WO2019128857A1 - 一种外源抗体基因拷贝数的检测方法和试剂盒 - Google Patents

一种外源抗体基因拷贝数的检测方法和试剂盒 Download PDF

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WO2019128857A1
WO2019128857A1 PCT/CN2018/122561 CN2018122561W WO2019128857A1 WO 2019128857 A1 WO2019128857 A1 WO 2019128857A1 CN 2018122561 W CN2018122561 W CN 2018122561W WO 2019128857 A1 WO2019128857 A1 WO 2019128857A1
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gene
copy number
antibody
exogenous
hinge region
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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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  • the invention belongs to the field of biotechnology and relates to detecting the copy number of a foreign antibody gene using a real-time PCR method.
  • the present invention relates to methods and kits for detecting the copy number of a foreign antibody gene using real-time PCR based on the heavy chain constant region of an IgG4 antibody.
  • An antibody also called immunoglobulin (Ig), which is produced by antigen-stimulated B cells to differentiate into plasma cells, can specifically bind to antigens and plays an important role in the human immune system.
  • the antibodies can be classified into five types, IgA, IgD, IgE, IgG, and IgM, depending on the difference in antigenicity of the heavy chain. Due to the high specificity and affinity of antibodies, many specific therapeutic antibody drugs have been developed for different indications.
  • IgG immunoglobulins in human serum
  • IgG has the longest serum half-life.
  • IgG can be further divided into four subtypes of IgG1, IgG2, IgG3 and IgG4.
  • the constant region sequences of each subtype are highly homologous, mainly showing the disulfide linkage between the heavy and light chains, the core CXXC sequence of the hinge region, and the CH2 interval to some extent.
  • the typical feature of the IgG4 subtype is that the monovalent is effective, avoids the formation of immune complexes, acts as a blocking antibody, avoids triggering mechanisms, and thus exhibits relatively "non-inflammatory" characteristics.
  • the selection of IgG4 subtypes is increasing in the development of therapeutic antibodies that do not require effector functions.
  • the monoclonal antibodies permbrolizumab and nivolumab which have been approved for marketing on the PD-1 target, are antibodies against the PD-1 receptor, all of which are human IgG4 monoclonal antibodies.
  • IgG4-based antibody drugs such as the antibody drug IPH-2101 for the KIR2D receptor and applied to the multiple myeloma indication, Reslizumab for the IL-5 target and for the asthma indication antibody drug are also in clinical research.
  • the CAR structure mainly includes the extracellular targeting recognition region and the hinge structure, the transmembrane region and the intracellular signal region, wherein the hinge region is mainly derived from CD8 or IgG4. Therefore, by establishing a method for detecting the IgG4 foreign antibody gene copy number, the foreign antibody copy number and the CAR copy number can be simultaneously detected, thereby evaluating the transduction efficiency of the exogenous antibody gene or CAR.
  • the inventors have obtained a set of primer pairs and probes through intensive research and creative labor. On the basis of this, the inventors obtained a copy number method and a kit for measuring the exogenous IgG4 antibody gene by real-time PCR.
  • the method or kit of the present invention is capable of determining the intracellular copy number of an antibody gene constructed based on the heavy chain constant region gene of an IgG4 antibody.
  • One aspect of the invention relates to a primer pair or a combination of primer pairs comprising:
  • Primer pair 1 primer pair set forth in SEQ ID NO: 1 and SEQ ID NO: 2;
  • Primer pair 2 primer pairs as shown in SEQ ID NO: 4 and SEQ ID NO: 5.
  • the primer pair of the present invention is designed based on the heavy chain constant region across exons, and the detection of the foreign antibody gene is versatile.
  • primer pair or combination of primer pairs may also be referred to as a primer combination.
  • the primer pair or combination of primer pairs is used to determine the foreign antibody gene copy number or for quality control of the foreign antibody
  • the exogenous antibody gene comprises a hinge region-CH3 gene of the exogenous antibody IgG4;
  • the exogenous antibody comprises the hinge region -CH3 of IgG4.
  • Another aspect of the invention relates to a probe or probe combination comprising:
  • Probe A the nucleic acid sequence thereof is shown in SEQ ID NO: 3;
  • Probe B the nucleic acid sequence of which is shown in SEQ ID NO: 6.
