WO2023013765A1 - Primer set for detecting chimeric antigen receptor - Google Patents
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
Definitions
- First-generation chimeric antigen receptors consist of the T-cell receptor subunit TCR ⁇ (CD3 ⁇ ) as the antigen-recognition site and the T-cell activation site.
- Second-generation CARs are composed of antigen recognition sites, co-stimulatory sites, CD3zeta, and the like.
- scFv single chain variable region fragment
- Costimulatory sites include the TNF receptor family, including 4-1BB, OX40, and CD27, and the CD28 family, including CD28 and inducible T cell costimulator (ICOS), and further CAR development is underway. Costimulatory sites have been introduced in hopes of enhancing CAR-T cell cytotoxicity, proliferation, and persistence.
- CAR-T cells do not require the intervention of human leukocyte antigen (HLA) to recognize and activate antigens via scFv, and are one of the cancer immune escape mechanisms. It is expected that even if there is down-modulation of HLA in cancer cells, the effect will be exhibited. CAR-T cells also exert antitumor effects by releasing cytotoxic substances such as TNF, perforin, and granzyme, and by inducing Fas-induced apoptosis of tumor cells.
- HLA human leukocyte antigen
- Item 1 A primer set for detecting a nucleic acid encoding a chimeric antigen receptor, comprising a primer having the nucleotide sequence of SEQ ID NO:1 and a primer having the nucleotide sequence of SEQ ID NO:2.
- Item 2 2.
- the primer set according to claim 1, wherein the nucleic acid comprises a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1 and a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:2.
- Item 3 Item 3.
- the primer set according to Item 1 or 2 wherein the chimeric antigen receptor comprises CD28 and CD3 ⁇ .
- Item 4 Item 4.
- the primer set according to Item 3, wherein the nucleotide sequence encoding CD28 comprises the nucleotide sequence of SEQ ID NO:5.
- Item 5 Item 5.
- the primer set according to Item 3 or 4, wherein the nucleotide sequence encoding CD3 ⁇ comprises the nucleotide sequence of SEQ ID NO:6.
- Item 6 Item 6.
- Item 7 Item 7.
- Item 8 Item 8.
- Item 9 A primer set for detecting a nucleic acid encoding a chimeric antigen receptor, comprising a primer consisting of the nucleotide sequence of SEQ ID NO:1 and a primer consisting of the nucleotide sequence of SEQ ID NO:2.
- Item 10 A kit for detecting a nucleic acid encoding a chimeric antigen receptor, comprising the primer set of any one of Items 1-9.
- Item 11 Item 11.
- the kit according to Item 10 further comprising a probe having the base sequence of SEQ ID NO:9.
- Item 12 Item 12.
- Item 13 The primer set according to any one of Items 1 to 9 or the kit according to any one of Items 10 to 12, for detecting a nucleic acid encoding the chimeric antigen receptor present in a biological sample of a subject.
- Item A1 A method for detecting a nucleic acid encoding a chimeric antigen receptor in a biological sample, comprising: extracting DNA from a biological sample, and using the extracted DNA as a template to perform PCR using a primer having the nucleotide sequence of SEQ ID NO: 1 and a primer having the nucleotide sequence of SEQ ID NO: 2; A method, including Item A2 The method according to item A1, wherein the nucleic acid comprises a base sequence complementary to the base sequence of SEQ ID NO:1 and a base sequence complementary to the base sequence of SEQ ID NO:2.
- Item A3 The method of paragraph A1 or A2, wherein said chimeric antigen receptor comprises CD28 and CD3 ⁇ .
- Item A4 The method according to item A3, wherein the base sequence encoding CD28 comprises the base sequence of SEQ ID NO:4.
- Item A5 The method according to item A3 or A4, wherein the base sequence encoding CD3 ⁇ comprises the base sequence of SEQ ID NO:5.
- Item A6 The method according to any one of paragraphs A1 to A5, wherein said PCR is quantitative PCR.
- Item A7 The method according to any one of items A1 to A6, further comprising contacting a probe having the base sequence of SEQ ID NO: 9 with the amplified product of the PCR.
- Item A8 The method according to any one of paragraphs A1 to A7, wherein the biological sample is blood.
- Item A9 The method according to Section A8, wherein the blood is blood collected from a patient to whom the chimeric antigen receptor T cells have been administered.
- Item A10 The method of Section A9, wherein said patient is in need of administration of T cells expressing chimeric antigen receptors comprising CD28 and CD3 ⁇ .
- Item 1B A primer set comprising a primer having the base sequence of SEQ ID NO: 1 and a primer having the base sequence of SEQ ID NO: 2.
- Item 2B The primer set of Section 1B, wherein said primer set is used to detect a nucleic acid encoding a chimeric antigen receptor.
- Item 3B Item 2B, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence and the nucleotide sequence of SEQ ID NO: 2 or its complementary nucleotide sequence.
- Item 4B The primer set of paragraph 2B or 3B, wherein said chimeric antigen receptor comprises CD28 and CD3 ⁇ .
- Item 5B Item 4B, wherein the CD28-encoding nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:5.
- Item 6B Item 4B or 5B, wherein the nucleotide sequence encoding CD3 ⁇ comprises the nucleotide sequence of SEQ ID NO:6.
- a primer set according to any one of Item 8B The primer set of any of claims 2B-7B, wherein said chimeric antigen receptor is for an antigen against multiple myeloma.
- Item 9B The primer set according to any one of claims 2B to 8B, wherein said nucleic acid is contained in one or more cells selected from PBMCs, T cells, NK cells, and NK-T cells.
- Item 10B A kit for detecting a nucleic acid encoding a chimeric antigen receptor, comprising a primer set according to any one of claims 1B-9B.
- Item 11B The kit according to claim 10B, further comprising a probe having the base sequence of SEQ ID NO: 9 and used for detecting or quantifying a nucleic acid encoding a chimeric antigen receptor.
- Item 12B The primer set according to any of claims 2B-9B or the kit according to claims 10B or 11B, wherein said chimeric antigen receptor is present in a biological sample.
- Item 13B A method for the quantitative detection of chimeric antigen receptor cells expressing (simultaneously/together) CD28 and CD3 ⁇ in a biological sample, comprising: (1) collecting a biological sample from a patient to whom chimeric antigen receptor cells expressing CD28 and CD3 ⁇ have been administered; (2) a step of extracting DNA from a biological sample; and (3) using the extracted DNA as a template, a primer having the nucleotide sequence of SEQ ID NO: 1, a primer having the nucleotide sequence of SEQ ID NO: 2, and a nucleotide sequence of SEQ ID NO: 9.
