JP4658926B2 - 肝癌治療後の再発予測判定薬 - Google Patents
肝癌治療後の再発予測判定薬 Download PDFInfo
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Description
また、本発明は、抗GPC3抗体を用いて被検試料中のGPC3を測定することを特徴とする肝癌治療後の再発予測方法を提供するものである。
本発明は、抗GPC3抗体を用いて被検試料中のGPC3を測定することにより肝癌治療後の再発を予測する方法である。
本発明で用いられる抗GPC3モノクローナル抗体はGPC3タンパク質に特異的に結合すればよく、その由来および形状を問わない。具体的には、マウス抗体、ラット抗体、ヒト抗体、キメラ抗体、ヒト型化抗体などの公知のモノクローナル抗体を用いることができる。又、支持体に固定される抗GPC3モノクローナル抗体と標識物質で標識される抗GPC3モノクローナル抗体はGPC3分子の異なるエピトープを認識することが好ましく、部位は特に制限されない。
まず、抗体取得の感作抗原として使用されるGPC3は、HuH6、HepG2細胞株等を培養してその上清に含まれる天然のGPC3を精製して、得ることができる。さらに、Lage,H.etal.,Gene 188(1997),151−156に開示されたGPC3(MXR7)遺伝子/アミノ酸配列を発現することによっても得ることができる。すなわち、GPC3をコードする遺伝子配列を公知の発現ベクター系に挿入して適当な宿主細胞を形質転換させた後、その宿主細胞中又は培養上清中から目的のヒトGPC3タンパク質を公知の方法で精製する。
次に、この精製GPC3を感作抗原として用いるが、GPC3の部分ペプチドを感作抗原として使用することもできる。この際、部分ペプチドはヒトGPC3のアミノ酸配列より化学合成により得ることもできるし、GPC遺伝子の一部を発現ベクターに組込んで得ることもでき、さらに天然のGPC3をタンパク質分解酵素により分解することによっても得ることができる。部分ペプチドとして用いるGPC3の部分および大きさは限られない。本発明においては、感作抗原として全長GPC3を用いるのが好ましい。
次に、形質転換された宿主細胞をin vitro又はin vivoで培養して目的とする抗体を産生させる。宿主細胞の培養は公知の方法に従い行う。例えば、培養液として、DMEM、MEM、RPMI1640、IMDMを使用することができ、牛胎児血清(FCS)等の血清補液を併用することもできる。
本発明において測定するGPC3は、ヘパラン硫酸などが付加されたGPC3タンパク質でも、GPC3コアタンパク質でもよい。
被検試料に含まれるGPC3の測定は、抗GPC3抗体を用いた免疫学的方法により測定される。免疫学的方法としては、例えば、ラジオイムノアッセイ、エンザイムイムノアッセイ、蛍光イムノアッセイ、発光イムノアッセイ、免疫沈降法、免疫比濁法、ウエスタンブロット、免疫染色、免疫拡散法などを挙げることができるが、好ましくはエンザイムイムノアッセイであり、特に好ましいのは酵素結合免疫吸着定量法(enzyme−linked immunosorbent assay:ELISA)(例えば、sandwich ELISA)である。ELISAなどの上述した免疫学的方法は当業者に公知の方法により行うことが可能である。
完全長ヒトGPC3 cDNAを含むプラスミドDNAを用い、flag付加可溶型GPC3、すなわちリコンビナントGPC3 cDNA発現プラスミドDNAを構築した。C末端側の疎水領域(564−580アミノ酸)を除くように設計した下流プライマー(5’−ATAGAATTCCACCATGGCCGGGACCGTGCGC−3’(配列番号1))とEcoRI認識配列、Kozak配列を加えた上流プライマー(5’−ATAGGATCCCTTCAGCGGGGAATGAACGTTC−3’(配列番号2))を用いてPCRを行った。得られたPCR断片(1711bp)をpCXND2−Flagにクローニングした。作製された発現プラスミドDNAをCHO細胞DXB11株へ導入し、500μg/mL Geneticin での選抜により、可溶型GPC3高発現CHO株を得た。
可溶性GPC3の産生細胞として、肝芽腫由来細胞株であるHuH6細胞を理研細胞バンクよりより購入し、10% FBS/ダルベッコの改変イーグル培地(Dulbecco’s Modified Eagle Medium,DMEM)(SIGMA CAT# D6429)/penicillin・streptomycin(GIBCO BRL CAT# 15140−122)で培養を行った。すなわち、直径150mmのディシュを用い、HuH6細胞の大量培養を行い、培養上清を回収し精製を行った。培養上清をDEAE Sepharose Fast Flow(Amersham CAT#17−0709−01)にチャージし、洗浄後、500mM NaClを含むバッファーにより溶出した。次に、Centriprep−10(Millipore CAT#4304)で濃縮後、Superdex 200 HR 10/30(Amersham CAT# 17−1088−01)によるゲルろ過にて精製することにより粗精製天然型GPC3を得た。
