JP2023063272A - 改変t細胞、医薬組成物、その製造方法、及びそれを使用する方法 - Google Patents
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Abstract
Description
単核細胞を含む体液を得る工程;
単核細胞を、IL-2及びゾレドロン酸を含む誘導性培養培地と接触させて、第1の培養細胞集団を得る工程;
第1の培養細胞集団を、IL-2を含む第1の培養培地と接触させて、第2の培養細胞集団を得る工程;並びに
第2の培養細胞集団を、IL-2及びIL-12を含む第2の培養培地と接触させて、第3の培養細胞集団を得る工程
を含む、方法を提供する。
天然に存在するT細胞又は従来のT細胞は、CD3HiTCRγ9HiTCRδ2Hi表現型を有する。1つの実施形態において、天然に存在するT細胞又は従来のT細胞は、Table 2(表2)において説明されるように、CD3HiTCRγ9HiTCRδ2HiCXCR4DimCXCR3Hi表現型を有する。
1つの実施形態において、本出願は、本明細書に記載される改変T細胞、及び薬学的に許容される担体又は賦形剤を含む医薬組成物を提供する。
1つの実施形態において、始原T細胞を同定し、改変T細胞を培養する方法は、図11において図示される。簡潔には、方法は、少なくとも、(a)単核細胞を含む体液を得る工程;(b)単核細胞を、IL-2及びゾレドロン酸を含む誘導性培養培地と接触させて、第1の培養細胞集団を得る工程;(c)第1の培養細胞集団を、IL-2を含む第1の培養培地と接触させて、第2の培養細胞集団を得る工程;並びに(d)第2の培養細胞集団を、IL-2及びIL-12を含む第2の培養培地と接触させて、第3の培養細胞集団を得る工程を含む。
IL-12の添加は、改変T細胞の表現型及び機能発達において主要な役割を果たす
単核細胞を3つの群において培養した(図1)。群Nにおいて、単核細胞を、60ng/mLのヒト組換えIL-2(hIL-2)、2%(v/v)のヒト血小板溶解物(HPL)及び5μMのゾレドロン酸の存在下、AIM-V培地中で3日間培養した。その後、細胞を、3日ごとに、120ng/mLのhIL-2及び4%(v/v)のHPLの存在下、AIM-V培地中で継代し、15日目に回収した。群Aにおいて、単核細胞を、60ng/mLのhIL-2、2%(v/v)のHPL及び5μMのゾレドロン酸の存在下、AIM-V培地中で3日間培養した。その後、細胞を、3日目及び6日目に120ng/mLのhIL-2及び4%(v/v)のHPLの存在下、AIM-V培地中で継代し、9日目及び12日目に120ng/mLのhIL-2、4%(v/v)のHPL及び8ng/mLのヒト組換えIL-12(hIL-12)の存在下、AIM-V培地中で継代し、15日目に回収した。群Bにおいて、単核細胞を、60ng/mLのhIL-2、2%(v/v)のHPL及び5μMのゾレドロン酸の存在下、AIM-V培地中で3日間培養した。その後、細胞を、3日目及び6日目に120ng/mLのhIL-2及び4%(v/v)のHPLの存在下、AIM-V培地中で継代し、9日目及び12日目に120ng/mLのhIL-2、4%(v/v)のHPL及び45ng/mLのヒト組換えIL-18(hIL-18)の存在下、AIM-V培地中で継代し、15日目に回収した。
後段階におけるIL-18の添加は、改変T細胞の表現型及び機能発達において主要な役割を果たす
単核細胞を2つの群において培養した。群Aにおいて(図1)、単核細胞を、60ng/mLのhIL-2、2%(v/v)のHPL及び5μMのゾレドロン酸の存在下、AIM-V培地中で3日間培養した。その後、細胞を、3日目及び6日目に120ng/mLのhIL-2及び4%(v/v)のHPLの存在下、AIM-V培地中で継代し、9日目及び12日目に120ng/mLのhIL-2、4%(v/v)のHPL及び2ng/mLのhIL-12の存在下、AIM-V培地中で継代し、15日目に回収した。群Cにおいて(図6)、0日目、3日目、6日目及び9日目における単核細胞の処理は、群Aにおける処理と同じであった。しかしながら、群Cにおける細胞は、12日目に120ng/mLのhIL-2、4%(v/v)のHPL、2ng/mLのhIL-12及び135、45又は15ng/mLのヒト組換えIL-18(hIL-18)の存在下、AIM-V培地中で継代し、15日目に回収した。培養細胞を、トリパンブルー色素排除を使用することによって計数し、CD69、CD11c、CXCR4及びCXCR3のmAbで染色した。試料を、取得し、Naviosフローサイトメーターにより分析した一方で、データ分析は、Kaluzaソフトウェア(Beckman Coulter社)により行った。
