TW201831518A - 抗herv-k套膜抗體及其用途 - Google Patents
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Abstract
本發明係關於一種新穎抗HERV-K套膜之抗體,其以未受到醣苷化或天然構型影響的保留區為標的,及其於診斷及/或治療的用途。
Description
本發明係關於一種新穎抗HERV-K套膜的抗體,其以未受到醣苷化(glycosylation)或天然構型影響的保留區為標的,及其於診斷及/或治療之用途。
肌肉萎縮性脊髓側索硬化症(Amyotrophic lateral sclerosis,ALS)疾病首先由法國神經學家讓-馬丁夏科特(Jean-Martin Charcot)描述,其名稱反映了皮質脊髓運動神經元的退化,該皮質脊髓運動神經元的下行軸突顯示改變的外側脊髓結構(側索硬化(lateral sclerosis)),以及與肌肉消瘦(肌萎縮(amyotrophy))有關之二次去神經支配有關的脊髓運動神經元的死亡(Taylor、Brown、及Cleveland 2016)。的確,ALS係一種進行性且最終致死性的神經退行性疾病,其由涉及計劃、控制及執行自主運動的大腦運動皮質、腦幹及脊髓中的運動神經元變性所引起。致死的結果通常發生在診斷後3-5年(Taylor、Brown、及Cleveland 2016)。ALS的盛行率大約於10萬例中達到5例,其反映該疾病的快速致命性(Taylor、Brown、及Cleveland 2016)。約10% 的ALS病例似乎是遺傳性地於家族中散播(遺傳性ALS),與特定的基因體突變有關。例如,約20%的家族性ALS與超氧化物歧化酶(superoxide dismutase,sod1)基因中的突變相關(Vucic and Kiernan 2009;Rosen 1993)。其他非家族性病例被分類於散發性ALS(90%的ALS病例)(Lagier-Tourenne and Cleveland 2009),意味著其發生並無家族史。
帕金森氏病(Parkinson’s disease)、亨廷頓舞蹈病(Huntington’s disease)、阿茲海默氏病(Alzheimer’s disease)、額顳葉變性(frontotemporal lobar degeneration,FTLD)和ALS等神經退行性疾病係與中樞神經系統中神經元細胞及神經膠質細胞之內外兩者的錯誤折疊蛋白質的蓄積有關(Polymenidou及Cleveland 2011)。此等錯誤折疊蛋白質聚集體係每種疾病的病理標誌且可以在啟動事件之後透過普里昂蛋白樣機制(prion-like mechanism)於細胞間傳播。一個廣泛持有的觀點為此等聚集體於疾病起始及進展中扮演關鍵角色,內源性蛋白質的錯誤折疊形式很可能潛在地透過增加聚集體內基本細胞組份的疏水性及/或隔離作用、產生氧化物種、蛋白酶體抑制及透過其他途徑而獲得毒性。另一種觀點則認為,大的聚集體並不代表毒性形式,而是防禦性細胞反應的最終產物,目的在於保護細胞免受大多數技術無法察覺的毒性較大的寡聚物種(Polymenidou及Cleveland 2011)。
來自ALS病患血清樣本但對於人類免疫缺陷 病毒(HIV)或人類T細胞白血病病毒(HTLV)外源性病毒為血清陰性之研究顯示,在50-60%的ALS樣本中,反轉錄酶(RT)活性水平與彼等HIV感染病患相當(MacGowan等人,2007;McCormick等人,2008;Andrews等人,2000;Steele等人,2005)。此與由於承認鼠類和人類兩者反轉錄病毒可引起類ALS症候群而數年來懷疑反轉錄病毒參與的事實一致(McCormick等人,2008)。於HIV陽性病患中類ALS失調可以以抗反轉錄病毒療法緩解(Moulignier等人,2001;von Giesen等人,2002)。此對於HIV感染病患的ALS症狀學有效,然而可能代表ALS病例的特殊亞類。
於ALS病患的一等親的血清中亦發現增加的RT活性,其導致推測RT活性可能源於人內源性反轉錄病毒(人類內源性反轉錄病毒(HERVs))中的遺傳活性拷貝,其代表我們的基因體的8%(Steele等人,2005)。
儘管如此,ALS中RT活性的偵測並未確定該酶的來源,但已顯示HERV-K於死後腦的涉及(Douville et al。2011)]。定序研究顯示與對照相比,ALS病患中7q34及7q36.1染色體位點(chromosomal loci)(分別對應HERV-K之HML-2及HML-3亞族)更常被表現(Douville及Nath 2014)。此外,近來已觀察到與對照相比,HERV-K gag-pol及env RNA兩者於來自ALS病患的腦中的表現顯著升高(Li等人,2015)。
HERV-K於人神經元培養中的表現造成神經元細胞毒性,如藉由於整個HERV-K基因體或僅 HERV-K-env基因轉染後以劑量依賴性方式減少的神經元數量以及軸突回縮所觀察到的。此暗示細胞內HERV-K-Env蛋白質可能有助於神經毒性。此已經藉由CRISP/casp9試驗證實,該試驗藉由VP64轉錄因子的LTR活化而允許HERV-K兩倍增加的表現(Li等人,2015)。HERV-K表現引起在皮質層神經元中表現HERV-K-env基因的轉基因小鼠體內運動皮質體積的喪失,此與使用離子化的鈣結合適配體分子1(ionized calcium-binding adapter molecule 1,Iba-1)小膠質細胞標記物測量的免疫反應性無關。行為分析顯示,HERV-K-env轉基因小鼠行走較短距離,休息較長時間,且在旋轉桿測試性能測試中下降得更快,顯示出痙攣狀態的證據,並增加了後肢的緊扣。除了此等運動功能障礙之外,轉基因小鼠發展出四肢及脊髓肌肉的深度缺陷,包括呼吸作用,其導致10個月內50%的死亡率(Li等人,2015)。
有趣地,HERV-K RT表現與來自ALS病患的神經元中TDP-43程度增加有相關,暗示了RT表現與疾病特徵性的其他異常細胞過程合併發生(Buratti及Baralle 2009;Geser等人,2009;Douville等人,2011)。此種於ALS中普里昂蛋白樣機制的證據目前涉及主要錯誤折疊的蛋白質SOD1及TDP-43(Polymenidou及Cleveland 2011)。最近,Li等人證實TDP-43可活化HERV-K-env於人類神經元中的表現,此與他們觀察到TDP-43能夠結合HERV-K之長末端重複(LTR)的區域 726-CCCTCTCCC-734一致(Li等人,2015)。他們亦顯示內源性TDP-43緘默化降低了HERV-K表現。此等結果最近藉由顯示正常TDP-43對活體外人類星形膠質細胞及神經元中HERV-K轉錄沒有影響而得到補充,而TDP-43於HERV-K啟動子之U5區域中具有結合位點。後一種結合隨著炎症而增強,例如,於腫瘤壞死因子(Tumor Necrosis Factor,TNFα)的存在下,或以蛋白酶體抑制(Manghera、Ferguson-Parry、及Douville 2016)。有趣地,相同研究顯示,當野生型(正常)TDP-43沒有時,TDP-43的聚集型的過度表現增進HERV-K病毒蛋白的表現及累積(Manghera、Ferguson-Parry、及Douville 2016)。此外,儘管ALS皮層神經元中存在增強的壓力顆粒(stress granule)及自噬反應(autophagic response)的證據,但此等細胞無法清除過量的HERV-K蛋白質累積。由於TDP-43啟動子含有干擾素調節因子(IRF1、IRF3)及核因子-κB(NFκB)的結合位點,因此TDP-43啟動子可能對干擾素及炎症相關轉錄因子有反應(Douville等人,2011)。
綜合考量,此等發現暗示內源性反轉錄病毒元件及HERV-K特別是參與了ALS的病理生理學,且可能是TDP43與此等蛋白質病變之間喪失聯結(Alfahad及Nath 2013)。因此ALS病患的神經元中的HERV-K套膜蛋白質表現可能促成神經變性及疾病致病機轉。
迄今,作為一種對症治療,利魯唑(Riluzole)仍然是唯一相對有效的藥物,且僅延長了病患的平均存 活時間3-6個月(Hardiman、van den Berg、及Kiernan 2011)。目前的治療方案係基於症狀管理及保存生活品質,提供多領域背景。有效治療的發現仍然是患此種快速致死性疾病病患急迫的需求(Hardiman、van den Berg、及Kiernan 2011)。
