CN108017716B - 包含C5aR胞内结构域的嵌合抗原受体、慢病毒载体、表达细胞及药物 - Google Patents
包含C5aR胞内结构域的嵌合抗原受体、慢病毒载体、表达细胞及药物 Download PDFInfo
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Abstract
本发明提供了一种包含C5aR胞内结构域的嵌合抗原受体、慢病毒载体、表达细胞及药物，所述包含C5aR胞内结构域的嵌合抗原受体，包含能够结合抗原的胞外结构域、跨膜结构域和至少一个胞内结构域，所述的至少一个胞内结构域是指C5aR胞内结构域，或与C5aR胞内结构域串联的信号传递区域的胞内结构域。本发明的嵌合抗原受体可以提高T17细胞亚群，消除调节T细胞的免疫抑制的作用，可以显著提高肿瘤靶细胞的体外杀伤效率，显著改善第二代CAR T细胞对肿瘤的杀伤效果，为CAR‑T细胞治疗领域提供一种新的思路和选择。
Description
技术领域
本发明属于肿瘤的细胞免疫治疗技术领域，具体涉及一种包含C5aR胞内结构域的嵌合抗原受体，还涉及包含编码C5aR胞内结构域的嵌合抗原受体的核酸和应用。
背景技术
嵌合抗原受体(CAR,Chimeric Antigen Receptors)T细胞是有望治愈肿瘤的里程碑事件,CAR-T技术是应用基因改造技术，将一段识别肿瘤抗原的单链抗体(single chainfragment variable,scFv)和胞内活化基序重组基因转染至T淋巴细胞上以达到更好的识别及杀伤肿瘤的作用。CAR-T分子通常包括胞外绞链区、跨膜区和胞内信号区。其中胞外绞链区是由单链抗体的重链和轻链可变区通过一条肽段连接而形成；跨膜区则主要来自于如CD8、CD28或4-1BB等分子的跨膜区；胞内信号区则主要来自于CD28、4-1BB、CD3zeta、CD27或OX-40等T细胞传导信号分子的胞内段嵌合体。高亲合力，高特异性的scFv决定CAR-T靶向某种抗原，一旦结合抗原，CAR的胞内段会传输活化以及共刺激信号给T细胞，激活T细胞进而有效靶向杀伤肿瘤细胞。T细胞的激活需要两种活化信号，即T细胞表面的TCR-CD3与MHC-I分子结合为活化的第一信号,决定T细胞对肿瘤细胞的杀伤活性；T细胞表面的共刺激分子与相应配体结合为活化的第二信号,决定T细胞增殖。简而言之,CAR-T细胞通过抗原-抗体识别模式对肿瘤细胞表面的特异分子进行识别,然后通过其胞内的信号传导进行激活、增殖并发挥细胞杀伤功能。
CAR分子的结构设计经历多代的研究发展。第一代CAR分子的结构包含识别肿瘤细胞表面抗原的scFv、跨膜结构域和激活T细胞的TCR复合物CD3zeta的胞内结构域。由于第一代CAR的信号域是单一信号分子，缺乏共刺激信号，第一代CAR-T细胞在体内存活时间短，对肿瘤细胞的杀伤力弱，疗效并不理想。为充分模拟人体内T淋巴细胞活化的双信号模型，增强第一代CAR分子激活T细胞的能力，开始出第二代CAR，第二代CAR主要是在胞内段结构域加入1个共刺激信号域，目前已经报导的共刺激配体主要集中在特异性结合T细胞上的关联共刺激分子的抗原呈递细胞，包括CD7、B7-1(CD80)、B7-2(CD86)、PD-L1、PD-L2、4-1BB、OX-40、ICOS-L、ICAM、CD30L、CD40、CD70、CD83、HLA-G、MICA、MICB、HEVM、淋巴毒素β受体、ILT3、ILT4、HVEM，进而增强其对肿瘤抗原的识别能力和T淋巴细胞杀伤力(MaQ,GomesEM,LoAS,etal.Advanced generation anti-prostate specific membrane antigen designer Tcells for prostate cancer immunotherapy[J].Prostate,2014,74(3)：286–296)。Savoldo等为6例B细胞淋巴瘤患者构建2种抗CD19-CAR-T细胞，一种CAR的信号域仅有单一信号分子ζ链(第一代CAR-T)，另一种则同时加入了ζ链和共刺激分子CD28(第二代CAR-T)，该研究体外培养及检测结果表明，在体外增殖、细胞因子分泌水平方面，含有CD28信号链的二代CAR-T细胞表现出显著优势。