技术领域technical field
本发明涉及基因工程、免疫学和肿瘤学领域，尤其是涉及一种利用CRISPR/Cas9技术敲除人PD-1基因构建可靶向MSLN(Mesothelin，间皮素)新型CAR-T细胞的方法及其应用。The present invention relates to the fields of genetic engineering, immunology and oncology, in particular to a method for knocking out the human PD-1 gene using CRISPR/Cas9 technology to construct a new type of CAR-T cell that can target MSLN (Mesothelin, mesothelin) and its application.
背景技术Background technique
随着工业化进程不断深入，大气污染、环境问题、食品安全以及人们生活方式的改变，促使癌症成为本世纪威胁人类健康的头号杀手和社会发展的主要障碍。癌症的免疫细胞生物治疗技术是一种新兴的、具有显著疗效的肿瘤治疗模式，它是运用基因工程技术对从病人体内采集的免疫细胞进行体外修饰、培养和扩增，再回输到病人体内，来激发和增强机体自身免疫功能，从而达到抑制肿瘤生长和治疗肿瘤的目的。细胞免疫治疗是继手术、放疗和化疗之后的第四大肿瘤治疗技术。在诸多癌症免疫细胞治疗方法中，近两年脱颖而出的是嵌合抗原受体(Chimeric antigen receptor,CAR)T细胞肿瘤靶向治疗(即CAR-T细胞治疗)。With the deepening of industrialization, air pollution, environmental problems, food safety and changes in people's lifestyles, cancer has become the number one killer threatening human health and the main obstacle to social development in this century. Immune cell biotherapy technology for cancer is an emerging tumor treatment model with significant curative effect. It uses genetic engineering technology to modify, culture and expand immune cells collected from patients in vitro, and then reinfuse them into patients. , to stimulate and enhance the body's own immune function, so as to achieve the purpose of inhibiting tumor growth and treating tumors. Cellular immunotherapy is the fourth largest tumor treatment technology after surgery, radiotherapy and chemotherapy. Among the many cancer immune cell therapy methods, the chimeric antigen receptor (Chimeric antigen receptor, CAR) T cell tumor targeting therapy (CAR-T cell therapy) has stood out in the past two years.
目前，CAR-T细胞免疫疗法在欧美及亚洲各国都正积极开展临床试验，其中美国开展临床试验项目最多，宾夕法尼亚大学医院、费城儿童医院、美国癌症研究院(NCI)、FredHutchinson癌症研究中心、纪念斯隆凯特琳癌症中心和西雅图儿童医院等是最早开展CAR-T细胞疗法的研究机构。传统国际制药巨头如诺华、葛兰素史克、辉瑞、强生等，以及新兴国际生物制药公司如Juno、Kite、Celgene、Cellectis、Bluebird都投入了大量资金和人力到CAR-T疗法的研发中，推动这一极具前景的疗法尽早进入到临床，从而造福癌症患者(Aservice of the U.S.National Institute of Health.http://clinicaltrials.gov)。At present, CAR-T cell immunotherapy is actively carrying out clinical trials in Europe, America and Asian countries, among which the United States has the most clinical trial projects, University of Pennsylvania Hospital, Children's Hospital of Philadelphia, National Cancer Institute (NCI), Fred Hutchinson Cancer Research Center, Memorial Sloan Kettering Cancer Center and Seattle Children's Hospital were the first research institutions to carry out CAR-T cell therapy. Traditional international pharmaceutical giants such as Novartis, GlaxoSmithKline, Pfizer, Johnson & Johnson, etc., and emerging international biopharmaceutical companies such as Juno, Kite, Celgene, Cellectis, and Bluebird have invested a lot of money and manpower in the research and development of CAR-T therapy, promoting This promising therapy enters the clinic as soon as possible to benefit cancer patients (A service of the U.S. National Institute of Health. http://clinicaltrials.gov).
CAR-T细胞疗法在晚期难治性白血病和淋巴瘤患者中进行的早期临床试验已经显示出非常振奋人心的结果。宾夕法尼亚大学研究人员在世界顶尖医学杂志《新英格兰医学杂志》上发表的一项临床试验结果显示，在接受了CTL019输注(靶向CD19抗原的CAR-T疗法)的30名复发性或难治性ALL受试者中，治疗后1个月有27名患者获得完全缓解，其中甚至包括15位已经接受过骨髓干细胞移植的患者。6个月时无反复生存率为67％(20人)，总体生存率为78％(23人)。另外还有1名患者2年随访时仍然持续完全缓解(Grupp SA et al,N EnglJ Med 2013,368(16):1509-18)。Early clinical trials of CAR-T cell therapy in patients with advanced refractory leukemia and lymphoma have shown very encouraging results. The results of a clinical trial published by researchers at the University of Pennsylvania in the New England Journal of Medicine, the world's top medical journal, showed that 30 relapsed or refractory patients who received CTL019 infusion (a CAR-T therapy targeting CD19 antigen) Among subjects with ALL, 27 patients achieved complete remission 1 month after treatment, including 15 patients who had received bone marrow stem cell transplantation. At 6 months, the recurrence-free survival rate was 67% (20 patients), and the overall survival rate was 78% (23 patients). Another patient remained in complete remission at the 2-year follow-up (Grupp SA et al, N EnglJ Med 2013,368(16):1509-18).
NCI开展的一项临床试验也展现了良好的结果，该试验入组了20名ALL患儿，在接受CAR-T细胞疗法后，14名达到了完全缓解；12名患儿的骨髓中已检测不到未成熟的白血病细胞，其中10名又接受了干细胞移植，至今无癌生存。研究结果还表明，CAR-T细胞疗法可帮助化疗无应答的患者顺利地过度到骨髓移植治疗。纪念斯隆凯特林癌症中心与NCI在成人患者中开展的1期临床试验也取得了类似的结果(Mazzarella L.2013 American Societyof Haematology meeting[J]2014,8:390)。A clinical trial conducted by NCI also showed good results. The trial enrolled 20 children with ALL, and after receiving CAR-T cell therapy, 14 achieved complete remission; Less than immature leukemia cells, 10 of them received stem cell transplantation, so far cancer-free survival. The findings also suggest that CAR-T cell therapy can help patients who do not respond to chemotherapy transition smoothly to bone marrow transplantation. The Phase 1 clinical trial conducted by Memorial Sloan Kettering Cancer Center and NCI in adult patients also achieved similar results (Mazzarella L. 2013 American Society of Haematology meeting[J]2014,8:390).
