CN114075291A - 一种靶向psma蛋白的抗体、嵌合抗原受体和药物及应用 - Google Patents
一种靶向psma蛋白的抗体、嵌合抗原受体和药物及应用 Download PDFInfo
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Abstract
本申请公开了一种靶向PSMA的抗体、嵌合抗原受体和药物及应用。所述抗体的重链可变区包括SEQ ID NO.1或5所示的氨基酸序列;轻链可变区包括SEQ ID NO.3或7所示的氨基酸序列。本发明以人PSMA蛋白作为抗原,结合噬菌体库展示技术,淘选得到2个新的scFv抗体。经实验验证,这两个抗体均具有结合PSMA蛋白的能力,采用该抗体制备的CAR‑T对PSMA蛋白呈阳性的前列腺癌靶细胞具有特异性的杀伤效果。利用这两个scFv抗体制备靶向PSMA蛋白的CAR‑T细胞对人前列腺癌细胞具有很好的杀伤作用,若联合使用雄激素剥夺疗法等方法进行协同作用,将极大增强对去势抵抗性前列腺癌的疗效。
Description
技术领域
本发明涉及细胞免疫治疗技术领域,具体说是一种靶向PSMA蛋白的抗体、嵌合抗原受体和药物及应用。
背景技术
前列腺癌是男性泌尿生殖系统最常见的肿瘤之一,全球发病率占男性恶性肿瘤第二位,全球死亡率占恶性肿瘤的第五位,是美国发病率最高的癌症,同时也是第二大致死癌症。数据显示,近10年以来,我国前列腺癌呈现高发趋势,大城市更成为“重灾区”,基于日趋老龄化的人口基数、年龄结构等特点,伴随前列腺癌发病率的升高,中国前列腺癌发病率上升幅度令人堪忧。
传统的前列腺癌治疗方法包括根治性前列腺切除、放疗、化疗和雄激素剥夺疗法(androgen deprivation therapy,ADT),即采用药物或手术降低体内雄激素水平。其中,雄激素剥夺疗法是不同阶段患者前列腺癌最主要的治疗方法,也叫内分泌治疗,因为雄激素能够促进肿瘤生长,而阻断治疗通过降低雄激素水平从而延缓疾病的进展。该疗法虽然具有一定疗效,但除了对心血管、性功能、代谢、心理和骨骼健康等方面有副作用以外,其对大部分患者的病情缓解仅能维持1至4年,然后患者对雄激素剥夺疗法产生抵抗,从而使癌细胞发展成为更具侵袭性的去势抵抗性前列腺癌(castration-resistant prostatecancer,CRPC),因为CRPC患者的预后极差,所以对这些患者的治疗是长期以来未满足的医疗需求。
免疫治疗是治疗前列腺癌的一种新兴疗法,免疫检查点抑制剂中的程序性死亡受体-1(programmed cell death-1,PD-1)/程序性死亡配体-1(programmed cell death-ligand1,PDL1)抑制剂可以通过激活自身的免疫系统,提高机体抗肿瘤免疫力,达到抑制和杀伤肿瘤细胞的作用,这为前列腺癌的免疫治疗提供新的方向。但是其单独治疗时,临床试验中的效果并不乐观,客观缓解(objective response,OR)率低,可能与前列腺癌内的浸润性淋巴细胞难以被激活以及前列腺癌组织中PD-L1的表达相对缺乏有关。Graff等2016年利用雄激素受体拮抗剂enzalutamide联合PD-1抑制剂pembrolizumab治疗免疫联合激素治疗mCRPC(metastatic castration resistant prostate cancer)的Ⅱ期临床试验,数据显示雄激素阻断治疗可以一定程度上增强免疫治疗疗效。虽然Wang DY等2018年综述了免疫检查点抑制剂在结肠炎、肝炎、神经毒性等方面存在不良反应,但有关激素阻断治疗联合免疫治疗的相关研究仍然有巨大的开拓前景。
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出现了完全缓解的结果,其治疗流程为: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、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细胞的细胞毒性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。
前列腺特异性膜抗原(prostrate specific membrane antigen,PSMA)在前列腺癌中高表达,是免疫治疗实体瘤的理想靶点,利用靶向PSMA的CAR-T细胞在肿瘤免疫治疗中极大的特异性、靶向性和较少的主要组织相容性复合物限制,Junghans R等(2016)和Zuccolotto G(2014)在临床前研究及临床实验中证明了CAR-T细胞的有效性和安全性,但其应用仍有局限,所以还需要进一步的研究和探索。
