CN105980411B - 抗体-药物缀合物和免疫毒素 - Google Patents
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Abstract
本发明涉及具有式(I)的缀合物:A‑(L‑D)p(I)或其药学上可接受的盐或溶剂化物,特别是抗体‑药物缀合物和免疫毒素,其中:A是选择性结合内皮糖蛋白的抗体;L是接头;D是包括溶细胞素或黑杆菌素‑b A链的药物;且p是1至10,并且涉及所述缀合物治疗性治疗肿瘤的用途。公开产生所述缀合物的方法和用于所述方法中的组分。
Description
发明领域
本发明涉及靶向内皮糖蛋白(Endoglin,ENG)的抗体-药物缀合物(ADC)和免疫毒素以及它们在医学中,例如在治疗某些癌症方面的用途。
发明背景
恶性上皮肿瘤是人类死亡的主要癌症相关病因。这些实体肿瘤常常展现显著间质反应,诸如所谓“促结缔组织增生性间质”或“反应性间质”,其占总体肿瘤质量的20-60%,并且特征在于存在大量间质细胞和致密细胞外基质(ECM)。新近研究已指示间质细胞的促肿瘤作用,如由血管细胞、免疫细胞、纤维母细胞、肌纤维母细胞、脂肪细胞和骨髓源性祖细胞所例示(1-6)。具体来说,常在包括乳腺癌、肺癌、结肠癌和胰腺癌的各种人类癌症的肿瘤相关间质内观察到可观数目的癌症相关纤维母细胞(CAF)(14,15)。在与间质的不同组分协调相互作用的情况下,CAF能够促进新血管生成和肿瘤生长;也已显示在癌症进展期间CAF对侵袭性肿瘤的发展和肿瘤侵袭性至关重要(16-25);CAF促进肿瘤细胞在远端器官中扩散和浸润,由此造成转移的形成。重要的是,也已指示间质细胞与全身性药物向肿瘤递送失败和出现抗药性的相关性(7-11)。对消除间质-肿瘤细胞相互作用并由此减弱肿瘤发生的细胞靶标和分子靶标的鉴定当前是转化肿瘤学中一个最重要的主题。实际上,靶向肿瘤周围间质是一种治疗代表超过90%的癌症患者死亡率的转移性肿瘤的相当新颖策略:迄今仅少许产品已获得治疗批准,它们中的大多数是抗血管生成药物(26)。然后鉴定和靶向肿瘤微环境内的其它新型分子为增加与基于间质的治疗方法组合的常规疗法的功效所必需,并且代表一种用于癌症和转移治疗的有效方法(12,13)。
基于单克隆抗体(MAb)的药物代表在对抗癌症方面的极大希望。这是因为它们允许治疗以精确和特异性方式针对分子水平。这些优势连同它们的商业吸引力(开发时间短暂、竞争力受局限以及一旦它们已被批准即可易于向其它癌症类型输出)一起已推动许多药物公司在开发新型基于抗体的分子方面,以及在从生物技术公司获得新型分子或技术的引入式授权方面大量投资。
然而,尽管治疗性抗体取得了临床成功,但靶向细胞表面肿瘤抗原的裸露MAb很少独自提供足够功效。为使MAb的低活性增加,新策略集中于使它们结合于毒性分子。植物和细菌毒素以及小化学治疗分子可为良好候选物,因为它们在极小量的情况下非常有效和有活性。
由于在过去数年期间进行的技术进步,用于治疗癌症的免疫毒素(IT)和抗体-药物缀合物(ADC)的领域近来已经历一场由药物公司进行的日益增长的开发活动,旨在解决它们最初呈现的关于免疫原性、不合需要的毒性、产量、半衰期和抗性的问题。
免疫缀合物由人、人源化或嵌合重组抗体共价连接于细胞毒性药物制得。这种结构的主要目标是将小细胞毒性剂(300至1000Da)的效力和肿瘤相关抗原靶向(TAA)MAb的高特异性结合。
Ab对于到达其表达必须局限在正常细胞中的抗原必须极具选择性。Ab也必须被高效内化至癌性细胞中。
选作效应物部分的细胞毒性剂必须仅在内化并向细胞质中释放之后杀灭细胞。最通常用于ADC中的有效载荷是DNA损害性药物,诸如卡奇霉素(calicheamicin)、倍癌霉素(duocarmicin);或微管靶向性化合物,如奥莉斯汀(auristatin)和美登木素(maitansinoid)。
Ab-细胞毒性剂接头被设计以达成全身稳定,并且在靶细胞内释放细胞毒性剂。
TAA常常是在患病组织中过度表达或至少充分表达以促进内化活化的细胞毒性的细胞膜蛋白质。理想的是,抗原在正常组织中呈现受局限表达,而在重要器官中表达较低或不存在表达。除此之外,肿瘤抗原必须由Ab以选择性方式以及以高亲和力识别。
新近研究表明被设计以在肿瘤-间质间期抑制TGF-β信号传导途径的治疗剂可防止癌症进展,从而改进预后和治疗。TGF-β共受体家族正作为作用于肿瘤或它的新血管系统的癌症治疗靶标而出现。作为II型TGF-β受体复合物的辅助蛋白的内皮糖蛋白(ENG,CD105)是这个家族的一部分,并且呈现以下特征:
●I型同二聚体膜蛋白
●在许多癌症类型中的细胞表面血管生成和新血管化相关蛋白
●在肿瘤微血管系统细胞中,具体来说在肿瘤的血管生成区域中过度表达
●在正常组织内皮中不表达
●乳腺转移相关的血浆水平
●内化
●从血流最优可及
已报道抗ENG抗体(例如scFv),并且描述它们在肿瘤间质靶向策略中的应用。已报道多柔比星上样的抗ENG免疫脂质体在体外(使用ENG+细胞)和在体内(在小鼠中)的特异性结合、细胞内化和抗肿瘤作用。已在小鼠肿瘤模型中评估抗ENG抗体和篦麻毒素(ricin)或天然黑杆菌素b(nigrin b)的ENG靶向缀合物(27-36)。
尽管取得这些进步,但仍然对用于治疗包括上皮肿瘤的肿瘤的其它治疗策略,以及对于用于此类治疗策略中的组分存在未满足的需要。本发明解决这些和其它需要。
发明简述
广泛来说,本发明涉及抗ENG抗体、其缀合物和用于抗体缀合物策略中的优化有效载荷。具体来说,本发明者已发现如本文所述的抗ENG抗体展现高度特异性结合以及快速和高效内化。此外,本发明者已发现可分离并在细菌宿主细胞中产生黑杆菌素(Nigrin)b的A链,其在缺少黑杆菌素-b B链下在缀合于单克隆抗体时仍然保留易位至细胞中的能力,并且展现细胞毒性活性。本文所述的黑杆菌素-b A链和/或溶细胞素(cytolysin)衍生物被有利地缀合于抗ENG抗体以用于治疗肿瘤。
因此,在第一方面,本发明提供一种具有式I的缀合物:
A-(L-D)p (I)
或其药学上可接受的盐或溶剂化物,
其中:
A是选择性结合内皮糖蛋白的抗体;
L是接头;
D是包括溶细胞素或黑杆菌素-b A链的药物;并且
p是1至10。
在根据本发明的这个和其它方面的一些情况下,A是选择性结合至人内皮糖蛋白的细胞外区域的单克隆抗体或其结合片段。在特定情况下,A可包含具有以下氨基酸序列的重链互补决定区1-3(CDRH1-3)和轻链互补决定区1-3(CDRL1-3):
(i)CDRH1:SEQ ID NO:7或其相较于SEQ ID NO:7的序列具有多达1或2个氨基酸取代的变体;
(ii)CDRH2:SEQ ID NO:8或其相较于SEQ ID NO:8的序列具有多达1或2个氨基酸取代的变体;
(iii)CDRH3:SEQ ID NO:9或其相较于SEQ ID NO:9的序列具有多达1或2个氨基酸取代的变体;
(iv)CDRL1:SEQ ID NO:10或其相较于SEQ ID NO:10的序列具有多达1或2个氨基酸取代的变体;
(v)CDRL2:SEQ ID NO:11或其相较于SEQ ID NO:11的序列具有多达1或2个氨基酸取代的变体;和
(vi)CDRL3:SEQ ID NO:12或其相较于SEQ ID NO:12的序列具有多达1或2个氨基酸取代的变体。
在某些情况下,CDRH1-3分别包含SEQ ID NO:7-9的氨基酸序列,并且CDRL1-3分别包含SEQ ID NO:10-12的氨基酸序列。
在某些情况下,A包含含与SEQ ID NO:5的全长序列具有至少90%、95%或99%序列同一性的氨基酸序列的重链可变区(VH)。
在某些情况下,A包含含SEQ ID NO:5的氨基酸序列的重链可变区(VH)。
在某些情况下,A包含含与SEQ ID NO:6的全长序列具有至少90%、95%或99%序列同一性的氨基酸序列的轻链可变区(VL)。具体来说,A可包含含SEQ ID NO:6的氨基酸序列的轻链可变区(VL)。
在某些情况下,A包含含与SEQ ID NO:3的全长序列具有至少90%、95%或99%序列同一性的氨基酸序列的重链。具体来说,A可包含含SEQ ID NO:3的氨基酸序列的重链。
在某些情况下,A包含含与SEQ ID NO:4的全长序列具有至少90%、95%或99%序列同一性的氨基酸序列的轻链。具体来说,A可包含含SEQ ID NO:4的氨基酸序列的轻链。
在某些情况下,A可为在结构上不同于本文例示的抗内皮糖蛋白抗体分子的竞争性结合抗内皮糖蛋白抗体。举例来说,A可为与在本文中标识为“A5”的抗人内皮糖蛋白IgG1抗体竞争结合固定的重组人内皮糖蛋白的抗内皮糖蛋白抗体分子。A5具有SEQ ID NO:3的重链氨基酸序列和SEQ ID NO:4的轻链氨基酸序列。
