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TWI726217B - 含有抗globo h抗體之抗體－藥物共軛物及其用途 - Google Patents

含有抗globo h抗體之抗體－藥物共軛物及其用途 Download PDF

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Publication number
TWI726217B
TWI726217B TW107120867A TW107120867A TWI726217B TW I726217 B TWI726217 B TW I726217B TW 107120867 A TW107120867 A TW 107120867A TW 107120867 A TW107120867 A TW 107120867A TW I726217 B TWI726217 B TW I726217B
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Taiwan
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antibody
globo
cancer
immunoconjugate
adc
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TW107120867A
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TW201905000A (zh
Inventor
李照斌
莊士賢
徐銓龍
陳怡仁
聶瑜瑾
魏文胤
吳佳城
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財團法人生物技術開發中心
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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Abstract

本發明提供包括抗Globo H抗體或其結合片段及治療劑或標記之免疫共軛物，其具有式：Ab-(L-D)m ，其中Ab係該抗Globo H抗體或其結合片段，L係連接基團或直接鍵，D係該治療劑或該標記，且m係1至8之整數。該抗體可為人類化抗體之單株抗體。本發明提供治療癌症之方法，該方法包括向需要此治療之個體投與醫藥學上有效量之免疫共軛物，該免疫共軛物含有針對Globo H之抗體或其結合片段及與該抗體共價共軛之治療劑。

Description

含有抗GLOBO H抗體之抗體－藥物共軛物及其用途
本發明係關於含有Globo H抗體之抗體-藥物共軛物及其在療法中之用途。
抗體-藥物共軛物(Antibody-drug conjugate，ADC)能夠提供用於治療諸如癌症之各種疾病或病況的靶向療法。ADC係包含抗體之複合分子，該抗體係經連接至諸如細胞毒性劑或藥物之生物活性劑。藉由組合抗體之獨特靶向與藥物之治療作用，抗體-藥物共軛物能夠區分正常細胞與癌細胞，從而使副作用降到最低。
ADC通常包含抗癌藥物(例如，細胞毒素)，該抗癌藥物係經偶合至可特異性靶向標記物(例如腫瘤標記物)之抗體。抗體在體內會一路追蹤此等蛋白質且將其自己結合於癌細胞表面。該抗體與該目標蛋白質(抗原)之間的結合會在腫瘤細胞中觸發信號，之後腫瘤細胞將ADC內化。ADC內化之後，細胞毒性藥物會被釋放出來並殺死癌細胞。因為該藥物具有特異性靶向，所以具有較低的副作用。
Globo H係屬於許多與腫瘤相關之碳水化合物抗原，是一種六醣，其可以在包括以下的各種上皮癌細胞表面上過度表現：乳癌細胞、結腸癌細胞、卵巢癌細胞、胰臟癌細胞、肺癌細胞及前列腺癌細胞。因此，Globo H係有前景的診斷/治療目標。
雖然針對Globo H之抗體係有用的，但是使用抗Globo H抗體用於經改良治療劑仍是有需要的。
本發明係關於含有Globo H抗體之抗體-藥物共軛物及其在療法中之用途。
本發明一個態樣係關於免疫共軛物。根據本發明一個實施例的免疫共軛物包括抗Globo H抗體或其結合片段及治療劑或標記，該共軛物具有式：Ab-(L-D)m ，其中Ab係抗Globo H抗體或其結合片段，L係連接基團或直接鍵，D係治療劑或標記，且m係1至8之整數。
根據本發明之任何實施例，Ab包含以下：重鏈可變域，其具有由HCDR1 (GYISSDQILN，SEQ ID NO:1)、HCDR2 (RIYPVTGVTQYXHKFVG，SEQ ID NO:2，其中X係任何胺基酸)及HCDR3 (GETFDS，SEQ ID NO:3)組成之三個互補區；及輕鏈可變域，其具有由LCDR1 (KSNQNLLX'SGNRRYZLV，SEQ ID NO:4，其中X'係F、Y或W，且Z係C、G、S或T)、LCDR2 (WASDRSF，SEQ ID NO:5)及LCDR3 (QQHLDIPYT，SEQ ID NO:6)組成之三個互補區。
