CN113122502A - 促实体瘤浸润的增强型cart细胞及其制备方法和细胞药物 - Google Patents
促实体瘤浸润的增强型cart细胞及其制备方法和细胞药物 Download PDFInfo
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Abstract
本发明公开了一种促实体瘤浸润的增强型CART细胞及其制备方法和细胞药物,涉及CART细胞技术领域。本发明公开的CART细胞含有第一核酸序列和第二核酸序列。该CART细胞对实体瘤的肿瘤组织具有更高的浸润能力,其对肿瘤细胞的杀伤能力更强,其具有广阔的临床应用前景,本发明为使用CART细胞治疗实体瘤提供了一种新的治疗思路和策略。
Description
技术领域
本发明涉及CART细胞技术领域,具体而言,涉及一种促实体瘤浸润的增强型CART细胞及其制备方法和细胞药物。
背景技术
过继性T细胞转移(Adoptive T cell transfer,ACT)是目前最有前景的免疫治疗方法,CD-19特异性的CAR-T细胞在治疗复发难治性急性淋巴细胞白血病中可出现完全缓解,其主要的治疗流程为:首先分离出患者自己的T细胞(或来自同种异体供者的T细胞),然后予以活化并进行基因修饰以获得嵌合抗原受体T细胞(chimeric antigen receptor Tcells,CAR-T),最后回输至患者体内。嵌合抗原受体由细胞外抗原识别结构域(通常是抗体单链可变片段scFv)与细胞内信号传导结构域(T细胞受体的CD3ζ链或同时引入一种或多种共刺激信号如CD28和4-1BB)相连接而成,其细胞外部分可使T细胞具有识别特异性抗原的能力。能够越过MHC限制性,可与其识别的抗原直接结合后便会通过信号传导结构域刺激T细胞增殖,同时激活T细胞的细胞毒作用并促进细胞因子分泌,最终消除带有该抗原的细胞,具有更好的特异性和持续性。
虽然CAR-T细胞在治疗血液瘤方面成效显著,但在治疗实体瘤上依旧面临着挑战,治疗效果并不理想。
鉴于此,特提出本发明。
发明内容
本发明的目的在于提供一种促实体瘤浸润的增强型CART细胞及其制备方法和细胞药物,本发明提供的CART细胞,其对实体瘤的肿瘤组织具有更高的浸润能力,提高了其对肿瘤细胞的杀伤能力,具有广阔的临床应用前景,为使用CART细胞治疗实体瘤提供了一种新的治疗思路和策略。
本发明是这样实现的:
第一方面,本发明实施例提供一种促实体瘤浸润的增强型CART细胞,所述CART细胞含有第一核酸序列和第二核酸序列;
所述第一核酸序列含有编码嵌合抗原受体的第一编码序列,所述嵌合抗原受体的抗原结合结构域能够靶向所述实体瘤;
所述第二核酸序列含有编码LIGHT蛋白的第二编码序列以及位于所述第二编码序列上游的信号肽编码序列;
所述第二编码序列由组成型启动子驱动表达。
本发明的发明人分析认为,目前,CART细胞对实体瘤的治疗效果不理想的一个主要原因在于:实体瘤特殊的脉管系统和基质屏障阻止T细胞浸润。肿瘤促生的异常血管使T细胞浸润特化血管高内皮微静脉High endothelial venules(HEV)丧失功能,功能不全的HEV降低对T细胞的浸润。同时,由肿瘤相关基质细胞cancer-associated stromal cells(CASCs)诱导形成的纤维结构影响T细胞的浸润和功能,Carcinoma-associatedfibroblasts(CAFs)作为CASCs的主要子集,还可通过分泌TGF-β等细胞因子抑制T细胞功能。
LIGHT蛋白(homologous to lymphotoxins,inducible,competes With HSVglycoprotein D for HVEM,expressed by T Lymphocyte、TNFSF14)是细胞因子TNF超家族的成员,是一种诱导型炎性细胞因子。本发明实施例提供的CART细胞利用组成型启动子驱动LIGHT蛋白持续大量地分泌表达,即过表达LIGHT蛋白,当CART细胞接触肿瘤细胞时,这些分泌表达的LIGHT蛋白,使CART细胞对实体瘤的浸润能力得到有效提高,进而提高了CART细胞杀伤肿瘤细胞的能力,该CART细胞具有广阔的临床应用前景,本发明为使用CART细胞治疗实体瘤提供了一种新的治疗思路和策略。
