JP2015536145A - 抗原特異的形質芽細胞の濃縮 - Google Patents
抗原特異的形質芽細胞の濃縮 Download PDFInfo
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
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- A61K39/4648—Bacterial antigens
- A61K39/46482—Clostridium, e.g. Clostridium tetani
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
Description
本出願は、2012年11月13日に出願された米国仮出願第61/725764号の利益を主張し、その全内容は本明細書に参照により援用される。
本出願は、ASCIIフォーマットで電子的に提出された配列表を含み、その全内容は本明細書に参照により援用される。前記ASCIIコピーは、2013年10月22日に作成され、P5500R1−WO_SL.txtと命名され、大きさは15,453バイトである。
本発明は抗原特異的形質芽細胞の濃縮のための方法に関する。該方法は、複数の抗原を認識する希少抗体を含む抗体の同定に有用である。
用語「形質芽細胞」は、形質細胞の前駆体である細胞を指す。
本発明の実施は、特に明記しない限り、当業者にとって既知であり、利用可能である細胞生物学、細胞培養、(組換え技術を含む)分子生物学、微生物学、生化学、及び免疫学の従来技術を用いる。そのような技術は、例えば、Molecular Cloning: A laboratory Manual, 第3版 (Sambrook et al., 2001) Cold Spring Harbor Press; Oligonucleotide Synthesis (P. Herdewijn, 編, 2004); Animal Cell Culture (R.I. Freshney, 編, 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F.M. Ausubel et al., 編, 1987); PCR: The Polymerase Chain Reaction (Mullis et al., 編, 1994); Current Protocols in Immunology (J.E. Coligan et al., 編, 1991); 及びShort Protocols in Molecular Biology (Wiley及びSons, 1999)などの文献に記載されている。細菌における抗体断片及びポリペプチドの発現については、例えば、米国特許第5648237号、第5789199号及び第5840523号に記載されている。(また、大腸菌における抗体断片の発現を説明している、Charlton, Methods in Molecular Biology, 248 巻 (B.K.C. Lo, 編, Humana Press, Totowa, NJ, 2003), 245-254頁も参照)
本発明の方法を用いて同定される抗体は、例えば米国特許第4816567号に記載されているような組換え方法及び組成物を用いて製造することができる。例えば、本発明に従って同定された抗体をコードする、単離された核酸分子が単離され得る。このような核酸は、抗体のVLを含むアミノ酸配列、及び/又は抗体のVHを含むアミノ酸配列(例えば、抗体の軽鎖及び/又は重鎖)をコードし得る。更なる実施態様において、そのような核酸を含む一又は複数のベクター(例えば、発現ベクター)が提供される。更なる実施態様において、そのような核酸を含む宿主細胞が提供される。そのような一実施態様において、宿主細胞は以下を含む(例えば、以下で形質転換されている):(1)抗体のVLを含むアミノ酸配列、及び抗体のVHを含むアミノ酸配列をコードする核酸を含むベクター、又は(2)抗体のVLを含むアミノ酸配列をコードする核酸を含む第一ベクター、及び抗体のVHを含むアミノ酸配列をコードする核酸を含む第二ベクター。一実施態様において、宿主細胞は、真核生物細胞、例えばチャイニーズハムスター卵巣(CHO)細胞、又はリンパ系細胞(例えば、Y0、NS0、Sp20細胞)である。
以下の実験は、SCID/ベージュマウスへのヒト末梢血単核細胞(PBMC)の脾臓内移植後のPBMCの増殖及び分化の影響を評価するために実施された。
採血7日前にDT(ジフテリアトキソイド及び破傷風トキソイド)ワクチンを受けた正常なヒトドナー由来のロイコパックを、Blood Centers of the Pacific(San Francisco,CA)から得た。標準的方法を用い、末梢血単核細胞(PBMC)をロイコパックから単離した。6−8週齢の雌SCID/ベージュマウスをCharles River Laboratories(Hollister,CA)より購入し、全米実験動物協会(American Association of Laboratory)の動物愛護ガイドライン(Animal Care guidelines)に従い、Genentech社にて飼養、保管した。実験研究はすべて、ジェネンテック実験動物研究の施設内動物管理使用委員会(the Institutional Animal Care and Use Committees of Genentech Lab Animal Research)の承認の下、AAALACi(国際実験動物ケア評価認証協会)認定施設内で、実験動物のケアと使用に関する指針及び関連法令、規則に従って実施された。健康なドナー由来のロイコパック又は血液は、書面によるインフォームド・コンセントが提出され、欧米の治験審査委員会(the Western Institutional Review Board)の倫理的承認を得た後、採取された。
