JP2021517581A - システイン改変抗体−毒素複合体(tdc)の部位特異的結合サイトのスクリーニング - Google Patents
システイン改変抗体−毒素複合体(tdc)の部位特異的結合サイトのスクリーニング Download PDFInfo
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- JP2021517581A JP2021517581A JP2020551558A JP2020551558A JP2021517581A JP 2021517581 A JP2021517581 A JP 2021517581A JP 2020551558 A JP2020551558 A JP 2020551558A JP 2020551558 A JP2020551558 A JP 2020551558A JP 2021517581 A JP2021517581 A JP 2021517581A
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Abstract
Description
実施例1:mcの合成
30ml氷酢酸に6−アミノカプロン酸3.9g(0.03mol)及び1.2eqの無水マレイン酸3.5g(0.036mol)を加えた。反応液を120℃で4〜6時間撹拌しながら反応させた。反応終了後、加熱を停止させ、室温まで自然に冷却した。60℃で減圧濃縮してほとんどの酢酸を除去した。得られた黄褐色粘稠液を水に入れ、さらに酢酸エチル20ml×3を加えて抽出し、有機層を合わせた。有機層を順に水、飽和食塩水で洗浄し、無水硫酸ナトリウムで乾燥させ、濾過し、濾液を減圧濃縮して黄褐色油状物を得、50ml水を加えて撹拌し、類白色固体が析出した後、濾過し、50℃で減圧乾燥させ、目的製品5.08gを得た。収率は80%であった。mp:89−92℃。m/z:212.2[M+H]+。1HNMR(400Mz,DMSO):13.21(br,1H,COOH)、6.75(s,2H,COCH=CHCO)、3.63(t,2H,J=7.2Hz,NCH2CH2)、2.42(t,2H,J=7.4Hz,CH2COOH)、1.52−1.68(m,4H,NCH2CH2CH2CH2)、1.30−1.42(m,2H,NCH2CH2CH2CH2)。
窒素ガスの保護下で50mlアセトニトリルに4.7g(22mmol)MC及び25g(22mmol)HOSuを加えた。4.5g(22mmol)DCCを25mlアセトニトリルに溶解し、内温を約0℃に保持しながら反応液にゆっくりと滴下した。反応液を0℃で2時間反応させた後、室温で一晩反応させた。濾過し、濾過ケーキをアセトニトリル10ml×3で洗浄し、濾液を減圧濃縮して乾燥させた。得られた油状物を室温で6時間減圧乾燥させ、淡褐色固体6.4gを得た。収率は95%であった。生成物を精製せずにそのまま次の反応に用いた。
m/z:309.2[M+H]+。1HNMR(400Mz,CDCl3):1〜2(m,6H,CCH2CH2CH2C)、2.68(t,2H,CH2CO),2.95(s,4H,COCH2CH2CO)、3.68(t,2H,CH2N)、6.81(s,2H,CH=CH)。
100mlTHFに10gのFmoc−Val及び3.4gのHOSuを加えた。6gのDCCを50mlアセトニトリルに溶解し、内温を約0℃に保持しながら反応液にゆっくりと滴下した。反応液を室温で24時間撹拌しながら反応させた。濾過し、濾過ケーキをTHFで洗浄し、濾液を減圧濃縮し、透明油状物を得た。油状物を精製せずにそのまま次の反応に用いた。m/z:437.4[M+H]+
20mlTHFに4.0g(1.05eq)のCit及び炭酸水素ナトリウムの水溶液60ml(NaHCO3 2g,1.05eq)を加えた。22.35mmolのFmoc−Val−OSuを60mlDMEに溶解し、さらに反応液に加えた。反応液を室温下で24時間撹拌しながら反応させた。反応終了後、反応系に15%クエン酸水溶液110mlを加え、EAで2回抽出し、有機層を合わせ、減圧濃縮して白色固体を得た。白色固体にメチルtert−ブチルエーテル100mlを加え、撹拌し、濾過し、濾過ケーキを40℃で4時間減圧乾燥させ、製品4.83gを得た。収率は65%であった。m/z:497.6(M+H)+。1HNMR(400Mz,DMSO):0.92(6H,m)、1.35〜1.65(4H,m)、2.10(1H,m)、3.01(2H,q)、3.99(1H,t)、4.01−4.45(2H,m)、4.45(2H,t)、5.46(2H,br)、6.03(1H,t)、7.20−8.02(8H,m)、8.25(1H,d)。
