CN114249811A - 一种特异识别癌/睾丸抗原hca587/magec2的t细胞受体及其应用 - Google Patents
一种特异识别癌/睾丸抗原hca587/magec2的t细胞受体及其应用 Download PDFInfo
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Abstract
本发明提供了特异识别癌/睾丸抗原HCA587/MAGEC2的T细胞受体(TCR)及其应用,所述TCR具有特异结合来源于HCA587/MAGEC2蛋白的抗原肽VIWEVLNAV‑HLA*0201复合物的特性,所述TCR包含α链的可变区和恒定区及β链的可变区和恒定区。经过所述TCR修饰的T细胞能特异识别癌/睾丸抗原HCA587/MAGEC2并被其激活,对HCA587/MAGEC2阳性且表达HLA‑A*0201的肿瘤细胞具有特异性杀伤作用,可作为TCR‑T细胞制剂使用,用于肿瘤患者的治疗或与其他药物联合治疗。
Description
技术领域
本发明属于基因工程与肿瘤免疫治疗领域,具体涉及一种特异识别癌/睾丸抗原HCA587/MAGEC2的T细胞受体及其应用。
背景技术
肿瘤免疫治疗是通过调动患者机体的免疫系统对抗肿瘤细胞,如何恢复和提高肿瘤患者体内免疫细胞和免疫分子对肿瘤细胞的杀伤能力是肿瘤免疫治疗的关键。目前肿瘤治疗除采用传统的手术治疗结合放疗和化疗及靶向治疗外,免疫治疗作为新兴的治疗手段显著提高了肿瘤患者的治疗效果。T细胞抗原受体修饰的T细胞(TCR-T)是最具治疗前景的免疫治疗手段之一,TCR-T细胞在患者体内可直接杀伤肿瘤细胞,可以避免肿瘤疫苗发挥作用的延迟效应,在临床试验中已经显示了良好的抗肿瘤作用。
T细胞利用其表面受体(TCR)特异识别抗原提呈细胞提呈的主要组织相容性复合物(MHC)与抗原肽的复合物,识别后发生活化、扩增,最后分化形成效应性T细胞发挥作用。TCR属于免疫球蛋白超家族中的一员,由α链和β链组成,两条链形成异源二聚体结构。α链和β链均由可变区和恒定区组成,可变区是TCR特异识别抗原的区域,可变区内有三个高变区(又称为互补决定区,即CDR区),其中CDR3区的氨基酸序列组成较CDR1和CDR2区显示了更大的变异性,因此CDR3区是决定TCR特异结合抗原的关键区域。
T细胞是机体内发挥抗肿瘤免疫的主要细胞群体,主要分为CD4+和CD8+T细胞。CD4+T细胞主要识别由抗原提呈细胞提呈的与MHC II类分子结合的抗原肽,传统观点认为这类细胞主要通过分泌细胞因子发挥作用,但目前研究认为一部分CD4+T细胞也可以直接发挥杀伤作用;CD8+T细胞主要识别由抗原提呈细胞提呈的与MHC I类分子结合的抗原肽,主要通过细胞毒作用杀伤肿瘤细胞。
癌/睾丸抗原HCA587/MAGEC2在正常组织中仅分布于睾丸,但在多种类型肿瘤中广泛分布,且在肿瘤的发生发展中发挥重要作用,抑制肿瘤细胞发生凋亡,促进肿瘤细胞的周期进程,并促进肿瘤细胞的体内转移,这些特点使其能够参与肿瘤恶性特征的维持。因此,癌/睾丸抗原HCA587/MAGEC2是肿瘤免疫治疗理想的靶点。
T细胞受体改造的T细胞(TCR-T)疗法是目前肿瘤免疫治疗的重要手段之一,该疗法通过基因工程手段改造患者自身的免疫细胞再回输到患者体内,通过这种免疫治疗方法可以增强患者机体的免疫细胞对肿瘤细胞的特异识别和杀伤能力。与同类型的其他免疫治疗方式,如CAR-T(嵌合性抗原受体改造T细胞疗法)、CAR-NK等相比较,TCR-T疗法在实体肿瘤治疗方面具有更加明显的优势,并且具有更广泛的应用空间。但是目前的临床治疗中有效靶点较少,从而限制了该疗法的应用范围。
