WO2017133222A1 - 检测car-t细胞的捕获探针、该细胞含量的检测方法及应用 - Google Patents

检测car-t细胞的捕获探针、该细胞含量的检测方法及应用 Download PDF

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WO2017133222A1
WO2017133222A1 PCT/CN2016/096469 CN2016096469W WO2017133222A1 WO 2017133222 A1 WO2017133222 A1 WO 2017133222A1 CN 2016096469 W CN2016096469 W CN 2016096469W WO 2017133222 A1 WO2017133222 A1 WO 2017133222A1
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car
ligand
cell
detecting
cells
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French (fr)
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鲁薪安
何霆
马列
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北京艺妙神州医疗科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1486—Counting the particles

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  • the present invention relates to the field of biotechnology, and in particular to a method for detecting a capture probe of a CAR-T cell, a method for detecting the content of the cell, and an application thereof.
  • CAR-T Chimeric Antigen Receptor T-Cell Immunotherapy
  • chimeric antigen receptor T cell immunotherapy is referred to as chimeric antigen receptor T cell immunotherapy.
  • CAR-T cells chimeric antigen receptor T cells
  • the method of gene transduction transfects a patient's T cells to express a chimeric antigen receptor.
  • the CAR-T treatment procedure involves collecting peripheral blood from the patient, isolating T cells, introducing CAR into T cells, quality control, and returning the cells to the patient.
  • CAR is a chimeric gene, which is expressed as a transmembrane protein.
  • the extracellular domain of the CAR expression product is a sequence derived from the Fab region of the antibody that recognizes cancer cell antigens, thereby ensuring that T cells perform precise clearance of target cells.
  • the intracellular region of the CAR expression product can rapidly activate the killing activity of the T cells, which in turn leads to apoptosis and lysis of the target cells.
  • CAR-T therapy in recent years, it has shown excellent therapeutic effects in many clinical trials and is a new potential for cancer treatment.
  • CAR-T therapy resulted in complete remission in 90% (27/30) of subjects (N Engl J Med 2014; 371:1507-17.).
  • CAR-T therapy resulted in complete remission in 88% (14/16) of subjects (Science Translational Medicine 2014; 6: 224).
  • CAR-T therapy is expected to become an important method for the treatment of many advanced cancers, with great clinical significance and market value.
  • mainstream clinical trials use real-time fluorescent quantitative PCR to detect the CAR content in the peripheral blood genome, and indirectly estimate the number of CAR-T cells.
  • the problem is that only the CAR that is successfully expressed can function, and the CAR content in the genome is not directly related to the expression level of CAR.
  • the number of copies of CAR in the genome is related to vector characteristics, virus titer, cell status, etc., which further leads to estimation of CAR-T cell errors by detecting CAR content in the peripheral blood genome. Therefore, accurate and rapid detection of CAR content is critical for the implementation, monitoring, and prognosis of the therapy.
  • the object of the present invention is to provide a capture probe for detecting CAR-T cells, which solves the problem that the detection result is inaccurate by detecting the CAR content in the genome and indirectly estimating the number of CAR-T cells in the prior art.
  • Technical problem is to provide a capture probe for detecting CAR-T cells, which solves the problem that the detection result is inaccurate by detecting the CAR content in the genome and indirectly estimating the number of CAR-T cells in the prior art.
  • a second object of the present invention is to provide a method for detecting the content of CAR-T cells by using the capture probe to solve the technical problem that the detection speed of the existing detection method is slow and the result is unreliable; the method can be implemented quickly and accurately.
  • the detection of CAR-T cells provides a strong guarantee for CAR-T monitoring and prognosis.
  • a third object of the present invention is to provide the use of the above-described capture probe to realize its application value in detecting CAR-T cell content and preparing a product for detecting CAR-T cell content, such as a corresponding kit.
  • the present invention provides a capture probe for detecting CAR-T cells, the capture probe comprising a ligand capable of binding to a CAR-T cell, and a fluorescent molecule bound to the ligand.
  • the capture probe mainly comprises a ligand that can recognize and bind to the CAR-T cell; since the ligand can be efficiently bound to the CAR-T cell, specifically to the CAR molecule (should be understood here)
  • the CAR molecule is the scFv domain of the expression product of the CAR gene, which can play a role in capturing CAR-T cells.
  • the ligand can also bind to fluorescent molecules and then flow cytometry. Quantify CAR-T cells quickly and accurately.
