技术领域technical field
本发明涉及生物医学领域中，一种扩增特异性CAR-T细胞的培养方法。The invention relates to a culture method for expanding specific CAR-T cells in the field of biomedicine.
背景技术Background technique
嵌合抗原受体(Chimeric Antigen Receptor，CAR)是基于T细胞受体的人工修饰融合蛋白，其由细胞外抗原识别结构域和细胞内信号传导结构域组成。嵌合抗原受体T细胞(CAR-T细胞)是将能识别某种肿瘤抗原的抗体的抗原结合部与CD3-ζ链或FcεRIγ的胞内部分在体外偶联为一个嵌合蛋白，通过基因转导的方法转染患者的T细胞，使其表达嵌合抗原受体(CAR)。嵌合抗原受体(CAR)由T细胞受体的胞外抗原结合区(scFv)、铰链区、中间的跨膜区及胞内信号区4个部分组成。目前，已有多种肿瘤抗原可用于胞外区识别，包括CD19、CD20、表皮生长因子受体(EGFR)和人类表皮生长因子受体2(HER-2)等。铰链区有CD8Hinge、CD28Hinge和较长的IgG1Fc或IgG4Fc等。中间的跨膜区有CD4、CD8和CD28等，将CAR结构锚定于T细胞膜上。细胞内结构域通常包含CD3ζ，CD28，4-1BB或OX40，用于增加T细胞活化。遗传修饰以表达CAR的T细胞可以直接识别CAR靶向的抗原，然后触发表达CAR特异性抗原的肿瘤细胞的T细胞活化，增殖，细胞因子分泌和细胞毒性。目前，CAR修饰的T细胞在治疗血液癌症，包括淋巴瘤，慢性淋巴细胞性白血病和急性淋巴细胞性白血病(ALL)方面取得了显著的成功。据报道，CD19靶向CAR-T细胞在所有患者中具有70％至90％的完全缓解率。可见，CAR-T细胞在肿瘤免疫治疗中的应用前景广阔，市场价值巨大。Chimeric Antigen Receptor (CAR) is an artificially modified fusion protein based on T cell receptors, which consists of an extracellular antigen recognition domain and an intracellular signaling domain. Chimeric antigen receptor T cells (CAR-T cells) are a combination of the antigen-binding portion of an antibody that can recognize a certain tumor antigen and the intracellular portion of CD3-ζ chain or FcεRIγ to form a chimeric protein in vitro. The transduction method transfects a patient's T cells to express a chimeric antigen receptor (CAR). The chimeric antigen receptor (CAR) consists of four parts: the extracellular antigen binding domain (scFv), the hinge region, the middle transmembrane region and the intracellular signal region of the T cell receptor. Currently, a variety of tumor antigens are available for extracellular domain recognition, including CD19, CD20, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2). Hinge region has CD8Hinge, CD28Hinge and longer IgG1Fc or IgG4Fc and so on. There are CD4, CD8 and CD28 in the middle transmembrane region, which anchor the CAR structure on the T cell membrane. The intracellular domain typically contains CD3ζ, CD28, 4-1BB or OX40 for increased T cell activation. T cells genetically modified to express a CAR can directly recognize the CAR-targeted antigen, and then trigger T cell activation, proliferation, cytokine secretion, and cytotoxicity of tumor cells expressing the CAR-specific antigen. Currently, CAR-modified T cells have achieved remarkable success in the treatment of blood cancers, including lymphoma, chronic lymphocytic leukemia, and acute lymphocytic leukemia (ALL). CD19-targeted CAR-T cells have been reported to have 70% to 90% complete remission rates in all patients. It can be seen that the application of CAR-T cells in tumor immunotherapy has broad prospects and huge market value.
CAR-T细胞治疗中，主要通过抗原-抗体识别模式对肿瘤细胞表面的特异分子进行识别，然后通过其胞内的信号传导进行激活、增殖并发挥细胞杀伤功能。因此，这种CAR特异性杀死肿瘤细胞机制，需要在CAR-T制备过程中，用CAR基因转导患者T细胞后，所获得的CAR-T特异性细胞越多越好，即在总T细胞中，转导了CAR基因的CAR-T细胞的比例(阳性率)越高越好，以达到更好的肿瘤细胞识别作用，激活靶细胞杀伤机制。In CAR-T cell therapy, specific molecules on the surface of tumor cells are recognized mainly through the antigen-antibody recognition mode, and then activated, proliferated, and cell killing functions through their intracellular signal transduction. Therefore, this mechanism of CAR-specific killing of tumor cells requires that in the process of CAR-T preparation, after transducing patient T cells with CAR gene, the more CAR-T-specific cells obtained, the better, that is, in the total T cells. In cells, the higher the proportion (positive rate) of CAR-T cells transduced with CAR gene, the better, in order to achieve better tumor cell recognition and activate the target cell killing mechanism.