連接基團L可為直接鍵，其中酬載(payload) D係與抗體或其結合片段直接連接(共軛)。連接基團可為蛋白質修飾或共軛中常用的任何連接基團，諸如短肽(例如，gly-gly-gly)、短有機分子連接基團(例如，SMCC，丁二醯亞胺基-4(N-順丁烯二醯亞胺基甲基)環己烷-1-甲酸酯)或類似連接基團。
酬載D可為治療劑，諸如細胞毒性劑。可用於本發明之實施例的細胞毒性劑之實例可包括類美登素(maytansinoid) (例如，DM1或DM4)、單甲基奧瑞他汀E (monomethyl auristatin E，MMAE)、單甲基奧瑞他汀F (monomethyl auristatin F，MMAF)、太平洋紫杉醇(paclitaxel)或類似物。
酬載D可為用於診斷或顯影之標記或試劑。顯影劑之實例可包括二伸乙三胺五乙酸(Diethylenetriaminepentaacetic acid，DTPA)或1,4,7,10-四氮雜環十二烷-1,4,7,10-四乙酸(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid，DOTA)。
根據本發明一些實施例，該抗體可為單株抗體，其可為人類化抗體。
本發明一個態樣係關於用以診斷出或顯影可表現Globo H之細胞或組織之方法。根據本發明一個實施例之方法包含向個體投與上述免疫共軛物。
本發明一個態樣係關於治療癌症之方法。根據本發明一個實施例之方法包含向需要癌症治療之個體投與醫藥學上有效量之上述免疫共軛物。該癌症係上皮細胞癌，諸如乳癌、結腸癌、卵巢癌、胰臟癌、肺癌或前列腺癌。
熟習此項技術者應瞭解，醫藥學上有效量視諸多因素而定，諸如患者病況、年齡、疾病狀態、投藥途徑等，且此有效量可基於常規實務之此等因素即可判定，無需過多的實驗。
本發明之其他態樣隨以下說明將變得顯而易見。
本發明之實施例係關於含有Globo H抗體之抗體-藥物共軛物及其在療法中之用途。Globo H係屬於許多與腫瘤相關之碳水化合物抗原，是一種六醣，其可以在包括以下的各種上皮癌細胞表面上過度表現：乳癌細胞、結腸癌細胞、卵巢癌細胞、胰臟癌細胞、肺癌細胞及前列腺癌細胞。因此，以針對Globo H之抗體為基礎的ADC係有用的診斷及/或治療劑。
然而，治療性抗體之快速內化或缺乏ADCC活性可能導致抗體無效以及產生抗性。因此，需要增強基於抗Globo H之治療劑的治療功效。一種方法係使酬載與抗Globo H抗體共軛(亦即，抗體-藥物共軛物)。藉由抗Globo H抗體與酬載共軛(亦即ADC)，本發明之實施例比未共軛抗Globo H抗體更有效，從而實現使用較少抗體。
根據本發明之實施例，Globo H抗體或其結合片段可與藥物、診斷劑或治療劑偶合。因此，如本文所用之術語「抗體-藥物共軛物」(ADC)可以指抗體部分(其可為全抗體或其結合片段)與酬載(其可為藥物、診斷劑或治療劑)偶合。
本發明ADC含有經設計用於治療或診斷用途的酬載。相比於未共軛Globo H抗體，此等ADC具有較佳生物活性且僅需較小的量即可達成所期望之作用。
本發明之實施例將藉由以下特定實例來說明。熟習此項技術者應瞭解，此等實例僅用於說明且其他修改及變化在不脫離本發明之範疇的情況下係可能的。實例
除非另有指示，否則各1 H NMR資料係在500 MHz下獲得。除非另外規定，否則本文中所用之縮寫如下： Bu：丁基；Bn：苯甲基；BOC：第三丁氧羰基；BOP：六氟磷酸苯并三唑-1-基氧基三/二甲胺基-鏻；DCC：二環己基碳化二亞胺；DMF：N,N-二甲基甲醯胺；DMAP：4-二甲胺基吡啶；EDC：1-(3-二甲胺基丙基)3-乙基碳化二亞胺鹽酸鹽；EtOAc：乙酸乙酯；Eq.：當量；HOBt：羥基苯并三唑；LAH：氫化鋰鋁；MeOH：甲醇；MHz：兆赫；MS(ES)：質譜分光光度計-電灑；NMP：N-甲基吡咯啶酮；Ph：苯基；Pr：丙基；TEA：三乙胺；THF：四氫呋喃；TLC：薄層層析；Tetrakis：肆(三苯基膦)鈀。實例 1. 製備抗 Globo H 抗體
根據本發明之實施例，用於產生抗Globo H抗體之通用方法包括獲得可產生針對Globo H之單株抗體的融合瘤。用於產生單株抗體之方法為此項技術中已知的且在此將不再贅述。簡言之，小鼠係以抗原(Globo H)與適當的佐劑一起進行激發。隨後，收集經免疫小鼠之脾細胞並與融合瘤融合。使用任何已知方法(諸如ELISA)，針對純系結合Globo H抗原之能力而鑑別出陽性純系。
