CN112457416B - 一种靶向bcma的嵌合抗原受体(car)及其应用 - Google Patents
一种靶向bcma的嵌合抗原受体(car)及其应用 Download PDFInfo
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Abstract
本发明属于生物制药技术领域，具体涉及一种靶向BCMA的嵌合抗原受体(CAR)及其应用，该嵌合抗原受体以靶向BCMA的单域抗体为抗原结合域，对CD3ζ结构域进行改造，缺失了部分ITAM1和ITAM3，保留原始ITAM2，并优化了ITAM2中部分氨基酸序列，可以提高CAR‑T内体存活时间，降低炎症因子表达水平，具有较好的临床应用前景。
Description
技术领域
本发明属于生物制药领域，具体涉及一种靶向BCMA的嵌合抗原受体(CAR)及其应用，尤其是涉及一种CD3ζ信号域经改造的嵌合抗原受体及其应用。
背景技术
嵌合抗原受体T细胞(CAR-T细胞)是指被高亲和性的嵌合抗原受体修饰的具有杀伤作用的T细胞，被基因修饰后的T细胞能够特异性地识别肿瘤抗原，识别后可激活T细胞使其大量增殖并通过免疫反应杀伤肿瘤细胞。CAR-T细胞技术的发展经历了一定的曲折和探索，第一代CAR-T细胞内只有单一信号分子，能够特异性识别靶抗原，但是缺乏协同刺激分子不能持续有效杀伤肿瘤细胞，所以无法充分激活是第一代CAR-T细胞的最主要缺陷，正因如此，第一代CAR-T细胞多用于理论研究，由于效果不佳这种新型细胞免疫疗法长期没有受到人们的关注。第二代CAR-T细胞借鉴了T细胞的双激活理论，在第一代的基础上增加了一个协同刺激分子，提高了第二代CAR-T细胞的增值分泌能力和持久性，从而增强了其抗癌效应。受到第二代CAR-T细胞成功的启发，研究人员尝试在第二代的基础上整合两个及以上的协同刺激分子，从而构建了第三代CAR-T细胞，提高了CART细胞的存活时间，进一步增强CAR-T细胞的抗癌效应。由于人体内存在多种抗肿瘤信号通路，研究人员试图将CAR-T细胞技术与其他肿瘤免疫疗法结合，故在第三代的基础上形成嵌合其他抗肿瘤信号通路的新型免疫治疗细胞，从而构成了第四代CAR-T细胞，在所选择的其他肿瘤信号通路中常用的包括IL-2、IL-12、TNF-α等抗肿瘤因子，以及PD-1、PD-L1和CTLA4等肿瘤免疫检查点。
多发性骨髓瘤(multiple myeloma，MM)是一种浆细胞肿瘤性疾病，主要特征是单克隆浆细胞的恶性增殖，它是一种常见的血液恶性肿瘤，在血液系统恶性肿瘤中占比约13％，MM的生存期很短，如果不进行任何治疗，进展期MM患者的中位生存期为6个月，即使接受一般抗肿瘤治疗，中位生存期也仅为3-5年，因此MM被认为是一种不可治愈的疾病。受到CAR-T技术在白血病和淋巴瘤治疗中成功的鼓舞，研究人员尝试将靶向CD19的CAR-T用于MM治疗，但是效果并不理想，这可能是由于CD19并非MM理想的治疗靶点，对此研究人员将目光聚焦于新型MM靶点——B细胞成熟抗原(B cell maturation antigen，BCMA)。BCMA即CD269，是肿瘤坏死因子超家族的成员(tumor necrosis factor receptor superfamilymember17，TNFRS17)，由185个氨基酸残基组成，属于III型跨膜蛋白，研究表明BCMA特异性表达于浆细胞，在其他正常体细胞中鲜有表达，这提示其可以作为MM的理想靶点。全球首个靶向BCMA的CAR-T临床试验由美国国家癌症研究所提出，其总体反应率为81％，完全缓解率为13％；随后蓝鸟公司开发出Bb2121，其是由慢病毒载体构建的鼠源二代靶向BCMA的CAR-T细胞，在临床试验中总体反应率85％，完全缓解率45％；我国的南京传奇公司开发了LCAR-B38M，是一种利用慢病毒载体构建的靶向BCMA双表位的鼠源二代CAR-T，在临床试验中总体有效率为88％，完全缓解达68％。
