CN117701556A - CAR genome virus vector copy number detection kit and application thereof - Google Patents

Abstract

The invention provides a CAR genome virus vector copy number detection kit and its application. Specifically, the present invention provides a pair of specific primers and probes respectively targeting the CAR sequence, the lentiviral backbone and the single-copy internal reference gene, and a kit constructed therefrom, which detects the sample CAR gene copy number and/or viral vector copy by calculating By using the method of relative quantification or absolute quantification of numbers, the persistence of CAR-T cells in the body can be evaluated, providing a basis for clinical treatment plans and efficacy evaluation. The detection method of the present invention has strong versatility, high sensitivity, good specificity, and wide linear range. One tube reaction can realize multiplex PCR detection, saves time, and has high practical value.

Description

CAR genome virus vector copy number detection kit and its application

Technical field

The invention belongs to the field of molecular biology, and specifically relates to a CAR genome virus vector copy number detection kit and its application.

Background technique

Chimeric antigen receptor (CAR)-T cell immunotherapy technology has become a hot topic in tumor treatment. This technology uses molecular biology methods to modify T cells at the genetic level, solving the problem of T cells recognizing tumor cells and activating their own functions at once. The method is to use artificially designed brand-new genetic engineering molecules to transfer the gene encoding the CAR molecule into T cells through a vector, so that the T cells express the CAR molecule. CAR-T cell therapy technology has brought revolutionary therapeutic effects and hope of cure to some patients. However, as a new type of living cell drug, CAR-T cell drugs are still an immature field of development. There is currently not enough data to evaluate their potential risks. Its preparation, quality control, and clinical application all present unprecedented challenges. and room for improvement.

Constructing CAR-T cells requires transfecting and integrating the CAR gene into the T cell genome through viral or non-viral systems. When the gene is expressed normally and a transmembrane CAR structure is formed on the cell membrane, CAR-T cells have the activity to recognize and kill target cells. Therefore, accurate detection of positive T cells expressing the CAR genome is a key to quality control of CAR-T drugs. key step. From the perspective of measuring gene safety, vector gene integration may bring harm to cells such as the activation of some proto-cancer cell genes or the inactivation of tumor suppressor genes, resulting in the risk of secondary infection with malignant tumors. Therefore, it is particularly urgent to detect the copy number of CAR genome that has been integrated into cells.

In summary, there is an urgent need in this field to develop CAR-positive genome copy number detection kits and detection methods with high accuracy, good specificity, convenient detection, stable results, and strong versatility to meet the needs of CAR-T drug quality control and clinical testing. Sample testing needs.

Contents of the invention

The purpose of the present invention is to provide a specific primer probe pair for CAR genome viral vector copy number detection and a kit constructed therefrom to meet the needs of CAR-T drug quality control and clinical sample detection.

Another object of the present invention is to provide the use of specific primer probe pairs and kits, as well as a method for detecting the copy number of CAR genome viral vectors.

In a first aspect of the present invention, a specific primer-probe pair is provided, the primer-probe pair includes: BBZ primer-probe pair, WPRE primer-probe pair and ACTIN primer-probe pair,

Wherein, the BBZ primer-probe pair includes: primer set 1 consisting of an upstream primer as shown in SEQ ID NO:1 and a downstream primer as shown in SEQ ID NO:2, and a probe sequence as shown in SEQ ID NO:7 1;

The WPRE primer-probe pair includes: primer set 2 consisting of an upstream primer shown in SEQ ID NO:3 and a downstream primer shown in SEQ ID NO:4, and probe sequence 2 shown in SEQ ID NO:8;

The ACTIN primer-probe pair includes: primer set 3 consisting of an upstream primer shown in SEQ ID NO:5 and a downstream primer shown in SEQ ID NO:6, and probe sequence 3 shown in SEQ ID NO:9.

In another preferred embodiment, the BBZ primer-probe pair specifically amplifies and binds to the BBZ sequence.

In another preferred embodiment, the WPRE primer-probe pair specifically amplifies and binds to the WPRE sequence.

In another preferred embodiment, the ACTIN primer-probe pair specifically amplifies and binds to the ACTIN sequence.

In another preferred embodiment, the probe sequence in the specific primer-probe pair is labeled with a fluorescent group at its 5' end and a quenching group at its 3' end.

In another preferred embodiment, the fluorescent group is selected from the following group: FAM, HEX, VIC, TET, JOE, ROX, NED, RED, TAMRA, CY3, CY5, CY5.5, CY7, or a combination thereof.

In another preferred embodiment, the quenching group is selected from the following group: MGB, TAMRA, BHQ1, BHQ2, BHQ3, Eclipse, Dabcyl, or a combination thereof.

In the second aspect of the present invention, there is provided a use of the primer-probe pair as described in the first aspect of the present invention for preparing a diagnostic reagent or kit, and the diagnostic reagent or kit is used for:

(i) Detect CAR gene copy number; and/or

(ii) Detection of viral vector copy number.

In another preferred example, the CAR gene is a BBZ sequence.

In another preferred embodiment, the viral vector is a lentiviral vector. Preferably, the viral vector copy number is the WPRE copy number.

In a third aspect of the present invention, a kit is provided, which includes: the primer-probe pair as described in the first aspect of the present invention and instructions.

In another preferred embodiment, the instructions indicate that the kit is used to detect the copy number of the CAR gene and/or the copy number of the viral vector, as well as the corresponding detection method.

