade water or saline. If the composition is administered by injection, an ampoule of sterile water or saline for injection may be provided so that the components can be mixed before administration.

[0105] Pharmaceutical compositions are sometimes provided as pharmaceutical packs or kits comprising one or more containers filled with a therapeutic composition of cells prepared by the methods described herein, either alone or together with such pharmaceutically acceptable carriers. In addition, one or more other prophylactic or therapeutic agents useful for the treatment of a disease may also be included in the pharmaceutical packs or kits. A pharmaceutical pack or kit may comprise one or more containers filled with one or more components of the pharmaceutical compositions described herein. Where necessary, a notice in the form prescribed by the governmental authority regulating the manufacture, use, or sale of a pharmaceutical or biologic product may be associated with such container(s), the notice reflecting the authority's approval for manufacture, use, or sale for human administration. A pharmaceutical pack or kit sometimes comprises one or more containers containing one or more other prophylactic and / or therapeutic agents useful for the treatment of a disease.

[0106] When cells are administered as immunotherapy after being enlarged ex vivo, the cells sometimes do not produce sufficient amounts of cytokines on their own. Cytokines that may be optionally added to the kits provided herein include, but are not limited to, TNF, IFNγ, interleukins, IL-1β, IL-4, IL-6, IL-7, IL-10, IL-12, IL-18, IL-21, CCL4 / RANTES, and TGFβ. In certain embodiments, the pharmaceutical compositions and / or kits provided herein may optionally include a second agent for co-administration with the compositions provided herein. The terms “co-administer,” “co-administers,” and “co-administration,” as used herein, mean that the agent is administered before, after, or in conjunction with the natural cell compositions provided herein.

[0107] In some embodiments, the second agent is an antibody that targets cancer cell antigens or infectious pathogens. The compositions, pharmaceutical compositions, and kits provided herein may be maintained at -4°C or below, or at approximately -75°C to approximately -80°C, as appropriate for storage or transport. Method for creating genetically modified natural cell compositions

[0108] The natural cell compositions provided herein can be genetically modified in a suitable manner (for example, by adding exogenous polynucleotides, which are genes or regulatory sequences, by mutating or deleting endogenous genes). Genetic modification can be carried out after obtaining the final composition, following activation and expansion, or after activation but before expansion of the cells. Non-limiting examples of methods for producing various genetic modifications are described below:

[0109] (a) CRISPR-CAS9 targeting suppression (permanent gene / locus deletion) Cells can be transfected with a DNA plasmid expressing both the CAS9 protein and a guide RNA (gRNA) specific to the target gene. gRNA-CAS9 mediated breaks in the genome can be repaired using the donor DNA plasmid, resulting in a specific deletion of the target gene and a permanent and complete loss of the gene-coding protein. Loss of protein expression can be verified using PCR (at the DNA level), Northern blot / FISH (at the RNA level), or any protein assay, such as Western blot or flow cytometry.

[0110] (b) CRISPR-CAS9 targeted expression (permanent gene / locular insertion) This method allows for the insertion of a target gene at a specific location in the cell genome. Cells can be transfected with a DNA plasmid expressing both a CAS9 protein and a guide RNA (gRNA) specific to the specific insertion site. gRNA-CAS9 mediated breaks in the genome can be repaired using a donor DNA plasmid containing the inserted target gene, flanked by the cell genome sequence on both sides of the DNA cut / double-strand break site, resulting in homologous recombination-mediated insertion of the target gene at a non-random, specific genomic location. Successful insertion and protein expression can be verified using PCR (at the DNA level), Northern blot / FISH (at the RNA level), or any suitable protein assay, such as Western blot or flow cytometry.

[0111] (c) RNA interference (permanent gene repression) via retrovirus / lentivirus / transposon-mediated transduction of shRNA / microRNA ShRNAs / microRNAs targeting specific genes / proteins of interest can be designed and cloned in retroviral / lentiviral / transposon vectors for stable integration into the cell genome. The vector can be transduced into cells, and successfully transduced cells can be selected using a selection marker encoded by the vector. ShRNA-mediated repression of the target gene can be evaluated, for example, using Northern blotting and protein assays.

[0112] (d) Lentivirus / γ-retrovirus-mediated random / multiple copy gene insertion Specific genes / proteins of interest can be designed and / or cloned in retroviral or lentiviral vectors for stable, random incorporation into the cellular genome. Cells can be transduced with the viral vector, and successfully transduced cells can be selected using a selection marker encoded by the vector. shRNA-mediated repression of the gene of interest can be evaluated using Northern blotting and any suitable protein assay, such as Western blotting or flow cytometry.

[0113] (e) Transposon-mediated random / multi-copy gene insertion The target specific gene / protein was designed and / or cloned in a mammalian transposon vector system, e.g., PiggyBac (SBI System Biosciences) or an equivalent. Cells can be co-transfected with a transposon vector and a transposase vector containing the target gene (cDNA) flanked by terminal inverted repeat (ITR) sequences. The transposase enzyme can mediate the transfer of the target gene to the TTAA chromosomal integration site. Cells that successfully transduced can be selected using vector-coding selection markers, if necessary. Successful insertion and protein expression can be verified using PCR (at the DNA level), Northern blot / FISH (at the RNA level), or any suitable protein assay, e.g., Western blot or flow cytometry.

[0114] (f) Direct transfection The mRNA encoding the target gene / protein can be directly transfected into cells. Transfection can be carried out using any established methodology, such as calcium chloride transfection, lipofection, Xfect, electroporation, sonoporation, and cell squeezing (e.g., introducing siRNA).

[0115] Treatment method Similarly, methods for treating cancer or infection are also provided, which involve administering to a subject in need of such treatment any of the compositions, pharmaceutical compositions, or kits provided herein in an amount effective for treating cancer or infection. The treatment may be administered in an autologous or allogeneic context. The donor of the sample from which the composition, pharmaceutical composition, or kit is prepared may be the recipient of the treatment. Often, the composition, pharmaceutical composition, or kit is prepared from a sample derived from one subject, and the treatment is administered to different subjects. In certain embodiments, the treatment may be administered on two or more separate days, and in certain embodiments, the treatment may be administered in multiple doses. In certain embodiments, the treatment may be administered at intervals of about two weeks to about four weeks, in doses ranging from about one unit to about 36 units or more. In some embodiments, the treatment is administered as a single unit dose once, twice, three, four, or up to five times daily, or once, two, three, four, five, six, seven, eight, nine, or ten times, or more times, over several days, weeks, or months, or every two days, or once, two, three, four, five, or six times per week. The treatment can be administered intravenously (IV), intrathecally, or intramuscularly (IM), intraperitoneally (IP), intrapleurally, or intraarticularly, or to the site of cancer or infection or its vicinity, with approximately 10 cells per kilogram of the subject's body weight. 4 From about 10 10 Between individuals, or approximately 10 cells per subject 6 From about 10 12 It is administered by injection or implantation in a unit dose per individual. In a particular embodiment, the unit dose is approximately 10 cells per subject. 10 Approximately 10 cells per kilogram of body weight of the subject. 8 It is an individual.

[0116] In certain embodiments of the treatment methods provided herein, the treatment is for cancer. In some embodiments, the cancer is a solid tumor. In several embodiments, the cancer is a blood cancer. In certain embodiments, the cancer is a blood cancer and the ratio of gamma-delta T cells to NK cells is greater than 1. In some embodiments, the cancer is a solid tumor and the ratio of NK cells to gamma-delta T cells is greater than 1. In certain embodiments of the treatment methods provided herein, the second agent is co-administered with a composition, pharmaceutical composition, or kit. In some embodiments, the second agent is an antibody that binds immune-specifically to a cancer-associated antigen.

[0117] In certain embodiments of the treatment methods provided herein, the treatment is for an infection. In some embodiments, the infection is characterized by the presence of a bacterial pathogen, a fungal pathogen, a viral pathogen, or a protozoan pathogen.

[0118] In certain embodiments of the methods provided herein, the composition, pharmaceutical composition, or kit contains a polyclonal population of gamma delta T cells (e.g., polyclonal with respect to V. delta 1 and V. delta 2 gamma delta T cells).

[0119] This technology will be further described with reference to the examples described herein. However, it should be understood that this technology is not limited to such examples. [Examples]

[0120] The embodiments described below illustrate a particular set of embodiments, but are not intended to limit the scope of this technology.

[0121] The following materials were used in certain methods described below (e.g., activation conditions, expansion conditions, phenotypic analysis).

[0122] The following monoclonal antibodies (mAbs) were produced at the Ospedale Pediatrico Bambino Gesu (OPBG), “Cell and Gene Therapy for Pediatric Tumor” laboratory:

[0123] c218 (IgG1, anti-CD56), c127 (IgG1, anti-CD16), AZ20 and F252 (IgG1 and IgM, anti-NKp30, respectively), BAB281 and KL247 (IgG1 and IgM, anti-NKp46, respectively), Z231 (IgG1, anti-NKp44), ECM217 and BAT221 (IgG2b and IgG1, anti-NKG2D, respectively), KRA236, GN18 and F5 (IgG1, IgG3 and IgM, anti-DNAM-1, respectively), EA4 (IgG2a, anti-CD18), MAR206, MA258 and QA196 (IgG1, IgG2b and IgM, anti-CD2, respectively), MA127 ON56 (IgG1 and IgG2b, anti-NTB-A, respectively), PP35, ST39 and CO54 (IgG1, IgG1a and IgM, anti-2B4, respectively), z27 (IgG1, anti-KIR3DL1 / S1), AZ158 (IgG2a, anti-KIR3DL1 / L2 / S1), z270, z199 and Y9 (IgG1, IgG2b and IgM, anti-NKG2A, respectively), 6A4 and A6 / 136 (IgG1 and IgM, anti-HLA-class I, respectively), D1 / 12 (IgG2a, anti-HLA-DR), 5A10 (IgG1, anti-PVR), L14 (IgG2a, anti-nectin-2), BAM195 (IgG1, anti-MICA).

[0124] F278(IgG1, anti-CD85j)mAb was donated by Dr. Daniela Pende, Istituto Giannina Gaslini, Genoa, Italy (see also, for example, Costa et al., Aids, 15:965-974 (2001)). Anti-NKG2C (IgG2b, clone 134522), anti-ULBP-1 (IgG2a, clone 170818), anti-ULBP-2 (IgG2a, clone 165903), anti-ULBP-3 (IgG2a, clone 166510), anti-CD34-APC (IgG1, clone QBEnd10), IgG-APC isotype control (IgG1 clone 11711), and anti-KIR2DL1-FITC or unconjugated (IgG1, clone 143211) mAbs were purchased from R&D System Inc (Abingdon, United Kingdom). Anti-KIR2DL1 / S1-Vioblue or -PE (IgG1, 11PB6 clone), anti-NKG2C-ViobrightFITC (REA205 clone), anti-KIR3DL1-Biotin or -FITC (IgG1, DX9 clone), anti-CD3-Viogreen (IgG2a, BW264 / 56 clone), anti-CD57-Vioblue (IgM, TB03 clone), anti-SIGLEC-7-Vioblue (REA214 clone), anti-NKp30-PE (IgG1, AF29-4D12 clone), anti-NKp46-PE (IgG1, 9E2 clone), anti-NKp44-PE (IgG1, 2.29 clone), anti-Biotin-PerCPVio700 (REA746 clone), REA-controlled VioBright FITC (clone REA293) and anti-PD-1 (IgG2b, PD1.3.1.3 clone) mAb were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).Anti-CD34 (IgG1, QBEnd10 clone), anti-NKG2A-PC7 (z199 clone), anti-KIR3DL1 / S1-PE (z27 clone), anti-KIR2DL2 / L3 / S2-PE (GL183 clone), anti-CD19-FITC (IgG1, J3-119 clone), anti-CD56-PC7 (IgG1, N901 clone), and IgG1-PC7 or -PE or -FITC isotype control (679.1Mc7 clone) mAbs were purchased from Beckman Coulter, Immunotech (Marseille, France). Anti-KIR2DL2 / L3-S2-FITC or unconjugated (IgG2b, CHL-clone), anti-CD107-PE (IgG1, H4A3 clone), anti-CD85j (IgG2b, GHI / 75 clone), anti-CD16-PerCpCy5.5 (IgG1, 3G8 clone), anti-CD56-BV510 (IgG2b, NCAM16.2 clone), IgG1-PE isotype control (clone MOPC-21) mAbs and Brillant Stain buffer were obtained from BD Bioscience Pharmingen (San Diego, CA). Anti-HLA-Bw6-FITC and anti-HLA-Bw4-FITC mAbs were purchased from ONE LAMBDA INC (Canoga Park, CA). Anti-human HLA-E (IgG1, 3D12 clone) and anti-human HLA-G (IgG1, MEM-G / 9 clone) mAbs were purchased from BioLegend (San Diego, CA) and Abnova (Taipei, Taiwan), respectively. Anti-NKG2D (IgG2a, 5C6 clone) and anti-HLA-C (IgG1, C-8 clone) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA).

[0125] Under expanded conditions, IL-2, IL-15, and OKT3 were obtained from Miltenyi Biotec (San Diego, CA, USA), as well as anti-CD2 and anti-NKp46 antibodies crosslinked or conjugated to beads.

[0126] (Example 1) Enrichment and expansion of specific innate cell-lytic immune cell (INNATE-K) populations derived from αβTCRneg cell populations This embodiment describes a process for enriching and expanding a specific innate cytolytic immune cell population (hereinafter referred to as the INNATE-K or BINATE cell population) consisting mainly of NK and γδTCR+ T cells, from a starting αβTCRneg cell population activated with antibodies against one or more immunoglobulin subfamily surface molecules under feeder-free culture conditions (see Figure 2 for an analysis of the composition of this expanded T cell population; the terms INNATE-K and BINATE may be used interchangeably herein and refer to the cell population considered herein, which is activated by a combination of an NCR antibody (e.g., anti-NKp46) and an LFA antibody (e.g., anti-CD2) and contains a mixture of NK cells and γδT cells).

[0127] 1. Perform large-scale leucopheresis in normal, healthy donors mobilized by G-CSF; or perform large-scale leucopheresis in normal, healthy donors that have not been mobilized, or use buffy coat.

[0128] 2. Density gradient cell separation using Ficoll® was performed with a SEPAX® device (Sepax Technologies, Inc., Newark DE) to remove erythrocytes, platelets, and granulocytes and obtain a mononuclear cell suspension; alternative methods for performing Ficoll® gradient separation include manual separation using a MILTENYI PRODIGY® (Miltenyi Biotec, San Diego, CA) or centrifuge.

[0129] 3. Mononuclear cells were subjected to αβT cell depletion using MILTENYI CLINIMACS® (Miltenyi Biotec, San Diego, CA) at clinical scale, according to the manufacturer's instructions, or subjected to laboratory-scale Miltenyi LS column separation.

[0130] 4. The obtained αβTCRneg cell population was placed under feeder-free culture conditions and activated with anti-CD2 and anti-NKp46 beads (NK Cell Activation / Expansion Kit (trademark) (Miltenyi Biotec, Inc., San Diego, CA, USA)) and 500 IU / mL human interleukin-2 (IL-2) (Miltenyi Biotec) according to the manufacturer's instructions.

[0131] 5. Place the activated αβTCRneg cell population in a 24-well plate at a rate of 0.25 × 10⁶ 6 At a cell seeding concentration of cells / ml, the cells were further cultured under feeder-free conditions using NK MACS® medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA)) supplemented with 5% AB serum and 500 IU / mL human interleukin-2 (IL-2), and then transferred to a T75 flask; alternative culture vessels may include a bioreactor (G-Rex® 25 ml, Wilson Wolf Manufacturing, St. Paul MN).

[0132] 6. Change the culture medium approximately every 3 days with fresh medium and fresh IL-2 at the concentrations described in #5, and raise the cells from 0.25 to 0.5 × 10⁶ 6 Seeds were seeded at a concentration of cells / ml. In certain variants of the method, the culture medium was changed approximately every 4 days. When seeding in a bioreactor, the culture medium may be changed approximately every 6 or 7 days until the target dose is reached.

