CN116376920A - Ceacam5靶向的car-t细胞的制备方法及应用 - Google Patents
Ceacam5靶向的car-t细胞的制备方法及应用 Download PDFInfo
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Abstract
本发明提供一种CEACAM5靶向的CAR‑T细胞的制备方法及应用。具体地，本发明提供了一种嵌合抗原受体（CAR），CAR的抗原结合结构域包括一个CEACAM5靶向的纳米抗体序列。本发明还提供了一种包含该CAR的CAR质粒、病毒载体、CAR‑T细胞，所述CAR质粒、病毒载体、CAR‑T细胞能够表达靶向CEACAM5的嵌合抗原受体。本发明的CAR‑T细胞可以用于CEACAM5阳性肿瘤的治疗。
Description
技术领域
本发明涉及生物技术和免疫治疗领域，更具体地说，涉及一种CEACAM5靶向的CAR-T细胞及其制备和应用。
背景技术
结直肠癌（Colorectal cancer, CRC）是最常见的恶性肿瘤之一，是全球癌症死亡的主要原因之一。有近25%的患者在首次就诊时就处于Ⅳ期状态，另有25-50%的患者虽然表现为早期疾病，但仍旧继续发展为转移性CRC。转移性CRC的预后仍然很差，美国和欧洲的5年中位生存率仅为18.5%。CRC的标准常规治疗方法是手术、化疗和放疗。嵌合抗原受体T（Chimeric antigen receptor T-cell, CAR-T）细胞是CRC治疗的新选择之一。CAR-T细胞由目标抗原特异性的抗体可变片段组成，这些片段融合到T细胞上，修饰的离体CAR-T细胞被重新注入患者体内并与靶抗原识别，实现到肿瘤部位的主动运输，并进一步体内扩增和长期持续存在，这种以主要组织相容性复合体非依赖性方式识别通过加CD28或4-1BB等共刺激结构域激活T细胞内部的信号通路，通过释放颗粒酶B、穿孔素、IL-2、IFN-γ等细胞因子以促进细胞毒性和肿瘤细胞凋亡。
实体瘤的CAR-T细胞疗法仍面临许多挑战。其中一个重要的问题是实体瘤中缺乏合适的CAR-T细胞靶抗原，在大多数人中，靶抗原也存在于健康组织中，导致非靶向毒性。癌胚抗原细胞粘附分子5（Carcinoembryonic antigen cell adhesion molecule 5,CEACAM5），也被称为CD66e或癌胚抗原（CEA），是属于CEA家族的高糖基化膜结合糖蛋白。CEACAM5长期以来一直被认为是一个有吸引力的结直肠癌靶点，因为它几乎只在结直肠肿瘤中以高抗原密度的形式高度表达，而在正常组织中表达有限。研究表明CEACAM5在超过80%的结直肠癌中表达。这使得CEACAM5成为针对CRC的新疗法的有希望的靶点。因此开发靶向CEACAM5的CAR-T细胞对于提高晚期CRC患者的免疫治疗效果具有巨大的应用前景。
CAR由细胞外区、铰链区、跨膜区和细胞内信号区四个组成部分构成。细胞外结构域具有灵活的剪接功能并决定抗原特异性，是识别肿瘤抗原的功能部件。CAR的靶向结构域主要基于通常是单克隆抗体的Fab或单链可变片段，因其紧凑的尺寸、高亲和力和特异性而被广泛使用。相较于传统的单链抗体片段，纳米抗体在开发各种形式的CAR-T方面具有独特的潜力。除了其天然的高结合能力外，纳米抗体在体内免疫原性、溶解度和稳定性方面也具有更有利的结构。纳米抗体由于缺乏导致机体产生免疫原性的接头，不会使机体产生免疫原性。通常来说，纳米抗体只需要对人源化过程进行微小的序列修正。另外，单链抗体片段会导致自发性活化诱导的CAR-T衰竭，这种自发性活化主要与构成CAR的单链抗体片段部分的框架区有关。这是CAR-T治疗实体瘤失败的重要原因之一。此外，纳米抗体避免了可变区和恒定区之间潜在的相互作用中断和疏水斑块的暴露。这种被破坏的相互作用和疏水残基可能严重影响溶解度和稳定性。基于这些优点，纳米抗体有望成为CAR-T设计中有潜力新选择。
基于以上背景，我们利用CEACAM5靶向纳米抗体的序列，研发了靶向CRC特异性靶点CEACAM5的CAR-T细胞，并在细胞层面和小鼠肿瘤模型中评估了其抗肿瘤活性。
发明内容
本发明提供一种靶向CEACAM5阳性细胞的CAR-T细胞的制备和应用，该CAR-T细胞能够表达靶向CEACAM5的嵌合抗原受体，特异性的识别和杀伤CEACAM5高表达的肿瘤细胞，适用于CRC的治疗。
具体地，本发明的内容包括：
第一方面，本发明提供一种靶向CEACAM5的纳米抗体的基因序列，具有的核苷酸序列如SEQ ID NO:2所示。