在某些情况下,A是选择性结合鼠内皮糖蛋白的细胞外区域的单克隆抗体或其结合片段。靶向鼠内皮糖蛋白的缀合物特别适用于例如采用各种癌症的充分表征的鼠模型进行的临床前测试。具体来说,A可包含具有以下氨基酸序列的重链互补决定区1-3(CDRH1-3)和轻链互补决定区1-3(CDRL1-3):
(i)CDRH1:SEQ ID NO:19或其相较于SEQ ID NO:19的序列具有多达1或2个氨基酸取代的变体;
(ii)CDRH2:SEQ ID NO:20或其相较于SEQ ID NO:20的序列具有多达1或2个氨基酸取代的变体;
(iii)CDRH3:SEQ ID NO:21或其相较于SEQ ID NO:21的序列具有多达1或2个氨基酸取代的变体;
(iv)CDRL1:SEQ ID NO:22或其相较于SEQ ID NO:22的序列具有多达1或2个氨基酸取代的变体;
(v)CDRL2:SEQ ID NO:23或其相较于SEQ ID NO:23的序列具有多达1或2个氨基酸取代的变体;和
(vi)CDRL3:SEQ ID NO:24或其相较于SEQ ID NO:24的序列具有多达1或2个氨基酸取代的变体。举例来说,CDRH1-3可分别包含SEQ ID NO:19-21的氨基酸序列,并且CDRL1-3可分别包含SEQ ID NO:22-24的氨基酸序列。
在某些情况下,A包含含与SEQ ID NO:17的全长序列具有至少90%、95%或99%序列同一性的氨基酸序列的重链可变区(VH),例如A可包含含SEQ ID NO:17的氨基酸序列的重链可变区(VH)。
在某些情况下,A包含含与SEQ ID NO:18的全长序列具有至少90%、95%或99%序列同一性的氨基酸序列的轻链可变区(VL),例如A可包含含SEQ ID NO:18的氨基酸序列的轻链可变区(VL)。
在某些情况下,A包含含与SEQ ID NO:15的全长序列具有至少90%、95%或99%或甚至100%序列同一性的氨基酸序列的重链。
在某些情况下,A包含含与SEQ ID NO:16的全长序列具有至少90%、95%或99%或甚至100%序列同一性的氨基酸序列的轻链。
在某些情况下,A可为在结构上不同于本文例示的抗内皮糖蛋白抗体分子的竞争性结合抗内皮糖蛋白抗体。举例来说,A可为与在本文中标识为“mE12”的抗鼠内皮糖蛋白IgG1抗体竞争结合固定的重组鼠内皮糖蛋白的抗内皮糖蛋白抗体分子。mE12具有SEQ IDNO:15的重链氨基酸序列和SEQ ID NO:16的轻链氨基酸序列。
根据本发明的这个和其它方面,D可为溶细胞素。在一些情况下,溶细胞素可为WO2008/138561 A1中公开的化合物,所述专利的整个内容以引用的方式明确并入本文(其中公开的化合物也被称为微管溶素(Tubulysine)衍生物)。可如WO 2008/138561中所述合成溶细胞素。在某些情况下,溶细胞素可如WO 2008/138561 A1的式I或式IV所定义。在某些情况下,溶细胞素可具有式IV:
其中:
R2(i)直接或间接连接于接头L或(ii)是H或是C1-C4烷基;
R6是C1-C6烷基;
R7是C1-C6烷基、CH2OR19或CH2OCOR20,其中R19是烷基,R20是C2-C6-烯基、苯基或CH2-苯基;
R9是C1-C6烷基;
R10是H、OH、O-烷基或O-乙酰基;
f是1或2;
R11具有以下结构:
其中
R21是H、OH、卤素、NH2、烷基氧基、苯基、烷基氨基或二烷基氨基;
R16是H或C1-C6-烷基基团;
R17(i)直接或间接连接于接头L或(ii)是CO2H、CO2R18、CONHNH2、OH、NH2、SH或任选取代的烷基、环烷基、杂烷基或杂环烷基基团,其中R18是任选取代的烷基、杂烷基或杂环烷基基团;并且
q是0、1、2或3;
并且其中术语“任选取代的”涉及其中一个或若干个H原子可被F、Cl、Br或I或OH、SH、NH2或NO2置换的基团;术语“任选取代的”进一步涉及可仅仅或另外被未取代的C1-C6烷基、C2C6烯基、C2-C6炔基、C1-C6杂烷基、C3-C10环烷基、C2-C9杂环烷基、C6-C10芳基、C1-C9杂芳基、C7-C12芳烷基或C2-C11杂芳烷基基团取代的基团。
在一些情况下,R2是至接头L的键。
在一些情况下,R17是C(O)X、CONHNHX、OX、NHX或SX,其中X是至接头L的键。
在一些情况下,接头L可进一步包含间隔基。
在一些情况下,间隔基具有2至30个原子的链长度。
在一些情况下,间隔基包含以下或由以下组成:亚烷基(即二价烷基)或亚杂烷基(即二价杂烷基)基团。
在一些情况下,间隔基包含以下或由以下组成:亚烷基或氧基亚烷基基团。
在一些情况下,间隔基包含以下或由以下组成:基团-(CH2)n-或-(OCH2CH2)n-,其中n≥1。
在一些情况下,间隔基包含以下或由以下组成:基团-(OCH2CH2)n-,其中n≥1。具体来说,n可为1至15、1至10、1至6、或2至5。举例来说,n可为3或4。
在一些情况下,间隔基包含一个与六个之间的乙二醇单元,例如三乙二醇。
在一些情况下,间隔基可直接连接于基团R17,或可经由桥连基团连接于基团R17。
在一些情况下,间隔基经由-C(O)X桥连基团连接于基团R17,其中X是至R17的键。
在一些情况下,R17是CONHNHX,并且间隔基经由-C(O)X桥连基团连接于基团R17,其中X代表间隔基与R17之间的键。
在一些情况下,R17是CONHNHX,并且间隔基是经由-C(O)X桥连基团连接于R17的-(OCH2CH2)n-,其中n=2、3或4。
在一些情况下,D包括具有以下结构的溶细胞素:
在一些情况下,D包括具有以下结构的溶细胞素:
在某些情况下,L包含用于连接于A的连接基团和蛋白酶可裂解部分。举例来说,L可包含缬氨酸-瓜氨酸单元。具体来说,L可包括马来酰亚胺基己酰基-缬氨酸-瓜氨酸-对氨基苯甲基氨基甲酸酯。
在一些情况下,使马来酰亚胺的双键与抗体A的半胱氨酸残基的硫醇基团反应以形成硫-碳键来实现接头L连接于抗体A。
在一些情况下,–L-D具有选自由以下组成的组的结构:
在某些情况下,–L-D可具有以下结构:
在某些情况下,–L-D可具有以下结构:
根据本发明的这方面和其它方面,p在一些情况下可位于1至5、例如1至4或1至3的范围内。在特定情况下,p可为1或2。在特定情况下,p可为3或4。
根据本发明的这个和其它方面,D可为黑杆菌素-b A链。优选地黑杆菌素-b A链缺少黑杆菌素-b B链。黑杆菌素-b A链可包含以下或由以下组成:SEQ ID NO:25的序列。
在某些情况下,黑杆菌素-b A链可或可已例如在细菌宿主细胞中重组产生。本发明者已惊人地发现尽管天然糖基化丧失或改变,诸如当在细菌宿主细胞中重组产生黑杆菌素-b A链时的情况,但黑杆菌素-b A链保留它的活性(例如细胞毒性和/或核糖体抑制活性)。
当本发明的缀合物包含黑杆菌素-b A链作为毒性有效载荷(即D)时,L可仅为在A上硫原子与D上硫原子之间的二硫键。因此,L可包括键(例如二硫键)或由键(例如二硫键)组成。
在第二方面,本发明提供一种用于医学中的如根据本发明的第一方面定义的缀合物。
在第三方面,本发明提供一种用于治疗哺乳动物受试者的肿瘤的方法中的如根据本发明的第一方面定义的缀合物。在某些情况下,缀合物用于治疗血液赘生物。在其它情况下,缀合物用于治疗实体肿瘤。具体来说,缀合物可用于治疗胰腺癌、尤文肉瘤(Ewingsarcoma)、乳腺癌、黑素瘤、肺癌、头颈癌、卵巢癌、膀胱癌或结肠癌。
在一些情况下,缀合物用于与一种或多种其它抗肿瘤药物同时、依序或分开施用。一种或多种其它抗肿瘤药物包括细胞毒性化学治疗剂或抗血管生成剂或免疫治疗剂。在一些情况下,一种或多种其它抗肿瘤药物包括吉西他滨(Gemcitabine)、凯素贝伐单抗(Abraxane bevacizumab)、伊曲康唑(itraconazole)、羧基酰胺基三唑、抗PD-1分子或抗PD-L1分子(例如尼鲁单抗(nivolumab)或派姆单抗(pembrolizumab))。
在第四方面,本发明提供一种治疗哺乳动物受试者的肿瘤的方法,其包括向有需要的所述受试者施用治疗有效量的如根据本发明的第一方面定义的缀合物。在一些情况下,方法可用于治疗血液赘生物。在其它情况下,方法可用于治疗实体肿瘤。具体来说,方法可用于治疗胰腺癌、尤文肉瘤、乳腺癌、黑素瘤、肺癌、头颈癌、卵巢癌、膀胱癌或结肠癌。
在第五方面,本发明提供溶细胞素制备抗体-药物缀合物的用途,其中所述抗体是内皮糖蛋白特异性抗体,例如根据本发明的第八方面的内皮糖蛋白特异性抗体。在一些情况下,溶细胞素可如根据本发明的第一方面所定义。在某一情况下,用途可为溶细胞素制备如根据本发明的第一方面定义的抗体-药物缀合物。
在第六方面,本发明提供一种缺少黑杆菌素-b B链的分离的黑杆菌素-b A链。黑杆菌素-b A链的氨基酸序列可包括以下或由以下组成:SEQ ID NO:25的序列。