因此,於治療ALS的有效治療劑仍有未滿足的需求。
本發明者等已開發了一種抗HERV-K套膜蛋白質之新穎抗體,其顯示出意想不到的性質。
本發明者等已顯示如本發明之抗體,命名為GN_mAb_Env_K01,為一種鼠類單株抗體(mAb),其選擇性結合至HERV-K-Env蛋白質的表面上的線狀抗原決定位SLDKHKHKKLQSFYP(SEQ ID NO:9)。GN_mAb_Env_K01為IgG2b/κ鼠亞型之一全長抗體。GN_mAb_Env_K01生物學活性已於ELISA及西方氏印漬術(Western Blotting)免疫試驗中證實。令人驚訝地,GN_mAb_Env_K01辨識固有的及變性的HERV-K-Env蛋白質兩者,但亦但也識別非醣苷化和醣苷化形式,而市售抗HERV-K-Env抗體無法偵測出醣苷化形式。
而且最出乎意料的是,標定的抗原決定位似乎與資料庫中描述的HERV-K env基因中的穩定胺基酸序列高度保留。無論涉及其套膜蛋白質之致病性表現的HERV-K拷貝為何,此賦予該抗體獨特的定位。此等未 預期的結果指出GN_mAb_Env_K01為用於標定HERV-K套膜蛋白質的獨創工具。他們亦顯示,即使對於本項技術領域中具通常知識者來說,選擇及獲得此種單株抗體亦不能藉由指定的免疫接種方案而預見或預測。
如此,本發明係關於一種辨識HERV-K套膜蛋白質之抗體,其中該抗體結合至抗原決定位SLDKHKHKKLQSFYP(SEQ ID NO:9)。
本發明亦係關於一種用於治療之辨識HERV-K套膜蛋白質之抗體,其中該抗體結合至抗原決定位SLDKHKHKKLQSFYP(SEQ ID NO:9)。
本發明亦係關於一種於治療肌肉萎縮性脊髓側索硬化症(ALS)(較佳為散發性ALS)之方法中使用的辨識HERV-K套膜蛋白質之抗體,其中該抗體結合至抗原決定位SLDKHKHKKLQSFYP(SEQ ID NO:9)。
於另一態樣,本發明係關於一種醫藥組成物,其包含如上定義之抗體及醫藥上可接受的賦形劑。
於另一態樣,本發明亦係關於一種於生物樣品中偵測HERV-K套膜蛋白質之方法,其包含使該生物樣品與如上述定義之抗HERV-K套膜抗體接觸的步驟。
於另一態樣,本發明亦係關於一種於病患診斷ALS之方法,特別是診斷散發性ALS,其包含使獲自該病患的生物樣品與如上述定義之抗HERV-K套膜抗體接觸的步驟。
[定義]
如本文所使用,本文使用的術語「治療」或 「處理」係指逆轉、緩解、抑制疾病或病況的進展、或預防該術語適用的疾病或病況,或逆轉、緩解、抑制疾病或病況的進展,或預防該術語適用的疾病或病況的一種或多種症狀。
如本文所使用,本文使用的術語「預防」係指防止尚未出現臨床症狀、使其臨床診斷之典型的損傷或生理失能的個體中發生疾病或病況。
如本文所使用,「抗體」或「免疫球蛋白」具有相同意義,並將在本發明中相等地使用。如本文使用之術語「抗體」係指免疫球蛋白分子及免疫球蛋白分子之免疫學上的活性部位,即,含有特異性結合抗原之抗原結合位的分子。如此,術語「抗體」不僅涵括完整的抗體分子,亦包括抗體片段,以及抗體的衍生物。
如本文所使用,詞句「抗體的片段」係指模擬高度變異區的此類抗體的一部分,諸如CDR(CDR-L1、CDR-L2、CDR-L3、CDR-H1、CDR-H2、CDR-H3)。根據本發明之抗體的片段保留了該抗體之結合親和力及特異性。此種片段係該抗體的功能等同物,且其在與該抗體實質上相同的抗原決定位結合。抗體片段之例包括但未限於重鏈、輕鏈、VL、VH、Fv、Fab、Fab'、F(ab)2及F(ab')2。
如本文所使用,詞句「抗體的衍生物」係指本發明之抗體的片段,較佳包括該抗體之至少一個CDR,較佳為該抗體之至少一個CDR3,稠合至至少一個不同於天然序列的序列(例如,另一物種的連結子序 列,...),該衍生物對HERV-K Env具有與本發明抗體相當的結合親和性及特異性。依據本發明之衍生物保留了該抗體的結合親和力及特異性。此種衍生物係該抗體的功能等同物,且其與該抗體實質上相同的抗原決定位結合。抗體的衍生物之例包括但未限於scFv、(scFv)2及雙價抗體(diabodies)。
於天然抗體,兩條重鏈(HC)藉由雙硫鍵相互連接,每條重鏈藉由雙硫鍵與輕鏈(LC)連接。有兩種類型的輕鏈,λ(l)及κ(k)。有五種主要的重鏈類型(或同型(isotype)),其決定抗體分子的功能活性:IgM、IgD、IgG、IgA及IgE。每條鏈含有不同的序列域。
典型地,輕鏈包括兩個結構域,即可變域(VL)及恆定域(CL)。重鏈包括四個結構域,一可變域(VH)及三個恆定域(CH1、CH2及CH3,統稱為CH)。輕鏈(VL)及重鏈(VH)兩者的可變區確定對抗原的抗原決定位特異的結合位點。輕鏈(CL)及重鏈(CH)之恆定區結構域賦予重要的生物學特性,諸如抗體鏈締合、分泌、經胎盤的運動(trans-placental mobility)、補體結合以及與Fc受體(FcR)的結合。Fv片段係免疫球蛋白之Fab片段的N端部分,且由一個輕鏈及一個重鏈的可變部分構成。抗體的特異性在於抗體結合位點與抗原的抗原決定位之間的結構互補性。抗體結合位點由主要來自高變異或互補決定區(CDR)的殘基組成。偶爾,來自非高變異區或框架區(FR)的殘基影響整個結構域結構並因此影響結合位點。互補決定區或CDR係指一起定義天然免疫球蛋白結合位 點的天然Fv區的結合親和力及特異性之胺基酸序列。免疫球蛋白之輕鏈及重鏈各具有三個CDR,分別命名為L-CDR1、L-CDR2、L-CDR3及H-CDR1、H-CDR2、H-CDR3。因此,抗原結合位點包括六個CDR,其包含來自重鏈及輕鏈V區之每一個的CDR組。框架區(FR)係指介於CDR之間的胺基酸序列。
如本文所使用,術語「嵌合抗體」係指包含來自任何物種(較佳小鼠)的抗體之VH域及VL域,以及人類抗體之CH域及CL域之抗體。
依據本發明,術語「人化抗體」係指具有來自人類抗體的可變區框架及恆定區,但保留來自任何物種(較佳為小鼠)之抗體的CDR。
術語「Fab」表示具有約50,000的分子量及抗原結合活性之抗體片段,其中在藉由以蛋白酶(木瓜蛋白酶)處理IgG獲得的片段中H鏈的N-末端側的約一半和整個L鏈藉由雙硫鍵結合在一起。
如本文所使用,術語「F(ab')2」係指具有分子量為約100,000及抗原結合活性之抗體片段,在藉由以蛋白酶(胃蛋白酶)處理IgG而獲得的片段中,其略大於藉由絞鏈區的雙硫鍵結合的Fab。
如本文所使用,術語「Fab'」係指具有分子量為約50,000及抗原結合活性之抗體片段,其藉由切斷F(ab')2之絞鏈區的雙硫鍵而獲得。
詞句「單鏈Fv」或「scFv」係指一種多肽,其係共價連接的VH::VL異二聚體,且通常從包括 由編碼肽的連接子連接之VH及VL編碼基因的基因融合體表達。「dsFv」係藉由雙硫鍵穩定的VH::VL異二聚體。二價及多價抗體片段可以藉由締合單價scFv自發地形成,或者可以藉由以肽連接子偶合單價scFv而產生,諸如二價sc(Fv)2。
術語「雙價抗體」係指具有兩個抗原結合位點之小抗體片段,該片段包含與同一多肽鏈中的輕鏈可變域(VL)連接的重鏈可變域(VH)(VH-VL)。藉由使用太短以致不能在同一鏈上的兩個域之間配對的連接子,此等域被迫與另一個鏈的互補域配對並產生兩個抗原結合位點。
如本文所使用,詞句「本發明之抗體」係指一種針對(即特異性結合)HERV-K套膜蛋白質(HERV-K Env)之抗體,較佳為針對抗K型人內源性逆轉錄病毒家族(the type-K human endogenous retrovirus family(HERV-K))之HERV-K套膜蛋白質,更佳為針對如SEQ ID NO:9所示之抗原決定位。
如本文所使用,本文使用之術語「生物樣品」係指於活體外評估之目的而獲得之任何生物樣品。於本發明,樣品或病患樣品可包含任何體液或疾病特異性組織及病灶,例如生檢。體液之例包括血液、血清、血漿、乳頭抽吸液、尿液、唾液、滑液及腦脊髓液(CSF)。
典型地,本發明之抗體保護人類神經元細胞免受由神經元暴露於HERV_K Env蛋白質引起的細胞毒性。
更特別地,本發明之抗體較佳展現下列功能特徵之一種或多種:- 其保留了暴露於HERV_K Env蛋白質的神經元之神經元功能活性;- 其保留了暴露於HERV_K Env蛋白質的神經元之細胞存活力;及/或- 其保留了暴露於HERV_K Env蛋白質的神經元之神經元形態。