  • the capture probe direct quantitative detection of CAR-T cells can be achieved, and the binding rate of the CAR-T cells can be increased by the preferred ligand, thereby accurately and rapidly achieving the quantitative detection of CAR-T cells; Detection of the CAR gene contained in the genome (the CAR content in the genome is not directly related to the expression level of CAR, and only the CAR that successfully expressed can play Compared with the way of indirectly estimating CAR-T cells, it also directly avoids estimation errors that may be caused by factors such as carrier characteristics, virus titer, and cell state.
  • the ligand comprises a first ligand and a second ligand
  • the first ligand is capable of binding to a CAR molecule scFv domain; and the first ligand is further capable of binding biotin; the second ligand is capable of recognizing the biotin, and the fluorescent molecule is capable of binding On the second ligand.
  • the ligand is further refined, the first ligand can bind to the scFv domain of the CAR molecule, and can also be stably labeled with biotin, and the second ligand can recognize biotin, thereby It acts as a capture and the second ligand can be labeled with fluorescent molecules for flow quantitative detection.
  • the first ligand comprises a protein capable of binding to a light chain or a heavychain of IgG; and/or the second ligand is a protein or polypeptide capable of binding biotin, the fluorescent molecule comprising FITC, PE Any of PE-CYx and APC.
  • the first ligand comprises Protein L, Protein A, Protein G, and variants of the several proteins; and/or the second ligand is streptavidin.
  • Protein L, Protein A, Protein G and their variants can bind to the light chain or heavy chain of IgG, so they can bind to the scFv domain of CAR molecule; streptavidin can be effectively combined Avidin molecules such as biotin.
  • a method for detecting CAR-T cell content based on flow detection comprising the steps of:
  • test sample is added to the ligand to which the fluorescent molecule is bound to be mixed, so that the ligand is bound to the CAR-T cell in the sample to be tested; then the supernatant is centrifuged and discarded, and the precipitate is retained; after the precipitate is resuspended Flow cytometry was performed to obtain CAR-T cell content.
  • a ligand to which a fluorescent molecule is previously bound is mixed with a sample to be tested and then bound to a CAR-T cell in the sample (specifically, a scFv domain of a CAR molecule), when a sufficient amount of the ligand is added, It can basically capture all CAR-T cells expressing CAR successfully. After centrifugation, the precipitate can be flow-detected after resuspension, so that the content of CAR-T cells in the sample can be quickly and accurately obtained.
  • the method specifically includes the following steps:
  • the temperature and time in different binding steps are specifically selected, and the washing operation is performed before resuspension, thereby improving the purity of the target substance in the precipitation and increasing the accuracy of the detection result.
  • the washing is carried out using a flow buffer.
  • the centrifugal force of the first centrifugation is 500-1000 g, and the time is 5-10 min; the centrifugal force of the second centrifugation is 500-1000 g, and the time is 5-10 min.
  • centrifugation In the process of centrifugation, the selection of the centrifugal conditions is crucial.
  • the proper centrifugal speed and temperature can ensure the solid-liquid separation effect while ensuring the integrity of the cell structure and preventing the cells from being improperly selected due to temperature and speed.
  • the structure caused damage.
  • the capture probe can achieve, its use in detecting CAR-T cell content and preparing products for detecting CAR-T cell content is also within the scope of the present invention, for example, based on the capture.
  • a capture probe for detecting CAR-T cells a method for detecting the content of the cell, and an application thereof have the beneficial effects of:
  • the capture probe and the method based on flow detection of CAR-T cells combined with the probe can quickly and accurately realize the content of CAR-T cells in the sample to be tested, and the whole detection method is simple and convenient, and is CAR-
  • the implementation, monitoring and prognosis of T therapy provide a strong guarantee
  • FIG. 1 is a result of detecting a CAR-T cell in vitro by a capture probe for detecting CAR-T cells according to an embodiment of the present invention
  • FIG. 2 is a detection result of detecting a CAR-T cell capture probe for a patient after returning CAR-T according to an embodiment of the present invention.
  • S11 providing a capture probe for detecting flow detection of CAR-T cells
  • the capture probe comprises a ligand protein (Protein L) that binds to CAR (expression product), which is bound to the fluorescently labeled molecule FITC (fluorescein isothiocyanate).
  • CAR expression product
  • FITC fluorescently labeled molecule
  • the cells to be detected are cultured CAR-T cells in vitro.
  • step S14 The product of step S13 is centrifuged and the supernatant is discarded, the precipitate is retained, and the precipitate is resuspended and subjected to flow detection.