发明内容
针对现有技术中存在的缺陷,本发明的目的在于提供一种靶向PSMA的抗体、嵌合抗原受体和药物及应用。
本发明提供的抗体具有可特异性识别与结合PSMA的抗原结合结构域,采用该抗体制备的嵌合抗原受体T细胞对PSMA蛋白呈阳性的靶细胞具有特异性的杀伤效果,可用于制备治疗或预防PSMA阳性的肿瘤药物。
为达到以上目的,本发明采取的技术方案是:
一种靶向PSMA的单链抗体1,包括重链可变区和轻链可变区,
所述重链可变区包括SEQ ID NO.1所示的氨基酸序列;
所述轻链可变区包括SEQ ID NO.3所示的氨基酸序列。
另一种靶向PSMA的单链抗体2,所述靶向PSMA的单链抗体包括重链可变区和轻链可变区,
所述重链可变区包括SEQ ID NO.5所示的氨基酸序列;
所述轻链可变区包括SEQ ID NO.7所示的氨基酸序列。
所述单链抗体1的编码基因,包括:SEQ ID NO.2和SEQ ID NO.4所示的核苷酸序列。
所述单链抗体2的编码基因,包括:SEQ ID NO.6和SEQ ID NO.8所示的核苷酸序列。
一种靶向PSMA的嵌合抗原受体,其特征在于:所述嵌合抗原受体包括按氨基端到羧基端方向依次顺序连接的抗原结合结构域、跨膜结构域和胞内结构域,
所述抗原结合结构域包括所述的靶向PSMA的单链抗体1和/或单链抗体2。
在上述嵌合抗原受体中,一种优选的实施方式为:所述靶向PSMA的单链抗体的重链可变区和轻链可变区之间由多肽铰链区连接,
和/或,所述抗原结合结构域的氨基端连接CD8α信号肽,
和/或,所述跨膜结构域来自CD8α,
和/或,所述抗原结合结构域与所述跨膜结构域之间由CD8铰链区连接,
和/或,所述胞内结构域包括4-1BB的胞内共刺激元件和CD3ζ的胞内结构域。
在上述嵌合抗原受体中,一种优选的实施方式为:所述多肽铰链区的氨基酸序列如SEQ ID NO.11所示,优选的,所述多肽铰链区的核苷酸编码序列如SEQ ID NO.10所示;
所述CD8α信号肽的核苷酸编码序列如SEQ ID NO.9所示;
所述跨膜结构域的核苷酸编码序列如SEQ ID NO.13所示;
所述CD8铰链区的核苷酸编码序列如SEQ ID NO.12所示;
所述4-1BB的胞内共刺激元件的核苷酸编码序列如SEQ ID NO.14所示;
所述CD3ζ的胞内结构域的核苷酸编码序列如SEQ ID NO.15所示。
本发明保护以上任一所述嵌合抗原受体的编码基因。
本发明保护含有以上任一所述基因的表达盒、重组质粒载体、重组菌或重组细胞;优选的,所述重组细胞为免疫细胞,更优选的,所述重组细胞细胞为CAR-T细胞。
以上所述的单链抗体1或2、以上任一所述的嵌合抗原受体、以上任一所述的基因、或所述的表达盒、重组质粒载体、重组菌或重组细胞在制备治疗和/或预防和/或诊断PSMA蛋白呈阳性的肿瘤细胞药物中的应用,优选的,所述肿瘤细胞为前列腺癌细胞。
本发明的有益效果如下:
本发明以人PSMA蛋白作为抗原,结合噬菌体库展示技术,淘选得到2个现有技术未曾报道的scFv抗体。经实验验证,该2个scFv抗体均具有结合PSMA蛋白的能力,采用该scFv抗体制备的嵌合抗原受体T细胞对PSMA蛋白呈阳性的前列腺癌靶细胞具有特异性的杀伤效果。利用这2个scFv抗体制备靶向PSMA蛋白的嵌合抗原受体T细胞对人前列腺癌细胞具有很好的杀伤作用,同时可联合使用雄激素剥夺疗法等方法进行协同作用,将极大增强对去势抵抗性前列腺癌的疗效。
附图说明
此处所说明的附图用来提供对本申请的进一步理解,构成本申请的一部分,本申请的示意性实施例及其说明用于解释本申请,并不构成对本申请的不当限定。在附图中:
图1为FACS检测淘选终止单克隆scFv与293T-ACE2细胞的结合活性。
图2为scFv纯化后PAGE鉴定结果。
图3为FACS检测scFv与293T-PSMA的结合活性。
图4为FACS检测scFv与LNCAP的结合活性。
图5为FACS检测scFv与RWPE-1的结合活性。
图6为PSMA-CAR的结构示意图。
图7为表达PSMA-CAR的慢病毒(Pbbz-HTS0101和Pbbz-HTS0103)滴度检测CAR阳性图。
图8为表达PSMA-CAR的T细胞(Pbbz-HTS0101 CAR-T和Pbbz-HTS0103CAR-T)CAR阳性图。
图9为表达PSMA-CAR的T细胞(Pbbz-HTS0101 CAR-T和Pbbz-HTS0103CAR-T)与靶细胞PC3-PSMA细胞共培养,CAR-T对靶细胞的杀伤效率图。