在可选的实施方式中,所述第二核酸序列位于所述第一核酸序列下游,所述组成型启动子位于所述第一核酸序列上游,所述第一编码序列和所述第二编码序列均由所述组成型启动子驱动表达。
将第一编码序列和第二编码序列置于同一组成型启动子下受其驱动表达,以及将第一核酸序列和第二核酸序列设置在一起即位于同一DNA链上,可以减少整个核酸序列的长度,降低CART细胞的基因组装载量,便于CART细胞的改造。当然,在其他的实施方式中,可以将第一编码序列和第二编码序列分别由两个独立的启动子驱动表达;甚至是将第一核酸序列和第二核酸序列分别置于不同的DNA链上或置于同一DNA链但第一核酸序列和第二核酸序列之间间隔有其他核酸序列,具有这些情形以及类似情形的CART细胞也是属于本发明的保护范围。
在可选的实施方式中,所述组成型启动子选自延伸生长因子-1α(EF-1α)、早期巨细胞病毒(CMV)启动子序列、类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、肌动蛋白启动子、肌球蛋白启动子和血红素启动子和肌酸激酶启动子中的任意一种。
但本发明的组成型启动子并不限于上述的启动子,使用其他类型的启动子驱动LIGHT蛋白的表达也是属于本发明的保护范围。
在可选的实施方式中,所述组成型启动子为EF-1α。
本发明的组成型启动子可以根据实际需求选择,其包括但不限于EF-1α,其只要能够驱动LIGHT蛋白大量表达即可。
这里的术语“大量表达”是指在该组成型启动子驱动下,CART细胞所分泌表达的LIGHT蛋白量高于没有该组成型启动子驱动下的表达量。
在可选的实施方式中,所述LIGHT蛋白为LIGHT全长蛋白或其功能性片段。
LIGHT蛋白可以是膜结合型、胞内游离型、胞外剪切型、或者其他各种具有活性的片段,只要其选自LIGHT全长蛋白且具有提高CART细胞杀伤肿瘤细胞的活性即可。
在可选的实施方式中,所述LIGHT蛋白为LIGHT全长蛋白的可溶性片段。
在可选的实施方式中,所述LIGHT蛋白的氨基酸序列如SEQ ID NO.15所示。
缺乏胞内段和跨膜区的溶解型LIGHT(SEQ ID NO.15)依然具有正常的生物学功能,组成性表达溶解型LIGHT的癌细胞无法成瘤,并且其在IFNγ的协同作用下,LIGHT杀伤肿瘤细胞的能力更强,更能提高CART细胞的肿瘤杀伤能力。
在可选的实施方式中,所述LIGHT蛋白融合VTP多肽。即第二编码序列编码LIGHT蛋白和VTP多肽。
在可选的实施方式中,所述VTP多肽的氨基酸序列如SEQ ID NO.17所示。
VTP多肽可以靶向肿瘤异常血管,通过VTP多肽的融合,其可以起到靶向运输的作用,将LIGHT蛋白带向肿瘤异常血管,从而促进肿瘤异常血管正常化,促进肿瘤部位淋巴样结构形成以引导CART细胞在此聚集与活化、调动自身免疫功能、重塑肿瘤微环境的抑制作用,从而改变CART细胞因无法穿透肿瘤组织及免疫抑制造成的效果局限性,增强CAR-T细胞对抗实体肿瘤的活性,极大的改善CART细胞在实体瘤治疗中的效果。
在可选的实施方式中,所述信号肽编码序列所编码的信号肽选自膜整合型信号肽或分泌型信号肽。
在可选的实施方式中,所述膜整合型信号肽选自CD8信号肽、CD28信号肽、GM-CSF信号肽、CD4信号肽和CD137信号肽中的任意一种。
在可选的实施方式中,所述分泌型信号肽选自IgG信号肽和细胞因子信号肽中的任意一种。
优选地,所述信号肽编码序列的碱基序列如SEQ ID NO.13所示。
在可选的实施方式中,所述嵌合抗原受体还具有跨膜结构域和共刺激信号传导区。
在可选的实施方式中,所述跨膜结构域选自如下蛋白分子中的至少一种的跨膜结构域:CD5、CD28、CD137、CD3ε、CD154、CD45、CD4、CD9、CD37、CD16、CD33、CD22、CD134和CD8α。
在可选的实施方式中,所述跨膜结构域为CD8α跨膜结构域。
在可选的实施方式中,所述共刺激信号传导区包含如下共刺激分子中至少一种的细胞内结构域:OX40、CD3γ、CD3δ、CD134、CD5、CD79a、CD137、ICD3ε、CD154、CD22、CD66d、CD2、CD28、CD4、CD5、CD79b、COS、4-1BB和CD3ζ。
在可选的实施方式中,所述共刺激信号传导区包括4-1BB的胞内共刺激元件和CD3ζ的胞内结构域。