目前，CAR-T细胞制备过程中，将CAR基因转导入T细胞的方法主要有：电穿孔法、化学介导法(如磷酸钙共沉淀法、脂质体转染法)、病毒介导法等。其中电穿孔法是利用高脉冲电压破坏细胞膜电位，使核酸通过膜上形成的小孔导入细胞。该方法适用性广，可转导DNA或RNA，然而缺点是所需核酸及细胞用量大，且细胞致死率极高，不是理想的CAR基因导入T细胞方法。化学介导法中，磷酸钙法是利用磷酸钙DNA复合物吸附细胞膜被细胞内吞，其操作简便但重复性差，并且不太适用于病人T细胞这种原代细胞的转染；脂质体法是利用带正电的脂质体与核酸带负电的磷酸基团形成复合物，脂质体上剩余的电荷与细胞膜上的唾液酸残基的负电荷结合或通过细胞内吞作用使核酸进入细胞，该方法使用简单，但有一定细胞毒性，并可在体内诱发强烈的抗炎反应，因此在很大程度上也限制了其应用。病毒介导的转染技术，可将外源基因整合到细胞基因组中，转染效率高，外源基因整合较稳定，是目前最常用的CAR-T细胞转导方式。CAR细胞制备中，通常用逆转录病毒或慢病毒，其中慢病毒感染法，既可以感染分裂期细胞又可以感染非分裂期细胞，在CAR-T临床试验制备中最为常用。上述CAR-T转染方法，目的均是将CAR基因转入T细胞，以期望获得高比例或高阳性率的CAR-T细胞，然而实际操作中，仍存在许多挑战和障碍，难以获得较高阳性率及特异性的CAR-T细胞。一方面，电穿孔法与化学介导法因细胞用量大或致死率高、有一定毒性等，不适于临床CAR-T细胞的制备；另一方面，逆转录病毒或慢病毒包装步骤繁琐，耗时长，工作量大，往往需要经验丰富的专业技术人员才能有效完成，并且重组病毒很难得到较高的滴度，这成为了CAR-T细胞制备中关键的制约因素；其次，因不同批次病毒包装时，转染所用工具细胞状态差异，不同技术人员操作差异等，包装的慢病毒滴度通常很不稳定、导致批次间差异较大，这些均成为了CAR-T细胞制备和应用研究的主要限速环节。At present, during the preparation of CAR-T cells, the main methods for transferring CAR genes into T cells include electroporation, chemical-mediated methods (such as calcium phosphate co-precipitation, lipofection), and virus-mediated methods. Wait. Among them, the electroporation method uses high pulse voltage to destroy the cell membrane potential, so that the nucleic acid is introduced into the cell through the pores formed on the membrane. This method has wide applicability and can transduce DNA or RNA. However, the disadvantage is that the required amount of nucleic acid and cells is large, and the cell lethality is extremely high, so it is not an ideal method for CAR gene introduction into T cells. Among the chemical-mediated methods, the calcium phosphate method uses calcium phosphate DNA complexes to adsorb the cell membrane and be endocytosed by the cells, which is easy to operate but has poor reproducibility, and is not suitable for transfection of primary cells such as patient T cells; liposomes The method is to use positively charged liposomes to form complexes with negatively charged phosphate groups of nucleic acids, and the remaining charges on the liposomes combine with the negative charges of sialic acid residues on the cell membrane or allow nucleic acids to enter through endocytosis. cells, this method is simple to use, but has certain cytotoxicity and can induce a strong anti-inflammatory response in vivo, so it also limits its application to a large extent. Virus-mediated transfection technology, which can integrate foreign genes into the cell genome, has high transfection efficiency and stable integration of foreign genes, and is currently the most commonly used CAR-T cell transduction method. In the preparation of CAR cells, retroviruses or lentiviruses are usually used. The lentivirus infection method, which can infect both dividing cells and non-dividing cells, is the most commonly used in the preparation of CAR-T clinical trials. The purpose of the above CAR-T transfection methods is to transfer the CAR gene into T cells in order to obtain a high proportion or high positive rate of CAR-T cells. However, in practice, there are still many challenges and obstacles, and it is difficult to obtain high Positive rate and specificity of CAR-T cells. On the one hand, electroporation and chemical-mediated methods are not suitable for the preparation of clinical CAR-T cells due to the large amount of cells, high lethality, and certain toxicity. It takes a long time and requires a lot of work, which often requires experienced professional technicians to complete it effectively, and it is difficult to obtain a high titer of recombinant virus, which has become a key constraint in the preparation of CAR-T cells; secondly, due to different batches of When the virus is packaged, the tools used for transfection are different in cell state, different technicians operate differently, etc. The titer of packaged lentivirus is usually very unstable, resulting in large differences between batches. These have become CAR-T cell preparation and application research. the main speed limit.
