JP2010512736A - ヒトサイトメガロウィルス(hmcv)に対する抗体 - Google Patents
ヒトサイトメガロウィルス(hmcv)に対する抗体 Download PDFInfo
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Abstract
Description
本発明は、ヒト細胞に感染するウィルスに特異的な生物学的活性を有する、ヒトB細胞から単離された新規抗体配列に関する。
ヒトサイトメガロウィルス(hCMV)は、広く蔓延し、高度に種特異的なヘルペスウィルスであり、免疫抑制された、または免疫的に未成熟な個人において著しい罹患および致死を引き起こす。
本発明は、hCMVと結合し、および中和し、ならびにhCMV感染またはhCMV関連疾患を検出、治療、阻害、予防および/または改善するために使用され得る新規抗体配列を提供する。
PCT/EP2005/056871に記載された方法によって、生物学的活性(例えば、ヒトまたはウィルス標的との結合および/または中和)を有する抗体を血液中に含む患者から得られる、アイソタイプ特異的ヒトB細胞の効率的不死化を、そのような生理活性を有する抗体を分泌する細胞のポリクローナル群を得るという観点から行うことができる。低細胞密度(例えば1ウェルあたり50、20細胞またはそれよりも少ない量)での一段階のクローニングの後に、広範囲のスクリーニングアッセイが、これらの方法によって獲得された継代培養の上清を使用して行われ得る。この方法によって、多くのレパートリーのIgG分泌継代培養が特徴付けられ、そして結果的に、抗原に対する所望の結合特異性および/または生物学的活性を有する多くのヒトモノクローナルIgGが同定され得る、不死化B細胞のポリクローナル群を得ることが可能である。
材料および方法
血清中にhCMVを中和するIgG抗体を有する、ヒト提供者の選択
これらのhCMV特異的アッセイを、PCT/EP2005/056871に記載されている通りに、または下記に要約された、文献記載の通りに行なった。
NT=抗体希釈の逆数[>50%阻害]x[(50%超の%阻害−50%)/(50%超の%阻害−50%未満の%阻害)]
hCMV特異的結合アッセイを、PCT/EP2005/056871に記載されている通りか、または製造業者によって指示された通りに行い、そしてCMV特異的IgG抗体の市販混合物(サイトテクト、バイオテスト)を用いて検証した。血清を、市販である、hCMVビリオンタンパク質と結合するヒトIgG特異的ELISA(BEIA−CMV IgG Quant;Bouty,cod.21465)、および同様に市販である、gB(AD2)hCMV IgG ELISA(CG3抗原バイオテストAG,cod.807035;図1A)を用いて試験した。
血清中にhCMV中和抗体が存在しているため、急性hCMV感染(CMV7)から回復した患者から、末梢血単核球(PBMC)を得た。CMV7患者由来のPBMCがその後にさらされるEBV不死化過程を、PCT/EP2005/056871の技術に従って行なった。短時間で、従来法のフィコール/ハイパックを用いた密度勾配遠心法によって、PBMCを末梢血から精製した。CD22陽性細胞を、製造業者によって記載された通りのバリオマックス(VarioMACS)技術(ミルテニー(Miltenyi)バイオテック社)を使用することによる、抗ヒトCD22被覆ビーズを用いて、新鮮なPBMC(>90%中純度)から単離した。精製された細胞を、CpG2006(コーリー(Coley)、1μg/ml)およびIL−2(ロシュ、200U/ml)の併用で刺激した。4日間の刺激の後、細胞を新鮮な細胞培地(RPMI−1640)で洗浄し、そしてB細胞を、製造業者の指示に従って、バリオマックス(VarioMACS)技術(ミルテニー(Miltenyi)バイオテック社)を使用することによる、抗ヒトIgG被覆ビーズを用いてIgG陽性細胞中で高度に濃縮した。
EBVにさらされてから15日後、hCMV中和活性を、上記のAD169/HELFを基盤としたマイクロ中和アッセイを用いて、増殖したポリクローナル細胞培養中で確認した。その後細胞を、CpG2006およびIL−2の非存在下、10%FCSおよび非必須アミノ酸(NEAA、100Xの原液から1Xに希釈された;ユーロクローン(EuroClone))を添加した100μlIMDL中の、放射線を照射した(30Gy)同種PBMC(50,000/ウェル)上に、20細胞/ウェルで蒔いた。全体で4224個の継代培養を製造し、そして2週間後、50μlの同じ培地を加えた。さらに1〜2週間の培養の後、増殖および凝集した細胞を有する細胞培養上清を、HELF細胞およびhCMV株AD169型hCMV中和アッセイを使用して同時に試験した。
血清中和抗体を誘導するものとして最もよく性質決定された(Qadri I et al,1992;Kropff B et al.,1993)、およびCG3抗原(図1A)中でクローン化された、hCMV抗原の一つである糖タンパク質BのAD2領域への結合に基づいて、ELISAに対する強い反応性(1:64希釈で陽性;サンプルは、IgG 抗gBが1/4またはそれ以上の希釈で存在する場合に陽性であると考えられる)と共に、ヒトPBMCを、血清中で著しいhCMV中和力価(1:105希釈で50%中和)を示すhCMV患者(CMV7)から得た。さらに、CMV7の血清は、hCMVビリオンタンパク質全体を使用した別のELISAにおいて陽性であって、74AU/mlの活性を示した(サンプルは、結果が少なくとも10AU/mlであるとき、抗hCMV IgGがある場合に陽性であると考えられる)。