CAR-T细胞的应用前景广泛，据不完全统计，目前全球已经有数百种CAR-T细胞在进行临床试验，但是CAR-T细胞治疗所引起的不良反应也逐渐受到人们的关注，常规的不良反应包括：细胞因子释放综合征(Cytokine Release Syndrome，CRS)，本质是CAR-T细胞识别靶细胞后自身被激活产生大量炎症因子，引起的全身炎症反应，参与CRS的炎症因子包括C反应蛋白、铁蛋白、IFN-γ、IL-1、IL-2Rα、IL-6、IL-8、IL-10、IL-15、GM-CSF、单核细胞趋化蛋白1(MCP-1)、肿瘤坏死因子受体P55(TNFR P55)、巨噬细胞炎性蛋白1B和可溶性gp130等，CRS临床上则表现为发热、乏力、肌肉酸痛等非特异性症状，严重时可导致患者死亡。免疫效应细胞相关神经毒性综合(Immune Effector Cell-associated NeurotoxicitySyndrome，ICANS)，是指免疫治疗后内源/注入性T细胞和/或其他免疫效应细胞激活或参与导致的，以中枢神经系统病理过程为特征的疾病，与CRS相比，目前ICANS发病机制仍未明确，据推测可能与大脑皮层代谢或血脑屏障改变有关。其他不良反应还包括，外周血细胞减少、B细胞功能缺陷、T细胞恶性转化等等，上述不良反应限制了CAR-T细胞技术的进一步临床应用。有研究表明，在靶向BCMA的CAR-T临床试验中，CRS的发生几率明显高于ICANS，以蓝鸟公司的Bb2121临床试验为例，总体CRS发生率76％，其中2例(6％)为严重CRS，而神经毒性发生率42％，1例(3％)为重症，可见CRS为治疗过程中最主要的不良反应。
为应对上述不良反应，研究人员开展了多方面的尝试，如将CAR-T细胞与其他药物联合使用，靶向不同抗原的CAR-T细胞共同治疗(如靶向CD19 CAR-T)，施用免疫抑制剂，调整给药计划和剂量等，还有人尝试改善嵌合抗原受体的结构，设置自杀系统以便实现可控的CAR-T细胞治疗。其中，对于胞内信号域的改造也受到了研究人员的关注，但是这方面研究较少，且大都处于初步阶段，难以指导如何基于胞内信号域的改变来降低不良反应，如专利文献CN111886242A中公开了CD3ζ多肽缺乏全部或部分的基于免疫受体酪氨酸的活化基序(ITAM)，有利于提高CAR-T细胞的活化程度，并且指明ITAM1对于CD3ζ的活性至关重要，当其缺失或突变后肿瘤杀伤效果将大为降低，但对于不良反应改善方面未作重点讨论；WO2018183385A1中公开了嵌合受体的信号传导部分包含ITAM的部分，例如半-ITAM，但是并未验证具体技术效果。
针对现有技术中的不足，本发明提供了一种新型嵌合抗原受体及其应用，包括信号肽、靶向BCMA的纳米抗体、铰链区、跨膜区、共刺激因子和胞内信号域，筛选出了新型高特异性的靶向BCMA的单域抗原结合域，并对CD3ζ结构域中的ITAM序列进行了改造和突变，在保留CAR-T细胞足够激活程度的同时，降低免疫因子的释放水平，缓解由于CAR-T细胞治疗而导致的不良反应，为开发新型的肿瘤免疫疗法提供了新思路。
发明内容
本发明的主要目的是提供一种靶向BCMA的新型嵌合抗原受体及其应用，以便提高CAR-T细胞治疗过程中肿瘤杀伤效果，降低不良反应，改善整体临床应用效果
本发明的详细技术方案如下：
提供了一种靶向BCMA的嵌合抗原受体，所述嵌合抗原受体包括信号肽、靶向BCMA的纳米抗体、铰链区、跨膜区、共刺激因子和胞内信号域，所述靶向BCMA的纳米抗体的氨基酸序列如SEQ ID NO:4所示,胞内信号域为修饰后的CD3ζ胞内信号域，其氨基酸序列如SEQID NO:5所示。
CD3ζ胞内信号域源自T细胞受体(T cell receptor，TCR)，TCR是由两条不同肽链构成的异二聚体，TCR的胞质区很短，无法独立传递信号，因此，以非共价形式与CD3形成复合物。每个TCR/CD3复合物，包括1条CD3γ链、1条CD3δ链、2条CD3ε链和2条CD3ζ链，2条CD3ζ链形成二聚体。CD3的胞质区含有多个ITAM(免疫受体酪氨酸激活基序，ImmunoreceptorTyrosine-based Activation Motif，ITAM)位点，其中γ、δ和ε胞质区分别含有1个ITAM，而ζ链含有3个ITAMs。ITAM是一段约18个氨基酸的比较保守的序列(YXXL/IX6-8YXXL/I)，通常认为序列中的两个酪氨酸(Y)的磷酸化在TCR信号传递中起着不可或缺的作用，CD3分子ITAM序列中酪氨酸的磷酸化后可激活包括PTKs(蛋白酪氨酸激酶，protein tyrosinekinases)途径在内的多种信号通路。通常认为CD3ζ链的3个ITAM均是不可或缺的，也有部分研究人员认为ITAM1对于保持CD3ζ链的生理活性至关重要，ITAM2和ITAM3则可以缺失或突变。本申请发明人在研究过程中，惊奇的发现，将ITAM1和ITAM3突变为仅包括部分YXXL序列，并将ITAM2中两个YXXL部分中的氨基酸序列进行调整或替换后，仍可保持CD3ζ链激活T细胞效应，并且能够有助于降低炎症因子的释放。
进一步的，所述跨膜区为CD28跨膜区，其氨基酸序列如SEQID NO:6所示。
