CN109971836A

CN109971836A - The method and kit of double fluorescent quantitative PCR measurement CAR copy number

Info

Publication number: CN109971836A
Application number: CN201711459675.2A
Authority: CN
Inventors: 钱其军; 金华君; 郝方元; 王超; 孙娟娟
Original assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Current assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Priority date: 2017-12-28
Filing date: 2017-12-28
Publication date: 2019-07-05
Also published as: WO2019129048A1

Abstract

The invention belongs to field of biotechnology, it is related to the method and kit and primer pair of double fluorescent quantitative PCR measurement CAR copy number.In particular it relates to be used for the primer pair of the method, in 3 primer pairs chosen from the followings any one, two or 3: primer pair 1, primer pair shown in SEQ ID NO:1 and SEQ ID NO:2；Primer pair 2, primer pair shown in SEQ ID NO:4 and SEQ ID NO:5；With primer pair 3, primer pair shown in SEQ ID NO:7 and SEQ ID NO:8.The present invention provides more accurate technical support for the quality control of CAR-T immune cell therapy and curative effect monitoring.Detection method of the invention can provide the accuracy and sensitivity detected close to substance fluorescence quantifying PCR method, but required amount of reagent, operating quantity save one times, significantly reduce testing cost and operation bring experimental error, improve detection efficiency and detection accuracy.

Description

The method and kit of double fluorescent quantitative PCR measurement CAR copy number

Technical field

The invention belongs to field of biotechnology, are related to the method and reagent of double fluorescent quantitative PCR measurement CAR copy number Box.In particular it relates to double fluorescent quantitative PCR measurement signaling zone containing CD28-CD3zeta and CD137-CD3zeta letter The method of the copy number of the second generation CAR or third generation CAR in number area.

Background technique

Chimeric antigen receptor T cell (Chimeric Antigen Receptor T-Cell, CAR-T) immunotherapy is mesh Most it is hopeful to cure one of the method for tumour in pre-neoplastic immunization therapy, is shown in the treatment of neoplastic hematologic disorder very actively Curative effect, the treated effect of the B acute lymphoblastic leukemia intractable to late recurrent can reach 90%, to chronic lymphatic Effective percentage > 50% of chronic myeloid leukemia and partial B cell lymthoma.On August 30th, 2017, U.S. FDA has approved Novartis Co., Ltd CAR-T therapy Kymriah (former name CTL-019) listing, clinical efficacy and clinical safety to CAR-T give sufficiently Certainly.CAR-T produces huge impetus to the application of CAR-T therapy in the howling success of blood tumor.CAR-T is treated at present The application of method has been not limited to the treatment of neoplastic hematologic disorder.

The preparation and Quality Control of CAR-T is to influence one of CAR-T Therapeutic safety and the key factor of therapeutic effect.CD28 and CD137 is two kinds of T cell costimulatory molecules, plays an important role to the activation of T cell, proliferation, is second generation CAR and the third generation The important component of CAR.

Real-time fluorescence quantitative PCR is that fluorophor is added in DNA amplification reaction, is accumulated using fluorescence signal, is supervised in real time Survey the variation of PCR product amount.Real-time fluorescence quantitative PCR is because of the spies such as its high sensitivity, reproducible, easy to operate, low in cost Point is widely used in detection field.It, can be from molecular level to the CAR in CAR-T using the method for real-time fluorescence quantitative PCR Copy number detection is carried out, the CAR copy number detection in the peripheral blood cells of patient is treated in the clinical Quality Control and CAR-T that are CAR-T Technical support is provided.Wherein, substance quantitative fluorescent PCR is one of most common method of quantitative PCR detection, in PCR reactant In system, primer, the probe of a target gene is added in an amplified reaction.

However, needing to detect two genes simultaneously in some experiments, for example, in addition to testing goal gene, it is also necessary to Reference gene is detected simultaneously.When detecting gene expression dose variation, by reference gene as reference, suppressor is standardized Expression quantity, avoid because of experimental error bring result inaccuracy existing for during applied sample amount and loading.In addition, in order to improve The accuracy of experimental result, the repetition that quantitative PCR generally requires each experiment sample to be arranged 3 or more.If one to be checked CAR-T sample need simultaneously testing goal gene and reference gene, then need to operate the reacting dose of 6 pipes even more multitube.Very Obviously, the task amount of dual-gene detection is at least twice of single-gene detection.In addition, when sample size is larger, reagent consumption It can be multiplied with operating quantity, and can also be risen at double by operation bring experimental error possibility.

Double fluorescent quantitative PCR uses different fluorophors by each gene, in same reaction system simultaneously Expand two target gene.In two genes of tube amplification, often cause Reverse transcriptase.

Therefore, it is still necessary to develop the method and Related product of new double fluorescent quantitative PCR measurement CAR copy number.

Summary of the invention

The present inventor passes through in-depth study and creative labor, has obtained a kind of primer pair and probe.It is basic herein On, the present inventor has obtained the method and kit of double fluorescent quantitative PCR measurement CAR copy number.Method or examination of the invention Agent box can measure the copy of CAR in the peripheral blood cells of the copy number of CAR or CAR-T treatment patient in the CAR-T of preparation Number, for assessing the transduction efficiency of CAR, controlling the quality of CAR-T or the external monitoring of clinical treatment.Thus provide following hairs It is bright:

One aspect of the present invention is related to the combination of a kind of primer pair or primer pair, in 3 primer pairs chosen from the followings Any one, two or 3:

Primer pair 1, primer pair shown in SEQ ID NO:1 and SEQ ID NO:2；

Primer pair 2, primer pair shown in SEQ ID NO:4 and SEQ ID NO:5；With

Primer pair 3, primer pair shown in SEQ ID NO:7 and SEQ ID NO:8.

