JP2011004751A - 免疫したゼノマウス(XenoMouse)に由来するヒト抗体 - Google Patents
免疫したゼノマウス(XenoMouse)に由来するヒト抗体 Download PDFInfo
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- JP2011004751A JP2011004751A JP2010171583A JP2010171583A JP2011004751A JP 2011004751 A JP2011004751 A JP 2011004751A JP 2010171583 A JP2010171583 A JP 2010171583A JP 2010171583 A JP2010171583 A JP 2010171583A JP 2011004751 A JP2011004751 A JP 2011004751A
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Abstract
【解決手段】抗原投与に応答して完全なヒト抗体を産生するように改変された、内因性の遺伝子座を欠損しているトランスジェニック動物に、特異的な抗原を投与することにより、この抗原に対する、単離された完全なヒト抗体を調製する方法。続いてさまざまな操作を行なうことにより、抗体そのものまたはその類似体を得ることができる。
【選択図】なし
Description
本発明は、免疫学の分野、特に、抗体の産生に関する。より具体的には、所望の抗体に対応する抗原を用いてトランスジェニック動物を免疫する段階を含む方法によって、該抗体を産生させることに関する。トランスジェニック動物は、内因性の抗体ではなく、ヒト抗体を産生するように改変されている。
参照として本明細書に組み入れられる、1994年2月3日に発行されたPCT出願国際公開公報第94/02602号(特許文献1)において、抗原投与に応答して、内因性の抗体ではなく完全なヒト抗体を産生するように改変された、ヒト以外のトランスジェニック動物の作出について詳しく説明されている。要約すると、トランスジェニック宿主動物において、免疫グロブリンの重鎖および軽鎖をコードする内因性の遺伝子座を不活化して、ヒト免疫グロブリンの重鎖および軽鎖をコードする遺伝子座をゲノムの中に挿入する。一般に、所望の改変形質をすべて備える動物は、改変すべき形質のすべてには足りない形質を有する中間的な動物を交配育種することによって得られる。本明細書で説明する好ましい態様におけるヒト以外の動物はマウスである。したがって、特に免疫原を投与したときに、これらの抗原に免疫特異的な、マウスの抗体でなく完全なヒト抗体を含む、ヒトの可変領域を有する抗体を産生するマウスについて説明する。
本発明は、少なくとも一つの工程に所望の抗原でヒト以外のトランスジェニック動物を免疫することが含まれる、ヒト抗体を産生する方法を提供することを目的とする。改変された動物は、内因性の抗体を産生できなくなるが、その代わりに、完全なヒト免疫グロブリンを分泌するB細胞を産生する。産生された抗体は、動物から直接的にまたはその動物由来の不死化B細胞から採取することができる。または、ヒトの可変領域を有する免疫グロブリンをコードする遺伝子を、抗体を直接採取するために回収し発現させるかまたは例えば一本鎖Fv分子のような抗体の類似体を得るために改変することができる。
免疫応答を刺激しそれにより該抗原に特異的な免疫グロブリンを分泌するB細胞をヒト以外の動物で産生させるような条件下で、内因性の免疫グロブリンの重鎖および軽鎖を実質的に産生できないがヒト免疫グロブリンを産生することができる該動物に、該抗原またはその免疫原部位を投与する段階、および
該免疫グロブリンまたは類似体を回収する段階。
〔2〕 回収段階に、動物からポリクローナル免疫グロブリンまたは類似体を回収することが含まれる、〔1〕記載の方法。
〔3〕 回収段階に以下の段階が含まれる、〔1〕記載の方法:
抗原を用いて免疫した動物のB細胞を不死化する段階、その結果できた不死化細胞を、該抗原に特異的な免疫グロブリンを分泌するものを得るためにスクリーニングする段階、および
a)該不死化B細胞によって分泌される免疫グロブリンを回収する段階、または
b)不死化B細胞から少なくとも免疫グロブリンをコードする遺伝子を回収し、選択的には該遺伝子を改変し、
免疫グロブリンもしくは類似体を産生するために、該遺伝子もしくはその改変遺伝子を発現させ、
該免疫グロブリンもしくは類似体を回収する段階。
〔4〕 回収段階に以下の段階が含まれる、〔1〕記載の方法:
動物の初期B細胞から免疫グロブリンをコードする遺伝子を回収する段階、
免疫グロブリンを発現する該遺伝子のライブラリーを作製する段階、
抗原にとって望ましい親和性を有する免疫グロブリンを得るために該ライブラリーをスクリーニングする段階、
免疫グロブリンをコードする遺伝子を回収する段階、
免疫グロブリンまたは類似体を産生させるために、回収した該遺伝子を発現させる段階、および該免疫グロブリンまたは類似体を回収する段階。
〔5〕 〔1〕記載の方法によって産生される免疫グロブリンまたは類似体をコードする塩基配列を含む組換えDNA分子。
〔6〕 コーディング塩基配列が、それを発現させることのできる調節配列に機能的に結合されている、〔5〕記載のDNA分子。
〔7〕 〔6〕記載のDNA分子を含むように改変された細胞または細胞系。
〔8〕 完全なヒト免疫グロブリンまたは類似体を産生させるために該コーディング塩基配列が発現されるような条件下で、〔7〕記載の細胞を培養すること、および該免疫グロブリンまたは類似体を回収することを含む、完全なヒト免疫グロブリンまたはその類似体を産生するための方法。
〔9〕 〔3〕記載の方法によって調製される遺伝子または改変遺伝子に相当する塩基配列を含むDNA分子。
〔10〕 コーディング塩基配列が、それを発現させることのできる調節配列に機能的に結合されている、〔9〕記載のDNA分子。
〔11〕 〔9〕記載のDNA分子を含むように改変された細胞または細胞系。
〔12〕 完全なヒト免疫グロブリンまたは類似体を産生させるために該コーディング塩基配列が発現されるような条件下で、〔11〕記載の細胞を培養すること、および該免疫グロブリンまたは類似体を回収することを含む、完全なヒト免疫グロブリンまたはその類似体を産生するための方法。
〔13〕 〔4〕記載の方法によって調製される、望ましい親和性を有するヒト免疫グロブリンをコードする塩基配列を含むDNA分子。
〔14〕 コーディング塩基配列が、それを発現させることのできる調節配列に機能的に結合されている、〔13〕記載のDNA分子。
〔15〕 〔13〕記載のDNA分子を含むように改変された細胞または細胞系。
〔16〕 完全なヒト免疫グロブリンまたは類似体を産生させるために該コーディング塩基配列が発現されるような条件下で、〔15〕記載の細胞を培養すること、および該免疫グロブリンまたは類似体を回収することを含む、完全なヒト免疫グロブリンまたはその類似体を産生するための方法。
