WO2017181552A1 - Anti ROBO1 CAR-T细胞及其制备和应用 - Google Patents

Anti ROBO1 CAR-T细胞及其制备和应用 Download PDF

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WO2017181552A1
WO2017181552A1 PCT/CN2016/092577 CN2016092577W WO2017181552A1 WO 2017181552 A1 WO2017181552 A1 WO 2017181552A1 CN 2016092577 W CN2016092577 W CN 2016092577W WO 2017181552 A1 WO2017181552 A1 WO 2017181552A1
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robo1
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李华顺
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李华顺
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Priority to US17/732,581 priority patent/US20220267731A1/en

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Definitions

  • the invention relates to the field of cell drug tumor therapy, in particular to an Anti ROBO1 CAR-T cell and its preparation and application.
  • the human T lymphocytes recognize the target cells through the T cell receptors on their surface. This recognition is specific, that is, a certain T lymphocyte recognizes only target cells having a specific antigen, and this specific antigen is After intracellular processing, it is presented to T lymphocytes under the action of special molecules.
  • antigen-presenting molecules are either present on the surface of antigen presenting cells or present on the surface of target cells.
  • cancer cells down-regulate the expression of antigen presenting molecules
  • the presented antigens have weak affinity with T cell receptors.
  • Chimeric Antigen Receptor consists mainly of two parts, one end of which is located outside the cell and can specifically recognize an antigen on the surface of cancer cells, and the other end of which contains a signal activation element (such as a T cell receptor). Zeta chain) acts to transmit signals to activate T cells.
  • Such T-lymphocytes (CAR-T cells) expressing CAR can prevent the T cell receptor from recognizing the restriction of target cells, thereby playing a role in targeting cancer cells.
  • CD19 is a good therapeutic target.
  • the CD19 CAR-T cells used in different clinical trials have some differences in composition and clinical design, but they are reported.
  • relapsed or refractory lymphocytic leukemia can achieve a response rate of 60-90%, and some patients achieve sustained remission, up to 2 years.
  • CAR-T In addition to blood system tumors, researchers have been working to extend CAR-T treatment to solid tumors. Clinical trials have shown that GD2-specific CAR-T has a certain effect on neuroblastoma, while aFR-specific CAR-T cells are specific for ovarian cancer, CAIX-specific CAR-T cells for renal cell carcinoma and PSMA. CAR-T cells showed no therapeutic effect on prostate cancer. Carl H June of the University of Pennsylvania reported the results of treatment of refractory and metastatic pancreatic ductal adenocarcinoma with mesothelin-specific CAR-T cells at the 2015 American Society of Clinical Oncology.
  • Robo1 is overexpressed in a variety of cancers, such as hepatocellular carcinoma, breast cancer, colon cancer, pancreatic cancer, prostate cancer, glioma, and the like. Ito et al. showed that Robo1 is abundantly expressed in hepatocellular carcinoma, but only a small amount is expressed in normal tissues, and 84.7% of liver cancer tissue samples are positively expressed. Therefore, Robo1 can be used as a new hepatocyte tumor-associated antigen. A potential therapeutic and diagnostic target. The results of the test showed that 80% of colon cancer patients had high expression of Robo1 mRNA, 45% of patients were normal tissue 4 times, and 15% of patients were 12 times normal tissues. Therefore, Robo1 can provide treatment for colon cancer.
  • the extracellular domain of ROBO1 is composed of the IG1-IG5 and FN3 domains, while the FN3 domain is distant from the cell membrane. The distance is the closest, so the ROBO1 molecule is the target, and it is better to select the FN3 region as the antigen.
  • the CAR-T cells constructed are in contact with the tumor cells expressing ROBO1, the distance will be pulled recently, and the killing effect is obtained. It will be even better.
  • the specific structure is shown in Figure 1.
  • the technical problem to be solved by the present invention is to provide an Anti ROBO1 CAR-T cell and a preparation and application thereof, which are methods for modifying and modifying T cells, so that the modified T cells can specifically recognize and kill tumors, and the method is prepared by the method. T cells have more efficient tumor killing activity.
  • one technical solution adopted by the present invention is to provide a CAR-T cell targeting the ROBO1 FN3 domain, which expresses SCFV-CD8 TM -4 in T cells. -1BB-CD3 ⁇ fusion protein.
