JP5838486B2 - 接合物質とコチニンとの接合体に抗−コチニン抗体が結合した複合体およびその用途 - Google Patents
接合物質とコチニンとの接合体に抗−コチニン抗体が結合した複合体およびその用途 Download PDFInfo
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- JP5838486B2 JP5838486B2 JP2014505086A JP2014505086A JP5838486B2 JP 5838486 B2 JP5838486 B2 JP 5838486B2 JP 2014505086 A JP2014505086 A JP 2014505086A JP 2014505086 A JP2014505086 A JP 2014505086A JP 5838486 B2 JP5838486 B2 JP 5838486B2
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Landscapes
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Description
1)接合物質をコチニンと接合させた接合体を調製する段階と、
2)抗−コチニン抗体を調製する段階と、
3)前記で調製した接合物質をコチニンと接合させた接合体および抗−コチニン抗体を結合させる段階とを含む方法によって調製することができる。
a)接合物質とコチニンとの接合体に抗−コチニン抗体が結合した複合体をコードする核酸分子をベクターに挿入するステップと、
b)前記ベクターを宿主細胞に導入するステップと、
c)前記宿主細胞を培養するステップとを含むことができる。
(1−1)抗−コチニンウサギscFvから抗体可変領域の増幅
ウサギの抗体可変領域(VLおよびVH)を増幅するために、抗−コチニンウサギscFv遺伝子(米国特許第8,008,448号参照)を鋳型とし、それぞれ60pmolのVL用の順方向および逆方向プライマー(配列番号:11および12)、およびVH用の順方向および逆方向プライマー(配列番号:13および14)を使用してポリメラーゼ連鎖反応(PCR)を行った。
ヒト軽鎖不変領域(Cκ)およびヒト重鎖不変領域(CH1−CH3)の抗体不変領域(Constant region)を増幅するために、20ngのpComb3XTTベクター(Barbas et al.、 Proc. Natl. Acad.Sci.、 USA、(1991)、 15:88(18)、7978−7982)を鋳型とし、それぞれ60pmolのCκ用順方向および逆方向プライマー(配列番号:15および16)およびCH1−CH3用の順方向および逆方向プライマー(配列番号:17および18)を使用したことを除いては、前記実施例(1−1)と同様の方法でPCRを行った後、増幅されたDNAのゲル電気泳動および精製を行った。
軽鎖を増幅するためのPCRを実行するために、前記実施例(1−1)および(1−2)において調製および精製したウサギの抗体軽鎖可変領域(VL)およびヒトの抗体軽鎖不変領域(Cκ)を重複伸長PCRによって結合させ軽鎖遺伝子を調製した。
重鎖を増幅するためのPCRを実行するために、前記実施例(1−1)および(1−3)において調製および精製した、ウサギの抗体重鎖可変領域(VH)およびヒトの抗体重鎖不変領域(CH1−CH3)を重複伸長PCRによって結合させ重鎖遺伝子を調製した。
前記実施例(1−3)において調製および精製した軽鎖PCR産物および前記実施例(1−4)において調製および精製した重鎖PCR産物を、それぞれ制限酵素HindIII/XbaI(NEB、米国)および制限酵素BamHI/NheI(NEB、米国)により切断して純粋分離した後、発現ベクター(pcDNA3.1) multiple clonining site(MCS)の部位に挿入した。
抗−コチニンウサギ/ヒトキメラIgG遺伝子を含む発現ベクターDNAにより、哺乳動物CHO DG44(インビトロジェン、米国)細胞を形質注入させた。形質注入された細胞を、100U/mlのペニシリンおよび100g/mlのストレプトマイシン(GIBCO、米国)を含むCD OptiCHO(登録商標)発現培地(GIBCO)に500μg/ml G418を添加し、37℃および135rpmの条件で培養した。培養液の上清液をLabscale TFFシステム(Millipore、米国)で濃縮して、タンパク質Aカラム(Repligen Co.、 米国)により精製した。精製された150KDaのIgGをクーマシー染色で確認し(図1参照)、その後の実験(試験例)に使用した。
(3−1)コチニンおよびペプチドの接合体