採血の7日前に季節性インフルエンザワクチンFluvirin(登録商標)(ノバルティス ロット#111796P1)を接収した正常ヒトドナー由来のロイコパックをBlood Centers of the Pacific(San Francisco,CA)より得た。標準的方法を用い、末梢単核細胞(PBMC)をロイコパックから単離した。6−8週齢の雌SCID/ベージュマウスをCharles River Laboratories(Hollister,CA)より購入し、全米実験動物協会(American Association of Laboratory)の動物愛護ガイドライン(Animal Care guidelines)に従い、Genentech社にて飼養、保管した。実験研究はすべて、ジェネンテック実験動物研究の施設内動物管理使用委員会の承認の下、AAALACi(国際実験動物ケア評価認証協会)認定施設内で、実験動物のケアと使用に関する指針及び関連法令、規則に従って実施された。健康なヒトドナー由来のロイコパック又は血液は、書面によるインフォームド・コンセントが提出され、欧米の治験審査委員会(the Western Institutional Review Board)の倫理的承認を得た後、採取された。
ヘマグルチニンH1及びH3交差反応性ヒト形質芽細胞(上に記載)は単一細胞選別され、約950個の形質芽細胞となった。単一形質芽細胞を、5%低IgGウシ胎児血清含有RPMIを50μl含むU字底型96ウェルマイクロウェルプレートに直接入れた。該プレートを600xgで5分間遠心分離し(Beckman Coulter,Brea,CA)、培地を吸引により注意深く除去した。細胞を再懸濁させ、同じ手順に従い、PBS 90μl中で二回洗浄した。
カッパ定常:CTCAGCGTCAGGGTGYTGCTGAG(配列番号2)
ラムダ定常:GGGTKTGGTSGTCTCCAC(配列番号3)
IGVH1aCAGGTGCAGCTGGTGCAGTCTGGGGC(配列番号4)
IGVH1bCAGGTCCAGCTGGTGCAGTCTGGGGC(配列番号5)
IGVH2CAGGTCACCTTGAAGGAGTCTGGTCC(配列番号6)
IGVH3GAGGTGCAGCTGGTGGAGTCTGGGGG(配列番号7)
IGVH4CAGGTGCAGCTGCAGGAGTCGGGCCC(配列番号8)
IGVH5GAGGTGCAGCTGGTGCAGTCTGG(配列番号9)
IGVH6CAGGTACAGCTGCAGCAGTCAGGTCC(配列番号10)
IGVH7CAGGTGCAGCTGGTGCAATCTGG(配列番号11)
IGKV1GHCATCCRGWTGACCCAGTCTC(配列番号12)
IGKV2GATRTTGTGATGACYCAGWCTC(配列番号13)
IGKV3GAAATWGTRWTGACRCAGTCTC(配列番号14)
IGKV4GACATCGTGATGACCCAGTCTCC(配列番号15)
IGKV5GAAACGACACTCACGCAGTCTC(配列番号16)
IGKV6GAWRTTGTGMTGACWCAGTCTC(配列番号17)
IGLV1CAGTCTGTGYTGACKCAGCCRCCCTC(配列番号18)
IGLV2CAGTCTGCCCTGACTCAGCCT(配列番号19)
IGLV3TCCTATGAGCTGACWCAGSHVCCCKC(配列番号20)
IGLV4CAGCCTGTGCTGACTCARTCVCCCTC(配列番号21)
IGLV5CAGCCTGTGCTGACTCAGCCAACTTC(配列番号22)
IGLV6AATTTTATGCTGACTCAGCCCCAC(配列番号23)
IGLV7CAGGCTGTGGTGACTCAGGAGCCC(配列番号24)
IGLV8CAGACTGTGGTGACCCAGGAGCC(配列番号25)
IGLV9CAGCCTGTGCTGACTCAGCCACC(配列番号26)
HC301.5定常GCAGCCCAGGGCSGCTGTGC(配列番号27)
カッパ102定常GCACACAACAGAGGCAGTTCCAG(配列番号28)
ラムダ202定常CTTGRAGCTCCTCAGAGGAG(配列番号29)
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGG(配列番号30)
sVH2:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGATCACCT(配列番号31)
sVH3vv:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAG(配列番号32)
sVH3gl:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGAGG(配列番号33)
