CN114846145A - 用于蛋白质表达的mRNA构建体及其用途 - Google Patents
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Abstract
本发明涉及mRNA或mRNA构建体,其包括:(a)编码靶蛋白或肽的区域,(b)在5′末端的β‑珠蛋白UTR区域,和(c)在3′末端插入BGH、BGH‑IRES或IRES‑BGH的区域,其中本发明的mRNA或mRNA构建体增强嵌合抗原受体或嵌合抗原受体蛋白(CAR蛋白)的细胞内表达、增强其表达稳定性、增强细胞内持久性、增加嵌合抗原受体的体内稳定性、和增加癌细胞的细胞毒性,因此可以有利地用于靶向实体癌的抗癌药物组合物和抗癌免疫疗法。
Description
技术领域
本发明涉及一种mRNA构建体,其能够稳定且高效地诱导靶蛋白的表达,同时保持靶蛋白的功能,并且本发明的mRNA构建体包括,在编码靶蛋白(或肽)的区域上游的5′-β-珠蛋白UTR,并且包括在其下游的BGH、BGH-IRES、或IRES-BGH区域。
本申请基于韩国知识产权局在2019年11月26日提交的第2019-0152938号韩国专利申请,并要求其优先权,该申请公开的内容通过引用整体合并于此。
背景技术
在几十年来,治疗癌症的方法一直在不断变化和进步。从1800年代到1900年代,通常实施诸如外科手术、化学疗法和放射疗法的方法,但是由于它们的局限性开始显现,近年来,作为免疫细胞疗法,已经在开发细胞疗法技术,其从身体提取免疫细胞,并且在将它们放回之前通过基因工程增强或修饰细胞。这种技术的公知实例包括肿瘤浸润淋巴细胞(TIL)、嵌合抗原受体(CAR)、T细胞受体(TCR)技术等,特别是,正在进行使用CAR(嵌合抗原受体)的研究和临床试验,CAR是使用基因重组工程的人工受体。然而,由于基因偶然被引入基因组的随机位置,使用上述基因工程的基因治疗或细胞治疗技术面临着从不良免疫反应到安全问题的多重挑战。在这点上,在使用DNA的转染的CAR-NK细胞(嵌合抗原受体自然杀伤细胞)中存在CAR基因整合到基因组中或发生突变的问题,因此,不能确保安全性。为了解决这些问题,已经尝试使用瞬时转染的CAR-NK细胞。然而,在使用mRNA的瞬时转染中,mRNA的不稳定性和低转染水平以及靶蛋白表达水平成为障碍,因此,还没有产生积极结果的研究。
发明内容
技术问题
为了解决上述问题,本发明的一个方面提供了mRNA构建体,其可以用于转染以使得靶蛋白(或肽)在细胞内无基因组突变地表达。
技术方案
根据本发明的一个方面,提供了mRNA构建体,其包括(a)编码靶蛋白或肽的区域,(b)靶蛋白编码区上游的5′-β-珠蛋白UTR区域,和(c)靶蛋白编码区下游的BGH、BGH-IRES或IRES-BGH区域。
根据本发明的一个实施方案,mRNA构建体可以用于瞬时转染。
根据本发明的另一个实施方案,靶蛋白(a)可以是嵌合抗原受体(CAR)。
根据本发明的另一个实施方案,CAR可以包括能够特异性结合靶的胞外域、穿透细胞膜的跨膜(TM)结构域、和诱导细胞内信号转导的胞内域。
根据本发明的另一个实施方案,所述胞外域可以是其中轻链和重链通过接头连接的抗体片段(ScFv),并且所述胞内域可以是CD28并且可以进一步包括DAP10和/或CD3z。
根据本发明的另一个实施方案,CAR的胞外域和TM结构域可以通过间隔区(spacer)连接,并且间隔区可以是Myc-铰链。
根据本发明的另一个实施方案,mRNA构建体可以在5′末端包括帽,并且可以在3′末端包括多聚(A)尾。
另外,本发明提供了包括mRNA构建体的碱基序列或其互补碱基序列的重组载体。
另外,本发明提供了一种瞬时转化体的制备方法,包括以下步骤:
(1)制备mRNA构建体或重组载体;以及
(2)将所述mRNA构建体或所述重组载体引入细胞中。