[0133] 7. At any time after the start of expansion, the obtained enriched specific innate immune cell lysates population (NK and γδTCR+ T cells) can be further enriched with respect to either pure NK or γδTCR+ T cells using CD3+ depletion or CD56+ depletion of the undesirable population, respectively, to obtain a pure cell population, which was further cultured as in step #5.

[0134] 8. At the end of the expansion, INNATE-K cells are cryopreserved in serum-free freezing medium containing 10% DMSO solution (CryoStor®, BioLife Solutions, Bothell WA).

[0135] Figure 1 illustrates the expansion of αβTCRneg cells as a function of the number of days after antibody activation and expansion under feeder-free culture conditions, as analyzed on day 10 of culture. The graph shows that the cells begin to achieve logarithmic expansion in approximately one week and continue to expand, producing a large number of cells suitable for successful off-the-shelf therapy. In Figure 1, activated αβTCRneg cells were cultured and expanded under feeder-free conditions. The total number of cells in the culture is shown day by day of expansion, and a significant increase in expansion is observed after 6 days of culture.

[0136] Figure 2 shows 70% NK cells and 28% γδTCR+T(GD) cells, a ratio suitable for therapeutic products. Further cell expansion may cause shifts; these two cell types were obtained in large quantities without contamination by other cells, even without feeder cell culture. Figure 2 illustrates the enrichment and phenotypes of specific innate immune cell-lysic cells (INNATE-K), NK, and γδTCR+T cells, as well as CD3+CD56+NKT cells and adaptive immune αβTCR+T cells, at various steps of the following examples of the manufacturing process.

[0137] a) The cell frequencies in the mononuclear cell fraction after Ficoll density gradient separation were 8% + 12% NK cells and 0.1% + 1% γδTCR+ T cells, 6% CD3+CD56+ NKT cells and 73% αβTCR+ T cells.

[0138] b) After the αβTCR+ T cell depletion step, the cell frequencies were 25%+15% NK cells and 3.2%+1.2% γδTCR+ T cells (of which 0.5% were CD56-CD3+ and 2.7% were CD56+CD3+) and less than 1% αβTCR+ T cells.

[0139] c) Ten days after activation and feeder-free culture expansion, the NK cell frequency was substantially enriched to 68%+23%, and γδTCR+ T cells were substantially enriched to 15.7%+5% (of which 12.2% were CD56-CD3+ and 3.5% were CD56+CD3+). Negligible levels of CD3+ adaptive immune cells were detected: αβTCR+ T cells were detected at 0.3%+0.5%, and NKT cells at 0.2%+0.3%.

[0140] Flow cytometry analysis (fluorescence-activated cell sorter, or FACS analysis) was performed on day 10 of cells co-expressing CD56(NK) surface antigen, CD16, and CD57. CD16 is an FCγ receptor III and can thus bind to the FC portion of IgG antibodies, mediating antibody-dependent cell-mediated cytotoxicity (ADCC) in antibody-bound target cells. CD16 plays a role in NK cell-mediated spontaneous cytotoxicity. The CD56+CD16+ subset is considered to be the most cytotoxic subset and constitutes the majority of NK cells (e.g., under physiological conditions). CD57 may be a marker for NK cells with poor proliferative capacity. Acquisition of CD57 on NK cells after IL-2 stimulation or co-culture with target cells correlates with the maturation of the CD56+ NK cell subset. This differentiation is accompanied by functional changes; compared to CD57- cells, CD57+ NK cells have poor proliferation in response to IL-2 and IL-15 and produce less IFN-γ in response to IL-12 and IL-18. In the case of CD56+ NK cells, 37% were CD16+ and 63% were CD16-; in the case of CD56+CD16- cells, 13% were CD57+ and 87% were CD57-; and in the case of CD56+CD16+ cells, 15% were CD57+ and 85% were CD57-. Flow cytometry phenotypic results showed that the cell composition contained enriched NK cells, which are highly cytotoxic NK cells in enlarged αβTCRneg cells, as evidenced by the co-expression of CD56(NK) and CD16. However, it also contained cells with an immature phenotype that has the potential to mature in vivo into more responsive and cytotoxic cells, as evidenced by the number of NK cells that do not express CD57 in both CD16+ and CD16-NK cells.

[0141] The frequency distribution of different γδ T cell subsets in 10-day samples of feeder-free cultured, expanded activated αβTCRneg cells was measured by flow cytometry. γδ T cells, in contrast to MHC-restricted αβ T cells, are capable of MHC-unrestricted recognition and lysis of diverse cancers, highlighting their potential for off-the-shelf allogeneic immunotherapy. Human γδ T cells can be divided into three main populations based on δ chain expression. The vast majority of circulating γδ T lymphocytes in healthy human adults, up to 50%–90%, express γδ2 chains. γδ T cells expressing γδ1 chains are most prominent in the inner epithelial layer of mucosal surfaces, where they are involved in maintaining the integrity of epithelial tissue when faced with injury, infection, or transformation; however, they can also appear in peripheral blood. γδ3 T cells constitute approximately 0.2% of circulating T cells, including CD4+, CD8+, and CD4-CD8- subsets. Although rare in peripheral blood, γδ3 T cells are usually expressed more highly in the liver. The percentage distribution of γδTCR+ T cells on day 10 (41% γδ1, 50% γδ2, 7% other γδ) is such that the proliferated γδ T cells are Vδ1, Vδ2, and Vδ1 neg Vδ2 neg The expression of a subset clearly indicates polyclonality. This polyclonality ensures the involvement of proliferated γδT cells in the spontaneous killing of tumors, altered cells, or infections.

[0142] Figure 3 illustrates the frequency and distribution of T cell lineage markers, CD4 (helper) and CD8 (cytotoxic), on γδTCR+ cells in a 10-day sample of feeder-free cultured, expanded, activated αβTCRneg cells, as analyzed by flow cytometry. Most γδ1 T cells did not express either CD4 or CD8, but a small percentage (19%) expressed the cytotoxic CD8 lineage marker, which has been described for the greatest tumor and infection killing and is consistent with the desirable innate cytolytic immune population. γδ2+ cells expressed small amounts of either CD4 or CD8, but nearly half of the "other γδTCR+ cells," mainly composed of γδ3 T cells, expressed the cytotoxic CD8 lineage marker.

[0143] Furthermore, flow cytometry phenotypic analysis data was obtained on day 8, and γδ T cells were obtained from feeder-free, expanded cultured activated αβTCRneg cells depleted using Miltenyi Biotec, Inc.'s αβT cell depletion reagent. Regarding the large-scale depleted cells, cultured CD3+ T cells on day 8 mainly expressed the γδ T cell receptor (αβTCR+ cells <0.05%). Phenotypic analysis was performed to analyze the co-expression of markers on γδ T cells. All cells were CD3+, with 31.8% being CD16+. Co-expression of CD57 and CD16 was observed on CD3+ γδ T cells. 10.7% co-expressed the CD57 and CD16 markers, thus representing the most mature cells. The majority were CD57- and CD16- (57%), thus representing immature cells. Expression of inhibitory and activating ligand receptors was analyzed. NKG2D is an activating receptor on the surface of NK cells. NKG2A dimerizes with CD94 to produce an inhibitory receptor. Cells lacked NKG2C expression, and 55% of cells co-expressed NKG2A and NKG2D. Phenotypic analysis was performed to analyze the co-expression of markers on γδ T cells in relation to CD56 co-expression. Of all γδ cells expressing CD3+ and CD56+, 29% co-expressed CD16. Co-expression of CD57 and CD16 was observed in CD3+CD56+γδ T cells. 5% co-expressed the CD57 and CD16 markers, thus representing the most mature cells. The majority (67%) were CD57- and CD16-, thus representing immature cells. The expression of inhibitory and activating ligand receptors was analyzed. The cells showed a lack of NKG2C expression on CD3+CD56+ cells, with the majority of cells (82%) expressing NKG2A, and 80% of CD3+CD56+ cells co-expressing NKG2A and NKG2D. These cells expressed the phenotypic markers most commonly associated with NK cells and represented an active innate immune cell population.

[0144] (Example 2) Expansion of transduced specific innate cell-lytic immune cells (INNATE-CAR) derived from αβTCRneg cell populations This example describes the expansion and production of a transduced population of specific innate cell-lysing immune cells (INNATE-CARs), including NK and γδTCR+ T cells, derived from activated culture of αβTCRneg cell populations under feeder-free culture conditions.

[0145] 1. Example 1: Aliquots of cells from the activated and cultured αβTCRneg cell population described in Step #5 were taken on day 4 (alternatively, aliquots can be taken between days 5 and 15) and placed on a human fibronectin-coated plate in a 0.25 × 10⁶ layer. 6 Cells were seeded in the presence of 500 IU / mL human interleukin-2 (IL-2) or 10 ng / mL IL-15 at a concentration of cells / mL (or RetroNectin® (Takara Bio USA, Mountain View CA) coated plates or VECTOFUSIN-1 (Miltenyi Biotec) transduction enhancer could be used), and transduced with gamma retrovirus supernatant containing a CD+19 chimeric antigen receptor construct containing a non-functional fragment of the CD34 surface antigen used as a marker for cell transduction.

[0146] 2.3 days later, the cells were detached and placed in NK MACS® medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA)) supplemented with 5% AB serum and 500 IU / mL human interleukin-2 (IL-2) in 24-well plates or T75 at a rate of 0.25 × 10⁶ 6 Further expansion was achieved with cell seeding concentrations of cells / ml; alternative culture vessels may include a bioreactor (G-Rex® 25ml, Wilson Wolf Manufacturing, St. Paul MN).

[0147] 3. The culture medium should be changed every 3 days with fresh medium and fresh IL-2 at the concentrations described in #6 of Example 1, and the cells should be 0.25-0.5 × 10⁶ 6Seeds were seeded at a concentration of cells / mL; when seeding in a bioreactor, the culture medium was changed every 7 days until the target dose was achieved.

[0148] 4. At any point after day 14, the INNATE-CAR population could be further enriched with respect to either NK-CAR or γδ-CAR T cells, and a pure cell population could be obtained using CD3+ depletion or CD56+ depletion of the undesirable population, respectively, which was then further cultured as in step #3.

[0149] 5. At the end of the expansion, freeze the cells in serum-free freezing medium containing 10% DMSO solution (CryoStor, BioLife Solutions, Bothell, WA).

[0150] Figure 4 illustrates the analysis of the growth rate of CAR.19 INNATE-K cells over 10 days. Similar to the expanded non-genetically modified population, the cultures demonstrated logarithmic growth beginning around one week, demonstrating the robust growth potential of off-the-shelf INNATE-CAR products. In Figure 4, activated INNATE-K cells were expanded by culture under feeder-free conditions and transduced on day 4 with a gamma retrovirus containing CD19 CAR and a non-functional CD34 marker.

[0151] Transduction of INNATE-CAR populations, NK cells, and γδTCR+ T cells was compared (with three different products). Specifically, %CAR.CD19 expression was compared in each of these cell populations using the CD34 marker incorporated into the CAR molecule. Both NK and γδTCR+ T populations showed significant transduction by the CAR.CD19 retroviral vector, as demonstrated by the analysis of the CD34 marker in the declared cell subsets. Transduction efficiencies were 33% and 30% on average for NK and γδTCR+ T cells, respectively. Flow cytometry analysis (fluorescence-activated cell sorter, or FACS analysis) performed on feeder-free NK cells that had not been transduced with the retroviral vector containing CAR.CD19, with 10 days of expansion, showed specificity of CD34+ staining on CD56+ cells. Flow cytometry (FACS) analysis was performed on feeder-free NK cells that were genetically modified 4 days after activation using a retroviral vector containing CAR.CD19 and then expanded for 10 days. The results showed that CAR expression levels in CD56+ cells were significantly higher after transduction, both in terms of the percentage of CD34+ cells and the mean fluorescence intensity of expression. Both features are associated with highly efficient tumor recognition. The same findings were also valid for the status of feeder-free γδTCR T cells present in the transduced and expanded INNATE population. In fact, γδTCR cell components were significantly transduced, as assessed by CD34 expression analysis in γδTCR T cells.

[0152] The INNATE-CAR platform is highly efficient in tumor recognition and removal, exhibiting a strong synergy between its innate activity and CAR-mediated functionality. Indeed, Figure 5 graphically illustrates data from co-culture cytotoxicity assays in which INNATE cells and INNATE-CAR.CD19 cells were co-cultured with CD19+ leukemia (221) or CD19+ lymphoma (Daudi) cell lines. The graph above shows the percentage of residual tumor after culture for different cell effectors. Non-transduced NK cells (INNATE-NK) killed 40% and 29% of leukemia and lymphoma tumor cells, respectively, while INNATE-K cells, characterized by a combined population of NK and γδTCR T cells, killed 88.4% and 96.1% of tumor cells, respectively. Transduction of CAR.CD19 into INNATE-NK cells (INNATE-NK-CAR.CD19) enabled the removal of 93% and 84% of tumor cells, respectively, while the INNATE-CAR.CD19 combination population killed almost all tumor cells (97% and 99%).

[0153] (Example 3) INNATE-NK cell alternative enrichment This example describes the enrichment process of INNATE-NK cells:

[0154] 1. NK cells were isolated from buffy coat using the NK DEPLETION KIT (trademark) (Miltenyi Biotec, Inc., San Diego, CA, USA).

[0155] 2. NK cells were activated with the NK Cell ACTIVATION / EXPANSION KIT (trademark) (Miltenyi Biotec, Inc., San Diego, CA, USA) and recombinant human IL-2 (500 U / ml).

[0156] 3.0 Day after activation, NK cells were cultured in 5% AB serum enriched medium (NK MACS® medium (#130-107-879 Miltenyi Biotec, Inc., San Diego, CA, USA)).

[0157] 4.4 to 15 days, aliquots of cells were placed in human fibronectin-coated plates at 0.25 × 10⁶ 6 The cells were subjected to transduction in the presence of 500 IU / mL human interleukin-2 (IL-2) at a concentration of 1 / ml.

[0158] 5. After 3 days, the cells were detached and seeded in 5% AB serum-enriched medium and 500 IU / mL human interleukin 2 (IL-2) at a cell seeding concentration of 0.25 × 10⁻¹⁴. 6 The concentration was further increased to 1 / ml.

[0159] 6. After transduction, NK cells were seeded in 24-well plates, and then seeded in T flasks or bioreactors.

[0160] 7. The culture medium should be changed every 3 days with fresh medium and fresh IL-2, and the cells should be changed to 0.25-0.5 × 10⁶ cells until the target cell count is reached. 6 Seeds were seeded at a concentration of cells / mL (if they were on a plate). Bioreactor cultures were changed every 7 days.

[0161] 8. On day 14, if there was more than 5% T cell contamination, non-transduced and CAR-transduced INNATE-NK cells were depleted again with respect to CD3+ cells and cultured further as in step #7 to obtain pure NK cells.

[0162] Exemplary highly enriched NK populations with more than 90% (>90%) innate immune cells were robustly expanded in both plates and bioreactors, as graphically illustrated in Figures 6–8. Figure 6 shows the total number of INNATE-NK cells expanded in a plate over several days. Figure 7 compares the total number of cells in INNATE-NK populations expanded in plates and bioreactors. Figure 8 shows the subset cell composition at different time points of in vitro expansion, demonstrating a significant purity level of INNATE-NK represented by more than 95% NK cells and negligible abundances of CD3+ T cells and NKT cells.

[0163] The level of CAR transduction efficiency was demonstrated using flow cytometry (FACS) analysis to evaluate the CD34 marker contained in the CAR.CD19 molecule. Non-transduced INNATE-NK cells expanded in a feeder-free environment did not express the CD34 marker. INNATE-NK cells were genetically modified on day 4 with a retroviral vector containing CAR.CD19. INNATE-NK CAR cells were significantly transduced, and the percentage of CAR+ cells in the NK population (CD56+) was high. Expression of the CAR molecule in INNATE-NK CAR cells remained stable during long-term in vitro culture, as shown in Figure 9.

