CN109055430A - 一种共表达il18和ccl19蛋白及靶向muc1基因car-t细胞的制备方法 - Google Patents
一种共表达il18和ccl19蛋白及靶向muc1基因car-t细胞的制备方法 Download PDFInfo
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Abstract
本发明涉及一种共同表达IL18和CCL19蛋白及靶向MUC1基因CAR‑T细胞的制备方法，包括将包含IL18和CCL19蛋白编码序列及靶向MUC1 CAR的慢病毒转染CD4+/CD8+阳性细胞；以及筛选并培养扩增转染后的T细胞以获得共表达IL18和CCL19蛋白及靶向MUC1基因的CAR‑T细胞。本发明通过表达第三细胞信号IL18和CCL19增强了CAR‑T细胞的增殖能力和生存周期，提高了CAR‑T细胞在实体瘤里的渗透作用，维持了CAR‑T细胞对实体瘤的杀伤作用。另外，本发明共表达的IL18和CCL19都由2A短肽连接，可以分别表达。
Description
技术领域
本发明涉及免疫细胞制备领域，具体地，本发明涉及一种共表达IL18和CCL19蛋白及靶向MUC1基因CAR-T细胞的制备方法。
背景技术
继手术、放疗、化疗后，肿瘤的免疫疗法已成为行之有效的治疗方式，近期收到广泛关注。其中嵌合抗原受体T细胞技术(Cheimeric Antigen Receptors-T cell,CAR-T)是一种新出现的过继性细胞疗法(Adoptive cell transfer therapy,ACT)，该技术将患者T细胞在体外修饰、活化和增殖后回输到患者体内，通过T细胞表达的特异性受体，靶向性的识别肿瘤细胞，并显示较强的杀伤活性和持久性。
嵌合抗原受体(CAR)由胞外抗体结合区、铰链区、跨膜区胞内信号传导区等构成，一般通过病毒转导或电穿孔，使CAR分子表达在T细胞表面形成CAR-T细胞。CAR分子的设计根据胞内信号区的不同经历了四代发展，其中第一代信号区仅包含CD3ζ；第二代信号区在第一代基础上加了一个共刺激因子，如CD28、4-1BB、OX40等；第三代在第一代基础上加了两个串联的共刺激因子；第四代包含IL12、IL18、CCL19等能改善微环境的细胞因子。靶向血液肿瘤方向的CD19的CAR-T细胞，部分研究中总体缓解率达90％以上。然而在部分血液肿瘤及大部分实体肿瘤中由于肿瘤微环境的抑制，效果不是很理想，第四代CAR-T在肿瘤微环境发挥的显著作用具有重要的临床应用价值。
细胞因子IL18是一种具有抗肿瘤活性的蛋白质，IL-18可作用于T、B以及NK细胞等效应细胞的增殖与分化，促进IFN-γ的表达和克里梅的释放，从而发挥其抗肿瘤功能。IL-18和IL12也可直接作用于CD8+T细胞和NK细胞，诱导其产生更多的IFN-γ、释放颗粒酶B、表达FasL，从而进一步杀伤肿瘤细胞。同时，IL18因其相对低毒的特性以及和传统疗法的的协同作用引人注目。
CCL19(Chemokine C-C motif ligand 19)属于CC类趋化因子，又称EB病毒诱导分子或人巨噬细胞炎性蛋白3β，主要在次级淋巴组织和器官如脾和淋巴结的T细胞中表达，趋化幼稚T细胞和成熟DC细胞。CCL19在抗肿瘤中起着关键作用，主要通过以下途径：诱导树突状细胞进入肿瘤组织；介导T细胞参与杀伤肿瘤细胞；肿瘤组织中表达的CCL19诱导免疫细胞浸润肿瘤，释放其他细胞因子；直接与CCR7受体结合，激活受体后通路抑制肿瘤增殖、侵袭及转移。CCL19招募免疫细胞到肿瘤部位，诱导抗原提呈和效应细胞活化，增强肿瘤局部多种免疫因子，克服肿瘤微环境的免疫抑制，在肿瘤的免疫治疗中趋化抗原提呈细胞等方面发挥重要作用。
此前的研究表明，淋巴组织中的网状成纤维细胞可以分泌趋化因子IL18以及CCR19，其中CCR19可以募集外周T细胞及DC细胞(树突状细胞)进入淋巴组织，而IL18在促进T细胞增殖同时可以维持T细胞稳定。通过将IL18和CCL19也装到CAR-T细胞之中，就能够在肿瘤治疗的同时招募更多的T细胞以及DC细胞来肿瘤组织中一起杀伤肿瘤。
发明内容
