JP2021516254A - 腫瘍抗原に対するl2a5抗体またはその機能的断片 - Google Patents
腫瘍抗原に対するl2a5抗体またはその機能的断片 Download PDFInfo
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Abstract
Description
1)STn発現と、ヒト癌細胞の発癌および転移能力ならびに癌開始細胞との関連(Okasaki et al.,2012)。
2)STnと、患者の予後不良、総生存率の低下および化学療法に対する応答の欠如との相関(Choi et al.,2000)、ならびに3)免疫防御からの回避(Carrascal et al.,2014)。
モノクローナル抗体(mAb)とは、単一のB細胞クローンによって産生される抗体を指す。MAbは、B細胞と骨髄腫細胞とのハイブリッドであるハイブリドーマ、または免疫グロブリン重鎖および軽鎖をコードする組換えDNAを発現し、したがって単一の特異的抗体を産生する細胞株によっても産生され得る。
各抗体は同じ抗原に2回結合する。すなわち、2価である。
シアリルTn:
NeuAcα−6GalNAcα/β1−
2,6−シアリルT:
Galβ1−3GalNAcα1−
│
NeuAcα2−6
ジシアリルT:
NeuAcα2−3Galβ1−3GalNAcα1−
│
NeuAcα2−6
2,6−シアロ−N−アセチルラクトサミン:
NeuAcα2−6Galβ1−4Glcβ1−を含む。
a)STnと、α2,6−結合シアル酸により末端化されたグリカンの群とに特異的に結合する抗体を用いて、腫瘍を有する可能性のある対象から得られた生物学的試料を染色する工程を含み、前述の染色は、STn、2,6−シアリルT、ジシアリルTまたは2,6−シアロラクトサミン(sialolactosamine)に対する抗体またはその機能的抗体断片もしくはプローブの特異的結合に適した条件下で行われ、
b)抗体の結合の存在または非存在は、細胞表面STn、2,6−シアリルT、ジシアリルTまたは2,6−シアロラクトサミンを発現する腫瘍細胞の存在または非存在を示す。
結腸直腸試料では、L2A5は癌組織と反応するが、非病理組織とも反応する。染色は基本的に腸細胞に局在するが、B72.3またはTKH2により得られた非特異的染色は杯細胞に存在する。結腸直腸試料では、L2A5を用いて、L2A5による異形成組織の弱い染色の存在を検出することができたが、染色の特異的な局在はなかった。
シアリルTn:
NeuAcα−6GalNAcα1/β1
2,6−シアリルT:
Galβ1−3GalNAcα1−
│
NeuAcα2−6
ジシアリルT:
NeuAcα2−3Galβ1−3GalNAcα1−AO
│
NeuAcα2−6
2,6−シアロラクトサミン:
NeuAcα2−6Galβ1−4Glcβ1
また、マイクロアレイに存在した他の抗原配列に対する結合は実質的に存在しない。結合された配列を図7に要約する。
以下の例は、本開示の様々な態様の単なる例示として提供されており、決して本開示を限定するものと解釈されるべきではない。それらは、抗体の特性評価、選択および産生に関する。
抗体産生−免疫化
抗体を産生する例示的な方法が提供されるが、他の任意の標準的な方法を使用することができる。
モノクローナル抗体(mAb)の産生は、ハイブリドーマ技術に従って行った。完全Freundアジュバント(Sigma−Aldrich)により1:1(V/V)で乳化したヒツジ顎下ムチン(OSM)10μgを用いて、6週齢の雌Balb/cマウス(Harlan,UK)を腹腔内免疫し、続いて不完全Freundアジュバント(Sigma Aldrich)により乳化したOSMを21日間間隔で2回追加注射した。マウスの頬から血液試料を採取し、ELISAによって、STn結合特異性について、採取した血清をスクリーニングした。血清が所望かつ特異的な免疫応答を示した場合、殺処分し脾臓を採取する3日前に、対応するマウスに対して最後のブースト注射を行う。
ELISA
