CN110418648B - 一种编码人GM-CSF与多串连表位融合的mRNA癌症疫苗 - Google Patents
一种编码人GM-CSF与多串连表位融合的mRNA癌症疫苗 Download PDFInfo
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Abstract
本发明提供了一种编码人GM‑CSF与多串连表位融合的mRNA癌症疫苗。编码人GM‑CSF与三个串连hTERT表位融合的pVec‑GM‑CSF‑hTes、编码人GM‑CSF与分别来自MUC1和Kras以及EGFR的三个串连表位融合的pVec‑GMKE以及编码人白细胞介素‑12的pVec‑hIL‑12被分别构建，并被作为模板用于产生相应的体外转录的mRNAs，进一步被混合作为mRNA癌症疫苗。该mRNA癌症疫苗含有用作免疫佐剂的人GM‑CSF、多个串连表位构成的多表位癌症抗原以及用于增强免疫治疗作用的hIL‑12。
Description
描述
发明背景
本发明涉及一种生物核酸mRNA疫苗，尤其是涉及一种编码人粒细胞巨噬细胞集落刺激因子(GM-CSF)与多串连表位融合的mRNA癌症疫苗。
通过刺激免疫系统来抗现有癌症的治疗性癌症疫苗是治疗癌症的最有效的药物，这是因为癌症疫苗可引起机体的免疫应答并产生免疫记忆。确保癌症疫苗成功的第一步是癌症疫苗的抗原设计。用于癌症疫苗的大多数抗原是肿瘤相关抗原(tumor-associatedantigens，TAAs)，例如人端粒酶逆转录酶(human telomerase reverse transcriptase，hTERT)、粘蛋白1(mucin 1，MUC1)、Kras和表皮生长因子受体(epidermal growth factorreceptor，EGFR)等。
端粒位于真核染色体的末端，是由串连重复非转录DNA序列(TTAGGG)和端粒结合蛋白组成的特殊“帽”结构。端粒的作用是维持染色体完整性和控制细胞分裂周期。染色体的端粒随着每次连续的细胞分裂而变短。当端粒收缩到一定程度时，细胞停止分裂并处于静止状态。端粒酶能够添加TTAGGG重复序列到染色体的末端。人端粒酶催化亚基为人端粒酶逆转录酶(human telomerase reverse transcriptase，hTERT)，其活性在正常细胞中被抑制，并且太低而不能被检测到。然而，在生殖细胞和干细胞中，特别是在大多数肿瘤细胞中(大于85％)，hTERT被激活并且能够大量表达。因此，hTERT是癌症治疗的理想靶标。
应用pGX0001，构建了编码含有两个突变位点的hTERT的phTERT DNA疫苗。证明电穿孔到体内的phTERT DNA疫苗可在动物体内打破免疫耐受并诱导各种强的细胞毒性应答[Yan J，et al.Cancer Immunol Res.2013；1(3)：179-89]。将构建的pGEM4Z/hTERT/A64和pGEM4Z/hTERT/LAMP/A64作为模板用于产生体外转录的mRNA，并被分别电穿孔转染到树突状细胞(dendritic cells，DC)。将DC-mRNA疫苗皮内接种到患有转移性前列腺癌的患者中。结果显示嵌合LAMP-hTERT疫苗可引起比未修饰的hTERT疫苗显著更高频率的hTERT特异性CD4+T细胞[Su Z，et al.J Immunol.2005；174(6)：3798-807]。编码hTERT基因的腺病毒疫苗(Ad-hTERT)可以引发靶向自体肿瘤细胞的强的CD8+细胞毒性T淋巴细胞(cytotoxic Tlymphocyte，CTL)应答，然而用于人体的腺病毒载体可能引起显著的副作用。此外，完整TAA能够引起强的抗癌免疫应答，但亦可能诱导免疫耐受或自身免疫应答。