sVH4:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGGTGCAGCTGCAGG(配列番号34)
sVH5:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGAGGTGCA(配列番号35)
sVH6:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGGTACAGC(配列番号36)
sVH7:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGGTGCA(配列番号37)
sVK1:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGACATCCAGATGACCCAGTCTCCATCCTCCCTG(配列番号38)
sVK2:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGATATTGTGATGACTCAGTCTCACTCTCCCTGC (配列番号39)
sVK3:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTG(配列番号40)
sVK4:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTG(配列番号41)
sVK5:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGAAACGACACTCACGCAGTCTCCAGC(配列番号42)
sVK6:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAGAAATTGTGCTGACTCAGTCTCCAGACTTTCG(配列番号3)
sVL1:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGTCTGTGYTGACKCAGCCRCCCTC(配列番号44)
sVL2:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGTCTGCCCTGACTCAGCCT(配列番号45)
sVL3:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCATCCTATGAGCTGACWCAGSHVCCCKC(配列番号46)
sVL4:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGCCTGTGCTGACTCARTCVCCCTC(配列番号47)
sVL5:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGCCTGTGCTGACTCAGCCAACTTC(配列番号48)
sVL6:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCAAATTTTATGCTGACTCAGCCCCAC(配列番号49)
sVL7:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGGCTGTGGTGACTCAGGAGCCC(配列番号50)
sVL8:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGACTGTGGTGACCCAGGAGCC(配列番号51)
wVL9:
CCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGCCTGTGCTGACTCAGCCACC(配列番号52)
重鎖定常:GCCAGGGGGAAGACCGATG(配列番号53)
カッハ゜定常:CTGGGATAGAAGTTATTCAGCAGGCACACAACAGAAGCAGTTCCAGATTTCAACTGCTC(配列番号54)
ラムタ゛定常:CTTGRAGCTCCTCAGAGGAG(配列番号29)
本発明の方法を用いて得られた各モノクローナル抗ヘマグルチニン抗体が種々のヘマグルチニンサブタイプに結合する能力を、ELISAによって次の通り調べた。種々のヘマグルチニン発現プラスミドが、上記293T細胞にトランスフェクトされた。これらには、H1N1/サウスカロライナ/1918インフルエンザAウイルスのヘマグルチニンH1、H3N2/パース/2009インフルエンザAウイルスのヘマグルチニンH3、H5N1/ベトナム/2004インフルエンザAウイルスのヘマグルチニンH5、及びH7N7/オランダ/2003インフルエンザAウイルスのヘマグルチニンH7が含まれた。2日後、細胞を50mMのトリス(pH8)、5mMのEDTA、150mMのNaCl、1%トリトンX−100プラスプロテアーゼ阻害剤カクテル(Roche)に溶解した。核を遠心分離により取り除き、得られた溶解物を−80℃で保存した。
本発明の方法を用いて同定された抗ヘマグルチニン抗体の、広範なヘマグルチニンサブタイプへの結合、並びにインフルエンザAグループ1及びグループ2ウイルス分離株パネルのインビトロ中和を引き起こす能力を下記の通り調べた。
各インフルエンザAウイルス菌株のヘマグルチニン(HA)サブタイプが図5に示されている。