依據本發明之抗體對細胞外HERV-K包膜細胞毒性的神經元功能活性的保護作用可以如本發明之實施例2中所說明進行活體外評估(參見要點2.1.3)。具體而言,可藉由以重組HERV-K Env蛋白質處理後記錄活體外人類神經元的自發電生理學活性來評估此種效果。因此,與未經該重組HERV-K Env蛋白質處理的人類神經元相比時,通常使用本發明之抗體治療使暴露於重組HERV-K Env蛋白質的人類神經元的自發活動恢復至少50%,尤其是至少60%,至少70%,至少80%,至少90%或至少95%。
於一些具體實施例,如實施例2所示(參見要點2.1.1),藉由分析重組HERV-K Env蛋白質處理後的神經元培養存活力,可以可選地或額外地活體外評估本發明抗體之保護作用。典型地,於此種具體實施例中,當與未以本發明之該抗體處理的人類神經元的存活力相比時,以本發明之抗體處理,與重組HERV-K Env蛋白質預培育的人類神經元的存活力提高至少20%。
於一些具體實施例,本發明之抗體的保護作用可以可選地或額外地活體外評估,藉由評估重組HERV-K Env蛋白質處理後的培養物中的人類神經元的神經元形態(例如軸突長度),例如實施例2說明者(參見要點2.1.2)。通常於此種具體實施例中,與未經本發明之該抗體處理的人類神經元的存活力相比,以本發明之抗體處理增加了與重組HERV-K Env蛋白質預培育的人類神經元的軸突長度至少20%。
於一具體實施例,本發明之抗體包含如SEQ ID No:1、SEQ ID No:2、SEQ ID No:3、SEQ ID No:4、SEQ ID No:5、及SEQ ID No:6中描述的6個CDR中的每一個。
於一具體實施例,本發明之抗體包含:-輕鏈,其中可變域包含CDR-L1的SEQ ID No:1、CDR-L2的SEQ ID No:2、及CDR-L3的SEQ ID No:3中描述的3個CDRs中的每一個;及-重鏈,其中可變域包含CDR-H1的SEQ ID No:4、CDR-H2的SEQ ID No:5、及CDR-H3的SEQ ID No:6中描述的3個CDRs中的每一個。
上述互補決定區(CDRs)被揭示於表1:
於一具體實施例,本發明之抗體、片段或衍生物包含:-如SEQ ID No:7所描述之輕鏈可變區(VL);及-如SEQ ID No:8所描述之重鏈可變區(VH)。
上述輕鏈及重鏈可變區揭示於表2:
於一具體實施例,本發明之抗體、片段或衍生物係選自由Fv、Fab、F(ab')2、Fab'、dsFv、scFv、sc(Fv)2、雙價抗體(diabody)、及由抗體片段形成的多特異性抗體(multispecific antibodies)所組成的群組。
於一較佳具體實施例,本發明之抗體為單株抗體。本發明之單株抗體為單價、雙價、多價、單特異性、雙特異性或多特異性。於另一具體實施例,針對HERV-K Env的抗體為一結合片段或結合物(conjugate)。例如本發明之抗體可與生長抑制劑、細胞毒性劑或前藥-活化酵素結合。
本發明之抗體之胺基酸修飾的另一類型對於改變抗體之原始醣苷化樣式可能為有用的。「改變」係指刪除一個或多個在抗體中發現的碳水化合物部分(carbohydrate moieties),及/或加入一個或多個不存在於 抗體中的醣苷化位點。抗體的醣苷化通常是N-連接的。「N-連接的」係指碳水化合物部分與天冬醯胺殘基的側鏈連接。三肽序列天冬醯胺-X-絲胺酸及天冬醯胺-X-蘇胺酸(其中X是除脯胺酸以外的任何胺基酸)用於將碳水化合物部分酵素性附著於天冬醯胺側鏈的識別序列。因此,多肽中此等三肽序列之任一個的存在產生潛在的醣苷化位點。藉由改變胺基酸序列以使其含有一個或多個上述三肽序列(對於N-連接的醣苷化位點)而方便地完成添加醣苷化位點於抗體。
於另一具體實施例,本發明之抗體為單株人化抗體,更佳為IgG4人化單株抗體。
該人化抗體可藉由獲得編碼CDR域的核酸序列來產生:將其插入至具有編碼與人抗體相同的重鏈恆定區及與人抗體相同的輕鏈恆定區的基因之動物細胞用表現載體中,並藉由將其導入動物細胞中來表現表現載體。人化抗體表現載體可為其中編碼抗體重鏈的基因及編碼抗體輕鏈的基因存在於不同載體上的類型或兩種基因存在於相同載體上的類型(串聯型)之任一種。關於構築人化抗體表現載體的容易性、導入動物細胞的容易性及在動物細胞中抗體H及L鏈表現水平之間的平衡,更佳為串聯型人化抗體表現載體。串聯型人化抗體表現載體之例包括pKANTEX93、pEE18等。基於習用重組DNA及基因轉染技術,製造人化抗體之方法於本領域中係廣為人知的。可使用本領域已知的多種技術將抗體人化,包括例如CDR移植、鑲貼(veneering)或表面重修 (resurfacing)以及鏈移動(chain shuffling)。用於製備此種抗體之一般重組DNA技術亦已知。
如此,本發明之一具體實施例係關於一種單株人化抗體,其包含:-輕鏈,其中可變域包含CDR-L1的SEQ ID No:1、CDR-L2的SEQ ID No:2、及CDR-L3的SEQ ID No:3中描述的3個CDRs中的每一個;及-重鏈,其中可變域包含CDR-H1的SEQ ID No:4、CDR-H2的SEQ ID No:5、及CDR-H3的SEQ ID No:6中描述的3個CDRs中的每一個。
[醫藥組成物]
本發明之另一目的係關於一種醫藥組成物,其包含有效劑量之針對HERV-K套膜蛋白質(HERV-K Env)之抗體及醫藥上可接受的賦形劑。
如上所述的本發明之任何治療劑可與醫藥上可接受的賦形劑及可任選用之緩釋基質如生物可降解聚合物加以組合以形成治療組成物。
「醫藥」或「醫藥上可接受的」係指於適當時投予於哺乳動物,特別是人類時,不產生不利反應、過敏反應或其他不良反應的分子實體及組成物。醫藥上可接受的載劑或賦形劑係指任何類型之無毒性固體、半固體或液體填充劑、稀釋劑、包封材料或製劑助劑。
醫藥組成物的形式、投予途徑、劑量及給藥方案(regimen)當然取決於待治療的病況、疾病的嚴重程度、病患的年齡、體重和性別等。
本發明的醫藥組成物可配製成局部、口服、鼻內、眼內、靜脈內、鞘內(直接於腦脊液中)、肌內或皮下投予等。
於一些具體實施例,配製用於鞘內投予的組成物可能為特別有利的。實際上,此種投予模式可允許在特定治療策略中短期施用非人抗體,諸如小鼠抗體或嵌合抗體。事實上,CNS之「免疫豁免(immune-privileged)」生理學允許經由全身途徑投予為不可能的耐受性。因此,鞘內注射模式為常見的神經及神經外科操作。例如,利妥昔單抗(rituximab)為一種小鼠-人類嵌合抗體,已被鞘內用於多發性硬化症及中樞神經系統淋巴瘤病患(參見著名的Bonnan M,Ferrari S,Bertandeau E,Demasles S,Krim E,Miquel M,Barroso B.「鞘內注射利妥昔單抗治療多發性硬化症:回顧支持未來試驗需要的證據。當代藥物靶體(Curr Drug Targets)」。2014;15(13):1205-14;Topping J,Dobson R,Lapin S,Maslyanskiy A,Kropshofer H,Leppert D,Giovannoni G,Evdoshenko E.「鞘內利妥昔單抗對多發性硬化症生物標誌物的影響」Mult Scler Relat Disord.2016 Mar;6:49-53;及Kadoch C,Li J,Wong VS,Chen L,Cha S,Munster P,Lowell CA,Shuman MA,Rubenstein JL.「補體激活及室內利妥昔單抗於復發性中樞神經系統淋巴瘤中的分佈」。Clin Cancer Res.2014 Feb 15;20(4):1029-41)。
較佳地,醫藥組成物含有媒液,其於能被注 射用製劑為醫藥上可接受的。此等可為特別是等滲的無菌鹽水溶液(磷酸一鈉或磷酸二鈉、氯化鈉、氯化鉀、氯化鈣或氯化鎂等或此等鹽之混合物),或乾燥的,特別是冷凍乾燥的組成物,其視情況當添加無菌水或生理鹽水時允許構成可注射溶液。
用於投予的劑量可以依各種參數進行調整,特別是作為使用的投予方式之函數、相關病理學、或可選用的所欲治療持續時間進行調整。
為了製備醫藥組成物,可將有效量之針對HERV-K套膜蛋白質(HERV-K Env)的抗體溶解或分散於醫藥上可接受的載劑或水性介質中。
適於注射使用的醫藥形式包括無菌水溶液或分散液;包括芝麻油、花生油或水性的丙二醇之製劑;及用於臨時製備無菌可注射溶液或分散液之無菌粉末。於所有情況下,該形式必須是無菌,且必須是存在容易可注射(syringability)程度的流動。其在製造和儲存條件下必須穩定,且必須防止微生物如細菌及真菌的污染作用。
