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CN113166274A - 一种嵌合抗原受体及应用 - Google Patents

一种嵌合抗原受体及应用 Download PDF

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CN113166274A
CN113166274A CN202180000930.5A CN202180000930A CN113166274A CN 113166274 A CN113166274 A CN 113166274A CN 202180000930 A CN202180000930 A CN 202180000930A CN 113166274 A CN113166274 A CN 113166274A
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dectin
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CN113166274B (zh
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王玮
魏于全
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Zeheng Biomedical Technology Chengdu Co ltd
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Sichuan University
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Abstract

提供一种嵌合抗原受体，包括胞外结构域、跨膜结构域和胞内结构域组成的共刺激信号结构域，该共刺激信号结构域包括编码反向dectin‑1的氨基酸全长或片段，可治疗多种实体肿瘤和血液恶性肿瘤。

Description

一种嵌合抗原受体及应用
优先权申请
本申请要求2020年2月13提交的中国发明专利申请【CN2020100907495】、名称为“一种嵌合抗原受体的共刺激信号结构域及其应用”的优先权，该优先权发明专利申请以引用方式全文并入。
技术领域
本发明属于生物医药领域，具体涉及一种嵌合抗原受体及应用。
背景技术
嵌合抗原受体(Chimeric Antigen Receptor，CAR)T细胞免疫疗法，作为一种非常有前景的肿瘤治疗策略，经历了一系列演化过程。第一代CAR介导的T细胞激活是通过CD3δ或FceRIg上的酪氨酸激活基序完成的。但第一代CAR改造T细胞的抗肿瘤活性在体内受到了限制，因T细胞增殖减少最终导致T细胞的凋亡。第二代CAR在胞内增加了一个新的共刺激信号，例如4-1BB或CD28。第二代CAR与第一代CAR相比，抗原特异性不变，T细胞增殖、细胞因子分泌增加，抗细胞凋亡蛋白分泌增加，细胞死亡延迟。然而，现有的CAR-T细胞免疫疗法目前仅在治疗血液恶性肿瘤方面取得了显著进展，而在治疗实体瘤领域的成功有限。
CAR-T细胞免疫疗法在治疗血液恶性肿瘤方面的进展包括非霍奇金淋巴瘤(NHL)，B细胞急性淋巴细胞白血病(ALL)，多发性骨髓瘤(MM)和慢性淋巴细胞白血病(CLL)。到目前为止，CD19特异性或BCMA特异性CAR-T的临床试验的客观缓解率(ORR)为48％～95％。2017年，美国食品药品监督管理局(US FDA)批准了两种针对B细胞淋巴瘤的CAR-T细胞产品，AxicabtageneCiloleucel(KTE-C19,Kite Pharma)和Tisagenlecleuce(CTL019,Novartis)。在Axicabtageneciloleucel的1/2期临床试验中，对108名难治性大B细胞淋巴瘤患者进行了为期2年的随访。结果显示，83％的患者达到客观缓解(OR)，58％的患者达到完全缓解(CR)，平均随访期为15.4个月(IQR 13.7-17.3)。这表明CAR-T细胞治疗可以维持长期缓解。但在实体瘤治疗中，CAR-T细胞疗法仍然面临许多挑战。例如缺乏适当的肿瘤特异性抗原，肿瘤微环境抑制以及CAR-T细胞定位和持久性不足。另外，持续的抗原暴露会导致CAR-T细胞衰竭，进而损害CAR-T细胞对肿瘤的有效性。因此，需要设计新的CAR来用于实体瘤的治疗。
Dectin-1，即CLEC-7A，是C型凝集素受体(CLRs)的一个新的亚组。Dectin-1包括细胞外结构域，跨膜结构域和细胞内结构域。Dectin-1的细胞外部分已被用作靶向真菌的CAR-T细胞的scFv。Dectin-1不仅主要在包括中性粒细胞，单核细胞，树突状细胞和巨噬细胞在内的髓样细胞上表达，而且在人的T细胞和B细胞的某些亚群上也表达。Dectin-1通过激活NK细胞在肿瘤生长和转移中起重要作用。Dectin-1还可以调节各种细胞反应，例如DC成熟，抗原呈递以及细胞因子和趋化因子的产生。此外，理论上，Dectin-1可以直接诱导先天免疫记忆，并影响CD8、CD4、T细胞和B细胞的发育。
综上，本发明实验团队设计将Dectin-1并入二代CAR结构中作为共刺激信号域，评估其改造的新型二代CAR-T细胞的功能和抗肿瘤活性。以解决上述问题中的至少一个。
发明内容
有鉴于此，本发明的目的之一在于提供一种新型的二代CAR结构。