进一步的，所述共刺激因子选自CD27、CD28、4-1BB、OX40、ICOS、B7-H3和/或它们的任何组合。
进一步的，所述共刺激因子选自CD28，其氨基酸序列如SEQ ID NO:8所示。
共刺激因子的选择对于CAR-T细胞发挥抗肿瘤作用也是至关重要的，最常用的共刺激因子为CD28和4-1BB，其中在T细胞活化方面，似乎CD28比常用的4-1BB更有效，但是由于其强烈的激活作用，也容易引发严重不良反应，在Juno公司开发的靶向CD19的以CD28为共刺激因子的CAR-T临床试验中，就曾出现了患者死亡的严重不良反应。通过研究CD28和CD3ζ链的结构特性，可知二者在结构上存在一定的相关性，可能存在相互干扰和相互促进的情况，故本申请中在选用CD28作为共刺激因子时，对CD3ζ链的结构进行了改造，可以清除其相关干扰的可能，进一步协调二者的活化效应，以便发挥协同治疗作用。
进一步的，所述铰链区其氨基酸序列如SEQ ID NO:7所示
进一步的，所述信号肽氨基酸序列如SEQID NO:9所示。
进一步的，所述嵌合抗原受体氨基酸序列如SEQID NO:10所示。
进一步的，所述嵌合抗原受体核苷酸序列如SEQID NO:11所示。
提供了一种表达载体，所述载体包括编码本申请中所述嵌合抗原受体的氨基酸的核苷酸或其核苷酸。
进一步的，所述表达载体为慢病毒载体。
提供了一种嵌合抗原受体T细胞，所述嵌合抗原受体T细胞表达本申请中所述的嵌合抗原受体。
提供了一种本申请中所述的嵌合抗原受体T细胞在制备抗肿瘤药物中的应用。
进一步的，所述的肿瘤为多发性骨髓瘤。
本发明的有益效果包括：
本发明提供了一种靶向BCMA的新型嵌合抗原受体结构及其应用，该嵌合抗原受体中以靶向BCMA的单域抗体为抗原结合域，可高度特异性结合目标抗原，而且单域抗体结构简单，可减低操作难度。对CD3ζ链结构进行改造，将ITAM1和ITAM3和突变为仅包括部分YXXL序列，并将ITAM2中两个YXXL部分中的氨基酸序列进行调整或替换后，仍可保持CD3ζ链激活T细胞效应，还有助于降低炎症因子的释放，避免细胞因子释放综合征等令人厌烦的不良反应。还优化了共刺激因子的选择方法，选用CD28与改良后的CD3ζ链进行配合，以便保证足够的T细胞活化程度，发挥良好的抗肿瘤效果。
附图说明
图1不同CD3ζ结构域的嵌合抗原受体结构图；
图2不同共刺激因子嵌合抗原受体结构图；
图3CD3ζ结构域改造对于肿瘤细胞杀伤效果的影响图；
图4不同共刺激因子对于肿瘤细胞杀伤效果的影响图；
图5小鼠肿瘤体积变化图；
图6CAR-T细胞体内存活时间图；
图7小鼠体内IL-6分泌水平图；
图8小鼠体内IL-10分泌水平图。
具体实施方式
以下非限制性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何形式限制本发明。凡基于本发明上述内容所实现的技术均应当属于本申请要求保护的范围之中。
以下实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂盒生物材料，如无特殊说明，均可从商业途径获得。
实施例1靶向BCMA的单域抗体的确定
利用人源BCMA蛋白免疫羊驼，通过噬菌体展示文库，筛选靶向BCMA的单域抗体，具体方法包括：
(一)羊驼抗原免疫。
将100μg人BCMA蛋白与100μl弗氏完全佐剂充分混合制成乳化混合物，选取健康成年单峰驼，采用该乳化混合物经背部皮下多点注射方式对羊驼进行免疫，第一次免疫和第二次免疫采用弗氏完全佐剂，后续免疫改用弗氏不完全佐剂与人BCMA蛋白混合而成的乳化混合物进行强化免疫，共免疫7-9次，免疫间隔时间为2周。各次免疫1周后，采集100ml外周血，通过ELISA检测抗血清效价，用浓度为0.5μg/ml的人BCMA蛋白包被检测板，每孔加入梯度稀释的抗血清，37℃孵育1.5h，洗涤3-5次，每孔加入1万倍稀释的辣根过氧化物酶标记的二抗，37℃孵育1h，洗涤3-5次后，加100μl TMB底物，37℃孵育15min，50μl 0.2M的H2SO4中止反应，OD 450nm测定吸光度。当抗血清效价达106以上时，构建抗体噬菌体展示文库。
(二)构建噬菌体展示文库。
采集免疫后的羊驼100mL外周血，使用淋巴细胞分离液分离获得外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)，提取PBMC总RNA，使用RT-PCR试剂盒,经由oligo(dT)反转录为cDNA，通过通用引物进行扩增，随后将羊驼的VHH基因片段经酶切纯化后，克隆至phagemid质粒，转化TG1细菌，经培养富集后，将含VHH基因片段的核酸连接至噬菌体载体上，然后经电击转化构建噬菌体文库。随机挑选48个克隆进行测序鉴定，结果显示，所构建噬菌体文库阳性率为98.2％，序列多样性为95.3％，有效插入率为97.5％，满足后续实验要求。