  • S21 providing a capture probe for detecting flow detection of CAR-T cells
  • the capture probe comprises a first ligand protein Protein L that binds to CAR (expression product), which is labeled with biotin; in addition, a second ligand protein (streptavidin) capable of recognizing biotin and A fluorescent molecule (PE, phycoerythrin) labeled on the streptavidin.
  • CAR expression product
  • streptavidin second ligand protein capable of recognizing biotin and A fluorescent molecule (PE, phycoerythrin) labeled on the streptavidin.
  • the sample to be tested is prepared by the following method:
  • PBMC mononuclear cells
  • biotin is as follows:
  • S31 providing a capture probe for detecting flow detection of CAR-T cells
  • the capture probe comprises a first ligand protein (Protein A) that binds to CAR (expression product), which is labeled with biotin; in addition, a second ligand protein (streptavidin) capable of recognizing biotin And a fluorescent molecule APC labeled on the streptavidin.
  • a first ligand protein Protein A
  • CAR expression product
  • streptavidin second ligand protein
  • step S22 It is the same as step S22 and will not be described here.
  • the capture probe comprises a first ligand protein (Protein L) that binds to CAR (expression product), which is labeled with biotin; in addition, a second ligand protein (streptavidin) capable of recognizing biotin And a fluorescent molecule PE-CY5 labeled on the streptavidin.
  • a first ligand protein Protein L
  • CAR expression product
  • streptavidin second ligand protein
  • step S22 It is the same as step S22 and will not be described here.
  • S51 providing a capture probe for detecting flow detection of CAR-T cells
  • the capture probe comprises a first ligand protein (Protein L) that binds to CAR (expression product), which is labeled with biotin; in addition, a second ligand protein (streptavidin) capable of recognizing biotin And the fluorescent molecule FITC labeled on the streptavidin.
  • a first ligand protein Protein L
  • CAR expression product
  • streptavidin second ligand protein
  • the cells to be detected are cultured CAR-T cells in vitro, and the following steps are specifically adopted:
  • PBMC Mononuclear cells
  • the centrifugal force for the first centrifugation was 800 g, and the centrifugation time was 8 min; the centrifugal force for the second centrifugation was 800 g, and the centrifugation time was 6 min.
  • S61 providing a capture probe for detecting flow detection of CAR-T cells
  • S62 providing a cell to be detected, wherein the cell to be detected expresses CAR;
  • the cells to be detected are cultured CAR-T cells in vitro and peripheral blood samples after patient CAR-T reinfusion.
  • the second precipitate is washed with a flow buffer, centrifuged, and the resulting precipitate is resuspended in a flow buffer and subjected to flow detection.
  • the wavelength of the flow detection is set according to the type of the fluorescent dye.
  • amino acid sequence of Protein L and the amino acid sequence of streptavidin are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • the CAR-T cell samples cultured in vitro were tested by the capture probe provided in Example 6 and the detection method thereof, and the culture samples of the control group, the 7th day, the 8th day, and the 10th day were respectively set.
  • the test results are shown in Figure 1.
  • the obtained cord blood mononuclear cells were counted, and the beads of the EUCD3/CD28 antibody were added in a ratio of 1:1, gently shaken for 20 min, and CD3-positive cells were obtained by using a magnetic stand.
  • the CD3 positive cells obtained in the step 2) were cultured using Lonza x-vivo 15 medium (containing cytokines such as IL-2 and inactivating AB plasma). After 48 hours, the cultured cells were infected with MOI (20-50) using a slow virus containing CAR. After 12 hours, the whole amount of liquid was changed and the culture was continued.
  • Lonza x-vivo 15 medium containing cytokines such as IL-2 and inactivating AB plasma.
  • quality control is required, including the following aspects: 1 detection of protoplasts; 2 detection of chlamydia; 3 detection of endotoxin; 4 detection of bacteria; 5 detection of fungi; 7CAR expression rate; 8T cell property detection; 9 in vitro killing ability detection.
  • the content of the CAR-T cells of the returned patient was detected by the capture probe provided in Example 6 and the detection method thereof, and the test samples were respectively set in the control group and the samples were returned for 8 days.
  • the test results are shown in Figure 2.
  • Cytokine storms are also called cytokine release syndrome.
  • T cells release many proteins (cytokines) when they kill other cells, such as bacterial viruses. They activate more immune cells to fight against pathogens. This positive feedback mechanism ensures rapid pathogens. Clear.
  • the method for rapidly and effectively detecting the CAR-T cell content provided by the present invention (the direct purpose is not to obtain a diagnosis result or a patient's health state, but to obtain intermediate information) is well-used for clinical use of a patient. monitor.