hTERT I540-548(ILAKFLHWL)是用于黑色素瘤免疫治疗的第一个hTERT表位疫苗，并已进入III期临床试验[Liu JP，et al.Biochim Biophys Acta.2010；1805(1)：35-42]。由hTERT蛋白残基611-626片段组成的16个氨基酸的hTERT多肽疫苗GV1001可以在癌症患者中引发广泛的抗hTERT CD4+T细胞应答[Inderberg-Suso EM，et al.Oncoimmunology2012；1(5)：670-686]。将包含hTERT540-548、hTERT572Y-580和hTERT865-873四聚体多抗原肽(multiple antigen peptides，MAP)的合成疫苗接种到动物中可以引发强的hTERT特异性细胞毒性T淋巴细胞(CTL)反应[Liao ZL，et al.Cancer Sci.2012；103(11)：1920-8]。将含有hTERT I540多肽(ILAKFLHWL)、hTERT R572Y多肽(YLFFYRKSV)、hTERT D988Y多肽(YLQVNSLQTV)、存活蛋白(survivin)Sur1M2多肽(LMLGEFLKL)和巨细胞病毒对照多肽N495(NLVPMVATV)的疫苗接种到移植自体干细胞的骨髓瘤患者后，能够引起强的T细胞恢复和持续减少调节性T细胞(regulatory T cells，Treg)[Rapoport AP，et al.Blood 2011；117(3)：788-97]。hTERT多肽疫苗例如GV1001疫苗在癌症治疗的一些临床试验中已经显示出有希望的结果，但是仍然不能在患有皮肤T细胞淋巴瘤的患者中诱导抗癌反应[SchlapbachC，et al.J Dermatol Sci.2011；62(2)：75-83]。此外，GV1001疫苗用于在化疗期间的胰腺癌患者不能改善患者的整体存活[Middleton G，et al.Lancet Oncol.2014；15(8)：829-40]。
粘蛋白1(Mucin 1，MUC1)大多数是含有连接到多肽核心的O-糖苷键的I型跨膜蛋白。在正常情况下，MUC1主要表达于腔上皮细胞或许多组织和器官的腺表面，呈现在上皮细胞的顶端表面。由于其在80-90％的肿瘤组织中的异常表达，因此MUC1成为抗癌治疗的潜在靶标[Pillai K，et al.Am J Clin Oncol.2015；38(1)：108-18]。然而，用在未经手术切除的III期非小细胞肺癌(non-small cell lung cancer，NSCLC)患者中的免疫治疗用MUC1(氨基酸残基130-154)多肽疫苗tecemotide未能在临床试验中改善NSCLC患者的存活[Butts C，et al.Lancet Oncol.2014；15(1)：59-68]。也许由单一肿瘤相关抗原(TAA)例如MUC1组成的疫苗用于NSCLC患者的免疫治疗可能是无效的[Xia W，et al.J ThoracDis.2014；6(10)：1513-20]。
与人类肿瘤相关的Ras基因家族包括Hras、Kras和Nras。其中，Kras对人类癌症有很大的影响，它就像身体的一个“开关”。在正常情况下，Kras可以调节细胞生长；在异常情况下，Kras引起细胞的连续生长并阻止细胞的自我毁坏。目前，一些靶向Kras的化疗药物已经进入临床使用，但这些药物易于耐药。一种潜在的方法是通过疫苗接种来治疗癌症。证明含有来自PANC细胞的完整抗原(具有Kras点突变)的树突细胞(DC)疫苗可以诱导良好的抗癌免疫应答，但是含有正常细胞组份的疫苗可能引起免疫耐受和自身免疫应答[Tan G，etal.Oncol Rep.2011；26(1)：215-21]。用Kras(12Val)突变多肽脉冲的DC疫苗可以促进成熟DC表面分子的表达并增强细胞毒性T淋巴细胞(CTL)应答，但不能实现强的抗癌免疫效果。因此，上述Kras(12Val)突变多肽脉冲的DC疫苗仍需要改进。
表皮生长因子受体(epidermal growth factor receptor，EGFR)是表皮生长因子(EGF)的受体。EGFR在正常上皮细胞的表面表达并在一些肿瘤细胞中异常表达。EGFR的过表达与肿瘤细胞转移、侵袭和预后不良有关。EGFR-酪氨酸激酶抑制剂如gefitinib和erlotinib用于具有EGFR突变的NSCLC患者，已证明其具有显著的临床活性。然而，大多数癌症患者可以产生耐药性。EGFR T790M突变(EGFR密码子790处的苏氨酸变为甲硫氨酸)是最普遍的药物抗性突变。含有EGFR T790M突变体的多肽疫苗用于NSCLC患者的免疫治疗，揭示靶向EGFR T790M突变体抗原的免疫治疗可能是用于治疗EGFR T790M突变的NSCLC患者的新选择[0fuji K，et al.Int J Oncol.2015；46(2)：497-504]。编码Kras突变基因的DNA疫苗可以引起对具有Kras突变的肿瘤的有效免疫应答，但对具有EGFR突变的肿瘤无效[WengTY，et al.Gene Ther.2014；21(10)：888-96]。此外，gefitinib和erlotinib用于治疗EGFR突变的NSCLC患者有效，但对于具有EGFR突变和Kras突变的NSCLC患者无效。如果同时靶向EGFR突变和Kras突变，癌症治疗的效果也许会倍增。