It is any in 3 probes chosen from the followings another aspect of the present invention relates to a kind of probe or probe combinations One, two or 3:

Probe A, nucleic acid sequence is as shown in SEQ ID NO:3；

Probe B, nucleic acid sequence is as shown in SEQ ID NO:6；With

Probe C, nucleic acid sequence is as shown in SEQ ID NO:9.

In one or more embodiments of the invention, the probe or probe combinations, wherein the 5 ' of the probe End is marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group；Preferably, the fluorescent reporter group be selected from FAM, Hex, VIC, ROX and Cy5；Preferably, the fluorescent quenching group is selected from BHQ1, TAMRA, JOE, BHQ2 and BHQ3.

The structural formula and connection type of FAM is as shown in following Formulas I.

The structural formula and connection type of BHQ1 is as shown in following Formula II.

The structural formula and connection type of Hex is as shown in following formula III.

The structural formula and connection type of TRAMA is as shown in following formula IV.

In one or more embodiments of the invention, the probe or probe combinations, wherein

The base of one or more positions in probe A and/or probe B modifies (i.e. probe A and/or probe B by lock nucleic acid In there are one or more bases to introduce one or more lock nucleic acid monomers)；

Preferably, any 1,2,3 or 4 in the 4th, the 7th, the 10th and the 13rd have been held in the 5 ' of probe A A base is modified by lock nucleic acid；And/or any 1 in the 4th, the 7th, the 10th and the 13rd, 2 have been held in the 5 ' of probe B A, 3 or 4 bases are modified by lock nucleic acid.

Fluorescent reporter group in probe A and the fluorescent reporter group in probe C be not identical, and the fluorescence in probe A Quenching group and the fluorescent quenching group in probe C be not identical；

And/or

Fluorescent reporter group in probe B and the fluorescent reporter group in probe C be not identical, and the fluorescence in probe B Quenching group and the fluorescent quenching group in probe C be not identical.

Double fluorescent quantitative PCR uses different fluorophors by each gene, in same reaction system simultaneously Expand two target gene.In two genes of tube amplification, often cause Reverse transcriptase.The present invention is suitably drawn by design Object, probe, CD28-CD3zeta or CD137-CD3zeta will not generate in certain detection range bright with reference gene Actin Aobvious Reverse transcriptase reacts (Fig. 9 B, 9C and Figure 10 C, 10D).

Another aspect of the invention is related to a kind of kit, and it includes primer pair of the invention and/or spies of the invention Needle.

In one or more embodiments of the invention, the kit, it includes:

Primer pair 1 and primer pair 3 and probe A and probe C；

And/or

Primer pair 2 and primer pair 3 and probe B and probe C；

Preferably, the kit also includes reagent needed for PCR reaction, such as Mg²⁺, reaction buffer, dNTP and Taq Enzyme.

Another aspect of the invention is related to a kind of method for measuring CAR copy number, includes the following steps:

(1) DNA of sample to be tested is extracted；

(2) use the CAR plasmid of signaling zone containing CD28-CD3zeta or the signaling zone containing CD137-CD3zeta as standard items Mother liquor extracts the T lymphocyte DNA not transduceed and carries out gradient dilution as background dna template, standard items series is made；

(3) using probe described in any one of primer pair and the present invention of the invention, to standard items series and to test sample The DNA of product carries out real-time fluorescence quantitative PCR；

(4) according to each standard items generate CD28-CD3zeta or CD137-CD3zeta gene Ct value with it is corresponding The Ct value of reference gene Actin obtains the difference △ Ct of target gene and the Ct value of reference gene Actin, uses △ Ct and mark The corresponding copy number log5 value of quasi- product draws standard curve, obtains the calculation formula of standard curve；

(5) by the Ct value of the CD28-CD3zeta or CD137-CD3zeta gene of the DNA of sample to be tested and corresponding internal reference The difference △ Ct of the Ct value of Gene A ctin, brings the calculation formula of the standard curve in step (4) into, obtains the CAR of sample to be tested Gene copy number；

Preferably, the sample to be tested in step (1) is the periphery of CAR-T or CAR-T feedback patient that CAR plasmid transduction is crossed Blood；

Preferably, the gradient dilution in step (2) is 5 times of gradient dilutions；

Preferably, the condition of the amplified reaction of real-time fluorescence quantitative PCR described in step (3) are as follows: 94 DEG C of 5min；94℃ 20s, 60 DEG C of 1min, totally 40 recycle.

Another aspect of the invention is related to the method that a kind of couple of CAR-T carries out quality control, includes the following steps:

A. CAR copy number is measured,

B. the CAR copy number required with immunization therapy is compared；

Wherein, measurement CAR copy number described in step A is using the heretofore described method for measuring CAR copy number.It is excellent Selection of land, the CAR contain " CD28-CD3zeta signaling zone " and/or " CD137-CD3zeta signaling zone ".Preferably, described CAR-T contains the CAR.

The production Quality Control or the monitored in vitro after CAR-T feedback that method of the invention can be used in CAR-T, such as examining Survey the CAR copy number in CAR-T or CAR-T treatment patient peripheral's haemocyte of preparation.It can be further used for assessing turning for CAR-T Efficiency, treatment curative effect or toxic side effect are led, or for assessing the CAR copy number in peripheral blood CAR-T and treatment curative effect or poison Correlation between side effect, so that clinical treatment adjusts therapeutic scheme in time.

