CN106749675B - 一种重组慢病毒及其应用 - Google Patents
一种重组慢病毒及其应用 Download PDFInfo
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Abstract
本发明涉及肿瘤的细胞免疫治疗领域，尤其涉及一种重组慢病毒及其应用，所述重组慢病毒包含嵌合抗原受体，所述嵌合抗原受体主要包括信号肽、抗原识别区、跨膜区、胞内共刺激信号传导域和CD3ζ信号传导域串联构成；其中，所述胞内共刺激信号传导域主要包括人TLR2胞内区。本发明制备的GPC3 CAT T细胞对肝癌细胞具有强烈的细胞杀伤效应，且高表达Th1细胞因子，极大程度刺激非CAR T细胞引起的肿瘤杀伤效应，可有效防止GPC3‑肿瘤细胞的逃逸和潜在复发威胁，且表达此嵌合抗原受体的T细胞能最大程度的杀伤肿瘤细胞，对正常组织的伤害程度很小，能突破肿瘤免疫抑制微环境，从而对实体瘤具有更好的疗效。
Description
技术领域
本发明涉及肿瘤的细胞免疫治疗领域，尤其涉及一种重组慢病毒及其应用，具体为以肿瘤特异靶点GPC3为基础的嵌合抗原受体T(CAR-T)细胞技术的构建方法及其在抗肿瘤治疗中的应用。
背景技术
GPC3(Glypican-3，磷脂酰肌醇蛋白聚糖3)基因定位于人染色体X26.10，参与了细胞分裂、生长、发育以及细胞对生长因子的反应等生命过程。GPC3的功能预期在生物体内的分布有密切关系，在人体的胚胎期，GPC3在胎盘、胚胎肝、胚胎肺和胚胎肾中都有表达。在成人正常组织中，在脑、肝脏、脾脏、胃、肠等均不表达，仅在卵巢、肺部及肾脏等部位有极低水平的表达(Hsu HC et al.,1997Cancer Reseach)。遗传性的GPC3基因突变缺失，Hedgehog信号通路失去负面调控，引起Simpson-Golabi-Behmel(SGBS)综合征(Capurro MI et al.,2008Dev Cell)。SGBS综合征是一种X染色体连锁遗传病，主要特征是巨舌、多指(趾)、肋骨及脊柱畸形、肌张力低下等，同时胚胎性肿瘤发生的危险性增加。
研究报道，GPC3在肝癌(Capurro M et al.,2003Gastroenterology)、肾细胞癌(Valsechi MC et al.,2014BMC Cancer)、卵巢癌(Maeda D et al.,2009Mod Pathol)、肺癌(Lin Q et al.,2012Mod Pathol)、黑色素瘤(Nakatsura T et al.,2004 Clin CancerRes)中特异性高表达。GPC3为肿瘤相关抗原，通过上调Wnt信号通路的自分泌和旁分泌产物，刺激肝癌细胞生长(Lai JP et al.,2010 Hepatology)。同时，GPC3还是一个潜在的肿瘤抑制基因。在卵巢癌细胞系中，GPC3基因表达常常会被甲基化沉默(Lin H et al.,1999Cancer Res)。通过基因表达分析，发现GPC3在肺腺癌(相对于正常肺癌细胞)、吸烟人群(相对于非吸烟人群)肺组织中高表达(Kim H et al.,2003Am J Respir Cell MolBiol)。GPC3在胰腺导管腺癌中高表达(相对于正常胰腺组织)，与肿瘤发生、发展、预后有关，是胰腺导管腺癌诊断的候选标记物(Yao H et al.,2016Cancer Biomark)。
作为检出早期肝癌的分子标志物，GPC3在肝癌中特异性呈高表达状态，在不典型增生结节及其它肿瘤中检测不到；且敏感性高，能够在肝癌发生的早期被检出。GPC3-AS1(The lncRNA glypican 3antisense transcript 1)，即GPC3反义转录子非编码长RNA，在肝癌中显著高表达，且其表达水平与GPC3表达、肿瘤生长、血管浸润、预后等有关联(Zhu XTet al.,2016FEBS J)。
GPC3参与肿瘤组织中的炎症反应，GPC3可上调F4/80+CD86+巨噬细胞数量，引起GPC3特异性CD8+T细胞免疫反应，延长小鼠生存期(Luo C et al.,2014Oncol Rep)。通过GPC3多肽疫苗，激活CTL细胞对GPC3高表达肝癌细胞的杀伤作用，治疗肝癌病人处于二期临床试验(Sawada Y et al.,2016Oncoimmunology)。临床研究报道，可利用GPC3特异性树突状细胞疫苗治疗肝癌，激活GPC3+肝癌细胞的识别杀伤(Tada F et al.,2012Int JOncol)。相对于共刺激因子组合CD28、CD28联合41BB，GPC3-41BB嵌合抗原受体T细胞具有更强的扩增和抗肿瘤潜能(Li W et al.,2016Hum Gene Ther)。