总之，DNA癌症疫苗也许会整合到宿主细胞的基因组中并产生插入突变，这是因为DNA癌症疫苗需要进入宿主细胞核并转录成mRNA，再被转运到细胞质中并翻译成相应的蛋白质。病毒为载体的癌症疫苗可能导致严重的副作用。肿瘤相关抗原(TAA)的完整序列可以引起强的抗癌免疫应答，但亦可能诱导免疫耐受和自身免疫应答。单一表位(或多肽)疫苗可能不会引起足够强的免疫应答，例如GV1001疫苗用于皮肤T细胞淋巴瘤的患者和在化疗期间的胰腺癌患者不能实现治疗效果。用单一表位疫苗例如MUC1多肽疫苗治疗NSCLC患者通常是无效的。多个表位例如hTERT I540/R572Y/D988Y混合疫苗，由多个表位(例如hTERT540-548、、572Y-580、865-873四聚体)构成的四聚体以及多个抗原多肽疫苗具有比单个表位更好的免疫治疗效果。新抗原癌症疫苗具有良好的免疫治疗效果。由于上述疫苗中缺乏强的免疫佐剂，因此癌症疫苗仍需要改进以实现所期望的免疫治疗效果。
发明内容
本发明的目的是提供一种编码人GM-CSF与多串连表位融合的mRNA癌症疫苗。
为了实现本发明的目的，pVec-GM-CSF-hTes、pVec-GMKE和pVec-hIL-12被分别构建，并被作为模板用于产生体外转录的mRNAs。将所获得的体外转录的mRNAs经电穿孔转染到细胞内，检测其表达，并进一步将其混合作为mRNA癌症疫苗。该mRNA癌症疫苗含有用作免疫佐剂的人GM-CSF、多个串连表位构成的多表位癌症抗原以及用于增强免疫治疗作用的hIL-12。
附图说明
图1.pVec-GM-CSF-hTes图
人GM-CSF(without a stop codon)-linker-three tandem hTERT epitopes(with a stop codon))被亚克隆到pVec的NheI和XhoI位点之间。
pVec-GM-CSF-hTes的全长核苷酸序列：3,930bp
GM-CSF-hTes：碱基801-1,391bp
图2.pVec-GMKE图
人GM-CSF与分别来自MUC1、Kras和EGFR的三个串连表位融合的GMKE被亚克隆到pVec的NheI和XhoI位点之间。
pVec-GMKE的全长核苷酸序列：3,966bp
GMKE：碱基801-1,427bp
图3.pVec-hIL-12图
SalI-hIL-12-NheI被亚克隆到pVec的XhoI和XbaI位点之间。
pVec-hIL-12的全长核苷酸序列：5,145bp
具体实施方式
本发明的目的是提供一种编码人粒细胞巨噬细胞集落刺激因子(granulocytemacrophage colony-stimulating factor，GM-CSF)与多串连表位融合的mRNA癌症疫苗。该疫苗系使用常规分子生物学技术通过以下步骤获得。
以pCMV-SPORT6-GM-CSF(购自Open Biosystems，GM-CSF GenBank登录号：BC108724)作为模板，使用根据Kozak序列设计为SEQ ID NO：1的正向引物和设计缺失人GM-CSF终止密码子(tga)并在3’端添加接头(SEQ ID NO：2)作为SEQ ID NO：3的反向引物，将通过聚合酶链反应(polymerase chain reaction，PCR)扩增获得的产物亚克隆到NheI和HindIII位点，并转化到top10化学感受态大肠杆菌细胞或DH5α感受态细胞中，获得pVec-NheI-GM-CSF(without a stop codon)-linker-HindIII。