因此，如能找到一种高效简便的方法，能有效提高CAR-T细胞的特异性及阳性率，则可以大大提高临床CAR-T细胞制备的效率，为CAR-T细胞的治疗效果提高保障。Therefore, if an efficient and simple method can be found, which can effectively improve the specificity and positive rate of CAR-T cells, the efficiency of clinical CAR-T cell preparation can be greatly improved, and the therapeutic effect of CAR-T cells can be improved.
发明内容SUMMARY OF THE INVENTION
本发明的所要解决的技术问题是如何提高CAR-T细胞的特异性及阳性率。The technical problem to be solved by the present invention is how to improve the specificity and positive rate of CAR-T cells.
为解决上述技术问题，本发明首先提供了扩增免疫细胞的方法，所述方法包括：In order to solve the above-mentioned technical problems, the present invention first provides a method for expanding immune cells, the method comprising:
1)向免疫细胞的培养体系中添加所述免疫细胞所识别的抗原，得到共培养体系；1) adding the antigen recognized by the immune cells to the culture system of the immune cells to obtain a co-culture system;
2)培养所述共培养体系，实现所述免疫细胞的扩增。2) Culturing the co-culture system to realize the expansion of the immune cells.
上述方法中，所述免疫细胞可为表达嵌合抗原受体的免疫细胞。In the above method, the immune cells may be immune cells expressing chimeric antigen receptors.
所述免疫细胞具体可为表达嵌合抗原受体的T细胞。The immune cells can specifically be T cells expressing chimeric antigen receptors.
上述方法中，所述抗原可为肿瘤抗原。In the above method, the antigen may be a tumor antigen.
进一步，所述抗原可为B细胞成熟抗原(BCMA)。Further, the antigen may be B cell maturation antigen (BCMA).
所述免疫细胞可为抗BCMA的CAR-T细胞。The immune cells can be anti-BCMA CAR-T cells.
上述方法中，所述共培养体系中所述免疫细胞与所述抗原的配比可为106个：1～100ng。进一步，所述共培养体系中所述免疫细胞与所述抗原的配比可为106个：5～50ng。In the above method, the ratio of the immune cells to the antigen in the co-culture system may be 106 : 1-100 ng. Further, the ratio of the immune cells to the antigen in the co-culture system may be 106 : 5-50 ng.
上述方法中，向免疫细胞的培养体系中添加所述免疫细胞所识别的抗原的次数和/或时间可根据具体情况调整，只要能实现提高所述免疫细胞的阳性率和/或特异性，均属于本发明的保护范围。In the above method, the number and/or time of adding the antigen recognized by the immune cells to the culture system of the immune cells can be adjusted according to the specific situation, as long as the positive rate and/or specificity of the immune cells can be improved. It belongs to the protection scope of the present invention.
所述扩增免疫细胞的方法在制备药物中的应用，也属于本发明的保护范围。The application of the method for expanding immune cells in the preparation of medicines also belongs to the protection scope of the present invention.
所述药物可用于治疗肿瘤，如多发性骨髓瘤。The medicament can be used to treat tumors, such as multiple myeloma.
本发明针对现有CAR-T细胞制备中，所得CAR-T细胞阳性率低而影响其有效性和特异性，提供了一种提高CAR-T细胞阳性率的扩增免疫细胞的方法，利用本发明的扩增免疫细胞的方法得到的CAR-T细胞的阳性率可提高4-6倍，并且该方法操作简便、高效、特异性强，实用性强。Aiming at the low positive rate of CAR-T cells obtained in the preparation of existing CAR-T cells, which affects its effectiveness and specificity, the present invention provides a method for expanding immune cells that improves the positive rate of CAR-T cells. The positive rate of CAR-T cells obtained by the invented method of expanding immune cells can be increased by 4-6 times, and the method is simple, efficient, specific and practical.