  • the method for preparing the CAR-T cell comprises the steps of:
  • step (3) separating human peripheral blood T lymphocytes, culturing and expanding, and infecting T lymphocytes by using the lentivirus obtained in the step (2), and expressing the SCFV-CD8 TM -4-1BB-CD3 ⁇ fusion protein by the T lymphocytes, thereby obtaining CAR-T cells.
  • the surface of the T lymphocyte expresses a molecule of the SCFV sequence, and the T cell has a T cell activation signal transmitted intracellularly by the 4-1BB-CD3 ⁇ molecule.
  • the SCFV-CD8 TM -4-1BB-CD3 ⁇ fusion protein in the amino acid sequence of SCFV as SEQ ID NO: 5 shown; the SCFV CD8 TM -4-1BB -CD3 ⁇ amino acid sequence of the fusion protein CD8 TM as the SEQ ID NO: 1 shown in FIG.
  • the 4-1BB in the -1BB-CD3 ⁇ fusion protein can be replaced with CD28, and the molecular sequence of the CD28 is shown in SEQ ID NO: 3.
  • the SCFV-CD8 TM -4-1BB-CD3 ⁇ fusion protein as the amino acid sequence of SEQ ID NO: 6 shown in FIG.
  • the use of the CAR-T cells for the preparation of a medicament for the treatment of tumors.
  • the CAR-T cell is used in the preparation of a medicament for the treatment of a tumor drug that highly expresses the ROBO1 molecule.
  • the beneficial effects of the present invention are: the Anti ROBO1 CAR-T cells of the present invention, and the preparation and application thereof, the ROBO1 antibody is used for the construction of CART cells, and the ROBO1 molecule is used as a target antigen, and the tumor cells are killed by the Anti ROBO1 CART cells. And as a cellular drug for the treatment of tumor diseases, it can be used for the treatment of ROBO1 molecules with high expression of tumors.
  • Figure 1 is a schematic view showing the structure of the ROBO1 molecule of the present invention.
  • Figure 2 is a diagram showing the lentiviral plasmid vector PRRLSIN-SCFV (anti ROBO1-FN3) of the present invention
  • Figure 3 is a flow chart of the MCF7/ROBO1 flow cytometry of the ROBO1-expressing engineering cell line of the present invention.
  • Figure 4 is a graph showing the results of the CAR-T in vitro killing experiment of the present invention.
  • Fig. 5 is a graph showing the in vitro killing effect of CAR-T cells under different effect-target ratio conditions of the present invention.
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • the gene synthesizes the SCFV (Anti ROBO1-FN3)-CD8-4-1BB-CD3 ⁇ fusion gene sequence, and the gene sequence is shown in SEQ ID NO: 7, and is ligated into the PRRSLIN vector by restriction enzyme transformation, and the upstream of the gene is the EP-1 ⁇ promoter. .
  • the vector was transformed into Stbl3 Escherichia coli strain, ampicillin was screened, positive clone was obtained, plasmid was extracted, and cloned by restriction enzyme digestion, and PRRLSIN-SCFV (anti ROBO1-FN3) lentiviral transfection vector was obtained, as shown in Fig. 2.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • Solution A 6.25 ml 2 x HEPES buffer buffer (5 large dishes packed together, the best effect).
  • Solution B A mixture of the following plasmids was added: 112.5 ug pRRLSIN-EF-ROBO1 (target plasmid); 39.5 ug pMD2.G (VSV-G envelop); 73 ug pCMVR8.74 (gag, pol, tat, rev); 625 ⁇ l 2M calcium ion solution. Total volume of solution A: 6.25 ml.
  • the solution B was thoroughly mixed, and while the solution A was gently vortexed, the solution A was added dropwise, and allowed to stand for 5-15 minutes. Gently swirl the above mixed solution of A and B, add dropwise to the Petri dish containing 293T cells, and gently shake the Petri dish to spread the mixture of DNA and calcium ions evenly. (Do not rotate the culture dish) and place it in the incubator for 16-18 hours. The fresh medium was replaced, the culture was continued, and the virus-containing supernatant was collected after 48 hours and 72 hours, respectively. Observed by fluorescence microscope. More than 95% of cells should show green fluorescence. 500 g, centrifuge at 10 ° C for 10 minutes. The PES membrane (0.45 ⁇ m) was filtered.