- a step of performing quantitative PCR by combining probes having A method for detecting chimeric antigen receptor cells comprising: Item 14B 13B.
- Item 15B A method for evaluating efficacy of chimeric antigen receptor cell therapy in a patient in need of chimeric antigen receptor cell therapy, comprising: (1) collecting a biological sample from a patient to whom chimeric antigen receptor cells expressing CD28 and CD3 ⁇ have been administered; (2) a step of extracting DNA from a biological sample; and (3) using the extracted DNA as a template, a primer having the nucleotide sequence of SEQ ID NO: 1, a primer having the nucleotide sequence of SEQ ID NO: 2, and a nucleotide sequence of SEQ ID NO: 9. and performing quantitative PCR to measure the amount of chimeric antigen receptor cells in the biological sample;
- a method, including Item 16B A method of determining a treatment strategy for a patient based on the results of the evaluation method of Section 15B.
- the nucleic acid of interest is a nucleic acid encoding a chimeric antigen receptor. In one embodiment, the nucleic acid of interest is a nucleic acid encoding a polypeptide in which CD28 and CD3 ⁇ are directly or indirectly linked.
- FIG. NGMC are non-transgenic T cells
- CAR-T are CAR-transgenic T cells. Quantitative PCR results measured at each blood sampling time point are shown.
- the nucleotide sequence of SEQ ID NO: 1 is CCACCCGCAAGCATTACC, which matches part of the nucleotide sequence encoding the co-stimulatory molecule CD28. Therefore, the primer having the base sequence of SEQ ID NO: 1 can bind (hybridize) to the complementary strand of the DNA strand encoding CD28.
- the base sequence of SEQ ID NO: 2 is CGCTCCTGCTGAACTTCACTCT, which is complementary to part of the base sequence encoding CD3 ⁇ . Therefore, the primer having the base sequence of SEQ ID NO: 2 can bind to the DNA strand encoding CD3 ⁇ .
- a DNA chain encoding a polypeptide in which CD28 and CD3 ⁇ are linked can be detected.
- a DNA chain encoding a polypeptide in which CD28 and CD3 ⁇ are linked by PCR using a combination (primer set) of a primer having the nucleotide sequence of SEQ ID NO: 1 and a primer having the nucleotide sequence of SEQ ID NO: 2. can be amplified, and the DNA strand can be detected using the presence of the amplification product as an index.
- the number of nucleotides constituting the primer having the base sequence of SEQ ID NO: 1 is arbitrary. In one embodiment, the number of nucleotides constituting the primer having the base sequence of SEQ ID NO: 1 is 15 or more and 25 or less, preferably 16 or more and 25 or less, and preferably 18 or more and 20 or less.
- the primer having the base sequence of SEQ ID NO: 1 is preferably complementary to the complementary strand of the DNA strand encoding CD28 as a whole.
- the number of nucleotides constituting the primer having the base sequence of SEQ ID NO:2 is arbitrary.
- the number of nucleotides constituting the primer having the base sequence of SEQ ID NO: 2 is 15 or more and 25 or less, preferably 18 or more and 25 or less.
- the primer having the base sequence of SEQ ID NO: 2 is preferably complementary to the DNA strand encoding CD3zeta as a whole.
- the number of constituent nucleotides is preferably set in the range of 18 or more and 25 or less for easy preparation for PCR. Nucleotide numbers below the above range do not exhibit sufficient specificity and result in decreased precision, including increased background. On the other hand, when the number of nucleotides exceeds, it is not desirable from a practical point of view because it increases the difficulty of utilization including an increase in design cost.
- the length of the constituent nucleotides is set in the range of 1:1 to 1.5, preferably 1, when comparing the length of the primer encoding CD28 and the length of the primer encoding CD3 ⁇ . : 1.1 to 1.3, more preferably 1:1.15 to 1.25.
- This nucleotide length is set according to the DNA strand to be recognized even when CD28 and CD3 ⁇ are arranged in reverse. Specifically, when the number of nucleotides constituting the primer having the nucleotide sequence of SEQ ID NO: 1 is 15, the number of nucleotides constituting the primer having the nucleotide sequence of SEQ ID NO: 2 is selected from 15 to 23.
- a primer having the nucleotide sequence of SEQ ID NO: 1 and a primer having the nucleotide sequence of SEQ ID NO: 2 can be created by artificial chemical synthesis or isolation.
- the nucleic acids (DNA strands) that can be detected by the primer set are arbitrary as long as each primer has a binding region.
- the DNA strand that can be detected by the primer set preferably has the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence and the nucleotide sequence of SEQ ID NO: 2 or its complementary nucleotide sequence.
- such a DNA strand may be a DNA strand encoding a chimeric antigen receptor comprising CD28 and CD3 ⁇ .
- the chimeric antigen receptor preferably comprises scFv, CD28, and CD3 ⁇ , which are preferably linked in this order, optionally via a linker, and constructed in the order scFv-CD28-CD3 ⁇ .
- a CAR construct it is preferable to use a set of primers having the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2 for its detection.
- the scFv amino acid sequence and the nucleotide sequence encoding it are arbitrary.
- the chimeric antigen receptor is preferably specific for multiple myeloma.
- Such chimeric antigen receptors preferably have an scFv that specifically binds to multiple myeloma.
- Such chimeric antigen receptors are disclosed, for example, in WO2017/026331.
- the polynucleotide encoding the light chain variable region (VL) that constitutes the scFv that specifically binds to multiple myeloma preferably has the following base sequence.
- a polynucleotide encoding a heavy chain variable region (VH) that constitutes scFv that specifically binds to multiple myeloma preferably has the following base sequence.
- the nucleotide sequence encoding the amino acid sequence of CD28 that constitutes the chimeric antigen receptor preferably includes the nucleotide sequence of SEQ ID NO:1.
- the nucleotide sequence encoding the CD28 amino acid sequence preferably includes the following nucleotide sequence.
- CD3 ⁇ (TCR ⁇ , CD247) that constitutes the chimeric antigen receptor preferably has a region encoded by a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:2.
- the nucleotide sequence encoding the CD3 ⁇ amino acid sequence preferably includes the following nucleotide sequence.
- VL and VH of scFv can be linked via an arbitrary linker.
- the nucleotide sequence encoding the linker connecting VL and VH preferably includes GGCTCCACTAGCGGTTCCGGCAAACCTGGCAGCGGAGAAGGCAGC (SEQ ID NO: 7).