本发明的目的在于克服现有技术中的瓶颈，一种共表达IL18和CCL19蛋白及靶向MUC1基因CAR-T细胞的制备方法，并证明了其在肿瘤治疗中的潜在应用。具体地说，发明提供一种靶向MUC1的CAR-T制备方法，通过共同表达IL18和CCL19，解除肿瘤微环境的免疫抑制，赋予T细胞更持久的肿瘤杀伤能力。
为实现上述目的，本发明采用如下技术方案：
本发明的第一个目的是提供一种CD4+/CD8+T细胞提取的方法，其中CD4+T细胞和CD8+T细胞分别由EasySeq试剂盒(STEMCELL)按对应说明书操作分别提取出来。
本发明的第二个目的是提供一种共表达IL18和CCL19蛋白及靶向MUC1的CAR慢病毒载体；
进一步地，所述CAR分子是利用pCDH载体依次串联人EF1α、CSF2RA嵌合受体信号肽、胞外抗体结合区、铰链区、跨膜区胞内信号传导区、T2A短肽连接的IL18以及CCL19和筛选基因CopGFP制备而成；
进一步地，胞内信号区为OX40-4-1BB-CD3ζ，OX40和4-1BB为嵌合受体共刺激分子；
优选地，所述铰链区为CD8hinge区域，所述跨膜区为CD8α；
进一步地，IL18和CCL19为第三信号分子；
更进一步地，MUC1 CAR pCDH载体质粒在辅助质粒pSPA2和vsv-G包膜质粒pMD2.G存在的情况下，待细胞长至80-90％，共转染HEK293T细胞，收集上清，浓缩后即可制成高质量的带有CAR分子的慢病毒。
最后，本发明第三个目的是提供一种共表达IL18和CCL19蛋白靶向MUC1 CAR基因的重组表达载体的宿主细胞；
进一步地，所述宿主细胞为T细胞或者含有T细胞的细胞群；
进一步地，所述带有MUC1 CAR分子的慢病毒与宿主细胞的moi值为1～5；
进一步地，所述宿主细胞转染后采用anti-CD3+CD28抗体磁珠激活，用于筛选并培养扩增转染后的T细胞以获得共表达IL18和CCL19蛋白靶向MUC1的CAR-T细胞。
在一个实施例中，将共表达IL18和CCL19的靶向MUC1的CAR-T细胞与表达MUC1(+)、MUC1(-)的肿瘤细胞系K562共孵育，结果显示共表达IL18和CCL19的靶向MUC1细胞能够在MUC1(+)靶细胞刺激的情况下大量的产生IFN-γ；但是却不能与MUC1(-)靶细胞共孵育时产生IFN-γ；同时非MUC1靶向的T细胞不能够在MUC1(+)靶细胞刺激下产生IFN-γ。该实施案例说明了共表达IL18和CCL19的靶向MUC1的CAR-T细胞有良好的靶向性，能够在MUC1靶点的刺激下将T细胞杀伤功能激活。
在另一个实施中，证明了在一定的效靶比(CAR-T细胞：靶细胞)下，MUC1 CAR-T细胞或共表达IL18和CCL19的MUC1 CAR-T细胞，均能够杀伤MUC1阳性的肿瘤细胞，且两者杀伤的能力差异不显著。该实例说明MUC1 CAR-T细胞能够有效杀伤MUC1阳性的肿瘤细胞，且共表达IL18和CCL19对MUC1 CAR-T细胞的活性影响较小。
在另一个实施中，针对MUC1阳性的小鼠模型，共表达IL18和CCL19的MUC1CAR-T细胞，
能够完全消除肿瘤细胞，可以使全部小鼠长期无瘤生存。而仅有IL18或CCL19或两者均不含有的MUC1 CAR-T细胞处理的小鼠模型，只有30％能够长期无瘤生存。
与现有技术相比，本发明具有以下有益效果：
目前，CAR-T技术在实体瘤领域的应用并不成功，一个重要的原因就是免疫微环境的免疫抑制效应，通过共表达IL18和CCL19蛋白，能够通过提高CAR-T细胞在实体瘤的渗透作用，解除肿瘤微环境的抑制功能；IL18和CCL19蛋白是分别通过2A短肽连接的，避免了融合蛋白之间的相互干扰，增加了临床应用的安全性。本发明所述的一种共表达IL18和CCL19蛋白靶向CAR-T细胞的制备方法，能够特异性解除免疫微环境的抑制，特异高效杀伤靶向肿瘤细胞。同时，募集外周T细胞及DC细胞进入淋巴组织，并且促进T细胞的增殖和稳定。
附图说明
图1为MUC1 CAR分子序列的说明示意图；
图2为慢病毒pCDH载体的基因结构的说明示意图；
图3为流式检测MUC1 CAR和18×19MUC1 CAR表达水平示意图；
图4为18×19MUC1 CAR-T细胞分泌IL18和CCL19表达水平；