  • the Beckman Coulter Ultra-clear SW28 centrifuge tubes were sterilized with 70% ethanol and sterilized under UV light for 30 minutes.
  • the filtered lentivirus-containing supernatant is transferred to a centrifuge tube.
  • the centrifuge tube was equilibrated with PBS, 25,000 rpm (82, 700 g), and centrifuged at 4 ° C for 2 hours. Carefully remove the tube, pour off the supernatant, and invert the tube to remove any residual liquid.
  • Embodiment 3 is a diagrammatic representation of Embodiment 3
  • the above 50 ml centrifuge tubes filled with blood were placed in a centrifuge for centrifugation. 400 g (2000 rpm), 10 min, the supernatant plasma was collected after centrifugation at room temperature, leaving a precipitate layer. The collected autologous plasma was inactivated at 56 ° C for 30 minutes. After standing at 4 ° C for 15 minutes, 900 g, 30 min, and centrifugation at 4 ° C, the supernatant was taken for use.
  • the above enriched blood cells were diluted to 30 ml/tube with physiological saline, and two new 50 ml centrifuge tubes were opened, and 15 ml of human lymphocyte separation liquid was added to each centrifuge tube.
  • the diluted blood cell solution is slowly added to the centrifuge tube containing the human lymphatic separation solution by a pipette, and tightened. Note that the blood should be added to the upper layer of the lymphatic separation solution, and the interface of the human lymphatic separation solution should not be broken. Put the added blood cell solution into the centrifuge and adjust to the minimum lifting rate, 400g (2000rpm), 20min, and centrifuge at room temperature. Two tubes of the middle white blood cell layer were collected in a 15 ml sterile centrifuge tube, 5 ml of physiological saline was added, and washed twice (400 g, 10 min centrifugation) to obtain peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • V-VIVO15 added autologous AB (FBS) concentration of 5%, IL-2 concentration of 40 ng / ml, and the isolated PBMC was diluted to 2 ⁇ 10 6 /ml with medium, taking 50ul flow
  • FBS autologous AB
  • the purity of T cells in PBMC was examined. 0 days, configure buffer1, add 1% FBS to PBS, shake the beads for 30s or shake it up and down for 5mins manually. According to the ratio of beads to T cells 3 ⁇ 1, take CD3/CD28beads and put them in 1.5ml EP tube, add 1ml Buffer1 to clean.
  • Beads then use a magnet to suck beads from the EP tube for 1 min, discard the wash solution, repeat twice, then re-suspend the beads to the original volume using the medium, mix the cells and beads, and add 2 ⁇ 10 6 PBMC/ML to the appropriate In the culture bottle.
  • fresh complete medium was added to adjust the cell density to 1 ⁇ 10 6 /ml to continue the culture. All cells were centrifuged, fresh medium was added, and the culture was continued.
  • Half a volume change was performed every 2-3 days to maintain a cell density of 0.5-1 x 10 6 /ml. After 10-12 days, the number of cells reached 10 9 grades, 400 g, and the cells were centrifuged at 5 min, and washed twice with pre-cooled PBS (400 g, 5 min). The cells were counted by a hemocytometer, and the cell group and the proportion of CART cells were detected by flow cytometry. The color change, cell density, and cell morphology of the medium were observed daily and recorded accordingly. Gradually expand the culture process and add the required amount of interleukin 2 to the total volume.
  • Embodiment 4 is a diagrammatic representation of Embodiment 4:
  • Embodiment 5 is a diagrammatic representation of Embodiment 5:
  • LDH release assay was used to detect the killing effect of Anti ROBO1-FN3-CART cells on engineered cell line MCF-1/ROBO1 and Robo1-expressing hepatoma cell line SMCC7721.
  • ELISA was used to detect LDH release.
  • the target cells were adjusted to 5 ⁇ 10 4 /ml with RPMI-1640 medium containing 5% calf serum.
  • Target cells were added to a 96-well cell culture plate, and 100 ⁇ l per well was added. The three effector cells naturally released the control well without adding the target cells, and only 100 ⁇ l of the culture solution was added.
  • Killing rate experimental group LDH (OD) / Max LDH release group (OD).