一种嵌合抗原受体，包括胞外结构域、跨膜结构域和胞内结构域，所述嵌合抗原受体的所述跨膜结构域和所述胞内结构域组成共刺激信号结构域，所述共刺激信号结构域包括编码反向dectin-1的氨基酸全长或片段。
进一步，所述胞外结构域包括靶向CD19或靶向HER2的单链抗体。
进一步，所述嵌合抗原受体还包括依次连接的CD8α铰链区、反向dectin-1跨膜结构域、反向dectin-1胞内信号域和CD3δ胞内信号域。
进一步，编码所述反向dectin-1氨基酸的序列如SEQ ID NO.3所示。
进一步，编码所述反向dectin-1跨膜结构域的氨基酸的序列如SEQ ID NO.2所示。
进一步，编码所述反向dectin-1胞内信号域的氨基酸序列如SEQ ID NO.1所示。
包含上述任一项所述的嵌合抗原受体的慢病毒载体。
进一步，所述慢病毒载体包括pCLK、psPAX2或pMD2.0G。
本发明的目的还在于提供一种根据上述新型的二代CAR改造的CAR-T细胞及其应用。
一种CAR-T细胞，表达上述嵌合抗原受体。
一种抗肿瘤药，含有上述CAR-T细胞和药学上允许添加的辅料和/或助剂。
进一步，所述肿瘤包括血液肿瘤和实体肿瘤。
进一步，所述肿瘤包括大B细胞淋巴瘤、B细胞淋巴瘤、非霍奇金淋巴瘤、髓细胞白血病、淋巴细胞白血病、乳腺癌、胃癌、食管癌或卵巢癌。
上述CAR-T细胞在制备抗肿瘤药物中的应用。
进一步，所述CAR-T细胞可以刺激效应细胞因子分泌。
进一步，所述效应细胞因子包括IFN-γ，TNF-α和IL-6。
进一步，所述CAR-T细胞可以刺激细中央记忆型T细胞分泌。
进一步，所述肿瘤包括血液肿瘤和实体肿瘤。
进一步，所述肿瘤包括大B细胞淋巴瘤、B细胞淋巴瘤、非霍奇金淋巴瘤、髓细胞白血病、淋巴细胞白血病、乳腺癌、胃癌、食管癌或卵巢癌。
本发明的目的还在于提供一种合成上述嵌合抗原受体的方法。
上述嵌合抗原受体的合成方法，其特征在于，包括以下步骤：(1)合成所述反向dectin-1跨膜结构域-反向dectin-1胞内结构域的基因序列或反向dectin-1跨膜结构域-CD3δ胞内信号域的基因序列；(2)根据靶点合成引物，再利用重叠PCR法合成所述嵌合抗原受体基因序列。
进一步，所述步骤(2)具体包括：先用引物F1、R1扩增所述胞外结构域和所述CD8α铰链区结合的基因序列，然后再用引物F2、R2扩增所述反向dectin-1跨膜结构域-CD3δ胞内信号域的基因序列，最后以所述胞外结构域和CD8α铰链区结合的基因序列和所述反向dectin-1跨膜结构域-CD3δ胞内信号域的基因序列为模板，以F1、R2为引物，合成所述嵌合抗原受体的基因序列。
进一步，所述引物F1如SEQ No.7所示，R1如SEQ No.8所示，F2如SEQ No.9所示，R2如SEQ No.10所示。
合成所述反向dectin-1跨膜结构域-反向dectin-1胞内结构域的基因序列的方法一致。
有益效果
本发明提供了一种以反向Dectin-1作为共刺激信号域的新型二代CAR结构以及其制备得到的CAR-T细胞。本发明的体内体外实验结果显示，新的CAR设计通过Dectin-1共刺激影响了T细胞功能，增强了多种细胞因子(包括IFN-γ，TNF-α和IL-6)的分泌和裂解能力，降低了衰竭力，增加了细胞扩增和显著的抗肿瘤活性。本发明提供的新型CAR-T细胞实验证明对多种实体肿瘤和血液恶性肿瘤有效。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍。显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其它的附图。
图1是CAR构建体和表达图：(A)嵌合受体的示意图，该受体包含与HER2或CD19结合的单链片段，并且在跨膜结构域和胞内域不同；(B)hH8-BBz CAR和hHD-Dz CAR以及h198-BBz CAR和h19D-Dz CAR在人T细胞的表面表达；
图2为CAR-T细胞的体外细胞因子产生和细胞毒性产生图，通过ELISA定量分析了来自四个不同CAR-T细胞或对照T细胞的上清液，细胞与目标阳性或阴性肿瘤细胞共培养过夜(E：T＝10：1或5：1)(n＝3/组)；通过单向方差分析进行统计分析，然后进行Tukey的事后分析；显着性认为P＜0.05：(A)IFN-γ,(B)IL-6和(C)TNF-α,(D)RTCA分析显示T细胞溶解能力(n＝4/组)；
图3为HER2特异性CAR-T细胞的表型、衰竭标记表达和CAR表达：(A)每个CAR-T细胞表型特征的流式细胞仪密度图：具有CD3或CD4或CD8或初始(TN)(CD45RA-/CD62L-)中央记忆T细胞(TCM)(CD45RO+/CD62L+)或效应记忆T细胞(TEM)(CD45RO+/CD62L-)；(B)每个CAR-T细胞抑制分子的流式细胞仪密度图：具有PD-1或LAG3或CTLA-4或TIM3的细胞；(C)通过流式细胞仪测量的hH8-BBz CAR-T细胞和hHD-Dz CAR-T细胞随时间的CAR表达；
图4为HER2特异性CAR-T细胞的体内抗肿瘤活性：(A)SK-OV-3-luc荷瘤模型的体内实时成像(n＝4/组)；(B)每组总体生存率的Kaplan-Meier分析；
图5为靶向HER2的二代嵌合抗原受体(RD-1)的结构示意图；
图6为传统二代CAR基因转染人的T细胞的表达检测图；
图7为新二代CAR(RD-1)基因转染人的T细胞的表达检测图；