  • the probe wherein 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, Hex, VIC, ROX and Cy5; preferably, the fluorescence quenching group is selected from the group consisting of BHQ1, TAMRA, JOE, BHQ2 and BHQ3.
  • the probe is for determining a foreign antibody gene copy number or for quality control of an exogenous antibody
  • the exogenous antibody gene comprises a hinge region-CH3 gene of the exogenous antibody IgG4;
  • the exogenous antibody comprises the hinge region -CH3 of IgG4.
  • a further aspect of the invention relates to a kit comprising the primer pair 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 for determining the copy number of a foreign antibody gene comprising the steps of:
  • the sample to be tested in the step (1) is a cell transduced with a hinge region-CH3 gene plasmid containing the exogenous antibody IgG4;
  • 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.
  • the present invention provides a method for detecting the copy number of an exogenous antibody based on the heavy chain constant region of an antibody IgG4.
  • the Ct value of the hinge region-CH3 exogenous antibody gene of each sample and the Ct value of the corresponding internal reference gene Actin were obtained and substituted into the constructed standard curve to obtain the absolute antibody gene and the absolute of Actin. Copy number.
  • the copy number content of the exogenous antibody per microgram of genome in the sample was obtained by normalization with 1 ⁇ g of genomic Actin copy number.
  • the assay method of the present invention can be used for copy number detection of a gene containing an exogenous antibody, and has versatility for detection of an antibody sequence containing an IgG4 hinge region-CH3 region. Can be used for exogenous antibody gene transfer to efficiency assessment, production quality control or in vitro monitoring.
  • a further aspect of the invention relates to a method of quality control of an exogenous antibody comprising the steps of:
  • the method for determining the copy number of the foreign antibody gene in the step A is the method for determining the copy number of the foreign antibody gene according to the present invention
  • the exogenous antibody comprises the hinge region -CH3 of IgG4.
  • 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 preparing a medicament for determining a copy number of a foreign antibody gene or for preparing a quality control for an exogenous antibody Use in medicine;
  • the exogenous antibody gene comprises a hinge region-CH3 gene of the exogenous antibody IgG4;
  • the exogenous antibody comprises the hinge region -CH3 of IgG4.
  • CAR Chimeric Antigen Receptor
  • TAA tumor associated antigen
  • CAR-T is a cell expressing the foreign antibody gene obtained by introducing a foreign antibody 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.
  • antibody also known as immunoglobulin (Ig) is produced by antigen-stimulated B-cell differentiation into plasma cells, a glycoprotein that specifically binds to an antigen.
  • exogenous antibody gene is an antibody gene sequence introduced into a target cell by a genetic engineering technique or a viral infection to express a corresponding antibody in a target cell.
  • IgG is an abbreviation for Immunoglobulin G (IgG) and is the major antibody component of serum, accounting for approximately 75% of serum Ig. 40%-50% of them are distributed in serum, and the rest are distributed in tissues. IgG is the only immunoglobulin that can pass through the placenta.
  • IgG4 is one of the IgG antibody protein subtypes, and IgG can be further classified into IgG1 (60%), IgG2 (25%), IgG3 (10%) and IgG4 (5) according to its distribution in serum. %) Four subtypes whose constant region sequences are highly homologous.
  • IgG4 antibody in this patent refers specifically to a human IgG4 antibody.
  • heavy chain constant region refers to two chains of a relatively large molecular weight of an antibody, referred to as heavy chains.
  • the approximately 110 amino acid sequence of the heavy chain near the N-terminus varies greatly, and the other partial amino acid sequences are relatively constant. Therefore, a region in which the amino acid sequence near the C-terminus of the Ig heavy chain is relatively stable is referred to as a constant region (C), which accounts for about 3/4 of the heavy chain.
  • hinge region is a domain that constitutes IgG, IgA, and IgD class immunoglobulin molecules, located between CH1 and CH2, joining the Fab and Fc segments.
  • the region is rich in proline, does not form an alpha helix, is prone to stretching and a certain degree of distortion, and is beneficial to the complementary binding between the antigen binding site of the antibody and the epitope.
  • hinge region-CH3 refers to the sequence from the hinge region to the CH3 region in the constant region of the heavy chain of the antibody, including the hinge region, the CH2 region, and the CH3 region.
  • the "hinge region-CH3” refers specifically to the sequence of the hinge region to the CH3 region in the heavy chain constant region of a human antibody.