图5为18×19MUC1 CAR-T细胞杀伤MUC1阳性细胞K562的效靶比示意图；
具体实施方式
本发明提供一种共表达IL18和CCL19，靶向MUC1的CAR-T细胞的制备方法，其包括CD4+/CD8+T细胞制备、共表达IL18和CCL19蛋白及靶向MUC1的CAR慢病毒载体的构建、共表达18×19CAR分子的慢病毒制备、慢病毒的转染T细胞、anti-CD3+CD28抗体磁珠激活并通过筛选培养获得MUC1 CAR-T细胞。
CD4+/CD8+T的制备
用EDTA抗凝管采集健康人外周血，加入等体积磷酸缓冲液PBS稀释，将外周血稀释液加入到等体积淋巴细胞分离液(Ficoll)的50ml离心管中，400g 30min离心；小心吸取淋巴分离液上的白膜层细胞，转入新的50ml离心管中，用PBS离心300g 10min洗涤3次，即可获得PBMC细胞；将PBMC细胞分别按照EasySeq试剂盒(EasySepTMHuman CD4+/CD8+T CellEnrichment Kit,STEMCELL)说明书操作即可获得CD4+和CD8+T细胞。
T细胞存储在含有20％人AB血清和10％DMSO的RPIM培养基中；T细胞复苏后，用含有30U/ml的T细胞培养基培养过夜；T细胞培养基(T cell culture medium,TCM)由以下组成：X-Vivo15(Lonza)，5％人AB血清(Valley Biomedical)，55μMβ-巯基乙醇，10mM N-乙酰L-半胱氨酸。
构建CAR分子到慢病毒载体pCDH
本发明中CAR分子是利用pCDH载体依次串联人EF1α、CSF2RA嵌合受体信号肽、胞外抗体结合区、铰链区、跨膜区胞内信号传导区和T2A短肽连接的筛选基因CopGFP制备而成，其信号域为OX40-4-1BB-CD3ζ，其分子结构见图2慢病毒pCDH结构示意图。
首先化学合成MUC1HMFG2scFv核酸片段至pUC57载体中，Not I/Xba I双酶切得到HMFG2片段；Not I/Xba I双酶切慢病毒载体pCDH；将线性pCDH载体与HMFG2片段使用T4连接酶链接，室温1～2小时，随后转化入TOP10感受态细胞，过夜培养后，挑取阳性单克隆扩增培养并提取质粒，测序验证其序列，正确的质粒命名为pCDH-EF1α-CSF2RA-MUC1scFv-CD8hinge-CD8α-OX40-4-1BB-CD3ζ-T2A-IL18-T2A-CCL19-CopGFP(以下简写为pCDH-18×19MUC1CAR)。
共表达IL18和CCL19的MUC1 CAR-T细胞的构建
CAR慢病毒的包装：首先使用无内毒素大提质粒试剂盒提取pCDH-18×19MUC1 CAR以及慢病毒系统辅助包装质粒pMD2.G和PSPAX2。转染前一天将2.0～3.0*107HEK 293T细胞铺至T75培养方瓶中，待细胞长至80％左右后用于转染。转染前将293T细胞培养基更换为10ml无血清培养基，使用转染试剂 HQ(Polyplus,France)将质粒共转染到293T细胞中，转染24小时后将细胞培养基更换为完全培养基DMEM+10％FBS。转然后48小时后收集上清，并加入15ml完全培养基，72小时后再收集上清，弃掉细胞。上清用0.45μm滤膜过滤，5000g离心1min去除杂质，随后40000g离心2小时将病毒沉淀，用0.1ml PBS重悬病毒。置于-80℃冰箱，冻存备用。
将转染后的病毒悬液加入到CD4+或CD8+T细胞中，37℃5％CO2培养24h后；将细胞与anti-CD3+CD28(Invitrogen)抗体磁珠以1:1的比例混合，并且用TCM将细胞浓度调整为1×106/ml，培养2-4h；加入含有18×19MUC1 CAR的慢病毒，培养24-36h；72h后利用流式细胞术检测GFP表达以检测CAR分子转导效率，结果见图3；磁珠在刺激4-6天后移除；将CD4+18×19MUC1 CAR-T细胞与CD8+18×19MUC1 CAR-T细胞按等浓度1:1比例混合，即可得到最终的共表达IL18和CCL19，靶向MUC1的CAR-T细胞。
18×19MUC1
CAR-T细胞活性测定
