CN108752482B - 携带截短或未截短的髓样细胞触发性受体信号结构的嵌合抗原受体及其应用 - Google Patents
携带截短或未截短的髓样细胞触发性受体信号结构的嵌合抗原受体及其应用 Download PDFInfo
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Abstract
本发明公开一种嵌合抗原受体(CAR),其包括:抗原结合结构域(scfv)和信号传导结构域,其中信号传导结构域包括第一传导结构域和第二传导结构域,第一传导结构域和第二传导结构域之间串联抗原结合结构域。本发明公开的CAR结构受到抗原刺激时分泌细胞因子水平极低,能够很好地保证临床应用安全性,即临床应用安全性更高;且体外抗原阳性肿瘤细胞杀伤能力更强,抗肿瘤活性更好。
Description
技术领域
本发明涉及肿瘤免疫治疗技术领域,具体涉及一种携带截短或未截短的髓样细胞触发性受体信号结构的嵌合抗原受体及其应用。
背景技术
嵌合抗原受体(CAR)是CAR-T的核心部件,利用配体结合结构域特性,CAR能针对所选择的免疫细胞重定向其特异性和反应性,因此赋予T细胞HLA非依赖的方式识别肿瘤抗原的能力,这使得经过CAR改造的T细胞相较于天然T细胞表面受体TCR能够识别更广泛的目标。CAR的基础设计中包括一个肿瘤相关抗原(tumor-associated antigen, TAA)结合区(通常来源于单克隆抗体抗原结合区域的scFV段),一个胞外铰链区,一个跨膜区和一个胞内信号区。
当前传统的CAR-T用于血液肿瘤疗效显著,但对于实体肿瘤疗效并不明显,限制其在临床的应用。从安全性而言,细胞因子释放综合症(CRS)为CAR-T细胞治疗的常见并发症,甚至可危及生命。CAR-T细胞输注到体内后,由于嵌合抗原受体与相应的肿瘤相关抗原特异性结合,CAR-T细胞被激活并开始增殖,引发细胞因子级联释放,介导多类免疫反应,从而引起发热、低血压、呼吸困难、凝血障碍、终末器官障碍等临床表现,即CRS,传统CAR-T受到抗原刺激分泌细胞因子水平直接影响到CRS发生的严重程度。从有效性而言,首先,由实体瘤相关的成纤维细胞(CAFs)组成的肿瘤基质为CAR-T细胞的浸润提供了一个物理屏障,CAFs还会分泌细胞外基质蛋白将T细胞从癌细胞中分离出来;其次,实体瘤肿瘤微环境的代谢环境不利于传统CAR-T细胞的持久性,因为一旦肿瘤形成被激活,肿瘤细胞就会停止通过氧化磷酸化产生ATP并转换为有氧糖酵解,导致肿瘤环境变得酸性,这就是所谓的“瓦氏效应”pH值从7.4降至6.5;最后,肿瘤微环境造成缺氧状态进一步产生免疫抑制,在低氧环境肿瘤细胞产生一种HIF1-ɑ分子,它通过吸引调控性T细胞(Tregs)到肿瘤部位来削弱传统CAR-T细胞抗肿瘤功能,Tregs抑制免疫反应,因此限制传统CAR-T对于实体肿瘤的治疗作用。
发明内容
本发明的目的之一在于提供一种安全性更好,疗效更加显著的携带髓样细胞触发性受体TREM1或TREM2信号结构的嵌合抗原受体(CAR)。
本发明的目的之二在于提供所述嵌合抗原受体的应用。
本发明的目的之三在于提供一种嵌合抗原受体的信号传导结构域。
本发明的目的之四在于提供所述信号传导结构域的应用。
本发明的上述目的的具体技术方案详述如下:
一种嵌合抗原受体(CAR),其包括:抗原结合结构域(scfv)和信号传导结构域,其中信号传导结构域包括第一传导结构域和第二传导结构域,第一传导结构域和第二传导结构域之间串联抗原结合结构域;所述的第一传导结构域可以为截短的或未截短的 TREM1或TREM2。
本发明所述的嵌合抗原受体中串联的第一传导结构域、抗原结合结构域和第二传导结构域,在抗原结合结构域特异性结合抗原后变成能够传递激活信号的多链形式,将激活信号传输到表达其的免疫细胞,实现免疫治疗的作用。
所述的嵌合抗原受体为多链结构嵌合抗原受体,其用第一传导结构域和第二传导结构域组成CAR的信号传导结构域,抗原结合结构域可特异性地结合至靶标中的配体并诱导免疫细胞的活化,产生免疫应答。
