CN107207613B - 含Toll样受体胞内结构域的嵌合抗原受体 - Google Patents
含Toll样受体胞内结构域的嵌合抗原受体 Download PDFInfo
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- CN107207613B CN107207613B CN201580000296.XA CN201580000296A CN107207613B CN 107207613 B CN107207613 B CN 107207613B CN 201580000296 A CN201580000296 A CN 201580000296A CN 107207613 B CN107207613 B CN 107207613B
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Abstract
本发明提供了一种嵌合抗原受体、其编码核酸和表达细胞,以及其在制备***的药物中的用途。所述嵌合抗原受体的胞内结构域至少包括Toll样受体1和/或Toll样受体2的胞内结构域。
Description
技术领域
本发明涉及肿瘤的细胞免疫治疗技术领域,具体地,涉及一种嵌合抗原受体,编码其的核酸和表达其的细胞,以及其在制备***的药物中的用途。
背景技术
嵌合抗原受体(CAR,Chimeric Antigen Receptors)T细胞是表面表达识别特定抗原并能传递信号的嵌合型受体的T细胞【1】。CAR T细胞通过表达嵌合抗原受体(CAR)分子而在抗肿瘤中发挥重要作用,CAR分子通常包括胞外段、跨膜区和胞内段:胞外段是由抗体重链和轻链可变区通过一条肽段相连接而形成的单链可变区(ScFv);胞内段是各种信号传导分子的胞内段嵌合体,包括CD3zeta、CD28、OX-40、4-1BB等;跨膜区则来自其他分子(如CD8,CD4,CD28和CD3zeta)的跨膜区。单链可变片段部分的基因分离自例如产生识别靶抗原的单克隆抗体的杂交瘤。表达CAR分子的T细胞独立于肿瘤细胞上的主要组织相容性抗原I型的表达而直接识别肿瘤细胞表面抗原,并且在同时激活T细胞,并因此表达CAR的T细胞可以有效杀伤肿瘤细胞。简言之,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。
目前,研究人员对CAR T的临床研究成果主要集中于血液肿瘤,如淋巴瘤、慢性淋系白血病、急性淋系白血病等。而近期在其它种类的肿瘤治疗的CAR T也有了一些进展,其中包括:抗5T4 CAR T治疗实体瘤,抗ROR-1 CAR T治疗实体瘤,抗PCSA CAR T治疗实体瘤,抗Mesothelin CAR T治疗胰腺间皮瘤,抗EGFRvIII CAR T治疗胶质瘤、成胶质细胞瘤,抗CD22 CAR T治疗B细胞肿瘤,抗L1CAM CAR T治疗成神经细胞瘤,抗MUC16&IL6 CAR T治疗卵巢癌等。综上,嵌合抗原受体T细胞免疫疗法已被证明能有效治疗各种肿瘤,包括化疗耐受和复发的白血病。但目前嵌合抗原受体T细胞治疗存在很多问题,如嵌合抗原受体T细胞在患者体内的杀伤功能,持久性,记忆性嵌合抗原受体T细胞的形成等。
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单链可变片段(scFv)、跨膜结构域和激活T细胞的TCR复合物CD3ζ的胞内结构域。由于第一代CAR的胞内段只有CD3ζ信号传递区域,没有共刺激信号,因而第一代CAR T细胞的功能存在很大缺陷,其在病人体内的扩增、持久性和效应功能等方面都表现出低水平。为了增强第一代CAR激活T细胞的能力的目的,已经开发出第二代CAR,第二代CAR在胞内加入了共刺激分子(如CD28、CD134(OX-40)、CD137(4-1BB)等)来源的胞内分子信号传递域。临床试验表明,第二代CAR T细胞在病人体内表现出较好的增殖、持久性和效应功能。第二代CAR T细胞的临床试验,大部分是抗CD19 CAR T对B细胞白血病的治疗。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。第三代CAR是为了进一步提高CAR T细胞疗法的疗效而开发的。第三代CAR的胞内段引入了两个共刺激分子的信号传递区域。通常情况下,一个共刺激信号是CD28胞内区域,另一个则是CD134、CD137或ICOS等的胞内信号传递区域。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都比第二代好。由此可知,现有技术中的CAR结构设计并非十分成熟,需要进一步针对T细胞活性激活、消除调节性T细胞免疫抑制作用和形成记忆性T细胞等方面,对CAR分子进行改良。
