CN116640218A - 一种靶向kras g12v单链抗体片段、嵌合抗原受体car及应用 - Google Patents
一种靶向kras g12v单链抗体片段、嵌合抗原受体car及应用 Download PDFInfo
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Abstract
本发明提高一种靶向KRAS G12V单链抗体片段、嵌合抗原受体CAR及应用,该发明基于KRAS G12V靶点,对TCR进行改造。保留TCR识别肿瘤特异性抗原的胞外信号区,和传统CAR结构中的胞外间隔区、跨膜区以及CD3ζ胞内信号域串联,使改造后的嵌合抗原受体CAR能够特异性识别HLA‑A*02:01递呈的KRAS G12V突变多肽。同时,借助CAR‑T/NK细胞治疗的优势,探索潜在的肿瘤治疗新方案,为临床试验奠定基础。
Description
技术领域
本发明涉及一种靶向KRAS G12V单链抗体片段、嵌合抗原受体CAR及应用,属于生物医药领域。
背景技术
细胞治疗技术已成为近年来最引人注目的领域之一,它在癌症、血液疾病、心血管疾病、糖尿病和神经系统疾病等领域均展示出良好的治疗前景,在各种疾病尤其在肿瘤免疫治疗中发挥了重要作用,目前已有十余款免疫细胞疗法上市。免疫细胞治疗虽然在血液及淋巴系统肿瘤中表现出良好的治疗效果,但在实体瘤治疗领域仍具有一定的局限性。目前临床试验中使用的多为肿瘤相关抗原(TAA),如间皮素(MSLN)、表皮生长因子受体(EGFR)、紧密连接蛋白Claudin18.2(CLDN18.2)等,但TAA在部分正常组织中也有表达,这给细胞疗法带来了脱靶风险和安全性问题。因此,实体瘤高特异性靶点的选择是亟需解决的首要问题。
肿瘤抗原分为肿瘤相关抗原(Tumor-associated antigens,TAA)和肿瘤特异性抗原(Tumor-specific antigen,TSA)。肿瘤相关抗原(TAA)指指肿瘤细胞和正常细胞组织均可表达的抗原,只是其含量在细胞癌变时明显增高,此类抗原只表现出量的变化而无严格的肿瘤特异性。肿瘤特异性抗原(TSA)是指肿瘤细胞特有的或只存在于某种肿瘤细胞而不存在于正常细胞的一类抗原。肿瘤新抗原(Neoantigen)是一种来源于非同义突变的肿瘤特异性抗原,是肿瘤免疫治疗中非常有吸引力的靶点。与TAA不同,TSA/新抗原是从肿瘤基因组的遗传改变中获得的突变肽,在肿瘤细胞中特异表达,在正常组织中不存在,因此显示出很高的肿瘤特异性并降低了非靶向毒性。基于新抗原的个性化免疫疗法可以激发针对肿瘤细胞的强大的抗肿瘤免疫反应。
RAS是第一个被发现的人类肿瘤基因,也是肿瘤中常见的突变基因,主要有三种亚型:HRAS、KRAS和NRAS。其中,KRAS是RAS家族中最主要的突变型,并且与22%的人类肿瘤相关,是人类三大致命性癌症,肺癌(17%),结直肠癌(33%)和胰腺癌(61%)中高频突变基因。在健康细胞中,KRAS是调节细胞生长的开关,在调节细胞增殖等多种细胞信号事件中发挥重要作用。然而,当KRAS基因发生突变时,KRAS会卡在“开启”位置,使细胞不受控制地生长并激活下游通路,导致细胞开始恶性增殖,引发癌症发生和转移。97%的KRAS突变发生在第12和13号密码子上,最常见的是KRAS G12C、G12D、G12V和G13D突变。其中G12V突变,已有临床研究证明其在多个癌种的免疫治疗中极具研究价值,是肿瘤免疫治疗的理想靶点。
