JP3951350B2 - Method for producing lymphocyte cells and immunotherapeutic agent - Google Patents
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- JP3951350B2 JP3951350B2 JP10159397A JP10159397A JP3951350B2 JP 3951350 B2 JP3951350 B2 JP 3951350B2 JP 10159397 A JP10159397 A JP 10159397A JP 10159397 A JP10159397 A JP 10159397A JP 3951350 B2 JP3951350 B2 JP 3951350B2
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Description
【0001】
【発明の属する技術分野】
本発明は、腫瘍細胞に細胞毒性(cytotoxicity)を有するリンパ球細胞亜集団、特にCD4+T細胞、CD8+T細胞、CD16+NK細胞及び/又はガンマー・デルタT細胞を、選択的に増殖し製造する方法並びに養子免疫療法に有用な免疫治療剤に関する。
【0002】
【従来の技術】
過去、癌治療を目的として、様々な免疫療法が試みられてきた。代表的な方法として、一つは、癌細胞に対する担癌患者自身の宿主免疫反応を高めることを期待して、特異的又は非特異的な免疫活性化物質が投与された。ある種、例えばインターフェロン、インターロイキン2(IL−2)、インターロイキン12(IL−12)等のサイトカイン類、PSK、OK−432、レンチナン等の免疫賦活物質の実験では癌治療に応用できる可能性が示唆されたが、目的の腫瘍抗原に対する免疫反応が強く誘導できないこと、基本的に低下している癌患者の免疫能を期待ほど高めることが出来ないこと、さらに投与された物質そのものの副作用が問題になったこと等の理由で、未だに確かな成功をみた例はない。
【0003】
他は、免疫細胞を体外に分離して刺激し免疫能を高め再び生体内に移入する方法で、免疫反応の低下している癌宿主の免疫反応に関わり無く直接的又は間接的に癌細胞を障害することが予想され、臨床試験が試みられ、ある程度の効果が確認された。このような治療に用いられる免疫的に活性のある細胞としては、リンフォカイン活性化キラー(LAK)細胞、腫瘍内浸潤リンパ球(TIL)細胞などが代表としてあげられる。
【0004】
特開昭62−116518号公報には、細胞セパレータを用いて患者の血液を4時間以内に10〜20リットル処理して、1回あたり約5×105〜5×1010個の単核球を集め、フィコール・ハイパックでリンパ球を分離し、IL−2で刺激してLAK細胞を得、最後に大量のIL−2と共にLAK細胞を患者に投与することが記載されている。その結果、一部の患者又は一部の癌種において、転移の部分的退縮等の効果が確認されている。しかし一方で、投与患者の大多数に発熱、悪寒、吐き気、嘔吐、呼吸困難、倦怠、紅班または皮膚発疹等のような比較的重篤な副作用も報告された。これらの方法の一つの大きな欠点は、治療に使用する細胞を大量に必要とすること、刺激後のリンパ球と同時に刺激物質を大量に投与することによる重篤な副作用が発現することであった。
【0005】
また特開平3−80076号公報では、少量のリンパ球を培養器に固相化した抗CD3抗体で刺激することにより、さらに抗CD3抗体とIL−2で刺激することにより、細胞数が出発したときの約100〜10000倍に増殖することが報告されている。また、これらのリンパ球と腫瘍を増大させる宿主の免疫を抑制するための薬剤を同時に投与した臨床試験を行っており、リンパ球を投与した患者の全てで腫瘍が50%以上縮小したことを報告している。しかしながら、この方法もリンパ球と同時に免疫を抑制する薬剤及び/又はIL−2を投与しているため、前記の方法と同様、副作用が発現することが類推される。
【0006】
インターロイキン15(IL−15)は、T細胞増殖因子として発見された新しいサイトカインであり(WO95/27722号明細書)、T細胞、NK細胞を活性化することが知られている。しかし、IL−2とは異なり、リンパ球系の細胞からは産生されず、むしろ単球内皮細胞、平滑筋細胞等の非リンパ球系の細胞から産生されることを特徴とする。また、T細胞以外の例えば血管内皮細胞などにも結合能を有する。IL−15は、IL−15受容体α鎖と共にIL−2受容体β及びγ鎖を介して情報伝達することもわかっている。しかしながら、IL−15のリンパ球に対する作用効果のIL−2との差異は明確になっていない。
【0007】
これに関連し、マウスに予めIL−2又はIL−15をその腹腔内に投与し、続いて125I標識牛血清アルブミン(BSA)を静脈内に投与すると、IL−2投与群で肺組織内の放射活性がIL−15の6倍に有意に上昇し、さらに、肺重量も有意に増加したことが報告され、これは、IL−2投与により血管漏出症候群(Vascular Leak Syndrome:VLS)及び肺浮腫が惹起されたことによることが確認されている(William Munger et al., Cellular Immunology,165巻、289〜293頁、1995年)。従って、IL−2は、IL−15より毒性が6倍高く、これは、ヒトの臨床試験で確認されている副作用の問題と関連があると思われる。
【0008】
【発明が解決しようとする課題】
