WO2019129055A1 - 一种锁核酸修饰的探针以及一种测定car拷贝数的方法 - Google Patents

一种锁核酸修饰的探针以及一种测定car拷贝数的方法 Download PDF

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WO2019129055A1
WO2019129055A1 PCT/CN2018/123890 CN2018123890W WO2019129055A1 WO 2019129055 A1 WO2019129055 A1 WO 2019129055A1 CN 2018123890 W CN2018123890 W CN 2018123890W WO 2019129055 A1 WO2019129055 A1 WO 2019129055A1
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car
probe
cd3zeta
copy number
nucleic acid
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PCT/CN2018/123890
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French (fr)
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钱其军
金华君
郝方元
王超
孙娟娟
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上海细胞治疗研究院
上海细胞治疗集团有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

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  • the invention belongs to the field of biotechnology, and relates to a locked nucleic acid modified probe and a method for determining CAR copy number.
  • the invention relates to a method for determining the copy number of a second generation CAR or a third generation CAR comprising a CD28-CD3zeta and CD137-CD3zeta signaling region using an LNA-TaqMan probe.
  • Chimeric Antigen Receptor T-Cell (CAR-T) immunotherapy is one of the most promising methods for tumor suppression in tumor immunotherapy.
  • the US FDA approved the marketing of Novartis's CAR-T therapy Kymriah (formerly known as CTL-019) and Kite Pharma's CAR-T therapy Yescarta (axicabtagene ciloleucel) for the treatment of children.
  • the listing of CAR-T drugs indicates the regulatory approval of the clinical efficacy and clinical safety of CAR-T.
  • the great success of CAR-T in hematoma has greatly contributed to the application of CAR-T therapy.
  • the current application of CAR-T therapy is not limited to the treatment of hematological tumors.
  • CD28 and CD137 are two T cell costimulatory molecules that play an important role in the activation and proliferation of T cells and are one of the important components of the second generation CAR and the third generation CAR.
  • Real-time quantitative PCR is the addition of a fluorescent group to a DNA amplification reaction, and the accumulation of fluorescent signals is used to monitor the change of the amount of PCR products in real time.
  • Real-time fluorescent quantitative PCR is widely used in the detection field because of its high sensitivity, good repeatability, simple operation and low cost.
  • Real-time quantitative PCR can be used to perform copy number detection on CAR in CAR-T at the molecular level, and provide technical support for clinical control of CAR-T and in vitro monitoring of clinical treatment.
  • the inventors have obtained a primer pair and a probe through intensive research and creative labor. On the basis of this, the inventors obtained a method and a kit for measuring CAR copy number by real-time PCR.
  • the method or kit of the present invention is capable of determining the copy number of CAR in the prepared CAR-T, or the copy number of CAR in peripheral blood cells of a CAR-T treated patient, for controlling the quality of CAR-T, and evaluating the transduction efficiency of CAR. Or in vitro monitoring of clinical treatment.
  • the following invention is thus provided:
  • One aspect of the invention relates to a primer pair or combination of primer pairs selected from any one or both of the following two primer pairs:
  • first and second in the above “first primer pair” and “second primer pair” are merely for the purpose of distinguishing, and do not have the meaning of order.
  • Another aspect of the invention relates to a probe or probe combination selected from any one or both of the following two probes:
  • the second probe has the nucleic acid sequence shown as SEQ ID NO: 6.
  • first and second in the above “first probe” and “second probe” are merely for the purpose of distinguishing, and do not have the meaning of order.
  • the probe is labeled with a fluorescent reporter group at the 5' end and a fluorescent quencher group at the 3' end; preferably, the fluorescent reporter group is selected from the group consisting of FAM And Hex, VIC, ROX and Cy5; preferably, the fluorescent quenching group is selected from the group consisting of BHQ1, TAMRA, JOE, BHQ2 and BHQ3.
  • the probe or probe combination wherein
  • the base of one or more positions in the first probe and/or the second probe is modified by a locked nucleic acid (ie, one or more bases in the first probe and/or the second probe are introduced into one or Multiple locked nucleic acid monomers);
  • any one, two, three or four bases of the 4th, 7th, 10th and 13th positions of the 5' end of the first probe are modified by a locked nucleic acid; and / Alternatively, any one, two, three or four bases of the 4th, 7th, 10th, and 13th positions at the 5' end of the second probe are modified with a locked nucleic acid. .
  • a further aspect of the invention relates to a kit comprising the primer pair of any of the invention, and/or the probe of any of the invention.
  • the kit comprises:
  • the kit further comprises reagents required for the PCR reaction, such as Mg 2+ , reaction buffer, dNTP and Taq enzyme.
  • reagents required for the PCR reaction such as Mg 2+ , reaction buffer, dNTP and Taq enzyme.
  • a further aspect of the invention relates to a method of determining a CAR copy number, comprising the steps of:
  • the sample to be tested in step (1) is a CAR-T or CAR-T transfused patient's peripheral blood transduced by a CAR plasmid;
  • the gradient dilution in step (2) is a 10-fold gradient dilution
  • the conditions of the amplification reaction of the real-time fluorescent quantitative PCR in the step (3) are: 94 ° C for 5 min; 94 ° C for 20 s, 60 ° C for 1 min, for a total of 40 cycles.