〔17〕 〔3〕の記載に従って調製される所望の抗原に対して完全なヒト免疫グロブリンを分泌する不死化B細胞。
〔18〕 〔17〕記載の細胞を培養すること、および該免疫グロブリンまたは類似体を回収することを含む、免疫グロブリンまたは類似体を産生するための方法。
〔19〕 〔1〕記載の方法によって産生される、完全なヒト免疫グロブリンまたは類似体。
〔20〕 所望の抗原が以下からなる群より選択される、〔19〕記載の免疫グロブリンまたはその類似体:
CD2、CD3、CD4、CD5、CD6、CD7、CD8、CD11a、b、c、CD13、CD14、CD18、CD19、CD20、CD22、CD23、CD27およびそのリガンド、CD28およびそのリガンドB7.1、B7.2、B73、CD29およびそのリガンド、CD30およびそのリガンド、CD40およびそのリガンドgp39、CD44、CD45およびアイソフォーム、CDw52(キャンパス(Campath)抗原)、CD56、CD58、CD69、CD72、CTLA-4、LFA-1、ならびにTCRからなる群より選択される白血球マーカー、
MHCクラスIまたはII、ルイスY抗原、SLex、SLey、SLea、およびSLebからなる群より選択される組織適合性抗原、
VLA-1、VLA-2、VLA-3、VLA-4、VLA-5、VLA-6、αVβ3、LFA-1、Mac-1、p150,95、αvβ1、gpIIbIIIa、αRβ3、ασβ4、αvβ5、αvβ6、およびαvβ7からなる群より選択されるインテグリン、
L-セレクチン、P-セレクチン、およびE-セレクチン、ならびにそれらのカウンターレセプターであるVCAM-1、ICAM-1、ICAM-2、およびLFA-3からなる群より選択されるセレクチン、
IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-14、およびIL-15からなる群より選択されるインターロイキン、
IL-1R、IL-2R、IL-3R、IL-4R、IL-5R、IL-6R、IL-7R、IL-8R、IL-9R、IL-10R、IL-11R、IL-12R、IL-13R、IL-14R、およびIL-15Rからなる群より選択されるインターロイキンレセプター、
PF4、RANTES、MIP1α、MCP1、NAP-2、Groα、Groβ、およびIL-8からなる群より選択されるケモカイン、
TNFアルファ、TGFベータ、TSH、VEGF/VPF、PTHrP、EGFファミリー、FGF、PDGFファミリー、エンドセリン、フィブロシン(F3F−1)、ヒト・ラミニン、およびガストリン解離ペプチド(GRP)からなる群より選択される増殖因子、
TNFアルファR、RGFベータR、TSHR、VEGFR/VPFR、FGFR、EGFR、PTHrPR、PDGFRファミリー、EPO-R、GCSF-R、およびその他の造血レセプターからなる群より選択される増殖因子レセプター、
IFNCαR、IFNβR、およびIFNλRからなる群より選択されるインターフェロンレセプター、
IgE、FceRI、およびFCERIIからなる群より選択されるIgおよびそのレセプター、
her2-neu、ムチン、CEA、およびエンドシアリンからなる群より選択される腫瘍抗原、
ハウスダストダニ抗原、lol p1(草)抗原、およびウルシオールからなる群より選択されるアレルゲン、
CMV糖蛋白質B、H、およびgCIII、HIV-1のエンベロープ糖蛋白質、RSVのエンベロープ糖蛋白質、HSVのエンベロープ糖蛋白質、HPVのエンベロープ糖蛋白質、肝炎ファミリーの表面抗原からなる群より選択されるウイルス蛋白質、
シュードモナスの内毒素およびオステオポンチン/ウロポンチン(osteopontin/uropontin)、ヘビ毒、クモ毒、およびハチ毒コノトキシン(conotoxin)からなる群より選択される毒素、
補体C3b、補体C4a、補体C4b-9、Rh因子、フィブリノーゲン、フィブリン、およびミエリン結合増殖阻害因子からなる群より選択される血液因子、ならびに
コレステロールエステル転移蛋白質、細胞膜結合基質メタロプロテアーゼ、およびグルタミン酸デカルボキシラーゼ(GAD)からなる群より選択される酵素。
〔21〕 所望の抗原が、ヒトIL-6、ヒトIL-8、ヒトTNFα、ヒトCD4、ヒトL-セレクチン、ヒトgp39、ヒトIgE、ヒトαVβ3、ヒトフィブロシン(F3F−1)、ヒト・ラミニン、ヒトPTHrp、および破傷風毒素C(TTC)からなる群より選択される、〔14〕記載の免疫グロブリンまたは類似体。
〔22〕 〔19〕〜〔21〕記載の免疫グロブリンまたは類似体をコードする塩基配列を含む組換えDNA分子。
〔23〕 コーディング塩基配列が、好ましくはそれを発現させることのできる調節配列に結合されている、〔22〕記載のDNA分子。
〔24〕 〔23〕記載のDNA分子を含むように改変された細胞または細胞系。
〔25〕 所望の抗原に特異的な免疫グロブリンまたは類似体を産生するために該塩基配列が発現されるような条件下で、〔24〕記載の細胞または細胞系を培養すること、および免疫グロブリンまたは類似体を回収することを含む、所望の抗原に特異的な免疫グロブリンまたは類似体を産生するための方法。
〔26〕 遷移状態類似体(transition state mimics)、白血球マーカー、組織適合性抗原、接着分子、インターロイキン、インターロイキンレセプター、ケモカイン、増殖因子、増殖因子レセプター、インターフェロンレセプター、免疫グロブリンおよびそのレセプター、腫瘍抗原、アレルゲン、ウイルス蛋白質、毒素、血液因子、酵素、ならびにその他の抗原であるガングリオシドGD3、ガングリオシドGB2、LMP1、LMP2、好酸球主要塩基性蛋白質、好酸球カチオン蛋白質、pANCA、アマドリ蛋白質、IV型コラーゲン、糖化脂質、λインターフェロン、A7、P-糖蛋白質、Fas(AFO-1)、および酸化LDLからなる群より選択される抗原と特異的に免疫反応する、ヒト抗体またはその類似体。
〔27〕 白血球マーカーが、CD2、CD3、CD4、CD5、CD6、CD7、CD8、CD11a、b、c、CD13、CD14、CD18、CD19、CD20、CD22、CD23、CD27およびそのリガンド、CD28およびそのリガンドB7.1、B7.2、B7.3、CD29およびそのリガンド、CD30およびそのリガンド、CD40およびそのリガンドgp39、CD44、CD45およびアイソフォーム、CDw52(キャンパス(Campath)抗原)、CD56、CD58、CD69、CD72、CTLA-4、LFA-1、ならびにTCRからなる群より選択され、
組織適合性抗原が、MHCクラスIまたはII、ルイスy抗原、SLex、SLey、Slea、およびSLebからなる群より選択され、