除白血病之外，CAR-T疗法对淋巴瘤也显示了喜人的疗效，NCI领导的CAR-T细胞临床试验招募了15名成人受试者，其中的绝大多数(9名)都患有晚期弥漫性大B细胞淋巴瘤(DLBCL)。据Kochenderfer博士及NCI的同事报道，该临床试验的大多数患者都达到了缓解，在7例可评估的DLBCL受试者中4例获得完全缓解，持续时间从9个月到22个月(Kochenderfer JN et al,Blood 2012,119(12):2709-20)。In addition to leukemia, CAR-T therapy has also shown promising effects on lymphoma. The NCI-led CAR-T cell clinical trial recruited 15 adult subjects, most of whom (9) had advanced stage Diffuse large B-cell lymphoma (DLBCL). According to Dr. Kochenderfer and colleagues at the NCI, the majority of patients in this clinical trial achieved remission, with 4 of 7 evaluable DLBCL subjects achieving complete remission, with durations ranging from 9 months to 22 months (Kochenderfer JN et al, Blood 2012, 119(12):2709-20).
虽然针对白血病和淋巴瘤的CAR-T细胞临床试验的疗效可以用革命性来形容，这也是投资者对这个技术充满希望的原因，但90％的癌症是实体瘤，所以实体瘤疗效是这个技术的一个关键门槛。最近，制药巨头诺华公司和美国宾州大学的初步研究表明，CAR-T疗法的安全性和有效性在实体瘤方面似乎同样出众。这一结果也将市场更广阔的实体瘤治疗领域展现在CAR-T疗法面前。在诺华-宾大此次进行的临床一期研究中，研究人员使分离获得的T细胞识别一种名为间皮素(mesothelin，MSLN)的蛋白，这种蛋白在多种实体瘤中都为高表达。研究人员在五名患者身上测试了这种名为CART-meso的新疗法，其中两人患有卵巢癌、两人患有上皮间质瘤(一种肺癌)、另有一人患有胰腺癌。这五名患者此前接受过多种疗法，均告无效。在此次临床一期研究中，尽管五位受试患者在接受治疗时出现了败血症、气短以及白细胞水平升高等副作用，但研究人员并未观察到重大的副作用事件发生。而直到患者接受T细胞回输后的第28天，患者体内仍然可以检测到这种修饰后T细胞的存在，同时还有迹象显示这些T细胞已经开始向肿瘤部位富集。负责这一研究的宾大教授Dr.JanosTanyi表示这些结果表明，对于实体瘤来说CAR-T疗法的安全性是有保证的。另一方面，影像学手段显示其中一名患者体内的实体瘤体积出现萎缩的迹象，而另外一名肺癌患者肺部的恶性细胞出现减少的迹象。这与之前CAR-T疗法在白血病等非实体瘤癌症方面的治疗效果有段距离。这一早期研究暗示，CAR-T疗法在实体瘤治疗方面或许还有更长的路要走(2015Workshop of Cancer and Immunotherapeutics)。更多种类的实体瘤以及更多的肿瘤表面特异性靶点抗原有待进一步确认。Although the efficacy of CAR-T cell clinical trials for leukemia and lymphoma can be described as revolutionary, which is why investors are full of hope for this technology, but 90% of cancers are solid tumors, so the efficacy of solid tumors is the key to this technology a key threshold. More recently, preliminary studies by pharmaceutical giant Novartis and the University of Pennsylvania have shown that the safety and efficacy of CAR-T therapy appears to be equally superior in solid tumors. This result also presents the broader solid tumor treatment field in front of CAR-T therapy. In the phase I clinical study conducted by Novartis-Penn University, the researchers made the isolated T cells recognize a protein called mesothelin (MSLN), which is present in many solid tumors. High expression. The researchers tested the new therapy, called CART-meso, on five patients, two with ovarian cancer, two with epithelial stromal tumor (a type of lung cancer), and one with pancreatic cancer. The five patients had previously received a variety of therapies, all of which failed. In this phase I clinical study, although five test patients experienced side effects such as sepsis, shortness of breath and elevated white blood cell levels during treatment, the researchers did not observe major side effects. And until the 28th day after the patient received T cell reinfusion, the presence of this modified T cell could still be detected in the patient's body, and there were signs that these T cells had begun to enrich the tumor site. Dr. Janos Tanyi, a professor at the University of Pennsylvania who is in charge of this research, said that these results show that the safety of CAR-T therapy for solid tumors is guaranteed. On the other hand, imaging methods showed signs of shrinkage in the volume of solid tumors in one of the patients, and signs of reduction in malignant cells in the lungs of the other lung cancer patient. This is a distance from the previous CAR-T therapy in the treatment of leukemia and other non-solid tumor cancers. This early study suggests that CAR-T therapy may have a longer way to go in the treatment of solid tumors (2015Workshop of Cancer and Immunotherapeutics). More types of solid tumors and more tumor surface-specific target antigens need to be further confirmed.