材料および方法
26A1継代培養の増殖および性質決定
26A1の最初の継代培養由来の細胞を、(10%FCSおよびNEAAを添加した)放射線を照射したIMDM培地中の同種PBMC上に増殖させ、そしてhCMV中和活性を、実施例1に記載の通りのhCMVマイクロ中和アッセイを使用して、この増殖段階の間に少なくとも2回確認した(表1参照)。
BEIA−CMV、gB型、およびgH型ELISAアッセイを、実施例1で記載した(図1および2)。HSVアッセイを、文献に従って行なった(Laquerre S et al,1998)。
初期26A1細胞培養の発現から得られた細胞培養の一定分量を、フュージョン・アンチボディーズ(Fusion Antibodies)社によって確立された技術に従って、26A1抗体の重鎖(VH)および軽鎖(VL)可変領域の配列決定のために使用した。(少なくとも50,000個の細胞を含む)凍結細胞のペレットを、全てのRNAを抽出するために使用した。対応するcDNAを、オリゴ(dT)プライマーを用いた逆転写によって製造した。IgG特異的プライマー混合物を使用してVH領域を、およびIgk/λプライマーの混合物を使用してVL領域を増幅するために、PCR反応を行なった。2つの増幅反応のPCR産物を、シーケンシング・ベクター(pCR2.1;インビトロジェン(Invitrogen))中のEco RI制限部位を使用してクローン化し、そしてTOP10 E.コリ細胞を形質転換するために使用した。
増殖し、およびIgGを分泌する細胞を含む継代培養の中で、それらの幾つかの細胞培養上清は、hCMV感染を中和する抗体を含んでいたが、ELISAによって試験された特定の組み換え抗原gBおよびgHに対して、著しい結合を示さなかった(図1および2)。特に、IgG1を分泌する26A1継代培養は、より強くそしてより再現可能なhCMV中和活性を示し、そしてそれは、より詳細な分子的および生物学的性質決定のために選択された。
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Claims (26)
- 配列番号8と少なくとも90%の同一性を有する配列を含むタンパク質。
- 配列番号6および/または配列番号7をさらに含む、請求項1記載のタンパク質。
- 配列番号5と少なくとも90%の同一性を有する配列を含む、請求項2記載のタンパク質。
- 配列番号11、配列番号12および配列番号13からなる群から選択される、一またはそれ以上の配列をさらに含む、請求項1〜3のいずれか一項に記載のタンパク質。
- 配列番号10と少なくとも90%の同一性を有する配列を含む、請求項4記載のタンパク質。
- 抗体、抗体断片、生理活性ペプチドまたは融合タンパク質である、請求項1〜5のいずれか一項に記載のタンパク質。
- 前記抗体がヒト組み換え抗体である、請求項6記載のタンパク質。
- 前記抗体が、配列番号15と少なくとも90%の同一性があるタンパク質配列を有する重鎖、および配列番号17と少なくとも90%の同一性があるタンパク質配列を有する軽鎖を含む、請求項7記載のタンパク質。
- 前記抗体断片が、可変性重鎖/軽鎖ヘテロ二量体または一本鎖可変断片である、請求項6記載のタンパク質。
- ヒトサイトメガロウィルス(hCMV)と結合し、および中和する、請求項1〜9のいずれか一項に記載のタンパク質。
- 26A1継代培養によって分泌される、ヒトIgG1抗体。
- hCMVの結合および中和において、請求項11記載のIgG1抗体と競合するタンパク質。
- 請求項1〜9のいずれか一項に記載のタンパク質である、請求項12記載のタンパク質。
- 請求項1〜13のいずれか一項に記載のタンパク質をコードする核酸。
- 配列番号4と少なくとも90%の同一性を有する配列を含む、請求項14記載の核酸。
- 配列番号9と少なくとも90%の同一性を有する配列をさらに含む、請求項15記載の核酸。
- 請求項14〜16のいずれか一項に記載の核酸を含むベクター。
- 請求項14〜17のいずれか一項に記載の核酸を含む、原核または真核宿主細胞。
- 請求項1〜12のいずれか一項に記載のタンパク質を分泌する、請求項18記載の宿主細胞。
- 請求項1〜12のいずれか一項に記載のタンパク質の産生のための、請求項14〜17のいずれか一項に記載の核酸、または請求項18もしくは19の宿主細胞の使用。
- hCMV感染症またはhCMV関連疾患の検出、治療、阻害、予防および/または寛解のための組成物の製造における、請求項1〜12のいずれか一項に記載のタンパク質の使用。
- 請求項1〜12のいずれか一項に記載のタンパク質、または請求項14〜17のいずれか一項に記載の核酸を含むhCMV感染症またはhCMV関連疾患のための、治療用、予防用または診断用組成物。
- 眼球投与用または局所投与用である、請求項22記載の組成物。
- 異なるhCMV中和抗体、静注用免疫グロブリン調製物および/または抗ウィルス化合物をさらに含む、請求項22または23記載の組成物。
- 請求項1〜13のいずれか一項に記載のタンパク質、または請求項14〜17のいずれか一項に記載の核酸の投与を含む、hCMV感染症またはhCMV関連疾患の治療、予防または診断方法。
- 異なるhCMV中和抗体、静注用免疫グロブリン調製物および/または抗ウィルス化合物の投与をさらに含む、請求項25記載の方法。
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Application Number | Priority Date | Filing Date | Title |