图8为CAR-T细胞(RD-1)靶向HER2阳性肿瘤细胞SKOV3和HER2阴性肿瘤细胞MDA-MB-468分泌IFN-γ的检测图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
所举实施例是为了更好地对本发明进行说明，但并不是本发明的内容仅局限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
需要说明的是，在本文中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者装置不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者装置所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括该要素的过程、方法、物品或者装置中还存在另外的相同要素。
如在本说明书中使用的，术语“大约”，典型地表示为所述值的+/-5％，更典型的是所述值的+/-4％，更典型的是所述值的+/-3％，更典型的是所述值的+/-2％，甚至更典型的是所述值的+/-1％，甚至更典型的是所述值的+/-0.5％。
在本说明书中，某些实施方式可能以一种处于某个范围的格式公开。应该理解，这种“处于某个范围”的描述仅仅是为了方便和简洁，且不应该被解释为对所公开范围的僵化限制。因此，范围的描述应该被认为是已经具体地公开了所有可能的子范围以及在此范围内的独立数字值。例如，范围
Figure GDA0003081949670000071
的描述应该被看作已经具体地公开了子范围如从1到3，从1到4，从1到5，从2到4，从2到6，从3到6等，以及此范围内的单独数字，例如1，2，3，4，5和6。无论该范围的广度如何，均适用以上规则。
“RD-1”即为反向Dectin-1(Reverse Dection-1),其氨基酸序列为“GLVVAIVLIVLCLIGLIVAILRWPPSAACSGKESVVAIRTNSQSDFHLQTYGDEDLNELDPHYEM”如SEQ NO.3所示；而Dectin-1的氨基酸序列为“MEYHPDLENLDEDGYTQLHFDSQSNTRTAVVSEKGSCAASPPWRLIAVILGILCLVILVIAVVLG”如SEQ NO.4所示。“RD-1胞内结构域”氨基酸序列为“RWPPSAACSGKESVVAIRTNSQSDFHLQTYGDEDLNELDPHYEM”，如SEQ NO.1所示；“RD-1跨膜结构域”氨基酸序列为“GLVVAIVLIVLCLIGLIVAIL”，如SEQ NO.2所示。发明人研究发现，在制备新型二代CAR时，采用反向Dectin-1作为共刺激结构域能高效地产生和保持Dectin-1的活性和功能(即能影响T细胞功能，增强多种细胞因子(包括IFN-γ，TNF-α和IL-6)的分泌和裂解能力，降低衰竭力，增加细胞扩增和显著的抗肿瘤活性)。因此在本文中，如无特别说明，在CAR结构中Dectin-1均是指的反向Dectin-1。
实施例一
1.材料与方法
细胞系：所有细胞系来源于ATCC。将K562细胞(髓细胞白血病)和NALM6细胞(淋巴细胞白血病)在RPMI-1640中与热灭活的10％胎牛血清(FBS)(PAN,Germany.Cat:ST30-3302)、青霉素(100U/mL)(Gibco,Thermo Fisher,Waltham,MA.Cat:SV30010)和链霉素(100ug/mL)(Gibco,Thermo Fisher,Waltham,MA.Cat:SV30010)一起培养。人癌细胞系SK-OV-3(卵巢囊腺癌)和MDA-MB-468(乳腺癌)在含有热灭活的10％FBS、青霉素(100U/mL)和链霉素(100ug/mL)的DMEM中培养。还工程化SK-OV-3使其表达荧光素酶。
2.构建编码CAR的质粒
抗CD19或抗HER2的CARs包括特异于CD19(克隆FMC63)或HER2(克隆4D5)的单链可变片段(scFv)。scFv之后是人CD8α铰链区，然后是人CD8α跨膜结构域(TM)，4-1BB或CD3δ胞内结构域(ICDs)。使用重叠PCR形成CARs序列。编码每个CAR序列的单个慢病毒质粒是用PCLK慢病毒载体进行双酶切构建的。构建的4条CARs序列如下：
1)hH8-BBZ:HER2 scFv-CD8α(hinge+TM)-4-1BB-CD3δICDs
2)hHD-DZ:HER2 scFv-CD8αhinge+Dectin-1(TM+cytoplasm)-CD3δICDs
3)h198-BBZ:CD19 scFv-CD8α(hinge+TM)-4-1BB-CD3δICDs
4)h19D-DZ:CD19 scFv-CD8αhinge+Dectin-1(TM+cytoplasm)-CD3δICDs
3.生产慢病毒颗粒
将购于ATCC的HEK-293T细胞(胚胎肾细胞)在DMEM中与热灭活的10％胎牛血清(FBS)(PAN,Germany.Cat:ST30-3302)，青霉素(100U/mL)和链霉素(100ug/mL)(Gibco,Thermo Fisher,Waltham,MA.Cat:SV30010)一起培养。
