JP6209206B2 - 上皮成長因子受容体の表面抗原上のエピトープおよびその用途 - Google Patents
上皮成長因子受容体の表面抗原上のエピトープおよびその用途 Download PDFInfo
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Description
[EGFRとGC1118のFabとの結合構造の究明]
本研究グループで開発したGC1118(KR 10−2011−0034914A 参照)のFab(fragment antigen binding)とEGFRとの結合構造を究明するため、EGFR/GC1118 Fab結合体結晶のX線回折データはBL26B2 beamline(Spring−8 institution、日本)を利用して獲得し、HKL2000ソフトウェア(HKL Research社、米国)により指標付け(indexing)およびスケーリング(scaling)して、分子置換(molecular replacement:MR)法によりEGFR/GC1118結合体の初期電子密度マップを得た。MR方法を利用するためには、EGFRと標的抗体との結合体と類似の構造を有するタンパク質の3次元構造データが必要であるが、EGFR/GC1118結合体構造の場合、EGFR/セツキシマブ結合体構造を利用した(PDB ID:1YY9)。EGFR/GC1118結合体の初期位相情報は、Molrepプログラム(http://www.ysbl.york.ac.uk/~alexei/molrep.html)を用いて得て、得た初期位相情報に基づいてCoot(Crystallographic Object-Oriented Toolkit,http://www.biop.ox.ac.uk/coot/)を用いてモデルビルディング作業を行い、Refmac5(http://www.ccp4.ac.uk/html/refmac5.html)およびPHENIX(Python-basedHierarchical ENvironment for Integrated Xtallography, http://www.phenix-online.org/)ソフトウェアを用いて精製を完了した。
[酵母細胞表面発現法を用いたGC1118のEGFR上のエピトープの検証]
構造モデルによって明らかになったGC1118に対するEGFRのエピトープ(R353番、F357番、H359番アミノ酸)を検証しようと、当該アミノ酸残基を、結合反応性のないグリシン(glycine)に置換された変異体を作製した後、酵母細胞表面に発現させた。以後、フローサイトメトリー(BD FACSCalibur(登録商標)、米国)を使用して、GC1118と酵母細胞の表面に発現させたEGFR変異体との結合を確認した結果、全てのEGFR変異体においてGC1118との結合力が減少したことを確認した。
ヒトEGFRの細胞外ドメイン(extracellular domain)をエンコードする遺伝子配列を鋳型とし、オーバーラップPCR方法を用いてEGFRアミノ酸配列(配列番号1を参照)のR353、F357、H359のいずれか1つ以上をグリシンに置換した表1に記載の変異体を作製した。
ヒトEGFR遺伝子配列を鋳型とし、AccuPower(登録商標)TLA PCR PreMix(Bioneer社、韓国)とEGFR遺伝子変異を誘導するように設計されたプライマーセット(表2)を使用してPCR反応をさせ、それぞれの変異体を共通に含む遺伝子片2つを合成した(約1150bp、720bp)。合成された2つの遺伝子を1%のアガロースゲルで電気泳動により分離した後、各DNAをキアゲンキット(Qiagen28706、ドイツ)で精製した。続いて、精製された2つの遺伝子セットを1つの鋳型として使用し、配列番号4〜19の配列を有するプライマー(表2参照)を用いて上記と同様の方法でオーバーラップPCR反応をさせ最終EGFR変異体の遺伝子を合成した(図5参照)。合成された遺伝子は、制限酵素NheI/MluI(New England BioLabs、米国)を使用して切断した。表2のプライマー名の後のForは順方向プライマーを、Revは逆方向プライマーを意味する。