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PCT/EP2006/069780 WO2007068758A1 (en) | 2005-12-16 | 2006-12-15 | Methods for obtaining immortalized antibody secreting cells |
EPPCT/EP2006/069780 | 2006-12-15 | ||
EP07110693 | 2007-06-20 | ||
EP07110693.4 | 2007-06-20 | ||
PCT/EP2007/064094 WO2008071806A1 (en) | 2006-12-15 | 2007-12-17 | Antibodies against human cytomegalovirus (hcmv) |
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JP2009540793A Expired - Fee Related JP5351041B2 (ja) | 2006-12-15 | 2007-12-17 | ヒトサイトメガロウィルス(hmcv)に対する抗体 |
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EP (1) | EP2126049B1 (ja) |
JP (1) | JP5351041B2 (ja) |
KR (1) | KR20090114364A (ja) |
CN (1) | CN101627115B (ja) |
AT (1) | ATE503769T1 (ja) |
AU (1) | AU2007331463B2 (ja) |
BR (1) | BRPI0720464A2 (ja) |
CA (1) | CA2672703C (ja) |
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PL (1) | PL2126049T3 (ja) |
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JP2023505514A (ja) * | 2019-12-04 | 2023-02-09 | トリノマブ バイオテック カンパニー リミテッド | 抗ヒトサイトメガロウイルス抗体及びその用途 |
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US7947274B2 (en) | 2007-01-04 | 2011-05-24 | Humabs, LLC. | Human cytomegalovirus neutralising antibodies and use thereof |
GB0700133D0 (en) | 2007-01-04 | 2007-02-14 | Humabs Llc | Human cytomegalovirus neutralising antibodies and use thereof |
WO2009024445A1 (en) * | 2007-08-22 | 2009-02-26 | Ribovax Biotechnologies Sa | Antibodies against human cytomegalovirus (hcmv) |
ES2682596T3 (es) | 2008-07-16 | 2018-09-21 | Institute For Research In Biomedicine | Anticuerpos neutralizantes del citomegalovirus humano y uso de los mismos |
US8703486B2 (en) * | 2008-09-23 | 2014-04-22 | UNIVERSITé LAVAL | Method for polyclonal immunoglobulin G production by human B cells |
MX2012007489A (es) | 2009-12-23 | 2012-11-29 | 4Antibody Ag | Miembros enlazadores para citomegalovirus. |
KR20130112879A (ko) * | 2010-09-29 | 2013-10-14 | 제넨테크, 인크. | 항체 조성물 및 사용 방법 |
EP2831119A1 (en) | 2012-03-28 | 2015-02-04 | F. Hoffmann-La Roche AG | Anti-hcmv idiotypic antibodies and uses thereof |
KR101581158B1 (ko) | 2014-03-26 | 2015-12-30 | 울산대학교 산학협력단 | 거대세포바이러스 실제 감염 여부를 진단하기 위한 거대세포바이러스의 감염 해석 모델 |
CA2943719A1 (en) * | 2014-05-19 | 2015-11-26 | F. Hoffmann-La Roche Ag | Method for production of polypeptides |
US10611800B2 (en) | 2016-03-11 | 2020-04-07 | Pfizer Inc. | Human cytomegalovirus gB polypeptide |
US11629172B2 (en) | 2018-12-21 | 2023-04-18 | Pfizer Inc. | Human cytomegalovirus gB polypeptide |