第二方面，本发明还提供一种CAR质粒，包括第一方面所述靶向CEACAM5的纳米抗体的基因序列（称为Nb41），能够表达靶向CEACAM5的嵌合抗原受体，该嵌合抗原受体包括靶向CEACAM5的信号肽、抗原结合结构域、跨膜结构域、胞内共刺激结构域和胞内结构域。
进一步地，所述信号肽选自CD8 leader的结构域，具的核苷酸序列如SEQ ID NO:1所示。
进一步地，所述跨膜结构域选自CD8 linker和CD8 TM结构域，具的核苷酸序列如SEQ ID NO:3所示。
进一步地，所述胞内共刺激结构域选自4-1BB的胞内共刺激结构域，具有的核苷酸序列如SEQ ID NO:4所示。
进一步地，所述胞内结构域选自CD3ζ的胞内结构域，具有的核苷酸序列如SEQ IDNO:5所示。
第三方面，本发明还提供一种CAR-T慢病毒，包括第二方面所述CAR载体。
第四方面，本发明还提供了一种CAR-T细胞，表达如第一方面所述的CAR基因，根据CEACAM5的抗原结合结构域和CEACAM5的特异性相互作用，设计了一种CAR-T细胞，该细胞能够靶向并杀灭CEACAM5高表达的肿瘤细胞，并用于CRC的免疫治疗，提高治疗效果，CRT细胞治疗后，小鼠的生存时间明显延长（观察至第24天）。
第五方面，本发明还提供了一种治疗结直肠癌的方法，包括给需要治疗的对象施用适量的本发明第二、三、四方面所述的质粒、病毒、细胞。
表1
| 结构域 | 序列 |
| CD8 leader | ATGGCCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCC |
| Nb41 | CAGTTGCAGCTCGTGGAGTCTGGTGGAGGCTTGGTGCAGGCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAAGCCTCTTCAGGATCAATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCAGCTATTACTAGTGCTGGTAGTACAAACTATGCAGATTTCGTGAAGGGCCGATTCACCATCTCCGCAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTACTGTAATACACCCTGGCCCGTAGGGAGGGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAACCA |
| CD8 linker & TM | ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC |
| 4-1BB | AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA |
| CD3ζ | AGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCCGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGAAGAGAGGAGTACGACGTGCTGGACAAGAGAAGAGGCAGAGACCCCGAGATGGGCGGCAAGCCCCAGAGAAGAAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGAGAAGAAGAGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCAGA |
附图说明
图1为本发明中CAR-T细胞表达质粒的构建。其中A为CAR-T细胞表达序列的图示；B为PCR检测表达片段及载体片段用高保真酶消化后线性化载体。
图2为本发明中CAR-T细胞的制备和鉴定。其中A为经过转染之后的CAR-T细胞的荧光及白光视野图片；B为流式细胞术检测经过转染之后的CAR-T细胞阳性细胞比。
图3为本发明中CAR-T细胞的细胞功能实验。其中A和B为B41-CAR-T细胞和Mock-CAR-T与LS174-T和HT-29共培养之后细胞杀伤效应图；C和D为不同肿瘤细胞与B41-CAR-T细胞和Mock-CAR-T细胞共培养之后细胞因子的分泌，****表示P < 0.0001。
图4为本发明中CAR-T细胞的体内肿瘤抑制实验。其中A为CAR-T细胞治疗皮下肿瘤小鼠在不同时间点肿瘤生物发光成像的图片；B为皮下肿瘤在不同时间点生物发光成像的荧光强度定量；C为皮下肿瘤在不同时间点测量得到的肿瘤体积变化曲线；D为不同组治疗肿瘤之后小鼠的生存曲线。
实施方式
以下实施例用于本发明，但不限制本发明的范围。若未特别指明，实施例均按照常规实验条件。
