CN117624381A - 一种治疗性的HPV mRNA疫苗及其应用 - Google Patents
一种治疗性的HPV mRNA疫苗及其应用 Download PDFInfo
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Abstract
本发明通过序列优化和设计得到了能稳定表达重组HPV抗原的mRNA,并基于该mRNA形成治疗性疫苗。所述mRNA可以编码重组的HPV E6E7融合多肽,免疫刺激因子以及利于融合多肽被呈递和加工的蛋白结构域。本发明进一步使用由式C的脂质化合物构成的脂质外层包裹所述的重组HPV抗原的mRNA分子内核,形成脂质纳米颗粒结构,将上述优化的重组HPV抗原mRNA分子准确递送至肿瘤微环境。
Description
本申请要求2022年11月22日提交的专利申请号为202211469571.0,发明名称为“一种治疗性的HPV mRNA疫苗及其应用”的在先申请的优先权。该在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于生物医药技术领域,尤其涉及一种治疗性的HPV mRNA疫苗、其制备方法及其应用。
背景技术
人乳头瘤病毒(Human papilloma virus,HPV)是一种嗜上皮组织的无包膜双链环状DNA病毒,由病毒衣壳蛋白L1、L2和核心单拷贝的病毒基因组DNA构成。L1蛋白在不同种类的HPV中高度保守,因此,HPV亚型定义为其L1基因序列与任何其他HPV亚型至少10%不同的完整基因组。约有200种HPV型别从人体中鉴定出来。根据致癌潜力,HPV分为高危型和低危型,高危型HPV主要引起子宫颈、肛门、生殖器癌,包括HPV16、18、31、33、35、39、45、51、52、56、58、59、68、73、82型等15种。
HPV基因组可以分为三个区:早期基因区(编码早期蛋白E1~E7)、晚期基因区(编码晚期蛋白L1、L2)和上游调节区URR。URR为非编码区,具有许多转录抑制因子和激活因子的结合位点,用于调控病毒复制和病毒蛋白表达。病毒感染细胞后,E1和E2蛋白启动并维持病毒复制。E6和E7通过靶向宿主细胞周期控制来驱动肿瘤发生。E7结合视网膜母细胞瘤基因产物pRb并使其失活,导致细胞增殖失控。在这种情况下,肿瘤抑制因子p53通常会诱导细胞凋亡;然而,E6与p53结合,导致其泛素化和随后的降解,从而抑制凋亡。E6和E7的协同作用导致HPV感染细胞的恶性转化和不受控制的肿瘤生长。E6和E7在癌前病变和浸润性病变中都有组成性表达,但在健康细胞中不存在,这使它们成为HPV诱发恶性肿瘤免疫治疗方法的理想靶点。
溶酶体相关膜蛋白-1(LAMP-1)是一种高度糖基化的溶酶体膜蛋白,属于溶酶体膜的主要蛋白成分。LAMP-1是I型跨膜蛋白,具有一个大的luminal domain,一个跨膜结构域和一个c端胞质尾部。
不稳定结构域(Destabilizing domains,DDs)是一类小蛋白质结构域,可与其他蛋白形成融合蛋白。在无配体的情况下,DD融合蛋白会迅速被泛素化标记,随后通过蛋白酶体途径降解。而Sheild-1可以特异性靶向DD融合蛋白,抑制DD序列诱导的蛋白降解过程,实现精准且可逆性调控蛋白表达。
Fms相关酪氨酸激酶3(Flt3)又叫CD135,在人体中由FLT3基因编码,在许多造血母细胞的表面表达,FLT3信号对造血干细胞和造血母细胞的正常发育至关重要。FLT3配体FLT3L可以促进造血前体干细胞向树突细胞发育,并促进树突细胞的存活和增强。小鼠实验证明FLT3L可以促进交叉提呈树突细胞亚群的扩增,增加肿瘤内浸润的抗原特异性CD8 T细胞数量。在病人临床试验中,FLT3L可以提高交叉提呈树突细胞在肿瘤部位的数目和活化,提高肿瘤特异性CD8 T细胞激活,提高对免疫检查点阻断治疗的效率。
OX40(CD134,TNFRSF4)最初被定义为T细胞活化标记物,后发现是具有共激活功能的NGFR/TNFR超家族成员。OX40为一种50kD的一型跨膜糖蛋白。主要在活化的效应T细胞(Teffs)和调节性T细胞(Tregs)上表达,也在NK T细胞,NK细胞和嗜中性粒细胞上表达。OX40与配体OX40L(CD252,TNFSF4)结合传递共刺激信号。OX40L为分子量为34kD的二型跨膜糖蛋白,可在抗原提呈细胞(APC),如:B细胞、树突细胞、巨噬细胞上表达;另外在其它细胞类型如Langerhans细胞、内皮细胞、平滑肌细胞、肥大细胞和NK细胞也可诱导表达。当OX40被其配体OX40L激活时,会引发一系列的免疫反应:增加效应T细胞和记忆T细胞的存活和扩增,增加细胞因子(例如IL-2、IL-4、IL-5、IFN-γ)的分泌;降低Tregs的免疫抑制活性,进一步放大T细胞活化效应。
除已获批上市了四种预防性HPV疫苗,目前也有多种治疗性HPV疫苗在研,种类包括细菌载体疫苗、病毒载体疫苗、多肽/蛋白疫苗、细胞疫苗和核酸疫苗。其中效果较好、进展较快的有美国Inovio公司针对HPV16和18的DNA疫苗VGX3100,以及韩国Genexine生物医药公司的针对HPV16、HPV18的DNA疫苗GX-188E,这两种疫苗在治疗HPV16/18阳性宫颈癌癌前病变的一期和二期临床实验中,均显示了良好的病毒清除效果和组织学逆转能力。从二期数据看,GX188E具有更好的临床效果。
VGX-3100是一种DNA疫苗,含有两个质粒的混合物,编码HPV16和18的优化共识E6和E7基因。在CIN 2/3女性的I期试验中,疫苗肌肉注射三次,然后进行电穿孔。在CIN 2/3患者中进行的随机双盲安慰剂对照IIb期试验显示,该试验对HPV16/18诱导的CIN有效。在49.5%的疫苗治疗患者中观察到组织病理学消退,而对照组为30.6%;治疗组和安慰剂组分别有40.2%和14.3%的受试者同时出现了组织病理学转归复合病毒学清除;在出现组织病理学转归的受试者中,治疗组比安慰剂组更可能出现病毒清除(82%vs 45%)。而在最新揭露的三期临床(REVEAL 1)中,第36周,HSIL的组织病理学消退与HPV-16和/或HPV-18的病毒学清除相结合的主要终点,治疗组的应答者百分比为23.7%(31/131),安慰剂组为11.3%(7/62)(p=0.022;百分比差异12.4%,95%CI:0.4,22.5)。结果虽然具有统计学意义,然而与二期临床的40.2%相比,组织学消退复合病毒学清除的患者比例显著降低。
目前,围绕VGX3100,开展了一系列临床实验。主要是针对HPV阳性的头颈鳞癌、口咽癌,宫颈癌等实体肿瘤。在临床给药的设计上,除了使用VGX3100以外,还使用了表达人IL12的质粒INO-9012,并联合PDL1抗体(或同步放化疗),从侧面表明单独使用VGX3100对肿瘤治疗效果可能有限,需要通过其他手段加强。
GX-188E是一种含有编码与外分泌信号肽、FLT3L以及HPV16和HPV18的E6和E7蛋白融合表达的质粒DNA疫苗。在针对HPV16/18阳性CIN3阳性人群的二期临床实验中,52%患者在第一次注射后20周内CIN3的组织病理学消退,67%的患者在第一次注射后36周表现出消退。该疫苗结合PD-1抗体Pembrolizumab治疗HPV16/18阳性的晚期宫颈癌的二期临床数据显示,完全缓解率为15%,ORR为42%,疾病控制率为58%,而仅仅使用PD-1抗体治疗晚期宫颈癌的ORR为14.3%。
DNA疫苗的缺点是:转染效率和免疫原性低,需要专门的疫苗接种设备和额外的佐剂来改善免疫反应。此外,DNA疫苗使用的剂量大,且需要进入到细胞核后才能表达,具有整合到细胞基因组上的风险。
因此,为了预防和治疗HPV感染引起的疾病,需开发更安全和高效的疫苗,具有增强的免疫原性以及高效安全的核酸递送系统。
目前,核酸的递送方法包括化学修饰、生物偶联技术、纳米载体技术、基于脂质的制剂、外泌体、球形核酸、DNA纳米结构、刺激-响应聚合物纳米材料等。其中,较为成熟的核酸递送载体是脂质纳米颗粒(LNP)。
该类脂质制剂的主要成分包括:阳离子/可电离脂质、辅助型脂质、胆固醇和聚乙二醇-脂质结合物。在这四种脂质成分中,阳离子脂质带电荷的头部能够与带负电的核酸结合,同时也能与细胞膜上的磷脂分子结合,在核酸包裹和膜融合过程中都发挥着关键的作用。考虑到永久性的阳离子脂质的潜在毒性,可电离阳离子脂质的脂质纳米颗粒存在更大的应用价值。
可电离阳离子脂质包括三个重要的结构组成部分:含胺基的亲水极性头部;疏水脂质链;负责连接极性头部和非极性尾部的连接链。目前,商业化的可电离阳离子脂质主要有MC3系列和用于新型冠状病毒mRNA疫苗的ALC-0315、SM-102。其中,MC3具有很强的肝靶向,对于可能具有潜在肝毒性的核酸制剂其应用将会受限,而且MC3是针对分子量较小的siRNA递送开发的,对于分子量较大的核酸制剂其负载量可能存在限制。ALC-0315和SM-102的递送效率仍需进一步提高。因此,在脂质制剂的发展及其在核酸药物递送等方面的应用,仍需开发出新的可电离的阳离子脂质,并筛选及优化新的纳米递送系统实现核酸的安全、高效递送。
发明内容
本发明的第一个方面是提供了一种HPV E6E7的融合多肽,其由以下部分组成:由HPV-16的E6蛋白的第1个-第95个氨基酸,HPV-16的E7蛋白的第1个-第78个氨基酸,HPV-16的E6蛋白的第81个-第158个氨基酸,HPV-16的E7蛋白的第64个-第98个氨基酸,以及HPV-18的E6蛋白的第1个-第95个氨基酸,HPV-18的E7蛋白的第1个-第78个氨基酸,HPV-18的E6蛋白的第81个-第158个氨基酸,HPV-18的E7蛋白的第51个-第105个氨基酸依次连接组成。
在一些实施方案中,所述E6E7融合多肽具有与SEQ ID NO:1所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的氨基酸序列,其中所述氨基酸序列具有抑制HPV E6、E7蛋白的致癌能力以及增强的免疫原性。在本发明的一个实施方式中,所述E6E7融合多肽具有如SEQ ID NO:1所示的氨基酸序列。可选的,所述E6E7融合多肽不含第1位的甲硫氨酸。
本发明还提供一种包含前述HPV E6E7融合多肽的融合蛋白,其包括以下部分:用于增强免疫活性的结构域、HPV E6E7融合多肽、自剪切序列和免疫共刺激因子。
所述融合蛋白中,所述用于增强免疫活性的结构域选自LAMP基因和DD结构域。
在一些实施方案中,LAMP结构域包含与SEQ ID NO:2所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的LAMP基因的luminal domain的氨基酸序列,以及与SEQ ID NO:3所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的LAMP基因的跨膜区和胞内区的氨基酸序列。在本发明的一个实施方式中,LAMP结构域具有如SEQ ID NO:2所示的氨基酸序列和SEQ ID NO:3所示的氨基酸序列。在所述融合蛋白中,HPV E6E7融合多肽的氨基酸序列位于SEQ ID NO:2所示的氨基酸序列和SEQ ID NO:3所示的氨基酸序列之间。
在一些实施方案中,DD结构域包含与SEQ ID NO:4所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的氨基酸序列。在本发明的一个实施方式中,DD结构域具有如SEQ ID NO:4所示的氨基酸序列。根据本发明,所述HPV E6E7融合多肽位于DD结构域的N端或者C端。在本发明的一个实施方式中,所述HPVE6E7融合多肽位于DD结构域的C端。
所述融合蛋白中,所述自剪切序列选自P2A、T2A、E2A和F2A,优选为P2A。
在一些实施方案中,自剪切序列包含与SEQ ID NO:5所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的氨基酸序列。在本发明的一个实施方式中,自剪切序列具有如SEQ ID NO:5所示的氨基酸序列。
所述融合蛋白中,所述免疫共刺激因子选自FLT3L和OX40L。
在一些实施方案中,FLT3L包含与SEQ ID NO:6所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的氨基酸序列。在本发明的一个实施方式中,FLT3L具有如SEQ ID NO:6所示的氨基酸序列。
在一些实施方案中,OX40L包含与SEQ ID NO:7或SEQ ID NO:20所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的氨基酸序列。在本发明的一个实施方式中,OX40L具有如SEQ ID NO:7所示的氨基酸序列。在本发明的一个实施方式中,OX40L具有如SEQ ID NO:20所示的氨基酸序列。
在一些实施方案中,为便于FLT3L的分泌,所述融合蛋白中还含有外分泌信号肽,所述信号肽选自IgE外分泌信号肽,或tPA外分泌信号肽。
在一些实施方案中,IgE外分泌信号肽包含与SEQ ID NO:8所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的氨基酸序列。在本发明的一个实施方式中,IgE外分泌信号肽具有如SEQ ID NO:8所示的氨基酸序列。
在一些实施方案中,tPA外分泌信号肽包含与SEQ ID NO:9所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的氨基酸序列。在本发明的一个实施方式中,tPA外分泌信号肽具有如SEQ ID NO:9所示的氨基酸序列。
在本发明的一些实施方式中,所述融合蛋白由以下部分组成:顺序连接的DD结构域、HPV E6E7融合多肽、自剪切序列和免疫共刺激因子。在本发明的一个实施方式中,所述融合蛋白由以下部分组成:顺序连接的DD结构域、HPV E6E7融合多肽、自剪切序列、外分泌信号肽和FLT3L。在本发明的一个实施方式中,所述融合蛋白由以下部分组成:顺序连接的DD结构域、HPV E6E7融合多肽、自剪切序列和OX40L。
在本发明的一个实施方式中,所述融合蛋白由以下部分组成:顺序连接的LAMP基因的luminal domain、HPV E6E7融合多肽、LAMP基因的跨膜区和胞内区、自剪切序列和免疫共刺激因子。在本发明的一个实施方式中,所述融合蛋白由以下部分组成:顺序连接的LAMP基因的luminal domain、HPV E6E7融合多肽、LAMP基因的跨膜区和胞内区、自剪切序列、外分泌信号肽和FLT3L。在本发明的一些实施方式中,所述融合蛋白由以下部分组成:顺序连接的LAMP基因的luminal domain、HPV E6E7融合多肽、LAMP基因的跨膜区和胞内区、自剪切序列和OX40L。
本发明的第二个方面是提供编码前述融合多肽和融合蛋白的多核苷酸。
一种编码前述HPV E6E7融合多肽的多核苷酸。所述多核苷酸是DNA或RNA。
一种编码前述包含HPV E6E7融合多肽的融合蛋白的多核苷酸。所述多核苷酸是DNA或RNA。
根据本发明,所述编码融合多肽和融合蛋白的多核苷酸序列是密码子优化后的序列。所述多核苷酸是mRNA。所述密码子优化后的序列是相对于野生型核苷酸序列包含至少一个同义核碱基的置换。
经过密码子优化后的序列可与某些组织靶标和/或宿主生物中的密码子频率相匹配以确保正确折叠,调整G/C含量以增加mRNA稳定性或减少二级结构。密码子优化方法在本领域中是已知的,包括来自GeneArt,GenSmart等。
在一些实施方案中,密码子优化后的编码前述融合蛋白的mRNA序列包含与SEQ IDNO:12-15任一所示的核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列。在本发明的一个实施方式中,密码子优化后的编码前述融合蛋白的mRNA序列具有如SEQ IDNO:12-15任一所示的核苷酸序列。
根据本发明,所述mRNA还可包括5’帽结构、5’UTR,3’UTR和poly-A尾。
5’帽结构:5’帽典型地是添加在mRNA分子的5’端的修饰的核苷酸(特别是鸟嘌呤核苷酸),也包括非典型的帽类似物。优选地,5’帽使用5’-5’-三磷酸酯键(也称为m7GpppN)添加。5’帽结构的另外实例包括甘油基,反向脱氧脱碱基残基(部分),4’,5’-亚甲基核苷酸,1-(β-D-赤式呋喃糖基)核苷酸,4’-硫代核苷酸,碳环核苷酸,1,5-脱水己糖醇核苷酸,L-核苷酸,α-核苷酸,修饰的碱基核苷酸,苏式戊呋喃糖基核苷酸,无环3’,4’-闭联核苷酸,无环3,4-二羟基丁基核苷酸,无环3,5-二羟基戊基核苷酸,3’-3’-反向核苷酸部分,3’-3’-反向脱碱基部分,3’-2’-反向核苷酸部分,3’-2’-反向脱碱基部分,1,4-丁二醇磷酸酯,3’-氨基磷酸酯,己基磷酸酯,氨基己基磷酸酯,3’-磷酸酯,3’硫代磷酸酯,二硫代磷酸酯或桥接或非桥接甲基膦酸酯部分。这些修饰的5’帽结构可用于本发明的情形中以修饰本发明的mRNA序列。在本发明的一些实施方式中,5’帽结构是CAP1(m7GpppN的相邻核苷酸的核糖的额外甲基化),CAP2(m7GpppN下游的第二核苷酸的核糖的额外甲基化),CAP3(m7GpppN下游的第三核苷酸的核糖的额外甲基化),CAP4(m7GpppN下游的第四核苷酸的核糖的额外甲基化)。
帽类似物:帽类似物是指具有帽功能的不可聚合二核苷酸,因为其促进翻译或定位,和/或当在RNA分子的5’端并入时阻止RNA分子的降解。不可聚合意为帽类似物将仅在5’端并入,因为其不具有5’三磷酸酯且因此无法通过模板依赖性RNA聚合酶在3’方向上延伸。帽类似物包括但不限于选自由以下组成的组的化学结构:m7GpppG、m7GpppA、m7GpppC;未甲基化帽类似物(例如,GpppG);二甲基化帽类似物(例如,m2,7GpppG)、三甲基化帽类似物(例如m2,2,7GpppG)、二甲基化对称帽类似物(例如,m7Gpppm7G)或抗反向帽类似物(例如,ARCA;m7,2’OmeGpppG、m7,2’dGpppG、m7,3’OmeGpppG、m7,3’dGpppG及它们的四磷酸酯衍生物)(Stepinski等,2001.RNA 7(10):1486-95)。
5’帽结构可使用帽类似物在化学RNA合成,或RNA体外转录(共同转录加帽)中形成,或可使用加帽酶(例如,可商购的加帽试剂盒)在体外形成。
在本发明的一个实施方式中,所述5’帽结构是Cap1结构。
根据本发明,所述5’UTR包含与SEQ ID NO:10所示的核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列。在本发明的一个具体实施方式中,所述5’UTR包含SEQ ID NO:10所示的核苷酸序列。
根据本发明,所述3’UTR包含与SEQ ID NO:11所示α2-珠蛋白3’UTR的片段至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列。在本发明的另一些实施方式中,所述3’UTR包含2个首尾相连的与SEQ ID NO:11所示的α2-珠蛋白3’UTR的片段至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列。在本发明的一个具体实施方式中,所述3’UTR包含2个首尾相连的SEQ ID NO:11所示的核苷酸序列。
根据本发明,所述poly-A尾的长度可以为50-200个核苷酸,优选为100-150个核苷酸,例如110-120个核苷酸,例如约110个核苷酸,约120个核苷酸,约130个核苷酸,约140个核苷酸,约150个核苷酸。
