WO2019129048A1 - 双重荧光定量pcr测定car拷贝数的方法和试剂盒 - Google Patents

双重荧光定量pcr测定car拷贝数的方法和试剂盒 Download PDF

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WO2019129048A1
WO2019129048A1 PCT/CN2018/123860 CN2018123860W WO2019129048A1 WO 2019129048 A1 WO2019129048 A1 WO 2019129048A1 CN 2018123860 W CN2018123860 W CN 2018123860W WO 2019129048 A1 WO2019129048 A1 WO 2019129048A1
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probe
car
cd3zeta
seq
primer pair
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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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Definitions

  • the invention belongs to the field of biotechnology and relates to a method and a kit for determining CAR copy number by dual fluorescent quantitative PCR.
  • the invention relates to a method for dual copy real-time PCR to determine the copy number of a second generation CAR or a third generation CAR comprising a CD28-CD3zeta signaling region and a CD137-CD3zeta signaling region.
  • Chimeric Antigen Receptor T-Cell (CAR-T) immunotherapy is one of the most promising methods for tumor suppression in tumor immunotherapy, and it shows very positive effects in the treatment of hematological tumors.
  • the effective rate can reach 90%, and the effective rate of chronic lymphocytic leukemia and some B cell lymphoma is >50%.
  • the US FDA approved Novartis's CAR-T therapy Kymriah (formerly known as CTL-019), which fully affirmed 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 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 CAR-T clinical quality control and CAR copy number detection in peripheral blood cells of CAR-T patients. .
  • single-quantitative quantitative PCR is one of the most commonly used methods for quantitative PCR detection. In the PCR reaction system, a primer and probe of a target gene are added in one amplification reaction.
  • Dual-quantitative PCR in which two genes of interest are simultaneously amplified in the same reaction system by using different fluorophores for each gene. Amplification of two genes in the same tube often leads to competitive inhibition.
  • 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 the determination of CAR copy number by dual fluorescence quantitative 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 evaluating the transduction efficiency of CAR and controlling the quality of CAR-T Or in vitro monitoring of clinical treatment.
  • One aspect of the invention relates to a primer pair or combination of primer pairs selected from any one, two or three of the following three primer pairs:
  • first, second and third in the above “first primer pair”, “second primer pair” and “third primer pair” are merely for the purpose of distinguishing, and not Have the meaning of order.
  • Another aspect of the invention relates to a probe or probe combination selected from any one, two or three of the following three probes:
  • the third probe has the nucleic acid sequence shown as SEQ ID NO: 9.
  • the probe or probe combination wherein the probe is labeled with a fluorescent reporter group at the 5' end and a fluorescent quencher group at the 3' end;
  • the fluorescent reporter group is selected from the group consisting of FAM, Hex, VIC, ROX and Cy5;
  • the fluorescence 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.
  • the probe or probe combination wherein
  • the fluorescent reporter group in the first probe is different from the fluorescent reporter group in the third probe, and the fluorescent quenching group in the first probe is different from the fluorescent quenching group in the third probe ;
  • the fluorescent reporter group in the second probe is not identical to the fluorescent reporter group in the third probe, and the fluorescent quencher group in the second probe is different from the fluorescent quencher group in the third probe .
  • Dual-quantitative PCR in which two genes of interest are simultaneously amplified in the same reaction system by using different fluorophores for each gene. Amplification of two genes in the same tube often leads to competitive inhibition.
  • CD28-CD3zeta or CD137-CD3zeta and the internal reference gene Actin do not produce a significant competitive inhibition reaction within a certain detection range (Fig. 9B, 9C and Figs. 10C, 10D).
  • a further aspect of the invention relates to a kit comprising a primer pair of the invention, and/or a probe 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 5-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-T, 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 be adjusted in time. Program.
  • CAR gene modification is performed on T cells, and methods such as lentiviral vector, retroviral vector, and non-viral vector are often used.
  • Each preparation method may have different requirements for the number of CAR copies in the prepared cells. For example, some need to achieve ⁇ 0.2 copies/cells as a criterion for determining whether the product meets the requirements of immunotherapy.
  • the CAR gene of each cell can have one or more copies. If the copy number is equal to 1, that is, each cell in the test sample has a CAR copy. If the copy number is equal to 0.2, the test sample contains approximately 0.2 CAR copies per cell. A fixed amount of CAR plasmid transduces a fixed amount of T cells. The higher the CAR copy number, the higher the average amount of CAR gene per cell and the higher the transduction efficiency.
  • the invention relates to the use of a primer pair according to the invention and/or a probe of the invention in the preparation of a medicament for the determination of CAR copy number or for the quality control of CAR-T.
  • 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.
  • the term “dual-quantitative quantitative PCR” refers to the simultaneous amplification of two genes in the same reaction tube, and the primers, probes and primers and probes of the reference gene are added at the same time.
  • the target gene and the internal reference gene are respectively selected from different fluorescent reporter groups, and the mutual interference of the fluorescent signals during detection can be avoided by the difference in wavelength of the probe fluorophore.
  • 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 transcripts 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 one transcription This and the corresponding protein sequence are NM_001561.5/NP_001552.2.
  • CD3zeta refers to human leukocyte differentiation antigen 3zeta, ID number 919 in NCBI GeneBank, and there are two transcripts 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 genes CD28 to CD3zeta in the CAR structure.
  • CD137-CD3zeta signal region refers to a gene sequence between genes CD137 to 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 present invention uses a dual-quantitative quantitative PCR method to detect and detect the internal reference gene Actin and CD28-CD3zeta or CD137-CD3zeta genes in the same tube, which can provide detection sensitivity close to single gene amplification, greatly reduce the use of reagent consumables, and reduce Detect costs while reducing errors due to operations and increasing reliability of results.
  • the primer set, the probe or the 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 the prepared CAR-T.
  • -T Quantitative detection of CAR copy number in peripheral blood cells of patients.
