CN108165536A - 一种重组溶瘤痘苗病毒及其制备方法与应用 - Google Patents
一种重组溶瘤痘苗病毒及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种重组溶瘤痘苗病毒及其制备方法与应用，所述病毒的胸腺嘧啶核苷激酶(TK)区包含SEQ ID NO.1所示的PD1全长抗体的编码序列。本发明将基因治疗的抑瘤效果与病毒治疗的溶瘤效应有效结合起来，制备了一种可高效表达PD1全长抗体基因的溶瘤痘苗病毒。在溶瘤病毒发挥溶瘤效应裂解肿瘤细胞的同时，大量表达PD1全长抗体，抑制T细胞表面PD1的活性，激活T细胞免疫应答，发挥双重的抗肿瘤效果。相对于单纯的基因疗法或者病毒疗法增强了其对恶性B细胞淋巴肿瘤的杀伤能力。采用病毒复制相关基因缺失的方式，缺失痘苗病毒基因组的TK区，确保病毒在异常增殖的肿瘤细胞内特异性复制，而在正常的细胞中则不能复制，大大增强了溶瘤痘苗病毒载体的安全性。
Description
技术领域
本发明属于基因工程学，免疫学和肿瘤学，涉及一种重组溶瘤痘苗病毒及其制备方法与应用。
背景技术
恶性淋巴瘤的发病率占整个癌症患者的3-5％，每年有452,000新增患者。大于90％的成人患者来源于成熟的B细胞，提示B细胞淋巴肿瘤是威胁人类健康的重要疾病之一。
免疫治疗是继手术，放疗，化疗，分子靶向治疗后又一种癌症治疗新方法。在各种癌症免疫治疗方法中，免疫检查点抑制剂是近年来获得突出疗效的药物之一。免疫检查点是预防免疫系统过度激活的抑制性通路，在被激活的免疫细胞表面存在一些蛋白如CTLA4，PD1等在过度免疫反应时起到“刹车”作用，然而在肿瘤发生时，这些蛋白因过度表达而持久地为免疫系统发出抑制信号，从而促进肿瘤的免疫逃逸。故免疫检查点抑制剂在癌症的免疫治疗中起着重要的作用。
PD-1/PD-L1信号通路是阻断免疫检查点策略的最为重要的靶点之一。在肿瘤发生时可导致T细胞表达PD1，多种类型癌症的肿瘤侵润淋巴细胞(TIL)也表达PD-1。同时研究表明多种癌细胞高表达PD-L1，包括淋巴瘤，白血病，恶性黑色素瘤和非小细胞肺癌(NSCLC)等。PD-1与癌细胞表面的PD-1配体(PD-L1或者PD-L2)结合抑制T细胞的活化，导致特异性T细胞耗竭或失能。
在B细胞淋巴肿瘤上，研究显示滤泡淋巴瘤(FL)的TIL和外周血T细胞上高表达PD1，且T细胞失能。另一方面，多种B细胞肿瘤，包括弥漫大B淋巴瘤(DLBCL)，原发纵膈大B淋巴瘤(PMBCL)以及睾丸大B淋巴瘤上均大量表达PD-L1。因此，有效地阻断PD-1/PD-L1信号通路，能够增强T细胞对B细胞淋巴肿瘤的杀伤。
新近，阻断免疫检查点的PD-1和PD-L1单抗和抑制剂已应用于I-III期临床研究。2015年6月，美国食品及药物管理局(FDA)批准PD-1单抗nivolumab作为一款治疗药物用于先前经过化疗的晚期NSCLC患者。I期临床研究表明，nivolumab治疗NSCLC患者的平均总生存期为14.9个月，1年和3年的生存率为42％和18％。
但是，免疫检查点抑制剂也有其局限性：(1)多数病人不能获得持久的完全反应；(2)PD-1单抗治疗后约25-30％患者出现严重免疫相关副作用(irAEs)；(3)PD-1/PD-L1通路在肿瘤微环境中的特点影响了PD-1单抗的疗效。联合治疗是克服免疫检查点抑制剂局限性的主要研究方向。目前正在研究的策略包括抗PD-1单抗与放化疗、小分子靶向药物、其他免疫检查点抑制剂或溶瘤病毒联合。
溶瘤病毒(Oncolytic virus，OV)疗法是今年来发展迅速的一种肿瘤生物治疗新方法。2015年10月，FDA批准安进公司的OV产品T-Vec用于治疗恶性黑色素瘤。12月，T-Vec又获批欧洲食品药品管理局，证明OV疗法是治疗癌症的一种非常有前景的新方法。溶瘤痘苗病毒(Oncolytic vaccinia virus,OVV)是近年来备受关注的一类溶瘤病毒。其优点在于：病毒稳定性好、致病性低、基因转染效力高、安全性良好。溶瘤痘苗病毒能选择性感染肿瘤细胞并在细胞内复制、杀死肿瘤细胞，而对正常组织和细胞的毒性很小。JX-594是第一个用于临床研究的溶瘤痘苗病毒，它缺失了胸腺嘧啶核苷激酶(TK)区并携带外源性基因GM-CSF，JX-594能在快速增殖的肿瘤细胞中复制，同时携带的GM-CSF能进一步刺激抗肿瘤免疫。Breitbach CJ等里程碑式的研究结果显示：JX-594静脉注射23例难治、复发的实体肿瘤，13例患者有效，1例患者获得部分缓解；表明OVV可以用于静脉注射，发挥全身抗肿瘤作用。
发明内容
本发明的发明人发现，现有的PD1的单抗在治疗B细胞淋巴肿瘤中存在局限性。为此，本发明提供一种重组溶瘤痘苗病毒及其制备方法与应用。
本发明的目的是通过以下技术方案实现的：一种重组溶瘤痘苗病毒，所述病毒的胸腺嘧啶核苷激酶(TK)区包含SEQ ID NO.1所示的PD1全长抗体的编码序列。