现有技术中以GPC3为肿瘤的靶点，主要通过GPC3肽段疫苗、特异性靶向GPC3过继性免疫、嵌合抗原受体T细胞等。而GPC3疫苗主要在肿瘤预防和肿瘤早期可起到一定的作用，而往往肝癌确诊的时候都是肝癌中晚期，GPC3疫苗往往效果不明显。GPC3抗原特异性的DC-CIK免疫细胞对肿瘤细胞的体内识别杀伤效果比较低，免疫细胞活性较弱。
CN 102180969 A公开了一种抗肝癌活性单克隆抗体及其应用。该单克隆抗体，是可以与GPC3抗原表位特异性结合的单克隆抗体；所述GPC3抗原表位的氨基酸序列为自GenBank accession Number为AAA98132.1的氨基端端第359-580位氨基酸残基。该发明制备了高效价、高特异、且具有免疫活性、并且对肿瘤细胞具有毒杀活性的鼠抗人GPC3-C单克隆抗体。GPC3单克隆抗体可有效抑制肝癌细胞生长和迁移，然而，GPC3抗体容易形成耐药，且能导致GPC3阴性肝癌细胞逃逸，埋下肿瘤复发的威胁。
CN 105949324 A公开了靶向GPC3的嵌合抗原受体及其用途，该嵌合抗原受体含有依次连接的CD8抗原的前导肽、抗GPC3单链抗体、人CD8α铰链区、人CD8α跨膜区、人41BB胞内区、人CD3ζ胞内区和任选的EGFR序列的含胞外结构域III和胞外结构域IV的片段。该嵌合抗原靶向效果并不十分理想，且肿瘤微环境会影响其治疗效果。
发明内容
针对目前CAR-T技术治疗肿瘤中靶向不十分理想，以及肿瘤微环境影响CAR-T技术治疗效果的情况，本发明提供一种重组慢病毒及其应用，本发明制备的GPC3CAT T细胞对肝癌细胞具有强烈的细胞杀伤效应，且高表达Th1细胞因子，极大程度刺激非CAR T细胞引起的肿瘤杀伤效应，可有效防止GPC3-肿瘤细胞的逃逸和潜在复发威胁。
为达此目的，本发明采用以下技术方案：
一方面，本发明提供一种嵌合抗原受体，所述嵌合抗原受体主要包括信号肽、抗原识别区、跨膜区、胞内共刺激信号传导域和CD3ζ信号传导域串联构成；
其中，所述胞内共刺激信号传导域主要包括人TLR2胞内区。
本发明中，通过在GPC3 CAR分子中引入人TLR2(TOLL LIKE RECEPTOR2)胞内信号域，构建成的GPC3 CAR T细胞，发现其免疫细胞因子分泌偏向于Th1类型，对肝癌细胞具有强烈的细胞杀伤效应，且高表达Th1细胞因子，极大程度刺激非CAR T细胞引起的肿瘤杀伤效应，可有效防止GPC3-肝癌细胞的逃逸和潜在复发威胁。
根据本发明，所述人TLR2胞内区的氨基酸序列如SEQ ID NO.1所示，所述人TLR2胞内区的核酸序列如SEQ ID NO.2所示。
所述人TLR2胞内区的氨基酸序列(SEQ ID NO.1)如下：
QAKRKPRKAPSRNICYDAFVSYSERDAYWVENLMVQELENFNPPFKLCLHKRDFIPGKWIIDNIIDSIEKSHKTVFVLSENFVKSEWCKYELDFSHFRLFDENNDAAILILLEPIEKKAIPQRFCKLRKIMNTKTYLEWPMDEAQREGFWVNLRAAIKS.
所述人TLR2胞内区的核酸序列(SEQ ID NO.2)如下：
5-CAGGCCAAAAGGAAGCCCAGGAAAGCTCCCAGCAGGAACATCTGCTATGATGCATTTGTTTCTTACAGTGAGCGGGATGCCTACTGGGTGGAGAACCTTATGGTCCAGGAGCTGGAGAACTTCAATCCCCCCTTCAAGTTGTGTCTTCATAAGCGGGACTTCATTCCTGGCAAGTGGATCATTGACAATATCATTGACTCCATTGAAAAGAGCCACAAAACTGTCTTTGTGCTTTCTGAAAACTTTGTGAAGAGTGAGTGGTGCAAGTATGAACTGGACTTCTCCCATTTCCGTCTTTTTGATGAGAACAATGATGCTGCCATTCTCATTCTTCTGGAGCCCATTGAGAAAAAAGCCATTCCCCAGCGCTTCTGCAAGCTGCGGAAGATAATGAACACCAAGACCTACCTGGAGTGGCCCATGGACGAGGCTCAGCGGGAAGGATTTTGGGTAAATCTGAGAGCTGCGATAAAGTCC-3.