  • the primer pair, probe or method of the present invention has a minimum effective detection concentration of 0.2 copies/cell for the CAR containing the CD28-CD3zeta signal region, and the lowest effective detection concentration for the CAR containing the CD137-CD3zeta signal region is 1. Copy/cell.
  • FIG. 1 Schematic diagram of a CAR plasmid map containing CD28-CD3zeta and a primer probe.
  • ITR is a transposon terminal repeat
  • TM is a transmembrane region
  • HyPB is a piggybac transposase.
  • FIG. 2 Schematic diagram of a CAR plasmid map containing CD137-CD3zeta and a primer probe.
  • ITR is a transposon terminal repeat
  • TM is a transmembrane region
  • HyPB is a piggybac transposase.
  • Figure 3A CD28-CD3zeta amplification product dissolution profile.
  • Figure 3B CD137-CD3zeta amplification product dissolution profile.
  • Figure 4A CD28-CD3zeta primer probe Amplification of CAR-T sample containing CD28-CD3zeta gene and control Control T cell sample, FAM channel.
  • Figure 4B CD28-CD3zeta primer probe Amplification of a CAR-T sample containing the CD28-CD3zeta gene and a control Control T cell sample, Hex channel.
  • Figure 5A CD137-CD3zeta primer, probe amplification of CAR-T sample containing CD137-CD3zeta gene and control Control T cell sample, FAM channel.
  • Figure 5B CD137-CD3zeta primer, probe amplification of CAR-T sample containing CD137-CD3zeta gene and control Control T cell sample, Hex channel.
  • Figure 6A Amplification plot of real-time PCR amplification of the CD28-CD3zeta standard, FAM channel.
  • Figure 6B Amplification plot of real-time PCR amplification of CD28-CD3zeta standards, HEX channel.
  • Figure 6C Standard curve of real-time fluorescent quantitative PCR amplification of CD28-CD3zeta standards.
  • Figure 7A Amplification plot of real-time PCR amplification of CD137-CD3zeta standards, FAM channel.
  • Figure 7B Amplification plot of real-time PCR amplification of CD137-CD3zeta standards, HEX channel.
  • Figure 7C Standard curve of real-time fluorescent quantitative PCR amplification of CD137-CD3zeta standards.
  • Figure 8A Reproducibility verification of amplified CD137-CD3zeta reaction system.
  • Figure 8B Reproducibility verification of amplified CD28-CD3zeta reaction system.
  • Figure 9A Comparison of amplification curves for CD28-CD3zeta preferred and non-preferred primer probe combination detection standards.
  • Figure 9B CD28-CD3zeta preferred primer probe combination detection standard, amplification curve of the internal reference gene Actin.
  • Figure 9C Amplification curve of the CD28-CD3zeta non-preferred primer probe combination detection standard, internal reference gene Actin.
  • Figure 10A Amplification curve of the CD137-CD3zeta non-preferred primer probe combination detection standard, gene CD137-CD3zeta.
  • Figure 10B Amplification profile of the CD137-CD3zeta preferred primer probe combination detection standard, gene CD137-CD3zeta.
  • Figure 10C Amplification curve of the gene Actin of the CD137-CD3zeta non-preferred primer probe combination detection standard.
  • Figure 10D Amplification profile of the gene Actin for the primer primer combination detection standard of CD137-CD3zeta.
  • Figure 11A Comparison of amplification of CAR-T samples containing the CD28-CD3zeta gene using a single gene amplification method and a dual fluorescence quantification method.
  • Figure 11B Comparison of amplification of CAR-T samples containing the CD137-CD3zeta gene using a single gene amplification method and a dual fluorescence quantification method.
  • CD28-CD3zeta signal region Fig. 1
  • CD137-CD3zeta signal region Fig. 2
  • the internal reference gene Actin designed primers and probes they were commissioned by Shanghai Jierui Biotechnology Co., Ltd. for synthesis.
  • 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.
  • 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: 11).
  • 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: 13).
  • Example 2 Using dual-quantitative PCR for CD28-CD3zeta containing regions and CD137-CD3zeta Regional CAR plasmid transduction samples for transduction efficiency assessment
  • Samples 36 cases of CAR plasmid electroporation T cell samples containing CD28-CD3zeta region, and 36 cases of CAR plasmid electroporation T cell samples containing CD137-CD3zeta region. Untransfected T cells were selected as control samples.
  • Reagent Cell Genomic DNA Extraction Kit (Tiangen Biochemical Co., Ltd.), TaqMan gene expression Master Mix reagent (ABI).
  • CAR DNA template Peripheral blood of CAR-T or CAR-T transfused patients transfected with CAR plasmid was taken, and cell DNA was extracted; as CD28-CD3zeta template and CD137-CD3zeta template, respectively.
  • the CAR plasmid containing the CD28-CD3zeta gene was 7092 bp, and the CAR plasmid containing the CD137-CD3zeta gene was 6057 bp.
  • Each standard contained 10 ng/ ⁇ l of the T cell genome.
  • a 5-fold gradient dilution standard was prepared separately (Table 2).
  • the CD28-CD3zeta standard ranges from 0.2 to 78125 copies/cell
  • the CD137-CD3Zeta standard ranges from 1-3125 copies/cell.
  • 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.
  • the amplification products of the CD28-CD3zeta and CD137-CD3zeta primers were subjected to melting curve analysis using Sybrgeen dye to verify the specificity of the primers.
  • a positive control was set up in the reaction by adding a standard containing the CD28-CD3zeta template or the CD137-CD3zeta template, a blank control, ie adding water without any DNA template.
  • the T cell genome that was not transfected with CAR was used as the background genome, using CD28-CD3zeta or CD137-CD3zeta primers, probe amplification, and the specificity of the primer probe combination.
  • Sensitivity detection A double fluorescence quantitative PCR reaction was carried out using a 5-fold gradient dilution standard. According to the difference ⁇ Ct between the Ct value of the CD28-CD3zeta or CD137-CD3zeta gene and the Ct value of the internal reference gene Actin, a standard curve was constructed with the log5 value of the corresponding standard copy number, and the detection range of the system was evaluated.