将共表达IL18和CCL19，靶向MUC1的CAR-T细胞与表达MUC1(+)、MUC1(-)的肿瘤细胞系共孵育，结果显示共表达IL18和CCL19的靶向MUC1细胞能够在MUC1(+)靶细胞刺激的情况下大量的产生IFN-γ；但是却不能与MUC1(-)靶细胞共孵育时产生IFN-γ；同时非MUC1靶向的T细胞不能够在MUC1(+)靶细胞刺激下产生IFN-γ。实验结果见图4。该实施案例说明了共表达IL18和CCL19的靶向MUC1的CAR-T细胞有良好的靶向性，能够在MUC1靶点的刺激下将T细胞杀伤功能激活。
18×19MUC1 CAR-T细胞杀伤MUC1靶细胞的能力
在另一个实施中，证明了在一定的效靶比(CAR-T细胞：靶细胞)下，MUC1 CAR-T细胞和18×19MUC1 CAR-T细胞均能够杀伤MUC1阳性的肿瘤细胞，且两者杀伤的能力差异不显著。该实例说明18×19MUC1 CAR-T细胞能够有效杀伤MUC1阳性的肿瘤细胞，且共表达IL18和CCL19对MUC1 CAR-T细胞的活性影响较小。
用5-竣基荧光素琥珀酰亚胺基(CFSE)将靶细胞染色18×19MUC1 CAR-T细胞或MUC1CAR-T细胞与MUC1阳性的K652细胞按以下效靶比混合培养：0.31:1,0.63:1,2.5:1,5:1,10:1。经过4小时共培养后，将细胞用PI试剂盒染色，同时设置对照组，对照组中的效应细胞为T淋巴细胞，空白组中不加任何效应细胞。使用TopCount NXT新一代微孔板闪烁发光计数仪对细胞杀伤进行检测，结果见图5。无论是否表达IL18和CCL19，CAR-T细胞均能够有效杀伤MUC1阳性K652细胞，但是对照组中的T细胞没有能够有效杀伤细胞。该实施案例说明了MUC1CAR-T具备明确的肿瘤细胞杀伤活性，同时也表明表达IL18和CCL19对CAR-T细胞的杀伤活性没有产生实质性的影响。
以上对本发明的具体实施例进行了详细描述，但其只作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。
序列表
<110> 杭州荣泽生物科技有限公司
<120> 一种共表达IL18和CCL19蛋白及靶向MUC1基因CAR-T细胞的制备方法
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accatattta ttataagtat gtataaagat agccagccta gaggtatggc tgtaactatc 300
tctgtgaagt gtgagaaaat ttcaactctc tcctgtgaga acaaaattat ttcctttaag 360
gaaatgaatc ctcctgataa catcaaggat acaaaaagtg acatcatatt ctttcagaga 420
agtgtcccag gacatgataa taagatgcaa tttgaatctt catcatacga aggatacttt 480
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tacatcgtga ggaacttcca ctaccttctc atcaaggatg gctgcagggt gcctgctgta 180
gtgttcacca cactgagggg ccgccagctc tgtgcacccc cagaccagcc ctgggtagaa 240
cgcatcatcc agagactgca gaggacctca gccaagatga agcgccgcag cagt 294
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tgtgttgcct ctggattcac tttcagtaac tactggatga actgggtccg ccagtctcca 180
gagaaggggc ttgagtgggt tgctgaaatt agattgaaat ctaataatta tgcaacacat 240
tatgcggagt ctgtgaaagg gaggttcacc atctcaagag atgattccaa aagtagtgtc 300