  • CD8 TM amino acid sequence of SEQ ID NO: 1 is:
  • sequence of 4-1BB SEQ ID NO: 2 is:
  • CD28 The sequence of CD28 is SEQ ID NO: 3:
  • the molecular sequence of CD3 ⁇ SEQ ID NO: 4 is:
  • SCFV Anti ROBO1-FN3 SEQ ID NO: 5 is:
  • SCFV Anti ROBO1-FN3-CD8 TM -4-1BB-CD3 ⁇ fusion protein amino acid sequence
  • SEQ ID NO: 6 is:
  • SCFV Anti ROBO1-FN3 -CD8 TM -4-1BB-CD3 ⁇ fusion protein nucleotide sequence of SEQ ID NO: 7 is:

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Abstract

提供一种嵌合抗原受体修饰T细胞的改造方法，将SCFV-CD8 TM-4-1BB-CD3ζ分子在T细胞内表达，该方法制备的CART细胞可特异性识别和结合ROBO1蛋白高表达的肿瘤细胞，可以应用于相应肿瘤疾病的预防和治疗。

Description

Anti ROBO1 CAR-T细胞及其制备和应用 技术领域
本发明涉及细胞药物肿瘤治疗领域，特别是涉及一种Anti ROBO1 CAR-T细胞及其制备和应用。
背景技术
人体的T淋巴细胞是通过其表面的T细胞受体来识别靶细胞的，这一识别具有特异性，即某一个T淋巴细胞只识别具有特定抗原的靶细胞，而且这种特定的抗原是在细胞内加工后，在特殊分子的作用下呈递给T淋巴细胞的。这种起抗原呈递作用的分子或者存在于抗原呈递细胞表面或者存在于靶细胞表面。至少有两方面的因素导致体内的T淋巴细胞不能很好地识别癌细胞：(1)癌细胞下调抗原呈递分子的表达，(2)被呈递的抗原与T细胞受体亲和力很弱。虽然癌症患者体内存在癌细胞高度特异性的T淋巴细胞，但是数量太少起不到治疗癌症的作用。基于这种情况，科学家提出了构建嵌合T细胞受体(现在一般称为嵌合抗原受体)的概念。嵌合抗原受体(Chimeric Antigen Receptor，CAR)主要由两部分构成，一端位于细胞外能够特异性识别癌细胞表面的某一抗原，另一端位于胞内含有信号激活元件(如T细胞受体的Zeta链)，起传递信号激活T细胞的作用。这样表达CAR的T淋巴细胞(CAR-T细胞)就能避免T细胞受体识别靶细胞的限制，从而起到靶向癌细胞的的杀伤作用。
目前，CAR-T治疗的临床试验正在迅速增长，其中大部分是对B细胞恶性肿瘤治疗的评估。大多数B细胞恶性肿瘤和正常B细胞表达CD19抗原，但是其他类型的细胞没有CD19，因此CD19是一个很好的治疗靶点。不同的临床试验所采用的CD19 CAR-T细胞在组成上有一定差别，临床设计上也不一样，不过都报道 了显著的效果，复发性或难治性淋巴细胞白血病的治疗能达到60-90％的反应率，一部分患者达到持续的缓解，最长的达到2年。虽然目前还不知道CD19 CAR-T治疗能达到多长时间的持续缓解，但是可以肯定的是这种免疫治疗已经给一些患者带来了以前所达不到的效果。
除了血液系统肿瘤外，研究者们一直在努力将CAR-T治疗扩展到实体瘤。临床试验显示，GD2特异性的CAR-T对神经母细胞瘤有一定的疗效，而aFR特异性的CAR-T细胞对卵巢癌，CAIX特异性的CAR-T细胞对肾细胞癌和PSMA特异性的CAR-T细胞对前列腺癌没有显示出治疗效果。宾州大学的Carl H June等在2015年美国临床肿瘤学年会上报告了用间皮素特异性的CAR-T细胞治疗难治的和转移的胰腺导管腺癌的结果，结果显示患者对CAR-T细胞具有很好的耐受性，没有出现细胞因子综合征，周血中能短时期内检测到CART细胞，有2名患者病情得到稳定。因此，CAR-T治疗实体瘤还处于早期阶段，还有许多问题需要解决。