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Abstract

提供了一种检测CAR-T细胞的捕获探针及其应用，所述捕获探针包括能够结合CAR-T细胞的配体以及结合在该配体上的荧光分子，该探针可快速准确地实现CAR-T细胞的检测。

Description

检测CAR-T细胞的捕获探针、该细胞含量的检测方法及应用 技术领域
本发明涉及生物技术领域，具体而言，涉及一种检测CAR-T细胞的捕获探针、该细胞含量的检测方法及应用。
背景技术
CAR-T，即Chimeric Antigen Receptor T-Cell Immunotherapy，全称为嵌合抗原受体T细胞免疫疗法。CAR-T细胞(嵌合抗原受体T细胞)是通过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。
患者的T细胞被“重编码”后，生成大量肿瘤特异性的CAR-T细胞。和其他免疫疗法类似，它的基本原理就是利用病人自身的免疫细胞来清除癌细胞，但是不同的是，这是一种细胞疗法，而不是一种药。
更为具体地，在实施的过程中，CAR-T治疗流程包括采集患者外周血、分离T细胞、将CAR导入T细胞、质量控制以及将细胞回输至患者。其中，CAR是一段嵌合基因，表达后为一个跨膜蛋白。CAR表达产物的胞外区是源自抗体Fab区的一段序列，可以识别癌细胞抗原，从而保证T细胞对靶细胞实施精准的清除。当CAR-T细胞识别靶细胞后，CAR表达产物的胞内区可以迅速激活T细胞的杀伤活性，进而导致靶细胞凋亡和裂解。
对于CAR-T疗法，近年来，其在多个临床试验中展现出了极佳的治疗效果，是非常具有潜力的癌症治疗新途径。在一项针对难治急性淋巴细胞白血病的临床试验中，CAR-T疗法使90％(27/30)的受试者得到完全缓解(N Engl J Med 2014；371:1507-17.)。在另一项针对成人难治急性淋巴细胞白血病的临床试验中，CAR-T疗法使88％(14/16)的受试者得到完全缓解(Science Translational Medicine 2014；6:224)。
此外，还有数十个针对多发性骨髓瘤、淋巴瘤、肺癌、肝癌、乳腺癌等疾病的CAR-T疗法正在临床试验中。未来，CAR-T疗法有望成为多种晚期癌症治疗的重要方法，临床意义和市场价值巨大。
然而，CAR基因能否表达、表达效率的高低、CAR-T细胞的活性和纯度都会直接影响CAR-T细胞对癌细胞的清除效率。临床研究证明，CAR-T细胞回输后在患者外周血中的增殖能力与疗效具有很强的相关性(ScienceTranslational Medicine 2015；7:303)。
目前，主流的临床试验采用实时荧光定量PCR的方法检测外周血基因组中的CAR含量，进而间接估算CAR-T细胞数量。但是，问题在于，只有成功表达的CAR才能发挥作用，而基因组中的CAR含量与CAR的表达水平并不直接相关。此外，CAR在基因组中的拷贝数与载体特征、病毒滴度、细胞状态等都有关系，会进一步导致通过检测外周血基因组中的CAR含量估算CAR-T细胞的误差。因此，准确、快速的检测CAR含量对于该疗法的实施、监测以及预后具有关键意义。
有鉴于此，特突出本发明。
发明内容
本发明的目的在于提供一种用于检测CAR-T细胞的捕获探针，以解决现有技术中通过检测基因组中CAR含量进而间接估算CAR-T细胞数量所存在的误差大，检测结果不准确的技术问题。
本发明的第二目的在于提供一种利用该捕获探针流式检测CAR-T细胞含量的方法，以解决现有检测方法检测速度慢，结果不可靠的技术问题；该方法可快速准确地实现CAR-T细胞的检测，为CAR-T监测、预后提供有力保障。
本发明的第三目的在于提供上述的捕获探针的用途，以实现其在检测CAR-T细胞含量、制备用于检测CAR-T细胞含量的产品(如相应的试剂盒)中的应用价值。
为了实现以上目的，本发明采用的技术方案如下：
本发明提供了一种检测CAR-T细胞的捕获探针，所述捕获探针包括能够结合在CAR-T细胞的配体，以及结合在该配体上的荧光分子。