tacctgcaaa tgaacaactt aagagctgaa gacactggca tttattactg tacctttggt 360
aactcctttg cttactgggg ccaagggacc acggtcaccg tctcctcagg tggaggcgga 420
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ctcaccacat cacctggtga aacagtcaca ctcacttgtc gctcaagtac tggggctgtt 540
acaactagta actatgccaa ctgggtccaa gaaaaaccag atcatttatt cactggtcta 600
ataggtggta ccaacaaccg agcaccaggt gttcctgcca gattctcagg ctccctgatt 660
ggagacaagg ctgccctcac catcacaggg gcacagactg aggatgaggc aatatatttc 720
tgtgctctat ggtacagcaa ccattgggtg ttcggtggag gaaccaaact gactgtccta 780
ggatccgaga ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840
cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900
agggggctgg acttcgcctg tgatttttgg gtgctggtgg tggttggtgg agtcctggct 960
tgctatagct tgctagtaac agtggccttt attattttct gggtgaggag taagaggagc 1020
aggctcctgc acagtgacta catgaacatg actccccgcc gccccgggcc cacccgcaag 1080
cattaccagc cctatgcccc accacgcgac ttcgcagcct atcgctccaa acggggcaga 1140
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gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 1260
aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 1320
gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1380
cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 1440
cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1500
ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1560
gcccttcaca tgcaggccct gccccctcgc gagggcagag gaagtcttct aacatgcggt 1620
gacgtggagg agaatcccgg ccctatggct gctgaaccag tagaagacaa ttgcatcaac 1680
tttgtggcaa tgaaatttat tgacaatacg ctttacttta tagctgaaga tgatgaaaac 1740
ctggaatcag attactttgg caagcttgaa tctaaattat cagtcataag aaatttgaat 1800
gaccaagttc tcttcattga ccaaggaaat cggcctctat ttgaagatat gactgattct 1860
gactgtagag ataatgcacc ccggaccata tttattataa gtatgtataa agatagccag 1920