本發明之抗體-藥物共軛物(ADC)可特異性靶向Globo H。此等ADC可使用任何與Globo H特異性結合的抗體。舉例而言，本發明ADC可以使用小鼠或人類化抗Globo H抗體或其scFv或Fab片段。例示性抗Globo H抗體可包含以下：重鏈可變域，其具有由HCDR1 (GYISSDQILN，SEQ ID NO:1)、HCDR2 (RIYPVTGVTQYXHKFVG，SEQ ID NO:2，其中X係任何胺基酸)及HCDR3 (GETFDS，SEQ ID NO:3)組成之三個互補區；及輕鏈可變域，其具有由LCDR1 (KSNQNLLX'SGNRRYZLV，SEQ ID NO:4，其中X'係F、Y或W，且Z係C、G、S或T)、LCDR2 (WASDRSF，SEQ ID NO:5)及LCDR3 (QQHLDIPYT，SEQ ID NO:6)組成之三個互補區。
根據本發明之實施例，抗體可為小鼠抗體。另一種選擇是，抗體可為嵌合抗體(例如，人類恆定區與小鼠可變區偶合)或人類化抗體(例如，小鼠CDR移植於人類免疫球蛋白之構架區上)或完全人類抗體。
單株抗體可藉由以下進行人類化：從融合瘤獲得CDR序列且將CDR序列選殖至人類構架序列中以產生人類化抗體。可使用此項技術中已知用於鑑別CDR序列之任何常見方法。本發明中之CDR區係用Kabat編號方案加以識別。首先，產生抗Globo H之融合瘤(例如小鼠GBH融合瘤)。此融合瘤可藉由用於產生單株抗體之標準方案產生。隨後例如使用TRIzol®試劑分離出融合瘤之總RNA。隨後例如使用第一股cDNA合成套組(Superscript III)及寡聚(dT20 )引子或Ig-3'恆定區引子，從總RNA合成cDNA。
隨後從cDNA選殖免疫球蛋白基因之重鏈及輕鏈可變區。舉例而言，藉由PCR，使用小鼠Ig-5'引子集(Novagen)，從小鼠GBH融合瘤cDNA擴增抗Globo H mAb之VH及VL可變區。該等PCR產物可使用CloneJetTM PCR選殖套組(Ferments)直接選殖至適合載體(例如pJET1.2載體)中。pJET1.2載體含有致死性插入序列段且僅在所需基因選殖至此致死性區域中時才會在選擇條件下存活。此有助於重組群落之選擇。最後，針對所需純系篩選重組群落，分離彼等純系之DNA並定序。可在國際ImMunoGeneTics資訊系統(IGMT)網站分析免疫球蛋白(IG)核苷酸序列。抗體表現及純化
為產生抗體，該等經分離之純系可在任何適合之細胞中表現。舉例而言，F293細胞(Life technologies)經表現抗Globo H mAb之質體轉染且培養7天。使用蛋白A親和管柱(GE)，從培養基純化出抗Globo H抗體。使用此項技術中已知的程序或根據製造商的說明，以Bio-Rad蛋白質分析套組測定蛋白質濃度及用12% SDS-PAGE進行分析。
根據本發明之實施例，可使用此等抗Globo H抗體中之任一者製備抗體-藥物共軛物(ADC)，如以下實例中所說明。實例 2. 製備抗 Globo H 抗體 - 生物素共軛物
Figure 02_image001
作為藥物與抗體偶合之實例，可使用生物素-抗生物素蛋白(avidin)系統來研究反應條件並說明ADC策略之可操作性。在此特定實例中，使用生物素類似物(亦即，生物素-N-丁二醯亞胺酯)作為偶合試劑與抗體上之胺基反應。該胺基可為抗體上之離胺酸殘基之側鏈。
簡言之，向抗Globo H單株抗體430 μL (2.7 mg/ml)於緩衝液(50 mM磷酸鉀、50 mM氯化鈉、2 mM EDTA；pH 6.5)中之溶液中緩慢添加3.8 μL Osu-生物素(生物素-O-丁二醯亞胺；20 mM，於DMSO中)。在氬氣下在室溫下攪拌反應混合物且分別攪拌2、4及16小時。使用配置有標稱分子量限值(NMWL)為30 kDa之Amicon Ultra-15離心過濾裝置在pH 7.4 PBS緩衝液中將抗體製劑進行脫鹽及濃縮，得到Globo H-生物素ADC2-1 。
Figure 02_image003
在替代方法中，該生物素類似物係與抗體上的半胱胺酸殘基之SH基偶合。如以上反應流程中所示，向抗Globo H單株抗體180 μL (5.0 mg/mL，於pH 7.4 PBS緩衝液中)之溶液中緩慢添加TCEP (5.0當量)且在37℃下攪拌1.0小時。隨後向反應混合物中添加生物素-順丁烯二醯亞胺(12當量)且在氬氣下在室溫下攪拌20小時。使用配置有NMWL為30 kDa之Amicon Ultra-15離心過濾裝置在pH 7.4 PBS緩衝液中將抗體製劑進行脫鹽及濃縮，得到Globo H-生物素ADC2-2 。實例 3. 製備抗 Globo H 抗體 -SMCC-DM1 共軛物
Figure 02_image005
在此實例中，ADC含有DM1，DM1係為了癌症療法而開發的類美登素。美登素(Maytansine)，一種苯并橋環巨環內酯(benzoansamacrolide)，係非常有效的靶向微管的化合物，其可誘導有絲分裂阻滯並在次奈莫耳(subnanomolar)濃度下殺死腫瘤細胞。DM1在微管之尖端結合以抑止微管之動態性，亦即，抑制微管之組裝。DM1是一種全身性毒性比美登素低的類美登素。在此實例中，SMCC-DM1係含有反應性連接基團SMCC之DM1，其用於與抗體反應以製造抗體藥物共軛物。SMCC-DM1可自商業來源獲得，諸如MedKoo Biosciences, Inc.或ALB Technology。