在本发明中，捕获探针主要包括可以识别并结合在CAR-T细胞的配体；由于该配体可以有效地结合在CAR-T细胞上，具体为结合到CAR分子(应理解，此处的CAR分子是CAR基因的表达产物)的scFv结构域上，进而可起到捕获CAR-T细胞的作用，另一方面由于该配体还可以与荧光分子结合，进而通过流式细胞仪即可快速准确地对CAR-T细胞定量。
通过该捕获探针，可实现CAR-T细胞的直接定量检测，而且通过优选配体，增大其与CAR分子的结合率，从而准确快速地实现CAR-T细胞的定量检测；与传统的通过基因组中所含CAR基因的检测(基因组中的CAR含量与CAR的表达水平并不直接相关，而只有成功表达的CAR才能发挥 作用)并间接估算CAR-T细胞的方式相比，还直接避免了载体特征、病毒滴度、细胞状态等因素可能会导致的估算误差。
优选的，所述配体包括第一配体和第二配体；
所述第一配体能够结合在CAR分子scFv结构域上；且第一配体还能结合有生物素；所述第二配体能够识别所述生物素，且所述荧光分子能够结合在所述第二配体上。
上述的方案中，对配体进行进一步的细化，第一配体其可以结合在CAR分子scFv结构域上，而且还可以稳定地被生物素标记，而第二配体可识别生物素，从而起到捕获作用，且第二配体可被荧光分子标记，以实现流式定量检测。
优选的，所述第一配体包括能够结合在IgG的light chain或者heavychain上的蛋白；和/或所述第二配体为能够结合生物素的蛋白或多肽，所述荧光分子包括FITC、PE、PE-CYx、APC中的任一种。
优选的，所述第一配体包括Protein L、Protein A、Protein G以及该几种蛋白的变体；和/或所述第二配体为链霉亲和素。
上述方案中，通过优选的配体蛋白、荧光分子从而实现捕获探针与被检测细胞更好的结合效果，以进一步提高检测的准确性。
其中，Protein L、Protein A、Protein G以及它们的变体，均能结合在IgG的light chain或者heavy chain上，因此能结合到CAR分子的scFv结构域上；链霉亲和素可以有效地结合生物素等亲和素分子。
一种基于流式检测CAR-T细胞含量的方法，包括以下步骤：
将待检样本中加入结合有荧光分子的配体进行混合，使得配体结合在待检样本中的CAR-T细胞上；然后离心并弃去上清，保留沉淀物；将沉淀物重悬后进行流式检测，获得CAR-T细胞含量。
在检测方法中，预先结合有荧光分子的配体与待检样本混合后结合在样本中的CAR-T细胞上(具体为CAR分子的scFv结构域上)，当所加的配体足够量时，其基本可以将表达CAR成功的所有CAR-T细胞捕获，离心后沉淀物经过重悬即可进行流式检测，进而可以快速准确地获知样本中CAR-T细胞的含量。
优选的，所述方法具体包括以下步骤：
1)在流式缓冲液中将待检样本与结合有生物素的第一配体混合，于2-8℃结合15-45分钟后进行第一次离心，得到第一沉淀物；
2)将第一沉淀物洗涤后利用流式缓冲液重悬，再加入结合有荧光分子的第二配体，并于2-8℃结合15-45分钟后进行第二次离心，得到第二沉淀物；
3)将第二沉淀物洗涤后离心，并将所得沉淀利用流式缓冲液重悬后进行流式检测。
上述具体的检测方法中，对不同结合步骤中的温度和时间进行了特定的选择，并且重悬之前进行了洗涤操作，从而提高沉淀中目标物质的纯度，增加检测结果的准确性。
优选的，在步骤2)和步骤3)中，所述洗涤均采用流式缓冲液进行。
优选的，所述第一次离心的离心力为500-1000g，时间为5-10min；所述第二次离心的离心力为500-1000g，时间为5-10min。
在离心的过程中，对于离心条件的选择是至关重要的，适当的离心速度和温度可保证固液分离效果的同时，还保证细胞结构的完整性，防止由于温度和速度选择不当而对细胞结构造成破坏。
本发明提供的捕获探针在检测CAR-T细胞含量中的应用。
本发明提供的捕获探针在制备用于检测CAR-T细胞含量的产品中的应用。
鉴于该捕获探针所能达到的技术效果，其在检测CAR-T细胞含量以及制备用于检测CAR-T细胞含量的产品中的应用也理应属于本发明的保护范围之内，例如依据该捕获探针制成的用于检测CAR-T细胞含量的试剂盒。
根据本发明提供的检测CAR-T细胞的捕获探针、该细胞含量的检测方法及应用，有益效果在于：
(1)该捕获探针以及结合该探针开展的基于流式检测CAR-T细胞含量的方法可以快速准确地实现待检样本中CAR-T细胞的含量，整个检测方法操作简便，为CAR-T疗法的实施、监测以及预后提供了有力保障；