一种重组溶瘤痘苗病毒的制备方法，其特征在于，包括以下步骤：
(1)合成人全长PD-1单克隆抗体，其基因序列如SEQ ID NO.1所示；
(2)扩增全长PD-1单克隆抗体，酶切后亚克隆至痘苗病毒穿梭质粒(pTK)的TK区中，构建出重组质粒pCB-aPD1。其中，aPD1基因由痘苗病毒的早/晚期启动子(P-se/l)控制。
(3)采用基因同源重组的方式，将pCB-aPD1质粒转染到已经被感染了野生型痘苗病毒的细胞中，使两者同源重组，产生重组痘苗病毒VV-aPD1。经筛选后，获得所述TK区包含SEQ ID NO.1所示的PD1全长抗体的编码序列的重组溶瘤痘苗病毒。
一种重组溶瘤痘苗病毒在制备治疗治疗B细胞淋巴肿瘤的药物中的应用。
本发明的有益效果是：
1、本发明将基因治疗的抑瘤效果与病毒治疗的溶瘤效应有效结合起来，制备了一种可高效表达PD1全长抗体基因的溶瘤痘苗病毒。在溶瘤病毒发挥溶瘤效应裂解肿瘤细胞的同时，大量表达PD1全长抗体，抑制T细胞表面PD1的活性，激活T细胞免疫应答，发挥双重的抗肿瘤效果。相对于单纯的基因疗法或者病毒疗法增强了其对恶性B细胞淋巴肿瘤的杀伤能力。
2、本发明采用病毒复制相关基因缺失的方式，缺失痘苗病毒基因组的TK区，确保病毒在异常增殖的肿瘤细胞内特异性复制，而在正常的细胞中则不能复制，大大增强了溶瘤痘苗病毒载体的安全性。
附图说明
图1a是pCB-aPD-1经ECORI和BglII双酶切后DNA凝胶电泳结果，1b是部分测序结果。
图2是流式检测pCB-aPD-1与活化的T细胞孵育后的PD-1表达，继而检测PD-1抗体活性，其中，左图为未经刺激的Jurkat细胞对照，右图为经ConA刺激后表达PD1抗原的Jurkat细胞。
图3是pCB-aPD1-GFP与WT在293A细胞中同源重组后的荧光图。
图4是OVV-aPD-1重组后，提取病毒基因组DNA通过PCR检测目的基因和野毒基因的存在。
图5是OVV-aPD-1以浓度梯度感染Raji细胞株后，流式检测GFP的表达效率。
图6是OVV-aPD-1以浓度梯度感染Raji细胞株后，通过检测集落形成和CCK8染色法检测OVV-aPD-1的细胞毒性，其中，b是对a的数据分析。
具体实施方式
本发明结合附图和实例作进一步的说明。除特别说明外，本发明可采用本领域常规技术。
1、人全长PD-1单克隆抗体的合成：
PD-1单克隆抗体的单链可变区序列参照施贵宝PD-1单抗nivolumab的序列，通过氨基酸推演和密码子优化得到我们设计的PD-1单链可变区序列，单抗的Fc段参照人IgG4的Fc段序列，采用化学合成的方法合成人PD-1抗体的重链和轻链，该重链和轻链基因由链接子IRES连接。PD-1单克隆抗体的全长基因序列如SEQ ID NO.1所示。
2、携带人PD-1单抗的溶瘤痘苗病毒穿梭质粒pCB-aPD1的构建与鉴定：
应用末端含有ECORI和BglII酶切位点的特异性PCR引物扩增出PD1全长抗体序列(含重链和轻链)，再经过ECORI和BglII双酶切后亚克隆至携带GFP基因的痘苗病毒穿梭质粒(pTK-GFP)的TK区中，构建出pCB-aPD1。酶切后的DNA凝胶电泳结果如图1a所示，从图中可以看出pCB-aPD1经酶切后产生2700bp的aPD1全长序列；1b是部分测序结果，结果证明所克隆的aPD1基因序列正确。aPD1基因由痘苗病毒的早/晚期启动子(P-se/l)控制。构建出的阳性克隆应用酶切、测序进行鉴定，选择鉴定正确的克隆进行质粒大量抽提，制备>4μg的高纯度质粒，-80℃保存备用。
其中，痘苗病毒穿梭质粒可参考房有荣,李红玉,陈科达,周文硕,&阎辉.(2012).Zeocin和gfp双筛选标记重组痘苗病毒载体的构建.国际流行病学传染病学杂志,39(3),148-152。
3、pCB-aPD-1表达PD-1抗体的检测
Jurkat细胞经ConA刺激后转导pCB-aPD-1，应用PE-anti Human IgG4(Fc)通过流式检测pCB-aPD-1表达PD-1抗体能力，结果如图2所示，说明pCB-aPD-1能表达11.9％的PD-1抗体。
4、OVV-a PD1病毒的包装与鉴定
采用基因同源重组的方式，通过脂质体转染将pCB-aPD1-GFP质粒转染到已经被感染了野生型痘苗病毒的293A细胞中，使两者同源重组，产生VV-aPD-1-GFP。包装的病毒经3-5轮药筛(霉酚酸，黄嘌呤与雌黄嘌呤)联合GFP显影筛选，嗜斑纯化，连续至少三轮“挑斑”，PCR鉴定，筛选出正确的阳性重组痘苗病毒。图3结果说明pCB-aPD1-GFP在293A细胞里成功与野生型痘苗病毒重组，产生病毒空斑。图4结果表明经多轮药筛产生的重组病毒携带PD1全长抗体的重链(1300bp)，轻链(704bp)和链接子IRES(650bp)序列，且无野毒污染。
5、OVV-aPD1对B细胞淋巴瘤细胞株的体外感染