嵌合抗原受体(chimeric antigen receptor,CAR)是一种人工合成的受体,能引导免疫细胞特异性地追踪识别和清除表达相关靶配体的肿瘤细胞,通常由能够识别肿瘤相关抗原的胞外结合域(一般来自单抗的抗原结合区的scFv片段)、铰链域、跨膜域和TCR分子的胞内信号域组成,具有高度靶向性。CAR以抗原依赖、非MHC(Major histocompatibilitycomplex)限制的方式结合肿瘤抗原,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能够识别更广泛的肿瘤细胞。
HLA-A*02等位基因在44%的高加索人群中表达,在中国人群中表达的比例高达37.7%,其中,HLA-A*02:01在中国人群中的比例超过10%。通过NetMHCpan-4.0生物信息学算法预测了HLA-A*02:01和RAS G12V突变蛋白的亲和力。传统的TCR-T可以识别由MHC递呈的胞内和胞外肿瘤抗原,对低水平突变的胞内抗原具有超敏感识别,但是有MHC限制性。而CAR-T/NK只能识别肿瘤细胞表面抗原。
发明内容
为解决上述技术问题,本发明第一个目的,提供一种靶向KRAS G12V单链抗体片段,包括重链可变区和轻链可变区,其中,重链可变区包括VH-CDR1、VH-CDR2、VH-CDR3轻链可变区包括VL-CDR1、VL-CDR2、VL-CDR3;
其中,VH-CDR1氨基酸序列为SEQ ID NO:3所示的序列,VH-CDR2氨基酸序列为SEQID NO:4所示的序列,VH-CDR3氨基酸序列为SEQ ID NO:5所示的序列;
SEQ ID NO:3:GSYIH;
SEQ ID NO:4:YIDPEYGYSRYADSVKG;
SEQ ID NO:5:DSDSDAMDV;
VL-CDR1氨基酸序列为SEQ ID NO:8所示的序列,VL-CDR2氨基酸序列为SEQ IDNO:9所示的序列,VL-CDR3氨基酸序列为SEQ ID NO:10(QQYYYPYPT)所示的序列。
SEQ ID NO:8:RASQDVNVAVA;
SEQ ID NO:9:SSSFLYS;
SEQ ID NO:10:QQYYYPYPT。
进一步地,所述重链可变区的全长氨基酸序列为SEQ ID NO:6所示的氨基酸序列;
SEQ ID NO:6:
ASEVQLESGGGLVQPGGSLLRSCAASGFNINGSYIHWVRQAPGKGLEWV AYIDPEYGYSRYADSVKGRFTISKNTSADTAYLQMNSLRATAEDVYYCSRDSD SDAMDVWGQGTLVTVSS;
或者如SEQ ID NO:7所示的氨基酸序列;
SEQ ID NO:7:
ASEVQLESGSGLVQPGGSLRLSCCASGFHNIGSYIHWVAQPAGKGLKWVR YIDPEYGYSRYADSVKGRFAISADMSKNTAYLQMNSLRAEDTAVYYYSRDSD SDAMDVWGQGTLVVVSS;
所述轻链可变区的全长氨基酸序列为SEQ ID NO:11所示的氨基酸序列;
SEQ ID NO:11:
DIQMTQSSSSLSASVGDRVTITCRASQDVNVAVAWYQQKPGKAPKLLIYSS SFLYSGVPSRFSGSRSGTDFTLSSLQTIPEDFATYYCQQYYYPYPTFGTGQKVEI KRT;
或者如SEQ ID NO:12所示的氨基酸序列。
SEQ ID NO:12:
DIQMTQMPSSLSASVGDRVTIACRASQDVNVAVAWYQQKPGKAPKLILYS SSFLYSGVPSRFSRFGSGQDFTTTISSLQPEDFATAYCQQYYYPYPTFGAGQKV EIKRT;
进一步地,所述重链可变区和轻链可变区由连接区连接,连接区由3个四肽多聚体组成,氨基酸序列如SEQ ID NO:13所示。
SEQ ID NO:13:GGGSGGGGSGGG。
