JP7173994B2 - リンパ球の製造方法 - Google Patents
リンパ球の製造方法 Download PDFInfo
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- JP7173994B2 JP7173994B2 JP2019567132A JP2019567132A JP7173994B2 JP 7173994 B2 JP7173994 B2 JP 7173994B2 JP 2019567132 A JP2019567132 A JP 2019567132A JP 2019567132 A JP2019567132 A JP 2019567132A JP 7173994 B2 JP7173994 B2 JP 7173994B2
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Description
[1]リンパ球を、
(a)ヒトフィブロネクチンのIII-1~3リピートを含む組換えポリペプチド、もしくは前記のIII-1~3リピートのアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入もしくは付加されたアミノ酸配列を含む組換えポリペプチド、
(b)ヒトフィブロネクチンのIII-8~10リピートを含む組換えポリペプチド、もしくは前記のIII-8~10リピートのアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入もしくは付加されたアミノ酸配列を含む組換えポリペプチド、及び
(c)ヒトフィブロネクチンのIII-12~14リピートを含む組換えポリペプチド、もしくは前記のIII-12~14リピートのアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入もしくは付加されたアミノ酸配列を含む組換えポリペプチド、の存在下で培養する工程を包含する、リンパ球の製造方法、
[2]リンパ球を、(a)、(b)及び(c)の組換えポリペプチドを同一分子内に含む組換えポリペプチドの存在下で培養する工程を包含する、[1]に記載の製造方法、
[3]組換えポリペプチドが、配列番号19に記載のアミノ酸配列を含む組換えポリペプチドであるか、あるいは配列番号19に記載のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入もしくは付加されたアミノ酸配列を含む組換えポリペプチドである、[2]に記載の製造方法、
[4]組換えポリペプチドが、配列番号31に記載のアミノ酸配列を含む組換えポリペプチドであるか、あるいは配列番号31に記載のアミノ酸配列において1もしくは数個のアミノ酸が置換、欠失、挿入もしくは付加されたアミノ酸配列を含む組換えポリペプチドである、[2]に記載の製造方法、
[5]組換えポリペプチドと抗CD3抗体の共存下でリンパ球の培養が実施される、[1]~[4]のいずれかに記載の製造方法、
[6]組換えポリペプチドの存在下でリンパ球を培養する工程が、組換えポリペプチドがコートされた固相とリンパ球とが接触した状態で実施される、[1]~[5]のいずれかに記載の製造方法、
[7]固相が、細胞培養用器材又は細胞培養用担体である、[6]に記載の製造方法、
[8]固相が、ディッシュ、プレート、フラスコ、バッグ、ビーズ、メンブレン又はスライドガラスである、[6]に記載の製造方法、
[9]リンパ球が、ヒト由来のリンパ球である、[1]~[8]のいずれかに記載の製造方法、
に関する。
<フィブロネクチン>
ヒト由来や哺乳動物由来のフィブロネクチンがよく研究されており、以下は、主にヒト由来血漿フィブロネクチンについての知見である。
本発明のリンパ球の製造方法は、リンパ球を組換えフィブロネクチンフラグメントであるポリペプチドの存在下で培養する工程を包含することを特徴とする。本明細書においてリンパ球とはリンパ球を含有する細胞集団を意味する。
メチオニン残基及び6個のヒスチジン残基から成るHis-tagをN末に有するFCH-296ポリペプチド(配列番号20)を、以下の操作により調製した。
以下の実施例で使用する培養器材に、抗ヒトCD3抗体(OKT3、タカラバイオ社製)及び実施例1で調製したHis-tag付きFCH-296を固定化した。固定化は24穴細胞培養プレート(ファルコン社製)に終濃度5μg/mLの抗ヒトCD3抗体と終濃度25μg/mLの上記His-tag付きFCH-296とを含むD-PBS(Promocell社製、C-40232)を0.4mL/ウェルずつ添加した後、37℃、5%CO2インキュベーターにて5時間以上静置して実施した。固定化プレートは使用直前に溶液を除去し、0.5mL/ウェルのD-PBSで2回洗浄した。また、対照として終濃度25μg/mLのCH-296(レトロネクチン:タカラバイオ社製)と終濃度5μg/mLの抗ヒトCD3抗体とを含むD-PBSでコートされたプレートも同様の方法で調製した。陰性対照にはコーティングをしていないプレートを用いた。
(1)細胞集団の拡大培養
インフォームド・コンセントの得られた健常人ドナー(TC0033)から常法に従い調製したヒト末梢血単核細胞(PBMC)を、終濃度200IU/mL IL-2(Proleukin、ニプロ社製)を添加したGT-T551培地(タカラバイオ社製、以下、GT-T551CMと称す)に1×105cells/mLになるように懸濁した。上記細胞を、2.8×105cells/ウェルでプレートにN=2で播種し、37℃、5%CO2で培養した(0日目)。