- CD28 and CD3 ⁇ can be linked via any linker.
- the nucleotide sequence encoding the linker that connects CD28 and CD3 ⁇ preferably contains GCGGGCCGCA.
- the polynucleotide encoding the chimeric antigen receptor preferably has a region encoding a signal peptide on the N-terminal side of the region encoding the scFv.
- the nucleotide sequence encoding the signal peptide is arbitrary, but preferably includes ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCTTCAGTCATAATGTCCAGAGGA (SEQ ID NO: 8).
- a polynucleotide encoding a chimeric antigen receptor comprising a signal peptide, scFv, CD28 and CD3 ⁇ can be those described in WO2017/026331 or Nature Medicine 23 12 2007 1436.
- Chimeric antigen receptors, nucleic acids encoding them, and T cells containing (or expressing) the nucleic acids can be prepared by known techniques.
- the nucleic acids detected by the primer set may be either single-stranded or double-stranded. In one embodiment, it is preferred that the nucleic acid is double-stranded. When the nucleic acid is double-stranded, one strand constituting the double strand has a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, and the other strand has a complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO: 2. It preferably has a base sequence.
- the nucleic acid detected by the primer set may be DNA or RNA, and in one embodiment, the nucleic acid is preferably DNA.
- the detection of the target nucleic acid using the primer set is preferably performed using PCR.
- the primer having the nucleotide sequence of SEQ ID NO: 1 is preferably used as a forward primer
- the primer having the nucleotide sequence of SEQ ID NO: 2 is preferably used as a reverse primer.
- quantitative PCR is preferred, although PCR can be used qualitatively.
- Quantitative PCR also called real-time PCR, is generally a method of temporally monitoring the process of generating amplified products in PCR. In the detection using quantitative PCR, the amplification product by PCR reaction is usually detected by optical means.
- the detection of the amplified product may be performed at or after the completion of the PCR reaction, or may be performed in parallel with the PCR reaction process.
- the detection of amplification products can be performed over time, for example. Detection (monitoring) over time can be, for example, continuous or discontinuous (intermittent). Also, by counting the number of PCR cycles at which the amplification product reaches a predetermined amount, the target nucleic acid contained in the sample can be quantified. Furthermore, it is also possible to quantify the copy number in a sample from a calibration curve calculated from standards.
- the nucleic acid detected or quantified using the primer set is preferably present in cells.
- the form of the nucleic acid is arbitrary, and for example, it may be in a state of being incorporated into a plasmid or into a state of being incorporated into genomic DNA.
- Cells containing the nucleic acid may be of any type, but are preferably one or more selected from, for example, PBMCs, T cells, NK cells, and NK-T cells.
- the nucleic acid or cells having the nucleic acid are preferably present in a biological sample.
- the biological sample may be of any type, but is preferably blood (for example, whole blood).
- the target nucleic acid is preferably detected using any probe that specifically recognizes the product amplified by PCR using the primer set.
- a probe is not particularly limited as long as it is a sequence present between the primer having the nucleotide sequence of SEQ ID NO: 1 and the primer having the nucleotide sequence of SEQ ID NO: 2.
- CACCACGCGACTTCGCAGCCTAT SEQ ID NO: 9
- an oligonucleotide having a base sequence complementary thereto can be made by any technique (eg, chemical synthesis).
- the fluorescent probe it is possible to select and use an appropriate commercially available one.
- fluorescein (FAM) can be used as a fluorescent probe, but it is not limited to this.
- NFQ non-fluorescent quenchers
- MGB minor groove binding
- the number of constituent nucleotides is arbitrary. In one embodiment, the number of nucleotides constituting the probe having the base sequence of SEQ ID NO:9 is preferably 20 to 25 or less.
- the base sequence distance between each primer and the probe is 5 to 25. It is set in the range of 1 nucleotide, preferably 5 to 15 nucleotides or less. More than 25 distances may result in increased reaction times, etc., while less than 5 may affect the degree of fluorescence of the probe.
- a kit for detecting a nucleic acid encoding a chimeric antigen receptor preferably contains the primer set.
- the kit may contain optional components, reagents (eg, PCR reagents), containers, equipment, etc., in addition to the primer set.
- the kit preferably further comprises the probes described above.
- PCR reagents include polymerases, dNTPs (deoxynucleoside triphosphates), intercalators or fluorescence-labeled probes, and buffers.
- a chimeric antigen receptor in a human biological sample can be detected and its amount can be quantified by quantitative PCR using the primer set. Specifically, (1) a step of collecting a biological sample from a patient to whom chimeric antigen receptor cells expressing CD28 and CD3 ⁇ were administered, (2) a step of extracting DNA from the biological sample, and (3) extraction. Using the obtained DNA as a template, a primer having the nucleotide sequence of SEQ ID NO: 1, a primer having the nucleotide sequence of SEQ ID NO: 2, and a probe having the nucleotide sequence of SEQ ID NO: 9 are combined to perform quantitative PCR.
- the nucleic acid encoding the chimeric antigen receptor in the biological sample can be quantitatively detected, and based thereon, the amount of chimeric antigen receptor cells in the biological sample can be determined. Therefore, in CAR-T cell therapy, a biological sample is collected from a subject after administration of CAR-T cells, and is used as a sample to measure the amount of nucleic acid encoding CAR. The kinetics (increase or decrease) can be assessed and also the efficacy of CAR-T cell therapy can be assessed. In addition, the evaluation results can be used to determine subsequent treatment strategies for patients, such as prediction of drug efficacy and appropriateness of drug administration.
- a patient in need of CAR-T cell therapy is, for example, a patient with cancer, and the cancer can be, for example, blood cancer or solid cancer.
- a hematologic cancer can be, for example, lymphoma, leukemia, or myeloma.
- the patient in need of CAR-T cell therapy is a human with multiple myeloma.
- Example 1 Extraction of CAR-T Cell-Derived gDNA Genomic DNA derived from CAR-T cells for preparing a standard curve is extracted from CAR-T cells according to the following procedure. This operation can be performed using a commercially available genomic DNA extraction kit (eg, DNeasy Blood & Tissue Kit), and the following procedure is exemplified.
- the CAR-T cells used in the examples were aITGB7 CAR-T cells, and the control substance was non-transgenic T cells (Takara Bio Inc.) (hereinafter also referred to as NGMC).
- the aITGB7 CAR to be introduced was obtained from Osaka University.
- the aITGB7 CAR employs the anti-integrin ⁇ 7 scFV-CD28-CD3 ⁇ .