为生产含有上清液的慢病毒，先用上述详述的质粒转染HEK-293T细胞：用适合的CAR编码质粒psPAX2和pMD2.0G(Invitrogen)。转染12小时后更换培养基。收集上清液并离心除去细胞碎片。过滤上清液，并通过在19,700rpm下超速离心2小时进行浓缩。弃去上清液。将所述慢病毒颗粒溶解在PBS培养基中，并将浓缩的慢病毒保存在-80℃。通过定量实时聚合酶链反应测量浓缩的慢病毒滴度(Q-RT-PCR)。
4.分离、转导、生产和扩增CAR-T细胞
用Ficoll-hypaque密度梯度法(Lonza,Cat:04-418Q)从健康献血者血液中分离外周血单个核细胞(PBMCs)，所有样本均经生物治疗国家重点实验室伦理委员会知情同意并批准后获得。
在含有5％人血清(Sigma-Aldrich,H4522)和100U/ml重组人IL-2(rhIL-2)(PeproTech,NJ,USA.Cat:200-02-10)的X-VIVO 15培养基(Sigma-Aldrich,Cat:10771)中培养的PBMC。用抗CD3/CD28磁珠(Gibco,Thermo Fisher,Waltham,MA.Cat:11131D)刺激PBMC。24小时后，将T细胞与感染复数(MOI)为5的慢病毒培养48小时，然后将细胞洗涤并在T细胞培养基中培养。通过流式细胞仪测定的CAR表达确定转导效率。
5.流式细胞检测
所有流式细胞仪检测均在新生细胞流式细胞仪(ACEA Biosicences,Inc.)上进行，并用新生细胞表达(ACEA Biosicences,Inc.)分析数据。
利用生物素-SP结合的亲和山羊抗鼠IgG(Cat:120962,Jackson ImmuneResearch)，F(ab’)2片段与PE-链霉亲和素(Cat:405203,BD Biosciences)特异性结合，评价其转导效率和相关CAR蛋白的表达。
以下抗体用于分化表型和衰竭标记分析：
anti-CD3-FITC(clone:HIT3a,Cat:300306,Biolegend)、anti-CD8-APC(clone:HIT8a,Cat:300912,Biolegen)、anti-CD4-PE(clone:RPA-T4,Cat:300508,Biolegend)、anti-CD45RO-PE(BD Biosciences,clone:UCHL1,Cat:555493)、anti-CD62L-APC(BDBiosciences,clone:DREG-56,Cat:559772)、anti-PD-1-APC(clone:EH12.2H7,Cat:329908,Biolegend)、anti-CTLA-4-APC(Cat:369612,Biolegen)、anti-LAG3-APC(Cat:369212,Biolegend)、anti-TIM3-APC(Cat:345012,Biolegend)。
6.体外细胞因子实验
将靶细胞(NALM6，K562，SK-OV-3或MDA-MB-468，以1×105个细胞/孔)接种到96孔板中，并在5％CO2中于37℃孵育过夜。之后，以效/靶比(E：T)为5或10的比例添加CAR-T细胞。通过转导效率将CAR-T细胞数量归一化(normalized)。与靶细胞共培养24小时后收集上清液。使用Invitrogen公司用于细胞因子测定的ELISA试剂盒(IFN-γCat:88-7316-88,TNF-αCat:88-7346-88,and IL-6Cat:88-7066-88)来定量检测IFN-γ，TNF-α和IL-6。
7.实时细胞毒性检测(Real-time cytotoxicity assays,RTCA)
通过实时细胞毒性测定法测量CAR-T细胞的细胞毒性作用(ACEA Bioscience,Inc.xCELLigence RTCA SP)。将SK-OV-3或MDA-MB-468细胞以1×104个细胞/孔的数量在E-plate 96检测板中培养约24小时。将CAR-T细胞(hH8-BBz和hHD-Dz)或对照T细胞(mock Tcells)以10的效/靶比添加到平板中，根据制造商提供的实验方法采集和分析数据(ACEA162Bioscience,Inc.RTCA Software 2.1)。
8.体内异种移植研究
采用6周大的雌性B-NSG小鼠(NOD-PrkdcscidIL2rgtm1/Bcgen)用于本研究。每只小鼠接受一次腹腔注射，注射2×106个SK-OV-3-luc细胞。
SK-OV-3-luc细胞是由SK-OV-3细胞工程化设计得到，其可以表达荧光素酶，在后续的肿瘤生物发光成像实验中通过荧光信号显示肿瘤的生长情况。
肿瘤生长3天后再次接受腹腔注射，注射1×107个CAR-T细胞(hH8-BBz或hHD-Dz或对照)。再过3天后，再进行一次将CAR-T细胞通过腹腔注射给每只小鼠。肿瘤的生物发光成像(BLI)在计划的第3天、第10天、第17天、第24天、第31天和第55天通过IVIS(活体成像系统)进行。肿瘤通量(光子/s/cm2/steradian)是通过测量肿瘤周围感兴趣区域(ROI)内的光子信号来量化的。利用活体图像软件(v2.50,Xenogen；Caliper Life Sciences)对BLI数据进行了演示。在每只小鼠死亡时建立生存分析数据。
9.数据
使用GraphPad Prism v6.01(GraphPad Software Inc.)和SPSS v17进行统计绘图和分析。数据表示为平均值±SD。单向方差分析用于在单一条件下3组的比较。
使用对数秩(Mantel-Cox)检验分析Kaplan-Meier生存数据。在需要标准化方差时对数据进行转换。符号表示统计显着性如下：*P<0.05；**P<0.01和***P<0.001。
