CN111393504A - 一种肝癌抗原组合及其应用、细胞毒性t淋巴细胞 - Google Patents
一种肝癌抗原组合及其应用、细胞毒性t淋巴细胞 Download PDFInfo
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Abstract
本发明提供了一种肝癌抗原组合及其应用、细胞毒性T淋巴细胞,属于生物医药技术领域,所述肝癌抗原的氨基酸序列如SEQ ID No.1~4所示。采用本发明提供的肝癌抗原组合共培养外周血单个核细胞,得到的细胞毒性T淋巴细胞,能够有效杀伤表达肝癌相关抗原的细胞。
Description
技术领域
本发明属于生物医药技术领域,尤其涉及一种肝癌抗原组合及其应用、细胞毒性T淋巴细胞。
背景技术
我国肝癌发病率最高,且死亡率高,随着精准治疗的推广,常规的化疗、放疗和靶向已无法满足肿瘤的个性化和多变性,因此,寻找个性化的肿瘤新抗原,培养识别个性化肿瘤新抗原的CTL(细胞毒性T淋巴细胞),将是彻底清除肿瘤的有效方法。
靶向肿瘤新抗原的CTL的培养,关键是如何选择肿瘤新抗原,目前常用的手段是,通过生物信息学方法,对肿瘤新抗原与HLA的亲和力进行预测,选取亲和力高的,作为备选的肿瘤新抗原,优点是快速且高通量,缺点是,基于算法脱离实验,结果不准确。
发明内容
有鉴于此,本发明的目的在于提供一种肝癌抗原组合及其应用、细胞毒性T淋巴细胞,采用本发明提供的肝癌抗原组合共培养外周血单个核细胞得到的细胞毒性T淋巴细胞,能够有效杀伤肝癌细胞。
为了实现上述发明目的,本发明提供了以下技术方案:
本发明提供了一种肝癌抗原组合,所述肝癌抗原的氨基酸序列如SEQ ID No.1~4所示。
本发明还提供了上述技术方案所述的肝癌抗原组合在制备细胞毒性T淋巴细胞中的应用。
优选的,所述应用包括:以每种肝癌抗原的终浓度为10~50μg/ml共培养外周血单个核细胞,得到细胞毒性T淋巴细胞。
优选的,所述每种肝癌抗原的终浓度为15~30μg/ml。
优选的,所述每种肝癌抗原的终浓度为20μg/ml。
本发明还提供了上述技术方案所述的肝癌抗原组合在制备治疗肝癌的药物中的应用。
本发明还提供了一种细胞毒性T淋巴细胞,采用上述技术方案所述的肝癌抗原组合共培养外周血单个核细胞,得到。
本发明还提供了上述技术方案所述的细胞毒性T淋巴细胞在制备治疗肝癌的药物中的应用。
本发明提供了一种肝癌抗原组合及其应用、细胞毒性T淋巴细胞,采用本发明提供的肝癌抗原组合共培养外周血单个核细胞,得到的细胞毒性T淋巴细胞,能够有效杀伤肝癌细胞。
附图说明
图1为1~26号多肽的筛选结果;
图2为27~50号多肽的筛选结果;
图3为流式分析细胞表型结果;
图4为CTL对靶细胞的杀伤作用结果。
具体实施方式
本发明提供了一种肝癌抗原组合,所述肝癌抗原的氨基酸序列如SEQ ID No.1~4所示,具体如下所示:
SEQ ID No.1:RRQNRCQFCR;
SEQ ID No.2:SYLTTKELR;
SEQ ID No.3:FRMQASSTF;
SEQ ID No.4:VMITMVCCAHR。
在本发明中,所述SEQ ID No.1~4对应的HLA等位基因是HLA-A*31:01。
本发明还提供了上述技术方案所述的肝癌抗原组合在制备细胞毒性T淋巴细胞中的应用。在本发明中,所述应用优选包括:以每种肝癌抗原的终浓度为10~50μg/ml共培养外周血单个核细胞,得到细胞毒性T淋巴细胞。本发明对培养得到细胞毒性T淋巴细胞的方法没有特殊限定,采用常规培养细胞毒性T淋巴细胞的方法即可。在本发明中,所述每种肝癌抗原的终浓度更优选为15~30μg/ml,最优选为20μg/ml。
本发明还提供了上述技术方案所述的肝癌抗原组合在制备治疗肝癌的药物中的应用。在本发明中,所述药物优选以肝癌抗原为唯一活性成分,还包括药学上可接受的辅料或辅助性成分,肝癌抗原的质量比优选为0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5,更优选为1:1:1:1:1:1:1:1:1:1。在本发明中,所述药物的剂型优选包括片剂、粉剂、颗粒剂、胶囊、口服液或缓释剂。本发明对所述肝癌抗原组合在上述剂型中的用量没有特殊限定,采用常规剂型中药物的用量即可。本发明对所述上述剂型使用的辅料或者辅助性成分的种类和含量没有特殊限定,采用常规即可。