作為游離鹼或藥理學上可接受的鹽類的活性化合物之溶液可於適合與界面活性劑如羥丙基纖維素混合的水中製備。分散液亦可於甘油、液體聚乙二醇、其混合物、及油中製備。於通常的儲存和使用條件下,此等製劑含有防腐劑以防止微生物生長。
於調配時,溶液將以與劑量調配物相容的方式並以治療有效的量投予。該製劑係易於以各種劑型投 予,例如上述可注射溶液的類型,但亦可以運用藥物釋放膠囊等。
較佳地,可以將本發明之針對HERV-K套膜蛋白質(HERV-K Env)的抗體配製於緩衝液中,於其中將其溶解,儲存並注射至病患。較佳地,該緩衝液包含20mM組胺酸、5%蔗糖及0.01%聚山梨醇酯20且呈現6.0的pH。
對於水溶液中的腸胃外投予,例如,溶液可被適當緩衝,且以充分的鹽水或葡萄糖首先使液體稀釋劑等滲。此等特定的水溶液特別適用於靜脈內、肌肉內、皮下及腹膜內投予。就此而言,根據本文揭示內容,可被運用的無菌水性介質將為本項領域中具通常知識者所知。例如,可以將一種劑量溶解於在1ml等滲NaCl溶液中,並將其加入到1000ml的皮下輸液(hypodermolysis fluid)或在建議的輸注位置注射(參見例如「Remington's Pharmaceutical Sciences」第15版,第1035-1038及1570-1580頁)。依被治療的病患的狀況,劑量的一些變動將必然會發生。無論如何,負責投予的人員將決定個別病患之適當劑量。
除了配製用於胃腸外投予例如靜脈內、鞘內或肌內注射的化合物之外,其他醫藥上可接受的形式包括例如,用於口服投予之錠劑或其他固體;時間釋放膠囊(time release capsules);及目前使用的任何其他形式。
[本發明之診斷方法]
於另一態樣,本發明亦係關一種於生物樣品中偵測HERV-K套膜蛋白質之方法,其包含使該生物樣 品與如上定義之抗HERV-K套膜抗體接觸的步驟。
於另一態樣,本發明亦係關於一種於病患中診斷ALS之方法,其包含使獲自該病患的生物樣品與如上定義之抗HERV-K套膜抗體接觸的步驟。
通常,生物樣品可為體液,諸如腦脊髓液。
[依據本發明之治療方法及監測方法]
於一態樣,本發明亦係關於一種治療罹患ALD的病患之方法,其包含投予該病患有效量之如上定義辨識HERV-K套膜蛋白質之抗體。
較佳地,該病患係罹患ALS,特別是散發性ALS。
通常,該抗體係鞘內、靜脈內或皮下投予。
本發明亦係關於一種治療罹患ALS的病患之方法,特別是散發性ALS,其包含下列步驟:1)藉由於生物樣品中偵測及/或定量HERV-K病毒來預測病患的預後;及然後2)若該步驟1)顯示人類內源性反轉錄病毒(HERV)K型的表現,則本發明之方法包含對該病患提供本發明之抗體的步驟。
本發明亦係關於一種偵測罹患ALS病患(特別是散發性ALS)對治療的反應之方法,該方法包含下列步驟:a.以根據本發明之抗體治療該病患;然後b.偵測及/或定量該病患之生物樣品中的HERV-K。
依據本發明,在監測對罹患ALS的病患的治療的反應之情況下,生物樣本可為體液諸如血液、腦脊髓液、尿液或於疾病特異性組織生檢中的樣品。
通常,偵測及/或定量步驟可依據本項技術領域中具通常知識者熟知的常規技術進行。通常,該步驟包含使病患之生物樣品與選擇性試劑諸如探針、引子、配位體或抗體接觸,因而偵測該樣品中最初感興趣的核酸或蛋白質的存在。
於一具體實施例,偵測及/或定量步驟可以如上定義之抗HERV-K抗體進行。
圖1:GN_mAb_Env_K01為IgG2b鼠類抗體且具有κ輕鏈。
藉由ELISA對來自抗鼠類免疫球蛋白捕獲的GN_mAb_Env_K01融合瘤(單株階段)的上清液1:10稀釋進行分型(isotyping)。以各種抗小鼠輕(A)或重(B)鏈抗體偵測顯示:GN_mAb_ENV_K01為IgG2b/κ鼠類抗體。結果繪製為重複值±SD的OD450nm平均值。
圖2:GN_mAb_Env_K01輕鏈及重鏈序列
提取來自融合瘤細胞的RNA並反轉錄成cDNA,其在以標定編碼鼠抗體重(A)和輕(B)鏈的cDNA的引子定序之前藉由PCR增幅。
(A)來自κ輕鏈的CDR1(粗體)、CDR2(劃底線)及CDR3(粗斜體)序列; (B)來自重鏈的CDR4(粗體)、CDR5(劃底線)及CDR6(粗斜體)序列;恆定鼠類IgG2序列(灰色斜體)。
圖3:GN_mAb_Env_K01結合至HERV-K-Env SLDKHKHKKLQSFYP抗原決定位。
展示來自HERV-K-Env(MyBiosource)(右)的每個肽的強度分佈(左)。此等具有一個殘基偏移的15個胺基酸的重疊肽顯示GN_mAb_Env_K01結合至線性SLDKHKHKKLQSFYP(SEQ ID NO:9)抗原決定位。結果表示為於使用的CCD相機獲得的信號強度(mAU),與標準96孔平盤ELISA判讀機類似。
圖4:藉由GN_mAb_ENV_K01於ELISA測偵醣苷化HERV-K Env。
於HEK細胞溶胞產物以各種稀釋度(1:25、1:50、1:100、1:200、1:400),使用GN_mAb_Env_K01(A)或來自AMSBIO的抗HERV-K-Env(B)(1μg/ml)作為ELISA中的一級抗體。與抗HERV-K-Env(AMSBio)相反,GN_mAb_Env_K01辨識醣苷化HERV-K-Env。結果繪製為重複值±SD的OD450nm平均值。
圖5:藉由GN_mAb_ENV_K01於ELISA中偵測非醣苷化HERV-K-Env。
在來自大腸桿菌的his-SUMO-HERV-K-Env重組蛋白質的1μg/ml ELISA中,使用GN_mAb_Env_K01或來自AMSBIO的抗HERV-K-Env(1μg/ml)作為一級抗體。兩種抗HERV-K-Env皆識別非醣苷化的HERV-K-Env蛋白質。結果繪製為重複值±SD的OD450nm平均值。
圖6:藉由以GN_mAB_ENV_K01的西方西方印漬術(Western Blotting)偵測HERV-K Env。
使用來自AMSBIO之抗HERV-K-Env(1μg/ml)(孔#1&2)或來自GN_mAb_Env_K01融合瘤(孔#3&4)之經稀釋(1:5)的上清液作為西方印漬術中的一級抗體。將0.2μg His-SUMO標記的HERV-K-Env沉積在孔# 1&3中,並將24.5μg來自HERV-K-Env轉染的HEK細胞的蛋白質提取物沉積在孔# 2&4中。非醣苷化的his-SUMO標記的HERV-K-Env蛋白質在75kDa被兩種抗體,以及高MW的多聚體及低MW的裂解片段偵測到。以GN_mAb_Env_K01僅偵測到醣苷化的HERV-K-Env(90kDa)。
MW:分子量。
圖7:GN_mAb_Env_K01(GN K01)抗體特異性保護人類神經元細胞免受細胞外HERV-K套膜蛋白質誘導的細胞毒性:細胞存活試驗。
以分化培養基(參見實施例2)及IgG樣品GN K01或對照非免疫IgG(Thermo產品# MA 1-10418)處理神經元培養物,於最終濃度為3ug/mL。預培育60分鐘後,加入重組HERV-K Env蛋白質(My BioSource,胺基酸90-632,目錄號MBS1391552)至最終濃度為100nM。將GN K01 Ig的一個樣品與HERV-K Env預培育30分鐘,然後一起添加至人類神經元。以GE INCell Analyzer 2000 BioImager觀察神經元培養物,在Env處理後5天的此實驗中,於各個時間點獲取每個孔的影像(每孔4個影像)。使用GE Investigator高內涵影像軟體確定神經元細胞計 數。直方圖條柱頂部的箭頭顯示GN_mAb_Env_K01(GN K01)抗體的結果。
圖8:GN_mAb_Env_K01(GN K01)抗體特異性保護人類神經元細胞免受細胞外HERV-K套膜蛋白質誘導的細胞毒性:軸突長度。
以分化培養基(如上述)及IgG樣品GN K01或對照非免疫IgG(Thermo產品# MA 1-10418)處理神經元培養物,於最終濃度為3ug/mL。預培育60分鐘後,加入重組HERV-K Env蛋白質(My BioSource,胺基酸90-632,目錄號MBS1391552)至最終濃度為100nM。以GE INCell Analyzer 2000 BioImager觀察神經元培養物,以在Env後處理後5天的該實驗中於各個時間點獲取每個孔的影像(每孔4個影像)。以GE Investigator高內涵影像軟體確定平均軸突纖維長度。
圖9:GN_mAb_Env_K01(GN K01)抗體特異性保護人類神經元細胞免受細胞外HERV-K套膜蛋白質誘導的細胞毒性:神經元整體電生理活動(neuron global electrophysiological activity)。