根据本发明的一个实施方式,步骤(2)可利用电穿孔,并可优选利用NEPA21系统。使用NEPA21系统的电穿孔可在110V至200V的条件下进行,优选在110V至140V的条件下进行,更优选在110V的条件下进行。
在上述制备方法中,细胞可以是植物细胞或动物细胞,但优选动物细胞,在动物细胞中可以是自然杀伤细胞(NK细胞)。
另外,本发明提供了包括mRNA构建体或重组载体的转染细胞。转染的细胞可以是瞬时转染的细胞。转染细胞可以通过上述制备方法制备,并表达染色体中没有DNA插入的靶蛋白(或肽)。靶蛋白(或肽)可以表达3天。
根据本发明的一个实施方案,NK细胞是原代NK细胞或NK细胞系,并且CAR-NK可经历经由NEPA21系统的转染。另外,上述转染可以在110V至200V的条件下进行。
同时,在其中上述制备方法中的细胞是NK细胞,且由引入细胞中的mRNA构建体或重组载体编码的靶蛋白是CAR的情况下,本发明的转染细胞制备方法可用作嵌合抗原受体自然杀伤(CAR-NK)细胞的制备方法,其中mRNA构建体或重组载体转染入细胞可利用NEPA21系统在110V至200V(优选地,对于NK宿主细胞为110V,对于原代NK细胞为200V)的条件下进行。
另外,本发明可以提供通过上述CAR-NK细胞制备方法制备的CAR-NK细胞,并且如此制备的CAR-NK细胞表达在染色体中没有DNA插入的嵌合抗原受体(CAR),因此具有高的体内稳定性。
此外,本发明提供了用于治疗癌症的药物组合物,其包括CAR-NK细胞。癌症可以是实体癌,并且实体癌可以是乳腺癌和/或肺癌。
另外,本发明提供一种癌症治疗方法,其包括将上述CAR-NK细胞施用于受试者的步骤。
此外,本发明提供CAR-NK细胞用于制备抗癌剂的用途。
有益效果
本发明的mRNA稳定,可以导入细胞内,长期表达功能性的靶蛋白,因此有望在转染技术、特别是瞬时转染技术中非常有用。此外,通过本发明的方法制备的转染的CAR-NK显示优异的癌细胞毒性,并且表达CAR而在NK细胞的基因组中没有基因插入,因此确保了体内安全性,并且因此可以有利地用于靶向实体癌的抗癌药物组合物和抗癌免疫疗法。
附图说明
图1显示CAR-mRNA的结构和CAR的结构域和基序构建体。
图2显示了转染优化的结果,和使用NEPA21系统建立NK92细胞转染条件的结果。
图3显示了转染优化的结果,和使用NEPA21系统建立原代NK细胞的转染条件的结果。
图4显示了在瞬时CAR NK92细胞和稳定CAR NK92细胞中随时间的CAR表达的表征结果。
图5显示关于瞬时CAR NK92细胞和稳定CAR NK92细胞随时间的癌细胞毒性的表征结果。
图6显示了构成CAR的载体构建体或结构域和基序,用于表征癌细胞毒性和含有DAP10的嵌合抗原受体的表达速率的实验,
图7,与图6相关,显示了当使用FACS转染CARmRNA时,CAR表达水平的表征取决于DAP10的存在。
图8显示在CAR结构中,具有两种共激活剂(CD28、DAP10)的第三代CAR和具有一种共激活剂(DAP10)的第二代CAR的CAR表达率和细胞毒性的差异结果。
图9显示了CAR结构中共激活剂DAP10和4-1BB之间CAR表达水平和细胞毒性差异的证实结果。
图10显示了CAR结构中共激活剂DAP10和2B4之间CAR表达水平和细胞毒性差异的证实结果。
图11A是设计具有或不具有5′-β-球蛋白UTR、3′-BGH和EMCV-IRES构建体的mRNA构建体的示意图,并且通过改变EMCV-IRES的正向/反向或5′/3′位置,图11B是mRNA构建体的制备过程的示意图。
图12是显示导入细胞的mRNA构建体1至7的时间依赖性回归比率的比较的图。
图13是比较和表征引入了mRNA构建体1至7的瞬时NK92细胞中的CAR蛋白的表达量的图。
图14是比较和表征引入了mRNA构建体1至7的瞬时NK92细胞在肺癌细胞系和乳腺癌细胞系中的毒性的图。
图15A是从含有与3′末端具有不同顺序BGH和IRES的mRNA的碱基序列相对应或互补的DNA序列的载体中获得的转录组的电泳。