(三)目标抗体的筛选
本实验中采用三轮筛选获得靶向BCMA的单域抗体，具体步骤为：
第一轮筛选：在ELISA板上包被天然的人BCMA蛋白，5μg/孔，4℃过夜；用PBST溶解BSA至浓度3％，300μL/孔，37℃封闭2h，洗板3次；每孔加入100μL的噬菌体库溶液，37℃孵育2h，洗板3次；加入甘氨酸缓冲液室温下轻摇10min，吸出洗脱液，然后加入的Tris-HCl缓冲液中和反应。将100μL洗脱液加入至5mL对数生长期的TG1菌种，37℃感染30min，随后加入含抗生素的培养基中，37℃过夜培养。取培养液4℃，4000rpm，离心15min，收集上清液加入5mLPEG/NaCl，冰浴30min，4℃，8000rpm，离心15min，弃去上清，用1ml的PBS重悬，4℃，12000rpm，离心10min，所得沉淀即为噬菌体抗体颗粒。
第二轮筛选：在ELISA板上包被天然的人BCMA，1μg/孔，洗板3次；用3％的脱脂奶粉，洗板3次；每孔加入100μL的噬菌体库溶液，37℃孵育2h，洗板5-8次；其余操作同第一轮筛选。
第三轮筛选：在ELISA板上包被天然的人BCMA，0.2μg/孔，洗板3次；用3％的BSA封闭，洗板3次；每孔加入100μL的噬菌体库溶液，37℃孵育2h，洗板5-8次；其余操作同第一轮筛选。
筛选完成后，用噬菌体感染TG1细菌，涂布于培养皿中，从培养后的平板上随机挑取50个单克隆，通过酶联免疫反应方法，以可溶解的人BCMA为抗原，筛选阳性克隆，进行测序鉴定。根据测序结果，选取12条序列用于后续实验。
(四)目标抗体的表达与分析
提取阳性克隆质粒并转化至大肠杆菌感受态细胞，以100mM IPTG诱导单域抗体蛋白表达，采用膜分离与树脂分离相互配合的方式纯化目标抗体蛋白。应用Fortebio生物分子相互作用平台检测亲和力，结果表明12个备选抗体中，有5个抗体与目标抗原的亲和力在3.58E-06至7.05E-08之间，选择其中亲和力最高的单域抗体用于构建嵌合抗原受体。
(五)单域抗体序列分析
测定上述BCMA单域抗体的氨基酸序列结构，如SEQID NO:4所示，将该氨基酸序列置于结构数据库中进行同源结构的搜索和抗体序列结构比对分析，确定其互补决定区(CDR区)，CDR1、CDR2、CDR3的氨基酸序列依次为SEQ ID NO:1-3所示。
实施例2CD3ζ胞内信号域结构设计
CD3ζ胞内信号域的野生型氨基酸序列如SEQID NO:12所示，其中包括三个ITAM结构域，其结构为YXXL/IX6-8YXXL/I，现有技术中通过序列结构与其活性的对应分析，通常认为这三个ITAM结构域对于CD3ζ链发挥其生理活性是必须的，尤其是其中两个酪氨酸(Y)的磷酸化在相关信号传递中起着不可或缺的作用。但是已有的研究结果表明，选用CD3ζ胞内信号域与CD28结合的CAR-T细胞中，由于T细胞的激活程度过高，往往会导致大量炎症因子的释放，引发令人生畏的细胞因子释放综合征。因此本申请发明人基于CD3ζ胞内信号域试图改造ITAM结构域，以便适当降低T的不当激活程度，从而调低炎症因子的释放水平。据此，设计了如下三个改造后的CD3ζ胞内信号域，分别为：
D1 CD3ζ：结构中ITAM2和ITAM3均仅保留一个“YXXL”结构，其氨基酸序列如SEQIDNO:13所示；
D2 CD3ζ：结构中ITAM1和ITAM3均仅保留一个“YXXL”结构，其氨基酸序列如SEQIDNO:14所示；
D3 CD3ζ：结构中ITAM2和ITAM3均仅保留一个“YXXL”结构，其氨基酸序列如SEQIDNO:15所示。
随后，有根据ITAM结构域中各个氨基酸的属性，进行了相关替换和突变，惊奇的发现，ITAM结构域中两个“YXXL”结构中间的6-8个氨基酸采用碱性氨基酸(H、R、K)或芳香族氨基酸(W、F、Y)等替换，在体内实验中的效果似乎更好，据此构建了：
D4 CD3ζ：结构中ITAM1和ITAM3仅保留一个“YXXL”结构，ITAM2中部份氨基酸进行了替换，其氨基酸序列如SEQID NO:5所示。
实施例3嵌合抗原受体结构设计