cctagaggta tggctgtaac tatctctgtg aagtgtgaga aaatttcaac tctctcctgt 1980
gagaacaaaa ttatttcctt taaggaaatg aatcctcctg ataacatcaa ggatacaaaa 2040
agtgacatca tattctttca gagaagtgtc ccaggacatg ataataagat gcaatttgaa 2100
tcttcatcat acgaaggata ctttctagct tgtgaaaaag agagagacct ttttaaactc 2160
attttgaaaa aagaggatga attgggggat agatctataa tgttcactgt tcaaaacgaa 2220
gacgagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctatg 2280
gccctgctac tggccctcag cctgctggtt ctctggactt ccccagcccc aactctgagt 2340
ggcaccaatg atgctgaaga ctgctgcctg tctgtgaccc agaaacccat ccctgggtac 2400
atcgtgagga acttccacta ccttctcatc aaggatggct gcagggtgcc tgctgtagtg 2460
ttcaccacac tgaggggccg ccagctctgt gcacccccag accagccctg ggtagaacgc 2520
atcatccaga gactgcagag gacctcagcc aagatgaagc gccgcagcag t 2571
Claims (8)
1.一种共同表达IL18和CCL19蛋白及靶向MUC1基因CAR-T细胞的制备方法，包括以下步骤：
准备CD4+/CD8+ T细胞；
准备共表达IL18和CCL19蛋白及靶向MUC1基因的嵌合抗原受体慢病毒；
将准备的慢病毒用于感染CD4+/CD8+ T细胞，antiCD3+CD28抗体激活T细胞扩增培养，通过荧光筛选实验以获得共表达IL18和CCL19蛋白及靶向MUC1基因的CAR-T细胞。
2.如权利要求1所述方法，其中CD4+/CD8+ T细胞的获得是通过EasySeq试剂盒按说明书操作，制备而成。
3.如权利要求1所述方法，其特征在于，所述表达的IL18和CCL19的蛋白序列由其核苷酸序列编码，其中IL18的核苷酸序列如SEQ ID No.1所示，CCL19的核苷酸序列如SEQ No.2所示。
4.如权利要求1所述方法，其特征在于，所述表达的IL18和CCL19蛋白都为单独表达蛋白，羧基端由一个T2A短肽核苷酸序列连接，序列如SEQ No.3所示。
5.如权利要求1所述的方法，其中共表达IL18和CCL19靶向MUC1的CAR是利用pCDH载体串联人EF1α启动子、CSF2RA嵌合受体信号肽、胞外抗体结合区、铰链区、跨膜区胞内信号传导区和T2A短肽连接的筛选基因CopGFP制备而成，其中CSF2RA-MUC1 scFv-CD8α-OX40-4-1BB-CD3ζ-IL18-CCL19核苷酸序列如SEQ No.4所示。
6.如权利要求5所述的方法，其中胞外抗原结合区为MUC1单链抗体scFv HMFG2，铰链区为CD8 Hinge，跨膜区为CD8α跨膜域，信号传导区为OX40-4-1BB-CD3ζ-IL18-CCL19，其中OX40和4-1BB为共刺激因子，IL18和CCL19为第三信号因子。
7.一种共表达IL18和CCL19蛋白及靶向MUC1基因CAR-T细胞的制备方法，包括
CD4+/CD8+细胞准备系统；
IL18和CCL19蛋白的共表达系统；
用于激活T细胞的偶联CD3+CD28抗体磁珠；以及
筛选培养系统，用于筛选并培养转后的T细胞用以获得共同表达IL18及CCL19的MUC1CAR-T细胞。
8.如权利要求1～7之一所述的共表达IL18和CCL19蛋白靶向CAR-T的方法，以及获得的分离的T细胞或细胞系或其次代培养物。
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