图10为表达PSMA-CAR的T细胞(Pbbz-HTS0101 CAR-T和Pbbz-HTS0103CAR-T)与靶细胞PC3-PSMA细胞共培养,CAR-T对靶细胞的杀伤IVIS图。
具体实施方式
一、抗体筛选及结合力验证:
使用过表达人PSMA抗原的293T细胞293T-PSMA对全人源抗体库进行淘选,使用经高强度淘选后的噬菌体侵染大肠杆菌TG1细胞,将培养上清中的scFv上清与293T-PSMA细胞共孵育,采用FACS筛选抗PSMA的单链抗体阳性克隆,将阳性克隆测序,对序列进行Germline分析和PTMs位点分析,从而得到抗PSMA的单链抗体序列。
进一步将以上纯化抗体在293T-PSMA外源细胞及前列腺癌细胞LNCAP,RWPE-1内源细胞中验证结合能力以后即可将此抗体序列应用于后续CAR的构建。
二、构建嵌合抗原受体的表达载体:
将以上获得的PSMA抗体序列设计成第二代CAR序列。然后,将其插入到慢病毒载体中,形成一个开放阅读框,构建成具有表达CAR的真核表达慢病毒载体。该PSMA抗体具有抗原结合位点的轻链可变区(VL)及重链可变区(VH),VL和VH由适当长度的多肽铰链区如甘氨酸-丝氨酸连接序列连接,可以选择性的识别和结合人PSMA抗原。CAR的信号转导区可选择如CD28、4-BB、CD3ζ等可以传递T细胞激活信号的区域,优选由可以在抗原结合后可以传递激活信号的T细胞4-BB胞内的共刺激元件和CD3ζ胞内区串联成的信号转导结构域,跨膜区由T细胞的CD8α跨膜区构成,用CD8α铰链区连接胞外scFv,在序列前端加上CD8α信号肽,构建成具有CD8α信号肽+PSMA scFv+CD8hinge+CD8αTM+4-BB+CD3ζ慢病毒表达系统,称为pELPS-PSMA-BBz。
三、嵌合抗原受体T细胞(CAR-T或CART)制备及功能评价:
使用慢病毒包装技术,将获得的慢病毒感染人T细胞制备得到PSMA-CAR-T,体外验证以上筛选得到的抗体制备的CAR-T对PSMA阳性肿瘤细胞的杀伤能力。
实施例1、抗体筛选
1、噬菌体库筛选
使用293T-PSMA细胞对全人源抗体库进行淘选。首先,使用293T细胞对建立的前列腺癌全人肿瘤特异性抗体库进行负淘选,具体步骤如下:
4℃孵育2小时,噬菌体的投入量为1012phage,离心后取上清,然后将上清加入到293T-PSMA的过表达细胞系中,4℃孵育2小时。离心后,使用PBS洗去未结合在细胞表面的噬菌体,再用0.1M的HCl-Glycine洗脱结合在细胞上的噬菌体,之后用Tris-HCl中和洗脱液,取部分噬菌体侵染对数生长期的大肠杆菌TG1,制备噬菌体用于下一轮淘选。逐渐增加每轮的筛选强度,富集度达到100倍以上时,终止淘选。
2、采用FACS筛选抗PSMA的单链抗体阳性克隆
1)挑选三轮淘选后的噬菌体侵染的TGI单克隆,接种于96孔板中,培养基为2YT/2%glucose/(100μg/ml Ampicilline)。
2)37℃、250rpm过夜培养后转接至新的培养基中,培养至对数生长期后4000rpm离心15min,使用2YT/(100μg/ml Ampicilline)/(1μM IPTG)培养基30℃过夜培养。
3)离心取scFv上清进行FACS鉴定克隆。每孔使用5e5个293T-PSMA细胞,加入50ulscFv上清,冰上孵育30分钟,之后PBS洗涤3次,再加入APC anti 6XHis的荧光二抗,避光冰上孵育30分钟,之后PBS洗涤3次,使用PBS重悬,进行流式细胞仪检测。
4)选择FACS中与二抗单染相比呈现阳性的克隆进行测序,发现很多的克隆序列相同,最终分析序列显示有2条germline差异较大的单链抗体序列,结果见图1。这两个scFv单链抗体分别是:抗PSMA单链抗体1(也标注为抗体HTS0101)和抗PSMA单链抗体2(也标注为抗体HTS0103)。
抗PSMA单链抗体1的重链可变区氨基酸序列如下:
EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYPLDDILTFPRVGTLDYWGQGTTVTVSS(SEQ ID NO.1);
抗PSMA单链抗体1重链可变区的核苷酸编码序列如下:
GAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATTACCCACTGGACGATATTTTGACTTTTCCTCGTGTAGGGACCCTTGACTACTGGGGCCAGGGGACCACGGTCACCGTCTCGAGT(SEQ ID NO.2);