STn発現タンパク質であるウシ顎下ムチン(BSM)に対するELISAにより、マウス血清滴定と、ハイブリドーマ上清のスクリーニングとを決定した。リン酸緩衝食塩水(PBS)に溶解した50μlのBSM(3μg/ml)により96ウェルプレートのウェルをコーティングし、4℃で一晩インキュベートした。シアリル化構造に対するスクリーニングしたハイブリドーマ上清の特異的結合を評価するために、シアリダーゼ緩衝液(10mM Na2HPO4、pH=6.0)で希釈した25mU/mlのクロストリジウム・パーフリンジェンス(Clostridium perfringens)(Roche)から得られた50μlのシアリダーゼをウェルのサブセットに加え、37℃で90分間インキュベートした。シアリダーゼ処理後、0.05%Tween 20(PBS−T)を含むPBSでプレートを3回洗浄した後、5%脱脂粉乳を用いて60分間ブロッキングした。PBS−Tで洗浄した後、希釈したマウス血清またはハイブリドーマ上清をウェルに加え、90分間インキュベートした。PBS−Tでプレートを4回洗浄した後、西洋ワサビペルオキシダーゼ(HRP)コンジュゲートヤギ抗マウスIg(1:1000)(BD Pharmingen)と60分間インキュベートした。追加の3回の洗浄工程の後、50μlのテトラメチルベンジジン(Thermofisher Scientific)基質を各ウェルに加え、プレートを暗所でインキュベートし、50μlの1M HClを加えて反応を停止させた。マイクロプレートリーダー上で450nmで光学密度を測定した。目的の抗体の最も高い力価を生じるマウスを融合のために選択した。ハイブリドーマ細胞の抗体産生をスクリーニングするために、同じ手順を行った。
フローサイトメトリーの調製および分析
STnおよび非STn発現親細胞を安定に発現するヒト膀胱細胞株およびヒト乳房細胞株を使用したフローサイトメトリーによって、抗体またはハイブリドーマ上清の結合を決定した。条件ごとに約3×105個の細胞を回収し、PBS緩衝液に再懸濁した。シアリル化構造に対するスクリーニングされたハイブリドーマ上清および抗体の特異的結合を評価するために、100mU/mlのシアリダーゼを用いて、37℃で90分間試料を処理した。シアリダーゼ処理後、細胞を洗浄し、抗STn mAb B72.3、3F1、TKH2、およびハイブリドーマ上清と4℃で30分間インキュベートした。続く洗浄工程を行い、暗所で15分間かけて、FITCコンジュゲート抗マウスIg(Dako;希釈1:10)を用いて一次抗体を検出した。洗浄後、各試料に対してフローサイトメーターを使用して、各試料からデータを取得した。
抗体産生−ハイブリドーマ技術
免疫したマウスから得られた脾細胞とSp2/0骨髄腫(ATCC,USA)細胞とを3:1の比で混合し、標準的なプロトコルを使用して、ポリエチレングリコール/ジメチルスルホキシドの存在下で融合させた。次いで、96ウェル平底マイクロプレート(Orange Scientific)に細胞を播種し、HAT(1×10−4Mヒポキサンチン、4×10−7Mアミノプテリン、1.6×10−5Mチミジン、Sigma−Aldrich)、10% FBS、2mM L−グルタミン、0.2mg/mlゲンタマイシン(Sigma−Aldrich)、1mMピルビン酸ナトリウム(Gibco)、1%(v/v)MEM非必須アミノ酸(Gibco)を補充したRPMI培地中で維持し、37℃で7〜12日間インキュベートした。BSMに反応する抗体を産生するハイブリドーマ細胞を増殖させ、間接ELISAによりスクリーニングし、少なくとも3回の限界希釈法によりクローニングして、安定な単一クローン細胞株を得た。HATを補充していない選択培地中で、選択したハイブリドーマを37℃で培養した。シアリル化構造、特にSTnに特異的な1つのハイブリドーマL2A5を選択し、4回の限界希釈によりクローニングした。
STn発現の免疫組織化学分析
地方倫理委員会に従って、15例の結腸直腸腫瘍(腺癌および腺腫)および15例の膀胱腫瘍(8例の膀胱切除術および7例の転移)を含む一連の30例を得た。さらに、腫瘍に隣接する正常な結腸直腸組織の5例を含めた。ビオチン/ストレプトアビジン系を使用して、免疫組織化学(IHC)によって、STnについてホルマリン固定パラフィン包埋(FFPE)組織をスクリーニングした。要約すると、キシレンを用いてFFPE組織切片を脱パラフィンし、段階的な一連のアルコール洗浄により再水和し、最大出力定格で5分間溶液を予熱した後、マイクロ波内で15分間クエン酸緩衝液pH6.0(Vector,Burlingame,USA)を使用して熱誘導抗原回収に供した。切片を0.3%過酸化水素(Merck KGaA,Darmstadt,Germany)と25分間インキュベートし、UV Block(登録商標)(Thermo Scientific,Fremont,USA)を用いてブロックし、抗STn mAb B72.3、TKH2(Kjeldsen et al.,1988)およびL2A5を入れた湿式チャンバー内で4℃で一晩インキュベートした。PBS−Tweenで洗浄した後、組織切片に二次抗体を加えてからストレプトアビジンとインキュベートした。3,3’−ジアミノベンジジン(ImmPACT(商標)DAB)(Vector,Burlingame,USA)と4分間インキュベートすることによりSTnを視覚化した。最後に、ヘマトキシリンを用いて核を1分間対比染色した。