CN117447604A - 靶向c-Met的嵌合抗原受体及应用 - Google Patents
靶向c-Met的嵌合抗原受体及应用 Download PDFInfo
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- CN117447604A CN117447604A CN202210880716.XA CN202210880716A CN117447604A CN 117447604 A CN117447604 A CN 117447604A CN 202210880716 A CN202210880716 A CN 202210880716A CN 117447604 A CN117447604 A CN 117447604A
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Abstract
本发明属于肿瘤治疗技术领域，具体公开一种靶向c‑Met的嵌合抗原受体及应用，包括依次连接的信号肽、靶向c‑Met的单链抗体、铰链区、跨膜区、共刺激信号区和信号转导区，单链抗体包括氨基酸序列如SEQ ID No.5、7、10、11、14、22、23、24、25所示的至少一条单链抗体，利用本发明上述靶向c‑Met的嵌合抗原受体构建的CAR‑T细胞，对c‑Met阳性肿瘤细胞具有特异性靶向作用，体内外杀伤作用显著，能够有效清除c‑Met阳性肿瘤细胞，在肿瘤治疗领域具有重要意义。
Description
技术领域
本发明属于肿瘤治疗技术领域，具体涉及一种靶向c-Met的嵌合抗原受体及应用。
背景技术
嵌合抗原受体T细胞(CAR-T)疗法已经被证实在血液瘤中取得了很好的疗效，目前已经有七款靶向血液瘤的CAR-T(Chimeric Antigen Receptor-T)产品获得批准。与血液瘤相比，CAR-T在实体瘤中的临床疗效并不理想。导致CAR-T在实体瘤中失败的主要原因之一为肿瘤细胞存在异质性，并非所有肿瘤细胞都表达同样的靶抗原，导致一部分肿瘤无法被CAR-T识别，而未被识别和杀伤的细胞生长起来导致肿瘤逃逸。因此，找到能够在大部分肿瘤细胞上，尤其是在引起转移的肿瘤细胞上也表达的靶点，能够提高CAR-T治疗的有效性；同时筛选出适当的单链抗体，也是保证CAR-T治疗实体瘤有药效和安全性的关键。
MET属于受体酪氨酸激酶(receptor tyrosine kinase,RTK)，是肝细胞生长因子(HGF)的受体。MET蛋白由高度糖基化的45kDa的α亚基和145kDa的跨膜型β亚基组成。在与HGF结合后，MET发生自磷酸化，3个酪氨酸残基(Y1230,Y1234,Y1235)被磷酸化。随后引起另外两个酪氨酸残基(Y1349,Y1356)的磷酸化，随后引起下游Ras/Raf/MAPK,PI3K/AKT/mTOR,和/或STAT3/5通路的激活。HGF主要由间质性细胞分泌，通过旁分泌的方式促进上皮来源细胞的生长和运动性。MET与EGFR,VEGFR,Wnt等通路形成复杂的信号网络，从而影响或引起癌症细胞的增殖、存活、迁移、侵袭、血管新生和转移。
根据Patrick C.Ma等人的报道，MET基因在100％肺癌病例(N＝32)中表达；其中61％的NSCLC病人强表达，25％的SCLC病人强表达。该团队之后检测了MET在不同癌症中的表达情况，结果显示MET在结肠癌、肾癌、乳腺癌、肺癌、卵巢癌等多种癌症中高表达。其中在结肠癌中97％病例表达MET、72％肺癌病例表达MET、85％肾癌病例表达MET。多篇报道均认为MET高表达与NSCLC的不良预后有关。MET的基因扩增(amplification)和磷酸化与EGFR-TKI耐药以及无进展生存期变短有关。
考虑到MET在多种肿瘤中的广泛高表达，以及与肿瘤发生、发展、转移的密切联系。本项目开发了一种靶向MET的CAR-T疗法，用于治疗c-Met阳性的实体瘤，已发表的文章及专利中，CAR结构中的单链抗体未经过体内和体外的大量筛选，所用抗体的疗效和安全性非最佳。且已发表的文章专利中无人鼠交叉反应的抗体，无法在小鼠模型中评估on target，offtumor毒性。
发明内容
本发明目的是提供一种靶向c-Met的嵌合抗原受体T细胞，包含多种不同序列的单链抗体、嵌合抗原受体、重组载体、CAR-T细胞及应用，用以解决CAR-T治疗实体瘤靶点少，抗体亲和力不优，毒性大，疗效差等问题，所述靶向c-MET的CAR-T细胞能够特异性的杀伤表达c-MET的肿瘤细胞，在体内能够有效抑制肿瘤的生长，部分抗体序列可以结合小鼠的c-MET,可用于体内安全性评估。发明的靶向c-Met的CAR-T产品能够治疗多种实体肿瘤，应用范围更广。