(2)该捕获探针应用到CAR-T细胞检测以及制备相应的检测产品后，可有效推动CAR-T疗法的实施，助于人类早日攻克癌症。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，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他的附图。
图1为本发明实施例提供的检测CAR-T细胞的捕获探针对体外培养CAR-T细胞的检测结果；
图2为本发明实施例提供的检测CAR-T细胞的捕获探针对患者回输CAR-T后的检测结果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将对本发明的技术方案进行清楚、完整的描述，基于本发明中的具体实施方式，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其他实施方式，都属于本发明所保护的范围。
实施例1
本实施例提供的用于检测CAR-T细胞的捕获探针以及利用该捕获探针基于流式检测CAR-T细胞含量的方法如下：
S11：提供检测流式检测CAR-T细胞的捕获探针；
该捕获探针包括结合CAR(表达产物)的配体蛋白(Protein L)，该蛋白结合有荧光标记分子FITC(异硫氰酸荧光素)。
S12：提供待检测细胞，该待检测细胞表达CAR；
该待检测细胞为体外培养CAR-T细胞。
S13：在流式缓冲液中将待检细胞样本中加入配体蛋白进行混合，使得配体结合在待检样本中的CAR-T细胞上；
S14：将步骤S13的产物离心并弃去上清，保留沉淀物，并将沉淀物重悬后进行流式检测。
实施例2
本实施例提供的用于检测CAR-T细胞的捕获探针以及利用该捕获探针基于流式检测CAR-T细胞含量的方法如下：
S21：提供检测流式检测CAR-T细胞的捕获探针；
该捕获探针包括结合CAR(表达产物)的第一配体蛋白Protein L，该蛋白上标记有生物素；此外，还包括能够识别生物素的第二配体蛋白(链霉亲和素)以及标记在该链霉亲和素上的荧光分子(PE，藻红蛋白)。
S22：提供待检测细胞，该待检测细胞表达CAR；
在该步骤中，待检样品通过以下方法制备：
1)从外周血中分离出单个核细胞(PBMC)；
2)从PBMC中分选出T细胞；
3)利用慢病毒转染体系对T细胞进行基因改造，制备多种不同靶点不同代数CAR-T。
S23：在流式缓冲液中将待检样本与结合有生物素的Protein L混合，于4℃结合30分钟后进行第一次离心，得到第一沉淀物；
S24：将第一沉淀物洗涤后利用流式缓冲液重悬，再加入结合有藻红蛋白的链霉亲和素，并于4℃结合30分钟后进行第二次离心，得到第二沉淀物；
S25：将第二沉淀物洗涤后离心，并将所得沉淀利用流式缓冲液重悬后进行流式检测。
在上述的实施例中，生物素的分子结构如下：
Figure PCTCN2016096469-appb-000001
实施例3
本实施例提供的用于检测CAR-T细胞的捕获探针以及利用该捕获探针基于流式检测CAR-T细胞含量的方法如下：
S31：提供检测流式检测CAR-T细胞的捕获探针；
该捕获探针包括结合CAR(表达产物)的第一配体蛋白(Protein A)，该蛋白上标记有生物素；此外，还包括能够识别生物素的第二配体蛋白(链霉亲和素)以及标记在该链霉亲和素上的荧光分子APC。
S32：提供待检测细胞，该待检测细胞表达CAR；
与步骤S22一致，在此不做赘述。
S33：在流式缓冲液中将待检样本与结合有生物素的Protein A混合，于8℃结合40分钟后进行第一次离心，得到第一沉淀物；
S34：将第一沉淀物洗涤后利用流式缓冲液重悬，再加入结合有APC的链霉亲和素，并于8℃结合40分钟后进行第二次离心，得到第二沉淀物；
S35：将第二沉淀物洗涤后离心，并将所得沉淀利用流式缓冲液重悬后进行流式检测。
实施例4
本实施例提供的用于检测CAR-T细胞的捕获探针以及利用该捕获探针基于流式检测CAR-T细胞含量的方法如下：
S41：提供检测流式检测CAR-T细胞的捕获探针；
该捕获探针包括结合CAR(表达产物)的第一配体蛋白(Protein L)，该蛋白上标记有生物素；此外，还包括能够识别生物素的第二配体蛋白(链霉亲和素)以及标记在该链霉亲和素上的荧光分子PE-CY5。