In another preferred embodiment, the primer-probe pairs are placed in separate containers.

In another preferred embodiment, the primer-probe pair is a dry powder or liquid preparation.

In another preferred example, the ratio of each primer-probe pair in the primer-probe pair is BBZ primer-probe pair: WPRE primer-probe pair: ACTIN primer-probe pair = 1-2:1-2:1, preferably 3:3:2, optimally 1:1:1.

In another preferred embodiment, the kit further includes:

(Z1) Enzyme reaction solution;

(Z2) Quantitative reference standards; and/or

(Z3)DNA dilution solution.

In another preferred embodiment, the enzyme reaction solution contains components selected from the following group: dNTP/dUTP Mix, Mg ²⁺ , Taq DNA Polymerase, thermosensitive UDG (thermosensitive uracil-DNA sugar basease, Heat-labileUDG), specific fluorescent quantitative PCR reference dye (Specific ROX Reference Dye), or a combination thereof.

In another preferred embodiment, the quantitative reference standard is a plasmid vector containing a CAR sequence, a lentiviral backbone sequence and a single-copy internal reference gene sequence.

In another preferred embodiment, the plasmid vector includes a single plasmid vector and/or a multi-plasmid vector.

In another preferred embodiment, the plasmid vector is selected from the following group: pUC57, pWPI, or a combination thereof.

In another preferred example, the plasmid vector is pUC57.

In another preferred embodiment, the quantitative reference standard is a gradient dilution standard series.

In another preferred example, the DNA diluent contains the following components: Tris, EDTA.

In a fourth aspect of the present invention, a non-diagnostic method for detecting CAR gene and/or viral vector copy number is provided, which method includes the following steps:

(1) Provide a sample to be tested and a certain amount of reference standards;

(2) The sample to be tested and the quantitative reference standard are subjected to PCR amplification using the primer probe pair as described in the first aspect of the present invention, thereby obtaining a CAR gene sequence, a viral vector sequence and/or a single copy of the internal reference gene. amplification product; and

(3) Calculate the copy number of the sample to be tested: According to the average Ct value of the quantitative reference standard, prepare a standard curve for the logarithmic copy number of each gene contained in it, and perform linear regression analysis. The Ct value of the test sample is substituted into the linear regression equation to calculate the CAR gene copy number, viral vector copy number and/or single copy internal reference gene copy number of the test sample.

In another preferred embodiment, the quantitative reference standard is derived from the kit as described in the third aspect of the present invention or is prepared by itself.

In another preferred example, in step (2), the PCR amplification includes the following steps:

(2a) Prepare standard PCR reaction solution, test sample PCR reaction solution and NTC PCR reaction solution respectively;

Among them, the standard PCR reaction solution contains the following components: 5 μL primer-probe mixture, 10 μL enzyme reaction solution, and 5 μL quantitative reference standard;

The test sample PCR reaction solution contains the following components: 5 μL primer-probe mixture, 10 μL enzyme reaction solution, and 5 μL sample to be tested;

The NTC PCR reaction solution contains the following components: 5 μL primer-probe mixture, 10 μL enzyme reaction solution, and 5 μL ddH ₂ O; and

(2b) Perform PCR amplification of the standard PCR reaction solution, test sample PCR reaction solution and NTC PCR reaction solution according to the following procedures:

The first stage: 37℃ digestion for 2 minutes, 95℃ pre-denaturation for 5 minutes, 1 cycle; the second stage: 95℃ denaturation for 15 seconds, 60℃ extension for 30 seconds, a total of 45 cycles;

Thus, amplification products containing CAR gene sequences, viral vector sequences, and/or single-copy internal reference genes are obtained.

In another preferred embodiment, the method further includes the steps of:

(4) Calculate the copy number of a single cell of the sample to be tested: the average number of CAR gene copies in a single cell = 2*CAR gene copy number/ACTIN gene copy number; the average number of viral vector copies in a single cell = 2*virus Vector copy number/ACTIN gene copy number.

In another preferred embodiment, the sample to be tested is derived from humans.

In another preferred embodiment, the sample to be tested is derived from a subject receiving cellular immunotherapy.

In another preferred embodiment, the sample to be tested is derived from a sample selected from the following group: body fluid samples (such as blood samples), cultured cell samples, tissue samples, exfoliated cell samples (such as cell fragments), secretion samples, or combination thereof.

In another preferred embodiment, the sample to be tested is obtained by extracting DNA from body fluid samples (such as blood samples), cultured cell samples, tissue samples, exfoliated cell samples (such as cell fragments), and secretion samples.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here.

Description of the drawings

Figure 1 shows the amplification curves of serially diluted ACTIN standards and their NTCs.

Figure 2 shows the amplification curves of serially diluted BBZ standards and their NTCs.

Figure 3 shows the amplification curves of serially diluted WPRE standards and their NTCs.

Figure 4 shows the comparative results of flow cytometry and real-time fluorescence quantitative PCR detection of positive Jurkat-CAR using R19m-AF647 antibody and the copy number detection kit of the present invention respectively.

Figure 5 shows the detection results of 1000ng genomic samples containing 4, 8, 20 and 40 copies.

Figure 6A and Figure 6B show the amplification curves of the internal reference gene ACTIN and BBZ target gene in the ST1-ST6 gradient standard, NTC and 006 samples respectively.