在可选的实施方式中,所述实体瘤选自肝癌、头颈癌、黑色素瘤、膀胱癌、胶质母细胞瘤、宫颈癌、肺癌、软骨肉瘤、甲状腺癌、肾癌、间皮瘤、骨肉瘤、胆管癌、卵巢癌、胃癌、膀胱癌、前列腺癌、脑膜瘤、胰腺癌、多发性鳞状细胞瘤、食管癌、肺小细胞癌、结直肠癌、乳腺癌、成神经管细胞瘤和乳腺癌中的任意一种。
在可选的实施方式中,所述实体瘤为前列腺癌;
需要说明的是,本发明的实体瘤包括但不限于肝癌、头颈癌、黑色素瘤、膀胱癌、胶质母细胞瘤、宫颈癌、肺癌、软骨肉瘤、甲状腺癌、肾癌、间皮瘤、骨肉瘤、胆管癌、卵巢癌、胃癌、膀胱癌、前列腺癌、脑膜瘤、胰腺癌、多发性鳞状细胞瘤、食管癌、肺小细胞癌、结直肠癌、乳腺癌、成神经管细胞瘤和乳腺癌,针对其他类型的实体瘤也是属于本发明的保护范围。
在可选的实施方式中,所述抗原结合结构域能够靶向前列腺癌的特异性膜抗原,所述抗原结合结构域选自Fab、Fab’、F(ab’)2和scFv中的任意一种。
在可选的实施方式中,所述抗原结合结构域为scFv。
需要说明的是,本发明实施例中的抗原结合结构域可以根据所要治疗的实体瘤类别进行选择,无论以何种scFv作为抗原结合结构域,其均是属本发明的保护范围。
在可选的实施方式中,所述抗原结合结构域的轻链可变区的氨基酸序列如SEQ IDNO.3所示,所述抗原结合结构域的重链可变区的氨基酸序列如SEQ ID NO.7所示,所述抗原结合结构域的重链可变区与轻链可变区之间的铰接区氨基酸序列如SEQ ID NO.5所示。
由SEQ ID NO.3、SEQ ID NO.5和SEQ ID NO.7构成的抗原结合结构域其抗原为前列腺特异性膜抗原,可以靶向前列腺癌。
需要说明的是,上述的第一核酸序列和第二核酸序列只要其编码相应的蛋白即可,其并没有特别的限制,在蛋白序列清楚的基础上,本领域技术人员是容易获得编码该蛋白的核酸序列的,因此,无论第一核酸序列和第二核酸序列的具体核苷酸序列如何变化,其只要能够编码上述的各自对应的蛋白,即属于本发明的保护范围。
第二方面,本发明实施例提供制备如前述实施方式任一项所述的促实体瘤浸润的增强型CART细胞的方法,其包括:使目标T细胞含有所述第一核酸序列和所述第二核酸序列。
需要说明的是,在本发明公开了上述CART细胞的基础上,本领域技术人员是采用本领域常规技术是容易制备出上述CART细胞的,因此,无论以何种方法制备出上述CART细胞,其均是本发明的保护范围。
第三方面,本发明实施例提供一种治疗实体瘤的细胞药物,其含有作为活性成分的如前述实施方式任一项所述的促实体瘤浸润的增强型CART细胞。
需要说明的是,本发明提供的CART细胞不仅可以单独作为活性药物应用治疗实体瘤,也可以与其他活性药物联合,用于治疗实体瘤,因此,将本发明提供的CART细胞与其他抗肿瘤药物联用也是属于本发明的保护范围。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为实施例1中第一核酸序列和第二核酸序列所含有的表达元件的结构示意图;图中,A第一核酸序列的结构示意图,B第一核酸和第二核酸连接的结构示意图。
图2为CJ-PSMA-LIGHT-VTP质粒载体骨架的结构示意图。
图3为慢病毒包装辅助质粒pMD2.G的结构示意图。
图4为慢病毒包装辅助质粒psPAX2的结构示意图。
图5为实施例1中的不同病毒感染293T细胞的阳性率检测结果。
图6为实施例1中的不同CART的CAR阳性率检测结果。
图7为实施例1中的不同CART的LIGHT表达水平。
图8为在不同效靶比下,不同CART的杀伤效率结果。
图9为在E:T为1:1的条件下,不同CART的分泌的LIGHT含量检测结果。
图10为不同CART在相同的培养体系中两周内的增殖情况。
图11为注射PSMA-CART或PSMA-CART+hLIGHT后,小鼠体内肿瘤大小的活体成像结果。
图12为注射PSMA-CART或PSMA-CART+hLIGHT治疗后,小鼠体内肿瘤直观大小。
图13为注射PBS或者hLIGHT后,小鼠体内肿瘤大小的活体成像结果。
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,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