在第七方面,本发明提供根据本发明的第六方面的分离的黑杆菌素-b A链制备免疫毒素的用途。在一些情况下,免疫毒素包含缀合和/或结合于所述分离的黑杆菌素-b A链的单克隆抗体。在一些情况下,免疫毒素包含选择性结合内皮糖蛋白的抗体,诸如单克隆抗体,例如人单克隆抗体。在一些情况下,免疫毒素包含根据本发明的第八方面的抗体。
在第八方面,本发明提供一种例如是人单克隆抗体的单克隆抗体,其:
(i)选择性结合至人内皮糖蛋白,并且其包含含SEQ ID NO:3的氨基酸序列的重链和含SEQ ID NO:4的氨基酸序列的轻链;或
(ii)选择性结合鼠内皮糖蛋白,并且其包含含SEQ ID NO:15的氨基酸序列的重链和含SEQ ID NO:16的氨基酸序列的轻链。
在第九方面,本发明提供一种用于医学中本发明的第八方面的抗体。
在第十方面,本发明提供一种本发明的第一方面的缀合物或一种本发明的第八方面的抗体,所述缀合物或所述抗体用于治疗炎性疾患(例如类风湿性关节炎)或眼病(例如糖尿病性视网膜病变或黄斑变性,诸如湿性年龄相关的黄斑变性)。
在第十一方面,本发明提供一种治疗哺乳动物受试者的炎性疾患(例如类风湿性关节炎)或眼病(例如类风湿性关节炎)或眼病(例如糖尿病性视网膜病变或黄斑变性,诸如湿性年龄相关的黄斑变性)的方法,其包括向有需要的所述受试者施用治疗有效量的本发明的第一方面的缀合物或本发明的第八方面的抗体。
在第十二方面,本发明提供根据本发明的第八方面的单克隆抗体制备抗体-药物缀合物或免疫毒素的用途。
在第十三方面,本发明提供一种包含载体的宿主细胞,所述载体包含编码至少一种具有选自由以下组成的组的氨基酸序列的多肽的多核苷酸:SEQ ID NO:1-6、13-18和25。在一些情况下,多核苷酸可包含SEQ ID NO:26的核酸序列。
在第十四方面,本发明提供一种用于产生根据本发明的第一方面的缀合物的方法,其包括:
(a)使选择性结合内皮糖蛋白的抗体衍生以引入至少一个巯基基团;以及
(b)使衍生化抗体与黑杆菌素-b A链(缺少黑杆菌素-b B链)上适当残基(例如半胱氨酸氨基酸)在允许在所述抗体与所述黑杆菌素-b A链之间形成二硫键连接,由此产生所述缀合物的条件下反应。方法可进一步包括纯化和/或分离缀合物的步骤(c)。
在一些情况下,步骤(a)可包括使抗体与4-琥珀酰亚胺基氧基羰基-α-甲基-α-(2-吡啶基-二硫代)甲苯(SMPT)、3-(2-吡啶基-二硫代丙酸)N-琥珀酰亚胺酯(SPDP)或4-巯基丁酰亚胺酸甲酯反应。
在第十五方面,本发明提供一种用于产生根据本发明的第一方面的缀合物的方法,其包括:
(a)使选择性结合内皮糖蛋白的抗体经由硫醇基团连接于接头;以及
(b)使溶细胞素通过溶细胞素分子上适当基团连接于接头。在一些情况下,使溶细胞素通过R2位或R17位连接于接头。可以任一顺序进行步骤(a)和(b)。在任选进一步骤(c)中,方法可包括纯化和/或分离缀合物。
本发明包括所述方面和优选特征的组合,例外之处是当这种组合是明确不允许的或被陈述应明确避免时。以下进一步详细并且参照随附实施例和附图来描述本发明的这些和其它方面和实施方案。
附图简述
图1显示用于结合重组小鼠ENG(aa 27-581)的mE12-IgG的ELISA结果。涂布的BSA作为阴性对照而包括。观察到与小鼠ENG的浓度依赖性结合;
图2显示对mE12-IgG与a)B16细胞(使用50μg/ml抗体),b)B16细胞(使用5μg/ml抗体),和c)作为阴性对照而包括的HT1080细胞的结合的流式细胞计量术分析。阴影化区域:单独细胞,无阴影化区域:与抗体一起孵育的细胞;
图3显示a)A5IgG1与固定的重组人ENG结合的ELISA。b)对A5-IgG与HT1080细胞的结合的流式细胞计量术分析;
图4显示重组黑杆菌素-b A链的MALDI-Tof概况。观察质量(Da):28546.55;预期质量(Da):28546.09;质量偏差:0.5;质量准确度:16ppm。
图5显示相对于天然(WT)黑杆菌素-b,在兔网织红细胞无细胞裂解物(RRL)中测试的重组黑杆菌素-b A链(recNgA)的核糖体失活蛋白(RIP)活性,(3a、3b、6c、9c)代表不同recNgA制剂
图6显示通过结晶紫存活力测定关于HT1080-FAP细胞系测试的recNgA细胞毒性(天然黑杆菌素–菱形;重组黑杆菌素-b A链–方块)
图7显示recNgA缀合物在RRL测定中的RIP活性,天然黑杆菌素b:菱形(◆);recNgA:方块(■);recNgA缀合物HPS-124-37-1(三角:▲),HPS-124-37-2(十字号:X)
图8显示通过vcPABA接头获得的溶细胞素缀合抗体的一般性抗体缀合物结构。溶细胞素可通过R1或R4(用箭头标识)连接
图9显示通过辨别显示仅膜染色(PM)、PM和细胞内染色、或仅细胞内染色的细胞(n=10-30)来对A5抗huENG IgG1内化的分析。
图10显示溶细胞素ADC对(A)野生型(WT)和(B)抗原(AG)表达性HT1080细胞的体外细胞毒性作用。通过在10-6至10-12M的浓度范围下孵育各化合物72小时之后进行结晶紫染色来证实细胞增殖阻滞。亲本TAM334溶细胞素用作不特异性细胞毒性的阳性对照。
图11显示recNgA和溶细胞素免疫缀合物的肿瘤生长抑制作用。(A)recNgA缀合物,以单一药剂形式(OMTX505)或与吉西他滨(OMTX505:GEM)组合施用;(B)TAM471(OMTX705-471)相对于TAM551(OMTX705-551)缀合物;(C)TAM471(OMTX705-471)和TAM553(OMTX705-553)相对于TAM558(OMTX705-558)缀合物。媒介物和GEM(吉西他滨):阴性和阳性对照组。
发明详述
在描述本发明时,以下术语将被采用,并且意图如以下指示所定义。
内皮糖蛋白
如所使用,“内皮糖蛋白”可为任何哺乳动物物种的内皮糖蛋白。在一些情况下,内皮糖蛋白是人内皮糖蛋白(也称为CD105、ENG或END),其氨基酸序列以UniProt登记号P17813(154版,日期是2013年11月13日)(SEQ ID NO:27)加以公开。在一些情况下,结合内皮糖蛋白的分子(例如抗体分子或其缀合物)可结合内皮糖蛋白的细胞外结构域的区域。人内皮糖蛋白的细胞外结构域包含全长人内皮糖蛋白的残基26-561。在一些情况下,内皮糖蛋白是鼠内皮糖蛋白(也称为CD105、MJ7/18抗原、ENG或END),其氨基酸序列以UniProt登记号Q63961(104版,日期是2013年11月13日)(SEQ ID NO:28)加以公开。鼠内皮糖蛋白的细胞外结构域包含全长鼠内皮糖蛋白的残基27-581。
缀合物
如本文所用,“缀合物”包括通过连接各分子形成的所得结构,并且明确包括抗体-药物缀合物(ADC)和免疫毒素(IT)。
选择性结合
术语选择性结合(selectively binds/selective binding)是指抗体或其结合片段以特异性方式结合预定分子(例如抗原)。举例来说,抗体或其结合片段可以至少约1x107M-1的亲和力结合内皮糖蛋白,例如其细胞外部分,并且结合预定分子的亲和力可比它结合除预定分子以外的分子的亲和力大至少两倍(例如大五倍或十倍)。
抗体分子
如本文关于本发明的所有方面所用,术语“抗体”或“抗体分子”包括天然或部分或完全合成产生的任何免疫球蛋白。术语“抗体”或“抗体分子”包括单克隆抗体(mAb)和多克隆抗体(包括多克隆抗血清)。抗体可为完整的,或源于完全抗体的片段(参见下文)。抗体可为人抗体、人源化抗体或非人来源的抗体。“单克隆抗体”是针对靶标分子的单一抗原位点或“决定簇”的均质高度特异性抗体群体。“多克隆抗体”包括针对靶标分子的不同抗原决定簇的异质抗体群体。术语“抗血清”是指从免疫的动物获得的含有抗体的血清。
已显示完整抗体的片段可执行结合抗原的功能。因此,在本文中提及抗体以及提及本发明的方法、阵列和试剂盒涵盖完全抗体,并且也涵盖包含抗体结合片段的任何多肽或蛋白质。结合片段的实例是(i)由VL、VH、CL和CH1结构域组成的Fab片段;(ii)由VH和CH1结构域组成的Fd片段;(iii)由单一抗体的VL和VH结构域组成的Fv片段;(iv)由VH结构域组成的dAb片段;(v)分离的CDR区;(vi)F(ab')2片段,即包含两个连接的Fab片段的二价片段;(vii)单链Fv分子(scFv),其中VH结构域和VL结构域由允许两个结构域缔合以形成抗原结合位点的肽接头连接;(viii)双特异性单链Fv二聚体(WO 93/11161)和(ix)“微型双功能抗体”,即通过基因融合构建的多价或多特异性片段(WO94/13804;58)。可通过并入连接VH结构域和VL结构域的二硫桥来使Fv、scFv或微型双功能抗体分子稳定。也可制备包含接合于CH3结构域的scFv的微型抗体。