美国免疫学家Janeway提出了模式识别理论,将天然免疫针对主要靶分子信号称作病原相关的分子模式(Pathogen-associated molecular pattern,PAMP);相对应的识别受体称为模式识别受体(pattern recognition receptor,PRR)。Toll样受体(TLR)是可特异性识别来源于不同微生物的、具有保守结构的分子,并激活内在免疫应答的PRR。Toll样受体是参与固有免疫的一类重要蛋白质分子,也是连接非特异性免疫和特异性免疫的桥梁。已经发现的人TLR有10种,分别为TLR1-10。大部分的TLR都是单独发挥作用的,除了TLR2可以分别与TLR1、TLR6形成二聚体,识别微生物外源分子和宿主内源分子。越来越多的研究显示,TLR信号在肿瘤发育和治疗中发挥着重要功能。因为一些TLR广泛表达于免疫细胞的细胞表面,而不同的TLR在这些细胞中发挥着不同的作用。研究人员发现,TCR激活的人体T细胞表达TLR2,不表达TLR4,TLR2为T细胞功能激活和记忆性T细胞维持提供共刺激信号【2】。TLR2对CD8阳性T细胞的作用:下调T细胞激活所需的TCR信号强度阈值,促使低TCR信号下的记忆性T细胞形成【7】。TLR1/TLR2激动剂可通过下调Foxp3表达,有效消除调节性T细胞功能【3、4、5】。进一步研究发现,TLR1/TLR2激动剂可通过减少调节性T细胞和上调细胞毒性T细胞的功能,对一般肿瘤达到治疗的效果【6】。
引用文献
1. Restifo,N.P.,Dudley,M.E.,and Rosenberg,S.A.(2012).Adoptiveimmunotherapy for cancer:harnessing the T cell response.Nature reviewsImmunology 12,269-281.
2. Komai-Koma,M.,L.Jones,G.S.Ogg,D.Xu,and F.Y.Liew.2004.TLR2isexpressed on activated T cells as a costimulatory receptor.Proc.Natl.Acad.Sci.USA101:3029–3034.
3. Takeuchi,O.,S.Sato,T.Horiuchi,K.Hoshino,K.Takeda,Z.Dong,R.L.Modlin,and S.Akira.2002.Cutting edge:role of Toll-like receptor 1inmediating immune response to microbial lipoproteins.J.Immunol.169:10–14.
4. Liu,H.,M.Komai-Koma,D.Xu,and F.Y.Liew.2006.Toll-like receptor2signaling modulates the functions of CD4+CD25+regulatory Tcells.Proc.Natl.Acad.Sci.USA103:7048–7053.
5. Sutmuller,R.P.,M.H.den Brok,M.Kramer,E.J.Bennink,L.W.Toonen,B.J.Kullberg,L.A.Joosten,S.Akira,M.G.Netea,and G.J.Adema.2006.Toll-likereceptor 2controls expansion and function of regulatory Tcells.J.Clin.Invest.116:485–494.
6. Zhang,Y.,Luo,F.,Cai,Y.,Liu,N.,Wang,L.,Xu,D.,and Chu,Y.2011.TLR1/TLR2agonist induces tumor regression by reciprocal modulation of effector andregulatory T cells.Journal of immunology 186,1963-1969.
7. Mercier,B.C.,Cottalorda,A.,Coupet,C.A.,Marvel,J.,and Bonnefoy-Berard,N.(2009).TLR2engagement on CD8T cells enables generation of functionalmemory cells in response to a suboptimal TCR signal.Journal of immunology182,1860-1867.
