CN111201240A - 特异性地结合muc1的抗体及其用途 - Google Patents
特异性地结合muc1的抗体及其用途 Download PDFInfo
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- CN111201240A CN111201240A CN201880026127.7A CN201880026127A CN111201240A CN 111201240 A CN111201240 A CN 111201240A CN 201880026127 A CN201880026127 A CN 201880026127A CN 111201240 A CN111201240 A CN 111201240A
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- antibody
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- cancer
- antigen
- muc1
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Abstract
本发明涉及特异性地结合粘蛋白1(MUC1)的抗MUC1抗体及其用途,并且更具体地,涉及抗MUC1抗体或其抗原结合片段,包含所述抗体或其抗原结合片段的抗体‑药物缀合物或双特异性抗体,一种包含所述缀合物或双特异性抗体的用于预防或治疗癌症的药物组合物,以及编码所述抗体或其抗原结合片段的核酸,均携带所述核酸的载体和宿主细胞,以及使用所述载体和宿主细胞制备抗MUC1抗体或其抗原结合片段的方法。根据本发明,特异性地结合MUC1的抗体或其抗原结合片段示出对MUC1的显著亲和力和结合力,并且其中抗体或其抗原结合片段与药物缀合的抗体‑药物缀合物可以特异性地结合表达MUC1的细胞,以特异性或选择性地高效转运所述药物。因此,根据本发明的抗MUC1抗体和抗体‑药物缀合物可以有效地应用于MUC1相关疾病例如癌症的治疗。
Description
技术领域
本发明涉及特异性地结合粘蛋白1(MUC1)的抗MUC1抗体及其用途,并且更具体地涉及抗MUC1抗体或其抗原结合片段,包含所述抗体或其抗原结合片段的抗体-药物缀合物或双特异性抗体,一种包含所述抗体或其抗原结合片段的用于预防或治疗癌症的药物组合物,编码所述抗体或其抗原结合片段的核酸,包含所述核酸的载体和宿主细胞,以及使用所述核酸制备抗MUC1抗体或其抗原结合片段的方法。
背景技术
粘蛋白1(MUC1)是跨膜糖蛋白,包括许多糖基化的细胞外结构域。MUC1在细胞表面上具有200nm至500nm的长度,并且位于正常上皮细胞的顶膜中。MUC1在许多器官的线性或腔上皮细胞中表达,所述器官例如乳房、胃、食道、胰腺、尿道、肺、肾和胆囊。在正常组织中,MUC1的带负电荷的碳水化合物形成物理屏障,用于保护基底上皮免于脱水、pH变化、花粉和微生物。
MUC1基因编码单个转录物。翻译后,MUC1在位于SEA结构域(海胆精子蛋白肠激酶和聚集蛋白结构域)的GSVVV基序中被自动切割。它们由两个肽片段组成,即N末端亚基(MUC1-N)和C末端亚基(MUC1-C)。
MUC1-N末端亚基具有可变数目的串联重复,并且由PTS(富含脯氨酸/苏氨酸/丝氨酸)结构域和SEA结构域组成。MUC1-C末端亚基由58个氨基酸的胞外结构域(ECD)、28个氨基酸的跨膜结构域(TMD)和72个氨基酸的胞质结构域(CD)组成。MUC1在细胞外结构域中引起广泛的O-连接的糖基化。取决于N-糖基化模式的程度,MUC1-C的大小为23kDa至25kDa,并且当N-糖基化缺陷时为17kDa。一旦首次产生MUC1,与细胞结合的部分和待释放至细胞外部的部分通过非共价相互作用结合。在这种情况下,切割是由称为“脱落酶”的酶引起的。通过刺激细胞因子如IFN-γ和TNF-α分离MUC1复合物。MUC1-N由酶释放,所述酶包括TNF-α转化酶(TACE)和基质金属蛋白酶(MMP)。这些酶将MUC1-C的ECD切割成两个片段。随着癌症的进展,细胞外片段从癌细胞分离并漂浮在身体的血液中,而细胞结合的片段连续地保持与癌细胞结合。MUC1对于癌细胞的生长是重要的,因为它通过结合与癌细胞增殖相关且存在于其他癌细胞中的细胞膜蛋白而发送连续细胞增殖信号,以在癌细胞增殖中起关键作用。此外,此片段与癌细胞具有相同的命运,直到癌细胞长好和消除,因此此片段充当癌症检测的良好靶标并且充当关于癌症消除的关键生物标记物。此外,与MUC1的其他部分不同,已知此片段是唯一未被糖基化的部分,并且被认为是区分癌细胞的MUC1与正常细胞的MUC1的独特部分。因此,诸位发明人基于以下观点开发了本发明的抗体:此片段与MUC1细胞结合并共享癌症的命运并且可以区分正常细胞的MUC1与癌细胞的MUC1,能够作为针对抗体的最佳抗原。
同时,通过接头将细胞毒性药物与抗体结合,获得抗体-药物缀合物(ADC)。由于单克隆抗体具有靶标特异性,抗体-药物缀合物中的药物可以被递送至表达抗原/靶标的肿瘤,所述抗原/靶标被具有选择性靶向能力的单克隆抗体识别。理想的是抗体-药物缀合物在给药后在血液中呈前药形式,应该是无毒的,并且当抗体结合靶肿瘤抗原并且然后内化到癌细胞中时,药物以活性形式释放并杀死癌细胞。
制备抗体-药物缀合物的最重要的关键点是确定抗体结合的靶标/抗原。具体地,抗体结合的靶标/抗原被认为主要在肿瘤细胞中表达并且理想地由癌细胞特异性地表达(过表达)的细胞表面蛋白。
在这些技术背景下,诸位发明人开发了一种特异性地结合癌细胞-MUC1的抗体,其识别与正常细胞中表达的MUC1不同的部分;并且诸位发明人发现结合“与细胞结合的部分”(该部分与癌细胞结合)并且因此基于ADC的特征共享癌细胞的命运以最大化ADC功效的抗MUC1-抗体以及包含所述抗体的抗体-药物缀合物能够特异性地结合表达MUC1的细胞,并且因此治疗由MUC1表达引起的疾病。基于此发现,完成本发明。
提供在此背景技术部分中公开的上述信息仅仅是为了更好地理解本发明的背景,并且因此它可不包含形成本发明所属领域的技术人员已知的现有技术的信息。
公开文本
技术问题
本发明的目的是提供抗体或其抗原结合片段,其特异性地结合MUC1的“与细胞结合的部分”;抗体-药物缀合物,其中药物与所述抗体或其抗原结合片段缀合;和包含所述抗体或其抗原结合片段的双特异性抗体。
本发明的另一个目的是提供用于产生抗MUC1抗体的杂交瘤(KCLRFBP00395)。
本发明的另一个目的是提供包含抗MUC1抗体或其抗原结合片段、抗体-药物缀合物或双特异性抗体的用于预防或治疗癌症的组合物,以及使用所述组合物治疗癌症的方法。
本发明的另一个目的是提供包含抗MUC1抗体或其抗原结合片段的用于诊断癌症的组合物,以及使用所述组合物诊断癌症的方法。
本发明的另一个目的是提供包含复合物的免疫原性组合物,在所述复合物中MUC1-C末端区域、MUC1的SEA结构域或MUC1的C末端细胞外结构域和CpG-DNA包封在脂质体中。
本发明的另一个目的是提供一种产生抗MUC1单克隆抗体的方法,所述方法包括用免疫原性组合物接种小鼠。
本发明的又另一个目的是提供编码抗MUC1抗体或其抗原结合片段的核酸,包含所述核酸的载体和宿主细胞,以及使用所述载体和宿主细胞产生抗MUC1抗体或其抗原结合片段的方法。
技术方案
为了实现上述目的,本发明提供了抗MUC1抗体或其抗原结合片段,其识别包含在MUC1的C末端细胞外结构域中的至少五个连续氨基酸的多肽。
优选地,抗MUC1抗体或其抗原结合片段包含六个互补决定区(CDR),其中抗体或其抗原结合片段包括至少一个选自以下的序列:SEQ ID NO:1的重链CDR1(GYTFTSYWMH);SEQID NO:2的重链CDR2(YINPGTGYIEYNQKFKD);SEQ ID NO:3的重链CDR3(STAPFDY);SEQ IDNO:4的轻链CDR1(KASQDIKSYLS);SEQ ID NO:5的轻链CDR2(YATRLAD);和SEQ ID NO:6的轻链CDR3(LQYDESPYT)。
本发明还提供了用于产生抗MUC1抗体的杂交瘤(KCLRFBP 00395)。
本发明还提供了抗体-药物缀合物或双特异性抗体,其包含抗MUC1抗体或其抗原结合片段。
本发明还提供了包含抗MUC1抗体或其抗原结合片段、抗体-药物缀合物或双特异性抗体的用于预防或治疗癌症的药物组合物,以及使用所述药物组合物治疗癌症的方法。
本发明还提供了抗MUC1抗体或其抗原结合片段用于预防或治疗癌症的用途。
本发明还提供了抗MUC1抗体或其抗原结合片段用于制备癌症预防剂或治疗剂的用途。
本发明还提供了包含抗MUC1抗体或其抗原结合片段的用于诊断癌症的组合物,以及使用所述组合物诊断癌症的方法。
本发明还提供了免疫原性组合物,其包含包封在脂质体中的(1)MUC1的C末端区域、MUC1的SEA结构域或MUC1的C末端细胞外结构域,和(2)CpG-DNA;以及用于产生抗MUC1单克隆抗体的方法,所述方法包括用免疫原性组合物接种小鼠。
本发明还提供了编码抗MUC1抗体或其抗原结合片段的核酸,包含所述核酸的载体和宿主细胞,以及使用所述载体和宿主细胞产生抗MUC1抗体或其抗原结合片段的方法。
附图说明
图1a是示出用rhMUC1-C蛋白免疫的小鼠中IgG总量的图。
图1b和图1c示出了筛选从免疫小鼠分离的杂交瘤细胞的结果,更特定地,图1b示出使用HAT培养基筛选的结果,并且图1c示出使用HT培养基筛选的结果。
图2a是示出对用杂交瘤细胞(hMUC1-1H7克隆)免疫的多只小鼠进行ELISA的结果的图,其示出rhMUC1-C特异性抗体的存在。
图2b示出通过SDS-PAGE和考马斯染色分析纯化的抗hMUC1单克隆抗体的结果。
图2c是示出通过ELISA确定抗MUC1抗体的同种型的结果的图。
图3a示出使用从乳腺癌细胞裂解物中的hMUC1-1H7克隆纯化的抗hMUC1单克隆抗体和抗MUC1-CT抗体的蛋白质印迹结果。
图3b示出在用从正常小鼠IgG或hMUC1-1H7克隆纯化的抗hMUC1单克隆抗体免疫沉淀MCF-7、MDA-MB-231、T47D和ZR75-1细胞的细胞裂解物后,使用抗MUC1-CT抗体的免疫印迹结果。
图3c示出用PNGase F处理T47D细胞裂解物以及然后使用从hMUC1-1H7克隆纯化的抗hMUC1单克隆抗体(以与使用其他抗体的情况进行比较)进行蛋白质印迹的结果。
图3d示出使用从hMUC1-1H7克隆纯化的抗hMUC1单克隆抗体对用PNGase F处理的T47D细胞裂解物的免疫沉淀、以及然后用抗MUC1-CT抗体或抗MUC1-CT2抗体进行免疫印迹的结果。
图4a示出使用抗MUC1-CT抗体、从hMUC1-1H7克隆纯化的抗hMUC1抗体或抗β-肌动蛋白抗体对胰腺癌细胞裂解物进行蛋白质印迹的结果。
图4b示出用从正常小鼠IgG或hMUC1-1H7克隆纯化的抗hMUC1单克隆抗体对胰腺癌细胞裂解物进行免疫沉淀、以及然后使用抗MUC1-CT抗体和抗hMUC1单克隆抗体进行免疫印迹的结果。
图5A是通过用从hMUC1-1H7克隆纯化的抗hMUC1抗体在4℃处理(表面)乳腺癌细胞、或通过用0.1%Triton X-100裂解细胞并且然后用抗体处理(细胞内)获得的荧光图像。
图5B示出通过用从荧光探测的hMUC1-1H7克隆纯化的抗hMUC1抗体处理乳腺癌细胞、并且然后在37℃下培养所述乳腺癌细胞6小时获得的荧光图像。
图6是通过在4℃从hMUC1-1H7克隆纯化的抗hMUC1抗体处理(表面)胰腺癌细胞、或通过用0.1%Triton X-100裂解细胞并且然后用抗体处理(细胞内)获得的荧光图像。
图7是通过用从荧光探测的hMUC1-1H7克隆纯化的抗hMUC1抗体处理胰腺癌细胞、并且然后在37℃下培养24小时获得的荧光图像。
图8a是示意性地示出重组表达质粒pFabE-hMUC1-1H7的切割图谱。
图8b示意性地示出了由重组表达质粒pFabE-hMUC1-1H7表达的重组蛋白。
图8c是通过用源自hMUC1-1H7克隆的抗hMUC1抗体的Fab片段处理MCF-7、MDA-MB-231、T47D和ZR75-1细胞而获得的荧光图像。
图9是示出当用源自hMUC1-1H7克隆的抗hMUC1抗体处理MDA-MB-231、T47D和ZR75-1细胞时细胞增殖变化的图。
图10a至图10h是通过将源自荧光标记的hMUC1-1H7克隆的抗hMUC1单克隆抗体静脉内给予患有乳腺癌的小鼠而获得的荧光图像。
图11a至图11c是通过将源自荧光标记的hMUC1-1H7克隆的抗hMUC1单克隆抗体静脉内给予患有胰腺癌的小鼠而获得的荧光图像。
图12a示出从异种移植小鼠模型中提取的肿瘤组织。
图12b是示出从接受源自hMUC1-1H7克隆的抗hMUC1单克隆抗体的异种移植小鼠模型中提取的肿瘤的大小((宽度2×长度)/2)的图。
图12c是示出从接受抗hMUC1单克隆抗体的异种移植小鼠模型中提取的肿瘤重量的图。
图12d是示出接受抗hMUC1单克隆抗体的异种移植小鼠模型的体重的图。
图13示出了鉴定MUC1蛋白是否在乳腺癌组织中表达的结果。
图14示出了鉴定MUC1蛋白是否在胰腺癌组织中表达的结果。
图15示出hMUC1-1H7抗体-药物缀合物在乳腺癌细胞系中的细胞毒性。
图16示出hMUC1-1H7抗体-药物缀合物对乳腺癌组织中癌细胞增殖的抑制作用。
图17示出通过ELISA鉴定人源化抗体(hMUC1-G3)和hMUC1-C的结合亲和力的结果。
图18示出了鉴定由hMUC1-G3抗体和hMUC1-1H7抗体识别的hMUC1-C的表位同源性的ELISA结果。
图19示出了鉴定hMUC1-G3抗体特异性地识别细胞中表达的hMUC1的流式细胞术结果。
图20示出流式细胞术的结果,所述流式细胞术鉴定hMUC1-G3抗体和hMUC1-1H7抗体以浓度依赖性方式识别在ZR75-1乳腺癌细胞中表达的hMUC1。
图21示出hMUC1-G3抗体-药物缀合物根据乳腺癌细胞系中MUC1表达细胞系(ZR75-1,T47D)和非MUC1表达细胞系(MDA-MB-231)中MUC1的表达而选择性地杀死乳腺癌细胞。
图22示出了hMUC1-G3抗体-药物缀合物在髓性白血病细胞系中的细胞毒性。
图23示出了在通过移植源自乳腺癌患者的TNBC组织产生的异种移植小鼠模型中hMUC1-G3抗体-药物缀合物选择性地抑制癌细胞的效果。
具体实施方式
除非另外定义,本文所用的全部技术和科学术语具有与本发明所属领域的技术人员所理解的意义相同的意义。通常,本文所用的命名法在本领域中为熟知的并且为通常使用的。
MUC1(粘蛋白1)通常在正常上皮的一侧(顶膜)上表达,并保护基底上皮免于干燥、pH变化、污染和微生物。然而,MUC1在各种人类癌症中异常高表达,参与糖基化程度的降低,在整个细胞表面均匀表达,并参与促进癌细胞的增殖、侵袭、转移和血管生成。因此,MUC1是癌症特异性疗法的靶标。
MUC1包括N末端亚基(MUC1-N)和C末端亚基(MUC1-C),其中MUC1-N和MUC1-C通过SEA结构域(海胆精子蛋白肠激酶和聚集蛋白结构域)内的切割位点中的自切割形成。SEA结构域有助于形成稳定的异二聚体复合物。当发生MUC1切割时,可以释放细胞外MUC1-N(MUC1的N-末端亚基)以分离许多抗MUC1-SEA抗体。迄今已知的大多数抗MUC1-SEA抗体已知靶向MUC1-N重复序列结构域(Prinssen等人1998;Gillespie等人2000)。由于MUC1-N并未直接发现于细胞表面上,而是在外周循环期间观察到,因此存在一个局限性,即仅有限的循环抗MUC1-N抗体可用于MUC1阳性肿瘤细胞。而且,难以选择MUC1-C(MUC1的C末端亚基)的表位。对此,原因在于,在大多数情况下,MUC1-C结构域是跨膜的或位于细胞质中。为了克服此问题,本发明提供了特异性地结合MUC1的新型抗体,所述抗体靶向在MUC1切割后保留在细胞表面上的MUC1-C末端区域(细胞外结构域)。
一方面,本发明涉及特异性地结合MUC1的抗MUC1抗体或其抗原结合片段,更具体地,涉及识别包含在MUC1的C-末端细胞外结构域内的至少五个连续氨基酸的多肽的抗MUC1抗体或其抗原结合片段。