請求項1及び2記載の発明は、毒性の低いIL−15を用いて、腫瘍細胞に対して細胞毒性を有するリンパ球を大量に増殖するリンパ球細胞の製造法を提供するものである。
また、請求項3記載の発明は、癌、感染症等の治療に用いられる養子免疫療法に好適な免疫治療剤を提供するものである。
【0009】
【課題を解決するための手段】
本発明は、リンパ球細胞を、抗CD3抗体及びインターロイキン15の存在下に培養することを特徴とするリンパ球細胞の製造法に関する。
また本発明は、リンパ球細胞を、固相化した抗CD3抗体及びインターロイキン15の存在下に培養する第1期培養工程、固相化した抗CD3抗体の非存在下かつインターロイキン15の存在下に培養する第2期培養工程の各工程を含むことを特徴とするリンパ球細胞の製造法に関する。
さらに本発明は、前記の製造法により得られたリンパ球細胞を含有してなる免疫治療剤に関する。
【0010】
【発明の実施の形態】
本発明で用いるリンパ球細胞としては、末梢血リンパ球、上皮性リンパ球、腫瘍内浸潤リンパ球、癌性腹水胸水浸潤リンパ球など生体に存在するあらゆるリンパ球が挙げられ、特に制限はされないが、末梢血リンパ球、特に採血直後の新鮮末梢血リンパ球が、採血患者への身体的負担、リンパ球分離の簡便さ、分離後のリンパ球の生存率の良さ等の点で好ましい。細胞の数としては、1×106〜50×106個程度であるのが好ましく、血液量では10〜20mlであるのが、量的に問題なく培養でき、また採血時の患者への負担も軽いので好ましい。
【0011】
上記リンパ球細胞は、抗CD3抗体の存在下に培養する。
ここでCD3とは、Tリンパ球の細胞表面に存在し、T細胞受容体(TCR)と共に標的抗原を認識する際に重要な分子である。CD3分子を介して刺激情報が細胞内に伝達されることにより、T細胞は増殖を開始する。同様な現象が、CD3分子に対する特異的抗体(抗CD3抗体)の刺激により起こる。
リンパ球細胞の刺激に用いる抗CD3抗体は、精製したCD3分子を用いて動物又は細胞に産生させることもできるが、安定性、コスト等に優れた市販品を用いることもできる。
【0012】
抗CD3抗体は固相化して用いるのが、増殖の効果が高いので好ましい。固相化には、抗CD3抗体を滅菌したリン酸緩衝液で1〜10μg/mlの濃度に希釈して用いるのが好ましい。抗体を固相化する器具としては、ポリスチレン等のプラスチック製の減菌済み細胞培養フラスコ等が好ましく、その大きさは適宜選択できる。固相化方法としては、前記抗CD3抗体の希釈液を固相化する器具に加え、2〜24時間、4〜37℃で静置する方法が好ましい。固相化後、使用時まで冷蔵庫(4℃)で保存することが好ましい。使用時には、液を除去し、好ましくは常温の、リン酸緩衝液等で洗浄する。
【0013】
また、本発明では培養にインターロイキン15(IL−15)を用いる。IL−15は、培養中1〜1000ng/mlの濃度となるように用いるのが好ましい。IL−15は市販されているものを用いることができる。IL−15は、水、生理食塩液、リン酸緩衝液、RPMI−1640、DMEM、IMDM、AIM−V等の一般に広く用いられる細胞培養液等に溶解して使用することができる。一度溶解したものは、活性の低下を防ぐため、冷蔵保存することが好ましい。
【0014】
リンパ球細胞の培養は、IL−15を含む培養液にリンパ球細胞を浮遊させ、抗CD3抗体を固相化した培養容器に入れ培養を開始するのが好ましい。培養液としては、リンパ球の培養に適したものであれば特に制限されず、血清等の生物由来の培養液、平衡塩類溶液にアミノ酸、ビタミン、核酸塩基などを加え、さらに必要に応じてウシ胎児血清やウシ血清アルブミンを加えた合成培地などが使用でき、RPMI−1640、AIM−V、DMEM、IMDM等が好ましいものとして挙げられ、RPMI−1640が特に好ましいものとして挙げられる。培養液は、正常ヒト血清を添加したものが増殖効果に優れ好ましい。
【0015】
これらの培地は市販品を用いることができる。培養は、一般的な細胞培養の方法に従うことができる。例えば、CO2インキュベータ内で行うことができる。CO2濃度は1〜10%、特に5%が好ましく、温度は、30〜40℃、特に37℃が好ましい。この培養は、日数に特に制限はないが、抗CD3抗体の刺激情報が細胞に伝達されることが前提となるため、2〜20日行うことが好ましく、3〜7日行うことが好ましい。この培養の期間内には、顕微鏡下で細胞の状態を観察し、適宜細胞数を計測しながら、培養液を適宜添加するのが好ましい。この培養では、通常、培養開始1〜2日目には目立った細胞の増殖はないが、3日目頃から細胞増殖が観察され、順調に増殖し出すと培養液がオレンジ色から黄色に変化するようになる。培養液の添加量は、添加前の培養中の液の液量に対して0.1〜5倍程度が好ましい。その添加割合は、培養液の劣化及びIL−15活性の低下を防ぐために、1〜7日に1回行うことが好ましい。
【0016】
本発明においては、上述の培養を第1期培養工程とし、固相化した抗CD3抗体の非存在下かつIL−15の存在下に培養する第2期培養工程を設けることが、リンパ球を大量に増殖・維持できるので好ましい。抗CD3抗体を固相化した培養容器で長期間培養を行うと、リンパ球の増殖が抑制されることがある。
第2期培養に用いる培養容器としては、細胞培養用のプラスチック製フラスコ、CO2ガス透過性のガス・パーミアブル・バッグ等を使用することができるが 、この工程ではリンパ球が急速に大量に増殖してくるため、2〜4日毎に培養器の数を増やしていくことが好ましい。細胞数の目安としては、5×105〜10×105個/mlになるように培養液の追加又は培養器の数の増加を行っていくことが好ましい。