  • CD28-CD3zeta or CD137-CD3zeta were extracted from three different concentrations of positive samples, respectively, using the corresponding primers and probes of the internal reference genes Actin and CD28/CD137-CD3zeta, and each standard was paralleled. Repeat 6 tubes. The measured CT values were statistically analyzed for differences within the group, and the repeatability of the reaction was determined by the coefficient of variation.
  • the specificity of the primers using the Sybrgeen dye showed that the peaks of the dissolution curves of the CD137-CD3ezta and CD28-CD3ezta amplification products were single, indicating that the primer specificity was good.
  • primers and probes for specifically amplifying the CD137-CD3ezta signaling region and the CD28-CD3zeta signaling region produced normal amplification curves, untransfected T cells ( The Ct value of the amplification curve of the control T cell group was close to that of the negative water, and the Control T cell sample of the internal reference gene Actin showed a normal amplification curve (Fig. 4B and Fig. 5B). It is shown that the CAR detection primers and probes provided by the invention do not have non-specific binding to normal T cell genomic fragments and have good specificity.
  • the detection range was 1 copy/cell - 3.125E + 03 copies per cell range ( Figures 7A, 7B and 7C).
  • the PCR reaction system mixture including primers and probes was freeze-thawed three times, and six CD28-CD3zeta positive samples and six CD137-CD3zeta positive samples were amplified each time, and the ⁇ Ct change was observed.
  • Tables 6 and 7 The results are shown in Tables 6 and 7 below.
  • the CAR plasmid electroporation samples containing the CD28-CD3zeta and CD137-CD3zeta genes were subjected to copy number detection using the aforementioned primers, probes and detection methods.
  • the test results showed that the copy number of CAR gene in different CAR-T samples was significantly different.
  • the copy number of the CD28-CD3zeta gene was from 1.03 to 1013.06 copies/cell, and the copy number of the CD137-CD3zeta gene was from 1.78 to 30.84 copies/cell (Table 8), and the copy number of the test sample was within the range of the standard curve.
  • Table 8 Double-quantitative PCR method for detection of CAR-T sample copy number containing CD28/CD137-CD3zeta
  • 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 double fluorescence quantitative PCR method of the invention can provide detection sensitivity close to single gene amplification, greatly reduce the use of reagent consumables, and reduce the detection cost;
  • the invention can detect the exogenous CAR gene by quantitative detection of the internal reference gene Actin and the exogenous gene CAR, 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.
  • Samples CAR standards containing a mixture of T-lymphocyte genome and CD28-CD3zeta signaling region, CAR samples containing a T lymphocyte genome and a CAR plasmid containing a CD137-CD3zeta signal region, mixed proportionally.
  • Reagent Cell Genomic DNA Extraction Kit (Tiangen Biochemical Co., Ltd.), TaqMan gene expression Master Mix reagent (ABI).
  • the prepared CAR standard containing cell genomic DNA and CD28-CD3zeta gene or CD137-CD3zeta gene was taken.
  • the reaction solution is prepared according to the reaction system of the double fluorescent quantitative PCR method, and the Ct value of the CD28-CD3zeta gene or the CD137-CD3zeta gene is obtained, and the amplified Ct value of the reference gene Actin of the corresponding sample is obtained, and the amplification curves of the two genes of the sample are obtained. Ct value.
  • both CD28-CD3zeta and CD137-CD3zeta standards were amplified using corresponding preferred primer probes (as in Table 1) and non-preferred primer probes, respectively.
  • the present inventors have designed a variety of primers and probes in the course of the research, and representative of these are representative non-preferred primer probes as comparative examples.
  • Non-preferred CD28-CD3zeta primer probes are:
  • CD28-F2 CTCCTGCACAGTGACTACATG (SEQ ID NO: 14);
  • CD28-R2 GAACTTCACTCTGGAGCGATAG (SEQ ID NO: 15);
  • CCCGCAAGCATTACCAGCCCTAT is represented as SEQ ID NO: 16.
  • Non-preferred CD137-CD3zeta primer probes are:
  • CD137-F2 TGGCTGTAGCTGCCGATTT (SEQ ID NO: 17);
  • CD137-R2 TCGTCCTAGATTGAGCTCGT (SEQ ID NO: 18);
  • TCTGCGCTCCTGCTGAACT is represented as SEQ ID NO: 19.
  • the designed primer probe was synthesized by Shanghai Jierui Biotechnology Co., Ltd.
  • the CAR plasmid containing the CD28-CD3zeta gene was 7092 bp, and the CAR plasmid containing the CD137-CD3zeta gene was 6057 bp.
  • Each standard contained 10 ng/ ⁇ l of the T cell genome. According to the calculation formula of the mass of the copy number in the step (3), a 5-fold gradient dilution standard was separately prepared. The standards were 0.2, 1, 5, 25, 125, 625, 3125, 15625, and 78125 copies/cell, respectively.
  • a quantitative PCR reaction system was prepared, which included the primers and probes in the step (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 referred to Table 3 above. Primer probe combinations (preferred) in Table 1 above and primer probe combinations (not preferred) in step (1) were used as controls, respectively.
  • Amplification comparisons were made using the corresponding preferred primer probes and non-preferred primer probes, respectively, using CD28-CD3zeta or CD137-CD3zeta standards as templates.
  • the CD28-CD3zeta primer probe combination provided by the present invention is well matched and has a better amplification curve effect. Comparing the preferred or non-preferred primer probes provided in the Examples, the Actin gene was able to produce a normal amplification curve within the detection range without significant competitive inhibition (Figure 9B and Figure 9C).
  • the CD137-CD3zeta primer probe combination provided by the present invention is well matched and has a better amplification curve effect.
  • both the CD137-CD3zeta preferred primer probe combination and the non-preferred primer probe combination provided by the present invention significantly affect the amplification of the internal reference gene Actin (Fig. 10C and Fig. 10D).