图15B显示了在3′末端以不同顺序BGH和IRES构成的mRNA构建体在细胞中的靶蛋白性能和靶蛋白表达效率的表征。
具体实施方式
本发明人研究了通过瞬时转染制备具有改善的治疗功效和确保安全性的CAR-NK细胞,并鉴定了结构稳定的mRNA构建体,当将其注射到细胞中时,可以长时间主动诱导功能性靶蛋白的表达,从而完成了本发明。
本发明提供了mRNA构建体,其包括:(a)编码靶蛋白或肽的区域;(b)在靶蛋白编码区上游的5′-β-珠蛋白UTR区;和(c)目标蛋白编码区下游的BGH、BGH-IRES、或IRES-BGH区。
在本发明中,术语“IRES(内部核糖体进入位点)”是指内部核糖体进入位点或核糖体结合位点,已知其在mRNA上形成环状结构并以非帽依赖性机制启动翻译。用其将IRES定位在两个或多个基因之间以制备单一载体,可在单一mRNA上同时表达两个基因中的每一个。在此背景下,为了诱导靶蛋白的表达而插入IRES区域被广泛地应用于遗传重组技术中。但是,本发明人确认,当IRES的环结构位于mRNA的3′UTR时,mRNA在结构上是稳定的,并且可以高效表达靶蛋白。特别是,发现在3′UTR包括IRES-BGH序列的RNA比包括BGH-IRES序列的RNA具有更高的结构一致性、稳定性和蛋白质表达效率。
本发明人在mRNA构建体研究中使用脑心肌炎病毒(EMCV)的IRES区域作为IRES。
同时,为了增强mRNA的稳定性和蛋白质表达效率,IRES区域的位置和顺序是重要的。本发明人确认,5′UTR中包括IRES区域的情况与3′UTR中包括IRES区域的情况相比,mRNA的稳定性和蛋白质的表达效率降低。另外,本发明人证实,即使当IRES位于3′UTR时,当IRES位于反向时,mRNA稳定性和蛋白质表达效率也比当IRES位于正向时低。由此可以确认,不仅IRES的环结构,而且环结构所处的位置(3′UTR)及其后面的BGH区域等的有机结合,都有助于mRNA稳定性和翻译效率的提高。
在本发明中,术语“BGH”是指牛生长激素(bGH),其是bGH的聚腺苷酸信号。BGH区域包括具有5′-β-珠蛋白UTR区域的聚腺苷酸化信号。本发明人通过实验证实,mRNA构建体中存在的5′-β-珠蛋白UTR区使mRNA稳定性增加约36%,位于3′UTR的BGH区使mRNA稳定性增加约20%。
本发明的mRNA构建体用于转染,优选瞬时转染,其在预定时间里表达靶蛋白而在宿主细胞的染色体中无基因插入。尽管mRNA的结构稳定对于蛋白质可以长时间高效表达很重要,但成功表达靶蛋白质固有功能的蛋白质可以被高效表达是实现转染最终目标的必要条件。
在这种情况下,本发明人通过用由上述mRNA构建体表达的作为靶蛋白的嵌合抗原受体(CAR)转染NK细胞制备了瞬时CAR-NK细胞,并且已经证实了瞬时CAR-NK细胞的癌细胞毒性。结果,可以证实mRNA构建体的蛋白表达率和用mRNA构建体转染的NK细胞的癌细胞毒性彼此成比例关系,并且本发明的mRNA构建体不影响靶蛋白质的固有功能。
在本发明中,术语“UTR(非翻译区)”也称为非翻译区,mRNA链中不作为蛋白质基因模板的部分,即mRNA的非翻译部分和非翻译部分,通常将编码区的上游称为5′UTR,将其下游称为3′UTR。
通常,在UTR中,存在5UTR(5非翻译区)和3UTR(3非翻译区),5UTR是指5′末端区,3UTR是指3′末端区。已知3UTR是真核生物的信使RNA(mRNA)中的重要调节元件,3′UTR是指有助于原核生物中mRNA的稳定性的翻译终止子。此外,据报道,mRNA中的非翻译区(UTR)在调节mRNA稳定性和mRNA翻译中起关键作用。
已知5′UTR基序,例如上游开放阅读框(uORF)或内部核糖体进入位点(IRES)参与基因调节,特别是翻译起始,但位于3′UTR的IRES的功能尚不清楚。
已知通过RNA结合蛋白和与其相互作用,UTR不仅影响mRNA稳定性和细胞内定位,而且影响翻译起始、延伸和终止。根据UTR中的特定基序,mRNA转换率可以增加或降低。