将这2种细胞同时回输入患者体内，定期检测细胞数量，结果表明，二代CAR-T细胞体内增殖速度及存活时间均优于一代CAR-T细胞(SavoldoB，RamosCA，LiuE，eta1.CD28costimulation improves expansion and persistence ofchimeric antigen receptor modified T cells in lymphoma patients[J].ClinInvest，2011，121(5)：1822–1826)。同样的研究也证实了含有CD137(4-1BB)信号链的二代CAR-T比一代CAR-T有更强的杀伤效应和持久性(Maude SL,Frey N,Shaw PA,etal.Chimeric antigen receptor T cells for sustained remissions in Leukemia.NEngL J M.2014；371:1507-1517)。虽然CAR-T细胞临床试验获得了疗效,但还存在进一步改善的空间，如进一步增强其激活、细胞杀伤功能，从而提高CAR-T疗法的疗效。第三代CAR-T则在第一代基础上整合了2个或2个以上共刺激分子信号，共刺激信号的不同组合可能影响CAR-T细胞的功能和疗效,研究表明,并不是所有的第三代CAR-T都比第二代好。第三代CAR-T已被用于套细胞淋巴瘤(MCL)及滤泡非霍奇金淋巴瘤(NHL)临床试验，但其并未显示出比第二代更优的临床结果。目前在急性白血病临床应用上较为成功的主要是第二代CAR-T技术，转染的一个共刺激分子主要是CD28或CD137(4-1BB)，另一个共刺激分子则是CD3zeta。由此可见，现有技术中CAR的结构设计并不是十分成熟，需要进一步针对增强T细胞杀伤功能、增强Th17细胞亚群，消除调节性T细胞的免疫抑制等方面对CAR分子进行改良。
在固有免疫应答及适应性体液免疫应答过程中，补体系统具有重要的生物学效应。体内补体固有成分在生理情况下以前体酶的形式存在，被激活后可产生活性分子片段，如C3a、C4a、C5a，从而发挥生物免疫学效应。在此活化过程中，最终合成攻膜复合物，攻击微生物或其他靶细胞，从而造成靶细胞的溶解破裂。而这些补体裂解产物主要通过与补体受体(CR)：C3aR、C5aR结合来发挥免疫学功能。补体与CD4+T细胞间存在密切的相互调节关系，我们已经证实C5aR补体途径促进Th17分化，同时抑制调节性T细胞的产生(Wang Y,Lai P,Chen X,et al.Attenuation of cGVHD by C5a/C5aR blockade is associated withincreased frequency of Treg[J].Sci Rep.2017；7(1):3603)。
目前国内外还没有使用C5aR与其他共刺激分子共同组合用于嵌合抗原受体的构建，更无法预期构建后的嵌合抗原受体效果如何。
发明内容
有鉴于此，本发明的目的之一在于提供包含C5aR胞内结构域胞的嵌合抗原受体；
本发明的目的之二在于提供包含C5aR胞内结构域的嵌合抗原受体的慢病毒载体；
本发明的之三在于提供所述包含C5aR胞内结构域的嵌合抗原受体或所述慢病毒载体在制备包含C5aR胞内结构域的嵌合抗原受体的免疫细胞中的应用；
本发明的目的之四在于提供利用所述包含C5aR胞内结构域的嵌合抗原受体或所述的慢病毒载体制备的表达包含C5aR胞内结构域的嵌合抗原受体的免疫细胞；
本发明的目的之五在于提供所述包含C5aR胞内结构域的嵌合抗原受体或所述的慢病毒载体在制备包含C5aR胞内结构域的嵌合抗原受体靶向肿瘤细胞的药物中的应用。
本发明通过以下技术方案实现上述目的：第一方面，本发明提供了一种嵌合抗原受体，其包含能够结合抗原的胞外结构域、跨膜结构域和至少一个胞内结构域。其中，“胞内结构域”是指已知在细胞中作为传输信号以引起生物过程的活化或抑制的结构域起作用的任何寡肽或多肽。而所述的至少一个胞内结构域是指C5aR胞内结构域，或C5aR胞内结构域串联其它信号传递区域如CD3ζ、CD28、4-1BB等的胞内结构域。