本实施例以前列腺癌为例,构建能够特异性靶向前列腺癌且分泌表达LIGHT-VTP蛋白的CART细胞。
1构建含表达嵌合抗原受体和LIGHT-VTP表达盒的质粒载体,表达盒上的各元件结构和位置关系参考图1,质粒载体骨架参考图2。
具体步骤如下:
由金斯瑞公司合成表达靶向前列腺特异性膜抗原的嵌合抗原受体的第一核酸序列,第一核酸序列(PSMA-CAR)包括:CD8α信号肽、PSMA单链抗体重链可变区、Linker1、PSMA单链抗体轻链、CD8铰链区、CD8α跨膜结构域、4-1BB的胞内共刺激元件和CD3ζ的胞内结构域(图1中A),将以上序列依次连接,在最前端引入Kozac序列和相应酶切位点。使用XbaI和SalI双酶切将第一核酸序列转移至质粒空载体(Carl June(CJ)团队的慢病毒转移载体),酶连后得到嵌合抗原受体表达载体CJ-PSMA-CAR,将其作为对照质粒。进一步地,以CJ-PSMA-CAR质粒为初始质粒,添加表达LIGHT-VTP的第二核酸序列,第一核酸序列与第二核酸序列之间用P2A连接,第二核酸序列(LIGHT-VTP)依次包括:分泌信号肽(SP)、LIGHT-VTP(图1中B)。得到的质粒命名为CJ-PSMA-LIGHT-VTP质粒(结构见图2)。
其中,表达靶向前列腺特异性膜抗原的嵌合抗原受体的第一表达盒的各元件序列如下:
CD8α信号肽(CD8αLeader)的碱基序列如SEQ ID NO.1所示:
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg。
PSMA单链抗体轻链可变区(PSMA-ScFv VL)的碱基序列如SEQ ID NO.2所示:
gacatcgtgatgacccagtccccctcctccctgtctgcctccgtgggcgacagagtgaccatcacatgcaaggcctcccaggattgtggcaccgccgtggactggtatcagcagaagcctggcaaggcccctaagctgctgatctactgggcctccaccagacacaccggcgtgcctgacagattcaccggctccggctctggcaccgacttcaccctgaccatctccagcctgcagcctgaggacttcgccgactacttctgccagcagtacaactcctaccctctgaccttcggcggaggcaccaagctggaaatcaaa;
PSMA单链抗体轻链可变区的氨基酸序列如SEQ ID NO.3所示:
DIVMTQSPSSLSASVGDRVTITCKASQDCGTAVDWYQQKPGKAPKLLIYWASTRHTGVPDRFT GSGSGTDFTLTISSLQPEDFADYFCQQYNSYPLTFGGGTKLEIK。
PSMA-ScFv VL与PSMA-ScFv VH的Linker的碱基序列如SEQ ID NO.4所示:
ggcggaggcggatcaggtggtggcggatctggaggtggcggaagc;
Linker的氨基酸序列如SEQ ID NO.5所示:
GGGGSGGGGSGGGGS。
PSMA单链抗体重链可变区(PSMA-ScFv VH)的碱基序列如SEQ ID NO.6所示:
gaagtgcagctggtgcagtctggcgccgaagtgaagaaacctggcgcctccgtgaagatctcctgcaagacctccggctacaccttcaccgagtacaccatccactgggtgaaacaggcctccggcaagggcctggaatggatcggcaacatcaaccctaacaacggcggcaccacctacaaccagaagttcgaggaccgggccaccctgaccgtggacaagtccacctccaccgcctacatggaactgtcctccctgcggtctgaggacaccgccgtgtactactgcgccgctggctggaacttcgactactggggccagggcaccacagtgacagtctcgagc;
PSMA单链抗体重链可变区(PSMA-ScFv VH)的氨基酸序列如SEQ ID NO.7所示:
EVQLVQSGAEVKKPGASVKISCKTSGYTFTEYTIHWVKQASGKGLEWIGNINPNNGGTTYNQKFEDRATLTVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSS。
CD8铰链区(CD8 hinge)的碱基序列如SEQ ID NO.8所示:
accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat。
CD8α跨膜结构域(CD8a-TM)的碱基序列如SEQ ID NO.9所示:
atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc。
4-1BB的胞内共刺激元件的碱基序列如SEQ ID NO.10所示:
aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg。
CD3ζ的胞内结构域的碱基序列如SEQ ID NO.11所示。
agagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc。
P2A的碱基序列如SEQ ID NO.12所示:
gccacaaacttctctctgctaaagcaagcaggtgatgttgaagaaaaccccgggcct。
分泌信号肽(SP)的碱基序列如SEQ ID NO.13所示:
atggaaactgatactttgctgctctgggtcctgctgctgtgggtccctggatcaacgggggac。
LIGHT的碱基序列如SEQ ID NO.14所示:
ggagagatggtcacccgcctgcctgacggacctgcaggctcctgggagcagctgatacaagagcgaaggtctcacgaggtcaacccagcagcgcatctcacaggggccaactccagcttgaccggcagcggggggccgctgttatgggagactcagctgggcctggccttcctgaggggcctcagctaccacgatggggcccttgtggtcaccaaagctggctactactacatctactccaaggtgcagctgggcggtgtgggctgcccgctgggcctggccagcaccatcacccacggcctctacaagcgcacaccccgctaccccgaggagctggagctgttggtcagccagcagtcaccctgcggacgggccaccagcagctcccgggtctggtgggacagcagcttcctgggtggtgtggtacacctggaggctggggagaaggtggtcgtccgtgtgctggatgaacgcctggttcgactgcgtgatggtacccggtcttacttcggggctttcatggtg;
LIGHT的氨基酸序列如SEQ ID NO.15所示:
GEMVTRLPDGPAGSWEQLIQERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV。
VTP的碱基序列如SEQ ID NO.16所示:
ggcggcggctgccggggccggagaagcaccggc。
VTP的氨基酸序列如SEQ ID NO.17所示:GGGCRGRRSTG。
2构建表达嵌合抗原受体和LIGHT-VTP的病毒
方法如下:利用大肠杆菌扩增上述CJ-PSMA-CAR和CJ-PSMA-LIGHT-VTP质粒以及慢病毒包装辅助质粒pMD2.G(见图3)和psPAX2(见图4),抽提质粒以后进行琼脂糖凝胶电泳及测序鉴定质粒的正确性。选择状态良好代数靠前的293T作为慢病毒包装细胞,利用转染试剂PEI将以上所述三种质粒转染293T细胞。转染在总体系为10mL的10cm培养皿中完成,每皿细胞转染混合物应使用无血清DMEM配制为1mL体系,使psPAX2质粒:pMD2.G质粒:CJ-PSMA-LIGHT-VTP质粒:PEI=5μg:3μg:5μg:50μl,室温混合转染混合物,静置20min后缓慢加入到已有9mL培养基的细胞密度达到70-80%的293T中,6-8h后更换新鲜培养基(DMEM+10%FBS+1%P/S)。分别在培养48h和72h时收获培养液上清,经超滤和超离浓缩后得到表达嵌合抗原受体和LIGHT-VTP的病毒,所得病毒命名为LIGHT-VTP-CAR病毒。以CJ-PSMA-CAR质粒(相较于LIGHT-VTP-CAR质粒,PSMA-CAR质粒缺乏表达LIGHT-VTP的第二表达盒)作为对照,同时参照以上方法进行转染与处理,命名为PSMA-CAR病毒。