  • the primers and probes of the present invention have detection versatility for detection of an exogenous antibody gene containing an IgG4 heavy chain constant region.
  • the detection method provided by the invention is convenient to operate, and the primers and probes provided have good specificity, high sensitivity, good reproducibility, and can be used for copying of foreign antibodies containing the IgG4 hinge region-CH3 sequence. Quantitative detection of the number.
  • the lowest effective detection concentration of the IgG4 exogenous antibody gene containing the hinge region-CH3 region of the present invention is 3.22E+03 copies/ ⁇ l.
  • Figure 1 Design location based on the IgG4 hinge region-CH3 gene primer probe.
  • FIG. 1 IgG4 hinge region-CH3 primer probe specific validation.
  • Figure 3A Standard amplification curve containing the exogenous IgG4 hinge region-CH3 gene.
  • Figure 3B Standard curve of the constructed exogenous IgG4 hinge region-CH3 gene.
  • Figure 4A Standard amplification curve for the internal reference gene Actin.
  • Figure 4B Standard curve of the constructed internal reference gene Actin.
  • Figure 5 Repeatability verification of the hinge region-CH3 primer probe.
  • the underlined portion is the nucleotide sequence of the 125 bp product (SEQ ID NO: 8)
  • Example 2 Transduction of a plasmid transduction sample containing the hinge region-CH3 gene using real-time PCR Rate assessment
  • Sample 8 plasmid electroporation T cell samples containing the IgG4 hinge region-CH3 gene. Two untransfected T cells were selected as control samples, which were designated as Control Sample 1 and Control Sample 2, respectively.
  • Reagent Cell Genomic DNA Extraction Kit (Tiangen Biochemical Co., Ltd.), TaqMan gene expression Master Mix reagent (ABI).
  • Extraction of exogenous antibody DNA template Take the plasmid-transduced cells containing the IgG4 hinge region-CH3 or the peripheral blood cells containing the IgG4 hinge region-CH3, and extract the cellular DNA; respectively, as the hinge region-CH3 gene and A detection template for the Actin gene.
  • the plasmid containing the hinge region-CH3 gene was 7092 bp, and the plasmid containing the Actin gene was 4091 bp.
  • Table 2 a 10-fold gradient dilution standard (Table 2) was prepared separately.
  • the range of exogenous antibody gene standards is 3.22E+03 copies/ ⁇ l to 3.22E+09 copies/ ⁇ l
  • the range of the internal reference gene Actin standard is 5.58E+03 copies/ ⁇ l to 5.58E+09. Copy/ ⁇ l.
  • a quantitative PCR reaction system was prepared, which included the primers and probes in Table 1.
  • the standard DNA template is subjected to an amplification reaction.
  • the reaction procedure was a two-step process: the first step was 95 ° C for 5 min; the second step, 95 ° C for 20 s, and 60 ° C for 1 min, for a total of 40 cycles.
  • the PCR reaction system is shown in Table 3 below.
  • T cells i.e., the aforementioned control sample
  • the genome of T cells which were not transfected with the IgG4 antibody gene was used as the background genome, and the specificity of the combination of the exogenous antibody gene primer, the probe amplification, and the primer probe was used.
  • a positive control was set up in the reaction by adding a standard containing the exogenous antibody template and a negative control, ie adding water without any DNA template.
  • Sensitivity detection Fluorescence quantitative PCR reaction was carried out using a 10-fold gradient dilution of the hinge-containing region-CH3 standard and the internal reference gene Actin standard, respectively. A standard curve of the exogenous antibody gene was constructed based on the Ct value of the hinge region-CH3 gene and the corresponding standard copy number, and the detection range of the system was evaluated.
  • a standard curve was constructed based on the Ct value of the Actin gene and the corresponding standard copy number, and the detection range of the system was evaluated.
  • the detection range was 3.22E + 09 copies / ⁇ l - 3.22E + 03 copies / ⁇ l ( Figure 3A and Figure 3B).
  • the cell sample electrotransformed with the antibody plasmid containing the hinge region-CH3 gene was subjected to copy number detection using the aforementioned primers, probes and detection methods.
  • the test results showed that the copy number of the exogenous antibody gene hinge region-CH3 in each electroporation sample was significantly different.
  • the copy number of the foreign antibody containing the hinge region-CH3 gene ranges from 8.54E+05 to 5.16E+06
  • the copy number of Actin is from 2.40E+05 to 5.15E+06 (Table 5).