酵母表面の発現ベクターpCTCON(Boder、ET et al. Nat Biotechnol1997、15(6):553-7)を、制限酵素NheI/MluIを使用して切断し、1%アガロースゲル電気泳動およびキアゲンキットで精製した。実施例2.1.1において準備したEGFR変異体のDNAとpCTCONとを混合し、T4 DNAligase(New England BioLabs社、米国)を添加して、25℃で2時間反応させた。前記ライゲーション混合物(ligation mixture)を大腸菌XL1−blue細胞(Stratagene、米国)にGene Pulser(登録商標)(Bio−Rad社、米国)を用いた電気穿孔法(electroporation)により形質転換させて、合計2mLの培地で1時間育てた後、カーベニシリン(carbenicillin)(Sigma社、米国)が含まれているLBアガー(agar)プレートに拡散した。以後、37℃において一晩培養した。
上記実施例2.1.2において培養したプレートにおいて、コロニーを50μg/mLのカーベニシリンが含まれているLB培地2mLにて一晩培養した後、キアゲンプラスミドミニキット(Qiagen27106、ドイツ)を用いて組換えプラスミドを分離し、プラスミドに挿入されたEGFR変異体のDNA塩基配列を確認した。塩基配列の決定に用いられたシーケンスプライマーは、配列番号20〜22で表示される配列を有するものを使用し(表3参照)、塩基配列の決定は、公知の方法に基づいてGenotech社(大田、韓国)に依頼して分析した。対照群として、human EGFRの塩基配列を用いた。EGFR変異体のDNA塩基配列を決定した後、決定されたDNA塩基配列をアミノ酸配列に翻訳するWebベースのプログラム(www.expasy.org:DNA to Protein translate tool)を使用して、アミノ酸配列に翻訳した。翻訳結果は、図6に示し、表1に記載された全てのEGFR変異体の遺伝子がpCTCONベクターに正しく挿入されていることを確認した。
2.2.1 EGFR変異体の酵母細胞の表面発現の過程
実施例2.1において確認されたpCTCON−EGFRを、Bio−Rad社製Gene Pulserを用いた電気穿孔法(electroporation)により、EBY100(出芽酵母Saccharomyces cerevisiae)酵母菌株に形質転換した(図1)。形質転換されたコロニーを選別培地であるSD−CAA(20g/Lのグルコース、6.7g/Lのアミノ酸のない酵母窒素原礎培地(yeast nitrogen base without amino acids)、5.4g/LのNa2HPO4、8.6g/LのNaH2PO4H2O、5g/Lカザミノ酸)において30℃で20時間液状培養した。以降EGFR変異体の酵母表面発現は、選別培地であるSG−CAA(20g/Lのガラクトース、6.7g/Lのアミノ酸のない酵母窒素原礎培地、5.4g/LのNa2HPO4、8.6g/LのNaH2PO4H2O、5g/Lのカザミノ酸)で30℃にて20時間の液状培養することにより誘導した。
EGFR変異体の酵母細胞の表面発現の有無は、BD FACSCalibur(登録商標)(Becton Dickinson社)を用いて確認した。約1x107個の発現された酵母を0.1mlのPBSB(りん酸緩衝生理食塩水、pH7.4、1mg/ml BSA)においてanti−c−myc 9E10 mAb(1:100希釈)と反応させて(25℃、30分)、PBSBで洗浄した。再び、R−フィコエリトリン結合ヤギ抗マウスIgG(1:25希釈)と氷にて15分間反応させPBSBにより洗浄した後、BD FACSCalibur(登録商標)を利用して分析した。分析の結果、EGFR変異体が酵母細胞表面によく発現していることを確認した(図7参照)。
酵母細胞の表面に発現されたEGFR変異体とGC1118間の結合をBD FACSCalibur(Becton Dickinson社)を用いて確認した。対照群の抗体としてセツキシマブ(Cetuximab)を使用した。約1x107個の発現された酵母を0.1mlのPBSB(りん酸緩衝生理食塩水、pH7.4、1mg/ml BSA)で各抗体1ugと反応させて(25℃、30分)、PBSBで洗浄した。次に、FITC結合抗ヒトIgG(1:50希釈)と氷にて15分間反応させPBSBで洗浄した後、BD FACSCaliburを利用して、平均蛍光強度を測定した。測定結果は、表4および図8に示した。表4から分かるように、構造によって明らかにした本発明のエピトープであるR353、F357、H359をグリシンに置換した変異体においてGC1118の結合が阻害され、この部位がGC1118のエピトープであることが証明された。