用限制性核酸内切酶SalI消化pYEX-BX encoding KAP123-flu(购自Addgene，质粒编号：24048)。随后，将通过1％琼脂糖凝胶电泳分离含有载体骨架的片段，用T4 DNA连接酶进行头尾连接，并转化top10化学感受态大肠杆菌细胞或DH5α感受态细胞，获得pYEX-BX载体。
包括I540-548(SEQ ID NO：4)、572Y-580(SEQ ID NO：5)和988Y-997(SEQ ID NO：6)的三个表位被选自hTERT(GenBank登录号：AF015950)。包括11个氨基酸(aa)接头(SEQ IDNO：7)和2个氨基酸接头(GlyGly)的两个接头被设计用于串连连接上述三个hTERT表位。
所设计的hTERT(I540-548)-11 aa-hTERT(572Y-580)-2 aa-hTERT(988Y-997)的氨基酸序列如SEQ ID NO：8，相应的核苷酸序列(包含起始密码子atg)为SEQ ID NO：9。
为了获得含有BamHI-hTERT(I540-548)-11 aa-hTERT(572Y-580)-2 aa-hTERT(988Y-997)-SalI的片段，合成并连接以下设计的寡核苷酸。
hTERT F1核苷酸序列如SEQ ID NO：10。
hTERT F2核苷酸序列如SEQ ID NO：11。
hTERT F3核苷酸序列如SEQ ID NO：12。
hTERT R1核苷酸序列如SEQ ID NO：13。
hTERT R2核苷酸序列如SEQ ID NO：14。
hTERT R3核苷酸序列如SEQ ID NO：15。
用BamHI和SalI消化2μg pYEX-BX，然后用碱性磷酸酶(calf intestinal，CIP，NewEngland Biolabs，Cat#：M0290S)去磷酸化并纯化。
将所有上述寡核苷酸(0.25μg/每个寡核苷酸)，2.5μl的10X反应缓冲液，2μl T4多核苷酸激酶(New England Biolabs，Cat#：M0201S)和直至总体积为25μl的适量水加入到PCR反应管中。混合后，将上述反应管在37℃下温育1小时进行磷酸化，接着在94℃下变性10分钟，在室温下退火30分钟，然后置于冰上10分钟。
将4μl上述退火的反应产物、等量的用BamHI和SalI消化并去磷酸化的pYEX-BX载体、2μl 10X T4连接缓冲液，1μl T4 DNA连接酶和直至总体积20μl的适量水置于PCR反应管中，在16℃温育过夜，然后转化top10化学感受态大肠杆菌细胞或DH5α感受态细胞，得到pYEX-BX-BamHI-hTERT(I540-548)-11 aa-hTERT(572Y-580)-2 aa-hTERT(988Y-997)-SalI。
将上述构建的载体pYEX-BX-BamHI-hTERT(I540-548)-11 aa-hTERT(572Y-580)-2aa-hTERT(988Y-997)-SalI作为模板，使用缺失起始密码子(atg)的正向引物如SEQ IDNO：16和添加终止密码子(tga)的反向引物如SEQ ID NO：17，将通过PCR扩增所获得的含有hTERT(I540-548)-11 aa-hTERT(572Y-580)-2 aa-hTERT(988Y-997)-stop codon(tga)的片段亚克隆到pVec-NheI-GM-CSF(without a stop codon)-linker-HindIII的HindIII和XhoI位点，并转化到top10化学感受态大肠杆菌细胞或DH5α感受态细胞中，获得pVec-NheI-GM-CSF(without a stop codon)-linker-HindIII-hTERT(I540-548)-11 aa-hTERT(572Y-580)-2 aa-hTERT(988Y-997)-stop codon(tga)-XhoI，称为pVec-GM-CSF-hTes。pVec-GM-CSF-hTes被保藏在美国典型培养物保藏中心(American Type Culture Collection，ATCC)，编号为PTA-122795。