  • the preferred primer probe combination has a lesser effect on the amplification of Actin than the non-preferred primer probe combination.
  • the copy number is 78125
  • the internal reference gene Actin in the non-preferred primer probe combination is unable to generate a normal amplification curve, and the Ct value cannot be detected.
  • the primer probe is combined with an amplification curve (Fig. 10C).
  • the preferred primer probe produces an Actin amplification curve that is significantly better than a non-preferred primer probe combination and produces a lower Ct value than the non-preferred primer probe combination.
  • amplification of the CD137-CD3zeta gene does not significantly affect the amplification of Actin.
  • Comparative Example 2 Comparison of double-quantitative quantitative PCR and single-plex quantitative PCR detection
  • Samples 10 CAR cell electroporation T cell samples containing CD28-CD3zeta region and 10 CAR plasmid electrotransfer T cell samples containing CD137-CD3zeta region.
  • Reagent Cell Genomic DNA Extraction Kit (Tiangen Biochemical Co., Ltd.), TaqMan gene expression Master Mix reagent (ABI).
  • the genomic DNA of the cell is extracted, and the reaction solution is prepared according to the reaction system of the double fluorescent quantitative PCR method, and the Ct value of the CD28-CD3zeta gene or the CD137-CD3zeta gene is obtained, and the Ct value of the internal reference gene Actin of the corresponding sample is obtained, and the sample is obtained.
  • the amplification curve and Ct value of the gene (primer, probe, and specific procedure are shown in Example 2).
  • the reaction system of single-quantitative real-time PCR is similar to the double-quantitative PCR reaction system.
  • only one of the CD28-CD3zeta or CD137-CD3zeta or the internal reference Actin gene is added to the corresponding primer probe. Then, the reaction system was supplemented with double distilled water.
  • the dual-fluorescence quantitative PCR method can be used to detect the target gene and the internal reference gene.
  • the double-fluorescence quantitative PCR method was used to amplify the internal reference gene Actin and CD28-CD3zeta genes or CD137-CD3zeta gene, and the difference between the Ct value obtained by the internal gene and the single-gene amplification method was small (Fig. 11A and Figure 11B). .
  • the results showed that among the ten CD28-CD3zeta samples randomly selected, the Ct values produced by the nine samples of single-quantitative PCR were less than 1 from the Ct values obtained by double-quantitative PCR, and ten CD137-CD3zeta were randomly detected. In the sample, the Ct value produced by six samples of single-quantitative PCR was less than 1 by the Ct value obtained by double-quantitative PCR.

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Abstract

本发明属于生物技术领域，涉及双重荧光定量PCR测定CAR拷贝数的方法和试剂盒以及引物对。具体地，本发明涉及用于所述方法的引物对，其选自如下的3个引物对中的任意一个、两个或者3个：第一引物对，SEQ ID NO:1和SEQ ID NO:2所示的引物对；第二引物对，SEQ ID NO:4和SEQ ID NO:5所示的引物对；和第三引物对，SEQ ID NO:7和SEQ ID NO:8所示的引物对。本发明为CAR-T免疫细胞治疗的质量控制和疗效监测提供了更准确的技术支持。本发明的检测方法可以提供接近单重荧光定量PCR方法检测的准确度和灵敏度，但所需试剂量、操作量节省了一倍，显著降低检测成本和操作带来的实验误差，提高检测效率和检测精度。

Description

双重荧光定量PCR测定CAR拷贝数的方法和试剂盒 技术领域
