JP6159011B2 - Cd9に対するハイブリドーマクローンおよびモノクローナル抗体 - Google Patents
Cd9に対するハイブリドーマクローンおよびモノクローナル抗体 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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Description
[0001]本出願は、2013年3月15日付けで出願された米国出願番号61/791,477に対して優先権を主張し、これらの全内容および開示は、参照により本明細書に組み入れらる。
[0002]同時に提出された、2014年3月10日に作製された「CD9Seq_List_ST25」と名付けられた1つの3キロバイトのASCII(テキスト)ファイルであるコンピューターで読取り可能なヌクレオチド/アミノ酸配列リストを、参照により本明細書にその全体を組み入れる。
[0003]本出願は、ハイブリドーマクローンおよびモノクローナル抗体に関し、より特定には、CD9タンパク質に対するハイブリドーマクローンおよびモノクローナル抗体、ならびに使用方法に関する。
[0036]あるいは、抗CD9抗体は、モノクローナル抗体であってもよい。例えばKohlerおよびMilstein(1975)Nature 256:495によって説明されている方法などのハイブリドーマ法を使用して、モノクローナル抗体を調製してもよい。ハイブリドーマ法において、典型的には、マウス、ハムスター、または他の適切な宿主動物を免疫剤で免疫化して、免疫剤に特異的に結合すると予想される抗体を生産させるか、またはそのような抗体を生産可能なリンパ球を発生させる。あるいは、このようなリンパ球は、インビトロで免疫化されてもよい。
[0048]ヒトで使用するために、本発明のマウスモノクローナル抗体をヒト化して、免疫原性を低下させてもよい。非ヒト(例えばマウス)抗体のヒト化された形態は、非ヒト免疫グロブリンから誘導された最小限の配列を含有するキメラ免疫グロブリン、免疫グロブリン鎖またはそれらのフラグメント(例えばFv、Fab、Fab’、F(ab’)2など、または抗体の他の抗原−結合部分配列)である。ヒト化抗体は、レシピエントの相補性決定領域(CDR)からの残基が、例えば望ましい特異性、親和性、および能力を有するマウス、ラットまたはウサギなどの非ヒト種のCDR(ドナー抗体)からの残基で置き換えられているヒト免疫グロブリン(レシピエント抗体)を包含する。場合によっては、ヒト免疫グロブリンのFvフレームワーク残基が、それに相当する非ヒト残基で置き換えられている。またヒト化抗体は、レシピエント抗体中、または移入されたCDRまたはフレームワーク配列中にも見出されない残基を含んでいてもよい。一般的には、ヒト化抗体は、少なくとも1つの、典型的には2つの可変ドメインの実質的に全部を含むと予想され、そのうちCDR領域の全部または実質的に全部は、非ヒト免疫グロブリンのCDR領域に相当し、FR領域の全部または実質的に全部は、ヒト免疫グロブリンのコンセンサス配列のFR領域に相当する。最適には、ヒト化抗体はまた、免疫グロブリンの定常領域(Fc)の少なくとも一部、典型的にはヒト免疫グロブリンの定常領域(Fc)の少なくとも一部も含むと予想される(Jonesら(1986)Nature 321:522;Riechmannら(1988)Nature 332:323;およびPresta(1992)Curr.Op.Struct.Biol.2:593)。
[0051]他の実施態様において、上記で説明したような抗体またはフラグメントを医薬的に許容されるキャリアー、希釈剤または添加剤と共に包含する医薬組成物が提供される。