反応フラスコにDCM/MeOH=2/1混合溶媒60mlを加え、さらに2g(4.2mmol)のFmoc−vc及び1.04g(2eq)のPABOHを加え、撹拌して一部を溶解した後、2.0g(2eq)のEEDQを加えた。反応系を室温の条件下で暗所で2.0日間撹拌しながら反応させた。反応終了後、40℃で減圧濃縮し、白色固体を得た。白色固体を収集し、メチルtert−ブチルエーテル100mlを加え、撹拌し、濾過し、濾過ケーキをメチルtert−ブチルエーテルで洗浄し、得られた白色固体を40℃で減圧乾燥させ、製品2.2gを得た。収率は約88%であった。m/z:602.6(M+H)+。1HNMR(400Mz,DMSO):0.95(6H,m)、1.45〜1.69(4H,m)、2.10(1H,m)、3.11(2H,m)、3.99(1H,m)、4.30(2H,d)、4.05〜−4.66(2H,m)、4.55(2H,d)、5.21(1H,t)、5.51(2H,br)、6.11(1H,t)、7.09−8.10(12H,m)、8.21(1H,d)、10.51(1H,br)。
10mlNMPに490mg(0.815mmol)のFmoc−vc−PABOHを加え、撹拌して溶解させ、さらにジエチルアミン2mlを加えた。室温下で24時間撹拌しながら反応させた。反応終了後、40℃で減圧濃縮し、得られた油状物に20mlDCMを加え、撹拌して晶析させ、濾過し、濾過ケーキをDCMで洗浄し、得られた固体を減圧乾燥し、製品277mgを得た。収率は90%であった。m/z:380.2(M+H)+。1HNMR(400Mz,DMSO):0.89(6H,m),1.31〜1.61(4H,m),1.82(1H,m),2.86(1H,m),2.89(2H,d),4.38(2H,d),4.44(1H,m),5.01(1H,br),5.35(2H,br),5.84(1H,br),7.14(2H,d),7.42(2H,d),8.08(1H,br),9.88(1H,br)。
10mlNMPに205mg(0.54mmol)のvc−PABOH及び184mg(1.1eq)のMC−OSuを加え、その後、室温で24時間撹拌しながら反応させた。反応終了後、40℃で減圧濃縮し、得られた油状物に20mlメチルtert−ブチルエーテルを加え、撹拌して晶析させた。濾過し、濾過ケーキをメチルtert−ブチルエーテルで洗浄し、製品310mgを得た。収率は100%であった。m/z:573.3(M+H)+。1HNMR(400Mz,DMSO):0.89(6H,m)、1.15−1.99(10H,m)、2.11(1H,m)、2.31(2H,t)、3.21(2H,m)、3.53(2H,t)、4.32(1H,t)、4.51(1H,m)、4.59(2H,br)、5.24(1H,br)、5.56(2H,br)、6.20(1H,br)、7.12(2H,s)、7.23(2H,d),7.58(2H,d)、7.94(1H,d),8.17(1H,d)、10.21(1H,br)。
窒素ガスの保護下で168.6mg(0.294mmol)のmc−vc−PABOHを5ml無水ピリジンに溶解し、反応系を約0℃に冷却した。PNP179mg(3eq)を5mlDCMに溶解し、それを反応系にゆっくりと加えた。約0℃に10分間保持した後、氷浴を取り外し、室温で3時間撹拌しながら反応させた。反応終了後、70mlのEA及び100mlの15%クエン酸水溶液を加え、有機層を分取した。有機層を順にクエン酸、水、飽和食塩水で洗浄し、さらに無水硫酸ナトリウムで乾燥させ、濾過し、濾液を減圧濃縮し、淡黄色油状物を得、メチルtert−ブチルエーテルで晶析させ、類白色固体86mgを得た。収率は40%であった。m/z:738(M+H)+。1HNMR(400Mz,CDCl3/CD3OD):0.84(6H,m)、1.11−1.84(10H,m)、2.05(1H,m)、2.15(2H,t)、3.09(2H,m)、3.32(2H,t)、4.12(1H,m)、4.38(1H,m)、5.15(2H,s)、6.61(2H,s)、6.84(1H,d),7.61(1H,d)、7.21(2H,d),7.50(2H,d)、7.61(2H,d),8.18(2H,d)、9.59(1H,br)。
2mlDMFに20mgのmc−vc−PAB−PNP(1.5eq)及び3mgのHOBTを加えた。室温でしばらく撹拌した後、13mgのMMAE、0.5mlのピリジン、25ulのDIEAを加えた。反応液を室温で2日間撹拌しながら反応させた。反応終了後、反応液をそのまま分取カラムで精製し、必要な成分を収集した後、濃縮して凍結乾燥させ、約10mg製品を得た。収率は約42%であった。m/z:1317.1(M+H)+。