- the aITGB7 CAR-T cells contain the nucleotide sequences of SEQ ID NOS:4-8. (1) Thaw CAR-T cells in a constant temperature water bath at 37°C. (2) Collect 1 ⁇ 10 7 cells. (3) Remove the supernatant after centrifugation (2000 ⁇ g, 5 minutes, room temperature). Then, perform tapping, add and mix 400 ⁇ L of PBS, and add 40 ⁇ L of Proteinase K. (4) Add 8 ⁇ L of RNase A (100 mg/mL), mix, and incubate at room temperature for about 2 minutes.
- Genomic DNA is extracted from blood samples according to the following procedure. This operation can be performed using a commercially available genomic DNA extraction kit (eg, DNeasy Blood & Tissue Kit), and the following procedure is exemplified, for example.
- a commercially available genomic DNA extraction kit eg, DNeasy Blood & Tissue Kit
- (1) Take 100 ⁇ L of thawed anticoagulant-treated blood. Add 20 ⁇ L of Proteinase K to this, and then add 100 ⁇ L of PBS. Prepare 3 bottles of this liquid for each individual sample (No. 1 to No. 3).
- qPCR Quantitative PCR is performed using commercially available quantitative PCR reagents (for example, LightCycler 480 Probe Master (manufactured by Roche Diagnostics Co., Ltd.). Hereinafter, the case of using LightCycler 480 Probe Master will be described as an example.
- H2O PCR-grade
- Table 3 shows the sequences of the primers and probes used for quantitative PCR.
- a StepOnePlus (trademark) real-time PCR system (Thermo Fisher Scientific Co., Ltd.) was used for PCR.
- Example 2 After administration of aITGB7 CAR-T cells to severely immunodeficient mice with tail vein transplantation of human multiple myeloma cells, aITGB7 CAR-T in mouse blood was detected using primers that specifically recognize aITGB7 CAR-T cells. Changes in cell amount were confirmed.
- AITGB7 CAR-T cells were used as the test substance, and NGMC was used as the control substance. All T cells were stored in the dark at -80°C.
- the multiple myeloma cell line used was MM.1S derived from human multiple myeloma. Cultivation was performed in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 Units/mL penicillin and 100 ⁇ g/mL streptomycin under conditions of 37° C., 5% CO 2 . Fetal bovine serum was inactivated by heating at 56°C for 30 minutes.
- NOG mice were used as severely immunodeficient mice. Twenty 5- to 6-week-old females were obtained, and food and water (autoclave-sterilized tap water) were freely available.
- aITGB7 CAR-T was administered at 1.33 ⁇ 10 5 cells/body. Serum-free RPMI 1640 medium was used at the time of transplantation. Since the PK test in NOG mice was performed at 6 time points, 3 mice were assigned to each time point, for a total of 18 mice. Blood sampling time points were 1 hour, 24 hours, 3 days, 1 week, 2 weeks, and 3 weeks after administration. The remaining two animals were bled without administration of aITGB7 CAR-T for preparation of whole blood samples for standard curve preparation.
- test substance 10 days after MM.1S cell tail vein transplantation, the test substance was administered to the tail vein, blood was collected from the mice at the designated time points, and quantitative PCR was performed on the DNA isolated from the whole blood. The amount of T was quantified. Specific conditions and procedures are as follows.
- Blood was collected from the retro-abdominal vena cava under isoflurane (Abbott Japan Co., Ltd.) inhalation anesthesia 1 hour, 24 hours, 3 days, 1 week, 2 weeks, and 3 weeks after administration. Immediately after the blood was collected, it was transferred to a heparin-filled tube, mixed by inversion, and allowed to stand on ice.
- isoflurane Abbott Japan Co., Ltd.
- Genomic DNA was extracted with NucleoSpinTM Blood (MACHEREY-NAGEL) from freeze-thawed aITGB7 CAR-T samples, NGMC samples, and blood samples derived from tumor-bearing mice administered with aITGB7 CAR-T. DNA content was measured with NanoDropTM and the primer set for aITGB7 CAR was used to assess the DNA content of aITGB7 CAR in each sample relative to the total genome content.
- NucleoSpinTM Blood MACHEREY-NAGEL
- Genomic DNA Extraction for aITGB7 CAR Specificity Analysis of Detection System Genomic DNA was extracted from freeze-thawed aITGB7 CAR-T and NGMC in order to analyze the amount of aITGB7 CAR DNA in each sample.
- Genomic DNA Extraction from Whole Blood Samples In order to analyze the amount of aITGB7 CAR DNA in each sample, genomic DNA was extracted from blood samples derived from tumor-bearing mice to which aITGB7 CAR-T was administered.
- the concentration of the extracted genomic DNA solution was measured.
- the extracted genomic DNA solution was stored on ice water until it was subjected to real-time PCR reaction.
- the sample extracted in the "extraction of genomic DNA for aITGB7 CAR specificity analysis of detection system” after measuring the DNA concentration, it was diluted to 1000 times with distilled water at a common ratio of 10 times.
- the primers and probes used for quantitative PCR were those listed in Table 3.
- the CAR gene PCR mixture was prepared by mixing 10 ⁇ L of PCR Master Mix (2X), 0.2 ⁇ L of Forward primer, 0.2 ⁇ L of Reverse primer, 0.5 ⁇ L of Probe and 1.1 ⁇ L of distilled water per reaction. . A necessary amount was prepared according to the number of samples to be measured. 12 ⁇ L of the PCR mixture was dispensed into each well of the PCR plate, and 8 ⁇ L of the corresponding DNA sample was added. Table 7 shows the conditions for real-time PCR.
- Calculation of the amount of aITGB7 CAR DNA Calculate the Ct value using the analysis software built into the real-time PCR device, calculate the ⁇ Ct value according to formula 1, and calculate the relative value of aITGB7 CAR DNA in the sample solution used for quantitative PCR (RQ : Relative Quantification) was calculated according to Equation 2.
- the RQ values of the calibration curve samples and the plasmid concentrations of the calibration curve samples were plotted to obtain a linear equation of an approximate straight line, and the aITGB7 CAR DNA concentration of each sample was calculated.
- the amount of aITGB7 CAR DNA present in a given volume of blood was calculated by further dividing by the total genome concentration isolated.
- FIG. 2 shows the results of quantitative PCR measured at each blood sampling time point. After administration of aITGB7 CAR-T at 1.33 ⁇ 10 5 cells/body, DNA of aITGB7 CAR was detected in whole blood from 1 hour later, and showed a maximum level 2 weeks after administration.