接着分子が、VLA-1、VLA-2、VLA-3、VLA-4、VLA-5、VLA-6、αVβ3、およびLFA-1、Mac-1、p150,95、αvβ1、gpIIbIIIa、αRβ3、ασβ4、αvβ5、αvβ6、およびαvβ7、L-セレクチン、P-セレクチン、およびE-セレクチン、ならびにそれらのカウンターレセプターであるVCAM-1、ICAM-1、ICAM-2、およびLFA-3からなる群より選択され、
インターロイキンが、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-14、およびIL-15からなる群より選択され、
インターロイキンレセプターが、IL-1R、IL-2R、IL-3R、IL-4R、IL-5R、IL-6R、IL-7R、IL-8R、IL-9R、IL-10R、IL-11R、IL-12R、IL-13R、IL-14R、およびIL-15Rからなる群より選択され、
ケモカインが、PF4、RANTES、MIP1α、MCP1、NAP-2、Groα、Groβ、およびIL-8からなる群より選択され、
増殖因子が、TNFアルファ、TGFベータ、TSH、VEGF/VPF、PthrP、EGFファミリー、FGF、PDGFファミリー、エンドセリン、フィブロシン(F3F−1)、ヒト・ラミニン、およびガストリン解離ペプチド(GRP)からなる群より選択され、
増殖因子レセプターが、TNFアルファR、RGFベータR、TSHR、VEGFR/VPFR、FGFR、EGFR、PTHrPR、PDGFRファミリー、EPO-R、GCSF-R、およびその他の造血レセプターからなる群より選択され、
インターフェロンレセプターが、IFNαR、IFNβR、およびIFNγRからなる群より選択され、
Igおよびそのレセプターが、IgE、FceRI、およびFCeRIIからなる群より選択され、
腫瘍抗原が、her2-neu、ムチン、CEA、およびエンドシアリンからなる群より選択され、
アレルゲンが、ハウスダストダニ抗原、lol p1(草)抗原、およびウルシオールからなる群より選択され、
ウイルス蛋白質が、CMV糖蛋白質B、H、およびGCIII、HIV-1のエンベロープ糖蛋白質、RSVのエンベロープ糖蛋白質、HSVのエンベロープ糖蛋白質、EBVのエンベロープ糖蛋白質、VZVのエンベロープ糖蛋白質、HPVのエンベロープ糖蛋白質、肝炎ファミリーの表面抗原からなる群より選択され、
毒素が、シュードモナスの内毒素およびオステオポンチン/ウロポンチン(osteopontin/uropontin)、ヘビ毒、クモ毒、およびハチ毒からなる群より選択され、
血液因子が、補体C3b、補体C5a、補体C5b-9、Rh因子、フィブリノーゲン、フィブリン、およびミエリン結合増殖阻害因子からなる群より選択され、
酵素が、コレステロールエステル転移蛋白質、膜結合基質メタロプロテアーゼ、およびグルタミン酸デカルボキシラーゼ(GAD)からなる群より選択される、〔26〕記載の抗体またはその類似体。
〔28〕 所望の抗原が、ヒトIL-6、ヒトIL-8、ヒトTNFα、ヒトCD4、ヒトL-セレクチン、ヒトgp39、ヒトIgE、および破傷風毒素C(TTC)からなる群より選択される、〔26〕記載の抗体またはその類似体。
〔29〕 所望の抗原がヒトIL-6である、〔19〕記載の抗体またはその類似体。
〔30〕 所望の抗原がヒトIL-8である、〔19〕記載の抗体またはその類似体。
〔31〕 所望の抗原がヒトTNFαである、〔19〕記載の抗体またはその類似体。
〔32〕 所望の抗原がヒトCD4である、〔19〕記載の抗体またはその類似体。
〔33〕 所望の抗原がヒトL-セレクチンである、〔19〕記載の抗体またはその類似体。
〔34〕 所望の抗原がヒトgp39である、〔19〕記載の抗体またはその類似体。
〔35〕 所望の抗原が破傷風毒素C(TTC)である、〔19〕記載の抗体またはその類似体。
〔36〕 所望の抗原がヒトIgEである、〔19〕記載の抗体またはその類似体。
〔37〕 所望の抗原がヒトαVβ3である、〔19〕記載の抗体またはその類似体。
〔38〕 所望の抗原がヒトフィブロシンである、〔19〕記載の抗体またはその類似体。
〔39〕 所望の抗原がヒトPTHrPである、〔19〕記載の抗体またはその類似体。
〔40〕 アゴニストまたは触媒である、〔26〕記載の抗体またはその類似体。
〔41〕 〔26〕〜〔40〕のいずれかに記載の抗体をコードする組換えDNA分子。
〔42〕 発現を可能にする調節配列に機能的に結合されている抗体またはその類似体をコードする塩基配列を有する、〔26〕〜〔40〕のいずれかに記載の抗体またはその類似体のための発現システムを含む組換えDNA分子。
〔43〕 〔42〕記載のDNA分子を含むように改変された組換え宿主細胞。
〔44〕 コード配列が発現されるような条件下で、〔43〕記載の細胞を培養すること、および産生された抗体または類似体を回収することを含む、抗体またはその類似体を産生するための方法。
〔45〕 ATCCアクセッション番号74367で識別されるyH1Cヒト重鎖YAC。
一般に、本発明に係る方法は、ヒト型の免疫特異的試薬が必要とされる抗原を、内因性の抗体ではなくヒト抗体を産生することができるよう遺伝的に改変されたヒト以外のトランスジェニック動物に投与することを含む。典型的には、この動物は、ゲノム中の内因性の重鎖および/またはカッパ軽鎖が不活化するように改変されているため、これらの内因性の遺伝子座は、抗原に応答して免疫グロブリンをコードする遺伝子を生成するために必要な再編成ができない。さらにこの動物は、少なくとも1個のヒト重鎖遺伝子座および少なくとも1個のヒト軽鎖遺伝子座をそのゲノムの中に安定的に備えており、それによって投与された抗原に応答してこの抗原に対する免疫特異的なヒト可変領域をコードする遺伝子を提供するために、ヒトの遺伝子座を再編成することができる。
所望の抗体を産生するための第一の段階は、抗原の投与である。この投与のための技術は常法であり、それには抗原そのものの性質に応じた適切な免疫化プロトコールおよび処方が含まれる。免疫原性を増強するために担体と共に抗原を投与すること、および/またはアジュバントを含む処方剤を用いること、および/または複数回の注射で投与すること、および/または免疫する経路を様々に変えることが必要となる可能性もある。このような技術は標準的なものであり、これらの最適化は、免疫特異的試薬が必要とされている特定の抗原の特徴に依存する。
CD2、CD3、CD4、CD5、CD6、CD7、CD8、CD11a、b、c、CD13、CD14、CD18、CD19、CD20、CD22、CD23、CD27およびそのリガンド、CD28およびそのリガンドであるB7.1、B7.2、B7.3、CD29およびそのリガンド、CD30およびそのリガンド、CD40およびそのリガンドであるgp39、CD44、CD45およびアイソフォーム、Cdw52(キャンパス(Campath)抗原)、CD56、CD58、CD69、CD72、CTLA-4、LFA-1、ならびにTCRなどの白血球マーカー、