100mgの10−O−Boc−SN−38を10ml乾燥ジクロロメタンで溶解させた後、25.6mg(1eq)DMAPを加え、0℃でトリホスゲンのジクロロメタン溶液(62mgトリホスゲンを2mlジクロロメタンで溶解させたもの)を滴下し、その後、引き続き0℃で2時間反応させ、減圧でジクロロメタンを除去し、10ml乾燥DMFで溶解させた後、144mgのmc−vc−PABOHを加え、室温で24時間撹拌し、分取液体クロマトグラフィーにより分離して41mgのmc−vc−PAB−SN−38を得た。二段階の総収率は19.7%であった。m/z:1063.2(M+H)+
FreestyleTM 293−F(Invitrogen)懸濁細胞を用いて2A1−HC−Cys254ins抗体を発現した。トランスフェクションの前日、6×105個/mLの密度で細胞を300mLのF17完全培地(FreestyleTM F17発現培地、Gibco社)を含む1L振盪フラスコに接種し、37℃、5%CO2、120rpmの条件下で細胞培養用シェーカーで一晩培養した。翌日にPEIで抗体発現プラスミドのトランスフェクションを行った(プラスミド:PEI=2:1)。トランスフェクションの翌日に、2.5%(v/v)でTN1フィード培地を加え、引き続き4日間培養した後、遠心分離し、上清を収集した。
この方法で得られた2A1−HC−Cys254ins抗体の濃度は2mg/mlであり、目的抗体モノマー(POI%)は90%を超えた。次の実験に用いた。
細胞で発現した2A1−HC−Cys357ins抗体をMabselect Sureにより精製し、低pHで溶離した直後、Tris溶液を加えて中和し、pH7.5のTris−HCl緩衝液に交換した。化合物mc−vc−PAB−MMAEは白色粉末であり、DMAに溶解して使用まで保存した。変異システイン残基上のマスキング基を除去するために、抗体を還元する必要がある。40倍の分子比で1MのDTT水溶液を2A1−HC−Cys357ins抗体溶液に加え、均一に混合した後、20℃で2時間反応させた。その後、サンプルのpHを5.0に調整し、SP Sepharose F.F.陽イオン交換クロマトグラフィーによりサンプル中のDTT及びマスキング基を除去した。その後、20倍の分子比でDHAA溶液をサンプルに加え、25℃で暗所で4時間反応させ、抗体鎖間ジスルフィド結合を再結合させた。そして、mc−vc−PAB−MMAE溶液を加え、mc−vc−PAB−MMAEと抗体変異システインを結合させ、均一に混合した後、25℃で2時間反応させた。反応終了後にSP Sepharose F.F.陽イオン交換クロマトグラフィーにより抗体分子に結合されていないmc−vc−PAB−MMAEを除去し、2A1−HC−Cys357ins−mc−vc−PAB−MMAE TDCサンプルを得た。
高速液体疎水クロマトグラフィー方法によりTDCサンプルを分析し、対応するピーク面積に基づいてDARを計算した。具体的な方法は以下の通りである。
カラム:Proteomix(登録商標)HICBu‐NP5(5μm,4.6x35mm)
移動相:A:2M硫酸アンモニウム,0.025M、pH7のリン酸緩衝液;B:0.025M、pH7のリン酸緩衝液;C:100%イソプロパノール
緩衝液Aで平衡化し、緩衝液Bと緩衝液Cでグラジエント溶離し、25℃、214nmで検出した。
TDC抗体骨格サンプルを37℃で保存し、0、7、14、21日目にそれぞれSEC−HPLCによりその凝集状況を分析した。具体的な方法は以下の通りである。
カラム:TSKgel SuperSW mAb HR(7.8mm×30cm)
移動相:0.1M硫酸ナトリウム;0.1M、pH6.7のリン酸緩衝液
25℃、280nmで検出した。
間接ELISA法により2A1−LC−Cys110ins−mc−vc−PAB−MMAE TDC、2A1−LC−Cys111ins−mc−vc−PAB−MMAE TDC、2A1−LC−Cys142ins−mc−vc−PAB−MMAE TDC和2A1−HC−Cys254ins−mc−vc−PAB−MMAE TDC、2A1−HC−Cys255ins−mc−vc−PAB−MMAE TDC、2A1−HC−Cys258ins−mc−vc−PAB−MMAE TDC、2A1−HC−Cys259ins−mc−vc−PAB−MMAE TDC、2A1−HC−Cys354ins−mc−vc−PAB−MMAE TDC、2A1−HC−Cys355ins−mc−vc−PAB−MMAE TDC、2A1−HC−Cys357ins−mc−vc−PAB−MMAE TDC、2A1−HC−Cys378ins−mc−vc−PAB−MMAE TDC、2A1−HC−Cys379ins−mc−vc−PAB−MMAE TDC、2A1−HC−Cys386ins−mc−vc−PAB−MMAE TDC、2A1−HC−Cys387ins−mc−vc−PAB−MMAE TDC、2A1−HC−Cys410ins−mc−vc−PAB−MMAE TDC及び2A1のEGFRvIIIに対する相対親和力を比較した。具体的な手順は以下の通りである。