【0017】
本発明の製造法によれば、主に、CD4+T細胞、CD8+T細胞、ガンマー・デルタT細胞及び/又はCD16+NK細胞が増殖してくる。さらに、これらの細胞の少なくとも一つは、腫瘍細胞に対して細胞障害性(cytotoxicity)を示す。つまり、新しいサイトカインであるIL−15の刺激により、少量のリンパ球から標的細胞を障害する活性を有するリンパ球集団が効率的かつ大量に誘導できる。
【0018】
ここでCD4+T細胞は、従来ヘルパー/インデューサーT細胞と言われ、標的となる細胞の組織適合性抗原(MHC)クラスII分子と抗原分子を認識する細胞である。また、CD8+T細胞は、従来キラー/サプレッサーT細胞と言われ、標的となる細胞の組織適合性抗原(MHC)クラスI分子と抗原分子を認識して攻撃する細胞である。また、CD16+NK(ナチュラルキラー)細胞は、表面抗原にCD16分子を発現している細胞で、標的細胞の組織適合性抗原に拘束される事無く標的細胞を攻撃する能力を有する細胞である。また、ガンマー・デルタT細胞は、少なくともCD3+CD16−の表面抗原を有し、T細胞受容体ガンマー・デルタ鎖を発現している細胞である。
【0019】
さらに、本発明の製造法により得られるリンパ球細胞は、IL−2受容体及びIL−12受容体を高発現している。リンパ球などの細胞が、サイトカインなどの細胞刺激物質の刺激情報を受けて細胞内に情報を伝達するには、それぞれの物質に特異的な受容体で情報を受け取る必要がある。つまり、細胞表面に受容体が存在すれば、その受容体に特異的に結合する物質の刺激を受け取ることが出来る。本発明により培養したリンパ球が、IL−2受容体及びIL−12受容体を高発現しているということは、本リンパ球がIL−15の刺激以外に少なくともIL−2及びIL−12に刺激を受け取り活性化されることを示しており、IL−15とこれらのサイトカインとの相加または相乗効果も期待される。さらに、IL−2受容体の一部を共有するIL−4、IL−7、IL−9等によっても同様の結果が得られる可能性を示している。従って、本発明により製造したリンパ球細胞は癌、各種感染症等の治療に有用な養子免疫療法に用いる免疫治療剤の有効成分とすることができる。また、研究用試薬として、種々の研究用途に用いることができる。
【0020】
免疫治療剤とする場合、その投与量は、1回あたりの細胞の量で106〜1012個の細胞が好ましい。投与形態としては、注射剤、点滴剤等の液体が好ましく 、前記細胞をヒト血清アルブミンを0.01〜5%となるように添加した生理食塩液に分散した注射剤又は点滴剤がより好ましい。投与方法としては、静脈への点滴又は静脈、動脈、局所等への注射が好ましい。投与する液量は、投与方法、投与する場所等に異なるが、50〜500ccとするのが好ましく、この液量に前記の量の細胞が含まれるようにするのが好ましい。投与頻度は1回/日〜1回/月とするのが好ましく、投与回数は少なくとも1回、好ましくは5回以上である。
【0021】
【実施例】
(1)抗CD3抗体固相化培養フラスコの調製
抗CD3抗体(OKT3,オルソ ファーマシューティカル コーポレイション製)をリン酸緩衝食塩水溶液(PBS)で5μg/mlの濃度に希釈し、表面積25cm2のフラスコに5ml注入した。この溶液をフラスコの底面にまんべんなく広げ、冷蔵庫(4℃)で一晩以上、使用時まで静置した。溶液を注入したフラスコは、使用前に常温のPBSで3回洗浄した。
【0022】
(2)IL−15を含む培養液(培地)の調整
RPMI−1640培地(シグマ社製)に、10%(体積/体積)ヒト正常血清(新鮮凍結血漿を37℃で解凍後、56℃、30分間加熱して非働化し、18000rpm、60分、4℃の遠心により沈殿物を除去し、さらに0.22μmのフィルターで濾過したもの)、100U/mlペニシリン(明治製菓株式会社製)、100γ/mlストレプトマイシン(明治製菓株式会社製)及びIL−15(イムノテック社製)50ng/mlを含むように調整した。
【0023】
(3)リンパ球の培養
ヘパリン処理をした20mlの注射器を用いて採血したヒト末梢血全血20mlに生理食塩液20mlを加え2倍に希釈し、密度勾配遠心分離の媒体であるFicoll-Paque(ファルマシア社製)4mlに希釈血液10mlを重層して1600rpm、25分、20℃の条件で遠心を行いリンパ球層を回収し、RPMI−1640培地で3回洗浄した。約2×107個のリンパ球が得られた。前記のIL−15を含む培養液中に細胞密度約2×106個/mlで懸濁させ、この細胞懸濁液約5mlを前記抗CD3抗体固相化培養フラスコ中に加えて培養を開始した。翌日(培養1日目)からIL−15を含む培養液を1ml添加し、この操作を培養4日目まで繰り返した。培養5日目に抗CD3抗体を固定化していない75cm2のフラスコに細胞浮遊液を移し、IL−15を含む培養液を約10ml添加した。これ以後、フラスコ1本当たりの細胞浮遊液の量は約30mlを目安とし、培養液のオレンジ色から黄色への色の変化、及び細胞数の増加(1〜10×105個/mlを目安)に応じて新しいフラスコに基本的に2分割して本数を増やし、IL−15を含む培地を約10ml添加した。この操作を繰り返し、リンパ球集団を8日間培養した。
【0024】
(4)培養リンパ球の表現型の測定