为了实现上述目标，本发明采用如下的技术方案：
本发明通过大量筛选后，提供一种靶向c-Met的单链抗体，单链抗体包括氨基酸序列如SEQ IDNo.5、7、10、11、14、22、23、24、25的至少一条单链抗体，通过构建数种CAR表达载体，将CAR表达载体包装成慢病毒载体，检测病毒滴度，将能成功包装成慢病毒的CAR慢病毒载体转染T细胞，制备成CAR-T，检测CAR-T细胞培养过程中CAR表达的稳定性、对人和鼠c-MET的识别能力，CAR-T细胞体外特异性杀伤表达c-Met的肿瘤细胞，CAR-T细胞体内抑制小鼠A549肿瘤生长。通过体内外大量筛选，得出能够特异性识别并有效杀伤c-MET阳性肿瘤的CAR分子的单链抗体。
在具体实施例中，氨基酸序列如SEQ IDNo.5、7、10、11、14、22、23、24、25所示的单链抗体能够特异性识别并有效杀伤c-MET阳性肿瘤。
可以理解，由于密码子的简并性，能够表达同一蛋白的核酸序列具有多种形式，用于实现本发明目的蛋白高效表达的核酸也应在本发明的保护范围内，具体的本发明筛选氨基酸序列部分列于下表1。
表1所筛选序列名称及其对应序列表位置
在一个具体实施例中，能有效杀伤c-MET阳性肿瘤靶的嵌合抗原受体，包括依次连接的靶向c-Met的单链抗体、铰链区、跨膜区、共刺激信号区和信号转导结构。
在一个具体实施例中，铰链区为选自以下至少一种蛋白：CD28、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154；
在一个具体实施例中，跨膜区为选自以下至少一种蛋白：T细胞受体的α、β或ζ链、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、ICOS、GITR、CD40、BAFFR、HVEM、SLAMF7、NKp80、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、ITGB7、TNFR2、DNAM1、SLAMF4、CD84、CD96、CEACAM1、CRTAM、Ly9、PSGL1、CD100、SLAMF6、SLAM、BLAME、SELPLG、LTBR、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D和NKG2C；
在一个具体实施例中，共刺激信号区为选自以下至少一种蛋白：CD28、OX-40、4-1BB/CD137、CD2、CD7、CD27、CD30、CD40、PD-1、ICOS、LFA-1(CD11a/CD 18)、CD3γ、CD3delta、CD3epsilon、CD247、CD276(B7-H3)、LIGHT、(TNFSF14),NKG2C,Igα(CD79a),DAP-10,Fcγ受体,MHCI类分子,TNF受体蛋白,免疫球蛋白,细胞因子受体,整合素,信号淋巴细胞激活分子(SLAM蛋白),激活NK细胞受体,BTLA,Toll配体受体,ICAM-1,B7-H3,CDS,ICAM-1,GITR,BAFFR,LIGHT,HVEM(LIGHTR),KIRDS2,SLAMF7,NKp80(KLRFl),NKp44,NKp30,NKp46,CD19,CD4,CD8alpha.、CD8beta、IL-2R beta、IL-2R gamma、IL-7R alpha、ITGA4、VLA1、CD49a、ITGA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a，LFA-1，ITGAM，CD1，ITGAX，CD11c，ITGB1，CD29，ITGB2，CD 18，LFA-1，ITGB7，NKG2D,TNFR2,TRANCE/RANKL,DNAMI(CD226),SLAMF4(CD244,2B4),CD84,CD96,CEACAM1,CRTAM,Ly9(CD229),CD160(BY55),PSGL1,CD100(SEMA4D),CD69、SLAMF6(NTB-A、Lyl08)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELLPG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD 19a、CD83特异性结合的配体；