本发明的嵌合抗原受体还可以包括铰链区，所述铰链区本领域技术人员可以根据实际情况进行选择，在此不做特殊限定，铰链区的存在不会对本发明的嵌合抗原受体的性能产生影响。
根据本发明，过表达特异性靶向GPC3的嵌合抗原受体(Chimeric AntigenReceptor，CAR)T细胞，可特异性识别GPC3+肿瘤抗原，杀伤肿瘤细胞，具有显著的体内外扩增和肿瘤杀伤作用，所述抗原识别区为特异性识别结合GPC3抗原的区域，优选为抗GPC3抗体胞外段。
根据本发明，所述信号肽为能够指导嵌合抗原受体跨膜转移的信号肽都是可行的，本领域技术人员可以根据需要选择本领域常规的信号肽，所述信号肽为人IgM信号肽或人CD8α信号肽，优选为人IgM信号肽。
优选地，所述的跨膜区为人CD28跨膜区或人CD8跨膜区，优选为人CD28跨膜区。
根据本发明，所述胞内共刺激信号传导域除了含有人TLR2胞内区以外，还包括人CD28胞内区、人4-1BB胞内区、人CD27胞内区、人CD28胞内区、人OX40胞内区、人CD30胞内区、人CD40胞内区、人PD-1胞内区、人ICOS胞内区、人淋巴细胞功能相关抗原1胞内区、人CD2胞内区、人CD7胞内区、人LIGHT胞内区、人NKG2C胞内区、人B7-H3胞内区或人CD83胞内区中的任意一种或至少两种的组合，所述组合例如可以是，优选为人4-1BB胞内区或CD28胞内区，在本发明中，所述完整的胞内共刺激信号传导域为人CD28胞内区或人4-1BB胞内区和人TLR2胞内区串联构成。
作为优选技术方案，所述嵌合抗原受体由人IgM信号肽、抗GPC3抗体、人CD28跨膜区、人4-1BB胞内区、人TLR2胞内区和人CD3ζ信号传导域串联构成，即IgM-GPC3-4-1BB-CD3ξ-TLR2(GTBBξ)；嵌合抗原受体由人IgM信号肽、抗GPC3抗体、人CD28跨膜区和胞内区、人TLR2胞内区和人CD3ζ信号传导域串联构成，即IgM-GPC3-CD28-CD3ξ-TLR2(GT28ξ)。
发明人发现，所述嵌合抗原受体由人IgM信号肽、抗GPC3抗体、人CD28跨膜区、人CD28或人4-1BB胞内区、人TLR2胞内区和人CD3ζ信号传导域串联构成，其对肿瘤的识别杀伤效果最好，可靶向GPC3-肿瘤细胞，有效抑制肝癌生长。
优选地，所述嵌合抗原受体IgM-GPC3-CD28-CD3ξ-TLR2的氨基酸序列如SEQ IDNO.3所示，所述嵌合抗原受体GT28ζ基因合成序列的氨基酸序列(SEQ ID NO.3)如下：
MLLLVTSLLLCELPHPAFLLIPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMHWVKQTPVHGLKWIGALDPKTGDTAYSQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRFYSYTYWGQGTLVTVSAAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQAKRKPRKAPSRNICYDAFVSYSERDAYWVENLMVQELENFNPPFKLCLHKRDFIPGKWIIDNIIDSIEKSHKTVFVLSENFVKSEWCKYELDFSHFRLFDENNDAAILILLEPIEKKAIPQRFCKLRKIMNTKTYLEWPMDEAQREGFWVNLRAAIKS.
优选地，所述嵌合抗原受体优选地，所述嵌合抗原受体IgM-GPC3-41BB-CD3ξ-TLR2的氨基酸序列如SEQ ID NO.4所示，所述嵌合抗原受体GTBBζ基因合成序列的的氨基酸序列(SEQ ID NO.4)如下：
MLLLVTSLLLCELPHPAFLLIPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMHWVKQTPVHGLKWIGALDPKTGDTAYSQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRFYSYTYWGQGTLVTVSAAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQAKRKPRKAPSRNICYDAFVSYSERDAYWVENLMVQELENFNPPFKLCLHKRDFIPGKWIIDNIIDSIEKSHKTVFVLSENFVKSEWCKYELDFSHFRLFDENNDAAILILLEPIEKKAIPQRFCKLRKIMNTKTYLEWPMDEAQREGFWVNLRAAIKS.