[0064]本発明の抗CD9抗体は、様々な有用性を有する。一実施態様において、抗CD9抗体は、様々な癌細胞またはエキソゾームの表面上でのCD9発現に関する診断または予後アッセイで使用でき、例えば、特異的な細胞、組織、または血清中でのその発現を検出することにおいて使用できる。例えば不均一または均一相のいずれかで行われる競合結合アッセイ、直接または間接サンドイッチアッセイ、および免疫沈降アッセイなどの、当業界公知の様々な診断および予後アッセイ技術を使用できる(Zola、Monoclonal Antibodies:A Manual of Techniques、CRCプレス社(CRC Press, Inc.)(1987)、147〜1581頁)。これらのアッセイで使用される抗体は、検出可能な部分で標識してもよい。検出可能な部分は、検出可能なシグナルを直接的または間接的のいずれかで生産することが可能であると予想される。抗体を検出可能な部分にコンジュゲートする当業界公知のあらゆる方法を採用でき、このような方法としては、Hunterら(1962)Nature、144:945;Davidら(1974)Biochemistry、13:1014;Painら(1981)J.Immunol.Meth.、40:219;ならびにNygren,J.Histochem. and Cytochem.、30:407(1982)によって説明された方法が挙げられる。
[0097]本発明のアッセイを実行するのに必要な試薬を含有する抗体キットが提供される。本キットは、1つまたはそれより多くの区画を包含していてもよく、これらの区画はそれぞれ、例えば:(a)上記で説明した本発明の構成要素のうち1つを含む第一の容器;および(b)以下に示すもの、すなわち:洗浄試薬、検出または競合アッセイのための対照としての抗体またはペプチドおよび/または組換えCD9タンパク質もしくはそれらのフラグメントの存在の検出が可能な試薬のうち1種またはそれより多くを含む1つまたはそれより多くの他の容器などの1つまたはそれより多くの容器を収容する。例えば、いくつかの実施態様において、本キットは、エキソゾーム精製キットまたは細胞単離キットとして設計されてもよい。
[00106]一般的なハイブリドーマ生産およびスクリーニングプロトコール
[00107]実験室での一般的なハイブリドーマ生産のため、加えてハイブリドーマのスクリーニングおよびサブクローニングのために、以下のプロトコールを使用した。
[00108]融合調製物:
[00109]融合の3〜4日前
[00110]1)10%のC−DMEM中に5×105細胞/ml(50ml)で骨髄腫細胞、P3×653(P3細胞)を含む2つのT−75または1つのT−225フラスコをセットアップした。融合の前日に新鮮な培地を添加した。加えて、この期間中に、融合に望ましいリンパ球を生産させるために使用した動物の静脈内にブースター注射を行った。さらに、動物からの組織の回収で使用されるあらゆる器具をオートクレーブした。
[00112]融合の日に、以下に示すような融合用培地を調製した:DMEM(LTI)128ml、HAT(50×;シグマ(Sigma))4ml、OPI(100×;シグマ)2ml、HEPES(1M;シグマ)2ml、グルタマックスI(Glutamax I)(100×;LTI)2ml、NCTC(シグマ)20ml、FBS(LTI)40ml、ペニシリン/ストレプトマイシン(LTI)2ml、ニュートリドーマ(Nutridoma)(BM)2.0ml。加えて、0.5mlの1MのHEPESを含む50mlのSF−DMEM(DMEM/HEPES)を調製した。その後、コニカルチューブに9.5mlのDMEM/HEPESと0.5mlDMSOとを添加した(DMEM/HEPES/DMSO)。
[00115]図2は、融合過程で採用できる工程を例示する。まず、(以下でより詳細に説明される通りに)抗原で予め免疫化したマウスを処分し、脾臓を摘出した。100mmの細胞培養中で10mlのDMEM/HEPES中に各脾臓を入れた。加えて、P3細胞を回収し、5〜20×107個の細胞が融合に使用されるように計数した。
[00120]一次スクリーニング