在一个具体实施例中，信号转导区为选自以下至少一种蛋白：2B4、4-1BB、TLA、CD2、CD22、CD27、CD28、CD30、CD3d、CD3d-ITAM、CD3e、CD3e-ITAM、CD3g、CD3g-ITAM、CD3z、CD3z-ITAM1、CD3z-ITAM2、CD3z-ITAM3、CD4、CD40、CD79a、CD79a ITAM、CD79b、CD79b ITAM、CD84、CD8a、CD8b、CRACC、CRTAM、CTLA-4、DAP10、DAP12、DAP12-ITAM、DNAM-1、DR3、FCER1G、FCER1G-ITAM、FCGR1A、FCGR2A、FCGR2B、FCGR3A、FCRL1、FCRL2、FCRL3、FCRL4、FCRL5、FCRL6、GITR、HVEM、ICOS、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL4、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、LAG3、LAT、LILRB1、LILRB2、Ly9、NKG2A、NKG2C、NKG2D、NKp30、NKp44、NKp46、OX40、PD-1、PILRB、SIRPa、SLAMF1、SLAMF6、SLAMF7、TIGIT、TIM1、TIM3、TLR1、TLR10、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9。
在一个更具体CAR结构实施例中，靶向c-Met的嵌合抗原受体，包括顺次连接的单链抗体、CD28铰链区、CD28跨膜区、CD28共刺激信号区和CD3ζ信号转导区。
CD28序列为GenBank登录号为NP_006130.1的蛋白质序列；
CD28铰链区由SEQ IDNo.29所示的核苷酸序列编码获得；
CD28跨膜区由SEQ IDNo.30所示的核苷酸序列编码获得；
CD28共刺激信号区由SEQ IDNo.31所示的核苷酸序列编码获得；
CD3ζ序列为GenBank上登录号为BAG36664.1的蛋白质序列；
CD3ζ信号转导区由SEQ IDNo.32所示的核苷酸序列编码获得。
可以理解，本发明的各段多肽(例如单链抗体、铰链区、跨膜结构域、共刺激结构域和信号转导区)可以独立地选自相同或者不同的种属来源，例如鼠(小鼠、大鼠)、兔、绵羊、山羊、马、鸡、牛、犬以及人，优选种属来源为人。
本发明一实施例的重组载体，其含有SEQ IDNo.5、7、10、11、14、22、23、24、25所示的至少一条单链抗体。
在一个具体示例中，上述重组载体选自逆转录病毒载体、慢病毒载体、腺病毒载体、腺病毒相关病毒载体或CRISPR/CAS质粒。优选地，上述重组载体为慢病毒载体。慢病毒载体可以将外源基因有效地整合到宿主染色体上，从而达到持久性表达目的序列的效果。在感染能力方面可有效地感染神经元细胞、肝细胞、肿瘤细胞、内皮细胞、干细胞等多种类型的细胞，从而达到良好的基因治疗效果。对于一些较难转染的细胞，如原代细胞、干细胞、不分化的细胞等，使用慢病毒载体，能大大提高目的基因的转导效率，且目的基因整合到宿主细胞基因组的几率大大增加，能够比较方便快捷地实现目的基因的长期、稳定表达。可以理解，载体的类型并不限于此，可根据具体需要进行调整。此外，载体还可包含基因工程中常用的调控元件，例如增强子、启动子等及其他表达控制元件(例如转录终止信号、或者多腺苷酸化信号和多聚U序列等)。
本发明一实施例的CAR-T细胞，其含有如上所述的重组载体所转化。
在一个具体示例中，上述CAR-T细胞的类型为辅助T细胞、细胞毒性T细胞、记忆T细胞、调节性T细胞、MAIT细胞和γδT细胞中的任意一种。可选地，上述CAR-T细胞的类型为CD3+T细胞、CD3+CD4+T细胞或CD3+CD8+T细胞。
本发明一实施例的药物组合物，包括如上所述的CAR-T细胞，以及药学上可接受的辅料。具体的本发明的CAR-T嵌合抗原受体可与其他药物联用，在手术、化疗、放疗、小分子药，抗体药中具体应用。
本发明达到的技术效果有：
1.体内外筛选了26个不同的抗体的单链可变区，筛选出SEQ IDNo.5、7、10、11、14、22、23、24、25所示的至少一条单链抗体适合用来制备CAR-T的序列
2.比较了体内外的药效，选择出药效最好的几种抗体，且与5D5相比药效更好，抗体序列有别于其他专利。
3.提供了四个人鼠交叉反应的抗体单链可变区序列，流式验证了对人和鼠靶抗原的结合，并且对表达人和鼠的靶细胞具有特异性杀伤，可以在体内评估安全性。
附图说明
图1 CAR结构示意图；
图2慢病毒滴度测定结果显示图；
图3病毒感染3天后CAR表达结果显示图；
图4病毒感染7天后CAR表达显示图；