进一步地,单链抗体可变区的氨基酸序列如SEQ ID NO:14所示或者至少80%的同源性序列;
SEQ ID NO:14:
DIQMTQSSSSLSASVGDRVTITCRASQDVNVAVAWYQQKPGKAPKLLIYSSSFLYSGVPSRFSGSRSGTDFTLSSLQTIPEDFATYYCQQYYYPYPTFGTGQKVEIKRTGGGSGGGGSGGGASEVQLESGGGLVQPGGSLLRSCAASGFNINGSYIHWVRQAPGKGLEWVAYIDPEYGYSRYADSVKGRFTISKNTSADTAYLQMNSLRATAEDVYYCSRDSDSDAMDVWGQGTLVTVSS;
或者如SEQ ID NO:15所示或者至少80%的同源性序列。
SEQ ID NO:15:
DIQMTQMPSSLSASVGDRVTIACRASQDVNVAVAWYQQKPGKAPKLILYSSSFLYSGVPSRFSRFGSGQDFTTTISSLQPEDFATAYCQQYYYPYPTFGAGQKVEIKRTGGGSGGGGSGGGASEVQLESGSGLVQPGGSLRLSCCASGFHNIGSYIHWVAQPAGKGLKWVRYIDPEYGYSRYADSVKGRFAISADMSKNTAYLQMNSLRAEDTAVYYYSRDSDSDAMDVWGQGTLVVVSS;
本发明的第二个目的,提供一种嵌合抗原受体CAR,包括抗原结合结构域、跨膜结构域、胞内信号结构域;其中抗原结构域包括信号肽、Myc标签和识别抗原的权利要求1-3所示的单链抗体片段。
进一步地,所述信号肽为CD8、GM-CSF、CD4、CD28、CD137、或其突变体/修饰体、或其组合。
进一步地,所述信号肽为CD8,氨基酸序列如SEQ ID NO:1所示。
SEQ ID NO:1:MALPVTALLLPLALLLHAARP;
进一步地,所述的Myc标签是一种来源于c-myc基因的表位标签,作为一种融合蛋白可融合至单链抗体片段的N端或C端,其氨基酸序列如SEQ ID NO:2所示。
SEQ ID NO:2:EQKLISEEDL。
进一步地,所述Myc标签融合在单链抗体片段的N端。
进一步地,所述抗原结合结构域和跨膜结构域通过铰链区相连,铰链区选自CD8α、CD28胞外结构域的衍生物、由IgG1或IgG4的FC部分或Fc部分的CH2/CH3结构域组成的基于IgG的铰链区、或DAP12。
进一步地,所述铰链区为CD8α,其氨基酸序列如SEQ ID NO:16所示。
SEQ ID NO:16:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD。
进一步地,所述跨膜结构域选自CD3ζ、CD8、CD28、NKG2D、2B4、DNAM1中的一种。
进一步地,所述跨膜结构域为CD28或CD8;其中,CD28跨膜区的氨基酸序列如SEQID NO:17所示;
SEQ ID NO:17:
FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS;
或/和
CD8跨膜区的氨基酸序列如SEQ ID NO:18所示。
SEQ ID NO:18:IYIWAPLAGTCGVLLLSLVITLYC。
进一步地,所述胞内信号结构域选自CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28家族、DAP10、DAP12、NKp44、NKG2D、TNFR家族或SLAM相关受体家族中的一种或多种。
进一步地,所述胞内信号结构域为4-1BB和CD3ζ的组合;其中,4-1BB的氨基酸序列如SEQ ID NO:19所示,CD3ζ的氨基酸序列如SEQ ID NO:20所示。
SEQ ID NO:19:
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL;
SEQ ID NO:20:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR。
本发明的第三个目的,提供一种表达上述嵌合抗原受体CAR的CAR-T细胞。
本发明的第四个目的,提供一种上述靶向KRAS G12V单链抗体片段、嵌合抗原受体CAR、CAR-T细胞在制备抗肿瘤药品或产品中的应用。
本发明所达到的有益技术效果:本发明提高一种靶向KRAS G12V单链抗体片段、嵌合抗原受体CAR及应用,基于KRAS G12V靶点,对TCR进行改造。保留TCR识别肿瘤特异性抗原的胞外信号区,和传统CAR结构中的胞外间隔区、跨膜区以及CD3ζ胞内信号域串联,使改造后的嵌合抗原受体CAR能够特异性识别HLA-A*02:01递呈的KRAS G12V突变多肽。同时,借助CAR-T/NK细胞治疗的优势,探索潜在的肿瘤治疗新方案,为临床试验奠定基础。
附图说明
图1KGV1 CAR和KGV2 CAR结构模式图;
图2流式检测KGV1 CAR-T和KGV2 CAR-T慢病毒感染效率;
图3阳性KGV1 CAR-T和KGV2 CAR-T分选效率;
图4KRAS G12V质粒、HLA-A*02:01质粒和Luciferase质粒结构示意图;
图5外源性靶细胞K562#4表达KRAS G12V突变和HLA*02:01的比例;
图6KGV1 CAR-T和KGV2 CAR-T对外源性靶细胞的杀伤;
图7外源性杀伤中细胞因子TNF-α和IFN-γ的分泌;
图8内源性靶细胞的表型鉴定;
图9KGV1 CAR-T和KGV2 CAR-T对内源性靶细胞的杀伤;
图10内源性杀伤中细胞因子TNF-α和IFN-γ的分泌。
具体实施方式
下面结合具体实施例对本发明作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案,而不能以此来限制本发明的保护范围。
下面结合附图和实施例对本发明专利进一步说明。
实施例一嵌合抗原受体CAR的序列设计与载体构建
CAR的信号肽、Myc标签、CD8α铰链区、CD8跨膜结构域、4-1BB和CD3ζ胞内结构域序列均来自公开数据库Addgene,单链抗体可变区(single-chain variable fragment,scFv)片段从实施例三所示噬菌体抗体突变文库中筛选获得。按照信号肽、Myc标签、靶向KRASG12V单链抗体片段、CD8α铰链区、CD8跨膜结构域、4-1BB和CD3ζ胞内信号结构域的顺序,连接到载体PCDH-EF1α的EcoR I和Sal I酶切位点之间构建主质粒,命名为KGV1 CAR和KGV2CAR,主质粒的结构模式图如图1所示。主质粒的具体构建过程可以为现有技术中的常用方法,在此不再赘述。
实施例二噬菌体抗体突变文库构建
从1000份健康人外周血中分离PBMC,提取mRNA并反转录为cDNA。基于已知抗体序列,利用易错PCR扩增出目的片段。PCR产物进行2%琼脂糖凝胶电泳后切下约500bp左右大小的目的条带,胶回收产物。将PCR产物和噬菌粒载体pCANTAB5E分别用SfiⅠ和NotⅠ双酶切,胶回收,按照载体:DNA bp比为1:3的比例加入T4连接酶进行连接,PCR仪16℃过夜连接。向100ul TG1感受态中加入5ul连接产物,冰上预冷,转入预冷后的电转杯中;调节电转仪电压2.5KV电击时间5ms,电击后,迅速加入0.9ml培养基,37℃振荡培养2小时。取10ul梯度稀释,涂布于SOBAG平板上,计算库容。随机挑取单克隆测序验证突变抗体库的多样性。
实施例三噬菌体抗体突变文库筛选