上記により得られた培養リンパ球5×105個を試験管に分取し、RPMI−1640培地を加え1回遠心後上清を除去して、直接染色の場合FITC(フルオレセインイソチオシアネート)またはPE(フィコエリスリン)標識モノクローナル抗体10μlを、間接染色の場合非標識モノクローナル抗体1μgをそれぞれ加え良く撹拌した。氷中で30分間静置の後、RPMI−1640培地で2回遠心洗浄した。間接染色の場合、次に適当に希釈したFITC標識ヒツジ抗マウス抗体又は抗ラット抗体(カッペル社製)10μlを加え氷中で30分間静置の後、RPMI−1640培地で2回遠心洗浄した。最後に0.5mlのRPMI−1640培地を加え、良く撹拌し、FACScan(ベクトン・デッキンソン社)で各抗体に反応した表面抗原を測定した。
【0025】
測定に用いたモノクローナル抗体は、直接染色には、FITC標識抗CD3抗体、FITC標識抗CD16抗体、FITC標識抗TCR−α/β抗体、FITC標識抗TCR−γ/δ抗体、FITC標識抗CD4+PE標識抗CD8抗体、PE標識抗CD56抗体(以上ベクトン・ディッキンソン社製)、間接染色には、ラット抗IL−2受容体α鎖抗体(抗CD25、セロテック社製)、マウス抗IL−2受容体β鎖抗体(Mik−β1、ニチレイ(株)製)、マウス抗IL−2受容体γ鎖抗体(AG184、ファーミンジェン社製)及びマウス抗IL−12受容体β1抗体(ホフマン・ラ・ロッシュ社製)及びFITC標識抗ラットIgG又はFITC標識抗マウスIgG(以上カッペル社製)を用いた。結果を表1に示す。
【0026】
【表1】
【0027】
表1のデータから示されることは、得られたリンパ球細胞の表現型はほとんどがCD3+CD4+及びCD3+CD8+のT細胞であり、T細胞受容体アルファー・ベータ鎖を有していることである。また、他に、少数のT細胞受容体ガンマー・デルタ鎖を有するT細胞及びCD16+NK細胞が存在した。また、これらのリンパ球は、IL−2受容体α鎖、β鎖、γ鎖及びIL−12受容体を強く発現しており、IL−15以外に例えばIL−2、IL−4、IL−7、IL−9等のIL−2受容体の一部を共有して情報を伝達するサイトカイン、及びIL−12等のIL−12受容体を介して情報を伝達するサイトカイン等の刺激により強く活性化されるであろう事は想像できる。つまり、IL−15とこれらサイトカインとの組み合わせによる、リンパ球活性化の相加または相乗効果が期待される。
【0028】
(5)培養腫瘍細胞に対する障害活性の測定
効果細胞として、培養して得られたリンパ球を、10%(体積/体積)牛胎児血清(FBS)添加RPMI−1640培地で3回洗浄し、同じ培地に浮遊させ、細胞数を数えた後96穴U底プレート(ファルコン社製)の最上段に200μlづつ播種し、以下2段目以降2倍づつ希釈して希釈段階を作製した。続いて、51Cr(ICN社製)を2時間標識した標的細胞(培養癌細胞株K562,Daudi及びRaji細胞)1×105個/mlを100μl(1×104個)づつ播種してリンパ球と混合し、5%CO2、37℃で4時間培養した。培養終了後、上清100μlを採取し、ガンマーカウンター(パッカード社製)で遊離した51Crの放射活性(cpm、試験遊離群)を測定した。細胞障害率(%)は、以下の式で算出した。
【数1】
なお、51Cr標識腫瘍細胞に3%トリトンX−100を加え細胞を溶解したものを最大遊離群、51Cr標識腫瘍細胞単独を自然遊離群とした。
【0029】
結果を図1に示す。これらリンパ球は、図1に示したように株化腫瘍細胞を効果的に障害したことから、養子免疫療法に用いるための効果的な細胞群であることが示された。
【0030】
(6)免疫治療剤の調整及びその使用方法
免疫治療剤
上記の方法で癌患者由来のリンパ球細胞を増殖して製造し、これを市販の分離剤(Lymphoprep,Nycomed社製)を用いて、そのプロトコルに従って分離精製する。得られる細胞2×109個を、ヒト血清アルブミン0.1%を含む生理食塩液200mlに懸濁して、点滴剤形態の免疫治療剤とすることができる。
免疫治療剤の使用方法
上記注射剤の全量を、癌患者に点滴静注する。
この操作を月1回の割合で繰り返すことにより、癌患者の癌を治癒したり、症状の進行の停止又は鈍化を図ることができる。
【0031】
【発明の効果】
請求項1及び2記載のリンパ球細胞の製造法によれば、養子免疫療法で用いるために十分な数と、腫瘍細胞を障害する活性を有する活性化リンパ球が少量の全血から出発して効率よく得られる。
請求項3記載の免疫治療剤は、腫瘍細胞を障害する活性を有する活性化リンパ球を多く含み、養子免疫療法に有用なものである。さらに、他のサイトカイン等の免疫刺激物質と組み合わせることにより、さらに活性化したリンパ球が効率よく得られるであろう事が示唆される。
【図面の簡単な説明】
【図1】本発明の製造法により得られたリンパ球(効果細胞)の癌細胞株(標的細胞)に対する細胞障害活性(Cytotoxicity)を示したグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for selectively proliferating and producing lymphocyte cell subpopulations having cytotoxicity to tumor cells, in particular CD4 + T cells, CD8 + T cells, CD16 + NK cells and / or gamma delta T cells, and adoptive immunization. The present invention relates to an immunotherapeutic agent useful for therapy.