发明内容
本发明的目的是针对上述要解决的问题,提供一种能够特异识别癌/睾丸抗原HCA587/MAGEC2的T细胞受体及其应用。
本发明提供了一种能特异识别HCA587/MAGEC2抗原的T细胞受体,所述T细胞受体能与VIWEVLNAV-HLA-A*0201复合物结合。
所述T细胞受体由α链和β链组成,所述β链的CDR3区的序列与SEQ ID NO:02序列至少具有75%及以上同源性且具有相同的生物学功能的氨基酸序列。
所述β链的CDR3区的氨基酸序列与SEQ ID NO:02所示的氨基酸序列相同。
所述β链的CDR3区的核酸序列为SEQ ID NO:01所示的核酸序列。
所述β链的可变区序列包含CDR3区的序列,所述可变区序列的氨基酸组成为SEQID NO:06所示的氨基酸序列。
所述β链的可变区的核酸序列为SEQ ID NO:05所示的核酸序列。
所述α链的CDR3区的序列与SEQ ID NO:04序列至少具有75%及以上同源性且具有相同的生物学功能的氨基酸序列。
所述的TCRα链的CDR3区的氨基酸序列与SEQ ID NO:04所示的氨基酸序列相同。
所述的TCRα链的CDR3区的核酸序列与SEQ ID NO:03所示的核酸序列相同。
所述α链的可变区序列包含CDR3区的序列,所述可变区序列的氨基酸组成为SEQID NO:08所示的氨基酸序列。
所述α链的可变区的核酸序列为SEQ ID NO:07所示的核酸序列。
本发明所述T细胞受体(TCR)包含α链和β链,二者形成异源二聚体结构。所述TCRα链和β链的恒定区为鼠源恒定区序列。
本发明包含一种载体,其特征为含有上述特异识别癌/睾丸抗原HCA587/MAGEC2的T细胞受体的核酸序列或氨基酸序列对应的优化密码子的核酸序列。
优选的,所述载体为慢病毒载体。
本发明提供一种细胞,所述细胞的表面表达上述的T细胞受体。
优选的,所述细胞为T细胞。
所述T细胞对表达癌/睾丸抗原HCA587/MAGEC2的HLA-A*0201+的肿瘤细胞具有特异杀伤作用。
一种T细胞在抗肿瘤药物制备中的应用及与其他药物的联合应用。
所述抗肿瘤药物及药物组合用于HLA-A*0201阳性肿瘤患者的治疗。
所述抗肿瘤药物及药物组合用于表达HCA587/MAGEC2抗原的肿瘤患者的治疗。
所述肿瘤患者的治疗方法,包括给予患者TCR-T细胞、免疫检查点抑制剂、靶向药物、化疗药物中的一种或几种组合。
附图说明
为了清楚的说明本发明中的实施例或现有技术的技术方案,将对实施例和现有技术方案使用的附图作简单描述。
图1.流式细胞仪分选特异识别HCA587/MAGEC2表位肽的CD8+T细胞
图2.DNA电泳鉴定5’RACE PCR获得的TCRα链和β链的可变区片段
图3.流式细胞术检测TCR在T细胞表面表达的阳性率
图4.流式细胞术检测识别HCA587/MAGEC2抗原肽的特异T细胞在CD8+T细胞中的比例
图5.ELISA方法检测TCR-T细胞与靶细胞孵育后IFN-γ分泌量
图6.LDH方法检测TCR-T细胞对靶细胞的杀伤作用
具体实施方式
为了更好的说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明做进一步说明。值得注意的是,基于简要明了的目的,对常规操作、步骤、试剂并未进行细致的描述。但应该理解,如未特别说明,对本领域的普通技术人员而言这些常规操作、步骤、试剂等是显而易见的。
实施例1:抗原特异性T细胞受体序列的确定