ペプチドとして、WKYMVm−NH2ペプチド(WKYMVm−NH2、配列番号:1)およびwkymvm−NH2ペプチド(wkymvm−NH2、配列番号:2)を使用した。WKYMVm−NH2およびwkymvm−NH2は、固相ペプチド合成(solid phase peptide synthesis)方法によって、ASP48Sペプチド自動合成器において合成した。その後、Vydac Everest C18カラム(250mm×22mm、10μm)を用いて逆相HPLC(reverse phase HPLC)で精製し、(95%より高い純度)、LC/MS(Agilent HP1100 series)でペプチドのサイズを確認した(95%より高い純度)。
アプタマーとして、AS1411DNAアプタマー(5’−dTTGGTGGTGGTGGTTGTGGTGGTGGTGG−3’、配列番号:3)、CRO26DNAアプタマー(5’−dCCTCCTCCTCCTTCTCCTCCTCCTCC−3’、配列番号:4)およびペガプタニブRNAアプタマー(5’−pCfpGmpGmpArpArpUfpCfpAmpGmpUfpGmpAmpAmpUfpGmpCfpUfpUfpAmpUfpAmpCfpAmpUfpCfpCfpGm3’−p−dT−3,配列番号:5)を使用した。
アブシキシマブ(Reopro)を利用して、コチニンおよびアブシキシマブの接合体(コチニン−アブシキシマブ)を調製した。
インスリン(配列番号:6)を使用して、コチニンおよびインスリンの接合体(コチニン−インスリン)を調製した。
前記実施例1で調製したコチニンと接合物質との接合体と、実施例2で調製した抗−コチニン抗体を含む複合体として、コチニン−WKYMVm−NH2/抗−コチニンIgG複合体を調製するために次のように実施した。
コチニンに対する抗−コチニンウサギ/ヒトキメラIgGの親和性は、BIAcore 3000(BIAcore AB、 Uppsala、 スウェデン)を使用して表面プラズモン共鳴技術(Surface plasmon Resonance、 SPR)より分析した。
コチニン−WKYMVm−NH2/抗−コチニンIgG複合体が、WKYMVm−NH2の細胞受容体であるFPR2に結合することを確認するためにフローサイトメトリー(flow cytometry)を行った。
(3−1)細胞内カルシウム(calcium)の濃度変化
細胞内のカルシウムの濃度([Ca2+]i)は、Fura−2/AMを利用したGrynkiewiczの方法(文献[Grynkiewicz G.et al.,J Biol Chem.,260,p3440−3450 1985]参照)を用いて測定した。
スーパーオキシドの発生は、スーパーオキシドに依存的なシトクロム(cytochrome)cの還元値を測ることにより測定することができる(文献[Bae et. al.,Blood,97,p2854−2862,2001]を参照)。
ヒト好中球を1×106/mlでRPMI1640培地に分注した後、マルチウェルチャンバー(multiwall chamber、Neuroprobe、米国)の上部ウェルに前記細胞懸濁液25μlを添加した(文献[Bae et al.、Blood、97、p2854−2862、2001]を参照)。マルチウェルチャンバーの上部ウェルは、WKYMVm−NH2(0nM、10nMまたは100nM)、コチニン−WKYMVm−NH2(0nM、10nMまたは100nM)およびコチニン−WKYMVm−NH2(0nM、10nMまたは100nM)/抗−コチニンIgG(モル比2:1)が含まれている下部ウェルと、3μmのポリ炭化水素フィルター(polyhydrocarbon filter)とによって分離されている。
4週〜6週齢の雄の野生型アルビノICRマウス(Institute of Cancer Research Center, ORIENT BIO Inc. 韓国)に、コチニン−WKYMVm−NH2(0.5mg/kg)、およびコチニン−WKYMVm−NH2(0.5mg/kg)/抗−コチニンIgG(10mg/kg)複合体をそれぞれ100μlのPBSで希釈し、尾静脈に注射した。
4週〜6週齢の雄の野生型アルビノICRマウス(ORIENT BIO Inc. 韓国)に、抗−コチニンIgG(10mg/kg)を100μlのPBSで希釈して尾静脈に注射した後、0、1、3、6、12時間目、および1、2、3、4、5、6、7、10、14、21、28日目の時に眼窩静脈叢採血を実施して末梢血を得た。各末梢血を、常温で30分間静置した後、800×gで15分間遠心分離し上清液のみを集めて血清を得て、血清中の抗−コチニンIgG量を測定するためにELISAを行った。
ICRマウスを2cm腹壁切開し盲腸を取り出して、回盲弁(ileocecal valve)直下にて25%の盲腸を結紮し、22ゲージ(gauge)の針で一回貫通した後大便が腹腔内に出てくるように処置した。腹壁の筋肉および皮膚層を縫合した。シャム−手術を施した(Sham−operated)マウス実験群は、腹壁を2cm切開して盲腸を取り出した後、何の処置も行わずに腹腔内に再び戻した。CLPを施したマウスを、各グループ当り20匹ずつ6グループに分け、CLPの後2時間後から12時間間隔で2日間、コチニン−WKYMVm−NH2(0.4mg/kgおよび0.04mg/kg)/抗−コチニンIgG(18mg/kgおよび1.8mg/kg)複合体の実験群、コチニン−WKYMVm−NH2(0.4mg/kg)実験群、WKYMVm−NH2単独(0.2mg/kg)実験群、抗−コチニンIgG(18mg/kg)単独実験群およびPBSビヒクル対照群(全100ulを投与)をそれぞれ尾静脈に注入し、飼育施設に入れて水と飼料を与え10日間観察した。続いて生存率を分析し、その結果を図11に示す。