此外，癌细胞在长期的进化过程中也发展出了一套自身的逃逸机制，可以躲过T细胞的摧毁，进而肆无忌惮地扩张。研究人员发现T细胞表面有一个重要的“接头”分子，他们把它称为“程序性死亡受体-1”或PD1(Programmed Death 1)，正常状态下，它都能明辨“敌我”，但当癌细胞表面出现“PD-L1”(Programmed Death Like 1)的时候，情况就变了。PD-L1蛋白正是PD1的配体，它们俩一旦结合便会向T细胞传递一种负向调控信号，诱导T细胞进入静息状态，让其无法识别癌细胞，并且使T细胞自身增殖减少或凋亡，有效解除机体的免疫反应，因此癌细胞可以毫不费力就“登堂入室”。值得一提的是，许多癌细胞表面都存在这种PD-L1蛋白，包括乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌、膀胱癌、胰腺癌、神经胶质瘤、黑色素瘤等。于是，科学家们开始思索如何能阻止PD1与PD-L1的结合，帮助T细胞恢复活性，正常识别癌细胞的威胁，进而全力攻击癌细胞。因此各大国际制药巨头纷纷致力于PD-1抑制剂和抗体药物的临床研究和开发。基于PD-1分子在肿瘤免疫逃逸过程中的重要作用，我们利用最新的基因组编辑技术CRISPR/Cas9，在表达靶向MSLN CAR分子的T细胞中将PD-1基因敲除，阻断癌细胞对T细胞的逃逸和抑制作用，使得CAR-T细胞能更加高效地识别和攻击癌细胞。In addition, cancer cells have also developed their own escape mechanism during the long-term evolution process, which can avoid the destruction of T cells and expand unscrupulously. The researchers discovered that there is an important "linker" molecule on the surface of T cells, which they call "programmed death receptor-1" or PD1 (Programmed Death 1). Under normal conditions, it can distinguish between "enemy and enemy". But when "PD-L1" (Programmed Death Like 1) appeared on the surface of cancer cells, the situation changed. PD-L1 protein is the ligand of PD1. Once they combine, they will send a negative regulatory signal to T cells, induce T cells to enter a resting state, make them unable to recognize cancer cells, and reduce the proliferation of T cells themselves. Or apoptosis, which effectively relieves the body's immune response, so cancer cells can "enter the room" without any effort. It is worth mentioning that this PD-L1 protein exists on the surface of many cancer cells, including breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, neuroglial cancer, etc. tumors, melanoma, etc. Therefore, scientists began to think about how to prevent the combination of PD1 and PD-L1, help T cells to restore their activity, recognize the threat of cancer cells normally, and then attack cancer cells with all their strength. Therefore, major international pharmaceutical giants are devoting themselves to the clinical research and development of PD-1 inhibitors and antibody drugs. Based on the important role of PD-1 molecules in the process of tumor immune escape, we used the latest genome editing technology CRISPR/Cas9 to knock out the PD-1 gene in T cells expressing MSLN-targeted CAR molecules, blocking cancer cells’ ability to The escape and inhibition of T cells enable CAR-T cells to recognize and attack cancer cells more efficiently.
发明内容Contents of the invention
本发明的第一个目的在于提供一种利用CRISPR/Cas9技术敲除人PD-1基因构建可靶向MSLN新型CAR-T细胞的方法。The first object of the present invention is to provide a method for using CRISPR/Cas9 technology to knock out the human PD-1 gene to construct a new type of CAR-T cells that can target MSLN.
本发明的第二个目的在于提供两种新型CAR-T细胞。The second object of the present invention is to provide two novel CAR-T cells.
本发明的第三个目的在于提供上述新型CAR-T细胞的应用。The third object of the present invention is to provide the application of the above-mentioned novel CAR-T cells.
为实现上述第一个目的，本发明采用以下内容：For realizing above-mentioned first object, the present invention adopts following content:
一种利用CRISPR/Cas9技术敲除人PD-1基因构建可靶向MSLN新型CAR-T细胞的方法，该方法利用CRISPR/Cas9技术通过使用携带人PD-1基因sgRNA和Cas9-HF核酸酶的病毒液同步感染CAR-T细胞来敲除人PD-1基因，得到可靶向MSLN新型CAR-T细胞；其中，人PD-1基因的mRNA序列由SEQ ID No.46至SEQ ID No.50所示的人PD-1基因外显子组成，靶向sgRNA序列由SEQ ID No.51至SEQ ID No.56所示的人PD-1基因靶向sgRNA序列PD-1-Tn组成；Cas9-HF核酸酶由序列表SEQ ID No.57至SEQ ID No.60所示的Cas9-HF-NLS-FLAG核酸酶序列组成。A method of using CRISPR/Cas9 technology to knock out the human PD-1 gene to construct a new type of CAR-T cell that can target MSLN. The method utilizes the CRISPR/Cas9 technology to carry human PD-1 gene sgRNA and Cas9-HF nuclease Virus fluid infects CAR-T cells synchronously to knock out the human PD-1 gene to obtain a new type of CAR-T cells that can target MSLN; wherein, the mRNA sequence of the human PD-1 gene ranges from SEQ ID No.46 to SEQ ID No.50 The shown human PD-1 gene exon composition, the targeting sgRNA sequence is composed of the human PD-1 gene targeting sgRNA sequence PD-1-Tn shown in SEQ ID No.51 to SEQ ID No.56; Cas9- The HF nuclease consists of the Cas9-HF-NLS-FLAG nuclease sequence shown in SEQ ID No.57 to SEQ ID No.60 in the sequence table.
进一步地，所述方法包括以下步骤：Further, the method includes the following steps:
1)构建携带人PD-1基因sgRNA寡聚核苷酸病毒载体1) Construction of viral vectors carrying human PD-1 gene sgRNA oligonucleotides
所述sgRNA在PD-1基因上的靶序列符合5’-N(20)NGG的序列排列规则，且所述sgRNA在PD-1基因上的靶序列位于基因的外显子；所述sgRNA在PD-1基因上的靶序列是唯一的，并且所述sgRNA在PD-1上的靶位点序列由SEQ ID No.51至SEQ ID No.56所示的人PD-1基因靶向sgRNA序列PD-1-Tn组成；合成sgRNA寡聚核苷酸双链，并退火，之后与线性化的慢病毒载体连接，获得携带PD-1基因sgRNA寡聚核苷酸病毒载体；The target sequence of the sgRNA on the PD-1 gene conforms to the sequence arrangement rule of 5'-N(20)NGG, and the target sequence of the sgRNA on the PD-1 gene is located in the exon of the gene; the sgRNA is in the The target sequence on the PD-1 gene is unique, and the target site sequence of the sgRNA on PD-1 is the human PD-1 gene targeting sgRNA sequence shown in SEQ ID No.51 to SEQ ID No.56 Composition of PD-1-Tn; synthesize sgRNA oligonucleotide double strands, anneal, and then connect with linearized lentiviral vector to obtain sgRNA oligonucleotide viral vector carrying PD-1 gene;
2)构建携带Cas9-HF核酸酶病毒载体2) Construction of viral vector carrying Cas9-HF nuclease
在Cas9核酸酶氨基酸序列上依次进行1-4个位点的突变，包括N497A、R661A、Q695A和Q926A，得到高保真Cas9-HF核酸酶，之后将高保真Cas9-HF克隆进慢病毒载体，获得Cas9-HF核酸酶病毒载体；On the amino acid sequence of Cas9 nuclease, mutate 1-4 sites sequentially, including N497A, R661A, Q695A and Q926A, to obtain high-fidelity Cas9-HF nuclease, and then clone high-fidelity Cas9-HF into lentiviral vector to obtain Cas9-HF nuclease viral vector;
3)转导T细胞3) Transduce T cells
按照MOI＝1-10的值将携带PD-1基因sgRNA和Cas9-HF核酸酶的病毒液同步感染CAR-T细胞，完成敲除人PD-1基因，获得敲除PD-1基因以及表达靶向MSLN CAR分子的T细胞，即新型CAR-T细胞。According to the value of MOI=1-10, the viral liquid carrying PD-1 gene sgRNA and Cas9-HF nuclease was synchronously infected into CAR-T cells to complete the knockout of human PD-1 gene, and obtain the knockout of PD-1 gene and expression target T cells directed to MSLN CAR molecules, that is, new CAR-T cells.