可溶性リコンビナントGPC3あるいは可溶性天然型GPC3を用いて、モルモットポリクローナル抗体の作製を行った。作製には、公知の方法を用いた。GPC3を、アジュバントを用いてエマルジョン化したものを皮下に投与し、免疫を行った。これを数回繰り返し、抗体価が上昇したのを確認した後、採血を行い、血清を得た後、硫安沈殿によりポリクローナル抗体を取得した。
Balb/Cマウス(CRL)5匹に、可溶性リコンビナントGPC3あるいは可溶性天然型GPC3を免疫した。初回免疫には免疫タンパク質を、可溶性天然型GPC3の場合は10μg/匹、可溶性リコンビナントGPC3の場合は100μg/匹となるように調製し、FCA(フロイント完全アジュバント(H37 Ra)、Difco(3113−60)、ベクトンディッキンソン(cat#231131))を用いてエマルジョン化したものを皮下に投与した。2週間後に可溶性天然型GPC3の場合は5μg/匹、可溶性リコンビナントGPC3の場合は50μg/匹となるように調製したものをFIA(フロイント不完全アジュバント、Difco(0639−60)、ベクトンディッキンソン(cat#263910))でエマルジョン化したものを皮下に投与した。以降1週間間隔で追加免疫を合計5回行った。最終免疫については、可溶性天然型GPC3の場合は5μg/匹、可溶性リコンビナントGPC3の場合は50μg/匹となるようにPBSに希釈し尾静脈内に投与した。GPC3コアタンパク質をコートしたイムノプレートを用いたELISAによりGPC3に対する血清中の抗体価が飽和しているのを確認後、マウスミエローマ細胞P3U1とマウス脾臓細胞を混合し、PEG1500(ロシュ・ダイアグノスティック、cat#783 641)により細胞融合を行った。96穴培養プレートに播種し、翌日よりHAT培地で選択後培養上清をELISAでスクリーニングした。陽性クローンについては限界希釈法によりモノクローン化した後、拡大培養を行い培養上清を回収した。ELISAによるスクリーニングは、GPC3コアタンパク質との結合活性を指標に行い、強い結合能を有する抗GPC3モノクローナル抗体を得た。
可溶性天然型GPC3より得られたハイブリドーマは、PPMX008及びPPMX009と命名した。また、可溶性リコンビナントGPC3より得られたハイブリドーマは、PPMX0010およびPPMX031と命名した。
なお、複数得られたハイブリドーマのうち以下に示すELISAにて高感度・特異性に優れたPPMX009をM18D04と命名し、独立行政法人産業技術総合研究所 特許生物寄託センターに寄託した(日本国 〒305−8566 茨城県つくば市東1丁目1番地1 中央第6、原寄託日;2004年9月7日、受託番号;FERM ABP−10325)。
抗体濃度はヤギ抗マウス IgG(gamma)(ZYMED CAT# 62−6600)とアルカリホスファターゼ−ヤギ抗マウス IgG(gamma)(ZYMED CAT# 62−6622)を用いたマウスIgGサンドイッチELISAを行い、市販の精製マウスIgG1抗体(ZYMED CAT#02−6100)をスタンダードとして定量した。
抗GPC3モノクローナル抗体のアイソタイピングは、ImmunoPure Monoclonal Antibody Isotyping Kit II(PIERCE CAT# 37502)を用い、方法は添付のマニュアルに従った。アイソタイピングの結果全てIgG1タイプであった。
血中のGPC3を測定するため、GPC3のサンドイッチELISA系を多数構築し、感度および特異性に優れた測定系を選択した。最も優れた結果は、ポリクローナル抗体であるPPMX042抗体を96ウェルプレートにコートし、M18D04抗体をビオチンで標識した系であった。尚、発色には高い測定感度を達成するためScytek社のTMBを用いた。
(1)対象および方法
実施例5で得られたELISA系(ポリクローナル抗体であるPPMX042抗体を96ウェルプレートにコートし、M18D04抗体をビオチンで標識した系)を用い、下記検体を測定した。
肝癌手術例67例(初発48例、再発19例)で、断端陰性かつ術前診断にて脈管浸潤および遠隔転移を認めなかった症例を対象とした(平均年齢64.2歳(26−88歳)、男:女=55:12例)。無再発生存に寄与する因子について、Cox比例ハザードモデルを用いて検討を行った。
(検討因子)年齢、性別、病因、腫瘍因子(最大腫瘍径、個数)、病理所見(分化度、背景肝、娘結節、脈管侵襲の有無)、術前、術後早期(1〜2週間)、術後中期(2週間〜2ヶ月後)GPC3値。
血清GPC3濃度の測定は、前記実施例5に記載の方法に従い実施した。