具体地,在本发明中,基于专利PCT/KR 2010/003879中披露的“包含脂质体包封的寡核苷酸和表位的免疫刺激组合物”的技术,使用在人粘蛋白1(CD227)全蛋白的1134个氨基酸中从961位氨基酸到1152位氨基酸(包括SEA结构域)总共表达192个氨基酸的抗原。粘蛋白1在多种人腺癌细胞中的表达高约100倍(Immunology,2018,225-240)。特定地,粘蛋白1在细胞中合成,并且然后在SEA结构域处切割成非共价结合的两个亚基,即包含SEA结构域的细胞结合部分和剩余部分。在正常细胞中,MUC1是高度糖基化的,但此部分在截去的部分中占主体,并且在细胞结合部分中未糖基化。在本发明中,基于这一事实,即使在切割后与细胞结合的190个氨基酸也用作在不经历糖基化的大肠杆菌中表达的抗原。
由于根据本发明的抗体是基于在大肠杆菌中表达的抗原产生的,所以它识别未糖基化的抗原。在本发明中,使用小鼠杂交瘤技术开发抗体,并且有趣的是,发现开发的抗体识别三级结构,而不是氨基酸序列的一级结构。
在本发明的一个实施方案中,发现当使用T47D和ZR75-1乳腺癌细胞系进行FACS时,MUC-1被特异性地识别,但未通过用以观察一级结构的蛋白质印迹识别通过细胞裂解获得的细胞裂解物。使用细胞裂解物和根据本发明的抗体,在免疫沉淀后检测抗原,然后进行蛋白质印迹,以验证三级结构的识别,这表明根据本发明的抗体特异性地识别三级结构。
在本发明中,MUC1的C末端细胞外结构域可以具有SEQ ID NO:10的氨基酸序列(SVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QS)。
如本文所用的术语“抗体”通常是指通过刺激免疫系统中的抗原产生的物质,并且其种类不受特别限制。抗体是指与特定抗原发生免疫反应的免疫球蛋白分子,并且是作为特异性地识别抗原的受体的蛋白质分子,并且包括多克隆抗体、单克隆抗体、全抗体和抗体片段中的全部。抗体可以是非天然产生的,例如,重组或合成产生的。抗体可以是动物抗体(例如,小鼠抗体)、嵌合抗体、人源化抗体或人抗体。抗体可以是单克隆抗体。除非另有说明,抗体还应理解为包括抗体的具有抗原结合能力的抗原结合片段。
在本说明书中,术语“互补决定区(CDR)”是指在抗体的可变区中赋予对抗原的结合特异性的位点。上述抗体的抗原结合片段可以是包含至少一个互补决定区的抗体片段。
在本发明中,抗MUC1抗体可以由杂交瘤hMUC1-1H7(KCLRF-BP-00395)产生。此外,抗MUC1抗体或其抗原结合片段具有由杂交瘤hMUC1-1H7(KCLRF-BP-00395)产生的抗体的互补决定区(CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2和CDR-L3)或重链可变区和轻链可变区。
一方面,根据本发明的抗MUC1抗体或其抗原结合片段包括六个互补决定区(CDR)。抗体或其抗原结合片段包括选自以下的至少一个:SEQ ID NO:1的重链CDR1(GYTFTSYWMH);SEQ ID NO:2的重链CDR2(YINPGTGYIEYNQKFKD);SEQ ID NO:3的重链CDR3(STAPFDY);SEQID NO:4的轻链CDR1(KASQDIKSYLS);SEQ ID NO:5的轻链CDR2(YATRLAD);和SEQ ID NO:6的轻链CDR3(LQYDESPYT)。
在本发明中,抗体或其抗原结合片段可包括:SEQ ID NO:1的重链CDR1;SEQ IDNO:2的重链CDR2;SEQ ID NO:3的重链CDR3;SEQ ID NO:4的轻链CDR1;SEQ ID NO:5的轻链CDR2;和SEQ ID NO:6的轻链CDR3。
在本发明中,抗体或其抗原结合片段的特征可在于其包括SEQ ID NO:22或24的重链可变区和SEQ ID NO:23或25的轻链可变区。更特定地,抗体或其抗原结合片段包括SEQID NO:22的重链可变区和SEQ ID NO:23的轻链可变区;或SEQ ID NO:24的重链可变区和SEQ ID NO:25的轻链可变区。
在本发明中,抗MUC1抗体或其抗原结合片段具有对MUC1的抑制作用。
MUC1基因编码一种转录物,并且在翻译后,MUC1蛋白在位于SEA结构域内的GSVVV基序的“G”中被自动切割。在本发明中,MUC1蛋白可以是人MUC1蛋白,并且例如可以具有GenBank登录号P15941(SEQ ID NO:7)的氨基酸序列,或与之具有至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%或至少99%的序列同源性。MUC1蛋白的细胞外结构域可以是人MUC1蛋白的细胞外结构域,例如,蛋白质片段(SEQ ID NO:8),所述蛋白质片段总共包含192个氨基酸,即GenBank登录号P15941(SEQ ID NO:7)的氨基酸序列中的从961位氨基酸到1152位氨基酸。MUC1蛋白的细胞外结构域包括SEA结构域。在一个例子中,SEA结构域是蛋白质片段(SEQ ID NO:9),其总共包含119个氨基酸,即GenBank登录号P15941(SEQ ID NO:7)的氨基酸序列中的从1034位氨基酸到1152位氨基酸。SEA结构域中存在的GSVVV基序的“G”是切割位点,并且当在此位点发生切割时,“G”后(C-末端方向)的位点是C-末端细胞外结构域(也称为“MUC1-C末端(位点)细胞外结构域”或“MUC1-C亚基”)(SEQID NO:10)(表1)。
[表1]
在本发明中,抗MUC1抗体或其抗原结合片段识别或特异性地结合包括在MUC1蛋白(例如SEQ ID NO:7)内的至少5个、至少7个、至少10个、至少12个、或优选至少15个氨基酸的多肽(表位),特定地,是MUC1蛋白的C末端细胞外结构域(例如SEQ ID NO:10)。抗MUC1抗体或其抗原结合片段识别和/或特异性地结合MUC1蛋白的细胞外结构域(例如SEQ ID NO:8)、MUC1蛋白的SEA结构域(例如SEQ ID NO:9)或MUC1蛋白的C末端细胞外结构域(例如SEQ IDNO:10)。
如本文所用,术语“MUC1特异性抗体”或“特异性地结合MUC1的抗体”是指与MUC1结合以引起对MUC1的生物学活性的抑制的抗体,并且可与“抗MUC1抗体”互换使用。
如本文所用,术语“抗MUC1抗体”是指动物抗体(例如小鼠抗体)、嵌合抗体(例如小鼠-人嵌合抗体)或人源化抗体,并且可以是单克隆抗体或多克隆抗体,例如单克隆抗体。术语“抗MUC1抗体”涵盖多克隆抗体和单克隆抗体两者,并且优选是单克隆抗体,并且可以具有全抗体形式。全抗体具有两条全长轻链和两条全长重链,并包括恒定区,其中每个轻链通过二硫键与相应的重链连接。
根据本发明的抗MUC1抗体的全抗体包括IgA、IgD、IgE、IgM和IgG形式,并且IgG包括亚型IgG1、IgG2、IgG3和IgG4。
全(whole(完整))IgG抗体具有包含两条全长轻链和两条全长重链的结构,其中每个轻链通过二硫键与相应的重链连接。抗体的恒定区分为重链恒定区和轻链恒定区。重链恒定区具有伽马(γ)、缪(μ)、阿尔法(α)、德耳塔(δ)和伊普西隆(ε)型,并且分为以下亚类:伽马1(γ1)、伽马2(γ2)、伽马3(γ3)、伽马4(γ4)、阿尔法1(α1)和阿尔法2(α2)。轻链恒定区具有卡帕(κ)和兰布达(λ)类型。
如本文所用,术语“重链”涵盖全长重链及其片段,所述全长重链包括可变区结构域(VH)、三个恒定区(CH1、CH2和CH3)和铰链,所述可变区结构域含有具有足够的可变区序列以赋予针对抗原的特异性的氨基酸序列。另外,如本文所用,术语“轻链”涵盖全长轻链及其片段,所述全长轻链包括可变区结构域(VL)、恒定区CL,所述可变区结构域包含具有足够的可变区序列以赋予针对抗原的特异性的氨基酸序列。
如本文所用,术语“互补决定区(CDR)”是指免疫球蛋白的重链和轻链的高变区的氨基酸序列。重链和轻链可各自具有三个CDR区(CDRH1、CDRH2、CDRH3和CDRL1、CDRL2、CDRL3)。CDR可提供主要的接触残基,以使抗体与抗原或表位结合。
同时,术语“特异性地结合”或“特异性地识别”具有与本领域技术人员通常已知的含义相同的含义,这意味着抗原和抗体通过特异性相互作用彼此进行免疫反应。
根据本发明的抗MUC1抗体的术语“抗原结合片段”是指抗MUC1抗体的具有结合抗原即MUC1的能力的片段,并且包括Fab、Fab'、F(ab')2、scFv、(scFv)2、scFv-Fc、Fv等。在本文中,术语“抗原结合片段”具有与术语“抗体片段”相同的含义,并且二者可互换使用。抗原结合片段可以是例如scFv、(scFv)2、Fab、Fab'或F(ab')2,但不限于此。在抗原结合片段中,Fab包括重链和轻链各自的可变区、轻链的恒定区和重链的第一恒定区(CH1),并且具有一个抗原结合位点。Fab'与Fab的不同之处在于,它还包括在重链的CH1结构域的C末端具有至少一个半胱氨酸残基的铰链区。F(ab')2抗体由铰链区中半胱氨酸残基之间的二硫键形成。Fv是仅具有重链可变区和轻链可变区的最小抗体片段,并且产生Fv片段的重组技术是本领域熟知的。双链Fv具有其中轻链可变区通过非共价键与重链可变区连接的结构,并且单链Fv具有其中重链可变区和轻链可变区经由肽接头通过共价键连接或直接在C末端连接以形成类似双链Fv的二聚体状结构的结构。抗原结合片段可以使用蛋白酶来获得(例如,Fab可通过用木瓜蛋白酶限制性切割全抗体来获得,并且F(ab′)2片段可通过用胃蛋白酶限制性切割全抗体来获得),并且可通过遗传重组技术来制备。
如本文所用,术语“铰链区”是指包括在抗体的重链中、介于CH1与CH2结构域之间、并为抗体中的抗原结合位点赋予柔性的区域。
抗MUC1抗体可以是单克隆抗体。可以根据本领域熟知的方法,例如通过噬菌体展示技术来制备单克隆抗体。可替代地,单克隆抗体可以使用抗MUC1抗体通过常规方法从小鼠产生。
同时,可以使用典型的ELISA(酶联免疫吸附测定)形式,基于与MUC1的结合能力来筛选单独的单克隆抗体。可以通过功能分析(例如用于测试结合物质上的分子相互作用的竞争性ELISA)和功能分析(例如基于细胞的测定)测试抑制活性。基于强抑制活性测试了所选单克隆抗体成员对MUC1的亲和力(Kd值)。
在根据本发明的抗MUC1抗体或其抗原结合片段中所含的轻链和重链的CDR1至CDR3中的至少一个,以及对MUC1抗原具有基本相同的结合能力和特异性的肽和适配体,属于根据本发明的抗MUC1抗体或其抗原结合片段的范围。
另一方面,本发明涉及产生抗MUC1抗体的杂交瘤。在本发明中,杂交瘤以保藏号KCLRF-BP-00395保藏。
本发明提供了由杂交瘤或其抗原结合片段产生的抗MUC1抗体。在另一个例子中,本发明提供了抗MUC1抗体或其抗原结合片段,包括由杂交瘤产生的抗MUC1抗体的重链互补决定区(CDR-H1、CDR-H2、CDR-H3或其组合)或轻链互补决定区(CDR-L1、CDR-L2、CDR-L3或其组合)或其组合;或由杂交瘤产生的抗MUC1抗体的重链可变区或轻链可变区或其组合。在这种情况下,可以通过任何常规方法来确定互补决定区,例如通过IMGT定义(http://www.imgt.org/IMGT_vquest/share/textes/)或Kabat定义(http://www.bioinf.org.uk/abs/)来确定,但不限于此。
在本发明中,抗MUC1抗体或其抗原结合片段特异性地识别MUC1的C末端细胞外结构域,从而MUC1-C末端细胞外结构域以比正常细胞中更高的水平表达,并且抗MUC1抗体或其抗原结合片段特异性地作用于较少糖基化的癌细胞或肿瘤细胞,并且可以识别/结合在完整表面以及在细胞的一侧表达的MUC1蛋白。另外,抗MUC1抗体或其抗原结合片段不仅结合MUC1蛋白(特别是MUC1-C末端细胞外结构域),而且将MUC1蛋白内化到细胞中(参见实施例9-8,实施例18),从而有效抑制MUC1介导的途径并使药理作用最大化。另外,抗MUC1抗体或其抗原结合片段的内化特性具有在作为抗体-药物缀合物(ADC)应用后将缀合的药物有效地递送到细胞中的优点。
另一方面,本发明涉及包含根据本发明的抗MUC1抗体或其抗原结合片段的嵌合抗原受体(CAR)-T细胞治疗剂和/或嵌合抗原受体(CAR)-天然杀伤细胞(NK)。CAR-T或CAR-NK中所含的抗MUC1抗体或其抗原结合片段的形式优选为scFv,但本发明不限于此。
另一方面,本发明涉及抗体-药物缀合物(ADC),其中药物与抗MUC1抗体或其抗原结合片段缀合。
在本发明的一个实施方案中,抗体-药物缀合物的药物是单甲基阿里他汀E(MMAE)。与表达MUC1的细胞结合后,抗体-药缀合物被内化到表达MUC1的肿瘤细胞中,并且MMAE通过蛋白水解切割而选择性地释放到靶标MUC1细胞中。当释放的MMAE与微管蛋白结合时,它会切割细胞内微管网络,诱导细胞周期停滞,并导致微管切割,并伴有细胞死亡(细胞凋亡)。
关于抗体-药物缀合物(ADC),抗癌药物应稳定地与抗原结合,直到将抗癌药物递送至靶标癌细胞为止。递送至靶标的药物应从抗体中释放出来并诱导靶细胞死亡。为此,药物应稳定地与抗体结合,并且同时,当在靶细胞中释放时,应展现出足够的细胞毒性以诱导靶细胞死亡。
在本发明中,抗MUC1抗体或其抗原结合片段与包括药物例如抗癌剂的细胞毒性物质彼此结合(例如,通过共价键、肽键等),并且因此,可以用作缀合物或融合蛋白(当细胞毒性物质和/或标记物质(标记物)是蛋白质时)。细胞毒性物质可以是对癌细胞特别是实体癌细胞有毒性的任何物质,并且可以是选自放射性同位素、细胞毒性化合物(小分子)、细胞毒性蛋白和抗癌药物的至少一种,但是本发明不限于此。所述细胞毒性蛋白选自蓖麻毒蛋白、皂草素、白树毒素、苦瓜毒蛋白、debouganin、白喉毒素、假单胞菌毒素等,但本发明不限于此。放射性同位素可以是选自131I、188Rh和90Y的至少一种,但是本发明不限于此。细胞毒性化合物可以选自倍癌霉素(duocarmycin)、一甲基阿里他汀E(MMAE)、一甲基阿里他汀F(MMAF)、N2’-二乙酰-N2'-(3-巯基-1-氧代丙基)美登素(DM1)、PBD(吡咯并苯二氮卓类)二聚体等,但不限于此。
在本发明中,抗体-药物缀合物可以根据本领域熟知的方法获得。
在本发明中,抗体-药物缀合物的特征可在于抗体或其抗原结合片段通过接头与药物结合。
在本发明中,接头可以是可切割的接头或不可切割的接头。
接头是用于将抗MUC1抗体与药物连接的位点。例如,接头允许药物在细胞内条件下以可切割的形式释放,即通过在细胞内环境中从抗体上切割接头来释放。
接头可以是肽接头,其可以被存在于细胞内环境中(例如在溶酶体或内体中)的切割剂切割,并且可以被细胞内肽酶或蛋白酶诸如溶酶体或内体蛋白酶切割。通常,肽接头具有至少两个氨基酸长度。切割剂可以包括组织蛋白酶B、组织蛋白酶D和纤溶酶,它们水解肽以将药物释放到靶细胞中。肽接头可以被在癌症组织中高表达的硫醇依赖性蛋白酶组织蛋白酶-B切割,并且可以是例如Phe-Leu或Gly-Phe-Leu-Gly接头。另外,肽接头可以是例如Val-Cit接头或Phe-Lys接头,其可以被细胞内蛋白酶切割。
在本发明中,可切割的接头对pH敏感,并且在一定pH值下可能对水解敏感。通常,pH敏感的接头是可以在酸性条件下水解的接头。可以在溶酶体中水解的酸不稳定性接头的例子可以包括腙、半卡巴腙、硫代半卡巴腙、顺式乌头酰胺、原酸酯、乙缩醛、缩酮等。
所述接头可以在还原条件下切割,并且可以是例如二硫键接头。可以使用N-琥珀酰亚胺基-S-乙酰硫代乙酸酯(SATA)、N-琥珀酰亚胺基-3-(2-吡啶基二硫代)丙酸酯(SPDP)、N-琥珀酰亚胺基-3-(2-吡啶基二硫代)丁酸酯(SPDB)和N-琥珀酰亚胺基-氧羰基-α-甲基-α-(2-吡啶基-二硫代)甲苯(SMPT)形成各种二硫键。
在本发明中,药物和/或药物接头可以通过抗体的赖氨酸随机缀合,或者可以通过当二硫键链还原时暴露的半胱氨酸缀合。在一些情况下,可以通过存在于基因工程化标签(例如肽或蛋白质)中的半胱氨酸来缀合接头-药物。基因工程化标签,例如肽或蛋白质,可以包括可以被例如类异戊二烯转移酶识别的氨基酸基序。肽或蛋白质通过间隔单元的共价键在肽或蛋白质的羧基末端具有缺失或在肽或蛋白质的羧基(C)末端具有添加。肽或蛋白质可以直接共价键合至氨基酸基序,或者可以通过与间隔单元的共价键连接至氨基酸基序。氨基酸间隔单元包括1至20个氨基酸,并且在它们之中优选为甘氨酸单元。
接头可以包括以多个拷贝存在于溶酶体中的β-葡萄糖醛酸苷接头,或被在一些肿瘤细胞中过表达的β-葡萄糖醛酸酶水解。与肽接头不同,此接头具有由于其高亲水性而与具有高疏水性的药物结合时增加抗体-药物组合物的溶解性的优点。
就此而言,在本发明中,可以使用在韩国专利公开号2015-0137015中披露的β-葡萄糖醛酸苷接头,例如包含自毁式基团的β-葡萄糖醛酸苷接头。