[EGFRに対するGC118とリガンドの競合的結合の分析]
GC1118および表5に記載の6種のEGFRリガンドがEGFRに対する結合部位が同一かまたは類似であるかを調べるために競合的結合の試験を行った。比較群の抗体としてセツキシマブを使用した。まず、2μg/mlでEGFRがコーティングされたプレートに一定濃度の抗体(1.5nM)と、さまざまな濃度のリガンドを同時に反応させる。この際、リガンドの濃度は、抗体:リガンドのモル比が1:1、1:10、1:102、1:103、1:104、1:105になるようにする。常温で30分間反応後、PBST(りん酸緩衝生理食塩水、pH7.4、0.05%Tween20)で5回洗浄する。その後、抗ヒトIgG(Fc特異的)ペルオキシダーゼ結合(Sigma A0170、米国)を室温で30分間反応させ、PBSTにより5回洗浄した後、TMB(3,3’、5,5’−テトラメチルベンジジン)マイクロウェルペルオキシダーゼ基質(KPL50−76−03、米国)溶液を100μLずつ各ウェルに添加した後、405nmでOD値を測定した。リガンド濃度を高めることによりGC1118のEGFRに対する結合力が減少すると、両反応物(抗体、リガンド)が競争しているということであり、これは両反応物のEGFRに対するエピトープが一致するか、非常に近接していることを意味する。抗体とリガンドとの競争状況を図9および表6に示した。その結果、GC1118がセツキシマブに比べEGF、TGF−a、BTC、EPR、HB−EGFの結合を阻害する能力が優れていることが分かる。
[上皮成長因子受容体リガンドによる細胞増殖の誘導抑制効果]
本発明のGC1118およびセツキシマブの上皮成長因子受容体リガンドによって誘導されたがん細胞の増殖抑制能を比較するために、大腸がん細胞株であるLS174T細胞株(ATCC、CL−188)を、細胞数が3,000になるように96ウェルマイクロプレート(Nunc社)に播いた。1日が経過した後、培養培地を除去した後、GC1118と比較抗体セツキシマブを、ウシ血清を含まない培養培地にそれぞれ0.1μg/ml、1μg/ml、10μg/mlの濃度で処理した。抗体処理と同時にEGFRリガンドを種類別に濃度(EGF;50ng/ml、TGF−a;100ng/ml、アンフィレグリン;100ng/ml、エピレグリン;300ng/ml、HB−EGF、50ng/ml、βセルリン;100ng/ml)により処理した後、3日間37℃のCO2インキュベーターで培養した。3日後、がん細胞の増殖程度を測定するためにMTS溶液(CellTiter96 Aqueous One solution Reagent、Promega)を96ウェルプレートにて培養されたがん細胞に40μlずつ添加してから、37℃のCO2インキュベーターで3時間の反応後、490nmにおいてOD値を測定した。
Claims (29)
- RGDSFTH(配列番号2)で表されるアミノ酸配列からなるEGFR上のエピトープ。
- RGDSFTHTP(配列番号3)の配列からなるEGFR上のエピトープ。
- 請求項1または2に記載のエピトープと担体(carrier)が結合された複合体。
- 前記担体は、ペプチド、血清アルブミン、免疫グロブリン、ヘモシアニン、および多糖体から選ばれる1つ以上であることを特徴とする、請求項3に記載の複合体。
- 請求項1または2に記載のエピトープをエンコードするポリヌクレオチド。
- 請求項5に記載のポリヌクレオチドを含む組み換えベクター。
- 前記エピトープが微生物細胞またはウイルス表面に発現するよう誘導するプロモーターまたはシグナルタンパク質をコードする配列を含むことを特徴とする、請求項6に記載の組み換えベクター。
- 請求項6または7に記載の組み換えベクターにより形質転換された組み換え微生物またはウイルス。
- 前記形質転換された組み換え微生物またはウイルスは、組み換え大腸菌、組み換え酵母、組み換えバクテリオファージから選ばれることを特徴とする、請求項8に記載の組み換え微生物またはウイルス。
- 請求項6乃至9のいずれか1項に記載の組み換えベクターを含む形質転換体、組み換え微生物またはウイルスを培養する段階を含む、配列番号2のアミノ酸配列または配列番号3のアミノ酸配列からなるエピトープを生産する方法。