本发明所述第二传导结构域为DAP12,第二传导结构域通过T2A与抗原结合结构域串联。
本发明所述的DAP12是一个跨膜结构域,广泛存在于自自然杀伤细胞、粒细胞、单核/巨噬细胞表面,用于传递活化信号,其核苷酸序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示。
本发明所述的T2A用于串联第二传导结构域和抗原结合结构域,其核苷酸序列如SEQ ID NO.3所示,氨基酸序列如SEQ ID NO.4所示。
本发明所述的第一传导结构域可以为截短的或未截短的TREM1或TREM2,其中,TREM1基因全长的核苷酸序列见NCBI,GenBank:NM_018643.4,氨基酸序列见NCBI,GenBank:NP_061113.1;TREM2基因全长的核苷酸序列见NCBI,Accession: NM_018965.3,氨基酸序列见NCBI,Accession:NP_061838.1。所述截短的TREM1或 TREM2表示其全长氨基酸序列C端的氨基酸序列。
为了提高本发明所述嵌合抗原受体的安全性和疗效,本发明还提供一种优选的第一传导结构域,其选自截短的TREM1氨基酸序列,命名为TREM1cut,本发明所述的TREM1cut为TREM1全长氨基酸序列C端40~90个氨基酸的多肽,优选为TREM1全长氨基酸序列C端50~85个氨基酸的多肽,更优选为TREM1全长氨基酸序列C端60~80个氨基酸的多肽,或者与前述多肽序列具有80%以上同一性的氨基酸序列,也或者与该序列具有85%以上同一性的氨基酸序列,也或者与该序列具有90%以上同一性的氨基酸序列,也或者与该序列具有95%以上同一性的氨基酸序列。
本发明还提供一种优选的第一传导结构域,为TREM1全长氨基酸序列C端80个氨基酸的多肽,氨基酸序列如SEQ ID NO.8所示,其核苷酸序列如SEQ ID NO.7所示。
本发明所述的抗原结合结构域可以根据不同的肿瘤目标进行本领域常规选择。
更具体的,本发明所述的嵌合抗原受体为DAP12、T2A、抗原结合结构域和第一传导结构域按顺序通过2-10个任意氨基酸进行串联。本发明的2-10个任意氨基酸的序列和个数对本申请所述的嵌合抗原受体功效没有明显影响,可以为任意的2-10个的氨基酸序列。
本发明所述的嵌合抗原受体可以使用例如逆转录病毒载体将编码嵌合抗原受体的核酸转移到免疫细胞如T细胞中,当嵌合抗原受体结合靶抗原时,第一传导结构域和抗原结合结构域分离形成激活信号传输到表达其的免疫细胞。
本发明还提供一种带有所述嵌合抗原受体的免疫细胞。
本发明还提供所述嵌合抗原受体或带有所述嵌合抗原受体的免疫细胞在肿瘤免疫治疗中的应用。
本发明提供一种信号传导结构域,包括第一传导结构域和第二传导结构域;所述第一传导结构域为截短的或未截短的TREM1或TREM2。
本发明所述的第一传导结构域和第二传导结构域,在抗原结合结构域特异性结合抗原后变成能够传递激活信号的多链形式,将激活信号传输到表达其的免疫细胞,实现免疫治疗的作用。
本发明所述第二传导结构域为DAP12。本发明所述的DAP12是一个跨膜结构域,广泛存在于自自然杀伤细胞、粒细胞、单核/巨噬细胞表面,用于传递活化信号,其核苷酸序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示。
本发明所述的第一传导结构域可以为截短的或未截短的TREM1或TREM2,其中,TREM1基因全长的核苷酸序列见NCBI,GenBank:NM_018643.4,氨基酸序列见NCBI,GenBank:NP_061113.1;TREM2基因全长的核苷酸序列见NCBI,Accession:NM_018965.3,氨基酸序列见NCBI,Accession:NP_061838.1。所述截短的TREM1或 TREM2表示其全长氨基酸序列C端的氨基酸序列。