本发明属于生物技术领域，涉及双重荧光定量PCR测定CAR拷贝数的方法和试剂盒。具体地，本发明涉及双重荧光定量PCR测定含CD28-CD3zeta信号区和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，其在血液肿瘤的治疗中显示出非常积极的疗效，对晚期复发难治性的B急性淋巴细胞白血病的治疗有效率可达到90％，对慢性淋巴细胞白血病和部分B细胞淋巴瘤的有效率>50％。2017年8月30日，美国FDA批准了诺华公司的CAR-T疗法Kymriah(曾用名CTL-019)上市，对CAR-T的临床疗效和临床安全性给予了充分肯定。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。
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CAR-T治疗患者的外周血细胞中的CAR拷贝数检测提供技术支持。其中，单重荧光定量PCR是定量PCR检测最常用的方法之一，其在PCR反应体系中，一次扩增反应加入一个目的基因的引物、探针。
然而，在一些实验中，需要同时检测两个基因，例如，除检测目的基因之外，还需要同时检测内参基因。在检测基因表达水平变化时，通过内参基因作为参照，标准化校正基因的表达量，避免因上样量和上样过程中存在的实验误差带来的结果不准确。另外，为了提高实验结果的准确性，定量PCR一般要求每个实验样本设置3个及以 上的重复。如果一个待检的CAR-T样本需要同时检测目的基因和内参基因，则需要操作6管甚至更多管的反应量。很明显，双基因检测的任务量至少是单基因检测的两倍。此外，当样本量较大时，试剂消耗量和操作量会成倍增加，而由操作带来的实验误差可能性也会成倍上升。
双重荧光定量PCR，即通过每个基因使用不同的荧光基团，在同一反应体系中同时扩增两个目的基因。在同管扩增两个基因，往往引起竞争性抑制。
因此，尚需要开发新的双重荧光定量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，本发明人得到了双重荧光定量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，用于评估CAR的转导效率、控制CAR-T的质量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3个引物对中的任意一个、两个或者3个：
SEQ ID NO:1和SEQ ID NO:2所示的第一引物对；
SEQ ID NO:4和SEQ ID NO:5所示的第二引物对；和
SEQ ID NO:7和SEQ ID NO:8所示的第三引物对。
其中，上述“第一引物对”、“第二引物对”和“第三引物对”中的“第一”、“第二”和“第三”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3个探针的中的任意一个、两个或者3个：
第一探针，其核酸序列如SEQ ID NO:3所示；
第二探针，其核酸序列如SEQ ID NO:6所示；和
第三探针，其核酸序列如SEQ ID NO:9所示。
其中，上述“第一探针”、“第二探针”和“第三探针”中的“第一”、“第二”和“第三”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，所述的探针或探针组合，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 PCTCN2018123860-appb-000001
BHQ1的结构式以及连接方式如下面的式II所示。
Figure PCTCN2018123860-appb-000002
Hex的结构式以及连接方式如下面的式III所示。
Figure PCTCN2018123860-appb-000003
TRAMA的结构式以及连接方式如下面的式IV所示。
Figure PCTCN2018123860-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。
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，
第一探针中的荧光报告基团与第三探针中的荧光报告基团不相同，并且第一探针中的荧光淬灭基团与第三探针中的荧光淬灭基团不相同；
和/或
第二探针中的荧光报告基团与第三探针中的荧光报告基团不相同，并且第二探针中的荧光淬灭基团与第三探针中的荧光淬灭基团不相同。
双重荧光定量PCR，即通过每个基因使用不同的荧光基团，在同一反应体系中同时扩增两个目的基因。在同管扩增两个基因，往往引起竞争性抑制。本发明通过设计合适的引物、探针，CD28-CD3zeta或CD137-CD3zeta与内参基因Actin在一定检测范围内不会产生明显的竞争性抑制反应(图9B、9C和图10C、10D)。
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：
第一引物对和第三引物对，以及第一探针和第三探针；
和/或
第二引物对和第三引物对，以及第二探针和第三探针；
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，提取未转导的T淋巴细胞DNA作为背景DNA模板进行梯度稀释，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)根据每个标准品产生的CD28-CD3zeta或CD137-CD3zeta基因的Ct值与对应的内参基因Actin的Ct值，获得目的基因与内参基因Actin的Ct值的差值△Ct，使用△Ct与标准品对应的拷贝数log5值绘制标准曲线，获得标准曲线的计算公式；
(5)将待测样本的DNA的CD28-CD3zeta或CD137-CD3zeta基因的Ct值与对应的内参基因Actin的Ct值的差值△Ct，带入步骤(4)中的标准曲线的计算公式，获得待测样品的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5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-T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。
目前，对T细胞进行CAR基因修饰，常采用慢病毒载体、逆转录病毒载体、非病毒载体等方法。每种制备方法对制备细胞中的CAR拷贝数要求可能不尽相同，例如有的需要达到≥0.2拷贝数/个细胞，作为产品是否达到免疫治疗要求的判断标准。
每个细胞的CAR基因可以有一个或多个拷贝。如果拷贝数等于1，即检测样本中近似每个细胞均有一个CAR拷贝。如果拷贝数等于0.2，即检测样本中近似为每细胞含0.2个CAR拷贝。固定量的CAR质粒转导固定量的T细胞，CAR拷贝数越高，平均每个细胞中含的CAR基因量就越高，转导效率就越高。
本发明在再一方面涉及本发明的引物对和/或本发明的探针在制备用于测定CAR拷贝数的药物或者用于对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)。
术语“双重荧光定量PCR”是指同一个反应管中同时进行两个基因的扩增反应，同 时加入目的基因的引物、探针和内参基因的引物、探针。目的基因和内参基因分别选用不同的荧光报告基团，通过探针荧光基团的波长差异，可以避免检测时荧光信号的相互干扰。
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个转录本及对应的蛋白序列，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个转录本及对应的蛋白序列，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个转录本及对应的蛋白序列，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 PCTCN2018123860-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 PCTCN2018123860-appb-000006