[0164] Figure 10 illustrates the increased expression of activation and cytolytic molecules after expansion of the INNATE-NK and INNATE-NK CAR populations, where data from flow cytometry analysis of INNATE-NK and INNATE-NK-CAR.19 cells are graphically presented. The analysis included evaluation of several NK markers associated with the activation and maturation of innate cells. In particular, CD16 was included as a basic marker for maturation analysis because it is associated with more mature NK subgroups that have high cytolytic activity but low proliferative capacity; signaling and co-stimulatory molecules for the activation and proliferation of the NK population were included, including CD2, LFA-1, NKp44, NKp30, and NKp46, and DNAM-1. All of these markers were significantly increased in the expanded populations of both INNATE-NK and INNATE-NK.CAR.19 cells, indicating that the cells were activated compared to circulating peripheral blood NK cells.

[0165] Figure 11 graphically illustrates the lack of exhaustion in feeder-free expanded INNATE-NK and INNATE-NK CAR cells. INNATE-NK and INNATE-NK-CAR.19 cells were generated and expanded as described in Example 3, and flow cytometry analysis was performed at days 20 and 60 to evaluate markers of cell maturity and exhaustion. LIR-1, a marker of innate cell inhibitory activity, showed a slight increase in the NK population at day 20 and an increase in both populations at day 60; NKG2c, a marker of signal-specific activity and maturation, remained unchanged throughout the culture period; CD57, a marker of maturation, is associated with decreased proliferative capacity and is thus expressed on the circulating NK population derived from the peripheral blood of healthy donors. Feeder-free expanded INNATE-NK cells at days 20 and 60 of in vitro expansion showed a significantly lower percentage of CD57+ NK cells, suggesting that these cells still retain proliferative capacity upon in vivo injection. Programmed cell death 1 molecule (PD-1) is not only a marker of decreased cellular potential and "exhaustion" in T cells, but has also recently been described as a checkpoint inhibitor in NK cells. Neither INNATE-NK nor INNATE-NK CAR cell populations showed the presence of PD-1+ cell subsets at either day 20 or day 60, indicating that the cells were not exhausted after long-term in vitro culture.

[0166] Figure 12 graphically illustrates cytotoxic co-culture assays with INNATE-NK or INNATE-NK-CAR.19, as well as four tumor cell lines: Column A: 221, CD19+ leukemia cell line; Column B: Daudi, CD19+ lymphoma cell line; Column C: BV173, CD19+ (variable expression) pre-B tumor cell line; and Column D: KARPAS, CD19- tumor cell line. Panel 1 of Figure 12 shows the reduction of CD19+ cells after in vitro co-culture with INNATE-NK (left subpanel) or INNATE-NK.CAR.19 (right subpanel) cells. The upper left section (framed section) in the INNATE-NK panel highlights the CD19+ tumor population after co-culture with unmodified INNATE-NK cells. The upper left section (framed section) in the INNATE-NK.CAR.19 panel highlights the residual CD19+ tumor population after co-culture with INNATE-NK.CAR.19 cells. INNATE-NK.CAR.19 cells were capable of significant tumor control against CD19+221 and DAUDI tumor targets, and residual tumor was negligible after 5 days of in vitro co-culture. BV173 showed higher resistance to recognition and removal, but genetic modification of INNATE-NK cells with CAR.CD19 significantly increased antitumor activity compared to unmodified INNATE-NK cells. The mean residual tumor values ​​observed in 10 different experiments are shown in Figure 12, Panel 2, for each co-culture experiment. Degranulation (analyzed by CD107a expression in NK cells after a short 3-hour in vitro co-culture) represents the cytolytic activity of INNATE-NK and INNATE-NK CAR effector cells, as shown in Figure 12, Panel 3 (columns A-D). In all cases, INNATE-NK.CAR.19 cells expressed greater cytolytic activity than INNATE-NK cells; in the negative control represented by KARPAS co-culture conditions, neither population expressed the CD107a degranulation marker. Figure 12, Panels 4 and 5 show the production of regulatory cytokines commonly produced in cases of lymphocyte cytolytic activity.All of these cytokines are significantly elevated during CD19 CAR T cell administration and are thought to likely play a role in cytokine release syndrome. For INNATE-NK and INNATE-NK CAR activity against CD19+ tumor cells, cytokine production of both IFNα and TNFα is 20–150 pg / ml / cell. 6 The levels were in the range of a few individuals, and many times smaller than those of adaptive T cell killing. Even when there was high targeted killing, the degree of cytokine secretion was not as large as that observed for CAR-T cells (typically 100 to 1,000 times or more), and therefore likely represents a less toxic treatment.

[0167] Figures 13–15 graphically illustrate that INNATE-NK and INNATE-NK-CAR- cells can exert significant antitumor activity against primary Bcp-ALL blasts. Figures 13 and 14 graphically illustrate the % specific lysis of primary tumor cells as a function of the ratio of effector (E) to target (T) for both INNATE-NK cell and INNATE-NK-CAR.19 cell populations in different experimental trials; the INNATE-NK-CAR.19 cell population was more effective across the entire E:T range. Figure 15 graphically illustrates the percentage of residual primary CD19+ tumors after co-culture with effector cells (INNATE-NK cell and INNATE-NK-CAR.19 cell populations) compared to a control condition in which primary CD19+ leukemic blasts were seeded in the absence of effector cells. Co-culturing with INNATE-NK cells resulted in approximately 18% residual tumor cells, while co-culturing with the INNATE-NK-CAR.19 cell population resulted in approximately 5% residual tumor cells.

[0168] In one example of an in vivo CD19+ lymphoma mouse model, each NGS mouse was equipped with 0.25 × 10⁶ tumor cells genetically modified with firefly luciferase so that they could be analyzed by biofluorescence analysis. 6 Individual intravenous administration was performed. Three days later, leukemia was shown to be established in the injected mice, and the mice were divided into 5 × 106 Mice were randomized into a treatment cohort to receive IV infusions of INNATE-NK or INNATE-NK-CAR.19. Animals were analyzed for the presence of luciferase-+ tumor cells throughout the experimental process. Data were compared to those from another experiment of the same design, but in this experiment, animals were given 5 × 10⁶ cells. 6 Individual unmodified adaptive T cells were administered.

[0169] (Example 4) INNATE-NK Test This example describes data showing that one dose of non-genetically modified INNATE-NK cells extended the time to death in animals compared to the same dose of adapted T cells. (Tumor cells 0.25 × 10⁻⁶) 6 Each individual was administered intravenously and allowed to expand for 3 days. 5 x 10 6 Individual INNATE-NK or INNATE-NK-CAR cells were administered intravenously, and the data were compared to those from experiments using 5M adaptive T cells. In mice treated with INNATE-NK CAR.19, tumor control was complete, no tumor biofluorescence was detected after day 11, and INNATE-NK-CAR19 demonstrated that the tumor was eradicated without recurrence until day 72 (end of the experiment). Importantly, INNATE-NK cells also did not exhibit a heterogeneous response (which had been observed at this point in the past with CAR T cells). The data also showed that one dose of non-modified INNATE-NK cells could extend the overall survival of animals compared to the same dose of adaptive T cells (comparison at day 50 and day 28, respectively). Figure 16 graphically illustrates the survival curves of animals from the above experiments treated with INNATE-NK and INNATE-NK-CAR.19 cells. The data curve for INNATE-NK.CAR.19 recipients demonstrated improved survival compared to recipients treated with non-modified INNATE-NK cells.

[0170] (Example 5) INNATE-NK cells for solid tumors This example demonstrates that INNATE-NK cells are a suitable platform for treating solid tumors. In this example, a neuroblastoma model was investigated. INNATE-NK and INNATE-NK CAR cells were generated as described in Example 3, Steps 1-8. INNATE-NK cells were genetically modified on day 4 with a retroviral vector containing a third-generation CAR specific to the GD2 antigen expressed by neuroblastoma tumor cells. The results demonstrated that the INNATE-NK CAR.GD2 generation showed significant levels of CAR expression in CD56+ cells when evaluated by flow cytometry (FACS) analysis with staining with an anti-idiotype antibody (1A7). The purity of the NK population in INNATE-NK CAR.GD2 production was evaluated in terms of a negligible percentage of CD3+ cells, while the level of CAR+ cells was evaluated as 1A7+ cells. CAR.GD2 expression in INNATE-NK CAR remained stable during long-term in vitro culture (measured on days 8, 15, 25, 31, and 35).

[0171] (Example 6) Evaluation of the role of innate immunity in solid tumor invasion This example evaluated the role of innate immunity in its ability to invade solid tumors. To assess this effect, 3-D neuroblastoma tumor cell spheroids were grown in MATRIGEL® culture, and then adapted T cells, INNATE-NK, or INNATE-NK-CAR.GD2 cells were introduced, and their invasiveness to the spheroids was evaluated. Specifically, a solid 3-D tumor model was created using genetically modified SH-SY5Y neuroblastoma tumor cells expressing green fluorescent protein (GFP), which were then cultured in vitro to form solid 3-D tumor spheres. Adapted peripheral blood T cells, unmodified INNATE-NK cells, and INNATE-NK.CAR.GD2 were labeled with a red fluorescent cell marker and incubated with the tumor spheres. The neurospheres were assayed using 3-D fluorescence microscopy in three planes to evaluate the cell position within the tumor spheres and their relative ability to invade the tumor. Confocal microscopy images of adaptive T cells (red) showed that although adaptive T cells were observed in all three planes, they were localized only to the outer surface of the tumor spheres (green), indicating that they were unable to actively invade the 3-D tumor and did not migrate to the center of the tumor. Further images showed that INNATE-NK cells (red) began to penetrate the surface of the tumor spheres, as observed in all three planes; INNATE-NK-CAR.GD2 cells also penetrated the surface of the tumor spheres, indicating that these cells possess the ability to migrate some distance into the tumor, as observed in all three planes.

[0172] (Example 7) Crosslinking or soluble activation As described in Example 1, cell populations were generated in culture after activation with a combination of cross-linked innate cytotoxic receptor (NCR) antibodies (e.g., anti-NKp46) and lymphocyte function-associated antigen (LFA) antibodies (e.g., anti-CD2) ("coated beads"; Miltenyi Biotec). The cell populations discussed herein, activated by the combination of NCR antibodies (e.g., anti-NKp46) and LFA antibodies (e.g., anti-CD2) and containing a mixture of NK cells and γδT cells, are referred to herein as the "BINATE" cell population (and in certain examples above as "INNATE-K" cells). In this example, the obtained BINATE population was analyzed after activation with either 1) a combination of crosslinked anti-NKp46 (clone 9E2 500 ng / ml; Miltenyi Biotec) and crosslinked anti-CD2 (OKT11 500 ng / ml), or 2) a combination of soluble anti-NKp46 (clone 9E2 500 ng / ml) and soluble anti-CD2 (clone OKT11 500 ng / ml). Crosslinking was achieved by coating the flask with the antibody combination in PBS for 24 hours before the start of the experiment, then decanting the antibody solution, and rinsing with PBS at the time of the experiment (referred to herein as coated plates).

[0173] 1. Buffy coats derived from normal, healthy donors were subjected to Ficoll® gradient cell separation by manual centrifugation (in certain variations of the methods described in this embodiment and other embodiments herein, normal, healthy donors were subjected to an apheresis procedure (apheresis, generally without Ficoll® gradient cell separation), and mononuclear cell products were collected according to a standard procedure, typically a Terumo Optia instrument). The mononuclear cells were subjected to αβT cell depletion using laboratory-scale Miltenyi LS column separation (in certain variations of the methods described in this embodiment and other embodiments herein, the apheresis products may be subjected to αβT cell depletion using a CLINIMAX separation device; further depletion may include CD19 B cell depletion; aliquots of αβT cell depleted mononuclear cells may be cryopreserved for later use for expansion). The resulting αβTCR-neg cell population was seeded in BINATE medium supplemented with 5% AB serum (i.e., NK MACS® medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA)) (or other media, such as Life Technologies, R&D Systems medium, CELLGENIX medium, etc., may be used with or without human serum)) and 500 IU / mL human interleukin-2 (IL-2) (Miltenyi Biotec) at a cell seeding concentration of 0.25-0.5 × 10⁻¹⁶. 6 At a concentration of 1 / ml, the cells were activated in feeder-free culture without additional activating reagents in 24-well coated plates, or in 24-well uncoated plates with a combination of 500 ng / ml soluble anti-NKp46 (clone 9E2) and 500 ng / ml anti-CD2 (clone OKT11) (or other concentrations of the activator may be used, or other agents may be added for activation).

[0174] 2. Activated αβTCRneg cell populations were seeded in BINATE medium and 500 IU / mL IL-2 at a cell seeding concentration of 0.25–0.5 × 10⁻¹⁴. 6Cells / ml in a T75 flask (or cell seeding concentration up to 1 × 10 in culture bags, flasks, or other larger culture vessels, e.g., bioreactors (G-Rex)). 6 The cultures were further expanded under feeder-free culture conditions at concentrations (which may range from up to cells / ml). Medium changes were performed every 3 days (less frequently (every 5-7 days) in the case of a bioreactor) using fresh BINATE medium and IL-2. In certain variations of the methods in this example and other examples herein, the cultured BINATE cells may be cryopreserved after 1, 2, 3, 4, or 5 weeks of expansion. In one example, the expanded BINATE cells were washed, concentrated, and serum-free cryopreservation medium (BIOLIFE) was added. The cryopreserved BINATE product may be thawed and re-seed for further culture as described herein, or thawed and injected, for example, into a patient with cancer or a viral infection.

[0175] 3. At the end of the 15-day expansion, BINATE cells were analyzed in detail with respect to NK and γδCD3+ T cell content and their subsets, such as CD56, and further analysis was performed for possible contaminating cell types, such as αβTCR+ T cells.

[0176] The phenotype of BINATE cells was reported as a percentage of the total cell population and showed no difference between activation on coated plates and activation with soluble antibodies. The NK population (CD56+CD3-) accounted for 89.1% of the final population when activated on coated plates, compared to 92% when activated with soluble antibodies. CD3+γδT cells accounted for 6.2% and 5.5% of the population under coated plate conditions and soluble antibody conditions, respectively. CD3+CD56+γδT cells and CD3+CD56-γδT cells accounted for 2.8% and 3.4% of the population in activated culture on coated plates, and 3.3% and 2.2% of the population in activated culture with soluble antibodies, respectively. The percentage of αβTCR+CD3+ T cells was negligible under both conditions, at 0.12% in activated on coated plates and 0.41% in activated with soluble antibodies.

[0177] Previous reports have shown that cross-linked antibodies may exhibit greater activity; however, the use of soluble antibodies is often preferred over cross-linked antibodies (e.g., when used on a surface such as a plate or beads) because it allows for better control of the culture, for example. Using a combination of soluble anti-NKp46 (clone 9E2, Miltenyi) and soluble anti-CD2 (clone OKT11), there was no difference compared to cross-linked conditions, and all experiments were subsequently performed using a soluble antibody activator.

[0178] (Example 8) Evaluation of activation and expansion using varying amounts of anti-NKp46 In separate experiments, different anti-NKp46 (Bab281) reagents were evaluated at three concentrations (10 ng / ml, 50 ng / ml, and 500 ng / ml) under two expanded conditions: 1) IL-2 (500 IU / mL human interleukin 2 (Miltenyi Biotec)) or 2) IL-15 (10 ng / mL human interleukin 15 (Miltenyi Biotec)).