将1.5mL上述突变抗体库接种到300mL培养基中,37℃振荡培养约1.5h,按5倍的体积加入辅助噬菌体M13KO7超染,37℃振荡培养约1h;4000rpm 15℃离心15min,去除培养基;加入200mL培养基(100μg/mL Amp,50μg/mL Kan)重悬细菌,37℃培养2h;10000rpm离心20min去除沉淀;上清加入40mL PEG/NaCl沉噬菌体,冰浴过夜;10000rpm离心20min,去掉上清;0.6mL培养基重悬噬菌体,4℃备用。ELISA微孔板包被His Bind Resin结合抗原蛋白,以靶蛋白为固定相,以噬菌体展示文库为流动相,经过2h共孵育后,洗去未结合的游离噬菌体,然后以酸洗脱下与靶分子结合吸附的噬菌体,洗脱的噬菌体感染TG1宿主菌后经繁殖扩增,进行下一轮洗脱。经过五轮的“吸附-洗脱-扩增”,富集特异性抗体。涂平板挑单克隆依次进行ELISA验证。最终成功筛选出两株靶向KRAS G12V的单链抗体。两株单链抗体可变区的氨基酸序列分别如SEQ ID NO:14和SEQ ID NO:15所示。
SEQ ID NO:14:
DIQMTQSSSSLSASVGDRVTITCRASQDVNVAVAWYQQKPGKAPKLLIYSSSFLYSGVPSRFSGSRSGTDFTLSSLQTIPEDFATYYCQQYYYPYPTFGTGQKVEIKRTGGGSGGGGSGGGASEVQLESGGGLVQPGGSLLRSCAASGFNINGSYIHWVRQAPGKGLEWVAYIDPEYGYSRYADSVKGRFTISKNTSADTAYLQMNSLRATAEDVYYCSRDSDSDAMDVWGQGTLVTVSS;
SEQ ID NO:15:
DIQMTQMPSSLSASVGDRVTIACRASQDVNVAVAWYQQKPGKAPKLILYSSSFLYSGVPSRFSRFGSGQDFTTTISSLQPEDFATAYCQQYYYPYPTFGAGQKVEIKRTGGGSGGGGSGGGASEVQLESGSGLVQPGGSLRLSCCASGFHNIGSYIHWVAQPAGKGLKWVRYIDPEYGYSRYADSVKGRFAISADMSKNTAYLQMNSLRAEDTAVYYYSRDSDSDAMDVWGQGTLVVVSS。
实施例四慢病毒制备
(1)将5×105个HEK293F细胞接种到30ml培养基摇瓶中,3天后细胞密度达到4.0–5.0×106cells/ml左右时,进行慢病毒包装。加入1.5ml不含抗生素和血清的培养基,按照1:1的比例依次加入37.5μg主质粒、37.5μg包装质粒混合物,轻轻吹打混匀;180μl转染试剂加到1.5ml不含抗生素和血清的培养基中,轻轻混匀;质粒和转染试剂混匀后室温孵育10min;将转染试剂-DNA混合物缓慢转移至摇瓶中,轻晃混匀,置于含5%CO2的37℃培养箱。
(2)转染48小时后收取上清培养液,3000rpm/min,4℃离心10min,弃沉淀,上清用0.45μm的滤膜过滤。
(3)按照1:4的比例依次加入病毒浓缩液和上述病毒上清滤液,病毒上清滤液缓慢流加,分层,10000g/min,4℃离心4h,进行病毒浓缩。弃上清,留病毒沉淀,按照病毒上清滤液:培养基为250:1的比例加入不含抗生素和血清的培养基,溶解沉淀,分装-80℃保存备用。
实施例五T细胞制备