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)本发明使用双重荧光定量PCR方法，能够同管扩增检测内参基因Actin与CD28-CD3zeta或CD137-CD3zeta基因，可以提供接近单基因扩增的检测灵敏度，大幅降低试剂耗材的使用，减少检测成本，同时减少因操作带来的误差，增加结果的可靠性。
(3)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者CAR-T治疗患者外周血细胞中CAR拷贝数的定量检测。
(4)本发明的引物对、探针或方法对含CD28-CD3zeta信号区的CAR最低的有 效检测浓度为0.2个拷贝/细胞，对含CD137-CD3zeta信号区的CAR最低的有效检测浓度为1个拷贝/细胞。
附图说明
图1：含CD28-CD3zeta的CAR质粒图谱、引物探针的示意图。ITR为转座子末端重复序列，TM为跨膜区，HyPB是piggybac转座酶。
图2：含CD137-CD3zeta的CAR质粒图谱、引物探针的示意图。ITR为转座子末端重复序列，TM为跨膜区，HyPB是piggybac转座酶。
图3A：CD28-CD3zeta扩增产物溶解曲线。
图3B：CD137-CD3zeta扩增产物溶解曲线。
图4A：CD28-CD3zeta引物探针扩增含CD28-CD3zeta基因的CAR-T样本与对照Control T细胞样本，FAM通道。
图4B：CD28-CD3zeta引物探针扩增含CD28-CD3zeta基因的CAR-T样本与对照Control T细胞样本，Hex通道。
图5A：CD137-CD3zeta引物、探针扩增含CD137-CD3zeta基因的CAR-T样本与对照Control T细胞样本，FAM通道。
图5B：CD137-CD3zeta引物、探针扩增含CD137-CD3zeta基因的CAR-T样本与对照Control T细胞样本，Hex通道。
图6A：实时荧光定量PCR扩增CD28-CD3zeta标准品的扩增曲线图，FAM通道。
图6B：实时荧光定量PCR扩增CD28-CD3zeta标准品的扩增曲线图，HEX通道。
图6C：实时荧光定量PCR扩增CD28-CD3zeta标准品的标准曲线图。
图7A：实时荧光定量PCR扩增CD137-CD3zeta标准品的扩增曲线图，FAM通道。
图7B：实时荧光定量PCR扩增CD137-CD3zeta标准品的扩增曲线图，HEX通道。
图7C：实时荧光定量PCR扩增CD137-CD3zeta标准品的标准曲线图。
图8A：扩增CD137-CD3zeta反应体系的重复性验证。
图8B：扩增CD28-CD3zeta反应体系的重复性验证。
图9A：CD28-CD3zeta优选和非优选引物探针组合检测标准品的扩增曲线对比。
图9B：CD28-CD3zeta优选引物探针组合检测标准品，内参基因Actin的扩增曲 线。
图9C：CD28-CD3zeta非优选引物探针组合检测标准品，内参基因Actin的扩增曲线。
图10A：CD137-CD3zeta非优选引物探针组合检测标准品，基因CD137-CD3zeta的扩增曲线。
图10B：CD137-CD3zeta优选引物探针组合检测标准品，基因CD137-CD3zeta的扩增曲线。
图10C：CD137-CD3zeta非优选引物探针组合检测标准品，基因Actin的扩增曲线。
图10D：CD137-CD3zeta优选引物探针组合检测标准品，基因Actin的扩增曲线。
图11A：使用单基因扩增方法与双重荧光定量方法对含CD28-CD3zeta基因的CAR-T样本扩增对比。
图11B：使用单基因扩增方法与双重荧光定量方法对含CD137-CD3zeta基因的CAR-T样本扩增对比。
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)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。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。
表1：引物序列和探针序列
Figure PCTCN2018123860-appb-000007
Figure PCTCN2018123860-appb-000008
CD28-CD3zeta信号区序列：
Figure PCTCN2018123860-appb-000009
其中下划线部分为117bp产物的核苷酸序列(SEQ ID NO:11)。
CD137-CD3zeta信号区序列：
Figure PCTCN2018123860-appb-000010
其中下划线部分为114bp产物的核苷酸序列(SEQ ID NO:13)。
实施例2：使用双重荧光定量PCR对含CD28-CD3zeta区域和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：36例含CD28-CD3zeta区域的CAR质粒电转T细胞样品，36例含CD137-CD3zeta区域的CAR质粒电转T细胞样品。选择未转染的T细胞作为对照样品。
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：LightCycler480荧光定量PCR检测仪(罗氏公司)。
2.实验方法
取36例含CD28-CD3zeta基因的CAR质粒电转T细胞样品和36例含CD137-CD3zeta基因的CAR质粒电转T细胞样品，分别抽提基因组。根据双重荧光定量PCR方法的反应体系配制反应液，获取CD28-CD3zeta基因或CD137-CD3zeta基因的Ct值及对应样本的内参基因Actin的Ct值，获得该样本两基因的Ct值差值△Ct。△Ct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 DNA模板的制备：取CAR质粒转导过的CAR-T或CAR-T回输患者的外周血，提取细胞DNA；分别作为CD28-CD3zeta模板和CD137-CD3zeta模板。
(2)标准品的制备：使用含CD28-CD3zeta信号区和CD137-CD3zeta信号区的CAR质粒分别制备标准品母液，提取未转导的T淋巴细胞DNA作为背景模板。
(3)外源基因拷贝数的质量的计算：假设①人T细胞基因组的用量为m ng，②包含外源基因的质粒长度为n bp，③单倍体T细胞基因组的大小为2.99╳10E9bp，④通常原代转基因T细胞外源基因全部为杂合体，外源基因会随机***T细胞基因组中，那么的转基因T细胞基因组中含有一个CAR外源基因拷贝数的质量为：m╳n╳÷(2╳2.99╳10 9bp)。
(4)含CD28-CD3zeta基因的CAR质粒为7092bp，含CD137-CD3zeta基因的CAR质粒为6057bp。每个标准品含T细胞基因组10ng/μl。根据(3)中的拷贝数的质量的计算公式，分别制备5倍梯度稀释标准品(表2)。其中，CD28-CD3zeta标准品的范围为0.2－78125个拷贝/细胞，CD137-CD3Zeta标准品的范围为1－3125个拷贝/细胞。
表2：CD137-CD3zeta标准品和CD28-CD3zeta标准品的制备
Figure PCTCN2018123860-appb-000011
Figure PCTCN2018123860-appb-000012
(5)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：CAR拷贝数检测的双重荧光定量PCR反应体系
Figure PCTCN2018123860-appb-000013
(6)探针特异性检测：
使用Sybrgeen染料，对CD28-CD3zeta和CD137-CD3zeta引物的扩增产物进行融解曲线分析，验证引物的特异性。