为了提高本发明信号传导结构域所形成的嵌合抗原受体的安全性和疗效,本发明还提供一种优选的第一传导结构域,其选自截短的TREM1氨基酸序列,命名为TREM1cut,本发明所述的TREM1cut为TREM1全长氨基酸序列C端40~90个氨基酸的多肽,优选为 TREM1全长氨基酸序列C端50~85个氨基酸的多肽,更优选为TREM1全长氨基酸序列 C端60~80个氨基酸的多肽,或者与前述多肽序列具有80%以上同一性的氨基酸序列,也或者与该序列具有85%以上同一性的氨基酸序列,也或者与该序列具有90%以上同一性的氨基酸序列,也或者与该序列具有95%以上同一性的氨基酸序列。
本发明还提供一种优选的第一传导结构域,为TREM1全长氨基酸序列C端80个氨基酸的多肽,氨基酸序列如SEQ ID NO.8所示,其核苷酸序列如SEQ ID NO.7所示。
本发明所述的信号传导结构域,其用跨膜受体DAP12结合第一传导结构域组成CAR的信号传导结构域,当CAR特异性地结合了靶标中的配体,可以诱导免疫细胞的活化,产生免疫应答。
本发明还提供所述信号传导结构域在嵌合抗原受体或肿瘤免疫治疗中的应用。
本发明所述的C端是指从C段第一个氨基酸开始截取的多肽,如C段40~90个氨基酸的多肽表示从C段第一个氨基酸开始到第40~90中的任意一个氨基酸为止的多肽。
本发明的有益效果:
(1)本发明所涉及的CAR结构受到抗原刺激时分泌细胞因子水平极低,能够很好地保证临床应用安全性,即临床应用安全性更高;
(2)本发明所涉及的CAR结构通过体外功能实验证实其对实体肿瘤的显著疗效,因此本发明不仅可以应用于血液肿瘤的治疗,而且可以拓展CAR-T在实体瘤治疗中的应用;
(3)本发明所涉及的CAR结构体外抗原阳性肿瘤细胞杀伤能力更强,抗肿瘤活性更好。
附图说明
图1是含有不同信号域结构的慢病毒载体;
图2是MSLN4CAR-T细胞感染慢病毒7天后通过流式细胞仪检测T细胞表面识别MSLN抗原的CAR结构的表达阳性率;
图3是CAR-T细胞感染慢病毒后细胞增殖情况;
图4是MSLN4CAR-T细胞在抗原刺激下IL-2的分泌情况;
图5是MSLN4CAR-T细胞在抗原刺激下IFN-γ的分泌情况;
图6是不同组的CAR-T在抗原刺激下IL-2,IFN-γ的分泌情况;
图7是不同组的CAR-T对抗原阳性肿瘤细胞株杀伤作用。
具体实施方式
下面将结合附图,对本发明的优选实施例进行详细的描述。实施例中未注明具体条件的实验方法,通常按照常规条件,例如分子克隆实验指南(第三版,J.萨姆布鲁克等著)中所述的条件或按照制造厂商所建议的条件。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。
实施例1、含有DAP12-T2A-scFv-TREM1cut这4个元件的CAR慢病毒的构建
为证明含有DAP12-TREM1cut胞内信号域的CAR-T细胞较以往已报道的含有 4-1BB-CD3ζ、DAP12-KIRS2和单DAP12刺激信号的CAR-T细胞更具有优势,因此需要分别构建含有不同刺激信号组合的病毒载体。本实施例中以靶向人间皮素(MSLN)的单链抗体为统一的胞外识别抗原的结构,分别需要构建如下5个嵌合抗原受体(图1):
MSLN(scfv)-CD8α-4-1BB-CD3ζ(MSLN1)
DAP12-T2A-MSLN(scfv)(MSLN2)
DAP12-T2A-MSLN(scfv)-KIRS2(MSLN3)
DAP12-T2A-MSLN(scfv)-TREM1cut(MSLN4)
DAP12-T2A-MSLN(scfv)-TREM1wt(MSLN5)
1、合成含有不同胞内刺激信号的靶向人间皮素的嵌合抗原受体的基因序列