舉例而言，向抗Globo H單株抗體500 μL (2.9 mg/mL)於緩衝液(50 mM磷酸鉀、50 mM氯化鈉、2 mM EDTA；pH 6.5)中之溶液中緩慢添加58 μL SMCC-DM1 (5 mM，於DMSO中)。在氬氣下在37℃下攪拌反應混合物且攪拌20小時。使用配置有NMWL為30 kDa之 Amicon Ultra-15離心過濾裝置在pH 7.4 PBS緩衝液中將抗體製劑進行脫鹽及濃縮，得到抗Globo H-SMCC-DM1 ADC3 。(DCBD16001)實例 4. 製備抗 Globo H 抗體 -SMCC-DM4 共軛物
Figure 02_image007
DM4係另一種美登素類似物。DM4亦為抑制處於有絲分裂之細胞增殖的有效靶向微管之化合物。本發明之一些實施例可以使用DM4。在此實例中，向抗Globo H單株抗體500 μL (2.9 mg/mL)於緩衝液(50 mM磷酸鉀、50 mM氯化鈉、2 mM EDTA；pH 6.5)中之溶液中緩慢添加58 μL SMCC-DM4 (5 mM，於DMSO中)。在氬氣下在37℃下攪拌反應混合物且攪拌20小時。使用配置有NMWL為30 kDa之Amicon Ultra-15離心過濾裝置在pH 7.4 PBS緩衝液中將抗體製劑進行脫鹽及濃縮，得到抗Globo H-SMCC-DM4 ADC4 。實例 5 . 製備抗 Globo H 抗體 -MMAE 共軛物
Figure 02_image009
單甲基奧瑞他汀E (MMAE)係一種抗惡性腫瘤藥(antineoplastic agent)；其藉由阻斷微管蛋白之聚合來抑制細胞分裂。其衍生自出現於海洋無殼軟體動物中之肽(尾海兔素(dolastatin))。已證明MMAE係ADC之有用酬載。
在此實例中，向抗Globo H單株抗體400 μL (5.0 mg/mL)於緩衝液(50 mM磷酸鉀、50 mM氯化鈉、2 mM EDTA；pH 6.5)中之溶液中緩慢添加40 μL OSu-MMAE (5 mM，於DMSO中)。在氬氣下在37℃下攪拌反應混合物且攪拌20小時。使用配置有NMWL為30 kDa之 Amicon Ultra-15離心過濾裝置在pH 7.4 PBS緩衝液中將抗體製劑進行脫鹽及濃縮，得到抗Globo H-MMAE ADC5 。實例 6. 製備抗 Globo H 抗體 -vc-MMAE 共軛物
Figure 02_image011
ADC中之連接基團會對生物活性具有顯著影響。舉例而言，活體內研究證實，經肽連接之共軛物會誘導經建立的腫瘤異種移植物之消退及治癒，其中治療指數高達60倍。此等共軛物說明了連接基團技術、藥物效力及共軛方法在開發用於癌症療法之安全及有效的mAb-藥物共軛物中的重要性。
本發明之一些實施例係關於經由溶酶體可裂解二肽纈胺酸-瓜胺酸(valine-citrulline，vc)連接於抗體之MMAE，已證明，該二肽可改良ADC功效。在此實例中，向抗Globo H單株抗體400 μL (5.0 mg/mL)於緩衝液(50 mM磷酸鉀、50 mM氯化鈉、2 mM EDTA；pH 6.5)中之溶液中緩慢添加40 μL OSu-vc-MMAE (5 mM，於DMSO中)。在氬氣下在37℃下攪拌反應混合物且攪拌20小時。使用配置有NMWL為30 kDa之Amicon Ultra-15離心過濾裝置在pH 7.4 PBS緩衝液中將抗體製劑進行脫鹽及濃縮，得到抗Globo H-vc-MMAE ADC6 。實例 7. 製備抗 Globo H 抗體 -MMAF 共軛物
Figure 02_image013
本發明之一些實施例係關於含有單甲基奧瑞他汀F (MMAF)之ADC，MMAF係MMAE之類似物。向抗Globo H單株抗體400 μL (5.0 mg/mL)於緩衝液(50 mM磷酸鉀、50 mM氯化鈉、2 mM EDTA；pH 6.5)中之溶液中緩慢添加40 μL OSu-MMAF (5 mM，於DMSO中)。在氬氣下在37℃下攪拌反應混合物且攪拌20小時。使用配置有NMWL為30 kDa之Amicon Ultra-15離心過濾裝置在pH 7.4 PBS緩衝液中將抗體製劑進行脫鹽及濃縮，得到抗Globo H-MMAF ADC7 。實例 8. 製備抗 Globo H 抗體 -4- 異硫氰基 - 苯基 -DTPA 共軛物
Figure 02_image015