图5 CAR-T与mouse c-Met结合效果图；
图6肿瘤细胞株c-Met表达检测结果显示图；
图7过表达人和鼠c-Met细胞株抗原表达显示图；
图8 CAR-T对靶细胞及IFN-r释放效果图；
图9人鼠交叉CAR-T杀伤靶细胞IFN-r释放效果图；
图10 CAR-T有效抑制A549异种移植瘤效果图。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述，并给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
下面将结合具体实施例以及附图对本发明的实施方案进行详细描述。
实施例1：质粒构建及病毒包装
1.1 CAR质粒构建
所有scFV抗体区域均通过基因合成获得，CAR剩余组件(按序列顺序包括CD28铰链区、CD28跨膜区、CD28共刺激信号区、CD3ζ信号转导区)作为一个片段进行基因合成，最后分别将scFV片段与CAR剩余组件片段以及慢病毒穿梭质粒载体pIMHEF进行Gibson assembly重组。重组后进行转化、涂板，挑取单克隆进行菌检以及测序，分析测序结果得到正确克隆，CAR分子的结构如图1所示。
1.2 c-Met过表达质粒构建
Human c-Met CDS及mouse c-Met CDS从商业化表达载体中PCR扩增(载体购买自义翘神州)，Flag tag通过PCR引物添加至CDS 3’端，Puro抗性基因从其他载体模板PCR获得，CDS片段及Puro抗性基因片段以及慢病毒穿梭质粒载体pIMHEF进行Gibson assembly重组。重组后进行转化、涂板，挑取单克隆进行菌检以及测序，分析测序结果得到正确克隆，本发明具体制备得到和使用的Human c-Met的氨基酸如SEQ IDNo.27所示，mouse c-Met的氨基酸如SEQIDNo.28所示。
构建步骤如下：
Human c-Met过表达载体：P21274-f1-F&P21274-f1-R1扩增hC-MET质粒获得完整hC-MET CDS序列并添加重组臂(片段274-1)，P21274-f1-F&P21274-f1-R2再次扩增上轮产物添加3XFLAG序列(片段274-2)，P21274-f2-F&P21007-f2-R扩增获得P2A+PUROresistance序列并添加重组臂(片段274-3)，片段274-2、274-3与载体进行重组。
Mouse c-Met过表达载体：P21275-f1-F&P21275-f1-R1扩增mC-MET质粒获得完整mC-MET CDS序列并添加重组臂(片段275-1)，P21275-f1-F&P21274-f1-R2再次扩增上轮产物添加3XFLAG序列(片段275-2)，P21274-f2-F&P21007-f2-R扩增获得P2A+PUROresistance序列并添加重组臂(片段275-3)，片段275-2、275-3与载体进行重组
PCR扩增所用引物序列如下表2所示：
表2引物序列
1.3慢病毒病毒包装
293F细胞使用专用的培养基加1％青霉素/链霉素双抗进行培养，取生长状态良好且培养时间小于22天的细胞进行病毒包装。用293F培养基分别配置PEI及辅助质粒PZ201/PZ202/PZ203+pLenti主质粒混合物，室温静置5～20分钟，移液管将PEI及质粒混合,晃动混匀,室温静置15min左右；混合好的转染混合物加入至培养293F的摇瓶中，细胞放回培养箱，37℃,5％C02,摇床110rpm/min进行培养。
包装细胞培养40-48h后，收集上清，500g离心10min，用0.45μm滤器过滤上清至50ml离心管中，包好封口膜，10000g，4℃离心2h，用200μl病毒保存液溶解沉淀后，取2ul进行滴度测定，剩余贴好标签，后置于-80℃保存。
1.4慢病毒滴度检测
1.4.1细胞感染：Jurkat-T细胞密度调整到为0.4M/ml；24孔板中每孔加入500ul，每一组病毒需3个孔，且每次实验需要准备两个空白对照孔；取2ul浓度的病毒原液，加入至198ul RPMI 1640+10％FBS+1％PS培养基中，后分别取2ul、10ul、50ul至三个布好Jurkat-T细胞的孔中，最后两个孔为阴性对照，轻摇混匀；放入37℃，5％CO2培养箱中培养48小时；
1.4.2：流式检测：每孔吸出100ul细胞悬液，吹打几下使得结团的细胞充分分散开，用含有2％FBS的PBS洗去培养基(一次)，100ul重悬细胞，并用对应抗体染色15分钟，流式检测。按阳性百分比计算病毒滴度，三组(2ul、10ul、50ul)结果取最小值作为最后结果；病毒的滴度检测如图2所示，217、222、223、229、230、232、234、235未能成功包装病毒，说明这些抗体序列不适合构建CAR分子。