另外,所述接头可以是例如不可切割的接头,并且所述药物能够仅通过水解抗体以产生例如氨基酸-接头-药物复合物的单个步骤而被释放。此类型的接头可以是硫醚基或马来酰亚胺基己酰基,并且可以保持血液中的稳定性。
在本发明中,药物可以是化学治疗剂、毒素、微小RNA(miRNA)、siRNA,shRNA或放射性同位素。可以将药物(作为具有药理作用的药剂)与抗体缀合。
化学治疗剂可以是细胞毒性剂或免疫抑制剂。特定地,化学治疗剂可以包括微管蛋白抑制剂、有丝分裂抑制剂、拓扑异构酶抑制剂、或能够用作DNA嵌入剂的化学治疗剂。化学治疗剂还可以包括免疫调节化合物、抗癌剂、抗病毒剂、抗细菌剂、抗真菌剂、驱虫剂或其组合。
例如,药物可以包括选自以下的至少一种:美登木素生物碱(maytansinoid)、阿里他汀、氨基蝶呤、放线菌素、博来霉素、沙利度胺、喜树碱、N8-乙酰基亚精胺、1-(2-氯乙基)-1,2-二甲基磺酰肼、埃斯培拉霉素(esperamycin)、依托泊苷、6-巯基嘌呤、尾海兔素、单端孢菌素、卡奇霉素、紫杉醇(taxol)、紫杉烷、紫杉醇(paclitaxel)、多西他赛、甲氨蝶呤、长春新碱、长春碱、多柔比星、美法仑、丝裂霉素A、丝裂霉素C、苯丁酸氮芥、杜卡霉素、L-天冬酰胺酶、巯基嘌呤、硫鸟嘌呤、羟基脲、阿糖胞苷、环磷酰胺、异环磷酰胺、亚硝基脲、顺铂、卡铂、丝裂霉素、达卡巴嗪、丙卡巴肼、拓扑替康、氮芥、环磷酰胺、依托泊苷、5-氟尿嘧啶、二氯乙基亚硝基脲(CNU)、伊立替康、喜树碱、博来霉素、伊达比星、道诺霉素、更生霉素、普卡霉素、米托蒽醌、天冬酰胺酶、长春瑞滨、苯丁酸氮芥、美法仑、卡莫司汀、洛莫司汀、白消安、苏消安、达卡巴嗪、依托泊苷、替尼泊苷、拓扑替康、9-氨基喜树碱、克雷斯托、三甲曲沙、霉酚酸、噻唑呋林、利巴韦林、5-乙炔基-1-β-D核糖呋喃糖基咪唑-4-甲酰胺(EICAR)、羟基脲、去铁胺、氟尿苷、多西氟尿啶、雷替曲塞、阿糖胞苷(ara C)、胞嘧啶阿拉伯糖苷、氟达拉滨、他莫昔芬、雷洛昔芬、甲地孕酮、戈舍瑞林、乙酸亮丙瑞林、氟他胺、比卡鲁胺、EB1089、CB1093、KH1060、维替泊芬、酞菁、光敏剂Pe4、脱甲氧基竹红菌素A、干扰素-α、干扰素-γ、肿瘤坏死因子、吉西他滨、万珂(Velcade)、revamid、沙利度胺、洛伐他汀、1-甲基-4-苯基吡啶鎓、星形孢菌素、放线菌素D、更生霉素、博来霉素A2、博来霉素B2、培洛霉素、表柔比星、吡柔比星、佐柔比星、米托蒽醌、维拉帕米和毒胡萝卜素、核酸酶和源自细菌或植物和动物的毒素,但是本发明不限于此。
在本发明中,药物可以具有选自以下的亲核基团:胺、硫醇、羟基、酰肼、肟、肼、硫代半卡巴腙、肼甲酸酯和芳基酰肼基团,所述亲核基团可以与接头上的亲电子基团和接头试剂反应形成共价键。
另一方面,本发明涉及包含抗MUC1抗体或其抗原结合片段的双特异性抗体。
双特异性抗体是指如下抗体,其中在抗体的两个臂中,一个臂包括根据本发明的抗MUC1抗体或其抗原结合片段,并且另一个臂包括与MUC1以外的抗原(优选癌症相关抗原或免疫检查点蛋白抗原、或免疫效应细胞相关抗原)特异性地结合的抗体或其抗原结合片段。
根据本发明的双特异性抗体中所包含的与抗MUC1抗体以外的抗体结合的抗原优选选自癌症相关抗原或免疫检查点蛋白抗原,包括Her2、EGFR、VEGF、VEGF-R、CD-20、MUC16、CD30、CD33、CD52、PD-1、PD-L1、CTLA4、BTLA4、EphB2、E-选择素、EpCam、CEA、PSMA、PSA、ERB3、c-MET等;并且优选选自免疫效应细胞相关抗原,包括TCR/CD3、CD16(FcγRIIIa)CD44、CD56、CD69、CD64(FcγRI)、CD89、CD11b/CD18(CR3)等,但本发明不限于此。
另一方面,本发明涉及用于预防和/或治疗与MUC1相关的疾病的药物组合物,其包含抗MUC1抗体或其抗原结合片段、抗体-药物缀合物或双特异性抗体。
MUC1相关疾病可以是与正常细胞相比与MUC1的表达或过表达、在MUC1的细胞的所有表面上的表达、和/或MUC1蛋白的糖基化减少相关的疾病,例如癌症。正常细胞可以是非肿瘤细胞。因此,MUC1相关疾病优选是癌症或肿瘤,但是所述疾病不限于此。
术语“癌症”或“肿瘤”通常是指或意指以不受控制的细胞生长/增殖为特征的哺乳动物的生理状况。
可以用本发明的组合物治疗的癌症或癌不受特别限制,并且包括实体癌和血液癌。这类癌症的例子包括但不限于皮肤癌(例如黑素瘤)、肝癌、肝细胞癌、胃癌、乳腺癌、肺癌、卵巢癌、支气管癌、鼻咽癌、喉癌、胰腺癌、膀胱癌、结直肠癌、结肠癌、宫颈癌、脑癌、前列腺癌、骨癌、甲状腺癌、垂体癌、肾癌、食道癌、胆道癌、睾丸癌、直肠癌、头颈癌、子宫颈癌、输尿管癌、骨肉瘤、神经母细胞瘤、纤维肉瘤、横纹肌肉瘤、星形细胞瘤和神经胶质瘤。
更优选地,癌症的特征在于表达MUC1蛋白,并且可以是乳腺癌、胰腺癌、前列腺癌、肺癌、甲状腺癌、胃癌、卵巢癌、结直肠癌、肝癌、胆囊癌、肾癌、子宫颈癌或膀胱癌,但不限于此。所述癌症可以是原发性癌症或转移性癌症。
MUC1相关疾病可以是非酒精性脂肪性肝炎(NASH)或TGF-β介导的免疫疾病,但不限于此。
在本发明的一个实施方案中,在用于预防和/或治疗癌症的药物组合物、方法和用途中,抗MUC1抗体或其抗原结合片段可以作为单一活性成分提供,可以与细胞毒性物质(例如抗癌剂)结合给予,或者可以按与细胞毒性物质(例如抗癌剂)缀合的抗体-药物缀合物(ADC)的形式提供。
另外,根据本发明的抗MUC1抗体或其抗原结合片段和包含所述抗MUC1抗体的药物组合物可以与常规治疗剂组合使用。即,根据本发明的抗MUC1抗体或其抗原结合片段和包含所述抗MUC1抗体或其抗原结合片段的药物组合物可以与常规治疗剂例如抗癌剂同时或依次使用。
另一方面,本发明涉及一种预防和/或治疗MUC1相关疾病的方法,所述方法包括向需要预防或治疗MUC1相关疾病的患者给予治疗有效量的抗MUC1抗体或其抗原结合片段或抗体-药物缀合物。预防和/或治疗MUC1相关疾病的方法可以进一步包括在给药前检查需要预防或治疗MUC1相关疾病的患者。
使用抗体缀合物以在组合物中局部递送药物使得可将药物递送至表达由抗MUC1抗体靶向的抗原的细胞。
药物组合物可以进一步包含药学上可接受的载体。药学上可接受的载体可以是一种通常用于制备药物的载体,并且可以包括选自以下的至少一种:乳糖、右旋糖、蔗糖、山梨糖醇、甘露糖醇、淀粉、阿拉伯胶、磷酸钙、藻酸盐、明胶、硅酸钙、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁、矿物油等,但本发明不限于此。另外,药物组合物还可包含选自以下的至少一种:稀释剂、赋形剂、润滑剂、润湿剂、甜味剂、调味剂、乳化剂、悬浮剂和防腐剂,它们通常用于制备药物组合物。
所述药物组合物可以口服或肠胃外给予。对于肠胃外给药,可以通过静脉内注射、皮下注射、肌内注射、腹膜内注射、皮内给药、鼻内给药、肺内给药、直肠内给药或局部给药至病变部位等来给予药物组合物。口服给药后,蛋白质或肽被消化,从而可以用活性药物包覆或配制口服组合物以保护所述蛋白质或肽在胃中不降解。另外,可以使用能够将活性物质递送至靶细胞的任何装置来给予组合物。
抗MUC1抗体或其抗原结合片段在药物组合物中的含量或剂量可以根据诸如配制方法、给药方法、以及年龄、体重、性别、病理状况、饮食、给药时间、给药间隔、给药途径、排泄率和患者反应性等因素而变化。例如,抗MUC1抗体或其抗原结合片段的日剂量可以在0.001至1,000mg/kg的范围内,特定地为0.01至100mg/kg,并且更特定地为0.1至50mg/kg,甚至更特定地为0.1至20mg/kg,但本发明不限于此。日剂量可以通过配制成具有单位剂量的单一剂型、以适当剂量配制或包装在多剂量容器中来制备。
药物组合物可以是在油或水性介质中的溶液、悬浮液、糖浆或乳液的形式,或者可以按提取物、粉剂、颗粒剂、片剂或胶囊剂的形式配制。药物组合物可以进一步包含分散剂或稳定剂。
接受药物组合物的患者可以是哺乳动物,包括灵长类动物(其包括人、猴子等)或者啮齿动物(其包括小鼠、大鼠等)。
在本说明书中,癌症的治疗可以指所有预防、改善或减轻癌症症状加重、或者部分或完全消除癌症(例如抑制癌细胞的增殖、癌细胞的死亡、或抑制癌细胞转移)的抗癌活动。
另一方面,本发明涉及抗MUC1抗体或其抗原结合片段用于预防或治疗癌症的用途。
另一方面,本发明涉及抗MUC1抗体或其抗原结合片段用于制备癌症的预防剂或治疗剂的用途。
抗MUC1抗体或其抗原结合片段特异性地结合MUC1蛋白,特别是结合MUC1-C末端细胞外结构域,并且因此可用于检测或鉴定MUC1蛋白或MUC1-C末端细胞外结构域。因此,另一方面,本发明涉及用于检测MUC1蛋白例如MUC1-C-末端细胞外结构域的组合物,其包含抗MUC1抗体或其抗原结合片段;以及用于检测MUC1的方法,所述方法包括用抗MUC1抗体或其抗原结合片段处理生物样品。
所述检测方法可以进一步包括在治疗之后鉴定抗原-抗体反应是否发生。在检测方法中,当检测到抗原-抗体反应时,可以确定在生物样品中存在MUC1,例如,MUC1-C-末端细胞外结构域。因此,所述检测方法可以进一步包括在所述鉴定之后,当检测到抗原-抗体反应时,确定所述生物样品中存在MUC1。所述生物样品可以选自从哺乳动物(例如人)(例如癌症患者)获得(分离)的细胞、组织、体液、其培养物等。
MUC1蛋白例如MUC1蛋白的C末端区域(或MUC1蛋白的SEA结构域或MUC1-C末端细胞外结构域)的表达与多种疾病(例如癌症)相关。因此,另一方面,本发明涉及用于检测或诊断MUC1蛋白相关疾病例如癌症的组合物,其包含抗MUC1抗体或其抗原结合片段;以及用于检测或诊断癌症的方法,或提供用于检测或诊断癌症的信息的方法,所述方法包括将抗MUC1抗体或其抗原结合片段给予从受试者分离的生物样品。
所述检测或诊断方法可以进一步包括在治疗之后鉴定抗原-抗体反应是否发生。在所述方法中,当检测到抗原-抗体反应时,可以确定MUC1相关疾病例如癌症存在于生物样品或作为生物样品来源的患者中。因此,所述方法可以进一步包括:在鉴定之后,当检测到抗原-抗体反应时,确定生物样品或作为生物样品来源的患者是患有MUC1相关疾病例如癌症的患者。所述生物样品可以选自从哺乳动物(例如人)(例如癌症患者)获得(分离)的细胞、组织、体液、其培养物等。
可以通过本领域已知的各种方法,例如,常规的酶反应、荧光、发光和/或放射检测,特别是通过选自免疫色谱、免疫组织化学、酶联免疫吸附测定(ELISA)、放射免疫测定(RIA)、酶免疫测定(EIA)、荧光免疫测定(FIA)、发光免疫测定(LIA)、蛋白质印迹、微阵列、免疫沉淀测定等的方法,鉴定抗原-抗体反应是否发生,但本发明不限于此。
在这种情况下,抗MUC1抗体或其抗原结合片段可以进一步包括标记物。标记物可以是选自放射性同位素、荧光物质、色原和染色物质中的至少一种。可以通过常规方法(例如,化学键如共价键、配位键或离子键)将标记物结合(连接)至抗体或抗原结合片段。抗体(或抗原结合片段)与标记物的结合可以根据本领域熟知的技术。
在本发明的一个实施方案中,通过从乳腺癌细胞系克隆MUC1的C末端区域并在Rosetta感受态细胞中表达MUC1的C末端区域来产生针对MUC1的单克隆抗体。将MUC1的C末端区域与CpG-DNA(例如MB-ODN 4531(O))配制在一起,并包封在DOPE:CHEMS复合物中,然后用于小鼠免疫。当用[MUC1的C末端区域]-[MB-ODN 4531(O)]-[DOPE:CHEMS]复合物免疫小鼠时,MUC1特异性单克隆抗体的产生显著增加。还通过将用[hMUC1-SEA]-[MB-ODN 4531(O)]-[DOPE:CHEMS]复合物免疫的小鼠获得的脾细胞与SP2/0细胞融合,获得了抗hMUC1-SEA单克隆抗体。
因此,另一方面,本发明涉及包含MUC1的C末端区域、MUC1的SEA结构域、或MUC1的C末端细胞外结构域的免疫原性组合物。另一个实施方案提供了一种包含复合物的免疫原性组合物,所述复合物包含包封在脂质体中的(1)MUC1的C末端区域、MUC1的SEA结构域、或MUC1的C末端细胞外结构域和(2)CpG-DNA。免疫原性组合物是指当注射(接种)到活生物体中时能够通过诱导免疫应答而产生抗体的组合物。
在本发明中,脂质体可以是阳离子脂质体,例如,摩尔比为1:0.5至1:2的二油酰基磷脂酰乙醇胺(DOPE)和胆甾醇半琥珀酸酯(CHEMS)的混合物,更特定地是1:0.67至1:1.5(DOPE:CHEMS)(例如摩尔比为约1:1)的混合物,或者可以是由所述混合物获得的产物(例如无溶剂脂质膜),但不限于此。
在本发明中,CpG-DNA是指包含总共10至20个核苷酸的寡脱氧核苷酸(ODN),包含一个或多个,例如一至三个CpG基序。在本发明的一个实施方案中,CpG-DNA可包括但不限于“AGCAGCGTTCGTGTCGGCCT”(SEQ ID NO:11)的核酸序列。
脂质体和寡脱氧核苷酸可以起佐剂的作用。佐剂有助于与免疫系统一起更容易地识别抗原,并改善免疫应答。细菌DNA含有合成的寡脱氧核苷酸(ODN)和未甲基化的CpG二核苷酸,其侧翼是特定的碱基序列,所述细菌DNA对B淋巴细胞、自然杀伤细胞、巨噬细胞和树突细胞具有重要的免疫调节作用。
已经示出阳离子脂质体(例如,脂质转染胺(lipofectamine)、和磷脂酰-β-油酰基-γ-棕榈酰乙醇胺(DOPE):胆固醇半琥珀酸酯(CHEMS))增加抗体产生、抗体递送和CTL应答。因此,包封在DOPE:CHEMS(1:1比例)复合物中的CpG-DNA(脂质体复合物(O))可以促进人和小鼠细胞中的有效免疫应答。
另一方面,本发明涉及产生抗MUC1-SEA单克隆抗体的方法,所述方法包括用MUC1的C末端区域、MUC1的SEA结构域、或MUC1的C-末端细胞外域结构域、或包含包封在脂质体内的(1)MUC1的C末端区域、MUC1的SEA结构域、或MUC1的C末端细胞外结构域和(2)CpG-DNA的复合物接种哺乳动物(例如小鼠)。制备抗MUC1-SEA单克隆抗体的方法可以进一步包括在接种后通过常规方法从小鼠血清中分离和纯化抗体。
另一方面,本发明涉及编码根据本发明的抗MUC1抗体的核酸。如本文所用,核酸可以存在于细胞或细胞裂解物中,或者可以按部分纯化的形式或按基本上纯的形式存在。当通过包括例如碱/SDS处理、CsCl显带、柱色谱、琼脂糖凝胶电泳和本领域熟知的其他技术的标准技术从其他细胞组分或其他污染物(例如从其他细胞的核酸或蛋白质)中纯化时,核酸可以是“分离的”或“基本上变成纯的”。本发明的核酸可以是例如DNA或RNA,并且可以包括或可以不包括内含子序列。
在本发明中,编码抗MUC1抗体的核酸可以包括选自SEQ ID NO:34至SEQ ID NO:45的至少一个序列。特定地,编码根据本发明的抗体的重链的多核苷酸序列由SEQ ID NO:34至39表示,和/或编码根据本发明的抗体的轻链的多核苷酸序列由SEQ ID NO:40至45表示。
另一方面,本发明涉及包含核酸的重组表达载体。为了表达根据本发明的抗MUC1抗体或其抗原结合片段,可以通过标准分子生物学技术(例如,使用表达所需抗体的杂交瘤进行PCR扩增或cDNA克隆)制备编码部分或全长轻链或重链的DNA,并且可以将DNA“可操作地连接”至转录和翻译控制序列并插入表达载体中。
如本文所用,术语“可操作地连接”可以意指将编码抗体的基因连接到载体中,使得载体内的转录和翻译控制序列起到调节抗体基因的转录和翻译的预期功能。选择表达载体和表达控制序列,以使其与用于表达的宿主细胞相容。将抗体的轻链基因和抗体的重链基因插入分开的载体中,或将两种基因插入同一个表达载体中。通过标准方法将抗体插入表达载体中(例如,将抗体基因片段和载体上的互补限制性酶切位点连接,或当不存在限制性酶切位点时进行平端连接)。在一些情况下,重组表达载体可以编码促进宿主细胞分泌抗体链的信号肽。可以将抗体链基因克隆到载体中,使得信号肽根据框架附接至抗体链基因的氨基末端。信号肽可以是免疫球蛋白信号肽或异源信号肽(即,源自除免疫球蛋白以外的蛋白质的信号肽)。另外,重组表达载体具有控制序列,所述控制序列控制宿主细胞中抗体链基因的表达。术语“控制序列”可以包括控制抗体链基因的转录或翻译的启动子、增强子、和其他表达控制元件(例如,聚腺苷酸化信号)。本领域技术人员将理解,可以通过选择不同的控制序列来改变表达载体的设计,所述控制序列的选择取决于多种因素,例如要转化的宿主细胞的选择和蛋白质的表达水平。