附图说明Description of drawings
图1为扩增特异性CAR-T细胞的培养方法流程图。Figure 1 is a flow chart of the culture method for expanding specific CAR-T cells.
图2为抗BCMA CAR-T细胞阳性率的检测结果，A-F的横坐标为CD3阳性细胞数，纵坐标表示CAR阳性细胞数,Q1+Q2是CAR的总阳性率，Q2+Q3是CD3的总阳性率，Q4是双阴性率，Q2是双阳性率。Figure 2 shows the detection results of the positive rate of anti-BCMA CAR-T cells. The abscissa of A-F is the number of CD3-positive cells, the ordinate is the number of CAR-positive cells, Q1+Q2 is the total positive rate of CAR, and Q2+Q3 is the total number of CD3 Positive rate, Q4 is double negative rate, Q2 is double positive rate.
具体实施方式Detailed ways
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.
下述实施例中的293T和293FT细胞为南京科佰生物科技有限公司产品。The 293T and 293FT cells in the following examples are products of Nanjing Kebai Biotechnology Co., Ltd.
实施例1、扩增CAR-T细胞的方法Example 1. Method for expanding CAR-T cells
本实施例以识别B细胞成熟抗原(B Cell Maturation Antigen，BCMA，CD269)的抗BCMA CAR-T细胞为例具体阐述了提高CAR-T细胞扩增效率的方法，流程图见图1，具体步骤如下：This example uses anti-BCMA CAR-T cells that recognize B Cell Maturation Antigen (BCMA, CD269) as an example to specifically describe the method for improving the expansion efficiency of CAR-T cells. The flowchart is shown in Figure 1. The specific steps as follows:
1、质粒的准备1. Plasmid preparation
采用无内毒素质粒大提试剂盒提取慢病毒包膜质粒pMD2.G、psPAX2以及含CAR基因的慢病毒表达质粒(pWPXLd-CAR-BCMA-EGFP质粒)和对照质粒pWPXLd-EGFP待用。其中，pMD2.G、psPAX2、pWPXLd-EGFP质粒均为addgene产品(http://www.addgene.org)；含CAR基因的慢病毒表达质粒(pWPXLd-CAR-BCMA-EGFP)由以下方式构建而成：Lentiviral envelope plasmids pMD2.G, psPAX2, lentiviral expression plasmid containing CAR gene (pWPXLd-CAR-BCMA-EGFP plasmid) and control plasmid pWPXLd-EGFP were extracted by endotoxin-free plasmid extraction kit. Among them, pMD2.G, psPAX2, pWPXLd-EGFP plasmids are all addgene products (http://www.addgene.org); the lentiviral expression plasmid containing CAR gene (pWPXLd-CAR-BCMA-EGFP) is constructed in the following way. to make:
将pWPXLd-EGFP的PmeI和SpeI识别序列间的DNA片段替换为序列表中SEQ ID NO.1所示的DNA片段，得到重组载体pWPXLd-CAR-BCMA-EGFP，pWPXLd-CAR-BCMA-EGFP能表达anti-BCMA scfv、CD8hinge、4-1BB、CD3ζ形成的融合蛋白，该融合蛋白质的序列为序列表中SEQ ID NO.2。Replace the DNA fragment between the PmeI and SpeI recognition sequences of pWPXLd-EGFP with the DNA fragment shown in SEQ ID NO.1 in the sequence listing to obtain the recombinant vector pWPXLd-CAR-BCMA-EGFP, which can express pWPXLd-CAR-BCMA-EGFP A fusion protein formed by anti-BCMA scfv, CD8hinge, 4-1BB and CD3ζ, the sequence of the fusion protein is SEQ ID NO.2 in the sequence listing.