以下の実験によりTDC細胞毒性活性を測定する。
TDCをEGFR過剰発現又はEGFRVIII発現のヒト腫瘍細胞培地に加え、細胞を72時間培養した後、細胞生存率を測定した。細胞のインビトロ実験により細胞生存率、細胞毒性及び本発明のTDC誘発性プログラム細胞死を測定した。
細胞増殖試験により抗体−毒素複合体のインビトロ薬効を測定した。CellTiter 96(登録商標)AqueousOne Solution Cell Proliferation Assayは市販品である(Promega Corp.,Madison,WI)。CellTiter 96(登録商標) AQueous One Solution Cell Proliferation Assay(a)は、比色法により細胞増殖及び細胞毒性実験における生細胞の数を測定する試薬である。この試薬は、新型のテトラゾール化合物[3−(4,5−dimethylthiazol−2−yl)−5−(3−carboxymethoxyphenyl)−2−(4−sulfophenyl)−2H−tetrazolium,inner salt;MTS]及び電子カップリング剤(phenazine ethosulfate;PES)を含む。PESは、増強された化学安定性を有するため、MTSと混合して安定した溶液を形成することができる。このような便利な「単一溶液」の形態は、第一世代CellTiter 96(登録商標)AQueous Assayをもとに改良したものである。CellTiter 96(登録商標)AQueous Assayに用いる電子カップリング剤PMSとMTS溶液は別々に提供される。MTS(Owen's reagent)は、細胞により着色されたフォルマザン生成物に生物学的に還元され、そのまま培地に溶解することができる(図1)。この変換は、代謝が活発な細胞における脱水素酵素により生成したNADPH又はNADHの作用下で達成される可能性がある。検出の際に、少量のCellTiter 96(登録商標)AQueous One Solution Reagentを培養プレートウェルの培地にそのまま加え、1−4時間インキュベートした後、マイクロプレートリーダーで490nmでの吸光度値を読み取ればよい。
所定量のTDCサンプルをヒトIgGが除去されたヒト血漿に加え、各TDCについて3チューブずつ準備し、37℃の水浴でインキュベートし、それぞれ0時間、24時間、96時間インキュベートした後、TDCサンプルを取り出し、各チューブにProteinA(MabSelect SuReTM LX Lot:#10221479 GE、PBS洗浄済み)100ulを加え、縦型混合器で2時間振盪しながら吸着させ、洗浄、溶離した後、インキュベートされたTDCを得た。所定時間インキュベートされたTDCサンプルをHIC−HPLC及びRP−HPLCにより検出し、サンプルの血漿安定性を判定した。
ヒト膵臓腺がん細胞BxPC3のヌードマウス皮下移植腫瘍モデルを構築し、2A1−HC−Cys357ins−mc−vc−PAB−MMAE等のTDC結合薬物のインビボ薬効を評価した。5×106でA431細胞を4〜6週齢のBALB/cヌードマウスの背中に皮下注射し、マウス腫瘍の平均サイズが約150〜200mm3まで増殖した後、6匹/群でランダムに群分けした。1日目に、2A1−HC−Cys357ins−mc−vc−PAB−MMAE(2A1−HC−Cys357ins TDC)、2A1−HC−Cys410ins−mc−vc−PAB−MMAE(2A1−HC−Cys410ins TDC、2A1−HC−Cys387ins−mc−vc−PAB−MMAE(2A1−HC−Cys387insTDC)、2A1−HC−Cys378ins−mc−vc−PAB−MMAE(2A1−HC−Cys378insTDC)を5mg/kgの用量で尾静脈に単回投与した(図6)。テータは、測定時の腫瘍平均体積±SEを示す。