另一方面,本发明涉及包含核酸或载体的宿主细胞。根据本发明的宿主细胞优选选自动物细胞、植物细胞、酵母、大肠杆菌(Escherichia coli)和昆虫细胞,但不限于此。
更特定地,根据本发明的宿主细胞可以是原核细胞,例如大肠杆菌(Escherichiacoli)、枯草芽孢杆菌(Bacillus subtilis)、链霉菌属物种(Streptomyces sp)、假单胞菌属物种(Pseudomonas sp)、奇异变形杆菌(Proteus mirabilis)或葡萄球菌属物种(Staphylococcus sp)。宿主细胞可以是选自真菌(例如曲霉属物种(Aspergillus sp))、酵母(例如巴斯德毕赤酵母(Pichia pastoris)、酿酒酵母(Saccharomyces cerevisiae)、裂殖酵母属物种(Schizosaccharomyces sp)、和粗糙脉孢菌(Neurospora crassa))的真核细胞,其他较低级的真核细胞,以及较高级的真核生物的细胞(例如来自昆虫的细胞)。
宿主细胞也可以源自植物或哺乳动物。优选地,有用的宿主细胞可以包括但不限于猴肾细胞(COS7)、NSO细胞、SP2/0、中国仓鼠卵巢(CHO)细胞、W138、幼仓鼠肾(BHK)细胞、MDCK、骨髓瘤细胞系、HuT 78细胞、HEK293细胞等。特别优选地,有用的宿主细胞是CHO细胞。
核酸或载体被转染到宿主细胞中。可以使用通常用于将外来核酸(DNA或RNA)引入原核或真核宿主细胞中的各种技术来进行“转染”,所述技术例如为电泳、磷酸钙沉淀、DEAE-葡聚糖转染或脂质转染。各种表达宿主/载体组合可以用于表达根据本发明的抗磷脂酰肌醇蛋白聚糖3抗体。用于真核宿主的合适的表达载体包含,例如,但不限于源自SV40、牛乳头瘤病毒、腺病毒、腺相关病毒、巨细胞病毒和逆转录病毒的表达控制序列。可以用于细菌宿主的表达载体包括从大肠杆菌(Escherichia coli)(E.coli)获得的细菌质粒(例如pET、pRSET、pBluescript、pGEX2T、pUC载体、col E1、pCR1、pBR322、pMB9及其衍生物)、具有广泛宿主范围的质粒(例如RP4)、可以由多种噬菌体λ衍生物(例如λgt10、λgt11和NM989)例示的噬菌体DNA、以及其他DNA噬菌体(例如M13和丝状单链DNA噬菌体)。可用于酵母细胞的表达载体包括2μ质粒及其衍生物。可用于昆虫细胞的载体是pVL 941。
另一方面,本发明涉及一种产生根据本发明的抗MUC1抗体或其抗原结合片段的方法,其包括培养宿主细胞以表达根据本发明的抗MUC1抗体或其抗原结合片段。
当将能够表达抗MUC1抗体或其抗原结合片段的重组表达载体引入哺乳动物宿主细胞中时,可以通过以下方式产生抗体:将宿主细胞培养足够长的时间以使所述抗体在宿主细胞中表达,更优选地将宿主细胞培养足够长的时间以使抗体分泌到培养基中。
在一些情况下,可以将表达的抗体与宿主细胞分离并纯化至同质。抗体的分离或纯化可以使用用于常规蛋白质的分离和纯化方法(例如色谱)进行。这样的色谱可以包括例如涉及蛋白A柱或蛋白G柱的亲和色谱、离子交换色谱、或疏水色谱。可以通过结合过滤、超滤、盐析、透析等进行色谱来分离和纯化抗体。
在下文中,将参考以下实施例更加详细地描述本发明。然而,对于本领域技术人员而言清楚的是,以下实施例仅出于说明本发明的目的来提供,而不应当解释为限制本发明的范围。
实施例1:重组hMUC1-C蛋白的产生
从MCF7细胞获得编码MUC1-C末端(以下称为“hMUC1-C蛋白”;包含192个氨基酸(从P15941的961位到1152位)的蛋白,其中1034-1152aa为MUC-SEA结构域)的人cDNA,并且然后使用以下引物组通过RT-PCR进行扩增:
有义引物:hMUC1 C-Nco I-S3 5'-CC ATG GCC TCA GGC TCT GCATC-3'(SEQ IDNO:12);以及
反义引物:hMUC1 C-Xho I-AS4 5'-CTC GAG AGA CTG GGC AGA GAA AGG AAA T-3'(SEQ ID NO:13)。
通过DNA测序鉴定获得的hMUC1-C末端蛋白编码序列,并且结果表明所述序列与GenBank登录号J05582.1(SEQ ID NO:26)的2954位核苷酸至3529位核苷酸的576个核苷酸的核苷酸序列相同。
实施例2:重组hMUC1-C(rhMUC1-C)蛋白的表达和纯化
将扩增的cDNA片段克隆到含有C末端His标签的表达载体pET-22b(Novagen,达姆施塔特,德国)中。将获得的质粒转化到大肠杆菌(Escherichia coli)RosettaTM(Invitrogen,卡尔斯巴德,加利福尼亚州)的感受态细胞中,并使用1mM异丙基-D-1-硫代吡喃葡萄糖苷(IPTG,Sigma-Aldrich,圣路易斯,密苏里州)在37℃诱导8小时。通过超声处理将获得的细胞溶解在冰相裂解缓冲液(50mM Tris-HCl,100mM NaCl,5mM EDTA,0.5%Triton X100,1μg/ml溶菌酶,蛋白酶抑制剂混合物)中。离心后,将包涵体部分与缓冲液B(100mM NaH2PO4,10mM Tris-HCl,8M尿素,pH 8.0)混合,并使用Ni-NTA琼脂糖(Qiagen,巴伦西亚,加利福尼亚州)系统纯化。将所得混合物加载到Ni-NTA柱上,并用洗涤缓冲液C(100mMNaH2PO4,10mM Tris-HCl,8M尿素,pH 6.3)洗涤。用洗脱缓冲液(100mM NaH2PO4,10mM Tris-HCl,8M尿素,pH 4.5)洗脱结合的蛋白,并通过SDS-PAGE和蛋白质印迹进行分析。使用抗His标签抗体(Santa Cruz)进行蛋白质印迹。
实施例3:脂质体复合物(O)的制备
MB-ODN 4531包含20个碱基,其中含有三个CpG基序(带下划线)(AGCAGCGTTCGTGTCGGCCT:SEQ ID NO:11)。CpG ODN(寡脱氧核苷酸)(以下称为“CpG-DNA4531(O)”)购自Samchully Pharm(韩国,首尔)和GenoTech(大田(Daejeon),韩国)。将磷脂酰-β-油酰基-γ-棕榈酰乙醇胺(DOPE)和胆固醇半琥珀酸酯(CHEMS)在乙醇中以1:1的摩尔比混合,并且然后用氮气蒸发以获得无溶剂的脂质膜。将无溶剂的脂质膜重悬于相同体积的含有50μg水性CpG-DNA 4531(O)和50μg rhMUC1-C蛋白(实施例2)的混合物中,并在室温下剧烈搅拌30分钟以产生共包封在DOPE和CHEMS复合物中的CpG-DNA 4531(O)和rhMUC1-C蛋白。“脂质体复合物(O)”(“Lipoplex(O)complex”)是指包封在DOPE和CHEMS复合物中的CpG-DNA 4531(O)。将pH调至7.0后,用超声发生器将rhMUC1-C蛋白和脂质体复合物(O)超声处理30秒,并且将所得溶液通过0.22μm过滤器过滤,并用液氮冷冻融化三次,并且然后用于以下实验。
实施例4:动物的制备
四周龄的雌性BALB/c小鼠(OrientBio公司,首尔,韩国)和BALB/cAnNCri-nu/nu小鼠(四周龄)购自Nara Biotech公司(首尔,韩国)。在无特定病原体的无菌条件下,将小鼠维持在20℃至25℃和32%至37%的湿度下。所有动物实验程序均已获得翰林大学(HallymUniversity)的机构动物护理和使用委员会(Institutional Animal Care and UseCommittee)的批准,并根据韩国国家兽医研究检疫服务处的《实验动物的护理和使用指南》进行。通过吸入异氟烷处死小鼠,并尽一切努力使疼痛最小化。
实施例5:小鼠的免疫
用脂质体复合物以10天间隔通过腹膜内注射对BALB/c小鼠免疫四次,所述脂质体复合物包括共包封在磷脂酰-β-油酰基-γ-棕榈酰乙醇胺:胆固醇半琥珀酸酯(DOPE:CHEMS复合物)中的具有50μg rhMUC1-C蛋白和50μg CpG-DNA 4531的复合物。
实施例6:抗原特异性Ig ELISA
通过ELISA测量rhMUC1-C特异性IgG的总量。在96孔免疫板上包被10μg/ml的rhMUC1-C蛋白(实施例2),并用PBS-T(含0.1%(v/v)Tween-20的PBS)中的牛血清白蛋白(BSA)封闭。用PBS-T稀释实施例5中免疫的小鼠血清、杂交瘤细胞培养上清液或纯化的抗体,并在室温下培养2小时。用PBS-T将板洗涤3次,并用山羊抗小鼠IgG HRP缀合的二抗培养1小时。使用TMB底物溶液A和B(1:1比例)(Kirkegaard and Perry Laboratories,盖瑟斯堡,马里兰州,美国)对板进行显色,使用Spectra Max 250酶标仪(Molecular Devices,森尼韦尔,美国)在450nm处测量吸光度,并进行比色测定。
实施例7:抗hMUC1单克隆抗体的制备和纯化
使用聚乙二醇(PEG,Sigma-Aldrich)将实施例5中用rhMUC1-C蛋白免疫的小鼠的脾细胞与SP2/0骨髓瘤细胞(ATCC)融合。培养融合细胞,并用次黄嘌呤-氨基蝶呤-胸苷(HAT,Sigma-Aldrich)培养基选择。通过ELISA测试所选杂交瘤细胞的培养上清液与rhMUC1-C蛋白的结合,并筛选出结合呈阳性的杂交瘤细胞,即,筛选出产生rhMUC1-C蛋白特异性抗体的杂交瘤细胞。根据标准杂交瘤技术筛选杂交瘤克隆(Yokoyama等人2006)。将获得的杂交瘤克隆(KCLRF-BP-00395;下文中,将杂交瘤细胞或其产生的抗体称为“hMUC1-1H7”)在次黄嘌呤-胸苷(HT)培养基中培养。对ELISA阳性杂交瘤细胞群体进行亚克隆。为了产生单克隆抗体,将杂交瘤细胞(hMUC1-1H7克隆)在初始阶段和腹膜内注射后10天经腹膜内注射到小鼠中。10天后,收集腹膜液,并以3,000rpm离心30分钟。使用蛋白A色谱(Repligen,沃尔瑟姆,马塞诸塞州),使用与rhMUC1-C蛋白结合的抗hMUC1单克隆抗体纯化上清液。
实施例8:抗hMUC1单克隆抗体的IgG同种型的鉴定
为了测量在实施例7中获得的抗hMUC1单克隆抗体的IgG同种型,在96孔免疫板(Nalgene Nunc International,Penfield,美国)上包被1μg/ml的hMUC1-C蛋白,并用含有1%BSA的PBS中的0.05%Tween-20(PBST)封闭。将抗hMUC1-C单克隆抗体添加到每个板的顶行,并将在PBST中的一系列1:4稀释物放置在下一行。将板在室温孵育2小时并用PBST洗涤。然后,将与辣根过氧化物酶(HRP:BD Pharmingen)缀合的抗小鼠全IgG、IgG1、IgG2a、IgG3b、IgG3抗体添加至每个孔(1:500稀释),并在室温下培养1小时。使用TMB底物溶液A和B(1:1比例,Kirkegaard and Perry Laboratories,盖瑟斯堡,马里兰州)分析总量,并使用SpectraMax 250酶标仪(Molecular Devices,森尼韦尔,加利福尼亚州,美国)测量450nm处的吸光度。
实施例9:抗hMUC1-SEA单克隆抗体的反应性测试
实施例9-1:细胞培养
人乳腺癌细胞系(MCF-7,MDA-MB-231)和胰腺癌细胞系(Capan-2,CFPAC-1)购自美国典型培养物保藏中心(ATCC,马纳萨斯,维吉尼亚州),以及人乳腺癌细胞系(T47D,ZR75-1)和胰腺癌细胞系(Capan-1,PANC-1)购自韩国细胞系库(Korean Cell Line Bank)(KCLB,首尔,韩国)。
将MCF-7细胞在补充有0.01mg/ml人重组胰岛素的伊格尔极限必需培养基中培养;将MDA-MB-231细胞在Leibovitz L-15培养基(Thermo Fisher Scientific)中培养;并且将T47D、ZR75-1和Capan-1细胞在RPMI-1640培养基(Thermo Fisher Scientific)中培养。将Capan-2细胞在McMcoy 5A培养基(Thermo Fisher Scientific)中培养,将CFPAC-1细胞在Iscove改良的达尔伯克培养基(IMDM,Thermo Fisher Scientific)中培养,并将PANC-1细胞在达尔伯克改良伊格尔培养基中(DMEM,Thermo Fisher Scientific)培养。本文使用的所有培养基均补充有10%(w/v)的胎牛血清(FBS,Thermo Fisher Scientific)、100U/ml的青霉素和100mg/ml的链霉素。将MCF-7、T47D、ZR75-1、Capan-1、Capan-2、CFPAC-1和PANC-1细胞在5%CO2存在的情况下于37℃培养,并将MDA-MB-231细胞在不存在CO2的情况下于37℃培养。
实施例9-2:用于测试的抗体的制备
从Abcam(剑桥,英国)获得市售的抗MUC1-CT抗体和抗MUC1-CT2抗体,以便通过蛋白质印迹和免疫沉淀检测来自细胞的MUC1。抗MUC1-CT抗体和抗MUC1-CT2抗体识别MUC1的胞质尾区。抗β-肌动蛋白抗体购自Sigma-Aldrich。
实施例9-3:蛋白质印迹分析
将细胞溶解在裂解缓冲液(20mM Tris-HCl,pH 8.0,150mM NaCl,5mM EDTA,100mMNaF,2mM Na3Vo4,蛋白酶抑制剂混合物,1%(w/v)NP-40)中并在4℃下以14,000rpm离心20分钟。在4%-12%Bis-Tris梯度凝胶(Thermo Fisher Scientific)中分离相同量的蛋白质,并在室温下转移至用PBS-T中3%(w/v)的BSA封闭的硝酸纤维素膜上保持1小时。将硝酸纤维素膜与抗hMUC1抗体(hMUC1-1H7;实施例7)、抗MUC1-CT抗体(Abacam,EPR1023)、抗MUC1-CT2抗体(Abcam,ab80952)、或抗β-肌动蛋白抗体(Sigma-Aldrich)在4℃孵育过夜。使用与辣根过氧化物酶和增强型化学发光试剂(Thermo Fisher Scientific)缀合的二抗测量免疫反应蛋白。
实施例9-4:免疫沉淀测定
将细胞裂解缓冲液(20mM Tris-HCl,pH 8.0,150mM NaCl,5mM EDTA,100mM NaF,2mM Na3Vo4,蛋白酶抑制剂混合物,1%(w/v)NP-40)与抗hMUC1抗体在4℃孵育过夜。将蛋白A珠粒添加到混合物中,并在4℃下培养1小时。对通过离心收集的免疫复合物进行洗涤,并通过蛋白质印迹分析。将膜与抗hMUC1抗体(hMUC1-1H7)、抗MUC1-CT抗体(Abacam,EPR1023)、抗MUC1-CT2抗体(Abcam,ab80952)、或抗β-肌动蛋白抗体(Sigma-Aldrich)一起培养。
实施例9-5:去糖基化测定
将来自T47D细胞的细胞裂解液用裂解缓冲液(0.5%(w/v)SDS,1%(w/v)β-巯基乙醇)提取,并在100℃下煮沸10分钟。然后,将样品与PNGase F(Elpis-Biotech,大田,韩国)在37℃下一起培养2小时,并在100℃下煮沸10分钟。将获得的样品用裂解缓冲液稀释,并用抗hMUC1单克隆抗体免疫沉淀。用抗MUC-CT抗体或抗MUC1-CT2抗体通过蛋白质印迹分析所得的免疫复合物。
实施例9-6:共聚焦显微镜检查
将细胞在12孔培养板中的涂覆有聚-L-赖氨酸的玻璃盖玻片上培养。对于细胞表面染色,将细胞用4%(w/v)多聚甲醛固定,用3%(w/v)BSA封闭,并在冰上用抗hMUC1抗体染色2小时。对于细胞内染色,将细胞用4%(w/v)多聚甲醛固定,用0.1%(w/v)Triton X-100透化,用3%(w/v)BSA封闭,并在室温下用抗hMUC1抗体染色2小时。用PBS-T洗涤后,将获得的细胞样品与缀合Alexa Fluor 488的二抗(Invitrogen,尤金,俄勒冈州)一起培养1小时。将细胞核用Hoechst 33258染色。将所有细胞样品固定在共聚焦激光扫描显微镜系统(CLSM,LSM 710,Carl Zeiss,耶拿(Jena),德国)上并用所述显微镜系统观察。
实施例9-7:内化测定
根据制造商的说明(Thermo Fisher Scientific),用DyLight 488标记抗hMUC1单克隆抗体。将乳腺癌细胞和胰腺癌细胞用DyLight 488标记的抗hMUC1抗体处理,并在37℃下孵育指定的时间。用CLSM(LSM 710,Carl Zeiss)检测从具有内化抗体的细胞产生的荧光信号。