合成依次含有自然杀伤激活受体(简称DAP12)、T2A、抗人间皮素的单链抗体scfv(简称MSLN(scfv)),髓样细胞触发性受体(简称TREM1wt),截短的髓样细胞触发性受体(简称TREM1cut),其结构如图1所示。其中DAP12的核苷酸序列如SEQ ID NO.1 所示,氨基酸序列如SEQ ID NO.2所示,T2A的核苷酸序列如SEQ ID NO.3所示,氨基酸序列如SEQ ID NO.4所示,抗人间皮素的单链抗体(MSLN)scfv的核苷酸序列如SEQ ID NO.5所示,氨基酸序列如SEQID NO.6所示,TREM1基因全长的核苷酸序列见NCBI, GenBank:AJ225109.1,氨基酸序列见NCBI,GenBank:CAB39168.1,TREM1cut的核苷酸序列如SEQ ID NO.7所示,氨基酸序列如SEQID NO.8所示,KIRS2的核苷酸和氨基酸序列见专利(用于过继免疫疗法的具有嵌合受体的靶向细胞毒性细胞,公开号: CN107580628A),CD8α-4-1BB-CD3ζ的核苷酸序列和氨基酸序列见专利(Methods for treatment of cancer,US 20130309258A1)。
2、构建表达嵌合抗原受体的慢病毒载体
pELNS Dap12-T2A-MSLN-KIRS2由南京卡提医学科技有限公司保存,或者根据文献(Enxiu Wang et al.Generation of Potent T-cell Immunotherapy for Cancer UsingDAP12-Based,Multichain,Chimeric Immunoreceptors.2015,Cancer ImmunologyResearch,3 (7):815)公开的方法进行构建,截短TREM1cut基因合成由南京金斯瑞生物科技公司合成并提供pUC19-TREM1cut质粒,将质粒pELNS Dap12-T2A-MSLN-KIRS2和 pUC19-TREM1cut通过NheI、SalI双酶切(购自Takara公司),酶切反应按说明书进行,获得pELNSDap12-T2A-MSLN片段约8900bp,截短的TREM1cut片段约243bp的DNA 片段,然后用回收试剂盒(Takara公司)进行DNA片段回收,具体方法见说明书,回收获得的pELNS Dap12-T2A-MSLN和TREM1cut基因,然后将目的片段TREM1cut和载体片段pELNS Dap12-T2A-MSLN通过T4连接酶(购自Takara公司)进行连接,获得表达嵌合抗原受体的慢病毒载体,命名为pELNS Dap12-T2A-MSLN-TREM1cut(简称MSLN4)。将5μL慢病毒载体MSLN4转化大肠杆菌TOP10感受态细胞中(购自南京安杰优生物科技有限公司),37℃培养16h后挑取单克隆,挑取的单克隆在37℃条件下培养12h后用质粒抽提试剂盒(购自Takara公司)抽提质粒,具体方法见说明书。
按照上述方法分别构建pELNS MSLN-CD8α-4-1BB-CD3ζ(简称MSLN1);pELNSDap12-T2A-MSLN(简称MSLN2);pELNS Dap12-T2A-MSLN-KIRS2(简称MSLN3); pELNS Dap12-T2A-MSLN-TREM1cut(简称MSLN4);pELNS Dap12-T2A-MSLN-TREM1wt (简称MSLN5)慢病毒载体。
3、慢病毒包装
本实施例包装慢病毒采用磷酸钙法,具体步骤如下:
(1)293T细胞隔天传代
每个T150细胞瓶种植5×106个细胞。48小时后,细胞数目应该达到20-25百万/瓶。
(2)293T细胞铺瓶
a)以1个T150细胞培养瓶为例,用约15ml的1×PBS轻柔地洗细胞两次
b)加入3ml0.25%胰酶-2.21mM EDTA
c)等到细胞脱落,加入12ml 10%(wt)FBS(购自Gibico)的DMEM培养基(购自corning)至已经脱落的细胞中
d)收集并将细胞转移至无菌离心管,1000rpm,离心10分钟
e)吸掉上清,将沉淀重悬于10ml 10%(wt)FBS的DMEM培养液中。
f)细胞计数,根据细胞浓度计算12×106个细胞所需要的体积