細胞で発現した2A1−HC−Cys357ins(配列番号:34)抗体をMabselect Sureにより精製し、低pHで溶離した直後、Tris溶液を加えて中和し、pH7.5のTris−HCl緩衝液に交換した。化合物mc−vc−PAB−MMAEは白色粉末であり、DMAに溶解して使用まで保存した。変異システイン残基上のマスキング基を除去するために、抗体を還元する必要がある。40倍の分子比で1MのDTT水溶液を2A1−HC−Cys357ins抗体溶液に加え、均一に混合した後、20℃で2時間反応させた。その後、サンプルのpHを5.0に調整し、SP Sepharose F.F.陽イオン交換クロマトグラフィーによりサンプル中のDTT及びマスキング基を除去した。その後、20倍の分子比でDHAA溶液をサンプルに加え、25℃で暗所で4時間反応させ、抗体鎖間ジスルフィド結合を再結合させた。そして、mc−vc−PAB−MMAE溶液を加え、mc−vc−PAB−MMAEと抗体変異システインを結合させ、均一に混合した後、25℃で2時間反応させた。反応終了後にSP Sepharose F.F.陽イオン交換クロマトグラフィーにより抗体分子に結合されていないmc−vc−PAB−MMAEを除去し、2A1−HC−Cys357ins−mc−vc−PAB−MMAE TDCサンプルを得た。
Claims (10)
- 抗体は、システイン部位特異的挿入抗体であり、
システイン挿入部位は、kappa/λ軽鎖定常領域軽鎖の第110位、第111位、第142位、IgG抗体重鎖定常領域重鎖の第254位、第255位、第258位、第259位、第354位、第355位、第357位、第378位、第379位、第386位、第387位、第410位から選択される1つ又は複数の部位を含む、システイン改変抗体−毒素複合体。 - システイン挿入部位のアミノ酸配列は、
LC−110ins:EIKRTCVAAPS
LC−111ins:IKRTVCAAPSV
LC−142ins:NNFYPCREAKV
HC−254ins:ISRTPCEVTCV
HC−255ins:SRTPECVTCVV
HC−258ins:PEVTCCVVVDV
HC−259ins:EVTCVCVVDVS
HC−354ins:PSRDECLTKNQ
HC−355ins:SRDELCTKNQV
HC−357ins:DELTKCNQVSL
HC−378ins:IAVEWCESNGQ
HC−379ins:AVEWECSNGQP
HC−386ins:GQPENCNYKTT
HC−387ins:QPENNCYKTTP
HC−410ins:KLTVDCKSRWQ
のうちの1つ又は複数を含む、請求項1に記載のシステイン改変抗体−毒素複合体。 - 高活性細胞毒素は、リンカーを介して挿入されたシステイン遊離チオール基(−SH)に部位特異的結合し、
リンカーと挿入されたシステインとが結合した抗体の軽鎖のアミノ酸配列は、EIKRTCVAAPS、IKRTVCAAPSV及びNNFYPCREAKVであり、
リンカーと挿入されたシステインとが結合した抗体の重鎖のアミノ酸配列は、ISRTPCEVTCV、SRTPECVTCVV、PEVTCCVVVDV、EVTCVCVVDVS、PSRDECLTKNQ、SRDELCTKNQV、DELTKCNQVSL、IAVEWCESNGQ、AVEWECSNGQP、GQPENCNYKTT、QPENNCYKTTP、KLTVDCKSRWQであり、
Cは、目的抗体の軽鎖又は重鎖に挿入されたシステインである、請求項1に記載のシステイン改変抗体−毒素複合体。 - 前記抗体軽鎖は、kappa又はλアイソタイプを含む、請求項1に記載のシステイン改変抗体−毒素複合体。
- 前記抗体重鎖は、IgG1、IgG2、IgG3又はIgG4アイソタイプを含む、請求項1に記載のシステイン改変抗体−毒素複合体。
- 前記システインは、チオール基を含む、請求項1に記載のシステイン改変抗体−毒素複合体。
- チオール基は、化学的に結合することができる、請求項6に記載のシステイン改変抗体−毒素複合体。
- 毒素/抗体比(DAR)は1.6−2.0又は3.2−4.0である、請求項1に記載のシステイン改変抗体−毒素複合体。
- 前記部位特異的結合の手順は以下の通りであり、
まず、還元剤で抗体を還元し、抗体上の改変したシステイン残基上のマスキングを除去し、陽イオン交換クロマトグラフィー又は限外濾過及び液体交換等の方式によりDTT及びマスキング基を除去し、
そして、酸化剤で抗体を酸化し、抗体の鎖間ジスルフィド結合を再結合させ、
最後に、payloadを加えて抗体上の改変したシステイン残基上の遊離チオール基に結合させ、陽イオン交換クロマトグラフィー又は限外濾過及び液体交換等の方式により抗体分子に結合していないpayloadを除去する、請求項1に記載のシステイン改変抗体−毒素複合体。
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