实施例10:抗hMUC1单克隆抗体的可变区的克隆
使用小鼠单克隆抗体同种型分型试剂盒(Dipstick格式,Bibco BRL或Roche,曼海姆,德国)培养抗hMUC1单克隆抗体(hMUC1-1H7;实施例7)。使用RNeasy Mini试剂盒(Qiagen)从杂交瘤细胞中提取总RNA,以产生cDNA。为了克隆从hMUC1-1H7产生的抗hMUC1单克隆抗体的重链可变区和轻链可变区(VH和VL),使用具有以下引物组的Vent聚合酶(NEB)扩增所得cDNA:
重链引物:IGG1(5'-GGA AGA TCT ATA GAC AGA TGG GGG TGT CGT TTT GGC-3';SEQ ID NO:14)和5'MH2(5'-CTT CCG GAA TTC SAR GTN MAG CTG SAG SAG TCW GG-3';SEQID NO:15)
轻链(κ链)的引物:3'Kc(5'-GGT GCA TGC GGA TAC AGT TGG TGC AGC ATC-3';SEQ ID NO:16)和5'Mk(5'-GG GAG CTC GAY ATT GTG MTS ACM CAR WCT MCA-3';SEQ IDNO:17)。
标准PCR反应进行25个循环。将所得的PCR产物直接连接至pGEM-T Easy载体(Promega)。通过DNA测序分析克隆的小鼠Ig插入物。
实施例11:抗hMUC1单克隆抗体的抗原结合片段(Fab)的表达
对编码抗hMUC1单克隆抗体(hMUC1-1H7;实施例7)的轻链可变区和重链可变区(VH和VL)的序列进行扩增,并分别使用SfiI和BstXI亚克隆到细菌表达载体FabE中(Jeon等人,Mol.Immunol.44:827-836(2007),Kwon等人,Oncol.Rep.18:513-517(2007)),并且将由此获得的重组表达质粒命名为“pFabE-hMUC1-1H7”(图8a)。
本文使用的引物如下。
用于扩增重链可变区编码序列(393bp)的引物:
正向引物:5'-ggc cca gcc ggc cat ggc cSA RGT NMA GCT GSA GSA GTC WGG-3'(SEQ ID NO:18);
反向引物:5'-GGC CGT GCT GGC CCC GAC AGA TGG GGG TGT CGT TTT GGC-3'(SEQ ID NO:19)。
用于扩增轻链可变区编码序列(396bp)的引物:
正向引物:5'-CCA TTG CAG TGG CAC TGG CTG GTT TCG CTA CCG TAG CAC AGGCAG CCG AYA TTG TGM TSA CMC ARW CTM CA-3'(SEQ ID NO:20);
反向引物:5'-CCA CCG TAC TGG CGG ATA CAG TTG GTG CAG CAT C-3'(SEQ IDNO:21)
通过限制性酶切分析和DNA测序鉴定重组表达质粒pFabE-hMUC1-1H7。将pFabE-hMUC1-1H7转化到TG1大肠杆菌(E.coli)细胞中,优化重组蛋白的表达,并使用抗His抗体通过蛋白质印迹鉴定pFabE-hMUC1-1H7。
为了大量产生和纯化重组Fab(pFabE-hMUC1-1H7),将500ml杂交瘤hMUC1-1H7克隆培养物用0.5mM IPTG处理,并在18℃下培养16小时。将培养溶液离心,将培养上清液收获,置于Ni-NTA亲和柱(Clontech)中,并使用VH-CH融合蛋白中的His标签分离适当折叠和组装的Fab蛋白。
实施例12:MTT测定
使用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑鎓溴化物(MTT,Sigma-Aldrich)溶液测量用抗hMUC1单克隆抗体(10μg/ml)处理5天的癌细胞的生长。在指定的时间,将MTT溶液添加到每个孔中,并将板在37℃下再培养4小时。除去介质后,将甲瓒晶体溶解在DMSO中。使用595nm分光光度计(参考波长为650nm)监测显色。
实施例13:体内生物分布成像
将50%(w/v)Matrigel中的5x106个Capan-2细胞皮下接种到4周龄雄性BALB/cAnCrj-nu/nu小鼠的右背侧中。对于乳腺癌细胞系注射,将17种β-雌二醇小球(美国,佛罗里达州,萨拉索塔(Sarasota),Innovative Research of America,小球,60天释放)皮下移植到小鼠中。第二天,将50%(w/v)Matrigel中的5x106个细胞(T47D,ZR75-1)皮下接种到小鼠的右背侧中。
当肿瘤体积达到平均约100mm3时,向小鼠静脉注射正常小鼠IgG-DyLight 755(5mg/kg)或抗hMUC1抗体-DyLight 755(5mg/kg)。然后,在0小时、24小时、48小时使用体内成像系统(IVIS 200,Xenogen公司,马萨诸塞州)监测抗体靶标图像。为了在检测体内抗hMUC1单克隆抗体的细胞内定位,静脉内注射DyLight 488标记的抗hMUC1抗体(5mg/kg)。两天后,收集肿瘤组织,并进行冷冻切片。用CLSM(LSM 710,Carl Zeiss)在肿瘤切片中检测到抗体的内化。
实施例14:胰腺癌小鼠模型的产生
为了进行异种移植分析,将50%Matrigel中的5x106个Capan-2细胞皮下接种到16只BALB/cAnCrj-nu/nu小鼠(n=8)的右背侧中。当肿瘤体积达到75mm3时,将小鼠随机分为两个处理组(每组八只小鼠),即PBS处理组和抗hMUC1单克隆抗体处理组。将抗体每周两次以10mg/kg的量在静脉内给予。使用卡尺以7天为间隔测量肿瘤直径,并使用以下式子计算肿瘤体积:(宽度2×长度)/2。用抗hMUC1单克隆抗体处理11周后,处死小鼠,并且然后对肿瘤称重。
实施例15:组织阵列和免疫组织化学
石蜡包埋的人乳腺癌组织切片购自ISU ABXIS(首尔,韩国)。用二甲苯处理组织切片30分钟以去除石蜡,用乙醇再水化,并与3%过氧化氢溶液一起孵育10分钟。抗原修复是在柠檬酸溶液(pH 6.0)中进行。将切片用正常马血清封闭30分钟,并在室温下与抗hMUC1抗体(1μg/载玻片)一起孵育2小时。将切片洗涤并与生物素化的抗小鼠IgG抗体(VectorLaboratories,伯林盖姆,加利福尼亚州)一起孵育1小时。然后,将组织切片洗涤,并与HRP-链霉亲和素一起孵育30分钟。使用3,3-二氨基联苯胺(DAB,Thermo Fisher Scientific)检测免疫反应性,并用苏木精(Muto Pure Chemicals,东京,日本)对组织切片进行复染。使用显微镜(Eclipse E-200,Nikon,东京,日本)检查所有图像。
实施例16:抗hMUC1单克隆抗体的表征
用PBS(对照)或rhMUC1-C和脂质体复合物(O)(n=4)免疫小鼠(参见实施例5),进行第三次免疫,收集小鼠血清,并且将rhMUC1-C特异性IgG的总量使用ELISA测量(参见实施例6)以鉴定针对rhMUC1-C的抗体的形成。结果示于图1a。在图1a中,对照代表接种PBS的组,而rhMUC1-C-1、-2、-3和-4代表单独的小鼠。从图1a可以看出,rhMUC1-C特异性IgG被显著诱导。
接下来,根据实施例7筛选从免疫小鼠中分离的杂交瘤细胞,并且结果示于图1b和图1c中。图1b示出了使用HAT培养基筛选的结果,并且图1c示出了使用HT培养基筛选的结果。在图1b和图1c中,#表示96孔板的数量,并且A-H表示板的水平部分的名称。从图1b和图1c的结果中,选择hMUC1-1H7克隆作为产生rhMUC1-C特异性单克隆抗体的杂交瘤。
选定的杂交瘤hMUC1-1H7细胞于2017年3月8日保藏在韩国首尔市钟路区Yeongeon-dong的韩国细胞系研究基金会(Korean Cell Line Research Foundation,KCLRF),保藏号为KCLRF-BP-00395。
接下来,从分离的杂交瘤hMUC1-1H7克隆中纯化抗hMUC1单克隆抗体,并使用SDS-PAGE和考马斯染色分析纯化的抗体。图2A示出了ELISA分析的结果,以确定将杂交瘤hMUC1-1H7克隆注射入小鼠腹膜腔后收集的腹水中rhMUC1-C特异性抗体的存在,其中对照血清是PBS接种组的结果。结果表明,在注射了hMUC1-1H7克隆的小鼠的腹水中存在rhMUC1-C特异性抗体,并且分离并纯化了rhMUC1-C特异性抗体。通过SDS-PAGE和考马斯染色来分析分离和纯化的抗hMUC1单克隆抗体,并且结果示于图2B。在图2B中,HC表示重链,并且LC表示轻链。
对几种IgG同种型进行ELISA以鉴定获得的抗MUC1抗体的同种型(参见实施例8),并且结果示于图2C中。从图2C中可以看出,纯化的抗MUC1抗体是IgG1类型。
实施例17:在乳腺癌细胞和胰腺癌细胞中的抗MUC1抗体特异性测试
为了鉴定获得的抗hMUC1单克隆抗体是否识别MUC1蛋白(在正常细胞中,MUC1蛋白包含由MUC1-N亚基结构域和MUC1-C亚基结构域构成的二聚体,并被高糖基化,但在癌细胞系中,MUC1蛋白主要由MUC1-C亚基结构域构成,并且被低糖基化),通过SDS-PAGE分离乳腺癌细胞(MCF-7、MDA-MB-231、T47D和ZR75-1)的细胞裂解物,并使用抗hMUC1单克隆抗体和抗MUC1-CT抗体进行蛋白质印迹(参见实施例9.4)。所获得的结果在图3A中示出。结果表明,抗MUC1-CT抗体检测到MCF-7、T47D和ZR75-1中的MUC1蛋白,但未检测到MDA-MB-231。另一方面,抗hMUC1单克隆抗体不能在所有细胞样品中识别MUC1蛋白。由于在MDA-MB-231细胞中未检测到MUC-1蛋白,所以此细胞系在所有测试中均用作阴性对照组。
为了确定抗hMUC1单克隆抗体是否可以识别完整(天然)状态的内源性MUC1蛋白,将MCF-7、MDA-MB-231、T47D和ZR75-1细胞的细胞裂解物用正常小鼠IgG或抗hMUC1单克隆抗体免疫沉淀,并使用抗MUC1-CT抗体进行免疫印迹(参见实施例9.5),并且结果示于图3B中。从图3b中可以看出,抗hMUC1单克隆抗体可以有效地免疫沉淀MCF-7、T47D和ZR75-1细胞中的在完整状态下的MUC1蛋白。
图3a的结果表明抗hMUC1单克隆抗体不识别在蛋白质印迹中使用的变性MUC1蛋白,而图3b的结果表明抗hMUC1单克隆抗体识别癌症中完整的MUC1蛋白,这指示抗hMUC1单克隆抗体识别MUC1蛋白的完整(固有)三级结构。
MUC1的细胞外结构域被密集糖基化到距细胞表面200至500nm。因此,确定抗hMUC1单克隆抗体是否能够识别去糖基化的蛋白核心(参见实施例9.6)。将T47D细胞裂解物用PNGase F处理,并使用抗MUC1-CT抗体、抗MUC1-CT2抗体、抗hMUC1单克隆抗体(实施例7)、或抗β-肌动蛋白抗体进行蛋白质印迹。结果示出于图3c中。从图3c可以看出,抗hMUC1单克隆抗体不能检测到MUC1蛋白。同时,图3D示出了用抗hMUC1单克隆抗体免疫沉淀经PNGase F处理的T47D细胞裂解物、并且然后用抗MUC1-CT或抗MUC1-CT2抗体对经PNGase F处理的T47D细胞裂解物进行免疫印迹的结果。从图3d可以看出,抗hMUC1单克隆抗体能够免疫沉淀T47D细胞中去糖基化的MUC1蛋白。这些结果证实,无论糖基化状态如何,抗hMUC1单克隆抗体都可以成功识别完整(天然)状态的MUC1蛋白。
为了测试抗hMUC1单克隆抗体是否可以识别胰腺癌细胞中完整的MUC1蛋白,通过SDS-PAGE分离了胰腺癌细胞(Capan-1、Capan-2、CFPAC-1和PANC-1)的细胞裂解物,使用抗MUC1-CT抗体、抗hMUC1抗体、或抗β-肌动蛋白抗体进行蛋白质印迹,并且结果示于图4a中。如在图4a中可见,抗hMUC1单克隆抗体不能在所有测试的细胞样品中识别MUC1蛋白。
同时,为了确定抗hMUC1单克隆抗体是否可以识别作为完整状态的固有MUC1蛋白,使用抗MUC1-CT抗体和抗hMUC1单克隆抗体对多种胰腺癌细胞(Capan-1、Capan-2、CFPAC-1和PANC-1)的细胞裂解物进行免疫沉淀和免疫封闭。所得结果示于图4b。从图4b可以看出,抗hMUC1单克隆抗体可以有效地免疫沉淀胰腺癌细胞(Capan-2和CFPAC-1)中完整的MUC1蛋白。
实施例18鉴定在细胞表面和细胞中MUC1蛋白由抗hMUC1单克隆抗体的识别和内化
为了鉴定抗hMUC1单克隆抗体是否可以识别并结合完整(未损伤)细胞中的MUC1蛋白,对乳腺癌细胞(MCF-7、MDA-MB-231、T47D和ZR75-1)和胰腺癌细胞(Capan-1、Capan-2、CFPAC-1和PANC-1)进行了免疫荧光染色,并通过共聚焦显微镜对结果进行了分析(参见实施例9-7)。
更特定地,将MCF-7、MDA-MB-231、T47D和ZR75-1细胞与抗hMUC1单克隆抗体或正常小鼠IgG在4℃(对于细胞表面MUC1蛋白)或室温(对于细胞内MUC1蛋白)下孵育,并且然后与缀合Alexa 488(绿色)的二抗孵育1小时,并且用Hoechst 33258(蓝色)将细胞核染色。用共聚焦显微镜(CLSM,LSM 710,Carl Zeiss,耶拿,德国)观察获得的荧光图像,并示于图5A中(比例尺:10μm)。此外,为了鉴定抗hMUC1单克隆抗体的内化,还用DyLight 488(绿色)标记的抗hMUC1单克隆抗体对MCF-7、MDA-MB-231、T47D、和ZR75-1细胞进行了染色,并在37℃下培养6小时。将细胞核用Hoechst 33258染色,并用共聚焦显微镜(CLSM,LSM 710,CarlZeiss,耶拿,德国)观察获得的荧光图像,并在图5B中示出(比例尺:10μm)。
另外,测试了抗hMUC1单克隆抗体与位于胰腺癌细胞的细胞表面上和细胞内区域中的MUC1蛋白的结合。将胰腺癌细胞系Capan-1、Capan-2、CFPAC-1和PANC-1细胞与抗hMUC1单克隆抗体或正常小鼠IgG在4℃(在细胞表面上)或室温(在细胞中)培养,并且和缀合的二抗一起培养1小时,并且将细胞核用Hoechst 33258染色。用共聚焦显微镜(CLSM,LSM 710,Carl Zeiss,耶拿,德国)观察获得的荧光图像,并在图6中示出(比例尺:10μm)。
从图5和图6所示的结果可以看出,抗hMUC1抗体在乳腺癌细胞(MCF-7、T47D和ZR75-1)和胰腺癌细胞(Capan-2和CFPAC-1)中明显使MUC1染色,并且MUC1位于细胞表面上和细胞内。
这些结果表明所述抗体识别MUC1 C末端亚基的细胞外区域。可以认为抗体作为治疗剂的功效更依赖于细胞内化。因此,将抗hMUC1抗体结合到DyLight 488上,并用抗体处理乳腺癌细胞和胰腺癌细胞6小时,并通过荧光成像鉴定抗体的细胞内化。用DyLight 488标记的抗hMUC1单克隆抗体处理胰腺癌细胞(Capan-1、Capan-2、CFPAC-1和PANC-1),并在37℃下培养0分钟、30分钟、1小时、3小时、6小时、12小时和24小时。将细胞核用Hoechst33258染色。用共聚焦显微镜(CLSM,LSM 710,Carl Zeiss,耶拿,德国)观察获得的荧光图像,并在图7中示出(比例尺:10μm)。从图5B可以看出乳腺癌中的细胞内化。
即,结果表明抗hMUC1单克隆抗体可以靶向并内化活细胞中的MUC1蛋白。
实施例19:抗hMUC1单克隆抗体的可变区的克隆结果
从产生抗hMUC1单克隆抗体的杂交瘤细胞(hMUC1-1H7)克隆抗hMUC1单克隆抗体的重链和轻链的可变区,并进行DNA测序(实施例10)。使用BLAST程序(http://www.ncbi.nlm.nih.gov)分析通过DNA测序鉴定的序列,以确定与已知序列的同源性。编码hMUC1-1H7抗体重链和轻链的可变区的cDNA(重链可变区编码cDNA:393bp;轻链可变区编码cDNA:396bp)与已知小鼠免疫球蛋白(IgG1)的重链和轻链的可变区的编码序列的序列同源性分别为80%-95%和93%-98%。
基于获得的DNA序列,通过已知方法(Kabat CDR定义)鉴定重链和轻链的CDR(表2)。重链和轻链的可变区序列示于下表3中。
[表2]
[表3]
实施例20:对于以下的测试:源自抗hMUC1单克隆抗体的重组Fab片段的表达的鉴定、以及重组Fab片段对癌细胞的MUC1蛋白的识别
使用表达载体pFabE在大肠杆菌(E.coli)中表达重组Fab(实施例11)。