为实现上述第二个目的，本发明采用以下内容：To achieve the above-mentioned second purpose, the present invention adopts the following content:
一种新型CAR-T细胞，该T细胞中PD-1基因被敲除并且表达靶向MSLN的CAR分子，该CAR分子由序列表SEQ ID No.1至SEQ ID No.22所示的序列组成；A novel CAR-T cell, in which the PD-1 gene is knocked out and expresses a CAR molecule targeting MSLN, the CAR molecule consists of the sequences shown in the sequence table SEQ ID No.1 to SEQ ID No.22 ;
其中，SEQ ID No.1至SEQ ID No.2为引导序列；Wherein, SEQ ID No.1 to SEQ ID No.2 are guide sequences;
SEQ ID No.3至SEQ ID No.12为scFv序列；SEQ ID No.3 to SEQ ID No.12 are scFv sequences;
SEQ ID No.13至SEQ ID No.14为CD8铰链区；SEQ ID No.13 to SEQ ID No.14 are CD8 hinge regions;
SEQ ID No.15至SEQ ID No.16为CD8跨膜区；SEQ ID No.15 to SEQ ID No.16 are CD8 transmembrane regions;
SEQ ID No.17至SEQ ID No.18为4-1BB胞内结构域；SEQ ID No.17 to SEQ ID No.18 are 4-1BB intracellular domains;
SEQ ID No.19至SEQ ID No.22为CD3ζ结构域。SEQ ID No.19 to SEQ ID No.22 are CD3ζ domains.
将所述CAR分子克隆进慢病毒载体，构建单一编码框的全长CAR序列表达框，利用EF1alpha启动子(序列表SEQ ID No.45)进行表达。The CAR molecule was cloned into a lentiviral vector to construct a full-length CAR sequence expression frame with a single coding frame, and expressed using the EF1alpha promoter (SEQ ID No. 45 in the sequence table).
一种新型CAR-T细胞，该T细胞中PD-1基因被敲除并且表达靶向MSLN的CAR分子，该CAR分子由序列表SEQ ID No.23至SEQ ID No.44所示的序列组成；A novel CAR-T cell, in which the PD-1 gene is knocked out and expresses a CAR molecule targeting MSLN, the CAR molecule consists of the sequences shown in the sequence table SEQ ID No.23 to SEQ ID No.44 ;
其中，SEQ ID No.23至SEQ ID No.24为引导序列；Wherein, SEQ ID No.23 to SEQ ID No.24 are guide sequences;
SEQ ID No.25至SEQ ID No.34为scFv序列；SEQ ID No.25 to SEQ ID No.34 are scFv sequences;
SEQ ID No.35至SEQ ID No.36为CD8铰链区；SEQ ID No.35 to SEQ ID No.36 are CD8 hinge regions;
SEQ ID No.37至SEQ ID No.38为CD8跨膜区；SEQ ID No.37 to SEQ ID No.38 are CD8 transmembrane regions;
SEQ ID No.39至SEQ ID No.40为4-1BB胞内结构域；SEQ ID No.39 to SEQ ID No.40 are 4-1BB intracellular domains;
SEQ ID No.41至SEQ ID No.44为CD3ζ结构域。SEQ ID No.41 to SEQ ID No.44 are CD3ζ domains.
将所述CAR分子克隆进慢病毒载体，构建单一编码框的全长CAR序列表达框，利用EF1alpha启动子(序列表SEQ ID No.45)进行表达。The CAR molecule was cloned into a lentiviral vector to construct a full-length CAR sequence expression frame with a single coding frame, and expressed using the EF1alpha promoter (SEQ ID No. 45 in the sequence table).
为实现上述第三个目的，本发明还公开了上述可靶向MSLN新型CAR-T细胞在制备用于治疗实体肿瘤的制剂中的应用，所述肿瘤为MSLN阳性肿瘤细胞。In order to achieve the above third objective, the present invention also discloses the application of the above-mentioned novel CAR-T cells that can target MSLN in the preparation of preparations for treating solid tumors, and the tumors are MSLN-positive tumor cells.
上述制备方法可以用来制备靶向ALK、CD171、CDK4/6、CEA、CLAN18(Claudin18)、CTLA-4、EGFR/EGFRvⅢ、GD2、GPC-3(Glypican-3)、FAP、GPC3、MET、MUC16、MUC1、HER2、IL-13、PD-1/PD-L1、PSMA、RANKL或VEGF/VEGFR/VEGFR2的新型CAR-T细胞。The above preparation method can be used to prepare targeting ALK, CD171, CDK4/6, CEA, CLAN18 (Claudin18), CTLA-4, EGFR/EGFRvⅢ, GD2, GPC-3 (Glypican-3), FAP, GPC3, MET, MUC16 , MUC1, HER2, IL-13, PD-1/PD-L1, PSMA, RANKL or VEGF/VEGFR/VEGFR2 novel CAR-T cells.