  • a further aspect of the invention relates to a method of quality control of a CAR-T comprising the steps of:
  • the CAR copy number is determined in the step A, and the method for determining the CAR copy number described in the present invention is used.
  • the CAR contains a "CD28-CD3zeta signal region" and/or a "CD137-CD3zeta signal region”.
  • the CAR-T contains the CAR.
  • the method of the invention can be used for production control of CAR-T or in vitro monitoring after CAR-T reinfusion, for example for detecting CAR copy number in peripheral blood cells of a prepared CAR-T or CAR-T treated patient. It can be further used to evaluate the transduction efficiency, therapeutic efficacy or side effects of CAR, or to evaluate the correlation between CAR copy number in peripheral blood CAR-T and therapeutic efficacy or side effects, so that clinical treatment can adjust the treatment plan in time.
  • a further aspect of the invention relates to a primer set according to the invention and/or a probe according to any one of the invention for use in the preparation of a medicament for the determination of CAR copy number or for the preparation of a medicament for quality control of CAR-T the use of.
  • the CAR contains a "CD28-CD3zeta signal region" and/or a "CD137-CD3zeta signal region”.
  • the CAR-T contains the CAR.
  • CAR Chimeric Antigen Receptor
  • TAA tumor associated antigen
  • CAR-T is a cell expressing the CAR gene obtained by introducing a CAR gene into a T cell by a technique of gene transduction/transfection. Such cells have the ability to recognize and attack tumor cells that express TAA on the corresponding cell surface.
  • second generation CAR The first generation CAR fused the immunoglobulin scFv and the Fc[epsilon]RI receptor or the intracellular domain of the CD3 complex to form a chimeric receptor.
  • the second generation of CAR is based on the first generation of CAR structure, adding a new co-stimulatory signal, such as CD28 or CD137.
  • third generation CAR Based on the second generation CAR structure, an additional co-stimulatory signal is added to allow the CAR to have two co-stimulatory factors (eg, both CD28 and CD137).
  • two co-stimulatory factors eg, both CD28 and CD137.
  • CD28 refers to human leukocyte differentiation antigen 28, also known as Tp44, which has an ID number of 940 in the NCBI GeneBank and has three mRNAs and corresponding protein sequences, respectively NM_001243077.1/NP_001230006.1, NM_001243078 .1/NP_001230007.1, NM_006139.3/NP_006130.1.
  • CD137 refers to human leukocyte differentiation antigen 137, also known as 4-1BB, which is the official name of TNFRSF9 (tumor necrosis factor receptor superfamily member 9) in NCBI GeneBank, ID number 3604, only 1 mRNA And the corresponding protein sequence is NM_001561.5/NP_001552.2.
  • CD3zeta refers to human leukocyte differentiation antigen 3zeta, ID number 919 in NCBI GeneBank, and there are two mRNAs and corresponding protein sequences, respectively NM_000734.3/NP_000725.1, NM_198053.2/NP_932170.1 .
  • CD28-CD3zeta signal region refers to a gene sequence between the gene CD28 and the gene CD3zeta in the CAR structure.
  • CD137-CD3zeta signal region refers to a gene sequence between the gene CD137 to the gene CD3zeta in the CAR structure.
  • locked nucleotide acid is one comprising one or more locked nucleic acid monomers (LNA monomer(s), ie [2'-O, 4'-C-methylene- ⁇ An oligonucleotide or oligonucleotide derivative of -D-ribofuranosyl monomer (s)]).
  • LNA monomer(s) locked nucleic acid monomers
  • the nucleic acid monomer is linked to the base in a manner as shown in the following Formula V, wherein B represents a base.
  • Primer pairs, probes or methods of the present invention have versatility for detection of second and third generation CAR detections containing the CD28-CD3zeta signal region and the CD137-CD3zeta signal region.
  • the primer pair, probe or method of the invention has good specificity, high sensitivity, good reproducibility of the reaction system, good stability, and can be used for quantitative detection or CAR of CAR copy number in CAR-T prepared.
  • Figure 1 Schematic diagram of the CAR plasmid map and primer probe design containing CD28-CD3zeta.
  • Figure 2 Schematic diagram of the CAR plasmid map and primer probe design containing CD137-CD3zeta.
  • Figure 3A Comparison of amplification curves for amplification of multi-concentration CD28-CD3zeta standards using primers for CD28-CD3zeta and corresponding Taqman probes and LNA-TaqMan probes.
  • Figure 3B Amplification curve comparison of amplification of multi-concentration CD137-CD3zeta standards using non-preferred CD137-CD3zeta primers and corresponding Taqman probes and LNA-TaqMan probes.
  • Figure 3C Comparison of amplification curves for amplification of multi-concentration CD137-CD3zeta standards using primers of the preferred CD137-CD3zeta and corresponding Taqman probes and LNA-TaqMan probes.
  • Figure 4A Amplification plot of CD28-CD3zeta primer and corresponding LNA-TaqMan probe amplified CD28-CD3zeta standard.