优选地，所述的嵌合抗原受体通过其编码的核酸序列转染到T细胞中表达。
优选地，所述转染的方式为通过病毒载体、真核表达质粒或mRNA序列中的任意一种或至少两种的组合转染到T细胞，优选为通过病毒载体转染到T细胞。
优选地，所述病毒载体为慢病毒载体、腺病毒载体或逆转录病毒载体中的任意一种或至少两种的组合，优选为慢病毒载体。
第二方面，本发明提供一种重组慢病毒，将包含如第一方面所述的嵌合抗原受体的病毒载体与包装辅助质粒pMD2.G和psPAX2共转染哺乳细胞得到的重组慢病毒。
第三方面，本发明提供一种组合物，所述组合物包括如第一方面所述的嵌合抗原受体和/或如第二方面所述的重组慢病毒。
第四方面，本发明提供如第一方面所述的嵌合抗原受体、如第二方面所述的重组慢病毒或如第三方面所述的组合物在制备嵌合抗原受体T细胞及其在肿瘤治疗药物中的应用。
优选地，所述肿瘤为GPC3阳性肿瘤。
根据本发明，所述嵌合抗原受体可特异性识别GPC3+肿瘤抗原，含有GPC3+肿瘤抗原的肿瘤细胞都可被识别和治疗，所述GPC3阳性肿瘤包括肝癌、卵巢癌、肺癌、黑色素瘤或肾细胞癌中的任意一种或至少两种的组合，其中，所述卵巢癌、肺癌、黑色素瘤或肾细胞癌中都含有GPC3+肿瘤抗原，其能够被所述嵌合抗原受体识别，其机理与肝癌类似，所述嵌合抗原受体也能够治疗卵巢癌、肺癌、黑色素瘤或肾细胞癌，所述GPC3阳性肿瘤优选为肝癌。
与现有技术相比，本发明具有如下有益效果：
(1)本发明的嵌合抗原受体具有高效肿瘤识别杀伤效应，可激活非CAR T细胞，体内T、NK、巨噬细胞等肿瘤免疫相关细胞的肿瘤免疫效应，靶向GPC3-肿瘤细胞，防治肿瘤免疫逃逸和复发威胁；
(2)本发明的嵌合抗原受体偏向于Th1型细胞因子分泌，可正反馈激活CAR T肿瘤免疫效应，还可在一定程度上减弱肿瘤微环境的肿瘤免疫抑制；
(3)本发明表达此嵌合抗原受体的T细胞能最大程度的杀伤肿瘤细胞，对正常组织的伤害程度很小，能突破肿瘤免疫抑制微环境，从而对实体瘤具有更好的疗效。
附图说明
图1为本发明实施例1中的嵌合抗原受体的合成基因序列图谱；
图2为本发明CAR GT28ξ慢病毒感染效率和CAR T细胞扩增；
图3本发明CAR GT28ξT细胞体外有效识别杀伤GPC3+肝癌细胞，其中，图3(A)为针对GPC3+的Huh7-GL肝癌细胞，图3(B)为针对GPC3+的HepG2-GL肝癌细胞，
为Mock-CarT，
为GPC3-Car T；
图4为本发明CAR-GT28ξT细胞的安全性验证，，
为Mock-Car T，
为GPC3-Car T；
图5为本发明CAR GT28ξT细胞体外抑制GPC3+肝癌生长，其中，图5(A)为GPC3+的Huh7-GL肝癌细胞，图5(B)为GPC3+的HepG2-GL肝癌细胞，
为Mock-Car T，
为GPC3-Car T；
图6为本发明GPC3抗原特异性激活的GT28ξCAR T细胞分泌Th1型细胞因子，其中，图6(A)为分泌的IL-2因子，图6(B)为分泌的INF-γ因子，“□”为Mock-Car T，“■”为GPC3-Car T；
图7为本发明CAR-GTBBξ、CAR-GT28ξ、CAR-G28BBξT细胞对GPC3+的HepG2-GL肝癌细胞的特异性识别杀伤。
具体实施方式
为更进一步阐述本发明所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本发明的技术方案，但本发明并非局限在实施例范围内。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1：嵌合抗原受体慢病毒载体的构建
(1)通过全基因合成人IgM信号肽、抗GPC3抗体胞外段、人CD28跨膜区、人41BB胞内区、人TLR2胞内区和人CD3ζ信号传导域，即CAR-GPC3-41BB-CD3ξ-TLR2(CAR-GTBBξ)，全基因合成人IgM信号肽、抗GPC3抗体胞外段、人CD28跨膜区和胞内区、人TLR2胞内区和人CD3ζ信号传导域，即CAR-GPC3-CD28-CD3ξ-TLR2(CAR-GT28ξ)，其序列如SEQ ID NO.4所示，再全基因合成CAR-CD19-CD28-CD3ξ-TLR2(Mock)核酸序列作为阴性对照，CAR-GPC3-41BB-CD28-CD3ξ(CAR-G28BBξ)核酸序列作为阳性对照其基因序列图谱如图1所示，合成的基因C端含限制性内切酶Pme1酶切位点及其保护碱基和N端含限制性内切酶Spe1酶切位点及其保护碱基；
(2)分别通过限制性内切酶Pme1和Spe1双酶切，凝胶电泳回收获得含粘性末端的合成DNA片段CAR-GTBBξ、CAR-GT28ξ、CAR-G28BBξ和Mock，将CAR-GTBBξ、CAR-GT28ξ、CAR-G28BBξ、Mock和含粘性末端的pWPXLd-eGFP慢病毒载体的线性化DNA；
(3)通过T4DNA连接酶(Invitrogent公司)，将线性化的pWPXLd-2A-EGFP与带粘性末端的合成的DNA片段CAR-GTBBξ、CAR-GT28ξ、Mock进行连接，获得CAR质粒转化载体pWPXLd-CAR-GTBBξ-2A-EGFP、pWPXLd-CAR-GT28ξ-2A-EGFP、pWPXLd-CAR-G28BBξ-2A-EGFP、pWPXLd-Mock-2A-EGFP。
实施例2：GT28ξCAR慢病毒包装
(1)在10cm培养皿中培养293T细胞，培养基为：DMEM高糖培养基+10％FBS(胎牛血清)+1％双抗(100×青霉素-链霉素混合溶液)；
(2)待150mm培养皿中的293T细胞密度达80-90％时，更换培养基：DMEM高糖培养基+1％FBS+1％双抗；
(3)更换培养基培养2-6小时后，用PEI分别将pWPXLd-CAR-EGFP质粒(pWPXLd-CAR-GTBBξ-2A-EGFP、pWPXLd-CAR-GT28ξ-2A-EGFP、pWPXLd-CAR-G28BBξ-2A-EGFP、pWPXLd-Mock-2A-EGFP)分别与慢病毒包装辅助质粒pMD2.G、psPAX2共同转导入293T细胞，加入试剂及剂量如下：
| 试剂 | 剂量 |
| pWPXLd-CAR-EGFP质粒 | 9μg |
| pMD2.G辅助质粒 | 3μg |
| psPAX2 | 12μg |
| PEI | 72μg |
(4)分别于转化后24、48和72小时，收集培养基上清，并加入新鲜培养基：DMEM高糖培养基+1％FBS+1％双抗；
(5)培养基上清收集完毕，将上清2500g离心0.5小时后；
(6)取离心上清，用0.45um过滤器过滤后，利用超高速离心机28000rpm离心1.5小时；
(7)超高速离心后，轻轻去除上清，加入200ul PBS，置于4℃12-16小时溶解，即得CAR慢病毒；
(8)病毒溶解后，收集病毒溶液分装于PCR管，冻存于-80℃待用。
实施例3：CAR T细胞的构建
(1)人体T细胞的分离纯化：通过Ficoll密度梯度法分离出人体血液中的单个核细胞，经红细胞裂解液裂解去除红细胞后，再通过MACS Pan-T磁珠分选出T细胞；