  • the numbers are all within the range of the standard curve.
  • Based on the amplification results of multiple control cell genomes an average of 3.49E+07 copies of Actin gene per ⁇ g of genome was included.
  • the copy number of the test sample ranged from 6.41E+06 to 5.21E+07 copies/ ⁇ g of genomic DNA.
  • the invention can detect the foreign antibody gene containing the hinge region-CH3 gene by using the detection of the internal reference gene Actin and the exogenous IgG4 antibody hinge region-CH3 gene, and can be used for the copy number detection of the foreign antibody gene, and further Used for transduction efficiency assessment, quality control and in vitro monitoring.
  • the primers and probes of the present invention have detection versatility for an exogenous antibody gene containing an IgG4 hinge region-CH3.

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Abstract

提供了一种外源抗体基因拷贝数的检测方法和试剂盒以及引物对。具体地，提供了一种引物对，其包含：引物对1，SEQ ID NO:1和SEQ ID NO:2所示的引物对；优选地，还包含引物对2，SEQ ID NO:4和SEQ ID NO:5所示的引物对。还提供了测定外源抗体基因的拷贝数的方法，进而为外源抗体基因的转导效率评估、质量控制、体外监测提供了更准确的技术支持。对含IgG4重链恒定区的外源抗体基因的检测具有通用性。

Description

一种外源抗体基因拷贝数的检测方法和试剂盒 技术领域
本发明属于生物技术领域，涉及使用荧光定量PCR方法检测外源抗体基因拷贝数。具体地，本发明涉及基于IgG4抗体重链恒定区，使用荧光定量PCR检测外源抗体基因拷贝数的方法和试剂盒。
背景技术
抗体，也叫免疫球蛋白(Ig)，其由抗原刺激B细胞分化成浆细胞所产生，能与抗原特异性结合，在人体免疫系统中起重要作用。根据重链抗原性的差异，可将抗体分IgA、IgD、IgE、IgG和IgM五种类型。由于抗体具有特异性高、亲和力强的特点，人们针对不同的适应症，已开发出学多特异性的治疗性抗体药物。
在人类血清的所有免疫球蛋白中，IgG的血清半衰期最长。基于实用性和功能上的考虑，目前开发的重组单克隆抗体均是IgG类型。根据其在血清的分布丰度，IgG可进一步分为IgG1、IgG2、IgG3和IgG4四种亚型。各亚型的恒定区序列高度同源，主要表现在重链和轻链之间的二硫键连接方式、铰链区的核心CXXC序列组成、CH2区间存在一定程度的差异。
在体内，IgG4亚型的典型特征是单价有效，能够避免形成免疫复合物，可以作为阻断抗体，避免触发效应机制，从而呈现出相对“非炎症性”特征。在一些不需要效应功能的治疗性抗体的开发中，IgG4亚型的选用越来越多。例如已被批准上市的，针对PD-1靶点开发的单克隆抗体permbrolizumab和nivolumab，它们都是抗PD-1受体的抗体，均为人源IgG4单克隆抗体。此外，针对KIR2D受体并且应用于多发性骨髓瘤适应症的抗体药物IPH-2101、针对IL-5靶点并且应用于哮喘适应症抗体药物的Reslizumab等基于IgG4的抗体药物也处于临床研究中。
当前CAR-T是肿瘤治疗领域中非常具有前景的技术，其在血液瘤中的疗效表现优异。2017年8月和2017年11月，美国食品和药物管理局先后批准了治疗复发或难治性急性淋巴细胞白血病的CAR-T药物Kymriah(CTL019)和针对特定淋巴癌的CAR-T药物Yescarta。目前，有相当数量的研究尝试将CAR与PD-1抗体基因或PD-L1抗体基因联合，构建在同一载体上并转导至T细胞，通过共表达PD-1抗体或PD-L1抗体以增强T细胞的杀伤效果，用于治疗实体瘤。