对于上述CAR分子，作为优选，所述抗原可以是肿瘤抗原，所述肿瘤抗原例如包括：5T4、α5β1-整联蛋白、707-AP、AFP、ART-4、B7H4、BAGE、β-联蛋白/m、Bcr-abl、MN/C IX抗体、CA125、CAMEL、CAP-1、CASP-8、CD4、CD19、CD20、CD22、CD25、CDC27/m、CD30、CD33、CD52、CD56、CD80、CDK4/m、CEA、CT、Cyp-B、DAM、EGFR、ErbB3、ELF2M、EMMPRIN、EpCam、ETV6-AML1、G250、GAGE、GnTV、Gp100、HAGE、HER-2/new、HLA-A*0201-R170I、HPV-E7、HSP70-2M、HST-2、hTERT(或hTRT)、iCE、IGF-1R、IL-2R、IL-5、KIAA0205、LAGE、LDLR/FUT、MAGE、MART-1/melan-A、MART-2/Ski、MC1R、Mesothelin、肌球蛋白/m、MUC1、MUM-1、MUM-2、MUM3、NA88-A、PAP、蛋白酶-3、p190minor bcr-abl、Pml/RARα、PRAME、PSA、PSM、PSMA、RAGE、RU1或RU2、SAGE、SART-1或SART-3、生存蛋白、TEL/AML1、TGFβ、TPI/m、TRP-1、TRP-2、TRP-2/INT2、VEGF、WT1、NY-Eso-1或NY-Eso-B等等；进一步优选地，所述肿瘤抗原为CD19。本发明专利所述抗原也可以是在自身免疫性疾病中出现的炎性细胞表面分子或导致自身免疫的TCR。
优选地，所述能够结合抗原的胞外结构域是指结合靶向抗原的抗体的单链可变片段。在具体实施方案中，上述CAR分子可以仅以C5aR胞内结构域作为其胞内结构域，还可以包含除了C5aR胞内结构域之外的一个或多个(例如2个或3个)其它胞内结构域。例如，在优选的实施方案中，除了C5aR胞内结构域之外，所述胞内结构域还包括4-1BB、CD3ζ胞内结构域；进一步优选地，所述C5aR胞内结构域配置在CD3ζ胞内结构域的C末端侧。
在一个具体实施方案中，所述胞内结构域为自N-末端侧开始依次连接的4-1BB胞内结构域、CD3ζ胞内结构域和C5aR胞内结构域。此外，本发明的嵌合抗原受体还涵盖其胞内结构域包括串联连接的两个或更多个胞内结构域的情形；以及，可供选择地，所述C5aR胞内结构域可以被配置在嵌合抗原受体胞内结构域的N-末端侧。在优选的具体实施方案中，所述嵌合抗原受体自N末端侧开始依次包括抗肿瘤抗原抗体的单链可变区作为胞外结构域，CD28分子的跨膜结构域和4-1BB胞内结构域，CD3ζ胞内结构域，C5aR胞内结构域。
C5aR胞内结构域含有如序列表1所示的核苷酸序列。嵌合抗原受体的编码核酸含有如序列表1所述的序列，但不局限于此系列，还应包括C5aR其他编码核酸。
第二方面，本发明提供了一种表达包含如上所述的嵌合抗原受体的慢病毒载体。
第三方面，本发明提供了一种嵌合抗原受体表达细胞，其中引入了如第一方面所述的嵌合抗原受体；优选地，所述细胞为T细胞或含有T细胞的细胞群。
本发明提供了一种制备如第三方面所述的嵌合抗原受体表达细胞的方法，其包括将如第一方面所述的嵌合抗原受体引入细胞的步骤；优选地，所述细胞为T细胞或含有T细胞的细胞群。
第四方面，本发明提供了一种如第一方面所述的嵌合抗原受体、如第二方面所述的慢病毒载体或如第三方面所述的嵌合抗原受体表达细胞在制备治疗肿瘤的药物中的用途。
优选地，所述肿瘤为实体瘤或血液瘤。
在本发明提供的该用途的具体实施例中，所述肿瘤为B-ALL。值得注意的是，本发明的CAR特征在于它包含C5aR胞内结构域作为其胞内结构域。所述C5aR胞内结构域包括具有相同功能的其变体。术语“变体”是指包含一个或几个至多个氨基酸的取代、缺失或添加的任何变体，条件是所述变体基本上保留原始序列所具有的相同功能。
本发明的嵌合抗原受体可以提高T17细胞亚群，消除调节T细胞的免疫抑制的作用，可以显著提高肿瘤靶细胞的体外杀伤效率，可显著改善第二代CAR T细胞对肿瘤的杀伤效果，为CAR-T细胞治疗领域提供一种新的思路和选择。
本发明首次通过实验证实C5aR可以作为嵌合抗原受体的胞内结构域，从而增强T细胞杀伤功能，提高T17细胞亚群，消除调节T细胞的免疫抑制的作用。
附图说明
图1a为含有C5aR胞内结构域组合的病毒载体示意图。
图1b为不含有C5aR胞内结构域组合的病毒载体示意图。
图2为GFP T、CAR19T和CAR19C5aR T细胞对表达CD19的NALM6-GL细胞的体外杀伤效应结果图，结果表明，CAR19C5aR T优于GFP T、CAR19T，图中NALM6表示NALM6-GL细胞。