  • Figure 4B Standard curve constructed from CD28-CD3zeta standard copy number and corresponding Ct values.
  • Figure 5A Amplification plot of CD137-CD3zeta primer and corresponding LNA-TaqMan probe amplified CD137-CD3zeta standard.
  • Figure 5B Standard curve constructed from CD137-CD3zeta standard copy number and corresponding Ct values.
  • Figure 6A CD28-CD3zeta primer and corresponding LNA-Taqman probe specific validation.
  • Figure 6B CD137-CD3zeta primer and corresponding LNA-Taqman probe specific validation.
  • Figure 7A Repeatability verification of the CD28-CD3zeta copy number detection system.
  • Figure 7B Repeatability verification of the CD137-CD3zeta copy number detection system.
  • Primers and probes were designed based on the CD28-CD3zeta signal region (Figure 1) and the CD137-CD3zeta signal region ( Figure 2).
  • the sequences of the primers and probes are shown in Table 1 below.
  • the CD28-CD3zeta and CD137-CD3zeta probes introduce a locked nucleic acid monomer, and the capitalized base is a locked nucleic acid modified base.
  • the designed primers and common probes were synthesized by Shanghai Jierui Biotechnology Co., Ltd., and the locked nucleic acid probes were synthesized by Nanjing Kingsray Biotechnology Co., Ltd.
  • CD28-CD3zeta signal region sequence CD28-CD3zeta signal region sequence:
  • the underlined portion is the nucleotide sequence of the 117 bp product (SEQ ID NO: 8).
  • CD137-CD3zeta signal region sequence CD137-CD3zeta signal region sequence:
  • the underlined portion is the nucleotide sequence of the 114 bp product (SEQ ID NO: 10).
  • Example 2 CAR using CD28-CD3zeta or CD137-CD3zeta regions using real-time PCR Plasmid transduction samples for transduction efficiency evaluation
  • Reagent Cell Genomic DNA Extraction Kit (Tiangen Biochemical Co., Ltd.), TaqMan gene expression Master Mix reagent (ABI).
  • CAR gene template CAR plasmid was electroporated into T cells, stimulated and activated by CD3 and CD28 stimulation factors, and expanded. About 5 ⁇ 10 5 T cells which were stimulated and activated for more than 3 days were used, and the DNA of the transduced T cells was extracted using a DNA extraction kit.
  • LNA-TaqMan probe sensitivity (minimum detection limit): using CD28-CD3zeta or CD137-CD3zeta corresponding primers, LNA-Taqman probe to separately amplify the gradient dilution standard, construct a standard curve, test The detection range of each reaction system.
  • LNA-TaqMan probe specificity The untransfected T cell genome was used as a background genome, and amplified using a primer corresponding to CD28-CD3zeta or CD137-CD3zeta, and a locked nucleic acid probe.
  • a positive control was set in the reaction by adding a standard containing the CD28-CD3zeta template or the CD137-CD3zeta template, and a negative control, ie adding water without any DNA template.
  • the CD28-CD3zeta-locked nucleic acid probe showed a better sensitivity than the normal probe with a ct value of about 2.5 (Fig. 3A, Table 4).
  • the use of a locked nucleic acid probe is more effective than an ordinary probe, and the probe binds better to the target fragment.
  • a set of primers for detecting a CD137-CD3zeta CAR template compared to the present invention :
  • Upstream primer TTACTGCAACCACAAACGGG (SEQ ID NO: 11);
  • Downstream primer CGCTCCTGCTGAACTTCACTC (SEQ ID NO: 12);
  • the normal amplification curve was generated using the CD137-CD3zeta primers and probes of the invention (Fig. 3C).
  • the difference in ct values produced by amplification of LNA-TaqMan and common TaqMan probes ranged from 0.39 to 0.66 (Table 4).
  • the results of real-time PCR showed that the standard DNA increased linearly with the gradient dilution CT value.
  • the standard curve for the construction of the CD28-CD3zeta template shows (Fig. 4A) that the linear relationship is good over the range of 1.49 x 10 8 copies/ ⁇ l to 1.49 x 10 2 copies/ ⁇ l.
  • both the CD28-CD3zeta and CD137-CD3zeta positive groups had normal amplification curves, but the amplification curves of the untransfected group (Control T group) were close to the negative water, indicating that the present invention provides The CD28-CD3zeta, CD137-CD3zeta primers and the corresponding LNA-Taqman probes do not bind to normal T cell genomic fragments and have good specificity.
  • the test results showed that the transduction efficiency of the CAR gene was significantly different between different samples.
  • the copy number of CD28-CD3zeta gene was from 8.47E+03 to 2.36E+09 copy number/ ⁇ l
  • the copy number of CD137-CD3zeta gene was from 3.07E+02 to 1.06E+05 copy number/ ⁇ l (Table 6). , are all within the range of the standard curve.
  • the primers and probes of the present invention have versatility for detection of second generation CAR and third generation CAR containing CD28-CD3zeta signal region and CD137-CD3zeta signal region;
  • the present invention uses a locked nucleic acid modified TaqMan probe to detect CAR copy number with high sensitivity, specificity and reproducibility of detection;
  • the invention can quantitatively detect the exogenous CAR gene, and can be used for the preparation of CAR-T quality control and CAR-T treatment of CAR copy number detection in peripheral blood cells of patients.