另外，CAR结构中主要包括胞外的靶向识别区和铰链结构、跨膜区和胞内信号区三大部分，其中铰链区主要来自CD8或IgG4。因此，通过建立检测IgG4外源抗体基因拷贝数方法，可以同时检测外源抗体拷贝数和CAR拷贝数，进而评估外源抗体基因或CAR的转导效率。
目前，亟需建立检测外源抗体基因特别是包含IgG4亚型的外源抗体的基因的检测方法。
发明内容
本发明人经过深入的研究和创造性的劳动，得到了一组引物对和探针。在此基础上，本发明人得到了荧光定量PCR测定外源IgG4抗体基因的拷贝数方法和试剂盒。本发明的方法或试剂盒能够测定基于IgG4抗体重链恒定区基因构建的抗体基因在细胞内的拷贝数。
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，其包含：
引物对1，SEQ ID NO:1和SEQ ID NO:2所示的引物对；
优选地，还包含
引物对2，SEQ ID NO:4和SEQ ID NO:5所示的引物对。
本发明的引物对基于重链恒定区跨外显子设计，对外源抗体基因的检测具有通用性。
所述引物对或引物对的组合，也可称为引物组合。
在本发明的一个或多个实施方案中，所述的引物对或引物对的组合，其用于测定外源抗体基因拷贝数，或者用于对外源抗体进行质量控制；
优选地，所述外源抗体基因含有外源抗体IgG4的铰链区-CH3基因；
优选地，所述外源抗体含有IgG4的铰链区-CH3。
本发明的另一方面涉及一种探针或探针组合，其包含：
探针A，其核酸序列如SEQ ID NO:3所示；
优选地，还包含
探针B，其核酸序列如SEQ ID NO:6所示。
在本发明的一个或多个实施方案中，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。
FAM的结构式以及连接方式如下面的式I所示。
Figure PCTCN2018122561-appb-000001
BHQ1的结构式以及连接方式如下面的式II所示。
Figure PCTCN2018122561-appb-000002
Hex的结构式以及连接方式如下面的式III所示。
Figure PCTCN2018122561-appb-000003
Figure PCTCN2018122561-appb-000004
TRAMA的结构式以及连接方式如下面的式IV所示。
Figure PCTCN2018122561-appb-000005
在本发明的一个或多个实施方案中，所述的探针，其用于测定外源抗体基因拷贝数，或者用于对外源抗体进行质量控制；
优选地，所述外源抗体基因含有外源抗体IgG4的铰链区-CH3基因；
优选地，所述外源抗体含有IgG4的铰链区-CH3。
本发明的再一方面涉及一种试剂盒，其包含本发明的引物对，和/或本发明中任一项所述的探针。
在本发明的一个或多个实施方案中，所述的试剂盒，其包含：
引物对1和探针A；
和/或
引物对2和探针B；
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外源抗体基因拷贝数的方法，包括如下步骤：
(1)提取待测样品的DNA；
(2)使用含IgG4铰链区-CH3基因的质粒和含Actin基因的质粒作为标准品母液， 分别制成外源抗体基因标准品系列和Actin标准品系列；
(3)使用本发明的引物对和本发明的探针，分别对外源抗体基因标准品系列、Actin标准品系列和待测样品的DNA进行时荧光定量PCR；
(4)根据外源抗体基因标准品系列产生的Ct值与对应拷贝数，含Actin标准品系列产生的Ct值与对应拷贝数，分别绘制标准曲线，获得计算公式；
(5)将待测样品的外源抗体基因和Actin基因的Ct值带入步骤(4)中的标准曲线的计算公式，获得待测样品的外源抗体基因拷贝数和Actin拷贝数；
(6)通过与每μg基因组含Actin的拷贝数进行校正，从而获得待测样品的每μg基因组中含外源抗体基因的拷贝数；
优选地，步骤(1)中的待测样品为含外源抗体IgG4的铰链区-CH3基因质粒转导过的细胞；
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。
本发明提供一种基于抗体IgG4重链恒定区检测外源抗体拷贝数的方法。通过同时扩增内参基因Actin，获得每个样本的铰链区-CH3外源抗体基因的Ct值与对应的内参基因Actin的Ct值，分别代入构建的标准曲线从而获得外源抗体基因和Actin的绝对拷贝数。通过与1μg基因组Actin拷贝数进行标准化，可获得该样本中每微克基因组中外源抗体的拷贝数含量。