- Example 3 Extraction of Genomic DNA From PBMCs obtained from human donors to whom aITGB7 CAR-T cells were administered, gDNA was extracted according to the protocol attached to QIAamp DNA Mini Kit (manufactured by QIAGEN). gDNA samples were stored at -20°C as needed. (1) 10 milliliters of PBS was transferred to a 15 mL tube. (2) PBMC were thawed in a 37° C. water bath and transferred to a 15 mL tube. (3) After centrifuging the tube (300 ⁇ g, 10 minutes, room temperature), the supernatant was removed. (4) After tapping, 1 mL of PBS was transferred to a 15 mL tube to suspend PBMCs.
- the column was set in a new collection tube (2 mL), and 500 ⁇ L of Buffer AW1 (manufactured by QIAGEN) was added.
- the tube was centrifuged at 6000 xg for 1 minute at room temperature.
- the column was set in a new collection tube (2 mL), and 500 ⁇ L of Buffer AW2 (manufactured by QIAGEN) was added.
- the tube was centrifuged at 20000 xg for 3 minutes at room temperature.
- the column was set in a new collection tube (2 mL).
- (21) The tube was centrifuged at 20000 xg for 1 minute at room temperature.
- Standard Solution A standard plasmid stock solution (1.00 ⁇ 10 8 copies/ ⁇ L, SS) was thawed at room temperature, and standard solutions C1 to C10 were prepared according to Table 9 below. Buffer AE was used as the NTC (no template control) sample.
- the standard plasmid is a linear plasmid having a partial DNA base sequence of aITGB7 CAR-T cells.
- test samples H, M, L, LL and BL were prepared according to Tables 10 and 11 below. prepared. Each gDNA extracted in 1 above was melted at room temperature and diluted with Buffer AE to prepare 50 ng/ ⁇ L of gDNA.
- H-1 to Hn, M-1 to Mn, L-1 to Ln, LL-1 to LL-n, and BL-1 to BL-n were prepared according to the number of donors n.
- PCR Master Mix A sufficient amount of PCR Master Mix was prepared on the day of use. The contents of one reaction are described below.
- the H2O (PCR grade) supplied with the LightCycler 480 Probe Master was used.
- LightCycler 480 Probe Master 10.0 ⁇ L 20 ⁇ Pre Mixed Primer and Probe 1.0 ⁇ L H2O ( PCR grade) 1.0 ⁇ L Total 12.0 ⁇ L
- the above "Pre Mixed Primer and Probe” includes a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1, a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2, and a probe having the nucleotide sequence of SEQ ID NO: 9 (5'-FAM-CACCACGCGACTTCGCAGCCTATC- NFQ-MGB-3') (FAM: Fluorescein, NFQ: Non-Fluorescent Quencher, MGB: Minor Groove Binder).
- Cp values, MSE, inverse regression values and sample concentrations were calculated using analysis software for real-time PCR systems (LightCyclerTM 480 software, Roche Diagnostics).
- C1-C10 were used to generate a standard curve. Acceptance criteria for calibration standards were as follows: (1) MSE less than 0.2. (2) The CV of the inverse regression value is less than 30% on at least 9 points out of 10 (C1 and C10 shall be less than 30%). (3) RE of the inverse regression value is within ⁇ 30% at least 9 points out of 10 (C1 and C10 shall be within ⁇ 30%). (4) The Cp value of NTC is 45.00 or clearly greater than the Cp value of C10.
- CV and RE were evaluated by analyzing H, M, L and LL with three replicates per level. Acceptance criteria for CV and RE were as follows: CV of QC sample is less than 30%. RE of QC sample value within ⁇ 30%.
- Freeze-Thaw Stability CV and RE were assessed by analyzing H and L with one replicate per level. QC samples were freeze-thawed 0, 1 and 3 times at ⁇ 20° C. before performing qPCR to confirm freeze-thaw stability. The melting operation was performed by standing the QC samples at RT. Acceptance criteria for freeze-thaw stability were as follows: QC samples have a CV less than 30%. The RE of the QC sample values is within ⁇ 30%.
- the present invention is not limited to the contents of the above examples.
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Abstract
Provided is a primer set that contains a primer having the base sequence of SEQ ID NO: 1 and a primer having the base sequence of SEQ ID NO: 2.
Description
キメラ抗原受容体の検出に関連する技術が開示される。
Techniques related to detection of chimeric antigen receptors are disclosed.
遺伝子導入したT細胞によるがん治療研究は、1990年代に始まり、2000年代に最初の臨床応用報告がなされた。第1世代のキメラ抗原受容体（chimeric antigen receptor：CAR）は、抗原認識部位とT細胞活性化部位としてT細胞受容体のサブユニットである TCRζ（CD3ζ）で構成されている。第2世代のCARは、抗原認識部位、共刺激部位、及びCD3ζなどで構成される。抗原認識部位は、抗体の重鎖と軽鎖のバリアブル領域をペプチドリンカーでつないだ単鎖可変領域フラグメント（scFv）が用いられる。共刺激部位には、4-1BB、OX40、CD27などのTNF受容体ファミリー、およびCD28、inducible T cell costimulator（ICOS）などのCD28ファミリーがあり、更なるCARの開発が進行中である。共刺激部位は、CAR-T細胞の細胞傷害活性と増殖、持続性の向上を期待して導入されている。
Cancer therapy research using transfected T cells began in the 1990s, and the first clinical application was reported in the 2000s. First-generation chimeric antigen receptors (CARs) consist of the T-cell receptor subunit TCRζ (CD3ζ) as the antigen-recognition site and the T-cell activation site. Second-generation CARs are composed of antigen recognition sites, co-stimulatory sites, CD3zeta, and the like. For the antigen recognition site, a single chain variable region fragment (scFv) is used, in which the heavy and light chain variable regions of an antibody are connected with a peptide linker. Costimulatory sites include the TNF receptor family, including 4-1BB, OX40, and CD27, and the CD28 family, including CD28 and inducible T cell costimulator (ICOS), and further CAR development is underway. Costimulatory sites have been introduced in hopes of enhancing CAR-T cell cytotoxicity, proliferation, and persistence.