[0002]
[Prior art]
In the past, various immunotherapy has been tried for the purpose of cancer treatment. As a typical method, one is a specific or non-specific immune activator administered in the hope of enhancing the host immune response of a cancer-bearing patient to cancer cells. Possibility that it can be applied to cancer treatment in certain types of experiments such as cytokines such as interferon, interleukin 2 (IL-2) and interleukin 12 (IL-12), and immunostimulatory substances such as PSK, OK-432 and lentinan It was suggested, however, that the immune response against the target tumor antigen cannot be strongly induced, that the immune ability of cancer patients who have basically declined cannot be increased as expected, and that the administered substance itself has side effects. There are no examples of solid success yet due to problems.
[0003]
The other is a method in which immune cells are isolated and stimulated outside the body to enhance immunity and transfer again into the body, directly or indirectly regardless of the immune response of the cancer host in which the immune response has decreased. Expected to be impaired, clinical trials were attempted and some effect was confirmed. Representative examples of the immunologically active cells used for such treatment include lymphokine-activated killer (LAK) cells and intratumoral infiltrating lymphocyte (TIL) cells.
[0004]
In Japanese Patent Application Laid-Open No. Sho 62-116518, 10-20 liters of a patient's blood is treated within 4 hours using a cell separator, and about 5 × 10 5 to 5 × 10 10 mononuclear cells are collected each time. , Separating lymphocytes with Ficoll Hipack, stimulating with IL-2 to obtain LAK cells, and finally administering LAK cells to patients with large amounts of IL-2. As a result, effects such as partial regression of metastasis have been confirmed in some patients or some cancer types. However, on the other hand, relatively serious side effects such as fever, chills, nausea, vomiting, dyspnea, malaise, erythema or skin rash were also reported to the majority of patients. One major drawback of these methods is that they require a large amount of cells to be used for treatment, and that serious side effects are caused by the administration of a large amount of stimulating substances simultaneously with the stimulated lymphocytes. .
[0005]
In JP-A-3-80076, the number of cells started by stimulating a small amount of lymphocytes with an anti-CD3 antibody immobilized on an incubator and further stimulating with an anti-CD3 antibody and IL-2. It has been reported to grow about 100 to 10000 times greater. In addition, we are conducting clinical trials in which these lymphocytes and a drug that suppresses host immunity that increases tumors are administered simultaneously, and report that all patients receiving lymphocytes have contracted tumors by 50% or more. is doing. However, since this method also administers a drug that suppresses immunity and / or IL-2 at the same time as lymphocytes, it is presumed that side effects will occur as in the above method.
[0006]
Interleukin 15 (IL-15) is a new cytokine discovered as a T cell growth factor (WO95 / 27722) and is known to activate T cells and NK cells. However, unlike IL-2, it is not produced from lymphocyte cells, but rather produced from non-lymphocyte cells such as monocyte endothelial cells and smooth muscle cells. In addition, it has binding ability to other than T cells such as vascular endothelial cells. IL-15 has also been shown to signal through the IL-2 receptor β and γ chains along with the IL-15 receptor α chain. However, the difference in the effect of IL-15 on lymphocytes from IL-2 is not clear.
[0007]
In this context, mice were pre-administered with IL-2 or IL-15 intraperitoneally, followed by intravenous administration of 125I-labeled bovine serum albumin (BSA), which resulted in intrapulmonary tissue in the IL-2 administration group. It was reported that the radioactivity was significantly increased 6-fold that of IL-15, and that the lung weight was also significantly increased. This was because IL-2 administration caused Vascular Leak Syndrome (VLS) and pulmonary edema. (William Munger et al., Cellular Immunology, 165, 289-293, 1995). Therefore, IL-2 is 6 times more toxic than IL-15, which seems to be related to the side effect problem identified in human clinical trials.