藉由孔(wells)中增加的尖峰率(spike rate)指出的電生理活動於活體外21天顯著增加,並藉由每日5分鐘記錄所有孔中的自發電活性來監測。於此時,以分化培養基(如上述)及GN K01或對照非免疫IgG(Thermo產品# MA 1-10418)處理人神經元培養物,於最終濃度3ug/mL。預培育60分鐘後,加入重組HERV-K Env蛋白質(My BioSource)至最終濃度為100nM。之後每天記錄 自發的電活動,從治療後24小時開始。於HERV-K Env暴露後24小時測定每個治療組的平均發射率(mean firing rate)。
直方圖條柱頂部的箭頭顯示GN_mAb_Env_K01(GN K01)抗體的結果。直方圖條柱頂部的箭頭顯示GN_mAb_Env_K01(GN K01)抗體的結果。
實施例1:GN_mAb_Env_K01抗體之開發及示性
1.材料及方法
1.1.單株抗體開發
1.1.1.免疫及免疫細胞回收
遵循來自Biotem公司的機密RAD(快速抗體發展(Rapid Antibody development))方案,以來自Mybiosource(MBS1391552)提供的大腸桿菌的his-SUMO標記的HERV-K-Env蛋白質(75kDa)免疫三隻雌性OF小鼠(Charles River)。
簡而言之,於第+10日(D+10),藉由於重組HERV-K Env蛋白質(MyBiosource,MBS1391552)或大腸桿菌溶胞產物作為陰性對照,藉由直接ELISA分析來自免疫小鼠的血液樣品。於D+13,犧牲免疫的小鼠,收集來自淋巴結的免疫細胞,並於244g離心7分鐘後以45ml Dulbecco's Modified Eagle's培養基(DMEM,SIGMA,D5671)洗滌三次,並重新懸浮於20ml DMEM(SIGMA, D5671)。依據1:3.9的比例,將來自免疫小鼠的免疫細胞(420×106個細胞)與骨髓瘤細胞(107×106個細胞)於指數生長期混合。將細胞於244g下離心7分鐘,並將沉澱物重新懸浮於1ml之作為融合劑的聚乙二醇(PEG)(SIGMA,P7181)。洗滌步驟後包括於108g離心12分鐘,將細胞再懸浮於10ml DMEM(SIGMA,D5671)、1X次黃嘌呤 胺基喋呤 胸腺嘧啶核苷(HAT,SIGMA,H0262-10VL)、20%胎牛血清(FCS,PAA,A15-251)、4mM L-麩醯胺酸(SIGMA,G7513)並於244g下離心7分鐘。將細胞再懸浮於DMEM(SIGMA,D5671)、1X HAT(SIGMA,H0262-10VL)、20%FCS(PAA,A15-251)、4mM L-麩醯胺酸(SIGMA,G7513)中並於室溫下儲存2小時。
1.1.2.融合
於D-1,免疫缺陷裸鼠(BIOTEM)注射含有20%FCS(PAA,A15-251)的5ml DMEM(SIGMA,D5671)及2+/-1分鐘後,收集來自腹膜液的巨噬細胞並於DMEM培養基(SIGMA,D5671)中培養。
已經選擇與P3X63Ag8骨髓瘤融合的綿羊紅細胞免疫的小鼠的BALB/c脾細胞,藉由BIOTEM進行示性及儲存。於D-10,此等骨髓瘤已於DMEM(SIGMA,D5671)-8-氮鳥嘌呤(AZA,SIGMA,A5284)-10%FCS(PAA,A15-251)中解凍及培養。
計數來自裸鼠的巨噬細胞並以104巨噬細胞/ml再懸浮於DMEM(SIGMA,D5671)、1X HAT(SIGMA, H0262-10VL)、20%FCS(PAA,A15-251)、4mM L-麩醯胺酸(SIGMA,G7513)、1%青黴素/鏈黴素(SIGMA,P0781)。然後,將使用作為生長因子的50μl巨噬細胞懸浮液(相當於500個巨噬細胞)接種於含有50μl融合瘤細胞懸浮液的96孔盤中。於37℃,5%CO2下培養此等細胞21日。
1.1.3.選殖
以DMEM(SIGMA,D5671)、HT(次黃嘌呤100μM,胸苷16μM-SIGMA H0137)、20%FCS(PAA,A15-251)、2%融合瘤增強補充劑(HES,SIGMA,H6020)、4mM L-麩醯胺酸(SIGMA,G7513)、1%青黴素/鏈黴素(SIGMA,P0781)解凍並於24孔盤中1週在37℃,5%CO2下培養融合瘤細胞。選殖前一天,將融合瘤細胞分開。
於D0,在培養基融合瘤懸浮液中於104、50、25、5及2.5細胞/ml系列稀釋後,以5、1及0.5細胞/200μl接種至96孔盤的孔中。於D+6,以新鮮DMEM(SIGMA,D5671)、HT(次黃嘌呤100μM,胸苷16μM-SIGMA H0137)、20%FCS(PAA,A15-251)、2%HES(SIGMA,H6020)、1%青黴素/鏈黴素(SIGMA,P0781)替換來自含細胞孔(通過以光學顯微鏡篩選選擇)的100μl上清液。
於對應於D+10的第一次ELISA篩選後,於24孔盤(0.5ml/孔)中培養抗HERV-K-Env陽性融合瘤。
在對應於D+14的第二次ELISA篩選後,將抗HERV-K Env陽性融合瘤於平盤或培養瓶(Corning)中培養,並將5個含有4個5×106細胞的小瓶在-196℃下冷凍(液態氮),於DMEM(SIGMA,D5671)、15%FCS(PAA, A15-251)、4mM L-麩醯胺酸(SIGMA,G7513)、1%HES(SIGMA,H6020)、1%青黴素/鏈黴素(SIGMA,P0781)、20%二甲基亞碸(DMSO,Sigma,D2650)培養基。
1.2.抗HERV-K-Env ELISA
以50μl的1μg/ml HERV-K Env蛋白質(Mybiosource,MBS1391552)、大腸桿菌溶胞產物(XL1-Blue MRF,Stratagene)、1X磷酸鹽緩衝食鹽水(PBS,BIOTEM)中的HEK細胞溶胞產物於室溫隔夜塗布Maxisorp 96錐形底部孔盤(NUNC,449824)。以[1X PBS+0.05%Tween20(VWR,28829.296)]洗滌緩衝液(300μl/孔)洗滌平盤。以[1X PBS+0.05%Tween20+2.5%牛奶(Regilait)]封阻緩衝液(150μl/孔)於室溫下封阻非特異性結合位點1小時。以[1X PBS+0.05%Tween20]洗滌緩衝液(300μl/孔)洗滌平盤。
抗體樣品已於[1X PBS+0.05%Tween20+0.5%BSA(VWR,1.12018.0100)稀釋緩衝液中稀釋。將抗體樣品或來自AMSB10的純化的抗HERV-K-Env(1μg/ml)(50μl/孔)於室溫下培育2小時。以[1X PBS+0.05%Tween20]洗滌緩衝液(300μl/孔)洗滌平盤三次,並與50μl/孔的多株過氧化物酶偶合的affiniPure F(ab)'2片段山羊抗小鼠IgG+IgM Jackson,115-036-068)(1/10000,在1X PBS+0.05%Tween20+0.5%BSA中)在室溫下培育1小時。將平盤洗滌三次,於室溫下以四甲基聯苯胺(TMB,Eurobio,52-00-01)基質溶液(50μl/孔)進行10分鐘的顯示(revelation)。以0.1M H2SO4(Merck,1.12080.1000)(50μl/孔)封阻反應。使用光密度(OD)讀取器(Dynex)於450nm處測量光密 度(OD)。
1.3.生產、純化、透析
將GN_mAb_Env_K01融合瘤細胞解凍並先於T75cm2中培養,然後在T300cm2組織培養瓶(Corning)中培養。最後,將10在12×106個細胞於500ml DMEM(SIGMA,D5671)、15%FCS(PAA,A15-251)、4mM L-麩醯胺酸(SIGMA,G7513)、1%HES(SIGMA H6020)、1%青黴素/鏈黴素(SIGMA,P0781)培養基之Hyperflask(Corning,10030),於37℃,5%CO2中培養10 +/-1日間。
將培養物上清液於244g離心7分鐘,並通過11μm尼龍網過濾器(SIGMA,NY1104700)蛋白質A層析管柱(GE Healthcare,Mab Select Xtra)過濾,以去礦物質水洗滌兩次並以5倍體積的1X PBS(Biotem)平衡。然後裝入0.5L不含細胞的培養上清液。該管柱以5倍體積的1X PBS洗滌。免疫球蛋白洗析於具有3.5 +/- 0.5體積的乙酸(SIGMA,A6283)的酸性pH下進行。以100μl之1M Tris pH8.8緩衝液(Biotem)中和含有免疫球蛋白的洗析級份(eluted fractions),並在4℃下儲存。