图3为GFP T、CAR19T和CAR19C5aR T细胞对表达CD19的Raji细胞的体外杀伤效应结果图，结果表明，CAR19C5aR T优于GFP T、CAR19T。
图4a及图4b为GFP T、CAR19T和CAR19C5aR T细胞对T17细胞亚群(CD4+IL17A+)，调节T细胞亚群(CD4+CD25+FoxP3+)的影响结果图，结果表明，CAR19C5aRT可以显著提高T17细胞亚群，消除对调节T细胞的免疫抑制。*P<0.05。
图5为GFP T、CAR19T和CAR19C5aRT细胞对NCG小鼠体内NALM6肿瘤细胞(CD19+)的负荷结果图，结果表明，CAR19C5aR T优于GFP T、CAR19T。*P<0.05。
图6为BLI图像显示NALM6细胞在NCG小鼠体内的负荷与部位。
图7为GFP T、CAR19T和CAR19C5aRT细胞对NCG小鼠生长期的影响结果图，结果表明，CAR19C5aR T较GFP T、CAR19T更能显著延长NCG小鼠生长期。*P<0.05。
具体实施方式
下面结合实施例，更具体地说明本发明的内容。应该理解，本发明的实施并不局限于下面的实施例，对本发明所做的任何形式上的变通和/或改变都落入本发明保护范围。实施例中未注明具体条件的实施方法，通常按照常规条件中所述的条件或按照制造厂商所建议的条件。
实施例1含有抗CD19ScFv-4-1BB-CD3ζ-C5aR(CAR19C5aR)质粒的制备
按如下步骤制备本发明携带含C5aR胞内结构域的嵌合抗原受体基因的质粒：
(1)通过基因合成、分子克隆等手段得到含抗CD19ScFv-4-1BB-CD3ζ(CAR19)的质粒pUC57-CAR19基因包含抗CD19单抗ScFV、CD28跨膜区和4-1BB、CD3ζ胞内区，即CD19ScFv-4-1BB-CD3ζ。
(2)通过内切酶Pmel和Sepl对所得到的pUC57-CAR19质粒进行酶切，获得CAR19基因，然后将CAR19基因连接至慢病毒载体pWPXLd-GFP，构建pWPXLd-CAR19-GFP。
(3)用内切酶NotI和SpeI对所得到的pWPXLd-CAR19-GFP质粒进行酶切，获得CAR19基因的胞内片段4-1BB-CD3ζ。
(4)用4-1BB-CD3ζ串联C5aR胞内信号传递结构域的cDNA作为模板，构建4条引物，通过重叠延伸PCR得到4-1BB-CD3ζ-C5aR。
(5)通过NotI和SpeI酶切，用4-1BB-CD3ζ-C5aR替换pUC57-CAR19中的4-1BB-CD3ζ，得到pUC57-CAR19C5aR质粒。
(6)最后，通过内切酶PmeI和SpeI酶切，用CAR19C5aR替换pWPXLD-CAR19-GFP中的CAR19，得到pWPXLd-CAR19C5aR-GFP质粒。(图1)
实施例2CAR质粒的慢病毒包装
使用实施例1制备的本发明CAR质粒以及相关对照质粒、通过慢病毒包装，获得分别表达GFP(空白对照)、CAR19-GFP(对照)、CAR19C5aR-GFP的三种慢病毒。本实施例2和3中，将含CAR质粒统一描述为为pWPXLd-CAR-GFP质粒，将过表达CAR的慢病毒统一描述为CAR慢病毒。
具体步骤如下:
(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-GFP二种质粒(即，分别包含CAR19、CAR19C5aR)或空白对照质粒pWPXLd-GFP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表1：
表1
(4)分别转化后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度12-16小时溶解，即得2种CAR慢病毒或空白对照GFP慢病毒；
(8)病毒溶解后，收集病毒分装于冻存管，冻存于-80℃待用。
实施例3使用包装的CAR病毒感染人体T细胞
(1)T细胞的分离纯化：通过Ficoll密度梯度法分离出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)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(产品来源：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的T细胞。