Figure 7 shows the qPCR curves of test samples 144, 156 and standards ST1-ST6.

Figure 8 shows the results of detection using single-plasmid and multi-plasmid standards respectively.

Detailed ways

After extensive and in-depth research and extensive screening, the inventor developed a CAR genome viral vector copy number detection kit for the first time. The detection kit of the present invention contains a quantitative reference standard. The probe-primer mixture of the kit integrates multiple pairs of primers and probes respectively targeting the CAR sequence, the lentiviral backbone and the single-copy internal reference gene. By comparing the detection sample with the standard By calculating the relevant parameters of the product, the detection of CAR genome copy number can be achieved with high accuracy, good specificity and stable results. Moreover, the detection method of the present invention is highly versatile and simple, and has high practical value. On this basis, the present invention was completed.

the term

In order that this disclosure may be more easily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below unless expressly stated otherwise herein.

As used herein, the terms "kit", "kit of the invention", "detection kit", "copy number detection kit", "CAR-T cell genome viral vector copy number detection kit", "CAR genome "Viral vector copy number detection kit" can be used interchangeably, and both refer to the kit constructed by the present invention for CAR-positive genome copy number detection, which consists of a probe-primer mixture containing 3 sets of specific primer-probe pairs, optionally Also included are enzyme reaction solutions, quantitative reference standards, and DNA diluents.

As used herein, the terms "standard," "plasmid standard," "standard plasmid," and "quantitative reference standard" are used interchangeably and all refer to the inclusion of a CAR sequence (BBZ sequence), a lentiviral backbone sequence (WPRE sequence) ) and a single-copy internal reference gene sequence (ACTIN sequence) plasmid can be gradiently diluted into a standard series as needed and used for standard control in kit testing.

As used herein, "CAR-positive genome" refers to CAR gene sequences that have been integrated into the genome of a cell.

The term "Chimeric Antigen Receptor" (CAR) is a chimeric gene composed of a single-chain antibody that recognizes a tumor associated antigen (TAA) on the tumor cell membrane and an intracellular signaling domain connected through a hinge region.

The term "CAR-T" refers to cells expressing the CAR gene obtained after introducing the CAR gene into T cells through gene transduction/transfection technology. This type of cells has the ability to recognize and attack tumor cells expressing TAA on the corresponding cell surface.

The term "second-generation CAR" refers to a second-generation CAR structure that adds a new co-stimulatory signal (such as CD28 or CD137, etc.) based on the first-generation CAR structure, where the first-generation CAR is an immunoglobulin A chimeric receptor formed by the fusion of scFv and the intracellular domain of the FcεRI receptor or CD3 complex.

The term "background genomic DNA" refers to the addition of a certain quality of untransfected CAR T cell or Jurkat cell genomic DNA during standard testing, which can simulate the complexity of the genome and achieve good consistency and complexity with clinical samples. , thereby improving the accuracy of detection.

The terms "sample" or "sample" as used herein interchangeably refer to material specifically associated with a subject from which specific information about the subject can be determined, calculated, or inferred. The sample may consist in whole or in part of biological material from the subject.

qPCR detection technology

Molecular biology detection of viral vector copy number has become a trend. Quantitative Real Time PCR (qPCR) is now the most common method. This method has the advantages of high sensitivity, good specificity, wide linear range and accurate quantification.

The Taqman probe method absolute quantitative qPCR experiment uses Taqman probes to mark and track the PCR products for real-time monitoring of the reaction. The products are analyzed using software that is compatible with them, and the initial concentration of the sample template to be tested can be calculated.

CAR-T cell genome viral vector copy number detection technology

(1) Commonly used methods of CAR introduction are lentivirus, retrovirus, electroporation, etc. Viruses have the ability to enter cells and transfer genetic information into cells, and are widely used in gene transduction. At present, the most commonly used method is lentivirus. The two autologous CAR-T products approved by the FDA achieve genetic modification through the lentiviral vector approach. The lentivirus carrying the CAR sequence will be integrated into the cell genome after infecting T cells. It also includes some backbone sequences of the lentiviral vector, including WPRE, psi, cppt and other element sequences. The extracted cellular genomic DNA will accordingly contain CAR gene DNA and lentiviral backbone sequences.

(2) In addition, CAR introduced through the electrotransposon system and SB (sleeping beauty) system can also achieve genome integration, and the extracted cellular genomic DNA will contain CAR copies.

(3) Use the plasmid containing the target detection sequence as a standard, and after gradient dilution, run the test on the machine at the same time as the detection sample to obtain the corresponding copy number of the CAR or lentiviral backbone sequence in the sample.

(4) The copy number of the target sequence in the unit gene DNA can be calculated based on the amount of DNA loaded.

(5) Detecting copy number can indirectly determine the infection efficiency of the virus and the positive rate of CAR cells.

Multiplex PCR and interference between primer pairs

The term "multiplex PCR" (multiplex Polymerase Chain Reaction, mPCR), also known as multiplex PCR, was first proposed by Chambehian' in 1988. Its basic principle is the same as that of conventional PCR. The difference is that more than one pair of primers are added to the multiplex PCR reaction system, and each pair of primers is combined with the corresponding part of the template to ultimately amplify more than one target DNA fragment.