本发明还提供了一种细胞毒性T淋巴细胞,采用上述技术方案所述的肝癌抗原组合共培养外周血单个核细胞,得到。在本发明中,采用肝癌抗原组合共培养外周血单个核细胞得到细胞毒性T淋巴细胞的方法与上述相同,在此不再赘述。在本发明中,采用肝癌抗原组合共培养外周血单个核细胞,得到的细胞毒性T淋巴细胞能够识别肝癌抗原组合,具有特异性杀伤作用并且提高杀伤作用,进而来治疗肝癌。
本发明还提供了上述技术方案所述的细胞毒性T淋巴细胞在制备治疗肝癌的药物中的应用。在本发明中,所述药物优选以细胞毒性T淋巴细胞为唯一活性成分,还包括药学上可接受的辅料或辅助性成分。在本发明中,所述药物的剂型优选包括片剂、粉剂、颗粒剂、胶囊、口服液或缓释剂。本发明对所述细胞毒性T淋巴细胞在上述剂型中的用量没有特殊限定,采用常规剂型中药物的用量即可。本发明对所述上述剂型使用的辅料或者辅助性成分的种类和含量没有特殊限定,采用常规即可。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
1.肝癌相关肿瘤新抗原:
患者刘某的肿瘤组织的全外显子测序结果,进行肿瘤新抗原分析(鼎成肽源生物信息技术有限公司),对48条抗原进行合成(南京金斯瑞多肽合成部);
表1 多肽序列及对应编号
2、共培养制备APC:
1)复苏冻存PBMC(2.5×107cells),以1640+10%FBS稀释冻存液,1500rpm 5min,以10mL 1640+10%FBS重悬,1×106cells/mL,37℃、5%CO2复苏培养24h;
2)配制多肽,1640+10%FBS+200U/mL IL-2+1600U/mL GM-CSF,多肽终浓度为50ng/mL,混匀后备用;
3)1500rpm 5min,离心收集细胞,以多肽溶液重悬细胞,密度调整至1×106cells/mL;
4)37℃、5%CO2培养24h后,细胞为APC,轻轻吹打细胞混匀备用;
3、共培养
1)离心收集PBMC,1640+10%FBS重悬,计数调整至1×106cells/mL;
2)按照100:1的比例加入APC,混匀,记为Day 0;
3)37℃、5%CO2培养48h后,每天按照总体家补加IL-2,终浓度为200U/mL;
4)培养至Day 21时,收集细胞即为CTL;
4、肿瘤新抗原的筛选:
1)收集CTL细胞,以1640+10%FBS重悬,计数调整至2×106cells/mL;
2)10mL cells+10mL 1640+10%FBS将细胞调整至1×106cells/mL,再加入8μL500U/μL IL-2,IL-2终浓度为200U/mL,再分至96孔板中(平底),200μL/孔;
3)每孔加入对应多肽,5μL 2μg/mL多肽;设置阳性对照OKT3167ng/mL、medium对照、CTL;
4)37℃5%CO2培养24h后,1500rpm离心10min,转移170μL上清至新的96孔板中;
5)为避免吸到细胞,再将96孔板进行1500rpm离心10min,转移110μL上清至新的96孔板;
6)以R&D ELISA试剂盒检测细胞上清中IFN-γ的释放量,以此来进行抗原的筛选。
5、靶细胞的构建
1)通过人工合成的方法,进行HLA-A3101因合成(金维智公司),合成的基因构建到pCDH表达载体上(插入位点为NheⅠ和Bam HⅠ);
2)上述得到的重组质粒转化感受态细胞,挑取单克隆,进行测序,选择测序结果正确的克隆,进行后续实验;
3)以天根质粒大提试剂盒(购自于天根生化科技(北京)有限公司)进行质粒的提取,方法按照厂家说明书操作。
4)慢病毒包装:复苏293T细胞,传两代后用于慢病毒包装;细胞转染:(T175培养瓶)细胞密度为2×107个/瓶;取一支15ml离心管(标记为A),将包装质粒4K(17μg)、6K(34μg)和目的质粒(51μg)加入1mL jetPRIME配套的Buffer中,轻轻地混匀。将jetPRIME(标记为B)缓慢滴加入A离心管中,加入同时匀速晃动A管,得到C溶液,之后静置10min;将T175中旧的培养基倒出,将18mL DMEM(不含抗生素和血清)加入C中,加入T175培养瓶中,置于37℃,5%CO2培养箱中培养。转染4-6h后,吸去含有转染复合物的培养基,更换为37℃预热的新鲜培养基。培养48h和72h并收集。