2、慢病毒的制备2. Preparation of lentivirus
通过293T或293FT细胞包装慢病毒，具体操作如下：Packaging lentivirus by 293T or 293FT cells, the specific operation is as follows:
①向DMEM高糖培养基中添加FBS(FBS在培养基中的质量百分比为10％)后培养293T细胞于10cm培养皿，待293T细胞汇合度达70-90％时，利用lipo2000(或PEI)将pWPXLd-CAR-BCMA-EGFP质粒、pMD2.G和psPAX2三个质粒转化293T细胞，每个10cm皿转化12ug的pWPXLd-CAR-BCMA-EGFP质粒，7.8ug的psPAX2，4.2ug的pMD2.G，48μl的lipo2000(或72μl的PEI)；①Add FBS to the DMEM high glucose medium (the mass percentage of FBS in the medium is 10%), and then culture 293T cells in a 10cm culture dish. When the confluence of 293T cells reaches 70-90%, use lipo2000 (or PEI) The pWPXLd-CAR-BCMA-EGFP plasmid, pMD2.G and psPAX2 plasmids were transformed into 293T cells, and each 10cm dish was transformed with 12ug of pWPXLd-CAR-BCMA-EGFP plasmid, 7.8ug of psPAX2, 4.2ug of pMD2.G, 48μl of lipo2000 (or 72μl of PEI);
②步骤①完成后，将转化后的体系正常培养，分别在培养24h、48h和72h收集三个时间段的培养上清液；将三个时间段的培养上清液分别经400g离心5分钟，弃沉淀，然后将离心得到的三种上清液分别通过0.45μm过滤器过滤，过滤后将滤液混合到一起，得到慢病毒液；将慢病毒液置于超级离心管中分别在超高速离心机上25000rpm、4℃离心2h使病毒沉淀，弃上清液，然后向每个超速离心管中加入100μl 4℃预冷的PBS或RPMI-1640无血清培养基重悬，即得到转染了pWPXLd-CAR-BCMA-EGFP质粒的慢病毒悬浮液，放置-80℃待用。② After step ① is completed, the transformed system is cultured normally, and the culture supernatants of the three time periods are collected at 24h, 48h and 72h respectively; the culture supernatants of the three time periods are centrifuged at 400g for 5 minutes respectively, Discard the precipitation, and then filter the three supernatants obtained by centrifugation through 0.45 μm filters, respectively. After filtering, the filtrates were mixed together to obtain a lentiviral solution; the lentiviral solution was placed in an ultracentrifuge tube and placed on an ultracentrifuge. Centrifuge at 25,000 rpm and 4 °C for 2 h to precipitate the virus, discard the supernatant, and then add 100 μl of 4 °C pre-cooled PBS or RPMI-1640 serum-free medium to each ultracentrifuge tube to resuspend, to obtain transfected pWPXLd-CAR - Lentiviral suspension of BCMA-EGFP plasmid, placed at -80°C until use.
按照上述方法，将pWPXLd-CAR-BCMA-EGFP质粒替换为对照质粒pWPXLd-EGFP，其他步骤均不变，得到转染了pWPXLd-EGFP的慢病毒悬液，放置-80℃待用。According to the above method, the pWPXLd-CAR-BCMA-EGFP plasmid was replaced with the control plasmid pWPXLd-EGFP, and other steps remained unchanged to obtain a lentiviral suspension transfected with pWPXLd-EGFP, which was placed at -80 °C for use.
3、T细胞的准备3. Preparation of T cells
通过Ficoll密度梯度法分离出健康人或多发性骨髓瘤患者血液中的单个核细胞(PBMC)。Mononuclear cells (PBMCs) were isolated from the blood of healthy people or patients with multiple myeloma by Ficoll density gradient method.
PBMC分离后，第一天先用添加了10％FBS的RPMI-1640培养基培养PBMC 12h，然后改用添加了10％FBS、50ng/ml的OKT3抗体(R&D system，MAB100)、50ng/ml的CD28抗体(MILTENYI，130-093-375)和10ng/ml IL-2(SIGMA，SPR3085)的RPMI-1640培养基激活T细胞并继续培养48h。After PBMC isolation, on the first day, PBMCs were cultured in RPMI-1640 medium supplemented with 10% FBS for 12 h, and then switched to OKT3 antibody (R&D system, MAB100) supplemented with 10% FBS, 50 ng/ml, and 50 ng/ml. CD28 antibody (MILTENYI, 130-093-375) and RPMI-1640 medium with 10 ng/ml IL-2 (SIGMA, SPR3085) activated T cells and continued to culture for 48 h.