关于抗体分子,术语“选择性结合”可在本文中用于指代其中特异性结合对的一个成员将不显示与除它的特异性结合配偶体以外的分子的任何显著结合的情况。当例如抗原结合位点对由许多抗原携带的特定表位具有特异性时,所述术语也是可适用的,在所述情况下,携带抗原结合位点的特异性结合成员将能够结合携带所述表位的各种抗原。
在根据本发明的一些情况下,抗体可为完全人抗体。
细胞毒性化学治疗剂
在根据本发明的任何方面的一些情况下,本发明的缀合物可与或用于与其它抗肿瘤药物一起施用(同时、依序或分开),所述抗肿瘤药物包括但不限于细胞毒性化学治疗剂或抗血管生成剂或免疫治疗剂。
细胞毒性化学治疗剂在本领域中是熟知的,并且包括抗癌剂,诸如:
烷基化剂,包括氮芥,诸如二氯甲基二乙胺(HN2)、环磷酰胺(cyclophosphamide)、异环磷酰胺(ifosfamide)、美法仑(melphalan)(L-溶肉瘤素(L-sarcolysin))和苯丁酸氮芥;10亚乙基亚胺和甲基三聚氰胺,诸如六甲基三聚氰胺、噻替派(thiotepa);alkylsulphonates,诸如白消安(busulfan);亚硝基脲,诸如卡莫司汀(BCNU)、洛莫司汀(CCNLJ)、司莫司汀(semustine)(甲基-CCN-U)和链脲佐菌素(streptozoein/streptozotocin);和三氮烯,诸如达卡巴嗪(decarbazine)(DTIC;二甲基三氮烯基咪唑甲酰胺);
抗代谢剂,包括叶酸类似物,诸如甲氨蝶呤(methotrexate)(氨甲蝶呤(amethopterin));嘧啶类似物,诸如氟尿嘧啶(5-氟尿嘧啶;5-FU)、氟尿苷(氟脱氧尿苷;FUdR)和阿糖胞苷(cytarabine)(胞嘧啶阿拉伯糖苷);和嘌呤类似物和相关抑制剂,诸如巯基嘌呤(6-巯基嘌呤;6-MP)、硫鸟嘌呤(6-硫鸟嘌呤;TG)和喷司他汀(pentostatin)(2'-脱氧柯福霉素(2'-deoxycofonnycin))。天然产物,包括长春花生物碱,诸如长春花碱(vinblastine,VLB)和长春新碱(vincristine);表鬼臼毒素(epipodophyllotoxin),诸如依托泊苷(etoposide)和替尼泊苷(teniposide);抗生素,诸如放线菌素D(dactinomycin/actinomycin D)、柔红霉素(daunorabicin)(道诺霉素(daunomycin);红比霉素(rubidomycin))、多柔比星(doxorubicin)、博莱霉素(bleomycin)、光神霉素(plicamycin)(米拉霉素(mithramycin))和丝裂霉素(mitomycin)(丝裂霉素Q);酶,诸如L-天冬酰胺酶;和生物响应调节剂,诸如干扰素alphenome。杂项药剂,包括铂配位络合物,诸如顺铂(cisplatin)(顺式DDP)和卡铂(carboplatin);蒽二酮,诸如米托蒽醌(mitoxantrone)和蒽环霉素(antbracycline);取代的脲,诸如羟基脲;甲基肼衍生物,诸如丙卡巴肼(procarbazine)(N-甲基肼,MIH);和肾上腺皮质抑制剂,诸如米托坦(mitotane)(o,p'-DDD)和胺鲁米特(aminoglutethimide);泰素(taxol)和类似物/衍生物;以及激素激动剂/拮抗剂,诸如氟他胺(flutamide)和他莫昔芬(tamoxifen)。另一优选细胞毒性剂是吉西他滨(Gemcitabine)另一优选细胞毒性剂是结合于人血清白蛋白的太平洋紫杉醇(Paclitaxel)
抗血管生成剂在本领域中是熟知的,并且包括抗癌剂,诸如贝伐单抗(bevacizumab)、伊曲康唑(itraconazole)和羧基酰胺基三唑。
免疫治疗剂在本领域中是已知的,并且包括例如抗程序化细胞死亡蛋白1(PD-1)抗体和抗程序化死亡配体1(PD-L1)抗体,包括尼鲁单抗(Nivolumab)(MDX1106)和派姆单抗(Pembrolizumab)(MK-3475)。
药物组合物
本发明的缀合物可与药学上可接受的赋形剂一起包含在药物组合物中。
药学上可接受的赋形剂可为进入药物组合物中,不激起次级反应,并且允许例如促进施用缀合物,增加它在体内的寿命和/或功效,或增加它在溶液中的溶解性的化合物或化合物的组合。这些药学上可接受的媒介物是熟知的,并且将由本领域技术人员随缀合物的施用模式而变加以改适。
在一些实施方案中,本发明的缀合物可以用于在施用之前复原的冻干形式提供。举例来说,冻干缀合物可在向个体施用之前于无菌水中复原,并且与盐水混合。
本发明的缀合物将通常以除缀合物之外,也可包含至少一种组分的药物组合物形式施用。因此,除缀合物之外,药物组合物也可包含为本领域技术人员所熟知的药学上可接受的赋形剂、载体、缓冲剂、稳定剂或其它物质。所述物质应无毒,并且不应干扰缀合物的功效。载体或其它物质的确切性质将取决于可为如下讨论的团注、输注、注射或任何其它适合途径的施用途径。
对于例如通过注射达成的静脉内施用,包含缀合物的药物组合物可呈无热原并具有适合pH、等张性和稳定性的胃肠外可接受的水溶液形式。本领域相关技术人员完全能够使用例如等张媒介物制备适合溶液,诸如氯化钠注射液,林格氏注射液(Ringer'sInjection)、乳酸林格氏注射液。可根据需要采用防腐剂、稳定剂、缓冲剂、抗氧化剂和/或其它添加剂,包括缓冲剂,诸如磷酸盐、柠檬酸盐和其它有机酸;抗氧化剂,诸如抗坏血酸和甲硫氨酸;防腐剂(诸如十八烷基二甲基苯甲基氯化铵;氯化六羟季铵;氯化苯甲烃铵;苄索氯铵;苯酚、丁醇或苯甲醇;对羟基苯甲酸烷酯,对羟基苯甲酸甲酯或对羟基苯甲酸丙酯;儿茶酚;间苯二酚;环己醇;3’-戊醇;和间甲酚);低分子量多肽;蛋白质,诸如血清白蛋白、明胶或免疫球蛋白;亲水性聚合物,诸如聚乙烯吡咯烷酮;氨基酸,诸如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸;单糖、二糖和其它碳水化合物,包括葡萄糖、甘露糖或糊精;螯合剂,诸如EDTA;糖,诸如蔗糖、甘露糖醇、海藻糖或山梨糖醇;成盐相对离子,诸如钠;金属络合物(例如Zn-蛋白质络合物);和/或非离子表面活性剂,诸如TWEENTM、PLURONICSTM或聚乙二醇(PEG)。
受试者
受试者可为人、伴侣动物(例如狗或猫)、实验室动物(例如小鼠、大鼠、兔、猪或非人灵长类动物)、家养或农场动物(例如猪、母牛、马或绵羊)。优选地,受试者是人。在一些情况下,受试者可为被诊断有例如上皮肿瘤、实体肿瘤或血液赘生物的癌症或分类为处于显现例如上皮肿瘤、实体肿瘤或血液赘生物的癌症的风险下的人。在某些情况下,受试者可为实验室动物,例如患有癌症的小鼠模型。在某些情况下,受试者可为已被诊断有诸如骨关节炎或类风湿性关节炎(RA)的炎性疾患或分类为处于显现诸如骨关节炎或类风湿性关节炎(RA)的炎性疾患的风险下的哺乳动物(例如人)。具体来说,受试者可为患有骨关节炎或RA的人。在某些情况下,受试者可为已被诊断有诸如糖尿病性视网膜病变或黄斑变性的眼病或分类为处于显现诸如糖尿病性视网膜病变或黄斑变性的眼病的风险下的哺乳动物(例如人)。
癌症
本文所述的抗ENG缀合物适用于治疗哺乳动物受试者的肿瘤。肿瘤可为实体肿瘤。具体来说,肿瘤可为胰腺癌、乳腺癌、黑素瘤、尤文肉瘤、肺癌、头颈癌、卵巢癌、膀胱癌或结肠癌。
炎性疾患
在根据本发明的一些情况下,抗ENG抗体或抗体药物缀合物可用于治疗炎性疾患。已报道ENG在骨关节炎和类风湿性关节炎中表达。参见例如Szekanecz,Z.等ClinicalImmunology and Immunopathology,1995,76,187–194;以及Leask A等,Arthritis&Rheumatism,2002,46,1857-1865。本发明者相信本文所述的抗ENG抗体和/或本文所述的其缀合物能够改善骨关节炎、类风湿性关节炎和/或骨关节炎或类风湿性关节炎的症状。
眼病
在根据本发明的一些情况下,抗ENG抗体或抗体药物缀合物可用于治疗眼病(例如糖尿病性视网膜病变或黄斑变性,诸如年龄相关的黄斑变性)。已报道ENG在某些眼疾患,包括黄斑变性和视网膜病变中表达。参见例如Tsutomu Yasukawa等,Curr Eye Res.2000,21,952-961;以及Abu El-Asrar AM等,Mediators Inflamm.2012,2012:697489;以及Malik RA等,J Cell Mol Med.2005,9:692-7。本发明者相信本文所述的抗ENG抗体和/或本文所述的其缀合物能够改善眼病和/或其症状(包括糖尿病性视网膜病变或黄斑变性,诸如年龄相关的黄斑变性)。
以下是通过举例方式呈现,并且不应解释为是对权利要求的范围的限制。
实施例
实施例1–产生抗ENG抗体