S42：提供待检测细胞，该待检测细胞表达CAR；
与步骤S22一致，在此不做赘述。
S43：在流式缓冲液中将待检样本与结合有生物素的Protein L混合，于2℃结合45分钟后进行第一次离心，得到第一沉淀物；
S44：将第一沉淀物洗涤后利用流式缓冲液重悬，再加入结合有PE-CY5的链霉亲和素，并于2℃结合45分钟后进行第二次离心，得到第二沉淀物；
S45：将第二沉淀物洗涤后离心，并将所得沉淀利用流式缓冲液重悬后进行流式检测。
实施例5
本实施例提供的用于检测CAR-T细胞的捕获探针以及利用该捕获探针基于流式检测CAR-T细胞含量的方法如下：
S51：提供检测流式检测CAR-T细胞的捕获探针；
该捕获探针包括结合CAR(表达产物)的第一配体蛋白(Protein L)，该蛋白上标记有生物素；此外，还包括能够识别生物素的第二配体蛋白(链霉亲和素)以及标记在该链霉亲和素上的荧光分子FITC。
S52：提供待检测细胞，该待检测细胞表达CAR；
该待检测细胞为体外培养CAR-T细胞，具体采用以下步骤实现：
从外周血中分离出单个核细胞(PBMC)，并从PBMC中分选出T细胞；利用逆转录病毒转染体系对T细胞进行基因改造，制备多种不同靶点不同代数CAR-T细胞。
S53：在流式缓冲液中将待检样本与结合有生物素的Protein L混合，于4℃结合35分钟后进行第一次离心，得到第一沉淀物；
S54：将第一沉淀物洗涤后利用流式缓冲液重悬，再加入结合有异硫氰酸荧光素的链霉亲和素，并于4℃结合30分钟后进行第二次离心，得到第二沉淀物；
S55：将第二沉淀物洗涤后离心，并将所得沉淀利用流式缓冲液重悬后进行流式检测。
在上述的实施例中，第一次离心的离心力为800g，离心时间为8min；第二次离心的离心力为800g，离心时间为6min。
实施例6
本实施例提供的用于检测CAR-T细胞的捕获探针以及利用该捕获探针基于流式检测CAR-T细胞含量的方法如下：
S61：提供检测流式检测CAR-T细胞的捕获探针；
与实施例5提供的捕获探针一致。
S62：提供待检测细胞，该待检测细胞表达CAR；
该待检测细胞为体外培养CAR-T细胞和患者CAR-T回输后的外周血样本。
S63：在流式缓冲液中将待检样本与结合有生物素的Protein L混合，于4℃结合35分钟后进行第一次离心(500-800g，5-10min)，得到第一沉淀物；
S64：将第一沉淀物以流式缓冲液洗涤后离心，再用流式缓冲液重悬，然后加入结合有异硫氰酸荧光素的链霉亲和素，并于4℃结合35分钟后进行再度离心(500-800g，5-10min)，得到第二沉淀物；
S65：将第二沉淀物以流式缓冲液洗涤后离心，并将所得沉淀利用流式缓冲液重悬后进行流式检测，流式检测的波长视荧光染料的种类设定。
另外，需要指出的是，在上述的所有实施例中，Protein L的氨基酸序列以及链霉亲和素的氨基酸序列表分别如SEQ ID NO:1和SEQ ID NO:2所示。
应用例1
以实施例6提供的捕获探针以及其检测方法对经过体外构建培养的CAR-T细胞样品进行检测，分别设置对照组、第7天、第8天以及第10天的培养样本。检测结果如图1所示。
通过图1可以看出，体外培养的CAR-T细胞，随着培养时间的增长，其CAR-T细胞含量持续稳定增大。
在该应用例中，体外构建培养的CAR-T细胞样品的具体制备以及培养方法如下：
1)单核细胞的制备
用移液管吸取DPBS或者生理盐水加入到采集的外周血中(1:1)，稀释血细胞，将血细胞稀释液缓慢加入装有淋巴细胞分离液(Ficoll或者 Histopaque-1077)的离心管中，800g离心20-30分钟后，吸取淋巴细胞分离液上方的白膜层细胞，转入一个新的离心管中，加入Lonza x-vivo 15无血清细胞培养基，离心后弃上清，保留离心管底部的细胞沉淀，即得到脐带血单核细胞。
2)将CD3阳性细胞从脐带血源的单核细胞中富集
将得到的脐带血单核细胞进行计数，按照1:1的比例加入欧联CD3/CD28抗体的beads，轻轻震荡20min，利用磁力架，得到CD3阳性的细胞。
3)制备CAR-T