将50μl(100μg)HCA587/MAGEC2表位肽VIWEVLNAV(杭州中肽有限公司合成)和50μl(20μg)CpG ODN-1826(上海生工生物工程有限公司)与100μl弗式佐剂(购自Sigma)混合后免疫HLA-A*0201转基因小鼠,共免疫2次,间隔1周,最后一次免疫后10天,取小鼠脾脏,制备单细胞悬液,与来源于HLA-A*0201转基因小鼠的DC细胞和VIWEVLNAV表位肽共同孵育,培养7天后,利用荧光标记CD8抗体(购自biolegend公司)和Pentamer(购自美国Proimmune公司)通过流式细胞术分选双阳性细胞群体(图1)。利用RNA提取试剂盒和5’RACE试剂盒(购自QIAGEN公司)提取总RNA,扩增特异性的TCRα链和β链的可变区序列(图2)。α链获得38个单克隆质粒,β链93个单克隆质粒。通过测序获得单克隆的核酸序列,并在国级免疫遗传学数据库IMGT进行逐一对比,α链有效序列29个,β链有效序列80个,通过对序列富集进一步分析,获得频率最高的α链和β链可变区序列,如SEQ ID NO:6和SEQ ID NO:8所示氨基酸序列。
实施例2:慢病毒载体的构建
基于慢病毒载体整合高效、表达持久的特点,选择第三代慢病毒质粒系统,表达质粒为pCDH-copGFP,辅助质粒为pSPAX2和pMD2.G。将实施例1中获得的T细胞受体α链和β链的可变区编码基因进行人工合成,二者之间插入2A蛋白的编码基因使TCR的α链和β链的编码基因在T细胞的表达水平一致,增加α链与β链配对的机会。将合成的HCA587/MAGEC2抗原特异的TCRα链与β链序列通过T4连接酶连接在表达载体pCDH-copGFP上,得到HCA587/MAGEC2抗原特异的TCR表达载体pCDH-TCR-GFP。将表达载体pCDH-TCR-GFP和包装载体pSPAX2及pMD2.G共转染HEK-293T细胞(购自中国医学科学院基础医学研究所基础医学研究中心),分别于48h和72小时收获病毒,4℃,2000g,离心10分钟,将上清液通过0.45μm滤膜过滤,加入到一个新的无菌离心管中,4℃,20000g高速离心2小时后吸弃上清,将沉淀用1ml无菌PBS缓冲液重悬,即为浓缩后的慢病毒悬液,置于-80℃保存。
实施例3:TCR-T细胞的制备
利用Ficoll密度梯度离心法分离人外周血单个核细胞(PBMC),通过贴壁法去除单核细胞,将淋巴细胞密度调整至1×106/mL,加入到预先包被了抗人CD3抗体(购自Biolegend公司)(3μg/mL,37℃孵育2h)的24孔细胞培养板中,再向培养体系中加入抗人CD28抗体(1μg/mL,购自Biolegend公司)和重组人IL-2(30U/ml,购自Peprotech公司),刺激T细胞非特异活化,培养48小时后取出部分细胞利用流式细胞仪检测T细胞各亚群的比例及活化T细胞的比例。将浓缩的HCA587/MAGEC2抗原特异的TCR慢病毒感染活化的T细胞,并加入重组人IL-2(100U/mL),流式细胞仪检测HCA587/MAGEC2抗原特异的TCR慢病毒感染T细胞后细胞膜表面TCR的表达情况,阳性率约为9%(图3)。慢病毒感染10天后进一步利用荧光标记的CD8抗体和合成的针对HCA587/MAGEC2表位肽的荧光标记的Dextramer(购于Immudex),通过流式细胞仪分析CD8和Dextramer双阳性的细胞群体约占35%(图4)。
实施例4:TCR-T细胞活性的检测
T2细胞是抗原处理相关转运体(TAP)缺陷的细胞系,表达HLA-A*0201分子,可以直接和HLA-A*0201限制性表位肽结合,将人工合成的HLA-A*0201限制性表位肽VIWEVLNAV 10μg/ml与T2细胞共同孵育,37℃,5%CO2,孵育2小时后,300g离心10分钟,弃上清,收集细胞沉淀,作为TCR-T细胞的靶细胞。