通过生物信息学方法检索并获得信号肽、CD8 hinge区、CD28跨膜区、CD28共刺激因子的氨基酸和核苷酸序列，其中CD28跨膜区氨基酸序列如SEQID NO:6所示，CD8 hinge区氨基酸序列如SEQID NO:7所示，CD28氨基酸序列如SEQID NO:8所示。为了便于分析比对，设计了4种CAR分子，其具体结构如图1所示，在上述CAR分子中分别选用D1 CD3ζ、D2 CD3ζ、D3CD3ζ和D4 CD3ζ作为胞内信号域，其中包含D4 CD3ζ的CAR分子氨基酸序列如SEQID NO:10所示，其核苷酸序列如SEQID NO:11所示。
为了验证共刺激因子的影响，本发明中又设计了包括4-1BB和ICOS共刺激因子元件的嵌合抗原受体结构，如图2所示，各组嵌合抗原受体分子中均采用D4 CD3ζ胞内信号域，其CAR05选用4-1BB为共刺激因子，CAR06选用ICOS为共刺激因子。
实施例4CAR-T细胞的制备
(一)含有嵌合抗原载体的载体制备
通过PCR法扩增并获得CAR 01-06核苷酸序列，通过引物设计在目标序列两端引入酶切位点，将目标基因片段与慢病毒载体质粒pCDH-CMV-MCS-EF1-GFP-T2A-Puro进行双酶切反应，37℃，孵育60min。酶切反应结束后，将所述核酸片段采用琼脂糖凝胶纯化(使用Taraka公司凝胶纯化试剂盒)，具体操作步骤按照试剂盒操作说明书进行。经凝胶纯化后的目标核酸分子，在T4连接酶的作用下16℃孵育10-12h，获得的连接产物通过电击转入DH5α感受态细胞中，将所获得的感受态细胞转入未添加抗生素的液体LB培养基中，37℃摇床200rpm，培养2h，然后4000rpm离心2min，然后离心重悬于新鲜LB培养基中，将所述菌液均匀涂布至含有抗生素的固体LB培养基中，37℃培养箱中培养过夜，挑取并鉴定阳性克隆，将阳性克隆菌株于-70℃冰箱中保存。
(二)慢病毒载体的制备
将测序正确并保存的菌液复苏并接种至20mL含抗生素的液体LB培养基37℃摇床200rpm，过夜培养，4000rpm离心收集菌体，采用Takara公司质粒提取试剂盒提取质粒，具体操作步骤按照试剂盒操作说明书进行。酶切鉴定，目标序列是否正确。同时，培养293T细胞(本实验室保存)，在37℃、5％CO2条件下稳定培养2-3代后，将106个293T细胞接种至六孔板，37℃、5％CO2条件下培养至对数生长期，更换新鲜培养基。按上述含有目标核酸质粒与VSVG包膜蛋白质粒按3:1的比例混合，后加入Opti-MEM培养基补足体积5mL，逐滴将质粒与转染试剂的混合溶液滴加至293T细胞培养液中，37℃、5％CO2条件下培养48h。
培养完成后，2500rpm离心20min收集上清，使用0.45μm滤膜和0.22μm滤膜依次过滤除去细胞碎片和其他杂质，含病毒上清液经过透析浓缩后，采用TCID50法检测上述病毒的滴度，结果显示携带有目标CAR基因的慢病毒滴度可达到3.4×107-2.5×109TCID50/mL。
(三)CAR-T细胞的制备
招募健康志愿者，采集100mL外周血，通过流式细胞仪法分离并获得外周血中的CD4+、CD8+T细胞，调整CD4+：CD8+比例为1:1，以备后续使用。将获得的T细胞接种于含10％FBS的RPMI1640培养基中，传代培养3-5代，以备达到稳定状态。
采用6孔板培养T细胞，每孔接种5.0×106个细胞，于37℃、5％CO2条件下培养对数生长期，然后分别加入携带有CAR01-06基因的慢病毒，37℃孵育3小时，而后弃去上清，采用新鲜培养基洗涤3-5次，然后37℃、5％CO2条件下培养继续培养5-7天，收集目标细胞用于后续实验。
实施例5CAR-T细胞体外抗肿瘤效果
为验证相关CAR-T细胞的抗肿瘤效果，首先在体外细胞实验中进行验证，以便进行初步筛选。在本发明细胞学实验中，选用人骨髓瘤细胞U266细胞作用实验对象。
(一)人骨髓瘤细胞U266的培养
取液氮中保存的人骨髓瘤细胞U266(本实验室保存)，于温水中快速复苏，然后加入RPMI1640培养基混合均匀后，2000rpm离心5min收集细胞，采用新鲜培养基洗涤3次，然后加入含10％FBS的RPMI1640培养基于37℃、5％CO2条件下培养3-5代，以便充分活化U266细胞。
(二)体外抗肿瘤活性的验证
将转染慢病毒后制备获得的CAR-T 01-04细胞，与U266细胞按照1:1等比例进行混合，采用PBS作为阴性对照，将上述各组混合细胞接种于6孔培养板中，5％CO2、37℃培养箱培养24小时后检测肿瘤细胞杀伤效果，具体检测方式为：混合培养完成后，每孔加入10μLCCK-8，37℃孵育2小时后，酶标仪检测450nm波长，测量OD值，杀伤率＝[1-(实验组OD值-效应细胞对照组OD值)/靶细胞对照组OD值]×100％。