发明内容
本发明的目的在于提供一种新的第三代嵌合抗原受体(CAR),以及编码其的核酸,表达其的细胞,其在制备***的药物中的用途。本发明的嵌合抗原受体在T细胞活性激活、消除调节性T细胞免疫抑制作用和形成记忆性T细胞等方面均具有相比于现有技术的明显优势。
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Toll样受体1和/或Toll样受体2胞内结构域,或Toll样受体1和/或Toll样受体2胞内结构域串联其它信号传递区域如CD3ζ、CD28、41BB等的胞内结构域。
所述Toll样受体(Toll-like receptors,简称TLR)1或Toll样受体2胞内结构域为胞内信号传递区域,也称为TIR结构域。Toll样受体是I型跨膜蛋白质,识别侵入体内的微生物进而激活免疫细胞的应答。被认为在先天性免疫***中起关键作用。Toll样受体是模式识别受体(pattern recognition receptors,PRR)的一类,识别与宿主不同的病原体分子。这些分子被统称为病原相关分子模式(pathogen-associated molecular patterns,PAMP),是最保守的免疫***的组成部分。Toll样受体1和Toll样受体都是Toll样受体家族的重要成员。
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、GnT-V、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、MUM-3、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或Mesothelin。本发明专利所提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Toll样受体1和/或Toll样受体2胞内结构域作为其胞内结构域,还可以包含除了Toll样受体1和/或Toll样受体2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Toll样受体1和/或Toll样受体2胞内结构域之外,所述胞内结构域还包括CD3ζ胞内结构域;进一步优选地,所述Toll样受体1和/或Toll样受体2胞内结构域配置在CD3ζ胞内结构域的C末端侧。
在进一步优选的实施方案中,除了Toll样受体1和/或Toll样受体2胞内结构域,CD3ζ胞内结构域之外,所述胞内结构域还包括CD28胞内结构域。
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CD28胞内结构域、CD3ζ胞内结构域和Toll样受体1和/或Toll样受体2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Toll样受体1和/或Toll样受体2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胞内结构域,CD3ζ胞内结构域,Toll样受体1和/或Toll样受体2胞内结构域。
第二方面,本发明提供了一种编码如第一方面所述的嵌合抗原受体的核酸。
第三方面,本发明提供了一种嵌合抗原受体表达细胞,其中引入了如第二方面所述的核酸;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,其包括将如第二方面所述的核酸引入细胞的步骤;优选地,所述细胞为T细胞或含有T细胞的细胞群。
第五方面,本发明提供了一种如第一方面所述的嵌合抗原受体、如第二方面所述的核酸或如第三方面所述的嵌合抗原受体表达细胞在制备***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或肺癌。
值得注意的是,本发明的CAR特征在于它包含Toll样受体1和/或Toll样受体2胞内结构域作为其胞内结构域。所述Toll样受体1和/或Toll样受体2的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。
有益效果
本发明通过在CAR分子中引入Toll样受体1和/或Toll样受体2胞内信号传递结构域,促进CAR T细胞的增殖和杀伤效应以及记忆性CAR T细胞的形成,进而提高肿瘤免疫效应和治疗效果。与现有技术中的CAR分子相比,本发明的CAR分子具有以下优势:1)促进CART细胞体内外的抗凋亡和增殖;2)显著提高了CAR T细胞的抗肿瘤功能;3)可降低调节性T细胞的免疫抑制作用;4)利于形成记忆性的CAR T细胞,抑制肿瘤复发;鉴于上述优势,本发明的CAR分子在抗肿瘤治疗中具有良好的临床应用前景。