如上所述,为了增强利用自然杀伤细胞(CAR-NK)的嵌合抗原受体的靶蛋白表达和mRNA构建体稳定性,添加如5′末端的β-珠蛋白基因的上游序列,和3′末端的多聚A序列和牛生长激素(BGH)序列的UTR,从而完成本发明。
同时,当本发明的mRNA构建体的靶蛋白是CAR时,当注射到NK细胞中时,显示高的癌细胞毒性,因此,本发明的mRNA构建体可以用于CAR-NK细胞的制备。在本发明中,其靶蛋白为CAR的mRNA构建体单独地称为“CARmRNA”。在本发明中,CARmRNA可以理解为包括增加来自mRNA的蛋白质表达率的功能区域。具体而言,CAR mRNA可以包括所有的诱导核糖体的区域、参与mRNA翻译起始或翻译促进的区域、参与mRNA核外转运的区域、参与与内质网膜结合的结构域、含有含内质网信号(含ER信号)序列的结构域和含有内质网信号序列的区域。
本发明提供了一种重组载体,其含有与上述mRNA对应或互补的DNA碱基序列。本发明的载体是表达载体,并且表达载体是指含有与插入物有效连接的必需调节元件的基因构建体,以便插入物可以在细胞内表达。上述表达载体可以利用标准重组DNA技术制备和纯化。表达载体只要是具有在原核生物和真核生物的各种宿主细胞中表达目的基因和生产目的蛋白的功能的载体,就没有特别的限制,优选是含有表达强活性和高表达量的启动子、且能够大量生产靶蛋白的载体。表达载体优选至少包括启动子、起始密码子、编码目的蛋白的基因和终止密码子,但不限于此。除了上述成分之外,表达载体可以包括编码信号肽的DNA、增强子序列、目的基因5和3侧的非翻译区、选择标记区、或克隆单元等,但不限于此。此外,表达载体是指其中一个或多个基本上含有编码序列的转录调节区和启动子可操作地连接在一起的表达盒,或包括表达盒的载体。靶蛋白,作为利用蛋白质分泌机制产生的作为靶的蛋白质、或本领域普通技术人员寻求大量生产的蛋白质,是指通过将编码相应蛋白质的多核苷酸插入重组表达载体中而能够在转化体中表达的任何蛋白质。在本发明中,靶蛋白也指嵌合抗原受体(CAR)、CAR蛋白、CAR的ScFv、抗体等。
此外,本发明提供了包括嵌合抗原受体(CAR)mRNA的NK细胞,其中NK细胞是嵌合抗原受体自然杀伤(CAR-NK)细胞,其可以是通过NEPA21系统用CAR mRNA或重组载体转染的细胞。
同时,转染或转导是指将外源DNA引入细胞。转染可以通过相应领域中已知的各种方法进行,例如磷酸钙-DNA共沉淀法、DEAE葡聚糖介导的转染法、聚凝胺介导的转染法、电穿孔法、显微注射法、脂质体融合法、脂转染胺和原生质体融合法。另外,转染是指通过感染的方式使用病毒或病毒载体颗粒将基因递送到细胞中。在本说明书中,转染和转导可以互换使用,并且优选地在更广泛的意义上解释为通过将外源基因递送至宿主细胞中的转化,并且通过转染或转导将外源基因引入其中的细胞称为转化体。
同时,当导入的外源基因是独立克隆的形式或插入宿主细胞的染色体DNA中时,转染被区分为稳定转染,而当不是时,转染被区分为瞬时转染。
本发明提供了一种用于转染的mRNA构建体,其在宿主细胞的染色体中没有DNA插入,没有突变的风险,并且其在体内几天内消失,因此具有高的体内稳定性,并且所述mRNA构建体尤其可以用于瞬时转染。
在本发明中,转化或转染通过使用NEPA21超级电穿孔仪(日本Negagene)的电穿孔进行。另外,本发明使用瞬时转染方法或瞬时转化方法。瞬时表达正迅速地用作快速哺乳动物蛋白生产的选择系统。瞬时转染的灵活性使得能够从本发明蛋白质的概念快速证实,并且可以同时或连续生产多种不同的蛋白质。在这方面,Rodrigo Vazquez-Lombardi et al.(Transient expression of human antibodies in mammalian cells,NatProtoc.2018Jan;13(1)99-117)报道瞬时哺乳动物表达系统能够以合理的成本产生100毫克/升或更多的重组抗体。