根据本发明,所述mRNA中的一个或多个核苷酸可以是经修饰的。例如,所述mRNA中的一个或多个核苷酸(例如所有核苷酸)可以各自独立替换为天然存在的核苷酸类似物或人工合成的核苷酸类似物,例如选自假尿苷(pseudouridine)、2-硫尿苷(2-thiouridine)、5-甲基尿苷(5-methyluridine)、5-甲基胞苷(5-methylcytidine)、N6-甲基腺苷(N6-methyladenosine)、N1-甲基假尿苷(N1-methylpseudouridine)、5-乙炔基尿苷(5-ethynyluridine)、假尿苷三磷酸(pseudo-UTP)、1-甲基-假尿苷三磷酸(N1-methyl-pseudo-UTP)、5-乙炔基尿苷三磷酸(5-ethynyl-UTP)、5-甲基胞苷三磷酸(5-methyl-CTP)等。
在本发明的一些实施方式中,所述mRNA包含与SEQ ID NO:16-19任一所示的核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列。在本发明的一个实施方式中,所述mRNA序列具有如SEQ ID NO:16-19任一所示的核苷酸序列。
本发明的第三个方面是提供一种载体,所述载体包含本发明第二方面所述的多核苷酸。所述载体包括质粒载体或病毒载体。在一些实施方案中,所述载体可以用于在体外通过转录制备本发明的mRNA。
在本发明的一个实施方式中,所述载体是体外转录用载体,其包含可操作性连接的编码5’UTR,3’UTR和poly-A尾的核苷酸序列。所述5’UTR包含与SEQ ID NO:10所示的核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列。所述3’UTR包含2个首尾相连的与SEQ ID NO:11所示的α2-珠蛋白3’UTR的片段至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列。所述poly-A尾的长度可以为50-200个核苷酸,优选为100-150个核苷酸,例如110-120个核苷酸,例如约110个核苷酸,约120个核苷酸,约130个核苷酸,约140个核苷酸,约150个核苷酸。
根据本发明,所述体外转录用载体,还包含编码所述融合蛋白的核苷酸序列。在本发明的一个实施方式中,所述编码所述融合蛋白的核苷酸序列是与SEQ ID NO:12-15任一所示核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列。
根据本发明,可使用常用的质粒作为载体。在本发明的一些实施方式中,质粒是psp73或pUC57-kana。
可采用本领域已知的方法制备本发明的mRNA,包括但不限于化学合成或体外转录等。在本发明的一些实施方式中,可以人工合成编码mRNA的核酸分子,将该核酸分子克隆到载体中,构建体外转录用质粒。将构建的质粒转化到宿主菌中培养扩增,提取质粒。将提取的质粒使用限制性内切酶酶切消化成线性分子。以制备的线性化质粒分子为模板,使用体外转录法制备mRNA。体外转录(IVT)系统通常包含转录缓冲液、三磷酸核苷酸(NTP)、RNase抑制剂和聚合酶。NTP可以选自但不限于天然和非天然(修饰的)NTP。聚合酶可以选自但不限于T7 RNA聚合酶、T3 RNA聚合酶和突变体聚合酶。可以在体外转录过程中添加帽结构类似物,直接得到具有帽结构的mRNA;也可以在体外转录结束后,使用加帽酶和二甲基转移酶给mRNA添加上帽结构。可采用本领域常规的方法纯化得到的mRNA,例如化学沉淀法、磁珠法、亲和层析法等。
本发明的序列如下:
SEQ ID NO:1:HPV1618 E6E7抗原序列:
MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTSEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPMARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNMHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQVMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ
SEQ ID NO:2:LAMP的luminal domain序列:
MAAPGSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMANFSAAFSVNYDTKSGPKNMTFDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNFTRNATRYSVQLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHMNNVTVTLHDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKYNVSGTNGTCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSEGTTVLLFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNAEEHVRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENS
SEQ ID NO:3:LAMP的跨膜区和胞内区序列:
MLIPIAVGGALAGLVLIVLIAYLVGRKRSHAGYQTI
SEQ ID NO:4:DD结构域氨基酸序列:
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKPE
SEQ ID NO:5:P2A自剪切序列:
ATNFSLLKQAGDVEENPGP
SEQ ID NO:6:FLT3L序列:
TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPGGGSGD
SEQ ID NO:7:OX40L序列:
MERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL
SEQ ID NO:8:IgE外分泌信号肽:
MDWTWILFLVAAATRVHS
SEQ ID NO:9:tPA外分泌信号肽:
MDAMKRGLCCVLLLCGAVFVSPS
SEQ ID NO:10:5’UTR
GGGACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGCCACC
SEQ ID NO:11:α2珠蛋白3’UTR
GCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUG
SEQ ID NO:12:082703A的ORF区(编码LAMP-HPV-LAMP~TM-P2A-tPA-FLT3L)
AUGGCCGCUCCGGGGAGUGCACGCCGGCCGUUGCUGUUACUUCUCCUGUUAUUGCUGCUGGGGCUCAUGCAUUGUGCCUCAGCAGCAAUGUUCAUGGUGAAGAACGGCAACGGCACGGCGUGCAUUAUGGCAAAUUUUUCGGCCGCUUUUAGUGUCAACUAUGACACUAAGAGCGGGCCGAAGAAUAUGACCUUCGACCUACCGUCGGACGCGACGGUAGUGUUGAACCGCUCGUCUUGUGGGAAGGAGAACACGAGCGACCCGUCCCUGGUGAUUGCGUUUGGGAGAGGUCAUACACUGACUCUCAACUUCACGCGUAACGCUACGCGUUACAGCGUGCAGUUGAUGAGUUUUGUGUAUAACCUCUCCGAUACGCACCUCUUUCCGAAUGCGUCAAGUAAGGAGAUCAAGACCGUGGAGAGCAUAACAGACAUACGAGCUGACAUCGACAAGAAGUAUCGUUGCGUCUCAGGGACACAGGUUCACAUGAACAAUGUGACUGUGACCCUGCACGACGCAACGAUACAGGCUUACUUGUCGAACAGCUCGUUUUCGAGGGGGGAGACUCGGUGUGAGCAGGACAGGCCGUCGCCCACGACGGCCCCUCCUGCUCCACCGAGUCCUUCCCCCUCGCCGGUCCCGAAGUCCCCGAGCGUUGACAAGUACAACGUCUCGGGGACUAACGGGACCUGUCUGUUAGCCUCUAUGGGCUUGCAGCUCAACUUGACGUAUGAGCGGAAAGACAACACCACGGUGACGCGGUUGCUCAACAUCAAUCCCAACAAGACGAGCGCGUCCGGGAGCUGCGGUGCGCAUCUGGUGACAUUGGAGUUACAUUCAGAAGGCACGACCGUGCUCCUCUUCCAGUUCGGCAUGAAUGCGAGUUCCUCACGGUUUUUCCUGCAGGGCAUUCAGCUGAACACGAUCCUGCCUGAUGCGCGGGACCCCGCGUUCAAGGCAGCGAACGGUUCGCUGAGGGCCCUGCAGGCGACCGUGGGGAACUCGUAUAAGUGCAAUGCGGAGGAGCACGUCCGCGUUACCAAAGCGUUCUCCGUCAACAUCUUUAAGGUCUGGGUACAGGCCUUUAAGGUUGAGGGAGGACAGUUUGGUAGCGUGGAGGAGUGCCUUCUGGAUGAGAACUCCAUGCACCAGAAGCGCACCGCAAUGUUCCAGGACCCCCAGGAGCGGCCGAGGAAGUUGCCUCAGCUGUGCACGGAGCUGCAGACUACAAUACACGACAUCAUCCUUGAGUGCGUGUAUUGUAAGCAGCAGCUCCUGCGCAGGGAGGUCUACGAUUUCGCCUUCAGGGACUUGUGCAUCGUAUACCGCGACGGUAAUCCGUACGCGGUAUGCGACAAGUGCCUGAAGUUCUAUUCGAAGAUCUCCGAGUACAGGCACUACUGUUACUCGCUGUAUGGGACAACGCUCAUGCACGGGGACACUCCCACGUUGCAUGAGUACAUGUUGGACCUACAGCCUGAGACAACUGACCUCUAUUGCUAUGAGCAGUUGAAUGACAGCAGCGAAGAGGAGGAUGAGAUAGAUGGGCCCGCCGGUCAGGCUGAGCCUGACCGGGCCCACUAUAACAUCGUCACUUUUUGCUGCAAGUGUGAUUCAACGCUCAGGCUCUGCGUGCAGAGCACACACGUGGACAUACGAACCUCAGAGUACAGGCACUACUGCUACUCGCUCUACGGCACGACAUUGGAGCAGCAGUACAACAAGCCCCUGUGUGACUUGUUGAUCCGCUGCAUCAAUUGCCAGAAGCCGCUGUGUCCUGAGGAGAAGCAGCGGCAUCUGGACAAGAAGCAGCGGUUCCACAACAUACGGGGGCGUUGGACUGGUCGCUGCAUGUCGUGCUGUAGAUCGAGUAGGACGCGCAGAGAGACGCAGCUGACGCUGCGUCUCUGUGUGCAGUCGACGCACGUAGACAUCCGCACUCUCGAGGACCUGCUUAUGGGGACGCUCGGGAUCGUGUGCCCGAUCUGCAGCCAGAAGCCUAUGGCGCGGUUCGAGGAUCCGACCCGGCGUCCCUAUAAGCUUCCGGACCUCUGUACUGAGCUCAACACCAGCUUGCAGGACAUAGAGAUCACCUGUGUGUACUGCAAGACGGUGUUAGAGCUCACAGAGGUCUUUGAGUUCGCCUUCAAGGACCUGUUCGUGGUUUAUCGAGACAGUAUCCCCCAUGCAGCCUGUCACAAGUGUAUUGACUUCUACUCCCGGAUACGGGAGUUGAGGCAUUACAGUGACAGCGUGUAUGGGGAUACUCUCGAGAAGCUCACGAACAUGCACGGUCCGAAGGCGACUCUGCAGGACAUAGUCCUGCAUCUGGAGCCCCAGAACGAGAUUCCGGUAGACCUGCUCUGCCAUGAGCAGCUAUCGGACUCCGAGGAGGAGAAUGAUGAGAUUGACGGUGUCAAUCAUCAGCAUUUGCCUGCUCGGAGGGCGGAGCCUCAGCGGCAUACAAUGCUCUGUAUGUGCUGCAAGUGCGAAGCGCGGAUCGAGCUGGUGGUGGAGAGCUACUCCGACAGUGUCUACGGUGACACUUUGGAGAAGCUCACCAACACCGGCUUGUACAAUCUGCUCAUUCGCUGCUUGCGGUGCCAGAAGCCAUUGAAUCCCGCUGAGAAGCUCCGCCACCUGAACGAGAAGCGGCGGUUUCACAAUAUCGCUGGCCACUACCGUGGCCAGUGUCAUUCGUGUUGCAACCGCGCUCGUCAGGAGCGCUUGCAGCGUCGCCGGGAGACCCAGGUGAUGUGCUGCAAGUGCGAAGCUCGUAUCGAGCUGGUCGUCGAGAGUUCUGCGGAUGAUCUACGUGCGUUCCAGCAGUUGUUCCUGAAUACUCUGAGCUUCGUAUGUCCCUGGUGUGCUUCGCAGCAGAUGCUGAUACCAAUCGCCGUAGGAGGGGCUUUGGCAGGAUUGGUGCUGAUCGUCCUUAUUGCAUACCUGGUCGGCAGAAAAAGGUCUCACGCCGGAUAUCAGACUAUCGCGACGAACUUCAGCUUACUGAAGCAGGCAGGCGACGUGGAGGAGAACCCCGGCCCCAUGGACGCAAUGAAGCGCGGCUUGUGCUGCGUUUUAUUGCUCUGUGGGGCCGUGUUCGUGUCACCGUCUAUAACCCAGGACUGUUCAUUUCAGCACAGCCCAAUCUCCUCAGACUUCGCCGUAAAGAUCCGCGAGUUGUCGGACUACCUGUUACAGGACUAUCCUGUGACGGUAGCCAGCAACUUGCAGGAUGAGGAGCUUUGCGGCGGUCUGUGGAGAUUGGUGCUGGCUCAGAGAUGGAUGGAACGGUUAAAGACGGUGGCGGGGAGCAAGAUGCAGGGGCUCUUGGAGCGAGUGAAUACCGAGAUUCACUUCGUUACCAAGUGCGCCUUCCAGCCCCCGCCGAGUUGUCUACGGUUUGUGCAGACCAACAUCAGUCGUCUCCUGCAGGAGACGAGUGAGCAGUUGGUCGCACUUAAACCGUGGAUAACUCGGCAGAAUUUCUCCCGUUGCCUGGAGCUUCAGUGUCAGCCUGACAGUUCGACGCUUCCCCCGCCCUGGUCCCCGAGGCCGUUGGAGGCGACGGCCCCAACGGCCCCGGGGGGCGGGUCGGGGGACUGA
SEQ ID NO:13:082704B的ORF区(编码LAMP-HPV-LAMP~TM-P2A-OX40L)
AUGGCCGCUCCGGGGAGUGCACGCCGGCCGUUGCUGUUACUUCUCCUGUUAUUGCUGCUGGGGCUCAUGCAUUGUGCCUCAGCAGCAAUGUUCAUGGUGAAGAACGGCAACGGCACGGCGUGCAUUAUGGCAAAUUUUUCGGCCGCUUUUAGUGUCAACUAUGACACUAAGAGCGGGCCGAAGAAUAUGACCUUCGACCUACCGUCGGACGCGACGGUAGUGUUGAACCGCUCGUCUUGUGGGAAGGAGAACACGAGCGACCCGUCCCUGGUGAUUGCGUUUGGGAGAGGUCAUACACUGACUCUCAACUUCACGCGUAACGCUACGCGUUACAGCGUGCAGUUGAUGAGUUUUGUGUAUAACCUCUCCGAUACGCACCUCUUUCCGAAUGCGUCAAGUAAGGAGAUCAAGACCGUGGAGAGCAUAACAGACAUACGAGCUGACAUCGACAAGAAGUAUCGUUGCGUCUCAGGGACACAGGUUCACAUGAACAAUGUGACUGUGACCCUGCACGACGCAACGAUACAGGCUUACUUGUCGAACAGCUCGUUUUCGAGGGGGGAGACUCGGUGUGAGCAGGACAGGCCGUCGCCCACGACGGCCCCUCCUGCUCCACCGAGUCCUUCCCCCUCGCCGGUCCCGAAGUCCCCGAGCGUUGACAAGUACAACGUCUCGGGGACUAACGGGACCUGUCUGUUAGCCUCUAUGGGCUUGCAGCUCAACUUGACGUAUGAGCGGAAAGACAACACCACGGUGACGCGGUUGCUCAACAUCAAUCCCAACAAGACGAGCGCGUCCGGGAGCUGCGGUGCGCAUCUGGUGACAUUGGAGUUACAUUCAGAAGGCACGACCGUGCUCCUCUUCCAGUUCGGCAUGAAUGCGAGUUCCUCACGGUUUUUCCUGCAGGGCAUUCAGCUGAACACGAUCCUGCCUGAUGCGCGGGACCCCGCGUUCAAGGCAGCGAACGGUUCGCUGAGGGCCCUGCAGGCGACCGUGGGGAACUCGUAUAAGUGCAAUGCGGAGGAGCACGUCCGCGUUACCAAAGCGUUCUCCGUCAACAUCUUUAAGGUCUGGGUACAGGCCUUUAAGGUUGAGGGAGGACAGUUUGGUAGCGUGGAGGAGUGCCUUCUGGAUGAGAACUCCAUGCACCAGAAGCGCACCGCAAUGUUCCAGGACCCCCAGGAGCGGCCGAGGAAGUUGCCUCAGCUGUGCACGGAGCUGCAGACUACAAUACACGACAUCAUCCUUGAGUGCGUGUAUUGUAAGCAGCAGCUCCUGCGCAGGGAGGUCUACGAUUUCGCCUUCAGGGACUUGUGCAUCGUAUACCGCGACGGUAAUCCGUACGCGGUAUGCGACAAGUGCCUGAAGUUCUAUUCGAAGAUCUCCGAGUACAGGCACUACUGUUACUCGCUGUAUGGGACAACGCUCAUGCACGGGGACACUCCCACGUUGCAUGAGUACAUGUUGGACCUACAGCCUGAGACAACUGACCUCUAUUGCUAUGAGCAGUUGAAUGACAGCAGCGAAGAGGAGGAUGAGAUAGAUGGGCCCGCCGGUCAGGCUGAGCCUGACCGGGCCCACUAUAACAUCGUCACUUUUUGCUGCAAGUGUGAUUCAACGCUCAGGCUCUGCGUGCAGAGCACACACGUGGACAUACGAACCUCAGAGUACAGGCACUACUGCUACUCGCUCUACGGCACGACAUUGGAGCAGCAGUACAACAAGCCCCUGUGUGACUUGUUGAUCCGCUGCAUCAAUUGCCAGAAGCCGCUGUGUCCUGAGGAGAAGCAGCGGCAUCUGGACAAGAAGCAGCGGUUCCACAACAUACGGGGGCGUUGGACUGGUCGCUGCAUGUCGUGCUGUAGAUCGAGUAGGACGCGCAGAGAGACGCAGCUGACGCUGCGUCUCUGUGUGCAGUCGACGCACGUAGACAUCCGCACUCUCGAGGACCUGCUUAUGGGGACGCUCGGGAUCGUGUGCCCGAUCUGCAGCCAGAAGCCUAUGGCGCGGUUCGAGGAUCCGACCCGGCGUCCCUAUAAGCUUCCGGACCUCUGUACUGAGCUCAACACCAGCUUGCAGGACAUAGAGAUCACCUGUGUGUACUGCAAGACGGUGUUAGAGCUCACAGAGGUCUUUGAGUUCGCCUUCAAGGACCUGUUCGUGGUUUAUCGAGACAGUAUCCCCCAUGCAGCCUGUCACAAGUGUAUUGACUUCUACUCCCGGAUACGGGAGUUGAGGCAUUACAGUGACAGCGUGUAUGGGGAUACUCUCGAGAAGCUCACGAACAUGCACGGUCCGAAGGCGACUCUGCAGGACAUAGUCCUGCAUCUGGAGCCCCAGAACGAGAUUCCGGUAGACCUGCUCUGCCAUGAGCAGCUAUCGGACUCCGAGGAGGAGAAUGAUGAGAUUGACGGUGUCAAUCAUCAGCAUUUGCCUGCUCGGAGGGCGGAGCCUCAGCGGCAUACAAUGCUCUGUAUGUGCUGCAAGUGCGAAGCGCGGAUCGAGCUGGUGGUGGAGAGCUACUCCGACAGUGUCUACGGUGACACUUUGGAGAAGCUCACCAACACCGGCUUGUACAAUCUGCUCAUUCGCUGCUUGCGGUGCCAGAAGCCAUUGAAUCCCGCUGAGAAGCUCCGCCACCUGAACGAGAAGCGGCGGUUUCACAAUAUCGCUGGCCACUACCGUGGCCAGUGUCAUUCGUGUUGCAACCGCGCUCGUCAGGAGCGCUUGCAGCGUCGCCGGGAGACCCAGGUGAUGUGCUGCAAGUGCGAAGCUCGUAUCGAGCUGGUCGUCGAGAGUUCUGCGGAUGAUCUACGUGCGUUCCAGCAGUUGUUCCUGAAUACUCUGAGCUUCGUAUGUCCCUGGUGUGCUUCGCAGCAGAUGCUGAUACCCAUCGCGGUGGGCGGCGCCCUCGCCGGGUUGGUGCUCAUCGUGUUGAUAGCCUACCUGGUGGGGCGCAAGCGCUCACACGCGGGGUAcCAGACAAUAGCCACCAACUUCUCCUUGCUCAAGCAGGCAGGGGACGUGGAGGAGAAUCCAGGUCCCAUGGAGCGGGUGCAGCCACUGGAGGAGAACGUCGGGAACGCUGCGAGACCGAGGUUCGAGCGGAAUAAGCUUCUGCUCGUAGCCUCGGUCAUCCAGGGUCUCGGCCUCCUCCUGUGCUUCACCUACAUCUGCUUGCACUUCAGUGCAUUGCAGGUGUCACACAGAUACCCCCGGAUUCAGAGUAUCAAGGUGCAGUUCACGGAGUACAAGAAGGAGAAGGGUUUCAUCCUCACUUCUCAGAAGGAGGAUGAGAUCAUGAAGGUCCAGAACAACUCCGUGAUCAUCAACUGCGAUGGAUUUUACCUUAUAAGUCUGAAAGGCUACUUCAGUCAGGAAGUUAACAUCUCUCUGCACUAUCAGAAGGAUGAGGAGCCCCUCUUCCAGCUGAAGAAGGUGCGGAGUGUUAACUCCCUGAUGGUAGCCUCUCUGACUUAUAAGGAUAAAGUCUAUCUCAACGUCACCACCGAUAACACGAGUCUGGAUGACUUUCAUGUUAACGGUGGUGAGUUGAUCUUGAUACAUCAGAAUCCGGGGGAGUUCUGUGUGUUAUGA
SEQ ID NO:14:082711的ORF区(编码DD-HPV-P2A-IgE-FLT3L)
AUGGGGGUUCAGGUAGAGACGAUCUCUCCAGGAGAUGGCAGAACCUUUCCUAAACGGGGGCAAACUUGCGUAGUUCACUAUACGGGCAUGCUGGAGGACGGGAAAAAAUUCGAUAGCAGUCGGGAUCGGAAUAAACCCUUUAAGUUUAUGCUCGGGAAGCAGGAAGUGAUUCGCGGGUGGGAAGAAGGCGUCGCUCAGAUGUCUGUUGGGCAAAGAGCAAAACUGACAAUCUCCCCCGACUACGCAUACGGGGCAACUGGUCACCCAGGAAUAAUCCCGCCUCACGCAACUCUGGUCUUCGAUGUCGAACUCUUGAAGCCGGAGCACCAGAAGCGCACCGCAAUGUUCCAGGACCCCCAGGAGCGGCCGAGGAAGUUGCCUCAGCUGUGCACGGAGCUGCAGACUACAAUACACGACAUCAUCCUUGAGUGCGUGUAUUGUAAGCAGCAGCUCCUGCGCAGGGAGGUCUACGAUUUCGCCUUCAGGGACUUGUGCAUCGUAUACCGCGACGGUAAUCCGUACGCGGUAUGCGACAAGUGCCUGAAGUUCUAUUCGAAGAUCUCCGAGUACAGGCACUACUGUUACUCGCUGUAUGGGACAACGCUCAUGCACGGGGACACUCCCACGUUGCAUGAGUACAUGUUGGACCUACAGCCUGAGACAACUGACCUCUAUUGCUAUGAGCAGUUGAAUGACAGCAGCGAAGAGGAGGAUGAGAUAGAUGGGCCCGCCGGUCAGGCUGAGCCUGACCGGGCCCACUAUAACAUCGUCACUUUUUGCUGCAAGUGUGAUUCAACGCUCAGGCUCUGCGUGCAGAGCACACACGUGGACAUACGAACCUCAGAGUACAGGCACUACUGCUACUCGCUCUACGGCACGACAUUGGAGCAGCAGUACAACAAGCCCCUGUGUGACUUGUUGAUCCGCUGCAUCAAUUGCCAGAAGCCGCUGUGUCCUGAGGAGAAGCAGCGGCAUCUGGACAAGAAGCAGCGGUUCCACAACAUACGGGGGCGUUGGACUGGUCGCUGCAUGUCGUGCUGUAGAUCGAGUAGGACGCGCAGAGAGACGCAGCUGACGCUGCGUCUCUGUGUGCAGUCGACGCACGUAGACAUCCGCACUCUCGAGGACCUGCUUAUGGGGACGCUCGGGAUCGUGUGCCCGAUCUGCAGCCAGAAGCCUAUGGCGCGGUUCGAGGAUCCGACCCGGCGUCCCUAUAAGCUGCCGGACCUCUGUACUGAGCUCAACACCAGCUUGCAGGACAUAGAGAUCACCUGUGUGUACUGCAAGACGGUGUUAGAGCUCACAGAGGUCUUUGAGUUCGCCUUCAAGGACCUGUUCGUGGUUUAUCGAGACAGUAUCCCCCAUGCAGCCUGUCACAAGUGUAUUGACUUCUACUCCCGGAUACGGGAGUUGAGGCAUUACAGUGACAGCGUGUAUGGGGAUACUCUCGAGAAGCUCACGAACAUGCACGGUCCGAAGGCGACUCUGCAGGACAUAGUCCUGCAUCUGGAGCCCCAGAACGAGAUUCCGGUAGACCUGCUCUGCCAUGAGCAGCUAUCGGACUCCGAGGAGGAGAAUGAUGAGAUUGACGGUGUCAAUCAUCAGCAUUUGCCUGCUCGGAGGGCGGAGCCUCAGCGGCAUACAAUGCUCUGUAUGUGCUGCAAGUGCGAAGCGCGGAUCGAGCUGGUGGUGGAGAGCUACUCCGACAGUGUCUACGGUGACACUUUGGAGAAGCUCACCAACACCGGCUUGUACAAUCUGCUCAUUCGCUGCUUGCGGUGCCAGAAGCCAUUGAAUCCCGCUGAGAAGCUCCGCCACCUGAACGAGAAGCGGCGGUUUCACAAUAUCGCUGGCCACUACCGUGGCCAGUGUCAUUCGUGUUGCAACCGCGCUCGUCAGGAGCGCUUGCAGCGUCGCCGGGAAACCCAGGUGAUGUGCUGCAAGUGCGAAGCUCGUAUCGAGCUGGUCGUCGAGAGUUCUGCGGAUGAUCUACGUGCGUUCCAGCAGUUGUUCCUGAAUACUCUGAGCUUCGUAUGUCCCUGGUGUGCUUCGCAGCAGGGCGGCGCCACGAACUUCUCUCUGUUAAAGCAAGCAGGAGACGUGGAAGAAAACCCCGGUCCUAUGGACUGGACCUGGAUUCUGUUCCUGGUGGCCGCCGCUACACGGGUGCACAGCACCCAGGACUGUUCAUUUCAGCACAGCCCAAUCUCCUCAGACUUCGCCGUAAAGAUCCGCGAGUUGUCGGACUACCUGUUACAGGACUAUCCUGUGACGGUAGCCAGCAACUUGCAGGAUGAGGAGCUUUGCGGCGGUCUGUGGAGAUUGGUGCUGGCUCAGAGAUGGAUGGAACGGUUAAAGACGGUGGCGGGGAGCAAGAUGCAGGGGCUCUUGGAGCGAGUGAAUACCGAGAUUCACUUCGUUACCAAGUGCGCCUUCCAGCCCCCGCCGAGUUGUCUACGGUUUGUGCAGACCAACAUCAGUCGUCUCCUGCAGGAGACGAGUGAGCAGUUGGUCGCACUUAAACCGUGGAUAACUCGGCAGAAUUUCUCCCGUUGCCUGGAGCUUCAGUGUCAGCCUGACAGUUCGACGCUUCCCCCGCCCUGGUCCCCGAGGCCGUUGGAGGCGACGGCCCCAACGGCCCCGGGGGGCGGGUCGGGGGACUGA
SEQ ID NO:15:082712的ORF区(编码DD-HPV-P2A-OX40L)
AUGGGGGUUCAGGUAGAGACGAUCUCUCCAGGAGAUGGCAGAACCUUUCCUAAACGGGGGCAAACUUGCGUAGUUCACUAUACGGGCAUGCUGGAGGACGGGAAAAAAUUCGAUAGCAGUCGGGAUCGGAAUAAACCCUUUAAGUUUAUGCUCGGGAAGCAGGAAGUGAUUCGCGGGUGGGAAGAAGGCGUCGCUCAGAUGUCUGUUGGGCAAAGAGCAAAACUGACAAUCUCCCCCGACUACGCAUACGGGGCAACUGGUCACCCAGGAAUAAUCCCGCCUCACGCAACUCUGGUCUUCGAUGUCGAACUCUUGAAGCCGGAGCACCAGAAGCGCACCGCAAUGUUCCAGGACCCCCAGGAGCGGCCGAGGAAGUUGCCUCAGCUGUGCACGGAGCUGCAGACUACAAUACACGACAUCAUCCUUGAGUGCGUGUAUUGUAAGCAGCAGCUCCUGCGCAGGGAGGUCUACGAUUUCGCCUUCAGGGACUUGUGCAUCGUAUACCGCGACGGUAAUCCGUACGCGGUAUGCGACAAGUGCCUGAAGUUCUAUUCGAAGAUCUCCGAGUACAGGCACUACUGUUACUCGCUGUAUGGGACAACGCUCAUGCACGGGGACACUCCCACGUUGCAUGAGUACAUGUUGGACCUACAGCCUGAGACAACUGACCUCUAUUGCUAUGAGCAGUUGAAUGACAGCAGCGAAGAGGAGGAUGAGAUAGAUGGGCCCGCCGGUCAGGCUGAGCCUGACCGGGCCCACUAUAACAUCGUCACUUUUUGCUGCAAGUGUGAUUCAACGCUCAGGCUCUGCGUGCAGAGCACACACGUGGACAUACGAACCUCAGAGUACAGGCACUACUGCUACUCGCUCUACGGCACGACAUUGGAGCAGCAGUACAACAAGCCCCUGUGUGACUUGUUGAUCCGCUGCAUCAAUUGCCAGAAGCCGCUGUGUCCUGAGGAGAAGCAGCGGCAUCUGGACAAGAAGCAGCGGUUCCACAACAUACGGGGGCGUUGGACUGGUCGCUGCAUGUCGUGCUGUAGAUCGAGUAGGACGCGCAGAGAGACGCAGCUGACGCUGCGUCUCUGUGUGCAGUCGACGCACGUAGACAUCCGCACUCUCGAGGACCUGCUUAUGGGGACGCUCGGGAUCGUGUGCCCGAUCUGCAGCCAGAAGCCUAUGGCGCGGUUCGAGGAUCCGACCCGGCGUCCCUAUAAGCUGCCGGACCUCUGUACUGAGCUCAACACCAGCUUGCAGGACAUAGAGAUCACCUGUGUGUACUGCAAGACGGUGUUAGAGCUCACAGAGGUCUUUGAGUUCGCCUUCAAGGACCUGUUCGUGGUUUAUCGAGACAGUAUCCCCCAUGCAGCCUGUCACAAGUGUAUUGACUUCUACUCCCGGAUACGGGAGUUGAGGCAUUACAGUGACAGCGUGUAUGGGGAUACUCUCGAGAAGCUCACGAACAUGCACGGUCCGAAGGCGACUCUGCAGGACAUAGUCCUGCAUCUGGAGCCCCAGAACGAGAUUCCGGUAGACCUGCUCUGCCAUGAGCAGCUAUCGGACUCCGAGGAGGAGAAUGAUGAGAUUGACGGUGUCAAUCAUCAGCAUUUGCCUGCUCGGAGGGCGGAGCCUCAGCGGCAUACAAUGCUCUGUAUGUGCUGCAAGUGCGAAGCGCGGAUCGAGCUGGUGGUGGAGAGCUACUCCGACAGUGUCUACGGUGACACUUUGGAGAAGCUCACCAACACCGGCUUGUACAAUCUGCUCAUUCGCUGCUUGCGGUGCCAGAAGCCAUUGAAUCCCGCUGAGAAGCUCCGCCACCUGAACGAGAAGCGGCGGUUUCACAAUAUCGCUGGCCACUACCGUGGCCAGUGUCAUUCGUGUUGCAACCGCGCUCGUCAGGAGCGCUUGCAGCGUCGCCGGGAAACCCAGGUGAUGUGCUGCAAGUGCGAAGCUCGUAUCGAGCUGGUCGUCGAGAGUUCUGCGGAUGAUCUACGUGCGUUCCAGCAGUUGUUCCUGAAUACUCUGAGCUUCGUAUGUCCCUGGUGUGCUUCGCAGCAGGGCGGCGCCACGAACUUCUCUCUGUUAAAGCAAGCAGGAGACGUGGAAGAAAACCCCGGUCCUAUGGAGCGGGUGCAGCCACUGGAGGAGAACGUCGGGAACGCUGCGAGGCCGAGGUUCGAGCGGAAUAAGCUGCUGCUCGUAGCCUCGGUCAUCCAGGGUCUGGGCCUCCUCCUGUGCUUCACCUACAUCUGCUUGCACUUCAGUGCAUUGCAGGUGUCACACAGAUACCCCCGGAUUCAGAGUAUCAAGGUGCAGUUCACGGAGUACAAGAAGGAGAAGGGUUUCAUCCUCACUUCUCAGAAGGAGGAUGAGAUCAUGAAGGUCCAGAACAACUCCGUGAUCAUCAACUGCGAUGGAUUUUACCUUAUAAGUCUGAAAGGCUACUUCAGUCAGGAAGUUAACAUCUCUCUGCACUAUCAGAAGGAUGAGGAGCCCCUCUUCCAGCUGAAGAAGGUGCGGAGUGUUAACUCCCUGAUGGUAGCCUCUCUGACUUAUAAGGAUAAAGUCUAUCUCAACGUCACCACCGAUAACACGAGUCUGGAUGACUUUCAUGUUAACGGUGGUGAGUUGAUCUUGAUACAUCAGAAUCCGGGGGAGUUCUGUGUGUUAUGA
SEQ ID NO:16:082703A的完整mRNA序列:
GGGACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGCCACCAUGGCCGCUCCGGGGAGUGCACGCCGGCCGUUGCUGUUACUUCUCCUGUUAUUGCUGCUGGGGCUCAUGCAUUGUGCCUCAGCAGCAAUGUUCAUGGUGAAGAACGGCAACGGCACGGCGUGCAUUAUGGCAAAUUUUUCGGCCGCUUUUAGUGUCAACUAUGACACUAAGAGCGGGCCGAAGAAUAUGACCUUCGACCUACCGUCGGACGCGACGGUAGUGUUGAACCGCUCGUCUUGUGGGAAGGAGAACACGAGCGACCCGUCCCUGGUGAUUGCGUUUGGGAGAGGUCAUACACUGACUCUCAACUUCACGCGUAACGCUACGCGUUACAGCGUGCAGUUGAUGAGUUUUGUGUAUAACCUCUCCGAUACGCACCUCUUUCCGAAUGCGUCAAGUAAGGAGAUCAAGACCGUGGAGAGCAUAACAGACAUACGAGCUGACAUCGACAAGAAGUAUCGUUGCGUCUCAGGGACACAGGUUCACAUGAACAAUGUGACUGUGACCCUGCACGACGCAACGAUACAGGCUUACUUGUCGAACAGCUCGUUUUCGAGGGGGGAGACUCGGUGUGAGCAGGACAGGCCGUCGCCCACGACGGCCCCUCCUGCUCCACCGAGUCCUUCCCCCUCGCCGGUCCCGAAGUCCCCGAGCGUUGACAAGUACAACGUCUCGGGGACUAACGGGACCUGUCUGUUAGCCUCUAUGGGCUUGCAGCUCAACUUGACGUAUGAGCGGAAAGACAACACCACGGUGACGCGGUUGCUCAACAUCAAUCCCAACAAGACGAGCGCGUCCGGGAGCUGCGGUGCGCAUCUGGUGACAUUGGAGUUACAUUCAGAAGGCACGACCGUGCUCCUCUUCCAGUUCGGCAUGAAUGCGAGUUCCUCACGGUUUUUCCUGCAGGGCAUUCAGCUGAACACGAUCCUGCCUGAUGCGCGGGACCCCGCGUUCAAGGCAGCGAACGGUUCGCUGAGGGCCCUGCAGGCGACCGUGGGGAACUCGUAUAAGUGCAAUGCGGAGGAGCACGUCCGCGUUACCAAAGCGUUCUCCGUCAACAUCUUUAAGGUCUGGGUACAGGCCUUUAAGGUUGAGGGAGGACAGUUUGGUAGCGUGGAGGAGUGCCUUCUGGAUGAGAACUCCAUGCACCAGAAGCGCACCGCAAUGUUCCAGGACCCCCAGGAGCGGCCGAGGAAGUUGCCUCAGCUGUGCACGGAGCUGCAGACUACAAUACACGACAUCAUCCUUGAGUGCGUGUAUUGUAAGCAGCAGCUCCUGCGCAGGGAGGUCUACGAUUUCGCCUUCAGGGACUUGUGCAUCGUAUACCGCGACGGUAAUCCGUACGCGGUAUGCGACAAGUGCCUGAAGUUCUAUUCGAAGAUCUCCGAGUACAGGCACUACUGUUACUCGCUGUAUGGGACAACGCUCAUGCACGGGGACACUCCCACGUUGCAUGAGUACAUGUUGGACCUACAGCCUGAGACAACUGACCUCUAUUGCUAUGAGCAGUUGAAUGACAGCAGCGAAGAGGAGGAUGAGAUAGAUGGGCCCGCCGGUCAGGCUGAGCCUGACCGGGCCCACUAUAACAUCGUCACUUUUUGCUGCAAGUGUGAUUCAACGCUCAGGCUCUGCGUGCAGAGCACACACGUGGACAUACGAACCUCAGAGUACAGGCACUACUGCUACUCGCUCUACGGCACGACAUUGGAGCAGCAGUACAACAAGCCCCUGUGUGACUUGUUGAUCCGCUGCAUCAAUUGCCAGAAGCCGCUGUGUCCUGAGGAGAAGCAGCGGCAUCUGGACAAGAAGCAGCGGUUCCACAACAUACGGGGGCGUUGGACUGGUCGCUGCAUGUCGUGCUGUAGAUCGAGUAGGACGCGCAGAGAGACGCAGCUGACGCUGCGUCUCUGUGUGCAGUCGACGCACGUAGACAUCCGCACUCUCGAGGACCUGCUUAUGGGGACGCUCGGGAUCGUGUGCCCGAUCUGCAGCCAGAAGCCUAUGGCGCGGUUCGAGGAUCCGACCCGGCGUCCCUAUAAGCUUCCGGACCUCUGUACUGAGCUCAACACCAGCUUGCAGGACAUAGAGAUCACCUGUGUGUACUGCAAGACGGUGUUAGAGCUCACAGAGGUCUUUGAGUUCGCCUUCAAGGACCUGUUCGUGGUUUAUCGAGACAGUAUCCCCCAUGCAGCCUGUCACAAGUGUAUUGACUUCUACUCCCGGAUACGGGAGUUGAGGCAUUACAGUGACAGCGUGUAUGGGGAUACUCUCGAGAAGCUCACGAACAUGCACGGUCCGAAGGCGACUCUGCAGGACAUAGUCCUGCAUCUGGAGCCCCAGAACGAGAUUCCGGUAGACCUGCUCUGCCAUGAGCAGCUAUCGGACUCCGAGGAGGAGAAUGAUGAGAUUGACGGUGUCAAUCAUCAGCAUUUGCCUGCUCGGAGGGCGGAGCCUCAGCGGCAUACAAUGCUCUGUAUGUGCUGCAAGUGCGAAGCGCGGAUCGAGCUGGUGGUGGAGAGCUACUCCGACAGUGUCUACGGUGACACUUUGGAGAAGCUCACCAACACCGGCUUGUACAAUCUGCUCAUUCGCUGCUUGCGGUGCCAGAAGCCAUUGAAUCCCGCUGAGAAGCUCCGCCACCUGAACGAGAAGCGGCGGUUUCACAAUAUCGCUGGCCACUACCGUGGCCAGUGUCAUUCGUGUUGCAACCGCGCUCGUCAGGAGCGCUUGCAGCGUCGCCGGGAGACCCAGGUGAUGUGCUGCAAGUGCGAAGCUCGUAUCGAGCUGGUCGUCGAGAGUUCUGCGGAUGAUCUACGUGCGUUCCAGCAGUUGUUCCUGAAUACUCUGAGCUUCGUAUGUCCCUGGUGUGCUUCGCAGCAGAUGCUGAUACCAAUCGCCGUAGGAGGGGCUUUGGCAGGAUUGGUGCUGAUCGUCCUUAUUGCAUACCUGGUCGGCAGAAAAAGGUCUCACGCCGGAUAUCAGACUAUCGCGACGAACUUCAGCUUACUGAAGCAGGCAGGCGACGUGGAGGAGAACCCCGGCCCCAUGGACGCAAUGAAGCGCGGCUUGUGCUGCGUUUUAUUGCUCUGUGGGGCCGUGUUCGUGUCACCGUCUAUAACCCAGGACUGUUCAUUUCAGCACAGCCCAAUCUCCUCAGACUUCGCCGUAAAGAUCCGCGAGUUGUCGGACUACCUGUUACAGGACUAUCCUGUGACGGUAGCCAGCAACUUGCAGGAUGAGGAGCUUUGCGGCGGUCUGUGGAGAUUGGUGCUGGCUCAGAGAUGGAUGGAACGGUUAAAGACGGUGGCGGGGAGCAAGAUGCAGGGGCUCUUGGAGCGAGUGAAUACCGAGAUUCACUUCGUUACCAAGUGCGCCUUCCAGCCCCCGCCGAGUUGUCUACGGUUUGUGCAGACCAACAUCAGUCGUCUCCUGCAGGAGACGAGUGAGCAGUUGGUCGCACUUAAACCGUGGAUAACUCGGCAGAAUUUCUCCCGUUGCCUGGAGCUUCAGUGUCAGCCUGACAGUUCGACGCUUCCCCCGCCCUGGUCCCCGAGGCCGUUGGAGGCGACGGCCCCAACGGCCCCGGGGGGCGGGUCGGGGGACUGAUUAAUUAAGCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGGCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGAAUUCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACUAG
SEQ ID NO:17:082704B的完整mRNA序列:
GGGACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGCCACCAUGGCCGCUCCGGGGAGUGCACGCCGGCCGUUGCUGUUACUUCUCCUGUUAUUGCUGCUGGGGCUCAUGCAUUGUGCCUCAGCAGCAAUGUUCAUGGUGAAGAACGGCAACGGCACGGCGUGCAUUAUGGCAAAUUUUUCGGCCGCUUUUAGUGUCAACUAUGACACUAAGAGCGGGCCGAAGAAUAUGACCUUCGACCUACCGUCGGACGCGACGGUAGUGUUGAACCGCUCGUCUUGUGGGAAGGAGAACACGAGCGACCCGUCCCUGGUGAUUGCGUUUGGGAGAGGUCAUACACUGACUCUCAACUUCACGCGUAACGCUACGCGUUACAGCGUGCAGUUGAUGAGUUUUGUGUAUAACCUCUCCGAUACGCACCUCUUUCCGAAUGCGUCAAGUAAGGAGAUCAAGACCGUGGAGAGCAUAACAGACAUACGAGCUGACAUCGACAAGAAGUAUCGUUGCGUCUCAGGGACACAGGUUCACAUGAACAAUGUGACUGUGACCCUGCACGACGCAACGAUACAGGCUUACUUGUCGAACAGCUCGUUUUCGAGGGGGGAGACUCGGUGUGAGCAGGACAGGCCGUCGCCCACGACGGCCCCUCCUGCUCCACCGAGUCCUUCCCCCUCGCCGGUCCCGAAGUCCCCGAGCGUUGACAAGUACAACGUCUCGGGGACUAACGGGACCUGUCUGUUAGCCUCUAUGGGCUUGCAGCUCAACUUGACGUAUGAGCGGAAAGACAACACCACGGUGACGCGGUUGCUCAACAUCAAUCCCAACAAGACGAGCGCGUCCGGGAGCUGCGGUGCGCAUCUGGUGACAUUGGAGUUACAUUCAGAAGGCACGACCGUGCUCCUCUUCCAGUUCGGCAUGAAUGCGAGUUCCUCACGGUUUUUCCUGCAGGGCAUUCAGCUGAACACGAUCCUGCCUGAUGCGCGGGACCCCGCGUUCAAGGCAGCGAACGGUUCGCUGAGGGCCCUGCAGGCGACCGUGGGGAACUCGUAUAAGUGCAAUGCGGAGGAGCACGUCCGCGUUACCAAAGCGUUCUCCGUCAACAUCUUUAAGGUCUGGGUACAGGCCUUUAAGGUUGAGGGAGGACAGUUUGGUAGCGUGGAGGAGUGCCUUCUGGAUGAGAACUCCAUGCACCAGAAGCGCACCGCAAUGUUCCAGGACCCCCAGGAGCGGCCGAGGAAGUUGCCUCAGCUGUGCACGGAGCUGCAGACUACAAUACACGACAUCAUCCUUGAGUGCGUGUAUUGUAAGCAGCAGCUCCUGCGCAGGGAGGUCUACGAUUUCGCCUUCAGGGACUUGUGCAUCGUAUACCGCGACGGUAAUCCGUACGCGGUAUGCGACAAGUGCCUGAAGUUCUAUUCGAAGAUCUCCGAGUACAGGCACUACUGUUACUCGCUGUAUGGGACAACGCUCAUGCACGGGGACACUCCCACGUUGCAUGAGUACAUGUUGGACCUACAGCCUGAGACAACUGACCUCUAUUGCUAUGAGCAGUUGAAUGACAGCAGCGAAGAGGAGGAUGAGAUAGAUGGGCCCGCCGGUCAGGCUGAGCCUGACCGGGCCCACUAUAACAUCGUCACUUUUUGCUGCAAGUGUGAUUCAACGCUCAGGCUCUGCGUGCAGAGCACACACGUGGACAUACGAACCUCAGAGUACAGGCACUACUGCUACUCGCUCUACGGCACGACAUUGGAGCAGCAGUACAACAAGCCCCUGUGUGACUUGUUGAUCCGCUGCAUCAAUUGCCAGAAGCCGCUGUGUCCUGAGGAGAAGCAGCGGCAUCUGGACAAGAAGCAGCGGUUCCACAACAUACGGGGGCGUUGGACUGGUCGCUGCAUGUCGUGCUGUAGAUCGAGUAGGACGCGCAGAGAGACGCAGCUGACGCUGCGUCUCUGUGUGCAGUCGACGCACGUAGACAUCCGCACUCUCGAGGACCUGCUUAUGGGGACGCUCGGGAUCGUGUGCCCGAUCUGCAGCCAGAAGCCUAUGGCGCGGUUCGAGGAUCCGACCCGGCGUCCCUAUAAGCUUCCGGACCUCUGUACUGAGCUCAACACCAGCUUGCAGGACAUAGAGAUCACCUGUGUGUACUGCAAGACGGUGUUAGAGCUCACAGAGGUCUUUGAGUUCGCCUUCAAGGACCUGUUCGUGGUUUAUCGAGACAGUAUCCCCCAUGCAGCCUGUCACAAGUGUAUUGACUUCUACUCCCGGAUACGGGAGUUGAGGCAUUACAGUGACAGCGUGUAUGGGGAUACUCUCGAGAAGCUCACGAACAUGCACGGUCCGAAGGCGACUCUGCAGGACAUAGUCCUGCAUCUGGAGCCCCAGAACGAGAUUCCGGUAGACCUGCUCUGCCAUGAGCAGCUAUCGGACUCCGAGGAGGAGAAUGAUGAGAUUGACGGUGUCAAUCAUCAGCAUUUGCCUGCUCGGAGGGCGGAGCCUCAGCGGCAUACAAUGCUCUGUAUGUGCUGCAAGUGCGAAGCGCGGAUCGAGCUGGUGGUGGAGAGCUACUCCGACAGUGUCUACGGUGACACUUUGGAGAAGCUCACCAACACCGGCUUGUACAAUCUGCUCAUUCGCUGCUUGCGGUGCCAGAAGCCAUUGAAUCCCGCUGAGAAGCUCCGCCACCUGAACGAGAAGCGGCGGUUUCACAAUAUCGCUGGCCACUACCGUGGCCAGUGUCAUUCGUGUUGCAACCGCGCUCGUCAGGAGCGCUUGCAGCGUCGCCGGGAGACCCAGGUGAUGUGCUGCAAGUGCGAAGCUCGUAUCGAGCUGGUCGUCGAGAGUUCUGCGGAUGAUCUACGUGCGUUCCAGCAGUUGUUCCUGAAUACUCUGAGCUUCGUAUGUCCCUGGUGUGCUUCGCAGCAGAUGCUGAUACCCAUCGCGGUGGGCGGCGCCCUCGCCGGGUUGGUGCUCAUCGUGUUGAUAGCCUACCUGGUGGGGCGCAAGCGCUCACACGCGGGGUAcCAGACAAUAGCCACCAACUUCUCCUUGCUCAAGCAGGCAGGGGACGUGGAGGAGAAUCCAGGUCCCAUGGAGCGGGUGCAGCCACUGGAGGAGAACGUCGGGAACGCUGCGAGACCGAGGUUCGAGCGGAAUAAGCUUCUGCUCGUAGCCUCGGUCAUCCAGGGUCUCGGCCUCCUCCUGUGCUUCACCUACAUCUGCUUGCACUUCAGUGCAUUGCAGGUGUCACACAGAUACCCCCGGAUUCAGAGUAUCAAGGUGCAGUUCACGGAGUACAAGAAGGAGAAGGGUUUCAUCCUCACUUCUCAGAAGGAGGAUGAGAUCAUGAAGGUCCAGAACAACUCCGUGAUCAUCAACUGCGAUGGAUUUUACCUUAUAAGUCUGAAAGGCUACUUCAGUCAGGAAGUUAACAUCUCUCUGCACUAUCAGAAGGAUGAGGAGCCCCUCUUCCAGCUGAAGAAGGUGCGGAGUGUUAACUCCCUGAUGGUAGCCUCUCUGACUUAUAAGGAUAAAGUCUAUCUCAACGUCACCACCGAUAACACGAGUCUGGAUGACUUUCAUGUUAACGGUGGUGAGUUGAUCUUGAUACAUCAGAAUCCGGGGGAGUUCUGUGUGUUAUGAUUaaUUaaGCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGGCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGAAUUCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACUAG
SEQ ID NO:18:082711的完整mRNA序列:
GGGACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGCCACCAUGGGGGUUCAGGUAGAGACGAUCUCUCCAGGAGAUGGCAGAACCUUUCCUAAACGGGGGCAAACUUGCGUAGUUCACUAUACGGGCAUGCUGGAGGACGGGAAAAAAUUCGAUAGCAGUCGGGAUCGGAAUAAACCCUUUAAGUUUAUGCUCGGGAAGCAGGAAGUGAUUCGCGGGUGGGAAGAAGGCGUCGCUCAGAUGUCUGUUGGGCAAAGAGCAAAACUGACAAUCUCCCCCGACUACGCAUACGGGGCAACUGGUCACCCAGGAAUAAUCCCGCCUCACGCAACUCUGGUCUUCGAUGUCGAACUCUUGAAGCCGGAGCACCAGAAGCGCACCGCAAUGUUCCAGGACCCCCAGGAGCGGCCGAGGAAGUUGCCUCAGCUGUGCACGGAGCUGCAGACUACAAUACACGACAUCAUCCUUGAGUGCGUGUAUUGUAAGCAGCAGCUCCUGCGCAGGGAGGUCUACGAUUUCGCCUUCAGGGACUUGUGCAUCGUAUACCGCGACGGUAAUCCGUACGCGGUAUGCGACAAGUGCCUGAAGUUCUAUUCGAAGAUCUCCGAGUACAGGCACUACUGUUACUCGCUGUAUGGGACAACGCUCAUGCACGGGGACACUCCCACGUUGCAUGAGUACAUGUUGGACCUACAGCCUGAGACAACUGACCUCUAUUGCUAUGAGCAGUUGAAUGACAGCAGCGAAGAGGAGGAUGAGAUAGAUGGGCCCGCCGGUCAGGCUGAGCCUGACCGGGCCCACUAUAACAUCGUCACUUUUUGCUGCAAGUGUGAUUCAACGCUCAGGCUCUGCGUGCAGAGCACACACGUGGACAUACGAACCUCAGAGUACAGGCACUACUGCUACUCGCUCUACGGCACGACAUUGGAGCAGCAGUACAACAAGCCCCUGUGUGACUUGUUGAUCCGCUGCAUCAAUUGCCAGAAGCCGCUGUGUCCUGAGGAGAAGCAGCGGCAUCUGGACAAGAAGCAGCGGUUCCACAACAUACGGGGGCGUUGGACUGGUCGCUGCAUGUCGUGCUGUAGAUCGAGUAGGACGCGCAGAGAGACGCAGCUGACGCUGCGUCUCUGUGUGCAGUCGACGCACGUAGACAUCCGCACUCUCGAGGACCUGCUUAUGGGGACGCUCGGGAUCGUGUGCCCGAUCUGCAGCCAGAAGCCUAUGGCGCGGUUCGAGGAUCCGACCCGGCGUCCCUAUAAGCUGCCGGACCUCUGUACUGAGCUCAACACCAGCUUGCAGGACAUAGAGAUCACCUGUGUGUACUGCAAGACGGUGUUAGAGCUCACAGAGGUCUUUGAGUUCGCCUUCAAGGACCUGUUCGUGGUUUAUCGAGACAGUAUCCCCCAUGCAGCCUGUCACAAGUGUAUUGACUUCUACUCCCGGAUACGGGAGUUGAGGCAUUACAGUGACAGCGUGUAUGGGGAUACUCUCGAGAAGCUCACGAACAUGCACGGUCCGAAGGCGACUCUGCAGGACAUAGUCCUGCAUCUGGAGCCCCAGAACGAGAUUCCGGUAGACCUGCUCUGCCAUGAGCAGCUAUCGGACUCCGAGGAGGAGAAUGAUGAGAUUGACGGUGUCAAUCAUCAGCAUUUGCCUGCUCGGAGGGCGGAGCCUCAGCGGCAUACAAUGCUCUGUAUGUGCUGCAAGUGCGAAGCGCGGAUCGAGCUGGUGGUGGAGAGCUACUCCGACAGUGUCUACGGUGACACUUUGGAGAAGCUCACCAACACCGGCUUGUACAAUCUGCUCAUUCGCUGCUUGCGGUGCCAGAAGCCAUUGAAUCCCGCUGAGAAGCUCCGCCACCUGAACGAGAAGCGGCGGUUUCACAAUAUCGCUGGCCACUACCGUGGCCAGUGUCAUUCGUGUUGCAACCGCGCUCGUCAGGAGCGCUUGCAGCGUCGCCGGGAAACCCAGGUGAUGUGCUGCAAGUGCGAAGCUCGUAUCGAGCUGGUCGUCGAGAGUUCUGCGGAUGAUCUACGUGCGUUCCAGCAGUUGUUCCUGAAUACUCUGAGCUUCGUAUGUCCCUGGUGUGCUUCGCAGCAGGGCGGCGCCACGAACUUCUCUCUGUUAAAGCAAGCAGGAGACGUGGAAGAAAACCCCGGUCCUAUGGACUGGACCUGGAUUCUGUUCCUGGUGGCCGCCGCUACACGGGUGCACAGCACCCAGGACUGUUCAUUUCAGCACAGCCCAAUCUCCUCAGACUUCGCCGUAAAGAUCCGCGAGUUGUCGGACUACCUGUUACAGGACUAUCCUGUGACGGUAGCCAGCAACUUGCAGGAUGAGGAGCUUUGCGGCGGUCUGUGGAGAUUGGUGCUGGCUCAGAGAUGGAUGGAACGGUUAAAGACGGUGGCGGGGAGCAAGAUGCAGGGGCUCUUGGAGCGAGUGAAUACCGAGAUUCACUUCGUUACCAAGUGCGCCUUCCAGCCCCCGCCGAGUUGUCUACGGUUUGUGCAGACCAACAUCAGUCGUCUCCUGCAGGAGACGAGUGAGCAGUUGGUCGCACUUAAACCGUGGAUAACUCGGCAGAAUUUCUCCCGUUGCCUGGAGCUUCAGUGUCAGCCUGACAGUUCGACGCUUCCCCCGCCCUGGUCCCCGAGGCCGUUGGAGGCGACGGCCCCAACGGCCCCGGGGGGCGGGUCGGGGGACUGAAAGCUUGCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGGCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGAAUUCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACUAG
SEQ ID NO:19:082712的完整mRNA序列:
GGGACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGCCACCAUGGGGGUUCAGGUAGAGACGAUCUCUCCAGGAGAUGGCAGAACCUUUCCUAAACGGGGGCAAACUUGCGUAGUUCACUAUACGGGCAUGCUGGAGGACGGGAAAAAAUUCGAUAGCAGUCGGGAUCGGAAUAAACCCUUUAAGUUUAUGCUCGGGAAGCAGGAAGUGAUUCGCGGGUGGGAAGAAGGCGUCGCUCAGAUGUCUGUUGGGCAAAGAGCAAAACUGACAAUCUCCCCCGACUACGCAUACGGGGCAACUGGUCACCCAGGAAUAAUCCCGCCUCACGCAACUCUGGUCUUCGAUGUCGAACUCUUGAAGCCGGAGCACCAGAAGCGCACCGCAAUGUUCCAGGACCCCCAGGAGCGGCCGAGGAAGUUGCCUCAGCUGUGCACGGAGCUGCAGACUACAAUACACGACAUCAUCCUUGAGUGCGUGUAUUGUAAGCAGCAGCUCCUGCGCAGGGAGGUCUACGAUUUCGCCUUCAGGGACUUGUGCAUCGUAUACCGCGACGGUAAUCCGUACGCGGUAUGCGACAAGUGCCUGAAGUUCUAUUCGAAGAUCUCCGAGUACAGGCACUACUGUUACUCGCUGUAUGGGACAACGCUCAUGCACGGGGACACUCCCACGUUGCAUGAGUACAUGUUGGACCUACAGCCUGAGACAACUGACCUCUAUUGCUAUGAGCAGUUGAAUGACAGCAGCGAAGAGGAGGAUGAGAUAGAUGGGCCCGCCGGUCAGGCUGAGCCUGACCGGGCCCACUAUAACAUCGUCACUUUUUGCUGCAAGUGUGAUUCAACGCUCAGGCUCUGCGUGCAGAGCACACACGUGGACAUACGAACCUCAGAGUACAGGCACUACUGCUACUCGCUCUACGGCACGACAUUGGAGCAGCAGUACAACAAGCCCCUGUGUGACUUGUUGAUCCGCUGCAUCAAUUGCCAGAAGCCGCUGUGUCCUGAGGAGAAGCAGCGGCAUCUGGACAAGAAGCAGCGGUUCCACAACAUACGGGGGCGUUGGACUGGUCGCUGCAUGUCGUGCUGUAGAUCGAGUAGGACGCGCAGAGAGACGCAGCUGACGCUGCGUCUCUGUGUGCAGUCGACGCACGUAGACAUCCGCACUCUCGAGGACCUGCUUAUGGGGACGCUCGGGAUCGUGUGCCCGAUCUGCAGCCAGAAGCCUAUGGCGCGGUUCGAGGAUCCGACCCGGCGUCCCUAUAAGCUGCCGGACCUCUGUACUGAGCUCAACACCAGCUUGCAGGACAUAGAGAUCACCUGUGUGUACUGCAAGACGGUGUUAGAGCUCACAGAGGUCUUUGAGUUCGCCUUCAAGGACCUGUUCGUGGUUUAUCGAGACAGUAUCCCCCAUGCAGCCUGUCACAAGUGUAUUGACUUCUACUCCCGGAUACGGGAGUUGAGGCAUUACAGUGACAGCGUGUAUGGGGAUACUCUCGAGAAGCUCACGAACAUGCACGGUCCGAAGGCGACUCUGCAGGACAUAGUCCUGCAUCUGGAGCCCCAGAACGAGAUUCCGGUAGACCUGCUCUGCCAUGAGCAGCUAUCGGACUCCGAGGAGGAGAAUGAUGAGAUUGACGGUGUCAAUCAUCAGCAUUUGCCUGCUCGGAGGGCGGAGCCUCAGCGGCAUACAAUGCUCUGUAUGUGCUGCAAGUGCGAAGCGCGGAUCGAGCUGGUGGUGGAGAGCUACUCCGACAGUGUCUACGGUGACACUUUGGAGAAGCUCACCAACACCGGCUUGUACAAUCUGCUCAUUCGCUGCUUGCGGUGCCAGAAGCCAUUGAAUCCCGCUGAGAAGCUCCGCCACCUGAACGAGAAGCGGCGGUUUCACAAUAUCGCUGGCCACUACCGUGGCCAGUGUCAUUCGUGUUGCAACCGCGCUCGUCAGGAGCGCUUGCAGCGUCGCCGGGAAACCCAGGUGAUGUGCUGCAAGUGCGAAGCUCGUAUCGAGCUGGUCGUCGAGAGUUCUGCGGAUGAUCUACGUGCGUUCCAGCAGUUGUUCCUGAAUACUCUGAGCUUCGUAUGUCCCUGGUGUGCUUCGCAGCAGGGCGGCGCCACGAACUUCUCUCUGUUAAAGCAAGCAGGAGACGUGGAAGAAAACCCCGGUCCUAUGGAGCGGGUGCAGCCACUGGAGGAGAACGUCGGGAACGCUGCGAGGCCGAGGUUCGAGCGGAAUAAGCUGCUGCUCGUAGCCUCGGUCAUCCAGGGUCUGGGCCUCCUCCUGUGCUUCACCUACAUCUGCUUGCACUUCAGUGCAUUGCAGGUGUCACACAGAUACCCCCGGAUUCAGAGUAUCAAGGUGCAGUUCACGGAGUACAAGAAGGAGAAGGGUUUCAUCCUCACUUCUCAGAAGGAGGAUGAGAUCAUGAAGGUCCAGAACAACUCCGUGAUCAUCAACUGCGAUGGAUUUUACCUUAUAAGUCUGAAAGGCUACUUCAGUCAGGAAGUUAACAUCUCUCUGCACUAUCAGAAGGAUGAGGAGCCCCUCUUCCAGCUGAAGAAGGUGCGGAGUGUUAACUCCCUGAUGGUAGCCUCUCUGACUUAUAAGGAUAAAGUCUAUCUCAACGUCACCACCGAUAACACGAGUCUGGAUGACUUUCAUGUUAACGGUGGUGAGUUGAUCUUGAUACAUCAGAAUCCGGGGGAGUUCUGUGUGUUAUGAAAGCUUGCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGGCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGAAUUCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACUAG
SEQ ID NO.20:鼠源OX40L氨基酸序列:
MEGEGVQPLDENLENGSRPRFKWKKTLRLVVSGIKGAGMLLCFIYVCLQLSSSPAKDPPIQRLRGAVTRCEDGQLFISSYKNEYQTMEVQNNSVVIKCDGLYIIYLKGSFFQEVKIDLHFREDHNPISIPMLNDGRRIVFTVVASLAFKDKVYLTVNAPDTLCEHLQINDGELIVVQLTPGYCAPEGSYHSTVNQVPL
SEQ ID NO.21:鼠源OX40LDNA序列:
ATGGAGGGGGAGGGGGTCCAGCCTCTAGACGAGAATCTAGAGAATGGATCCCGTCCCCGCTTCAAGTGGAAGAAGACGCTGCGCCTGGTTGTGAGTGGAATCAAAGGAGCAGGCATGCTCCTTTGTTTCATTTACGTCTGTCTTCAACTGAGTAGCAGCCCCGCGAAGGACCCGCCCATCCAGCGGCTTCGCGGGGCTGTTACTCGGTGTGAAGACGGACAGCTCTTCATCTCCTCCTATAAGAACGAGTACCAGACGATGGAGGTGCAGAACAACTCGGTCGTCATTAAGTGTGACGGCCTCTACATCATCTATCTCAAGGGCTCGTTCTTTCAGGAGGTGAAGATTGATCTTCACTTCCGTGAAGATCACAACCCCATCAGTATCCCGATGCTCAACGATGGGCGTCGGATCGTGTTCACGGTCGTGGCGAGCCTTGCCTTCAAGGACAAGGTTTACCTGACCGTGAACGCGCCCGACACCCTTTGTGAGCATCTGCAGATAAATGATGGGGAGCTGATTGTGGTACAGCTCACTCCGGGCTACTGTGCTCCGGAGGGGTCGTACCACAGTACAGTTAACCAGGTGCCGCTCTGA
本发明的第四个方面是提供一种脂质纳米颗粒,所述脂质纳米颗粒包含本发明第二个方面所述的多核苷酸,以及脂质化合物。在一些实施方案中,所述的多核苷酸可以与脂质化合物一同制备成脂质纳米颗粒,再制备成治疗性疫苗。在一些实施方案中,脂质化合物与mRNA的质量比为(5-20):1。
本发明的第五个方面是提供一种疫苗或疫苗组合物,其包含本发明第二个方面所述的多核苷酸。在本发明的一些实施方式中,所述疫苗或疫苗组合物中,所述多核苷酸以脂质纳米颗粒为载体。
在本发明的一个实施方式中,所述疫苗或疫苗组合物,其包含本发明第四个方面所述的脂质纳米颗粒以及药学上可接受的赋形剂,和/或免疫佐剂。
根据本发明,所述疫苗或疫苗组合物中,当以脂质纳米颗粒为载体时,mRNA位于脂质纳米颗粒中,脂质纳米颗粒含有占其总体脂质分子30-60mol%的式C的可电离阳离子脂质分子,5-30mol%的中性脂质分子,30-50mol%的胆固醇类脂质分子,0.4-10mol%的PEG化的脂质分子;优选含有30-55mol%的式C的可电离阳离子脂质分子,8-18mol%中性脂质分子,32-50mol%的胆固醇类脂质分子,0.5-2.5mol%的PEG化的脂质分子。
式C其中每个n3都彼此独立,可以相同或不同,每个n3选自1~8的整数,每个m3都彼此独立,可以相同或不同,每个m3选自0~8的整数;优选,每个n3选自4~8的整数,每个m3选自4~8的整数;优选,每个n3都彼此相同,每个m3都彼此相同。示例性的式C化合物如下:
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式C化合物优选如下所示化合物:
Ⅱ-37:
中性脂质分子可以选自式E所示的磷脂酰胆碱类化合物式F所示的磷脂酰乙醇胺类化合物其中Ra、Rb、Rc、Rd独立的选自直链或支链的C10-30烷基,直链或支链的C10-30烯基,优选为CH3(CH2)17CH2-、CH3(CH2)15CH2-、CH3(CH2)13CH2-、CH3(CH2)11CH2-、CH3(CH2)9CH2-、CH3(CH2)7CH2-、CH3(CH2)7-CH=CH-(CH2)7-、CH3(CH2)4CH=CHCH2CH=CH(CH2)7-、CH3(CH2)7-CH=CH-(CH2)9-。在本发明的一些实施方式中,所述中性脂质分子选自DOPE和DSPC。
胆固醇类脂质分子可以选自胆固醇、5-十七基间苯二酚和胆固醇半琥珀酸酯。在本发明的一些实施方式中,所述胆固醇类脂质分子选自胆固醇。
PEG化的脂质分子包含脂质部分和基于PEG的聚合物部分,表示为“脂质部分-PEG-数均分子量”,所述脂质部分是二酰基甘油或二酰基甘油酰胺,选自二月桂酰甘油、二肉豆蔻酰甘油、二棕榈酰甘油、二硬脂酰甘油、二月桂基甘油酰胺、二肉豆蔻基甘油酰胺、二棕榈酰甘油酰胺、二硬脂酰甘油酰胺、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸乙醇胺;PEG的数均分子量为约130至约50,000,例如约150至约30,000,约150至约20,000,为约150至约15,000,为约150至约10,000,为约150至约6,000,为约150至约5,000,为约150至约4,000,为约150至约3,000,为约300至约3,000,为约1,000至约3,000,为约1,500至约2,500,例如约2000。在本发明的一些实施方式中,所述PEG化的脂质分子选自DSPE-PEG2000和DMG-PEG2000。
在本发明的一些实施方式中,所述疫苗或疫苗组合物是冻干制剂。
所述冻干制剂复溶后形成的液体制剂中,所述脂质纳米颗粒的粒径在1-1000nm,多分散性指数≤0.5,电动电位在-45~60mV;优选,所述脂质纳米颗粒的粒径在20-500nm,多分散性指数≤0.5,电动电位在-45~60mV;进一步优选,所述脂质纳米颗粒的粒径在45-300nm,多分散性指数≤0.5,电动电位在-45~50mV;更优选,所述脂质纳米颗粒的粒径在60-300nm,多分散性指数≤0.3,电动电位在-45~50mV。
发明人通过研究发现,冻干制剂复溶后的液体制剂中的脂质纳米颗粒的粒径会比冻干前的粒径有所增大,增大的范围不会超过100nm,通常小于80nm,推测原因可能是冻干过程中和/或复溶过程中存在脂质纳米颗粒的融合等。因此可以根据复溶后制剂中所要求的脂质纳米颗粒的粒径大小,来调整冻干前制剂中脂质纳米颗粒的粒径。
所述冻干制剂中进一步含有冻干保护剂。所述冻干保护剂可以是本领域中常用的冻干保护剂,包括但不限于蔗糖、海藻糖和麦芽糖。在本发明的一些实施方式中,所述冻干保护剂是蔗糖、海藻糖和麦芽糖中的一种或任意两种的混合物或三种的混合物。在本发明的一个实施方式中,所述冻干保护剂是蔗糖。所述冻干保护剂的含量占冻干制剂复溶后的液态制剂的5-30w/v%,优选为8-20w/v%,例如5.5w/v%,6w/v%,6.5w/v%,7w/v%,7.5w/v%,8w/v%,8.5w/v%,9w/v%,9.5w/v%,10w/v%,10.5w/v%,11w/v%,11.5w/v%,12w/v%,12.5w/v%,13w/v%,13.5w/v%,14w/v%,14.5w/v%,15w/v%,15.5w/v%,16w/v%,16.5w/v%,17w/v%,17.5w/v%,18w/v%,18.5w/v%,19w/v%,19.5w/v%,20.5w/v%,21w/v%,21.5w/v%,22w/v%,22.5w/v%,23w/v%,23.5w/v%,24w/v%,24.5w/v%,25w/v%,25.5w/v%,26w/v%,26.5w/v%,27w/v%,27.5w/v%,28w/v%,28.5w/v%,29w/v%,29.5w/v%等。
所述冻干制剂中可以进一步含有缓冲盐。所述缓冲盐可以是本领域中常用的缓冲盐,包括但不限于Tris,Hepe,EDTA,柠檬酸-柠檬酸盐,乙酸-乙酸盐,磷酸盐-磷酸氢盐,碳酸盐-碳酸氢盐等。所述缓冲盐的含量占冻干制剂复溶后的液态制剂的0.05-1w/v%,优选为0.1-0.5w/v%。冻干制剂复溶后的液体制剂的pH值可以为4-9。对于静脉注射用液体制剂,通常更优选pH为6-8。对于非静脉注射用液体制剂,例如肌肉注射用,可以容忍相对较宽的pH值。
所述冻干制剂中可以进一步含有渗透压调节剂。所述渗透压调节剂可以是本领域中常用的渗透压调节剂,包括但不限于氯化钠、氯化钾、氯化钙、葡萄糖、磷酸盐、枸橼酸盐等。所述渗透压调节剂的用量使得冻干制剂复建后的液态制剂为0.5~3个等渗浓度的溶液。对于静脉注射用液体制剂,通常更优选为0.9~1.5个等渗浓度的溶液。对于非静脉注射用液体制剂,例如肌肉注射用,其等渗浓度范围可以在较大范围内被身体容忍,一般可以是0.5~3个等渗浓度的溶液。
在使用前,可以采用本领域已知的方法,通过使用注射用溶液将所述冻干制剂复溶成液态制剂,然后进行注射给药。所述注射用溶液可以是注射用水、0.9%注射用氯化钠溶液、或者注射用葡萄糖溶液。
所述疫苗或疫苗组合物的冻干制剂,可以采用本领域已知的冻干技术,在获得包含本发明所述脂质纳米颗粒的液态组合物后,冻干所述液态组合物以得到冻干制剂。
在本发明的一些实施方式中,制备含有脂质纳米颗粒的液体组合物的方法包括:1)制备含有式C化合物、中性脂质分子、胆固醇类脂质分子、PEG化的脂质分子的有机相;2)制备含有活性成分的水相;3)将有机相和水相进行混合,制备脂质纳米颗粒混悬液;4)将所述混悬液浓缩后,加入冻干保护剂,或进一步加入缓冲盐和/或渗透压调节剂等。所述步骤1)中,优选,使用无水乙醇或乙醇的水溶液作为溶剂。所述步骤2)中,优选使用水作为溶剂。所述步骤3)中,优选有机相和水相的体积比为1:2~4。
在本发明中,步骤4)中浓缩得到的混悬液在加入冻干保护剂等物质后,就是冻干前液体制剂,其体积与冻干制剂复溶后形成的液体制剂的体积优选是相同的,这样,加入到混悬液中的冻干保护剂、缓冲盐和/或渗透压调节剂等辅料的含量,与冻干制剂复溶后形成的液体制剂中各物质的含量是相同的。
在本发明的一些实施方式中,冻干过程包括预冻、一次干燥和二次干燥。优选所述预冻为-40℃至-60℃保持1-12h;所述一次干燥为在真空度为0.02mbar-0.4mbar条件下,-30℃至-55℃保持10-80h;所述二次干燥为在真空度为0.02mbar-0.4mbar条件下,4℃至20℃保持4-30h。
本发明的第六个方面是提供,本发明第二方面所述的多核苷酸,或者第三方面所述的载体,或者第四方面所述的脂质纳米颗粒,或者第五方面所述的疫苗或疫苗组合物,在制备用于预防和治疗由人乳头瘤病毒引发的疾病的药物或疫苗中的用途。具体的,所述疾病包括但不限于肿瘤、肛门和生殖器疣、赘疣等。更具体的,所述肿瘤是宫颈癌。
根据本发明,所述药物或疫苗单独或与第二药剂组合施用。所述第二药剂是肿瘤治疗药物,例如检查点抑制剂(例如抗PD-1抗体、抗PD-L1抗体和/或抗CTLA-4抗体等),肿瘤化疗剂(包括但不限于:烷化剂、氮芥类、塞替派类、亚硝脲类、甲基磺酸酯类、铂类化合物、丝裂霉素等,具体如:氮芥、苯丁酸氮芥、环磷酰胺、异环磷酰胺、塞替派、卡莫司汀、司莫司汀、白消安、顺铂、奥沙利铂、卡铂、草酸铂、丝裂霉素等;影响核酸合成的药物,例如二氢叶酸还原酶抑制剂、胸腺核苷合成酶抑制剂、嘌呤核苷合成酶抑制剂、核苷酸还原酶抑制剂、DNA多聚酶抑制剂,具体如:甲氨蝶呤、5-FU、FT-207、卡培他滨、6-巯基嘌呤、6-TG、羟基脲、阿糖胞苷、吉西他滨、培美曲塞等;作用于核酸转录的药物,例如放线菌素D、柔红霉素、阿霉素、表阿霉素、阿克拉霉素、光辉霉素等;作用于DNA复制的拓扑异构酶I抑制剂,例如伊立替康、拓扑替康、羟基喜树碱等;作用于有丝分裂M期干扰微管蛋白合成的药物,例如紫杉醇、多西他赛、长春花碱、长春新碱、长春瑞滨、鬼臼碱类、高三尖杉酯碱等),血管生成抑制剂(包括但不限于:抑制血管内皮生长因子的作用的那些抑制剂,例如来那度胺、沙利度胺、抗血管内皮细胞生长因子抗体例如贝伐单抗,VEGF受体酪氨酸激酶抑制剂凡德他尼(vandetanib)、瓦他拉尼(vatalanib)、舒尼替尼等)。