將IgG純化的級分於4℃在0.5ml微透析膠囊Quixsep®(Roth,H448-1)上以10kDa SnakeSkin透析管,22mm(Thermofischer,68100)在1X PBS中透析2小時並於4℃於Vivaspin 20(30Kda)(Sartorius,ref)上藉由離心濃縮。於0.22μm Minisart®過濾器(Sartorius,ref)上過濾抗體,並藉由分光光度計於280nm處測量蛋白質濃度。
1.4.藉由SDS PAGE凝膠電泳之純度分析
將預先稀釋於Laemmli緩衝液(Biotem)中的抗體(5μl,0.2μg/μl)於95℃加熱5分鐘,並以5% SDS-PAGE於含有集集膠體(stacking gel)的13.5%十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳(SDS-PAGE)中分離。凝膠在90V下運行30分鐘,然後在120V下運行2小時。使用著色溶液(Biotem)在攪拌下進行蛋白質檢測1小時。攪拌下將凝膠用脫色溶液(Biotem)洗滌1小時。
1.5.分型
1.5.1.藉ELISA分型
室溫下以50μl之1μg/ml抗鼠類免疫球蛋白(Clinisciences,1010-01)塗覆maxisorp 96錐形底部孔盤(NUNC,449824)隔夜。以[1X PBS+0.05%Tween20]洗滌緩衝液(300μl/孔)洗滌平盤。以[1X PBS+0.05%Tween20+2.5%牛奶]封阻緩衝液(150μl/孔)於室溫下封阻非特異性結合位點1小時。以[1X PBS+0.05%Tween20]洗滌緩衝液(300μl/孔)洗滌平盤。融合瘤上清液於[1X PBS+0.05%Tween20+0.5%BSA]稀釋緩衝液中1:10稀釋。樣品(50μl/孔)在室溫下培育2小時。以[1X PBS+0.05%Tween20]洗滌緩衝液(300μl/孔)洗滌平盤一次,並與50μl/孔之過氧化物酶結合的山羊抗小鼠重鏈(IgA、IgG1、IgG2a、IgG2b、IgG3、IgM)(Clinisciences,5300-05)(於1X PBS+0.05%Tween20+0.5%BSA中1/2000)於室溫下培育1小時。將平盤洗滌一次,並於室溫下以TMB(Eurobio,52-00-01)基質溶液(50μl/孔)進行10分鐘的顯示。反應以0.1M H2SO4(Merck,1.12080.1000)終止 溶液(50μl/孔)封阻。使用光密度(OD)讀取器(Dynex)於450nm處測量光密度(OD)。
1.5.2.藉由側流免疫分析(Lateral-flow Immunoassay)之分型
以側流免疫分析(LFIA)(ThermoFisher,26179)示性輕鏈(κ或λ)。
1.6.HEK轉染
以1μg之HERV-K-Env(登錄號AY037928.1)表現質體轉染人類胚胎細胞(1.106個細胞/mL)。於37℃,8%CO2,120rpm攪拌下培養轉染的細胞。
1.7.西方氏印漬術分析
在2X Laemmli緩衝液(SIGMA,S3401)中稀釋12.5ng/μl的重組HERV-K-Env蛋白質(Mybiosource)及1.5μg/μl來自HEK轉染的細胞的蛋白質溶胞產物(1:1)並於90℃加熱5分鐘。然後,將32μl樣品裝載到8-16%十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳(SDS-PAGE,Novex,EC60452BOX)上。於1X Tris-甘胺酸SDS運行緩衝液(Novex,LC2675)中以160mA運行凝膠1小時。於1X Tris-甘胺酸轉移緩衝液(Novex,LC3675)中將蛋白質轉移到0.2μm硝酸纖維素膜(Biorad,162-0146)後,以[1XPBS+5%牛奶(La Vie Claire)]封阻緩衝液於室溫下在旋轉平台上將膜封阻1小時。將來自GN_mAb_Env_K01融合瘤的細胞上清液在[1X PBS+1%牛奶]抗體稀釋液中稀釋1:5並作為一級抗體經由培育1小時。然後將膜於[1X PBS+0.05%Tween20(SIGMA,P7949)]洗滌緩衝液中 洗滌三次5分鐘,並與1:1000稀釋的HRP結合的山羊抗小鼠IgG抗體(Jackson,115035-146)培育30分鐘。依據所提供的方案,將膜洗滌三次,並以比色反應(Opti 4-CN,Biorad,170-8235)偵測感興趣的蛋白質。
1.8.抗原決定位作圖
抗原決定位作圖於Pepscan Presto BV(Zuidersluisweg 2,8243RC Lelystad,荷蘭)進行。
1.8.1.肽庫合成
為了重建目標分子的抗原決定位而合成肽庫。藉由以專有的親水聚合物製劑接枝而得胺基官能化的聚丙烯載體,隨後使用二環己基碳化二亞胺(DCC)與N-羥基苯并三唑(HOBt)與三級丁氧基羰基-六亞甲二胺(BocHMDA)反應,隨後使用三氟乙酸(TFA)裂解Boc-基團。藉由客制改良JANUS液體處理站(JANUS liquid handling stations)(Perkin Elmer),使用標準Fmoc-肽合成於胺基官能化固體撐體上合成肽。使用Pepscan的專有在支架上的化學連接肽技術(Chemically Linked Peptides on Scaffolds,CLIPS)完成結構模擬物的合成。CLIPS技術允許將肽構成單環、雙環、三環、片狀折疊、螺旋狀折疊及其組合。CLIPS模板與半胱胺酸殘基偶合。肽中多個半胱胺酸的側鏈與一個或兩個CLIPS模板偶合。例如,將P2 CLIPS(2,6-雙(溴甲基)吡啶)的0.5mM溶液溶解於碳酸氫銨(20mM,pH7.8)/乙腈(1:3(v/v))中。此溶液被添加到肽陣列上。CLIPS模板將與存在於肽陣列的固相結合肽(具有3μl孔的455孔盤)中的兩個半胱胺酸的 側鏈結合。將肽陣列在溶液中輕輕搖晃30至60分鐘,同時完全覆蓋於溶液中。最後,以過量的H2O充分洗滌肽陣列,並於70℃於含有1%SDS/0.1%β-巰基乙醇於PBS(pH 7.2)的瓦解-緩衝液(disrupt-buffer)進行超音波處理30分鐘,然後在H2O中超音波處理另45分鐘。帶有肽的T3 CLIPS以類似的方式製備,但現以三個半胱胺酸製備。
1.8.2.ELISA篩選
於基於Pepscan的ELISA中試驗抗體與每種合成肽的結合。將肽陣列與一級抗體溶液(在Pepscan緩衝液中1μg/ml的GN_mAb_Env_K01)於4℃培育隔夜。洗滌後,肽陣列於25℃下與1/1000稀釋的兔抗小鼠IgG(H+L)HRP結合物抗體(Southern Biotech;表4)一起培育1小時。洗滌後,添加過氧化物酶基質2,2'-連氮基-二-3-乙基苯并噻唑啉磺酸鹽(ABTS)及20μl/ml的3%H2O2。一小時後,測量顏色的發展。使用電荷耦合裝置(charge coupled device,CCD)-照相機及圖像處理系統對顯色進行量化。從CCD相機獲得的值範圍為0到3000mAU,與標準的96孔平盤ELISA讀數器相似。
1.9.定序
使用PureLink RNA Mini Kit(Life technologies,12183018A)自融合瘤細胞提取並純化高品質RNA並於瓊脂糖凝膠上控制。自純化的總RNA開始,然後Superscript酶(Invitrogen,18064022)被用於合成第一股互補DNA(cDNA),隨後使用HotStar HiFidelity Polymerase Kit(Qiagen,202602)及特異性標定編碼鼠抗體重鏈及輕鏈的cDNA的退化性引子(degenerate primers)(Biotem)進行聚合酶鏈式反應)。PCR產物於瓊脂糖凝膠上進行控制並定序(雙股定序)。使用專用生物訊息工具(Blast-ClustalW)分析生成的序列的組裝及品質控制並被轉譯成肽序列。
1.10.Biotem緩衝液組成物
2.結果
使用能夠偵測單株抗體的不同重鏈及輕鏈的特異性抗免疫球蛋白抗體,吾人已顯示藉由抗κ輕鏈抗體(圖1A)及抗IgG2b重鏈抗體(圖1B)偵測GN_mAb_Env_K01。
將GN_mAb_Env_K01重鏈及輕鏈進行定序,顯示它們的三個CDR區(圖2)並確認IgG2b重鏈及κ輕鏈兩者。