载体pFabE包含两个克隆位点BstX I和Sfi I,其中插入了轻链恒定区(CL)、重链恒定区(CH1)、轻链可变区(VL)和重链可变区(VH)。重组表达质粒pFabE-hMUC1-1H7是通过依次亚克隆hMUC1-1H7的VL和VH序列而构建的(图8a)。重组质粒在大肠杆菌(E.coli)中的LacZ启动子的控制下诱导VL-CL融合蛋白和VH-CHl融合蛋白的双顺反子表达。VL-CL融合蛋白包括N末端OmpA标签和C末端Pre-S1标签,而VH-CH1融合蛋白包括N末端pelB标签和C末端His标签(图8b)。由于重组VH-CH1融合蛋白包含His标签,因此使用Ni-NTA亲和柱色谱进行大肠杆菌(E.coli)的大规模培养和从培养上清液中纯化hMUC1-1H7的重组Fab。
为了确定纯化的重组Fab(下文称为“重组Fab-hMUC1-1H7”)是否可以识别癌细胞的MUC1,对乳腺癌细胞(MCF-7、MDA-MB-231、T47D和ZR75-7)进行免疫荧光染色。将乳腺癌细胞MCF-7、MDA-MB-231、T47D和ZR75-1与重组Fab-hMUC1-1H7各自一起培养,并且然后与缀合Alexa 488的二抗一起培养,并且将细胞核用Hoechst 33258染色。用共聚焦显微镜(CLSM,LSM710,Carl Zeiss,耶拿,德国)观察获得的荧光图像,并在图8c中示出(比例尺:10μm)。
从图8c的共聚焦图像可以看出,重组Fab-hMUC1-1H7在乳腺癌细胞(MCF-7、T47D和ZR75-1)中清楚地染色了MUC1,指示重组Fab-hMUC1-1H7识别乳腺癌细胞的MUC1。
实施例21:抗hMUC1单克隆抗体对乳腺癌细胞增殖的抑制作用
MUC1在各种类型的癌组织中过表达,以促进细胞增殖。为了确定抗hMUC1单克隆抗体作为癌症治疗剂的有效性,检查了抗hMUC1单克隆抗体对乳腺癌细胞增殖的作用。
用抗hMUC1抗体(10μg/ml;实施例7)或正常小鼠IgG1(10μg/ml)处理MDA-MB-231、T47D和ZR75-1细胞,并通过MTT测定检查所述抗体对细胞增殖的作用(实施例12)。
所得结果示于图9。在图9中,对照是未用抗体处理的组。如图9所示,与用IgG处理的对照组相比,发现抗hMUC1抗体的处理导致T47D和ZR75-1细胞的增殖显著延迟。另一方面,抗hMUC1抗体没有改变MDA-MB-231细胞的增殖。这些结果证明抗hMUC1单克隆抗体对表达MUC1的癌细胞具有选择性的抗癌作用。
实施例22:鉴定注射到乳腺癌和胰腺癌中的抗hMUC1单克隆抗体的体内定位
为了证实抗hMUC1单克隆抗体的体内功效,将DyLight 755标记的正常IgG(5mg/kg)或DyLight 755标记的抗hMUC1单克隆抗体(5mg/kg)静脉内给予至具有T47D、ZR75-1或Capan-2肿瘤的BALB/C nu/nu小鼠,并进行了全身荧光成像(实施例13)。通过在0小时、24小时和48小时测量荧光的总通量(光子/秒)来定量标记抗体的分布。
所获得的结果示于图10a至图10h(乳腺癌)和图11a至图11c(胰腺癌)中。
图10a至图10d示出了通过皮下注射作为乳腺癌细胞的T47D细胞在小鼠中诱导肿瘤的结果,并且图10e至图10h示出了通过皮下注射作为乳腺癌细胞的ZR75-1细胞在小鼠中诱导肿瘤的结果。在图10b和图10f中,使用实时IVIS成像系统200对解剖的小鼠进行成像。图10c和图10g示出了抗体在各种分离的器官和肿瘤中分布的结果。图10d和图10h示出了具有用DAPI染色的细胞核的肿瘤切片的共聚焦显微镜检查的结果(比例尺:10μm)。
图10a、图10b、图10e、图10f、图11a和图11b的结果表明抗hMUC1抗体特异性地定位在肿瘤区域中。此外,图10c和图10g的结果表明,当分离肿瘤和其他器官时,抗hMUC1抗体仅位于肿瘤组织中,而不影响其他重要器官。
此外,结果示出,DyLight标记的抗hMUC1单克隆抗体在肿瘤切片中示出清晰的染色结果,而DyLight标记的正常IgG没有这种作用。此外,图10d、图10h和图11c的共聚焦图像进一步示出抗hMUC1单克隆抗体位于肿瘤特异性细胞中。
这些结果表明,抗hMUC1单克隆抗体可用于在动物模型中特异性地靶向乳腺癌和胰腺癌。
实施例23:在异种移植小鼠模型中测试抗hMUC1单克隆抗体的抗癌作用
使用异种移植小鼠模型在体内测试了靶向MUC1的单克隆抗体对胰腺癌细胞生长的作用。首先,将Capan-2细胞皮下注射到NORs小鼠(n=8)的右背侧中,以使肿瘤生长。当肿瘤大小达到75mm3时,每周两次静脉内给予抗hMUC1单克隆抗体,并监测肿瘤大小11周。根据实施例14进行异种移植小鼠模型的制备和肿瘤大小测试。
在异种移植小鼠模型测试中,PBS给药组的八只小鼠中有两只死亡,并且抗hMUC1单克隆抗体给药组的八只小鼠中有一只死亡。
从异种移植小鼠模型中提取的肿瘤组织示于图12a,并且肿瘤的大小((宽度2×长度)/2)、其重量和小鼠的体重分别示于图12b、图12c和图12d。从图12a至图12d可以看出,抗hMUC1单克隆抗体的给药抑制了胰腺肿瘤的进展,并且抗体治疗没有不利地影响受试者的体重。
实施例24:测试MUC1蛋白在人乳腺癌和胰腺癌组织中的表达
通过免疫组织化学研究了MUC1蛋白是否在乳腺癌和胰腺癌组织中表达。与作为对照组的正常组织相比,测试了乳腺癌组织和胰腺癌组织。结果示于图13(乳腺癌)和图14(胰腺癌)中。从图13和图14可以看出,MUC1在大多数乳腺癌组织和胰腺癌组织中表达,而MUC1在正常乳腺组织中不表达。
乳腺癌组织和胰腺癌组织中MUC1表达的免疫组织化学分析结果分别示于表4(乳腺癌组织)和表5(胰腺癌组织)中。
[表4]
[表5]
如表4所示,对51个乳腺癌样品的分析结果表明,所有样品中的18%在超过75%的肿瘤细胞中呈MUC1阳性。14%和25%的癌细胞样品分别在50%-74%和11%-49%的肿瘤细胞中呈MUC1表达呈阳性。但是,有12%的乳腺癌样品不表达MUC1。
另外,如表5所示,在33个胰腺癌样品的情况下,3%的样品在75%或更多的肿瘤细胞中呈MUC1阳性。9.1%和48.5%的癌症样品分别在50%-74%和11%-49%的肿瘤细胞中呈MUC1表达阳性。
这些结果表明MUC1表达可用于乳腺癌的诊断和治疗。
实施例25:鉴定抗hMUC1小鼠抗体(1H7)ADC的细胞毒性
ADC是在Levene Biopharma(美国)中通过将MMAE与1H7抗体结合来产生。制备的ADC中MMAE与抗体之比(药物与抗体之比,DAR)为5.80。MDA-MB-231、T47D和ZR75-1的三种细胞系用于1H7-ADC的细胞毒性。将每种细胞系培养在96孔板中,并且开始培养后24小时,以预定浓度处理1H7-ADC,如图15所示。1H7-ADC处理后72小时,使用CCK-8试剂盒(Dojindo,美国)比较细胞存活率。根据提供的手册进行CCK-8处理,并使用I3X酶标仪(MolecularDevices,美国)测量吸光度。用1H7-ADC处理的T47D和ZR75-1细胞的存活率显著低于未使用1H7-ADC处理的对照组的存活率。相比之下,用1H7-ADC处理的MDA-MB-231细胞的存活率与对照组的存活率没有显著差异。这些结果意味着1H7-ADC对表达MUC1的癌细胞具有选择性的抗癌作用。
实施例26:抗hMUC1单克隆抗体(1H7)-ADC对源自患者的乳腺癌组织中的癌症增殖的抑制作用
在动物模型中鉴定了实施例25中提及的1H7-ADC对癌症增殖的抑制作用。将购自Jung-ah Biotech(首尔,韩国)的过表达MUC1的源自患者的乳腺癌组织(TNBC)移植到NRGA小鼠中,并且当移植到小鼠的乳腺癌组织生长超过一定大小(100-200mm3)时,通过血管注射向小鼠给予1H7-ADC。如图16所示,接受1H7-ADC的小鼠的癌组织大小随时间减小,而没有接受1H7-ADC的对照小鼠的癌组织大小随时间按比例增加。结果指示1H7-ADC具有在源自表达MUC1的患者的癌组织中在组织水平上抑制癌细胞生长的作用。
实施例27:基于1H7的人源化抗体的产生
基于1H7抗体产生了人源化抗体,将所述人源化抗体用于鉴定通过ADC产生在动物模型中的细胞毒性和抗癌作用。人源化抗体由Osong High-Tech Medical IndustryPromotion Foundation(Osong,韩国)和Fusion Antibodies(英国)的新药开发支持中心生产。通过使用1H7抗体的相同抗原识别位点并改变可变区序列而不是抗原识别位点,产生了六个重链序列和六个轻链序列。每个氨基酸序列示于表6中。
[表6]
实施例28:测量人源化抗体对hMUC1-C的结合亲和力
通过ELISA方法测量实施例27中制备的抗hMUC1-C人源化抗体与hMUC1-C蛋白结合的亲和力。通过用制备的抗hMUC1-C人源化抗体以相同浓度包被96孔免疫板并用超级封闭溶液封闭板,来进行ELISA的亲和力分析。然后,将MBP-hMUC1-C蛋白稀释并添加到包被有抗hMUC1-C人源化抗体的免疫板上,并使其在37℃的培养箱中静置以诱导结合。将板用洗涤溶液洗涤三次,并与缀合HRP的抗MBP抗体反应。使用TMB底物溶液使反应继续进行,并使用2NHCl终止反应。然后,通过I3X酶标仪(Molecular Devices,美国)测量通过抗原-抗体结合反应获得的吸光度值,并进行比色测定。图17示出了包被的人源化抗体对MBP-hMUC1-C的结合亲和力的差异。
实施例29:鉴定抗hMUC1-C人源化抗体与抗hMUC1-C小鼠抗体(1H7)之间的表位同源性
通过竞争性ELISA鉴定了产生的抗hMUC1-C人源化抗体和抗hMUC1-C小鼠抗体的表位同源性。将MBP-hMUC1-C蛋白稀释并以各种浓度包被在96孔免疫板上。然后,将生物素标记的50nM抗hMUC1-C人源化抗体和抗hMUC1-C小鼠抗体从5μM连续稀释,同时添加至包被有MBP-hMUC1-C蛋白的板中,并静置以诱导反应。以恒定浓度处理HRP标记的链霉亲和素,以检测抗hMUC1-C人源化抗体标记的生物素。用于鉴定亲和力的TMB底物溶液用于比色分析。使用I3X酶标仪(Molecular Devices,美国)测量并分析反应的吸光度。结果示于图18中,并且吸光度随着没有生物素标记的抗hMUC1-C小鼠抗体的浓度增加而降低,这表明抗hMUC1-C人源化抗体和抗hMUC1-C小鼠抗体识别相同的表位。
实施例30:抗hMUC1-C人源化抗体与hMUC1-C的细胞结合模式分析(FACS分析)
进行FACS分析以分析已知表达hMUC1的细胞系中抗hMUC1人源化抗体与hMUC1-C的细胞结合模式。将每种细胞系在培养基中培养,并将预定数量的细胞分到对应的试管中。然后,将细胞用4%多聚甲醛固定,离心并用FACS分析溶液洗涤一次。用抗hMUC1人源化抗体处理所制备的细胞系,并在4℃下培养,以使相应的人源化抗体与细胞结合。然后,将细胞用FITC标记的抗人IgG抗体处理,并使用BD FACS Canto(BD,美国)进行FACS分析。分析结果示于图19,并与作为对照物质的人IgG的结果进行了比较。由于在已知表达hMUC1的三种细胞系中抗hMUC1人源化抗体与hMUC1-C之间的结合,荧光值增加,并且在不表达hMUC1的MDA-MB-231细胞系中荧光值未改变。这指示产生的抗hMUC1人源化抗体特异性地识别细胞中表达的hMUC1。
FACS分析示出,当用各种浓度的抗hMUC1-C人源化抗体(G3)和抗hMUC1-C小鼠抗体(1H7)处理发现表达hMUC1的ZR75-1细胞系时,荧光值取决于抗体的浓度而增加。分析结果示于图20。
实施例31:鉴定抗hMUC1人源化抗体ADC(G3-ADC)的细胞毒性
实施例31-1:鉴定乳腺癌细胞系中的细胞毒性
ADC是在Alteoxen(大田,韩国)通过将MMAE与抗hMUC1人源化抗体结合来产生。产生的ADC中MMAE与抗体之比(DAR)为4.8。使用MDA-MB-231(MUC1-,HER2-)、T47D(MUC1+,HER2+)和ZR75-1(MUC1+,HER2+)的三种细胞系确定所产生ADC的细胞毒性。将每个细胞系在96孔板中培养。培养开始后24小时,用MUC1-ADC(G3-ADC)和作为对照组的Kadcyla(HER2-ADC)以相应的浓度处理细胞。ADC处理后72小时,使用CellTiterGlo试剂盒(Promega,美国)比较了细胞存活率。根据手册进行CellTiterGlo处理方法,并使用I3X酶标仪(MolecularDevices,美国)测量吸光度。用G3-ADC处理的T47D和ZR75-1细胞的生长比例显著低于未用G3-ADC处理的对照组的生长比例(图21)。另一方面,用ADC处理的MDA-MB-231细胞的存活率与对照组的存活率没有显著差异。这些结果指示G3-ADC对表达MUC1的癌细胞具有选择性抑制作用。与G3-ADC相比,用作对照组的Kadcyla具有较低的细胞毒性,这被认为是由于测试细胞中Her2的表达水平较低。
实施例31-2:鉴定在髓性白血病细胞系中的细胞毒性
使用与实施例31-1相同的抗hMUC1人源化抗体ADC鉴定了髓性白血病细胞系中的细胞毒性。本文使用的髓性白血病细胞系是两种类型即K562(MUC1+)和KG-1(MUC1-)株系。在96孔板中培养每种细胞系,并且在开始培养后24小时,用抗hMUC1人源化抗体ADC以相应的浓度处理细胞。在用抗hMUC1人源化抗体ADC处理后72小时,通过MTT测定比较细胞存活率(活力,%)。用抗hMUC1人源化抗体ADC处理的细胞的存活率显著低于未处理的对照组的存活率。此结果示于图22中,并且在两种细胞系中仅在发现表达MUC1的K562细胞系中发现了细胞毒性。
实施例32:抗hMUC1-C人源化抗体-ADC对源自患者的乳腺癌组织中的癌症增殖的抑制作用
在动物模型中鉴定了实施例31中提及的抗hMUC1-C人源化抗体-药物缀合物对癌症增殖的抑制作用。将从Jung-ah Biotech(首尔,韩国)购买的源自过表达MUC1的患者的乳腺癌组织(TNBC)移植到总共24只NRGA小鼠中。当移植到小鼠中的乳腺癌组织生长超过一定大小(100-200mm3)时,将它们随机选择并分成4组(n=5)。对应的组是阴性对照组,给予Kadcyla(5mg/kg)的组和给予抗hMUC1-G3-ADC(5mg/kg和10mg/kg)的组,每周一次总计三次对它们通过血管注射给药。从图23可以看出,接受抗hMUC1-C人源化抗体-药物缀合物的小鼠的癌组织大小减小,而未接受抗hMUC1-C人源化抗体-药物缀合物的对照组小鼠的癌组织大小未显示出统计学显著性差异。这些结果指示,抗hMUC1-C人源化抗体-药物缀合物具有抑制表达MUC1的癌组织、特别是TNBC乳腺癌组织的生长的作用。在所测试的任何动物中均未观察到统计学上显著的体重变化,并且发现癌细胞生长以浓度依赖性方式被有效抑制。统计分析通过双因素方差分析进行。
保藏号
保管机构:韩国细胞系研究基金会
保藏号:KCLRFBP00395
保藏日期:2017年3月8日
实用性
根据本发明,与MUC1或其抗原结合片段特异性地结合的抗体展现出对MUC1的优异的亲和力和结合能力,并且抗体-药物缀合物(其中药物与抗体或其抗原结合片段缀合)通过与表达MUC1的细胞特异性地结合而有效地且特异性地或选择性地递送药物。因此,根据本发明的抗MUC1抗体和抗体-药物缀合物可以有效地应用于治疗MUC1相关疾病,例如癌症。
尽管已经详细描述了本发明的具体配置,但是本领域技术人员应理解,将本说明书作为优选实施方案提供,并且不应解释为限制本发明的范围。因此,本发明的实质范围由提交的所附权利要求及其等同物限定。
序列表自由文本
附有电子文件。
<110> 株式会社菲特伦
<120> 特异性地结合MUC1的抗体及其用途
<130> PP-B2039
<150> KR 10-2017-0035622
<151> 2017-03-21
<160> 26
<170> KoPatentIn 3.0
<210> 1
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> 重链可变区CDR1
<400> 1
Gly Tyr Thr Phe Thr Ser Tyr Trp Met His
1 5 10
<210> 2
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 重链可变区CDR2
<400> 2