本发明的多核苷酸(例如mRNA)可以以任何可用途径施用,包括但不限于肿瘤内、肠内、胃肠道、硬膜外、口服、透皮、硬脑膜外(硬膜外)、脑内(进入大脑)、脑室内(进入脑室)、表皮(应用于皮肤上)、皮内(进入皮肤本身)、皮下(皮肤下方)、鼻腔施用(通过鼻子)、静脉内(进入静脉),腹膜内(进入腹膜)、动脉内(进入动脉)、肌内(进入肌肉)、心内(进入心脏)、骨内输注(进入骨髓)、鞘内(进入椎管)、腹膜内(输注或注射进入腹膜)、膀胱内输注、玻璃体内(通过眼睛)、海绵体内注射(进入阴茎根部)、阴道内施用、子宫内、羊膜外施用、透皮(通过完整皮肤扩散用于全身分布)、经粘膜(通过粘膜扩散)、吹入(鼻吸)、舌下、阴唇下、灌肠、滴眼液(到结膜上)或滴耳液。在一些实施方案中,本发明的mRNA通过胃肠外施用(例如,包括皮下、静脉内、腹膜内、肿瘤内、肌肉内、关节内、滑膜内、胸骨内、鞘内、肝内、病灶内和颅内注射或输注技术)、心室内、口服、吸入喷雾、局部、直肠、鼻腔、口腔、阴道或通过植入的储库。在一个特定的实施方案中,本发明的多核苷酸(例如mRNA)肿瘤内施用。
本发明的第七个方面是提供,本发明第二方面所述的多核苷酸,或者第三方面所述的载体,或者第四方面所述的脂质纳米颗粒,或者第五方面所述的疫苗或疫苗组合物,在制备免疫增强剂中的用途。优选的,所述免疫增强剂用于激活T细胞和/或NK细胞。优选的,所述激活T细胞包括诱导T细胞增殖;更优选的,所述激活T细胞包括诱导肿瘤中的T细胞浸润或增加肿瘤浸润T细胞的数量。
本发明还提供下列方法。
一种治疗人乳头瘤病毒引发的疾病的方法,所述方法包括向有需要的治疗对象施用本发明第一方面所述的多核苷酸,或者第二方面所述的载体,或者第三方面所述的脂质纳米颗粒,或者第四方面所述的疫苗或疫苗组合物。所述疾病包括但不限于肿瘤、肛门和生殖器疣、赘疣等。更具体的,所述肿瘤是宫颈癌。
一种增强受试者机体免疫的方法,所述方法包括向有需要的治疗对象施用本发明第一方面所述的多核苷酸,或者第二方面所述的载体,或者第三方面所述的脂质纳米颗粒,或者第四方面所述的疫苗或疫苗组合物。所述增强机体免疫是激活T细胞和/或NK细胞。优选的,所述激活T细胞包括诱导T细胞增殖;更优选的,所述激活T细胞包括诱导肿瘤中的T细胞浸润或增加肿瘤浸润T细胞的数量。
本发明的式C可电离脂质化合物可以采用本领域已有的方法进行合成,例如,使一当量或一当量以上的胺与一当量或一当量以上的环氧基封端化合物在合适的条件下反应形成。可电离脂质化合物的合成是在有或无溶剂的情况下进行,并且所述合成可在25-100℃范围内的较高温度下进行。可任选纯化制得的可电离脂质化合物。
在本发明的一些实施方式中,本发明的可电离脂质化合物可以采用如下的一般制备方法进行制备。
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步骤1:还原
在还原剂的存在下,将化合物A1的羧基还原成羟基以获得化合物A2。还原剂的实例包括但不限于氢化铝锂、二异丁基氢化铝等。反应所用溶剂的实例包括但不限于醚类(如乙醚、四氢呋喃和二氧六环等),卤代烃(如氯仿、二氯甲烷和二氯乙烷等),烃类(如正戊烷、正己烷、苯和甲苯等),及这些溶剂的两种或以上形成的混合溶剂。
步骤2:氧化
在氧化剂的存在下,将化合物A2的羟基氧化成醛基以获得化合物A3。氧化剂的实例包括但不限于2-碘酰基苯甲酸(IBX)、氯铬酸吡啶(PCC)、二氯铬酸吡啶盐(PDC)、戴斯-马丁氧化剂、二氧化锰等。反应所用溶剂的实例包括但不限于卤代烃(如氯仿、二氯甲烷和二氯乙烷等),烃类(如正戊烷、正己烷、苯和甲苯等),腈类(例如乙腈等),及这些溶剂的两种或以上形成的混合溶剂。
步骤3:卤代-还原
首先在酸性条件下,将化合物A3的醛α-氢与卤代试剂发生卤代反应以获得α-卤代醛中间体,然后在还原剂存在下,将α-卤代醛的醛基还原成羟基以获得化合物A4。提供酸性条件的实例包括但不限于DL-脯氨酸。卤代试剂的实例包括但不限于N-氯代丁二酰亚胺(NCS)和N-溴代丁二酰亚胺(NBS)。还原剂的实例包括但不限于硼氢化钠、氰基硼氢化钠和三乙酰氧基硼氢化钠。
步骤4:环氧化
将化合物A4在碱的存在下进行分子内的亲核取代反应以获得环氧化合物A5。碱的实例包括但不限于碱金属的氢氧化物或氢化物,例如氢氧化钠、氢氧化钾和氢化钠。反应所用溶剂的实例包括但不限于二氧六环和水的混合物。
步骤5:开环反应
使化合物A5与胺(例如N,N-二(2-氨基乙基)甲胺)发生开环反应以获得最终化合物。反应用溶剂的实例包括但不限于乙醇、甲醇、异丙醇、四氢呋喃、三氯甲烷、己烷、甲苯、乙醚等。
所述制备方法中的原料A1可以商购也可以采用常规方法合成。
本发明的脂质纳米颗粒还可以采用本领域已知的其他方法进行制备。这些方法包括(但不限于)单一和双重乳液溶剂蒸发、溶剂萃取、相分离、纳米沉淀、微流控、简单和复杂凝聚等。
术语说明:
“和/或”将被视为具有或不具有另一个的两个指定特征或组件中的每一个的具体公开。因此,在诸如“A和/或B”之类的短语中使用的术语“和/或”旨在包括“A和B”、“A或B”、“A”(单独)和“B”(单独)。同样地,在诸如“A、B和/或C”的短语中使用的术语“和/或”旨在涵盖以下方面中的每一个:A、B和C;A、B或C;A或C;A或B;B或C;A和C;A和B;B和C;A(单独);B(单独);和C(单独)。
“包括”和“包含”具有相同的含义,旨在是开放的并且允许但不要求包括额外的元件或步骤。当在本文中使用术语“包括”或“包含”时,因此也包括和公开了术语“由......组成”和/或“基本上由……组成”。
在本说明书和权利要求书中,核苷酸通过其通常接受的单字母代码来指代。除非另有说明,否则核苷酸序列以5’至3’方向从左向右书写。核碱基在本文中由IUPAC-IUB生物化学命名委员会推荐的通常已知的单字母符号表示。因此,A代表腺嘌呤,C代表胞嘧啶,G代表鸟嘌呤,T代表胸腺嘧啶,U代表尿嘧啶。技术人员将理解,本文公开的密码子中的T碱基存在于DNA中,而T碱基在相应RNA中将被U碱基取代。例如,本文公开的DNA形式的密码子-核苷酸序列,例如载体或体外翻译(IVT)模板,其T碱基在其相应的转录mRNA中转录为U碱基。在这一方面,密码子优化的DNA序列(包含T)和它们相应的mRNA序列(包含U)都被认为是本公开的密码子优化的核苷酸序列。本领域技术人员还将理解,可以通过用非天然碱基替换一个或多个碱基来产生等同的密码子图谱。
术语“核酸序列”、“核苷酸序列”或“多核苷酸序列”可互换使用,并且是指连续的核酸序列。序列可以是单链或双链的DNA或RNA,例如mRNA。
“编码…的核苷酸序列”是指编码多肽的核酸(例如,mRNA或DNA分子)编码序列。编码序列可以进一步包括与调控元件可操作地连接的起始和终止信号,所述调控元件包括能够指导在施用核酸的个体或哺乳动物的细胞中的表达的启动子和多腺苷酸化信号。
同源性:如本文所用,术语“同源性”是指聚合物分子之间的总体相关性,例如,在核酸分子(例如DNA分子和/或RNA分子)之间和/或在多肽分子之间。通常,术语“同源性”意味着两个分子之间的进化关系。因此,两个同源的分子将具有共同的进化祖先。在本公开的背景下,术语同源性包括同一性和相似性。
在一些实施方案中,如果分子中至少25%,30%,35%,40%,45%,50%,55%,60%,65%,70%,75%,80%,85%,90%,95%,96%,97%,98%,99%或100%的单体是相同的(完全相同的单体)或相似(保守置换),聚合物分子被认为是彼此“同源的”。术语“同源的”必然是指至少两个序列(多核苷酸或多肽序列)之间的比较。
同一性:如本文所用,术语“同一性”是指聚合物分子之间,例如多核苷酸分子(例如DNA分子和/或RNA分子)之间和/或多肽分子之间的整体单体保守性。例如,可以通过以进行最佳比较目的比对两个序列来进行两个多核苷酸序列的百分同一性的计算(例如,可以在第一和第二核酸序列之一或两个中引入空位用于最佳比对和非相同的序列可以对于比较目的放弃。在某些实施方案中,为了比较目的而比对的序列的长度是参考序列长度的至少30%,至少40%,至少50%,至少60%,至少70%,至少80%,至少90%,至少95%或100%。然后比较相应核苷酸位置的核苷酸。当第一序列中的位置被与第二序列中的相应位置相同的核苷酸占据时,那么分子在该位置是相同的。两个序列之间的百分同一性是序列共有的相同位置的数目的函数,考虑到需要引入以实现两个序列的最佳比对的空位的数量和每个空位的长度。可以使用数学算法完成序列的比较和两个序列之间的百分同一性的确定。当比较DNA和RNA时,胸腺嘧啶(T)和尿嘧啶(U)可被认为是等同的。
合适的软件程序可从各种来源获得,例如Bl2seq,Needle,Stretcher,Water或Matcher等。
术语“编码区”和“编码区域”是指多核苷酸中的开放阅读框(ORF),其在表达时产生多肽或蛋白质。
“可操作地连接”是指两个或更多个分子,构建体,转录物,实体,部分等之间的功能性连接。
结构域:如本文所用,当提及多肽时,术语“结构域”是指具有一个或多个可识别的结构或功能特征或性质(例如,结合能力,用作蛋白质-蛋白质相互作用的位点)的多肽的基序。
表达:如本文所用,核酸序列的“表达”是指一个或多个以下事件:(1)从DNA序列产生mRNA模板(例如,通过转录);(2)mRNA转录物的加工(例如,通过剪接,编辑,5'帽形成和/或3'末端加工);(3)将mRNA翻译成多肽或蛋白质;和(4)多肽或蛋白质的翻译后修饰。
“烷基”是指从含有1到30个碳原子的烃部分通过去除单个氢原子得到的饱和烃基。烷基的实例包括(但不限于)甲基、乙基、丙基、异丙基、正丁基、仲丁基、叔丁基、正戊基、异戊基、新戊基、正己基、正庚基、正辛基、正癸基、正十一烷基和正十二烷基。
“烯基”表示从具有至少一个碳-碳双键的烃部分通过去除单个氢原子得到的单价基团。烯基包括例如乙烯基、丙烯基、丁烯基、1-甲基-2-丁烯-1-基等。
“药学上可接受的赋形剂”是指除本文所述多核苷酸(例如IL-12mRNA)之外的任何成分,并且在患者中具有基本上无毒和非炎性的性质,包括但不限于任何和所有溶剂、分散介质或其他液体载体、分散或悬浮助剂、稀释剂、制粒和/或分散剂、表面活性剂、等渗剂、增稠剂或乳化剂、防腐剂、粘合剂、润滑剂、着色剂、甜味剂或调味剂、稳定剂、抗氧化剂、抗微生物剂或抗真菌剂、摩尔渗透压浓度调节剂、pH调节剂、缓冲剂、螯合剂、冷冻保护剂和/或填充剂,如适合于所需的特定剂型的。用于配制药物组合物的各种赋形剂和用于制备组合物的技术是本领域已知的。示例性抗微生物剂或抗真菌剂包括但不限于苯扎氯铵,苄索氯铵,对羟基苯甲酸甲酯,对羟基苯甲酸乙酯,对羟基苯甲酸丙酯,对羟基苯甲酸丁酯,苯甲酸,羟基苯甲酸,苯甲酸钾或苯甲酸钠,山梨酸钾或钠,丙酸钠,山梨酸等,以及它们的组合。示例性防腐剂包括但不限于,维生素A,维生素C,维生素E,β-胡萝卜素,柠檬酸,抗坏血酸,丁基化羟基苯甲醚,乙二胺,十二烷基硫酸钠(SLS),十二烷基醚硫酸钠(SLES)等,以及它们的组合。控制pH的示例性缓冲液可包括但不限于磷酸钠,柠檬酸钠,琥珀酸钠,组氨酸(或组氨酸-HCl),苹果酸钠,碳酸钠等,和/或其组合。示例性的冷冻保护剂包括但不限于甘露醇,蔗糖,海藻糖,乳糖,甘油,右旋糖等,以及它们的组合。示例性填充剂可包括但不限于蔗糖,海藻糖,甘露糖醇,甘氨酸,乳糖,棉子糖及其组合。
附图说明
图1:ELISA法检测本发明重组HPV抗原1(LAMP-E6E7-FLT3L,082703A)转染A549和HEK293细胞后FLT3L的表达水平。
图2:本发明重组HPV抗原1(LAMP-E6E7-FLT3L,082703A)与GX188E分别转染细胞后FLT3L表达水平的比较。
图3:FACS法检测本发明重组HPV抗原2(LAMP-E6E7-OX40L,082704B)转染HEK293细胞后不同时间OX40L的表达水平。
图4:FACS法检测本发明重组HPV抗原2(LAMP-E6E7-OX40L,082704B)转染A549细胞后OX40L的表达水平。
图5:Luc法检测本发明重组HPV抗原2(LAMP-E6E7-OX40L,082704B)转染HEK293细胞后上清液中OX40L的功能水平。
图6:ELISA法检测本发明重组HPV抗原3(DD-E6E7-FLT3L,082711)转染A549和HEK293细胞后FLT3L的表达水平,其中1为A549细胞的结果,2为HEK293细胞的结果。
图7:ELISA法检测本发明重组HPV抗原3(DD-E6E7-FLT3L,082711)转染A549细胞后不同时间FLT3L的表达水平。
图8:本发明重组HPV抗原3(DD-E6E7-FLT3L,082711)与GX188E体外翻译表达HPVE6水平的比较。
图9:本发明重组HPV抗原3(DD-E6E7-FLT3L,082711)与GX188E分别转染HEK293细胞后FLT3L表达水平的比较。
图10:ELISA法检测本发明重组HPV抗原4(DD-E6E7-OX40L,082712)转染A549细胞后OX40L的表达水平。
图11:FACS法检测本发明重组HPV抗原4(DD-E6E7-OX40L,082712)转染A549和HEK293细胞后OX40L的表达水平。
图12:ELISA法检测本发明重组HPV抗原4(DD-E6E7-OX40L,082712)转染A549细胞后不同时间OX40L的表达水平。
图13:Luc法检测本发明重组HPV抗原4(DD-E6E7-OX40L,082712)转染HEK293细胞后上清液中OX40L的功能水平。
图14:本发明重组HPV抗原4(DD-E6E7-OX40L,082712)与GX188E体外翻译表达HPVE6水平的比较。
图15:接受本发明重组HPV抗原1~4、GX188E的LNP免疫后小鼠体内TC-1肿瘤生长曲线比较。
图16:接受本发明重组HPV抗原1~4、GX188E的LNP免疫后小鼠脾细胞中HPV抗原特异的CTL细胞比例比较。
图17:可电离脂质II-37和C14-113分别形成的包裹mRNA的LNP的细胞转染效率对比。
图18:MTT法测定II-37-LNP和C14-113-LNP的细胞毒性。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解,下列实施例仅为示例性地说明和解释本发明,而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明,以下实施例中使用的原料和试剂均为市售商品,或者可以通过已知方法制备。所述实验方法是本领域常规的分子生物学方法,可以参照本领域的分子生物学实验手册或试剂盒产品说明书的指引进行操作。
实施例1:本发明重组HPV抗原1(简称“LAMP-E6E7-FLT3L”,以下也以代号“082703A”表示)的DNA及mRNA构建
1.1制备DNA和mRNA构建体
将设计而成的具有SEQ ID NO:1所示氨基酸序列的E6E7融合多肽、具有SEQ IDNO:2所示氨基酸序列的LAMP基因的luminal domain、具有SEQ ID NO:3所示氨基酸序列的LAMP基因的跨膜区和胞内区、具有SEQ ID NO:5所示氨基酸序列的自剪切肽2A序列以及具有SEQ ID NO:9所示氨基酸序列的tPA外分泌信号肽和具有SEQ ID NO:6所示氨基酸序列的FLT3L基因胞外区的编码序列,进行全基因合成,克隆到用于mRNA体外转录的载体pIVT4K上。该质粒包含有SEQ ID NO:10所示的5’UTR、2个首尾相连的SEQ ID NO:11所示3’UTR、以及120A polyA结构。
同时,参考文献报道,合成GX188E对照的编码序列,克隆到载体pIVT4K上。该mRNA编码tPa信号肽-FLT3L胞外区-HPV抗原的融合蛋白。
该构建体用于后续体外转录反应和加帽反应,制备mRNA。
1.2体外转录
按照实施例1.1制备的相应的DNA质粒,首先使用speI内切酶将质粒线性化,以线性化的质粒为模板,使用T7 RNA聚合酶体外转录制备mRNA。然后用氯化锂沉淀法纯化制备mRNA(以下也称为082703AmRNA),其序列如SEQ ID NO:16所示。
实施例2:本发明重组HPV抗原1(LAMP-E6E7-FLT3L)的转染表达验证
2.1mRNA细胞转染
将实施例1中制备的mRNA转染到细胞内以检测其生物学功能。A549细胞和HEK293细胞分别以2.5×105cells/well和4.5×105cells/well的密度铺到六孔板中,24h后,细胞单层密度达到80%左右。将082703A mRNA以0.5μg/ml的浓度进行转染,转染试剂为Lipofectamine Messenger MAX Reagent。为提高转染效率,在转染前2h将正常培养基替换成不含血清的F-12K培养基,转染后5h再次更换培养基为正常培养基。
2.2细胞上清Flt3L表达水平检测
转染48h后,收集细胞上清,使用ELISA试剂盒检测上清中Flt3L的表达量。结果表明,与NC组相比,转染082703A的mRNA在两种细胞系中Flt3L的表达水平显著升高(P<0.001)(图1)。这意味着本发明制备的08270A mRNA在A549和HEK293细胞内均可以高效表达Flt3L,这就使得该疫苗可以引起更强的免疫应答反应。
实施例3:本发明重组HPV抗原1(LAMP-E6E7-FLT3L)与GX188E的Flt3L表达水平比较
3.1mRNA细胞转染
HEK293细胞以4.5×105cells/well的密度铺到六孔板中,24h后,细胞单层密度达到80%左右。将本发明重组HPV抗原1(LAMP-E6E7-FLT3L)的mRNA和GX188E mRNA均以1.5μg/ml的浓度进行转染,转染试剂为Lipofectamine Messenger MAX Reagent。为提高转染效率,在转染前2h将正常培养基替换成不含血清的F-12K培养基,转染后5h再次更换培养基为正常培养基。再5h后,其中一组HPV抗原1(LAMP-E6E7-FLT3L)转染细胞加入25μM氯喹。
3.2重组HPV抗原1(LAMP-E6E7-FLT3L)与GX188E Flt3L表达水平比较
转染24h后,收集细胞上清,使用ELISA试剂盒检测上清中Flt3L的表达量。结果表明与GX188E mRNA转染组相比,重组HPV抗原1(LAMP-E6E7-FLT3L)及重组HPV抗原1(LAMP-E6E7-FLT3L)+氯喹实验组中的Flt3L表达水平明显更高(P<0.05),但重组HPV抗原1(LAMP-E6E7-FLT3L)和重组HPV抗原1(LAMP-E6E7-FLT3L)+氯喹两组之间的Flt3L表达水平无明显差异(图2)。这表明,相比于GX188E,本发明的重组HPV抗原1(LAMP-E6E7-FLT3L)可显著增加HEK293细胞内的Flt3L表达水平,从而进一步显著提高交叉提呈树突细胞在肿瘤部位的数目和活化,最终提高肿瘤特异性CD8 T细胞激活以及对免疫检查点阻断治疗的效率。
实施例4:本发明重组HPV抗原2(简称“LAMP-E6E7-OX40L”,以下也以代号“082704B”表示)的DNA及mRNA构建
4.1制备DNA和mRNA构建体
将设计而成的编码SEQ ID NO:1所示氨基酸序列的E6E7融合多肽、SEQ ID NO:2所示氨基酸序列的LAMP基因的luminal domain、编码SEQ ID NO:3所示氨基酸序列的LAMP基因的跨膜区和胞内区、以及编码SEQ ID NO:5所示氨基酸序列的自剪切肽2A和SEQ ID NO:7所示氨基酸序列的OX40L蛋白的DNA,进行全基因合成,克隆到用于mRNA体外转录的载体pIVT4K上。该构建体用于后续体外转录反应和加帽反应,制备mRNA。
4.2体外转录
按照4.1制备的相应的DNA质粒,首先使用speI内切酶将质粒线性化,以线性化的质粒为模板,使用T7RNA聚合酶体外转录制备mRNA。然后用氯化锂沉淀法纯化制备mRNA(以下也称为082704B mRNA),其序列如SEQ ID NO:17所示。
同样的,使用鼠源OX40L序列(氨基酸序列如SEQ ID NO.20,DNA序列如SEQ IDNO.21所示)替代重组HPV抗原2中人源OX40L序列,构建用于小鼠实验的鼠源重组HPV抗原2替代物(简称“LAMP-E6E7-mOX40L”,以下也以代号“082704Bm”表示),制备mRNA用于小鼠体内抗肿瘤验证。