再発症例は30例でリンパ節再発、遠隔転移、断端周囲再発をそれぞれ1例、27例は肝内異所性再発であった。観察期間は0.5〜32ヶ月、平均観察期間は9.0ヶ月であった。6ヶ月間の無再発生存率は73%であった。図1に、術前(A)、術後1〜2週間後(B)および術後2週間〜2ヶ月後(C)のGPC3値が1ng/mL未満の患者およびGPC3値が1ng/mL以上の患者の無再発生存率を比較した結果を示す。統計解析は、Log−rank testで行った。その結果、術前、術後早期および術後中期のいずれの場合にもGPC3濃度が1ng/mL未満の患者の再発率は、1ng/mL以上の患者の再発率よりも有意に高かった。(Log−rank testで術前P=0.0443、術後早期P=0.0415、術後中期P=0.0007。)従って、その結果、GPC3濃度測定は手術後再発の予測に有用であり、肝癌治療後の再発の予測因子として血清GPC3が有用なマーカーとなりうることが判明した。
実施例6にて解析した症例のうち他の腫瘍マーカーであるAFP、PIVKA−IIおよびAFP−L3分画を測定した症例の一部を表1に示した。いずれの症例も術後のGPC3濃度がcut off値0.4以上を示した症例である。他の腫瘍マーカーのうち、AFPに関しては、症例834において2回目のTAE後、症例287においては全般にcut off値20以上を示すものの全体的に低値であった。PIVKA−IIにおいては症例975の術後でcut off値40以上を示すものの、他の症例では低値であった。AFP−L3分画においては症例184の3回目TAE後および症例975の再発時にcut off値15以上を示したに過ぎなかった。これら、症例からも他の腫瘍マーカーでは肝癌再発の予知が不可能であるにもかかわらず、GPC3では再発が予知できることが示された。
Claims (6)
- 抗GPC3抗体を含有する肝癌治療後の再発予測判定薬。
- 抗GPC3抗体が、抗可溶性GPC3抗体である請求項1記載の予測判定薬。
- 抗GPC3抗体を用いて被検試料中のGPC3を測定することを特徴とする肝癌治療後の再発予測方法。
- 抗GPC3抗体が、抗可溶性GPC3抗体である請求項3記載の予測方法。
- 被検試料が、血液、血清又は血漿である請求項3又は4記載の予測方法。
- 支持体に固定した抗GPC3抗体と標識物質で標識された抗GPC3抗体を用いる請求項3〜5のいずれか1項記載の予測方法。
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US20070087005A1 (en) | 2005-10-14 | 2007-04-19 | Lazar Gregory A | Anti-glypican-3 antibody |
WO2013070468A1 (en) | 2011-11-08 | 2013-05-16 | The Trustees Of The University Of Pennsylvania | Glypican-3-specific antibody and uses thereof |
EP4119947A1 (en) * | 2012-12-21 | 2023-01-18 | Chugai Seiyaku Kabushiki Kaisha | Gpc3-targeting drug which is administered to patient responsive to gpc3-targeting drug therapy |
TWI693073B (zh) * | 2012-12-21 | 2020-05-11 | 日商中外製藥股份有限公司 | 對gpc3標的治療劑療法為有效之患者投與的gpc3標的治療劑 |
EP3141603A4 (en) | 2014-05-08 | 2017-12-27 | Chugai Seiyaku Kabushiki Kaisha | Gpc3-targeted therapeutic agent for administration to patients for whom gpc3-targeted therapeutic agent therapy is effective |
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US11376326B2 (en) | 2015-07-01 | 2022-07-05 | Chugai Seiyaku Kabushiki Kaisha | GPC3-targeting therapeutic agent which is administered to patient for whom the GPC3-targeting therapeutic agent is effective |
JP6292564B2 (ja) * | 2016-03-10 | 2018-03-14 | シスメックス株式会社 | 肝細胞がん患者の再発リスク予測を補助する方法、装置、コンピュータプログラム製品及びキット |
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