The term "interference between primer pairs" refers to the fact that multiplex PCR experiments do not simply mix multiple pairs of specific primers into one reaction system. The reason why multiplex PCR is difficult is that the amplification conditions of multiple targets are incompatible, and each target needs the cooperation of other primers. Multiplex PCR requires specific amplification of multiple sites in the same reaction system, so pairing between primers and competitive amplification will affect the amplification effect. If the appropriate reaction system and reaction conditions can be selected, the amplification effect of multiplex PCR is expected to be improved.

BBZ

As used herein, the term "BBZ" refers to the gene sequence between gene 4-1BB and gene CD3zeta in the CAR structure.

The "BBZ" sequence includes the fusion of two genes, "BB" and "Z", where "BB" refers to human leukocyte differentiation antigen 137, also known as 4-1BB. Its official name in NCBI GeneBank is TNFRSF9 (tumor necrosis factor Receptor superfamily member 9), ID number is 3604, has only one mRNA and corresponding protein sequence, which is NM_001561.5/NP_001552.2; and "Z" refers to human leukocyte differentiation antigen 3zeta, the ID in NCBI GeneBank The number is 919, and there are two mRNA and corresponding protein sequences, namely NM_000734.3/NP_000725.1 and NM_198053.2/NP_932170.1.

WPRE

The term "WPRE" refers to the post-transcriptional regulation element (WPRE) derived from the Woodchuck hepatitis virus, which is beneficial to increasing protein expression.

WPRE is a commonly used element in lentiviral vectors. It will be integrated into the cell genome along with the lentiviral and CAR gene sequences. Therefore, it can be used to evaluate the copy number of the lentiviral vector in the genome, and also indirectly represents the copy number of the CAR gene sequence.

primer probe pair

As used herein, the terms "primer-probe pair", "specific primer-probe pair" and "specific primer-probe pair of the invention" are used interchangeably and refer to the specific primer pair sequence (including upstream primers and downstream primers, collectively The combination of primer set) and the corresponding probe sequence can be a dry powder preparation or a liquid preparation, also called a "probe-primer mixture".

In the present invention, there are three different sets of primer-probe pairs, namely BBZ primer-probe pair, WPRE primer-probe pair and ACTIN primer-probe pair.

The BBZ primer-probe pair includes primer set 1 and probe sequence 1, where primer set 1 includes an upstream primer as shown in SEQ ID NO:1 and a downstream primer as shown in SEQ ID NO:2; probe sequence 1 is as SEQ ID NO:7 shown.

The WPRE primer-probe pair includes primer set 2 and probe sequence 2, where primer set 2 includes an upstream primer as shown in SEQ ID NO:3 and a downstream primer as shown in SEQ ID NO:4; probe sequence 2 is as SEQ ID NO:8 shown.

The ACTIN primer-probe pair includes primer set 3 and probe sequence 3, where primer set 3 includes an upstream primer as shown in SEQ ID NO:5 and a downstream primer as shown in SEQ ID NO:6; probe sequence 2 is as SEQ ID Shown in NO:9.

Preferably, the probe sequence in the primer-probe pair of the present invention is labeled with a fluorescent group at its 5' end and a quenching group at its 3' end, and the fluorescent group is selected from the following group: FAM, HEX, VIC, TET, JOE, ROX, NED, RED, TAMRA, CY3, CY5, CY5.5, CY7, or a combination thereof; the quenching group is selected from the following group: MGB, TAMRA, BHQ1, BHQ2, BHQ3, Eclipse, Dabcyl , or a combination thereof.

Typically, the 5' end of the probe sequence 1 of the present invention is labeled with the fluorescent group FAM, and the 3' end is labeled with the quenching group BHQ1; the 5' end of the probe sequence 2 of the present invention is labeled with the fluorescent group CY5, The 3' end is labeled with the quenching group BHQ2; the 5' end of the probe sequence 3 of the present invention is labeled with the fluorescent group HEX, and the 3' end is labeled with the quenching group BHQ1.

The primer-probe pair provided by the invention can realize multiple simultaneous detection of BBZ sequence, WPRE sequence and ACTIN sequence. The primer-probe pair of the present invention can be used to prepare a diagnostic reagent or kit for detecting CAR gene copy number and/or viral vector copy number.

Test kit of the present invention

The present invention also provides a kit for detecting CAR gene copy number and/or viral vector copy number. The kit includes: the primer-probe pair as described in the first aspect of the present invention and instructions; the instructions indicate the The above kit is used to detect CAR gene copy number and/or viral vector copy number, as well as corresponding detection methods.

Preferably, the primer-probe pairs are placed in separate containers and are dry powder or liquid preparations.

Preferably, the kit further includes: (Z1) enzyme reaction solution; (Z2) quantitative reference standard; and/or (Z3) DNA dilution, wherein the quantitative reference standard is a protein containing a CAR sequence, a lentiviral backbone The plasmid vector (including single plasmid vector and/or multi-plasmid vector) of the sequence and single-copy internal reference gene sequence can be serially diluted when used. Typically, the plasmid vector is selected from the group consisting of pUC57, pWPI, or a combination thereof.

Preferably, the enzyme reaction solution may contain components selected from the following group: dNTP/dUTP Mix, Mg ²⁺ , Taq DNA polymerase (Taq DNA Polymerase), thermosensitive UDG (thermosensitive uracil-DNA glycosylase, Heat-labile UDG), Specific ROX Reference Dye, or a combination thereof. The DNA diluent contains the components Tris and EDTA.