如图3所示，在本发明中所构建的CAR-T 01-04细胞均能有效发挥肿瘤细胞抑制作用，相比于PBS阴性对照组，各个CAR-T治疗组均能较大程度的发挥抗肿瘤作用，这说明了本发明中所筛选并获得的新型靶向BCMA单域抗体能够有效结合目标抗原，介导抗肿瘤作用的发挥。在各个CAR-T治疗组中CAR-T 03的肿瘤细胞杀伤率最低，说明仅保留完整的ITAM3，难以有效激活T细胞，而保留ITAM1或ITAM2的CAR-T均展现了较强的抗肿瘤活性，说明保留在氨基酸序列结构中距离靠前的ITAM1对于T细胞的活化是必要的，而且与完全突变“YXXL”做法不同的是，在保留其中之一YXXL序列的“半突变”ITAM中，保留完整的ITAM2相比于保留完整的ITAM1似乎能够获得更高的抗肿瘤效果。经ITAM2中间氨基酸改造的CAR-T 04也展示出了与保留有天然ITAM2的CAR-T 02类似的细胞杀伤效果。鉴于上述结果，本发明中以对U266细胞杀伤活性较高的CAR-T 02和CAR-T 04作为候选药物。
(三)共刺激因子选择的影响
为验证不同共刺激因子对嵌合抗原受体活性的影响，本发明中验证了含有CD28、4-1BB和ICOS作为共刺激因子时，对U266细胞的杀伤效果，具体实施和检测方式同上述做法。
如图4所示，在不同共刺激因子构成的CAR-T细胞中，采用ICOS、4-1BB为共刺激因子的CAR-T细胞杀伤效率相似，其中4-1BB似乎略高于ICOS，但是二者的细胞杀伤率均明显低于采用CD28的CAR-T 04，这说明本发明中所采用的改造后的CD3ζ结构域更适合于CD28配合使用，二者在T细胞活化过程中能够实现协同效果，相比于其他常规共刺激因子，这种组合更能够强烈的发挥T细胞激活作用。
实施例6CAR-T细胞体内抗肿瘤效果
由于体外环境与体内环境具有较大的差异，通常细胞学实验仅能够作为初步的实验证据，因此为了进一步验证上述CAR-T细胞的抗肿瘤效果，本实施例以Balb/c小鼠移植瘤模型(负载人骨髓瘤细胞U266细胞)验证CAR-T细胞的体内抗肿瘤效果。
(一)动物模型制备
选取6-10周龄BALB/C小鼠，于实验室洁净环境中饲养。复苏并培养U266细胞，具体方法同上述细胞学实验中做法，当U266细胞传代稳定后，调整细胞浓度使其达到1×106个/mL。每只小鼠采用皮下注射方式注射U266细胞，每只注射1mL细胞悬液。然后每天观察小鼠生长状态和肿瘤组织发展情况，约2-3周后可见明显瘤块出现，说明肿瘤小鼠模型建立成功。
(二)小鼠肿瘤体积检测
将上述荷瘤小鼠随机分为5组，每组10只小鼠，分别采用CAR-T 01-04(尾静脉注射1×106个细胞)和生理盐水(阴性对照)进行治疗，分别于治疗后2周和4周测量肿瘤体积，结果如图5所示，所有CAR-T治疗组均可抑制肿瘤的增长，其中CAR-T 04的抑制效果最为明显，然后依次为CAR-T 01、CAR-T 02和CAR-T 03，其中CAR-T 03的治疗效果最差，可能是由于其CD3ζ中的ITAM1和ITAM2均已被破坏，难以有效激活T细胞，进而难以发挥其抗肿瘤效果；而CAR-T 01组与CAR-T 02组的治疗效果类似，但与体外细胞实验结果有一定的差异，推测这可能是由于体内、体外肿瘤微环境的差异造成的，相比于体外细胞培养环境，体内环境更加复杂，肿瘤的发生和发展过程受到多种细胞信号通路的调控，故体内外实验中可能存在一定误差，但总体实验趋势仍在可接受范围内。此外，上述两组的抗肿瘤效果明显弱于CAR-T04组，可能是由于该CAR-T强烈诱导炎症因子的表达(参见图7、图8)，虽然在初期可有效抑制肿瘤生长，然而剧烈的免疫反应导致了整体机能的下降，反而不利用长期的抗肿瘤过程，CAR-T 04组能够实现相对温和而持久的抗肿瘤效果。
(三)CAR-T细胞体内存活期实验
尽管CAR-T 02和CAR-T 04的体外抗肿瘤活性类似，但是在体内实验中，CAR-T 04却展现出了更好的效果，发明人推测这可能与其体内半衰期有关，故检测了上述两种细胞的体内存活情况。采用流式细胞仪技术检测小鼠血清中的活性CAR-T细胞浓度，以便验证该疗法的维持时间，为简化实验步骤选用在体内和体外实验中均展示出较高抗肿瘤活性的CAR-T 02和CAR-T 04进行实验，每周检测一次，共检测4周。
如图6所示，从体内的存活情况来看，CAR-T 04在体内的存活时间似乎更长，CAR-T02在第2周达到峰值，然后开始快速减退，而CAR-T 04则在第3周达到峰值，虽然后续时间中也开始出现浓度降低迹象，但其降低幅度较小，可长时间维持较高水平，从而发挥持久抗肿瘤效果。
(四)免疫因子分泌情况