(5)CART细胞扩增：将CART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，CART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2种CART细胞(分别简称CAR19-GFP、CAR19C5aR-GFP)或空白对照T细胞(GFP-T)的比例。检测对Th17细胞亚群(CD4+IL17A+)，调节T细胞亚群(CD4+CD25+FoxP3+)的影响。
调节T细胞亚群检测方法1.准备效应细胞(GFP-T、CAR19-GFP或CAR19C5aR-GFP T细胞)；加入表面抗体5ul CD4-PERCP、5ulCD25-PE及相对应同型抗体，4℃避光孵育30min，加入1ml PBS洗涤，300g离心5min，去除上清，加入破膜固定剂(破膜剂：稀释液＝1:3，新鲜配制)1ml，4℃避光孵育35min。2.35min后加入2ml破膜缓冲液(缓冲液：纯水＝1:9)，混匀，300g离心5min。3.去上清，加入FoxP3抗体5ul，4℃避光孵育30min。4.30min后加入2mlPBE洗涤，300g离心5min。5.每管加入400ulPBS重悬上机检测。
Th17细胞亚群检测方法：1.准备效应细胞(GFP-T、CAR19-GFP或CAR19C5aR-GFP T细胞)，加入表面抗体5ul CD4-PERCP，混匀，避光，孵育30min。2.加入100ul破膜剂A，室温孵育15min。3.加入3ml PBS+0.1％NaN3+5％FBS.4.300g，5min离心，去上清，加入缓冲液B，混匀，加入5ul IL-17A APC-Cy7，孵育20min。5.加入3ml PBS+0.1％NaN3+5％FBS.300g，5min离心，去上清，400-500ulPBE重悬上机检测。
实施例4CART细胞体外识别杀伤肿瘤的效应
将实施例3制备的GFP T(空白对照)、CAR19-GFP T(对照)、CAR19C5aR-GFP T细胞以不同比例分别与5x104的肿瘤细胞混合，加入到96孔U型板中，每组设3个复孔，并设单独加肿瘤细胞组作为阳性对照，250g离心5min后，置于37度5％CO2培养箱共培养24h；体外比较GFP T、CAR19-GFP T、CAR19C5aR-GFP T细胞对血液肿瘤的识别杀伤功能时，肿瘤细胞选用NALM6-GL、Raji二种白血病或淋巴瘤细胞系。
CFSE/PI双染细胞毒性检测方法评估定量评估杀伤效率：1.准备效应细胞(GFP T(空白对照)、CAR19-GFP T(对照)、CAR19C5aR-GFP T细胞)和靶细胞，计数，将CART细胞的GFP％用同一批次的WT细胞进行校准，使各组GFP％大小相近；用1640+10％FBS+1％P/S培养基稀释T细胞8x106/ml。2.所需量的靶细胞(Nalm6细胞、Raji细胞)，1ml 1640培养基重悬，加入5umol CSFE，37℃避光孵育20min后，加入10ml培养基终止5min，离心，去上清，靶细胞稀释为5x105/ml。3.将T细胞进行倍比稀释，用排枪在V型底96孔板内预先加入100ul新鲜培养基，第一排不加，然后第一排加入T细胞100ul，第二排加入T细胞100ul，然后用排枪吹打第二排5次以上，吸取100ul放入第三排，以此类推，将T细胞顺着排数进行倍比稀释，全部孔内加入100ul靶细胞，使T细胞与靶细胞以不同比例混合(16:1，8:1，4:1，2:1，1:1，1:2，1:4)。4.将混合好的细胞放培养箱内培养24小时。5.将96孔板内的细胞吸取，离心。6.用4℃的PBS洗涤一次，加入100ul 1x IP Buffer，再加入5ul PI，避光孵育15min后，再加入200ul1xIP Buffer，1小时内上机检测。7.杀伤率计算：以不加T细胞的靶细胞孔作为参照，杀伤百分比＝目的孔CSFE+PI+％-对照孔CSFE+PI+％。结果表明，CAR19C5aRT和对表达CD19的肿瘤靶细胞的体外杀伤效率都显著高于CAR19T细胞(见图2和图3)。
实施例5CAR19C5aR T细胞体内识别杀伤肿瘤