CAR-T細胞は、scFvを介して抗原を認識して活性化するために、ヒト白血球型抗原（human leukocyte antigen：HLA）の介在を必要とせず、がん免疫逃避機構の1つであるがん細胞のHLAのダウンモジュレーションがあったとしてもその効果が発揮されることが期待されている。また、CAR-T細胞は、細胞傷害性物質であるTNF、パーフォリン、グランザイムなどを放出したり、腫瘍細胞のFas誘導性アポトーシスを誘導したりすることで抗腫瘍効果を発揮する。
CAR-T cells do not require the intervention of human leukocyte antigen (HLA) to recognize and activate antigens via scFv, and are one of the cancer immune escape mechanisms. It is expected that even if there is down-modulation of HLA in cancer cells, the effect will be exhibited. CAR-T cells also exert antitumor effects by releasing cytotoxic substances such as TNF, perforin, and granzyme, and by inducing Fas-induced apoptosis of tumor cells.
日本では、2019年に再発または難治性のCD19陽性のB細胞性急性リンパ芽球性白血病（B-cell acute lymphoblastic leukemia：B-ALL）および再発または難治性のCD19陽性のびまん性大細胞型B細胞リンパ腫（diffuse large B-cell lymphoma：DLBCL）を対象としたCAR-T療法について製造販売承認が付与されている（非特許文献１）。それ以降、CD19をはじめとする複数の抗原を対象としたCAR-T療法の開発が進められており、生体内におけるCAR-Tの増殖及び消失の経時変化を定量に評価することで、投与量、治療効果又は副作用とCAR-T体内動態の関連を明らかにすることが期待されている。
In Japan, relapsed or refractory CD19-positive acute lymphoblastic leukemia (B-ALL) and relapsed or refractory CD19-positive diffuse large cell type B were reported in 2019. Manufacturing and marketing approval has been granted for CAR-T therapy for diffuse large B-cell lymphoma (DLBCL) (Non-Patent Document 1). Since then, the development of CAR-T therapy targeting multiple antigens, including CD19, has progressed. , is expected to clarify the relationship between therapeutic effects or side effects and CAR-T pharmacokinetics.
CAR-T細胞療法に関する開発が活発に行われる中、CARの検出方法を提供することが一つの課題である。
Among the active development of CAR-T cell therapy, one of the challenges is to provide a method for detecting CAR.
上記課題を解決すべく鋭意研究を重ね、特定のプライマーを用いることにより、CARを検出できることを見出し、下記に代表される発明が提供される。
After extensive research to solve the above problems, it was found that CAR can be detected by using specific primers, and the following inventions are provided.
項１
配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーを含む、キメラ抗原受容体をコードする核酸を検出するためのプライマーセット。
項２
前記核酸が、配列番号１の塩基配列と相補的な塩基配列及び配列番号２の塩基配列と相補的な塩基配列を含む、請求項１に記載のプライマーセット。
項３
前記キメラ抗原受容体がＣＤ２８及びＣＤ３ζを含む、項１又は２に記載のプライマーセット。
項４
前記ＣＤ２８をコードする塩基配列が配列番号５の塩基配列を含む、項３に記載のプライマーセット。
項５
前記ＣＤ３ζをコードする塩基配列が配列番号６の塩基配列を含む、項３又は４に記載のプライマーセット。
項６
前記検出が定量ＰＣＲを用いて行われる、項１～５のいずれかに記載のプライマーセット。
項７
前記キメラ抗原受容体が、多発性骨髄腫に対するキメラ抗原受容体である、項１～６のいずれかに記載のプライマーセット。
項８
前記多発性骨髄腫に対するキメラ抗原受容体が、インテグリンβ７を標的としたキメラ抗原受容体である、項７に記載のプライマーセット。
項９
配列番号１の塩基配列からなるプライマー及び配列番号２の塩基配列からなるプライマーを含む、キメラ抗原受容体をコードする核酸を検出するためのプライマーセット。
項１０
項１～９のいずれかに記載のプラマーセットを含む、キメラ抗原受容体をコードする核酸を検出するためのキット。
項１１
配列番号９の塩基配列を有するプローブを更に含有する、項１０に記載のキット。
項１２
前記プローブが、配列番号９の塩基配列からなる、項１１に記載のキット。
項１３
被検体の生体試料中に存在する前記キメラ抗原受容体をコードする核酸を検出するための、項１～９のいずれかに記載のプライマーセット又は項１０～１２のいずれかに記載のキット。 Item 1
A primer set for detecting a nucleic acid encoding a chimeric antigen receptor, comprising a primer having the nucleotide sequence of SEQ ID NO:1 and a primer having the nucleotide sequence of SEQ ID NO:2.
Item 2
2. The primer set according to claim 1, wherein the nucleic acid comprises a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1 and a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:2.
Item 3
Item 3. The primer set according to Item 1 or 2, wherein the chimeric antigen receptor comprises CD28 and CD3ζ.
Item 4
Item 4. The primer set according to Item 3, wherein the nucleotide sequence encoding CD28 comprises the nucleotide sequence of SEQ ID NO:5.
Item 5
Item 5. The primer set according to Item 3 or 4, wherein the nucleotide sequence encoding CD3ζ comprises the nucleotide sequence of SEQ ID NO:6.
Item 6
Item 6. The primer set according to any one of Items 1 to 5, wherein the detection is performed using quantitative PCR.
Item 7
Item 7. The primer set according to any one of Items 1 to 6, wherein the chimeric antigen receptor is a chimeric antigen receptor for multiple myeloma.
Item 8
Item 8. The primer set according to Item 7, wherein the chimeric antigen receptor for multiple myeloma is a chimeric antigen receptor targeting integrin β7.
Item 9
A primer set for detecting a nucleic acid encoding a chimeric antigen receptor, comprising a primer consisting of the nucleotide sequence of SEQ ID NO:1 and a primer consisting of the nucleotide sequence of SEQ ID NO:2.
Item 10
A kit for detecting a nucleic acid encoding a chimeric antigen receptor, comprising the primer set of any one of Items 1-9.
Item 11
Item 11. The kit according toItem 10, further comprising a probe having the base sequence of SEQ ID NO:9.
Item 12
Item 12. The kit according to Item 11, wherein the probe consists of the base sequence of SEQ ID NO:9.
Item 13
The primer set according to any one of Items 1 to 9 or the kit according to any one ofItems 10 to 12, for detecting a nucleic acid encoding the chimeric antigen receptor present in a biological sample of a subject.