(2)将分选出来的T细胞用培养基：AIM-V培养基+5％FBS+青霉素100U/ml+链霉素0.1mg/ml稀释至细胞浓度2.5×106个/mL待用；
(3)通过包被CD2、CD3、CD28抗体的磁珠(德国美天旎)刺激T细胞，即包被磁珠与T细胞以1:2比例混合，T细胞最终密度应为5×106个/mL/cm2。混合后，置于37℃、5％CO2培养箱培养刺激48小时；
(4)慢病毒转染T细胞：将激活的T细胞-磁珠混合液中的磁珠通过磁场作用去除，300g离心5min，去上清，用新鲜培养基重悬，分别加入表达CAR和GFP(空白对照)慢病毒(病毒加入量为MOI＝10)后，加入8μg/mL的polybrene和300IU/mL IL-2，置于37℃，5％CO2培养箱培养24h后，300g离心5min，去上清，用含300IU/mL IL-2的新鲜培养基重悬，即得过表达CAR质粒的T细胞；
(5)CAR T细胞扩增：将CAR T细胞密度维持在1×106个/mL左右，每2-3天进行一次半量换液，两周后，CAR T细胞数可扩增100倍，GFP阳性的细胞为转染成功的细胞，GFP阳性比例通过流式进行检测，即得到CAR-GTBBξT、CAR-GT28ξT、CAR-G28BBξT、Mock T细胞或阴性对照(未经处理)T胞的比例，结果如图2所示，可以看出加入TLR2胞内区的嵌合抗原受体T细胞具有较高的转染率，且可促进T细胞更好的扩增。
实施例4：其他胞内共刺激信号传导域的CAR T细胞的构建
(1)通过全基因合成人CD8α信号肽、抗GPC3抗体胞外段、人CD28跨膜区、胞内共刺激信号传导域、人TLR2胞内区和人CD3ζ信号传导域，其中胞内共刺激信号传导域为人CD27胞内区、人CD28胞内区、人OX40胞内区、人CD30胞内区、人CD40胞内区、人PD-1胞内区、人ICOS胞内区、人淋巴细胞功能相关抗原1胞内区、人CD2胞内区、人CD7胞内区、人LIGHT胞内区、人NKG2C胞内区、人B7-H3胞内区或人CD83中的任意一种，共合成14个嵌合抗原受体，合成的基因C端含限制性内切酶Pme1酶切位点及其保护碱基和N端含限制性内切酶Spe1酶切位点及其保护碱基；
(2)分别通过限制性内切酶Pme1和Spe1双酶切，凝胶电泳回收获得含粘性末端的合成DNA片段和含粘性末端的pWPXLd-eGFP慢病毒载体的线性化DNA；
(3)通过T4 DNA连接酶(Invitrogent公司)，将线性化的pWPXLd-2A-EGFP与带粘性末端的合成的DNA片段连接，获得CAR质粒转化载体。
慢病毒的包装步骤同实施例2，CAR T细胞的构建步骤同实施例3，可以制备得到不同胞内共刺激信号传导域的14个CAR T细胞。
实施例5：CAR-GT28ξT细胞的体外肿瘤杀伤
(1)将实施例2制备的CAR-GT28ξT、Mock T细胞与的GPC3+肝癌细胞分别以1：3、1：1、3：1、5：1比例混合，加入到96孔U型板中，每组设3个复孔，250g离心5min后，置于37度5％CO2培养箱共培养18h；
(2)体外评估CAR-GT28ξT细胞对GPC3+肝癌细胞的识别杀伤功能，肿瘤细胞分别选用Huh7-GL、HepG2-GL带荧光素酶的人肝癌细胞系；
(3)荧光素酶(Luciferase)定量杀伤效率评估方法：CAR T细胞与肿瘤细胞共培养(实验对照组为肿瘤细胞单独培养)后18小时，在96孔细胞培养板中加入100μl/孔的荧光素酶底物(1×)，将细胞重悬混匀，立即通过多功能酶标仪测定RLU(relative light unit)，测定时间设为1秒。
杀伤比例计算公式：100％×(对照孔读数-实验孔读数)/对照孔读数(不加细胞的空白组读数可以忽略)；
其结果如图3(A)-图3(B)所示，结果表明，CAR-GT28ξT细胞对GPC3+的Huh7-GL、HepG2-GL肝癌细胞具有显著的体外杀伤效率。
CAR-G28BBξT细胞对GPC3+的Huh7-GL、HepG2-GL肝癌细胞的杀伤效果与CAR-GT28ξT细胞类似，而选择其他胞内共刺激信号传导域的14个CAR-T细胞虽然对GPC3+的Huh7-GL、HepG2-GL肝癌细胞具有杀伤效果，但效果不如CAR-G28BBξT细胞和CAR-GT28ξT细胞。
实施例6：CAR T细胞安全性试验
(1)将实施例2制备的CAR-GT28ξT、Mock T细胞与GPC3-肿瘤细胞分别以1：3、1：1、3：1、5：1比例混合，加入到96孔U型板中，每组设3个复孔，250g离心5min后，置于37度5％CO2培养箱共培养18h；
(2)体外评估CAR-GT28ξT细胞对GPC3-肿瘤细胞的非特异性识别杀伤作用，GPC3-肿瘤细胞分别选用A549-GL带荧光素酶的人肺腺癌细胞系；
(3)荧光素酶(Luciferase)定量杀伤效率评估方法：CAR T细胞与肿瘤细胞共培养(实验对照组为肿瘤细胞单独培养)后18小时，在96孔细胞培养板中加入100μl/孔的荧光素酶底物(1×)，将细胞重悬混匀，立即通过多功能酶标仪测定RLU(relative light unit)，测定时间设为1秒。
杀伤比例计算公式：100％×(对照孔读数-实验孔读数)/对照孔读数(不加细胞的空白组读数可以忽略)；
其结果如图4所示，结果表明，CAR-GT28ξT细胞对GPC3-的A549-GL肺癌细胞没有非特异性杀伤效应。
CAR-G28BBξT细胞对GPC3-的A549-GL肺癌细胞也没有非特异性杀伤效应。
实施例7：CAR GT28ξT细胞的体内肿瘤杀伤
(1)将细胞数为1×105的GPC3+肝癌细胞移植入NOD/SCID IL2rg-/-免疫缺陷小鼠体内，构建肝癌异种移植小鼠模型；
(2)体内评估CAR-GT28ξT细胞对GPC3+肝癌细胞生长的抑制作用，肿瘤细胞分别选用Huh7-GL、HepG2-GL带荧光素酶的人肝癌细胞系；
(3)肿瘤移植7、14天后，分别在肝癌异种移植小鼠模型中静脉注射细胞数为2×106的CAR-GT28ξT、Mock T细胞，共两个实验组，每组设置五个重复；
(3)于肿瘤移植后第7、10、13、16、19、21天，用游标卡尺分别量取两个实验组小鼠皮下肿瘤块大小，并记录，绘制肿瘤生长曲线图；
结果如图5(A)-图5(B)所示，结果表明，CAR-GT28ξT细胞在体内可识别杀伤GPC3+肝癌细胞Huh7-GL、HepG2-GL，有效抑制肝癌生长。
CAR-G28BBξT细胞也可在体内可识别杀伤GPC3+肝癌细胞Huh7-GL、HepG2-GL，有效抑制肝癌生长。
实施例8：激活的CAR GT28ξT细胞细胞因子分泌
(1)将实施例2制备的CAR-GT28ξT、Mock T细胞(阴性对照，非特异性靶向GPC3)与的GPC3+肝癌细胞分别以1：1比例混合，加入到96孔U型板中，每组设3个复孔，250g离心5min后，置于37度5％CO2培养箱共培养18h；
(2)细胞因子分泌评估中GPC3+肿瘤细胞分别选用Huh7-GL、HepG2-GL带荧光素酶的人肝癌细胞系，GPC3-肿瘤细胞选用A549-GL带荧光素酶的人肺癌细胞系；