本发明的测定方法能够用于含外源抗体基因的拷贝数检测，对含IgG4铰链区-CH3区的抗体序列具有检测的通用性。可用于外源抗体基因转到效率评估，生产质控或体外监测。
本发明的再一方面涉及一种对外源抗体进行质量控制的方法，包括下述步骤：
A.测定外源抗体基因的拷贝数，
B.评估外源抗体基因转导效率或与免疫治疗要求的外源抗体基因拷贝数进行比较；
其中，步骤A中所述测定外源抗体基因拷贝数采用本发明所述的测定外源抗体基因拷贝数的方法；
优选地，所述外源抗体含有IgG4的铰链区-CH3。
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外源抗体基因拷贝数的药物或者制备用于对外源抗体进行质量控制的药物中的用途；
优选地，所述外源抗体基因含有外源抗体IgG4的铰链区-CH3基因；
优选地，所述外源抗体含有IgG4的铰链区-CH3。
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”是将外源抗体基因通过基因转导/转染的技术导入T细胞后获得的表达该外源抗体基因的细胞。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“抗体”也叫免疫球蛋白(Ig)，是由抗原刺激B细胞分化成浆细胞所产生的，一种能与抗原特异性结合的糖蛋白。
“外源抗体基因”是通过基因工程技术或病毒感染等途径引入靶细胞中的抗体基因序列，从而在靶细胞表达相应抗体。
术语“IgG”是免疫球蛋白G(Immunoglobulin G，IgG)的缩写，是血清主要的抗体成分，约占血清Ig的75％。其中40％－50％分布于血清中，其余分布在组织中。IgG是唯一可以通过胎盘的免疫球蛋白。
术语“IgG4”是IgG抗体蛋白亚型中的一种，IgG可根据其在血清中的分布丰度进一步分为IgG1(60％)，IgG2(25％)，IgG3(10％)和IgG4(5％)四种亚型，它们的恒定区序列高度同源。本专利术语“IgG4抗体”特指人源的IgG4抗体。
术语“重链恒定区”是指抗体分子量较大的两条链，被称为重链。重链靠近N端的约110个氨基酸序列变化很大，其他部分氨基酸序列相对恒定。因此，将Ig重链中靠近C端的氨基酸序列相对稳定的区域，称为恒定区(constant region，C)，约占重链的3/4。
术语“铰链区”是组成IgG、IgA和IgD类免疫球蛋白分子的一种结构域，位于CH1和CH2间，连接Fab和Fc段。该区富含脯氨酸，不形成α螺旋，易发生伸展及一定程度扭曲，有利于抗体的抗原结合部位与抗原表位间的互补性结合。
术语“铰链区-CH3”是指抗体重链恒定区中铰链区至CH3区的序列，包括铰链区、 CH2区和CH3区。所述“铰链区-CH3”特别是指人抗体重链恒定区中铰链区至CH3区的序列。
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)本发明的引物和探针对含IgG4重链恒定区的外源抗体基因检测具有检测的通用性。
(2)本发明提供的检测方法操作方便，提供的引物和探针检测的特异性好，灵敏性高，反应体系可重复性好，能够用于含IgG4铰链区-CH3序列的外源抗体拷贝数的定量检测。
(3)本发明对含铰链区-CH3区的IgG4外源抗体基因最低的有效检测浓度为3.22E+03个拷贝/μl。
附图说明
图1：基于IgG4铰链区-CH3基因引物探针设计位置。
图2：IgG4铰链区-CH3引物探针特异性验证。
图3A：含外源IgG4铰链区-CH3基因的标准品扩增曲线。
图3B：构建的外源IgG4铰链区-CH3基因的标准曲线。
图4A：含内参基因Actin的标准品扩增曲线。
图4B：构建的内参基因Actin的标准曲线。
图5：铰链区-CH3引物探针的重复性验证。
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
基于人源IgG4重链恒定区铰链区-CH3(图1)以及内参基因Actin设计引物和探针，委托上海捷瑞生物科技有限公司合成。引物和探针的序列如下面的表1所示。
表1：引物序列和探针序列
Figure PCTCN2018122561-appb-000006
人源IgG4铰链区-CH3序列：
Figure PCTCN2018122561-appb-000007
其中下划线部分为125bp产物的核苷酸序列(SEQ ID NO:8)
实施例2：使用荧光定量PCR对含铰链区-CH3基因的质粒转导样本进行转导效 率评估
1.样品、试剂及仪器