抗PSMA单链抗体1的轻链可变区氨基酸序列如下:
EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLGWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTGFTFGPGTKVEIK(SEQ ID NO.3);
抗PSMA单链抗体1轻链可变区的核苷酸编码序列如下:
GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGGTTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACAGGATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA(SEQ ID NO.4);
抗PSMA单链抗体2的重链可变区氨基酸序列如下:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYASHWVRQAPGKGLEWVSAISGSGSGSTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAPNYEDEKLDDYFRVTLYWGQGTTVTVSS(SEQ ID NO.5);
抗PSMA单链抗体2重链可变区的核苷酸编码序列如下:
GAAGTGCAGCTGCTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTACCTTTAGCAGCTATGCGAGCCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGAGCGCGATTAGCGGCAGCGGCAGCGGCAGCACCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACAGCAAAAACACCCTGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCGCGCGCGATGCGCCGAACTATGAAGATGAAAAACTGGATGATTATTTTCGCGTGACCCTGTATTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ ID NO.6);
抗PSMA单链抗体2的轻链可变区氨基酸序列如下:
EIVLTQSPATLSLSPGERATLSCRASQSVSSGWALQQPGQSAQRLIYLDANRASTVIDAFSGSGPGTDFTLTISSLVPAEFVGVYYCARATTQAQGKTFGPGTKVEIK(SEQ ID NO.7);
抗PSMA单链抗体2轻链可变区的核苷酸编码序列如下:
GAAATTGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGAGCCCGGGCGAACGCGCGACCCTGAGCTGCCGCGCGAGCCAGAGCGTGAGCAGCGGCTGGGCGCTGCAGCAGCCGGGCCAGAGCGCGCAGCGCCTGATTTATCTGGATGCGAACCGCGCGAGCACCGTGATTGATGCGTTTAGCGGCAGCGGCCCGGGCACCGATTTTACCCTGACCATTAGCAGCCTGGTGCCGGCGGAATTTGTGGGCGTGTATTATTGCGCGCGCGCGACCACCCAGGCGCAGGGCAAAACCTTTGGCCCGGGCACCAAAGTGGAAATTAAA(SEQ ID NO.8);
3、筛选所得2条scFv的表达纯化
取小摇之后的菌液,按照1:100的比例接种,37℃、220rpm培养。当OD在0.5左右时,室温8000rpm离心5分钟,获得菌液沉淀,30℃IPTG诱导培养12小时左右后收集表达上清进行纯化。具体纯化步骤如下:
1)轻轻颠倒翻转瓶子几次,使介质混合均匀;
2)吸取一定量的介质加入到柱子中,柱高约3-4厘米,等介质沉降至底部时加上封口;
3)放干储存液,加入10mL平衡缓冲液平衡层析介质;
4)使用离心瓶离心,4000rpm,4℃离心10min,将上清转移到新的离心瓶中,12000rpm,4℃,离心30min;
5)上样,流速控制为1mL/min;
6)25mL 10mM咪唑洗涤缓冲液洗涤柱子,流速控制为1mL/min;
7)25mL 20mM咪唑洗涤缓冲液洗涤柱子,流速控制为1mL/min;