本发明具有以下优点：The present invention has the following advantages:
本发明应用CRISPR/Cas9技术将CAR-T细胞中的人PD-1基因敲除，阻断肿瘤细胞对T细胞的逃逸和抑制作用，得到可靶向MSLN新型CAR-T细胞，该新型CAR-T细胞能更有效地靶向攻击肿瘤细胞，可用来制备用于治疗实体肿瘤的制剂，尤其是表达MSLN阳性肿瘤细胞的制剂。本发明的制备方法步骤简单、获得的CAR-T细胞对肿瘤细胞的杀伤率高。The present invention uses CRISPR/Cas9 technology to knock out the human PD-1 gene in CAR-T cells, block the escape and inhibition of tumor cells on T cells, and obtain a new type of CAR-T cells that can target MSLN. T cells can target and attack tumor cells more effectively, and can be used to prepare preparations for treating solid tumors, especially preparations expressing MSLN-positive tumor cells. The preparation method of the present invention has simple steps, and the obtained CAR-T cells have a high rate of killing tumor cells.
附图说明Description of drawings
下面结合附图对本发明的具体实施方式作进一步详细的说明。The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings.
图1是KD-020 CAR分子的表达。Figure 1 is the expression of KD-020 CAR molecules.
图2是KD-021 CAR分子的表达。Figure 2 is the expression of KD-021 CAR molecules.
图3是Cas9-HF核酸酶及PD-1蛋白的表达。Figure 3 is the expression of Cas9-HF nuclease and PD-1 protein.
图4是KD-020 CAR-T细胞对Ovcar3细胞的杀伤率。Figure 4 shows the killing rate of Ovcar3 cells by KD-020 CAR-T cells.
图5是KD-021 CAR-T细胞对BxPC-3细胞的杀伤率。Figure 5 shows the killing rate of KD-021 CAR-T cells on BxPC-3 cells.
具体实施方式detailed description
为了更清楚地说明本发明，下面结合优选实施例对本发明做进一步的说明。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。In order to illustrate the present invention more clearly, the present invention will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not limit the protection scope of the present invention.
实施例Example
一种利用CRISPR/Cas9特异性敲除人PD-1基因构建靶向MSLN的新型CAR-T细胞的方法，包括以下步骤：A method for constructing a novel CAR-T cell targeting MSLN by using CRISPR/Cas9 to specifically knock out the human PD-1 gene, comprising the following steps:
1)携带PD-1基因sgRNA寡聚核苷酸病毒载体的构建1) Construction of viral vectors carrying PD-1 gene sgRNA oligonucleotides
sgRNA在PD-1基因上的靶序列符合5’-N(20)NGG的序列排列规则，且sgRNA在PD-1基因上的靶序列位于基因的外显子；sgRNA在PD-1基因上的靶序列是唯一的，并且sgRNA在PD-1上的靶位点序列由SEQ ID No.51至SEQ ID No.56所示的人PD-1基因靶向sgRNA序列PD-1-Tn组成；The target sequence of the sgRNA on the PD-1 gene conforms to the sequence arrangement rules of 5'-N(20)NGG, and the target sequence of the sgRNA on the PD-1 gene is located in the exon of the gene; the target sequence of the sgRNA on the PD-1 gene The target sequence is unique, and the target site sequence of the sgRNA on PD-1 consists of the human PD-1 gene targeting sgRNA sequence PD-1-Tn shown in SEQ ID No.51 to SEQ ID No.56;
人PD-1基因的mRNA序列由SEQ ID No.46至SEQ ID No.50所示的人PD-1基因外显子组成；The mRNA sequence of the human PD-1 gene consists of human PD-1 gene exons shown in SEQ ID No.46 to SEQ ID No.50;
合成sgRNA寡聚核苷酸双链(生工生物工程(上海)股份有限公司合成)，混合后于95℃退火5分钟，之后与线性化的慢病毒载体lentiGuide-Puro连接，获得携带PD-1基因sgRNA寡聚核苷酸病毒载体；Synthesize sgRNA oligonucleotide double strands (synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.), anneal at 95°C for 5 minutes after mixing, and then connect with linearized lentiGuide-Puro to obtain PD-1-carrying Gene sgRNA oligonucleotide viral vector;
2)携带Cas9-HF核酸酶病毒载体的构建2) Construction of viral vector carrying Cas9-HF nuclease
在Cas9核酸酶氨基酸序列上依次进行1-4个位点的突变，包括N497A、R661A、Q695A和Q926A，得到高保真Cas9-HF核酸酶，由序列表SEQ ID No.57至SEQ ID No.60所示的Cas9-HF-NLS-FLAG核酸酶序列组成，之后将高保真Cas9-HF克隆进慢病毒载体lentiCas9-Blast，获得Cas9-HF核酸酶病毒载体；On the amino acid sequence of Cas9 nuclease, mutations of 1-4 sites are sequentially carried out, including N497A, R661A, Q695A and Q926A, to obtain high-fidelity Cas9-HF nuclease, from SEQ ID No.57 to SEQ ID No.60 in the sequence table The Cas9-HF-NLS-FLAG nuclease sequence composition shown, and then the high-fidelity Cas9-HF is cloned into the lentiCas9-Blast lentiviral vector to obtain the Cas9-HF nuclease viral vector;
3)转导T细胞3) Transduce T cells
按照MOI＝1-10的值将携带PD-1基因sgRNA和Cas9-HF核酸酶的病毒液同步感染靶向MSLN的CAR-T细胞(记为KD-020 CAR-T细胞和KD-021 CAR-T细胞)，完成敲除人PD-1基因，获得敲除PD-1基因以及表达靶向MSLN的CAR分子T细胞，即得到敲除基因后的新型CAR-T细胞。According to the value of MOI=1-10, the virus fluid carrying PD-1 gene sgRNA and Cas9-HF nuclease was synchronously infected with CAR-T cells targeting MSLN (referred to as KD-020 CAR-T cells and KD-021 CAR-T cells and KD-021 CAR-T cells). T cells), complete the knockout of the human PD-1 gene, obtain the knockout of the PD-1 gene and the expression of CAR molecule T cells targeting MSLN, that is, obtain the new type of CAR-T cells after the knockout gene.