实施例2：CAR-T细胞制备
使用CD3分选磁珠从人外周血单个核细胞(PBMC)中分离T细胞。将分选出的T细胞加入到提前包被了CD3、CD28抗体的孔板中，激活T细胞。T细胞激活24～72h后加入CAR表达慢病毒进行感染(MOI＝3～10),同时制备未感染病毒的unT细胞，使用X-VIVO15培养基+5％胎牛血清+青霉素100U/mL+链霉素0.1mg/mL+300IU/mL IL-2进行培养；感染慢病毒的24h后，300g离心5min，去上清，培养基重悬T细胞即为CAR-T细胞；
实施例3：CAR对human c-Met结合及CAR表达稳定性
为了检测不同CAR-T是否能结合human c-Met抗原，及CAR表达是否稳定，在感染慢病毒3d后及7d，unT和各组CAR-T细胞各取两份，PBS清洗后，用human c-Met-His蛋白各组细胞孵育15分钟，清洗后用anti His的流式抗体进行标记15分钟，用流式细胞仪进行检测。其中使用的human c-Met-His蛋白购自ACRO，PE-anti His及APC-anti His抗体购自Biolegend。
检测结果如图3及图4所示，207、208不能与human c-Met抗原结合，216、219、220、221、224、225、226、227、228、231、233、273、309、310、311、312均能够结合。大部分的CAR-T细胞在培养过程中CAR的表达相对稳定，荧光强度变化不大，CAR阳性比例有一定程度的增加。216及221两组CAR阳性率低，且在培养第7天时，CAR表达的荧光强度明显减弱，阳性率下降。该结果表明，并非所有抗体序列都适合用来制备CAR-T,这也体现了我们大量筛选的必要性。
实施例4：CAR-T细胞与mouse c-Met抗原结合检测
为了检测不同CAR-T是否能够交叉识别人和鼠的c-Met抗原，将unT和各组CAR-T细胞与mouse c-Met-His蛋白孵育15分钟，清洗后用anti His的流式抗体进行标记15分钟，用流式细胞仪进行检测。其中使用的mouse c-Met-His蛋白购自义翘神州，PE-anti His及APC-anti His抗体购自biolegend。
检测结果如图5所示，309、310、311、312四组CAR-T能够与mouse c-Met抗原结合，其他的CAR-T均不能够结合。该四组CAR-T可以用于在小鼠中评估靶向c-Met的on target，off tumor毒性。
实施例5：肿瘤细胞株c-Met表达检测
肿瘤细胞A549及MIACaPa2用胰蛋白酶消化，各准备两管细胞，每管5E5细胞，用PBS+2％FBS清洗，重悬至100ulPBS+2％FBS中，分别加入5ul isotype抗体及FITC-anti humanc-Met抗体，孵育15分钟后PBS+2％FBS清洗，重悬后上流式细胞仪检测，抗体购自Thermofisher。
结果如图6所示，A549表达c-MET，作为抗原阳性靶细胞；MIACaPa2不表达c-MET，作为抗原阴性靶细胞。
实施例6：K562过表达human c-Met、mouse c-Met细胞株构建
K562细胞用RPMI1640+10％FBS培养，取状态良好的K562细胞分成三份，每份1E6细胞在六孔板中培养，分别加入过表达human c-Met、mouse c-Met的慢病毒载体(MOI＝3)进行感染、以未加病毒作为对照，制备K562-human c-Met、K562-mouse c-Met、K562细胞株，病毒感染2d后检测抗原表达。
检测结果如图7所示，K562-human c-Met、K562-mouse c-Met分别表达humanc-Met抗原及mouse c-Met抗原，对照K562细胞不表达这两种抗原。
实施例7：CAR-T细胞能够特异性杀伤表达c-Met抗原的肿瘤靶细胞
为了检测不同CAR-T对c-Met阳性肿瘤的特异性杀伤。将1E5CFSE标记的A549细胞或MIA PaCa-2分别铺于96孔板中，X-VIVO15+5％FBS培养基体积为100ul，次日用unT细胞将各组CAR-T CAR阳性率调成一致，对应96孔板中加入1E5CAR阳性细胞，unT组加入的量与CAR-T组细胞总数一致，体积为100ul。CAR-T细胞与肿瘤细胞孵育24h后，离心取上清ELISA检测上清中IFN-r的释放,用同等体积的PBS清洗剩余细胞，胰酶消化靶细胞并用相同体积的培养基终止消化，合并收集细胞及液体，同等流速收集30ul体积下靶细胞的总数，计算杀伤效率，ELISA试剂盒从R&D购买。