Currently, CAR gene modification is carried out to T cell, frequently with slow virus carrier, retroviral vector, non-viral load The methods of body.Every kind of preparation method may be not quite similar to the CAR copy number requirement prepared in cell, such as some need reaches Whether >=0.2 copy number/cell, the judgment criteria of immunization therapy requirement is reached as product.

The CAR gene of each cell can have one or more copies.It is if copy number is equal to 1, i.e., close in detection sample There is a CAR copy like each cell.It if copy number is equal to 0.2, i.e., is approximately every cell containing 0.2 in detection sample CAR copy.The T cell of the CAR plasmid transduction fixed amount of fixed amount, CAR copy number is higher, the CAR contained in average each cell Gene dosage is higher, and transduction efficiency is higher.

The present invention is related to primer pair of the invention and/or probe of the invention in preparation for measuring CAR in another further aspect The drug of copy number or for CAR-T carry out quality control drug in purposes.Preferably, the CAR contains " CD28-CD3zeta signaling zone " and/or " CD137-CD3zeta signaling zone ".Preferably, the CAR-T contains the CAR.

Part term of the present invention is explained below.

Term " Chimeric antigen receptor " (Chimeric Antigen Receptor, CAR) is swollen on tumor cell film The single-chain antibody of tumor related antigen (tumor associated antigen, TAA) is connected with intracellular signal domain by hinge area The mosaic gene of composition.

Term " CAR-T " is should by the expression obtained after gene transfer/transfection technological sourcing T cell by CAR gene The cell of CAR gene.This kind of cell, which has, to be identified and attacks the ability for expressing the tumour cell of corresponding cell surface TAA.

Term " second generation CAR ": first generation CAR is by immunoglobulin scFv and Fc ε RI receptor or CD3 compound knot intracellular It merges to form Chimerical receptor in structure domain.Second generation CAR is based on first generation CAR structure, increases a new costimulatory signal, such as CD28 or CD137 etc..

Term " third generation CAR ": on the basis of two generation CAR structures, an additional costimulation letter is further increased Number, so that CAR is gathered around simultaneously, there are two costimulating factor (such as while possessing CD28 and CD137).

Term " double fluorescent quantitative PCR " refers to the amplified reaction in the same reaction tube while carrying out two genes, together When be added the primer of target gene, the primer of probe and reference gene, probe.Target gene and reference gene select difference respectively Fluorescent reporter group can be interfered with each other to avoid fluorescence signal when detection by the wavelength difference of fluorescence probe group.

Herein, " CD28 " refers to human leukocytes differentiation antigen 28, also known as Tp44, its ID in NCBI GeneBank Number be 940, have 3 transcripts and corresponding protein sequence, respectively NM_001243077.1/NP_001230006.1, NM_ 001243078.1/NP_001230007.1, NM_006139.3/NP_006130.1.

Herein, " CD137 " refers to human leukocytes differentiation antigen 137, also known as 4-1BB, its official in NCBI GeneBank The entitled TNFRSF9 (A member of the TNF receptor family 9) in side, ID number 3604, only 1 transcript and corresponding Protein sequence is NM_001561.5/NP_001552.2.

Herein, " CD3zeta " refers to human leukocytes differentiation antigen 3zeta, and the ID number in NCBI GeneBank is 919, There are 2 transcripts and corresponding protein sequence, respectively NM_000734.3/NP_000725.1, NM_198053.2/NP_ 932170.1。

In the present invention, term " CD28-CD3zeta signaling zone " refers to the base in CAR structure between gene C D28 to CD3zeta Because of sequence.

In the present invention, term " CD137-CD3zeta signaling zone " refers in CAR structure between gene C D137 to CD3zeta Gene order.

In the present invention, term " lock nucleic acid (locked nucleotide acid, LNA) " is a kind of comprising one or more Lock nucleic acid monomer (LNA monomer (s), i.e. [2'-O, 4'-C-methylene- β-D-ribofuranosyl monomer (s)] oligonucleotides or oligonucleotide derivative).Lock nucleic acid is introduced in conventional TaqMan probe, probe and mesh can be improved Sequence affinity, increase the Tm value of probe.

In one preferred embodiment of the invention (such as embodiment 1), the connection type of lock nucleic acid monomer and base As shown in following Formula V, wherein B indicates base.

Specific connection type (the LNA- of base T, C (5- methylation), G that are modified in lock nucleic acid monomer and probe sequence T, LNA-5-Me-C and LNA-G) respectively as shown in following Formula IV, VII and VIII.

Advantageous effect of the invention

The present invention achieves at least one in following technical effect:

(1) primer pair of the invention, probe or method are to signaling zone containing CD28-CD3zeta and CD137-CD3zeta signal The second generation and the third generation CAR detection in area have the versatility of detection.

(2) present invention uses double fluorescent quantitative PCR method, being capable of tube amplification detection reference gene Actin and CD28- CD3zeta or CD137-CD3zeta gene can provide the detection sensitivity expanded close to single-gene, and reagent consumption is greatly reduced The use of material reduces testing cost, while reducing because operating bring error, increases the reliability of result.

(3) specificity of primer pair of the invention, probe or method is good, and sensitivity is high, reaction system favorable repeatability, surely It is qualitative good；It can be used in the quantitative detection of CAR copy number or CAR-T in the CAR-T of preparation to treat in patient peripheral's haemocyte The quantitative detection of CAR copy number.

(4) primer pair of the invention, probe or the method effective detection minimum to the CAR of the signaling zone containing CD28-CD3zeta Concentration be 0.2 copy/cell, the effective detectable concentration minimum to the CAR of the signaling zone containing CD137-CD3zeta be 1 copy/ Cell.