附图说明
图1显示GFP T、CAR19 T、CAR19T1 T和CAR19T2 T细胞对不表达CD19的K562-GL细胞的体外杀伤效应。
图2显示GFP T、CAR19 T、CAR19T1 T和CAR19T2 T细胞对表达CD19的K562-CD19-GL细胞的体外杀伤效应。
图3显示GFP T、CAR19 T、CAR19T1 T和CAR19T2 T细胞对表达CD19的NALM6-GL细胞的体外杀伤效应。
图4显示GFP T、CAR19 T、CAR19T1 T和CAR19T2 T细胞对表达CD19的REH-GL细胞的体外杀伤效应。
图5显示GFP T、CAR19 T、CAR19T1 T和CAR19T2 T细胞分别与K562GL或K562-CD19-GL细胞共培养18h后,上清中IL-2的分泌水平。
图6显示CARMeso和CARMesoT1经慢病毒转染T细胞的效率。
图7显示图6中的经慢病毒转染的T细胞对A549GL细胞的体外杀伤效应;野生型T细胞,以及针对CD22分子的CAR T细胞作为对照。
图8显示GFP、CAR19、CAR19T1、CAR19T2经慢病毒分别转染T细胞的效率。
图9显示图8中的经慢病毒转染的T细胞对NALM6细胞在免疫缺陷小鼠体内形成的肿瘤大小的影响;
图10显示图8中的经慢病毒转染的T细胞对NALM6细胞在免疫缺陷小鼠体内形成的肿瘤重量的影响。
图11显示图6中经慢病毒转染的T细胞对A549-GL细胞在免疫缺陷小鼠体内形成肿瘤重量的影响。
具体实施方式
为便于理解本发明,本发明列举实施例如下。本领域技术人员应该明了,所述实施例仅仅是帮助理解本发明,不应视为对本发明的具体限制。
一般性方法
总的来说,在分别用于治疗血液瘤(急性B淋系白血病,B-ALL)的抗人CD19嵌合抗原受体和实体瘤(肺癌)的抗人Mesothelin嵌合抗原受体的胞内段中***TLR1和/或TLR2信号传递结构域序列,构建四种新型嵌合抗原受体:抗CD19 ScFv-CD28-CD3ζ-TLR1/TLR2和抗Mesothelin ScFv-CD28-CD3ζ-TLR1/TLR2,以下分别将四种嵌合抗原受体简称为:CAR19T1、CAR19T2、CARMesoT1、CARMesoT2,并通过体内、外实验验证表达上述CAR分子的CAR T细胞的增殖功能、肿瘤杀伤功能和记忆性CAR T的形成。
CAR质粒的构建
如上所述,CAR分子包括胞外区域、跨膜区域、胞内区域,因此,以下实施例所用CAR质粒的构建步骤包括:
首先,通过基因合成获得CAR质粒所需各基因DNA,包括:抗CD19抗体免疫球蛋白重链和轻链的可变区域序列、抗Mesothelin抗体免疫球蛋白重链和轻链的可变区域序列、CD28跨膜信号区域序列、TLR1信号传递区域序列、TLR2信号传递区域序列、CD3ζ信号传递区域序列;
然后,通过酶切、连接等步骤将所需要的上述合成的各基因序列串联起来,即获得本发明的新型CAR分子,其序列结构分别如下:
CAR19T1:抗CD19抗体免疫球蛋白重链和轻链的可变区域(胞外区域)、CD28跨膜信号区域(跨膜区域)、CD3ζ信号传递区域+TLR1信号传递区域;
CAR19T2:抗CD19抗体免疫球蛋白重链和轻链的可变区域(胞外区域)、CD28跨膜信号区域(跨膜区域)、CD3ζ信号传递区域+TLR2信号传递区域;
CAR19(CAR19T1/T2的对照组):抗CD19抗体免疫球蛋白重链和轻链的可变区域(胞外区域)、CD28跨膜信号区域(跨膜区域)、CD3ζ信号传递区域;
CARMesoT1:抗Mesothelin抗体免疫球蛋白重链和轻链的可变区域(胞外区域)、CD28跨膜信号区域(跨膜区域)、CD3ζ信号传递区域+TLR1信号传递区域;
CARMesoT2:抗Mesothelin抗体免疫球蛋白重链和轻链的可变区域(胞外区域)、CD28跨膜信号区域(跨膜区域)、CD3ζ信号传递区域+TLR1信号传递区域;
CARMeso(CARMesoT1/T2的对照组):抗Mesothelin抗体免疫球蛋白重链和轻链的可变区域(胞外区域)、CD28跨膜信号区域(跨膜区域)、CD3ζ信号传递区域。