[0008]
[Problems to be solved by the invention]
The inventions according to claims 1 and 2 provide a method for producing lymphocyte cells that proliferate a large amount of lymphocytes having cytotoxicity against tumor cells using IL-15 having low toxicity.
The invention described in claim 3 provides an immunotherapeutic agent suitable for adoptive immunotherapy used for the treatment of cancer, infectious diseases and the like.
[0009]
[Means for Solving the Problems]
The present invention relates to a method for producing lymphocyte cells, which comprises culturing lymphocyte cells in the presence of an anti-CD3 antibody and interleukin 15.
The present invention also provides a first phase culture step in which lymphocyte cells are cultured in the presence of an immobilized anti-CD3 antibody and interleukin 15, the absence of the immobilized anti-CD3 antibody and the presence of interleukin 15. The present invention relates to a method for producing lymphocyte cells, comprising each step of a second phase culture step to be cultured below.
Furthermore, the present invention relates to an immunotherapeutic agent comprising lymphocyte cells obtained by the above production method.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
Examples of lymphocyte cells used in the present invention include all lymphocytes present in a living body such as peripheral blood lymphocytes, epithelial lymphocytes, intratumoral infiltrating lymphocytes, and cancerous ascites pleural effusion infiltrating lymphocytes. Peripheral blood lymphocytes, particularly fresh peripheral blood lymphocytes immediately after blood collection, are preferable in terms of physical burden on blood collection patients, ease of lymphocyte separation, good survival rate of lymphocytes after separation, and the like. The number of cells is preferably about 1 × 10 6 to 50 × 10 6 , and the blood volume is 10 to 20 ml, which can be cultivated without any problem in quantity, and the burden on the patient at the time of blood collection Is also preferable because it is light.
[0011]
The lymphocyte cells are cultured in the presence of an anti-CD3 antibody.
Here, CD3 is a molecule that exists on the cell surface of T lymphocytes and recognizes a target antigen together with a T cell receptor (TCR). T cells start to proliferate when stimulation information is transmitted into the cell via the CD3 molecule. A similar phenomenon occurs upon stimulation of a specific antibody (anti-CD3 antibody) against the CD3 molecule.
The anti-CD3 antibody used for the stimulation of lymphocyte cells can be produced in animals or cells using purified CD3 molecules, but commercially available products having excellent stability and cost can also be used.
[0012]
It is preferable to use the anti-CD3 antibody in a solid phase because of its high proliferation effect. For immobilization, the anti-CD3 antibody is preferably diluted with a sterilized phosphate buffer to a concentration of 1 to 10 μg / ml. The instrument for immobilizing the antibody is preferably a plastic sterilized cell culture flask or the like made of polystyrene or the like, and its size can be appropriately selected. As the solid phase immobilization method, a method of adding the anti-CD3 antibody diluted solution to the solid phase immobilization apparatus and allowing to stand at 4 to 37 ° C. for 2 to 24 hours is preferable. After solid-phase formation, it is preferably stored in a refrigerator (4 ° C.) until use. At the time of use, the solution is removed and washed with a phosphate buffer solution or the like, preferably at room temperature.
[0013]
In the present invention, interleukin 15 (IL-15) is used for the culture. IL-15 is preferably used at a concentration of 1-1000 ng / ml during the culture. What is marketed can be used for IL-15. IL-15 can be used by dissolving in generally used cell culture media such as water, physiological saline, phosphate buffer, RPMI-1640, DMEM, IMDM, AIM-V and the like. Once dissolved, it is preferably stored refrigerated to prevent a decrease in activity.
[0014]
The lymphocyte cells are preferably cultured by suspending the lymphocyte cells in a culture solution containing IL-15 and placing the cells in a culture vessel in which an anti-CD3 antibody is solid-phased. The culture solution is not particularly limited as long as it is suitable for lymphocyte culture, and amino acids, vitamins, nucleobases, etc. are added to a culture solution derived from a living organism such as serum or a balanced salt solution, and if necessary, bovine. A synthetic medium to which fetal serum or bovine serum albumin is added can be used, and RPMI-1640, AIM-V, DMEM, IMDM and the like are preferable, and RPMI-1640 is particularly preferable. A culture medium to which normal human serum is added is preferable because of its excellent proliferation effect.
[0015]
Commercially available products can be used for these media. The culture can follow general cell culture methods. For example, it can be performed in a CO2 incubator. The CO 2 concentration is preferably 1 to 10%, particularly 5%, and the temperature is preferably 30 to 40 ° C, particularly 37 ° C. Although this culture is not particularly limited in the number of days, it is preferably performed for 2 to 20 days, and preferably for 3 to 7 days, since it is premised on the stimulation information of the anti-CD3 antibody being transmitted to the cells. During the culture period, it is preferable to appropriately add a culture solution while observing the state of the cells under a microscope and measuring the number of cells as appropriate. In this culture, there is usually no noticeable cell growth on the first or second day of the culture, but cell growth is observed from the third day, and when the culture starts to grow smoothly, the culture solution changes from orange to yellow. To come. The addition amount of the culture solution is preferably about 0.1 to 5 times the amount of the solution during the culture before the addition. In order to prevent the deterioration of the culture solution and the decrease in IL-15 activity, the addition ratio is preferably performed once every 1 to 7 days.