MHCクラスIまたはII、ルイスY抗原、Slex、Sley、Slea、およびSlebなどの組織適合性抗原、
VLA-1、VLA-2、VLA-3、VLA-4、VLA-5、VLA-6、LFA-1、Mac-1、αVβ3、およびp150,95などのインテグリンを含む接着分子、
L-セレクチン、E-セレクチン、およびP-セレクチンなどのセレクチン、ならびにそれらのカウンターレセプターであるVCAM-1、ICAM-2、およびLFA-3、
IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-14、およびIL-15などのインターロイキン、
IL-1R、IL-2R、IL-3R、IL-4R、IL-5R、IL-6R、IL-7R、IL-8R、IL-9R、IL-10R、IL-11R、IL-12R、IL-13R、IL-14R、およびIL-15などのインターロイキンレセプター、
PF4、RANTES、MIP1α、MCP1、IP-10、ENA-78、NAP-2、Groα、Groβ、およびIL-8などのケモカイン(chemokine)、
TNFアルファ、TGFベータ、TSH、VEGF/VPF、PTHrP、EGFファミリー、FGF、PDGFファミリー、エンドセリン、フィブロシン(F3F−1)、ラミニン、およびガストリン解離ペプチド(GRP)などの増殖因子、
TNFアルファR、RGFベータR、TSHR、VEGFR/VPFR、FGFR、EGFR、PTHrPR、PDGFRファミリー、EPO-R、GCSF-R、および他の造血レセプターなどの増殖因子レセプター、
IFNαR、IFNβR、IFNγRなどのインターフェロンレセプター、
Ig、ならびに、IGE、FceRIおよびFceRIIなどのIgレセプター、
her2-neu、ムチン、CEA、およびエンドシアリンなどの腫瘍抗原、
ハウスダストダニ抗原、lol p1(草)抗原、およびウルシオールなどのアレルゲン、
CMV糖蛋白質B、H、およびgCIII、HIV-1のエンベロープ糖蛋白質、RSVのエンベロープ糖蛋白質、HSVのエンベロープ糖蛋白質、EBVのエンベロープ糖蛋白質、VZVのエンベロープ糖蛋白質、HPVのエンベロープ糖蛋白質、肝炎ファミリーの表面抗原などのウイルス蛋白質、
シュードモナスの内毒素およびオステオポンチン/ウロポンチン(osteopontin/uropontin)、ヘビ毒、クモ毒、およびハチ毒などの毒素、
補体C3b、補体C5a、補体C5b-9、Rh因子、フィブリノーゲン、フィブリン、およびミエリン結合増殖阻害因子などの血液因子、
コレステロールエステル転移蛋白質、細胞膜結合基質メタロプロテアーゼ、およびグルタミン酸デカルボキシラーゼ(GAD)などの酵素、ならびに
ガングリオシドGD3、ガングリオシドGM2、LMP1、LMP2、好酸球主要塩基性蛋白質、PTHrp、エオシン好性カチオン蛋白質、pANCA、アマドリ(Amadori)蛋白質、IV型コラーゲン、糖化脂質、γインターフェロン、A7、P-糖蛋白質とFas(AFO-1)、および酸化LDLを含むその他の抗原。
8週〜20週齢のゼノマウスを3匹から5匹、週齢を合わせて、フロイント不完全アジュバントに乳状化した50μgのヒトIL-6を初回免疫として腹腔内投与することにより免疫し、以後の注射では、完全フロイントアジュバントに入れて免疫した。マウスは、2〜3週間の間隔で6回注射した。血清の滴定濃度は、二回目の投与後に決定し、その後も各投与後に決定した。注射後6日〜7日目に、後延髄叢から採血した。この血液は、血清を分離して採集する前に室温に約2時間おいて凝固させてから、4℃で少なくとも2時間インキュベートした。
ヒトIL-6の代わりに組換えヒトTNFα(1回の注射につき5μg)を用いたこと以外は、実施例1で説明したようにして、免疫および血清の調製を行なった。ELISAプレートの最初のコーティングに、コーティング用緩衝液中1μg/mlの組換えヒトTNFαを100μl/ウェル用いたこと以外は、実施例1で説明したようにして、ELISAを行なった。
トランスフェクションされた組換え細胞上のヒトCD4ζを用いて、以下のようにして、ヒトCD4抗原を表面蛋白質として調製した。ヒトCD4ζは、CD3複合体の成熟ζ鎖のCD4細胞外ドメイン、CD4膜通過ドメイン、および31〜142残基目に相当する細胞質ドメインを含む。「ロバーツ(Roberts)ら、Blood (1994) 84:2878」で説明されているヒトCD4ゼータ(F15 LTR)を、「フィナー(Finer)ら、Blood (1994) 83:43」に記載のカット(Kat)高効率形質導入法を用いて、「カラン(Callan), M.,ら、Proc. Natl. Acad. Sci. USA (1993) 90:10454」に記載されているラットの好塩基球性白血病細胞系RBL-2H3に導入した。要約すると、ウェルあたり106個のRBL-2H3細胞を、750μlのDMEMlow+20% FBS(ギブコ(Gibco))および16μg/mlのポリブレン中で、等量のプロウイルス上清とともに、37℃、5%CO2で2時間培養した。培地1mlを取り除いて750μlの感染用培地およびレトロウイルス上清を各ウェルに加え、この培養液を一晩インキュベートした。細胞を洗浄し、十分な細胞が選別に利用できるようになるまで、DMEMlow+10% FBS中で増殖させた。FACSTARプラス(ベクトンディキンソン(Becton Dickinson))を用いて、CD4ゼータを導入したRBL-2H3細胞を選別した。マウス抗ヒトCD4 PE抗体で、ヒトCD4に対する細胞の染色を行ない、上位2〜3%の発現細胞を選抜した。
マウスプレB細胞300.19をLAM-1 cDNA(LAM-1は、L-セレクチンをコードする遺伝子である)でトランスフェクションして得られた高発現クローンであるC51細胞の表面提示蛋白質として(テッダー(Tedder)ら、J. Immunol. (1990) 144:532)または同じようにトランスフェクションされたCHO細胞を用いて、抗原を調製した。抗-Leu-8抗体を標識に用いた蛍光活性化細胞選別法を用いて、トランスフェクションされた細胞を選別した。
ELISAのために、トランスフェクションした細胞を96穴プレートに接種し、細胞数により1日〜2日間細胞が単層になるまで増殖させ、集密状態になったときにELISAに用いた。冷却1×PBSによる最初の洗浄によって細胞を固定し、その後固定液(5%氷酢酸、95%エタノール)を加えた。プレートを−25℃で5分間インキュベートして、プレートシーラーでシールすることにより、この温度で保存することができる。