为验证OVV-aPD1对B细胞淋巴瘤细胞株的体外感染，选取Raji细胞株，OVV-aPD1分别以5，10，20MOI的浓度梯度感染Raji细胞株，利用流式细胞仪检测OVV-aPD1对Raji细胞株的感染效率，结果如图5所示，OVV-aPD1对Raji细胞株的感染效率在20MOI时达到24.28％。
6、OVV-a PD1对B细胞淋巴瘤细胞株的体外杀伤
为验证OVV-a PD1对B细胞淋巴瘤细胞株的体外杀伤，OVV-aPD1分别以5，10，20MOI的浓度梯度感染Raji细胞株72小时，利用集落形成实验和CCK8染色法检测OVV-a PD1对Raji细胞株的生长抑制作用，结果如图6a和6b所示，OVV-a PD1对Raji细胞株的生长抑制作用呈浓度依赖，在20MOI时细胞呈小且分散集落生长，细胞毒性作用达到65％，严重影响细胞增殖。
序列表
<110> 浙江大学
<120> 一种重组溶瘤痘苗病毒及其制备方法与应用
<160> 1
<170> SIPOSequenceListing 1.0
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<212> DNA
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gaattctcac ttgcccaggg acagggacag gcttttctga gtataatggt tgtgcagtgc 60
ttcgtgcatc acggagcagg agaacacgtt gccctcctgc catctagact tatccacggt 120
cagccggcta tacagaaaga agctgccatc ggagtccagc acgggtggtg tggtcttgta 180
attgttctcg ggctggccat tggactccca ctccacagcg atgtcagatg gatagaagcc 240
cttcaccaga caggtcaggc tcacctggtt ctttgtcatc tcctcctggg aagggggcag 300
ggtgtacacc tgtggctccc taggctggcc cttggcctta gagattgtct tctcgatgct 360
ggatggcagg cccttattgc tcaccttgca cttatactcc ttgccgttca gccagtcctg 420
atgcagcacg gtcagcacgc tcaccacccg gtatgtggag ttaaactgct cctccctagg 480
cttggtctta gcattgtgca cctccacgcc atccacatac cagttgaact gcacctctgg 540
atcctcctgg gacacgtcca ccaccacgca ggtcacctca ggtgtccgag agatcatcag 600
tgtgtcctta ggctttggag gaaacaggaa cacggaaggt cctcccagga actcaggagc 660
aggacaaggt gggcaaggag gtccgtactt agactccacc ctcttatcca ccttggtatt 720
gctaggctta tggtccacgt tgcaggtata tgtcttggtg cccagagagc tggagggcac 780
tgtcaccaca gagctcaggc tgtacaggcc ggaagactgc agcacagcag gaaaggtgtg 840
cacgccggat gtcagggcgc cggagttcca gctcacggtc actggctcag ggaaataatc 900
cttcaccaga cagcccagag cggcggtgct ctcggatgta gacctgctgc agggagccag 960
tgggaacacg gaggggccct ttgtgctggc gctggacacg gtcaccagtg tgccctggcc 1020
ccagtaatcg tcattggtag cacaatagta cacggctgta tcctcggccc tcagagagtt 1080
catctgcaga aacagggtat tcttggagtt gtccctagag attgtgaatc tgcccttcac 1140
ggaatcggca tagtacctct tagagccgtc gtaccagatc acagccaccc actccagtcc 1200
cttgccagga gcctgtctca cccagtgcat gccggagtta gagaaggtga tgccgctggc 1260
cttgcagtcc agccgcaggg acctgcctgg ctgcaccacc ccccctcctg attcgaccag 1320
