CN111647070B - T细胞受体或其抗原结合片段及应用 - Google Patents
T细胞受体或其抗原结合片段及应用 Download PDFInfo
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Abstract
本发明公开了T细胞受体或其抗原结合片段及应用,编码T细胞受体的核酸,包含T细胞受体的核酸的载体以及经修饰的细胞及其制备治疗肿瘤的产品和筛选检测抗原、制备疫苗中的应用。
Description
技术领域
本发明本发明属于细胞免疫学、基因工程领域,涉及T细胞受体或其抗原结合片段及应用。
背景技术
T细胞受体基因工程改造的T细胞(T cell receptorgene engineered Tcells,TCR-T)疗法是以修饰T细胞为基础的肿瘤过继免疫治疗方法,可以凭借其对肿瘤特异性抗原的高亲和性识别能力,在体内发挥较强的抗肿瘤免疫效应。根据目前开展的相关临床试验,已经证明了TCR-T细胞疗法在治疗恶性黑色素瘤、骨髓瘤等恶性肿瘤中有显著疗效,具有很大的发展潜力。
TCR是T细胞表面的特征性标志,介导识别提呈的抗原。TCR分子属于免疫球蛋白超家族,是由两条不同的肽链组成的异二聚体,每条肽链分为胞外区(包括结合抗原的可变区及与之相连的恒定区)、跨膜区和胞质区。肽链氨基端在细胞外,羧基端在细胞内。其中可变区氨基端肽链的氨基酸顺序变化较大,保守区近膜端肽链的氨基酸顺序变化较小。TCR可分为αβTCR和γδTCR两类,在外周血中,90%~95%的T细胞表达的TCR由α和β两条链组成。采取基因转载体将目标TCR的α链和β链基因插入的方式,制备具有特定肿瘤抗原特性的TCR-T细胞。外源基因所表达的TCR以非共价键形式结合CD3分子,形成表达于细胞膜上的TCR-CD3复合物,进一步识别MHC-抗原肽复合物。细胞内原有的信号转导结构诱发T细胞活化,然后对靶细胞进行特异性杀伤。
免疫疗法的飞速发展为人类对抗肿瘤带来了新希望,然而TCR-T细胞临床免疫治疗也面临一些挑战,提高TCR-T细胞治疗的有效性和安全性成为目前研究的重点。
发明内容
本发明涉及T细胞受体或其抗原结合片段,并且涉及用于治疗应用、诊断应用的用途。
本发明的一个方面涉及T细胞受体或其抗原结合片段,其包含Vα和/或Vβ,其中所述Vα、所述Vβ或二者包含多个互补决定区(CDR)。
在一个实施方案中,所述的T细胞受体或其抗原结合片段包含SEQ ID NO.7所示的VβCDR3氨基酸序列。在另一个实施方案中,所述的T细胞受体或其抗原结合片段包含SEQ IDNO.8所示的VβCDR3氨基酸序列。在另一个实施方案中,所述的T细胞受体或其抗原结合片段包含SEQ ID NO.9所示的VβCDR3氨基酸序列。在另一个实施方案中,所述的T细胞受体或其抗原结合片段包含SEQ ID NO.10所示的VβCDR3氨基酸序列。在另一个实施方案中,所述的T细胞受体或其抗原结合片段包含SEQ ID NO.11所示的VβCDR3氨基酸序列。
在一个实施方案中,所述T细胞体或其抗原结合片段还包含SEQ ID NO.1的VαCDR1氨基酸序列,SEQ ID NO.2的VαCDR2氨基酸序列,SEQ ID NO.3的VαCDR3氨基酸序列。
在一个实施方案中,所述T细胞体或其抗原结合片段还包含如SEQ ID NO.5所示的VβCDR1氨基酸序列,如SEQ ID NO.6所示的VβCDR2氨基酸序列。