用无血清培基培养(购自达科为公司)将TCR-T细胞调整为2×105/ml,96孔板,每孔100μl;靶细胞(负载抗原肽的T2细胞)调整细胞浓度为2×104/ml,每孔加100μl,37℃,5%CO2,孵育24小时,取培养上清,通过人IFN-γELISA试剂盒(购自达科为公司)检测TCR-T细胞分泌的IFN-γ量,可见负载抗原肽的T2细胞可激活TCR-T细胞,产生的IFN-γ显著高于对照组(图5)。
将TCR-T细胞调整为2×105/ml,96孔板,每孔加100μl按效靶比10∶1,5∶1,1∶1的比例,调整加入靶细胞(负载抗原肽的T2细胞)的量,每孔加50μl,37℃,5%CO2,孵育6小时后,将50μl上清转移至一新板中,加50μl检测底物,室温孵育30分钟,加终止液50μl(CytoTox96 Non-Radioactive Cytotoxicity Assay,购于Promega公司),测量490NM的吸光度值,计算TCR-T细胞对靶细胞的杀伤活性(图6)。
Claims (10)
1.一种针对癌/睾丸抗原HCA587/MAGEC2基因的特异性T细胞受体(TCR),其特征在于,能够和HCA587/MAGEC2衍生的抗原肽VIWEVLNAV与HLA-A*0201形成的复合物发生特异性结合。
2.根据权利要求1所述的识别癌/睾丸抗原HCA587/MAGEC2的特异性T细胞受体,其特征在于,包含TCR的α链和β链,所述的TCRβ链的CDR3区的氨基酸序列与SEQ ID NO:02所示的序列具有至少75%及以上同源性且功能相同的氨基酸序列;或者所述TCRβ链的CDR3区的序列为SEQ ID NO:02所示的氨基酸序列。
3.根据权利要求2所述的特异识别HCA587/MAGEC2的T细胞受体,其特征在于,所述的TCRβ链可变区序列包含CDR3区序列,所述可变区氨基酸序列为SEQ ID NO:06所示的序列。
4.根据权利要求1或2或3所述的特异识别癌/睾丸抗原HCA587/MAGEC2的T细胞受体,其特征在于,所述TCRα链的CDR3区的氨基酸序列与SEQ ID NO:04所示的序列具有至少75%及以上同源性且功能相同的氨基酸序列;或者所述TCRα链的CDR3区的序列为SEQ ID NO:04所示的氨基酸序列。
5.根据权利要求书4所述的特异识别癌/睾丸抗原HCA587/MAGEC2的T细胞受体,其特征在于,所述的TCRα链可变区序列包含CDR3区,所述可变区的序列为SEQ ID NO:08所示的氨基酸序列。
6.根据权利要求书1或2或3或4或5所述的特异识别癌/睾丸抗原HCA587/MAGEC2的T细胞受体,其特征为由TCRα链和β链形成的异源二聚体结构。
7.一种载体,其特征为所述载体包含具有编码所述的特异识别癌/睾丸抗原HCA587/MAGEC2的T细胞受体的核酸序列或所述氨基酸序列对应的优化密码子的核酸序列。
8.权利要求7所述载体,为慢病毒载体,包含特异识别癌/睾丸抗原HCA587/MAGEC2的T细胞受体序列。
9.一种细胞,特征为所述细胞的细胞膜上表达所述的特异识别癌/睾丸抗原HCA587/MAGEC2的T细胞受体。
10.一种特异识别癌/睾丸抗原HCA587/MAGEC2的T细胞受体的应用,其特征为用于制备能够杀伤表达HLA-A*0201分子且癌/睾丸抗原HCA587/MAGEC2表达阳性的肿瘤细胞。
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