实施例5:本发明重组HPV抗原2(LAMP-E6E7-OX40L)的转染表达验证
5.1mRNA细胞转染HEK293细胞
将实施例4中制备的mRNA转染到细胞内以检测其表达。HEK293细胞以2×105cells/well的密度铺到12孔板中,16h后,细胞单层密度达到90%左右。将重组HPV抗原2(LAMP-E6E7-OX40L)的mRNA以0.5μg/ml的浓度进行转染,转染试剂为LipofectamineMessenger MAX Reagent。在转染后2h添加氯喹(0uM、30uM、50uM)、亮肽素(0uM、10uM、30uM),刺激反应。
5.2细胞不同时间点OX40L流式检测比较
转染5h、10h、24h、48h后,收集细胞,应用直接免疫荧光标记法,将转染细胞和对照NC细胞离心,用FACS buffer(含2%FBS的PBS溶液)重悬细胞,细胞浓度为1×106cells/ml,取100μl细胞悬液加入离心管,分别加入2μl anti-human CD252(OX40L)Antibody流式检测抗体,以及相应的同型对照。4℃避光染色30min。每管加入1ml FACS Buffer洗细胞,弃上清,加入200μl FACS buffer,流式分析检测OX40L的表达。结果表明,使用PE anti-humanCD252(OX40L)Antibody流式检测抗体染色,与NC组相比,转染重组HPV抗原2(LAMP-E6E7-OX40L)组中OX40L的表达水平显著升高,检测细胞中OX40L的表达量24h效果最佳(图3)。
5.3mRNA细胞转染A549细胞
将实施例4中制备的mRNA转染到细胞内以检测其生物学功能。A549细胞以2×105cells/well的密度铺到六孔板中,16h后,细胞单层密度达到80%左右。将重组HPV抗原2(LAMP-E6E7-OX40L)的mRNA以1.0μg/ml的浓度进行转染,转染试剂为LipofectamineMessenger MAX Reagent。在转染后2h添加50μM氯喹,刺激反应。
5.4细胞OX40L表达水平流式检测
转染42h后,收集细胞,应用直接免疫荧光标记法,将转染细胞和对照NC细胞离心,用FACS buffer(含2%FBS的PBS溶液)重悬细胞,细胞浓度为1×106cells/ml,取100μl细胞悬液加入离心管,分别加入2μl anti-human CD252(OX40L)Antibody流式检测抗体,以及相应的同型对照。4℃避光染色30min。每管加入1ml FACS Buffer洗细胞,弃上清,加入200μlFACS buffer,流式分析检测OX40L的表达。结果表明,与NC组相比,转染重组HPV抗原2(LAMP-E6E7-OX40L)组OX40L的表达水平显著升高(图4)。
实施例6:本发明重组HPV抗原2(LAMP-E6E7-OX40L)的生物活性检测
6.1mRNA细胞转染
OX40/NFkB-Luc/HEK293细胞以6×104cells/well,50μl/well,铺到96孔板中,铺板24h后,将082704BmRNA以5.0μg/ml的浓度加入至OPTI-MEM培养基中,再加入OPTI-MEM培养基稀释的转染试剂,转染试剂为Lipofectamine Messenger MAX Reagent,静置5min后,用OPTI-MEM培养基以2倍梯度稀释后,以50μl/well加入至细胞中;将OX40L蛋白以150ng/ml的浓度加入至OPTI-MEM培养基中,用OPTI-MEM培养基以2倍梯度稀释后,以50μl/well加入至细胞中。
6.2重组HPV抗原2(LAMP-E6E7-OX40L)生物活性表达水平
培养6h后,加入和细胞培养上清等体积的Bright-GloTMLuciferase Assay System检测液,用酶标仪进行检测。实验表明,重组HPV抗原2(LAMP-E6E7-OX40L)的生物学活性较高,转染浓度为10μg/ml时得到的荧光信号值约等于OX40L浓度为75ng/ml的荧光信号值(图5)。本发明的重组HPV抗原2(LAMP-E6E7-OX40L)在OX40/NFkB-Luc/HEK293细胞中表达的OX40L具有生物活性,从而进一步会引发一系列的免疫反应,增加效应T细胞和记忆T细胞的存活和扩增,增加细胞因子(例如IL-2、IL-4、IL-5、IFN-γ)的分泌;降低Tregs的免疫抑制活性,进一步放大T细胞活化效应。
实施例7:本发明重组HPV抗原3(简称“DD-E6E7-FLT3L”,以下也以代号“082711”表示)的DNA及mRNA构建
7.1制备DNA和mRNA构建体
将设计而成的编码SEQ ID NO:4所示氨基酸序列的DD结构域、编码SEQ ID NO:1所示氨基酸序列的E6E7融合多肽、以及编码SEQ ID NO:5所示氨基酸序列的自剪切肽2A序列、编码SEQ ID NO:8所示氨基酸序列的IgE外分泌信号肽和具有SEQ ID NO:6所示氨基酸序列的FLT3L基因胞外区的核酸序列,依次串联,进行全基因合成,将合成的基因片段克隆到用于mRNA体外转录的载体pIVT4K上,得到082711质粒。该构建体用于后续体外转录反应和加帽反应,制备mRNA。
7.2体外转录
按照实施例7.1制备的相应的DNA质粒,首先使用speI内切酶将质粒线性化,以线性化的质粒为模板,使用T7 RNA聚合酶体外转录制备mRNA。然后用氯化锂沉淀法纯化制备mRNA(以下也称为082711mRNA),其序列如SEQ ID NO:18所示。
实施例8:本发明重组HPV抗原3(DD-E6E7-FLT3L)的转染表达验证
8.1 mRNA细胞转染
将实施例7中制备的mRNA转染到细胞内以检测其生物学功能。A549细胞和HEK293细胞分别以2.5×105cells/well和4.5×105cells/well的密度铺到六孔板中,24h后,细胞单层密度达到80%左右。将重组HPV抗原3(DD-E6E7-FLT3L)的mRNA以0.5μg/ml的浓度进行转染,转染试剂为Lipo MessengerMAX。为提高转染效率,在转染前2h将正常培养基替换成不含血清的F-12K培养基,转染后5h再次更换培养基为正常培养基。
8.2细胞上清Flt3L表达水平检测
转染48h后,收集细胞上清,使用ELISA试剂盒检测上清中Flt3L的表达量。结果表明,与NC组相比,转染重组HPV抗原3(DD-E6E7-FLT3L)的两种细胞系中Flt3L的表达水平显著升高(P<0.001)(图6)。这意味着本发明制备的082711mRNA在A549和HEK293细胞内均可以高效表达Flt3L,这就使得该疫苗可以引起更强的免疫应答反应。
8.3转染后不同时间点Flt3L表达水平检测
为检测重组HPV抗原3(DD-E6E7-FLT3L)在转染A549细胞后不同时间点的表达水平,在重组HPV抗原3(DD-E6E7-FLT3L)转染后5h、10h、24h、48h收集细胞上清,使用ELISA试剂盒检测上清中FLT3L的表达量。结果表明,随着重组HPV抗原3(DD-E6E7-FLT3L)转染后的时间延长,细胞上清中FLT3L的表达水平逐渐升高,在48h时细胞长期中可达到19.1ng/ml的浓度(图7)。
实施例9:体外翻译反应验证重组HPV抗原3(DD-E6E7-FLT3L)表达HPV E6蛋白功能
9.1体外翻译反应
为了验证本发明是否可以表达HPV E6蛋白,将重组HPV抗原3(DD-E6E7-FLT3L)的mRNA经过体外翻译反应获得相应的蛋白。体外翻译反应是借助于兔网织红细胞裂解液,该裂解液含有蛋白合成所必需的细胞成分,通过该反应反应可以迅速获得重组HPV抗原3(DD-E6E7-FLT3L)mRNA翻译的蛋白,从蛋白水平验证该发明的生物学功能。
9.2检测并比较重组HPV抗原3(DD-E6E7-FLT3L)与GX188E的HPV E6的表达水平
将上述体外翻译反应的产物按体积比加入6×Loading buffer,混匀后进行变性,随后每个样品进行Western Blot实验以检测该序列是否可以翻译HPV E6蛋白。结果表明,重组HPV抗原3(DD-E6E7-FLT3L)的mRNA经体外翻译反应后的产物可以大量表达HPV E6,且表达量高于GX188E mRNA体外翻译产物(图8)。而这意味着相比于GX188E,本发明可获得的有效剂量更低。
实施例10:重组HPV抗原3(DD-E6E7-FLT3L)与GX188E的FLT3L的表达水平比较
10.1mRNA细胞转染
HEK293细胞以4.5×105cells/well的密度铺到六孔板中,24h后,细胞单层密度达到80%左右。将重组HPV抗原3(DD-E6E7-FLT3L)的mRNA和GX188E mRNA均以1.5μg/ml的浓度进行转染,转染试剂为Lipo MessengerMAX。为提高转染效率,在转染前2h将正常培养基替换成不含血清的F-12K培养基,转染后5h再次更换培养基为正常培养基。再5h后,其中一组重组HPV抗原3(DD-E6E7-FLT3L)转染细胞加入10μMShield-1。
10.2重组HPV抗原3(DD-E6E7-FLT3L)与GX188E的FLT3L表达水平比较
转染24h后,收集细胞上清,使用ELISA试剂盒检测上清中FLT3L的表达量。结果表明,与GX188E mRNA转染组相比,重组HPV抗原3(DD-E6E7-FLT3L)及重组HPV抗原3(DD-E6E7-FLT3L)+Shield-1实验组中的FLT3L表达水平明显更高(P<0.05),但重组HPV抗原3(DD-E6E7-FLT3L)及重组HPV抗原3(DD-E6E7-FLT3L)+Shield-1两组之间FLT3L表达水平无明显差异(图9)。这表明,相比于GX188E,本发明的重组HPV抗原3(DD-E6E7-FLT3L)可显著增加HEK293细胞内的FLT3L表达水平,从而进一步显著提高交叉提呈树突细胞在肿瘤部位的数目和活化,最终提高肿瘤特异性CD8T细胞激活以及对免疫检查点阻断治疗的效率。
实施例11:本发明重组HPV抗原4(简称“DD-E6E7-OX40L”,以下也以代号“082712”表示)的DNA及mRNA构建
11.1制备DNA和mRNA构建体
将设计而成的编码SEQ ID NO:4所示氨基酸序列的DD结构域、编码SEQ ID NO:1所示氨基酸序列的E6E7融合多肽、编码SEQ ID NO:5所示氨基酸序列的自剪切肽2A、编码SEQID NO:7所示氨基酸序列的OX40L蛋白的核酸序列,依次串联,进行全基因合成后,将合成的基因片段克隆到用于mRNA体外转录的载体pIVT4K上,得到082712质粒。该构建体用于后续体外转录反应和加帽反应,制备mRNA。
11.2体外转录
按照实施例11.1制备的相应的DNA质粒,首先使用speI内切酶将质粒线性化,以线性化的质粒为模板,使用T7 RNA聚合酶体外转录制备mRNA。然后用氯化锂沉淀法纯化制备mRNA(以下也称为082712mRNA),其序列如SEQ ID NO:19所示。
同样的,使用鼠源OX40L序列替代重组HPV抗原4中人源OX40L序列,构建用于小鼠实验的鼠源重组HPV抗原4替代物(简称“DD-E6E7-mOX40L”,以下也以代号“082712m”表示),制备mRNA用于小鼠体内抗肿瘤验证。
实施例12:本发明重组HPV抗原4(DD-E6E7-OX40L)的转染表达验证
12.1mRNA细胞转染
将实施例11中制备的mRNA转染到细胞内以检测其生物学功能。A549细胞和HEK293细胞分别以2.0×105cells/well的密度铺到六孔板中,24h后,细胞单层密度达到80%左右。将重组HPV抗原4(DD-E6E7-OX40L)的mRNA以1.0μg/ml的浓度进行转染,转染试剂为Lipofectamine Messenger MAX Reagent。在2h后,其中一组重组HPV抗原4(DD-E6E7-OX40L)转染细胞加入1μM shield-1。
12.2细胞上清OX40L表达水平ELISA检测
转染42h后,收集细胞上清,使用ELISA试剂盒检测上清中OX40L的表达量。结果表明,与NC组相比,转染重组HPV抗原4(DD-E6E7-OX40L)在A549细胞系中OX40L的表达水平显著升高,说明本发明提供的重组HPV抗原4(DD-E6E7-OX40L)mRNA在A549可以高效表达OX40L(图10)。
12.3细胞OX40L表达水平流式检测
转染42h后,收集细胞,应用直接免疫荧光标记法,将转染细胞和阴性对照细胞离心,用FACS buffer(含2%FBS的PBS溶液)重悬细胞,细胞浓度为1×106cells/ml,取100μl细胞悬液加入离心管,分别加入2μlanti-human CD252(OX40L)Antibody流式检测抗体,以及相应的同型对照。4℃避光染色30min。每管加入1ml FACS Buffer洗细胞,弃上清,加入200μl FACS buffer,流式分析检测OX40L的表达。结果表明,与NC组相比,转染重组HPV抗原4(DD-E6E7-OX40L)的两种细胞系中OX40L的表达水平显著升高(图11),当OX40被其配体OX40L激活时,会引发一系列的免疫反应,增加效应T细胞和记忆T细胞的存活和扩增,增加细胞因子的分泌,进一步放大T细胞活化效应。
实施例13:重组HPV抗原4(DD-E6E7-OX40L)不同时间点表达水平比较
13.1 mRNA细胞转染
A549细胞以2.5×105cells/well,2ml/well的密度铺到六孔板中,18h后,细胞单层密度达到80%左右。将重组HPV抗原4(DD-E6E7-OX40L)mRNA以1.5μg/ml的浓度进行转染,转染试剂为Lipofectamine Messenger MAX Reagent。为提高转染效率,在转染前2h将正常培养基替换成不含血清的MEM培养基,转染后5h再次更换培养基为正常培养基。同时,其中一组重组HPV抗原4(DD-E6E7-OX40L)转染细胞加入1μM shield-1。
13.2重组HPV抗原4(DD-E6E7-OX40L)不同时间点表达水平比较
转染24h后,收集细胞上清,使用ELISA试剂盒检测上清中OX40L的表达量。结果表明于转染后48h表达最高(图12)。
实施例14:重组HPV抗原4(DD-E6E7-OX40L)的生物活性检测
14.1 mRNA细胞转染
OX40/NFkB-Luc/HEK293细胞以3×104cells/well的密度铺到96孔板中,铺板24h后,将重组HPV抗原4(DD-E6E7-OX40L)的mRNA均以3.0μg/ml的浓度梯度稀释后进行转染,转染试剂为Lipofectamine Messenger MAX Reagent。
14.2重组HPV抗原4(DD-E6E7-OX40L)的生物活性表达水平比较
培养24h后,加入和细胞培养上清等体积的Bright-GloTMLuciferase AssaySystem检测液,用酶标仪进行检测。重组HPV抗原4(DD-E6E7-OX40L)的生物学活性高,转染浓度为625ng/ml时得到的荧光信号值约等于OX40L浓度为75ng/ml的荧光信号值(图13)。本发明的重组HPV抗原4(DD-E6E7-OX40L)在OX40/NFkB-Luc/HEK293细胞中表达OX40L具有生物活性,从而进一步会引发一系列的免疫反应,增加效应T细胞和记忆T细胞的存活和扩增,增加细胞因子(例如IL-2、IL-4、IL-5、IFN-γ)的分泌;降低Tregs的免疫抑制活性,进一步放大T细胞活化效应。
实施例15:体外翻译反应验证重组HPV抗原4(DD-E6E7-OX40L)表达HPV E6蛋白功能
15.1体外翻译反应
为了验证本发明是否可以表达HPV E6蛋白,将重组HPV抗原4(DD-E6E7-OX40L)mRNA经过体外翻译反应获得相应的蛋白。体外翻译反应是借助于兔网织红细胞裂解液,该裂解液含有蛋白合成所必需的细胞成分,通过该反应可以迅速获得082712mRNA翻译蛋白,从蛋白水平验证该发明的生物学功能。
15.2检测并比较重组HPV抗原4(DD-E6E7-OX40L)与GX188E的HPV E6表达水平
将上述体外翻译反应的产物按体积比加入6×Loading buffer,混匀后进行变性,随后每个样品进行Western Blot实验以检测该序列是否可以翻译HPV E6蛋白。结果表明,重组HPV抗原4(DD-E6E7-OX40L)的mRNA经体外翻译反应后的产物可以大量表达HPV E6,且表达量高于GX188E mRNA体外翻译产物(图14)。而这意味着相比于GX188E,该发明可获得的免疫原性更强。
实施例16本发明的重组HPV抗原1-4的活性实验
LNP-mRNA制剂
称取化合物II-37、CHOL、DSPC、DMG-PEG2000,将每种脂质于适当容器中,无水乙醇或95%乙醇将其充分溶解备用。按照II-37/DSPC/CHOL/DMG-PEG2000=45:15:38.5:1.5(摩尔比)将脂质溶液按比例混合均匀,作为有机相,将前述制备的本发明的HPV mRNA(或其鼠源替代物)配置成水溶液为水相pH=4。
水相与有机相体积比例为3:1混合,在微流控平台(PNI Ignite)上制备得到纳米颗粒混悬液。将得到的纳米颗粒悬浮液经100KDa超滤离心管离心过滤,纯化浓缩,将浓缩后的液体与蔗糖溶液混合,进行分装,于-80℃条件下冻存。
将制备所得的新鲜的脂质纳米颗粒(LNP-mRNA)进行检测,检测指标包括采用激光纳米粒度仪检测的粒径(Diameter)、PDI、电位(Zeta Pttential),采用多功能酶标仪结合RiboGreen RNA试剂盒检测包封率(EE)。制备得到的脂质纳米颗粒理化质控数据如下表所示:
可以看到所获得包载抗原mRNA的纳米颗粒PDI均小于0.15,粒径小于100nm,表面电位在10-60mV,包封率均大于97%。
LNP-mRNA在小鼠体内抗肿瘤活性以及免疫原性评估
取4~6周龄的雌性C57BL/6小鼠,通过肌肉注射方式进行HPV mRNA-LNP或者生理盐水空白对照接种,接种剂量设置为20μg/只/次,接种时间为第0天(d0)、第5天(d5)、第10天(d10),每组10只小鼠。于第15天(d15),所有小鼠右侧颈背部皮下注射5×105TC-1肿瘤细胞,随后每周测量2次肿瘤体积,并记录测量值。于day40天时,将动物实施安乐死。取脾及肿瘤组织,保存在预冷的PBS中,用于后续的检测分析。所有实验组的小鼠,均未能观测到有肿瘤形成,而生理盐水组肿瘤正常生长(图15)。
将脾脏研磨分离出脾淋巴细胞,分别使用1640培养基悬浮,加入HPV16 E749-57(RAHYNIVTF)抗原肽至终浓度为1μg/ml,接种脾细胞2×105cells/孔于活化的鼠IFN-γELISPOT预包被板,于37℃,5%CO2培养箱培养24h后,按照鼠IFN-γELISPOT检测试剂盒说明书检测脾细胞对E749-57(RAHYNIVTF)抗原肽刺激的应答情况。统计数据显示,所有的HPVmRNA LNP均能诱导出强大的HPV抗原特异的细胞免疫反应,与GX188E-LNP对照组相比,本发明公开的四个抗原能诱导出更多的抗原特异CTL细胞(图16)。
实施例17可电离脂质II-37的合成
亚麻油醇(a2)的合成:在0℃下,向950mL四氢呋喃中加入LiAlH4(7.20g)、亚麻油酸(50g,a1),之后混合物在25℃下搅拌2h。薄层色谱法(TLC)显示反应完成后,向反应液依次加入水(7.2mL),NaOH水溶液(7.2mL,质量分数为15%)和水(21.6mL)淬灭,并加入适量Na2SO4搅拌15分钟后通过布氏漏斗过滤并用乙酸乙酯洗涤滤饼,收集滤液并蒸发浓缩,得到目标产物亚麻油醇(a2)47.4g。
1H NMR(400MHz,CDCl3):δ5.27-5.44(m,4H),3.63(t,J=6.63Hz,2H),2.77(t,J=6.44Hz,2H),1.97-2.12(m,4H),1.57-1.63(m,1H),1.20-1.46(m,18H),0.83-0.95(m,3H)