ADC除了治療之外，亦可用於診斷及/或顯影。本發明之一些實施例係關於能夠與目標分子特異性結合的顯影試劑。舉例而言，為了顯影，ADC之酬載可含有用於結合所選擇的金屬(諸如放射性過渡金屬)之螯合官能基。此項技術中已知多種用於診斷及/或顯影用途的螯合官能基，諸如二伸乙三胺五乙酸(DTPA)或1,4,7,10-四氮雜環十二烷-1,4,7,10-四乙酸(DOTA)。DTPA係具有二伸乙基三胺主鏈及5個羧基的胺基多羧酸。DTPA可視為EDTA之放大版本。此處吾人將使用DTPA作為實例。熟習此項技術者應瞭解，在不脫離本發明之範疇的情況下亦可使用其他螯合劑。
在此實例中，向抗Globo H單株抗體400 μL (5.0 mg/mL)於緩衝液(10 mM磷酸鈉；pH 8.0)中之溶液中緩慢添加40 μL 4-異硫氰基-苯基-DTPA (5 mM，於DMSO中)。在氬氣下在37℃下攪拌反應混合物且攪拌20小時。使用配置有NMWL為30 kDa之Amicon Ultra-15離心過濾裝置在pH 7.4 PBS緩衝液中將抗體製劑進行脫鹽及濃縮，得到抗Globo H-硫脲-4-苯基-DTPA ADC8 。實例 9. 製備抗 Globo H 抗體 -N- 苯基己二醯二胺 -DTPA 共軛物
Figure 02_image017
在此實例中，DPTA經由連接基團己二醯二胺連接於抗體。向抗Globo H單株抗體400 μL (5.0 mg/mL)於緩衝液(10 mM磷酸鈉；pH 8.0)中之溶液中緩慢添加40 μL 6-OSu-N-苯基己醯胺-DPTA (5 mM，於DMSO中)。在氬氣下在37℃下攪拌反應混合物且攪拌20小時。使用配置有NMWL為30 kDa之Amicon Ultra-15離心過濾裝置在pH 7.4 PBS緩衝液中將抗體製劑進行脫鹽及濃縮，得到抗Globo H-N-苯基己醯胺-DTPA ADC9 。實例 10. SDS-PAGE
本發明之各種ADC可藉由此項技術中已知之技術進行分析，諸如SDS-PAGE及HPLC。舉例而言，從實例1及3獲得之抗Globo H mAb及抗Globo H ADC3 (DCBD16001)之溶液藉由使用4-12%非還原性及還原性SDS-PAGE凝膠、繼之以庫馬斯亮藍(Coomassie brilliant blue)染色來分析。圖1顯示，ADC (DCB16001)在非還原條件及還原條件下保留適當抗體結構，亦即適當分子量。實例 11. PLRP-HPLC
本發明之各種ADC亦可藉由HPLC來分析。圖2顯示，共軛反應基本上係完全的且僅留下剩餘量之Globo H抗體及SMCC-DM1。實例 12. 酬載偶合分析
評估藥物:抗體比(DAR)對於監測目標抗體上之酬載共軛效率係重要的。藥物:抗體比會影響抗Globo H ADC產物之治療功效。液相層析-質譜法(Liquid chromatography-mass spectrometry，LC-MS)係用於測定離胺酸連接之抗體-藥物共軛物(ADC)的藥物:抗體比(DAR)及載藥量分佈(drug load distribution)的精選方法。峰面積百分比表示特定載藥ADC種類的相對分佈。隨後使用峰面積百分比資訊及載藥數目計算加權平均DAR。
圖3說明本發明ADC (抗Globo H-SMCC-DM1 ADC3 . (DCBD16001))之MS分析的一個實例，其指示各種數目之藥物與抗體連接的分佈，其中最豐富種類係具有1至8個藥物與抗體連接。此樣品中之平均藥物:抗體比(DAR)係4.07。多個藥物複本與一個抗體連接將確保藥物更有效地遞送至細胞中。實例 13. 結合親和力
本發明ADC之結合親和力可藉由此項技術中已知之任何適合方法評定，諸如BIAcore或ELISA。在此實例中，使用BIAcore來量測本發明ADC之親和力。
簡言之，製備抗Globo H ADC之流動溶液以進行結合動力學研究。將配體Globo H固定在CM5晶片上：首先，在固定緩衝液(10 mM乙酸鈉，pH 4.5)中，將配體(Globo H-胺)稀釋至6 mg/mL。一般在25℃下使用5 μL/min之流速固定。用於固定之試劑係提供在胺偶合套組中。活化：EDC/NHS 7分鐘。固定：流動時間720秒。失活：1.0 M乙醇胺，pH 8.5，7分鐘。此過程將在感測器晶片CM5上產生約200 RU之反應結合水準。
隨後，如下進行單循環動力學分析：Biacore單循環動力學(single-cycle kinetics，SCK)方法具有用以獲得動力學資料之軟體。選擇操作(Run)：方法。參數設定如下：資料收集速率：1 Hz；偵測模式：雙重；溫度：25℃；濃度單位：nM；緩衝液A：HBS-EP+緩衝液。選擇啟動(Start up)且將複製數改為3。選擇啟動循環(Startup cycle)且參數設定如下：類型：低樣品消耗；接觸時間：150秒；解離時間：420秒；流速：50 μL/min；流動路徑：兩個。選擇樣品循環(Sample cycle)且參數設定如下：類型：單循環動力學；濃度/循環：5；接觸時間：150秒；解離時間：420秒；流速：50 μl/min；流動路徑：兩個。