样品：8例含IgG4铰链区-CH3基因的质粒电转T细胞样品。选择2例未转染的T细胞作为对照样品，分别命名为对照样品1和对照样品2。
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：荧光定量PCR检测仪(ABI公司)。
2.实验方法
取8例含IgG4铰链区-CH3基因的外源抗体质粒电转T细胞样品，抽提基因组。根据荧光定量PCR方法的反应体系配制反应液，获取抗体基因的Ct值及对应样本的内参基因Actin的Ct值。将两基因获取的Ct值代入各自的标准曲线，计算各基因的拷贝数。
具体步骤如下：
(1)外源抗体DNA模板的抽提制作：取含IgG4铰链区-CH3的质粒转导过的细胞或含IgG4铰链区-CH3的外周血细胞，提取细胞DNA；分别作为铰链区-CH3基因和Actin基因的检测模板。
(2)标准品的制备：使用含IgG4铰链区-CH3的质粒制备外源抗体基因标准品母液，提取含内参基因Actin的质粒作为内参基因标准品母液。
(3)含铰链区-CH3基因的质粒为7092bp，含Actin基因的质粒为4091bp。根据表2，分别制备10倍梯度稀释标准品(表2)。其中，外源抗体基因标准品的范围为3.22E+03个拷贝/μl～3.22E+09个拷贝/μl，内参基因Actin标准品的范围为5.58E+03个拷贝/μl～5.58E+09个拷贝/μl。
表2：外源抗体基因标准品和Actin标准品的制备
Figure PCTCN2018122561-appb-000008
(4)实时荧光定量PCR检测：配制定量PCR反应体系，该反应体系中包含了表1中的引物和探针。对标准品DNA模板进行扩增反应。反应程序为两步法：第一步95℃5min；第二步，95℃20s，60℃1min，共进行40个循环。PCR反应体系如下面的表3所示。
表3：外源抗体基因拷贝数检测的荧光定量PCR反应体系
Figure PCTCN2018122561-appb-000009
Figure PCTCN2018122561-appb-000010
(5)探针特异性检测：
使用未转染含IgG4抗体基因的T细胞(即前述对照样品)的基因组作为背景基因组，使用外源抗体基因引物、探针扩增，引物探针组合的特异性。反应中设置一个阳性对照，即添加含外源抗体模板的标准品，一个阴性对照，即添加不含任何DNA模板的水。
(6)灵敏性检测：使用10倍梯度稀释的含铰链区-CH3标准品和内参基因Actin标准品分别进行荧光定量PCR反应。根据铰链区-CH3基因的Ct值与对应标准品拷贝数构建外源抗体基因的标准曲线，评估体系检测范围。
根据Actin基因的Ct值与对应标准品拷贝数构建标准曲线，评估体系检测范围。
(7)重复性检测：分别抽取含铰链区-CH3基因三个不同浓度的阳性样本，使用铰链区-CH3对应的引物、探针扩增，每个标准品平行重复6管。测得的CT值进行组内差异的统计学分析，以变异系数情况来确定该反应的重复性。
3.实验结果
3.1特异性验证
如图2所示，用于特异性扩增铰链区-CH3的引物和探针，检测的阳性样本产生正常的扩增曲线，未转染的T细胞(对照样本)的扩增曲线Ct值与阴性水接近，显示本发明提供的铰链区-CH3检测引物、探针能够与含外源抗体IgG4铰链区-CH3区的序列特异性地结合，不会与正常的T细胞基因组片段发生非特异性结合，具有良好的特异性。
3.2标准曲线
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。
含铰链区-CH3标准曲线的回归方程：y＝-3.858ln(x)+47.217，R2＝0.999。
检测范围为3.22E+09个拷贝/μl—3.22E+03个拷贝/μl(图3A和图3B)。
Actin标准曲线的回归方程：y＝-3.7ln(x)+47.279，R2＝0.997(图4A和图4B)，检测范围为5.58E+09个拷贝/μl—5.58E+03个拷贝/μl。
3.3重复性
采用变异系数CV(％)评价重复性结果。使用3个不同浓度的铰链区-CH3样本和Actin样本进行重复性验证，每个样本设置6个重复。
结果显示，同一样本重复管的CT值都较为接近(图5)，变异系数CV小于5％(表4)。表明本方法结果稳定，有良好的重复性。
表4：铰链区-CH3反应体系的重复性验证
Figure PCTCN2018122561-appb-000011
3.4外源抗体基因转导细胞样本检测结果