5)慢病毒转染3T3细胞:使用DMEM配制含有7%FBS的全细胞培养基,记为DMEM。使用DMEM把3T3细胞浓度调整到5X10^5/ml,加入6孔板,每孔1mL,37℃培养箱孵育过夜。弃去6孔板中DMEM培养基,使用1mLAIM-V-P混匀病毒液加入对应各孔中。培养3天后转入T75培养瓶中,加入嘌呤霉素进行筛选;筛选6天后,得到稳定表达HHL-A3101的3T3细胞,标记为3T3-HLA;
6、杀伤实验——LDH法
1)收集靶细胞3T3和3T3-HLA,1000rpm离心5min;
2)以PBS洗一遍,1000rpm离心5min;
3)加入多肽溶液,进行有效新抗原组合的负载,37℃2-12h,优选4h;
4)多肽溶液:多肽溶液以1640培养基进行配制,每种多肽的终浓度为20μg/mL;
5)1000rpm离心5min,除去多余溶液,并以PBS洗2次;
6)以1640+2%FBS重悬后,计数调整至8×104cells/mL,分至96孔板中(U型底),50μL/孔,备用;
7)收集CTL细胞,1000rpm离心5min;
8)以PBS洗一遍,1000rpm离心5min;
9)以1640+2%FBS重悬细胞计数后,分至96孔板中(U型底),50μL/孔,设置效靶比为40:1、20:1、10:1、5:1、2.5:1和1.25:1;
10)37℃5%CO2共孵育20h后,进行LDH检测。
7、流式分型
1)收集CTL,1000rpm离心5min;
2)1×106cells/管,加入CD3、CD4、CD8和CD56的抗体,室温避光30min;
3)PBS洗两次,1000rpm离心5min;
4)PBS重悬,上机检测。
结果:
1、肝癌相关肿瘤新抗原的筛选
多肽分为两组1~26号多肽为一组,27~50号为一组进行筛选,筛选结果见图1~2,IFN-γ的释放水水平超过对照的,认为是有效的新抗原,有效的新抗原见表2。
表2 有效的多肽抗原序列及对应编号
多肽编号 | HLA Allele | MT Epitope Seq |
1 | HLA-A*31:01 | RRQNRCQFCR |
7 | HLA-A*31:01 | SYLTTKELR |
11 | HLA-A*31:01 | FRMQASSTF |
17 | HLA-A*31:01 | VMITMVCCAHR |
2、以筛选到有效新抗原(表2)进行组合,每种多肽抗原的终浓度为20μg/mL进行CTL的培养,流式分析细胞表型,结果如图3所示,NKT(CD56+CD3+)比例为23.1%,90%以上为T细胞,其中82.8%为CD8+T细胞。
3、CTL对靶细胞的杀伤作用
经过抗原刺激培养的CTL,结果见图4,对负载抗原多肽的靶细胞比只负载HLA的靶细胞具有更高的杀伤效率,说明,培养的CTL,可以识别此抗原组合,且具有特异的杀伤作用,可以作为肝癌治疗的首选抗原组合。
由以上实施例可以得出,采用本发明提供的肝癌抗原组合共培养得到CTL后,对负载抗原多肽的靶细胞具有更高的杀伤效率,进而可治疗肝癌。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
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Claims (8)
1.一种肝癌抗原组合,其特征在于,所述肝癌抗原的氨基酸序列如SEQ ID No.1~4所示。
2.权利要求1所述的肝癌抗原组合在制备细胞毒性T淋巴细胞中的应用。
3.根据权利要求2所述的应用,其特征在于,所述应用包括:以每种肝癌抗原的终浓度为10~50μg/ml共培养外周血单个核细胞,得到细胞毒性T淋巴细胞。
4.根据权利要求3所述的应用,其特征在于,所述每种肝癌抗原的终浓度为15~30μg/ml。
5.根据权利要求4所述的应用,其特征在于,所述每种肝癌抗原的终浓度为20μg/ml。
6.权利要求1所述的肝癌抗原组合在制备治疗肝癌的药物中的应用。
7.一种细胞毒性T淋巴细胞,其特征在于,采用权利要求1所述的肝癌抗原组合共培养外周血单个核细胞,得到。
8.权利要求7所述的细胞毒性T淋巴细胞在制备治疗肝癌的药物中的应用。
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CN112694519B (zh) * | 2021-01-06 | 2022-09-09 | 南方科技大学 | 抗原、核酸、表达载体、抗原呈递细胞和细胞毒性t细胞及其应用 |
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