Detailed description of the invention

Fig. 1: the schematic diagram of CAR plasmid map, primed probe containing CD28-CD3zeta.ITR is transposon ends repetition Sequence, TM are transmembrane region, and HyPB is piggybac transposase.

Fig. 2: the schematic diagram of CAR plasmid map, primed probe containing CD137-CD3zeta.ITR is transposon ends repetition Sequence, TM are transmembrane region, and HyPB is piggybac transposase.

Fig. 3 A:CD28-CD3zeta amplified production solubility curve.

Fig. 3 B:CD137-CD3zeta amplified production solubility curve.

Fig. 4 A:CD28-CD3zeta primed probe amplification the gene containing CD28-CD3zeta CAR-T sample with compare Control T cell sample, the channel FAM.

Fig. 4 B:CD28-CD3zeta primed probe amplification the gene containing CD28-CD3zeta CAR-T sample with compare Control T cell sample, the channel Hex.

Fig. 5 A:CD137-CD3zeta primer, probe amplification gene containing CD137-CD3zeta CAR-T sample with compare Control T cell sample, the channel FAM.

Fig. 5 B:CD137-CD3zeta primer, probe amplification gene containing CD137-CD3zeta CAR-T sample with compare Control T cell sample, the channel Hex.

Fig. 6 A: real-time fluorescence quantitative PCR expands the amplification curve diagram of CD28-CD3zeta standard items, the channel FAM.

Fig. 6 B: real-time fluorescence quantitative PCR expands the amplification curve diagram of CD28-CD3zeta standard items, the channel HEX.

Fig. 6 C: the canonical plotting of real-time fluorescence quantitative PCR amplification CD28-CD3zeta standard items.

Fig. 7 A: real-time fluorescence quantitative PCR expands the amplification curve diagram of CD137-CD3zeta standard items, the channel FAM.

Fig. 7 B: real-time fluorescence quantitative PCR expands the amplification curve diagram of CD137-CD3zeta standard items, the channel HEX.

Fig. 7 C: the canonical plotting of real-time fluorescence quantitative PCR amplification CD137-CD3zeta standard items.

Fig. 8 A: the repeatability verifying of amplification CD137-CD3zeta reaction system.

Fig. 8 B: the repeatability verifying of amplification CD28-CD3zeta reaction system.

Fig. 9 A:CD28-CD3zeta is preferably compared with the amplification curve of not preferred primer combination of probe examination criteria product.

The preferred primer combination of probe examination criteria product of Fig. 9 B:CD28-CD3zeta, the amplification curve of reference gene Actin.

The amplification of the not preferred primer combination of probe examination criteria product of Fig. 9 C:CD28-CD3zeta, reference gene Actin is bent Line.

The not preferred primer combination of probe examination criteria product of Figure 10 A:CD137-CD3zeta, the expansion of gene C D137-CD3zeta Increase curve.

The preferred primer combination of probe examination criteria product of Figure 10 B:CD137-CD3zeta, the amplification of gene C D137-CD3zeta Curve.

The not preferred primer combination of probe examination criteria product of Figure 10 C:CD137-CD3zeta, the amplification curve of Gene A ctin.

The preferred primer combination of probe examination criteria product of Figure 10 D:CD137-CD3zeta, the amplification curve of Gene A ctin.

Figure 11 A: using single-gene amplification method with double fluorescent quantitative method to the CAR-T of the gene containing CD28-CD3zeta Sample amplification comparison.

Figure 11 B: using single-gene amplification method with double fluorescent quantitative method to the CAR- of the gene containing CD137-CD3zeta The amplification comparison of T sample.

Specific embodiment

Below by specific embodiment combination attached drawing, invention is further described in detail.Those skilled in the art will It will be appreciated that following case study on implementation is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.In case study on implementation not Particular technique or condition person are indicated, described technology or conditions are (such as with reference to J. Pehanorm cloth Shandong according to the literature in the art Gram equal to write, " Molecular Cloning:A Laboratory guide " that Huang Peitang etc. is translated, the third edition, Science Press) or according to product description into Row.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.

Embodiment 1: the design and synthesis of primer and probe

Based on CD28-CD3zeta signaling zone (Fig. 1) and CD137-CD3zeta signaling zone (Fig. 2) and reference gene Actin design primer and probe, the synthesis of commission Shanghai JaRa Biotechnology Co., Ltd.The sequence of primer and probe is for example following Shown in table 1.Wherein CD28-CD3zeta and CD137-CD3zeta probe introduces lock nucleic acid monomer, and the base of letter capitalization is The base of lock nucleic acid modification.

Table 1: primer sequence and probe sequence

CD28-CD3zeta signal region sequence:

CCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTT CTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCC GCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCA GACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGT TTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACA ATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGG CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCC TCGCTGATAA(SEQ ID NO:10)

Wherein underscore part is the nucleotide sequence (SEQ ID NO:11) of 117bp product.

CD137-CD3zeta signal region sequence:

AAACGGGGCAGAAAGAAGCTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGA TGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACG CCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTG GACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGA ACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACG ATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO:12)

Wherein underscore part is the nucleotide sequence (SEQ ID NO:13) of 114bp product.

Embodiment 2: using double fluorescent quantitative PCR to the region containing CD28-CD3zeta and the region CD137-CD3zeta CAR plasmid transduction sample carries out transduction efficiency assessment

1. sample, reagent and instrument

Sample: the CAR plasmid electricity in 36 regions containing CD28-CD3zeta turns T cell sample, and 36 contain CD137-CD3zeta The CAR plasmid electricity in region turns T cell sample.Select the T cell of untransfected as control sample.