使用前述的引物、探针和检测方法，对含铰链区-CH3基因的抗体质粒电转的细胞样本进行拷贝数检测。检测结果显示，每个电转样本中外源抗体基因铰链区-CH3的拷贝数有较大差异。其中，含铰链区-CH3基因的外源抗体拷贝数从8.54E+05至5.16E+06范围，Actin的拷贝数从2.40E+05至5.15E+06(表5)，检测样本各基因拷贝数均位于标准曲线范围内。根据多个对照细胞基因组的扩增结果，平均每μg基因组含Actin基因3.49E+07拷贝。Ig4铰链区-CH3经过与Actin基因标准化后，检测样本的拷贝数范围为6.41E+06—5.21E+07拷贝/μg基因组DNA。
表5：含人源IgG4铰链区-CH3基因样本的检测
Figure PCTCN2018122561-appb-000012
Figure PCTCN2018122561-appb-000013
由上述可见：
本发明通过对内参基因Actin和外源IgG4抗体铰链区-CH3基因的检测，能够对含铰链区-CH3基因的外源抗体基因进行绝对定量检测，可用于外源抗体基因的拷贝数检测，进而用于转导效率评估、质量控制和体外监测。
本发明的引物和探针对含IgG4铰链区-CH3的外源抗体基因具有检测通用性。
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 (10)

  1. 引物对，其包含：
    引物对1，SEQ ID NO:1和SEQ ID NO:2所示的引物对；
    优选地，还包含
    引物对2，SEQ ID NO:4和SEQ ID NO:5所示的引物对。
  2. 探针，其包含：
    探针A，其核酸序列如SEQ ID NO:3所示；
    优选地，还包含
    探针B，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. 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3中任一权利要求所述的探针。
  5. 根据权利要求4所述的试剂盒，其包含：
    引物对1和探针A；
    和/或
    引物对2和探针B；
    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。
  6. 一种测定外源抗体基因拷贝数的方法，包括如下步骤：
    (1)提取待测样品的DNA；
    (2)使用含人源IgG4铰链区-CH3基因的质粒和含Actin基因的质粒作为标准品母液，分别制成外源抗体基因标准品系列和Actin标准品系列；
    (3)使用权利要求1的引物对和权利要求2或3所述的探针，分别对外源抗体基因标准品系列、Actin标准品系列和待测样品的DNA进行实时荧光定量PCR；
    (4)根据每个外源抗体标准品系列产生的Ct值与对应拷贝数，含Actin标准品系列产生的Ct值与对应拷贝数，分别绘制标准曲线，获得计算公式；
    (5)将待测样品的外源抗体基因和Actin基因的Ct值带入步骤(4)中的标准曲线的计算公式，获得待测样品的外源抗体基因拷贝数和Actin拷贝数；
    (6)通过与每μg基因组含Actin的拷贝数进行校正，获得待测样品的每μg基因组中含外源抗体基因的拷贝数；
    优选地，步骤(1)中的待测样品为含外源抗体IgG4的铰链区-CH3基因质粒转导过的细胞；
    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。
  7. 一种对外源抗体进行质量控制的方法，包括下述步骤：
    A.测定外源抗体基因的拷贝数，
    B.评估转导效率或与免疫治疗要求的外源抗体基因拷贝数进行比较；
    其中，步骤A中所述测定外源抗体基因拷贝数采用权利要求6中所述的测定外源抗体基因拷贝数的方法；
    优选地，所述外源抗体含有IgG4的铰链区-CH3。
  8. 权利要求1所述的引物对和/或权利要求2至3中任一权利要求所述的探针在制备用于测定外源抗体基因拷贝数的药物或者制备用于对外源抗体进行质量控制的药物中的用途；
    优选地，所述外源抗体基因含有外源抗体IgG4的铰链区-CH3基因；
    优选地，所述外源抗体含有IgG4的铰链区-CH3。
  9. 根据权利要求1所述的引物对，其用于测定外源抗体基因拷贝数，或者用于对外源抗体进行质量控制；
    优选地，所述外源抗体基因含有外源抗体IgG4的铰链区-CH3基因；
    优选地，所述外源抗体含有IgG4的铰链区-CH3。
  10. 根据权利要求2至3中任一权利要求所述的探针，其用于测定外源抗体基因拷贝数，或者用于对外源抗体进行质量控制；
    优选地，所述外源抗体基因含有外源抗体IgG4的铰链区-CH3基因；
    优选地，所述外源抗体含有IgG4的铰链区-CH3。
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