人结直肠癌细胞系（HT-29，LS174-T）和人T淋巴细胞瘤细胞（Jurkat）人胚胎肾细胞293（HEK293）细胞购买自ATCC。4周龄雌性Balb/c裸鼠购自广州市言诚生物科技有限公司。慢病毒包装质粒（pCDH-CMV、psPAX2、pMD2.G质粒）购买自美国Addgene公司。限制性核酸内切酶（BamHI、XbaI）购买自New England Biolabs。克隆相关试剂（Phanta® Max Super-Fidelity DNA Polymerase、ClonExpress® MultiS重组克隆试剂盒、胶回收试剂盒、质粒小提试剂盒等）购买自南京诺唯赞生物科技有限公司。ELISA试剂盒（IFN-γ、IL2）购买自Absin公司。Lipofectamine™ 3000转染试剂购买自Invitrogen公司。Opti-MEM减血清培养基购买自Thermo Fisher公司。增强型CCK-8试剂盒购买自碧云天生物技术公司。D-Luciferin荧光素钾盐购自PerkinElmer。荧光素酶报告基因慢病毒由Igenebio定制。
实施例1 基因表达载体的构建
首先按照基因序列合成基因片段，以合成片段为模板，利用引物设计软件CEDesign，输入插入片段及pCDH-CMV载体序列，生成引物，核对之后送公司合成引物，利用PCR反应合成插入基因片段，将反应产物经过凝胶电泳分离，把目的条带切胶回收基因片段。基因片段载体选择Xbal、BamHI限制性核酸内切酶将其环形载体线性化，将反应产物经过凝胶电泳分离，把目的条带切胶回收基因片段。配置重组反应体系将目的基因片段重组到线性化载体当中，将重组产物转化到DH5α感受态细胞中，涂布到平板上，过夜培养后，挑取其中5个克隆扩大培养之后送测序鉴定重组结果，测序结果正确的载体即为pCDH-Nb41-CD8α-4-1BB-CD3ζ（B41 CAR）载体。设计的对照载体（Mock-CAR）除不包含纳米抗体Nb41序列之外其他均与pCDH-Nb41-CD8α-4-1BB-CD3ζ载体相同。
我们选择pCDH-CMV作为慢病毒构建的载体。通过BamHI、XbaI双酶切可以把pCDH-CMV载体切成线性化载体，分子量为6232 bp，经过酶切之后的线性化载体的琼脂糖凝胶电泳的分子量大小符合预期（图1B，Lane 6-8）。B41-CAR序列由CD8信号肽、Nb41纳米抗体序列、跨膜区和4-1BB共刺激区、CD3ζ信号区构成（图1A）。Nb41为靶向CEACAM5的纳米抗体序列，用于识别肿瘤细胞表面的CEACAM5抗原。作为对照，设计了没有Nb41靶向序列的Mock-CAR（图1A）。通过模板进行PCR扩增产物大小都符合预期（B41-CAR 1900 bp，Mock-CAR由两个片段重组而成，分别为850 bp和680 bp，图1B）。线性化载体与目的基因片段酶连重组后得到部分阳性转化克隆，经测序正确之后继续扩增提取质粒得到CAR-T表达质粒。
实施例2 CAR-T细胞的构建
复苏HEK-293细胞，传代培养接种到10 cm培养皿，至观察细胞密度约50％时即可进行转染。按照7.5 μg psPAX质粒、2.5 μg pMD2.G质粒、7.5 μg B41-CAR或者Mock-CAR质粒的比例与375 μL opti-MEM和15 μL P3000配置DNA稀释液（A液），375 μL opti-MEM与15μL lipo3000配置lipo3000稀释液（B液），将A液、B液轻轻混匀静置之后缓缓滴加到HEK-293细胞的opti-MEM培养基中。转染后6 h换新鲜培液，48 h收集第一次的病毒上清，4 ℃保存。72 h收集第二次的病毒上清，4 ℃保存。然后两批病毒上清混合起来，3000 g离心10 min去除细胞碎片，0.45 mm滤器除菌病毒液加入到离心之后的Jurkat细胞中，转染Jurkat细胞后24 h更换细胞培养基，72 h之后可以在荧光显微镜下观察绿色荧光。用3 μg/mL嘌呤霉素筛选阳性表达的细胞，经过1-2周的抗性筛选之后，存活的细胞及为阳性表达的细胞，改用含1.5 μg/mL嘌呤霉素的培养基继续培养细胞。
通过脂质体将pxPAX2、pMD2.G和目的质粒转染HEK-293细胞，收获病毒液用于感染目的细胞，经过筛选之后获得细胞稳株。Jurkat细胞是一种永生的人类白血病T细胞系，广泛用于检测T细胞的激活和信号机制。在这里，我们在人类Jurkat T细胞中评估CAR-T体内外功能。如图2A所示，在荧光显微镜下，经过转染并筛选之后的Jurkat细胞能够观察到明显的eGFP绿色荧光，表明CAR-T细胞稳株的成功构建。