为比较CAR19T、CAR19C5aR T细胞的体内识别杀伤肿瘤的效应，将同等数量(5×105)的NALM6细胞(表达luciferase)分别通过尾静脉7只NCG(NOD/SCID IL2rg-/-)免疫缺陷小鼠体内；NALM6细胞移植后第2天和第8天(肿瘤细胞移植当天为第0天)，将5×106个T细胞(四组：GFP T、CAR19T、CAR19C5aR T，每组注射6只小鼠)静脉注射入已移植NALM6细胞的NSI免疫缺陷小鼠体内。由于NALM6表达luciferase，通过腹腔注射底物D-luciferin 200ul，计时5分钟后，放入体内成像附带的麻醉室内，通过异氟烷进行麻醉，然后将麻醉好的小鼠放入成像仪内进行成像，通过自动曝光模式进行成像，成像后将小鼠放回笼子内饲养，分数数据结果表明，CAR19C5aR T能显著降低NCG小鼠体内NALM6肿瘤细胞的负荷(见图5和6)，并继续观察小鼠生存期，直接死亡，最终结果CAR19C5aR T能显著延长NCG小鼠生长期(图7)。提示加入C5aR的胞内结构域后，则可显著改善第二代CAR T细胞(CAR19T)对肿瘤的杀伤效果。通过以上实验结果，对比实验组和对照组CAR T对肿瘤识别杀伤功能的强弱，验证了C5aR胞内信号传导结构域对CAR T体内、外肿瘤杀伤功能的改善。
申请人声明通过上述实施实例来说明本发明的产品、用途及其使用方式，但本发明并不局限于上述详细用途或使用方式，即不意味着本发明必须依赖上述详细用途和使用方式才能实验。所属技术领域的人员应该明了，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。
序列表
<110> 赖沛龙
<120> 包含C5aR胞内结构域的嵌合抗原受体、慢病毒载体、表达细胞及药物
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 410
<212> DNA
<213> Artificial
<220>
<221> misc_feature
<400> 1
cttgggcagg agggaccttc gatcctcggg gagcccagga gaccagaaca tgaactcctt 60
caattatacc acccctgatt atgggcacta tgatgacaag gataccctgg acctcaacac 120
ccctgtggat aaaacttcta acacgctgcg tgttccagac atcctggcct tggtcatctt 180
tgcagtcgtc ttcctggtgg gagtgctggg caatgccctg gtggtctggg tgacggcatt 240
cgaggccaag cggaccatca atgccatctg gttcctcaac ttggcggtag ccgacttcct 300
ctcctgcctg gcgctgccca tcttgttcac gtccattgta cagcatcacc actggccctt 360
tggcggggcc gcctgcagca tcctgccctc cctcatcctg ctcaacatgt 410
Claims (4)
1.包含C5aR胞内结构域的嵌合抗原受体，包含能够结合抗原的胞外结构域、跨膜结构域和胞内结构域，其特征在于，所述胞内结构域为自N末端侧开始依次连接的4-1BB胞内结构域、CD3ζ胞内结构域和C5aR胞内结构域，编码所述C5aR胞内结构域的核苷酸序列如序列1所示，所述能够结合抗原的胞外结构域是CD19 ScFv，跨膜结构域是CD28。
2.一种表达如权利要求1所述的嵌合抗原受体的慢病毒载体。
3.一种表达嵌合抗原受体的细胞，其特征在于：引入了权利要求1所述的嵌合抗原受体。
4.一种预防或治疗肿瘤的药物，包含有如权利要求1所述的嵌合抗原受体、或如权利要求2所述的慢病毒载体或如权利要求3所述的细胞。
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| PCT/CN2018/075858 WO2019134214A1 (zh) | 2018-01-02 | 2018-02-08 | 包含C5aR胞内结构域的嵌合抗原受体、慢病毒载体、表达细胞及药物 |
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