Methods for detecting CAR gene and/or viral vector copy number

The invention also provides a method for detecting CAR gene and/or viral vector copy number, which method includes the following steps:

(1) Provide a sample to be tested and a certain amount of reference standards;

(2) The sample to be tested and the quantitative reference standard are subjected to PCR amplification using the primer probe pair as described in the first aspect of the present invention, thereby obtaining a CAR gene sequence, a viral vector sequence and/or a single copy of the internal reference gene. amplification product; and

(3) Calculate the copy number of the sample to be tested: According to the average Ct value of the quantitative reference standard, prepare a standard curve for the logarithmic copy number of each gene contained in it, and perform linear regression analysis. The Ct value of the test sample is substituted into the linear regression equation to calculate the CAR gene copy number, viral vector copy number and/or single-copy internal reference gene copy number of the test sample.

Wherein, in step (2), the PCR amplification includes the following steps:

(2a) Prepare standard PCR reaction solution, test sample PCR reaction solution and NTC PCR reaction solution respectively;

Among them, the standard PCR reaction solution contains the following components: 5 μL primer-probe mixture, 10 μL enzyme reaction solution, and 5 μL quantitative reference standard;

The test sample PCR reaction solution contains the following components: 5 μL primer-probe mixture, 10 μL enzyme reaction solution, and 5 μL sample to be tested;

The NTC PCR reaction solution contains the following components: 5 μL primer-probe mixture, 10 μL enzyme reaction solution, and 5 μL ddH ₂ O; and

(2b) Perform PCR amplification of the standard PCR reaction solution, test sample PCR reaction solution and NTC PCR reaction solution according to the following procedures:

The first stage: 37℃ digestion for 2 minutes, 95℃ pre-denaturation for 5 minutes, 1 cycle; the second stage: 95℃ denaturation for 15 seconds, 60℃ extension for 30 seconds, a total of 45 cycles;

Thus, amplification products containing CAR gene sequences, viral vector sequences, and/or single-copy internal reference genes are obtained.

Preferably, the method further includes the steps of:

(4) Calculate the copy number of a single cell of the sample to be tested: the average copy number of the CAR gene in a single cell = 2*CAR gene copy number/ACTIN gene copy number; the average copy number of the viral vector in a single cell = 2*virus Vector copy number/ACTIN gene copy number.

The main advantages of the present invention include:

(1) The specific primer-probe pair of the present invention and the kit constructed therefrom provide a new method that takes both quality control and risk assessment into consideration for the immature CAR-T cell drug field to meet the current needs for CAR in the field. -T requirements for drug quality control, clinical sample testing, etc.

(2) The specific primer-probe pair of the present invention and the kit constructed therefrom are designed to target the CAR structure and the lentiviral backbone respectively. The two synergistically can detect most CAR-T on the market, and the product is universal. Strong sex.

(3) The specific primer-probe pair of the present invention and the kit constructed therefrom have consistent primer-probe amplification efficiency for the CAR structure and lentivirus backbone, which not only increases versatility but also ensures the accuracy and reliability of the results.

(4) The detection kit of the present invention contains the calibration internal reference ROX, and the ROX concentration is optimized, so there is no need to adjust the usage amount according to different machine models.

(5) The detection kit of the present invention introduces the Heat-labile UDG anti-pollution system to effectively prevent PCR product contamination.

(6) The detection kit of the present invention is simple to operate, convenient for detection, and has a wide range of sample loading (10-500ng), thereby reducing operating errors and increasing the stability of the results.

(7) The detection kit of the present invention has strong anti-interference ability, and the elution buffer of the multiple extraction reagents does not affect the PCR amplification efficiency.

(8) The detection kit of the present invention adopts multiplex fluorescence PCR, and one tube reaction can realize multiple detections, saving time, cost and precious samples.

(9) The detection kit of the present invention has the advantages of high sensitivity, good specificity, and wide linear range, and can perform relative quantitative and absolute quantitative detection for accurate quantification.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. Suggested conditions. Unless otherwise stated, percentages and parts are by weight.

Example 1 Development of CAR-T cell genome virus vector copy number detection kit

1. The kit includes: probe and primer mixture, enzyme reaction solution, quantitative reference standard and DNA diluent. The probe and primer mixture includes 3 sets of primer-probe pairs: BBZ primer-probe pair, WPRE primer-probe pair and ACTIN primer-probe pair. For, and the ratio of each set of primer-probe pairs is BBZ primer-probe pair: WPRE primer-probe pair: ACTIN primer-probe pair = 1-2:1-2:1, preferably 3:3:2, optimally 1:1:1.

Each set of primer-probe pairs includes 1 primer set and 1 probe sequence. For example, the BBZ primer-probe pair includes primer set 1 and probe sequence 1. There are 3 primer sets in the probe-primer mixture (primer set 1, 2, and 3). ) and three probe sequences (probe sequences 1, 2, and 3).

The specific sequence information is shown in Table 1:

Table 1

2. Preparation of quantitative reference standards:

The quantitative reference standards in the kit are prepared using the following general method: the CAR sequences corresponding to the three sets of primer probes, the lentiviral backbone sequence and the single-copy internal reference gene sequence are constructed into two plasmids by gene synthesis, one of which is Including CAR sequence, lentivirus backbone sequence, and another plasmid including a single copy of the internal reference gene sequence. The corresponding single clone bacterial liquid is amplified and the endotoxin-free ultrapure plasmid is extracted. After quantification, doubling dilution is performed according to the molecular weight of the plasmid as a standard. It is also used to confirm the linear range, minimum detection limit and other parameters of the kit.