实施例二
1.具有Dectin-1共刺激信号域的新型CAR结构
在本发明中，将靶向CD19或HER2表位的scFv域与Dectin-1 TM，Dectin-1和CD3δICD结合，设计了新型的第二代CAR构建体(图1A)。用最流行的第二代CAR结构(带有4-1BB和CD3δICDs的人源CD8αTM)的部分生成了另外两个CAR结构。所有构建的CAR在T细胞表面表达(图1B)。关于抗HER2的CARs，hH8-BBz(4-1BB)CAR表达率为49.61％，hHD-Dz(Dectin-1)CAR表达率为46.17％。关于抗CD19的CARs，h198-BBz(4-1BB)CAR表达率是92.95％，h19D-Dz(Dectin-1)CAR表达率是95.07％。尽管抗HER2 CAR的表达低于抗CD19 CAR，但在包含相同共刺激信号域的抗HER2或抗CD19CAR中，其表达水平上没有明显差异。
2.Dectin-1共刺激对新型CAR-T细胞效应功能的影响
通常，可以通过细胞因子的分泌来评估CAR-T细胞后来的效应子功能。因此，在暴露于表达HER2或CD19的肿瘤细胞后，评估Dectin-1作为共刺激信号分子对新CAR-T细胞释放细胞因子的影响。
在靶向阳性细胞系(SK-OV-3和NALM6)中吸收Dectin-1信号结构域后，抗HER2或抗CD19 CAR-T细胞释放的效应细胞因子(包括IFN-γ，TNF-α和IL-6)显着增加(图2A，2B和2C)。本研究中的抗HER2 CAR-T细胞未显示出针对MD-MB-468(阴性细胞系)的细胞因子产生显着增加(图2A，2B和2C)。在SK-OV-3细胞系中，hHD-Dz CAR-T细胞比hH8-BBz CAR-T细胞显示更高的IFN-γ水平。相反，hH8-BBz CAR-T细胞分泌更多的TNF-α(图2A，2B和2C)。在NALM6细胞系中，h19D-Dz CAR-T细胞产生的IFN-γ和TNF-α水平与h198-BBz CAR-T细胞相似(图2A，2B和2C)。
进一步研究了抗HER2 CAR-T细胞的细胞毒性功能，以说明抗原结合(antigenengagement)和CAR-T细胞的活化。在将抗HER2 CAR-T细胞与SK-OV-3或MDA-MB-468肿瘤细胞系共培养后，观察到hHD-Dz和hH8-BBz CAR-T细胞均能有效裂解SK-OV-3肿瘤细胞。hHD-Dz CAR-T细胞显示出比hH8-BBz CAR-T细胞强得多的裂解细胞毒性功能(图2D)。
3.抗HER2 CAR-T细胞的表型和衰竭标记表达
在细胞扩增7天后，分析了抗HER2 CAR-T细胞和对照T细胞的表型和衰竭标记表达(图3)。
hHD-Dz，hH8-BBz CAR-T和对照T细胞显示出相似的CD4+或CD8+T细胞百分比(图3A)。尽管在hHD-Dz和hH8-BBz CAR-T细胞中观察到了相当比例的效应记忆T(TEM，CD45RO+CD62L-)，但hHD-Dz CAR-T细胞比hH8-BBz CAR-T细胞显示出更多的中央记忆T细胞(TCM，CD45RO+CD62L+)(图3A)。
本发明还评估了抗HER2 CAR-T细胞(图3B)抑制性受体的表达模式，包括PD-1、CTLA-4、TIM3和LAG3。
结果表明，hHD-Dz CAR-T细胞中的PD-1或LAG3阳性细胞比hH8-BBz CAR-T细胞中少约10％。就TIM3和CTLA-4阳性细胞而言，在hHD-Dz和hH8-BBz CAR-T细胞中观察到了相似的百分比(图3B)。
本发明还进一步探讨了抗HER2 CAR表达的时程。尽管在96小时内CAR表达下降，但在另外48小时后CAR表达达到90％以上(图3C)。
总结起来，以上结果表明，新型CAR-T细胞中的Dectin-1信号域可能导致明显的表型和衰竭标记表达，并提高T细胞的增殖潜能。
4.抗HER2 CAR-T细胞的体内抗肿瘤活性
使用携带异种移植SK-OV-3-luc肿瘤细胞的NSG小鼠，本发明进一步研究了抗HER2CAR-T细胞(hH8-BBz或hHD-Dz CAR-T细胞)的体内抗肿瘤活性。评价了总生存期和肿瘤体积(图4)。与对照T细胞治疗的小鼠相比，抗HER2 CAR-T细胞治疗使肿瘤进展延迟(图4A)，抗HER2 CAR-T细胞组的荷瘤小鼠中位生存期至少翻了一番(图4B)。对数秩(Mantel-Cox)测试证明，抗HER2 CAR-T细胞组和对照T细胞组之间的存活率存在统计学上的显着差异。此外，hHD-Dz CAR-T细胞组中的100％小鼠在第55天还存活，而hH8-BBz CAR-T组显示出更长的总生存期(图4B)。
实施例三
本实施例还提供一种嵌合抗原受体的共刺激信号结构域，所述共刺激信号结构域包含RD-1胞内区。
进一步，所述共刺激信号结构域包含CD3δ。
进一步，所述RD-1胞内区包含含有SEQ No.1的氨基酸序列或与所述SEQ No.1不低于90％同一性的氨基酸序列。