Reagent: cell genomic dna extraction agent box (Tiangeng biochemical corp), TaqMan gene expression Master Mix reagent (ABI company).

Instrument: LightCycler480 fluorescent quantitative PCR detector (Roche Holding Ag).

2. experimental method

The CAR plasmid electricity of 36 genes containing CD28-CD3zeta is taken to turn T cell sample and 36 bases containing CD137-CD3zeta The CAR plasmid electricity of cause turns T cell sample, extracts genome respectively.It is prepared according to the reaction system of double fluorescent quantitative PCR method Reaction solution obtains the Ct value of CD28-CD3zeta gene or CD137-CD3zeta gene and the reference gene Actin of corresponding sample Ct value, obtain the Ct value difference value △ Ct of two gene of sample.△ Ct substitutes into standard curve, calculates the absolute copy of each sample Number.

Specific step is as follows:

(1) preparation of CAR DNA profiling: the CAR-T or CAR-T for taking CAR plasmid transduction to cross feed back the peripheral blood of patient, mention Take cell DNA；Respectively as CD28-CD3zeta template and CD137-CD3zeta template.

(2) preparation of standard items: the CAR matter of signaling zone containing CD28-CD3zeta and CD137-CD3zeta signaling zone is used Grain prepares standard items mother liquor respectively, extracts the T lymphocyte DNA not transduceed as background template.

(3) calculating of the quality of copy number of foreign gene: assuming that 1. the dosage of human T-cell's genome is m ng, 2. include The plasmid length of foreign gene is n bp, and 3. the size of monoploid T cell genome is 2.99 × 10E9 bp, 4. usually primary The all heterozygotes of transgenic T cells foreign gene, foreign gene can in radom insertion T cell genome, then transgenosis T Quality containing a CAR copy number of foreign gene in cellular genome are as follows: m × n × ÷ (2 × 2.99 × 10⁹bp)。

(4) the CAR plasmid of the gene containing CD28-CD3zeta is 7092bp, and the CAR plasmid of the gene containing CD137-CD3zeta is 6057bp.Each standard items genome containing T cell 10ng/ μ l.According to the calculation formula of the quality of the copy number in (3), respectively Prepare 5 times of gradient dilution standard items (table 2).Wherein, the range of CD28-CD3zeta standard items be 0.2-78125 copy/carefully Born of the same parents, the range of CD137-CD3Zeta standard items are 1-3125 copy/cell.

The preparation of table 2:CD137-CD3zeta standard items and CD28-CD3zeta standard items

(5) real-time fluorescence quantitative PCR detects: preparing quantitative PCR reaction system, contains in table 1 in the reaction system Primer and probe.Standard items DNA profiling is carried out amplification reaction.Response procedures are two-step method: 95 DEG C of 5min of the first step；Second Step, 95 DEG C of 20s, 60 DEG C of 1min carry out 40 circulations altogether.PCR reaction system is as shown in Table 3 below.

The double fluorescent quantitative PCR reaction system of table 3:CAR copy number detection

(6) probe specificity detects:

Using Sybrgeen dyestuff, the amplified production of CD28-CD3zeta and CD137-CD3zeta primer is melted The specificity of primer is verified in tracing analysis.

One positive control of setting in reaction, i.e. addition template containing CD28-CD3zeta or CD137-CD3zeta template Standard items, the water that a blank control, i.e. addition are free of any DNA profiling.

Use the T cell genome of untransfected CAR as background genes group, uses CD28-CD3zeta or CD137- CD3zeta primer, probe amplification, the specificity of primer combination of probe.

(7) sensitivity detects: carrying out double fluorescent quantitative PCR reaction using the standard items of 5 times of gradient dilutions.According to The difference △ Ct of the Ct value of the Ct value and reference gene Actin of CD28-CD3zeta or CD137-CD3zeta gene, with corresponding mark The log5 value of quasi- product copy number constructs standard curve, the detection range of evaluation system.

(8) repeatability detection: the positive sample of tri- various concentrations of CD28-CD3zeta or CD137-CD3zeta is extracted respectively This, uses corresponding primer, the probe amplification of reference gene Actin and CD28/CD137-CD3zeta, each standard items respectively It is parallel to repeat 6 pipes.The CT value measured carries out the statistical analysis of group difference, and the weight of the reaction is determined with coefficient of variation situation Renaturation.

(9) Detection of Stability: by comprising PCR reaction system mixture freeze thawing 3 times including primer, probe, 6 are expanded every time A CAR-T sample and 6 CAR-T samples containing CD137-CD3zeta containing CD28-CD3zeta, observation △ Ct variation.

3. experimental result

3.1 specificity verification

As shown in Figure 3A and Figure 3B, it is shown using specificity verification of the Sybrgeen dyestuff to primer, CD137-CD3ezta It is single with CD28-CD3ezta amplified production solubility curve peak, show that primer specificity is good.

As shown in Fig. 4 A and Fig. 5 A, it to be used for specific amplification CD137-CD3ezta signaling zone and CD28-CD3zeta signal The primer and probe in area, the positive sample of detection generate normal amplification curve, T cell (control T cell) group of untransfected Amplification curve Ct value and negative water are close, while reference gene Actin detection Control T cell sample has normal amplification curve (Fig. 4 B and Fig. 5 B).Show that CAR detection primer provided by the invention, probe will not occur with normal T cell genomic fragment Non-specific binding has good specificity.

3.2 standard curve

Fluorescent quantitative PCR result shows that standard items DNA is linearly incremented by with gradient dilution CT value.