组织病理学检测显示Robo1在多种癌症中过表达，如肝细胞癌、乳腺癌、结肠癌、胰腺癌、前列腺癌、神经胶质瘤等。Ito等的研究显示Robo1在肝细胞癌中大量表达，而在正常组织中只有少量表达，且84.7％的肝癌组织样本为阳性表达，因此Robo1可以作为一种新的肝细胞肿瘤相关抗原，是一种潜在的治疗和诊断靶标。
Figure PCTCN2016092577-appb-000001
等的检测结果显示，80％的结肠癌患者的癌组织高度表达Robo1mRNA，45％的患者是正常组织4倍，15％的患者是正常组织的12倍，因此Robo1可以为结肠癌的治疗提供了一个潜在靶点。通过比较胰腺导管癌和其周围良性组织，He等发现Robo1在癌组织中表达上调，而这种表达上调可能与胰腺癌细胞的淋巴转移相关。Huang等的研究也表明Robo1与结肠癌的迁移有关。
ROBO1分子胞外段由IG1-IG5和FN3结构域组成，而FN3结构域距离细胞膜 的距离最近，因此以ROBO1分子为靶点，选择FN3区域作为抗原是比较好的选择，以此构建的CAR-T细胞与表达ROBO1分子的肿瘤细胞接触时，距离会被拉的最近，杀伤效果也会更优异，具体结构见图1。
发明内容
本发明主要解决的技术问题是提供一种Anti ROBO1 CAR-T细胞及其制备和应用，是修饰和改造T细胞的方法，使改造后的T细胞能够特异性识别和杀伤肿瘤，该方法制备的T细胞具备更高效的肿瘤杀伤活性。
为解决上述技术问题，本发明采用的一个技术方案是：提供一种以ROBO1 FN3结构域为靶点的CAR-T细胞，所述CAR-T细胞是在T细胞内表达SCFV-CD8TM-4-1BB-CD3ζ融合蛋白。
在本发明一个较佳实施例中，所述CAR-T细胞的制备方法包括步骤为：
(1)合成和扩增SCFV-CD8TM-4-1BB-CD3ζ融合蛋白基因，将所述SCFV-CD8TM-4-1BB-CD3ζ融合蛋白基因克隆到慢病毒表达载体上；
(2)利用慢病毒包装质粒和步骤(1)得到的慢病毒表达载体质粒感染293T细胞，包装和制备慢病毒；
(3)分离人外周血T淋巴细胞，培养扩增，利用步骤(2)得到的慢病毒感染T淋巴细胞，使所述T淋巴细胞表达SCFV-CD8TM-4-1BB-CD3ζ融合蛋白，得到CAR-T细胞。
在本发明一个较佳实施例中，所述T淋巴细胞的表面表达所述SCFV序列的分子，所述T细胞的胞内由所述4-1BB-CD3ζ分子传递T细胞活化信号。
在本发明一个较佳实施例中，所述SCFV-CD8TM-4-1BB-CD3ζ融合蛋白中所述SCFV的氨基酸序列为如SEQ ID NO：5所示；所述SCFV  CD8TM-4-1BB-CD3ζ融合蛋白中所述CD8TM的氨基酸序列如SEQ ID NO：1所示。
在本发明一个较佳实施例中，所述SCFV-CD8TM-4-1BB-CD3ζ融合蛋白中所述4-1BB的氨基酸序列如SEQ ID NO：2所示；所述SCFV-CD8TM-4-1BB-CD3ζ融合蛋白中的所述4-1BB能替换为CD28，所述CD28的分子序列如SEQ ID NO：3所示。
在本发明一个较佳实施例中，所述SCFV-CD8TM-4-1BB-CD3ζ融合蛋白中所述CD3ζ的氨基酸序列如SEQ ID NO：4所示；所述T细胞来源于人外周血T淋巴细胞中。
在本发明一个较佳实施例中，所述SCFV-CD8TM-4-1BB-CD3ζ融合蛋白的氨基酸序列如SEQ ID NO：6所示。
在本发明一个较佳实施例中，其特征在于，所述CAR-T细胞在制备治疗肿瘤药物中的应用。
在本发明一个较佳实施例中，所述CAR-T细胞在制备治疗高表达ROBO1分子的肿瘤药物中的应用。
本发明的有益效果是：本发明的Anti ROBO1 CAR-T细胞及其制备和应用，将ROBO1抗体用于CART细胞的构建，并提出以ROBO1分子为靶抗原，利用Anti ROBO1 CART细胞杀伤肿瘤细胞，并作为细胞药物用于肿瘤类疾病的治疗，可以用于ROBO1分子高表达肿瘤的治疗。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图，其中：
图1是本发明的ROBO1分子的结构示意图；
图2是本发明的慢病毒质粒载体PRRLSIN-SCFV(anti ROBO1-FN3)图；