在一个实施方案中,所述T细胞体或其抗原结合片段包含如SEQ ID NO.4所示的Vα氨基酸序列和/或如SEQ ID NO.12所示的Vβ氨基酸序列。在另一个实施方案中所述T细胞体或其抗原结合片段包含如SEQ ID NO.4所示的Vα氨基酸序列和/或如SEQ ID NO.13所示的Vβ氨基酸序列。在另一个实施方案中所述T细胞体或其抗原结合片段包含如SEQ ID NO.4所示的Vα氨基酸序列和/或如SEQ ID NO.14所示的Vβ氨基酸序列。在另一个实施方案中所述T细胞体或其抗原结合片段包含如SEQ ID NO.4所示的Vα氨基酸序列和/或如SEQ ID NO.15所示的Vβ氨基酸序列。在另一个实施方案中所述T细胞体或其抗原结合片段包含如SEQ IDNO.4所示的Vα氨基酸序列和/或如SEQ ID NO.16所示的Vβ氨基酸序列。
本发明的另一个方面涉及一种免疫动员单克隆T细胞受体,包含可溶性的述T细胞体或其抗原结合片段,可溶性的述T细胞体或其抗原结合片段如前所述。
在一个实施方案中,免疫动员单克隆T细胞受体是将亲和性单克隆T细胞受体(mTCR)靶向与治疗作用机制(如,抗CD3scFv)组合的双功能性蛋白。优选的,免疫动员单克隆T细胞受体包含抗CD3scFv。
本发明的另一个方面提供了一种核酸,所述核酸编码前述的T细胞受体或其抗原结合片段。
在其它相关实施例中,核酸可以是编码经修饰的TCR的多核苷酸的变异体。多核苷酸变异体与编码本文所述的经修饰的TCR的核酸序列可以具有实质一致性。举例来说,多核苷酸可以是使用本文所述的方法(例如,使用标准参数的BLAST分析,如下文所描述),与参考多核苷酸序列(例如编码本文所述的TCR的序列)相比包含至少70%序列一致性、优选至少75%、80%、85%、90%、95%、96%、97%、98%或99%或更高序列一致性的多核苷酸。本领域的技术人员将认识到,这些值可以适当地调整,以便通过考虑密码简并、氨基酸类似性、阅读框架定位等,确定由两个核苷酸序列编码的蛋白质的相应一致性。
典型地,多核苷酸变异体将含有一个或多个取代、添加、缺失和/或插入,优选使得由变异的多核苷酸编码的TCR的结合亲和力相对于由本文阐述的多核苷酸序列编码的TCR并不实质上降低。
本领域的普通技术人员应了解,作为遗传密码简并的结果,存在许多编码如本文所述的TCR的核苷酸序列。这些多核苷酸中的一些与编码结合到例如相同抗原的经修饰的TCR的天然或初始多核苷酸序列的核苷酸序列具有最小序列一致性。尽管如此,本发明明确地涵盖由于密码子使用差异而改变的多核苷酸。在某些实施例中,具体涵盖已经经过密码子优化以用于哺乳动物表达的序列。
如本文所用,“核酸”或“核酸分子”或“多核苷酸”是指任何脱氧核糖核酸(DNA)、核糖核酸(RNA)、寡核苷酸,例如通过聚合酶链式反应(PCR),或通过体外翻译产生的片段,以及由任何连接、断裂、核酸内切酶作用或核酸外切酶作用产生的片段。在某些实施方案中,通过PCR产生本公开的核酸。核酸可以由作为天然存在的核苷酸(诸如脱氧核糖核苷酸和核糖核苷酸)的单体、天然存在的核苷酸的类似物(例如天然存在的核苷酸的α-对映体形式)或两者的组合构成。经修饰的核苷酸可以具有糖部分或嘧啶或嘌呤碱基部分的修饰或置换。核酸单体可以通过磷酸二酯键或这类键的类似物连接。磷酸二酯键的类似物包括硫代磷酸酯、二硫代磷酸酯、硒代磷酸酯、二硒代磷酸酯、苯胺基硫代磷酸酯、苯胺磷酸酯、氨基磷酸酯等。核酸分子可以是单链或双链。
本发明的另一个方面提供了一种载体,所述载体包含前述的核酸。
载体是能够在宿主细胞中自主地复制并且可以接受外源DNA的核酸分子。载体携有其自身的复制起点;一个或多个可以用于插入外源DNA的限制性核酸内切酶的独特识别位点;以及通常可选标记,例如编码抗生素抗性的基因,和通常用于表达插入的DNA的识别序列(例如,启动子)
本发明的再一个方面提供了一种经修饰的细胞,所述细胞包括如前所述的T细胞受体或其抗原结合片段,或其嵌合抗原受体。