两种特异CAR分子，分别记为KD-020 CAR分子和KD-021 CAR分子。其中，KD-020CAR分子由序列表SEQ ID No.1至SEQ ID No.22所示的序列组成；其中，SEQ ID No.1至SEQ IDNo.2为引导序列；SEQ ID No.3至SEQ ID No.12为scFv序列；SEQ ID No.13至SEQ ID No.14为CD8铰链区；SEQ ID No.15至SEQ ID No.16为CD8跨膜区；SEQ ID No.17至SEQ ID No.18为4-1BB胞内结构域；SEQ ID No.19至SEQ ID No.22为CD3ζ结构域。KD-021CAR分子由序列表SEQ ID No.23至SEQ ID No.44所示的序列组成；其中，SEQ ID No.23至SEQ ID No.24为引导序列；SEQ ID No.25至SEQ ID No.34为scFv序列；SEQ ID No.35至SEQ ID No.36为CD8铰链区；SEQ ID No.37至SEQ ID No.38为CD8跨膜区；SEQ ID No.39至SEQ ID No.40为4-1BB胞内结构域；SEQ ID No.41至SEQ ID No.44为CD3ζ结构域。将CAR分子克隆进慢病毒载体，构建单一编码框的全长CAR序列表达框，利用EF1 alpha启动子(序列表SEQ ID No.45)进行表达。Two kinds of specific CAR molecules are respectively denoted as KD-020 CAR molecule and KD-021 CAR molecule. Among them, the KD-020CAR molecule is composed of the sequences shown in the sequence table SEQ ID No.1 to SEQ ID No.22; wherein, SEQ ID No.1 to SEQ ID No.2 is the guide sequence; SEQ ID No.3 to SEQ ID No.12 is the scFv sequence; SEQ ID No.13 to SEQ ID No.14 is the CD8 hinge region; SEQ ID No.15 to SEQ ID No.16 is the CD8 transmembrane region; SEQ ID No.17 to SEQ ID No. 18 is the 4-1BB intracellular domain; SEQ ID No.19 to SEQ ID No.22 are CD3ζ domains. The KD-021CAR molecule consists of the sequences shown in the sequence table from SEQ ID No.23 to SEQ ID No.44; wherein, SEQ ID No.23 to SEQ ID No.24 are guide sequences; SEQ ID No.25 to SEQ ID No .34 is the scFv sequence; SEQ ID No.35 to SEQ ID No.36 is the CD8 hinge region; SEQ ID No.37 to SEQ ID No.38 is the CD8 transmembrane region; SEQ ID No.39 to SEQ ID No.40 It is a 4-1BB intracellular domain; SEQ ID No.41 to SEQ ID No.44 are CD3ζ domains. The CAR molecule was cloned into a lentiviral vector to construct a full-length CAR sequence expression frame with a single coding frame, and expressed using the EF1 alpha promoter (SEQ ID No.45 in the sequence table).
对获得的KD-020 CAR-T细胞和KD-021 CAR-T细胞进行体外活性分析In vitro activity analysis of the obtained KD-020 CAR-T cells and KD-021 CAR-T cells
1)T细胞分离培养1) T cell isolation and culture
通过密度梯度离心分离新鲜的外周血单核细胞(PBMC,peripheral bloodmononuclear cell)；利用偶联anti-CD3和anti-CD28抗体的paramagnetic beads(Dynabeads ClinExVivo CD3/CD28,Invitrogen,Camarillo,CA,USA)富集CD3+细胞，磁珠与细胞比例为3：1；细胞稀释到TNC(total nucleated cell)浓度为(10-30)×106/mL，在培养皿中与磁珠在室温共孵育2-3小时；利用Magnetic particles concentrator(MPC)(Invitrogen)富集CD3+细胞；含有CD3+的细胞在培养皿中重悬于培养液(OpTmizerTM CTSTMT-Cell Expansion SFM,Life Technologies)中，终浓度为1×106cells/mL。在37℃、5％CO2培养箱中培养2天。Fresh peripheral blood mononuclear cells (PBMC, peripheral bloodmononuclear cell) were isolated by density gradient centrifugation; paramagnetic beads (Dynabeads ClinExVivo CD3/CD28, Invitrogen, Camarillo, CA, USA) coupled with anti-CD3 and anti-CD28 antibodies were used to enrich Collect CD3+ cells, the ratio of magnetic beads to cells is 3:1; the cells are diluted to a TNC (total nucleated cell) concentration of (10-30)×106 /mL, and co-incubated with magnetic beads in a culture dish at room temperature for 2-3 hours; use Magnetic particles concentrator (MPC) (Invitrogen) to enrich CD3+ cells; cells containing CD3+ were resuspended in culture medium (OpTmizerTM CTSTM T-Cell Expansion SFM, Life Technologies) in a culture dish, and the final concentration was 1 ×106 cells/mL. Incubate for 2 days in a 37°C, 5% CO2 incubator.
2)病毒液的制备2) Preparation of virus liquid
将KD-020 CAR分子或KD-021 CAR分子、人PD-1基因sgRNA以及高保真Cas9-HF核酸酶DNA片段分别克隆进慢病毒穿梭载体；包装载体按照Lenti-X Packaging Single Shots(Takara)说明书进行制备。所有载体进行高纯度无内毒素抽提，共转染Lenti-X 293T细胞，转染后6h更换为完全培养基，培养48h和72h后，分别收集富含慢病毒颗粒的细胞上清液，病毒上清液通过超离心浓缩病毒。KD-020 CAR molecules or KD-021 CAR molecules, human PD-1 gene sgRNA, and high-fidelity Cas9-HF nuclease DNA fragments were respectively cloned into lentiviral shuttle vectors; packaging vectors followed the instructions of Lenti-X Packaging Single Shots (Takara) Prepare. All vectors were extracted with high purity and without endotoxin, co-transfected into Lenti-X 293T cells, and replaced with complete medium 6 hours after transfection. After 48 hours and 72 hours of culture, the cell supernatants rich in lentiviral particles were collected, and the virus The supernatant was concentrated for virus by ultracentrifugation.