[0016]
In the present invention, the above-mentioned culture is used as a first phase culture step, and a second phase culture step of culturing in the absence of immobilized anti-CD3 antibody and in the presence of IL-15 is provided. It is preferable because it can be proliferated and maintained in large quantities. When culturing for a long time in a culture vessel in which an anti-CD3 antibody is immobilized, lymphocyte proliferation may be suppressed.
As the culture vessel used for the second phase culture, a plastic flask for cell culture, a CO 2 gas permeable gas permeable bag, etc. can be used. In this process, lymphocytes grow rapidly and in large quantities. Therefore, it is preferable to increase the number of incubators every 2 to 4 days. As a standard of the number of cells, it is preferable to add a culture solution or increase the number of incubators so as to be 5 × 10 5 to 10 × 10 5 cells / ml.
[0017]
According to the production method of the present invention, CD4 + T cells, CD8 + T cells, gamma delta T cells and / or CD16 + NK cells mainly proliferate. In addition, at least one of these cells is cytotoxic to tumor cells. That is, by stimulating IL-15, which is a new cytokine, a lymphocyte population having an activity of damaging target cells from a small amount of lymphocytes can be induced efficiently and in large quantities.
[0018]
Here, CD4 + T cells are conventionally referred to as helper / inducer T cells, and are cells that recognize histocompatibility antigen (MHC) class II molecules and antigen molecules of target cells. CD8 + T cells are conventionally referred to as killer / suppressor T cells, and are cells that recognize and attack histocompatibility antigen (MHC) class I molecules and antigen molecules of target cells. A CD16 + NK (natural killer) cell is a cell that expresses a CD16 molecule as a surface antigen and has the ability to attack the target cell without being restricted by the target cell's histocompatibility antigen. A gamma delta T cell is a cell having at least a CD3 + CD16− surface antigen and expressing a T cell receptor gamma delta chain.
[0019]
Furthermore, lymphocyte cells obtained by the production method of the present invention highly express IL-2 receptor and IL-12 receptor. In order for cells such as lymphocytes to receive stimulation information from a cell stimulating substance such as a cytokine and transmit information into the cell, it is necessary to receive information through a receptor specific to each substance. In other words, if a receptor exists on the cell surface, a stimulus of a substance that specifically binds to the receptor can be received. The fact that the lymphocytes cultured according to the present invention highly express IL-2 receptor and IL-12 receptor means that this lymphocyte is at least IL-2 and IL-12 in addition to stimulation by IL-15. It has been shown to be stimulated and activated, and an additive or synergistic effect between IL-15 and these cytokines is also expected. Furthermore, it is possible that similar results can be obtained by IL-4, IL-7, IL-9 and the like sharing a part of the IL-2 receptor. Therefore, the lymphocyte cells produced according to the present invention can be used as an active ingredient of an immunotherapeutic agent used in adoptive immunotherapy useful for the treatment of cancer, various infectious diseases and the like. Moreover, it can be used for various research uses as a research reagent.
[0020]
When used as an immunotherapeutic agent, the dose is preferably 10 6 to 10 12 cells per cell. The dosage form is preferably a liquid such as an injection or infusion, and more preferably an injection or infusion in which the cells are dispersed in a physiological saline solution to which human serum albumin is added at 0.01 to 5%. As the administration method, intravenous drip or injection into veins, arteries, and the like is preferable. The amount of the liquid to be administered varies depending on the administration method, the place to administer, etc., but is preferably 50 to 500 cc, and it is preferable that the amount of cells is included in this liquid amount. The frequency of administration is preferably 1 time / day to 1 time / month, and the frequency of administration is at least once, preferably 5 times or more.
[0021]
【Example】
(1) Preparation of anti-CD3 antibody-immobilized culture flask Anti-CD3 antibody (OKT3, manufactured by Ortho Pharmaceutical Corporation) was diluted with phosphate buffered saline solution (PBS) to a concentration of 5 μg / ml, and a flask with a surface area of 25 cm 2 . 5 ml was injected. This solution was spread evenly on the bottom of the flask and allowed to stand in the refrigerator (4 ° C.) for more than one night until use. The flask into which the solution was injected was washed three times with normal temperature PBS before use.
[0022]
(2) Preparation of a culture solution (medium) containing IL-15 10% (volume / volume) human normal serum (fresh frozen plasma was thawed at 37 ° C., 56 ° C., to RPMI-1640 medium (manufactured by Sigma) Heated for 30 minutes to inactivate, removed precipitate by centrifugation at 18000 rpm for 60 minutes at 4 ° C., and filtered through a 0.22 μm filter), 100 U / ml penicillin (manufactured by Meiji Seika Co., Ltd.), 100γ / ml streptomycin (manufactured by Meiji Seika Co., Ltd.) and IL-15 (manufactured by Immunotech) were adjusted to contain 50 ng / ml.