图3是本发明的高表达ROBO1的工程细胞株MCF7/ROBO1流式检测结果
图；
图4是本发明的CAR-T体外杀伤实验结果图；
图5是本发明的不同效靶比条件下CAR-T细胞体外杀伤效果图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
实施例一：
慢病毒表达载体制备：
基因合成SCFV(Anti ROBO1-FN3)-CD8-4-1BB-CD3ζ融合基因序列，基因序列如SEQ ID NO：7所示，通过酶切转化连接到PRRSLIN载体中，基因上游为EP-1α启动子。载体转化Stbl3大肠杆菌菌株，氨苄青霉素筛选，获得阳性克隆，提取质粒，酶切鉴定克隆，获得PRRLSIN-SCFV(anti ROBO1-FN3)慢病毒转染载体，见图2。
实施例二：
慢病毒制备：
(1)转染前24小时，以每皿约8×106将293T细胞接种至15cm培养皿中。确保转染时细胞在80％左右的汇合度且均匀分布于培养皿中。
(2)准备溶液A和溶液B
溶液A：6.25ml 2×HEPES buffer缓冲液(5个大皿一起包装的量，效果最好)。
溶液B：分以加入以下质粒的混合物：112.5ug pRRLSIN-EF-ROBO1(target plasmid)；39.5ug pMD2.G(VSV-G envelop)；73ug pCMVR8.74(gag,pol,tat,rev)；625μl 2M钙离子溶液。溶液A总体积：6.25ml。
充分混匀溶液B，轻轻涡旋溶液A的同时，逐滴加入溶液A，静置5-15分钟。轻轻涡旋上述A和B的混合溶液，逐滴加入含293T细胞的培养皿中，轻轻前后晃动培养皿使DNA与钙离子的混合物均匀分布。(不要旋转培养皿)放置于培养箱中培养16-18小时。更换新鲜培养基，继续培养，分别在48小时和72小时后收集含病毒的上清液。荧光显微镜观察。95％以上的细胞都应该显示绿色荧光。500g，25℃离心10分钟。PES膜(0.45μm)过滤。以70％乙醇消毒贝克曼库尔特Ultra-clear SW28 centrifuge tubes，并置于紫外灯下消毒30分钟。将已过滤的含慢病毒的上清液转移至离心管中。在离心管底部小心铺上一层20％蔗糖(每8ml上清液加1ml蔗糖)。以PBS平衡离心管，25000rpm(82,700g)，4℃离心2小时。小心取出离心管，倒掉上清液，倒置离心管去掉残余液体。加入100μl PBS，密封离心管，在4℃放置2小时，每20分钟轻轻涡旋一次，500g离心1分钟(25℃)，收集病毒上清。冰上冷却后，置于-80℃保存。
实施例三：
Anti ROBO1-FN3-CART细胞制备：
取0.5ml血进行快速的病原微生物检测，排除HBV、HCV、HDV和HEV、HIV-1/2、梅毒螺旋体及寄生虫等微生物感染；无菌条件下，用肝素瓶采血50ml(肝素抗凝)，立即(4℃，24小时内)送至细胞制备实验室，保证此过程无病原微生物污染。得到患者血液后，在GMP制备室，用酒精棉球擦拭肝素瓶表面 进行消毒后放入生物安全柜。预先打开2个50ml离心管，将血液转入两个50ml离心管中，旋紧。将上述装好血液的两个50ml离心管放入离心机离心。400g(2000rpm)，10min，室温离心后收集上层血浆，留下沉淀层。收集的自体血浆经56℃、30分钟灭活。4℃放置15分钟后，900g，30min，4℃离心，取上清备用。将上述富集的血细胞用生理盐水稀释至30ml/管，打开2个新的50ml离心管，每个离心管分别加入15ml人淋巴细胞分离液。用移液管把稀释后的血细胞液缓缓加入到盛有人淋巴分离液的离心管中，旋紧。注意血液要加到淋巴分离液的上层，勿打破人淋巴分离液的界面。将加好的血细胞液放入离心机，调至最小的升降速率，400g(2000rpm)，20min，常温离心。收集两管的中层白细胞层于一支15ml无菌离心管中，加入5ml生理盐水，洗两次(400g，10min离心)，得外周血单核细胞(PBMC)。配置完全生长培养基，V-VIVO15添加自体AB(FBS)浓度为5％，IL-2浓度为40ng/ml，将分离得到的PBMC用培养基稀释成2×106/ml，取50ul流式检测PBMC中T细胞的纯度。