进一步，所述共刺激信号结构域还包含CD3,CD4,CD8,FcR,DAP10,DAP12,CD27,CD28,CD137,CD134,ICOS,OX40,CD30,CD40,PD-1,LFA-1,CD2,CD7,LIGHI,NKG2C,B7-H3,特异结合CD83的配体，CDS,ICAM-1,GITR,BAFFR,HVEM(IGHTR),SLAMF7,NKp80 KLRE1,CD160,CD19,CD83,IL-2Rβ,IL-Rγ,IL-7Rα,ITGA4,VLA1,CD49α,ITGA4,IA4,CD49D,ITGA6,LA-6,CD49f,ITGAD,CD11d,ITGAE,CD103,ITGAL,CD11α,LFA-1,ITGAM,CDIIB,ITGAX,CD11c,ITGB1,CD29,ITGB2,CD18,LFA-1.ITGB7 TAFR2,TRANCE/RANKL,DNAM1(CD226),SLAMF4(CD244,2B4),CD84,CD96(Tactile),CEACAM1 CRTAM,Ly9(CD229),D160(BY55),PSGL1,CD100(SEMA4D),CD69,SLAMF6(NTB-A,Ly108 SLAM(SLAMF1,CD150,IPO-3),BLAME(SLAME8),SELPLG(CD162),LTBR,LAT,GADS,SLP-76PAG/Cbp,NKp4,NWKp30,NKp46,NKG2D中的一种或多种。
本实施例还提供一种包含前述的共刺激信号结构域的嵌合抗原受体。
进一步，所述嵌合抗原受体包括依次连接的抗原识别域、CD8α铰链区、跨膜区、RD-1胞内区和CD3δ。
进一步，所述跨膜区包含的氨基酸序列能够将所述嵌合抗原受体锚定在细胞膜上。
进一步，所述跨膜区包含RD-1跨膜区。
进一步，所述RD-1跨膜区包含含有SEQ No.2氨基酸序列或与所述SEQ No.2不低于90％同一性的氨基酸序列。
优选地，所述抗原识别域是HER2结合域。
进一步，所述嵌合抗原受体包含含有SEQ No.5的氨基酸序列。
优选地，所述抗原识别域是CD19结合域。
进一步，所述的嵌合抗原受体包含含有SEQ No.6的氨基酸序列。
本实施例还提供一种前述的嵌合抗原受体的合成方法，所述方法包括以下步骤：步骤(1)合成RD-1-CD3δ基因序列；步骤(2)合成嵌合抗原受体基因序列。
进一步，所述步骤(2)具体包括：先用引物F1、R1扩增抗原识别域和CD8α铰链区结合的基因序列，然后再用引物F2、R2扩增所述RD-1-CD3δ基因序列，最后以所述抗原识别域和CD8α铰链区结合的基因序列和所述RD-1-CD3δ基因序列为模板，以F1、R2为引物，合成所述嵌合抗原受体的基因序列。
进一步，所述引物F1如SEQ No.7所示，R1如SEQ No.8所示，F2如SEQ No.9所示，R2如SEQ No.10所示。
合成的(anti-HER2 scFV)-(CD8αhinge)-(RD-1TM+Cytoplasmic)-(CD3δ)的氨基酸序列为SEQ No.5。
本实施例还提供一种前述的嵌合抗原受体与表达载体构建的重组质粒载体。
进一步，所述表达载体为pCLK载体。
本实施例还提供了前述的重组质粒载体的构建方法，所述构建方法包括以Mlu I和Spe I为酶切位点，连接所述抗原受体基因与所述表达载体，从而获得所述重组质粒载体。
本实施例还提供了一种前述嵌合抗原受体修饰的免疫细胞。
进一步，所述免疫细胞为T淋巴细胞。
进一步，所述免疫细胞还包括B淋巴细胞、K淋巴细胞和NK淋巴细胞中的一种或多种。
本实施例还提供了一种获取前述的免疫细胞的方法，所述方法包括以下步骤：以Mlu I和Spe I为酶切位点，连接所述嵌合抗原受体基因与表达载体，得重组质粒载体；将所述重组质粒载体与包装质粒共同转入培养细胞中培养，获得重组病毒颗粒；再用所述重组病毒颗粒对免疫细胞进行修饰。
进一步，所述修饰的方法包括采用病毒载体或非病毒载体系统。
进一步，所述病毒载体系统包括逆转录病毒载体、慢病毒载体、腺病毒载体、腺相关病毒载体和仙台病毒载体中的一种或多种。
进一步，所述非病毒载体系统包括转座子系统、CRISPR基因编辑系统、TALEN系统、脂质体转染系统和电转染系统中的一种或多种。
本发明目的在于还提供一种前述免疫细胞在制备抗肿瘤药物中的应用。
进一步，所述抗肿瘤药物组合物，其特征在于，还包括化学药物。
进一步，所述化学药物包括环磷酰胺和/或氟达拉滨。
进一步，所述抗肿瘤药物组合物为抗乳腺癌和/或抗卵巢癌药物。
本实施例还提供了一种包含前述嵌合抗原受体的组合物，所述组合物还包括与所述共刺激信号结构域结合的β-葡聚糖受体、Syk、CR3和CD11b中的一种或多种。
本实施例中获得CAR基因所用的引物情况如表1。
表1获得CAR基因所用的引物
Figure GDA0003081949670000171
Figure GDA0003081949670000181
实施例四
PCR获得全长抗原为HER2的CAR基因：
(1)第一步，基因合成：合成RD-1(TM+cytoplasmic)-CD3δ片段全长。
(2)第二步，合成引物，并利用重叠PCR方法得到(anti-HER2 scFV)-(CD8αhinge)-(RD-1TM+Cytoplasmic)-(CD3δ)融合片段，其中具体包括以下两个步骤：
(2.1)利用引物F1、R1扩增(anti-HER2 scFV)-(CD8αhinge)；利用F2、R2扩增(RD-1TM+Cytoplasmic)-(CD3δ)。
(2.2)再以(anti-HER2 scFV)-(CD8αhinge)和(RD-1TM+Cytoplasmic)-(CD3δ)为模版，F1、R2为引物，通过PCR获得全长CAR基因。
合成的(anti-HER2 scFV)-(CD8αhinge)-(RD-1TM+Cytoplasmic)-(CD3δ)的氨基酸序列为SEQ NO.5.