配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーを含む、キメラ抗原受容体をコードする核酸を検出するためのプライマーセット。
項２
前記核酸が、配列番号１の塩基配列と相補的な塩基配列及び配列番号２の塩基配列と相補的な塩基配列を含む、請求項１に記載のプライマーセット。
項３
前記キメラ抗原受容体がＣＤ２８及びＣＤ３ζを含む、項１又は２に記載のプライマーセット。
項４
前記ＣＤ２８をコードする塩基配列が配列番号５の塩基配列を含む、項３に記載のプライマーセット。
項５
前記ＣＤ３ζをコードする塩基配列が配列番号６の塩基配列を含む、項３又は４に記載のプライマーセット。
項６
前記検出が定量ＰＣＲを用いて行われる、項１～５のいずれかに記載のプライマーセット。
項７
前記キメラ抗原受容体が、多発性骨髄腫に対するキメラ抗原受容体である、項１～６のいずれかに記載のプライマーセット。
項８
前記多発性骨髄腫に対するキメラ抗原受容体が、インテグリンβ７を標的としたキメラ抗原受容体である、項７に記載のプライマーセット。
項９
配列番号１の塩基配列からなるプライマー及び配列番号２の塩基配列からなるプライマーを含む、キメラ抗原受容体をコードする核酸を検出するためのプライマーセット。
項１０
項１～９のいずれかに記載のプラマーセットを含む、キメラ抗原受容体をコードする核酸を検出するためのキット。
項１１
配列番号９の塩基配列を有するプローブを更に含有する、項１０に記載のキット。
項１２
前記プローブが、配列番号９の塩基配列からなる、項１１に記載のキット。
項１３
被検体の生体試料中に存在する前記キメラ抗原受容体をコードする核酸を検出するための、項１～９のいずれかに記載のプライマーセット又は項１０～１２のいずれかに記載のキット。 Item 1
A primer set for detecting a nucleic acid encoding a chimeric antigen receptor, comprising a primer having the nucleotide sequence of SEQ ID NO:1 and a primer having the nucleotide sequence of SEQ ID NO:2.
Item 2
2. The primer set according to claim 1, wherein the nucleic acid comprises a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1 and a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:2.
Item 3
Item 3. The primer set according to Item 1 or 2, wherein the chimeric antigen receptor comprises CD28 and CD3ζ.
Item 4
Item 4. The primer set according to Item 3, wherein the nucleotide sequence encoding CD28 comprises the nucleotide sequence of SEQ ID NO:5.
Item 5
Item 5. The primer set according to Item 3 or 4, wherein the nucleotide sequence encoding CD3ζ comprises the nucleotide sequence of SEQ ID NO:6.
Item 6
Item 6. The primer set according to any one of Items 1 to 5, wherein the detection is performed using quantitative PCR.
Item 7
Item 7. The primer set according to any one of Items 1 to 6, wherein the chimeric antigen receptor is a chimeric antigen receptor for multiple myeloma.
Item 8
Item 8. The primer set according to Item 7, wherein the chimeric antigen receptor for multiple myeloma is a chimeric antigen receptor targeting integrin β7.
Item 9
A primer set for detecting a nucleic acid encoding a chimeric antigen receptor, comprising a primer consisting of the nucleotide sequence of SEQ ID NO:1 and a primer consisting of the nucleotide sequence of SEQ ID NO:2.
A kit for detecting a nucleic acid encoding a chimeric antigen receptor, comprising the primer set of any one of Items 1-9.
Item 11
Item 11. The kit according to
Item 12
Item 12. The kit according to Item 11, wherein the probe consists of the base sequence of SEQ ID NO:9.
Item 13
The primer set according to any one of Items 1 to 9 or the kit according to any one of
項Ａ１
生体試料中のキメラ抗原受容体をコードする核酸を検出する方法であって、
生体試料からＤＮＡを抽出すること、及び
抽出したＤＮＡを鋳型として、配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーを用いてＰＣＲを行うこと、
を含む、方法。
項Ａ２
前記核酸が、配列番号１の塩基配列と相補的な塩基配列及び配列番号２の塩基配列と相補的な塩基配列を含む、項Ａ１に記載の方法。
項Ａ３
前記キメラ抗原受容体がＣＤ２８及びＣＤ３ζを含む、項Ａ１又はＡ２に記載の方法。
項Ａ４
前記ＣＤ２８をコードする塩基配列が配列番号４の塩基配列を含む、項Ａ３に記載の方法。
項Ａ５
前記ＣＤ３ζをコードする塩基配列が配列番号５の塩基配列を含む、項Ａ３又はＡ４に記載の方法。
項Ａ６
前記ＰＣＲが定量ＰＣＲである、項Ａ１～Ａ５のいずれかに記載の方法。
項Ａ７
前記ＰＣＲの増幅産物に配列番号９の塩基配列を有するプローブ接触させることを更に含む、項Ａ１～Ａ６のいずれかに記載の方法。
項Ａ８
前記生体試料は血液である、項Ａ１～Ａ７のいずれかに記載の方法。
項Ａ９
前記血液は、キメラ抗原受容体Ｔ細胞が投与された患者から採取された血液である、項Ａ８に記載の方法。
項Ａ１０
前記患者がＣＤ２８及びＣＤ３ζを含むキメラ抗原受容体を発現するＴ細胞の投与を必要とする、項Ａ９に記載の方法。 Item A1
A method for detecting a nucleic acid encoding a chimeric antigen receptor in a biological sample, comprising:
extracting DNA from a biological sample, and using the extracted DNA as a template to perform PCR using a primer having the nucleotide sequence of SEQ ID NO: 1 and a primer having the nucleotide sequence of SEQ ID NO: 2;
A method, including
Item A2
The method according to item A1, wherein the nucleic acid comprises a base sequence complementary to the base sequence of SEQ ID NO:1 and a base sequence complementary to the base sequence of SEQ ID NO:2.
Item A3
The method of paragraph A1 or A2, wherein said chimeric antigen receptor comprises CD28 and CD3ζ.
Item A4
The method according to item A3, wherein the base sequence encoding CD28 comprises the base sequence of SEQ ID NO:4.
Item A5
The method according to item A3 or A4, wherein the base sequence encoding CD3ζ comprises the base sequence of SEQ ID NO:5.
Item A6
The method according to any one of paragraphs A1 to A5, wherein said PCR is quantitative PCR.
Item A7
The method according to any one of items A1 to A6, further comprising contacting a probe having the base sequence of SEQ ID NO: 9 with the amplified product of the PCR.
Item A8
The method according to any one of paragraphs A1 to A7, wherein the biological sample is blood.
Item A9
The method according to Section A8, wherein the blood is blood collected from a patient to whom the chimeric antigen receptor T cells have been administered.
Item A10
The method of Section A9, wherein said patient is in need of administration of T cells expressing chimeric antigen receptors comprising CD28 and CD3ζ.