The regression equation of CD28-CD3zeta standard curve: y=-0.3956x+1.8151, R2=0.9941 (Fig. 6 A, figure 6B and Fig. 6 C), detection range is 0.2-7.812E+04 copy/cell.

The regression equation of CD137-CD3zeta standard curve: y=-0.3854x+2.1448, R2=0.9916.

Detection range is in 1 copy/cell -3.125E+03 copy/cell context (Fig. 7 A, Fig. 7 B and Fig. 7 C).

3.3 repeated

Repeated result is evaluated using coefficient of variation CV (%).Using 3 various concentrations CD28-CD3zeta sample and CD137-CD3zeta sample carries out repeated verifying, and 6 repetitions are arranged in each sample.

The results show that either Actin, CD28-CD3zeta or CD137-CD3zeta, the CT of same sample repeating pipe Value is all closer to (Fig. 8 A and Fig. 8 B), and coefficient of variation CV is less than 4% (table 4, table 5).Show that this method result is stablized, has good Repeatability.

The repeatability verifying of table 4:CD28-CD3zeta reaction system

The repeatability verifying of table 5:CD137-CD3zeta reaction system

3.4 stability

By comprising PCR reaction system mixture freeze thawing 3 times including primer, probe, 6 CD28-CD3zeta are expanded every time Positive sample and 6 CD137-CD3zeta positive samples, observation Δ Ct variation.As a result as shown in following table 6 and table 7.

The results show that reaction system passes through thawing, the Δ Ct value difference of each sample is different to be respectively less than 1, most numerical example Δ Ct value less than 0.5.

The repeatability verifying of table 6:CD28-CD3zeta reaction system

The repeatability verifying of table 7:CD137-CD3zeta reaction system

3.5CAR-T pattern detection result

Using primer above-mentioned, probe and detection method, to the gene containing CD28-CD3zeta and CD137-CD3zeta CAR plasmid electricity sample transfer this progress copy number detection.Testing result shows, in different CAR-T samples the copy number of CAR gene have compared with Big difference.From 1.03 to 1013.06 copies of copy number/cell of CD28-CD3zeta gene, CD137-CD3zeta gene copy Number from 1.78 to 30.84 copies/cell (table 8), the copy number for detecting sample are respectively positioned in standard curve range.

Table 8: double fluorescent quantitative PCR method is detected containing CD28/CD137-CD3zeta's

CAR-T sample copy number

From the foregoing, it can be seen that:

The second generation of primer and probe of the invention to signaling zone containing CD28-CD3zeta and CD137-CD3zeta signaling zone CAR and third generation CAR has the versatility of detection；

The present invention uses double fluorescent quantitative PCR method, is capable of providing the detection sensitivity expanded close to single-gene, substantially The use of reagent consumptive material is reduced, testing cost is reduced；

The present invention can carry out external source CAR gene exhausted by the detection to internal reference Gene A ctin and foreign gene CAR To quantitative detection, the CAR copy number in the control of CAR-T mass and CAR-T treatment patient peripheral's haemocyte that can be used for preparing is examined It surveys.

Comparative examples 1

1. sample, reagent and instrument

Sample: the CAR mark that the CAR plasmid of genome containing T lymphocyte and CD28-CD3zeta signaling zone mixes in proportion The CAR standard items that the CAR plasmid of quasi- product, genome containing T lymphocyte and CD137-CD3zeta signaling zone mixes in proportion.

2. experimental method

Take the CAR standard containing cell genomic dna and CD28-CD3zeta gene or CD137-CD3zeta gene of preparation Product.Reaction solution is prepared according to the reaction system of double fluorescent quantitative PCR method, obtains CD28-CD3zeta gene or CD137- The Ct value of CD3zeta gene, and the amplification Ct value of the reference gene Actin of sample is corresponded to, obtain the amplification of two gene of sample Curve and Ct value.As control, CD28-CD3zeta and CD137-CD3zeta standard items are preferably drawn using corresponding respectively Physical prospecting needle (such as table 1) and not preferred primed probe are expanded.

Specific step is as follows:

(1) design, synthesis of not preferred primed probe:

The present inventor devises a variety of primers, probe in the course of the research, enumerates here wherein representational not preferred Primed probe embodiment as a comparison.

Not preferred CD28-CD3zeta primed probe is respectively as follows:

CD28-F2:CTCCTGCACAGTGACTACATG(SEQ ID NO:14)；

CD28-R2:GAACTTCACTCTGGAGCGATAG(SEQ ID NO:15)；

CD28 Taqman probe 2:

5 ' FAM-CCCGCAAGCATTACCAGCCCTAT-3 ' TAMRA, wherein

CCCGCAAGCATTACCAGCCCTAT is expressed as SEQ ID NO:16.

Not preferred CD137-CD3zeta primed probe is respectively as follows:

CD137-F2:TGGCTGTAGCTGCCGATTT(SEQ ID NO:17)；

CD137-R2:TCGTCCTAGATTGAGCTCGT(SEQ ID NO:18)；

CD137 Taqman probe 2:

5 ' FAM-TCTGCGCTCCTGCTGAACT-3 ' BHQ1, wherein

TCTGCGCTCCTGCTGAACT is expressed as SEQ ID NO:19.

The primed probe of design transfers to Shanghai JaRa biotech firm to synthesize.