末梢血を、正常な任意提供者から100ユニット/mlヘパリンで採集した。約3.5mlの血液で、等量の一段階ポリモルフ・グラジエント(One-step Polymorph Gradient)(アキュレイトケミカル(Accurate Chemical), Westbury, NY)の上を覆い、20℃、450×gで30分間遠心した。好中球分画を除去して、DPBS/2% FBSで2回洗浄した。
(1)C51細胞(L-セレクチンを発現する)で免疫したゼノマウスから採取した抗血清、
(2)陰性対照として、ヒトgp39を発現する細胞で免疫したゼノマウスから採取した抗血清。
gp39(CD40に対するリガンド)は、活性化されたヒトCD4 T細胞で発現される。本実施例にしたがって組換えgp39で免疫したゼノマウス(登録商標)の血清には、gp39に免疫特異的な完全なヒト抗体が含まれていた。
本実施例において調製される抗体は、破傷風毒素で免疫したゼノマウスのB細胞を不死化して得たハイブリドーマによって分泌された。免疫化のプロトコールは、実施例1に示したものと同じで、腹腔内注射により初回免疫するために完全フロイントアジュバントに乳濁させた50μgの破傷風毒素を用い、以後は不完全フロイントアジュバントに取り込んだ抗原で腹腔内注射を行なった。マウスは、2〜3週間の間隔で合計4回注射した。
ゼノマウス-2(登録商標)の群を、ラトクリファら(Ratcliffeら、J. Immunol. Methods 127:109 (1990))の説明に従って、BTGに結合したPTHrp(1-34)または4分岐MAP(多重抗原性ペプチドシステム)として合成されたPTHrp(1-34)のいずれかを用いて腹腔内注射により免疫した。抗原は、CFA(完全フロイントアジュバント)に乳濁させ、動物1匹あたり25μgの用量を2週間の間隔で腹腔内注射し、2回注射した後採血した。この採血で得た血清を、前記で説明したようにしてELISAで解析した。
免疫および血清の調製は、ヒト組換えIL-8を免疫原として使用したこと以外、実施例1に説明されたところに従った。
8週〜10週齢のゼノマウス(登録商標)4〜6匹のグループを用いて、免疫およびハイブリドーマ作製を行なった。初回免疫には、完全フロイントアジュバント(CFA、シグマ(Sigma))に乳濁させた25μgの組換えヒトIL-8(バイオソースインターナショナル(Biosource International), CA, USA)を腹腔内投与してゼノマウス(登録商標)を免疫した。以後の注射はすべて、不完全フロイントアジュバント(IFA、シグマ(Sigma))に取り込ませた抗原によって行なった。ハイブリドーマ作製のために脾臓の供与体として用いた動物に対しては、リン酸緩衝食塩水(PBS)に入れた抗原の最終投与を、融合の4日前に行なった。免疫したゼノマウス(登録商標)の血清滴定濃度について、2回目の抗原投与の後に解析を開始し、その後抗原投与する度に解析した。注射後6日〜7日目に、後延髄叢から試験採血した。この血液は、血清を分離して採集する前に、室温に約2時間おいて凝固させてから4℃で少なくとも2時間インキュベートした。
予め抗原で免疫したゼノマウス(登録商標)から採取した脾臓細胞を、ガルフールら(Galfre, G.ら、Methods in Enzymology 73:3〜46, (1981) )の説明に従って、bcl-2(NSO-bcl2)でトランスフェクションした非分泌性NSOミエローマ細胞と融合させた。要約すると、融合は、洗浄した脾臓細胞とミエローマ細胞とを5:1の割合で混合し、800×gの遠心分離でゆっくりと沈澱させることにより行った。上清を完全に除去した後、細胞に、1mlの50%PEG/DMSO(分子量1500のポリエチレングリコール、10%DMSO、シグマ(Sigma))を1分間以上かけて加えて処理し、この混合液をさらに1分間インキュベートし、そして、2mlのDMEMで2分間以上かけて徐々に希釈してから、8mlのDMEMで3分以上かけてさらに希釈した。この処理は、37℃でゆっくり撹拌を続けながら行った。融合後、細胞を、37℃、空気中10% CO2で培養するために、HATを含み、L-グルタミン、ペニシリン/ストレプトマイシンを添加した15%FCS DMEMに再懸濁した。この細胞を、平底の微量滴定用96穴プレートに接種した。培養物は、HT添加培地に移植するまでの2週間、HAT添加培地で維持した。ハイブリッド細胞の増殖を見るために定期的に培養液を調べ、上述したような抗原特異的なELISAで、ヒトμ鎖、ヒトγ2鎖、およびヒトκ鎖の存在について一次スクリーニング解析を行うために、ハイブリドーマを含むウェルの上清を回収した。陽性の培養液を48穴プレートに移植し、集密状態になったら24穴プレートに移植した。ヒトμ鎖、ヒトγ2鎖、およびヒトκ鎖の存在に関する抗原特異的なELISAで、上清を試験した。
これらの抗体の動力学的パラメータ、特に、オン・オフの速度および解離定数(KD)を決定するために、バイオコア(BIOcore)装置(ファルマシア(Pharmacia))で解析した。バイオコア(BIOcore)装置は、抗原でコートした金のチップへの抗体の結合を測定するために、プラスモンの共鳴を利用する。
バイオコア(BIOcore)の試薬および仕組み:
IL-8に特異的な完全なヒトモノクローナル抗体の解離ならびに結合速度および見かけ上の親和性定数の決定。
d[AB]/dt=ka[A][B]-kd[AB]
ここで、Bは表面に固定されており、Aは一定の濃度Cで注入される。この反応は、複合体[AB]の濃度の測定値であり、濃度の項はすべて、バイオコア(BIOcore)の反応単位(RU)として表すことができる。
dR/dt−kaC(Rmax−R)−kdR
ここで、dR/dtは、シグナルの変化速度であり、Cは解析物質の濃度、Rmaxは解析物質の最大結合能力をRUで表したもの、Rは時間tにおけるシグナルをRUで表したものである。この解析において、kaおよびkdの値は、センサーの表面上の固定化リガンドの濃度とは無関係である。製造業者によって提供されるソフトウェアのBIA評価2.1(BIA evaluation 2.1)を用いて、解離速度(kd)および結合速度(ka)を決定した。固定化IL-8を含む表面へのハイブリドーマ上清の注入が完了した後、一定の緩衝液流速45μl/分で10分間続く解離相の間、解離速度定数を測定した。結合相は、流速45μl/分で1.25分以上続くが、このデータは、この前に決定されたkd値を用いたモデルにおいて適合する。異なる濃度の抗IL-8ハイブリドーマ上清について、結合を試験し動力学的なデータを解析するときには、異なるレベルの固定化リガンドを有する少なくとも2つの表面を用いた。これら2つの表面で決定された動力学的定数を表4に示す。親和性は、多様であり、7×10−11から2×10−9 Mの範囲で決定された。これは、正常なマウスから得たマウスモノクローナル抗体の親和性と全く変わらない値である。