T细胞来源于健康人外周血,在8个50ml离心管中预先加入恢复至室温的15ml淋巴细胞分离液,取100ml肝素抗凝血与生理盐水按比例1:1稀释混匀,用移液管将25ml稀释后的血沿管壁缓慢加于淋巴细胞分离液之上,650g/min,20℃,水平离心20min,为保证分离效果,需将离心机的升速、降速调至最低。离心后可看见管内分为四层,自上而下依次为血浆、白膜层PBMC、淋巴细胞分离液、粒细胞和红细胞,将最上层血浆层吸弃,余下约1ml,用吸管沿管壁旋转吸出白膜层的PBMC,移入另一50ml离心管,补加生理盐水,470g,离心10min。弃上清,若底部红色物质较多可加入红细胞裂解液,裂解红细胞。裂解完成之后补加生理盐水,300g/min离心10min。弃上清,加入生理盐水洗涤一次,300g/min离心5min。弃上清,加入37℃预热的1640完全培养基,重悬细胞,计数并计算细胞活力。通过此密度梯度离心法分离出PBMC,然后加入浓度为200ng/ml的细胞刺激因子CD3、CD28激活T细胞,24h后加入10ng/ml的IL-7、20ng/ml的IL-15和20ng/ml的IL-21继续扩增培养。
实施例六CAR-T细胞的制备
采用慢病毒系统来实现CAR-T细胞的制备。将激活的T细胞加入MOI=10的慢病毒浓缩液进行病毒转导,并加入5μg/ml Polybrene助染剂,24小时后换液继续培养。利用流式细胞术检测到慢病毒感染效率分别为77.45%和76.01%,结果如图2所示。
实施例七阳性CAR-T细胞的分选
通过Myc-Tag(9B11)Mouse mAb(PE Conjugate)流式抗体(购自Cell SignalingTechnology公司)和Anti-PE MicroBeads(购自Miltenyi Biotec公司)磁珠分选出阳性的CAR-T细胞。具体实施方式为:每1000万细胞中加入5μlMyc-Tag(9B11)Mouse mAb(PEConjugate)抗体,4℃孵育30min。PBS洗三次,然后加入80μl分选buffer和20μl Anti-PEMicroBeads,4℃孵育15min。PBS洗三次后过LS柱。从LS柱上洗脱下来的细胞即为阳性CAR-T细胞。分选后的KGV1 CAR-T和KGV2 CAR-T的阳性率达到98%以上,结果如图3所示。
实施例八外源性表达KRAS G12V突变和HLA*02:01靶细胞的构建与验证
选择K562细胞作为外源性靶细胞,因其不表达KRAS和内源性HLA,分别将KRASG12V和HLA*02:01质粒通过慢病毒感染的方式稳转进K562细胞,72h检测表达,培养十四天后成为稳转系,流式分选出双阳性细胞,扩大培养并流式鉴定表型,结果如图5所示。其中,KRAS G12V质粒、HLA*02:01质粒和Luciferase质粒结构示意图如图4所示。同时,将表达荧光素酶的转座子质粒电转进双阳性细胞,用于后续杀伤功能实验的检测,命名为K562#4,结果如表1所示。
表1K562-luc细胞及K562#4-luc细胞的萤火虫荧光素酶检测
实施例九内源性表达KRAS G12V突变和HLA*02:01靶细胞的鉴定
选择胰腺癌细胞系CFPAC-1作为内源性靶细胞,通过测序和基因组PCR验证其内源性KRAS G12V突变和HLA*02:01均有表达。同时选择胰腺癌细胞系BxPC-3作为对照靶细胞,测序和基因组PCR验证其没有内源性KRAS G12V突变和HLA*02:01的表达。同时选择胰腺癌细胞系PANC-1作为对照靶细胞,测序和基因组PCR验证其有内源性KRAS G12D突变而无内源性KRAS G12V突变,以及少量HLA*02:01表达,结果如图9、表2和表3所示。内源性靶细胞的表型鉴定如图8所示。KRAS测序的引物序列如表4所示。
表2三种胰腺癌细胞株KRAS测序结果
表3三种胰腺癌细胞株HLA-A等位基因测序结果
表4KRAS测序的引物序列
实施例十CAR-T对外源性表达KRAS G12V和HLA-A*02:01肿瘤细胞的体外功能研究结果分析