为了验证本发明中所提供的CAR-T细胞是否能够有效降低炎症因子分泌情况，采用ELISA试剂盒检测血清中各个验证因子的分泌水平，选择CAR-T细胞治疗3周的小鼠血清作为研究对象，分别检测了小鼠血清中IL-6和IL-10的水平。
结果如图7和图8所示，采用CAR-T治疗后小鼠体内IL-6和IL-10的表达水平均大幅度提高，这说明一方面在免疫细胞疗法中，炎症因子释放将难以避免；另一方面，适当炎症因子的释放也有助于增强抗肿瘤效果。在IL-6的表达中，CAR-T 01表达水平最高，其下依次为CAR-T 04、CAR-T 02和CAR-T 03，可见保留完成ITAM2的CAR-T 02和CAR-T 04可引起相对低水平的IL-6释放，而CAR-T 03组中IL-6水平最低，这可能是由于该中嵌合抗原受体中的CD3ζ中的ITAM1、ITAM2均被破坏，难以有效激活T细胞，故呈现了低水平的IL-6，这与细胞杀伤实验的效果是一致的。在IL-10的表达中，类似的，CAR-T 01表达水平仍最高，当CAR-T 03的表达水平较低，其上依次为CAR-T 04和CAR-T 02，这可能是由于不同炎症因子的释放和调控机制不同所致。综上可以知晓，选用经过改造后的CD3ζ与CD28配合使用，可有效降低IL-6、IL-10等炎症因子的表达，从而抑制CRS的发生，这在临床应用上具有积极意义。
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305 310 315 320
Glu Leu Arg Lys Trp Lys Phe Tyr His Arg Tyr Ser Glu Ile Gly Met
325 330 335
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
340 345 350
Leu Ser Thr Ala Thr Lys Asp Thr His Met Gln Ala Leu Pro Pro Arg
355 360 365
<210> 11
<211> 1104
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atggcgctgc cggtgaccgc gctgctgctg ccgctggcgc tgctgctgca tgcggcgcgc 60
ccggaacagc aggtgcagct ggtggaaagc ggcggcggcc tggtgcaggg cggcggcagc 120
ctgcgcctga gctgcgtggc gagcgcgtgc ggcagctatt ataccagcca ggtgccgtgg 180
tttcgccagg cgccgggcaa agaaagcgtg ccggtggcgg cgatttttcg caacagctgg 240
ccgtggcgca tttataccac ctatagctgg ccgatgagcg tgaaaggccg ctttaccatt 300
agccgcgatc aggcggcgaa caccgtgtat ctgcagatga acagcctgaa accggaagat 360
accgcggtgt attattgcca tcgcatgtat accaccatta ttgtgccgca ggcgtgcaac 420
tggggccagg gcacccaggt gaccgtgagc ggcaccacca ccccggcgcc gcgcccgccg 480
accccggcgc cgaccattgc gagccagccg ctgagcctgc gcccggaagc gtgccgcccg 540
gcggcgggcg gcgcggtgca tacccgcggc ctggattttg cgtgcgattt ttgggtgctg 600
gtggtggtgg gcggcgtgct ggcgtgctat agcctgctgg tgaccgtggc gtttattatt 660
ttttgggtgc gcagcaaacg cagccgcctg ctgcatagcg attatatgaa catgaccccg 720
cgccgcccgg gcccgacccg caaacattat cagccgtatg cgccgccgcg cgattttgcg 780
gcgtatcgca gccgcgtgaa atttagccgc agcgcggatg cgccggcgta tcagcagggc 840
cagaaccagc tgtataacga actgaacctg ggccgccgcg aagaagataa acgccgcggc 900