在一个实施方案中,T细胞受体的抗原结合片段包含单链TCR。
在一个实施方案中,嵌合抗原受体为TCR-CAR。
在一个实施方案中,所述细胞为人类免疫细胞。优选的,所述免疫细胞为T细胞、NK细胞或NK-T细胞。
术语“T细胞”是在胸腺中成熟并产生T细胞受体(TCR)的免疫系统细胞。T细胞可以是原始的(不暴露于抗原;与TCM相比,CD62L、CCR7、CD28、CD3、CD127和CD45RA的表达增加,且CD45RO的表达降低)、记忆性T细胞(TM)(经历抗原且长寿命)和效应细胞(经历抗原,具有细胞毒性)。TM可进一步分为中央记忆性T细胞(TCM,与原始T细胞相比,CD62L、CCR7、CD28、CD127、CD45RO和CD95的表达增加,且CD54RA的表达降低)和效应记忆T细胞(TEM,与原始T细胞或TCM相比,CD62L、CCR7、CD28、CD45RA的表达降低,且CD127的表达增加)的亚群。效应T细胞(TE)是指经历抗原的CD8+细胞毒性T淋巴细胞,其相比TCM具有降低的CD62L、CCR7、CD28表达,并且对颗粒酶和穿孔素呈阳性。其他示例性T细胞包括调节性T细胞,诸如CD4+CD25+(Foxp3+)调节性T细胞和Treg17细胞,以及Tr1、Th3、CD8+CD28-和Qa-1限制性T细胞。
本发明的另一个方面提供了一种组合物,所述组合物包含:
前述的T细胞受体或其抗原结合片段;或
前述的免疫动员单克隆TCR;或
前述的核酸;或
前述的载体;或
前述的细胞。
在一个实施方案中,所述组合物还包括医学上可接受的载剂。医药学上可接受的载剂用以将本文公开用于实践本发明的化合物调配成适用于全身性投药的剂量的用途属于本发明的范围内。通过恰当选择载剂和适合制造实践,本发明的组合物、尤其调配成溶液的组合物可以在肠胃外,例如通过静脉内注射而投与。适当化合物可以容易使用本领域中熟知的医药学上可接受的载剂调配成适用于口服投药的剂量。此类载剂使本发明的化合物能调配成片剂、丸剂、胶囊、液体、凝胶、糖浆、浆液、悬浮液等,用于由待治疗的患者口服摄取。
本发明的另一个方面提供了前述的T细胞受体或其抗原结合片段、免疫动员单克隆TCR、核酸、载体、细胞或组合物在制备治疗肿瘤的产品中的应用。
如本发明所用,“治疗”是指用于获得有益或所要结果、包括并且优选临床结果的方法。治疗可以指改善疾病或病况的症状或延迟疾病或病况的进展。
本发明的另一个方面提供了前述的T细胞受体或其抗原结合片段、免疫动员单克隆TCR、核酸、载体、细胞或组合物在筛选检测抗原、制备疫苗中的应用。
本发明的TCR和/或具有与TCR可变区类似(大于90%一致性)的一级结构并且维持对于标靶配位体的高亲和力的结合片段可以调配成中性或盐形式的疫苗。医药学上可接受的盐包括(但不限于)用无机酸(例如,盐酸或磷酸)和有机酸(例如,乙酸、草酸、酒石酸或马来酸)形成的酸加成盐(用肽的游离氨基形成)。用游离羧基形成的盐也可以来源于无机碱,例如,钠、钾、铵、钙或铁氢氧化物;和有机碱,例如,异丙胺、三甲胺、2-乙基氨基-乙醇、组氨酸和普鲁卡因。
附图说明
图1是TCR-T杀伤功能检测图。
具体的实施方式
下面结合附图和实施例对本发明作进一步详细的说明。以下实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1高通量测序筛选TCR序列
1、RNA提取及质控
收集急性髓系白血病患者的外周血样本,患者无甲肝,乙肝,丙肝,戊肝,艾滋病,梅毒,淋病和结核感染。