本発明の方法を使用して得られた抗ヘマグルチニン抗体の特性を更に明らかにした。mAb 39.29(mAb 39.29 NC v1)の、マウスにおけるインフルエンザAウイルス感染に対するインビボ有効性試験が以下の通り実施された。DBA/2Jマウス(Jackson Lab,Bar Harbor,ME)を、インフルエンザ培地(DMEM、0.2% BSA、2μg/mL TPCKで処理したトリプシン)中、最小LD100量に希釈した種々のインフルエンザAウイルス株50μに経鼻感染させた。マウス当たり40PFUで使用したH1N1 A/プエルトリコ/8/1934(Genentech;IC50 2.0nM);マウス当たり3PFUで使用したH3N2 A/香港/1/1968(ViraPur,San Diego,CA;IC50 45.1nM);マウス当たり1.5x104PFUで使用したH3N2 A/ポート・チャーマーズ/1/1973 (ViraPur,San Diego,CA;IC50 2.2nM);及びマウス当たり2x102PFUで使用したH3N2 A/愛知/2/1968(ViraPur,San Diego,CA;IC50 35nM)を含む、様々なインビトロIC50値を示す4つの異なるインフルエンザAウイルス株が、この一連の実験で使用された。mAb 39.29の静脈内投与に先立って、インフルエンザウイルス感染を72時間進行させた。
Claims (19)
- 抗原に特異性を有する形質芽細胞を濃縮した形質芽細胞の集団を得る方法であって、ドナーから末梢血単核細胞(PBMC)を得ることと、該PBMCを、PBMC/抗原プレミックスを得るために抗原又はその断片もしくは一部分と接触させることと、該PBMCを免疫不全動物の脾臓に移植することと、及び該免疫不全動物の脾臓から形質芽細胞を単離することとを含み、その結果抗原に特異性を有する形質芽細胞を濃縮した形質芽細胞の集団を得る、方法。
- PBMCの免疫不全動物の脾臓への移植前に、PBMCを洗浄して過剰又は非結合抗原をPBMC/抗原プレミックスから除去することを更に含む、請求項1に記載の方法。
- ドナーが、抗原又はその一部の断片に予め曝されているドナーである、請求項1に記載の方法。
- 形質芽細胞の単一細胞選別を更に含む、請求項1に記載の方法。
- 形質芽細胞の抗原特異的単一細胞選別を更に含む、請求項1に記載の方法。
- 形質芽細胞から免疫グロブリンをクローニングすることを更に含む、請求項1に記載の方法。
- 抗原特異的形質芽細胞を濃縮するための方法であって、ドナーから末梢血単核細胞(PBMC)を得ることと、該PBMCを、PBMC/抗原プレミックスを得るために少なくとも一種の抗原と接触させることと、該PBMCを免疫不全動物の脾臓に移植することと、及び該免疫不全動物の脾臓から形質芽細胞を単離することとを含み、その結果抗原特異的形質芽細胞を濃縮する、方法。
- PBMCの免疫不全動物の脾臓への移植前に、PBMCを洗浄して過剰又は非結合抗原をPBMC/抗原プレミックスから除去することを更に含む、請求項7に記載の方法。
- ドナーが、抗原又はその一部の断片に予め曝されているドナーである、請求項7に記載の方法。
- 形質芽細胞の単一細胞選別を更に含む、請求項7に記載の方法。
- 形質芽細胞の抗原特異的単一細胞選別を更に含む、請求項7に記載の方法。
- 形質芽細胞から免疫グロブリンをクローニングすることを更に含む、請求項7に記載の方法。
- PBMCを、複数の異なる抗原と接触させることを更に含む、請求項7に記載の方法。
- 抗原に特異性を有する抗体を産生することができる形質芽細胞を同定する方法であって、ドナーから末梢血単核細胞(PBMC)を得ることと、該PBMCを、PBMC/抗原プレミックスを得るために少なくとも一種の抗原と接触させることと、該PBMCを免疫不全動物の脾臓に移植することと、該免疫不全動物の脾臓から形質芽細胞を単離することと、及び単離された形質芽細胞から抗原特異的細胞を選別することとを含み、その結果抗原特異性を有する抗体を産生できる形質芽細胞を同定する、方法。
- PBMCの免疫不全者の脾臓への移植前に、PBMCを洗浄して過剰又は非結合抗原をPBMC/抗原プレミックスから除去することを更に含む、請求項14に記載の方法。
- 単離された形質芽細胞から免疫グロブリンをクローニングすることを更に含む、請求項14に記載の方法。
- 複数の抗原に特異性を有する抗体を産生することができる形質芽細胞を同定する方法であって、ドナーから末梢血単核細胞(PBMC)を得ることと、該PBMCを、PBMC/抗原プレミックスを得るために複数の異なる抗原と接触させることと、該PBMCを免疫不全動物の脾臓に移植することと、該免疫不全動物の脾臓から形質芽細胞を単離することと、及び単離された形質芽細胞から抗原特異的細胞を選別することとを含み、その結果抗原特異性を有する抗体を産生できる形質芽細胞を同定する、方法。
- PBMCの免疫不全者の脾臓への移植前に、PBMCを洗浄して過剰又は非結合抗原をPBMC/抗原プレミックスから除去することを更に含む、請求項17に記載の方法。
- 単離された形質芽細胞から免疫グロブリンをクローニングすることを更に含む、請求項17に記載の方法。
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RU2015116927A (ru) | 2017-01-10 |
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