同时,本发明的药物组合物包括药学上或药理学上可接受的载体。本发明的药学或药理学上可接受的载体通常用于制剂中,并且本发明的药物组合物除了上述成分外,还可以包括悬浮液、防腐剂等。适当的药理学上可接受的载体或制剂在Remington’sPharmaceutical Sciences(第19版,1995)中有详细描述。
本发明的药物组合物,当以给药于受试者的形式使用时,可以在使用前通过制药领域的常规方法,例如静脉内注射、皮下注射、肌内注射等配制成适于给药于患者体内的单位剂型的制剂。
本发明的药物组合物的优选剂量可以根据患者的状态、体重、疾病的严重程度、药物形式、给药途径和时间而变化,但是可以由本领域技术人员适当地选择。
本发明的药物组合物可以通过注射安瓿使用。注射安瓿可以在临用前与注射液混合制备,对于注射液,可以使用盐水溶液、葡萄糖、甘露醇、林格氏溶液等。如此制备的本发明的药物组合物或制剂可以通过使用相应领域中常用的给药方法,以与用于移植和其它用途的细胞的混合物的形式给药。活性成分的实际给药量应根据其它因素,如待治疗的疾病、疾病的严重程度、给药途径、患者的体重、年龄和性别来确定。
以下,通过实施例更详细地说明本发明。然而,提供以下实施例仅用于说明本发明,因此不应解释为限制本发明的范围。
〈实施例1〉CARmRNA结构
本发明中使用的嵌合抗原受体(CAR),由识别靶的胞外域、诱导细胞内信号转导(细胞信号传导)的胞内域、连接胞外域并赋予柔性的间隔区(Myc-铰链)和穿过细胞膜的跨膜(TM)结构域组成。胞外域由通过接头连接的重链和轻链组成的ScFv组成,并且胞内域采用CD28和DAP10作为共激活因子,并且是由负责细胞毒性信号传导的CD3z组成的第三代CAR构建体,如图1所示。
〈实施例2〉转染的优化
为了建立最佳转染条件,在1×106个细胞中使用2μg GFP-mRNA,将电压从110V改变到200V的同时,从使用NEPA21系统进行的转染观察GFP表达(图2),结果,随着电压增加,观察到较低的细胞生存力。在NK92细胞系(NK细胞系、自然杀伤细胞系)中,在110V、125V和140V观察到90%或更高的GFP表达,在CAR-mRNA转染效率评价中,在110V观察到最高的细胞存活力和表达率。将1×106个NK92中每个细胞的mRNA用量从2μg增加到5μg会导致表达水平增加,并且在转染后3天内观察到CAR表达。在基于此放大后,当以1×107个的每细胞20μgmRNA转染时,表达水平良好,并且发现在转染后持续3天,这与1×106细胞中的持续时间相同。
使用NEPA21系统,将5μg GFP-mRNA转染到1×106个原代NK细胞中,所述细胞通过用氢化可地松和细胞因子(IL15,IL21)处理并老化从脐带血中除去CD3而获得的单核细胞7天或更长时间而获得。结果,在190V和200V的条件下显示了高转染效率(图3)。如上所示,在CAR-mRNA转染效率的评价中,在200V观察到最高效率。
〈实施例3〉在瞬时CARNK92细胞和稳定CAR NK92细胞中时间依赖性CAR表达的表征
使用NEPA21系统,将具有识别CEACAM6的抗CEACAM6 scFv、针对抗原的可替宁标记的抗体和能够识别其的抗可替宁scFv的CARmRNA转染到NK92中,并且检查CAR表达随时间的分布和瞬时CAR-NK细胞的细胞毒性。检查了就可替宁和CEACAM6而言,根据scFV序列在NK92细胞中CAR表达水平。结果,抗CEACAM6-CAR在mRNA引入后多至48小时仍保持90%或更高的高表达水平,且抗可替宁CAR在mRNA引入后多至16小时仍显示70%或更高的表达水平,且表达持续多至2天。此外,使用EGFR的缀合分子,测量可替宁-CAR-NK细胞的细胞溶解活性。结果发现,当CAR mRNA转染后蛋白质表达高时,细胞毒性高,并且细胞毒性随着其后表达水平的降低而降低(图4和图5)。
〈实施例4〉证实含DAP10嵌合抗原受体表达水平和癌细胞毒性的提高