In this example, the CAR sequence, lentiviral backbone sequence and single-copy internal reference gene sequence information are as follows:

(1) CAR sequence: CAR BBZ sequence (SEQ ID NO: 10)

GGGCAGAAAGAAGCTCCGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG AAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCC

(2) Lentivirus backbone sequence: LV WPRE sequence (SEQ ID NO: 11)

AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACC TGTCAGCTCCTTTCCGGGACTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTG CGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCCGCCTCCCCGC

(3) Single copy internal reference gene sequence: ACTIN sequence (SEQ ID NO: 12):

GCTGGGTGGGGCAGCCCCGGGAGCGGGCGGGAGGCAAGGGCGCTTTCTCTGCACAGGAGCCTCCCGGTTTCCGGGGTGGGGGCTGCGCCCGTGCTCAGGGCTTCTTGTCCTTTCCTTCCCAGGGCGTGGGCATGGGTCAGAAGGATTCCTATGTGGGCGACGAGGCCCAGAGCAAGAGAGGCATCCTCACCCTGAAGTACCCCATCGAGCACGGCATCGTCACCAACTGGGACGACATGGAGAAAATCTGGCACC ACACCTTCTACAATGAGCTGCGTGTGGCTCCCGAGGAGCACCCCGTGCTGCTGACCGAGGCCCCCCTGAACCCCAAGGCCAACCGCGAGAAGATGACCCAGGTGAGTGGCCCGCTACCTCTTCTGGTGGCCGCCTCCCT

In this example, the above sequences were separately constructed into the pUC57 vector by gene synthesis, single clones were selected for sequencing verification, and the corresponding single clone bacterial liquid was amplified and then the endotoxin-free supernatant was extracted using a Qiagen extraction kit. For pure plasmids, after quantification, calculate the copy number of each plasmid based on the molecular weight of the plasmid, and use DNA diluent to dilute it so that the copy number of each plasmid is 2*10^n/μL (where n is a positive integer, n=1, 2, 3 , 4, 5, 6, 7...) as the plasmid standard, that is, the quantitative reference standard in the kit.

Example 2 Linear performance analysis of CAR-T cell genome virus vector copy number detection kit

In order to analyze the linear performance of three sets of primer-probe pairs of ACTIN, BBZ and WPRE, in this example, the inventor selected the standards of ACTIN, BBZ and WPRE respectively, and diluted the above standards to 2*10. ^6copies/μL, 2*10^5copies/μL... 2*10^1copies/μL, six 10-fold diluted standards, based on using water as NTC (notemplate control) control, the above-mentioned ACTIN, BBZ and WPRE Doubling diluted standards were used for qPCR amplification.

As shown in Figure 1-3, the amplification curves of ACTIN, BBZ and WPRE standards and their NTC are shown respectively, where A-F are 2*10^6copies/μL, 2*10^5copies/μL...2*10 ^1copies/μL Amplification curves of six 10-fold diluted standards, while G and H are both NTC amplification curves.

According to the results shown in Figure 1-3, it is shown that each set of primer-probe pairs of multiplex fluorescence PCR has good linearity (R ² ≥ 0.998), and the amplification efficiency is close, both are >90% and <110%, which shows that this method The three sets of primer-probe pairs invented can meet the requirements of multiplex PCR for specific amplification of multiple sites in the same reaction system, and can achieve better amplification effects.

The above results also show that the kit of the present invention has good specificity, which is specifically reflected in the fact that even at a high cycle number of 45, there is still no amplification curve in the control using water as NTC.

According to the analysis of the linear performance of the kit in this example, it is shown that each set of primer-probe pairs used for multiplex fluorescent PCR in the kit of the present invention not only has good linearity and close amplification efficiency, but also can meet the needs of clinical applications. Samples, especially copy number detection at the baseline point (without CAR-T infusion) and long-term post-infusion (when CAR-T does not exist or has very low levels in the blood), so that when the baseline point is reached, the need for CAR-T infusion can be met requirements for accurate analysis of cellular kinetics.

Example 3 Accuracy Performance Analysis of CAR-T Cell Genome Virus Vector Copy Number Detection Kit

Under the premise of ensuring that the total cell number is the same (5*10^6), the inventor diluted the positive Jurkat-CAR (J-C) with negative Jurkat cells into samples of different proportions (50%, 25%, 12.5%...0.18% , 0.018% and 0.0018%), and then use the R19m-AF647 antibody and the copy number detection kit of the present invention to perform flow cytometry and real-time fluorescence quantitative PCR (genomic DNA extraction) detection respectively.

As shown in Figure 4, both flow cytometry and real-time fluorescence quantitative PCR detection have obtained a good linear relationship, and the results of the two detection methods are very consistent, indicating that the kit and its detection method of the present invention can achieve high accuracy.

Example 4 Sensitivity performance analysis of CAR-T cell genome virus vector copy number detection kit

Add 4, 8, 20 and 40 copies of standard plasmids to 1000ng of extracted genomic DNA of Jurkat cells (or blood cells) without CAR transduction, adjust the volume to 20μL with DNA diluent, and take 5μL as a sample Use the kit to perform PCR reaction and set up 3 duplicate wells.