始原細胞の調製
40mLの末梢血を、健康なボランティアから、K2EDTAを含有する真空管に収集した。血液試料を、等体積の事前に温めたリン酸緩衝食塩水(PBS)(Biological Industries社、イスラエル)と混合した。40mLの希釈された末梢血アリコートを、50mLの遠心分離管に入れ、10mLの事前に温めたFicoll-Paque(商標)PREMIUMを加えた。遠心分離管を、930×g、室温で30分間遠心分離した。界面層の単核細胞を収集し、PBSで1回洗浄した。単核細胞を、ヒト血小板溶解物及びサイトカインカクテルの存在下、AIM-Vとともに培養した。
改変T細胞の培養
実施例3からの単核細胞を、以下の通り培養した。
(a)l×l06/mLの単核細胞を、0日目に、AIM-V培地、HPL(濃度:2%v/v)、IL-2(濃度:60ng/mL)及びゾレドロン酸(濃度:5μM)を含む誘導性培養培地と接触させる。
(b)誘導性培養培地を、3日目に、AIM-V培地、HPL(濃度:4%v/v)及びIL-2(濃度:120ng/mL)を含む第1の培養培地と交換し、6日目に、回収し、細胞の半分を遠沈し、続いて細胞ペレットを、AIM-V培地、HPL及びIL-2を含む第1の培養培地で2×l06/mL細胞に再懸濁させる。
(c)9日目及び12日目に、工程(b)の細胞の半分を回収及び遠沈し、続いて細胞ペレットを、AIM-V培地、HPL(濃度:4%v/v)、IL-2(濃度:120ng/mL)及びIL-12(濃度:2ng/mL)を含む第2の培養培地で2×l06/mL細胞に再懸濁させる。
(d)15日目に、工程(c)の培養細胞の全てを収集する。
(a)l×l06/mLの単核細胞を、0日目に、AIM-V培地、HPL(濃度:2%v/v)、IL-2(濃度:60ng/mL)及びゾレドロン酸(濃度:5μM)を含む誘導性培養培地と接触させる。
(b)3日目に、工程(a)の細胞の半分を回収及び遠沈し、続いて細胞ペレットを、AIM-V培地、HPL(濃度:4%v/v)及びIL-2(濃度:120ng/mL)を含む第1の培養培地で2×l06/mL細胞に再懸濁させる。
(c)培地を、6日目に、AIM-V培地、HPL及びIL-2を含む第1の培養培地と交換する。
(d)9日目に、工程(c)の細胞の半分を回収及び遠沈し、続いて細胞ペレットを、AIM-V培地、HPL(濃度:4%v/v)、IL-2(濃度:120ng/mL)及びIL-12(濃度:2ng/mL)を含む第2の培養培地で2×l06/mL細胞に再懸濁させる。
(e)培地を、12日目に、AIM-V培地、HPL、IL-2及びIL-12を含む第2の培養培地と交換する。
(f)15日目に、工程(e)の培養細胞の全てを収集する。
(a)l×l06/mLの単核細胞を、0日目に、AIM-V培地、HPL(濃度:2%v/v)、IL-2(濃度:60ng/mL)及びゾレドロン酸(濃度:5μM)を含む誘導性培養培地と接触させる。
(b)3日目及び6日目に、工程(a)の細胞の半分を回収及び遠沈し、続いて細胞ペレットを、AIM-V培地、HPL(濃度:4%v/v)及びIL-2(濃度:120ng/mL)を含む第1の培養培地で2×l06/mL細胞に再懸濁させる。
(c)9日目に、工程(b)の細胞の半分を回収及び遠沈し、続いて細胞ペレットを、AIM-V培地、HPL(濃度:4%v/v)、IL-2(濃度:120ng/mL)及びIL-12(濃度:2ng/mL)を含む第2の培養培地で2×l06/mL細胞に再懸濁させる。
(d)12日目に、工程(c)の細胞の半分を回収及び遠沈し、続いて細胞ペレットを、AIM-V培地、HPL(濃度:4%v/v)、IL-2(濃度:120ng/mL)、IL-12(濃度:2ng/mL)及びIL-18(濃度:45ng/mL)を含む第3の培養培地で2×l06/mL細胞に再懸濁させる。
(e)15日目に、工程(d)の培養細胞の全てを収集する。