将步骤2)中得到的CD3阳性细胞利用Lonza x-vivo 15培养基(含IL-2等细胞因子以及灭活AB血浆)进行培养。48小时后，利用含CAR的慢病毒以MOI(20-50)对培养的细胞进行感染。12小时后，进行全量换液，继续培养。
4)CAR-T质量控制
在CAR-T培养过程以及细胞制品制备时，需对其进行质量控制，包含如下几个方面：①支原体检测；②衣原体检测；③内毒素检测；④细菌检测；⑤真菌检测；⑥病毒残留检测；⑦CAR表达率；⑧T细胞性质检测；⑨体外杀伤能力检测。
应用例2
以实施例6提供的捕获探针以及其检测方法对回输患者的CAR-T细胞的含量进行检测，检测样本分别设置对照组、回输8天样本。检测结果如图2所示。
从图2可以看出，回输CAR-T8天之后，患者体内所含的CAR-T细胞较对照组相比，CAR-T细胞含量增长明显。
CAR-T疗法在应用时，病人接受CAR-T疗法也潜在的临床风险-细胞因子风暴。细胞因子风暴也叫细胞因子释放综合征。产生的原因是T细胞在杀死其它细胞，比如细菌病毒的时候会释放很多蛋白(细胞因子)，它们可激活更多的免疫细胞来一起对抗病原体，这种正反馈机制保证了对病原体的快速清除。
然而由于CAR-T杀癌细胞实在是太快太有效，于是会瞬间在局部产生超大量的细胞因子，并引起惊人的免疫反应(有时临床表现就是病人高烧不退)，如果控制不好，则病人存在很大的风险。因此通过本发明提供的这种快速有效地检测CAR-T细胞含量(直接目的并不是为了获得诊断结果或者患者的健康状态，而是获取中间信息)的方法，可很好的用于患者的临床监控。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种用于检测CAR-T细胞的捕获探针，其包括能够结合在CAR-T细胞的配体，以及结合在该配体上的荧光分子。
  2. 根据权利要求1所述的捕获探针，其中，所述配体包括第一配体和第二配体；
    所述第一配体能够结合在CAR分子scFv结构域上；且第一配体还能结合有生物素；所述第二配体能够识别所述生物素，且所述荧光分子能够结合在所述第二配体上。
  3. 根据权利要求2所述的捕获探针，其中，所述第一配体包括能够结合在IgG的light chain或者heavy chain上的蛋白；
    和/或所述第二配体为能够结合生物素的蛋白或多肽，所述荧光分子包括FITC、PE、PE-CYx、APC中的任一种。
  4. 根据权利要求3所述的捕获探针，其中，所述第一配体包括Protein L、Protein A、Protein G以及它们的变体；
    和/或所述第二配体为链霉亲和素。
  5. 一种基于流式检测CAR-T细胞含量的方法，其包括以下步骤：
    将待检样本中加入结合有荧光分子的配体进行混合，使得配体结合在待检样本中的CAR-T细胞上；然后离心并弃去上清，保留沉淀物；将沉淀物重悬后进行流式检测，获得CAR-T细胞含量。
  6. 根据权利要求5所述的检测CAR-T细胞含量的方法，其中，所述方法具体包括以下步骤：
    1)在流式缓冲液中将待检样本与结合有生物素的第一配体混合，于2-8℃结合15-45分钟后进行第一次离心，得到第一沉淀物；
    2)将第一沉淀物洗涤后利用流式缓冲液重悬，再加入结合有荧光分子的第二配体，并于2-8℃结合15-45分钟后进行第二次离心，得到第二沉淀物；
    3)将第二沉淀物洗涤后离心，并将所得沉淀利用流式缓冲液重悬后进行流式检测。
  7. 根据权利要求6所述的检测CAR-T细胞含量的方法，其中，在步骤2)和步骤3)中，所述洗涤均采用流式缓冲液进行。
  8. 根据权利要求6或7所述的检测CAR-T细胞含量的方法，其中，所述第一次离心的离心力为500-1000g，时间为5-10min；所述第二次离心的离心力为500-1000g，时间为5-10min。
  9. 根据权利要求1-4中任一项所述的捕获探针在检测CAR-T细胞含量中的应用。
  10. 根据权利要求1-4中任一项所述的捕获探针在制备用于检测CAR-T细胞含量的产品中的应用。
PCT/CN2016/096469 2016-02-04 2016-08-24 检测car-t细胞的捕获探针、该细胞含量的检测方法及应用 WO2017133222A1 (zh)

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