先前已描述从合成抗体噬菌体文库分离的抗ENG scFv(29)。使一种针对人ENG的细胞外区域、称为“A5”(29)的scFv和一种针对鼠ENG的细胞外区域、称为“mE12”(31)的scFv转变成全长IgG,以用于随后表征研究和产生免疫毒素和ADC。所有scFv都在大肠杆菌(E.coli)中产生,并且通过IMAC纯化,使用被开发用于抗体产生的Lonza GS表达载体pEE6.4和pEE14.4在哺乳动物细胞(CHO)中产生IgG。scFv原料的特征概述于表1中。
表1:抗体、特异性、子类和用作原料的载体
所有scFv都在大肠杆菌TG1中通过细菌产生,并且通过IMAC从1L培养物的周质提取物纯化。
产生对应于全长IgG1抗体的质粒,并且将其转染至CHO细胞中以在Lonza的CHO表达系统中以约1mg/L细胞培养物的产率产生抗体(实验室规模)。通过蛋白A色谱法来从细胞培养上清液纯化抗体。通过SDS-PAGE和尺寸排阻色谱法表征纯化蛋白质。通过使用重组ENG以及用HRP缀合的抗人IgG抗体检测结合的抗体进行ELISA来分析生物活性。通过使用ENG-表达小鼠B16细胞进行流式细胞计量术来分析细胞结合。
结果:
产生(并且测序)的质粒:
A5 IgG1:pEE14.4 A5-IgG1 OCMTX003p(人抗huENG IgG1)
mE12 IgG1:pEE14.4 mE12-IgG1 OCMTOO4p(人抗muENG IgG1)
实施例2-表征抗ENG抗体
以下分别显示抗人ENG IgG1 A5(A5-IgG1)重链(HC)和轻链(LC)的氨基酸序列:
抗人内皮糖蛋白A5-IgG1-HC:
导致ADCC和CDC缺乏的突变以粗斜体显示(也参见WO99/58572)
信号序列被加框显示
VH结构域被加下划线;CDRH1-H3以粗体显示并加弯曲下划线。
抗人内皮糖蛋白A5-IgG1-LC:
aa 214
加工的HC的分子量 23,113
理论pI 7.76
信号序列被加框
VL结构域被加下划线;CDRL1-L3以粗体显示并加弯曲下划线。
A5-IgG1-HC–无信号序列:
A5-IgG1-LC-无信号序列:
A5-VH:
A5-VL:
A5-CDRH1:
SYAMS(SEQ ID NO:7)
A5-CDRH2:
AIYGSDGDTTY(SEQ ID NO:8)
A5-CDRH3:
VFYTAGFDY(SEQ ID NO:9)
A5-CDRL1:
RASQSISSSLN(SEQ ID NO:10)
A5-CDRL2:
AASSLQS(SEQ ID NO:11)
A5-CDRL3:
QQAPAKPPT(SEQ ID NO:12)
完全A5-IgG的参数如下:
抗鼠内皮糖蛋白mE12-IgG1-HC:
导致ADCC和CDC缺乏的突变以粗斜体显示
信号序列被加框显示
VH结构域被加下划线
抗鼠内皮糖蛋白mE12-IgG1-LC:
aa 212
MW(加工) 22,516
计算pI 6.69
信号序列被加框显示
VL结构域被加下划线
mE12-IgG1-HC–无信号序列:
mE12-IgG1-LC–无信号序列:
mE12-VH:
mE12-VL:
mE12-CDRH1:
SYGMH(SEQ ID NO:19)
mE12-CDRH2:
RINSDGSSTSYADSVKG(SEQ ID NO:20)
mE12-CDRH3:
ATGTWVMS(SEQ ID NO:21)
mE12-CDRL1:
QGDSLRSYYAS(SEQ ID NO:22)
mE12-CDRL2:
GKNNRPS(SEQ ID NO:23)
mE12-CDRL3:
NSRDSSGTV(SEQ ID NO:24)
在ELISA中分析纯化抗muENG mE12和抗huENG A5抗体与重组ENG的结合,并且使用流式细胞计量术分析(FACS)对所述抗体进行分析。显示mE12抗muENG(图1和2)和A5抗huENGIgG1(图3)的亲和力结果,并且概述于表2中。图1显示mE12-IgG与重组小鼠ENG(aa27-581),但不与阴性对照(BSA)的浓度依赖性结合。图2显示对mE12-IgG与a)B16细胞(使用50μg/ml抗体),b)B16细胞(使用5μg/ml抗体),和c)作为阴性对照而包括的HT1080细胞的结合的流式细胞计量术分析。图3证明根据a)ELISA和b)流式细胞计量术分析,A5-IgG以浓度依赖性方式结合人ENG。
对于按比例扩大,将抗体构建体克隆于GS双重载体(pEE14.4)中。将DNA质粒转化、扩增并短暂转染至CHOK1SV细胞中以在200ml的体积下进行表达评估。在第二步骤中,在5-10L大规模培养物中短暂表达抗体。使用一步蛋白A色谱法纯化澄清的培养物上清液。使用浓度是1mg/ml的纯化物质,以及内部人抗体作为对照样品,通过SE-HPLC、SDS-PAGE和LAL进行产物质量分析。
纯化蛋白质样品经0.2μm过滤器过滤,并且通过SE-HPLC色谱图加以分析。mE12-IgG被纯化至>98.8%。A5-IgG被纯化至>90%。内毒素水平是<0.5EU/mg。
所有纯化蛋白质都通过SDS-PAGE在还原性和非还原性条件下分析(数据未显示)。
通过SDS-PAGE和尺寸排阻色谱法来表征纯化蛋白质A5-IgG和mE12-IgG。通过使用重组小鼠/人ENG以及用HRP缀合的抗人IgG抗体检测结合的抗体进行ELISA来分析生物活性。通过使用ENG阳性HT1080和ENG表达小鼠eEnd2进行流式细胞计量术来分析细胞结合。使用zetasizer nano,通过动态光散射来测定熔点。使用Attana A100,通过QCM来测定亲和力。通过间接免疫荧光共焦显微术来对可透化的细胞进行内化研究,用FITC标记的二级抗体检测结合和内化的抗体。
以实验室规模与更大规模均成功生产全长IgG1纯化的抗体以用于产生免疫缀合物。抗体性质的概述显示于表2中。抗体保留它们的特异性,如通过ELISA和流式细胞计量术实验所示。如通过QCM测定的亲和力与亲本抗体的亲和力相当。QCM测量结果指示亲合力效应有助于高亲和力结合。在不同IgG之间存在热稳定性差异(64-72℃)。
初步内化研究指示针对表达人ENG的细胞的A5-IgG的快速细胞内化。实际上在30分钟内,几乎90%的A5-IgG存在于HT1080细胞内。
表2:抗体性质的概述
图9显示抗huENG A5IgG1的内化。
实施例3–黑杆菌素-b A链
为避免可被释放在血流中的游离毒素的副作用以及为降低RIP毒素的潜在免疫原性(如以篦麻毒素广泛描述),克隆并在细菌中表达黑杆菌素b的酶结构域即A链。本发明者假设如果在细菌中产生的A链能够保留它的活性,那么它将不能够进入细胞,除非缀合于诸如抗体的载体分子。
产生
在考虑细菌表达的密码子优化的情况下合成黑杆菌素-b A链,并且将合成的基因克隆于两种不同载体黑杆菌素_pET30b-3和黑杆菌素_pET33b-1(+/-His标签)中以在两种不同大肠杆菌菌株,即大肠杆菌BLR(DE3)和大肠杆菌HMS174(DE3)中表达。不同培养基用于检查不同表达条件。使用Capto Q色谱法和高效SP琼脂糖建立纯化方法。在5mg/ml下,在1XPBS(pH7.4)、0.5mM DTT、10%甘油中配制纯化的重组黑杆菌素-b A链(recNgA)。内毒素水平是<1EU/mg黑杆菌素,并且呈单体形式的纯度>99%。
Eldman N末端测序揭示recNgA的N末端对应于预期序列。
重组黑杆菌素-b A链氨基酸序列:
MIDYPSVSFNLDGAKSATYRDFLSNLRKTVATGTYEVNGLPVLRRESEVQVKSRFVLVPLTNYNGNTVTLAVDVTNLYVVAFSGNANSYFFKDATEVQKSNLFVGTKQNTLSFTGNYDNLETAANTRRESIELGPSPLDGAITSLYHGDSVARSLLVVIQMVSEAARFRYIEQEVRRSLQQATSFTPNALMLSMENNWSSMSLEIQQAGNNVSPFFGTVQLLNYDHTHRLVDNFEELYKITGIAILLFRCSSPSND(SEQ ID NO:25)
重组黑杆菌素-b A链具有以下特征:
氨基酸的数目:256
分子量:28546.0
理论pI:5.45
编码重组黑杆菌素-b A链的核苷酸序列如下:
材料
-黑杆菌素_pET30b-3遗传构建体。
-大肠杆菌(Migula)Castellani和Palmers BLR(DE3)
-培养基:自诱导培养基(AIM)
-提取培养物缓冲液:甘氨酸/NaOH 10mM、亮抑酶肽(Leupeptine)1μg/ml、胃酶抑素(Pepstatine)1μg/ml,pH 9.5。
-提取上清液缓冲液:Tris-HCl 50mM、NaCl 200mM、MgCl2 2mM、亮抑酶肽1μg ml-1、胃酶抑素1μg ml-1、溶菌酶0.1mg ml-1,pH8.0。
-透析溶液:柠檬酸/NaOH 25mM,pH 5.0。-Capto Q FPLC:平衡缓冲液A:甘氨酸/NaOH 50mM pH 9.5。洗脱缓冲液B:甘氨酸/NaOH 50mM(pH 9.5)、NaCl 1M。
-从Capto Q步骤汇集的级分(+80ml提取液)。