g)将细胞和25ml的10%(wt)FBS的DMEM培养液合并,放入T150细胞瓶中,轻摇,使得细胞均匀分布到细胞瓶底37℃,5%CO2培养箱中培养过夜。
(3)细胞转染
观察细胞,细胞密度大约达到80%-90%,此时就可以开始转染
a)在转染前30-60分钟,轻柔吸掉培养液。
b)混合质粒DNA和氯化钙溶液,以一个T150瓶为例,需要28ug pRSV.rev(购自Invitrogen公司),28ug pGAG-Pol(购自Invitrogen公司),11ug pVSVG(购自Invitrogen公司),23ug重组慢病毒表达质粒pELNS Dap12-T2A-MSLN-TREM1cut,加入到1.5ml氯化钙溶液中,混匀。
c)将1.5ml BBS溶液加入到15ml无菌离心管中,用1ml枪头把DNA-氯化钙溶液混匀后滴加到BBS溶液中,迅速混匀15-20下,室温孵育25-30分钟。
d)用5ml移液管把DNA-氯化钙-BBS混合物(购自上海碧云天生物技术有限公司)均匀逐滴加到T150瓶中。在含5%二氧化碳的37℃细胞培养箱内培养,6h换液。
e)6h后换液。轻轻晃动培养板数次以充分悬浮一些磷酸钙沉淀,吸去含磷酸钙沉淀的培养液,加入20ml新鲜的5%(wt)FBS的DMEM培养液,继续培养。
(4)初次收集病毒上清
a)将前一天转染的293T细胞培养上清收集到离心管,1000rpm离心5分钟,标记,暂存于4℃冰箱。
b)将事先预热的20ml 5%(wt)FBS的DMEM培养基加入细胞瓶中,37℃细胞培养箱继续培养过夜。
(5)第二次收集病毒上清液(48h/第四天)。
(6)过滤上清
将两次收集的上清液集中在一起,用0.45μm的滤膜过滤去除细胞碎片。
(7)病毒浓缩
4℃,12000-24000rpm离心过夜
(8)病毒储存
离心后,倾倒全部上清,加入新鲜5%(wt)FBS的DMEM培养基重悬,进行病毒分装,迅速存放于-80℃冰箱备用
(9)慢病毒滴度测定
a)病毒感染293T细胞
感染前将293T细胞铺至24孔板中,取已纯化浓缩病毒200μL加到293T细胞中, 24h后用含10%FBS(wt)的DMEM培养基换液,感染72h后于1200r/min条件下离心 5min以收集细胞,抽提基因组。
b)抽提基因组
基因组抽提试剂盒为购自于Takara公司,按试剂盒说明书操作。
c)qPCR测定病毒滴度
反应体系如下:Probe qPCR Mix 12.5μL(购自Takara),上游引物0.5μL(由南京金斯瑞合成),下游引物0.5μL(由南京金斯瑞合成),探针1μL(由南京金斯瑞合成),模板2μL,灭菌水8.5μL,反应体系为25μL,反应条件按照说明书设置,反应结束后,用分析软件分析数据,根据标准曲线计算病毒滴度。计算结果显示,病毒滴度为1.3×106TU/ml。
实施例2、病毒感染T细胞
1、T细胞的分离活化及病毒感染
(1)人外周血单核细胞的分离
用含有抗凝剂的采血管采集外周血约10ml,室温(18-25℃)自然沉降约30min,收集上层血浆,将收集的上层血浆于5000r/min条件下离心10min,按体积比1:1加到淋巴细胞分离液(购自天津市灏洋生物制品科技有限责任公司)上,梯度离心,3000r/min,离心30min,离心后,离心管由上之下分层:第一层为血浆层;第二层为淋巴细胞白膜层;第三层为透明分离液层;第四层红细胞层。吸取淋巴细胞白膜层,并用PBS洗涤2次,两次离心以1500r/min,离心10min,PBS重悬细胞,加入5%自体血浆+300IU/ml重组人IL-2+KBM581完全培养基培养人外周血单核细胞。
(2)慢病毒感染T淋巴细胞
用含5%自体血浆+300IU/ml重组人IL-2+KBM581完全培养基培养新制备的单个核细胞PBMC,IL-2购自R&D Systems,KBM581购自Corning,第0天加入CD3/CD28 Dynabeads免疫磁珠(购自invitrogen)活化T细胞,前3天进行慢病毒感染,加入0.25MOI 对应的慢病毒载体,未感染的T淋巴细胞作为空白对照,48h后将培养基更换为含有5%自体血浆+300IU/ml重组人IL-2+KBM581完全培养基,继续培养7-9天。