3)敲除人PD-1基因的KD-020 CAR-T细胞或KD-021 CAR-T细胞的制备3) Preparation of KD-020 CAR-T cells or KD-021 CAR-T cells knocking out the human PD-1 gene
培养T细胞至浓度为(1-2)×106/ml；将状态良好的细胞接种到24孔板，使细胞浓度为1×105/ml细胞，接种细胞数量因细胞的生长速度而略有不同，一般是保证第二天进行病毒感染的时候细胞汇合率介于50-70％之间；第二天将适量的携带KD-020 CAR分子或KD-021CAR分子以及人PD-1基因sgRNA的病毒液和携带高保真Cas9-HF核酸酶的病毒液一同加入细胞培养瓶后，封好口，放入平角离心机后，低速(500g-1000g/min)离心1h，然后放入培养箱中37℃培养。感染后48小时后可以进行下一步的功能实验。Cultivate T cells to a concentration of (1-2)×106 /ml; inoculate the cells in good condition into a 24-well plate, so that the cell concentration is 1×105 /ml cells, and the number of inoculated cells varies slightly due to the growth rate of the cells There are differences. Generally, it is ensured that the cell confluence rate is between 50-70% when the virus is infected the next day; the next day, an appropriate amount of KD-020 CAR molecule or KD-021CAR molecule and human PD-1 gene sgRNA will be carried Add the virus liquid and the virus liquid carrying high-fidelity Cas9-HF nuclease together to the cell culture bottle, seal the mouth, put it in a flat-angle centrifuge, centrifuge at a low speed (500g-1000g/min) for 1 hour, and then put it in the incubator Incubate at 37°C. The next functional experiment can be carried out 48 hours after infection.
4)利用流式细胞术分析检测CAR分子的表达4) Detection of the expression of CAR molecules by flow cytometry analysis
离心细胞，用PBS清洗两次后重悬于FACS液中(含0.1％叠氮化钠和0.4％BSA的PBS)；将Biotin-labeled polyclonal goat anti-mouse-F(ab)2抗体(anti-Fab,JacksonImmunoresearch)加入细胞悬液中，4℃孵育30分钟；清洗细胞两次后，加入phycoerythrin(PE)-labeled streptavidin(BD Pharmingen)，室温30分钟；BD FacsCanto II用于获取染色细胞，FlowJo用于分析结果。如图1所示，对照组为感染空载体病毒液的T细胞，几乎检测不到CAR分子的表达；而在感染KD-020 CAR分子病毒液的T细胞中，CAR分子的表达率达到47.7％。如图2所示，对照组为感染空载体病毒液的T细胞，几乎检测不到CAR分子的表达；而在感染KD-021 CAR分子病毒液的T细胞中，CAR分子的表达率达到64.7％。Cells were centrifuged, washed twice with PBS and resuspended in FACS solution (PBS containing 0.1% sodium azide and 0.4% BSA); Biotin-labeled polyclonal goat anti-mouse-F(ab)2 antibody (anti- Fab, Jackson Immunoresearch) was added to the cell suspension and incubated at 4°C for 30 minutes; after the cells were washed twice, phycoerythrin (PE)-labeled streptavidin (BD Pharmingen) was added for 30 minutes at room temperature; BD FacsCanto II was used to obtain stained cells, and FlowJo used to analyze the results. As shown in Figure 1, the control group was T cells infected with empty vector virus fluid, and the expression of CAR molecules was almost undetectable; while in T cells infected with KD-020 CAR molecule virus fluid, the expression rate of CAR molecules reached 47.7% . As shown in Figure 2, the control group was T cells infected with empty vector virus fluid, and the expression of CAR molecules was almost undetectable; while in T cells infected with KD-021 CAR molecule virus fluid, the expression rate of CAR molecules reached 64.7% .
5)Cas9-HF的表达及PD-1基因敲除的验证5) Expression of Cas9-HF and verification of PD-1 gene knockout
首先利用western blotting检测了Cas9-HF核酸酶的表达。感染病毒后的细胞经过离心、清洗以后在含有蛋白酶抑制剂的RIPA裂解液中裂解1小时，高速离心后得到的裂解上清液与4x LDS上样缓冲液混合后在10％Bis-Tris预制胶(Life technologies)中进行分离，然后通过电转将蛋白转移到PVDF膜上。PVDF膜在含5％脱脂奶粉的TBST中封闭一小时，在1：1000的兔抗HA抗体中4℃孵育过夜。用TBST清洗膜3次，用1：5000偶联HRP的羊抗兔IgG二抗室温孵育1小时。用ECL Plus发光液孵育5分钟后，在Tanon 9500机器中曝光。如图3所示，相对于没有感染病毒液的对照T细胞而言，在感染Cas9-HF核酸酶病毒液的T细胞中有很强的Cas9-HF蛋白表达(图3中A)。First, the expression of Cas9-HF nuclease was detected by western blotting. After centrifugation and washing, the cells infected with the virus were lysed in RIPA lysate containing protease inhibitors for 1 hour, and the lysed supernatant obtained after high-speed centrifugation was mixed with 4x LDS loading buffer and run on 10% Bis-Tris precast gel (Life technologies), and then the protein was transferred to PVDF membrane by electroporation. PVDF membranes were blocked in TBST containing 5% skimmed milk powder for one hour, and incubated overnight at 4°C in 1:1000 rabbit anti-HA antibody. The membrane was washed 3 times with TBST, and incubated with 1:5000 HRP-conjugated goat anti-rabbit IgG secondary antibody for 1 hour at room temperature. After 5 minutes of incubation with ECL Plus luminescence solution, exposure was performed in a Tanon 9500 machine. As shown in Figure 3, compared with the control T cells not infected with the virus fluid, there is a strong Cas9-HF protein expression in the T cells infected with the Cas9-HF nuclease virus fluid (A in Figure 3).