-SP琼脂糖HP FPLC:平衡缓冲液A:柠檬酸25mM,pH 4.0。洗脱缓冲液B:柠檬酸25mM(pH 4.0)、NaCl 1M。
方法
在1L形式的具有30μg ml-1卡那霉素的自诱导培养基(AIM)中培养持有表达黑杆菌素_pET30b-3的大肠杆菌BLR(DE3)。在生长约3-4小时之后,通过乳糖活化和葡萄糖消耗来触发蛋白质表达。接着,使温度降低至20℃,持续过夜。
对于提取,初始将各细胞沉淀再混悬于80ml提取缓冲液/培养物L中,并且在8℃下在振荡下孵育30分钟之后进行3个在1100-110巴下崩解7分钟的循环。接着提取物经受在15,900g、8℃下离心60分钟。上清液是纯化的原料。
Capto Q FPLC:将从8l培养物提取的160ml产物上样至160ml Capto Q中,并且使用4CV平衡缓冲液平衡并用15CV平衡缓冲液洗涤。分三步进行洗脱:在1.5mS/cm下15CV(7.6%B);在23.8mS/cm下20CV(18.9%B);20CV 100%B。
在以下条件下进行透析:于25mM柠檬酸/NaOH(pH 5.0)中的4x5L浴内透析650ml产物,截断值6-8000Da。透析因子约3500,<24小时。在透析之后,在20,500g和8℃下离心30分钟使得可溶性级分与不溶性级分分离。在透析前和透析后均对总级分和可溶性级分进行SDS-PAGE(10μl上样于SDS-PAGE上)。将洗脱液透析至PBS(pH7.4)中,并且使用2x20cm2EKV过滤器进行过滤。
SP琼脂糖HP:将610ml在25mM柠檬酸(pH 5.0)中透析的Capto Q汇集物上样至采用4CV平衡缓冲液的240ml高效SP琼脂糖中,并且用15CV平衡缓冲液洗涤并以25Cv梯度至20%B;4CV 100%B步骤洗脱。
在PBS(pH 7.4)、0.5mM DTT中透析从SP琼脂糖HP步骤汇集的级分(5x4L浴,汇集级分是950mL,在0.97mg/ml下)。截断值是6-8000Da,透析因子是约3130,时间>24小时。之后,在20,55g和8℃下离心30分钟使得可溶性级分与不溶性级分分离。之后添加10%甘油。
尺寸排阻色谱法和质谱测定法分析证明所得重组黑杆菌素-b A链(recNgA)的单体和纯化状态(图4)。
进行稳定性研究以评估pH和温度对黑杆菌素-b A链蛋白自身和它的活性的影响。recNgA在5至9的范围内的pH下,以及在存在或不存在甘油(10至45%)下是稳定的(数据未显示)。
活性
测试重组黑杆菌素-b A链在兔网织红细胞无细胞裂解物中的核糖体失活蛋白(RIP)活性:所得IC50值类似于天然黑杆菌素-b,并且在2.5至25pM范围内(参见图5)。因此,来自黑杆菌素-b,在细菌中以重组蛋白形式表达的A链维持它的酶活性,从而支持糖基化不为黑杆菌素-b A链的RIP活性所需。
如果在(-80℃)下冷冻储存以及低于3个冷冻-融化循环,那么RecNgA在兔网织红细胞无细胞裂解物中保留它的活性(未显示)。
通过基于结晶紫的存活力测定来测试recNgA对细胞培养物的细胞毒性活性。缺乏用以在细胞内易位的B链的recNgA呈现的毒性活性比天然黑杆菌素-b小100至1000倍,如图6中所示。天然黑杆菌素b显示IC50≈2x10-8M(类似于先前公布的数据,参见37),而recNgA显示IC50≈2x10-6M。
先前公布的研究显示在RRL测定中,天然黑杆菌素b呈现的RIP活性高于篦麻毒素,而它在细胞中或在体内的毒性小得多(约30-10,000倍)(参见表3中的IC50和LD50值)。
在移除B链后,篦麻毒素A链在RRL测定与细胞毒性测定两者中均丧失活性。出乎意料的是,本发明中首次产生的黑杆菌素b A链仅在细胞细胞毒性测定中丧失活性,而它相对于天然黑杆菌素b在RRL测定中甚至增加。这些数据表明在篦麻毒素的情况下,移除B链不仅影响A链的结合和易位,而且也影响它的RIP活性,而对于黑杆菌素b A链来说,情况不是这样,所述黑杆菌素b A链相对于它的天然对应物保留乃至增加它的RRL活性。因此,在RRL中,黑杆菌素b A链的活性比篦麻毒素A链大50倍。
因此,在缀合后,黑杆菌素b A链缀合物比篦麻毒素A链缀合物(nM范围)呈现更高细胞毒性活性(IC50在pM范围内)(未显示)。
表3:篦麻毒素和黑杆菌素b(天然和A链)的体外和体内活性数据。
(本发明者的自有数据–黑杆菌素b A链;也参见Ferreras J.M.等,Toxins,3:420,2011;Svinth M.等,BBRC,249:637,1998)
实施例4–使黑杆菌素-b A链缀合于抗ENG抗体
对于含有RIP以展现最大细胞毒性的免疫缀合物,RIP必须以完全活性形式从靶向性载体释放,此需要避免空间位阻(38)。二硫键是符合这个准则的唯一连接类型(39,40)。这个键允许使用用于引入游离巯基基团的试剂进行缀合,所述试剂诸如3-(2-吡啶基-二硫代丙酸)N-琥珀酰亚胺酯(SPDP)和4-琥珀酰亚胺基氧基羰基-α-甲基-α-(2-吡啶基-二硫代)甲苯(SMPT)。由通过位阻化二硫化物接头共价结合于毒素的mAb组成的免疫毒素,常被标注为第二代免疫毒素的免疫毒素,是稳定的,长寿的,并且显示对靶细胞的有效细胞毒性(41)。
SPDP已用于制备含有黑杆菌素b的免疫毒素(IT)(36,42)。此外,SMPT保护二硫键免遭由硫醇盐阴离子攻击,从而改善连接的体内稳定性(43,44)。
材料
-重组黑杆菌素b A链于PBS(pH 7.4)、10%甘油、0,5mM DTT、4.92gl-1中,储存在5℃下。
-5,5'-二硫代-双-(2-硝基苯甲酸)
-GE PD MiniTrap G-10脱盐柱。
-0.2μm 28mm无菌Minisart过滤器。
-Sciclone ALH 3000工作站。
-Sarstedt 96孔平底微型测试板,参考号82.1581。
方法
二硫苏糖醇(DTT,克莱兰氏试剂(Cleland′s reagent))是一种将用于使蛋白质样品中存在的硫醇基团游离的氧化还原剂。一旦所述基团已被游离并且因此可用于反应,即将添加5,5'-二硫代-双(2-硝基苯甲酸)(埃尔曼试剂(Ellman reagent))。埃尔曼试剂二硫桥将被裂解,并且2个所得硫基-硝基苯甲酸盐分子(TNB)将在硫醇基团位点处连接于蛋白质。为滴定TNB,将在λ=412nm(即DTT不被吸收,从而赋予硫醇基团浓度所处的波长)下获取吸光度值。这些值与由蛋白质在λ=280下的吸光度获取的蛋白质浓度的比例将产生每个蛋白质分子的游离硫醇基团的数目。
如下进行直接硫醇滴定:
将204μl recNg b A溶解于796μl 20mM磷酸盐、250mM NaCl、1mM EDTA(pH 7.0)(测定缓冲液)中(1.0033gl-1=最终浓度)。将埃尔曼试剂在3gl-1下溶解于0.2M磷酸盐中。对于两种缓冲液,均以1.61比1质量比例添加磷酸二氢钠和磷酸氢二钠。在室温下调整PH,并且过滤缓冲液。制备100ml埃尔曼缓冲液和500ml测定缓冲液。将埃尔曼试剂完全再混悬而非称重。
在室温下在4.8mM DTT存在下孵育recNgA样品30分钟。接着在柱中纯化recNgbA样品,并且等分前10ml洗脱液(V=0.5ml)。获取等分试样的A280,并且混合两个最浓等分试样。再次获取A280。添加10μl 3gl-1DTNB,并且在2分钟之后测量A412(n=1),使用在测定缓冲液中以相同浓度稀释的埃尔曼试剂作为空白(nb=3)。读数属于0.1-3AU线性范围。在涡旋之后,将蛋白质溶液吸移恰好处于弯月面下方。每孔吸移100μl。
这个研究的结果显示属于recNgA的单一半胱氨酸残基的硫醇基团是游离的,并且可用于反应,而非由它的三级结构阻断。这将允许recNgbA使用需要位阻化链间二硫键的接头加以缀合。
明确确定的是含有核糖体失活蛋白的免疫缀合物仅在毒素分子以完全活性形式从靶向性载体释放时展现最大细胞毒性。RIP分子从载体分离为避免空间位阻以及允许毒素有效易位至细胞质中所需(38)。目前,二硫键是似乎符合这些准则的唯一连接类型(40)。
两个不同蛋白质大分子的导致异二聚体形成的偶联要求各蛋白质在混合它们以进行反应之前被修饰。在2型RIP的A链的情况下,修饰限于还原性裂解连接分子的活性(A)链和结合(B)链的天然半胱氨酸残基。
对于IgG分子,这是不可能的,因为半胱氨酸残基参与维持蛋白质的三级和/或四级结构,以致不可能使它们还原而不损失特定蛋白质功能。此外,推测一些半胱氨酸残基并非在空间上可及,如由每个免疫球蛋白必须产生10个硫醇基团以达成最优缀合于活化的RIP所证明(45)。
出于这些原因,在大多数IgG分子中,使用异双官能试剂化学插入硫醇基团,并且已开发若干方法来产生异缀合物,从而避免形成同聚物或使同聚物的形成降至最少。在大多数情况下,用于引入硫醇基团的试剂与氨基基团反应,从而形成酰胺键或脒键。氨基基团是反应性的,充足的,并且以有限方式对于大多数蛋白质来说是可消耗的。也就是说,有限数目的氨基基团可被修饰而不减弱蛋白质的生物活性(40)。