[0179] 1. Buffy coat cells derived from normal, healthy donors were subjected to gradient cell separation using Ficoll®, performed by manual centrifugation. Mononuclear cells were subjected to αβT cell depletion using laboratory-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was divided into 0.25-0.5 × 10⁶ wells in a 24-well plate. 6 At a cell seeding concentration of cells / ml, cells were cultured in BINATE medium and under feeder-free conditions using IL-2 or IL-15, with three concentrations of 500 ng / ml anti-CD2 (clone OKT11) and anti-NKp46 (Bab281): one combination of 10 ng / ml, 50 ng / ml, or 500 ng / ml.

[0180] 2. The activated αβTCRneg cell population was seeded in BINATE medium with 500 IU / mL IL-2 or 10 ng / mL IL-15 at a cell seeding concentration of 0.25–0.5 × 10⁶. 6 Cells / ml in a T75 flask (or cell seeding concentration in a culture bag, flask, or other larger culture vessel, e.g., a bioreactor (G-Rex) of 1 × 10⁶ cells / ml) 6 The culture was further expanded under feeder-free conditions at concentrations up to a range of up to 1 / ml. Medium changes were performed every 3 days (less frequently (every 5-7 days) in a bioreactor) using fresh BINATE medium and IL-2 or IL-15 in plates or flasks.

[0181] 3. At the end of the 15-day expansion, BINATE cells were analyzed for NK and γδCD3+ T cell content and their subsets, such as CD56, and further analysis was performed for possible contaminating cell types, such as αβTCR+ T cells.

[0182] The phenotype of BINATE cells was reported as a percentage of the total cell population, and there were no significant differences among the three concentrations of anti-NKp46 (Bab281) when the culture was expanded by IL-15. The NK population (CD56+CD3-) comprised 84.5%, 87%, and 87.2% for 500 ng / ml anti-NKp46 (Bab281), 50 ng / ml anti-NKp46 (Bab281), and 10 ng / ml anti-NKp46 (Bab281), respectively. The γδTCR CD3+ population comprised 11.4%, 8.7%, and 8.1%, respectively, while the CD3+56+ population comprised 7.40%, 5.40%, and 5.30%, respectively. αβTCR+ cells were virtually undetectable at all concentrations. [Table 1]

[0183] (Example 9) Marker evaluation of activated and enlarged cells In different experiments, the binate phenotypes under two expansion conditions (i.e., IL-2 and IL-15) after activation were compared using a combination of a 10 ng / ml anti-NKp46 (Bab281) antibody of Good Manufacturing Practice (GMP) quality and a 500 ng / ml anti-CD2 (OKT11) antibody of GMP quality.

[0184] 1. Buffy coat cells derived from normal, healthy donors were subjected to gradient cell separation using Ficoll®, performed by manual centrifugation. Mononuclear cells were subjected to αβT cell depletion using laboratory-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was divided into 0.25-0.5 × 10⁶ wells in a 24-well plate. 6Cells were cultured under feeder-free conditions using BINATE medium containing 500 ng / ml anti-CD2 (OKT11) and 10 ng / ml anti-NKp46 (Bab281) (or other concentrations of the activator may be used, or other agents may be added for activation) and either 500 IU / ml IL-2 or 10 ng / ml IL-15 at a cell seeding concentration of cells / ml.

[0185] 2. The activated αβTCRneg cell population was seeded in BINATE medium with 500 IU / mL IL-2 or 10 ng / mL IL-15 at a cell seeding concentration of 0.25–0.5 × 10⁶. 6 Cells / ml in a T75 flask (or cell seeding concentration in a culture bag, flask, or other larger culture vessel, e.g., a bioreactor (G-Rex) of 1 × 10⁶ cells / ml) 6 The culture was further expanded under feeder-free conditions at concentrations up to a range of up to 1 / ml. Medium changes were performed every 3 days (less frequently (every 5-7 days) in a bioreactor) using fresh BINATE medium and IL-2 or IL-15 in plates or flasks.

[0186] 3. At the end of the 20-day culture expansion, BINATE cells were analyzed in detail for the content of native, cytotoxic, and exhausted cells and their subsets, including markers such as CD56, CD16, and CD57, and additional analyses were performed for possible contaminating cell types, such as αβTCR+ T cells.

[0187] The total BINATE cell population contained 92% and 84.3% CD56+ native cells after expansion with IL-15 and IL-2, respectively. The population contained 33% and 12% CD56+CD16+ cytotoxic native cells, 76% and 59.6% CD8+ cytotoxic native cells, and 99% and 96.5% CD56+CD57- immature native cells after culture expansion with IL-15 and IL-2, respectively. No detectable abTCR+ T cells were observed after culture expansion with either IL-15 or IL-2. CD4+, CD8+, and CD4-CD8- populations of NK and γδT cells are provided in Table 2 below. [Table 2-1] [Table 2-2]

[0188] (Example 10) Evaluation of cells expanded under quiescent culture and bioreactor culture conditions. In separate experiments, we compared binate and bioreactor culture conditions under two expansion conditions (IL-2 and IL-15) after activation using combinations of GMP-quality 50 ng / ml anti-NKp46 (Bab281) antibody and GMP-quality 50 ng / ml anti-CD2 (OKT11) antibody.

[0189] 1. Buffy coat cells derived from normal, healthy donors were subjected to gradient cell separation using Ficoll®, performed by manual centrifugation. Mononuclear cells were subjected to αβT cell depletion using laboratory-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was divided into 0.25-0.5 × 10⁶ wells in a 24-well plate. 6Cells were placed in feeder-free culture conditions using BINATE medium containing a combination of 50 ng / ml anti-CD2 (OKT11) and 50 ng / ml anti-NKp46 (Bab281) (or other concentrations of the activator may be used, or other agents may be added for activation) and either 500 IU / ml IL-2 or 10 ng / ml IL-15 at a cell seeding concentration of cells / ml.

[0190] 2. The activated αβTCRneg cell population was seeded in BINATE medium with 500 IU / mL IL-2 or 10 ng / mL IL-15 at a cell seeding concentration of 0.25–0.5 × 10⁶. 6 Cells / ml in a T75 flask or G-Rex benchtop bioreactor (or cell seeding concentration in a culture bag, flask, or other larger culture vessel, e.g., 1 × 10 in a bioreactor (G-Rex)). 6 The culture was further expanded under feeder-free conditions at concentrations up to a range of up to 1 / ml. Medium changes were performed every 3 days (less frequently (every 5-7 days) in a bioreactor) using fresh BINATE medium and IL-15 in plates or flasks.

[0191] 3. The activated αβTCRneg cell population was cultured in a GRex culture device using BINATE medium and 10 ng / ml IL-15, with a cell seeding concentration of 1 × 10⁶. 6 The culture was further expanded at a rate of cells / ml under feeder-free culture conditions. The culture medium was changed every 7 days using fresh BINATE medium and IL-15.

[0192] The total BINATE expansion is shown as total cell number in Figure 17. The expansion of activated αβTCRneg cells in BINATE medium supplemented with either IL-2 or IL-15 in flasks was equivalent over a period of 41 days. Similarly, the expansion of the activated αβTCRneg cell population with IL-15 in the bioreactor during the first 21 days was equivalent to the expansion with IL-15 in flasks.

[0193] (Example 11) Evaluation of Alloreactivity of Activated and Expanded Cells In separate experiments, the alloreactivity of activated BINATE cell populations expanded in either IL-2 or IL-15 was evaluated.

[0194] 1. Gradient cell separation using Ficoll™ was performed on buffy coats from normal and healthy donors by manual centrifugation separation. Mononuclear cells were subjected to αβ T cell depletion using laboratory-scale Miltenyi LS column separation. The resulting αβ TCRneg cell population was placed in feeder-free culture conditions using BINATE medium at a cell seeding concentration of 0.25 - 0.5×10 6 cells / ml with a combination of 50 ng / ml anti-CD2 (OKT11) and 50 ng / ml anti-NKp46 (Bab281) (alternatively, other concentrations of activators may be used, or other agents may be added for activation).

[0195] 2. The activated αβ TCRneg cell population was further cultured and expanded under feeder-free culture conditions in a T75 flask at a cell seeding concentration of 0.25 - 0.5×10 6 cells / ml using BINATE medium and either 500 IU / mL IL-2 or 10 ng / ml IL-15. Medium exchange for flasks was performed every 3 days using fresh BINATE medium and IL-2 or IL-15.

[0196] 3. BINATE cells and stimulator cells derived from PBMCs of three random healthy donors (HD) were tested in a one-way mixed lymphocyte reaction (MLR). Stimulator cells derived from the three HDs were pooled and used after irradiation with radiation (30 Gy). BINATE cells were seeded at 100 μl / well (1×10 5 cells) together with the same number of stimulator cells in 200 μl of BINATE medium in a 96-well flat-bottom microtiter plate. After 5 days of incubation, 18 kBq 3H-thymidine was pulsed for 12 hours and harvested on a glass fiber filter. The dried filter was counted on a Microbeta Trilux 1450 counter (Wallac, Perkin Elmer). The results were expressed as SI (cpm by antigen / cpm of background). BINATE cells without cytokine addition were used as negative controls, and positive controls were represented by BINATE cells seeded with cytokines (IL-2 or IL-15).

[0197] Mixed lymphocyte co-culture was analyzed for 3 H thymidine incorporation (CPM), an indicator of proliferation. In the IL-15 experiment, the negative control consisted of unstimulated BINATE cells not supplemented with IL-15, and 3 H thymidine incorporation was 1144, indicating essentially no proliferation. Irradiated pooled healthy donor cells (HD) 3 had H thymidine incorporation of 1194, also indicating no proliferation. The positive control included proliferation stimulation by IL-15, and 3 H thymidine incorporation was 15558, a 14-fold increase compared to the negative control. Activated BINATE cells expanded in IL-15 3 had H thymidine incorporation of 942, essentially no difference from the background.

[0198] In the IL-2 experiment, the negative control included unstimulated BINATE cells not supplemented with IL-2, and 3 H thymidine incorporation was 834, indicating essentially no proliferation. Irradiated pooled human donor cells (HD) 3 had H thymidine incorporation of 1194, also indicating no proliferation. The positive control included proliferation stimulation by IL-2, and 3 H thymidine incorporation was 18834, a 22-fold increase compared to the negative control. Activated BINATE cells expanded in IL-15 3 had H thymidine incorporation of 1245, essentially no difference from the background control.

[0199] The absence of an MLR response may be due to the lack of αβTCR+ T cells, supporting the absence of alloreactivity in the native BINATE cell population. The absence of alloreactivity is clinically significant because it supports the lack of the cells' ability to produce a graft-versus-host response.

[0200] (Example 12) Evaluation of cell proliferation and phenotype under different magnification conditions In separate experiments, the proliferative capacity and binate phenotype after activation with GMP-quality anti-NKp46 (Bab281) antibody (50 ng / ml) (Caprico) and GMP-quality anti-CD2 (OKT11) antibody (50 ng / ml) (Caprico) were compared under five different expansion conditions.

[0201] 1. Buffy coat cells derived from normal, healthy donors were subjected to gradient cell separation using Ficoll®, performed by manual centrifugation. Mononuclear cells were subjected to αβT cell depletion using laboratory-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was divided into 0.25-0.5 × 10⁶ wells in a 24-well plate. 6 At cell seeding concentrations of cells / ml, a combination of 50 ng / ml anti-CD2 (OKT11) and 50 ng / ml anti-NKp46 (Bab281) (or other concentrations of the activator may be used, or other agents may be added for activation), as well as the following expansion adjuvants: a.500IU / mL IL-2(Miltenyi Biotec) b.10ng / ml IL-15(Miltenyi Biotec) c. A combination of 500 IU / mL IL-2 and 10 ng / mL IL-15 (IL-2 / IL-15) d. Combination of 500 IU / mL IL-2 and 10 ng / mL OKT3 clone anti-CD3 antibody (Miltenyi Biotec) (IL-2 / OKT3) e. A combination of IL-15 10 ng / mL and IL-2 500 IU / mL and OKT3 10 ng / mL (IL-2 / IL-15 / OKT3) The cells were placed under feeder-free culture conditions using BINATE medium containing one of the following:

[0202] 2. The activated αβTCRneg cell population was seeded in BINATE medium at a cell seeding concentration of 0.25-0.5 × 10⁻⁶. 6 Cells / ml in a T75 flask (or cell seeding concentration in a culture bag, flask, or other larger culture vessel, e.g., a bioreactor (G-Rex) of 1 × 10⁶ cells / ml) 6 Further culture expansion was performed under feeder-free conditions using one of the five expansion conditions listed above (which may range up to cells / ml). Medium changes were performed with fresh BINATE medium every 3 days for plates or flasks (less frequently (every 5-7 days) for bioreactors), and the same expansion conditions were used during the initial culture activation and expansion phases.

[0203] Total BINATE expansion is shown as total cell number in Figure 18. Expansion of activated αβTCRneg cells in BINATE medium supplemented with either IL-2, IL-15, or IL-2 / OKT3 in flasks was equivalent over 40 days. Expansion of activated αβTCRneg cells in BINATE medium supplemented with either IL-2 / IL-15 or IL-2 / IL-15 / OKT3 in flasks was equivalent over a period of 26 days, but thereafter proliferation slowed, and the total cell number was lower compared to the other three groups from culture 26 to 41 days.

[0204] As shown in Table 3 below, at days 7, 20, and 30 of culture expansion, BINATE cells from each of the five conditions were determined to be NK or γδT cells by flow cytometry. As shown in Table 4, cells from each of the conditions at days 7, 20, and 30 were similarly analyzed by flow cytometry for specific markers of innate, cytotoxic, and exhaustion on the cells, as well as immature expression and the use of specific VdTCR in γδT cells. [Table 3-1] [Table 3-2] [Table 4]

[0205] Low PD-1 expression, an indicator of cell exhaustion, was observed. On day 30, under IL-15 augmentation, expression was 2.7% in the total BINATE population, 2% in CD56+CD3-NK cells, 10.6% in CD3+CD56-γδT cells, and 17.05% in CD3+CD56+γδT cells, demonstrating a lack of exhaustion. Under IL-2 augmentation, PD-1 expression was 14.2% lower in the total BINATE population as determined by flow cytometry, with CD56+CD3-NK cells expressing 2% PD-1, and CD3+CD56-γδT cells and CD3+CD56+γδT cells expressing 62.9% and 78.3%, respectively.

[0206] (Example 13) CD107a cytotoxicity 30-day cells from each of the five expansion conditions of Example 12 were co-cultured with tumor cell lines including IMR32, SH-SY5Y (neuroblastoma cell line), and K562 (B-cell leukemia cell line). After 3 days of culture, CD10, a measure of cytotoxic granule release, was measured. Expression of 7a was measured by flow cytometry analysis in basal BINATE cells and BINATE cells co-cultured with each tumor cell line. Table 5 shows the difference in tumor-stimulated expression after subtracting the baseline values ​​for each tumor cell line and each BINATE subset (CD56+CD3-NK cells, CD3+CD56+γδT cells, and CD3+CD56-γδT cells) for each of the five expanded conditions (IL-2, IL-2 / OKT3, IL-15, IL-2 / IL-15, and IL-2 / IL-15 / OKT3). Statistical significance is reported as p-values ​​by t-tests. [Table 5-1] [Table 5-2]

[0207] (Example 14) Evaluation of phenotypic markers under different magnification conditions Cells at day 20 from three expansion conditions (IL-2, IL-15, and IL-2 / OKT3) in Example 12 were analyzed for specific activation, cytotoxicity, and exhaustion markers. Figure 19 shows the phenotypic analysis for each marker type (activation, cytotoxicity, and exhaustion / immaturity) under the IL-15 expansion condition. Figure 20 shows the phenotypic analysis for each marker type (activation, cytotoxicity, and exhaustion / immaturity) under the IL-2 expansion condition. Figure 21 shows the phenotypic analysis for each marker type (activation, cytotoxicity, and exhaustion / immaturity) under the IL-2 / OKT3 expansion condition. Under all conditions, BINATE cells generally showed high activation and cytotoxicity, as well as low exhaustion.