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，一个空白对照，即添加不含任何DNA模板的水。
使用未转染CAR的T细胞基因组作为背景基因组，使用CD28-CD3zeta或CD137-CD3zeta引物、探针扩增，引物探针组合的特异性。
(7)灵敏性检测：使用5倍梯度稀释的标准品进行双重荧光定量PCR反应。根据CD28-CD3zeta或CD137-CD3zeta基因的Ct值与内参基因Actin的Ct值的差值△Ct，与对应标准品拷贝数的log5值构建标准曲线，评估体系的检测范围。
(8)重复性检测：分别抽取CD28-CD3zeta或CD137-CD3zeta三个不同浓度的阳性样本，分别使用内参基因Actin和CD28/CD137-CD3zeta的对应的引物、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。
(9)稳定性检测：将包含引物、探针在内的PCR反应体系混合物冻融3次，每次扩增6个含CD28-CD3zeta的CAR-T样本和6个含CD137-CD3zeta的CAR-T样本，观察△Ct变化。
3.实验结果
3.1特异性验证
如图3A和图3B所示，使用Sybrgeen染料对引物的特异性验证显示，CD137-CD3ezta和CD28-CD3ezta扩增产物溶解曲线峰单一，表明引物特异性好。
如图4A和图5A所示，用于特异性扩增CD137-CD3ezta信号区和CD28-CD3zeta信号区的引物和探针，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T细胞)组的扩增曲线Ct值与阴性水接近，同时内参基因Actin检测Control T细胞样本有正常扩增曲线(图4B和图5B)。显示本发明提供的CAR检测引物、探针不会与正常的T细胞基因组片段发生非特异性结合，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。
CD28-CD3zeta标准曲线的回归方程：y＝-0.3956x+1.8151，R2＝0.9941(图6A、图6B和图6C)，检测范围为0.2—7.812E+04拷贝/细胞。
CD137-CD3zeta标准曲线的回归方程：y＝-0.3854x+2.1448，R2＝0.9916。
检测范围为1拷贝/细胞—3.125E+03拷贝/个细胞范围内(图7A、图7B和图7C)。
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CD28-CD3zeta样本和 CD137-CD3zeta样本进行重复性验证，每个样本设置6个重复。
结果显示，无论是Actin，CD28-CD3zeta还是CD137-CD3zeta，同一样本重复管的CT值都较为接近(图8A和图8B)，变异系数CV小于4％(表4、表5)。表明本方法结果稳定，有良好的重复性。
表4：CD28-CD3zeta反应体系的重复性验证
Figure PCTCN2018123860-appb-000014
表5：CD137-CD3zeta反应体系的重复性验证
Figure PCTCN2018123860-appb-000015
3.4稳定性
将包含引物、探针在内的PCR反应体系混合物冻融3次，每次扩增6个CD28-CD3zeta阳性样本和6个CD137-CD3zeta阳性样本，观察△Ct变化。结果如下面的表6和表7所示。
结果显示，反应体系经过多次冻融，每个样本的△Ct值差异均小于1，大多数样本的△Ct值小于0.5。
表6：CD28-CD3zeta反应体系的重复性验证
Figure PCTCN2018123860-appb-000016
Figure PCTCN2018123860-appb-000017
表7：CD137-CD3zeta反应体系的重复性验证
Figure PCTCN2018123860-appb-000018
3.5CAR-T样本检测结果
使用前述的引物、探针和检测方法，对含CD28-CD3zeta和CD137-CD3zeta基因的CAR质粒电转样本进行拷贝数检测。检测结果显示，不同CAR-T样本中CAR基因的拷贝数有较大差异。CD28-CD3zeta基因的拷贝数从1.03至1013.06拷贝/细胞，CD137-CD3zeta基因拷贝数从1.78至30.84拷贝/细胞(表8)，检测样本的拷贝数均位于标准曲线范围内。
表8：双重荧光定量PCR方法检测含CD28/CD137-CD3zeta的CAR-T样本拷贝数
Figure PCTCN2018123860-appb-000019
Figure PCTCN2018123860-appb-000020
Figure PCTCN2018123860-appb-000021
Figure PCTCN2018123860-appb-000022
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；
本发明使用双重荧光定量PCR方法，能够提供接近单基因扩增的检测灵敏度，大幅降低试剂耗材的使用，减少检测成本；
本发明通过对内参基因Actin和外源基因CAR的检测，能够对外源CAR基因进行绝对定量检测，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。
对照实施例1
1.样品、试剂及仪器
样品：含T淋巴细胞基因组和CD28-CD3zeta信号区的CAR质粒按比例混合的CAR标准品，含T淋巴细胞基因组和CD137-CD3zeta信号区的CAR质粒按比例混合的CAR标准品。
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：LightCycler480荧光定量PCR检测仪(罗氏公司)。
2.实验方法
取制备的含细胞基因组DNA及CD28-CD3zeta基因或CD137-CD3zeta基因的CAR 标准品。根据双重荧光定量PCR方法的反应体系配制反应液，获取CD28-CD3zeta基因或CD137-CD3zeta基因的Ct值，及对应样本的内参基因Actin的扩增Ct值，获得该样本两基因的扩增曲线及Ct值。作为对照，CD28-CD3zeta和CD137-CD3zeta标准品均分别使用对应的优选的引物探针(如表1)和非优选引物探针进行扩增。
具体步骤如下：
(1)非优选的引物探针的设计、合成：
本发明人在研究过程中设计了多种引物、探针，这里列举其中有代表性的非优选引物探针作为对比实施例。
非优选的CD28-CD3zeta引物探针分别为：
CD28-F2:CTCCTGCACAGTGACTACATG(SEQ ID NO:14)；
CD28-R2:GAACTTCACTCTGGAGCGATAG(SEQ ID NO:15)；
CD28 Taqman probe 2:
5’FAM-CCCGCAAGCATTACCAGCCCTAT-3’TAMRA，其中，
CCCGCAAGCATTACCAGCCCTAT表示为SEQ ID NO:16。
非优选的CD137-CD3zeta引物探针分别为：
CD137-F2:TGGCTGTAGCTGCCGATTT(SEQ ID NO:17)；
CD137-R2:TCGTCCTAGATTGAGCTCGT(SEQ ID NO:18)；
CD137 Taqman probe 2:
5’FAM-TCTGCGCTCCTGCTGAACT-3’BHQ1，其中
TCTGCGCTCCTGCTGAACT表示为SEQ ID NO:19。
设计的引物探针交由上海捷瑞生物公司合成。
(2)标准品的制备：使用含CD28-CD3zeta信号区和CD137-CD3zeta信号区的CAR质粒分别制备标准品母液，提取未转导的T淋巴细胞DNA作为背景模板。
(3)外源基因拷贝数的质量的计算：假设①人T细胞基因组的用量为m ng，②包含外源基因的质粒长度为n bp，③单倍体T细胞基因组的大小为2.99╳10E9bp，④通常原代转基因T细胞外源基因全部为杂合体，外源基因会随机***T细胞基因组中，那么转基因T细胞基因组中含有一个CAR外源基因拷贝数的质量为：m╳n╳÷(2╳2.99╳10 9bp)。