- 請求項1乃至5のいずれか1項に記載のエピトープ、前記エピトープを含む複合体、または前記エピトープをエンコードするポリヌクレオチドを含むがんワクチン組成物。
- 体内に注入の際、抗体形成を増進する役割をする、薬学的に許容可能な免疫補助剤(adjuvant)をさらに含むことを特徴とする、請求項11に記載のがんワクチン組成物。
- 前記免疫補助剤は、アルミニウム塩(Al(OH)3,ALPO4)、スクアレン(squalene)、ソルビタン(sorbitane)、ポリソルベート80(polysorbate 80)、CpG、リポソーム、コレステロール、MPL(monophosphoryl lipid A)およびGLA(glucopyranosyl lipid A)から選ばれる1つ以上であることを特徴とする、請求項12に記載のがんワクチン組成物。
- 前記ポリヌクレオチドは、薬学的に許容可能なベクターに含まれた形態であることを特徴とする、請求項11に記載のがんワクチン組成物。
- 前記薬学的に許容可能なベクターは、ウイルス性ベクターまたは非ウイルス性ベクターであることを特徴とする、請求項14に記載のがんワクチン組成物。
- 配列番号2のアミノ酸配列または配列番号3のアミノ酸配列からなるエピトープ、前記エピトープを含む複合体、または前記エピトープをエンコードするポリヌクレオチドを用いた配列番号2または配列番号3に基づくエピトープに特異的に結合する抗体、またはその抗体のエピトープに対する結合機能を保有している断片を生産する方法。
- 前記抗体は、ポリクローナル抗体、またはモノクローナル抗体であることを特徴とする、請求項16に記載の方法。
- 配列番号2のアミノ酸配列または配列番号3のアミノ酸配列からなるエピトープ、前記エピトープを含む複合体、または前記エピトープをエンコードするポリヌクレオチドを動物に接種して、接種された動物から配列番号2のアミノ酸配列または配列番号3のアミノ酸配列からなるエピトープに特異的に結合する抗体を選別して生産することを特徴とする、請求項16に記載の方法。
- ヒト型化(humanization)段階または脱免疫化(deimmunization)段階をさらに含むことを特徴とする、請求項18に記載の方法。
- 前記ヒト型化段階は、ヒト抗体のFR(framework)に動物から生産された抗体のCDR配列を移植(grafting)する段階を含むことを特徴とする、請求項19に記載の方法。
- 親和度(affinity)を高めたり、免疫原性(immunogenecity)を低下させるために、1つ以上のアミノ酸配列を置換、挿入、欠失する段階をさらに含むことを特徴とする、請求項20に記載の方法。
- 前記動物は、ヒト由来の配列と同一の抗体を生産できるように形質転換された動物であることを特徴とする、請求項18に記載の方法。
- 前記形質転換された動物は、形質転換されたマウスであることを特徴とする、請求項22に記載の方法。
- ディスプレイ技術を利用することを特徴とする、請求項16に記載の方法。
- 前記ディスプレイ技術は、ファージディスプレイ(phage display)、細菌ディスプレイ(bacteria display)、またはリボソームディスプレイ(ribosome display)の中から選択されるいずれかであることを特徴とする、請求項24に記載の方法。
- 前記ディスプレイに用いられるライブラリ(library)は、ヒト由来抗体の配列を有するように設計されたことを特徴とする、請求項24または25に記載の方法。
- 前記配列番号2のアミノ酸配列または配列番号3のアミノ酸配列からなるエピトープ、または前記エピトープを含む複合体を利用して、配列番号2のアミノ酸配列または配列番号3のアミノ酸配列からなるエピトープに特異的に結合する抗体のみを選別(panning)する段階を含むことを特徴とする、請求項24乃至26のいずれか一項に記載の方法。
- 請求項1項乃至5項のいずれか一項の記載によるエピトープ、前記エピトープを含む複合体、または前記エピトープをエンコードするポリヌクレオチドを含むがん診断用組成物。
- 請求項1項乃至5項のいずれか一項の記載によるエピトープ、前記エピトープを含む複合体、または前記エピトープをエンコードするポリヌクレオチドを含むがん診断用キット。
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