将CAR-T细胞与K562#4肿瘤靶细胞以1:2,1:1,2:1,5:1,10:1的效靶比共培养,培养24小时后用Luciferase标记活的靶细胞检来计算杀伤效率,并检测细胞因子IFN-γ和TNF-α的表达情况。空白K562细胞作为对照。
结果显示,两种靶细胞的杀伤率随着效靶比的升高逐渐上升。K562细胞是不表达KRAS G12V和HLA-A*02:01的双阴性细胞,与对照组相比,总体不具有统计学差异(P>0.05),且两种CAR-T细胞的杀伤能力相似。而对于同时表达KRAS G12V和HLA-A*02:01的双阳性细胞K562#4来说,两种CAR-T细胞的杀伤效率在各效靶比均明显高于对照组,差异具有统计学意义(P<0.05),且两种CAR-T细胞的杀伤能力相当,结果如图6所示。
此外,我们还检测了上清中细胞因子的释放量(浓度)。在双阴性细胞K562中,尽管细胞因子的释放量随着效靶比的升高而升高,但升高幅度小,且三个实验组之间不存在明显差异。但是在双阳性细胞K562#4中,TNF-α和IFN-γ的含量随着效靶比的升高显著增加,且两个CAR-T细胞组与对照T细胞组之间的细胞因子浓度存在显著差异(P<0.05),说明K562#4细胞能够激活两种CAR-T细胞。对比两个靶细胞组也可发现,双阳性细胞K562#4的TNF-α和IFN-γ含量远远超过K562组,结果如图7所示。由该体外功能实验可知,我们设计使用的两种CAR均能够使T细胞特异性识别外源性表达的靶抗原并发挥杀伤作用,且靶向的抗原通过特异性激活能够识别其的T细胞,刺激T细胞释放更多的细胞因子,不仅能促使T细胞迅速扩增,还能强化T细胞的杀伤效应。
实施例十一CAR-T对内源性表达KRAS G12V和HLA-A*02:01肿瘤细胞的体外功能研究结果分析
将CAR-T细胞与三种胰腺癌细胞以1:5,1::2,1:1,2:1,5:1,10:1的效靶比共培养,培养24小时后用CCK8标记活的靶细胞来计算杀伤效率,以及试剂盒检测上清中细胞因子IFN-γ和TNF-α的表达。
对照组反映了T细胞对靶细胞固有的杀伤能力,对三种靶细胞的杀伤率随着效靶比的升高而缓慢升高。尽管KGV1 CAR-T细胞和KGV2 CAR-T细胞对PANC-1和BxPC-3细胞的杀伤能力也随着效靶比的升高而缓慢上升,但与对照组的杀伤接近,三者之间不存在统计学差异(P>0.05),即KGV1 CAR-T细胞和KGV2 CAR-T细胞对PANC-1和BxPC-3仅发挥了T细胞的固有杀伤效能。在CFPAC-1细胞组中,两种CAR-T细胞的杀伤能力显著高于对照组(P<0.05),在效靶比10:1时几乎能完全清除所有的靶细胞,杀伤率接近100%,结果图9所示。也就是说,KGV1 CAR-T和KGV2 CAR-T通过HLA-A*02:01递呈的KRAS G12V多肽的特异性识别使T细胞活化,赋予了T细胞固有杀伤属性以外的杀伤效力。
细胞因子的结果总体而言,每种细胞因子的含量都随着效靶比的升高而升高。其中IFN-γ的含量变化有着极其明显的趋势。CFPAC-1细胞组的两种CAR-T细胞释放的IFN-γ远高于对照组,其余组的CAR-T细胞释放的IFN-γ与对照组类似,且CFPAC-1细胞的CAR-T细胞组IFN-γ释放量远高于另外两个胰腺癌细胞的CAR-T细胞组(P<0.05),结果如图10所示。
因此,我们设计使用的两种CAR均能够使T细胞特异性识别内源性的靶抗原、活化T细胞并发挥杀伤作用。由于T细胞激活产生了更多的细胞因子,不仅能反馈式地促使T细胞迅速扩增,还能强化T细胞的杀伤效应。
以上已以较佳实施例公布了本发明,然其并非用以限制本发明,凡采取等同替换或等效变换的方案所获得的技术方案,均落在本发明的保护范围内。
Claims (10)