为了评估构成信号传导胞内域的每种组分的功能和效应,制备了不含胞外域的ΔEcto-TM-10z和具有抗EGFR活性的亲和体作为胞外域的ZEGFR-CAR。对于ZEGFR-CAR,构建了以pSK载体作为背景的具有ITAM基序和CD28的TM基序的ZEGFR-TM载体、具有加入了DAP10的ZEGFR-TM-10载体或具有加入了CD3z的ZEGFR-TM-z载体、以及具有DAP10和CD3z的ZEGFR-TM-10z载体(图6)。由此,合成CAR mRNA,并使用FACS测量NK92细胞中的CAR表达(图7)。对于具有DAP10和CD3z两者的ZEGFR-TM-10z,CAR表达水平与ZEGFR-TM-10相似。比较具有两个共激活基序(如CD28和DAP10)的第三代CAR构建体与仅具有一个激活基序DAP10的第二代CAR构建体,发现CAR的表达水平和表达回归比相似,在转染CAR mRNA后8h细胞毒性相似,在转染后25h,第二代CAR构建体显示更高的细胞毒性(图8)。此外,当使用4-1BB基序代替DAP10基序时,在由两个共激活结构域组成的第三代CAR中,使用DAP10基序的ZEGFR-10z比具有4-1BB基序的ZEGFR-BBz显示出更好的CAR表达水平和持续时间,并且对肺癌细胞系A549和乳腺癌细胞系AU565的细胞毒性与ZEGFR-10z相似或更高(图9)。接着,当使用2B4基序代替DAP10基序时,使用DAP10基序的ZEGFR-10z比使用2B4基序的ZEGFR-2B4z显示出更好的CAR表达水平和持续时间,并且使用ZEGFR-10z在CAR mRNA转染后8h和25h对乳腺癌细胞系AU565细胞的细胞毒性也更高(图10)。
〈实施例5〉增强mRNA的结构稳定性和靶蛋白表达的UTR表征
5-1.mRNA构建体的设计和制备
为了增强mRNA的结构稳定性和靶蛋白的表达,设计了具有各种5′UTR和3′UTR修饰的mRNA构建体1-7(图11A),通过获得转录组制备mRNA构建体1-7,所述转录组来自具有T7启动子区并编码每个mRNA构建体的载体(图11B)。表1和图11A显示了设计的mRNA构建体,并且实验中使用的mRNA构建体包括5′帽和3′多聚A尾。与mRNA构建体中所含的每个区域互补的DNA序列示于下表2中。
[表1]
构建体编号 | 名称 | mRNA大小 | 排名 |
1 | ZE-BBz | 1286bp | 1 |
2 | ZE-BBz-BGH | 1519bp | 3 |
3 | 5U-ZE-BBz | 1345bp | 2 |
4 | 5U-ZE-BBz-BGH | 1578bp | 4 |
5 | 5U-ZE-BBz-IRES-BGH | 2164bp | 5 |
6 | 5U-ZE-BBz-reIRES-BGH | 2164bp | 5 |
7 | 5U-IRES-ZE-BBz-BGH | 2164bp | 5 |
[表2]
5-2.mRNA稳定性的评价
在1×107个NK92细胞中,在实施例2中鉴定的优化条件下诱导20μg mRNA构建体1至7的转染,并随时间监测引入NK92细胞中的mRNA水平。
为了根据5′-β-珠蛋白UTR、3′-BGH或EMCV-IRES的存在/不存在,以及EMCV-IRES的5′/3′位置或正向/反向来评价CARmRNA的稳定性,鉴定了随着时间引入细胞中的mRNA的减少比率(图12(a))。
结果,在转染后4小时,即转化的早期,评估mRNA构建体的存在量,作为参考(1),5U-ZE-BBz显示mRNA稳定性比ZE-BBz提高36.9%,5U-ZE-BBz-BGH显示mRNA稳定性比ZE-BBz-BGH提高35.9%,ZE-BBz-BGH显示mRNA稳定性比ZE-BBz提高20.2%,5U-ZE-BBz-BGH显示mRNA稳定性比5U-ZE-BBz提高19.1%,同时含有5′-β-珠蛋白和3′-BGH的U-ZE-BBz-BGH显示mRNA稳定性比ZE-BBz提高56.1%。因此,可以确认5′-β-珠蛋白和3′-BGH的存在增强了mRNA的稳定性(图12(b)和图12(c))。