(9Z,12Z)-十八碳-9,12-二烯醛(a3)的合成:在室温下,向170mL乙腈中加入亚麻油醇(25.0g,a2)和2-碘酰基苯甲酸(39.4g),之后混合物在85℃下搅拌4h。反应液通过布氏漏斗过滤并用二氯甲烷洗涤滤饼,收集滤液并蒸发浓缩,得到目标产物(9Z,12Z)-十八碳-9,12-二烯醛(a3)24.0g。
1H NMR(400MHz,CDCl3):δ9.76(t,J=1.76Hz,1H),5.25-5.43(m,4H),2.76(t,J=6.17Hz,2H),2.41(td,J=7.33,1.87Hz,2H),2.04(q,J=6.84Hz,4H),1.56-1.68(m,2H),1.22-1.36(m,14H),0.88(t,J=6.73Hz,3H)
(9Z,12Z)-2-氯代-十八碳-9,12-二烯-1-醇(a4)的合成:在0℃下,向246mL乙腈中加入(9Z,12Z)-十八碳-9,12-二烯醛(43.0g,a3),DL-脯氨酸(5.62g)和N-氯代丁二酰亚胺,然后在0℃下搅拌2h。反应完成后,用无水乙醇(246mL)稀释反应液,再加入硼氢化钠(8.8g),之后在0℃下搅拌4h。向反应混合物中加水(120mL)淬灭,并用甲基叔丁基醚萃取,合并有机相后用饱和食盐水洗涤,硫酸钠干燥之后过滤、蒸发浓缩,得到目标产物(9Z,12Z)-2-氯代-十八碳-9,12-二烯-1-醇(a4,46g),直接用于下一步。
1H NMR(400MHz,CDCl3):δ5.25-5.51(m,4H),3.97-4.07(m,1H),3.79(dd,J=12.01,3.63Hz,1H),3.59-3.70(m,1H),2.67-2.90(m,2H),1.96-2.15(m,5H),1.64-1.82(m,1H),1.20-1.49(m,15H),0.89(br t,J=6.75Hz,3H)
2-[(7Z,10Z)-十六碳烷-7,10-二烯]环氧乙烷(a5)的合成:在室温下,向450mL1,4-二氧六环中加入(9Z,12Z)-2-氯代-十八碳-9,12-二烯-1-醇(45g,a4)和氢氧化钠水溶液(120g氢氧化钠溶于585mL水),滴加完毕后混合物在35℃下搅拌2h。TLC显示反应完成后,反应液通过分液漏斗分离,并用饱和食盐水洗涤,硫酸钠干燥后过滤并蒸发浓缩,然后通过快速过柱法用石油醚/乙酸乙酯洗脱来纯化残余物,得到目标产物2-[(7Z,10Z)-十六碳烷-7,10-二烯]环氧乙烷(a5)29.11g。
1H NMR(400MHz,CDCl3):δ5.27-5.46(m,4H),2.87-2.98(m,1H),2.70-2.85(m,3H),2.46(dd,J=5.00,2.75Hz,1H),1.94-2.21(m,4H),1.24 -1.58(m,17H),0.78-1.00(m,3H)
II-37的合成:在室温下,向10mL乙醇中加入2-[(7Z,10Z)-十六碳烷-7,10-二烯]环氧乙烷(5g)和N,N-二(2-氨基乙基)甲胺(739mg),之后混合物在90℃下搅拌36h。反应液蒸发浓缩,然后通过快速过柱法用二氯甲烷/甲醇洗脱来纯化残余物,得到粗产物II-37(4g)。再次通过快速过柱法用二氯甲烷/甲醇纯化目标产物,得到II-37(2.2g)。
1H NMR(400MHz,CDCl3):δ5.27-5.44(m,12H),3.48-3.79(m,3H),2.63-3.00(m,12H),2.16-2.61(m,12H),2.05(q,J=6.80Hz,12H),1.18-1.57(m,51H),0.89(t,J=6.88Hz,9H)
ESI-MS:m/z 910.8[M+H]+,911.8[M+2H]+,912.8[M+3H]+
可电离脂质有两个主要作用:结合核酸和允许核酸分子在细胞中释放。脂质的pKa是一个重要因素,因为脂质需要在低pH值下带正电荷才能与核酸结合,但在中性pH值下不带电荷,才能使形成的LNP不会引起毒性。通过TNS染料结合试验测定可电离脂质Ⅱ-37的pKa在6.81。
II-37和其结构类似物分子C14-113的对比
C14-113的结构式为:
分别使用II37和C14-113制备脂质纳米颗粒,具体摩尔配比为:II-37:DSPC:CHOL:DMG-PEG2000=45:15:38.5:1.5;C14-113:DSPC:CHOL:DMG-PEG2000=45:15:38.5:1.5;N/P比为10:1。以乙醇溶液作为有机相溶解脂质,把Lucferase mRNA(LucRNA)溶于pH=4的水溶液作为水相。按照水相和有机相的体积比为3:1,在纳米药物制造仪(加拿大PNI公司,Ignite型号)上用微流控技术制备得到纳米颗粒混悬液。制备完后再超滤浓缩得到最终的LucRNA-LNP脂质纳米颗粒,保存于2-8℃备用。
使用Zetasizer Pro纳米粒度电位仪(马尔文帕纳科)进行LucRNA-LNP粒径和Zeta电位的表征。制备得到的脂质纳米颗粒理化质控数据如下表所示:
样本信息 | 粒径(nm) | PDI | Zeta电位 |
mRNA-LNP(II-37-LNP) | 136.68 | 0.14 | 20.07 |
mRNA-LNP(C14-113-LNP) | 152.65 | 0.12 | 24.1 |
用多功能酶标仪(BioTek,型号为SLXFATS)荧光素报告基因法来检测制备的LucRNA-LNP HEK293T细胞的转染效率,转染的LucRNA量分别为0.5μg、1.0μg、2.0μg。体外转录LucRNA的方法如下:HEK293T细胞铺板,细胞密度为2.5×105个细胞/mL,当细胞融合度为30%-50%进行转染。阴性对照为不添加LucRNA-LNP的细胞培养基。将制备的脂质纳米颗粒转染细胞293T,了解蛋白的表达情况,结果如图17所示,在转染的mRNA量相同的情况下,II-37(图中以II-37-LNP表示)制备的脂质纳米颗粒携带mRNA转染细胞之后,细胞中蛋白表达量远远高于C14-113的,说明II-37制成的脂质纳米颗粒的细胞转染效率很高。
此外,采用MTT法测定II-37-LNP和C14-113-LNP的细胞毒性,考察载体剂量、作用时间等因素对正常细胞(293T)细胞增殖的影响。结果如图18所示,II-37(图中以II-37-LNP表示)制备的脂质纳米颗粒携带mRNA转染细胞48小时之后,在较高的剂量(2μg/mL)时依然保持较好细胞活性,说明II-37制成的脂质纳米颗粒的细胞毒性很低。
采用包括II-37在内的本发明的式C化合物,与中性脂质分子、胆固醇、PEG化的脂质分子一同,按照本实施例中的方法,制备包载本发明所述抗原1-4的mRNA的脂质纳米颗粒及其制剂,所述制剂用于前述活性实验验证。
本发明从提升HPV E6和E7的肿瘤治疗效果,减少其对正常细胞的毒性这两点出发,通过序列优化和设计得到了能稳定表达HPV E6和E7的mRNA治疗性疫苗,而且该mRNA更稳定,蛋白表达增加,错误折叠率降低。此外,本发明使用由以II-37为代表的式C的脂质化合物构成的脂质外层包裹编码产生抗原的mRNA分子内核,形成纳米颗粒结构,将上述优化后的mRNA分子准确递送至肿瘤微环境。本发明使用的脂质纳米颗粒的配方细胞毒性低,相较其他同类分子具有更好的mRNA递送效力,能够保障原位注射后mRNA顺利进入肿瘤细胞进行表达,从而增强疫苗的疗效。
以上,对本发明的实施方式进行了说明。但是,本发明不限定于上述实施方式。凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (11)
1.一种HPV E6E7的融合多肽,其特征在于,由以下部分组成:由HPV-16的E6蛋白的第1个-第95个氨基酸,HPV-16的E7蛋白的第1个-第78个氨基酸,HPV-16的E6蛋白的第81个-第158个氨基酸,HPV-16的E7蛋白的第64个-第98个氨基酸,以及HPV-18的E6蛋白的第1个-第95个氨基酸,HPV-18的E7蛋白的第1个-第78个氨基酸,HPV-18的E6蛋白的第81个-第158个氨基酸,HPV-18的E7蛋白的第51个-第105个氨基酸依次连接组成;
优选,所述E6E7融合多肽具有与SEQ ID NO:1所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的氨基酸序列;
优选,所述E6E7融合多肽具有如SEQ ID NO:1所示的氨基酸序列;
可选的,所述E6E7融合多肽不含第1位的甲硫氨酸。
2.一种包含HPV E6E7融合多肽的融合蛋白,其特征在于包括以下部分:用于增强免疫活性的结构域、HPV E6E7融合多肽、自剪切序列和免疫共刺激因子,所述HPV E6E7融合多肽如权利要求1所述;
优选,所述用于增强免疫活性的结构域选自LAMP基因和DD结构域;
优选,所述LAMP结构域包含与SEQ ID NO:2所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的LAMP基因的luminal domain的氨基酸序列,以及与SEQ ID NO:3所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的LAMP基因的跨膜区和胞内区的氨基酸序列,在所述融合蛋白中,HPV E6E7融合多肽的氨基酸序列位于LAMP基因的luminal domain的氨基酸序列和LAMP基因的跨膜区的氨基酸序列之间;优选,LAMP基因的luminal domain具有如SEQ ID NO:2所示的氨基酸序列,LAMP基因的跨膜区和胞内区具有SEQ ID NO:3所示的氨基酸序列;
优选,DD结构域包含与SEQ ID NO:4所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的氨基酸序列;优选,DD结构域具有如SEQID NO:4所示的氨基酸序列;
优选,所述自剪切序列选自P2A、T2A、E2A和F2A;
优选,自剪切序列包含与SEQ ID NO:5所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的氨基酸序列;优选,自剪切序列具有如SEQ ID NO:5所示的氨基酸序列;
优选,所述免疫共刺激因子选自FLT3L和OX40L;
优选,所述FLT3L包含与SEQ ID NO:6所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的氨基酸序列;优选,FLT3L具有如SEQ ID NO:6所示的氨基酸序列;
优选,OX40L包含与SEQ ID NO:7或SEQ ID NO:20所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的氨基酸序列;优选,OX40L具有如SEQ ID NO:7所示的氨基酸序列,或,OX40L具有如SEQ ID NO:20所示的氨基酸序列;
优选,所述融合蛋白中还含有外分泌信号肽,所述信号肽选自IgE外分泌信号肽,或tPA外分泌信号肽;
优选,IgE外分泌信号肽包含与SEQ ID NO:8所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的氨基酸序列;优选,IgE外分泌信号肽具有如SEQ ID NO:8所示的氨基酸序列;
优选,tPA外分泌信号肽包含与SEQ ID NO:9所示的氨基酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%或约100%同一性的氨基酸序列;优选,tPA外分泌信号肽具有如SEQ ID NO:9所示的氨基酸序列。
3.如权利要求2所述的融合蛋白,其特征在于,由以下部分组成:顺序连接的DD结构域、HPV E6E7融合多肽、自剪切序列和免疫共刺激因子,或者,顺序连接的LAMP基因的luminaldomain、HPV E6E7融合多肽、LAMP基因的跨膜区和胞内区、自剪切序列和免疫共刺激因子;
优选,所述融合蛋白由以下部分组成:顺序连接的DD结构域、HPV E6E7融合多肽、自剪切序列、外分泌信号肽和FLT3L;
优选,所述融合蛋白由以下部分组成:顺序连接的DD结构域、HPV E6E7融合多肽、自剪切序列和OX40L;
优选,所述融合蛋白由以下部分组成:顺序连接的LAMP基因的luminal domain、HPVE6E7融合多肽、LAMP基因的跨膜区和胞内区、自剪切序列、外分泌信号肽和FLT3L;
优选,所述融合蛋白由以下部分组成:顺序连接的LAMP基因的luminal domain、HPVE6E7融合多肽、LAMP基因的跨膜区和胞内区、自剪切序列和OX40L。
4.一种编码权利要求2或3所述融合蛋白的多核苷酸,其特征在于,所述多核苷酸是DNA或RNA;
优选,所述多核苷酸是mRNA,其包含与SEQ ID NO:12所示的核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列;优选,其包含如SEQ ID NO:12所示的核苷酸序列;
优选,所述多核苷酸是mRNA,其包含与SEQ ID NO:13所示的核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列;优选,其包含如SEQ ID NO:13所示的核苷酸序列;
优选,所述多核苷酸是mRNA,其包含与SEQ ID NO:14所示的核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列;优选,其包含如SEQ ID NO:14所示的核苷酸序列;
优选,所述多核苷酸是mRNA,其包含与SEQ ID NO:15所示的核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列;优选,其包含如SEQ ID NO:15所示的核苷酸序列。
5.如权利要求4所述的多核苷酸,其特征在于,所述mRNA还包括5’UTR,3’UTR和poly-A尾;
优选,所述5’UTR包含与SEQ ID NO:10所示的核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列;
优选,所述3’UTR包含1个与SEQ ID NO:11所示α2-珠蛋白3’UTR的片段至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列;或者,2个及以上首尾相连的、与SEQ ID NO:11所示的α2-珠蛋白3’UTR的片段至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列;
优选,所述poly-A尾的长度为50-200个核苷酸,优选为100-150个核苷酸;
优选,所述多核苷酸进一步包含5’帽,优选,所述5’帽为CAP1。
6.如权利要求4或5所述的多核苷酸,其特征在于,其具有与SEQ ID NO:16所示的核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列;优选,其具有SEQ IDNO:16所示的核苷酸序列;
或者,其具有与SEQ ID NO:17所示的核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列;优选,其具有SEQ ID NO:17所示的核苷酸序列;
或者,其具有与SEQ ID NO:18所示的核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列;优选,其具有SEQ ID NO:18所示的核苷酸序列;
或者,其具有与SEQ ID NO:19所示的核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列;优选,其具有SEQ ID NO:19所示的核苷酸序列。
7.一种体外转录载体,其特征在于,其包含可操作性连接的编码5’UTR,3’UTR和poly-A尾的核苷酸序列;所述5’UTR包含与SEQ ID NO:10所示的核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列;所述3’UTR包含2个首尾相连的、与SEQ ID NO:11所示的α2-珠蛋白3’UTR的片段至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%同源性的核苷酸序列;所述poly-A尾的长度为50-200个核苷酸;
优选,所述体外转录载体还包含编码权利要求2或3所述的融合蛋白的多核苷酸,所述融合蛋白的多核苷酸的序列与SEQ ID NO:12或SEQ ID NO:13或SEQ ID NO:14或SEQ IDNO:15所示核苷酸序列至少60%,至少70%,至少75%,至少80%,至少85%,至少90%,至少95%,至少96%,至少97%,至少98%,至少99%或约100%的同源性。
8.一种疫苗组合物,其特征在于,包含权利要求4-6任一项所述的多核苷酸;优选,所述疫苗组合物还含有药学上可接受的赋形剂,和/或免疫佐剂;优选,所述疫苗组合物用于治疗HPV引发的病症。
9.如权利要求8所述的疫苗组合物,其还含有脂质纳米颗粒,mRNA位于脂质纳米颗粒中,脂质纳米颗粒含有占其总体脂质分子30-60mol%的式C的可电离阳离子脂质分子,5-30mol%的中性脂质分子,30-50mol%的胆固醇类脂质分子,0.4-10mol%的PEG化的脂质分子;
式C其中每个n3都彼此独立,可以相同或不同,每个n3选自1~8的整数,每个m3都彼此独立,可以相同或不同,每个m3选自0~8的整数;优选,每个n3选自4~8的整数,每个m3选自4~8的整数;优选,每个n3都彼此相同,每个m3都彼此相同;
优选,式C为
优选,中性脂质分子选自式E所示的磷脂酰胆碱类化合物式F所示的磷脂酰乙醇胺类化合物其中Ra、Rb、Rc、Rd独立的选自直链或支链的C10-30烷基,直链或支链的C10-30烯基,优选为CH3(CH2)17CH2-、CH3(CH2)15CH2-、CH3(CH2)13CH2-、CH3(CH2)11CH2-、CH3(CH2)9CH2-、CH3(CH2)7CH2-、CH3(CH2)7-CH=CH-(CH2)7-、CH3(CH2)4CH=CHCH2CH=CH(CH2)7-、CH3(CH2)7-CH=CH-(CH2)9-;
优选,胆固醇类脂质分子选自胆固醇、5-十七基间苯二酚和胆固醇半琥珀酸酯;
优选,PEG化的脂质分子包含脂质部分和基于PEG的聚合物部分,表示为“脂质部分-PEG-数均分子量”,所述脂质部分是二酰基甘油或二酰基甘油酰胺,选自二月桂酰甘油、二肉豆蔻酰甘油、二棕榈酰甘油、二硬脂酰甘油、二月桂基甘油酰胺、二肉豆蔻基甘油酰胺、二棕榈酰甘油酰胺、二硬脂酰甘油酰胺、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸乙醇胺;PEG的数均分子量为130~50,000,优选150~30,000,进一步优选150~10,000,更优选300~3,000,特别优选1,500~2,500;
优选,脂质分子总质量和mRNA的质量比为5-20:1;
优选,含有30-55mol%的式C的可电离阳离子脂质分子,8-18mol%中性脂质分子,32-50mol%的胆固醇类脂质分子,0.5-2.5mol%的PEG化的脂质分子。
10.权利要求4-6任一项所述的多核苷酸或权利要求8-9任一项所述的疫苗组合物在制备预防或治疗HPV引发的疾病的药物或疫苗中的用途;优选,所述疾病选自肿瘤、肛门和生殖器疣、赘疣;优选,所述肿瘤是宫颈癌。
11.权利要求4-6任一项所述的多核苷酸或权利要求8-9任一项所述的疫苗组合物在制备免疫增强剂中的用途;优选的,所述免疫增强剂用于激活T细胞和/或NK细胞;优选的,所述激活T细胞包括诱导T细胞增殖;更优选的,所述激活T细胞包括诱导肿瘤中的T细胞浸润或增加肿瘤浸润T细胞的数量。
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