As shown in Figure 5, it was found that the three wells of the 1000ng genome sample containing 20 and 40 copies all formed amplification curves, but under the condition of 8 copies, it was impossible to achieve the amplification curves in all three wells, and the other 4 copies of 1000ng There is no amplification curve in the three reaction wells of the genomic sample, so the sensitivity of the kit can reach 20 copies/μg genomic DNA.

Example 5 Specific performance analysis of CAR-T cell genome virus vector copy number detection kit

As shown in Figure 6A and Figure 6B, Figure 6A is the amplification curve of the internal reference gene ACTIN, and Figure 6B is the amplification curve of the BBZ target gene. The inventors respectively detected the ST1-ST6 gradient standard, NTC and 006 samples (note :006 sample is the amplification curve of ACTIN and BBZ in Jurkat cell genome sample without CAR transduction), it was found that the CAR-T cell genome virus vector copy number detection kit of the present invention:

(1) Can specifically amplify CAR containing the target BBZ structure;

(2) There is no amplification curve in NTC;

(3) The untransduced Jurkat cell genome sample (006 sample) has no amplification curve.

The above results show that the kit of the present invention has strong specificity and establishes a relatively stable internal reference system.

Example 6 Detection Example of CAR-T Cell Genome Virus Vector Copy Number Detection Kit

(1)Main instruments and equipment:

Biological safety cabinet, ABI7500 fluorescence quantitative PCR, desktop centrifuge, vortex suspension, Nanodrop ultra-micro spectrophotometer.

(2)Main reagents and consumables:

Cell genome extraction kit (QIAGEN), ABI fluorescence quantitative PCR instrument 96-well plate 4306737, ABI 96-well plate optical film 4311971, Q113-02 AceQ U+Probe Master Mix, TE Buffer.

(3) Specific steps for detecting viral vector copy number in CAR-T cell genome:

①Extract samples: Take the CAR-Jurkat cell sample suspension and the uninfected Jurkat cell suspension in a sterile environment, use the QIAGEN cell genome extraction kit to extract the genomes of the CAR-Jurkat cell sample and the uninfected Jurkat cell, respectively. The extracted genome was analyzed for DNA concentration and purity using a Nanodrop instrument.

② Preparation of PCR reaction solution: Prepare multiple tubes of PCR reaction solution, including standard PCR reaction solution, test sample PCR reaction solution and NTC PCR reaction solution. The PCR reaction volumes are all 20 μL. The systems of each tube of PCR reaction solution are as follows: :

(i) Gradient dilution of standard PCR reaction solution:

(a) 5 μL primer-probe mixture (can be a primer-probe liquid preparation, or a primer-probe mixture prepared from a primer-probe dry powder preparation);

(b) 10 μL enzyme reaction solution; and

(c) 5 μL of gradient diluted plasmid standards ST1-ST6 (that is, the quantitative reference standard in the kit);

Among them, the plasmid standard (2*10^7 copies/μL) is diluted with a DNA diluent containing a certain concentration of Tris and EDTA.

Use the above-mentioned plasmid standard containing BBZ, WPRE and ACTIN sequences as the standard stock solution, use untransfected T cell genomes to dilute the standard stock solution, and prepare 6 gradient plasmid standard series with 10-fold gradient dilution. The sequence copy number information contained in the plasmid standards is shown in Table 2.

Table 2

(ii) Test sample PCR reaction solution:

(a) 5 μL primer-probe mixture (same as above);

(b) 10 μL enzyme reaction solution; and

(c) 5 μL extracted DNA samples (numbers 144 and 156 are whole blood genomic DNA samples before and after treatment with WuXi Giant Norezil (relma-cel), of which 144 is the baseline sample after relma-cel infusion, and 156 is a whole blood genomic DNA sample taken at a certain time after infusion).

(iii)NTC PCR reaction solution

(a) 5 μL primer-probe mixture (same as above);

(b) 10 μL enzyme reaction solution; and

(c) 5 μL ddH ₂ O.

③On-machine inspection:

(a) The first stage, digestion at 37°C for 2 minutes, pre-denaturation at 95°C for 5 minutes, 1 cycle;

(b) The second stage is denaturation at 95°C for 15 seconds and extension at 60°C for 30 seconds, a total of 45 cycles.

④Test sample data analysis:

Draw a qPCR standard curve chart based on the on-machine detection data of the test samples and standards ST1-ST6.

As shown in Figure 7, the qPCR standard curves ST1, ST2...ST6 show that the kit has good linearity and amplification performance; the WPRE gene of the 144 baseline sample does not form an amplification curve, indicating that the viral vector has not yet been integrated into the cell at this time In the genome, it is consistent with the theoretical results of the baseline sample; while sample 156 after infusion of relma-cel for a certain period of time has an obvious and consistent amplification curve, indicating that the viral vector and CAR gene sequence have been integrated into the cell genome, and the infusion is effective.

The above results show that the kit of the present invention has high specificity and stability, and can judge whether the viral vector and CAR gene sequence have been integrated into the cell genome based on the amplification curve, thereby making effective decisions on clinical treatment plans and their efficacy. sexual assessment. Based on the calculation of copy number, quantitative detection can also be performed, thereby achieving a quantitative presentation of CAR-positive genomes during treatment.