抗原为HER2的CAR的重组质粒载体的构建：
(1)第一步，基因合成：合成RD-1(TM+cytoplasmic)-CD3δ片段全长。
(2)第二步，设计引物，并利用重叠PCR方法得到(anti-HER2 scFV)-(CD8αhinge)-(RD-1TM+Cytoplasmic)-(CD3δ)融合片段，其中具体包括以下两个步骤：
(2.1)利用引物F1、R1扩增(anti-HER2 scFV)-(CD8αhinge)；利用F2、R2扩增(RD-1TM+Cytoplasmic)-(CD3δ)；
(2.2)以(anti-HER2 scFV)-(CD8αhinge)和(RD-1TM+Cytoplasmic)-(CD3δ)为模版，F1、R2为引物，PCR获得全长CAR基因。
(3)以Mlu I和Spe I为酶切位点，连接通过上述步骤合成的CAR基因及PCLK载体，获得CAR-PCLK质粒载体。
T淋巴细胞的病毒转染及目的基因的表达检测：
(1)将具有嵌合抗原受体基因新的二代CAR基因(anti-HER2 scFV)-(CD8αhinge)-(RD-1TM+Cytoplasmic)-(CD3δ)(结构示意图如图5)，传统二代CAR基因(anti-HER2 scFV)-(CD8αhinge+TM)-(4-1BB)-(CD3δ)(结构示意图如图5)分别装入慢病毒载体PCLK中，并与两个辅助载体psPAX2和pMD2.G共同转染T细胞，分别包装获得病毒颗粒，经离心浓缩后获得高浓度的慢病毒载体。
(2)利用密度梯度离心法，分离得到淋巴细胞，以CD3抗体(1ug/ml)和IL-2(100IU/ml)刺激淋巴细胞。一天后收集淋巴细胞进行病毒转染，培养淋巴细胞48小时，收集转染的淋巴细胞。
(3)收集到的经过病毒转染的淋巴细胞，用识别抗体Fab片段的特异性抗体，进行流式检测。
如图6为传统二代CAR基因转染人T细胞的表达检测，一抗为生物素标记的山羊抗小鼠Fab单克隆抗体，二抗为PE标记的抗streptavidin流式抗体。第一泳道为转染空载病毒的T细胞，第二泳道为转染CAR基因的T细胞。(图2中hH8-BBz为传统二代CAR基因(anti-HER2scFV)-(CD8αhinge+TM)-(4-1BB)-(CD3δ)；)。
如图7为新二代CAR基因转染人T细胞的表达检测，一抗为生物素标记的山羊抗小鼠Fab单克隆抗体，二抗为PE标记的抗streptavidin流式抗体。第一泳道为转染空载病毒的T细胞，第二泳道为转染CAR基因的T细胞。(图3中hHD-Dz为新二代CAR基因(anti-HER2scFV)-(CD8αhinge)-(RD-1TM+Cytoplasmic)-(CD3δ))。
在相同MOI(感染复数)病毒转染效力下，经流式细胞仪检测证实，新的二代CAR分子和传统二代CAR分子在淋巴细胞表面均高效表达。
对表达HER2的肿瘤细胞的杀伤作用：
传统二代CAR-T细胞：(anti-HER2 scFV)-(CD8αhinge+TM)-(4-1BB)-(CD3δ)-T细胞
新二代CAR-T细胞：(anti-HER2 scFV)-(CD8αhinge)-(RD-1TM+Cytoplasmic)-(CD3δ)-T细胞
(1)利用RTCA(RealTime Cellular Analysis,实时无标记细胞分析技术)分别检测两种T细胞对HER2阳性细胞SKOV3(人卵巢癌细胞)肿瘤细胞杀伤效力检测，其检测结果如图2D。(图2D中hH8-BBz为传统二代CAR-T细胞；hHD-Dz为新二代CAR-T细胞)。
(2)利用ELISA分别检测两种CAR T细胞向HER2阳性肿瘤细胞SKOV3(人卵巢癌细胞)和HER2阴性肿瘤细胞MDA-MB-468(人乳腺癌细胞)分泌细胞因子情况,其检测分析结果如图8。图8清晰显示，新二代CAR T比传统二代CAR T向HER2阳性肿瘤细胞SKOV3分泌的细胞因子((IFNγ)情况要更多。(图8中hH8-BBz为传统二代CAR基因(anti-HER2 scFV)-(CD8αhinge+TM)-(4-1BB)-(CD3δ)；hHD-Dz为新二代CAR基因(anti-HER2 scFV)-(CD8αhinge)-(RD-1TM+Cytoplasmic)-(CD3δ))。
总结
典型的CAR主要由三个关键部分组成，包括识别抗原的单链可变片段(scFv)，铰链和跨膜结构域(TM)，例如CD3，CD28或CD8蛋白；以及细胞内信号域(ICDs)，例如CD3δ或FcRγ。CAR包含一个或多个细胞内共刺激信号域，例如CD28、4-1BB，CD27，OX40，ICOS，DAP10，IL-15Rα，MyD88/CD40和TLR2，以传输激活信号。
已经证明，不同的TM和/或ICDs会影响T细胞扩增，存续和其他功能。最近，已经在患有实体瘤(例如转移性结直肠癌和肉瘤)的患者中测试了几种第二代CAR。与治疗血液恶性肿瘤所获得的结果相比，这些试验的结果令人振奋。因此，探索不同的共刺激域可能提供一种提高实体瘤中CAR-T细胞抗肿瘤效力的新方法。
在本发明中，我们的实验团队将靶向CD19或HER2的scFv结构域与4-1BB或dectin-1信号ICD偶联，以构建四个不同的第二代CAR。实验数据显示，在体外和体内实验中，新的CAR设计均通过Dectin-1共刺激影响了T细胞功能，例如增强了细胞因子分泌和裂解能力，降低了衰竭力，增加了细胞扩增和显著的抗肿瘤活性。