4週〜6週齢の雄の野生型ICRマウスに、コチニン−ペガプタニブ(0.135mg/kg)およびコチニン−ペガプタニブ(0.135mg/kg)/抗−コチニンIgG(1mg/kg)複合体をそれぞれ100μlのPBSに希釈し尾静脈に注射した。0、0.5、1、1.5、および2時間後に眼窩静脈叢採血を施しそれぞれの末梢血を得て、常温で30分間静置した後、800×gで15分間遠心分離し、上清液のみを集めて血清を得た後、ELISAにより分析した。
(8−1)コチニン−アプタマー/抗−コチニンIgG細胞表面のヌクレオリン(nucleolin)に対するコチニン−AS1411/抗−コチニンIgG複合体の結合
Raji(ヒトバケットリンパ腫、American Type Culture Collection、米国)細胞を各ウェル当たり1×105個の細胞で分注した後、コチニン−AS1411(1、10、50および100nM)/抗−コチニンIgG(100nM)複合体を50ul/sampleの量で処理し、4℃で20分間反応させた。その後の分析バッファー(1%FBSおよび0.02%アジ化ナトリウム(NaN3)in PBS)により2回洗浄した後、FITCが標示されたモノクローナル抗−ヒトFc特異的IgG(Thermo Fisher Scientific)を分析バッファーに1:100で希釈し各ウェルに添加し、4℃で15分間反応させた。
Raji細胞をPBSで3回洗浄した後、1mlの溶解バッファー(20mM Tris−Cl、pH7.5、150mM NaCl、1%Triton X−100、0.25mM合成デキストローズコンプリート培地(synthetic dextrose complete medium)、1mM PMSF(phenylmethanesulfonyl fluoride)、1μg/mLアプロチニン(aprotinin)、1μg/mLロイペプチン(leupeptin)、および1μg/mlのペプスタチン(pepstatin)A)を入れ、音速ディスメンブレーターモデル500(sonic Dismembratormodel500、Thermo Fisher Scientific)機械を用いて放出セッティング(output setting)7の条件で10秒ずつ3回音波処理(sonication)をした。17,000×gで10分間遠心分離し上清液のみを集め、ブラッドフォード分析(bradford assay、 Bio−Rad、米国)を行い、濃度を測定した。
Raji細胞溶解物1mgを、コチニン−AS1411(40nM)/抗−コチニンIgG(20nM)複合体、コチニン−CRO26(40nM)/抗−コチニンIgG(20nM)複合体、およびコチニン−AS1411(40nM)/非特異的抗体(20nM)複合体とともに4℃で一晩中回転(rotation)させながら放置した。
96−ウェルELISAプレートの各ウェルをPBSに溶解した50ngのヒトVEGFを用いて4℃で一晩中コーティングしPBSBによりブロッキングした。100nMコチニン−ペガプタニブ/50nMの抗−コチニン抗体複合体をPBSBに希釈した後、10倍ずつ希釈してコーティングされた各ウェルに50μlずつ添加した。続いて、常温で1時間放置し、PBS−Tで洗浄した。この際比較のために、VEGFに対する抗体である100nMベバシズマブ(bevacizumab)を前記複合体の代わりに用いて、上記と同じ実験を繰り返した。
(9−1)コチニン−アブシキシマブ/抗−コチニンIgG複合体、コチニン−アブシキシマブ、およびアブシキシマブの反応性を確認するための酵素−リンクされた酵素結合免疫吸着測定法(Enzyme−linked immunosorbent assay)
0.001nM〜1000nMのアブシキシマブおよびコチニン−アブシキシマブを用いて、インテグリンアルファ2bベータ3に対する反応性を測定した。この時、1uMの抗−コチニンIgG抗体を使用した。
0、5nMおよび50nMのコチニン−アブシキシマブを、0、0.1μMと1μMの抗−コチニン抗体で活性化されたヒト血小板に処理した後、抗−ヒトFc−FITC(Thermo Fisher Scientific、米国)を1:75に希釈した後、血小板に処理し20分間培養した。続いて得られた細胞を、前記試験例8と同様にフローサイトメトリーにより分析した。
(10−1)コチニン−インスリン/抗−コチニン抗体複合体のMCF−7およびSK−Br−3細胞に対する特異的結合試験