5-3.翻译稳定性的评价
随后,为了评价依赖于5′-β-珠蛋白UTR、3′-BGH和EMCV-IRES构建体的存在以及EMCV-IRES的正向/反向或5′/3′位置的CAR mRNA翻译稳定性,鉴定了转染诱导后4、8、18和24小时的CAR蛋白表达降低水平(图13)。
结果,在转染后诱导8小时的CAR蛋白表达水平,此时CAR蛋白表达作为参考(1)是最高的,ZE-BBz-BGH显示蛋白表达水平比ZE-BBz提高28.4%,5U-ZE-BBz-BGH显示蛋白表达水平比5U-ZE-BBz提高24.3%,5U-ZE-BBz-IRES-BGH显示蛋白表达水平比5U-ZE-BBz-BGH提高33.3%。因此,可以确认3′-BGH和3′-EMCV-IRES有助于提高蛋白质表达水平。
同时,与3′-EMCV-IRES的存在增强了蛋白质表达的事实相反,当通过反向插入EMCV-IRES且EMCV-IRES位于5′从而修饰翻译的环结构时,蛋白质表达水平急剧下降。
5-4.细胞溶解活性的评价
随后,为了表征瞬时NK92细胞中功能性CAR蛋白的表达水平,通过用NK92细胞处理肺癌细胞系A549和乳腺癌细胞系AU565来评估细胞溶解活性(图14A和图14B)。
结果,在瞬时NK92细胞中,观察到对应于CAR蛋白表达水平的细胞溶解活性,并且一系列响应,响应于此的IFNγ和TNFα分泌也增加。观察到CAR蛋白的表达水平和细胞溶解活性在5U-ZE-BBz-BGH和5U-ZE-BBz-IRES-BGH中是高的,特别是在5U-ZE-BBz-IRES-BGH中最高。
5-5.IRES-BGH与BGH-IRES的比较
鉴定了位于3′UTR的IRES和BGH序列的影响。
首先,如下述表3所示设计mRNA,用图12所示的方法制备mRNA,进行电泳。结果,可以确认,对于在3′UTR具有BGH-IRES序列的mRNA,在mRNA中形成了没有和具有IRES的单独转录组,而对于具有IRES-BGH的mRNA,形成了在3′UTR具有IRES的单个mRNA。由此结果可以确认,由于IRES的复杂的二维结构,当在BGH之后跟随IRES时,会发生IRES与mRNA的分离(图15A)。
[表3]
构建体编号 | 名称 |
1 | UTR-COT-MH-10Z-IRES-BGH-多聚A |
2 | UTR-COT-MH-10Z-BGH-IRES-多聚A |
此外,CAR蛋白的表达水平和癌细胞的细胞毒性通过利用以上通过实施例2中所述的方法制备的mRNA构建体转染NK92细胞来鉴定。结果,确认了具有IRES-BGH序列的mRNA高效表达CAR蛋白,与具有BGH-IRES序列的mRNA相比,显示出更高的癌细胞毒性(图15B)。
<110> 韩国生命工学研究院(KOREA RESEARCH INSTITUTE OF BIOSCIENCE ANDBIOTECHNOLOGY)
<120> 用于蛋白质表达的mRNA构建体及其用途
<130> M19-6451
<150> KR 10-2019-0152938
<151> 2019-11-26
<160> 3
<170> KoPatentIn 3.0
<210> 1
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 5'-UTR (B-珠蛋白)
<400> 1
acatttgctt ctgacatagt tgtgttgact cacaacccca gaaacagaca tcc 53
<210> 2
<211> 215
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 3'-UTR (BGH)
<400> 2
gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 60
ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 120
cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 180
gaggattggg aagacaatag caggcatgct gggga 215
<210> 3
<211> 580
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 3'-UTR (EMCV-IRES)
<400> 3
tccccctctc cctccccccc cccctaacgt tactggccga agccgcttgg aataaggccg 60
gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc 120
ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa 180
aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag 240
acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg 300
cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg 360
ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa 420
caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg 480
gtgcacatgc tttacatgtg tttagtcgag gttaaaaaac gtctaggccc cccgaaccac 540
ggggacgtgg ttttcctttg aaaaacacgt tgataatttg 580
Claims (13)
1.一种mRNA构建体,包括:(a)编码靶蛋白或肽的区域;(b)靶蛋白编码区上游的5′-β-珠蛋白UTR区;和(c)靶蛋白编码区下游的BGH、BGH-IRES、或IRES-BGH区。
2.如权利要求1所述的mRNA构建体,其中所述靶蛋白或肽(a)是嵌合抗原受体(CAR)。
3.如权利要求2所述的mRNA构建体,其中所述CAR包括能够特异性结合靶的胞外域、穿透细胞膜的跨膜结构域和诱导细胞内信号转导的胞内域。
4.如权利要求3所述的mRNA构建体,其中所述胞外域是抗体片段(ScFv),其中轻链和重链通过接头连接,并且所述胞内域是CD28。
5.如权利要求4所述的mRNA构建体,其中所述胞内域进一步包括DAP10和/或CD3z。
6.一种重组载体,其包括与权利要求1至5中任一项的mRNA构建体的碱基序列对应或互补的DNA碱基序列。
7.一种转化体的制备方法,包括以下步骤:(1)制备权利要求1至5任一项所述的mRNA构建体;(2)将mRNA构建体引入宿主细胞。
8.根据权利要求7的转化体制备方法,其中步骤(2)是在110V-140V的条件下通过NEPA21电穿孔进行的。
9.一种CAR-NK细胞的制备方法,包括以下步骤:(a)制备权利要求2的mRNA构建体;和(b)将所述mRNA构建体引入NK细胞中。
10.如权利要求9所述的CAR-NK细胞制备方法,其中所述步骤(b)的所述NK细胞是原代NK细胞,并且将所述mRNA构建体向所述NK细胞中的所述引入通过NEPA21电穿孔在190V至200V的条件下进行。
11.一种通过权利要求9或权利要求10的方法制备的CAR-NK细胞,其中所述细胞的特征在于,表达CAR蛋白3天。
12.一种用于治疗癌症的药物组合物,其包括权利要求11所述的CAR-NK细胞作为活性成分。
13.如权利要求12所述的药物组合物,其中所述癌症是实体癌。
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