実施例3-(1)で得られた10日目の細胞集団を、0.1%ウシ血清アルブミン(シグマ社製)を含むPBS(以下、0.1%BSA/PBSと記載)で洗浄した。0.1%BSA/PBSに細胞集団を懸濁し、FITC標識マウス抗ヒトCD8抗体、RD-1標識マウス抗ヒトCD4抗体、PC-5標識マウス抗ヒトCD3抗体のカクテル抗体(ベックマンコールター社製)を添加し抗体反応を行った。その後、0.1%BSA/PBSで細胞集団を2回洗浄し、再度0.1%BSA/PBSに懸濁した。この細胞集団をフローサイトメトリー(FC-500、ベックマンコールター社製)に供し、各々の細胞集団のCD3陽性かつCD4陽性の細胞(CD3+CD4+)率を算出した。細胞表面マーカー測定の結果を表2に示す。なお、全細胞中のCD3陽性率はすべての試験区で94%以上であった。
(1)細胞集団の拡大培養
実施例3と同じ健常人ドナー(TC0033)及び異なる健常人ドナー(TC0071)から調製したPBMCを用いて、実施例3-(1)と同様の方法で細胞培養を行った。0日目の細胞数を1としたときの、4日目、7日目、及び10日目の細胞数を表3に示す。
実施例4-(1)で得られた10日目の細胞集団の細胞表面マーカーを、実施例3-(2)と同様の方法で測定した。結果を表4に示す。なお、全細胞中のCD3陽性率はすべての試験区で94%以上であった。
(1)細胞集団の拡大培養
健常人ドナー(TC0033及びTC0071)から調製したPBMCを、終濃度200IU/mL IL-2を添加したGT-T551CMに1×105cells/mLになるように懸濁した。上記細胞を、2.8×105cells/ウェルでプレートにN=2で播種し、37℃、5%CO2で培養した(0日目)。0日~4日目までの培養には、抗ヒトCD3抗体と、FCH-296ポリペプチド(His-tag付き、配列番号20)又は9アミノ酸をN末に挿入したFCH-296ポリペプチド(配列番号31)をコーティングしたプレートを用いた。細胞継代は4日目及び7日目に行った。4日目には、コーティングをしていない12穴細胞培養プレートに2.612mL/ウェルのGT-T551CMと0.358mL/ウェルの細胞懸濁液を混合して培養を継続した。7日目には、コーティングをしていない12穴細胞培養プレートに1.485mL/ウェルのGT-T551CMと1.485mL/ウェルの細胞懸濁液を混合して培養を継続した。陰性対照には、培養0日目から、コーティングをしていないプレートを用いた。なお、9アミノ酸をN末に挿入したFCH-296ポリペプチドは、SP Sepharose(登録商標) Fast Flow(GE Healthcare社製)等の通常のカラムを用いて常法により調製した。
10日目の細胞集団の細胞表面マーカーを、実施例3-(2)と同様の方法で測定した。結果を表5に示す。なお、全細胞中のCD3陽性率はすべての試験区で94%以上であった。
実施例5-(1)で得られた10日目の細胞集団を、0.1%ウシ血清アルブミン(シグマ社製)を含むPBS(以下、0.1%BSA/PBSと記載)で洗浄した。0.1%BSA/PBSに細胞集団を懸濁し、RD-1標識IgG1マウス抗ヒト2H4(CD45RA)抗体(ベックマンコールター社製)及びFITC標識IgG2Aマウス抗ヒトCCR7抗体(R&D社製)、を添加し抗体反応を行った。その後、0.1%BSA/PBSで細胞集団を2回洗浄し、再度0.1%BSA/PBSに懸濁した。この細胞集団をフローサイトメトリーに供し、各々の細胞集団のナイーブ細胞比率(CD45RA+CCR7+)を算出した。測定の結果を図2に示す。
様々なN末配列をもつFCH-296を調製した。すなわち、N末9アミノ酸欠失のFCH-296(配列番号25)、N末6アミノ酸欠失のFCH-296(配列番号26)、N末5アミノ酸欠失のFCH-296(配列番号27)、N末3アミノ酸欠失のFCH-296(配列番号28)、FCH-296(配列番号19)、N末3アミノ酸挿入のFCH-296(配列番号29)、N末6アミノ酸挿入のFCH-296(配列番号30)、N末11アミノ酸挿入のFCH-296(配列番号32)、N末12アミノ酸挿入のFCH-296(配列番号33)、N末14アミノ酸挿入のFCH-296(配列番号34)、N末15アミノ酸挿入のFCH-296(配列番号35)、N末HKRHEEGH挿入のFCH-296(配列番号36)、N末HKRH挿入のFCH-296(配列番号37)、N末HH挿入のFCH-296(配列番号38)、及びN末HHH挿入のFCH-296(配列番号39)を調製した。
SEQ ID NO:2 ; Partial region of fibronectin named III-2
SEQ ID NO:3 ; Partial region of fibronectin named III-3
SEQ ID NO:4 ; Partial region of fibronectin named III-4
SEQ ID NO:5 ; Partial region of fibronectin named III-5
SEQ ID NO:6 ; Partial region of fibronectin named III-6
SEQ ID NO:7 ; Partial region of fibronectin named III-7
SEQ ID NO:8 ; Partial region of fibronectin named III-8