具体的には、SK−Br−3またはMCF−7細胞(インスリン受容体陽性乳癌細胞株)(ATCC、米国)(1×106/反応)をFACSバッファー(PBS、1%FBSおよび0.02%アジ化ナトリウム)により3回洗浄した。様々な濃度(0nM〜5000nM)のコチニン−インスリン25μl、および抗−コチニン抗体(1000nM)またはシナジス(1000nM)25μlを、FACSバッファー中において常温で45分間適用した。このとき、シナジスは、陰性抗体対照群として使用した。FACSバッファーで2回洗浄した後、FACSバッファー50μl中で2次抗体である抗−ヒトFc−FITC 1μg/mlを適用し、癌の条件下の常温において30分間放置した。次いで細胞をFACSバッファーで2回洗浄し、PBS100μl中において再懸濁した。2つの細胞株を、FACScan蛍光活性化細胞分析装置(BD Biosciences)を用いてフローサイトメトリーで蛍光分析を行った。その結果を図20Aに示す。
補体依存細胞毒性分析を行うために、コチニン−インスリン/抗−コチニン抗体複合体を細胞可視インジケータ(cell viability indicator)のWST−1(タカラ)を用いてテストした。
ヒト補体C5(complement component C5)に結合力を有する免疫グロブリンエクリズマブ(eculizumab)のアミノ酸配列(US6,355,245 B1)から、VH、VL配列のみを用いてscFvを組み合わせて、これをcotinibody(コチニンに結合する抗体ScFv)の最初のscFvに位置するようにした。このベクターを293F細胞株に形質転換させタンパク質を発現させた後、細胞培養液から抗体を精製するためにプロテインAアガロース(protein A agarose)を用いたアフィニティークロマトグラフィー(affinity chromatography)を行った。ヒト血清を1/10、1/20、1/40、1/80(レーン1、2、3、4)希釈したもの20μLと、精製されたヒト補体C5 100ng(レーン5)とをSDS−PAGE実施し、電気泳動を行ったタンパク質をニトロセルローズメンブレンに伝導させた。ヒト補体C5−cotinibodyタンパク質を、2μg/mLになるように5%スキムミルク入りトリス緩衝生理食塩液(skim milk in Tris−buffered saline)に希釈した後、メンブレンに接触した状態で4℃、16時間反応させた。メンブレンは、0.2%Tween20が含まれたTBS溶液をもって洗浄し、horseradish peroxidaseが結合されたコチニンを1/1000希釈した溶液を、常温で2時間メンブレンと反応させた。その後、メンブレンを0.2%Tween20が含まれているTBS溶液により洗浄し、化学発光(chemiluminescent)物質を処理した後フィルムに感光した。ヒト補体C5−cotinibodyが選択的にヒト補体C5に結合することを確認した(図23)。
Claims (13)
- コチニンと接合物質との接合体に抗−コチニン抗体が結合した複合体であって、
前記接合物質がペプチド、アプタマー、抗体、ホルモン、および化学物質からなる群より選択されることを特徴とする複合体。 - 前記接合物質は、WKYMVm−NH2ペプチド、AS1411アプタマー、ペガプタニブ、アブシキシマブおよびインスリンからなる群より選択されることを特徴とする、請求項1に記載の複合体。
- 前記接合物質は、コチニンとPEGリンカーまたはアミノC6リンカーで接続されることを特徴とする、請求項1に記載の複合体。
- 前記接合体は、抗−コチニン抗体の抗原結合部位に結合されたことを特徴とする、請求項1に記載の複合体。
- 前記抗−コチニン抗体は、前記抗体と、Fab、ScFvおよびドメイン抗体から選択される抗体分節と、前記抗体および抗体分節とを構成成分として含んでいる複合タンパク質(fusion protein)からなる群より選択されることを特徴とする、請求項1に記載の複合体。
- 前記接合物質の生体内半減期(in vivo half−life)を増加させることを特徴とする、請求項1に記載の複合体。
- 補体依存性毒性(complement−mediated cell cytotoxicity、CDC)を有することを特徴とする、請求項1に記載の複合体。
- 抗体依存性細胞毒性(antibody−dependent cell cytotoxicity、ADCC)を有することを特徴とする、請求項1に記載の複合体。
- 生体外生物学的分析方法(in vitro biological assay)において、コチニンと接合物質との接合体を分析ツールとして用いることを特徴とする方法であって、
前記接合物質がペプチド、アプタマー、抗体、ホルモン、および化学物質からなる群より選択されることを特徴とする方法。 - 前記生体外の生物学的分析方法は、フローサイトメトリー、ウェスタンブロット分析法、免疫沈降分析法および酵素−リンクされた免疫化学分析法からなる群より選択されることを特徴とする、請求項9に記載の方法。