PBS中の5%BSAで1:5、1:5および1:3にそれぞれ希釈した抗STn mAb B72.3、TKH2およびL2A5ハイブリドーマ培養上清を使用して、STn発現を評価した。陽性対照切片および陰性対照切片を並行して試験した。一次抗体の非存在下で陰性対照切片を行った。陽性対照としてSTn+腫瘍組織を使用した。腫瘍細胞内の褐色発色産物の顕微鏡的存在によって抗STn TKH2抗体の免疫反応性が観察された場合、腫瘍を陽性として分類した。STn発現およびL2A5染色は、2名の独立した観察者が二重盲検下で評価し、経験豊富な病理学者が検証した。意見の相違があった場合は常にスライドを再検討し、合意に達した。抗体特異性を評価するために、過酸化水素とのインキュベーション後にシアリダーゼ処理を行い、STn抗原からシアル酸を除去することにより、抗体による認識を阻害した。したがって、この酵素処理後の陽性染色(37℃で4時間、0.2U/mL)は非特異的であると考えられた。
ウエスタンブロット(WB)
製造業者の指示に従って、膜タンパク質抽出キットを使用して細胞株から膜タンパク質を単離した。製造業者の推奨に従って、タンパク質アッセイキットを使用して、得られたタンパク質の量を推定した。膜タンパク質抽出物(50μg)、またはSTn(1μg)(BSMおよびMUC1 STn−IgG)を含有する精製タンパク質を変性させ、8%勾配アクリルアミドゲル上にロードし、還元条件下でSDS−PAGE電気泳動に供し、標準手順に従ってフッ化ポリビニリデン(PVDF)メンブレン(Amersham Hybond P 0.2μm PVDF、GE Healthcare Life Sciences)に電気泳動的に転写した。TBS Tween 0.1%(TBS−T)中の10%脱脂粉乳を用いてメンブレンを1時間ブロックした後、TBS−Tで希釈した一次抗体抗STn B72.3、3F1またはL2A5上清と4℃で一晩インキュベートした。TBS−Tで洗浄した後、TBS−Tで1:2500に希釈したHRPコンジュゲートヤギ抗マウスIgを1時間使用して、標識タンパク質を明らかにした。洗浄後、Lumi−Lightウエスタンブロット基質(Roche)によって標識タンパク質を明らかにし、次いでX線フィルムに露光した。
mRNA単離およびcDNA合成
RNA単離に1×106個〜5×106個のハイブリドーマ細胞を使用した。細胞を300xgで5分間遠心分離し、上清を廃棄した。細胞ペレットをPBSで洗浄し、製造業者の指示に従って、GenElute(商標)Mammalian Total RNA Miniprepキット(Sigma−Aldrich)を使用して全RNAを単離した。Nanodropを使用して、抽出された全RNAを定量し、High−Capacity cDNA転写キット(Applied Biosystems)に記載されているように、最大2μgを逆転写に使用した。25℃で10分間、その後37℃で120分間および85℃で5秒間の熱サイクル条件を使用して、cDNA合成を行った。反応を最後に保持し、4℃に冷却した。
抗体配列決定−scFv断片
プライマー対VH Forward(TTTTTGGATCCSARGTNMAGCTGSAGSAGTCWGG)/VH Reverse(ATTGGGACTAGTTTCTGCGACAGCTGGATT)およびVL Forward(TTTTTGAATTCTGAYATTGTGMTSACMCARWCTMCA)/VL Reverse(TTTTTGGGCCCGGATACAGTTGGTGCAGCATC)を使用して、cDNAからL2A5 MAbの可変重鎖(VH)ドメインおよび可変軽鎖(VL)ドメインを増幅した。PCRはいずれも、Advantage HF 2 PCRキット(Clontech)を使用して行った。94℃で3分間の初期溶融、続いて95℃で45秒、70℃で1分間および68℃で2分間の熱サイクル条件を使用した。次いで、反応を68℃で5分間保持し、4℃に冷却した。製造業者のプロトコルに従って、精製したPCR産物をさらにpGEM−Teasy(Promega)クローニングベクターにクローニングした。製造業者のプロトコルに従って、QIAGEN plasmid plus midi キット(QIAGEN)を使用してプラスミドを単離した。pGEM−Teasyベクター用のT7プロモータープライマーを使用して、Seqlab(Gottingen,Germany)が配列決定を行った。
これらの組成物は、臨床用途および診断用途のために一貫した品質で製造することができる。
抗体ドメイン
配列分析ツールIgBLASTを使用してIMGT Vドメイン描写システム(international ImMunoGeneTics database;http://imgt.cines.fr)を検索して、L2A5 mAbのFRドメインおよびCDRドメインのアミノ酸配列をコードする可変重鎖(VH)ヌクレオチド配列および可変軽鎖(VL)ヌクレオチド配列を決定した。