(3)共培养18h后，取不同实验组的共培养上清，通过ELISA检测上清中的Th1型细胞因子(IL2和IFNγ)的表达水平；
结果如图6(A)-图6(B)所示，结果表明，CAR-GT28ξT细胞在GPC3抗原的特异性激活下，细胞因子IL-2、IFNγ的表达水平显著高于Mock T细胞。
实施例9：不同胞内区的CAR-GPC3 T细胞的体外肿瘤杀伤
(1)将实施例2制备的CAR-GTBBξ、CAR-GT28ξ、CAR-G28BBξ(阳性对照)、Mock(阴性对照)T细胞与的GPC3+肝癌细胞分别以1：3、1：1、3：1比例混合，加入到96孔U型板中，每组设3个复孔，250g离心5min后，置于37度5％CO2培养箱共培养18h；
(2)体外评估CAR-GT28ξT细胞对GPC3+肝癌细胞的识别杀伤功能，肿瘤细胞分别选用HepG2-GL带荧光素酶的人肝癌细胞系；
(3)荧光素酶(Luciferase)定量杀伤效率评估方法：CAR T细胞与肿瘤细胞共培养(实验对照组为肿瘤细胞单独培养)后18小时，在96孔细胞培养板中加入100μL/孔的荧光素酶底物(1×)，将细胞重悬混匀，立即通过多功能酶标仪测定RLU(relative light unit)，测定时间设为1秒。
杀伤比例计算公式：100％×(对照孔读数-实验孔读数)/对照孔读数(不加细胞的空白组读数可以忽略)；
其结果如图7所示，结果表明，对HepG2的杀伤效果CAR-GTBBξ>CAR-GT28ξ>CAR-G28BBξ，相对于对照组Mock有明显的杀伤作用，表明共刺激信号分子TLR2胞内区可有效提高嵌合抗原受体T细胞对肿瘤细胞的特异性识别杀伤作用。
综上所述，本发明的嵌合抗原受体偏向于Th1型细胞因子分泌，可正反馈激活CART肿瘤免疫效应，还可在一定程度上减弱肿瘤微环境的肿瘤免疫抑制，本发明表达此嵌合抗原受体的T细胞能最大程度的杀伤肿瘤细胞，对正常组织的伤害很小，能突破肿瘤免疫抑制微环境，从而对实体瘤具有更好的疗效。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 深圳市体内生物医药科技有限公司
<120> 一种重组慢病毒及其应用
<130> 2016
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<170> PatentIn version 3.3
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<213> 人工合成序列
<400> 2
caggccaaaa ggaagcccag gaaagctccc agcaggaaca tctgctatga tgcatttgtt 60
tcttacagtg agcgggatgc ctactgggtg gagaacctta tggtccagga gctggagaac 120
ttcaatcccc ccttcaagtt gtgtcttcat aagcgggact tcattcctgg caagtggatc 180
attgacaata tcattgactc cattgaaaag agccacaaaa ctgtctttgt gctttctgaa 240
aactttgtga agagtgagtg gtgcaagtat gaactggact tctcccattt ccgtcttttt 300
gatgagaaca atgatgctgc cattctcatt cttctggagc ccattgagaa aaaagccatt 360
ccccagcgct tctgcaagct gcggaagata atgaacacca agacctacct ggagtggccc 420
atggacgagg ctcagcggga aggattttgg gtaaatctga gagctgcgat aaagtcc 477
<210> 3
<211> 645
<212> PRT
<213> 人工合成序列
<400> 3
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Val Val Met Thr Gln Thr Pro Leu Ser
20 25 30
Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser
35 40 45
Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
50 55 60
Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn
65 70 75 80
Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
85 90 95
Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val
100 105 110
Tyr Phe Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Ser Gly
115 120 125
Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
145 150 155 160
Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Thr Phe Thr Asp Tyr Glu Met His Trp Val Lys Gln Thr Pro Val His
180 185 190
Gly Leu Lys Trp Ile Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
195 200 205
Tyr Ser Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln
245 250 255
Gly Thr Leu Val Thr Val Ser Ala Ala Ala Ala Ile Glu Val Met Tyr
260 265 270
Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His
275 280 285
Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser
290 295 300
Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr
305 310 315 320
Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys
325 330 335
Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg
340 345 350
Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp
355 360 365
Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
370 375 380
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
385 390 395 400
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
405 410 415
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
420 425 430
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
435 440 445
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
450 455 460
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
465 470 475 480
Gln Ala Leu Pro Pro Arg Gln Ala Lys Arg Lys Pro Arg Lys Ala Pro
485 490 495
Ser Arg Asn Ile Cys Tyr Asp Ala Phe Val Ser Tyr Ser Glu Arg Asp
500 505 510
Ala Tyr Trp Val Glu Asn Leu Met Val Gln Glu Leu Glu Asn Phe Asn
515 520 525
Pro Pro Phe Lys Leu Cys Leu His Lys Arg Asp Phe Ile Pro Gly Lys
530 535 540
Trp Ile Ile Asp Asn Ile Ile Asp Ser Ile Glu Lys Ser His Lys Thr
545 550 555 560
Val Phe Val Leu Ser Glu Asn Phe Val Lys Ser Glu Trp Cys Lys Tyr
565 570 575
Glu Leu Asp Phe Ser His Phe Arg Leu Phe Asp Glu Asn Asn Asp Ala
580 585 590
Ala Ile Leu Ile Leu Leu Glu Pro Ile Glu Lys Lys Ala Ile Pro Gln
595 600 605
Arg Phe Cys Lys Leu Arg Lys Ile Met Asn Thr Lys Thr Tyr Leu Glu
610 615 620
Trp Pro Met Asp Glu Ala Gln Arg Glu Gly Phe Trp Val Asn Leu Arg
625 630 635 640
Ala Ala Ile Lys Ser
645
<210> 4
<211> 687
<212> PRT
<213> 人工合成序列
<400> 4
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Val Val Met Thr Gln Thr Pro Leu Ser
20 25 30
Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser
35 40 45
Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
50 55 60
Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn
65 70 75 80
Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
85 90 95
Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val
100 105 110
Tyr Phe Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Ser Gly
115 120 125
Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
145 150 155 160
Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Thr Phe Thr Asp Tyr Glu Met His Trp Val Lys Gln Thr Pro Val His
180 185 190
Gly Leu Lys Trp Ile Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
195 200 205
Tyr Ser Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser
210 215 220
Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser
225 230 235 240
Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln
245 250 255
Gly Thr Leu Val Thr Val Ser Ala Ala Ala Ala Ile Glu Val Met Tyr
260 265 270
Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His
275 280 285
Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser
290 295 300
Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr
305 310 315 320
Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys
325 330 335
Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg
340 345 350
Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp
355 360 365
Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
370 375 380
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
385 390 395 400