2、T细胞中CAR阳性率的检测
将培养至第7天的已感染病毒的T细胞,1200r/min,离心5min,弃尽上清以收集细胞,用含有体积分数1%FBS的PBS溶液重悬细胞,并将细胞调整密度为1×105个/ml,加入生物素标记羊抗鼠F(ab)2(Jackson ImmunoResearch公司),再加入Streptavidin-PE(BDBiosciences公司),4℃孵育15min,PBS溶液洗涤2次,上流式细胞仪进行检测,结果显示经过7天的培养,CAR-T细胞CAR的阳性率:MSLN1病毒感染组阳性率41%,MSLN2 病毒感染组阳性率52%,MSLN3病毒感染组阳性率59%,MSLN4病毒感染组阳性率20% (图2),MSLN5病毒感染组阳性率49%。
实施例3、病毒感染CAR-T细胞对细胞增殖的影响
各组病毒感染完T细胞后,将T细胞用含体积分数5%自体血浆+300IU/ml重组人IL-2+KBM581完全培养基,每1-2天计数一次。然后观察T淋巴细胞生长情况,结果如图3所示。结果表明细胞在感染表达CAR的病毒后,依然能够形成典型的增殖克隆团,通过对细胞进行计数,绘制细胞增殖曲线可见感染MSLN4CAR-T细胞增殖与MSLN1、 MSLN2、MSLN3、MSLN5CAR-T增殖相似,比未感染病毒的T细胞(图3中NTD)增殖能力稍弱。
实施例4、检测病毒感染CAR-T细胞的细胞因子分泌
(1)细胞因子检测采用Elisa的方法,使用R&D公司试剂盒进行。
(2)标准品的稀释:准备1ml离心管7支,依次编号号码,先在各离心管中加入标准品稀释液500μL,然后取原浓度标准品500μL加入到1只已编好号的离心管中,充分混匀,再在该离心管中取500μL加入第二支离心管中,充分混匀;再在该离心管中取 500μL加入第三只离心管中,充分混匀;再在该离心管中取500μL加入第四只离心管中,充分混匀;再在该离心管中取500μL加入第五只离心管中,充分混匀;再在该离心管中取500μL加入第六只离心管中,充分混匀;再在该离心管中取500μL加入第七只离心管中,充分混匀。
(3)在酶标包被板上设标准品孔,依次加入不同浓度的标准品100μL,每个浓度 2-3个平行孔。
(4)加样:分别设置空白孔(空白对照孔用水代替,酶标试剂及生物素标记的抗体操作照旧)、待测样品孔,在酶标包被板上待测样品孔中先加样品100μL,加样将样品加于酶标板孔底部,尽量不触及孔壁,轻轻晃动混匀
(5)孵育:室温放置孵育2h
(6)洗涤:弃去液体,甩干,每孔加200μL洗涤液,静止30s后弃去,如此重复 3次,拍干
(7)加抗体:酶标包被板上加入100μL检测抗体
(8)孵育:同操作(5)
(9)洗涤:同操作(6)
(10)加标记:每孔加入100μL辣根过氧化物酶标记链霉亲和素
(11)孵育:避光室温放置孵育20min
(12)洗涤:同操作(6)
(13)显色:每孔加入显色液100μL,轻轻震荡混匀,避光室温放置孵育20min
(14)终止:每孔加入终止液50μL,终止反应
(15)测定:以空白值校零,450nm波长依序测量各孔的吸光度(OD值),测定应在加入终止液后15min内进行。
选择抗原表达水平具有差异的靶细胞与MSLN4CAR-T共培养,检测MSLN4CAR-T 受到抗原刺激产生响应作用分泌IL-2和IFN-γ水平,靶细胞选择OVCAR3(MSLN高表达),293T(MSLN阴性),以此来显示出MSLN4CAR-T在受到MSLN抗原刺激时所特异性分泌出IL-2和IFN-γ,结果反映出MSLN4CAR对于抗原表达水平具有差异的靶细胞产生了不同的响应作用。MSLN4的CAR-T在MSLN高表达靶细胞OVCAR3共培养时显著分泌IFN-γ和IL-2(图4和图5),表明MSLN4CAR对于抗原阳性的肿瘤细胞具有响应作用。