检测结果如图8所示，unT细胞对A549及MIA Capa2均无杀伤作用。216、219、220、221、224、225、226、227、228、231组细胞对A549有杀伤,对MIACapa2细胞无杀伤作用。表明各组CAR-T细胞能够特异性杀伤c-Met阳性的肿瘤细胞株。
实施例8：人鼠交叉反应CAR-T能够杀伤人和鼠c-Met阳性靶细胞
为了检测不同人鼠交叉反应CAR-T对human c-Met及mouse c-Met阳性肿瘤的特异性杀伤。将2E5K562细胞、K562-human c-Met或K562-mouse c-Met分别铺于U底96孔板中，X-VIVO15+5％FBS培养基体积为100ul，用unT细胞将各组CAR-T CAR阳性率调成一致，对应96孔板中加入1E5CAR阳性细胞，unT组加入的量与CAR-T组细胞总数一致，体积为100ul。CAR-T细胞与肿瘤细胞孵育24h后，离心取上清。ELISA检测上清中IFN-r的释放,ELISA试剂盒由R&D购买。
检测结果如图9所示，unT细胞对各肿瘤细胞均无杀伤作用。309、310、311、312CAR-T组细胞对K562-human c-Met及K562-mouse c-Met均有杀伤,上清中释放IFN-r，但对K562细胞无杀伤作用。表明人鼠交叉反应CAR-T细胞能够特异性杀伤humnan c-Met及mouse c-Met阳性的肿瘤细胞株。
实施例9：c-Met CAR-T能有效抑制A549异种移植瘤
人源非小细胞肺癌细胞A549细胞，连续培养小于十代，5E6细胞混合matrigel皮下接种M-NSG小鼠，11天后，用unT调整各组CAR-T阳性率一致，尾静脉注射2E6CAR阳性的CAR-T细胞，unT细胞注射量与CAR-T细胞总量一致。连续观察小鼠体重(Body weigth，BW)及肿瘤体积(Tumor Volume,TV)，在注射CAR-T第8天，233组显示出明显毒性，小鼠死亡；224组第30天小鼠体重下降，可能由GvHD导致；其余组小鼠体重未见明显下降。
从图10及表3中可看出，219、221、224、225、228组CAR-T显示出明显的肿瘤抑制效果，相对肿瘤体积(Relative Tumor Volume,RTV)较unT组明显降低。来源于5D5抗体的273组抑瘤效果不明显，与专利US10800840相比，在A549的肿瘤模型中药效较差，由于不同的模型对CAR-T的敏感性不同。220、227、231、233、226组抑瘤效果不明显。这些结果也表明，本发明筛选出了安全性及有效性更高的抗体序列及CAR结构。
表3 A549异种移植瘤相对肿瘤体积(RTV)
Claims (9)
1.一种靶向c-Met的嵌合抗原受体，其特征在于，包括依次连接的靶向c-Met的单链抗体、铰链区、跨膜区、共刺激信号区和信号转导区，所述单链抗体包括氨基酸序列如SEQ IDNo.5、7、10、11、14、22、23、24、25所示的至少一条单链抗体。
2.根据权利要求2所述的一种靶向c-Met的嵌合抗原受体，其特征在于，
所述铰链区为选自以下至少一种蛋白：CD28、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154；
和/或所述跨膜区为选自以下至少一种蛋白：T细胞受体的α、β或ζ链、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、ICOS、GITR、CD40、BAFFR、HVEM、SLAMF7、NKp80、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、ITGB7、TNFR2、DNAM1、SLAMF4、CD84、CD96、CEACAM1、CRTAM、Ly9、PSGL1、CD100、SLAMF6、SLAM、BLAME、SELPLG、LTBR、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D和NKG2C；