pVec-GM-CSF-hTes的hTERT(I540-548)-11 aa-hTERT(572Y-580)-2 aa-hTERT(988Y-997)的核苷酸序列如SEQ ID NO：18。pVec-GM-CSF-hTes中的GM-CSF(without astop codon)-linker-HindIII-hTERT(I540-548)-11 aa-hTERT(572Y-580)-2 aa-hTERT(988Y-997)或GM-CSF-hTes的核苷酸序列如SEQ ID NO：19，相应的氨基酸序列如SEQ IDNO：20。pVec-GM-CSF-hTes的全长核苷酸序列已由Genewiz公司测序，如SEQ ID NO：21。
选自MUC1(GenBank登录号：J05582)的MUC1表位(aa 130-154)的氨基酸序列如SEQID NO：22，相应的核苷酸序列如SEQ ID NO：23。
选自Kras(GenBank登录号：M54968)的Kras 12 Val表位(aa 5-17)的氨基酸序列如SEQ ID NO：24，相应的核苷酸序列如SEQ ID NO：25。
选自EGFR(GenBank登录号：GU255993)的EGFR T790M表位(aa 788-798)的氨基酸序列如SEQ ID NO：26，相应的核苷酸序列如SEQ ID NO：27。
用于串连连接上述表位的接头的氨基酸序列为Gly Gly，相应的核苷酸序列为ggaggt。
设计的MUC1(aa 130-154)-2 aa-Kras 12 Val(aa 5-17)-2 aa-EGFR T790M(aa788-798)的氨基酸序列如SEQ ID NO：28，相应的核苷酸序列如SEQ ID NO：29。此外，将终止密码子(tga)添加到3’端。
将含有MUC1(aa 130-154)-2 aa-Kras 12 Val(aa 5-17)-2 aa-EGFR T790M(aa788-798)-stop codon(tga)的插入物逐步亚克隆到pVec-NheI-GM-CSF(without a stopcodon)-linker-HindIII的HindIII和XhoI位点，并转化到top10化学感受态大肠杆菌细胞或DH5α感受态细胞中。
以pVec-NheI-GM-CSF(without a stop codon)-linker-HindIII作为模板，使用上述正向引物如SEQ ID NO：1以及反向引物如SEQ ID NO：30，将通过PCR扩增获得的产物亚克隆到pVec-NheI-GM-CSF(without a stop codon)-linker-HindIII的NheI和XhoI位点，并转化top10化学感受态细胞或DH5α感受态细胞，获得pVec-NheI-GM-CSF(without a stopcodon)-linker-HindIII-MUC1a-XhoI。
以上述获得的pVec-NheI-GM-CSF(without a stop codon)-linker-HindIII-MUC1a-XhoI为模板，使用上述正向引物如SEQ ID NO：1以及反向引物如SEQ ID NO：31，将通过PCR扩增获得的产物亚克隆到pVec-NheI-GM-CSF(without a stop codon)-linker-HindIII的NheI和XhoI位点，并转化top10化学感受态细胞或DH5α感受态细胞，得到pVec-NheI-GM-CSF(without a stop codon)-linker-HindIII-MUC1-XhoI。
以上述获得的pVec-NheI-GM-CSF(without a stop codon)-linker-HindIII-MUC1-XhoI为模板，使用上述正向引物如SEQ ID NO：1以及反向引物如SEQ ID NO：32，将通过PCR扩增获得的产物亚克隆到pVec-NheI-GM-CSF(without a stop codon)-linker-HindIII的NheI和XhoI位点，并转化top10化学感受态细胞或DH5α感受态细胞，得到pVec-NheI-GM-CSF(without a stop codon)-linker-HindIII-MUC1-2 aa-Kras 12 Val-2aa-XhoI。