(4)含CD28-CD3zeta基因的CAR质粒为7092bp，含CD137-CD3zeta基因的CAR质粒为6057bp。每个标准品含T细胞基因组10ng/μl。根据步骤(3)中的拷贝数的质量的计算公式，分别制备5倍梯度稀释标准品。标准品分别为0.2、1、5、25、125、625、 3125、15625、78125个拷贝/细胞。
(5)实时荧光定量PCR检测：配制定量PCR反应体系，该反应体系包含了步骤(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反应体系参考上面的表3。分别使用前面表1中的引物探针组合(优选)和步骤(1)中引物探针组合(非优选)作为对照。
(6)获取CD28-CD3zeta基因或CD137-CD3zeta基因的Ct值，及对应样本的内参基因Actin的扩增Ct值，获得该样本两基因的扩增曲线及Ct值。
3.实验结果
使用CD28-CD3zeta或CD137-CD3zeta标准品作为模板，分别使用对应的优选引物探针和非优选引物探针进行扩增对比。
通过图9A可见，本发明提供的CD28-CD3zeta引物探针组合匹配良好，有更好的扩增曲线效果。对照实施例中提供的优选或非优选引物探针，在检测范围内Actin基因均能产生正常的扩增曲线，不会有明显的竞争性抑制反应(图9B和图9C)。
通过图10A和图10B可知，本发明提供的CD137-CD3zeta引物探针组合匹配良好，有更好的扩增曲线效果。但在拷贝数大于3125时，本发明的提供的CD137-CD3zeta优选引物探针组合和非优选引物探针组合均会明显影响内参基因Actin的扩增(图10C和图10D)。但明显地，优选的引物探针组合对Actin的扩增影响弱于非优选引物探针组合。在拷贝数为78125时，非优选引物探针组合中内参基因Actin已无法产生正常扩增曲线，无法检测到Ct值，优选引物探针组合有扩增曲线(图10C)。在拷贝数15625时，优选的引物探针产生的Actin扩增曲线明显优于非优选的引物探针组合，产生的Ct值也低于非优选的引物探针组合。在拷贝数在3125及其以下的标准品中，无论是优选引物探针还是非优选引物探针，CD137-CD3zeta基因的扩增均不会明显影响Actin的扩增。
对照实施例2：双重荧光定量PCR与单重荧光定量PCR检测效果比对
1.样品、试剂及仪器
样品：10例含CD28-CD3zeta区域的CAR质粒电转T细胞样品，10例含CD137-CD3zeta区域的CAR质粒电转T细胞样品。
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：LightCycler480荧光定量PCR检测仪(罗氏公司)。
2.实验方法
抽提细胞基因组DNA，根据双重荧光定量PCR方法的反应体系配制反应液，获取CD28-CD3zeta基因或CD137-CD3zeta基因的Ct值，及对应样本的内参基因Actin的扩增Ct值，获得该样本两基因的扩增曲线及Ct值(使用的引物、探针、具体步骤见实施例2)。
单重荧光定量PCR(单基因的荧光定量)的反应体系与双重荧光定量PCR反应体系相似，单次反应仅添加CD28-CD3zeta或CD137-CD3zeta或内参Actin基因中的一个基因对应配套的引物探针，然后用双蒸水补足反应体系。
3.实验结果
使用双重荧光定量PCR方法，可以同管检测目的基因和内参基因。如表9、表10所示，使用双重荧光定量PCR方法扩增内参基因Actin和CD28-CD3zeta基因或CD137-CD3zeta基因，获得的Ct值与单基因扩增方法获得的Ct值差异较小(图11A和图11B)。。
表9：使用单重荧光PCR和双重荧光PCR方法对含CD28-CD3zeta信号区的CAR拷贝数检测
Figure PCTCN2018123860-appb-000023
Figure PCTCN2018123860-appb-000024
表10：使用单重荧光PCR和双重荧光PCR方法对含CD137-CD3zeta信号区的CAR拷贝数检测
Figure PCTCN2018123860-appb-000025
Figure PCTCN2018123860-appb-000026
结果显示，在随机检测的十份CD28-CD3zeta样本中，九份样本单重荧光定量PCR的产生的Ct值与双重荧光定量PCR获得的Ct值差异小于1，在随机检测的十份CD137-CD3zeta样本中，有六份样本单重荧光定量PCR的产生的Ct值与双重荧光定量PCR获得的Ct值差异小于1。
结果表明，使用双重荧光定量PCR方法同时扩增内参基因Actin和CD28/CD137-CD3zeta基因，可以获得接近单基因扩增的检测准确度。
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3个引物对中的任意一个、两个或者3个：
    SEQ ID NO:1和SEQ ID NO:2所示的第一引物对；
    SEQ ID NO:4和SEQ ID NO:5所示的第二引物对；和
    SEQ ID NO:7和SEQ ID NO:8所示的第三引物对。
  2. 探针，其选自如下的3个探针的中的任意一个、两个或者3个：
    第一探针，其核酸序列如SEQ ID NO:3所示；
    第二探针，其核酸序列如SEQ ID NO:6所示；和
    第三探针，其核酸序列如SEQ ID NO:9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；
    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。
  5. 根据权利要求3或4所述的探针，其中，
    第一探针中的荧光报告基团与第三探针中的荧光报告基团不相同，并且第一探针中的荧光淬灭基团与第三探针中的荧光淬灭基团不相同；
    和/或
    第二探针中的荧光报告基团与第三探针中的荧光报告基团不相同，并且第二探针中的荧光淬灭基团与第三探针中的荧光淬灭基团不相同。
  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：
    第一引物对和第三引物对，以及第一探针和第三探针；
    和/或
    第二引物对和第三引物对，以及第二探针和第三探针；
    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，提取未转导的T淋巴细胞DNA作为背景DNA模板进行梯度稀释，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，对标准品系列和待测样品的DNA分别进行实时荧光定量PCR；
    (4)根据每个标准品产生的CD28-CD3zeta或CD137-CD3zeta基因的Ct值与对应的内参基因Actin的Ct值，获得目的基因与内参基因Actin的Ct值的差值△Ct，使用△Ct与标准品对应的拷贝数log5值绘制标准曲线，获得标准曲线的计算公式；
    (5)将待测样本的DNA的CD28-CD3zeta或CD137-CD3zeta基因的Ct值与对应的内参基因Actin的Ct值的差值△Ct，带入步骤(4)中的标准曲线的计算公式，获得待测样品的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5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，其其用于测定CAR拷贝数，或者用于对CAR-T进行质量控制。
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