将以上六种CAR分子核酸序列分别***第二代慢病毒表达载体pWPXLd-GFP中,分别构建pWPXLd-CAR19T1-GFP、pWPXLd-CAR19T2-GFP、pWPXLd-CAR19-GFP、pWPXLd-CARMesoT1-GFP、pWPXLd-CARMesoT2-GFP和pWPXLd-CARMeso-GFP质粒。
具体实施例
实施例1
CAR19T1、CAR19T2质粒的制备
按如下步骤制备本发明的携带含TLR1或/和TLR2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CAR19基因(SEQ ID NO.1)的质粒pUC57-CAR19,CAR19基因包含抗CD19单抗ScFv(SEQ ID NO.15)、CD28跨膜区和胞内区、CD3ζ胞内区,即CD19ScFv28Z。
(2)通过内切酶PmeI和SpeI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中,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28Z。
(4)用28Z串联TLR1胞内信号传递结构域的cDNA作为模板,构建4条引物(SEQ IDNO.2-5),通过重叠延伸PCR得到28ZT1(SEQ ID NO.9);同样地,用28Z串联TLR2胞内信号传递区域的cDNA作为模板,构建另外4条引物(SEQ ID NO.2,6,7,8),通过重叠延伸PCR得到28ZT2(SEQ ID NO.10)。
(5)通过NotI和SpeI酶切,用28ZT1或28ZT2替换pUC57-CAR19中的28Z,分别得到pUC57-CAR19T1或pUC57-CAR19T2质粒。
(6)最后,通过内切酶PmeI和SpeI酶切,用CAR19T1或CAR19T2替换pWPXLD-CAR19-GFP中的CAR19,分别得到pWPXLd-CAR19T1-GFP或pWPXLd-CAR19T2-GFP质粒。
TLR1和TLR2的胞内信号传递结构域分别称为TIR1(其核苷酸序列如SEQ ID NO.11所示)和TIR2(其核苷酸序列如SEQ ID NO.12所示),TIR1为TLR1蛋白最C端的162个氨基酸(a.a.625-786,序列如SEQ ID NO.13所示),TIR2为TLR2蛋白最C端的159个氨基酸(a.a.626-784,序列如SEQ ID NO.14所示)。
实施例2 CARMesoT1、CARMesoT2、CAR22质粒的制备
通过基因合成得到Mesothelin单抗scFv结构域序列(SEQ ID NO.17),通过内切酶PmeI和NotI酶切,用Mesothelin单抗scFv结构域分别替换pWPXLd-CAR19T1-GFP和pWPXLd-CAR19T2-GFP中的CD19单抗scFv结构域,分别得到pWPXLd-CARMesoT1-GFP或pWPXLd-CARMesoT2-GFP。
此外,CARMesoT1/T2的阴性对照为CAR22质粒,其质粒结构为:慢病毒表达载体pWPXLd-抗CD22抗体免疫球蛋白重链和轻链的可变区域(胞外区域)-CD28跨膜信号区域(跨膜区域)-CD3ζ信号传递区域-GFP;其制备方法为:通过基因合成抗CD22抗体免疫球蛋白重链和轻链的可变区域(即SEQ ID NO.16),并通过酶切黏贴替代CAR19质粒中的抗CD19抗体免疫球蛋白重链和轻链的可变区域,即得CAR22质粒(pWPXLd-CAR22-GFP)。
实施例3 CAR质粒的慢病毒包装
使用实施例1、2所制备的本发明CAR质粒以及相关对照质粒,通过慢病毒包装,获得分别过表达GFP(空白对照)、CAR19T1-GFP、CAR19T2-GFP、CAR19-GFP(对照)、CARMesoTLR1-GFP、CARMesoTLR2-GFP、CARMeso-GFP(对照)、CAR22-GFP(阳性对照)的8种慢病毒。本实施例3和4中,将含CAR的质粒统一描述为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六种质粒(即,分别包含CAR19T1、CAR19T2、CAR19、CARMesoT1、CARMesoT2、CARMeso)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:
④分别于转化后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6种CAR慢病毒或空白对照GFP慢病毒;