以上述获得的pVec-NheI-GM-CSF(without a stop codon)-linker-HindIII-MUC1-2aa-Kras 12 Val-2 aa-XhoI作为模板，使用上述正向引物如SEQ ID NO：1以及反向引物如SEQ ID NO：33，将通过PCR扩增获得的产物亚克隆到pVec-NheI-GM-CSF(without astop codon)-linker-HindIII的NheI和XhoI位点，并转化top10化学感受态细胞或DH5α感受态细胞，获得pVec-NheI-GM-CSF(without a stop codon)-linker-HindIII-MUC1(aa130-154)-2 aa-Kras 12 Val(aa 5-17)-2 aa-EGFR T790M(aa 788-798)-stop codon(tga)-XhoI，称为pVec-GMKE。pVec-GMKE被保藏在美国典型培养物保藏中心(ATCC)，编号为PTA-122796。
pVec-GMKE中的GM-CSF(without a stop codon)-linker-HindIII-MUC1(aa 130-154)-2 aa-Kras 12 Val(aa 5-17)-2 aa-EGFR T790M(aa 788-798)或GMKE的氨基酸序列如SEQ ID NO：34，相应的核苷酸序列如SEQ ID NO：35。pVec-GMKE的全长核苷酸序列已由Genewiz公司测序，如SEQ ID NO：36。
将用SalI和NheI消化pORF-hIL-12G2(InvivoGen)获得的人白细胞介素-12(hIL-12)基因亚克隆到pVec的XhoI和XbaI位点，获得pVec-hIL-12。pVec-hIL-12的全长核苷酸序列如SEQ ID NO：37。
分别扩增上述获得的pVec-GM-CSF-hTes、pVec-GMKE和pVec-hIL-12，用Qiaprepspin miniprep kit(Qiagen，目录号：27106)纯化，用限制性核酸内切酶SpeI消化，获得相应的线性化质粒DNA。取少量上述SpeI酶切的质粒DNA于1％琼脂糖凝胶电泳检测各质粒DNA是否完全线性化。通过以下方案纯化每个获得的线性化质粒DNA。将100μl SpeI酶切的质粒DNA反应溶液与500μl缓冲液PB的混合物转移到离心柱中，离心30秒并弃去流出物(流过物)。将750μl缓冲液PE加入到上述离心柱中，离心30秒，排出流出液，然后再次离心1分钟。将上述离心柱置于干净的微量离心管中，向离心柱中加入30μl水，静置1分钟，离心1分钟。少量纯化的线性化质粒DNA用于测定DNA浓度，并调节其浓度为0.5-1μg/μl。
使用HiScribeTM T7High Yield RNA Synthesis Kit(New England Biolabs，Cat#：E2040S)和3’-O-Me-m7G(5’)ppp(5’)G RNA Cap Structure Analog(ARCA，NewEngland Biolabs，Cat#：S1411S)，通过以下步骤分别产生体外转录的GM-CSF-hTes mRNA、GMKE mRNA和hIL-12mRNA。
在室温下，将下列试剂分别加入到1.5ml微量离心管中。
| 无核酸酶水 | xμl | |
| 10×反应缓冲液 | 2μl | |
| ATP(100mM) | 2μl | 10mM最终 |
| UTP(100mM) | 2μl | 10mM最终 |
| CTP(100mM) | 2μl | 10mM最终 |
| GTP(20mM) | 2μl | 2mM最终 |
| ARCA(40mM) | 4μl | 8mM最终 |
| 模板DNA(线性化) | xμl | 1μg |
| T7RNA聚合酶混合物 | 2μl | |
| 总反应体积 | 20μl |
在充分混合和离心3-5秒后，将上述反应管在37℃下温育2小时。为了除去模板DNA，向上述反应管中加入70μl无核酸酶的水，10μl 10X DNase I缓冲液和2μl DNase I(New England Biolabs，Cat#：M0303S)，在37℃下温育15分钟。