Tyr Ile Asn Pro Gly Thr Gly Tyr Ile Glu Tyr Asn Gln Lys Phe Lys
1 5 10 15
Asp
<210> 3
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 重链可变区CDR3
<400> 3
Ser Thr Ala Pro Phe Asp Tyr
1 5
<210> 4
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> 轻链可变区CDR1
<400> 4
Lys Ala Ser Gln Asp Ile Lys Ser Tyr Leu Ser
1 5 10
<210> 5
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 轻链可变区CDR2
<400> 5
Tyr Ala Thr Arg Leu Ala Asp
1 5
<210> 6
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 轻链可变区CDR3
<400> 6
Leu Gln Tyr Asp Glu Ser Pro Tyr Thr
1 5
<210> 7
<211> 1255
<212> PRT
<213> 人工序列
<220>
<223> MUC1
<400> 7
Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr
1 5 10 15
Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly
20 25 30
Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser
35 40 45
Thr Glu Lys Asn Ala Val Ser Met Thr Ser Ser Val Leu Ser Ser His
50 55 60
Ser Pro Gly Ser Gly Ser Ser Thr Thr Gln Gly Gln Asp Val Thr Leu
65 70 75 80
Ala Pro Ala Thr Glu Pro Ala Ser Gly Ser Ala Ala Thr Trp Gly Gln
85 90 95
Asp Val Thr Ser Val Pro Val Thr Arg Pro Ala Leu Gly Ser Thr Thr
100 105 110
Pro Pro Ala His Asp Val Thr Ser Ala Pro Asp Asn Lys Pro Ala Pro
115 120 125
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
130 135 140
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
145 150 155 160
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
165 170 175
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
180 185 190
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
195 200 205
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
210 215 220
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
225 230 235 240
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
245 250 255
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
260 265 270
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
275 280 285
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
290 295 300
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
305 310 315 320
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
325 330 335
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
340 345 350
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
355 360 365
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
370 375 380
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
385 390 395 400
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
405 410 415
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
420 425 430
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
435 440 445
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
450 455 460
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
465 470 475 480
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
485 490 495
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
500 505 510
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
515 520 525
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
530 535 540
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
545 550 555 560
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
565 570 575
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
580 585 590
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
595 600 605
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
610 615 620
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
625 630 635 640
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
645 650 655
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
660 665 670
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
675 680 685
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
690 695 700
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
705 710 715 720
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
725 730 735
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
740 745 750
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
755 760 765
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
770 775 780
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
785 790 795 800
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
805 810 815
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
820 825 830
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
835 840 845
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
850 855 860
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
865 870 875 880
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
885 890 895
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
900 905 910
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
915 920 925
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Asn
930 935 940
Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr Ser
945 950 955 960
Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn Gly
965 970 975
Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro Phe
980 985 990
Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser His
995 1000 1005
Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Ser Val Pro Pro
1010 1015 1020
Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly Val
1025 1030 1035 1040
Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn Ser
1045 1050 1055
Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp
1060 1065 1070
Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly
1075 1080 1085
Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu Thr
1090 1095 1100
Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr Gln
1105 1110 1115 1120
Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile
1125 1130 1135
Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser
1140 1145 1150
Gly Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val Cys
1155 1160 1165
Val Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys
1170 1175 1180
Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg
1185 1190 1195 1200
Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly
1205 1210 1215
Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val
1220 1225 1230
Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val
1235 1240 1245
Ala Ala Thr Ser Ala Asn Leu
1250 1255
<210> 8
<211> 192
<212> PRT
<213> 人工序列
<220>
<223> MUC1细胞外结构域
<400> 8
Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn Gly
1 5 10 15
Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro Phe
20 25 30
Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser His
35 40 45
Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Ser Val Pro Pro
50 55 60
Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly Val
65 70 75 80
Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn Ser
85 90 95
Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp
100 105 110
Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly
115 120 125
Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu Thr
130 135 140
Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr Gln
145 150 155 160
Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile
165 170 175
Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser
180 185 190
<210> 9
<211> 119
<212> PRT
<213> 人工序列
<220>
<223> MUC1 SEA结构域
<400> 9