在本发明中，我们证实了先前研究的结果。显示具有4-1BB共刺激信号域的CAR-T细胞功能增强。有趣的是，hHD-Dz CAR-T细胞的体外T细胞功能(例如增加的细胞因子产量)与基于4-1BB的细胞相当，并且两者均优于对照T细胞。至于HER2特异性CAR-T细胞，hHD-DzCAR-T细胞的IFN-γ分泌高于hH8-BBz，提示可能是Th1表型。hH8-BBz CAR-T细胞释放更多的TNF-α，与Th1/Th2表型一致。抗CD19 CAR-T细胞的相似细胞因子产生模式表明，与采用的具体共刺激信号传导域无关，表型是具有可比性的。在RTCA(实时细胞毒性检测)中，我们阐明了hHD-Dz CAR-T细胞优于hH8-BBz CAR-T细胞的细胞毒性能力。以上结果表明，Dectin-1协同刺激为治疗实体瘤的CAR-T细胞免疫疗法提供了一种新的机制。
由于免疫抵抗和T细胞耗竭，CAR-T细胞疗法治疗实体瘤的最大挑战之一是抑制肿瘤微环境。然而，研究已经证实，不同T细胞表型可能在抗肿瘤免疫中发挥重要作用，例如，在过继免疫疗法中，TCM细胞远比TEM细胞重要。在本发明中，我们发现hHD-Dz CAR-T细胞中更多的TCM和显著的耗竭标记物表达表达可能表明新的CAR-T细胞可通过实体瘤中的Dectin-1共刺激信号转导受到肿瘤免疫抑制微环境的影响较小。
此外，我们通过建立肿瘤异种移植模型中的Dectin-1或4-1BB共刺激证明了第二代CAR-T细胞显著的抗肿瘤作用。但是，此处具有不同共刺激信号域的CAR在体内存活率方面显示出离散的抗肿瘤活性趋势。hHD-Dz CAR-T细胞在早期时间点显示出增强的效应子功能，而hH8-BBz CAR-T细胞则显示出较晚的抗肿瘤活性。这些观察结果表明，不同的共刺激信号域可能导致不同的T细胞表型。。
上面结合附图对本发明的实施例进行了描述，但是本发明并不局限于上述的具体实施方式，上述的具体实施方式仅仅是示意性的，而不是限制性的，本领域的普通技术人员在本发明的启示下，在不脱离本发明宗旨和权利要求所保护的范围情况下，还可做出很多形式，这些均属于本发明的保护之内。
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<110> 四川大学
<120> 一种嵌合抗原受体及应用
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<213> artificial sequence
<400> 10
ggactagttt agcgaggggg 20
7

Claims (21)

1.一种嵌合抗原受体，包括胞外结构域、跨膜结构域和胞内结构域，其特征在于，所述嵌合抗原受体的所述跨膜结构域和所述胞内结构域组成共刺激信号结构域，所述共刺激信号结构域包括编码反向dectin-1的氨基酸全长或片段。
2.如权利要求1所述的嵌合抗原受体，其特征在于，所述胞外结构域包括靶向CD19或靶向HER2的单链抗体。
3.如权利要求2所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包括依次连接的CD8α铰链区、反向dectin-1跨膜结构域、反向dectin-1胞内信号域和CD3ζ胞内信号域。
4.如权利要求1所述的嵌合抗原受体，其特征在于，编码所述反向dectin-1氨基酸的序列如SEQ ID NO.3所示。
5.如权利要求3所述的嵌合抗原受体，其特征在于，编码所述反向dectin-1跨膜结构域的氨基酸的序列如SEQ ID NO.2所示。
6.如权利要求3所述的嵌合抗原受体，其特征在于，编码所述反向dectin-1胞内信号域的氨基酸序列如SEQ ID NO.1所示。
7.包含权利要求1-6任一项所述的嵌合抗原受体的慢病毒载体。
8.如权利要求7所述的慢病毒载体，其特征在于，所述慢病毒载体包括pCLK、psPAX2或pMD2.0G。
9.一种CAR-T细胞，其特征在于，表达权利要求1-6任一项所述的嵌合抗原受体。
10.一种抗肿瘤药，其特征在于，含有权利要求9所述的CAR-T细胞和药学上允许添加的辅料和/或助剂。
11.如权利要求10所述的抗肿瘤药，其特征在于，所述肿瘤包括血液肿瘤和实体肿瘤。
12.如权利要求10所述的抗肿瘤药，其特征在于，所述肿瘤包括大B细胞淋巴瘤、B细胞淋巴瘤、非霍奇金淋巴瘤、髓细胞白血病、淋巴细胞白血病、乳腺癌、胃癌、食管癌或卵巢癌。
13.权利要求9所述的CAR-T细胞在制备抗肿瘤药物中的应用。
14.如权利要求13所述的应用，其特征在于，所述CAR-T细胞可以刺激效应细胞因子分泌。
15.如权利要求14所述的应用，其特征在于，所述效应细胞因子包括IFN-γ，TNF-α和IL-6。
16.如权利要求13所述的应用，其特征在于，所述CAR-T细胞可以刺激细中央记忆型T细胞分泌。
17.如权利要求13所述的应用，其特征在于，所述肿瘤包括血液肿瘤和实体肿瘤。
18.如权利要求13所述的应用，其特征在于，所述肿瘤包括大B细胞淋巴瘤、B细胞淋巴瘤、非霍奇金淋巴瘤、髓细胞白血病、淋巴细胞白血病、乳腺癌、胃癌、食管癌或卵巢癌。
19.权利要求1-6任一项所述的嵌合抗原受体的合成方法，其特征在于，包括以下步骤：(1)合成所述反向dectin-1跨膜结构域-反向dectin-1胞内结构域的基因序列或反向dectin-1跨膜结构域-CD3ζ胞内信号域的基因序列；(2)根据靶点合成引物，再利用重叠PCR法合成所述嵌合抗原受体基因序列。
20.如权利要求19所述的合成方法，其特征在于，所述步骤(2)具体包括：先用引物F1、R1扩增所述胞外结构域和所述CD8α铰链区结合的基因序列，然后再用引物F2、R2扩增所述反向dectin-1跨膜结构域-CD3ζ胞内信号域的基因序列，最后以所述胞外结构域和CD8α铰链区结合的基因序列和所述反向dectin-1跨膜结构域-CD3ζ胞内信号域的基因序列为模板，以F1、R2为引物，合成所述嵌合抗原受体的基因序列。
21.如权利要求20所述的合成方法，其特征在于，所述引物F1如SEQ No.7所示，R1如SEQNo.8所示，F2如SEQ No.9所示，R2如SEQ No.10所示。
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