最通常使用的引入游离巯基基团的试剂是3-(2-吡啶基-二硫代丙酸)N-琥珀酰亚胺酯(SPDP)和4-琥珀酰亚胺基氧基羰基-α-甲基-α-(2-吡啶基-二硫代)甲苯(SMPT),其通过与氨基基团反应以形成中性酰胺来将2-吡啶基二硫化物基团引入蛋白质中;以及4-巯基丁酰亚胺酸甲酯(2-亚氨基硫杂环戊烷,特劳特氏试剂(Traut’s reagent)),其引入巯基丁酰亚胺基基团,反应以形成带电荷的脒,由此保持衍生化氨基酸的正电荷(40;44)。
SPDP和SMPT引入位阻化二硫键,而2-亚氨基硫杂环戊烷–SH必须通过使它与5,5’-二硫代双-2-硝基苯甲酸(埃尔曼氏试剂)反应加以保护。
与埃尔曼氏试剂的反应也用于快速测量蛋白质巯基基团(45,46)。
SMPT具有连接于与二硫键邻近的碳原子的甲基基团和苯环,其保护二硫键免遭由硫醇盐阴离子攻击,由此改进连接的体内稳定性(43,44)。
基于这些数据,IgG蛋白可用SMPT修饰,此不显著影响分子在以下条件下的抗原结合性质,即使它们改变蛋白质在反应位点中的电荷。
在一个研究中,本发明者研究遵循缀合方案(参见40),在使用SMPT:mAb摩尔比6进行衍生化之后,使用2种不同recNgA:mAb摩尔比2.5和3.5使IgG1与recNgA缀合。通过尺寸排阻色谱法在Sephacryl S200上进行纯化(参见41)。
在所述条件下,免疫毒素主要是连接于一个或两个毒素分子的抗体的混合物,其中存在高分子量组分(连接于若干RIP蛋白的IgG)以及游离和聚合物RIP(在recNgA的情况下是二聚体)和游离抗体。因此,仔细纯化被认为获得纯产物是所需的。
体外活性测试
通过评估在兔网织红细胞无细胞裂解物(RRL)测定中的RIP活性来对如上所述制备的缀合物进行活性测试。结果呈现于图7中。
对天然黑杆菌素-b或recNgA获得的IC50值在2.5pM范围内,并且缀合物的那些IC50值是类似的,并且在1-0.5pM范围内,甚至高于天然黑杆菌素-b阳性对照,从而显示抗体缀合不影响recNgA的酶促活性。
实施例5–溶细胞素与抗ENG抗体的缀合
用于缀合研究的溶细胞素选自以上显示的一般结构(式IV)。这些结构展现针对不同癌细胞系的活性(nM至pM范围)。
各种接头系统可被使用并连接于分子的R2位或R17位。
包括vcPABA接头和抗ENG抗体的溶细胞素缀合物的一般性概述显示于图8中(在图8中描绘的结构中,溶细胞素与vcPABA接头的连接位点在R1位或R4位处–图11中使用的R1和R4编号系统不同于例如在权利要求中使用的R基团编号系统;图11的R1意在对应于权利要求中的R2,并且图11的R4对应于权利要求的R17)。
vcPABA(缬氨酸-瓜氨酸-PABC)蛋白酶可裂解接头先前已用于ADC分子布妥昔单抗维多汀(Brentuximab Vedotine)中,所述ADC分子由Seattle Genetics和Takeda开发,并且近来由FDA和EMEA以批准(分别是2011年,和2012年11月)。在这个ADC中,vcPABA已在它的游离NH2处偶联于马来酰亚胺己酰基以用于基于硫醇而缀合在mAb(cAC10抗CD30抗体)上。在另一侧,vcPABA已通过它的COOH缀合于来自Seattle Genetics的奥莉斯汀细胞毒性药物(MMAE)。(参见48)
本发明者已使用这个接头(马来酰亚胺己酰基-vcPABA)通过基于硫醇的与马来酰亚胺己酰基的反应来缀合抗ENG抗体,并且在另一端,使溶细胞素细胞毒性分子通过它的环状哌啶与vcPABA缀合(溶细胞素的R1位或R4位显示于图8中)。
合成马来酰亚胺基-val-cit-PABOCO-微管溶素/溶细胞素-TAM461:
将TAM461和TAM465在干燥条件下溶解于无水DMF中,并且用HOBt和DIPEA处理所得溶液。在室温下搅拌反应物18小时。浓缩反应混合物,并且所得油状物通过使用2-6%甲醇:DCM进行柱色谱法加以纯化以产生35mg(64%)呈白色固体状的TAM467。ESI-MS:m/z=1371[M+H]。
合成马来酰亚胺基-val-cit-PABOCO-微管溶素/溶细胞素-TAM470:
将TAM470和TAM466在干燥条件下溶解于无水DMF中,并且用HOBt和DIPEA处理所得溶液。在室温下搅拌反应物18小时,接着用TLC分析,指示反应完成。浓缩反应混合物,并且所得油状物用使用4-12%甲醇:DCM的柱色谱法纯化以产生56mg TAM471(产率:62%)。ESI-MS:1384.6[M+1]。
进行体外活性测试。通过微管抑制测定评估功能活性,而通过结晶紫存活力测定来测定细胞毒性活性。
产生溶细胞素-接头衍生物
根据图11中呈现的一般结构合成不同溶细胞素-接头衍生物,其中vcPABA接头单独或与乙二醇间隔基(EG;n=1至3)一起添加在R1位(TAM467、TAM551)或R4位(TAM471、TAM553、TAM558)中或被乙二醇基团(n=3)取代(TAM552)。相应化学结构呈现于表4中。
表4:溶细胞素-接头衍生物的化学结构
通过微管蛋白聚合抑制测定(TPI;微管蛋白聚合测定试剂盒;Cytoskeleton,目录号BK011P)以及在HT1080细胞上的细胞增殖阻滞(CPA;结晶紫)来评估各新型衍生物的微管抑制活性和细胞毒性活性。计算IC50,并且结果呈现于表5中。
表5:溶细胞素-接头衍生物的微管抑制活性和细胞细胞毒性活性。(ND:未测出)
亲本溶细胞素TAM334的体外活性与当前用于产生抗体-药物缀合物的其它有效载荷,诸如奥莉斯汀(MMAE)或美登木素(DM1-DM4)在相同范围内。如对于来自微管溶素A家族的其它化合物所预期以及先前所述,在添加接头后,相对于亲本化合物TAM334,溶细胞素的细胞细胞毒性活性降低。此外,在两种测定中,TAM467衍生物均呈现显著最低活性。所有衍生物都在缀合中用于产生ADC分子,并且在体外和在体内以比较方式进行评估以选择最具活性的溶细胞素-接头衍生物。
ADC的缀合和化学表征
遵循非位点特异性缀合方法,使各新产生的衍生物在半胱氨酸残基上缀合于单克隆IgG1人抗体。为了这个目的,使一批抗体还原并与各衍生物反应。测试不同TCEP比率以达到3-4的最优DAR,小于10%的游离抗体和药物。最优缀合条件如下:TCEP=2.5和3.57硫醇水平埃尔曼氏试剂。接着在G25Sephadex上纯化缀合物,并且通过尺寸排阻色谱法(SEC)分析以测定它们的纯度,以及通过疏水性相互作用色谱法(HIC)和聚合液体反相色谱法(PLRP)分析以测定DAR、游离抗体的含量和不同ADC物质的分布概况(0-8药物/mAb)。通过紫外检测方法在280nm下评估游离药物的含量。确定化学分析结果(未显示),并且ADC的生物化学特征显示于表6中。
表6:不同ADC分子的化学特征的概述
各种药物产生不同聚集水平。具体来说,ADC HPS157-039-002(TAM551)已在DAR=3.08下显示最高聚集水平,留有22.4%未缀合抗体。与TAM467的初步缀合也显示高聚集水平:在DAR 3.27下,SEC纯度已仅为67%,有16%游离药物(数据未显示)。这些数据表明对于这个类型的溶细胞素分子,vcPABA接头在R1位中不是最优的。
体外评估溶细胞素缀合物通过增殖阻滞测定(结晶紫染色)来在体外比较评估溶细胞素ADC分子。结果呈现于图10中,并且IC50值呈现于表7中。
表7:在增殖阻滞测定中获得的IC50值(nM)
化合物 | HT1080-WT | HT1080-AG(+) |
TAM334 | 1.04 | 0.77 |
ADC-471(HPS-157-039-001) | 5.6 | 10.33 |
ADC-551(HPS-157-039-002) | 964 | 552 |
ADC-553(HPS-157-039-004) | 90 | 108 |
ADC-558(HPS-157-039-005) | 555 | 0.96 |
相对于R4位(ADC-471),单独vcPABA接头定位于R1位中(ADC-551)产生具有小得多的体外细胞毒性活性的缀合物(图10;表7)。此外,向R4位中的vcPABA接头中增加作为间隔基的乙二醇基团的数目(ADC-471(n=0)相对于ADC-553(n=1)和ADC-558(n=3))显示使体外抗原特异性细胞毒性活性增加(图10)。实际上,尽管ADC-471和ADC-553显示较低和不显示抗原特异性细胞毒性活性(分别是10nM和100nM IC50范围),其中在野生型(WT)HT1080细胞与抗原(AG)表达HT1080细胞之间无差异,但ADC-558呈现1nM范围特异性细胞毒性活性,其中在AG HT1080细胞与WT HT1080细胞之间的特异性比率是500。
实施例6–评估缀合物的体内抗肿瘤作用