Pro Gln Leu Ser Thr Gly Val Ser Phe Phe Phe Leu Ser Phe His Ile
1 5 10 15
Ser Asn Leu Gln Phe Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr
20 25 30
Tyr Gln Glu Leu Gln Arg Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr
35 40 45
Lys Gln Gly Gly Phe Leu Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly
50 55 60
Ser Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn
65 70 75 80
Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala
85 90 95
Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser Asp Val Pro
100 105 110
Phe Pro Phe Ser Ala Gln Ser
115
<210> 10
<211> 55
<212> PRT
<213> 人工序列
<220>
<223> MUC1-C细胞外结构域
<400> 10
Ser Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn
1 5 10 15
Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala
20 25 30
Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser Asp Val Pro
35 40 45
Phe Pro Phe Ser Ala Gln Ser
50 55
<210> 11
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> CpG-DNA
<400> 11
agcagcgttc gtgtcggcct 20
<210> 12
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> sense 引物
<400> 12
ccatggcctc aggctctgca tc 22
<210> 13
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 反义引物
<400> 13
ctcgagagac tgggcagaga aaggaaat 28
<210> 14
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 14
ggaagatcta tagacagatg ggggtgtcgt tttggc 36
<210> 15
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 15
cttccggaat tcsargtnma gctgsagsag tcwgg 35
<210> 16
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 16
ggtgcatgcg gatacagttg gtgcagcatc 30
<210> 17
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 17
gggagctcga yattgtgmts acmcarwctm ca 32
<210> 18
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 前向引物
<400> 18
ggcccagccg gccatggccs argtnmagct gsagsagtcw gg 42
<210> 19
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 反向引物
<400> 19
ggccgtgctg gccccgacag atgggggtgt cgttttggc 39
<210> 20
<211> 71
<212> DNA
<213> 人工序列
<220>
<223> 前向引物
<400> 20
ccattgcagt ggcactggct ggtttcgcta ccgtagcaca ggcagccgay attgtgmtsa 60
cmcarwctmc a 71
<210> 21
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 反向引物
<400> 21
ccaccgtact ggcggataca gttggtgcag catc 34
<210> 22
<211> 116
<212> PRT
<213> 人工序列
<220>
<223> hMUC1-1H7_重链可变区
<400> 22
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Gly Thr Gly Tyr Ile Glu Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Ser Thr Ala Pro Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 23
<211> 108
<212> PRT
<213> 人工序列
<220>
<223> hMUC1-1H7_轻链可变区
<400> 23
Asp Ile Val Ile Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Trp Lys Ser Pro Lys Thr Leu Ile
35 40 45
Tyr Tyr Ala Thr Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser
65 70 75 80
Asp Asp Thr Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Ser Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 24
<211> 116
<212> PRT
<213> 人工序列
<220>
<223> G3_重链可变区
<400> 24
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Gly Thr Gly Tyr Ile Glu Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Ser Thr Ala Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 25
<211> 108
<212> PRT
<213> 人工序列
<220>
<223> G3_轻链可变区
<400> 25
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Asp Ile Lys Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Tyr Ala Thr Arg Leu Ala Asp Gly Ile Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Tyr Asp Glu Ser Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 26
<211> 576
<212> DNA
<213> 人工序列
<220>
<223> hMUC1-C
<400> 26
gcctcaggct ctgcatcagg ctcagcttct actctggtgc acaacggcac ctctgccagg 60
gctaccacaa ccccagccag caagagcact ccattctcaa ttcccagcca ccactctgat 120
actcctacca cccttgccag ccatagcacc aagactgatg ccagtagcac tcaccatagc 180
tcggtacctc ctctcacctc ctccaatcac agcacttctc cccagttgtc tactggggtc 240
tctttctttt tcctgtcttt tcacatttca aacctccagt ttaattcctc tctggaagat 300
cccagcaccg actactacca agagctgcag agagacattt ctgaaatgtt tttgcagatt 360
tataaacaag ggggttttct gggcctctcc aatattaagt tcaggccagg atctgtggtg 420
gtacaattga ctctggcctt ccgagaaggt accatcaatg tccacgacgt ggagacacag 480
ttcaatcagt ataaaacgga agcagcctct cgatataacc tgacgatctc agacgtcagc 540
gtgagtgatg tgccatttcc tttctctgcc cagtct 576
Claims (27)
1.一种抗MUC1抗体或其抗原结合片段,识别包含在MUC1的C末端细胞外结构域中的至少五个连续氨基酸的多肽。
2.根据权利要求1的抗MUC1抗体或其抗原结合片段,其中所述MUC1的C末端细胞外结构域具有SEQ ID NO:10所示的氨基酸序列。
3.根据权利要求1的抗MUC1抗体或其抗原结合片段,包含:
SEQ ID NO:1的重链CDR1;SEQ ID NO:2的重链CDR2;SEQ ID NO:3的重链CDR3;以及
SEQ ID NO:4的轻链CDR1;SEQ ID NO:5的轻链CDR2;和SEQ ID NO:6的轻链CDR3。
4.根据权利要求3的抗MUC1抗体或其抗原结合片段,包含:
SEQ ID NO:22或24的重链可变区;和
SEQ ID NO:23或25的轻链可变区。
5.根据权利要求4的抗MUC1抗体或其抗原结合片段,包含:
SEQ ID NO:22的重链可变区和SEQ ID NO:23的轻链可变区;或
SEQ ID NO:24的重链可变区和SEQ ID NO:25的轻链可变区。
6.一种用于产生抗MUC1抗体的杂交瘤,其保藏号为KCLRF-BP-00395。
7.一种抗体-药物缀合物,其中药物与根据权利要求1的抗MUC1抗体或其抗原结合片段缀合。
8.根据权利要求7的抗体-药物缀合物,其中所述抗MUC1抗体或其抗原结合片段通过接头与所述药物连接。
9.根据权利要求8的抗体-药物缀合物,其中所述接头是可切割的接头或不可切割的接头。
10.根据权利要求7的抗体-药物缀合物,其中所述药物是化学治疗剂、毒素、微小RNA(miRNA)、siRNA、shRNA或放射性同位素。
11.一种双特异性抗体,其包含根据权利要求1的抗MUC1抗体或其抗原结合片段。
12.根据权利要求11的双特异性抗体,其中所述双特异性抗体包含抗MUC1抗体或其抗原结合片段,以及能够结合癌相关抗原或免疫检查点蛋白抗原或免疫效应细胞特异性靶分子的抗体。
13.一种用于预防或治疗癌症的药物组合物,其包含根据权利要求1至5中任一项的抗MUC1抗体或其抗原结合片段、根据权利要求7至10中任一项的抗体-药物缀合物、或根据权利要求11至12中任一项的双特异性抗体。
14.根据权利要求13的用于预防或治疗癌症的药物组合物,其中所述癌症表达MUC1蛋白。
15.根据权利要求14的用于预防或治疗癌症的药物组合物,其中所述癌症是乳腺癌、胰腺癌、前列腺癌、肺癌、甲状腺癌、胃癌、卵巢癌、结直肠癌、肝癌、胆囊癌、肾癌、宫颈癌或膀胱癌。
16.一种用于诊断癌症的组合物,其包含根据权利要求1至5中任一项的抗MUC1抗体或其抗原结合片段。
17.根据权利要求16的用于诊断癌症的组合物,其中所述癌症表达MUC1蛋白。
18.一种包含复合物的免疫原性组合物,所述复合物包含包封在脂质体中的(1)MUC1的C末端区域、MUC1的SEA结构域或MUC1的C末端细胞外结构域,和(2)CpG-DNA。
19.根据权利要求18的免疫原性组合物,其中所述脂质体是以1:0.5至1:2的摩尔比混合的二油酰磷脂酰乙醇胺(DOPE)和胆甾醇半琥珀酸酯(CHEMS)的混合物。
20.根据权利要求18的免疫原性组合物,其中所述CpG-DNA是包含具有一至三个CpG基序的总共10至20个核苷酸的寡脱氧核苷酸。
21.一种产生抗MUC1-SEA单克隆抗体的方法,其包括用根据权利要求18至20中任一项的免疫原性组合物接种小鼠。
22.一种核酸,其编码根据权利要求1至5中任一项的抗MUC1抗体或其抗原结合片段。
23.一种重组表达载体,其包含根据权利要求22的核酸。
24.一种用根据权利要求23的重组表达载体转化的宿主细胞。
25.根据权利要求24的宿主细胞,其中所述宿主细胞选自动物细胞、植物细胞、酵母、大肠杆菌和昆虫细胞。
26.根据权利要求25的宿主细胞,其中所述宿主细胞选自猴肾细胞(COS7)、NSO细胞、SP2/0细胞、中国仓鼠卵巢(CHO)细胞、W138、幼仓鼠肾(BHK)细胞、MDCK、骨髓瘤细胞系,HuT78细胞、HEK293细胞、大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Escherichia coli)、链霉菌属物种(Streptomyces sp)、假单胞菌属物种(Pseudomonas sp)、奇异变形杆菌(Proteus mirabilis)、葡萄球菌属物种(Staphylococcus sp)、曲霉属物种(Aspergillussp),巴斯德毕赤酵母(Pichia pastoris)、酿酒酵母(Saccharomyces cerevisiae)、裂殖酵母属物种(Schizosaccharomyces sp)和粗糙脉孢菌(Neurospora crassa)。
27.一种产生抗MUC1抗体或其抗原结合片段的方法,所述方法包括培养根据权利要求24至26中任一项的宿主细胞。
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PE20230602A1 (es) * | 2020-03-18 | 2023-04-10 | Biomodifying Llc | Anticuerpos anti-sea de muc1 |
KR20220092432A (ko) * | 2020-12-24 | 2022-07-01 | 주식회사 엘지화학 | 뮤신 1에 특이적인 폴리펩티드 및 이의 이용 |
JP2024529506A (ja) * | 2021-07-30 | 2024-08-06 | アール.ピー.シェーラー テクノロジーズ,エルエルシー | ネクチン-4に特異的な抗体および抗体複合体、並びにそれらの使用方法 |
WO2023027534A1 (ko) | 2021-08-27 | 2023-03-02 | 주식회사 펩트론 | 신규 항 muc1항체 및 이의 용도 |
CN114028539B (zh) * | 2021-09-13 | 2024-04-26 | 北京大学 | 粘蛋白1在抑制冠状病毒中的应用 |
AU2022434346A1 (en) * | 2022-01-24 | 2024-07-25 | Cyron Therapeutics Co., Ltd. | Antibody specifically binding to muc-1 and uses thereof |
WO2024183635A1 (en) * | 2023-03-03 | 2024-09-12 | Beigene, Ltd. | Muc1 and cd16a antibodies and methods of use |
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