選擇再生(Regeneration)且參數設定如下：再生溶液：10 mM甘胺酸pH2.0/1.5 (v/v=1)；接觸時間：45秒；流速：30 μL/min；流動路徑：兩個。選擇樣品之複本(Copy)且參數設定如上。製備樣品：將分析物抗體DCBPR1101在操作緩衝液中稀釋至200 nM。從200 nM樣品製備濃度系列：將200 μL之200 nM溶液與200 μL操作緩衝液混合，得到100 nM溶液。繼續連續稀釋以獲得以下：200、100、50、25及12.5 nM。製備樣品且根據支架位置(Rack Position)定位。根據製備操作方案(Prepare Run Protocol)，確保一切正確，且點選開始(Start)以開始實驗。使用由Biacore T100評估軟體2.0提供之預定模型(1:1結合)進行親和力結合曲線擬合。
表1彙總了本發明之一個ADC (DCBD16001)的BIAcore分析結果。此等結果顯示，本發明ADC仍然能夠與抗原(Globo H)特異性結合。表 1
Figure 107120867-A0304-0001
 實例14. 細胞毒性分析
含有細胞毒性酬載之本發明ADC可用於殺死目標細胞，諸如殺死癌細胞。為此分析，從ATCC獲得MCF-7、HCC-1428、BT-474。所有細胞株在適合培養基中於37℃下在5% CO2 之含濕氣培育箱氛圍中培養。所有細胞株繼代培養至少三代，將細胞種植於96孔黑色平底培養盤(所有細胞株均為10,000個細胞/100微升/孔)中，且使細胞於37℃下在5% CO2 之含濕氣氛圍中進行細胞黏附隔夜。
ADC係從1 μg/μl儲備溶液製備且在細胞接種之後24小時稀釋至適當的操作濃度。首先用PBS將20 mM測試物品稀釋成最高操作濃縮物。用DMSO對此最高操作濃縮物進行八點連續三倍稀釋，且隨後另外用培養基稀釋100×。偵測8種濃縮物，最終濃度在100 nM至0.046 nM (DM-1)或66.67 nM至0.030 nM (DBCD16001、DCBPR1101)之範圍內。最終PBS濃度係1%。隨後移除培養基且替換成含有不同濃度之游離DM1、DBCD16001、DCBPR1101及200微升/孔的新鮮培養基且培育細胞72小時。在室溫下預解凍Cell Titer-Glo試劑48小時。在此時間點之後，移除培養基且在室溫下向孔中添加100微升/孔的Cell Titer-Glo試劑(Promega G7571，批次0000182872)，持續10分鐘，且使用CLARIOstar®高效單色器多模式微量培養板讀取器來量測發光信號。關於所有細胞分析，使用GraphPad Prism 6三參數曲線擬合來產生劑量反應曲線。
此等分析之結果顯示於下表2中。顯然，相比於抗原陰性細胞(亦即BT-474)，本發明ADC (亦即DCBD16001)可有效殺死抗原(Globo H)表現細胞(MCF-7及HCC-1428)。表 2
Figure 107120867-A0304-0002
 實例 15. 內化分析
抗體經受體介導之內化作用可提供細胞特異性藥物遞送。內化係某些使用ADC之靶向療法所需要的。此項技術中已知，ADC內化係抗體依賴性及酬載依賴性的。亦即，並非所有抗體都能為ADC提供遞送機制。類似地，不同酬載在相同抗體上會具有顯著不同的內化效率。
抗Globo H ADC及抗Globo H抗體之內化及降解可藉由流動式細胞測量術量測。在此研究中可使用兩種方法，直接在抗體上進行螢光標記或使用經螢光共軛之二級抗體來偵測內化後留在細胞表面上之初級抗體。
簡言之，以1×105 個細胞/孔接種MCF7或HCC 1428乳癌細胞。接著，使0.5-1 mg經螢光標記之抗Globo H抗體或抗Globo H ADC在FACS緩衝液中在4℃下經受100 μl目標細胞(細胞密度1×106 /ml)作用1小時，以實現抗Globo H抗體或抗Globo H ADC與細胞表面目標之特異性結合。在培育之後，用FACS緩衝液洗滌細胞三次以移除未結合抗體。隨後在37℃下在5% CO2 下培育細胞以進行抗體內化。
在不同時間點，用胰蛋白酶解離細胞且用經螢光共軛抗人類IgG二級抗體染色5分鐘，隨後使用Beckman流動式細胞測量術系統進行分析。關於經間接標記之抗體或經間接螢光標記之二級抗體之內化及降解的量測，如相比於4℃對照組(作為抗體內化背景)，藉由Beckman流動式細胞測量術軟體分析在各時間點之螢光強度及細胞結合百分比。各實驗以三重複方式進行。
為了藉由螢光顯影研究抗Globo H ADC及抗Globo H抗體之內化，使用標準濾光器組態在DeltaVision® Core顯微鏡上對細胞進行顯影。簡言之，MCF-7或HCC1428目標細胞以約2×105 個細胞/孔在8孔玻璃蓋玻片底培養皿(Nunc)上繼代培養。在附著之後，細胞在37℃下與10 nM抗Globo H抗體或與Alexa-488共軛之抗Globo H ADC一起培育隔夜。在內化之前，用Rab5-mCherry或LAMP1-mApple質體轉染細胞以便用螢光標記物LysoTracker進行核內體及溶酶體標記。對細胞進行洗滌且在Deltavision反褶積顯微鏡(deconvolution microscope)上顯影，以確定Alexa 488及Alexa594共定位。