標的モジュールへの核酸のクローニング
記載されている通り(Cartellieri et al 2016)であるが、CDR領域をLA25核酸配列に置き換えて、抗STn標的モジュール(TM)を行った。標準的なクロム放出アッセイを使用して、T細胞媒介腫瘍殺傷を測定した。ベクター対照(EGFPマーカータンパク質のみをコードするベクター骨格)、UniCAR Stop構築物(細胞内シグナル伝達ドメインを欠く)またはα−E5B9シグナル伝達構築物(UniCAR 28/ζ)(Mitwasi 2017)のいずれかを移植したT細胞と、MDA−MB−231細胞株およびMCR STn+細胞株をインキュベートした。80nMの抗STn TM(a−STn TM)の存在下または非存在下で、5:1のエフェクター対標的(E:T)の比で、それぞれ遺伝子操作されたT細胞とともに両細胞株を24時間培養した。
インビボ抗腫瘍活性
MDA−MB−231 STn細胞を形質導入してホタルルシフェラーゼ(Luc)を発現させ、MDA−MB−231 STn−Luc細胞を得た。記載されている通り(Cartellieri et al 2016)であるが、CDR領域をLA25核酸配列に置き換えて、抗STn標的モジュール(TM)を行った。マウス1匹当たり、1.5×106個の腫瘍細胞と、1×106個のUniCAR 28/ζ T細胞および10μgの抗STn TMとを混合した。MDA−MB−231 STn−Luc細胞(1.5×106)を単独で、またはTMを含まない1×106個のUniCAR 28/ζ T細胞と混合して、未処理対照として使用した。それぞれの混合物を雌NMRI−Foxn1nu/Foxn1nuマウスに皮下注射し、それぞれ5匹のマウスからなる3群の動物を得た。200μLのD−ルシフェリンカリウム塩(15mg/mL)の腹腔内注射を0日目に開始し、続いて1日目、3日目、6日目および8日目に行った後に、麻酔したマウスの発光イメージングを10分間行った。
Claims (8)
- 抗体、その機能的抗体断片またはプローブであって、該抗体、その機能的抗体断片またはプローブが、L2A5モノクローナル抗体に由来し、かつSTnとα2,6−結合シアル酸により末端化されたグリカンの群とに特異的に結合し、
a)軽鎖L−CDR1、L−CDR2 L−CDR3が、それぞれ配列番号6、8および10であり、
b)重鎖H−CDR1、H−CDR2、H−CDR3が、それぞれ配列番号12、14および16である
相補性決定領域をそれぞれ含む可変軽鎖ドメインおよび可変重鎖ドメインをコードする
ことを特徴とする抗体、その機能的抗体断片またはプローブ。 - α2,6−結合シアル酸により末端化されたグリカンの群が、STn、2,6−シアリルTまたはジシアリルTまたは2,6−シアロラクトサミンを含むことを特徴とする、請求項1に記載の抗体。
- 抗体、その機能的抗体断片またはプローブが、キメラであるかヒト化されていることを特徴とする、請求項1又は2に記載の抗体。
- 抗体、その機能的抗体断片またはプローブが、グリコシル化部位でグリカン変化に供されることを特徴とする、請求項1から3のいずれか一項に記載の抗体。
- a)STnと、α2,6−結合シアル酸により末端化されたグリカンの群とに特異的に結合する抗体をコードするヌクレオチド配列を用いて、腫瘍を有する可能性のある対象から得られた生物学的試料を染色する工程を含み、前述の染色が、STn、2,6−シアリルT、ジシアリルTまたは2,6−シアロラクトサミンをコードするヌクレオチド配列によりコードされる抗体またはその機能的抗体断片もしくはプローブの特異的結合に適した条件下で行われ、
b)抗体をコードするヌクレオチド配列の結合の存在または非存在が、細胞表面STn、2,6−シアリルT、ジシアリルTまたは2,6−シアロラクトサミンを発現する腫瘍細胞の存在または非存在を示す
ことを特徴とする、請求項1から4のいずれか一項に規定される抗体、その機能的抗体断片またはプローブを使用して、対象の腫瘍を検出する方法。 - 生物学的試料が、単離された細胞、または組織、または腫瘍由来タンパク質を含むことを特徴とする、請求項5に記載の方法。
- 腫瘍を治療するために使用されることを特徴とする、請求項1から4のいずれか一項に規定される抗体、その機能的抗体断片またはプローブの使用。
- 医薬組成物が薬学的に許容される担体をさらに含むことを特徴とする、請求項1から4のいずれか一項に規定される抗体、その機能的抗体断片またはプローブを含む医薬組成物。
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