⑧病毒溶解后,收集病毒溶液分装于PCR管,冻存于-80℃待用。
实施例4使用包装的CAR慢病毒感染人体T细胞
①T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(7种)CAR的T细胞。
⑤CAR T细胞扩增:将CAR T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,CAR T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6种CAR T细胞(分别简称CAR19T1-GFP、CAR19T2-GFP、CAR19-GFP、CARMesoTLR1-GFP、CARMesoTLR2-GFP、CARMeso-GFP)或空白对照T细胞(GFP-T)的比例。
实施例5 CAR T细胞体外识别杀伤肿瘤的效应
将实施例4制备的GFP T(空白对照)、CAR19T1 T、CAR19T2T和CAR19 T(对照),或GFP T、CARMesoT1 T、CARMesoT2 T和CARMeso T(对照)细胞以不同比例分别与1x104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18h;
体外比较GFP T、CAR19 T、CAR19T1 T、CAR19T2 T细胞对血液瘤的识别杀伤功能时,肿瘤细胞选用NALM6-GL(GFP+Luciferase)、REH-GL、K562-GL和K562-CD19-GL四种带荧光素酶的白血病或淋巴瘤细胞系;体外验证GFP T、CARMeso T、CARMesoT1 T、CARMesoT2 T细胞对实体瘤的识别杀伤功能时,肿瘤细胞选用表达Mesothelin的A549-GL带荧光素酶的人肺腺癌细胞系。
荧光素酶(Luciferase)定量杀伤效率评估方法:CAR T细胞与肿瘤细胞共培养(实验对照组为肿瘤细胞单独培养)后18小时,在96孔细胞培养板中加入100μl/孔的荧光素酶底物(1×),将细胞重悬混匀,立即通过多功能酶标仪测定RLU(relative light unit),测定时间设为1秒。杀伤比例计算公式:100%×(对照孔读数-实验孔读数)/对照孔读数(不加细胞的空白组读数可以忽略);其结果如图1-4所示。
结果表明,CAR19T1 T和CAR19T2 T细胞对表达CD19的肿瘤靶细胞的体外杀伤效率都显著高于CAR19 T细胞,在E:T(即Effector效应T细胞与Target靶细胞的比例)很小的情况下,即肿瘤靶细胞远大于效应T细胞,CAR19T1 T和CAR19T2 T细胞也能表现出很强的肿瘤杀伤活性(见图1-4)。
通过ELISA检测GFP T、CAR19 T、CAR19T1 T、CAR19T2 T细胞分别与K562GL或K562-CD19-GL细胞共培养18h后上清中的IL-2水平,结果如图5所示;图5结果显示,CAR19T2 T细胞分泌IL-2的水平高于CAR19 T细胞,这说明TLR2的胞内结构域的加入能提高CAR T细胞的IL-2分泌,即,可促进T细胞增殖。
另外,在CAR T细胞比例很低的情况下(如图6所示),CARMesoT1 T细胞对表达Mesothelin的靶细胞的体外杀伤效率也显著高于CARMeso T细胞(见图7)。
实施例6 CAR19T1/T2 T细胞体内识别杀伤肿瘤
为比较GFP T、CAR19 T、CAR19T1 T和CAR19T2 T细胞的体内识别杀伤肿瘤的效应,将同等数量(2×105)的NALM6细胞分别通过皮下移植至16只NSI(NOD/SCID IL2rg-/-)免疫缺陷小鼠体内;NALM6细胞移植后第2天和第9天(肿瘤细胞移植当天为第0天),将2×106个T细胞(四组:GFP T、CAR19 T、CAR19T1 T、CAR19T2,每组注射4只小鼠,阳性细胞比例如图8所示)静脉注射入已移植NALM6细胞的NSI免疫缺陷小鼠体内;第33天,将所有小鼠安乐处死,取肿瘤量大小并称重。
结果表明,CAR19T1 T和CAR19T2 T都能显著抑制皮下NALM6细胞的生长,而CAR19T细胞组跟GFP T细胞组相比,肿瘤重量则没有显著变化(图9,10)。
另一方面,也说明CAR19 T虽然能对血液性肿瘤具有良好的杀伤效果,但对皮下实体瘤的杀伤效果不佳;而加入TLR1或TLR2的胞内结构域后,则可显著改善第二代CAR T细胞对实体瘤的杀伤效果。
实施例7 CARMesoT1/T2 T细胞体内识别杀伤肿瘤