Comparative example 1

In order to study the impact of the amount of genome loading on the amplification efficiency of the kit and the accuracy of copy number detection, the inventor extracted the genome from CAR-Jurkat cells containing a certain proportion of CAR-Jurkat cells, which was quantified to 118.3ng/μL. Adjust the concentration of the Elution Buffer in the extraction kit to 100ng/μL and use it as sample JC; then dilute it 8 times with ddH ₂ O to 12.5ng/μL and use it as sample JC1:8.

Take 5 μL of each sample JC and JC1:8 (that is, the loading amounts of the two samples JC and JC1:8 are 500ng and 62.5ng respectively) according to the method in Example 6 for on-machine testing together with the standard and NTC. The standard The amplification curve has very good linearity and there is no non-specific amplification in NTC. Three duplicate wells are set up for each sample. Samples JC and JC1:8 are used to calculate the copy number results of the corresponding genes based on the standard.

Table 3 Copy number detection before and after sample dilution 8 times

As shown in Table 3, it was found that the copy number ratios of BBZ, ACTIN and WPRE genes before and after dilution were 8.173, 7.96 and 8.13 respectively, which were all very close to the dilution factor of 8.

The above results show that the kit of the present invention has a wide loading capacity, and the quantitation is basically accurate at loading volumes of 500ng and 62.5ng, so that the kit has a wide loading range and can avoid the need to dilute the sample for loading. It may cause operational errors, and the Elution Buffer has no effect on the reaction, indicating that the kit has strong anti-interference ability.

In addition, it can also be seen from Table 3 that the copy number ratio of BBZ and WPRE calculated based on the standard product in the same sample is about 1.1 times, indicating that the copy numbers of the two genes have good consistency and the values are very close. WPRE is a slow The universal elements of the viral skeleton can represent the CAR copy number in cells, thus increasing the universal usability of the copy number detection kit, and can detect samples containing BBZ structures and/or WPRE elements, making the kit of the present invention more commercially valuable. .

Comparative example 2

In order to study the impact of single plasmid (that is, all target genes are constructed on one plasmid through gene synthesis) and multiple plasmids (each target gene sequence is constructed on different plasmids) as standards on the linearity and sensitivity of the detection, the inventor used a single plasmid The plasmid and multi-plasmid standards were gradient diluted into 6 gradient standard concentrations of ST1, ST2...ST6 and ST1-D, ST2-D...ST6-D. The corresponding concentrations are 2*10^6 copies/μL. , 2*10^5 copies/μL... 2*10^1 copies/μL, together with the NTC sample, perform loading and detection according to the reaction system in Example 6.

The detection results are shown in Figure 8. It can be seen that the linear relationship of the kit using single plasmid or multi-plasmid standard is very good, and the latter has higher sensitivity. Therefore, in the present invention, a multi-plasmid method is used to prepare the standard and obtain Highly sensitive detection results.

All documents mentioned in this application are incorporated by reference in this application to the same extent as if each individual document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims (10)

1. A specific primer-probe pair, wherein the primer-probe pair comprises: BBZ primer probe pair, WPRE primer probe pair and ACTIN primer probe pair,

wherein, the BBZ primer probe pair comprises: a primer set 1 composed of an upstream primer shown as SEQ ID NO. 1 and a downstream primer shown as SEQ ID NO. 2, and a probe sequence 1 shown as SEQ ID NO. 7;

the WPRE primer probe pair comprises: a primer set 2 composed of an upstream primer shown as SEQ ID NO. 3 and a downstream primer shown as SEQ ID NO. 4, and a probe sequence 2 shown as SEQ ID NO. 8;

the ACTIN primer pair comprises: an upstream primer shown as SEQ ID NO. 5 and a downstream primer shown as SEQ ID NO. 6, and a probe sequence 3 shown as SEQ ID NO. 9.

2. The primer probe pair of claim 1, wherein the probe sequences in the specific primer probe pair are labeled with a fluorescent group at their 5 'end and a quenching group at their 3' end.

3. The primer pair of claim 2, wherein the fluorescent moiety is selected from the group consisting of: FAM, HEX, VIC, TET, JOE, ROX, NED, RED, TAMRA, CY3, CY5, CY5.5, CY7, or combinations thereof.

4. The primer pair of claim 2, wherein the quenching group is selected from the group consisting of: MGB, TAMRA, BHQ1, BHQ2, BHQ3, eclipse, dabcyl, or combinations thereof.

5. Use of the primer pair of claim 1 for preparing a diagnostic reagent or kit for:

(i) Detecting the CAR gene copy number; and/or

(ii) The viral vector copy number is detected.

6. A kit, comprising: the primer pair of claim 1 and instructions.

7. The kit of claim 6, wherein the primer pair is a dry powder or a liquid formulation.

8. The kit of claim 6, wherein the ratio of each primer pair of the primer pair is a BBZ primer pair: WPRE primer probe pair: ACTIN primer pair = 1-2:1-2:1, preferably 3:3:2, most preferably 1:1:1.

9. The kit of claim 6, further comprising:

(Z1) an enzyme reaction solution;

(Z2) a quantitative reference standard; and/or

(Z3) DNA dilution;

wherein the quantitative reference standard is a plasmid vector comprising a CAR sequence, a lentiviral backbone sequence, and a single copy internal reference gene sequence.

10. The kit of claim 9, wherein the enzyme reaction solution comprises a component selected from the group consisting of: dNTP/dUTP Mix, mg ²⁺ Taq DNA polymerase, thermosensitive UDG, specific fluorescent quantitative PCR reference dye, or a combination thereof.