[0208] (Example 15) Evaluation of in vitro tumor killing In different experiments, we compared in vitro tumor killing of BINATE cells under two expanded conditions (IL-2 and IL-15) after activation using combinations of 10 ng / ml anti-NKp46 (Bab281) antibody and 500 ng / ml anti-CD2 (OKT11) antibody.

[0209] 1. Buffy coat cells derived from normal, healthy donors were subjected to gradient cell separation using Ficoll®, performed by manual centrifugation. Mononuclear cells were subjected to αβT cell depletion using laboratory-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was divided into 0.25-0.5 × 10⁶ wells in a 24-well plate. 6 Cells were placed in feeder-free culture conditions using BINATE medium containing a combination of 500 ng / ml anti-CD2 (OKT11) and 10 ng / ml anti-NKp46 (Bab281) (or other concentrations of the activator may be used, or other agents may be added for activation) and either 500 IU / ml IL-2 or 10 ng / ml IL-15 at a cell seeding concentration of cells / ml.

[0210] 2. The activated αβTCRneg cell population was seeded in BINATE medium with 500 IU / mL IL-2 or 10 ng / mL IL-15 at a cell seeding concentration of 0.25-0.5 × 10⁻¹⁴. 6 Cells / ml in a T75 flask (or cell seeding concentration in a culture bag, flask, or other larger culture vessel, e.g., a bioreactor (G-Rex) of 1 × 10⁶ cells / ml) 6 The culture was further expanded under feeder-free conditions at concentrations up to a range of up to 1 / ml. Medium changes were performed every 3 days (less frequently (every 5-7 days) in a bioreactor) using fresh BINATE medium and IL-2 or IL-15 in plates or flasks.

[0211] 3. After the 20-day culture expansion, BINATE cells were placed in tumor co-culture experiments for 5-7 days. The experiments shown in Table 6 below involved culturing leukemia cell lines in 6-well culture dishes with 2 ml of medium per culture well at a 1:1 ratio of effector to target (i.e., 0.5 M BINATE cells (effector) together with 0.5 M tumor cells (target)).

[0212] The experiments shown in Table 7 below involved culturing solid tumor cell lines (neuroblastoma, sarcoma, and colon tumor lines) in 6-well culture dishes with 2 ml of medium per culture well at a 2:1 ratio of effector to target (i.e., 0.5 M BINATE cells (effectors) together with 0.25 M tumor cells (targets)). Co-culture of tumor and effector cells was performed in the absence of cytokines. Co-cultures were evaluated by microscopy and flow cytometry on days 3–7.

[0213] Examination of residual tumors in co-culture experiments, as shown in Table 6, demonstrated that activated BINATE cells, expanded in the presence of either IL-2 or IL-15, successfully eradicated 50% or more of tumor cells in all cell lines, and more than 75% of AML cell lines in 4-5 of them. Table 6 shows that short-term co-culture of all cell lines resulted in BINATE cells eradicating all 221 leukemia cells, >80% of Daudi leukemia cells, and up to 50% of Karpas cell lines, which are often considered resistant to spontaneous elimination. [Table 6]

[0214] Table 7 shows that when examining residual tumors in co-culture experiments, activated BINATE cells expanded with either IL-2 or IL-15 successfully eradicated neuroblastoma tumors in short-term co-culture experiments. While IL-2-enhanced BINATE cells were not tested in co-culture with colon cancer, IL-15-enhanced BINATE cells showed activity against sarcoma and colon cancer. [Table 7]

[0215] (Example 16) Evaluation of in vitro transduction efficiency In separate experiments, to assess the stability of transduction, the early and late in vitro transduction efficiencies of BINATE cells using retroviral constructs encoding third-generation GD2 chimeric antigen receptors (CARs) were compared after activation with a combination of 10 ng / ml anti-NKp46 (Bab281) antibody and 500 ng / ml anti-CD2 (OKT11) antibody.

[0216] 1. Buffy coat cells derived from normal, healthy donors were subjected to gradient cell separation using Ficoll®, performed by manual centrifugation. Mononuclear cells were subjected to αβT cell depletion using laboratory-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was divided into 0.25-0.5 × 10⁶ wells in a 24-well plate. 6 Cells were cultured under feeder-free conditions using BINATE medium and 500 IU / ml IL-2, with a cell seeding concentration of cells / ml, containing a combination of 500 ng / ml anti-CD2 (OKT11) and 10 ng / ml anti-NKp46 (Bab281) (or other concentrations of the activator may be used, or other agents may be added for activation).

[0217] 2. For the production of any BINATE.CAR as described herein, aliquots of cells from an activated, cultured, expanded αβTCRneg cell population are collected on days 4–15 and subjected to 0.25 × 10⁶ of aliquots in the presence of gamma retrovirus supernatant containing a specific chimeric antigen receptor construct. 6The cells were seeded at a concentration of cells / ml on human fibronectin-coated plates or alternatively on culture bags (Gobain). After 3 to 4 days, the cells were washed and the process, which may include either expansion or cryopreservation, was continued for non-transduced cells as well. Expansion conditions may include 500 IU / mL human interleukin-2 (IL-2) or 10 ng / mL IL-15, but may include any combination of the conditions mentioned herein. The chimeric antigen receptor (CAR) constructs used herein include CAR.GD2, CAR.123, and CAR.CD19, which contain non-functional markers, e.g., fragments of the CD34 surface antigen or mutant CD19 fragments (see Example 25 for a description of a particular construct). In certain variants of the methods herein, VECTOFUSION-1 (Miltenyi Biotec) may be used instead of RETRONECTIN to aid retroviral transfection, and genetic modification may include genetic modification of other CAR constructs of any selected antigen or modification by other molecules. In this example, the activated αβTCRneg cell population on day 5 was placed in a human fibronectin-coated plate (or alternatively, a culture bag (Gobain)) in a 0.25 × 10⁶ container. 6 The GD2 chimeric antigen receptor (CAR) construct was placed in the presence of gamma retrovirus supernatant at a concentration of cells / ml. The GD2 construct contained a variant fragment of the CD34 receptor as a selection and tracking marker.

[0218] 3. After 3 days, the transduced cells were washed and seeded in BINATE medium and 500 IU / mL IL-2 at a cell seeding concentration of 0.25-0.5 × 10⁶. 6 Cells / ml in a T75 flask (or cell seeding concentration in a culture bag, flask, or other larger culture vessel, e.g., a bioreactor (G-Rex) of 1 × 10⁶ cells / ml) 6 The culture was further expanded under feeder-free conditions at concentrations up to a range of up to 1 / ml. Medium changes were performed every 3 days (less frequently, every 5-7 days, for plates or flasks) using fresh BINATE medium and IL-2.

[0219] 4. Three and fifteen days after transduction, the BINATE cell subset and the VdTCR subset of γδT cells were analyzed for the expression of the mutant CD34 receptor (an indicator of transduction efficiency). On days 3 and 15, the transduction efficiencies of CD56+CD3-NK cells were 82.4% and 82.4%, respectively. On days 3 and 15, the transduction efficiencies of CD3+56+ cells were 43.2% and 62.4%, respectively. On days 3 and 15, the transduction efficiencies of CD3+CD56-γδT cells were 37.3% and 56.8%, respectively. The genetic modification remained stable throughout the culture period, which meant a highly activated population enabling high transduction and persistent integration.

[0220] In similar experiments, transduction of the BINATE subpopulation demonstrated high transduction of the GD2 CAR construct, as well as polyclonal transduction of both Vd1 and Vd2 TCR γδT cells. CD56+CD3-NK cells were transduced at 69%, while CD3+γδT cells were transduced at 39%. The Vd1 TCR population had a transduction efficiency of 67%, and the Vd2 TCR population had a transduction efficiency of 34%.

[0221] (Example 17) Evaluation of GD2 CAR proliferation after in vitro transduction. In separate experiments, the proliferative capacity of BINATE cells after in vitro transduction with a retroviral construct encoding a third-generation GD2 CAR, following activation with a combination of 50 ng / ml anti-NKp46 (Bab281) antibody and 50 ng / ml anti-CD2 (OKT11) antibody, was compared with that of unmodified BINATE cells under IL-2 and IL-15-mediated augmentation conditions.

[0222] 1. Buffy coat cells derived from normal, healthy donors were subjected to gradient cell separation using Ficoll®, performed by manual centrifugation. Mononuclear cells were subjected to αβT cell depletion using laboratory-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was divided into 0.25-0.5 × 10⁶ wells in a 24-well plate. 6 Cells were cultured under feeder-free conditions using BINATE medium and 500 IU / ml IL-2 or 10 ng / ml IL-15, with a cell seeding concentration of cells / ml, containing a combination of 50 ng / ml anti-CD2 (OKT11) and 50 ng / ml anti-NKp46 (Bab281) (or other concentrations of the activator may be used, or other agents may be added for activation).

[0223] 2. Activated non-genetically modified αβTCRneg cell populations were seeded in BINATE medium with 500 IU / mL IL-2 or 10 ng / mL IL-15 at a cell seeding concentration of 0.25–0.5 × 10⁶. 6 Cells / ml in a T75 flask (or cell seeding concentration in a culture bag, flask, or other larger culture vessel, e.g., a bioreactor (G-Rex) of 1 × 10⁶ cells / ml) 6 The culture was further expanded under feeder-free conditions at concentrations up to a range of up to 1 / ml. Medium changes were performed every 3 days (less frequently (every 5-7 days) in a bioreactor) using fresh BINATE medium and IL-2 or IL-15 in plates or flasks.

[0224] 3. On day 5, the activated αβTCRneg cell population was cultured in human fibronectin-coated plates or culture bags (Gobain) at a rate of 0.25 × 10⁶ 6 The GD2 chimeric antigen receptor (CAR) construct was placed in the presence of gamma retrovirus supernatant at a concentration of cells / ml. The GD2 construct contained a variant fragment of the CD34 receptor as a selection and tracking marker.

[0225] 4.5 days later, the transduced cells were washed and seeded in BINATE medium with 500 IU / mL IL-2 or 10 ng / mL IL-15 at a cell seeding concentration of 0.25-0.5 × 10⁶. 6 Cells / ml in a T75 flask (or cell seeding concentration in a culture bag, flask, or other larger culture vessel, e.g., a bioreactor (G-Rex) of 1 × 10⁶ cells / ml) 6 The culture was further expanded under feeder-free conditions at concentrations up to a range of up to 1 / ml. Medium changes were performed every 3 days (less frequently (every 5-7 days) in a bioreactor) using fresh BINATE medium and IL-2 or IL-15 in plates or flasks.

[0226] 5. Cell counts were recorded every 7 days for the BINATE and BINATE.CARGD2 populations.

[0227] Unmodified BINATE cells and BINATE.CARGD2 cells were enlarged before transduction (represented by vertical lines in Figure 22). Enlargement after transduction is represented by diagonal lines in Figure 22 for different conditions. There was no significant difference between genetically modified and unmodified cells, and there was no difference in total cell number enlargement induced by IL-2 or IL-15.

[0228] (Example 18) Evaluation of in vitro toxicity of solid tumors In separate experiments, we compared in vitro killing of solid tumors under two expansion conditions (IL-2 and IL-15) after transduction of activated BINATE with a retroviral construct encoding a third-generation GD2 CAR.

[0229] 1. Buffy coat cells derived from normal, healthy donors were subjected to gradient cell separation using Ficoll®, performed by manual centrifugation. Mononuclear cells were subjected to αβT cell depletion using laboratory-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was divided into 0.25-0.5 × 10⁶ wells in a 24-well plate. 6Cells were placed in feeder-free culture conditions using BINATE medium and 500 IU / ml IL-2 or 10 ng / ml IL-15, with a cell seeding concentration of cells / ml, and a combination of 500 ng / ml anti-CD2 (OKT11) and 10 ng / ml anti-NKp46 (Bab281) (or other concentrations of the activator may be used, or other agents may be added for activation).

[0230] 2.5 Day 2. Activated αβTCRneg cell population was subjected to 0.25 × 10⁶ saturation in the presence of gamma retrovirus supernatant containing a GD2 chimeric antigen receptor construct. 6 The cells were placed in human fibronectin-coated plates or culture bags (Gobain) at a concentration of cells / ml. The GD2 constructs contained variant fragments of the CD34 receptor as selection and tracking markers.

[0231] 3. After 3 days, the transduced cells were washed and seeded in BINATE medium with 500 IU / mL IL-2 or 10 ng / mL IL-15 at a cell seeding concentration of 0.25-0.5 × 10⁶. 6 Cells / ml in a T75 flask (or cell seeding concentration in a culture bag, flask, or other larger culture vessel, e.g., a bioreactor (G-Rex) of 1 × 10⁶ cells / ml) 6 The culture was further expanded under feeder-free conditions at concentrations up to a range of up to 1 / ml. Medium changes were performed every 3 days (less frequently, every 5-7 days, for plates or flasks) using fresh BINATE medium and IL-2 or IL-15.

[0232] 4. At the end of the 20-day culture expansion, BINATE cells were placed in a tumor co-culture experiment for 5–7 days. The experiment involved culturing 2 ml of medium / well in a 6-well culture dish at a 1:1 ratio of effector to target (i.e., 0.5 M BINATE cells or BINATE.CAR.GD2 cells (effector) together with 0.5 M tumor cells (target)). Co-culture of tumor and effector cells was performed in the absence of cytokines. The co-culture was evaluated by microscopy and flow cytometry on days 3–7.

[0233] Table 8 shows the tumors, unmodified BINATE IL-2 or BINATE IL-15, or CAR.GD2 modified BINATE IL-2 or CAR.GD2 modified BINATE This section presents data from co-cultures regarding the percentage of residual tumor after co-culture with either IL-15. Residual tumor in the control group was set to 100%. Both BINATE IL-2 and BINATE IL-15 showed significant tumor killing. Similarly, CAR.GD2 modified BINATE IL-2 and BINATE IL-15 also showed significant killing. [Table 8]

[0234] (Example 19) Evaluation of in vitro sterilization of myeloid leukemia cell lines In separate experiments, the inclusion of myeloleukemia cell lines in long-term co-culture after transduction of activated BINATE with a retroviral construct encoding third-generation CD123CAR We evaluated the in vitro killing effect.

[0235] 1. Buffy coat cells derived from normal, healthy donors were subjected to gradient cell separation using Ficoll®, performed by manual centrifugation. Mononuclear cells were subjected to αβT cell depletion using laboratory-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was divided into 0.25-0.5 × 10⁶ wells in a 24-well plate.6 Cells were cultured under feeder-free conditions using BINATE medium and 10 ng / ml IL-15, with a cell seeding concentration of cells / ml, and a combination of 50 ng / ml anti-CD2 (OKT11) and 50 ng / ml anti-NKp46 (Bab281) (or other concentrations of the activator may be used, or other agents may be added for activation).

[0236] 2.5 days later, activated αβTCRneg cell populations were subjected to 0.25 × 10⁶ steps in the presence of gamma retrovirus supernatant containing a CD123 chimeric antigen receptor (CAR) construct. 6 The cells were placed on human fibronectin-coated plates at a concentration of cells / ml. The CD123 construct contained a variant fragment of the CD19 receptor as a selection and tracking marker.

[0237] 3. After 3 days, the transduced cells were washed and seeded in BINATE medium and 10 ng / mL IL-15 at a cell seeding concentration of 0.25-0.5 × 10⁶. 6 Cells / ml in a T75 flask (or cell seeding concentration in a culture bag, flask, or other larger culture vessel, e.g., a bioreactor (G-Rex) of 1 × 10⁶ cells / ml) 6 The culture was further expanded under feeder-free conditions at concentrations up to a range of up to 1 / ml. Medium changes were performed every 3 days (less frequently, every 5-7 days, for plates or flasks) using fresh BINATE medium and IL-15.