为比较CAR22 T、CARMesoT1 T和CARMesoT2 T细胞的体内识别杀伤肿瘤的效应,将同等数量(1×105)的A549-GL细胞分别移植至12只NSI(NOD/SCID IL2rg-/-)免疫缺陷小鼠后肢腹股沟处皮下;NALM6细胞移植后第7天和第14天(肿瘤细胞移植当天为第0天),分别将2×106和1×106个T细胞(三组:CAR22 T、CARMesoT1 T和CARMesoT2 T,每组注射4只小鼠,阳性细胞比例如图6所示)静脉注射入已移植NALM6细胞的NSI免疫缺陷小鼠体内;第68天,将所有小鼠安乐处死,取肿瘤并称重记录。
结果表明,CARMeso T和CARMesoT1 T细胞均能抑制皮下肿瘤的生长,而CARMesoT1T细胞比CARMeso T细胞的抑制效果更佳(图11)。
通过以上检测结果,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TLR1和TLR2信号传递结构域对CAR T体内、外肿瘤杀伤功能的改善。
申请人声明,本发明通过上述实施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和使用方式,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施。所属技术领域的技术人员应该明了,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。
Claims (16)
1.一种嵌合抗原受体,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,所述至少一个胞内结构域包括Toll样受体1和/或Toll样受体2胞内结构域;
其中,所述抗原是肿瘤抗原,所述胞内结构域包括Toll样受体1的氨基酸序列如SEQ IDNO.13所示,所述胞内结构域包括Toll样受体2的氨基酸序列如SEQ ID NO.14所示;
所述能够结合抗原的胞外结构域是结合抗原的抗体的单链可变片段,所述胞内结构域还包括CD3ζ胞内结构域;
所述胞内结构域进一步包括CD28胞内结构域。
2.根据权利要求1所述的嵌合抗原受体,其特征在于,所述Toll样受体1和/或Toll样受体2胞内结构域配置在CD3ζ胞内结构域的C末端侧。
3.根据权利要求1所述的嵌合抗原受体,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CD28胞内结构域、CD3ζ胞内结构域和Toll样受体1和/或Toll样受体2胞内结构域。
4.根据权利要求1所述的嵌合抗原受体,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CD3ζ胞内结构域、Toll样受体1和/或Toll样受体2胞内结构域和CD28胞内结构域。
5.根据权利要求1所述的嵌合抗原受体,其特征在于,所述胞内结构域包括串联连接的三个或更多个胞内结构域。
6.根据权利要求1所述的嵌合抗原受体,其特征在于,所述Toll样受体1和/或Toll样受体2胞内结构域配置在C-末端侧。
7.根据权利要求1所述的嵌合抗原受体,其特征在于,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胞内结构域,CD3ζ胞内结构域,Toll样受体1和/或Toll样受体2胞内结构域。
8.根据权利要求7所述的嵌合抗原受体,其特征在于,所述肿瘤抗原为CD19或Mesothelin抗原。
9.编码如权利要求1-8任一项所述的嵌合抗原受体的核酸。
10.一种嵌合抗原受体表达细胞,其中引入了如权利要求9所述的核酸。
11.根据权利要求10所述的嵌合抗原受体表达细胞,其特征在于,所述细胞为T细胞或含有T细胞的细胞群。
12.制备如权利要求10所述的嵌合抗原受体表达细胞的方法,其包括将如权利要求9所述的核酸引入细胞的步骤。
13.根据权利要求12所述的方法,其特征在于,所述细胞为T细胞或含有T细胞的细胞群。
14.如权利要求1-8任一项所述的嵌合抗原受体、如权利要求9所述的核酸或如权利要求10或11所述的嵌合抗原受体表达细胞在制备***的药物中的用途。
15.根据权利要求14所述的应用,其特征在于,所述肿瘤为实体瘤或血液瘤。
16.根据权利要求15所述的应用,其特征在于,所述肿瘤为B-ALL或肺癌。
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