IL-8のインビボでの一次的な機能は、好中球を誘引して活性化することである。好中球は、AレセプターおよびBレセプターと名づけられた2つの異なるIL-8のレセプターを表面に発現させている。完全なヒト抗体がIL-8の活性を中和できるか否かを判定するために、ヒト好中球を用いて、2つの異なるインビトロ解析を行なった。一つ目の解析方法では、放射性標識したIL-8が好中球のIL-8レセプターに結合することを、抗体によってブロックできるか否かを試験した。二番目の解析方法では、抗体によって、IL-8に誘導された好中球応答、すなわち好中球の表面におけるインテグリンMac-1の上昇制御がブロックされるか否かを試験した。Mac-1には、2本のペプチド鎖、CD11bとCD18とが含まれる。典型的には、抗CD11b抗体が検出のために用いられる。
好中球の単離:
IL-8レセプター結合解析:
好中球CD11b(Mac-1)発現解析:
表5に示すように、6つの抗体のうち5つがCD11bの上昇制御をある程度ブロックし、5つのうち3つが完全に遮断した。
すべての配列は、ヒトVHおよびヒトVκファミリーに特異的なプライマー(マークス(Marks)ら、1991; Euro. J. Immunol. 21:985〜991)、ならびにヒトγ2定常領域に特異的なプライマー(MG-40d; 5'-GCTGAGGGAGTAGAGTCCTGAGGACTGT-3';配列番号21)またはヒトκ定常領域に特異的なプライマー(HKP2;グリーン(Green)ら、1994; Nature Genetics 7:13〜21)を用いて、ハイブリドーマD1.1、K2.2、K4.2、およびK4.3から調製されたRNAのRT-PCR反応によって生成されたPCR断片のダイレクトシークエンシングによって得られた。図16A〜Hにおいて、4つのクローンの両鎖を配列決定し、完全な配列になるよう解析した。すべての配列は、「V BASE 配列ディレクトリ」(トムリンソン(Tomlinson)ら、MRC Centre for Protein Engineering, Cambridge, UK)に対するアラインメントによって解析した。可変領域とJ(結合)領域とを括弧[ ]で示した。「N」を含む塩基は、作成された配列の中で不確かなものであることを示している。
サッカロマイセス・セレビシアエ(S. cerevisiae)に含まれているyH1Cは、米国メリーランド州ロックビル20852、パークローンドライブ(Parklawn Drive)12301にある米国標準培養株コレクション(American Type Culture Collection)(「ATCC」)に、1996年4月26日に寄託し、ATCC寄託番号74367を与えられた。このYACの寄託は、例示の目的でのみなされたものであり、本発明の請求内容を実施するためにこの寄託物が必要であるということを出願人が認めていることを意味するのではない。
Claims (44)
- 所望の抗原に特異的なヒト免疫グロブリンまたはその類似体を産生するための、以下の段階を含む方法:
免疫応答を刺激しそれにより該抗原に特異的な免疫グロブリンを分泌するB細胞をヒト以外の動物で産生させるような条件下で、内因性の免疫グロブリンの重鎖および軽鎖を実質的に産生できないがヒト免疫グロブリンを産生することができる該動物に、該抗原またはその免疫原部位を投与する段階、および
該免疫グロブリンまたは類似体を回収する段階。 - 回収段階に、動物からポリクローナル免疫グロブリンまたは類似体を回収することが含まれる、請求項1記載の方法。
- 回収段階に以下の段階が含まれる、請求項1記載の方法:
抗原を用いて免疫した動物のB細胞を不死化する段階、その結果できた不死化細胞を、該抗原に特異的な免疫グロブリンを分泌するものを得るためにスクリーニングする段階、および
a)該不死化B細胞によって分泌される免疫グロブリンを回収する段階、または
b)不死化B細胞から少なくとも免疫グロブリンをコードする遺伝子を回収し、選択的には該遺伝子を改変し、
免疫グロブリンもしくは類似体を産生するために、該遺伝子もしくはその改変遺伝子を発現させ、
該免疫グロブリンもしくは類似体を回収する段階。 - 回収段階に以下の段階が含まれる、請求項1記載の方法:
動物の初期B細胞から免疫グロブリンをコードする遺伝子を回収する段階、
免疫グロブリンを発現する該遺伝子のライブラリーを作製する段階、
抗原にとって望ましい親和性を有する免疫グロブリンを得るために該ライブラリーをスクリーニングする段階、
免疫グロブリンをコードする遺伝子を回収する段階、
免疫グロブリンまたは類似体を産生させるために、回収した該遺伝子を発現させる段階、および該免疫グロブリンまたは類似体を回収する段階。 - 請求項1記載の方法によって産生される免疫グロブリンまたは類似体をコードする塩基配列を含む組換えDNA分子。
- コーディング塩基配列が、それを発現させることのできる調節配列に機能的に結合されている、請求項5記載のDNA分子。
- 請求項6記載のDNA分子を含むように改変された細胞または細胞系。
- 完全なヒト免疫グロブリンまたは類似体を産生させるために該コーディング塩基配列が発現されるような条件下で、請求項7記載の細胞を培養すること、および該免疫グロブリンまたは類似体を回収することを含む、完全なヒト免疫グロブリンまたはその類似体を産生するための方法。
- 請求項3記載の方法によって調製される遺伝子または改変遺伝子に相当する塩基配列を含むDNA分子。
- コーディング塩基配列が、それを発現させることのできる調節配列に機能的に結合されている、請求項9記載のDNA分子。
- 請求項9記載のDNA分子を含むように改変された細胞または細胞系。
- 完全なヒト免疫グロブリンまたは類似体を産生させるために該コーディング塩基配列が発現されるような条件下で、請求項11記載の細胞を培養すること、および該免疫グロブリンまたは類似体を回収することを含む、完全なヒト免疫グロブリンまたはその類似体を産生するための方法。
- 請求項4記載の方法によって調製される、望ましい親和性を有するヒト免疫グロブリンをコードする塩基配列を含むDNA分子。
- コーディング塩基配列が、それを発現させることのできる調節配列に機能的に結合されている、請求項13記載のDNA分子。
- 請求項13記載のDNA分子を含むように改変された細胞または細胞系。
- 完全なヒト免疫グロブリンまたは類似体を産生させるために該コーディング塩基配列が発現されるような条件下で、請求項15記載の細胞を培養すること、および該免疫グロブリンまたは類似体を回収することを含む、完全なヒト免疫グロブリンまたはその類似体を産生するための方法。
- 請求項3の記載に従って調製される所望の抗原に対して完全なヒト免疫グロブリンを分泌する不死化B細胞。
- 請求項17記載の細胞を培養すること、および該免疫グロブリンまたは類似体を回収することを含む、免疫グロブリンまたは類似体を産生するための方法。
- 請求項1記載の方法によって産生される、完全なヒト免疫グロブリンまたは類似体。