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Abstract

一种锁核酸修饰的探针以及一种测定CAR拷贝数的方法以及引物对。用于上述方法的引物对，选自如下的2个引物对中的任意一个或两个：SEQ ID NO:1和SEQ ID NO:2所示的第一引物对；和SEQ ID NO:4和SEQ ID NO:5所示的第二引物对。

Description

一种锁核酸修饰的探针以及一种测定CAR拷贝数的方法 技术领域
本发明属于生物技术领域，涉及一种锁核酸修饰的探针以及一种测定CAR拷贝数的方法。具体地，本发明涉及使用LNA-TaqMan探针测定含CD28-CD3zeta和CD137-CD3zeta信号区的第二代CAR或第三代CAR的拷贝数的方法。
背景技术
嵌合抗原受体T细胞(Chimeric Antigen Receptor T-Cell，CAR-T)免疫疗法是目前肿瘤免疫治疗中最有希望治愈肿瘤的方法之一。2017年8月和10月，美国FDA先后批准了诺华公司的CAR-T疗法Kymriah(曾用名CTL-019)和Kite Pharma公司的CAR-T疗法Yescarta(axicabtagene ciloleucel)上市，分别用于治疗儿童、年轻成年患者急性淋巴细胞性白血病和罹患特定类型的大B细胞淋巴瘤。CAR-T药物的上市，表明监管部门对CAR-T的临床疗效和临床安全性的认可。CAR-T在血液瘤的极大成功对CAR-T疗法的应用产生了巨大的推动作用。目前CAR-T疗法的应用已经不局限于血液肿瘤的治疗。
CAR-T的制备与质控是影响CAR-T治疗安全性和治疗效果的关键因素之一。CD28和CD137是两种T细胞共刺激分子，对T细胞的活化、增殖有重要作用，是第二代CAR和第三代CAR的重要组成部分之一。
实时荧光定量PCR是在DNA扩增反应中加入荧光基团，利用荧光信号积累，实时监测PCR产物量的变化。实时荧光定量PCR因其灵敏度高、重复性好、操作简单、成本低廉等特点，在检测领域广泛使用。利用实时荧光定量PCR的方法，可以从分子水平对CAR-T中的CAR进行拷贝数检测，为CAR-T的临床质控和临床治疗的体外监测提供技术支持。
目前，尚需要开发新的荧光定量PCR测定CAR拷贝数的方法和相关产品。
发明内容
本发明人经过深入的研究和创造性的劳动，得到了一种引物对和探针。在此基础上，本发明人得到了荧光定量PCR测定CAR拷贝数的方法和试剂盒。本发明的方法或试剂盒能够测定制备的CAR-T中CAR的拷贝数，或CAR-T治疗患者的外周血细 胞中CAR的拷贝数，用于控制CAR-T的质量、评估CAR的转导效率或临床治疗的体外监控。由此提供了下述发明：
本发明的一个方面涉及一种引物对或引物对的组合，其选自如下的2个引物对中的任意一个或两个：
SEQ ID NO:1和SEQ ID NO:2所示的第一引物对；和
SEQ ID NO:4和SEQ ID NO:5所示的第二引物对。
其中，上述“第一引物对”和“第二引物对”中的“第一”和“第二”仅仅是为了指代上的区分，并不具有次序的含义。
本发明的另一方面涉及一种探针或探针组合，其选自如下的2个探针的中的任意一个或两个：
第一探针，其核酸序列如SEQ ID NO:3所示；和
第二探针，其核酸序列如SEQ ID NO:6所示。
其中，上述“第一探针”和“第二探针”中的“第一”和“第二”仅仅是为了指代上的区分，并不具有次序的含义。
在本发明的一个或多个实施方案中，所述探针的5’端标记有荧光报告基团，3’端标记有荧光淬灭基团；优选地，所述荧光报告基团选自FAM、Hex、VIC、ROX和Cy5；优选地，所述荧光淬灭基团选自BHQ1、TAMRA、JOE、BHQ2和BHQ3。
FAM的结构式以及连接方式如下面的式I所示。
Figure PCTCN2018123890-appb-000001
BHQ1的结构式以及连接方式如下面的式II所示。
Figure PCTCN2018123890-appb-000002
Hex的结构式以及连接方式如下面的式III所示。
Figure PCTCN2018123890-appb-000003
TRAMA的结构式以及连接方式如下面的式IV所示。
Figure PCTCN2018123890-appb-000004
在本发明的一个或多个实施方案中，所述的探针或探针组合，其中，
第一探针和/或第二探针中的一个或多个位置的碱基被锁核酸修饰(即第一探针和/或第二探针中有一个或多个碱基引入了一个或多个锁核酸单体)；
优选地，第一探针的5’端起第4位、第7位、第10位和第13位中的任意1个、 2个、3个或4个碱基被锁核酸修饰；和/或，第二探针的5’端起第4位、第7位、第10位和第13位中的任意1个、2个、3个或4个碱基被锁核酸修饰。。
本发明的再一方面涉及一种试剂盒，其包含本发明任一项所述的引物对，和/或本发明任一项所述的探针。
在本发明的一个或多个实施方案中，所述的试剂盒，其包含：
第一引物对和第一探针；
和/或
第二引物对和第二探针；
优选地，所述试剂盒还包含PCR反应所需的试剂，例如Mg 2+、反应缓冲液、dNTP和Taq酶。