在5mL Ficoll中缓慢加入外周血,离心2000rpm,15min。吸取中间白膜层,加入0.9%生理盐水,计数单个核细胞数量,离心1000rpm,5min,磁珠分选T细胞。采用TRIzol法对处理的细胞样本进行RNA的提取,对提取后的RNA质量使用Qubit RNA HS Assay kits,经2100生物分析仪(安捷伦)检测RNA完整性。2、反转录及文库制备
用Repertoire Analysis Kit(Human TCR alpha,beta)进行建库。将所有试剂置于冰上解冻,在配置mix之前,混匀和离心这些试剂。在冰上配置杂交引物需用的mix(表1)。充分混匀后离心,加热反转录引物杂交mix,65℃5min,冰上孵育至少2min。在冰上准备配制反转录mix(表2),混匀后离心mix。
表1反应体系
组分 | 体积 |
RT dNTP Mixture | 1μl |
RT Primer H TCRα/β | 1μl |
RNA样本(100pg-1000ng) | 最多到6.2μl |
Nuclease-free water | 补足至8.2μl |
表2反转录体系
将11.8μl的杂交buffer加到上一步反应的杂交RNA中混匀,然后离心,在PCR仪(T100,Bio-RAD)上运行以下程序(表3):
表3反应条件
3、第一步PCR
准备第一步PCR需用的试剂(表4),并在冰上解冻,混匀后瞬时离心,在冰上配制第一步PCR反应的mix,配置好的mix用移液器混匀,然后离心。
表4第一步PCR反应体系
每管中加入41μl的第一步PCR mix,加入9μl的第一链cDNA产物中混匀,然后离心,在PCR仪(T100,Bio-RAD)上运行以下程序:
表5第一步PCR反应条件
4、第二步PCR
准备第二步PCR需用的试剂(表6),并在冰上解冻,混匀后瞬时离心,在冰上配制第二步PCR反应的mix,配置好的mix用移液器混匀,然后离心。
表6第二步PCR反应体系
组分 | 体积 |
PCR buffer | 25μl |
PCR dNTP Mixture | 10μl |
Nuclease-free water | 3μl |
DNA polymerase | 1μl |
总体积 | 39μl |
准备一个新的PCR管,每管中加入1μl的第二步PCR引物,hTCR α#1-18(编号:A1-A18),或者hTCR β#1-18(编号:B1-B18)。加入39μl第二步反应的mix和10μl的第一步PCR产物混匀,然后离心,在PCR仪(T100,Bio-RAD)上运行以下程序:
表7第二步PCR反应条件
5、第三步PCR
准备第三步PCR需用的试剂在冰上解冻(表9),混匀后瞬时离心。在冰上配制第二步PCR反应的mix,配置好的mix用移液器混匀,然后离心。
表8第三步PCR反应体系
组分 | 体积 |
PCR buffer | 25μl |
PCR dNTP Mixture | 10μl |
3rd universal primer | 2.5μl |
Nuclease-free water | 6.5μl |
DNA polymerase | 1μl |
总体积 | 45μl |
准备一个新的PCR管,每管中加入45μl的第三步PCR mix,加入5μl的第二步PCR产物中,用手指肚弹匀,然后离心,在PCR仪(T100,Bio-RAD)上运行以下程序:
表9第三步PCR反应条件
6、琼脂糖凝胶电泳
配置1.5%的琼脂糖胶,用1*TAE buffer和核酸染料(北京金博益),DNA marker上样量为2μl,加1μl的6*loading buffer到PCR产物中,点样。100V电泳30-45min。使用凝胶到成像系统(1708195,Bio-RAD),判断产物大小。hTCRɑ/β片段在650bp左右。