[0023]
(3) Culture of
[0024]
(4) Measurement of phenotype of cultured lymphocytes 5 × 10 5 cultured lymphocytes obtained as described above are collected in a test tube, RPMI-1640 medium is added, and the supernatant is removed after centrifugation once. In the case of staining, 10 μl of FITC (fluorescein isothiocyanate) or PE (phycoerythrin) labeled monoclonal antibody was added, and in the case of indirect staining, 1 μg of unlabeled monoclonal antibody was added and stirred well. After standing for 30 minutes in ice, it was washed twice by centrifugation with RPMI-1640 medium. In the case of indirect staining, 10 μl of an appropriately diluted FITC-labeled sheep anti-mouse antibody or anti-rat antibody (manufactured by Kappel) was added, and the mixture was allowed to stand in ice for 30 minutes, followed by centrifugal washing twice with RPMI-1640 medium. Finally, 0.5 ml of RPMI-1640 medium was added, stirred well, and the surface antigen reacted with each antibody was measured with FACScan (Becton Dickinson).
[0025]
For direct staining, the monoclonal antibody used for the measurement was FITC-labeled anti-CD3 antibody, FITC-labeled anti-CD16 antibody, FITC-labeled anti-TCR-α / β antibody, FITC-labeled anti-TCR-γ / δ antibody, FITC-labeled anti-CD4 + PE label. Anti-CD8 antibody, PE-labeled anti-CD56 antibody (manufactured by Becton Dickinson), for indirect staining, rat anti-IL-2 receptor α chain antibody (anti-CD25, manufactured by Serotech), mouse anti-IL-2 receptor β Chain antibody (Mik-β1, manufactured by Nichirei Co., Ltd.), mouse anti-IL-2 receptor γ-chain antibody (AG184, manufactured by Farmingen) and mouse anti-IL-12 receptor β1 antibody (Hoffman La Roche) And FITC-labeled anti-rat IgG or FITC-labeled anti-mouse IgG (manufactured by Kappel). The results are shown in Table 1.
[0026]
[Table 1]
[0027]
What is shown from the data in Table 1 is that the lymphocyte cell phenotype obtained is mostly CD3 + CD4 + and CD3 + CD8 + T cells and has T cell receptor alpha-beta chains. In addition, there were other T cells with a small number of T cell receptor gamma delta chains and CD16 + NK cells. These lymphocytes strongly express IL-2 receptor α chain, β chain, γ chain and IL-12 receptor. In addition to IL-15, for example, IL-2, IL-4, IL- 7. Strongly active by stimulating cytokines that transmit information by sharing a part of IL-2 receptors such as IL-9 and cytokines that transmit information via IL-12 receptors such as IL-12 I can imagine what will be done. That is, an additive or synergistic effect of lymphocyte activation is expected by a combination of IL-15 and these cytokines.
[0028]
(5) Measurement of damaging activity on cultured tumor cells As cells, the lymphocytes obtained by culturing were washed 3 times with RPMI-1640 medium supplemented with 10% (volume / volume) fetal bovine serum (FBS), and the same After suspending in the medium and counting the number of cells, 200 μl was seeded on the top of a 96-well U-bottom plate (Falcon) and diluted 2 times thereafter to prepare a dilution stage. Subsequently, 1 × 10 5 / ml of target cells (cultured cancer cell lines K562, Daudi and Raji cells) labeled with 51Cr (ICN) for 2 hours are seeded in 100 μl (1 × 10 4 ) lymphocytes. And cultured at 37 ° C. for 4 hours with 5% CO 2 . After completion of the culture, 100 μl of the supernatant was collected, and the radioactivity (cpm, test free group) of 51Cr released with a gamma counter (manufactured by Packard) was measured. The cytotoxic rate (%) was calculated by the following formula.
[Expression 1]
The 51 Cr-labeled tumor cells added with 3% Triton X-100 and the cells were lysed were defined as the maximum free group, and the 51 Cr-labeled tumor cells alone as the natural free group.
[0029]
The results are shown in FIG. Since these lymphocytes effectively damaged the established tumor cells as shown in FIG. 1, it was shown that these lymphocytes are effective cell groups for use in adoptive immunotherapy.
[0030]
(6) Preparation of immunotherapeutic agent and method of use thereof Immunotherapeutic agent Proliferated lymphocyte cells derived from cancer patients by the above method, using a commercially available separating agent (Lymphoprep, manufactured by Nycomed), Separate and purify according to the protocol. 2 × 10 9 cells obtained can be suspended in 200 ml of physiological saline containing 0.1% human serum albumin to obtain an immunotherapeutic agent in the form of an instillation.
Method of using immunotherapeutic agent The whole amount of the above injection is intravenously administered to cancer patients.
By repeating this operation once a month, the cancer of the cancer patient can be cured, or the progression of symptoms can be stopped or slowed down.
[0031]
【The invention's effect】
According to the method for producing lymphocytes according to claims 1 and 2, a sufficient number for use in adoptive immunotherapy and activated lymphocytes having an activity of damaging tumor cells start from a small amount of whole blood. Obtained efficiently.
The immunotherapeutic agent according to claim 3 contains many activated lymphocytes having an activity of damaging tumor cells, and is useful for adoptive immunotherapy. Furthermore, it is suggested that more activated lymphocytes may be efficiently obtained by combining with other immunostimulatory substances such as cytokines.
[Brief description of the drawings]
FIG. 1 is a graph showing cytotoxicity (cytotoxicity) of lymphocytes (effect cells) obtained by the production method of the present invention against cancer cell lines (target cells).
Claims (7)
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