[00121]まず、コーティング緩衝液(50mMのトリス−Cl、pH9.5)中のおよそ0.5μg/mlの対象のタンパク質 (例えばグルタチオンにコンジュゲートされた配列番号2の野生型CD9CD9ポリペプチド)を1ウェルあたり25μlで、2つの384ウェルプレートをコーティングした。これらの2つの384ウェルプレートを4℃で一晩インキュベートした。
[00124]実験方法
[00125]CD9に対するモノクローナル抗体の生産
[00126]モノクローナル抗体(mAb)を、以下の改変を施した以外はこれまでに述べられたようにして生成した(Azorsaら(1999)J Immunol Methods 229:35〜48):雌Balb/cマウス(6〜8週齢)に、PBS中の3〜500万個の洗浄した生きたLoVo結腸直腸癌細胞(ATCC CCL−229)を、2週間のインターバルで3回腹膜内注射し、それに続いてPBS中の3〜500万個の洗浄した生きたLoVo結腸直腸癌細胞を連続3日注射した。LoVo細胞は、高レベルのCD9を発現することがわかっている。脾細胞を単離して、上記で説明したようにPEG:DMSO(50:5、%v、シグマ−アルドリッチ(Sigma-Aldrich))を使用して骨髄腫細胞株P3×653に融合させた。20%のFBS(インビトロジェン(Invitrogen))、2mMのグルタマックスI(インビトロジェン)、25mMのHepes、1×HAT(シグマ−アルドリッチ)、ペニシリン/ストレプトマイシン、および0.5×ニュートリドーマ−CS(ロシュ(Roche)、ニュージャージー州ブランチバーグ)が補充されたDMEM:NCTC−109(90:10、%v、インビトロジェン、カリフォルニア州カールスバッド)培地中の96ウェルプレートに、融合した細胞を植え付けた。ハイブリドーマコロニーをELISAでスクリーニングし、これを限界希釈で2回サブクローニングした。Z9.1およびZ9.2と名付けられた抗CD9mAbを含有する2つのハイブリドーマクローンからの組織培養上清を収集して、0.02%アジ化ナトリウムを用いて4℃で保存した。
[00129]インキュベート後、研究者は、スクリーニングされた細胞が確実に単一起源のクローンになるようにスクリーニング前に2回の限界希釈を行った。以下の手法を2回繰り返し、そのうち第一の限界希釈は20−HT培地を使用し、第二の限界希釈は20−HY培地を使用した。
[00132]モノクローナル抗体を、Azorsaら(1999)J Immunol Methods 229:35〜48でこれまでに述べられたようにしてスクリーニングした。簡単に言えば、CD9(配列番号2)からの細胞外ドメイン2(EC2)を発現する組換えグルタチオン(GST)コンストラクトを使用して、抗CD9モノクローナル抗体(親クローンおよび限界希釈により得られた関連するサブクローンを包含する、クローンZ9.1およびZ9.2)の結合特異性を評価した。GST−CD9−EC2組換えタンパク質および対照を96ウェルのELISAプレートで平板培養し、分泌されたZ9.1およびZ9.2モノクローナル抗体(抗CD9)を含有するZ9.1およびZ9.2ハイブリドーマ細胞およびサブクローンからの上清で別々に処置した。上清をそのまま室温で1時間インキュベートした。ELISAプレートのウェルをPBS−トゥイーンで3回洗浄した。洗浄後、二次抗体(ジャクソン・イムノリサーチ(Jackson Immunoresearch)からの、5μg/mlのホースラディッシュ−ペルオキシダーゼにコンジュゲートしたヤギ抗マウスFc)を各ウェルに添加し、室温で1時間インキュベートした。プレートのウェルを再度PBS−トゥイーンで3回洗浄した。洗浄後、50μlの基質(ピアースからのOPD)を5〜10分かけて添加した。25μlの2MのH2SO4の添加によって反応を止めた。各ウェル中の得られた溶液の吸光度を490nmで読み取った。
[00134]子宮頚癌細胞株HeLaは、CD9を発現することが知られている。HeLa細胞を十分な密集度になるまで増殖させ、回収し、従来の溶解緩衝液を使用して溶解させた。得られた溶解産物を非還元アクリルアミドゲルで泳動した。溶解産物を探索するために、抗CD9抗体(Z9.1)を含有するハイブリドーマ上清を1:10に希釈して使用した。当業界公知の技術を使用してウェスタンブロットを行った。