- 所望の抗原が以下からなる群より選択される、請求項19記載の免疫グロブリンまたはその類似体:
CD2、CD3、CD4、CD5、CD6、CD7、CD8、CD11a、b、c、CD13、CD14、CD18、CD19、CD20、CD22、CD23、CD27およびそのリガンド、CD28およびそのリガンドB7.1、B7.2、B73、CD29およびそのリガンド、CD30およびそのリガンド、CD40およびそのリガンドgp39、CD44、CD45およびアイソフォーム、CDw52(キャンパス(Campath)抗原)、CD56、CD58、CD69、CD72、CTLA-4、LFA-1、ならびにTCRからなる群より選択される白血球マーカー、
MHCクラスIまたはII、ルイスY抗原、SLex、SLey、SLea、およびSLebからなる群より選択される組織適合性抗原、
VLA-1、VLA-2、VLA-3、VLA-4、VLA-5、VLA-6、αVβ3、LFA-1、Mac-1、p150,95、αvβ1、gpIIbIIIa、αRβ3、ασβ4、αvβ5、αvβ6、およびαvβ7からなる群より選択されるインテグリン、
L-セレクチン、P-セレクチン、およびE-セレクチン、ならびにそれらのカウンターレセプターであるVCAM-1、ICAM-1、ICAM-2、およびLFA-3からなる群より選択されるセレクチン、
IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-14、およびIL-15からなる群より選択されるインターロイキン、
IL-1R、IL-2R、IL-3R、IL-4R、IL-5R、IL-6R、IL-7R、IL-8R、IL-9R、IL-10R、IL-11R、IL-12R、IL-13R、IL-14R、およびIL-15Rからなる群より選択されるインターロイキンレセプター、
PF4、RANTES、MIP1α、MCP1、NAP-2、Groα、Groβ、およびIL-8からなる群より選択されるケモカイン、
TNFアルファ、TGFベータ、TSH、VEGF/VPF、PTHrP、EGFファミリー、FGF、PDGFファミリー、エンドセリン、フィブロシン(F3F−1)、ヒト・ラミニン、およびガストリン解離ペプチド(GRP)からなる群より選択される増殖因子、
TNFアルファR、RGFベータR、TSHR、VEGFR/VPFR、FGFR、EGFR、PTHrPR、PDGFRファミリー、EPO-R、GCSF-R、およびその他の造血レセプターからなる群より選択される増殖因子レセプター、
IFNCαR、IFNβR、およびIFNλRからなる群より選択されるインターフェロンレセプター、
IgE、FceRI、およびFCERIIからなる群より選択されるIgおよびそのレセプター、
her2-neu、ムチン、CEA、およびエンドシアリンからなる群より選択される腫瘍抗原、
ハウスダストダニ抗原、lol p1(草)抗原、およびウルシオールからなる群より選択されるアレルゲン、
CMV糖蛋白質B、H、およびgCIII、HIV-1のエンベロープ糖蛋白質、RSVのエンベロープ糖蛋白質、HSVのエンベロープ糖蛋白質、HPVのエンベロープ糖蛋白質、肝炎ファミリーの表面抗原からなる群より選択されるウイルス蛋白質、
シュードモナスの内毒素およびオステオポンチン/ウロポンチン(osteopontin/uropontin)、ヘビ毒、クモ毒、およびハチ毒コノトキシン(conotoxin)からなる群より選択される毒素、
補体C3b、補体C4a、補体C4b-9、Rh因子、フィブリノーゲン、フィブリン、およびミエリン結合増殖阻害因子からなる群より選択される血液因子、ならびに
コレステロールエステル転移蛋白質、細胞膜結合基質メタロプロテアーゼ、およびグルタミン酸デカルボキシラーゼ(GAD)からなる群より選択される酵素。 - 所望の抗原が、ヒトIL-6、ヒトIL-8、ヒトTNFα、ヒトCD4、ヒトL-セレクチン、ヒトgp39、ヒトIgE、ヒトαVβ3、ヒトフィブロシン(F3F−1)、ヒト・ラミニン、ヒトPTHrp、および破傷風毒素C(TTC)からなる群より選択される、請求項14記載の免疫グロブリンまたは類似体。
- 請求項19〜21記載の免疫グロブリンまたは類似体をコードする塩基配列を含む組換えDNA分子。
- コーディング塩基配列が、好ましくはそれを発現させることのできる調節配列に結合されている、請求項22記載のDNA分子。
- 請求項23記載のDNA分子を含むように改変された細胞または細胞系。
- 所望の抗原に特異的な免疫グロブリンまたは類似体を産生するために該塩基配列が発現されるような条件下で、請求項24記載の細胞または細胞系を培養すること、および免疫グロブリンまたは類似体を回収することを含む、所望の抗原に特異的な免疫グロブリンまたは類似体を産生するための方法。
- 遷移状態類似体(transition state mimics)、白血球マーカー、組織適合性抗原、接着分子、インターロイキン、インターロイキンレセプター、ケモカイン、増殖因子、増殖因子レセプター、インターフェロンレセプター、免疫グロブリンおよびそのレセプター、腫瘍抗原、アレルゲン、ウイルス蛋白質、毒素、血液因子、酵素、ならびにその他の抗原であるガングリオシドGD3、ガングリオシドGB2、LMP1、LMP2、好酸球主要塩基性蛋白質、好酸球カチオン蛋白質、pANCA、アマドリ蛋白質、IV型コラーゲン、糖化脂質、λインターフェロン、A7、P-糖蛋白質、Fas(AFO-1)、および酸化LDLからなる群より選択される抗原と特異的に免疫反応する、ヒト抗体またはその類似体。
- 白血球マーカーが、CD2、CD3、CD4、CD5、CD6、CD7、CD8、CD11a、b、c、CD13、CD14、CD18、CD19、CD20、CD22、CD23、CD27およびそのリガンド、CD28およびそのリガンドB7.1、B7.2、B7.3、CD29およびそのリガンド、CD30およびそのリガンド、CD40およびそのリガンドgp39、CD44、CD45およびアイソフォーム、CDw52(キャンパス(Campath)抗原)、CD56、CD58、CD69、CD72、CTLA-4、LFA-1、ならびにTCRからなる群より選択され、
組織適合性抗原が、MHCクラスIまたはII、ルイスy抗原、SLex、SLey、Slea、およびSLebからなる群より選択され、
接着分子が、VLA-1、VLA-2、VLA-3、VLA-4、VLA-5、VLA-6、αVβ3、およびLFA-1、Mac-1、p150,95、αvβ1、gpIIbIIIa、αRβ3、ασβ4、αvβ5、αvβ6、およびαvβ7、L-セレクチン、P-セレクチン、およびE-セレクチン、ならびにそれらのカウンターレセプターであるVCAM-1、ICAM-1、ICAM-2、およびLFA-3からなる群より選択され、
インターロイキンが、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-14、およびIL-15からなる群より選択され、