2.1.GN_mAb_Env_K01辨識來自HERV-K-Env表面單元的線性抗原決定位SLDKHKHKKLQSFYP。
在高度嚴格條件下使用來自Mybiosource的截短型HERV-K-Env蛋白質序列(無信號肽及截短的跨膜域抗原決定位作圖試驗中的一組529個肽,顯示結合至具SLDKHKHKKLQSFYP核心序列的線性肽的GN-mAb-Env_K01抗體(圖3,左圖)。該抗原決定位含於HERV-K-Env蛋白質的細胞外域內(圖3右圖),對應於Dewannieux等人描述的HERV-K-Env蛋白質的298-312區域(Dewannieux、Blaise和Heidmann 2005)。自美國國家生物技術訊息中心(NCBI)的Blastp中將此抗原決定位揭示,顯示其為一個高度保留的抗原決定位,其係與HERV-K-Env序列對應的前100個命中點為100%同一性的高度保留的抗原決定位(數據未顯示)。
2.2.GN_mAb_Env_K01辨識於天然條件下醣苷化及非醣苷化HERV-K-Env蛋白質。
我們進一步分析GN_mAb_Env_K01識別醣苷化RV-K-Env的能力。於此目的,以編碼HERV-K-Env蛋白質的質體轉染人類胚腎(HEK)細胞。儘管以各種緩衝液作了數次嘗試,但我們未能提取可溶性HERV-K-Env蛋白 質(數據未顯示)。然而,來自轉染的HEK細胞溶胞產物的醣苷化HERV-K-Env蛋白質之此種不溶性級份可能顯示GN_mAb_Env_K01藉由ELISA特異性識別醣苷化的HERV-K-Env抗原(圖4,左圖)。相反地,來自AMSBIO的典型商用抗-HERV-K-Env mAb(HERM-1821-5,IgG2b)(圖4,右圖)未檢測到醣苷化HERV-K-Env。
目前的結果指出,GN_mAb_Env_K01於ELISA中具有生物學活性,且確認SLDKHKHKKLQSFYP抗原決定位於天然條件下可以獲得。
雖然來自AMSBIO的GN_mAb_Env_K01及抗HERV-K-Env mAb(HERM-1821-5,IgG2b)識別來自大腸桿菌的天然his-SUMO標記的重組HERV-K-Env(圖5),與來自AMSBIO的抗-HERV-K-mA mAb對應物相比,似乎GN_mAb_Env_K01給予出具有高得多的偵測,當兩者以相同濃度(1μg/ml)測試時(圖5)。此證明GN_mAb_Env_K01對於HERV-K Env具有更高的親和力。
如圖6所示,GN_mAb_Env_K01及來自AMSBIO的抗HERV-K-Env mAb(HERM-1821-5,IgG2b)識別來自大腸桿菌之變性的his-SUMO標記的重組HERV-K-Env,如以75kDa信號所觀察的。
重要的是,GN_mAb_Env_K01亦偵測到在90kDa處觀察到的來自HEK轉染細胞的變性HERV-K-Env醣苷化蛋白質,而以來自AMSBIO的抗-HERV-K-Env抗體偵測不到信號。除了先前以非變性蛋白質的ELISA結果, GN_mAb_Env_K01在西方氏印漬術為生物學上活性的。因此SLDKHKHKKLQSFYP抗原決定位亦可用於變性條件。
4.結論
本報告揭示,於小鼠免疫、抗體篩選及單株融合瘤選擇後,開發識別HERV-K-Env SLDKHKHKKLQSFYP抗原決定位的鼠類單株抗體(命名為GN_mAb_EnvK-01),且顯示出意想不到的性質。
與來自AMSBIO的另一種抗HERV-K mAb Env(HERM-1821-5,IgG2b)的生物學比較證實:儘管它們具有類似的起源(鼠類)、同型(IgG2b,κ)及蛋白質標的(HERV-W-Env蛋白質),由於識別醣苷化和醣苷化蛋白質兩者,GN_mAb_EnvK-01具有優勢,同時在天然和變性條件下均表現出高親和力。此外,GN_mAb_EnvK-01抗體針對數據庫中描述的無數種及各式各樣的拷貝之HERV-K套膜序列中穩定及保留的抗原決定位。
因此GN_mAb_EnvK-01為一有用的工具,不僅用於針對HERV-K Env蛋白質之免疫分析,而且用於針對HERV-K Env蛋白質之治療目的,例如ALS。其在HERV-K拷貝中的穩定抗原決定位序列、它的高親和力及其與天然醣苷化形式的有效結合,為滿足對於具有例如ALS的病患之有價值的治療的重要要件,其中在各種HERV-K拷貝中似乎被顯著地表現。
實施例2:GN_mAb_Env_K01(GN K01)抗體有效地中和HERV-K套膜對人類神經元細胞的神經毒
性。
1.材料及方法
1.1.人類神經元細胞
如(Efthymiou、Shaltouki等人,2014)所述,製備衍生自神經元培養的人類神經幹細胞(NSCs)。簡言之,將NSCs以7500至10,000個細胞/cm2分開至以0.002%聚-L-鳥胺酸(Sigma,St.Louis,MO)及10μg/mL層黏連蛋白(Life Technologies)塗覆的96孔盤,並鋪盤後24小時添加神經元分化培養基。分化培養基含有具GlutaMax的DMEM/F12、1.8%牛血清白蛋白(BSA)、1×StemPro hESC補充物(均來自LifeTechnologies)、10ng/mL腦衍生的神經營養因子(BDNF)及神經膠質細胞系衍生的神經營養因子(GDNF,R & D Systems,Minneapolis,MN),且細胞隔日接受新鮮培養基及生長因子。於神經毒性試驗中利用活體外7-12日的神經元。
1.2神經毒性試驗:
1.2.1.神經元形態及存活力
人類神經元培養物(每孔15-20,000個細胞),穩定地表現Td-番茄螢光蛋白質以標記細胞及過程,如上述接種於96孔盤並維持於5%CO2、37℃濕潤組織培養箱中。以分化培養基(如上述)及IgG樣品GN K01或對照非免疫IgG(Thermo產品# MA 1-10418)處理神經元培養物,最終濃度為3μg/mL。預培育60分鐘後,添加重組HERV-K Env蛋白質(My BioSource,胺基酸90-632,目錄號MBS1391552)至最終濃度為100nM。將GN K01 Ig的一 個樣品與HERV-K Env預培育30分鐘,然後一起添加至人類神經元。以GE INCell Analyzer 2000 BioImager觀察神經元培養物,而於處理後24、48、72h的不同時間點獲取每個孔的影像(每孔4個影像)。以GE Investigator 1.93分析軟體達成此等培養物之高通內涵影像/分析。定量每個樣品的神經元存活力、軸突長度及其他形態參數。以Graph Pad Prism 7.02描述數據。
1.2.2電生理分析
使用Axion Maestro微電極陣列(MEA)分析進行電生理分析。
48孔t-MEA盤被用於人類神經元培養物的分析。此等盤每孔含有16個主動記錄電極。將200,000個神經元施加於t-MEA盤的每孔,並維持於37℃在5%CO2的濕潤組織培養箱中。電生理學活性,藉由於孔中增加的尖峰率而注意到,於活體外21日顯著增加且藉由記錄每日5分鐘之所有孔中的自發電活性來監測。於此點,人類神經元培養物以分化培養基(上述)處理並以IgG樣品GN K01或對照非-免疫IgG(Thermo產品# MA 1-10418)處理,於最終濃度3ug/Ml。預培育60分鐘後,添加重組HERV-K Env蛋白質(My BioSource)至最終濃度100nM。於此等情形,首先添加該抗體至無Env的細胞,如此重製擴散到其腦組織中的治療性抗體的存在的經治療病患的情況。之後,加入活性蛋白質(未與抗體預培育,非「預中和且未添加作為非活性蛋白質」),因而在細胞外空間中重製細胞外分泌的病原性蛋白質的表現。將GN K01 Ig的一個樣品與HERV-K Env預培育30分鐘,然後一起添加至人類神經元。之後每日記錄自發電活動,自治療後24小時開始。以Axion Axis軟體完成電活動的定量。確定每次治療的參數,諸如尖峰數(number of spikes)、平均發射率及爆發數(number of bursts)。
2.結果
2.1.細胞外HERV-K套膜蛋白質對人類神經元細胞為毒性且其毒性經GN_mAb_Env_K01(GN K01)抗體特異性地抑制
2.1.1.神經元存活性
以100nM重組HERV-K Env蛋白質處理的人類神經元培養物顯示出顯著的神經毒性,導致接下來的數日內顯著的神經元細胞損失。HERV-K Env的作用在暴露於HERV-K Env蛋白質後5天定量。我們觀察到與Env處理的神經元相比,Env+GN K01 Ig(3μg/mL)處理的神經元具有增加的存活。當在將HERV-K套膜蛋白質加入培養基後5日被分析時,以Env加上3μg/mL之非免疫IgG對照抗體處理的細胞展現相似的毒性。以GN K01 3μg/mL處理的神經元,於HERV-K Env之前或與Env預培預後然後施用於神經元培養皆顯示更有存活力的神經元,因而確認GN K01抗體於中和HERV-K Env蛋白質毒性的效力(圖7)。