8)5mL 250mM咪唑洗脱缓冲液洗涤柱子,流速控制为1mL/min,收集洗脱液;
9)浓缩,将洗脱液加入到超滤管中,1500rpm,4℃离心10min,浓缩scFv;
10)PAGE鉴定及浓度测定;
对2个样品的部分scFv进行纯化后均得到了0.5mg以上的样品,纯化结果见图2所示。
4、FACS检测scFv的293T-PSMA外源细胞结合EC50活性
每孔使用5×105个293T-PSMA细胞,加入稀释的scFv,起始浓度45ug/ml,3倍稀释,稀释12个梯度,细胞每孔加入50ul抗体,冰上孵育30分钟,之后PBS洗涤3次,再加入APCanti 6XHis的荧光二抗,避光冰上孵育30分钟,之后PBS洗涤3次,使用PBS重悬,进行流式细胞仪检测。检测结果如图3所示。
5、FACS检测scFv的LNCAP,RWPE-1内源细胞结合EC50活性
每孔使用5×105个LNCAP或RWPE-1细胞,加入稀释的scFv,起始浓度45ug/ml,3倍稀释,稀释12个梯度,细胞每孔加入50ul抗体,冰上孵育30分钟,之后PBS洗涤3次,再加入APC anti 6XHis的荧光二抗,避光冰上孵育30分钟,之后PBS洗涤3次,使用PBS重悬,进行流式细胞仪检测,结果如图4和图5所示。
实施例2、构建含表达嵌合抗原受体的质粒载体
由金斯瑞公司全基因合成表达靶向前列腺特异性膜抗原的嵌合抗原受体的表达盒(如图6所示):PSMA-CAR包括:CD8α信号肽、PSMA单链抗体重链可变区、Linker、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,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双酶切将表达盒转移至慢病毒包装穿梭质粒(上海邦耀生物科技有限公司),酶连后得到两个嵌合抗原受体表达载体pELPS-PSMA-BBz-HTS0101及pELPS-PSMA-BBz-HTS0103。其中,表达靶向前列腺特异性膜抗原的嵌合抗原受体的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.9所示:
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG;
抗PSMA单链抗体1轻链可变区(PSMA-ScFv VL)的碱基序列如SEQ ID NO.4所示;
抗PSMA单链抗体1轻链可变区的氨基酸序列如SEQ ID NO.3所示;
抗PSMA单链抗体1重链可变区(PSMA-ScFv VH)的碱基序列如SEQ ID NO.2所示;
抗PSMA单链抗体1重链可变区的氨基酸序列如SEQ ID NO.1所示;
抗PSMA单链抗体2轻链可变区的碱基序列如SEQ ID NO.8所示;
抗PSMA单链抗体2轻链可变区的氨基酸序列如SEQ ID NO.7所示;
抗PSMA单链抗体2重链可变区的碱基序列如SEQ ID NO.6所示;
抗PSMA单链抗体2重链可变区的氨基酸序列如SEQ ID NO.5所示;
PSMA-ScFv VL与PSMA-ScFv VH的Linker的碱基序列如SEQ ID NO.10所示:
GGCGGAGGCGGATCAGGTGGTGGCGGATCTGGAGGTGGCGGAAGC;
Linker的氨基酸序列如SEQ ID NO.11所示:
GGGGSGGGGSGGGGS;
CD8铰链区(CD8 hinge)的碱基序列如SEQ ID NO.12所示:
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT;
CD8α跨膜结构域(CD8a-TM)的碱基序列如SEQ ID NO.13所示:
ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC;
4-1BB的胞内共刺激元件的碱基序列如SEQ ID NO.14所示:
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG;
CD3ζ的胞内结构域的碱基序列如SEQ ID NO.15所示:
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC。
实施例3、构建表达嵌合抗原受体的病毒