7、用AGENCOURT AMPure XP(BEACKMAN)磁珠纯化
准备nuclease-free 0.2mL的8连管,按照表10稀释文库。
表10稀释体系
组分 | 体积 |
第三步PCR产物 | 30μl |
Nuclease-free water | 20μl |
总体积 | 50μl |
配置DNase-free 70%乙醇(sigma)。磁珠使用前室温平衡30min,将22.5μl的磁珠加到稀释后文库中,用移液器混匀至少10次。室温孵育5min,然后简单的离心。将8连管放置在96孔磁力架上磁吸5min,观察液体和磁珠分离。将所有的上清转移到新的8连管中。再次混匀未使用过的磁珠,加17.5μl的磁珠到上清中,用移液器混匀至少10次。室温孵育5min,然后简单离心。将8连管放在96孔磁力架上磁吸5min,观察液体和磁珠分离。弃85μl的上清,保留5μl的上清以免吸到磁珠。不要将磁珠与磁力架分离。加200μl的70%的乙醇漂洗。室温30s,弃酒精,重复酒精洗涤步骤。弃所有上清,加30μl的10mM Tris-HCl到每个PCR管中,用移液器混匀至少10次。室温孵育2min然后简单离心。将离心管放置在磁力架上室温5min,分离液体和磁珠。将上清20μl转移至新的PCR管中。
8、文库浓度定量及质控
测定文库浓度用Qubit dsDNA HS kit(英潍捷基),使用设备Qubit 2.0(life),取2μl的样本用于测定。通常文库浓度集中在5-40ng/μl。如果样本浓度≥1.72ng/μl可用于Miseq测序。
9、文库目标片段质控
人的TCRα/β文库目标片段集中在650bp,使用1.5%的琼脂糖凝胶电泳对文库片段进行质控,用1*TAE buffer和核酸染料(北京金博益),DNA marker上样量为2μl,加1μl的6*loading buffer到PCR产物中,点样。100V电泳30-45min。使用凝胶到成像系统(1708195,Bio-RAD),判断产物大小。
10、文库混库及质控
文库混样,是按此批次浓度最低的文库为标准,浓度最低文库乘以10μl为基准纳克数,其他文库按照此纳克数进行取样,确保每个文库的总量是一致的,混样后按照纳克与摩尔数之间的换算,稀释到8.58ng/μl(20nM,650bp)。
11、高通量测序
文库使用Miseq(Illumina)进行2×300bp双端测序,对上述混合完成的文库再次使用设备Qubit 2.0(life)进行文库定量,最终稀释到10pM,与10pM denatured PhiX进行混合(PhiX占比30%),混合后的600μl进行上机测序。
12、数据分析
采用MiXCR进行初级分析,可以对高度个性化的TCR和免疫球蛋白序列进行分析。MiXCR可以针对不同的数据类型在参数上进行调整,并对分析结果和输出进行优化。进一步高级分析多个多样性指数,如香农指数(Shannon)、辛普森指数(Simpson)、Inverse-Simpson指数和基尼指数(Gini)等。采用VDJtools用于二级TCR profiling分析以及多样性评估。
13、噬菌体展示技术筛选高亲和力的TCR序列。
14、结果
通过噬菌体展示技术筛选出高亲和力的TCR,其α链可变区的氨基酸序列如SEQ IDNO.4所示,其中CDR1-3的序列分别如SEQ ID NO.1-3所示;其β链可变区的氨基酸序列如SEQID NO.12-16任一项所示,其中,CDR1-2的序列分别如SEQ ID NO.5-6所示,CDR3的序列如SEQ ID NO.7-11任一项所示。
实施例2 TCR-T的构建