病毒滴度检测
方法如下:
选择状态良好的293T检测病毒滴度,在24孔板中接种500μl密度为4*10^5/mL的细胞,待细胞贴壁后,添加不同梯度体积的浓缩病毒液,培养48h后将细胞消化,使用CAR可以识别结合的生物素化的PSMA蛋白在4度与细胞共孵育50min后清洗,然后用可以和生物素结合的APC-链霉亲和素SA在4度染30min,染色完毕后清洗装管,使用流式仪检测CAR阳性率,选择阳性率恰当的病毒体积计算病毒滴度,滴度计算公式:滴度(TU/mL)=(2×105×CAR阳性率)/病毒体积。
滴度检测结果如图5所示,滴度检测按照上述滴度检测方法,细胞接板贴壁以后,对照病毒PSMA-CAR与LIGHT-VTP-CAR病毒分别设置2μl和5μl两个体积梯度,为避免非特异性染色带来的假阳性,需设置CTRL进行CAR阳性圈门,落入APC阳性门内即为CAR阳性细胞,所示比例数值即为CAR阳性率。由图5结果可以看出,2μl的PSMA-CAR浓缩病毒感染20万293T可达到91.5%阳性率,5μl对应阳性率为97.7%;2μl的LIGHT-VTP-CAR病毒感染20万293T可达到93.5%的阳性率,5μl对应阳性率为97%,因为阳性率过高无法反应病毒真实的滴度,所以均选用2μl的体积计算滴度,对照病毒PSMA-CAR的滴度可达9.15×107TU/mL,而LIGHT-VTP-CAR病毒滴度为9.35×107TU/mL。
3构建表达嵌合抗原受体和LIGHT-VTP的T细胞
方法如下:
使用淋巴分离液从人体血液当中分离得到PBMC,然后使用CD4、CD8磁珠分选法分离T细胞,经CD3/CD28复合物激活48h后,即可使用包装好的PSMA-CAR和LIGHT-VTP-CAR病毒按照MOI=10离心感染2h,24h后更换成新鲜的培养基(XVIVO+10%FBS+IL-2),两种CART细胞分别命名为PSMA-CART细胞和LIGHT-VTP-CART细胞。感染换液48h后按照滴度检测同类方法检测以上两种CART的CAR表达水平。
结果见图6显示,LIGHT-VTP-CART和PSMA-CART的CAR阳性率分别为58.9%和61.6%。
LIGHT-VTP的表达检测:
对于LIGHT-VTP-CAR,不仅需要检测其CAR的表达,还需验证是否具有表达LIGHT-VTP的能力,所以收集上述两种在相同培养体系条件下的CART细胞(阳性率调整一致)48h的上清,通过ELISA验证LIGHT的表达。
结果如图7显示,ELISA的检测结果显示,LIGHT-VTP-CART细胞在2×106个细胞/mL浓度下分泌的LIGHT浓度最高可达1630pg/mL,而PSMA-CART在相同培养浓度下最高可达1281pg/mL,说明本发明实施例成功构建出过表达LIGHT-VTP的CAR-T细胞。
实验例1
LIGHT-VTP增强了CART对肿瘤细胞的杀伤效果
以CAR阳性率调整一致的LIGHT-VTP-CART和PSMA-CART作为效应细胞,PC3-PSMA(人前列腺癌细胞系,利用慢病毒稳定表达PSMA和荧光素酶)作为靶细胞,首先在低吸附孔板中加入等量的靶细胞(2万),按效靶比(效应细胞:靶细胞)4:1、2:1、1:1加入相应数量的CART效应细胞,同时做不同梯度只有靶细胞的孔(0、1、2、4、6、8、16万)作为标准曲线。由于靶细胞可以表达荧光素酶,经过12h的共孵育(cc:co-culture),加入底物后,吸光值与靶细胞数量成线性关系,可以做出标曲,计算剩余靶细胞数量,从而计算出杀伤效率Lysis(%)=(起始靶细胞数-剩余靶细胞数)/起始靶细胞数,以杀伤效率Lysis(%)为纵坐标,不同的效靶比(E:T)为横坐标,得到如图8结果,可知PSMA-CART和LIGHT-VTP-CART对人前列腺癌细胞杀伤效果明显,且随着效靶比的增加,杀伤效果也逐步上升,但LIGHT-VTP-CART对人前列腺癌细胞杀伤效果明显好于PSMA-CART,说明过表达LIGHT-VTP的CART细胞可以增强其对肿瘤细胞的杀伤能力。