使用RNeasy mini试剂盒(Qiagen，Cat#：74104)，通过以下步骤分别纯化体外转录的GM-CSF-hTes mRNA、GMKE mRNA和hIL-12mRNA。取20-30μl用无核酸酶水稀释的体外转录的mRNA，并转移到微量离心管(无核酸酶)中，将350μl含有1％β-巯基乙醇(β-mercaptoethanol，β-ME)的缓冲液RLT加入到上述管中。用移液管充分混合后，向上述管中加入等体积的70％乙醇。混合后，将上述混合物转移到离心柱中，离心和排出流出物。将700μl缓冲液RW1加入到上述离心柱中，离心和排出流出物。将500μl缓冲液RPE加入到上述离心柱中，离心并排出流出物，重复两次。再次离心1分钟后，将上述离心柱转移到干净的微量离心管(无核酸酶)中，并将30μl不含核酸酶的水加入上述离心柱中，静置1分钟，然后离心1分钟。应用Nanodrop分光光度计测定纯化的体外转录mRNA浓度，并通过1％甲醛琼脂糖凝胶电泳检测其质量。
将纯化的体外转录的GM-CSF-hTes mRNA(5μg)、GMKE mRNA(5μg)和hIL-12mRNA(5μg)分别加入到0.2cm比色皿中，在350V和500μs的条件下电穿孔1X106细胞(如小鼠细胞系)。随后加入细胞生长培养液，在5％CO2、37℃下培养36小时，然后收集上述细胞上清液。
应用人GM-CSF酶联免疫吸附测定(enzyme-linked immunosorbent assay，ELISA)试剂盒(eBioscience，Cat#：88-8337-22)通过以下步骤测定收集的电穿孔法使GM-CSF-hTes mRNA和GMKE mRNA转染细胞的上清液中表达的GM-CSF。
用100μl 1X包被缓冲液以1∶250比例稀释的捕获抗体包被ELISA板每个孔，密封并在4℃下温育过夜。
第二天，弃去过量的捕获抗体溶液，并用洗涤缓冲液[含有0.05％Tween-20的1X磷酸盐缓冲盐水(phosphate-buffered saline，PBS)]洗涤上述ELISA板3次，每次至少1分钟并拍干，将200μl的1X ELISA/ELISPOT Diluent加入到上述板的每个孔中，然后在室温下温育1小时。
按照前述方法洗涤上述ELISA板1次。向每个孔中加入100μl 1X ELISA/ELISPOTDiluent稀释的标准人GM-CSF或100μl收集的细胞上清液，然后密封并在室温下温育2小时。
根据上述方法洗涤上述板3至5次。向每个孔中加入100μl的1X ELISA/ELISPOTDiluent稀释的检测抗体，然后密封并在室温下温育1小时。
根据前述方法洗涤上述板3至5次。向每个孔中加入100μl的1X ELISA/ELISPOTDiluent稀释的亲和素-辣根过氧化物酶(horseradish peroxidase，HRP)，然后密封并在室温下温育30分钟。
根据上述方法洗涤板5至7次。向每个孔中加入100μl的1X四甲基联苯胺(tetramethylbenzidine，TMB)溶液，在室温下温育15分钟。
然后向上述ELISA板的每个孔中加入50μl 2M H2SO4终止溶液。使用微量板读数器在450nm测量光密度(optical density，OD)值来确定在细胞上清液中表达的人GM-CSF的浓度。
实验结果显示，电穿孔法使GM-CSF-hTes mRNA转染的细胞和使GMKE mRNA转染的细胞均可以表达人GM-CSF。
按照前述方法，应用人IL-12ELISA试剂盒(eBioscience，Cat#：88-7126-88)检测收集的电穿孔法使体外转录的hIL-12mRNA转染细胞的上清液中表达的人IL-12。
用100μl 1X包被缓冲液以1∶250比例稀释的捕获抗体包被ELISA板每个孔，密封并在4℃下温育过夜。
在弃去含有捕获抗体的包被溶液后，用洗涤缓冲液(含有0.05％Tween-20的1XPBS)漂洗3次，每次至少1分钟，拍干，加入200μl的1X ELISA/ELISPOT Diluent到上述板的每个孔中，然后在室温下温育1小时。
根据前述方法，洗涤上述板。向每个孔中加入100μl的1X ELISA/ELISPOT Diluent稀释的标准人IL-12或100μl收集的上清液，然后密封并在室温下温育2小时。
根据前述方法洗涤板3至5次。向每个孔中加入100μl的1X ELISA/ELISPOTDiluent稀释的检测抗体，然后密封并在室温下温育1小时。