[0238] 4. At the end of the 20-day culture expansion, BINATE cells and BINATE.CAR123 were placed in a tumor co-culture experiment for 7 days or more. The experiment was conducted in a 6-well culture dish with a 1:1 ratio of effector to target in 2 ml medium / well (i.e., 0.5 × 10⁶). 6 Individual BINATE or BINATE.CAR123 cells (effectors) are 0.5 × 10 6The study involved culturing tumor cells (targets) together. Co-culture of tumor and effector cells was performed in the absence of cytokines. The co-cultures were evaluated by microscopy and flow cytometry on days 3–7.

[0239] BINATE.CD123 cells were transduced with 65% efficiency. Myeloid leukemia cell lines evaluated in co-culture included THP1, MOLM3, and OCI AML cell lines. Data included the percentage of residual tumor after co-culture of tumor with either unmodified BINATE or BINATE.CAR.CD123. Residual tumor in the control was measured as 100%. BINATE cells failed to eradicate myeloid leukemia cells due to persistent growth of residual tumor after initial short-term elimination. BINATE.CAR.CD123 cells were able to successfully eliminate tumors and maintain tumor-free culture in co-culture with THP1 and MOLM3 cell lines, and were >70% of the control in OCI AML cell lines.

[0240] (Example 20) Evaluation of in vivo killing of leukemia cells In separate experiments, we evaluated the in vivo killing of leukemia cells after transduction of activated BINATE cells with a retroviral construct encoding second-generation CD19 CAR.

[0241] 1. Gradient cell separation was performed using Ficoll®, a manual centrifugation method, on buffy coat derived from normal, healthy donors. Mononuclear cells were subjected to laboratory-scale Miltenyi LS column separation and NK enrichment using the Miltenyi NK selection kit. NK and γδT cell populations were separated into 0.25–0.5 × 10⁶ well plates. 6 Cells were cultured under feeder-free conditions using BINATE medium and 500 IU / ml IL-2, containing a combination of 500 ng / ml anti-CD2 antibody (clone LT2, Miltenyi) and 10 ng / ml anti-NKp46 (or other concentrations of the activator may be used, or other agents may be added for activation) at a cell seeding concentration of cells / ml.

[0242] 2.5 days later, the activated population was subjected to 0.25 × 10⁶ measurements in the presence of gamma retrovirus supernatant containing a CD19 chimeric antigen receptor construct. 6 The cells were placed on human fibronectin-coated plates at a concentration of cells / ml. The CD19 construct contained a variant fragment of the CD34 receptor as a selection and tracking marker.

[0243] 3. After 3 days, the transduced cells were washed and seeded in BINATE medium and 500 IU / mL IL-2 at a cell seeding concentration of 0.25-0.5 × 10⁶. 6 Cells / ml in a T75 flask (or cell seeding concentration in a culture bag, flask, or other larger culture vessel, e.g., a bioreactor (G-Rex) of 1 × 10⁶ cells / ml) 6 The cultures were further expanded under feeder-free conditions at concentrations (which could range up to cells / ml). Medium changes were performed every 3 days (less frequently, every 5-7 days, for plates or flasks) using fresh BINATE medium and IL-2. At the end of expansion, the BINATE cells were washed and formulated in PBS solution for IV administration to mice.

[0244] Heterogeneous in vivo leukemia model NOD / SCID IL-2Rγnull (NSG) xenograft mice were injected with DAUDI cells, and the antitumor effect of CAR transdermal cells was evaluated in vivo. The mouse experiments were conducted in accordance with international ethical requirements, EU requirements, and national requirements, and were approved by the Italian Ministry of Health (N°88 / 2016-PR). NGS mice (5 weeks old; The Jackson Laboratory, USA) were injected with firefly luciferase-labeled Daudi cells (FF-Daudi) (0.25 × 10⁶). 6 The mice were inoculated with 50 x 10 units on day 3. 6 Individual BINATE cells or BINATE.CAR19 cells were injected via IV on day 0 and subjected to weekly bioluminescence imaging (IVIS System, Perkin Elmer, USA).

[0245] Three doses of BINATE cells or BINATE.CD19 cells were injected into an animal model on day 0. Non-genetically modified BINATE cells were able to produce a partial response, and mice survived until day 42. BINATE cells genetically modified with CAR.CD19 demonstrated a dose-effect response, with the lowest dose being 1 × 10⁶ 6 While BINATE cells could sustain animals for one week longer than non-transduced cells, 5 × 10 6 individual or 10 x 10 6 Animals administered individual BINATE.CAR.CD19 cells could not maintain the cells for as long as those given to other animals. This model was supported by IL-2 subcutaneous injections twice weekly. Unlike CAR-T CD19 genetically modified αβT cells, the animals survived until sacrifice on day 90 and showed no evidence of cytokine release syndrome (CRS) or allogeneic or heterogeneous reactivity despite IL-2 administration, supporting improved safety of native cells. After the animals were sacrificed on day 90 and tissues were evaluated for the presence of human CD45+ cells (BINATE) in peripheral blood and liver, %NK and γδT subsets were examined in each tissue, respectively. As shown in Figure 23, BINATE cells were maintained in both blood and tissue, demonstrating the potential for survival and expansion of BINATE cell populations in an in vivo environment.

[0246] (Example 21) Evaluation of in vivo killing of solid tumors (neuroblastoma) In separate experiments, we evaluated the in vivo killing of solid tumors and neuroblastomas after transduction of activated BINATE cells with a retroviral construct encoding third-generation GD2 CAR.

[0247] 1. Buffy coat cells derived from normal, healthy donors were subjected to gradient cell separation using Ficoll®, performed by manual centrifugation. Mononuclear cells were subjected to αβT cell depletion using laboratory-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was divided into 0.25-0.5 × 10⁶ wells in a 24-well plate. 6Cells were cultured under feeder-free conditions using BINATE medium and 10 ng / ml IL-15, with a cell seeding concentration of cells / ml, and a combination of 50 ng / ml anti-CD2 (OKT11) and 50 ng / ml anti-NKp46 (Bab281) (or other concentrations of the activator may be used, or other agents may be added for activation).

[0248] 2.5 Day 2. Activated αβTCRneg cell population was subjected to 0.25 × 10⁶ saturation in the presence of gamma retrovirus supernatant containing a GD2 chimeric antigen receptor construct. 6 The GD2 constructs were placed in human fibronectin-coated plates at a concentration of 1 / ml. The GD2 constructs contained variant fragments of the CD34 receptor as selection and tracking markers.

[0249] 3. After 3 days, the transduced cells were washed and seeded in BINATE medium and 10 ng / mL IL-15 at a cell seeding concentration of 0.25-0.5 × 10⁶. 6 Cells / ml in a T75 flask (or cell seeding concentration in a culture bag, flask, or other larger culture vessel, e.g., a bioreactor (G-Rex) of 1 × 10⁶ cells / ml) 6 The culture was further expanded under feeder-free conditions at concentrations up to a range of cells / ml. The culture medium was changed every 3 days (less frequently in the case of a bioreactor, every 5-7 days). On day 20, the cells were washed and formulated in PBS for administration.

[0250] Heterogeneous in vivo neuroblastoma model The antitumor effect of CAR-transduced cells was evaluated in vivo by injecting SH-SY5Y cells into NOD / SCID IL-2Rγnull (NSG) xenograft mice. The mouse experiments were conducted in accordance with international, EU, and national ethical requirements and were approved by the Italian Ministry of Health (N°88 / 2016-PR). NGS mice (5 weeks old; The Jackson Laboratory, USA) were injected with firefly luciferase-labeled SH-SY5Y cells (0.75 × 10⁶). 6 The mice were inoculated intraperitoneally with 30 × 10 units on day 3. 6BINATE cells or BINATE.CARGD2 cells were injected intraperitoneally in three doses every two weeks and subjected to weekly bioluminescence imaging (IVIS System, Perkin Elmer, USA).

[0251] Non-genetically modified BINATE cells showed a partial response with disappearance of peripheral tumor masses around day 18, but the mice subsequently showed local progression. Genetically modified BINATE cells with CAR.CDGD2 demonstrated significant removal of peripheral tumor masses, but failed to kill local lesions. This model was supported by 2x weekly subcutaneous injections of IL-2.

[0252] (Example 22) In vitro sterilization of solid tumors (neuroblastoma) and evaluation of ADCC antibodies by NK cells In separate experiments, in vitro killing of solid tumors and neuroblastomas by native NK cells or native NK.CAR.GD2 cells was evaluated.

[0253] 1. Gradient cell separation was performed using Ficoll®, a manual centrifugation method, on buffy coat derived from normal, healthy donors. Mononuclear cells were subjected to laboratory-scale Miltenyi LS column separation and NK enrichment using the Miltenyi NK selection kit. The resulting population was divided into 0.25–0.5 × 10⁶ well plates in 24-well plates. 6 Cells were cultured under feeder-free conditions using BINATE medium and 500 IU / ml IL-2, containing a combination of 500 ng / ml anti-CD2 antibody (clone LT2, Miltenyi) and 10 ng / ml anti-NKp46 (or other concentrations of the activator may be used, or other agents may be added for activation) at a cell seeding concentration of cells / ml.

[0254] 2.5 days later, the activated population was subjected to 0.25 × 10⁶ measurements in the presence of gamma retrovirus supernatant containing a GD2 chimeric antigen receptor (CAR) construct. 6The cells were placed on human fibronectin-coated plates at a concentration of 1 / ml. The GD2 constructs contained variant fragments of the CD34 receptor as selection and tracking markers.

[0255] 3. After 3 days, the transduced cells were washed and seeded in BINATE medium and 500 IU / mL IL-2 at a cell seeding concentration of 0.25-0.5 × 10⁶. 6 Cells / ml in a T75 flask (or cell seeding concentration in a culture bag, flask, or other larger culture vessel, e.g., a bioreactor (G-Rex) of 1 × 10⁶ cells / ml) 6 The culture was further expanded under feeder-free conditions at concentrations up to a range of up to 1 / ml. Medium changes were performed every 3 days (less frequently, every 5-7 days, for plates or flasks) using fresh BINATE medium and IL-2.

[0256] 4. At the end of the 20-day culture expansion, BINATE cells and BINATE.CARGD2 cells were placed in a tumor co-culture experiment for 7 days or more. The experiment was conducted in a 6-well culture dish with 2 ml of medium / well at a 1:1 ratio of effector to target (i.e., 0.5 × 10⁶). 6 0.5 x 10 Natural NK or Natural NK.CAR.GD2 (effects pedal) 6 The study involved culturing individual SH-SY5Y tumor cells (target) together. Anti-GD2 antibody (10 μg, 14.G2a monoclonal antibody) was added to each co-culture group, and the ADCC activity of activated cells was evaluated. Co-culture of tumor and effector cells was performed in the absence of cytokines. The co-cultures were evaluated by microscopy and flow cytometry on days 3-7.

[0257] The data included the percentage of residual tumor after co-culturing tumors with unmodified native NK cells or native NK.CAR.GD2 cells, in or out of the presence of the 14.G2a ADCC antibody. Residual SH-SY5Y tumor in the control group was measured as 99.5%. Addition of the 14.G2a antibody to SH-SY5Y tumor cells did not result in any difference compared to the control group (98.5%). Addition of unmodified activated native NK cells to SH-SY5Y tumor cells resulted in a residual tumor percentage of 22%. Addition of the 14G.2a antibody to a combination of native NK cells and SH-SY5Y tumor cells resulted in a lower residual tumor percentage (14.5%). When native NK.CAR.GD2 cells were added to SH-SY5Y tumor cells, a residual tumor percentage of 40.9% was achieved. When native NK.CAR.GD2 cells, SH-SY5Y tumor cells, and 14.G2a antibody were co-cultured, the residual tumor percentage decreased to 8.7%. In the presence of cells expressing the FcγIII receptor, such as activated native cells, the ADCC activity mediated by the antibody enhanced tumor killing.

[0258] (Example 23) In separate experiments, the effect of cytokine switching during expansion on cell content was evaluated.

[0259] 1. Buffy coats derived from normal, healthy donors were subjected to gradient cell separation using Ficoll®, performed by manual centrifugation.

[0260] 2. Mononuclear cells were subjected to αβT cell depletion using laboratory-scale Miltenyi LS column separation.

[0261] 3. The obtained αβTCRneg cell population was placed in a feeder-free culture environment using NK MACS® medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA)) supplemented with 5% AB serum (or other media, such as R&D Systems medium or CELLGENIX medium, may be used).

[0262] 4. Activation was achieved by either a combination of GMP-purified 10 ng / ml anti-NKp46 (clone Bab281) and 10 ng / ml anti-CD2 (clone OKT11) or 50 ng / ml anti-NKp46 (clone Bab281) and 50 ng / ml anti-CD2 (clone OKT11) by Caprico (or other concentrations of the activator may be used, or other agents may be added for activation). The activated αβTCRneg cell population was 0.25 × 10⁶ in a 24-well plate. 6 At a cell seeding concentration of cells / ml, use NK MACS® medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA)) supplemented with 5% AB serum (or other media, such as R&D Systems medium or CELLGENIX medium, may be used) under the following conditions: a. 500 IU / mL Human Interleukin-2 (IL-2) (Miltenyi Biotec) b. 10 ng / ml Human Interleukin-15 (IL-15) (Miltenyi Biotec) c. Combination of 500 IU / mL IL-2 and 10 ng / mL IL-15 d. Combination of 500 IU / mL IL-2 and 10 ng / mL OKT3 (Miltenyi Biotec) e. A combination of 10 ng / mL IL-15 and 500 IU / mL IL-2 and 10 ng / mL OKT3 After culturing under feeder-free conditions using one of the methods, the culture was transferred to a T75 flask.

[0263] 5. The culture medium was changed every 3 days (less frequently in the case of a bioreactor, every 5-7 days) using fresh medium and one of the conditions listed above.

[0264] 6. Dissolve the cells in 0.25-0.5 × 10 6 cells / ml (or cell seeding concentration is 1 × 10 in culture bags, flasks, or other larger culture vessels, e.g., bioreactors (G-Rex)) 6Seeds were sown at a concentration (which may range from up to 1 / ml).

[0265] 7. Furthermore, cells initially expanded with IL-2 for the first 25 days were then washed and supplemented with IL-15 until the end of the culture (day 30). Cells initially expanded with IL-15 were washed on day 25 and supplemented with IL-2 until the end of the culture on day 30 (referred to as cytokine switching conditions). In some cases, cells initially expanded with IL-2 for the first 20 days were then washed and supplemented with IL-15 until the end of the culture (day 30). Cells initially expanded with IL-15 were washed on day 20 and supplemented with IL-2 until the end of the culture on day 30. Alternative combinations or other cytokines, such as IL-7, IL-12, IL-18, IL-21, OKT3, OKT11, and anti-NCR, may be used as switching conditions.

[0266] 8. At the end of the expansion, BINATE cells were analyzed for NK and γδCD3+ T cell content and characterized by on-cellular receptor expression via flow cytometry.

[0267] In this example, a single cytokine was compared to a switching condition (IL-2 versus switching IL-2 to IL-15). When comparing IL-2 with IL-2 / IL-15, the number of γδT cells increased with the addition of IL-15 compared to IL-2 alone (70% vs. 50%, respectively). When comparing IL-15 with IL-15 / IL-2, the NK population increased under the switching condition compared to IL-15 alone (90% vs. 80%, respectively). Since both IL-2 and IL-15 act through interaction with the IL-2 receptor on cells, the data indicate that the expansion of a population favorable to the initial expansion condition by a particular cytokine is enhanced by switching to another cytokine.

[0268] (Example 24) Isolation of pure NK cells or pure γδT cells This example describes the process for isolating a pure NK cell population and a pure γδT cell population. A flowchart illustrating a modified version of this process is shown in Figure 24.

[0269] 1. Perform gradient cell separation using Ficoll® by manual centrifugation on buffy coat derived from a normal, healthy donor; Percoll gradient, SEPAX, Cobe2991, or eltriation systems may also be used. Alternatively, apheresis, umbilical cord blood units, bone marrow, or body fluids, such as ascites, CSF, or pleural fluid, may be used as a cell source with or without gradient centrifugation.