选取以上E:T为1:1的12h共孵育上清使用ELISA检测LIGHT的表达,结果如图9所示,接受抗原刺激的LIGHT-VTP-CART分泌了更多的LIGHT(在相同的培养浓度(1×106个细胞/mL)下,LIGHT-VTP-CART分泌LIGHT浓度为423.75pg/mL,PSMA-CART分泌LIGHT浓度为223.75pg/mL,CTRL-T为68.75pg/mL),进一步确认了是LIGHT-VTP的作用增强了LIGHT-VTP-CART的功能。
实验例2
LIGHT-VTP增强了CART的增殖
为了验证LIGHT促进T细胞增殖的能力,将阳性率一致的各20万LIGHT-VTP-CART和PSMA-CART在相同的培养体系中培养,通过计数仪进行绝对计数追踪两种不同CART在两周内的增殖情况。结果如图10所示,LIGHT-VTP-CART具有明显的增殖优势。
实验例3
LIGHT联合CART促进肿瘤消退
为了初步判断LIGHT与CART结合的抗肿瘤效果,使用两者共同注射的方式在前列腺癌NSG老鼠模型中进行实验。将PC3-PSMA细胞系(Luciferase标记)注射至小鼠皮下,使用游标卡尺与小鼠活体成像技术追踪肿瘤的体积,当达到一定大小时,设置以下四组实验:PBS、hLIGHT、PSMA-CART、PSMA-CART+hLIGHT。PSMA-CART通过尾静脉只输注一次(3.5×106个细胞/只),PBS通过尾静脉只输注一次(100ul PBS/只),hLIGHT(重组人全长LIGHT,100ng/mL)通过瘤内注射每周注射一次,每次25ul。治疗后每周通过活体成像2次连续追踪40天。如图11所示(PSMA-CART(上)与PSMA-CART+hLIGHT(下)组),从活体成像的结果可知,通过尾静脉注射CART并且在瘤内注射hLIGHT的联合治疗方式有效的减小了肿瘤的体积(图示为活体成像图:Radiance(p/sec/cm2/sr))。图12所示为42天时对应小鼠的肿瘤大小,可以看出,PSMA-CART+hLIGHT组的肿瘤体积明显小于PSMA-CART组。
图13为对照组,只注射PBS或者hLIGHT,由图可知,老鼠在追踪过程中肿瘤未变小,且在34天时都已经死亡,说明hLIGHT单独治疗前列腺癌NSG老鼠模型(高度免疫缺陷)没有效果。暗示LIGHT发挥作用需要借助免疫细胞。
综上,大多数四期实体瘤在被确诊时,原发瘤中没有或只有很少浸润的T淋巴细胞,这就是患者对免疫检查点抑制剂或者其他免疫组合疗法响应率低的原因,而且在各类癌症的治疗中肿瘤浸润淋巴细胞(TIL)的存在与良好的预后都密切相关。因此TIL是免疫疗法的关键因素,“热”肿瘤指的就是出现淋巴细胞浸润和炎症的肿瘤,“冷”肿瘤指的就是没有淋巴细胞浸润和炎症的肿瘤。TIL已经成为了肿瘤治疗中重要的评分标准和治疗依据。对于大多数是“冷”肿瘤的实体瘤而言,使淋巴细胞尤其是T细胞进入肿瘤组织即浸润是CART细胞接近肿瘤细胞的第一步,是CART细胞充分发挥功能的首要条件。本发明实施例以实体瘤中典型“冷”肿瘤前列腺癌为切入点,及第二代靶向PSMA的CAR-T技术为基础,设计和构建一个可以分泌LIGHT的靶向PSMA的新型“浸润型”CART细胞,并且该LIGHT融合了肿瘤异常血管靶向多肽VTP,即LIGHT-VTP。所以由CAR-T细胞分泌的LIGHT-VTP可以特异性地靶向肿瘤异常血管,从而促进肿瘤异常血管正常化,并且可以进一步给肿瘤“加热”,促进肿瘤部位淋巴样结构形成以引导CAR-T细胞在此聚集与活化、调动自身免疫功能、重塑肿瘤微环境的抑制作用,从而改变CART细胞因无法穿透肿瘤组织及免疫抑制造成的效果局限性,增强CART细胞对抗实体肿瘤的活性,在LIGHT-VTP协同作用下,极大的改善CAR-T在前列腺癌等实体瘤治疗中的效果。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 华东师范大学
上海邦耀生物科技有限公司
<120> 促实体瘤浸润的增强型CART细胞及其制备方法和细胞药物
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Claims (10)
1.一种促实体瘤浸润的增强型CART细胞,其特征在于,
所述CART细胞含有第一核酸序列和第二核酸序列;