接下来利用western blotting检测PD-1蛋白在CAR-T细胞中的表达情况。感染病毒后的细胞经过离心、清洗以后在含有蛋白酶抑制剂的RIPA裂解液中裂解1小时，高速离心后得到的裂解上清液与4x LDS上样缓冲液混合后在10％Bis-Tris预制胶(Lifetechnologies)中进行分离，然后通过电转将蛋白转移到PVDF膜上。PVDF膜在含5％脱脂奶粉的TBST中封闭一小时，在1：1000的兔抗人PD-1抗体中4℃孵育过夜。用TBST清洗膜3次，用1：5000偶联HRP的羊抗兔IgG二抗室温孵育1小时。用ECL Plus发光液孵育5分钟后，在Tanon9500机器中曝光。如图3所示，相对对照组，PD-1的蛋白表达量在敲除组有明显的下降(图3中B和C)。Next, western blotting was used to detect the expression of PD-1 protein in CAR-T cells. After centrifugation and washing, the cells infected with the virus were lysed in RIPA lysate containing protease inhibitors for 1 hour, and the lysed supernatant obtained after high-speed centrifugation was mixed with 4x LDS loading buffer and run on 10% Bis-Tris precast gel (Lifetechnologies), and then transfer the protein to PVDF membrane by electroporation. PVDF membranes were blocked in TBST containing 5% nonfat dry milk for one hour and incubated overnight at 4°C in 1:1000 rabbit anti-human PD-1 antibody. The membrane was washed 3 times with TBST, and incubated with 1:5000 HRP-conjugated goat anti-rabbit IgG secondary antibody for 1 hour at room temperature. After incubation with ECL Plus luminescent solution for 5 minutes, it was exposed in Tanon9500 machine. As shown in Figure 3, compared with the control group, the protein expression level of PD-1 was significantly decreased in the knockout group (B and C in Figure 3).
6)KD-020 CAR-T细胞杀伤测试6) KD-020 CAR-T cell killing test
利用7-AAD/CFSE细胞毒性测试试剂盒评估KD-020 CAR-T细胞对肿瘤细胞OVCAR-3的杀伤力。CSFE标记肿瘤细胞后，以每孔2×104的细胞铺于培养板中，分别以10：1、5：1和1：1的比例将KD-020 CAR-T细胞加入肿瘤细胞中，培养20小时后离心去上清，细胞沉淀清洗之后用7AAD染色，BD FacsCanto II用于获取染色细胞，FlowJo用于分析结果。如图4所示，相对于不感染病毒和感染空载体病毒液的对照组，KD-020 CAR-T细胞对靶细胞OVCAR-3有明显的杀伤作用，并且在KD-020 CAR-T细胞中敲除人PD-1基因能显著提高该杀伤率。The 7-AAD/CFSE cytotoxicity test kit was used to evaluate the lethality of KD-020 CAR-T cells on tumor cell OVCAR-3. After the tumor cells were labeled with CSFE, 2×104 cells per well were spread on the culture plate, and KD-020 CAR-T cells were added to the tumor cells at the ratio of 10:1, 5:1 and 1:1, respectively, and cultured. After 20 hours, the supernatant was removed by centrifugation, and the cell pellet was washed and stained with 7AAD. BD FacsCanto II was used to obtain the stained cells, and FlowJo was used to analyze the results. As shown in Figure 4, KD-020 CAR-T cells had a significant killing effect on the target cell OVCAR-3 compared with the control group not infected with the virus and infected with the empty vector virus fluid, and the KD-020 CAR-T cells Knocking out the human PD-1 gene can significantly increase the killing rate.
7)KD-021 CAR-T细胞杀伤测试7) KD-021 CAR-T cell killing test
利用7-AAD/CFSE细胞毒性测试试剂盒评估KD-021 CAR-T细胞对肿瘤细胞BxPC-3的杀伤力。CSFE标记肿瘤细胞后，以每孔2×104的细胞铺于培养板中，分别以10：1、5：1和1：1的比例将KD-021 CAR-T细胞加入肿瘤细胞中，培养20小时后离心去上清，细胞沉淀清洗之后用7AAD染色，BD FacsCanto II用于获取染色细胞，FlowJo用于分析结果。如图5所示，相对于不感染病毒和感染空载体病毒液的对照组，KD-021 CAR-T细胞对靶细胞BxPC-3有明显的杀伤作用，并且在KD-021 CAR-T细胞中敲除人PD-1基因能显著提高该杀伤率。The 7-AAD/CFSE cytotoxicity test kit was used to evaluate the lethality of KD-021 CAR-T cells on tumor cell BxPC-3. After the tumor cells were labeled with CSFE,2 ×104 cells per well were spread on the culture plate, and KD-021 CAR-T cells were added to the tumor cells at the ratio of 10:1, 5:1 and 1:1, respectively, and cultured. After 20 hours, the supernatant was removed by centrifugation, and the cell pellet was washed and stained with 7AAD. BD FacsCanto II was used to obtain the stained cells, and FlowJo was used to analyze the results. As shown in Figure 5, KD-021 CAR-T cells had a significant killing effect on the target cell BxPC-3 compared with the control group not infected with the virus and infected with the empty vector virus fluid, and the KD-021 CAR-T cells Knocking out the human PD-1 gene can significantly increase the killing rate.
本发明的CAR分子能特异性靶向识别MSLN阳性的肿瘤细胞。本发明的Cas9-HF核酸酶能有效降低CRISPR/Cas9基因组编辑过程中的脱靶现象。利用本发明方法敲除PD-1基因后得到的靶向MSLN新型CAR-T细胞可用于评估对实体瘤癌细胞的靶向杀伤力，对正常细胞的副作用，以及用于卵巢癌、胰腺癌及肺癌、乳腺癌等其他各类实体瘤病症。The CAR molecule of the present invention can specifically target and recognize MSLN-positive tumor cells. The Cas9-HF nuclease of the present invention can effectively reduce the off-target phenomenon in the process of CRISPR/Cas9 genome editing. The novel CAR-T cells targeting MSLN obtained after knocking out the PD-1 gene by the method of the present invention can be used to evaluate the targeted lethality of solid tumor cancer cells, the side effects on normal cells, and to be used for ovarian cancer, pancreatic cancer and Lung cancer, breast cancer and other solid tumor diseases.
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定，对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动，这里无法对所有的实施方式予以穷举，凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。Apparently, the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those of ordinary skill in the art can also make It is impossible to exhaustively list all the implementation modes here, and any obvious changes or changes derived from the technical solutions of the present invention are still within the scope of protection of the present invention.
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| Date | Code | Title | Description |
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| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
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| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication | Application publication date:20170308 |