利用大肠杆菌扩增上述pELPS-PSMA-BBz-HTS0101及pELPS-PSMA-BBz-HTS0103作为两个目的质粒以及慢病毒包装辅助质粒pMD2.G和psPAX2,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:pELPS-PSMA-BBz-HTS0101或pELPS-PSMA-BBz-HTS0103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上述嵌合抗原受体的慢病毒,所得病毒命名为Pbbz-HTS0101及Pbbz-HTS0103。
病毒滴度检测:
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4×105/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°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。
滴度检测按照上述滴度检测方法,细胞接板贴壁以后,Pbbz-HTS0101及Pbbz-HTS0103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。根据图7流式结果,2μl的Pbbz-HTS0101浓缩病毒感染20万293T可达到93.8%阳性率,5μl对应阳性率为97.7%;2μl的Pbbz-HTS0103病毒感染20万293T可达到93.8%的阳性率,5μl对应阳性率为94.8%,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,病毒Pbbz-HTS0101的滴度可达9.38x107TU/mL,而Pbbz-HTS0103病毒滴度为9.48x107TU/mL。
实施例4、构建表达嵌合抗原受体的T细胞
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,使用含有IL-2 200IU/ml的完全培养基培养,经CD3/CD28复合物激活48h后,即可使用包装好的Pbbz-HTS0101及Pbbz-HTS0103病毒按照MOI=10 1800rpm离心感染2h,24h后更换成新鲜的培养基(XVIVO+10%FBS+200IU/ml IL-2),两种CAR-T细胞分别命名为Pbbz-HTS0101 CAR-T细胞和Pbbz-HTS0103 CAR-T细胞。
感染换液48h后按照上述滴度检测同类方法检测以上两种CAR-T的CAR表达水平,结果如图8所示。
实施例5、体外验证PSMA-CAR-T对PSMA阳性肿瘤细胞的杀伤效果
以Pbbz-HTS0101 CAR-T和Pbbz-HTS0103 CAR-T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E:T(效应细胞:靶细胞)5:1、2.5:1、1.25:1、0.63:1加入相应数量的CAR-T效应细胞,同时做不同梯度只有靶细胞的孔(0.5、1、1.5、2、2.5、3、5万)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。
结果:如图9和图10所示,表达PSMA-CAR的T细胞对人前列腺癌细胞系的杀伤效率效靶比的升高而升高,效靶比E:T为5:1时杀伤效率最高,两种表达PSMA-CAR的T细胞对肿瘤细胞的杀伤效率最高,都达到了100%,效靶比E:T为0.63:1时,两种表达PSMA-CAR的T细胞对肿瘤细胞的杀伤效率也都可以达到70%。另外,在效靶比E:T为2.5:1和1.25:1时,Pbbz-HTS0103CAR-T细胞对肿瘤细胞的杀伤效率明显高于Pbbz-HTS0101 CAR-T细胞,且在效靶比E:T为2.5:1时,杀伤效率就可以达100%,这说明抗体HTS0103靶向抗原PSMA的效果好于HTS0101。
本说明书中未作详细描述的内容属于本领域专业技术人员公知的现有技术。以上所述仅为本申请的实施例而已,并不用于限制本申请。对于本领域技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原理之内所作的任何修改、等同替换、改进等,均应包含在本申请的权利要求范围之内。
序列表
<110> 中国人民解放军海军军医大学第一附属医院
<120> 一种靶向PSMA的抗体、嵌合抗原受体和药物及应用
<130> JH-CNP201296
<160> 15
<170> PatentIn version 3.5
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gaggtgcaac tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60