第三天，将刺激后的T细胞重悬并400g离心5min，去上清，用含10ug/ml polybrene的RPMI1640培养基重悬并铺板至六孔板中，每孔2ml，含2×106个细胞，然后在不同孔中加入步骤2得到的一种慢病毒，根据慢病毒滴度，MOI设为20，然后将细胞置于37℃5％CO2培养箱中培养24h，重悬细胞，400g 5min离心去上清液后换上新鲜的含10％FBS、50ng/ml IL-2和50ng/ml IL-7的RPMI-1640培养基，37℃5％CO2培养箱中培养至第7天，初步获得抗BCMACAR-T细胞，期间每隔一天对T细胞半量换培养液并计数，此期间可荧光观察及流式检测EGFP及CAR的表达率。On the third day, the stimulated T cells were resuspended and centrifuged at 400g for 5min, the supernatant was removed, resuspended in RPMI1640 medium containing 10ug/ml polybrene and plated into a six-well plate, 2ml per well, containing 2×106 Then add a lentivirus obtained in step 2 to different wells, set the MOI to 20 according to the lentivirus titer, then place the cells in a 37°C 5% CO2 incubator for 24 hours, resuspend the cells, After centrifugation at 400g for 5min, the supernatant was removed and replaced with fresh RPMI-1640 medium containing 10% FBS, 50ng/ml IL-2 and 50ng/ml IL-7, and cultured in a 5%CO2 incubator at 37°C until the seventh day. On the next day, anti-BCMACAR-T cells were initially obtained. During the period, half of the T cells were replaced with medium and counted every other day. During this period, the expression rates of EGFP and CAR can be detected by fluorescence observation and flow cytometry.
4、抗原刺激扩增抗BCMA CAR-T细胞4. Antigen stimulation to expand anti-BCMA CAR-T cells
初步获得抗BCMA CAR-T细胞后，接步骤3用含10％FBS、50ng/ml IL-2和50ng/mlIL-7的RPMI-1640培养基继续培养细胞，期间每隔一天，分别向培养体系中添加在培养体系中的浓度为10ng/ml的BCMA抗原(ACRO BIOSYSTEMS，BCA-H522y)，保持细胞培养密度为1×106个/ml，每2～3天进行细胞计数及半量换液，培养第14天，收获CAR-T细胞并用流式细胞仪检测CAR-T细胞阳性率，所用试剂为APC标记的CD3抗体(EBIOSCIENCE，17-0036-72)，生物素化的羊抗鼠Fab抗体(Jackson Immunoresearch，115-066-072)，羊IgG抗体(ABCAM，37373)，生物素化的羊IgG抗体(ABCAM，37376)，小鼠IgG抗体(碧云天，A7028)，PE标记的链霉亲和素(BD，554061)。添加抗原实验设两个重复实验，分别记为第一组和第二组。After the initial acquisition of anti-BCMA CAR-T cells, continue to culture cells in RPMI-1640 medium containing 10% FBS, 50ng/ml IL-2 and 50ng/ml IL-7 in step 3. BCMA antigen (ACRO BIOSYSTEMS, BCA-H522y) was added to the culture system at a concentration of 10 ng/ml to maintain the cell culture density at 1×106 cells/ml, and the cells were counted every 2 to 3 days and the medium was changed in half. On the 14th day of culture, CAR-T cells were harvested and the positive rate of CAR-T cells was detected by flow cytometry. The reagents used were APC-labeled CD3 antibody (EBIOSCIENCE, 17-0036-72), biotinylated goat anti-mouse Fab antibody (Jackson Immunoresearch, 115-066-072), goat IgG antibody (ABCAM, 37373), biotinylated goat IgG antibody (ABCAM, 37376), mouse IgG antibody (Biyuntian, A7028), PE-labeled streptavidin and Su (BD, 554061). The antigen addition experiment consisted of two repeated experiments, which were recorded as the first group and the second group.
利用不添加BCMA抗原培养步骤3初步获得的抗BCMA CAR-T细胞，培养至第14天作为不添加抗原对照实验。The anti-BCMA CAR-T cells initially obtained in step 3 were cultured without the addition of BCMA antigen, and cultured to the 14th day as a control experiment without the addition of antigen.