- コチニンと接合物質との接合体に抗−コチニン抗体が結合した複合体の製造方法であって、
前記接合物質をコチニンと結合させた複合体に前記抗−コチニン抗体を結合させることを含み、
前記接合物質がペプチド、アプタマー、抗体、ホルモン、および化学物質からなる群より選択されることを特徴とする、製造方法。 - 前記接合物質の生体内半減期(in vivo half−life)を増加させることを特徴とする、請求項11に記載の製造方法。
- 前記接合物質は、WKYMVm−NH 2 ペプチド、AS1411アプタマー、ペガプタニブ、アブシキシマブおよびインスリンからなる群より選択されることを特徴とする、請求項11に記載の製造方法。
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CN104321342B (zh) | 2012-03-15 | 2017-12-22 | 首尔大学校产学协力团 | Gremlin‑1抗体 |
KR102157701B1 (ko) * | 2015-08-17 | 2020-09-21 | 서울대학교산학협력단 | 항-코티닌 항체가 연결된 키메라 항체 수용체 및 이의 용도 |
US11167037B2 (en) | 2016-06-21 | 2021-11-09 | Seoul National University R&Db Foundation | Antibody drug conjugate platform using bispecific antibody |
CN110475857B (zh) | 2017-01-05 | 2023-07-18 | 韩国生命工学研究院 | 表达抗-可替宁嵌合抗原受体的天然杀伤细胞 |
KR102122546B1 (ko) * | 2017-01-05 | 2020-06-15 | 한국생명공학연구원 | 항-코티닌 키메릭 항원 수용체를 발현하는 자연살해 세포 |
BR112019019630A2 (pt) | 2017-05-19 | 2020-04-14 | Philip Morris Products Sa | teste diagnóstico para distinguir o status de tabagismo de um sujeito |
CN109679981A (zh) * | 2019-02-21 | 2019-04-26 | 武汉大学 | 一种甲酰基肽定向进化噬菌体及其制备方法与应用 |
KR102506295B1 (ko) | 2020-08-28 | 2023-03-08 | 국립암센터 | 디그옥시제닌에 대한 인간화 항체 및 이의 용도 |
KR102506288B1 (ko) | 2020-09-07 | 2023-03-06 | 국립암센터 | 디그옥시제닌에 대한 항체를 포함하는 복합체, 및 이들의 용도 |
EP4384224A1 (en) * | 2021-08-13 | 2024-06-19 | GlaxoSmithKline Intellectual Property Development Limited | Cytotoxicity targeting chimeras |
CN118103074A (zh) * | 2021-08-13 | 2024-05-28 | 葛兰素史克知识产权发展有限公司 | 用于ccr2表达细胞的细胞毒性靶向嵌合体 |
WO2023161876A1 (en) * | 2022-02-25 | 2023-08-31 | Glaxosmithkline Intellectual Property Development Limited | Cytotoxicity targeting chimeras for cxcr3-expressing cells |
WO2023161879A1 (en) * | 2022-02-25 | 2023-08-31 | Glaxosmithkline Intellectual Property Development Limited | Cytotoxicity targeting chimeras for fibroblast activation protein-expressing cells |
WO2023161875A1 (en) * | 2022-02-25 | 2023-08-31 | Glaxosmithkline Intellectual Property Development Limited | Cytotoxicity targeting chimeras for prostate specific membrane antigen-expressing cells |
WO2023161878A1 (en) * | 2022-02-25 | 2023-08-31 | Glaxosmithkline Intellectual Property Development Limited | Cytotoxicity targeting chimeras for folate receptor-expressing cells |