圖4A及圖4B顯示此等研究之結果。結果指示，本發明之ADC可被表現Globo H之細胞(例如，MCF-7 (圖4B)及HCC-1428細胞(圖4A))內化。實例 16. 活體內 PK
此研究使用Meso Scale Discovery (MSD)電化學發光(Electrochemiluminescent，ECL)法對BALB/c小鼠樣品中之DCBD16001進行藥物動力學分析。抗體MSD分析可量測共軛及未共軛抗體兩者，如圖5中所說明。如此實例或總量抗體分析中所說明，培養盤塗有山羊抗人類IgG，其可捕捉所有人類化抗體(共軛及未共軛)。關於共軛抗體分析，培養盤係塗有針對酬載(藥物)之抗體，諸如圖5中所示之抗美登素抗體。
以1 mg/kg之劑量經由尾部靜脈對BALB/c小鼠投藥。隨後在不同時間點獲得血液樣品以藉由MESO QuickPlex SQ 120方法測定BALB/c小鼠中之DCBD16001之濃度。DCBD16001之藥物動力學參數藉由非室體分析(noncompartmental analysis)，使用6.3版Phoenix™ WinNonlin程式來分析。
圖6顯示活體內藥物動力學研究之結果且下表3彙總了PK研究之結果。總量抗體MSD分析：量測共軛及未共軛抗體兩者。共軛抗體MSD分析：僅量測共軛抗體。DCBD16001之活體內半衰期係約127小時，其相比於已知治療性抗體之活體內半衰期而言相對較短，表明若出現嚴重副作用，則DCBD16001可被較快排出。表 3
Figure 107120867-A0304-0003
 實例 17. 抗 Globo H ADC 之異種移植模型
本發明之一些實施例係關於使用ADC中之抗體作為歸巢劑/靶向劑進行診斷、顯影及治療疾病的方法。該抗體部分將與其目標抗原特異性結合，而酬載將提供診斷/顯影或治療試劑。可能的診斷/顯影試劑可包括例如螢光部分或放射性探針，而治療試劑可包括例如細胞毒性劑或免疫調節劑(例如，CD3)。
在此特定實例中，係評定本發明ADC治療癌症之能力。簡言之，從BioLasco Taiwan Co., LTD.購得六至七週齡雄性CB.17 SCID小鼠，且檢疫一週。在實驗期內，將5隻小鼠圈養在一個籠中。所有動物均以12-小時光/12-小時暗循環託管於Da-Hu動物研究室中19-25℃下。動物可以自由地隨意取食嚙齒動物顆粒狀食物及水。動物研究之實驗方案係經實驗動物照護及使用委員會(Institutional Animal Care and Use Committee)、DCB審查及批准。
在注射腫瘤細胞前7天，給小鼠接種17-β-雌二醇集結粒(0.18 mg/集結粒，60天釋放；Innovative Research of America, USA)。在對數期生長期間收集用於植入的HCC1428乳癌細胞且將其再懸浮於磷酸鹽緩衝鹽水(phosphate buffered saline，PBS)中。在各小鼠之側腹皮下(s.c.)注射0.15 mL 50%基質膠溶液(Matrigel solution) (BD Biosciences, MA, USA)中之1×107 個HCC1428細胞。當平均腫瘤體積達到250 mm3 時，將小鼠隨機分成8組。第1組接受PBS，充當對照組，用於計算腫瘤生長抑制率。第2組至第5組分別按以下接受DCBD16001：3 mg/kg (mpk)每週兩次、10 mpk每週兩次、20 mpk每週一次及在第1天及第11天各30 mg/kg。第6組接受未共軛抗體PR1101 (無酬載)，10 mpk每週兩次。第7組中之小鼠接受同型抗體，10 mpk每週兩次，充當陰性對照組。第8組中之小鼠接受太平洋紫杉醇，10 mpk每週一次，充當陽性治療對照組。經媒劑、DCBD16001、未共軛抗體及同型抗體處理之小鼠係藉由靜脈內注射，而太平洋紫杉醇係藉由腹膜內注射給予。
腫瘤體積使用測徑規每週量測三次且使用下式估算：腫瘤體積 = (w 2 ×l )/2，其中w = 腫瘤之寬度且l = 直徑長度(mm)。腫瘤生長抑制(tumor growth inhibition，TGI)之百分比使用下式計算：TGI% = [1 - (T/C)] × 100%，其中T及C分別表示處理組及對照組之平均腫瘤體積。
給動物稱重，每週三次，直至研究完成。體重變化按相比於初始體重之體重增加百分比計算。
圖7說明實驗方案及治療方案。在此實驗中，使用HCC1428細胞，其係乳癌細胞株。此等細胞會在其表面上表現Globo H。