[00136]モノクローナル抗CD9抗体のスクリーニング
[00137]まず、抗CD9モノクローナル抗体を生産する選択されたハイブリドーマクローンおよび関連するサブクローンの特異性を評価するために実験を行った。特に、これらのクローンはCD9を発現するLoVo結腸直腸細胞を使用して開発されたため、確認実験を行って、CD9のEC2ドメイン(すなわち、配列番号2)に対する特異性があることを確認した。図3で例示したように、親ハイブリドーマ細胞(親Z9.1および親Z9.2と名付けられた)の上清に含有されていた抗体は、配列番号2に結合することを示した。加えて、Z9.1の一部のサブクローン(59.6F10から始まる条件)およびZ9.2の一部のサブクローン(59.6E11から始まる条件)はいずれも、CD9のEC2ドメインへの結合が成功したことを示した。特に、クローン59.6F10.A4および59.6E11.E8は、陽性対照モノクローナル抗体(ALMA1)と同じかまたはそれよりも高い程度のCD9のEC2ドメインへの結合を示した。これらの2つのクローンをさらにサブクローニングして、Z9.1およびZ9.2ハイブリドーマサブクローンを得た。したがって、このデータから、Z9.1およびZ9.2モノクローナル抗体は、CD9のEC2ドメインに結合する(配列番号2)ことが示された。
Claims (13)
- Z9.1、ATCC受入番号PTA−121025およびZ9.2、ATCC受入番号PTA−121024からなる群より選択される、単離されたハイブリドーマ細胞株によって生産されたモノクローナル抗体。
- 前記モノクローナル抗体が、配列番号1のアミノ酸配列に特異的に結合する、請求項1に記載のモノクローナル抗体。
- 前記抗体が、IgG1、カッパ鎖アイソタイプである、請求項1に記載のモノクローナル抗体。
- 前記抗体が標識されている、請求項1に記載のモノクローナル抗体。
- 前記抗体が、ビオチン標識、蛍光標識、酵素標識、補酵素標識、化学発光標識、および放射性同位体標識からなる群より選択される1種またはそれより多くの標識で標識されている、請求項4に記載のモノクローナル抗体。
- 前記モノクローナル抗体が、配列番号2のアミノ酸配列に特異的に結合する、請求項1に記載のモノクローナル抗体。
- モノクローナル抗体の作製方法であって、該方法は、Z9.1、ATCC受入番号PTA−121025およびZ9.2、ATCC受入番号PTA−121024からなる群より選択されるハイブリドーマ細胞株を提供すること(ここで該ハイブリドーマ細胞株は、ヒト野生型CD9タンパク質に特異的なモノクローナル抗体を生産する)、およびモノクローナル抗体の生産が可能な条件下で選択されたハイブリドーマ細胞株を培養することを含む、上記方法。
- 前記モノクローナル抗体に標識をカップリングすることをさらに含む、請求項7に記載の方法。
- 前記標識が、ビオチン標識、蛍光標識、酵素標識、補酵素標識、化学発光標識、および放射性同位体標識からなる群より選択される、請求項8に記載の方法。
- 前記モノクローナル抗体が、配列番号2のアミノ酸配列に特異的に結合する、請求項7に記載の方法。
- Z9.1、ATCC受入番号PTA−121025およびZ9.2、ATCC受入番号PTA−121024からなる群より選択されるハイブリドーマ細胞株によって生産されたモノクローナル抗体を含有する少なくとも1つの容器を含む、エキソゾーム精製キット。
- 前記モノクローナル抗体が、標識されている、請求項11に記載のエキソゾーム精製キット。
- 前記抗体が、ビオチン標識、蛍光標識、酵素標識、補酵素標識、化学発光標識、および放射性同位体標識からなる群より選択される1種またはそれより多くの標識で標識されている、請求項12に記載のエキソゾーム精製キット。
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