实施例3 流式细胞术检测Jurkat细胞中CAR的表达效率
收集Jurkat CAR-T细胞，1000 rpm离心3 min，弃去培养基，用PBS洗涤细胞，去除培养基中的酚红以免影响影响结果。PBS重悬细胞，用多色流式细胞仪分析，通道选择B525-FITC，结果用Cytexpert软件分析。
通过流式细胞术检测CAR的阳性表达效率，如图2B所示，CAR-T细胞阳性细胞比达到63.72 ± 3.47%。
实施例4 Jurkat CAR-T细胞的细胞杀伤实验
为了验证CAR-T细胞的功能，我们选择了CEACAM5高表达的LS174-T和HT-29肠癌细胞分别铺板于96孔板之上，每孔约5000个细胞，每组3个副孔。将Jurkat CAR-T细胞及为转染空质粒的Jurkat细胞计数，按照效应细胞与靶细胞按1:1、2:1、4:1、8:1、16:1的比例加入到96孔板内与CAR-T细胞进行共孵育。对贴壁细胞进行3次洗涤，加入含10 μL的CCK-8和90μL的DMEM培养液，37 ℃孵育2-4 h，使用酶标仪测量每个孔在450 nm处的吸光度，并使用公式归一化细胞活力。
结果显示，B41-CAR-T细胞可以特异性杀伤LS174-T和HT-29肿瘤细胞，而且这种细胞杀伤效果与细胞效靶比（Effector to target ratio）成正相关，而LS174-T和HT-29细胞与Mock-CAR-T细胞共孵育之后没有观察到明显的细胞死亡（图3A，B）。这些结果表明B41-CAR-T细胞对CEACAM5阳性肿瘤细胞系有很强的杀伤效率。
实施例5 ELISA检测CAR-T细胞分泌细胞因子的含量
将CAR-T细胞与靶细胞LS174-T和HT-29按照效靶比8:1的比例共培养24小时，收集共培养上清液，用ELISA的方法检测共培养过程中细胞因子的分泌。结果表明相较于Mock-CAR-T细胞，B41-CAR-T细胞中具有抗肿瘤活性的细胞因子IFN-γ、IL-2的分泌明显升高（P < 0.0001）（图3C，D）。这表明说明B41-CAR-T细胞对靶细胞的识别依赖于细胞表面CEACAM5的表达，通过细胞因子的分泌介导了细胞杀伤作用。
实施例6 CAR-T细胞体内治疗效果研究
为了验证CAR-T细胞体内治疗效果，通过慢病毒液转染CEACAM5阳性的LS174-T细胞构建LS174-T-luc细胞稳株。在小鼠右侧大腿根部接种5×106 抗性筛选后的LS174-T-luc细胞建立LS174-T-luc皮下肿瘤模型。一周后每天用卡尺测量肿瘤的大小，并使用以下等式计算肿瘤体积：肿瘤体积=（宽度）2×长度/2。当肿瘤体积长到约为200-300 mm3，将小鼠随机分为3组（n = 6）：CAR-T细胞组，Mock组，PBS组。通过尾静脉注射CAR-T细胞（1×107），注射时间点分别为第0天和第6天。记录小鼠的体重及肿瘤大小变化，通过生物发光成像（BLI）动态监测肿瘤大小。记录的时间点为第0、3、6、10、13、20、26、34天。向小鼠注射D-荧光素钾盐工作液并用异氟醚麻醉，然后对小鼠进行成像以检测荧光素酶信号，BLI信号由IVIS成像系统分析。小鼠在第35天被安乐死。同时在以下任何一种情况下，动物被视为达到实验终点并安乐死：肿瘤大小超过1.4 cm3；出现腹水；体重下降>20%；或有任何痛苦的身体征兆。
实验结果显示，B41-CAR-T细胞能够显著抑制LS174-T-luc皮下肿瘤的生长（图4A）。肿瘤荧光曲线和体积变化曲线表明B41-CAR-T细胞能够显著抑制肿瘤生长，并且Mock-CAR-T治疗组与PBS对照组治疗组之间并无显著差异（图4B，C）。生存曲线显示B41-CAR-T细胞治疗能够明显延长小鼠的生存时间（观察至第24天，图4D）。这些结果表明B41-CAR-T细胞有较强的体内肿瘤抑制效应。
Claims (5)
1.一种核苷酸序列，其特征在于，所述核苷酸序列编码CEACAM5靶向纳米抗体，所述核苷酸的结构域由CD8 leader、Nb41、CD8 linker & TM、4 1BB、CD3ζ组成。
2.一种CAR质粒载体，其特征在于，所述CAR质粒包括如权利要求1所述CEACAM5靶向纳米抗体核苷酸序列。
3.一种CAR-T病毒载体，其特征在于，包括如权利要求2所述CAR质粒载体。
4.一种CAR-T细胞，其特征在于，表达如权利要求1所述的CEACAM5靶向纳米抗体。
5.权利要求1-4任一项所述的核苷酸序列、质粒、病毒载体、CAR-T细胞在制备用于治疗肿瘤的药物中的应用，所述肿瘤为CEACAM5阳性。
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