本发明的再一方面涉及一种测定CAR拷贝数的方法，包括如下步骤：
(1)提取待测样品的DNA；
(2)使用含CD28-CD3zeta信号区或含CD137-CD3zeta信号区的CAR质粒作为标准品母液，制成标准品系列；
(3)使用本发明的引物对和本发明中任一项所述的探针，对标准品系列和待测样品的DNA进行实时荧光定量PCR；
(4)根据每个标准品获得Ct值与标准品对应的拷贝数，绘制CD28-CD3zeta和CD137-CD3zeta标准曲线，将待测样品的DNA的Ct值代入对应的标准曲线，得到待测样品的CD28-CAR或CD137-CAR基因拷贝数；
优选地，步骤(1)中的待测样品为CAR质粒转导过的CAR-T或CAR-T回输患者的外周血；
优选地，步骤(2)中的梯度稀释为10倍梯度稀释；
优选地，步骤(3)中所述实时荧光定量PCR的扩增反应的条件为：94℃5min；94℃20s，60℃1min，共40个循环。
本发明的再一方面涉及一种对CAR-T进行质量控制的方法，包括下述步骤：
A.测定CAR拷贝数，
B.与免疫治疗要求的CAR拷贝数进行比较；
其中，步骤A中所述测定CAR拷贝数采用本发明中所述的测定CAR拷贝数的方法。优选地，所述CAR含有“CD28-CD3zeta信号区”和/或“CD137-CD3zeta信号区”。优选地，所述CAR-T含有所述CAR。
本发明的方法能够用于CAR-T的生产质控或CAR-T回输后的体外监测，例如用于检测制备的CAR-T或CAR-T治疗患者外周血细胞中的CAR拷贝数。可进一步用于评估CAR的转导效率、治疗疗效或毒副作用，或者用于评估外周血CAR-T中的CAR拷贝数与治疗疗效或毒副作用间的相关性，以便临床治疗及时调整治疗方案。
本发明的再一方面涉及本发明的引物对和/或本发明中任一项所述的探针在制备用于测定CAR拷贝数的药物或者制备用于对CAR-T进行质量控制的药物中的用途。优选地，所述CAR含有“CD28-CD3zeta信号区”和/或“CD137-CD3zeta信号区”。优选地，所述CAR-T含有所述CAR。
下面对本发明涉及的部分术语进行解释。
术语“嵌合抗原受体”(Chimeric Antigen Receptor,CAR)是识别肿瘤细胞膜上肿瘤相关抗原(tumor associated antigen,TAA)的单链抗体和胞内信号域通过铰链区相连构成的嵌合基因。
术语“CAR-T”是将CAR基因通过基因转导/转染的技术导入T细胞后获得的表达该CAR基因的细胞。这类细胞具有识别并攻击表达相应细胞表面TAA的肿瘤细胞的能力。
术语“第二代CAR”：第一代CAR将免疫球蛋白scFv和FcεRI受体或CD3复合物胞内结构域融合形成嵌合受体。第二代CAR基于第一代CAR结构，增加了一个新的共刺激信号，如CD28或CD137等。
术语“第三代CAR”：在二代CAR结构的基础上，进一步增加了一个额外的共刺激信号，使得CAR同时拥有两个共刺激因子(如同时拥有CD28和CD137)。
本文中，“CD28”指人白细胞分化抗原28，又称为Tp44，它在NCBI GeneBank中的ID号为940，有3条mRNA及对应的蛋白序列，分别为NM_001243077.1/NP_001230006.1，NM_001243078.1/NP_001230007.1，NM_006139.3/NP_006130.1。
本文中，“CD137”指人白细胞分化抗原137，又称为4-1BB，它在NCBI GeneBank的官方名称为TNFRSF9(肿瘤坏死因子受体超家族成员9)，ID号为3604，只有1 条mRNA及对应的蛋白序列，为NM_001561.5/NP_001552.2。
本文中，“CD3zeta”指人白细胞分化抗原3zeta，在NCBI GeneBank中的ID号为919，有2条mRNA及对应的蛋白序列，分别为NM_000734.3/NP_000725.1，NM_198053.2/NP_932170.1。
本发明中，术语“CD28-CD3zeta信号区”是指CAR结构中基因CD28到基因CD3zeta间的基因序列。
本发明中，术语“CD137-CD3zeta信号区”是指CAR结构中基因CD137到基因CD3zeta间的基因序列。
本发明中，术语“锁核酸(locked nucleotide acid，LNA)”是一种包含一个或多个锁核酸单体(LNA monomer(s)，即[2'-O,4'-C-methylene-β-D-ribofuranosyl monomer(s)])的寡核苷酸或寡核苷酸衍生物。在常规的TaqMan探针中引入锁核酸，能提高探针与目的序列的亲和力，增加探针的Tm值。
在本发明一个优选的实施方式(例如实施例1)中，锁核酸单体与碱基的连接方式如下面的式V所示，其中，B表示碱基。
Figure PCTCN2018123890-appb-000005
锁核酸单体与探针序列中被修饰的碱基T、C(5-甲基化)、G的具体连接方式(LNA-T，LNA-5-Me-C以及LNA-G)分别如下面的式VI、VII和VIII所示。