圖8顯示活體內異種移植腫瘤治療研究之結果。如所示，10 mg/kg、20 mg/kg及30 mg/kg之本發明ADC (DCBD16001)明顯比未共軛抗體(抗Globo H抗體)更有效。結果指示本發明ADC之有效性。明顯改良的功效係因為該經偶合酬載，其可以增強細胞毒性及/或改良藥物動力學。
引人注意的是，在第1天及第11天用30 mpg DCBD16001處理(總劑量60 mg/kg)係最有效的，明顯比10 mg/kg每週兩次(總劑量60 mg/kg)更有效。此結果指示，在較高的單一注射劑量及較長的投藥時間間隔(10天)下，ADC更有效。在相同的總劑量下，較長的投藥時間間隔(例如，10天)產生較佳的結果，這一事實出人意料。這表明，對於抗Globo H ADC而言，較高的Cmax將比較高的AUC更重要。
以下事實證實腫瘤生長抑止係由增強的腫瘤細胞殺死或腫瘤細胞生長抑制引起：不同處理組中之小鼠的體重沒有明顯變化，如圖9中所示。
以上實例清楚地說明了獲得並表徵本發明ADC之各種方法，以及本發明ADC在治療癌症方面的有效性。即使本發明之實施例用有限數目之實例加以說明，熟習此項技術者仍應瞭解，其他變化及修改在不脫離本發明之範疇的情況下係可能的。因此，本發明之範疇應僅受所附申請專利範圍限制。
圖1顯示根據本發明一個實施例的ADC (DCB16001)之SDS-PGAE凝膠分析。結果顯示，ADC在非還原條件及還原條件下保留適當抗體結構，亦即適當分子量。
圖2顯示HPLC曲線，指示Globo H抗體與SMCC-DM1之間的共軛反應基本上係完全的且僅留下剩餘量之Globo H抗體及SMCC-DM1。
圖3說明本發明ADC (DCB16001)之MS分析的一個實例，其指示各種數目之藥物與抗體連接的分佈，其中最豐富種類是具有1至8個藥物與抗體連接。此樣品中平均藥物:抗體比(drug-to-antibody ratio，DAR)係4.07。
圖4A及圖4B顯示ADC內化之螢光顯影結果。結果指示，本發明之ADC可被表現Globo H之細胞(例如，MCF-7 (圖4B)及HCC-1428細胞(圖4A))內化。
圖5顯示說明Meso Scale Discovery (MSD)電化學發光分析的示意圖，其係用於量測總量抗Globo H抗體及共軛抗Globo H抗體以進行DCBD16001之活體內藥物動力學研究。
圖6顯示DCBD16001之活體內藥物動力學研究結果。
圖7顯示在癌症異種移植模型中使用本發明ADC之治療方案。
圖8顯示HCC-1428異種移植模型中之腫瘤生長抑制。
圖9顯示小鼠在圖8之實驗中之處理期間的體重變化。
Figure 12_A0101_SEQ_0001
Figure 12_A0101_SEQ_0002
Figure 107120867-A0304-11-0002-1

Claims (10)

  1. 一種與Globo H特異性結合之免疫共軛物，其包含：抗Globo H抗體或其結合片段，及治療劑或標記，其中該免疫共軛物具有式：Ab-(L-D)m，其中Ab係該抗Globo H抗體或其結合片段，L係連接基團或直接鍵，D係該治療劑或該標記，且m係1至8之整數；其中該抗體包含以下：重鏈可變域，其具有由HCDR1(GYISSDQILN，SEQ ID NO：1)、HCDR2(RIYPVTGVTQYXHKFVG，SEQ ID NO：2，其中X係任何胺基酸)及HCDR3(GETFDS，SEQ ID NO：3)組成之三個互補區；及輕鏈可變域，其具有由LCDR1(KSNQNLLX'SGNRRYZLV，SEQ ID NO：4，其中X'係F、Y或W，且Z係C、G、S或T)、LCDR2(WASDRSF，SEQ ID NO：5)及LCDR3(QQHLDIPYT，SEQ ID NO：6)組成之三個互補區。
  2. 如請求項1之免疫共軛物，其中該抗體係單株抗體。
  3. 如請求項1之免疫共軛物，其中該抗體係人類化抗體。
  4. 如請求項1至3中任一項之免疫共軛物，其中該治療劑係細胞毒性劑。
  5. 如請求項4之免疫共軛物，其中該細胞毒性劑係類美登素1(maytansinoid 1，DM1)、類美登素4(DM4)、單甲基奧瑞他汀E(monomethyl auristatin E，MMAE)、單甲基奧瑞他汀F(monomethyl auristatin F，MMAF)或太平洋紫杉醇(paclitaxel)。
  6. 如請求項1至3中任一項之免疫共軛物，其中D係診斷或顯影試劑。
  7. 一種用於診斷或顯影可表現Globo H之細胞或組織之組合物，其包含如請求項6之免疫共軛物。
  8. 一種用於治療癌症之醫藥組合物，其包含如請求項1至5中任一項之免疫共軛物。
  9. 如請求項8之醫藥組合物，其中該癌症係上皮細胞癌。
  10. 如請求項9之醫藥組合物，其中該癌症係乳癌、結腸癌、卵巢癌、胰臟癌、肺癌或前列腺癌。
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