cgcgatccgg aaatgggcgg caaaccgcgc cgcaaaaacc cgcaggaagg cctgtataac 960
gaactgcgca aatggaaatt ttatcatcgc tatagcgaaa ttggcatgaa aggcgaacgc 1020
cgccgcggca aaggccatga tggcctgtat cagggcctga gcaccgcgac caaagatacc 1080
catatgcagg cgctgccgcc gcgc 1104
<210> 12
<211> 112
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 13
<211> 104
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
65 70 75 80
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
85 90 95
His Met Gln Ala Leu Pro Pro Arg
100
<210> 14
<211> 104
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Asp
20 25 30
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
35 40 45
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
50 55 60
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
65 70 75 80
Gly His Asp Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
85 90 95
His Met Gln Ala Leu Pro Pro Arg
100
<210> 15
<211> 104
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Asp
20 25 30
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
35 40 45
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
50 55 60
Glu Ala Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
65 70 75 80
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
85 90 95
His Met Gln Ala Leu Pro Pro Arg
100
Claims (7)
1.一种靶向BCMA的嵌合抗原受体，其特征在于，所述嵌合抗原受体包括信号肽、靶向BCMA的单域抗体、铰链区、跨膜区、共刺激因子和胞内信号域，所述靶向BCMA的单域抗体的互补决定区（CDR区）氨基酸序列依次为：SEQ ID NO:1所示CDR1，SEQ ID NO:2所示CDR2和SEQ ID NO:3所示CDR3，胞内信号域为修饰后的CD3ζ胞内信号域，其氨基酸序列如SEQ IDNO:5所示。
2.根据权利要求1所述的嵌合抗原受体，其特征在于，所述单域抗体的氨基酸序列如SEQ ID NO:4所示。
3.根据权利要求1所述的嵌合抗原受体，其特征在于，所述跨膜区为CD28跨膜区，其氨基酸序列如SEQ ID NO:6所示；所述铰链区其氨基酸序列如SEQ ID NO:7所示；所述共刺激因子选自CD28，其氨基酸序列如SEQ ID NO:8所示，所述信号肽氨基酸序列如SEQ ID NO:9所示。
4.根据权利要求1所述的嵌合抗原受体，其特征在于，其氨基酸序列如SEQ ID NO:10所示。
5.根据权利要求1所述的嵌合抗原受体，其特征在于，其核苷酸序列如SEQ ID NO:11所示。
6.一种嵌合抗原受体T细胞，其特征在于，所述嵌合抗原受体T细胞表达权利要求1-5任一项所述的嵌合抗原受体。
7.权利要求6中所述的嵌合抗原受体T细胞在制备抗肿瘤药物中的应用，其特征在于：所述的肿瘤为多发性骨髓瘤。
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