根据上述方法洗涤板3至5次。将100μl的1X ELISA/ELISPOT Diluent稀释的抗生物素蛋白-HRP加入到上述板的每个孔中，密封并在室温下温育30分钟。
根据上述方法洗涤平板5至7次，向每个孔中加入100μl的1×TMB溶液，在室温下温育15分钟。
然后向上述板的每个孔中加入50μl 2M H2SO4终止溶液。使用微量板读数器测量450nm处的OD值来测定细胞上清液中表达的人IL-12的浓度。
实验结果显示电穿孔法使体外转录hIL-12mRNA转染的细胞能够表达人IL-12。
使用基本局部比对搜索工具(basic local alignment search tool，BLAST)获得查询序列和受试者之间的百分比同一性。
编码人GM-CSF与多串连表位融合的mRNA癌症疫苗的组份包括GM-CSF-hTes mRNA、GMKE mRNA和hIL-12mRNA。该mRNA癌症疫苗含有用作免疫佐剂的人GM-CSF、多个串连表位构成的多表位癌症抗原以及用于增强免疫治疗作用的hIL-12。因此，该mRNA癌症疫苗将是用于癌症免疫治疗，特别是靶向非小细胞肺癌(NSCLC)患者的非常有效的疫苗。
Claims (10)
1.一种包括GM-CSF-hTes mRNA、GMKE mRNA和hIL-12 mRNA的癌症疫苗，其中GM-CSF-hTes mRNA是以核酸序列为SEQ ID NO：21的pVec-GM-CSF-hTes作为模板通过体外转录产生的；GMKE mRNA是以核酸序列为SEQ ID NO：36的pVec-GMKE作为模板通过体外转录产生的；hIL-12 mRNA是以核酸序列为SEQ ID NO：37的pVec-hIL-12作为模板通过体外转录产生的。
2.根据权利要求1所述的癌症疫苗，其特征在于pVec-GM-CSF-hTes中的hTERT(I540-548)-11 aa-hTERT(572Y-580)-2 aa-hTERT(988Y-997)的氨基酸序列为SEQ ID NO：8。
3.根据权利要求1所述的癌症疫苗，其特征在于pVec-GM-CSF-hTes中的hTERT(I540-548)-11 aa-hTERT(572Y-580)-2 aa-hTERT(988Y-997)的核苷酸序列为SEQ ID NO：18。
4.根据权利要求1所述的癌症疫苗，其特征在于pVec-GM-CSF-hTes中的GM-CSF-hTes的氨基酸序列为SEQ ID NO：20。
5.根据权利要求1所述的癌症疫苗，其特征在于pVec-GM-CSF-hTes中的GM-CSF-hTes的核苷酸序列为SEQ ID NO：19。
6.根据权利要求1所述的癌症疫苗，其特征在于pVec-GMKE中的MUC1(aa 130-154)-2aa-Kras 12 val(aa 5-17)-2 aa-EGFR T790M(aa 788-798)的氨基酸序列为SEQ ID NO：28。
7.根据权利要求1所述的癌症疫苗，其特征在于pVec-GMKE中的MUC1(aa 130-154)-2aa-Kras 12 val(aa 5-17)-2 aa-EGFR T790M(aa 788-798)的核苷酸序列为SEQ ID NO：29。
8.根据权利要求1所述的癌症疫苗，其特征在于pVec-GMKE中的GMKE的氨基酸序列为SEQ ID NO：34。
9.根据权利要求1所述的癌症疫苗，其特征在于pVec-GMKE中的GMKE的核苷酸序列为SEQ ID NO：35。
10.根据权利要求1所述的癌症疫苗，其特征在于pVec-hIL-12中的人白细胞介素-12cDNA被亚克隆在pVec的限制性核酸内切酶XhoI和XbaI位点之间，以获得pVec-hIL-12。
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| US20190076460A1 (en) | 2019-03-14 |
| CN110418648A (zh) | 2019-11-05 |
| WO2018156106A1 (en) | 2018-08-30 |
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