0天，配置buffer1，PBS添加1％的FBS，将beads振荡30s或手动上下摇匀5min，根据beads与T细胞比例3比1的比例取出CD3/CD28beads置于1.5ml EP管中，添加1mlBuffer1清洗beads，之后使用磁铁从EP管外吸beads 1min，弃洗液，重复两次，再使用培养基将beads重悬到原体积，将细胞和beads混合后按2×106 PBMC/ML加到合适的培养瓶中。第二天将细胞密度调整至3-5×106/ml，按virus vector：cell＝1:5比例添加virus vector，同时添加polybrene 4ug/ml和40ng/ml IL-2。4h之后，补加新鲜的完全培养基将细胞密度调整至1×106/ml继续培养。将所有的细胞离心，加入新鲜的培养基，继续培养。每隔2-3天进行半量换液，维持细胞密度在0.5-1×106/ml。10-12天，细胞数量达到109级别，400g，5min离心得免疫细胞，再用预冷的PBS洗涤两遍(400g，5min)。用血球计数板计数，流式细胞仪检测 细胞类群，CART细胞比例。每天观察培养基的颜色变化、细胞密度、细胞形态并作相应记录。逐步扩大培养过程中，加入总体积所需的白细胞介素2。
实施例四：
工程细胞株的构建及检测：
(1)用于构建高表达Robo1 FN3工程细胞株慢病毒的制备(具体制备方法见实施例二中的方法)；
(2)MCF细胞的侵染：侵染前一天，接种50万个MCF7细胞于6孔板中，待第二天细胞长到80％时，加入包装好的500ul的ROBO1病毒于6孔板中，同时设置对照细胞(不添加病毒)，12-16小时后换液，侵染3天后，流式分选Robo1的阳性细胞；
(3)工程细胞株的检测：取分选的Robo1的阳性细胞2万个，400g，5min，再用预冷的PBS洗涤两遍，加入2.5ul的Robo1的抗体(Biolegend)避光孵育20min，离心，再用预冷的PBS洗涤1遍，100ul PBS重悬细胞，上流式检测Robo1的表达(见图3),实验结果证明工程细胞株构建成功，可作为靶细胞用于后续杀伤实验。
实施例五：
Anti ROBO1-FN3-CART细胞体外活性检测
LDH释放法检测Anti ROBO1-FN3-CART细胞对工程细胞株MCF-1/ROBO1和高表达Robo1的肝癌细胞系SMCC7721细胞的杀伤效应，ELISA方法检测LDH释放。
(1)用含5％小牛血清的RPMI-1640培养液将靶细胞调整到5×104/ml。
(2)在96孔细胞培养板中加入靶细胞，每孔加100μl。3个效应细胞自然释放对照孔不加靶细胞，只加100μl培养液。
(3)向各孔加100μl效应细胞，效应细胞与靶细胞的比例50:1；25:1；10:1；5:1；1:1。自然释放孔不加效应细胞只加100μl培养液，效应细胞与靶细胞共孵育6小时，每个实验置三个复孔。
(4)最大释放孔中(阳性对照)加10μl Lysis Solution(10×)，孵育45min-60min，每个实验置三个复孔。
(5)取上述3和4中待测样品和对照样品各50ul，加入新鲜的96孔酶标板中，再加入assay buffer和substrate mix，避光30min。
(6)加入50ul stop solution。
(7)490nm或492nm处测吸光度值，在1小时内测完。
(8)杀伤率＝实验组LDH(OD)/Max LDH释放组(OD)。
(9)计算公式：杀伤效率＝(experimental-effector spontaneous-target spontaneous)/(target maximum-target spontaneous)×100％。
实验结果显示，制备的Anti ROBO1-FN3-CART细胞能够显著杀伤高表达ROBO1的靶细胞株MCF-7/ROBO1和SMCC7721，不同比例的ROBO1 CAR-T与target cells共孵育4小时后，ELISA实验结果显示，随着E:T比例的增加，细胞杀伤效率也逐渐增加(见图5)，显微成像显示肿瘤细胞发生明显死亡(图4)。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书内容所作的等效结构或等效流程变换，或直接或间接运用在其它相关的技术领域，均同理包括在本发明的专利保护范围内。