2.1.2.神經元軸突長度
平行分析HERV-K Env對平均軸突長度的影響,與Env或Env+對照非免疫Ig處理的神經元相比,GN K01 (3μg/mL)添加至暴露於Env的神經元顯著地增加軸突長度(圖8)。
2.1.3.神經元功能活動
以Axion Maestro MEA系統進行電生理研究以評估是否HERV-K Env處理造成對自發電活動的功能性改變,其為正常神經活動的主要特徵。於48孔MEA盤上植入神經元培養物21日後,將培養物於分化培養基(如上述)並與GN K01或對照非免疫IgG(Thermo產品# MA 1-10418)培育,以最終濃度3μg/mL。預培育60分鐘後,添加重組HERV-K Env蛋白質(My BioSource,目錄號MBS1391552)至最終濃度為100nM。將GN K01 Ig的一個樣品與HERV-K Env預培育30分鐘,然後一起添加至人類神經元。處理後24小時開始記錄自發電活動。HERV-K處理後24小時,Env處理後尖峰數和平均發射率降低40%。暴露於HERV-K Env加對照非免疫IgG處理的神經元顯示尖峰數和平均發射率降低約30%。最有趣地,暴露於HERV-K Env加上GN K01的神經元顯示尖峰數及平均發射率相似於與僅具對照培養基培育的孔,因而顯示完全抑制HERV-K Env於整體的神經元功能活性上的病源性效果(圖9)。
3.結論
因此GN K01抗體特異性之效力及其針對神經元暴露於HERV_K Env蛋白質的病原性後果之有利效果藉由下列顯著處而被證實:(i)細胞死亡的抑制;(ii)神經元細胞形態及軸突長度的維持;及(iii)神經功能活性的全 部恢復,於HERV-K Env蛋白質之病原存在下以電生理活動測量,與使用不相關的對照抗體或不使用抗體的相同的HERV-K Env暴露相比。
因此,(i)顯示了在具有散發性ALS的病患的腦實質(brain parenchyma)內改變的神經元中特異性檢測到HERV-K Env蛋白質後,以及(ii)在概念證明此HERV-K Env蛋白質單獨驅動致病性之後,已經藉由表現編碼該獨特蛋白質的HERV-K env基因的轉基因小鼠中再生的散發性ALS的臨床和組織學證據提出(Li,Lee等。2015年),此GN K01抗體之特異性效力,如本發明者等於相關人類神經元細胞及功能態樣上證實,證明於ALS中它的治療價值,特別是於散發性ALS。
此種抗體的特異性活性似乎中和(即:治療)HERV-K套膜(Env)蛋白質的致病作用,其本身顯示與具有散發性ALS的病患腦中的ALS特殊病徵的神經元損傷有關且再現相同的神經元改變,當添加到培養的神經元中時,或當在小鼠中表現作為單一轉基因時,以及與ALS中相同的臨床徵兆。此外,於HERV-K Env病原性蛋白質存在下GN K01抗體之添加顯示GN K01與HERV-K Env重組蛋白質於活體外的預培育之更顯著的效力,其顯示其在生理條件下的最佳功效,因此於治療應用中。
由先前知識,HERV_K蛋白質毒性不僅涉及轉染的目標細胞中或來自轉基因動物的神經元中的細胞內表現,而且亦涉及分泌的細胞外HERV-K蛋白質對天 然(非HERV-K轉染或過表現)神經元的致病性。因此,本發明之抗體亦保護人類神經元細胞免於由神經元暴露於分泌的及/或細胞外的HERV_K Env蛋白質引起的細胞毒性的旁分泌分佈擴散。隱含地,此亦適用於表現或過表現HERV-K env編碼基因的神經元中產生的HERV-K Env蛋白質引起自分泌細胞毒性。
作為結論,依據本發明,特異性標定HERV-K套膜蛋白質的抗體,可中和於下述觀察到的病理生理學性質:ALS,特別是散發性ALS,或再現ALS特徵的HERV-K env小鼠轉基因模型。
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Claims (9)
- 一種辨識HERV-K套膜蛋白質之抗體,其中該抗體結合至如SEQ ID NO:9所示之抗原決定位。
- 如請求項1之抗體,其中:i)該辨識HERV-K套膜蛋白質之抗體包含如SEQ ID No:1、SEQ ID No:2、SEQ ID No:3、SEQ ID No:4、SEQ ID No:5及SEQ ID No:6所描述的6個CDRs的每一者。
- 如請求項1或2之抗體,其中:i)該辨識HERV-K套膜蛋白質之抗體包含- 如SEQ ID No:7所描述之輕鏈可變區(VL);及- 如SEQ ID No:8所描述之重鏈可變區(VH)。
- 如上述請求項中任一項之抗體,其中該抗體為單株抗體,較佳為鼠類單株抗體、嵌合單株抗體或人化單株抗體。
- 如上述請求項中任一項之抗體,其用於治療。
- 如上述請求項中任一項之抗體,其用於一種治療肌肉萎縮性脊髓側索硬化症(ALS)之方法中,較佳為散發性ALS。
- 一種醫藥組成物,其包含如請求項1至4中任一項之抗體、及醫藥上可接受的賦形劑。
- 一種於生物樣品中偵測HERV-K套膜蛋白質之方法,其包含使該生物樣品與如請求項1至4中任一項定義的抗HERV-K套膜抗體接觸的步驟。
- 一種於病患中診斷ALS之方法,其包含使獲自該病患的生物樣品與如請求項1至4中任一項定義的抗HERV-K套膜抗體接觸的步驟。
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FR2865403B1 (fr) * | 2004-01-23 | 2009-06-12 | Biomerieux Sa | Composition pour le traitement d'une pathologie associee a la msrv/herv-w |
US20090297530A1 (en) * | 2006-05-22 | 2009-12-03 | Feng Wang-Johanning | Herv-k antigens, antibodies, and methods |
FR2912314B1 (fr) * | 2007-02-09 | 2012-08-03 | Geneuro Sa | Composition pharmaceutique comprenant des anticorps diriges contre l'enveloppe de herv-w. |
WO2013059426A1 (en) * | 2011-10-21 | 2013-04-25 | The Regents Of The University Of California | Human endogenous retrovirus peptides, antibodies to the peptides, and methods of use thereof |
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EA039460B1 (ru) | 2022-01-28 |
IL267955B1 (en) | 2024-09-01 |
UA126677C2 (uk) | 2023-01-11 |
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TWI793098B (zh) | 2023-02-21 |
AU2018210388C1 (en) | 2024-09-19 |
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US10981977B2 (en) | 2021-04-20 |
AU2018210388A1 (en) | 2019-08-01 |
EA201991736A1 (ru) | 2019-12-30 |
BR112019014773A8 (pt) | 2020-06-09 |
IL267955A (en) | 2019-09-26 |
EP3570878A1 (en) | 2019-11-27 |
EP3351265A1 (en) | 2018-07-25 |
US20200308258A1 (en) | 2020-10-01 |
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