结果如图2所示，图2中A为不添加抗原对照实验中第5天抗BCMA CAR-T细胞的阳性率(即表达抗BCMA的CAR-T细胞占总CAR-T细胞的百分比)，B为不添加抗原对照实验中第14天抗BCMA CAR-T细胞的阳性率，C、E为添加抗原实验中第5天抗BCMA CAR-T细胞的阳性率，D、F为添加抗原实验中第14天抗BCMA CAR-T细胞的阳性率。结果显示，不添加抗原对照实验中，抗BCMA CAR-T细胞在第5天的阳性率为4.49％，在第14天的阳性率为10.5％；添加抗原实验中，第一组，抗BCMA CAR-T细胞在第5天的阳性率为4.49％(图2中C)，在第14天的阳性率为46.8％(图2中D)；第二组，抗BCMA CAR-T细胞在抗原刺激前(第5天)的阳性率为12.2％(图2中E)，抗原刺激后(第14天)的阳性率为68.2％(图2中F)，是未经抗原刺激的4-6倍。表明，利用本发明的扩增CAR-T细胞的方法可以提高CAR-T细胞的阳性率，说明CAR-T细胞的有效性和特异性得到了极大提高。扩增后的CAR-T细胞，可用于体内外功能检测、杀瘤效果评估级临床应用。The results are shown in Figure 2. In Figure 2, A is the positive rate of anti-BCMA CAR-T cells on the 5th day in the control experiment without adding antigen (that is, the percentage of CAR-T cells expressing anti-BCMA to total CAR-T cells), B is the positive rate of anti-BCMA CAR-T cells on the 14th day in the control experiment without antigen added, C and E are the positive rate of anti-BCMA CAR-T cells on the 5th day in the experiment with added antigen, D and F are the positive rate of anti-BCMA CAR-T cells in the experiment with added antigen Positive rate of anti-BCMA CAR-T cells on day 14. The results showed that in the control experiment without adding antigen, the positive rate of anti-BCMA CAR-T cells was 4.49% on day 5 and 10.5% on day 14; in the experiment adding antigen, the first group, anti-BCMA CAR-T cells - The positive rate of T cells on day 5 was 4.49% (C in Figure 2), and the positive rate on day 14 was 46.8% (D in Figure 2); in the second group, anti-BCMA CAR-T cells were stimulated with antigen The positive rate before (day 5) was 12.2% (E in Figure 2), and the positive rate after antigen stimulation (day 14) was 68.2% (F in Figure 2), which was 4-6 times that without antigen stimulation . It is shown that using the method for expanding CAR-T cells of the present invention can improve the positive rate of CAR-T cells, indicating that the effectiveness and specificity of CAR-T cells have been greatly improved. The expanded CAR-T cells can be used for in vitro and in vivo function detection and tumoricidal effect evaluation-level clinical applications.
<110> 深圳华大生命科学研究院<110> Shenzhen BGI Life Science Research Institute
<120> 一种扩增特异性CAR-T细胞的培养方法<120> A culture method for expanding specific CAR-T cells
<160> 2<160> 2
<170> PatentIn version 3.5<170> PatentIn version 3.5
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ccagaggtgc agctgctgga atctggcgga ggactggtgc agcctggcgg ctctctgaga 120ccagaggtgc agctgctgga atctggcgga ggactggtgc agcctggcgg ctctctgaga 120
ctgtcttgtg ccgccagcgg cttcaccttc agcagctacc ctatgagctg ggtgcgccag 180ctgtcttgtg ccgccagcgg cttcaccttc agcagctacc ctatgagctg ggtgcgccag 180
gcccctggta aaggtttgga atgggtttct gctattggtg gttcaggtgg ttggagttat 240gcccctggta aaggtttgga atgggtttct gctattggtg gttcaggtgg ttggagttat 240
tatgccgatt ctgttaaggg tagattcacc atttctagag acaactctaa gaacaccttg 300tatgccgatt ctgttaaggg tagattcacc atttcagag acaactctaa gaacaccttg 300
tacttgcaaa tgaactcctt gagagctgaa gatactgctg tttattactg tgctagatac 360tacttgcaaa tgaactcctt gagagctgaa gatactgctg tttattactg tgctagatac 360
tggccaatgg attcttgggg tcaaggtact ctggtcaccg tctcctcagg tggaggaggt 420tggccaatgg attcttgggg tcaaggtact ctggtcaccg tctcctcagg tggaggaggt 420
agcggaggag gcgggagcgg tggaggtggc tctgagatcg tgctgacaca gagccctggc 480agcggaggag gcgggagcgg tggaggtggc tctgagatcg tgctgacaca gagccctggc 480
accctgagcc tgtctcctgg tgaaagagct actttgtctt gttggttgtc tcaatctgtt 540accctgagcc tgtctcctgg tgaaagagct actttgtctt gttggttgtc tcaatctgtt 540
tcctctactt acttggcttg gtatcaacaa aaaccaggtc aagctccaag attattgatc 600tcctctactt acttggcttg gtatcaacaa aaaccaggtc aagctccaag attattgatc 600
tacgatgctt cttctagagc accaggtatt ccagatagat tttctggttc tggttccggt 660tacgatgctt cttctagagc accaggtatt ccagatagat tttctggttc tggttccggt 660
actgatttca ctttgactat ctctagattg gaaccagaag atttcgctgt ttactactgc 720actgatttca ctttgactat ctctagattg gaaccagaag atttcgctgt ttactactgc 720
caacaatact ctgagtggcc attgactttt ggtcaaggta caaaggttga aatcaaaacc 780caacaatact ctgagtggcc attgactttt ggtcaaggta caaaggttga aatcaaaacc 780
acgacgccag cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc 840acgacgccag cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc 840
ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag tgcacacgag ggggctggac 900ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag tgcacacgag ggggctggac 900
ttcgcctgtg atatctacat ctgggcgccc ttggccggga cttgtggggt ccttctcctg 960ttcgcctgtg atatctacat ctgggcgccc ttggccggga cttgtggggt ccttctcctg 960
tcactggtta tcacccttta ctgcaaacgg ggcagaaaga aactcctgta tatattcaaa 1020tcactggtta tcacccttta ctgcaaacgg ggcagaaaga aactcctgta tatattcaaa 1020
caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 1080caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 1080
ccagaagaag aagaaggagg atgtgaactg cgcgtgaagt tcagcaggag cgcagacgcc 1140ccagaagaag aagaaggagg atgtgaactg cgcgtgaagt tcagcaggag cgcagacgcc 1140
cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 1200cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 1200
gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 1260gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 1260
aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1320aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1320
tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1380tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1380
cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1440cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1440
cctcgctaa 1449cctcgctaa 1449
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His Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly LeuHis Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
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Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly PheVal Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
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Thr Phe Ser Ser Tyr Pro Met Ser Trp Val Arg Gln Ala Pro Gly LysThr Phe Ser Ser Tyr Pro Met Ser Trp Val Arg Gln Ala Pro Gly Lys
50 55 60 50 55 60
Gly Leu Glu Trp Val Ser Ala Ile Gly Gly Ser Gly Gly Trp Ser TyrGly Leu Glu Trp Val Ser Ala Ile Gly Gly Ser Gly Gly Trp Ser Tyr
65 70 75 8065 70 75 80
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn SerTyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
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Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp ThrLys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
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Ala Val Tyr Tyr Cys Ala Arg Tyr Trp Pro Met Asp Ser Trp Gly GlnAla Val Tyr Tyr Cys Ala Arg Tyr Trp Pro Met Asp Ser Trp Gly Gln
115 120 125 115 120 125
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly GlyGly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140 130 135 140
Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro GlyGly Ser Gly Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly
145 150 155 160145 150 155 160
Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Trp LeuThr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Trp Leu
165 170 175 165 170 175
Ser Gln Ser Val Ser Ser Thr Tyr Leu Ala Trp Tyr Gln Gln Lys ProSer Gln Ser Val Ser Ser Thr Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
180 185 190 180 185 190
Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Ser Arg Ala ProGly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Ser Arg Ala Pro
195 200 205 195 200 205
Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe ThrGly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
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Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr CysLeu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
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Gln Gln Tyr Ser Glu Trp Pro Leu Thr Phe Gly Gln Gly Thr Lys ValGln Gln Tyr Ser Glu Trp Pro Leu Thr Phe Gly Gln Gly Thr Lys Val
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Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala ProGlu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
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Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg ProThr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
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Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys AspAla Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
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Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu LeuIle Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
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Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu LeuSer Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu
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Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln GluTyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
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Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly CysGlu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
355 360 365 355 360 365
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr GlnGlu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380 370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg GluGln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met GlyGlu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415 405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu LeuGly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430 420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys GlyGln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445 435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu SerGlu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460 450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu ProThr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480465 470 475 480
Pro ArgPro Arg
| Application Number | Priority Date | Filing Date | Title |
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| CN201711458709.6ACN109971715A (en) | 2017-12-28 | 2017-12-28 | A culture method for expanding specific CAR-T cells |
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| CN201711458709.6ACN109971715A (en) | 2017-12-28 | 2017-12-28 | A culture method for expanding specific CAR-T cells |
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| CN109971715Atrue CN109971715A (en) | 2019-07-05 |
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| CN201711458709.6APendingCN109971715A (en) | 2017-12-28 | 2017-12-28 | A culture method for expanding specific CAR-T cells |
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