(3) calculating of the quality of copy number of foreign gene: assuming that 1. the dosage of human T-cell's genome is m ng, 2. include The plasmid length of foreign gene is n bp, and 3. the size of monoploid T cell genome is 2.99 × 10E9 bp, 4. usually primary The all heterozygotes of transgenic T cells foreign gene, foreign gene is understood in radom insertion T cell genome, then transgenosis T is thin Quality containing a CAR copy number of foreign gene in born of the same parents' genome are as follows: m × n × ÷ (2 × 2.99 × 10⁹bp)。

(4) the CAR plasmid of the gene containing CD28-CD3zeta is 7092bp, and the CAR plasmid of the gene containing CD137-CD3zeta is 6057bp.Each standard items genome containing T cell 10ng/ μ l.According to the calculation formula of the quality of the copy number in step (3), 5 times of gradient dilution standard items are prepared respectively.Standard items, which are respectively 0.2,1,5,25,125,625,3125,15625,78125, to be copied Shellfish/cell.

(5) real-time fluorescence quantitative PCR detects: preparing quantitative PCR reaction system, which contains in step (1) Primer and probe.Standard items DNA profiling is carried out amplification reaction.Response procedures are two-step method: 95 DEG C of 5min of the first step；Second Step, 95 DEG C of 20s, 60 DEG C of 1min carry out 40 circulations altogether.PCR reaction system refers to table 3 above.Respectively before use in table 1 Primer combination of probe (preferably) and step (1) in primer combination of probe it is (not preferred) as control.

(6) the Ct value of CD28-CD3zeta gene or CD137-CD3zeta gene, and the reference gene of corresponding sample are obtained The amplification Ct value of Actin obtains the amplification curve and Ct value of two gene of sample.

3. experimental result

Use CD28-CD3zeta or CD137-CD3zeta standard items as template, uses corresponding preferred primer respectively Probe and not preferred primed probe carry out amplification comparison.

By Fig. 9 A as it can be seen that CD28-CD3zeta primer combination of probe provided by the invention matching is good, there is better expansion Increase J curve effectJ.The preferred or not preferred primed probe provided in comparative examples, Actin gene can produce in detection range Raw normal amplification curve does not have apparent Reverse transcriptase reaction (Fig. 9 B and Fig. 9 C).

By Figure 10 A and Figure 10 B it is found that CD137-CD3zeta primer combination of probe provided by the invention matching is good, have Better amplification curve effect.But when copy number is greater than 3125, the preferred primer of the CD137-CD3zeta of offer of the invention is visited Needle combination and not preferred primer combination of probe can significantly affect the amplification (Figure 10 C and Figure 10 D) of reference gene Actin.But it is bright Aobvious ground, preferred primer combination of probe are weaker than not preferred primer combination of probe to the amplification of Actin influence.It is in copy number When 78125, reference gene Actin can not generate normal amplification curve in not preferred primer combination of probe, can not detect Ct Value, preferably primer combination of probe has amplification curve (Figure 10 C).In copy number 15625, what preferred primed probe generated Actin amplification curve is substantially better than not preferred primer combination of probe, and the Ct value of generation is also below not preferred primed probe group It closes.In copy number in 3125 and its standard items below, either preferred primed probe or not preferred primed probe, The amplification of CD137-CD3zeta gene will not significantly affect the amplification of Actin.

Comparative examples 2: double fluorescent quantitative PCR is compared with substance fluorescence quantitative PCR detection effect

1. sample, reagent and instrument

Sample: the CAR plasmid electricity in 10 regions containing CD28-CD3zeta turns T cell sample, and 10 contain CD137-CD3zeta The CAR plasmid electricity in region turns T cell sample.

2. experimental method

Cell genomic dna is extracted, reaction solution is prepared according to the reaction system of double fluorescent quantitative PCR method, is obtained The Ct value of CD28-CD3zeta gene or CD137-CD3zeta gene, and correspond to the amplification Ct of the reference gene Actin of sample Value obtains the amplification curve and Ct value of two gene of sample (primer that uses, probe, specific steps are shown in embodiment 2).

The reaction system and double fluorescent quantitative PCR reaction system of substance quantitative fluorescent PCR (monogenic fluorescent quantitation) Similar, the gene pairs that single reaction is only added in CD28-CD3zeta or CD137-CD3zeta or internal reference Actin gene is answered Matched primed probe, then supplies reaction system with distilled water.

3. experimental result

It, can be with pipe testing goal gene and reference gene using double fluorescent quantitative PCR method.Such as table 9,10 institute of table Show, expands reference gene Actin and CD28-CD3zeta gene or CD137-CD3zeta using double fluorescent quantitative PCR method Gene, the Ct value difference that the Ct value and single-gene amplification method of acquisition obtain are different smaller (Figure 11 A and Figure 11 B).

Table 9: using substance fluorescent PCR and double fluorescent PCR method to the CAR copy number of the signaling zone containing CD28-CD3zeta Detection

Table 10: the CAR of the signaling zone containing CD137-CD3zeta is copied using substance fluorescent PCR and double fluorescent PCR method Number detection

The results show that in ten parts of CD28-CD3zeta samples of random detection, nine parts of sample substance quantitative fluorescent PCRs The Ct value difference that the Ct value and double fluorescent quantitative PCR of generation obtain is different less than 1, in ten parts of CD137-CD3zeta of random detection In sample, the Ct value difference for having the Ct value of the generation of six parts of sample substance quantitative fluorescent PCRs to obtain with double fluorescent quantitative PCR is different small In 1.

The result shows that expanding reference gene Actin and CD28/CD137- simultaneously using double fluorescent quantitative PCR method CD3zeta gene can obtain the accuracy in detection expanded close to single-gene.