Figure PCTCN2018123890-appb-000006
Figure PCTCN2018123890-appb-000007
发明的有益效果
本发明取得了如下技术效果中的至少一项：
(1)本发明的引物对、探针或方法对含CD28-CD3zeta信号区和CD137-CD3zeta信号区的第二代和第三代CAR检测具有检测的通用性。
(2)本发明的引物对、探针或方法的特异性好，灵敏性高，反应体系可重复性好，稳定性好；能够用于制备的CAR-T中CAR拷贝数的定量检测或CAR-T治疗患者外周血细胞中CAR拷贝数的定量检测。
(3)本发明对含CD28-CD3zeta信号区的CAR最低的有效检测浓度为1.49×10 2拷贝/μl，对含CD137-CD3zeta信号区的CAR最低的有效检测浓度为1.52×10 2拷贝/μl。
附图说明
图1：含CD28-CD3zeta的CAR质粒图谱、引物探针设计示意图。
图2：含CD137-CD3zeta的CAR质粒图谱、引物探针设计示意图。
图3A：使用CD28-CD3zeta的引物及对应的Taqman探针和LNA-TaqMan探针扩增多浓度CD28-CD3zeta标准品的扩增曲线对比。
图3B:使用非优选的CD137-CD3zeta引物及对应的Taqman探针和LNA-TaqMan探针扩增多浓度CD137-CD3zeta标准品的扩增曲线对比。
图3C：使用优选的CD137-CD3zeta的引物及对应的Taqman探针和LNA-TaqMan探针扩增多浓度CD137-CD3zeta标准品的扩增曲线对比。
图4A：CD28-CD3zeta引物及对应的LNA-TaqMan探针扩增CD28-CD3zeta标准品的扩增曲线图。
图4B：根据CD28-CD3zeta标准品拷贝数及对应的Ct值构建的标准曲线。
图5A：CD137-CD3zeta引物及对应的LNA-TaqMan探针扩增CD137-CD3zeta标准品的扩增曲线图。
图5B：根据CD137-CD3zeta标准品拷贝数及对应的Ct值构建的标准曲线。
图6A：CD28-CD3zeta引物及对应的LNA-Taqman探针特异性验证。
图6B：CD137-CD3zeta引物及对应的LNA-Taqman探针特异性验证。
图7A：CD28-CD3zeta拷贝数检测体系的重复性验证。
图7B：CD137-CD3zeta拷贝数检测体系的重复性验证。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。本领域技术人员将会理解，下面的实施案例仅用于说明本发明，而不应视为限定本发明的范围。实施案例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：引物和探针的设计与合成
基于CD28-CD3zeta信号区(图1)和CD137-CD3zeta信号区(图2)设计引物和探针。引物和探针的序列如下面的表1所示。其中CD28-CD3zeta和CD137-CD3zeta探针引入了锁核酸单体，字母大写的碱基为锁核酸修饰的碱基。
设计的引物和普通探针交由上海捷瑞生物科技有限公司合成，锁核酸探针交由南京金斯瑞生物科技有限公司合成。
表1：引物序列和探针序列
Figure PCTCN2018123890-appb-000008
Figure PCTCN2018123890-appb-000009
Figure PCTCN2018123890-appb-000010
CD28-CD3zeta信号区序列：
Figure PCTCN2018123890-appb-000011
其中下划线部分为117bp产物的核苷酸序列(SEQ ID NO:8)。
CD137-CD3zeta信号区序列：
Figure PCTCN2018123890-appb-000012
其中下划线部分为114bp产物的核苷酸序列(SEQ ID NO:10)。
实施例2：使用荧光定量PCR对含CD28-CD3zeta或CD137-CD3zeta区域的CAR 质粒转导样本进行转导效率评估
1.样品、试剂及仪器
样品：10例含CD28-CD3zeta区域的CAR质粒电转T细胞样本和10例含CD137-CD3zeta区域的CAR质粒电转T细胞样本。选择未转染的细胞作为对照样本。
试剂：细胞基因组DNA抽提试剂盒(天根生化公司)，TaqMan gene expression Master Mix试剂(ABI公司)。
仪器：ABI7500荧光定量PCR检测仪(ABI公司)。
2.实验方法
取10例含CD28-CD3zeta基因的CAR质粒电转T细胞样品和10例含CD137-CD3zeta基因的CAR质粒电转T细胞样品，分别抽提基因组。取100ng用以检测。根据提供的荧光定量PCR方法反应体系配制反应液，获取CD28-CD3zeta或CD137-CD3zeta的Ct值。Ct值代入标准曲线，计算各样本的绝对拷贝数。
具体步骤如下：
(1)CAR基因模板的制备：CAR质粒经电转方式进入T细胞，经过CD3和CD28刺激因子包被刺激、活化，扩大培养。取刺激活化3天以上的T细胞约5×10 5个，使用DNA抽提试剂盒提取转导T细胞的DNA。