和/或所述共刺激信号区为选自以下至少一种蛋白：CD28、OX-40、4-1BB/CD137、CD2、CD7、CD27、CD30、CD40、PD-1、ICOS、LFA-1(CD11a/CD 18)、CD3γ、CD3 delta、CD3 epsilon、CD247、CD276(B7-H3)、LIGHT、(TNFSF14),NKG2C,Igα(CD79a),DAP-10,Fcγ受体,MHCI类分子,TNF受体蛋白,免疫球蛋白,细胞因子受体,整合素,信号淋巴细胞激活分子(SLAM蛋白),激活NK细胞受体,BTLA,Toll配体受体,ICAM-1,B7-H3,CDS,ICAM-1,GITR,BAFFR,LIGHT,HVEM(LIGHTR),KIRDS2,SLAMF7,NKp80(KLRFl),NKp44,NKp30,NKp46,CD19,CD4,CD8alpha、CD8beta、IL-2R beta、IL-2R gamma、IL-7R alpha、ITGA4、VLA1、CD49a、ITGA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a，LFA-1，ITGAM，CD1，ITGAX，CD11c，ITGB1，CD29，ITGB2，CD18，LFA-1，ITGB7，NKG2D,TNFR2,TRANCE/RANKL,DNAMI(CD226),SLAMF4(CD244,2B4),CD84,CD96,CEACAM1,CRT AM,Ly9(CD229),CD160(BY55),PSGL1,CD100(SEMA4D),CD69、SLAMF6(NTB-A、Lyl08)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELLPG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD 19a、CD83特异性结合的配体；
和/或所述信号转导区为选自以下至少一种蛋白：2B4、4-1BB、TLA、CD2、CD22、CD27、CD28、CD30、CD3d、CD3d-ITAM、CD3e、CD3e-ITAM、CD3g、CD3g-ITAM、CD3z、CD3z-ITAM1、CD3z-ITAM2、CD3z-ITAM3、CD4、CD40、CD79a、CD79a-ITAM、CD79b、CD79b-ITAM、CD84、CD8a、CD8b、CRACC、CRTAM、CTLA-4、DAP10、DAP12、DAP12-ITAM、DNAM-1、DR3、FCER1G、FCER1G-ITAM、FCGR1A、FCGR2A、FCGR2B、FCGR3A、FCRL1、FCRL2、FCRL3、FCRL4、FCRL5、FCRL6、GITR、HVEM、ICOS、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL4、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、LAG3、LAT、LILRB1、LILRB2、Ly9、NKG2A、NKG2C、NKG2D、NKp30、NKp44、NKp46、OX40、PD-1、PILRB、SIRPa、SLAMF1、SLAMF6、SLAMF7、TIGIT、TIM1、TIM3、TLR1、TLR10、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9。
3.根据权利要求2所述的一种靶向c-Met的嵌合抗原受体，其特征在于，包括顺次连接的单链抗体、CD28铰链区、CD28跨膜区、CD28共刺激信号区和CD3ζ信号转导结构。
4.根据权利要求3所述的一种靶向c-Met的嵌合抗原受体，其特征在于，所述CD28铰链区由如SEQ IDNo.29所示的核苷酸序列编码获得；
和/或CD28跨膜区由如SEQ IDNo.30所示的核苷酸序列编码获得；
和/或CD28共刺激信号区由如SEQ IDNo.31所示的核苷酸序列编码获得；
和/或CD3ζ信号转导结构由如SEQ IDNo.32所示的核苷酸序列编码获得。
5.一种靶向c-Met的重组载体，其特征在于，包括权利要求1-4任一所述靶向c-Met的嵌合抗原受体的基因和初始载体。
6.根据权利要求5所述的一种靶向c-Met的重组载体，其特征在于，所述初始载体为慢病毒载体。
7.一种CAR-T细胞，其特征在于，由权利要求5或6所述的重组载体转染T细胞获得。
8.一种药物组合物，其特征在于，包括权利要求7所述的CAR-T细胞，以及药学上可接受的辅料。
9.权利要求1-4任一所述的靶向c-Met的嵌合抗原受体、权利要求6或7所述的重组载体、权利要求7所述的CAR-T细胞，权利要求8所述的药物组合物在制备用于治疗c-MET阳性肿瘤药物中的应用。
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