Although a specific embodiment of the invention has obtained detailed description, it will be understood to those of skill in the art that.Root According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these change in guarantor of the invention Within the scope of shield.Full scope of the invention is given by the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110>Shanghai cell therapy research institute

Shanghai cell therapy Engineering Technical Research Centre Group Co., Ltd

<120>method and kit of double fluorescent quantitative PCR measurement CAR copy number

<130> IDC170180

<160> 19

<170> PatentIn version 3.2

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<212> DNA

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ctcctgcaca gtgactacat g 21

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gaacttcact ctggagcgat ag 22

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ccgcaagcat taccagcc 18

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aaggaggatg tgaactgaga 20

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gcatgggtca gaaggattcc tat 23

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tgtagaaggt gtggtgccag att 23

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tcgagcacgg catcgtcacc aa 22

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<223>CD28-CD3zeta signal region sequence

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cccttttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 60

gtggccttta ttattttctg ggtgaggagt aagaggagca ggctcctgca cagtgactac 120

atgaacatga ctccccgccg ccccgggccc acccgcaagc attaccagcc ctatgcccca 180

ccacgcgact tcgcagccta tcgctccaga gtgaagttca gcaggagcgc agacgccccc 240

gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 300

tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 360

aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 420

agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 480

ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 540

cgctgataa 549

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ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat 60

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aaacggggca gaaagaagct cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactgagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 180

cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 240

cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 300

tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 360

gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 420

gacacctacg acgcccttca catgcaggcc ctgccccctc gc 462

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aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agacgccccc gcgtaccagc 60

agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag tacg 114

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Claims

1. primer pair, in 3 primer pairs chosen from the followings any one, two or 3:

Primer pair 1, primer pair shown in SEQ ID NO:1 and SEQ ID NO:2；

Primer pair 2, primer pair shown in SEQ ID NO:4 and SEQ ID NO:5；With

Primer pair 3, primer pair shown in SEQ ID NO:7 and SEQ ID NO:8.

2. probe, in 3 probes chosen from the followings any one, two or 3:

Probe A, nucleic acid sequence is as shown in SEQ ID NO:3；

Probe B, nucleic acid sequence is as shown in SEQ ID NO:6；With

Probe C, nucleic acid sequence is as shown in SEQ ID NO:9.

3. probe according to claim 2, wherein 5 ' ends of the probe are marked with fluorescent reporter group, 3 ' end labels There is fluorescent quenching group；

Preferably, the fluorescent reporter group is selected from FAM, Hex, VIC, ROX and Cy5；

Preferably, the fluorescent quenching group is selected from BHQ1, TAMRA, JOE, BHQ2 and BHQ3.

4. probe according to claim 2 or 3, wherein

The base of one or more positions in probe A and/or probe B is modified by lock nucleic acid；

Preferably, any 1,2,3 in the 4th, the 7th, the 10th and the 13rd or 4 alkali have been held in the 5 ' of probe A Base is modified by lock nucleic acid；And/or any 1,2,3 in the 4th, the 7th, the 10th and the 13rd have been held in the 5 ' of probe B A or 4 bases are modified by lock nucleic acid.

5. probe according to claim 3 or 4, wherein

Fluorescent reporter group in probe A and the fluorescent reporter group in probe C be not identical, and the fluorescent quenching in probe A Group and the fluorescent quenching group in probe C be not identical；

And/or

Fluorescent reporter group in probe B and the fluorescent reporter group in probe C be not identical, and the fluorescent quenching in probe B Group and the fluorescent quenching group in probe C be not identical.

6. a kind of kit, it includes any claims in primer pair described in claim 1 and/or claim 2 to 5 The probe.

7. kit according to claim 6, it includes:

Primer pair 1 and primer pair 3 and probe A and probe C；

And/or

Primer pair 2 and primer pair 3 and probe B and probe C；

8. a kind of method for measuring CAR copy number, includes the following steps:

(1) DNA of sample to be tested is extracted；

(2) use the CAR plasmid of signaling zone containing CD28-CD3zeta or the signaling zone containing CD137-CD3zeta as standard items mother Liquid extracts the T lymphocyte DNA not transduceed and carries out gradient dilution as background dna template, standard items series is made；

(3) using probe described in any claim in primer pair and claim 2 to 5 described in claim 1, to standard The DNA of strain column and sample to be tested carries out real-time fluorescence quantitative PCR respectively；

(4) according to the Ct value of the CD28-CD3zeta or CD137-CD3zeta gene of each standard items generation and corresponding internal reference The Ct value of Gene A ctin obtains the difference △ Ct of target gene and the Ct value of reference gene Actin, uses △ Ct and standard items Corresponding copy number log5 value draws standard curve, obtains the calculation formula of standard curve；

(5) by the Ct value of the CD28-CD3zeta or CD137-CD3zeta gene of the DNA of sample to be tested and corresponding reference gene The difference △ Ct of the Ct value of Actin, brings the calculation formula of the standard curve in step (4) into, obtains the CAR gene of sample to be tested Copy number；

Preferably, the sample to be tested in step (1) is the peripheral blood of CAR-T or CAR-T feedback patient that CAR plasmid transduction is crossed；

Preferably, the condition of the amplified reaction of real-time fluorescence quantitative PCR described in step (3) are as follows: 94 DEG C of 5min；94 DEG C of 20s, 60 DEG C 1min, totally 40 circulations.

9. the method that a kind of couple of CAR-T carries out quality control, includes the following steps:

A. CAR copy number is measured,

B. the CAR copy number required with immunization therapy is compared；

Wherein, measurement CAR copy number described in step A is using the method for measuring CAR copy number described in claim 8.

10. probe described in any claim is used in preparation in primer pair described in claim 1 and/or claim 2 to 5 Purposes in the drug of the drug of measurement CAR copy number or preparation for carrying out quality control to CAR-T.