WO2023161877A1 (en) * | 2022-02-25 | 2023-08-31 | Glaxosmithkline Intellectual Property Development Limited | Cytotoxicity targeting chimeras for integrin avb6-expressing cells |
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GB8506102D0 (en) * | 1985-03-08 | 1985-04-11 | Baylor College Medicine | Monoclonal antibodies |
EP0194158A3 (en) | 1985-03-08 | 1988-02-24 | Baylor College of Medicine | Anti-nicotine and anti-cotinine antibodies (monoclonal and other), and their uses in nicotine and cotinine assays |
US5500375A (en) * | 1993-04-13 | 1996-03-19 | Serex, Inc. | Integrated packaging-holder device for immunochromatographic assays in flow-through or dipstick formats |
US6074642A (en) | 1994-05-02 | 2000-06-13 | Alexion Pharmaceuticals, Inc. | Use of antibodies specific to human complement component C5 for the treatment of glomerulonephritis |
GB0031079D0 (en) * | 2000-12-20 | 2001-01-31 | Smithkline Beecham Plc | Vaccine |
KR100352030B1 (en) * | 2002-01-10 | 2002-09-11 | Orion Corp | Gum composition for removing nicotine |
CN101120021A (zh) * | 2004-12-31 | 2008-02-06 | 基因技术公司 | 结合br3的多肽及其用途 |
US8008448B2 (en) | 2007-03-14 | 2011-08-30 | National Cancer Center | Cotinine neutralizing antibody |
US9745574B2 (en) * | 2009-02-04 | 2017-08-29 | Rxi Pharmaceuticals Corporation | RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality |
JP5927194B2 (ja) * | 2010-09-24 | 2016-06-01 | マリンクロッド エルエルシー | 治療ナノキャリアおよび/または診断ナノキャリアのターゲティングのためのアプタマーコンジュゲート |
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KR20140027219A (ko) | 2014-03-06 |
KR101648960B1 (ko) | 2016-08-22 |
AU2012243512B2 (en) | 2016-04-07 |
US20140056926A1 (en) | 2014-02-27 |
WO2012141554A3 (ko) | 2013-01-10 |
EP2700653B1 (en) | 2019-07-24 |
CN103476798A (zh) | 2013-12-25 |
EP2700653A2 (en) | 2014-02-26 |
CN103476798B (zh) | 2017-04-05 |
KR20160105959A (ko) | 2016-09-08 |
WO2012141554A8 (ko) | 2013-10-24 |
KR101827962B1 (ko) | 2018-02-13 |
AU2012243512A1 (en) | 2013-11-21 |
WO2012141554A2 (ko) | 2012-10-18 |
EP2700653A4 (en) | 2014-11-12 |
US9867886B2 (en) | 2018-01-16 |
JP2014517819A (ja) | 2014-07-24 |
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