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gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagagattac 300
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tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180
tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240
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agcgcgcagc gcctgattta tctggatgcg aaccgcgcga gcaccgtgat tgatgcgttt 180
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agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
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cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
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Claims (10)
1.一种靶向PSMA的单链抗体,其特征在于,所述靶向PSMA的单链抗体包括重链可变区和轻链可变区,
所述重链可变区包括SEQ ID NO.1所示的氨基酸序列;
所述轻链可变区包括SEQ ID NO.3所示的氨基酸序列。
2.一种靶向PSMA的单链抗体,其特征在于,所述靶向PSMA的单链抗体包括重链可变区和轻链可变区,
所述重链可变区包括SEQ ID NO.5所示的氨基酸序列;
所述轻链可变区包括SEQ ID NO.7所示的氨基酸序列。
3.权利要求1所述单链抗体的编码基因,其特征在于:所述基因包括:SEQ ID NO.2和SEQ ID NO.4所示的核苷酸序列。
4.权利要求2所述单链抗体的编码基因,其特征在于:所述基因包括:SEQ ID NO.6和SEQ ID NO.8所示的核苷酸序列。
5.一种靶向PSMA的嵌合抗原受体,其特征在于:所述嵌合抗原受体包括按氨基端到羧基端方向依次顺序连接的抗原结合结构域、跨膜结构域和胞内结构域,
所述抗原结合结构域包括权利要求1和/或2所述的靶向PSMA的单链抗体。
6.如权利要求5所述的嵌合抗原受体,其特征在于:所述靶向PSMA的单链抗体的重链可变区和轻链可变区之间由多肽铰链区连接,
和/或,所述抗原结合结构域的氨基端连接CD8α信号肽,
和/或,所述跨膜结构域来自CD8α,
和/或,所述抗原结合结构域与所述跨膜结构域之间由CD8铰链区连接,
和/或,所述胞内结构域包括4-1BB的胞内共刺激元件和CD3ζ的胞内结构域。
7.如权利要求6所述的嵌合抗原受体,其特征在于:所述多肽铰链区的氨基酸序列如SEQ ID NO.11所示,优选的,所述多肽铰链区的核苷酸编码序列如SEQ ID NO.10所示;
所述CD8α信号肽的核苷酸编码序列如SEQ ID NO.9所示;
所述跨膜结构域的核苷酸编码序列如SEQ ID NO.13所示;
所述CD8铰链区的核苷酸编码序列如SEQ ID NO.12所示;
所述4-1BB的胞内共刺激元件的核苷酸编码序列如SEQ ID NO.14所示;
所述CD3ζ的胞内结构域的核苷酸编码序列如SEQ ID NO.15所示。
8.权利要求5-7中任一所述嵌合抗原受体的编码基因。
9.含有权利要求3、4或8所述基因的表达盒、重组质粒载体、重组菌或重组细胞;优选的,所述重组细胞为免疫细胞,更优选的,所述重组细胞为CAR-T细胞。
10.权利要求1或2所述的单链抗体、权利要求5-7中任一所述的嵌合抗原受体、权利要求3、4或8所述的基因、或权利要求9所述的表达盒、重组质粒载体、重组菌或重组细胞在制备治疗和/或预防和/或诊断PSMA蛋白呈阳性的肿瘤细胞药物中的应用,优选的,所述肿瘤细胞为前列腺癌细胞。
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