序列表：
CD8TM的氨基酸序列SEQ ID NO：1为：
Figure PCTCN2016092577-appb-000002
4-1BB的序列SEQ ID NO：2为：
Figure PCTCN2016092577-appb-000003
CD28的序列SEQ ID NO：3为：
Figure PCTCN2016092577-appb-000004
CD3ζ的分子序列SEQ ID NO：4为：
Figure PCTCN2016092577-appb-000005
SCFV(Anti ROBO1-FN3)的序列SEQ ID NO：5为：
Figure PCTCN2016092577-appb-000006
SCFV(Anti ROBO1-FN3)-CD8TM-4-1BB-CD3ζ融合蛋白氨基酸的序列SEQ ID NO：6为：
Figure PCTCN2016092577-appb-000007
SCFV(Anti ROBO1-FN3)-CD8TM-4-1BB-CD3ζ融合蛋白核苷酸的序列SEQ ID NO：7为：
Figure PCTCN2016092577-appb-000008
Figure PCTCN2016092577-appb-000009

Claims (9)

  1. 一种Anti ROBO1 CAR-T细胞，其特征在于，所述CAR-T细胞是在T细胞内表达SCFV-CD8TM-4-1BB-CD3ζ融合蛋白。
  2. 根据权利要求1所述的Anti ROBO1 CAR-T细胞，其特征在于，所述CAR-T细胞的制备方法包括步骤为：
    (1)合成和扩增SCFV-CD8TM-4-1BB-CD3ζ融合蛋白基因，将所述SCFV-CD8TM-4-1BB-CD3ζ融合蛋白基因克隆到慢病毒表达载体上；
    (2)利用慢病毒包装质粒和步骤(1)得到的慢病毒表达载体质粒感染293T细胞，包装和制备慢病毒；
    (3)分离人外周血T淋巴细胞，培养扩增，利用步骤(2)得到的慢病毒感染T淋巴细胞，使所述T淋巴细胞表达SCFV-CD8TM-4-1BB-CD3ζ融合蛋白，得到CAR-T细胞。
  3. 根据权利要求1所述的Anti ROBO1 CAR-T细胞，其特征在于，所述T淋巴细胞的表面表达所述SCFV序列的分子，所述T细胞的胞内由所述4-1BB-CD3ζ分子传递T细胞活化信号。
  4. 根据权利要求1或2所述的Anti ROBO1 CAR-T细胞，其特征在于，所述SCFV-CD8TM-4-1BB-CD3ζ融合蛋白中所述SCFV的氨基酸序列为如SEQ ID NO：5所示；所述SCFV-CD8TM-4-1BB-CD3ζ融合蛋白中所述CD8TM的氨基酸序列如SEQ ID NO：1所示。
  5. 根据权利要求1或2所述的Anti ROBO1 CAR-T细胞，其特征在于，所述SCFV-CD8TM-4-1BB-CD3ζ融合蛋白中所述4-1BB的氨基酸序列如SEQ ID NO：2所示；所述SCFV-CD8TM-4-1BB-CD3ζ融合蛋白中的所述4-1BB能替换为CD28，所述CD28的分子序列如SEQ ID NO：3所示。
  6. 根据权利要求1或2所述的Anti ROBO1 CAR-T细胞，所述SCFV-CD8TM-4-1BB-CD3ζ融合蛋白中所述CD3ζ的氨基酸序列如SEQ ID NO：4所示；所述T细胞来源于人外周血T淋巴细胞中。
  7. 根据权利要求1或2所述的Anti ROBO1 CAR-T细胞，其特征在于，所述SCFV-CD8TM-4-1BB-CD3ζ融合蛋白的氨基酸序列如SEQ ID NO：6所示。
  8. 根据权利要求1或2所述的Anti ROBO1 CAR-T细胞，其特征在于，所述CAR-T细胞在制备治疗肿瘤药物中的应用。
  9. 根据权利要求8所述的Anti ROBO1 CAR-T细胞，其特征在于，所述CAR-T细胞在制备治疗高表达ROBO1分子的肿瘤药物中的应用。
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