[0270] 2. Mononuclear cells are used directly, or after gradient separation, or subjected to further cell selection, such as Ficoll® MNC, antibody depletion, positive selection of NK cells, negative selection of NK cells, negative selection of γδT cells, and / or positive selection of CD3+ cells. In certain examples, mononuclear cells are subjected to positive or negative selection of CD56+ NK cells using a Miltenyi magnetic bead-based method or other cell selection techniques. Alternatively, cells may be subjected to positive selection of γδTCR+ cells using a Miltenyi magnetic bead-based technique or other cell selection techniques. Alternatively, cells may be subjected to CD3+ cell depletion using a Miltenyi magnetic bead-based technique or other cell selection techniques.

[0271] 3. Next, the obtained cell population is activated in feeder-free culture using BINATE medium (NK MACS™ medium #130-107-879 Miltenyi Biotec, Inc., San Diego, CA, USA, supplemented with 5% AB serum (or other media, such as R&D Systems medium or CELLGENIX medium, may be used)) or other animal component-free culture media, with or without human serum supplementation, such as In Vivo, R&D Systems, or Cellgenix, in the presence of soluble or cross-linked anti-LFA and anti-NCR antibodies.

[0272] 4. Cell expansion is 0.25–5 × 10 in a T75 flask. 6 Cell seeding concentration of cells / ml (or cell seeding concentration of 1 × 10 in culture bags, flasks, or other larger culture vessels, e.g., bioreactor (G-Rex)) 6 This is done by supplementing with cytokines such as IL-2, IL-15, IL-12, IL-18, and combinations thereof, at a rate that can range from up to 1 / ml. The culture medium is changed every 3 days (less frequently (every 5-7 days) in the case of a bioreactor) with growth medium and cytokines.

[0273] 5. The cultures obtained on days 7, 20, 30, 45, and 60 may contain a binate population of NK and γδT cells, which are then cryopreserved for administration. Alternatively, the obtained cell population may undergo a further expansion cell selection step, such as NK CD56+ depletion, to obtain a pure γδT cell population. Alternatively, the cells may be subjected to positive removal or negative selection of γδT cells to obtain a pure NK population, or positive selection of CD16 to produce a highly enriched ADCC FcyIII receptor+ population. Alternatively, CD16 selection may be performed on the ADCC γδT cell population after NK depletion of the cells.

[0274] (Example 25) Retrovirus constructs This example describes the retroviral vectors CARGD2, CARCD123, and CARCD19 used in a specific example described above.

[0275] Vector skeleton The construct described below contains an SFG skeleton, which uses a retroviral vector based on Moloney's mouse leukemia virus (MoMLV). All env and gag-pol sequences except the packaging sequence (psi) were removed. As a result, the vector is replication incompetent.

[0276] Producing cell lines Packaging cell lines for producing the following retroviral constructs were prepared in the laboratory environment of OPBG, Cell and Gene Therapy for Pediatric Tumor Institute, using a dedicated laminar flow hood and CO2 incubator, as well as gamma-irradiated Pharmagrade FBS (EuroClone, catalog ECS017). The product was prepared using non-animal-derived materials, with the exception of 2L (lot EUS0131906GI). The packaging cell line was generated from a human-based 293VEC RD114-producing cell line deposited under cGMP received from BioVec Pharma. In particular, the 293VEC RD114 cell line was selected as the packaging cell line because it is of human origin and enables the production of high vector titers suitable for large-scale clinical-grade production.

[0277] CARGD2 The iC9-CARGD2.CD28.41bb.CD3 zeta (OPBG-91 vector) retroviral vector was constructed at the Ospedale Pediatrico Bambino Gesu (OPBG), “Cell and Gene Therapy for Pediatric Tumor” laboratory. A bicistronic vector was used to enable the simultaneous expression of two transgenes, namely inducible caspase 9 (iC9) and CARGD2 (iC9-CARGD2.CD28.41bb.CD3 zeta). Single-cell cloning was performed, and the 293VEC RD114 clone (i.e., OPBG-91-7), which produced the highest titer (using PCR analysis for the presence of the vector in the supernatant), was expanded, deposited at Officina Farmaceutica OPBG, tested for sterility and mycoplasma, and then used for retroviral production under cGMP conditions.

[0278] In the iC9 component, the catalytic domain of the intracellularly expressed Casp9 protein is fused to the drug-binding domain derived from human FK506-binding protein (FKBP12) with the F36V mutation. To transduce an activation signal after antigen binding, a CAR molecule based on a single-strand fusion VH-VL region of the human antigen GD2-specific monoclonal antibody 14.G2A, in frame with the CD28 transmembrane domain and its endodomain, the 4.1bb costimulatory domain, and the CD3ζ cytoplasmic domain, was cloned in a retroviral vector after the iC9 sequence was included in the gene cassette to improve the safety aspects of the approach.

[0279] Certain functional and structural components related to the expression and activity of iC9.CARGD2.CD28.41bb.CD3z are summarized in Table 9 below and listed herein:

[0280] • 5'LTR - The retroviral long-term repeat sequence at the 5' end of the vector (functions as a promoter sequence).

[0281] • ψ - Retroviral capsidation signal (psi; required for packaging RNA into virion particles).

[0282] • SA - Splice acceptor site.

[0283] • iCasp9 - Inducible caspase-9 expression cassette. iCasp9 consists of a human FK506-binding protein (FKBP12) with the F36V mutation, linked to a modified CARD domain-deleted human caspase-9 via a 6-amino acid Gly-Ser linker.

[0284] • FKBP12-F36V - An engineered FK506-binding protein containing the F36V mutation to optimize binding affinity to AP1903. The FKBP12-F36V protein domain serves as the drug-binding / oligomerization domain for linked therapeutic proteins. FKBP12-F36V functions as a regulator of caspase-9. In the absence of AP1903, iCasp9 has minimal activity; when AP1903 binds to FKBP12-F36V, it promotes dimerization, juxtaposing two caspase-9 molecules and initiating apoptosis. Thus, the FKBP12-F36V portion functionally replaces the endogenous dimerization / activation module (caspase activation and recruitment domain; CARD) of caspase-9 that mediates Apaf-1-related oligomerization.

[0285] • Linker - A synthetic Ser-Gly-Gly-Gly-Ser-Gly peptide linker used to fuse the switching regulatory factor sequence to caspase-9.

[0286] • Caspase-9 - Human caspase-9 cDNA sequence (a key pro-apoptotic regulator) and therapeutic component of the construct (a regulated suicide gene). The endogenous dimerization / activation module (caspase activation and recruitment domain; CARD) was deleted to reduce spontaneous Apaf1 binding and thus reduce background killing.

[0287] • 2A - Encodes a synthetic 20-amino acid peptide derived from the Thosea Asigna insect virus, which functions as a cleavable linker between the caspase-9 protein and the CAR protein.

[0288] • Signal peptides - Short amino acid sequences that enable the precise movement of secreted proteins from the endoplasmic reticulum to the cell membrane.

[0289] • CAR - A CAR molecule based on a single-strand fusion VH-VL region of the human antigen GD2-specific monoclonal antibody 14.G2A, in frame with the CD28™ and costimulatory domains, 4.1bb costimulatory domain and CD3ζ cytoplasmic domain.

[0290] • 3'LTR - A retroviral long-term repeat sequence at the 3' end of the vector (functions as a terminator / polyadenylation sequence). [Table 9-1] [Table 9-2]

[0291] The reference electron vector sequence was assembled by combining the DNA sequence files of each component of the vector construct. Since retroviral genomes are RNA-based, sequencing analysis was performed on the plasmid DNA used for transfection into the 293VEC cell line (the first step in retroviral product preparation). Bidirectional sequencing was performed on the entire OPBG-91 vector in Ospedale Pediatrico Bambino Gesu. The sequencing runs were assembled using SnapGene software. No mismatched bases were identified compared to the theoretical reference electron sequence.

[0292] CARCD123 The ΔCD19-2A-CAR-CD123-ΔCD34.CD8.41bb.CD3 zeta (OPBG-242 vector) retroviral vector was constructed at the Ospedale Pediatrico Bambino Gesu (OPBG), “Cell and Gene Therapy for Pediatric Tumor” laboratory. A bicistronic vector (ΔCD19-2A-CAR-CD123-ΔCD34.CD8.41bb.CD3 zeta) was used to enable the simultaneous expression of two transgenes, namely ΔCD19 and CARCD123. Single-cell cloning was performed, and the clone producing the highest titer (using PCR analysis for the presence of the vector in the supernatant) was expanded as described above and deposited.

[0293] ΔCD19 represents the extracellular domain of human CD19 ligated to its transmembrane region. It has a dual function to aid in the selection and phenotypic characterization of genetically modified cells. For transduction of the activation signal after antigen binding, a CAR molecule based on a single-strand fusion VH-VL region of the human antigen CD123-specific monoclonal antibody 7G3, in frame with the CD8 transmembrane domain and its endodomain, the 4.1bb costimulatory domain, and the CD3ζ cytoplasmic domain, was cloned after a gene expression cassette containing the ΔCD19 sequence via the use of the 2A sequence in a retroviral vector.

[0294] Certain functional and structural components for ΔCD19-2A-CAR-CD123-ΔCD34.CD8.41bb.CD3z expression and activity are summarized in Table 10 below and listed herein:

[0295] • 5'LTR - The retroviral long-term repeat sequence at the 5' end of the vector (functions as a promoter sequence).

[0296] • ψ - Retroviral capsidation signal (psi; required for packaging RNA into virion particles).

[0297] • SA - Splice acceptor site.

[0298] • △CD19 - Contains optimized human extracellular and transmembrane domains.

[0299] • 2A - Encodes a synthetic 20-amino acid peptide derived from the Thosea Asigna insect virus.

[0300] • Signal peptides - Short amino acid sequences that enable the precise movement of secreted proteins from the endoplasmic reticulum to the cell membrane.

[0301] • Contains a short peptide derived from human CD34 that helps detect transduced CAR+ T cells.

[0302] • CAR - A CAR molecule based on a single-strand fusion VH-VL region of the human antigen CD123-specific monoclonal antibody 7G3, in frame with the CD8™ and costimulatory domains, 4.1bb costimulatory domain and CD3ζ cytoplasmic domain.

[0303] • 3'LTR - A retroviral long-term repeat sequence at the 3' end of the vector (functions as a terminator / polyadenylation sequence). [Table 10]

[0304] The reference electron vector sequence was assembled by combining the DNA sequence files of each component of the vector construct. Since retroviral genomes are RNA-based, sequencing analysis was performed on the plasmid DNA used for transfection into the 293VEC cell line (the first step in retroviral product preparation). Bidirectional sequencing was performed on the entire OPBG-242 vector in Ospedale Pediatrico Bambino Gesu. The sequencing runs were assembled using SnapGene software. No mismatched bases were identified compared to the theoretical reference electron sequence.

[0305] CARCD19 The therapeutic retroviral construct SFG-iC9-Car.CD19.41bb encodes a synthetic ligand-induced human caspase-9 cDNA ligated to a single strand of the fusion VH-VL region of a monoclonal antibody specific to the mouse antigen CD19,4.1bb costimulatory domain. The assembly is identical to CARGD2 described above, except that it is second generation. The retroviral vector was constructed at the EU Cell and Gene Therapy Laboratory. Single-cell cloning was performed, and the clone producing the highest titer (using PCR analysis for the presence of the vector in the supernatant) was amplified, deposited in a cGMP facility, and, after sterility and mycoplasma testing, used for retroviral production under cGMP conditions.

[0306] The following are some specific functional and structural components for SFG-iC9-Car.CD19.41bb expression and activity:

[0307] • 5'LTR - The retroviral long-term repeat sequence at the 5' end of the vector (functions as a promoter sequence).

[0308] • ψ - Retroviral capsidation signal (psi; required for packaging RNA into virion particles).

[0309] • SA - Splice acceptor site.

[0310] • iCasp9 - Inducible caspase-9 expression cassette. iCasp9 consists of a human FK506-binding protein (FKBP12) with the F36V mutation, linked to a modified CARD domain-deleted human caspase-9 via a 6-amino acid Gly-Ser linker.

[0311] • FKBP12-F36V - An engineered FK506-binding protein containing the F36V mutation to optimize binding affinity to AP1903. The FKBP12-F36V protein domain serves as the drug-binding / oligomerization domain for linked therapeutic proteins. FKBP12-F36V functions as a regulator of caspase-9. In the absence of AP1903, iCasp9 has minimal activity; when AP1903 binds to FKBP12-F36V, it promotes dimerization, juxtaposing two caspase-9 molecules and initiating apoptosis. Thus, the FKBP12-F36V portion functionally replaces the endogenous dimerization / activation module (caspase activation and recruitment domain; CARD) of caspase-9 that mediates Apaf-1-related oligomerization.

[0312] • Linker - A synthetic Ser-Gly-Gly-Gly-Ser-Gly peptide linker used to fuse the switching regulatory factor sequence to caspase-9.

[0313] • Caspase-9 - Human caspase-9 cDNA sequence (a key pro-apoptotic regulator) and therapeutic component of the construct (a regulated suicide gene). The endogenous dimerization / activation module (caspase activation and recruitment domain; CARD) was deleted to reduce spontaneous Apaf1 binding and thus reduce background killing.

[0314] • 2A - Encodes a synthetic 20-amino acid peptide derived from the Thosea Asigna insect virus, which functions as a cleavable linker between the caspase-9 protein and the CAR protein.

[0315] • Signal peptides - Short amino acid sequences that enable the precise movement of secreted proteins from the endoplasmic reticulum to the cell membrane.

[0316] • CAR - A CAR molecule based on a single-strand fusion of the VH-VL region with a monoclonal antibody specific to the 4.1bb costimulatory domain of the mouse antigen CD19.

[0317] • 3'LTR - A retroviral long-term repeat sequence at the 3' end of the vector (functions as a terminator / polyadenylation sequence).

[0318] (Example 26) Examples of certain non-restrictive embodiments The following are non-limiting examples of certain embodiments of this technology.

[0319] A method for producing a composition comprising a population of cells enriched with respect to A1.NK cells and gamma-delta T cells, Obtain a sample containing cells from one or more subjects; Depleting alpha-beta T cells from a sample under conditions that generate a depleted cell population including NK cells and gamma-delta T cells; and Exposing the depleted cell population to activation conditions comprising (a) contact with at least one exogenous polypeptide that immunospecifically binds to cell adhesion polypeptides, and (b) at least one exogenous polypeptide that immunospecifically binds to polypeptides different from cell adhesion polypeptides and is expressed on the surface of one or more types of cells in the sample population; and Exposing a depleted cell population to expansion conditions that include contacting the depleted cell population with at least one auxiliary polypeptide, thereby generating a composition containing a population of cells enriched with respect to NK cells and gamma delta T cells. A method that includes this.

[0320] A1.1. A method for producing a composition comprising a population of cells enriched with respect to NK cells and gamma-delta T cells, Obtain a sample containing cells from one or more subjects; Activation conditions comprising contacting the sample with (a) at least one exogenous polypeptide that immunospecifically binds to cell adhesion polypeptides, and (b) at least one exogenous polypeptide that immunospecifically binds to a polypeptide different from the cell adhesion polypeptide and is expressed on the surface of one or more types of cells in a sample population, wherein (a) or (b) is soluble, or (a) and (b) are soluble; and Exposing a sample to expansion conditions that include contacting the sample with at least one auxiliary polypeptide, thereby generating a composition containing a population of cells enriched with respect to NK cells and gamma delta T cells. A method that includes this.

[0321] A2. The method according to Embodiment A1 or A1.1, wherein at least one auxiliary polypeptide is selected such that the amount of NK cells compared to the amount of gamma delta T cells in the population is dependent on the amount and / or type of at least one auxiliary polypeptide.