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
405 410 415
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
420 425 430
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
435 440 445
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
450 455 460
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
465 470 475 480
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
485 490 495
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
500 505 510
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
515 520 525
Gln Ala Lys Arg Lys Pro Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr
530 535 540
Asp Ala Phe Val Ser Tyr Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn
545 550 555 560
Leu Met Val Gln Glu Leu Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys
565 570 575
Leu His Lys Arg Asp Phe Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile
580 585 590
Ile Asp Ser Ile Glu Lys Ser His Lys Thr Val Phe Val Leu Ser Glu
595 600 605
Asn Phe Val Lys Ser Glu Trp Cys Lys Tyr Glu Leu Asp Phe Ser His
610 615 620
Phe Arg Leu Phe Asp Glu Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu
625 630 635 640
Glu Pro Ile Glu Lys Lys Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg
645 650 655
Lys Ile Met Asn Thr Lys Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala
660 665 670
Gln Arg Glu Gly Phe Trp Val Asn Leu Arg Ala Ala Ile Lys Ser
675 680 685
Claims (11)
1.一种特异针对GPC3-肝癌的嵌合抗原受体，其特征在于，所述嵌合抗原受体是由人IgM信号肽、抗GPC3抗体、人CD28跨膜区和胞内区、人TLR2胞内区和人CD3ζ信号传导域串联构成的IgM-GPC3-CD28-CD3ξ-TLR2嵌合抗原受体，所述嵌合抗原受体IgM-GPC3-CD28-CD3ξ-TLR2的氨基酸序列如SEQ ID NO.3所示。
2.一种特异针对GPC3-肝癌的嵌合抗原受体，其特征在于，所述嵌合抗原受体是由人IgM信号肽、抗GPC3抗体、人CD28跨膜区、人41BB胞内区、人TLR2胞内区和人CD3ζ信号传导域串联构成的IgM-GPC3-41BB-CD3ξ-TLR2嵌合抗原受体，所述嵌合抗原受体IgM-GPC3-41BB-CD3ξ-TLR2的氨基酸序列如SEQ ID NO.4所示。
3.根据权利要求1或2所述的嵌合抗原受体，其特征在于，所述的嵌合抗原受体通过其编码的核酸序列转染到T细胞中表达。
4.根据权利要求3所述的嵌合抗原受体，其特征在于，所述转染的方式为通过病毒载体、真核表达质粒或mRNA序列中的任意一种或至少两种的组合转染到T细胞。
5.根据权利要求4所述的嵌合抗原受体，其特征在于，通过病毒载体转染到T细胞。
6.根据权利要求5所述的嵌合抗原受体，其特征在于，所述病毒载体为慢病毒载体、腺病毒载体或逆转录病毒载体中的任意一种或至少两种的组合。
7.根据权利要求6所述的嵌合抗原受体，其特征在于，所述病毒载体为慢病毒载体。
8.一种重组慢病毒，其特征在于，将包含如权利要求1-7中任一项所述的嵌合抗原受体的病毒载体与包装辅助质粒pMD2.G和psPAX2共转染哺乳细胞得到的重组慢病毒。
9.一种组合物，其特征在于，所述组合物包括如权利要求1-7中任一项所述的嵌合抗原受体和/或如权利要求8所述的重组慢病毒。
10.如权利要求1-7中任一项所述的嵌合抗原受体、如权利要求8所述的重组慢病毒或如权利要求9所述的组合物在制备嵌合抗原受体T细胞及其在肿瘤治疗药物中的应用。
11.根据权利要求10所述的应用，其特征在于，所述肿瘤为GPC3阳性肿瘤，所述GPC3阳性肿瘤为肝癌。
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| US20230071098A1 (en) * | 2018-07-17 | 2023-03-09 | Noile-Immune Biotech, Inc. | Anti-gpc3 single-chain antibody-containing car |
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| CN109913422A (zh) * | 2017-12-13 | 2019-06-21 | 苏州康聚生物科技有限公司 | 一种包含肿瘤抗原识别受体的免疫细胞及其应用 |
| CN107982538B (zh) * | 2017-12-26 | 2021-10-22 | 深圳市体内生物医药科技有限公司 | 一种药物组合物及其应用 |
| CN107904282A (zh) * | 2017-12-28 | 2018-04-13 | 北昊干细胞与再生医学研究院有限公司 | 检测pd‑1敲除的或低表达的t细胞对肿瘤细胞杀伤性的方法 |
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| CN112390894A (zh) * | 2019-08-12 | 2021-02-23 | 广东东阳光药业有限公司 | 嵌合抗原受体及其应用 |
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