インターロイキンレセプターが、IL-1R、IL-2R、IL-3R、IL-4R、IL-5R、IL-6R、IL-7R、IL-8R、IL-9R、IL-10R、IL-11R、IL-12R、IL-13R、IL-14R、およびIL-15Rからなる群より選択され、
ケモカインが、PF4、RANTES、MIP1α、MCP1、NAP-2、Groα、Groβ、およびIL-8からなる群より選択され、
増殖因子が、TNFアルファ、TGFベータ、TSH、VEGF/VPF、PthrP、EGFファミリー、FGF、PDGFファミリー、エンドセリン、フィブロシン(F3F−1)、ヒト・ラミニン、およびガストリン解離ペプチド(GRP)からなる群より選択され、
増殖因子レセプターが、TNFアルファR、RGFベータR、TSHR、VEGFR/VPFR、FGFR、EGFR、PTHrPR、PDGFRファミリー、EPO-R、GCSF-R、およびその他の造血レセプターからなる群より選択され、
インターフェロンレセプターが、IFNαR、IFNβR、およびIFNγRからなる群より選択され、
Igおよびそのレセプターが、IgE、FceRI、およびFCeRIIからなる群より選択され、
腫瘍抗原が、her2-neu、ムチン、CEA、およびエンドシアリンからなる群より選択され、
アレルゲンが、ハウスダストダニ抗原、lol p1(草)抗原、およびウルシオールからなる群より選択され、
ウイルス蛋白質が、CMV糖蛋白質B、H、およびGCIII、HIV-1のエンベロープ糖蛋白質、RSVのエンベロープ糖蛋白質、HSVのエンベロープ糖蛋白質、EBVのエンベロープ糖蛋白質、VZVのエンベロープ糖蛋白質、HPVのエンベロープ糖蛋白質、肝炎ファミリーの表面抗原からなる群より選択され、
毒素が、シュードモナスの内毒素およびオステオポンチン/ウロポンチン(osteopontin/uropontin)、ヘビ毒、クモ毒、およびハチ毒からなる群より選択され、
血液因子が、補体C3b、補体C5a、補体C5b-9、Rh因子、フィブリノーゲン、フィブリン、およびミエリン結合増殖阻害因子からなる群より選択され、
酵素が、コレステロールエステル転移蛋白質、膜結合基質メタロプロテアーゼ、およびグルタミン酸デカルボキシラーゼ(GAD)からなる群より選択される、請求項26記載の抗体またはその類似体。 - 所望の抗原が、ヒトIL-6、ヒトIL-8、ヒトTNFα、ヒトCD4、ヒトL-セレクチン、ヒトgp39、ヒトIgE、および破傷風毒素C(TTC)からなる群より選択される、請求項26記載の抗体またはその類似体。
- 所望の抗原がヒトIL-6である、請求項19記載の抗体またはその類似体。
- 所望の抗原がヒトIL-8である、請求項19記載の抗体またはその類似体。
- 所望の抗原がヒトTNFαである、請求項19記載の抗体またはその類似体。
- 所望の抗原がヒトCD4である、請求項19記載の抗体またはその類似体。
- 所望の抗原がヒトL-セレクチンである、請求項19記載の抗体またはその類似体。
- 所望の抗原がヒトgp39である、請求項19記載の抗体またはその類似体。
- 所望の抗原が破傷風毒素C(TTC)である、請求項19記載の抗体またはその類似体。
- 所望の抗原がヒトIgEである、請求項19記載の抗体またはその類似体。
- 所望の抗原がヒトαVβ3である、請求項19記載の抗体またはその類似体。
- 所望の抗原がヒトフィブロシンである、請求項19記載の抗体またはその類似体。
- 所望の抗原がヒトPTHrPである、請求項19記載の抗体またはその類似体。
- アゴニストまたは触媒である、請求項26記載の抗体またはその類似体。
- 請求項26〜40のいずれかに記載の抗体をコードする組換えDNA分子。
- 発現を可能にする調節配列に機能的に結合されている抗体またはその類似体をコードする塩基配列を有する、請求項26〜40のいずれかに記載の抗体またはその類似体のための発現システムを含む組換えDNA分子。
- 請求項42記載のDNA分子を含むように改変された組換え宿主細胞。
- コード配列が発現されるような条件下で、請求項43記載の細胞を培養すること、および産生された抗体または類似体を回収することを含む、抗体またはその類似体を産生するための方法。
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CA2219361A1 (en) | 1996-10-31 |
JP2005336200A (ja) | 2005-12-08 |
DE69637481T2 (de) | 2009-04-09 |
EP1709970A1 (en) | 2006-10-11 |
EP1978033A2 (en) | 2008-10-08 |
JP4772092B2 (ja) | 2011-09-14 |
KR20050085971A (ko) | 2005-08-29 |
DE69637481D1 (de) | 2008-05-15 |
JPH11505523A (ja) | 1999-05-21 |
JP2008253268A (ja) | 2008-10-23 |
CA2761116A1 (en) | 1996-10-31 |
JP2013074891A (ja) | 2013-04-25 |
JP2009039127A (ja) | 2009-02-26 |
ATE390933T1 (de) | 2008-04-15 |
WO1996033735A1 (en) | 1996-10-31 |
US20120117669A1 (en) | 2012-05-10 |
US20090149637A1 (en) | 2009-06-11 |
JP2006115839A (ja) | 2006-05-11 |
CA2219361C (en) | 2012-02-28 |
US20130117871A1 (en) | 2013-05-09 |
KR100654645B1 (ko) | 2007-04-04 |
EP1978033A3 (en) | 2008-12-24 |
JP2007167070A (ja) | 2007-07-05 |
JP2009136290A (ja) | 2009-06-25 |
EP0822830B1 (en) | 2008-04-02 |
EP0822830A4 (en) | 2003-03-19 |
EP0822830A1 (en) | 1998-02-11 |
JP2010057507A (ja) | 2010-03-18 |
JP4312259B2 (ja) | 2009-08-12 |
ES2304786T3 (es) | 2008-10-16 |
KR19990008096A (ko) | 1999-01-25 |
AU5632296A (en) | 1996-11-18 |
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