[0322] A2.1. The method according to Embodiment A1, A1.1, or A2, wherein at least one auxiliary polypeptide increases or decreases the amount of NK cells compared to gamma delta T cells in a cell population after contacting a depleted cell population with at least one auxiliary polypeptide.

[0323] A3. The method according to any one of Embodiments A1, A1.1, A2, or A2.1, wherein the activation conditions do not include serum derived from a non-human animal.

[0324] A4. The method according to any one of Embodiments A1 to A3, wherein the expansion condition does not include serum derived from a non-human animal.

[0325] A5. The method according to any of Embodiments A1 to A4, wherein the activation conditions do not include feeder cells.

[0326] A6. The method according to any of Embodiments A1 to A5, wherein the expansion conditions do not include feeder cells.

[0327] A7. The method according to any one of embodiments A1 to A6, wherein the sample is selected from peripheral blood, liver tissue, epithelial tissue, bone marrow, and umbilical cord blood.

[0328] A8. The method according to Embodiment A7, wherein the sample is peripheral blood.

[0329] A9. The method according to Embodiment A8, wherein the peripheral blood sample is a processed sample.

[0330] A10. The method according to Embodiment A7, wherein the sample is umbilical cord blood.

[0331] A11. The method according to Embodiment A10, wherein the umbilical cord blood sample is a processed sample.

[0332] The method according to any one of embodiments A1 to A11, wherein the exogenous polypeptide in A12.(b) is immunospecifically bound to the NK cell activating receptor, the gamma-delta T cell activating receptor, or both.

[0333] A13. The method according to Embodiment A12, wherein the receptor is NKp30, NKp44, or NKp46.

[0334] A14. The method according to embodiment A13, wherein the receptor is NKp46.

[0335] The method according to any one of embodiments A1 to A14, wherein the exogenous polypeptide in A15.(a) is immunospecifically bound to CD2.

[0336] A16. The method according to any one of Embodiments A1 to A15, wherein the exogenous polypeptide in A16(a) or (b), or in (a) and (b), is an antibody or an antigen-binding fragment thereof.

[0337] A17. The method according to any one of embodiments A1 to A16, wherein at least one of (a) or (b) is soluble.

[0338] The method according to any one of embodiments A1 to A17, wherein both of the exogenous polypeptides in A17.1.(a) and (b) are soluble.

[0339] The method according to any one of Embodiments A1 to A16, wherein the exogenous polypeptide in A18.(a) or the exogenous polypeptide in (b) is bound to a substrate.

[0340] A19. The method according to any one of Embodiments A1 to A18, wherein the activation condition includes contacting a sample or depleted cell population with at least two exogenous polypeptides.

[0341] A20. The method according to Embodiment A19, wherein a first exogenous polypeptide is immunospecifically bound to CD2 and a second exogenous polypeptide is immunospecifically bound to NKp46.

[0342] A21. The method according to Embodiment A19 or A20, wherein the first exogenous polypeptide and / or the second exogenous polypeptide is an antibody or an antigen-binding fragment thereof.

[0343] A22. The polypeptide components under the activation conditions are (a) Exogenous polypeptides that immunospecifically bind to cell adhesion polypeptide CD2; and (b) Unlike the exogenous polypeptide in (a), this is an exogenous polypeptide that binds immunospecifically to NKp46. The method according to any one of embodiments A1 to A21, which is essentially composed of or consists of.

[0344] A23. The method according to any one of embodiments A1 to A22, wherein the auxiliary polypeptide is a polypeptide that binds immunospecifically to cytokines and / or receptors on gamma-delta T cells.

[0345] A24. The method according to Embodiment A23, wherein the expansion condition includes contacting a sample or depleted cell population with at least one co-opposite polypeptide that is a cytokine, and optionally with a co-opposite polypeptide that binds immunospecifically to a receptor on gamma delta T cells.

[0346] A25. The method according to Embodiment A24, wherein the cytokine is interleukin (IL).

[0347] A26. The method according to any one of embodiments A23 to A25, wherein at least one auxiliary polypeptide comprises IL-2, IL-4, IL-15, or any combination thereof.

[0348] A27. Magnification conditions for the sample or depleted cell population (a) IL-2 polypeptide, and polypeptides that, if necessary, immunospecifically bind to receptors on gamma-delta T cells; (b) IL-15 polypeptide; or (c) IL-2 polypeptide and IL-15 polypeptide, and polypeptides that, if necessary, immunospecifically bind to receptors on gamma delta T cells. A method according to any one of embodiments A23 to A26, which includes bringing into contact with the

[0349] A28. The method according to any one of embodiments A23 to A27, wherein the receptor on gamma-delta T cells is CD3.

[0350] A29. The method according to any one of embodiments A23 to A28, wherein the polypeptide that immune-specifically binds to a receptor on a gamma-delta T cell is an antibody or an antigen-binding fragment thereof.

[0351] A30. Magnification conditions, the sample (a) IL-2 polypeptide; (b) IL-15 polypeptide; (c) IL-2 polypeptide and IL-15 polypeptide; (d) An antibody that binds immunospecifically to IL-2 polypeptide and CD3; or (e) Antibodies that bind immunospecifically to IL-2 polypeptide, IL-15 polypeptide, and CD3. The method according to Embodiment A29, which includes bringing into contact with the

[0352] A31. The method according to Embodiment A30, wherein the antibody that binds immunospecifically to CD3 is OKT3.

[0353] A32. The method according to any one of embodiments A1 to A31, wherein the activation and expansion conditions are carried out simultaneously or sequentially in any order.

[0354] A33. At least one exogenous polypeptide can also function as an auxiliary polypeptide; or At least one auxiliary polypeptide can also function as an exogenous polypeptide; or The method according to any one of Embodiments A1 to A32, wherein at least one exogenous polypeptide can also function as an auxiliary polypeptide, and at least one auxiliary polypeptide can also function as an exogenous polypeptide.

[0355] A34.(i) The expansion condition includes contacting the sample or depleted cell population with IL-2 polypeptide; (ii) The method according to any of Embodiments A30 to A33, wherein the resulting cell population enriched with respect to NK cells and gamma delta T cells contains approximately 25-30% NK cells and approximately 70-75% gamma delta T cells.

[0356] A35.(i) The expansion condition includes contacting the sample or depleted cell population with IL-15 polypeptide; (ii) The method according to any of Embodiments A30 to A33, wherein the resulting cell population enriched with respect to NK cells and gamma delta T cells comprises approximately 80-99% NK cells and approximately 1-20% gamma delta T cells.

[0357] A36.(i) The expansion condition includes contacting the sample or depleted cell population with an antibody that immunospecifically binds to IL-2 polypeptide and CD3; (ii) The method according to any of Embodiments A30 to A33, wherein the resulting cell population enriched with respect to NK cells and gamma delta T cells contains approximately 40-45% NK cells and approximately 55-60% gamma delta T cells.

[0358] A37. The conditions for expansion are, Exposing a sample or depleted cell population to a first set of conditions containing one or more auxiliary polypeptides to result in a first cell population containing a first ratio of NK cells to gamma delta T cells; and The method according to any of Embodiments A1 to A36, comprising contacting a first cell population with a second set of conditions comprising one or more auxiliary polypeptides to yield a second cell population having a desired final ratio of NK cells to gamma delta T cells, wherein the first set of conditions is different from the second set of conditions.

[0359] A38. The method according to Embodiment A37, wherein the first cell population is washed before contact with the second set of conditions.

[0360] A39. The first set of conditions includes IL-2, and the second set of conditions includes IL-15; The first set of conditions includes IL-15, and the second set of conditions includes IL-2; The first set of conditions includes antibodies that bind immunospecifically to IL-2 and CD3, and the second set of conditions includes IL-15; or The method according to Embodiment A37 or A38, wherein the first set of conditions comprises antibodies that immunospecifically bind to IL-15 and CD3, and the second set of conditions comprises antibodies that immunospecifically bind to IL-2 and CD3.

[0361] A40. The method according to Embodiment A39, wherein the antibody that binds immunospecifically to CD3 is OKT3.

[0362] A41. Depleting alpha and beta T cells from the sample before exposing it to activation and expansion conditions, thereby generating a depleted cell population; and To expose depleted cell populations to conditions for activation and expansion. It further includes, The method according to any one of Embodiments A1.1 and A2-A40, wherein a composition is obtained comprising a population of cells enriched with respect to NK cells and gamma delta T cells.

[0363] A41.1. The method according to any one of Embodiments A1 to A41, wherein, prior to activation and expansion, the sample or depleted cell population is not exposed to conditions that select for NK cells or gamma-delta T cells, nor to conditions that deplete cells other than alpha-beta T cells.

[0364] A41.2. The method of Embodiment A41.1, wherein the sample or depleted cell population is not exposed to conditions that deplete CD3+ cells before activation and expansion.

[0365] A42. The method according to any one of Embodiments A1 to A41.1, wherein the cells of the sample or depleted cell population do not contain exogenous nucleic acids before, during, or after activation and expansion.

[0366] A43. The method according to any of Embodiments A1 to A41.1, wherein the cells of the sample or depleted cell population do not contain tumor necrosis factor receptors, chimeric antigen receptors (CARs), myeloid differentiation primary response proteins, or exogenous nucleic acids encoding innate immune signaling adapters before, during, or after activation and expansion.

[0367] A44. The method according to any of Embodiments A1 to A43, wherein the cells of the sample or depleted cell population are not genetically modified before, during, or after activation and expansion.

[0368] A45. The method according to any one of embodiments A1 to A44, further comprising subjecting a population of cells enriched with respect to NK cells and gamma delta T cells to a treatment which thereby depletes gamma delta cells, wherein the resulting population is essentially composed of or consists of NK cells.

[0369] A46. The method according to any one of embodiments A1 to A44, further comprising subjecting a population of cells enriched with respect to NK cells and gamma delta T cells to a treatment thereby depleting NK cells, wherein the resulting population is essentially composed of or consists of gamma delta T cells.

[0370] The method according to any one of embodiments A1 to A45, further comprising subjecting a population of cells enriched with respect to NK cells and gamma delta T cells to positive selection for NK cells, thereby obtaining a population of cells essentially consisting of or comprising NK cells.

[0371] A48. The method according to any one of embodiments A1 to A44 and A46, further comprising subjecting a population of cells enriched with respect to NK cells and gamma delta T cells to positive selection for gamma delta cells, thereby obtaining a population of cells essentially consisting of or comprising gamma delta cells.

[0372] A49. The method according to any one of Embodiments A1 to A48, wherein the expansion conditions include incubation of the sample or depleted cell population in feeder cell-free medium for about 1 to 10 weeks, thereby obtaining a composition containing an expanded cell population enriched with respect to NK cells and gamma delta T cells.

[0373] A50. The method according to Embodiment A49, wherein the culture conditions include incubation of the sample or depleted cell population in feeder cell-free medium for approximately 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 30, 35, 40, 45, 50, 55, or 60 days or more, or incubation for approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.

[0374] A51. The method according to any one of embodiments A1 to A50, wherein a population of cells enriched with respect to NK cells and gamma delta T cells is expanded by more than approximately 2 log over 30 days under expansion conditions.

[0375] A52. The method according to Embodiment A51, wherein the cell population is expanded by more than approximately 3 log over 30 days under expansion conditions.

[0376] A53. The method according to any one of embodiments A1 to A52, wherein the enlarged cell population enriched with respect to NK cells and gamma delta T cells is free from exhausted cells.

[0377] A54. The method according to any one of embodiments A1 to A53, wherein the enlarged cell population enriched with respect to NK cells and gamma delta T cells does not contain exhausted cells after 60 days of enlargement conditions.

[0378] A55. The method according to any one of Embodiments A1 to A54, wherein less than 5%, less than 4%, less than 3%, or less than 2% of NK cells in an enlarged cell population enriched with respect to NK cells and gamma delta T cells contain the PD-1 marker, and / or about 20%, 15%, 10%, or less than that of total cells in an enlarged population or gamma delta T cells in an enlarged population contain the PD-1 marker.

[0379] A56. Enriched cell populations with respect to NK cells and gamma delta T cells were identified as the following activation markers as a percentage of the total cell number in the population: (a) 90% or more of KIR5; (b) 10% or more of SIGLEC-7; (c) 60% or more KIR3D51; (d) 10% or more of KIR2DL1; (e) 25% or more of NKp30, NKp44, or NKp46; (f) 35% or more NKG2D; (g) 90% or more DNAM1; (h) 85% or more NTBA; (i) 95% or more of CD2; and (j) 55% or more KIR3DS1 The method according to any of embodiments A1 to A55, which includes one or more of the above.

[0380] A57. The method according to any one of embodiments A1 to A56, wherein the cell population enriched with respect to NK cells and gamma delta T cells comprises 80% or more innate immune cells.

[0381] A58. The method according to Embodiment A57, wherein between approximately 70% and approximately 100% of the cells, or at least approximately 70%, 71%, 7...

Claims

1. A method for producing a composition containing 70% to 75% γδ T cells derived from a sample containing cells from one or more subjects, (a) Depleting αβT cells from the sample under conditions that generate a depleted cell population including NK cells and γδT cells; (b) Exposing the depleted cell population to activation conditions which include contacting (a) an antibody or antigen-binding fragment thereof that immunospecifically binds to the cell adhesion molecule polypeptide CD2, and (b) an antibody or antigen-binding fragment thereof that immunospecifically binds to NKp46; and (c) Exposing the depleted cell population to expansion conditions including contact with IL-2, thereby generating a composition containing 70-75% γδ T cells. A method that includes this.

2. The method according to claim 1, wherein the composition comprises 30% or less of NK cells.

3. The method according to claim 1 or 2, wherein the expansion condition further comprises contacting the depleted cell population with an antibody or an antigen-binding fragment thereof that is immune-specifically bound to CD3.

4. The method according to any one of claims 1 to 3, wherein the expansion condition comprises incubation of the depleted cell population for 1 to 6 weeks in the presence of IL-2 and / or anti-CD3 antibodies.

5. The method according to any one of claims 1 to 4, wherein the activation conditions include (a) an antibody or an antigen-binding fragment thereof that immunospecifically binds to the cell adhesion molecule polypeptide CD2, and (b) incubation of the depleted cell population for 1 hour to 1 week in the presence of an antibody or an antigen-binding fragment thereof that immunospecifically binds to NKp46.

6. The method according to any one of claims 1 to 5, wherein 1% or less of the total cells in the composition are αβT cells.

7. The method according to any one of claims 1 to 6, wherein depleting αβT cells from the sample includes contacting the sample with an antibody that binds immunospecifically to αβT cell receptors.

8. The method according to any one of claims 1 to 7, wherein the antibody or its antigen-binding fragment that binds immunospecifically to CD2 is a monoclonal anti-CD2 antibody, and / or the antibody or its antigen-binding fragment that binds immunospecifically to NKp46 is a monoclonal anti-NKp46 antibody.

9. The method according to claim 8, wherein the anti-CD2 antibody and / or anti-NKp46 antibody is soluble.

10. The method according to any one of claims 1 to 9, wherein the activation conditions and / or expansion conditions do not include feeder cells.

11. The method according to any one of claims 1 to 10, wherein the activation condition and / or expansion condition does not include a bisphosphonate.

12. The method according to claim 11, further comprising subjecting the depleted cell population to genetic modification conditions before exposing the depleted cell population to activation conditions.

13. The method according to claim 11, further comprising exposing the depleted cell population to a widening condition, and then subjecting the depleted cell population to a genetic modification condition.

14. The method according to claim 12 or 13, wherein the gene modification includes introducing an exogenous polynucleotide encoding a chimeric antigen receptor into the cell.

15. The method according to any one of claims 1 to 14, wherein the activation condition and the expansion condition are carried out simultaneously.

16. The method according to any one of claims 1 to 14, wherein the activation condition and the expansion condition are carried out sequentially.

17. The method according to any one of claims 1 to 16, wherein the sample is peripheral blood, liver tissue, bone marrow, epithelial tissue, or umbilical cord blood.

Citation Information

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