在先前选择用于抗原表达的胰腺癌的患者源性异种移植物小鼠模型(PAXF-736)中评估两种类型的免疫缀合物(recNgA缀合物和溶细胞素缀合物)的体内抗肿瘤作用。
进行剂量范围研究以限定待用于功效研究中的最大耐受剂量(未显示)。对于recNgA免疫缀合物,发现最高耐受剂量是0.5mg/kg,而在2.5mg/kg直至20mg/kg的剂量下,独立于所用衍生物施用溶细胞素缀合物,而无任何重量减轻或毒性作用。
接着一周一次历经5周腹膜内施用免疫缀合物。每2-3天测量肿瘤体积和体重。媒介物治疗的和吉西他滨治疗(150mg/kg)的PDX小鼠分别用作阴性和阳性对照组。结果显示于图11中。
在胰腺癌的PDX鼠模型中,recNgA免疫缀合物(OMTX505)在0.5mg/kg的剂量下呈现高体内抗肿瘤功效(60%)(图11A)。当与吉西他滨组合时,它甚至显示100%肿瘤生长抑制和肿瘤消退。
根据体外结果(参见图10和表7),单独vcPABA接头定位于R1位中(OMTX705-551)产生不具有体内抗肿瘤活性的缀合物(图11B)。
支持体外数据的是,向R4位中的vcPABA接头中增加作为间隔基的乙二醇基团的数目(OMTX705-471(n=0)相对于OMTX705-553(n=1)和OMTX705-558(n=3))显示使体内抗肿瘤作用增加(图11C)。OMTX705-471和OMTX705-553不显示任何体内抗肿瘤作用,而OMTX705-558在胰腺癌的PDX小鼠模型中在2.5mg/kg剂量下呈现40%肿瘤生长抑制作用。
根据这些数据,recNgA和TAM558分子被选作用于抗ENG缀合物的最佳有效载荷。
实施例7–尤文肉瘤模型
肿瘤细胞可塑性使得某些类型的高度恶性肿瘤细胞能够去分化,并且衔接塑性多能胚胎样表型,其使得它们能够在肿瘤进展期间‘改适’并逃脱常规治疗策略。新近研究证明ENG表达与尤文肉瘤中的肿瘤细胞可塑性相关联,并且这与尤文肉瘤患者的存活期恶化显著相关。ENG水平降低的尤文肉瘤显示体内肿瘤生长降低。因此,这个研究描绘ENG在侵袭性肿瘤的肿瘤细胞可塑性和进展方面的重要作用(51)。
本发明者假设抗ENG单克隆抗体、IT和ADC在治疗尤文肉瘤方面具有治疗潜力。
已开发用于体外研究的14个尤文肉瘤细胞系模型,以及它们的用于筛选和表征用以治疗尤文肉瘤的治疗性分子的相应异种移植物模型。已确认ENG在所有14个细胞系以及已被检察的所有人尤文肉瘤患者样品(n=10)中表达。
本文引用的所有参考文献都以引用的方式整体以及出于所有目的并入本文,所述引用的程度就如同已特定地和个别地指示将各单独出版物或专利或专利申请以引用的方式整体并入一般。
本文所述的特定实施方案是通过举例方式,而非通过限制方式提供。本文任何副标题皆仅为了方便起见而包括,并且不应解释为以任何方式限制本公开。
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1.一种人单克隆抗体,其选择性结合人内皮糖蛋白,并且其包含含SEQ ID NO:3的氨基酸序列的重链和含SEQ ID NO:4的氨基酸序列的轻链。
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- 2014-02-06 GB GB201402009A patent/GB201402009D0/en not_active Ceased
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2015
- 2015-02-04 EP EP15705779.5A patent/EP3102606B1/en active Active
- 2015-02-04 WO PCT/EP2015/052342 patent/WO2015118031A2/en active Application Filing
- 2015-02-04 HU HUE15705779A patent/HUE040517T2/hu unknown
- 2015-02-04 US US15/116,419 patent/US10004812B2/en active Active
- 2015-02-04 ES ES15705779T patent/ES2701188T3/es active Active
- 2015-02-04 DK DK15705779.5T patent/DK3102606T3/en active
- 2015-02-04 JP JP2016550786A patent/JP6616776B2/ja active Active
- 2015-02-04 KR KR1020167021450A patent/KR102342934B1/ko active IP Right Grant
- 2015-02-04 CN CN201580007513.8A patent/CN105980411B/zh active Active
- 2015-02-04 CA CA2937600A patent/CA2937600C/en active Active
- 2015-02-04 PL PL15705779T patent/PL3102606T3/pl unknown
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WO2008138561A1 (en) * | 2007-05-10 | 2008-11-20 | R & D Biopharmaceuticals Gmbh | Tubulysine derivatives |
WO2012135517A2 (en) * | 2011-03-29 | 2012-10-04 | Immunogen, Inc. | Preparation of maytansinoid antibody conjugates by a one-step process |
WO2012149412A2 (en) * | 2011-04-29 | 2012-11-01 | Immunogen Inc. | Anti-endoglin (cd105) antibodies, immunoconjugates, and uses thereof |
Non-Patent Citations (2)
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In vitro and in vivo effects of an anti-mouse endoglin (CD105)–immunotoxin on the early stages of mouse B16MEL4A5 melanoma tumours;Raquel Mun˜oz;《Cancer Immunol Immunother》;20130301;第62卷(第3期);第541-551页 * |
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Also Published As
Publication number | Publication date |
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KR20160113623A (ko) | 2016-09-30 |
EP3102606A2 (en) | 2016-12-14 |
HUE040517T2 (hu) | 2019-03-28 |
PL3102606T3 (pl) | 2019-05-31 |
DK3102606T3 (en) | 2018-12-17 |
GB201402009D0 (en) | 2014-03-26 |
WO2015118031A3 (en) | 2015-10-01 |
JP6616776B2 (ja) | 2019-12-04 |
US10004812B2 (en) | 2018-06-26 |
CN105980411A (zh) | 2016-09-28 |
ES2701188T3 (es) | 2019-02-21 |
US20170007714A1 (en) | 2017-01-12 |
EP3102606B1 (en) | 2018-09-12 |
JP2017506234A (ja) | 2017-03-02 |
KR102342934B1 (ko) | 2021-12-24 |
CA2937600A1 (en) | 2015-08-13 |
CA2937600C (en) | 2023-01-03 |
WO2015118031A2 (en) | 2015-08-13 |
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