SEQ ID NO:9 ; Partial region of fibronectin named III-9
SEQ ID NO:10 ; Partial region of fibronectin named III-10
SEQ ID NO:11 ; Partial region of fibronectin named III-11
SEQ ID NO:12 ; Partial region of fibronectin named III-12
SEQ ID NO:13 ; Partial region of fibronectin named III-13
SEQ ID NO:14 ; Partial region of fibronectin named III-14
SEQ ID NO:15 ; Partial region of fibronectin named CS-1
SEQ ID NO:16 ; Fibronectin fragment named 120k-fr
SEQ ID NO:17 ; Fibronectin fragment named CH-271
SEQ ID NO:18 ; Fibronectin fragment named CH-296 (RetroNectin)
SEQ ID NO:19 ; Fibronectin fragment named FCH-296
SEQ ID NO:20 ; His-tag FCH-296
SEQ ID NO:21 ; N-terminal 9a.a. deletion of III-1
SEQ ID NO:22 ; N-terminal 6a.a. deletion of III-1
SEQ ID NO:23 ; N-terminal 5a.a. deletion of III-1
SEQ ID NO:24 ; N-terminal 3a.a. deletion of III-1
SEQ ID NO:25 ; N-terminal 9a.a. deletion of FCH-296
SEQ ID NO:26 ; N-terminal 6a.a. deletion of FCH-296
SEQ ID NO:27 ; N-terminal 5a.a. deletion of FCH-296
SEQ ID NO:28 ; N-terminal 3a.a. deletion of FCH-296
SEQ ID NO:29 ; N-terminal 3a.a. insertion of FCH-296
SEQ ID NO:30 ; N-terminal 6a.a. insertion of FCH-296
SEQ ID NO:31 ; N-terminal 9a.a. insertion of FCH-296
SEQ ID NO:32 ; N-terminal 11a.a. insertion of FCH-296
SEQ ID NO:33 ; N-terminal 12a.a. insertion of FCH-296
SEQ ID NO:34 ; N-terminal 14a.a. insertion of FCH-296
SEQ ID NO:35 ; N-terminal 15a.a. insertion of FCH-296
SEQ ID NO:36 ; N-terminal HKRHEEGH insertion of FCH-296
SEQ ID NO:37 ; N-terminal HKRH insertion of FCH-296
SEQ ID NO:38 ; N-terminal HH insertion of FCH-296
SEQ ID NO:39 ; N-terminal HHH insertion of FCH-296
Claims (7)
- リンパ球を、配列番号19に記載のアミノ酸配列を含む組換えポリペプチド、あるいは配列番号19に記載のアミノ酸配列において1~10個のアミノ酸が置換、欠失、挿入もしくは付加されたアミノ酸配列を含む組換えポリペプチドであって、リンパ球を増殖させる機能、リンパ球の機能を維持する機能、またはリンパ球を誘導する機能を保持する組換えポリペプチド、の存在下で培養する工程を包含する、リンパ球の製造方法。
- リンパ球を 、配列番号31に記載のアミノ酸配列を含む組換えポリペプチド、あるいは配列番号31に記載のアミノ酸配列において1~10個のアミノ酸が置換、欠失、挿入もしくは付加されたアミノ酸配列を含む組換えポリペプチドであって、リンパ球を増殖させる機能、リンパ球の機能を維持する機能、またはリンパ球を誘導する機能を保持する組換えポリペプチド、の存在下で培養する工程を包含する、リンパ球の製造方法。
- 組換えポリペプチドと抗CD3抗体の共存下でリンパ球の培養が実施される、請求項1または2に記載の製造方法。
- 組換えポリペプチドの存在下でリンパ球を培養する工程が、組換えポリペプチドがコートされた固相とリンパ球とが接触した状態で実施される、請求項1~3のいずれか1項に記載の製造方法。
- 固相が、細胞培養用器材又は細胞培養用担体である、請求項4に記載の製造方法。
- 固相が、ディッシュ、プレート、フラスコ、バッグ、ビーズ、メンブレン又はスライドガラスである、請求項4に記載の製造方法。
- リンパ球が、ヒト由来のリンパ球である、請求項1~6のいずれか1項に記載の製造方法。
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