(2)标准品的制备：分别使用含CD28-CD3zeta和CD137-CD3zeta的CAR质粒作为标准品母液，使用未转染的T细胞基因组来稀释CAR质粒，以10倍梯度稀释的制备7个梯度的标准品系列，如下面的表2。
表2：含CD28/CD137-CD3zeta信号区的CAR标准品制备
Figure PCTCN2018123890-appb-000013
Figure PCTCN2018123890-appb-000014
(3)LNA-Taqman探针与普通Taqman探针灵敏性对比：定量PCR反应体系列于表3，普通探针与锁核酸探针检测的标准品检测相同的系列梯度标准品。扩增反应条件为：94℃5min；94℃20s，60℃1min，共40个循环。
表3：荧光定量PCR反应体系
Figure PCTCN2018123890-appb-000015
(3)LNA-TaqMan探针灵敏性(最低检测限)：使用CD28-CD3zeta或CD137-CD3zeta对应的引物、LNA-Taqman探针分别对梯度稀释的标准品进行扩增反应，构建标准曲线，检测各反应体系的检测范围。
(4)LNA-TaqMan探针特异性：使用未转染的T细胞基因组作为背景基因组，使用CD28-CD3zeta或CD137-CD3zeta对应的引物、锁核酸探针扩增。
反应中设置一个阳性对照，即添加含CD28-CD3zeta模板或CD137-CD3zeta模板的标准品，一个阴性对照，即添加不含任何DNA模板的水。
(5)LNA-TaqMan探针体系的重复性：抽取CD28-CD3zeta三个浓度梯度的标准品1.49×10 7拷贝数/μl，1.49×10 5拷贝数/μl,1.49×10 3拷贝数/μl，使用对应的引物、LNA-Taqman探针扩增，每个标准品平行重复5管；抽取CD137-CD3zeta三个浓度梯度的标准品1.52×10 8拷贝数/μl，1.52×10 6拷贝数/μl，1.52×10 4拷贝数/μl，使用对 应引物、LNA-Taqman探针扩增，每个标准品平行重复5管。测得的CT值进行组内和组间差异的统计学分析，以变异系数情况来确定该反应的重复性。
3.实验结果
(1)TaqMan探针和LNA-TaqMan探针灵敏性对比
通过CD28-CD3zeta的扩增曲线对比，CD28-CD3zeta锁核酸探针比普通探针的ct值小2.5左右(图3A，表4)，显示出更好的灵敏性。使用锁核酸探针比普通探针的扩增曲线效果更好，探针与目的片段结合更好。
一组与本发明对比的检测CD137-CD3zeta CAR模板的引物：
上游引物：TTACTGCAACCACAAACGGG(SEQ ID NO:11)；
下游引物：CGCTCCTGCTGAACTTCACTC(SEQ ID NO:12)；
普通探针：5'FAM-CACATCCTCCTTCTTCTTC-3'BHQ1，其中序列CACATCCTCCTTCTTCTTC表示为SEQ ID NO:13。
相同条件下的实验结果显示，本发明的LNA-TaqMan探针具有更强的结合能力，检测标准品有正常的扩增曲线。而普通探针无法产生正常的扩增曲线，结合效率极低，锁核酸修饰后的探针与目的序列结合能力更强，可以正常使用(图3B)。
使用本发明的CD137-CD3zeta引物和探针能产生正常的扩增曲线(图3C)。LNA-TaqMan和普通TaqMan探针扩增产生的ct值差异在0.39～0.66范围内(表4)。
表4：锁核酸探针与普通探针检测标准品模板的Ct值差异
Figure PCTCN2018123890-appb-000016
Figure PCTCN2018123890-appb-000017
(2)灵敏性与标准曲线的建立
荧光定量PCR结果显示，标准品DNA随着梯度稀释CT值呈线性递增。CD28-CD3zeta模板构建的标准曲线显示(图4A)，在1.49×10 8拷贝数/μl—1.49×10 2拷贝数/μl范围内，线性关系良好。CD28-CD3zeta标准曲线的回归方程：y＝-3.807log 10(X)+41.135，R2＝0.995(图4B)。
CD137-CD3zeta标准曲线的回归方程：y＝-3.964log 10(x)+42.245，R2＝0.997。有效检测的范围为1.52×10 8—1.52×10 2拷贝数/μl(图5A和图5B)。
(3)引物、探针特异性验证
如图6A和图6B所示，CD28-CD3zeta和CD137-CD3zeta阳性组均有正常的扩增曲线，但未转染组(Control T组)的扩增曲线与阴性水接近，显示本发明提供的CD28-CD3zeta、CD137-CD3zeta引物及对应的LNA-Taqman探针不会与正常的T细胞基因组片段结合，有良好的特异性。
(4)反应体系重复性验证
采用变异系数CV(％)评价重复性结果。使用3个不同浓度样本的5次CT值都较为接近(图7A和图7B)。变异系数CV小于5％(表5)，表明本方法结果稳定，有良好的重复性。
表5：LNA-TaqMan探针实时定量PCR检测CD28/CD137-Zeta标准品Ct值批内变异