1.靶向KRAS G12V单链抗体片段,其特征在于:包括重链可变区和轻链可变区,其中,重链可变区包括VH-CDR1、VH-CDR2、VH-CDR3轻链可变区包括VL-CDR1、VL-CDR2、VL-CDR3;
其中,VH-CDR1氨基酸序列为SEQ ID NO:3所示的序列,VH-CDR2氨基酸序列为SEQ IDNO:4所示的序列,VH-CDR3氨基酸序列为SEQ ID NO:5所示的序列;
VL-CDR1氨基酸序列为SEQ ID NO:8所示的序列,VL-CDR2氨基酸序列为SEQ ID NO:9所示的序列,VL-CDR3氨基酸序列为SEQ ID NO:10所示的序列。
2.根据权利要求1所述靶向KRAS G12V单链抗体片段,其特征在于:所述重链可变区的全长氨基酸序列为SEQ ID NO:6所示的氨基酸序列;
或者如SEQ ID NO:7所示的氨基酸序列;
所述轻链可变区的全长氨基酸序列为SEQ ID NO:11所示的氨基酸序列;
或者,如SEQ ID NO:12所示的氨基酸序列;
所述重链可变区和轻链可变区由连接区连接,连接区由3个四肽多聚体组成,氨基酸序列如SEQ ID NO:13所示;优选地,单链抗体可变区的氨基酸序列如SEQ ID NO:14所示或者至少80%的同源性序列;
或者如SEQ ID NO:15所示或者至少80%的同源性序列。
3.一种嵌合抗原受体CAR,其特征在于:包括抗原结合结构域、跨膜结构域、胞内信号结构域;其中抗原结构域包括信号肽、Myc标签和识别抗原的权利要求1-3所示的单链抗体片段。
4.根据权利要求3所述的嵌合抗原受体CAR,其特征在于:所述信号肽为CD8、GM-CSF、CD4、CD28、CD137、或其突变体/修饰体、或其组合;优选地,所述信号肽为CD8,氨基酸序列如SEQ ID NO:1所示。
5.根据权利要求3所述的嵌合抗原受体CAR,其特征在于:所述的Myc标签是一种来源于c-myc基因的表位标签,作为一种融合蛋白可融合至单链抗体片段的N端或C端,其氨基酸序列如SEQ ID NO:2所示;优选地,所述Myc标签融合在单链抗体片段的N端。
6.根据权利要求3所述的嵌合抗原受体CAR,其特征在于:所述抗原结合结构域和跨膜结构域通过铰链区相连,铰链区选自CD8α、CD28胞外结构域的衍生物、由IgG1或IgG4的FC部分或Fc部分的CH2/CH3结构域组成的基于IgG的铰链区、或DAP12;优选地,所述铰链区为CD8α,其氨基酸序列如SEQ ID NO:16所示。
7.根据权利要求3所述的嵌合抗原受体CAR,其特征在于:所述跨膜结构域选自CD3ζ、CD8、CD28、NKG2D、2B4、DNAM1中的一种;优选地,所述跨膜结构域为CD28或CD8;其中,CD28跨膜区的氨基酸序列如SEQ ID NO:17所示;
或/和
CD8跨膜区的氨基酸序列如SEQ ID NO:18所示。
8.根据权利要求3所述的嵌合抗原受体CAR,其特征在于:所述胞内信号结构域选自CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28家族、DAP10、DAP12、NKp44、NKG2D、TNFR家族或SLAM相关受体家族中的一种或多种;优选地,所述胞内信号结构域为4-1BB和CD3ζ的组合;其中,4-1BB的氨基酸序列如SEQ ID NO:19所示,CD3ζ的氨基酸序列如SEQ ID NO:20所示。
9.一种表达权利要求3-8所述的嵌合抗原受体CAR的CAR-T细胞。
10.根据权利要求1-2所述的靶向KRAS G12V单链抗体片段、权利要求3-8所述的嵌合抗原受体CAR、权利要求9所述的CAR-T细胞在制备抗肿瘤药品或产品中的应用。
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