T細胞の機能性の決定
「死滅」アッセイ
実施例4からの改変T細胞の細胞傷害性の評価を、PanToxiluxキット(OncoImmunin, Inc.社)によって行った。ヒト慢性骨髄性白血病(CML)細胞株のK562は、標的細胞としての役割を果たし、最適な濃度下のTFL4で50分間染色した。TFL4標識化標的細胞及び改変T細胞とカスパーゼ基質との共培養は、37℃で20分である。細胞を、回収し、フローサイトメトリーによりTFL4+基質+のシグナルを分析した。カスパーゼに対する陽性細胞は、死滅を示す。
実施例4からの改変T細胞の抗原提示の活性の評価を混合リンパ球反応(MLR)によって行った。レスポンダー細胞(CD25- PBMC)を富化し、CellTrace(商標)Violet cell増殖キット(Invitrogen社)で染色した。CellTrace Violet標識化CD25- PBMC及び改変T細胞の共培養は、37℃で5日間である。hIL-2及びhIL-15を、1日目及び3日目に添加して、TCR会合の閾値を低減させた。次いで、細胞を、回収し、フローサイトメトリーによりCellTrace Violet希釈パターンを分析した。
Claims (25)
- CD3HiTCRγ9HiTCRδ2HiCXCR4Hiの表現型を含む改変T細胞。
- CD69DimCD11cHiCXCR3Dimの表現型を更に含む、請求項1に記載の改変T細胞。
- IFN-γHiの表現型を更に含む、請求項1に記載の改変T細胞。
- (a)請求項1に記載の改変T細胞;及び
(b)薬学的に許容される担体又は賦形剤
を含む医薬組成物。 - がん細胞を処置する方法であって、
有効量の請求項1に記載の改変T細胞を含む組成物を、それを必要とする対象に投与する工程
を含む、方法。 - 有効量が、用量あたり約l×103~約l×109個細胞である、請求項5に記載の方法。
- 改変T細胞が、自己又は同種異系である、請求項5に記載の方法。
- 改変T細胞が、末梢血、臍帯血又は骨髄に由来する、請求項5に記載の方法。
- 改変T細胞をインビトロで増殖させる工程を更に含む、請求項5に記載の方法。
- 改変T細胞を培養する方法であって、
単核細胞を含む体液を得る工程;
単核細胞を、IL-2及びゾレドロン酸を含む誘導性培養培地と接触させて、第1の培養細胞集団を得る工程;
第1の培養細胞集団を、IL-2を含む第1の培養培地と接触させて、第2の培養細胞集団を得る工程;並びに
第2の培養細胞集団を、IL-2及びIL-12を含む第2の培養培地と接触させて、第3の培養細胞集団を得る工程
を含む、方法。 - 単核細胞が、末梢血、臍帯血又は骨髄に由来する、請求項10に記載の方法。
- 誘導性培養培地、第1の培養培地及び第2の培養培地が、造血細胞培地を更に含む、請求項10に記載の方法。
- 造血細胞培地が、AIM-V培地を含む、請求項12に記載の方法。
- 誘導性培養培地、第1の培養培地及び第2の培養培地が、血清タンパク質を更に含む、請求項12に記載の方法。
- 血清タンパク質が、ヒト血小板溶解物を含む、請求項14に記載の方法。
- 単核細胞が、誘導性培養培地と約1~3日間接触する、請求項10に記載の方法。
- 第1の培養細胞集団が、第1の培養培地と約1~6日間接触する、請求項10に記載の方法。
- 第2の培養細胞集団が、第2の培養培地と約1~3日間接触する、請求項10に記載の方法。
- 第2の培養培地と接触させた後、第3の培養細胞集団を、IL-2、IL-12及びIL-18を含む第3の培養培地と接触させて、第4の培養細胞集団を得る工程を更に含む、請求項10に記載の方法。
- 第3の培養培地が、造血細胞培地を更に含む、請求項19に記載の方法。
- 造血細胞培地が、AIM-V培地を含む、請求項20に記載の方法。
- 第3の培養培地が、血清タンパク質を更に含む、請求項20に記載の方法。
- 血清タンパク質が、ヒト血小板溶解物を含む、請求項22に記載の方法。
- 第3の培養細胞集団が、第3の培養培地と約1~3日間接触する、請求項19に記載の方法。
- CD3HiTCRγ9HiTCRδ2HiCXCR4Hiの表現型を有する改変T細胞を単離する工程を更に含む、請求項10に記載の方法。
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