Figure PCTCN2018123890-appb-000018
Figure PCTCN2018123890-appb-000019
(5)含CD28-CD3zeta和CD137-CD3zeta的CAR样本检测结果
检测结果显示，CAR基因的转导效率在不同样本间有较大差异。检测样本中，CD28-CD3zeta基因的拷贝数从8.47E+03至2.36E+09拷贝数/μl，CD137-CD3zeta基因拷贝数从3.07E+02至1.06E+05拷贝数/μl(表6)，均位于标准曲线范围内。
表6：含CD28-CD3zeta和CD137-CD3zeta的CAR样本检测结果
Figure PCTCN2018123890-appb-000020
Figure PCTCN2018123890-appb-000021
由上述可见：
本发明的引物和探针对含CD28-CD3zeta信号区和CD137-CD3zeta信号区的第二代CAR和第三代CAR具有检测的通用性；
本发明使用锁核酸修饰的TaqMan探针检测CAR拷贝数具有检测的高灵敏性、特异性和可重复性；
本发明能够对外源CAR基因进行定量检测，可用于制备的CAR-T质量控制和CAR-T治疗患者外周血细胞中的CAR拷贝数检测。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (12)

  1. 引物对，其选自如下的2个引物对中的任意一个或两个：
    SEQ ID NO:1和SEQ ID NO:2所示的第一引物对；和
    SEQ ID NO:4和SEQ ID NO:5所示的第二引物对。
  2. 探针，其选自如下的2个探针的中的任意一个或两个：
    第一探针，其核酸序列如SEQ ID NO:3所示；和
    第二探针，其核酸序列如SEQ ID NO:6所示。
  3. 根据权利要求2所述的探针，其中，所述探针的5’端标记有荧光报告基团，3’端标记有荧光淬灭基团；
    优选地，所述荧光报告基团选自FAM、Hex、VIC、ROX和Cy5；
    优选地，所述荧光淬灭基团选自BHQ1、TAMRA、JOE、BHQ2和BHQ3。
  4. 根据权利要求2或3所述的探针，其中，
    第一探针和/或第二探针中的一个或多个位置的碱基被锁核酸修饰。
  5. 根据权利要求4所述的探针，其中，
    第一探针的5’端起第4位、第7位、第10位和第13位中的任意1个、2个、3个或4个碱基被锁核酸修饰；
    和/或，
    第二探针的5’端起第4位、第7位、第10位和第13位中的任意1个、2个、3个或4个碱基被锁核酸修饰。
  6. 一种试剂盒，其包含权利要求1所述的引物对，和/或权利要求2至5中任一权利要求所述的探针。
  7. 根据权利要求6所述的试剂盒，其包含：
    第一引物对和第一探针；
    和/或
    第二引物对和第二探针；
    优选地，所述试剂盒还包含PCR反应所需的试剂，例如Mg 2+、反应缓冲液、dNTP和Taq酶。
  8. 一种测定CAR拷贝数的方法，包括如下步骤：
    (1)提取待测样品的DNA；
    (2)使用含CD28-CD3zeta信号区或含CD137-CD3zeta信号区的CAR质粒作为标准品母液，制成标准品系列；
    (3)使用权利要求1所述的引物对和权利要求2至5中任一权利要求所述的探针，对标准品系列和样品的DNA进行实时荧光定量PCR检测；
    (4)根据每个标准品的Ct值及其对应的拷贝数，分别绘制CD28-CD3zeta基因和CD137-CD3zeta基因的标准曲线；将检测样本的DNA的Ct值代入对应的标准曲线，得到待测样品的CAR基因拷贝数；
    优选地，步骤(1)中的待测样品为CAR质粒转导过的CAR-T或CAR-T回输患者的外周血；
    优选地，步骤(2)中的梯度稀释为10倍梯度稀释；
    优选地，步骤(3)中所述实时荧光定量PCR的扩增反应的条件为：94℃5min；94℃20s，60℃1min，共40个循环。
  9. 一种对CAR-T进行质量控制的方法，包括下述步骤：
    A.测定CAR拷贝数，
    B.与免疫治疗要求的CAR拷贝数进行比较；
    其中，步骤A中所述测定CAR拷贝数采用权利要求8中所述的测定CAR拷贝数的方法。
  10. 权利要求1所述的引物对和/或权利要求2至5中任一权利要求所述的探针在制备用于测定CAR拷贝数的药物或者制备用于对CAR-T进行质量控制的药物中的用途。
  11. 根据权利要求1所述的引物对，其用于测定CAR拷贝数，或者用于对CAR-T 进行质量控制。
  12. 根据权利要求2至5中任一权利要求所述的探针，其用于测定CAR拷贝数，或者用于对CAR-T进行质量控制。
PCT/CN2018/123890 2017-12-28 2018-12-26 一种锁核酸修饰的探针以及一种测定car拷贝数的方法 WO2019129055A1 (zh)

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