所述第一核酸序列含有编码嵌合抗原受体的第一编码序列,所述嵌合抗原受体的抗原结合结构域能够靶向所述实体瘤;
所述第二核酸序列含有编码LIGHT蛋白的第二编码序列以及位于所述第二编码序列上游的信号肽编码序列;
所述第二编码序列由组成型启动子驱动表达。
2.根据权利要求1所述的促实体瘤浸润的增强型CART细胞,其特征在于,所述第二核酸序列位于所述第一核酸序列下游,所述组成型启动子位于所述第一核酸序列上游,所述第一编码序列和所述第二编码序列均由所述组成型启动子驱动表达。
3.根据权利要求1或2所述的促实体瘤浸润的增强型CART细胞,其特征在于,所述组成型启动子选自EF-1α、早期巨细胞病毒启动子序列、类人猿病毒40早期启动子、小鼠乳癌病毒、人免疫缺陷病毒(HIV)长末端重复启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、肌动蛋白启动子、肌球蛋白启动子和血红素启动子和肌酸激酶启动子中的任意一种;
优选地,所述组成型启动子为EF-1α。
4.根据权利要求1或2所述的促实体瘤浸润的增强型CART细胞,其特征在于,所述LIGHT蛋白为LIGHT全长蛋白或其功能性片段;
优选地,所述LIGHT蛋白为LIGHT全长蛋白的可溶性片段;
优选地,所述LIGHT蛋白的氨基酸序列如SEQ ID NO.15所示。
5.根据权利要求4所述的促实体瘤浸润的增强型CART细胞,其特征在于,所述LIGHT蛋白融合VTP多肽;
优选地,所述VTP多肽的氨基酸序列如SEQ ID NO.17所示。
6.根据权利要求1或2所述的促实体瘤浸润的增强型CART细胞,其特征在于,所述信号肽编码序列所编码的信号肽选自膜整合型信号肽或分泌型信号肽;
优选地,所述膜整合型信号肽选自CD8信号肽、CD28信号肽、GM-CSF信号肽、CD4信号肽和CD137信号肽中的任意一种;
优选地,所述分泌型信号肽选自IgG信号肽和细胞因子信号肽中的任意一种;
优选地,所述信号肽编码序列的碱基序列如SEQ ID NO.13所示。
7.根据权利要求1或2所述的促实体瘤浸润的增强型CART细胞,其特征在于,所述嵌合抗原受体还具有跨膜结构域和共刺激信号传导区;
优选地,所述跨膜结构域选自如下蛋白分子中的至少一种的跨膜结构域:CD5、CD28、CD137、CD3ε、CD154、CD45、CD4、CD9、CD37、CD16、CD33、CD22、CD134和CD8α;
优选地,所述跨膜结构域为CD8α跨膜结构域;
优选地,所述共刺激信号传导区包含如下共刺激分子中至少一种的细胞内结构域:OX40、CD3γ、CD3δ、CD134、CD5、CD79a、CD137、ICD3ε、CD154、CD22、CD66d、CD2、CD28、CD4、CD5、CD79b、COS、4-1BB和CD3ζ;
优选地,所述共刺激信号传导区包括4-1BB的胞内共刺激元件和CD3ζ的胞内结构域。
8.根据权利要求1或2所述的促实体瘤浸润的增强型CART细胞,其特征在于,所述实体瘤选自肝癌、头颈癌、黑色素瘤、膀胱癌、胶质母细胞瘤、宫颈癌、肺癌、软骨肉瘤、甲状腺癌、肾癌、间皮瘤、骨肉瘤、胆管癌、卵巢癌、胃癌、膀胱癌、前列腺癌、脑膜瘤、胰腺癌、多发性鳞状细胞瘤、食管癌、肺小细胞癌、结直肠癌、乳腺癌、成神经管细胞瘤和乳腺癌中的任意一种;
优选地,所述实体瘤为前列腺癌;
优选地,所述抗原结合结构域能够靶向前列腺癌的特异性膜抗原,所述抗原结合结构域选自Fab、Fab’、F(ab’)2和scFv中的任意一种;
优选地,所述抗原结合结构域为scFv;
优选地,所述抗原结合结构域的轻链可变区的氨基酸序列如SEQ ID NO.3所示,所述抗原结合结构域的重链可变区的氨基酸序列如SEQ ID NO.7所示,所述抗原结合结构域的重链可变区与轻链可变区之间的铰接区氨基酸序列如SEQ ID NO.5所示。
9.制备如权利要求1-8任一项所述的促实体瘤浸润的增强型CART细胞的方法,其特征在于,其包括:使目标T细胞含有所述第一核酸序列和所述第二核酸序列。
10.一种治疗实体瘤的细胞药物,其特征在于,其含有作为活性成分的如权利要求1-8任一项所述的促实体瘤浸润的增强型CART细胞。
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