ctgcacctgt gagtggacac cagttgcggt tgcgaccaga aacagaatga tacatgacca 1380
tcccatggaa ggtcgtctcc ttgtgggttg tggcaagctt atcatcgtgt ttttcaaagg 1440
aaaaccacgt ccccgtggtt cggggggcct agacgttttt ttaacctcga ctaaacacat 1500
gtaaagcatg tgcaccgagg ccccagatca gatcccatac aatggggtac cttctgggca 1560
tccttcagcc ccttgttgaa tacgcttgag gagagccatt tgactctttc cacaactatc 1620
caactcacaa cgtggcactg gggttgtgcc gcctttgcag gtgtatctta tacacgtggc 1680
ttttggccgc agaggcacct gtcgccaggt ggggggttcc gctgcctgca aagggtcgct 1740
acagacgttg tttgtcttca agaagcttcc agaggaactg cttccttcac gacattcaac 1800
agaccttgca ttcctttggc gagaggggaa agacccctag gaatgctcgt caagaagaca 1860
gggccaggtt tccgggccct cacattgcca aaagacggca atatggtgga aaataacata 1920
tagacaaacg cacaccggcc ttattccaag cggcttcggc cagtaacgtt aggggggggg 1980
gagggagagg ggcgtcaaca ttctccacgg ttgaaacttt ttgtgactgg tgaggacaga 2040
ccctggtgag tgacttcaca agcatacacc ttgtgcttct cgtaatcggc cttggacagt 2100
gtcagggtgc tggacagaga atatgtgcta tccttggagt cctgctcggt cacagactcc 2160
tggctattgc cggactgcag agcgttatcc accttccact gcaccttggc ctcccgaggg 2220
tagaaattgt tcagcaggca caccacgctg gctgtgccgg acttcagctg ctcgtcggaa 2280
gggggaaaga tgaacacgga gggagcggcc acggttctct tgatctccac ctttgtgccc 2340
tggccaaagg tccgtggcca attagagctc tgctggcaat agtacacggc gaaatcctca 2400
ggctccaggg aagagattgt cagggtaaag tctgtgccag atccgcttcc ggagaaccta 2460
gcggggatgc cggtagctct gttagaggcg tcgtagatca gcagcctggg agcctggcct 2520
ggcttctgct gataccaggc caggtaggag gacacggact ggctagcccg gcagctcagg 2580
gtggccctct ctcctgggga cagagacaga gttgcggggc tctgagtcag cacaatctct 2640
gaatgcacac cagttgcggt tgcgaccagg aacaggatga tacaggacca tcccatagat 2700
ct 2702
Claims (3)
1.一种重组溶瘤痘苗病毒，其特征在于，所述病毒的胸腺嘧啶核苷激酶(TK)区包含SEQID NO.1所示的PD1全长抗体的编码序列。
2.一种权利要求1所述的重组溶瘤痘苗病毒的制备方法，其特征在于，包括以下步骤：
(1)合成人全长PD-1单克隆抗体，其基因序列如SEQ ID NO.1所示；
(2)扩增全长PD-1单克隆抗体，酶切后亚克隆至痘苗病毒穿梭质粒(pTK)的TK区中，构建出重组质粒pCB-aPD1。其中，aPD1基因由痘苗病毒的早/晚期启动子(P-se/l)控制。
(3)采用基因同源重组的方式，将pCB-aPD1质粒转染到已经被感染了野生型痘苗病毒的细胞中，使两者同源重组，产生重组痘苗病毒VV-aPD1。经筛选后，获得所述TK区包含SEQID NO.1所示的PD1全长抗体的编码序列的重组溶瘤痘苗病毒。
3.一种权利要求1所述的重组溶瘤痘苗病毒在制备治疗治疗B细胞淋巴肿瘤的药物中的应用。
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