1、利用高通量测序和数据分析结果,全合成相关序列,通过XbaI/SalI限制内切酶切点,连接到pCDH载体中。
2、TCR-T细胞制备
采集健康人外周血,在Ficoll中缓慢加入外周血,离心2000rpm,15min。吸取中间白膜层,加入0.9%生理盐水,计数单个核细胞数量,离心1000rpm,5min。磁珠分选T细胞。同时加入CD3/CD28抗体偶联磁珠刺激,24h及48h后加入病毒感染2次,病毒感染时加入IL-2,培养3-20天,获得TCR-T细胞。
实施例3 TCR-T杀伤功能检测
从本研究中所述的序列构建的TCR-T中任选一个进行功能检测。
计数靶细胞(K562)和阴性参照靶细胞(Daudi),分别标记Celltrace far red和CFSE染色。计数TCR-T(TCR的Vβ可变区的氨基酸序列如SEQ ID NO.12所示)细胞作为效应细胞。按照计划效靶比(3:1,9:1,18:1),重悬效应细胞和靶细胞至相应浓度。圆底96孔板中每个效靶比分别铺入铺入组1效应细胞、组2靶细胞和组3效应细胞+靶细胞,每组4个复孔。培养2-24h,收细胞样本,分别收管A:组1效应细胞+组2靶细胞,管B:组3效应细胞+靶细胞,多聚甲醛液固定,流式上机。作Killing Rate与E:T Ratio曲线图。其中,
结果,如图1所示,相比对照组,本申请的TCR-T具有较好的杀伤效果。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出,对于本领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也将落入本发明权利要求的保护范围内。
序列表
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Claims (11)
1.一种T细胞受体或其抗原结合片段,其特征在于,包含如SEQ ID NO.1所示的VαCDR1氨基酸序列,如SEQ ID NO.2所示的VαCDR2氨基酸序列,如SEQ ID NO.3所示的VαCDR3氨基酸序列;和包含如SEQ ID NO.5所示的VβCDR1氨基酸序列,如SEQ ID NO.6所示的VβCDR2氨基酸序列,如SEQ ID NO.7所示的VβCDR3氨基酸序列,包含如SEQ ID NO.4所示的Vα氨基酸序列和包含如SEQ ID NO.12所示的Vβ氨基酸序列。
2.一种免疫动员单克隆T细胞受体,包含可溶性的根据权利要求1所述的T细胞受体或其抗原结合片段。
3.根据权利要求2所述的免疫动员单克隆T细胞受体,其特征在于,所述免疫动员单克隆T细胞受体包含抗CD3scFv。
4.一种核酸,其特征在于,编码权利要求1所述的T细胞受体或其抗原结合片段。
5.一种载体,其特征在于,包含权利要求4所述的核酸。
6.一种经修饰的细胞,其特征在于,包括权利要求1所述的T细胞受体或其抗原结合片段,或其嵌合抗原受体。
7.根据权利要求6所述的细胞,其特征在于,T细胞受体的抗原结合片段包含单链TCR。
8.根据权利要求6所述的细胞,其特征在于,所述嵌合抗原受体为TCR-CAR。
9.根据权利要求6所述的细胞,其特征在于,所述细胞为人类免疫细胞。
10.根据权利要求9所述的细胞,其特征在于,所述免疫细胞为T细胞、NK细胞或NK-T细胞。
11.一种组合物,其特征在于,包含:
权利要求1所述的T细胞受体或其抗原结合片段;或
权利要求2或3所述的免疫动员单克隆TCR;或
权利要求4所述的核酸;或
权利要求5所述的载体;或
权利要求6-10任一项所述的细胞。
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