TW202413391A (zh) | 2020-06-21 | 2024-04-01 | 美商輝瑞股份有限公司 | 人巨細胞病毒糖蛋白B(gB)多肽 |
CN115947869B (zh) * | 2022-11-28 | 2023-12-12 | 广州佰芮慷生物科技有限公司 | 一种靶向人巨细胞病毒的嵌合抗原受体、car-nk细胞及用途 |
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CN1445243A (zh) * | 2002-12-13 | 2003-10-01 | 中国疾病控制中心病毒病预防控制所 | 抗人巨细胞病毒人源中和性基因工程Fab抗体 |
AU2004215125B2 (en) * | 2003-02-26 | 2011-01-06 | Institute For Research In Biomedicine | Monoclonal antibody production by EBV transformation of B cells |
WO2006124269A2 (en) | 2005-05-16 | 2006-11-23 | Amgen Fremont Inc. | Human monoclonal antibodies that bind to very late antigen-1 for the treatment of inflammation and other disorders |
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2007
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- 2007-12-17 AU AU2007331463A patent/AU2007331463B2/en not_active Ceased
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- 2007-12-17 KR KR1020097014818A patent/KR20090114364A/ko not_active Application Discontinuation
- 2007-12-17 CA CA2672703A patent/CA2672703C/en not_active Expired - Fee Related
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2009
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- 2009-07-14 NO NO20092671A patent/NO20092671L/no not_active Application Discontinuation
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2023505514A (ja) * | 2019-12-04 | 2023-02-09 | トリノマブ バイオテック カンパニー リミテッド | 抗ヒトサイトメガロウイルス抗体及びその用途 |
JP7458484B2 (ja) | 2019-12-04 | 2024-03-29 | チューハイ トリノマブ ファーマシューティカル カンパニー リミテッド | 抗ヒトサイトメガロウイルス抗体及びその用途 |
Also Published As
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ATE503769T1 (de) | 2011-04-15 |
ES2363992T3 (es) | 2011-08-22 |
EA018701B1 (ru) | 2013-10-30 |
EA200970586A1 (ru) | 2009-12-30 |
KR20090114364A (ko) | 2009-11-03 |
EP2126049B1 (en) | 2011-03-30 |
EP2126049A1 (en) | 2009-12-02 |
CN101627115A (zh) | 2010-01-13 |
WO2008071806A1 (en) | 2008-06-19 |
CN101627115B (zh) | 2013-05-29 |
US8153129B2 (en) | 2012-04-10 |
BRPI0720464A2 (pt) | 2014-01-14 |
IL199366A (en) | 2015-03-31 |
DK2126049T3 (da) | 2011-07-18 |
CA2672703C (en) | 2016-11-22 |
PL2126049T3 (pl) | 2011-09-30 |
JP5351041B2 (ja) | 2013-11-27 |
PT2126049E (pt) | 2011-07-04 |
AU2007331463B2 (en) | 2014-03-27 |
DE602007013618D1 (de) | 2011-05-12 |
CA2672703A1 (en) | 2008-06-19 |
NO20092671L (no) | 2009-09-03 |
AU2007331463A1 (en) | 2008-06-19 |
US20100040602A1 (en) | 2010-02-18 |
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