另一方面,比较MSLN1、MSLN2、MSLN3、MSLN4、MSLN5CAR-T与OVCAR3 共培养时,各组分泌IL-2和IFN-γ细胞因子水平。结果如图6所示,MSLN4CAR-T分泌水平较MSLN2和MSLN5CAR-T组有显著提升(由于MSLN2CAR-T抗肿瘤效果很弱,故没有分泌细胞因子),而较MSLN1和MSLN3分泌量有一定程度的下降。体外细胞因子分泌结果表明MSLN4CAR-T和MSLN5CAR-T产生更低水平的细胞因子,是可能提高临床应用安全性的。
实施例5、病毒感染CAR-T细胞体外杀伤效果评估
(1)分别培养靶细胞OVCAR3细胞(MSLN高表达细胞株)、293T(MSLN阴性细胞株)和效应细胞MSLN1、MSLN2、MSLN3、MSLN4、MSLN5组CAR-T细胞。
(2)收集靶细胞和效应细胞,1500rpm/min,离心5min,弃上清
(3)用10%FBS+1640完全培养基重悬靶细胞和效应细胞
(4)利用实时细胞分析系统(RTCA),在E-Plate16的空中加入50μL 1640培养基
(5)利用RTCA检测基线,确定所选孔接触正常
(6)设置效靶比为0:1、1:1、5:1、10:1
(7)取出E-Plate16,按照效靶比,在每孔中加入混合均匀的靶细胞悬液100μL,使每孔种细胞数目为104cells/100μL。
(8)将E-Plate16置于培养箱中,以37℃,5%CO2条件下,过夜放置
(9)第二天,将E-Plate16取出,加入50μL相应的效应细胞,计算加入效应细胞8h后的杀伤率。
检测结果如图7所示。MSLN4的CAR-T对MSLN抗原阳性肿瘤细胞杀伤作用明显优于其余四组,特别是明显高于MSLN2和MSLN5组。图6和图7结果综合显示,体外杀伤实验结果表明截短的髓样细胞触发性受体,即TREM1cut作第一信号传导结构域在提高CAR安全性的基础上具备很强的抗肿瘤活性,该CAR信号区设计有利于临床应用。
最后说明的是,以上优选实施例仅用以说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
序列表
<110> 南京卡提医学科技有限公司
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Claims (7)
1.一种嵌合抗原受体,其特征在于包括:抗原结合结构域和信号传导结构域,其中信号传导结构域包括第一传导结构域和第二传导结构域,第一传导结构域和第二传导结构域之间串联抗原结合结构域;所述的第一传导结构域的氨基酸序列如SEQ ID NO.8所示;所述第二传导结构域为DAP12;第二传导结构域通过T2A与抗原结合结构域串联。
2.根据权利要求1所述的嵌合抗原受体,其特征在于,所述的嵌合抗原受体中串联的第一传导结构域、抗原结合结构域和第二传导结构域,在抗原结合结构域特异性结合抗原后变成能够传递激活信号的多链形式,将激活信号传输到表达其的免疫细胞,实现免疫治疗的作用。
3.根据权利要求1所述的嵌合抗原受体,其特征在于,所述氨基酸序列如SEQ ID NO.2所示,DAP12核苷酸序列如SEQ ID NO.1所示;所述T2A氨基酸序列如SEQ ID NO.4所示,核苷酸序列如SEQ ID NO.3所示。
4.根据权利要求3所述的嵌合抗原受体,其特征在于,所述的第一传导结构域核苷酸序列如SEQ ID NO.7所示。
5.根据权利要求1所述的嵌合抗原受体,其特征在于,所述的嵌合抗原受体为DAP12、T2A、抗原结合结构域和第一传导结构域按顺序通过2-10个任意氨基酸进行串联。
6.带有权利要求1所述的嵌合抗原受体的免疫细胞。
7.权利要求1~5任一项所述的嵌合抗原受体或权利要求6所述的免疫细胞在制备用于肿瘤免疫治疗药物中的应用。
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