生体試料中のキメラ抗原受容体をコードする核酸を検出する方法であって、
生体試料からＤＮＡを抽出すること、及び
抽出したＤＮＡを鋳型として、配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーを用いてＰＣＲを行うこと、
を含む、方法。
項Ａ２
前記核酸が、配列番号１の塩基配列と相補的な塩基配列及び配列番号２の塩基配列と相補的な塩基配列を含む、項Ａ１に記載の方法。
項Ａ３
前記キメラ抗原受容体がＣＤ２８及びＣＤ３ζを含む、項Ａ１又はＡ２に記載の方法。
項Ａ４
前記ＣＤ２８をコードする塩基配列が配列番号４の塩基配列を含む、項Ａ３に記載の方法。
項Ａ５
前記ＣＤ３ζをコードする塩基配列が配列番号５の塩基配列を含む、項Ａ３又はＡ４に記載の方法。
項Ａ６
前記ＰＣＲが定量ＰＣＲである、項Ａ１～Ａ５のいずれかに記載の方法。
項Ａ７
前記ＰＣＲの増幅産物に配列番号９の塩基配列を有するプローブ接触させることを更に含む、項Ａ１～Ａ６のいずれかに記載の方法。
項Ａ８
前記生体試料は血液である、項Ａ１～Ａ７のいずれかに記載の方法。
項Ａ９
前記血液は、キメラ抗原受容体Ｔ細胞が投与された患者から採取された血液である、項Ａ８に記載の方法。
項Ａ１０
前記患者がＣＤ２８及びＣＤ３ζを含むキメラ抗原受容体を発現するＴ細胞の投与を必要とする、項Ａ９に記載の方法。 Item A1
A method for detecting a nucleic acid encoding a chimeric antigen receptor in a biological sample, comprising:
extracting DNA from a biological sample, and using the extracted DNA as a template to perform PCR using a primer having the nucleotide sequence of SEQ ID NO: 1 and a primer having the nucleotide sequence of SEQ ID NO: 2;
A method, including
Item A2
The method according to item A1, wherein the nucleic acid comprises a base sequence complementary to the base sequence of SEQ ID NO:1 and a base sequence complementary to the base sequence of SEQ ID NO:2.
Item A3
The method of paragraph A1 or A2, wherein said chimeric antigen receptor comprises CD28 and CD3ζ.
Item A4
The method according to item A3, wherein the base sequence encoding CD28 comprises the base sequence of SEQ ID NO:4.
Item A5
The method according to item A3 or A4, wherein the base sequence encoding CD3ζ comprises the base sequence of SEQ ID NO:5.
Item A6
The method according to any one of paragraphs A1 to A5, wherein said PCR is quantitative PCR.
Item A7
The method according to any one of items A1 to A6, further comprising contacting a probe having the base sequence of SEQ ID NO: 9 with the amplified product of the PCR.
Item A8
The method according to any one of paragraphs A1 to A7, wherein the biological sample is blood.
Item A9
The method according to Section A8, wherein the blood is blood collected from a patient to whom the chimeric antigen receptor T cells have been administered.
Item A10
The method of Section A9, wherein said patient is in need of administration of T cells expressing chimeric antigen receptors comprising CD28 and CD3ζ.
項１Ｂ
配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーを含む、プライマーセット。
項２Ｂ
前記プライマーセットが、キメラ抗原受容体をコードする核酸を検出するために用いられる、項１Ｂに記載のプライマーセット。
項３Ｂ
前記核酸が、配列番号１の塩基配列またはその相補的な塩基配列及び配列番号２の塩基配列またはその相補的な塩基配列からなる、項２Ｂに記載のプライマーセット。
項４Ｂ
前記キメラ抗原受容体がＣＤ２８及びＣＤ３ζを含む、項２Ｂ又は３Ｂに記載のプライマーセット。
項５Ｂ
前記ＣＤ２８をコードする塩基配列が配列番号５の塩基配列を含む、項４Ｂに記載のプライマーセット。
項６Ｂ
前記ＣＤ３ζをコードする塩基配列が配列番号６の塩基配列を含む、項４Ｂ又は５Ｂに記載のプライマーセット。
項７Ｂ
前記検出が定量ＰＣＲを用いて行われるものであって、配列番号１の塩基配列を有するプライマーがフォワードプライマー、配列番号２の塩基配列を有するプライマーがリバースプライマーとして使用される、請求項２Ｂ～６Ｂのいずれかに記載のプライマーセット。
項８Ｂ
前記キメラ抗原受容体が、多発性骨髄腫に対する抗原に対するキメラ抗原受容体である、請求項２Ｂ～７Ｂのいずれかに記載のプライマーセット。
項９Ｂ
前記核酸が、ＰＢＭＣ、Ｔ細胞、ＮＫ細胞、及びＮＫ－Ｔ細胞から選択される一種以上の細胞に含まれる、請求項２Ｂ～８Ｂのいずれかに記載のプライマーセット。
項１０Ｂ
請求項１Ｂ～９Ｂのいずれかに記載のプライマーセットを含む、キメラ抗原受容体をコードする核酸を検出するためのキット。
項１１Ｂ
更に、配列番号９の塩基配列を有するプローブを含有し、キメラ抗原受容体をコードする核酸を検出又は定量するために使用される、請求項１０Ｂに記載されるキット。
項１２Ｂ
前記キメラ抗原受容体が生体試料中に存在する、請求項２Ｂ～９Ｂのいずれかに記載のプライマーセット又は請求項１０Ｂ若しくは１１Ｂに記載のキット。
項１３Ｂ
生体試料中のＣＤ２８及びＣＤ３ζを（同時／一緒に）発現するキメラ抗原受容体細胞を定量的に検出するための方法であって、
（１）ＣＤ２８及びＣＤ３ζを発現するキメラ抗原受容体細胞が投与された患者から、生体試料を採取する工程、
（２）生体試料からＤＮＡを抽出する工程、及び
（３）抽出したＤＮＡを鋳型として、配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーと、配列番号９の塩基配列を有するプローブを組み合わせて、定量ＰＣＲを行う工程、
を含む、キメラ抗原受容体細胞の検出方法。
項１４Ｂ
前記患者が多発性骨髄腫の治療を必要とする、請求項１３Ｂに記載の方法。
項１５Ｂ
キメラ抗原受容体細胞による治療を必要とする患者における、キメラ抗原受容体細胞による治療奏功性を評価方法であって、
（１）ＣＤ２８及びＣＤ３ζを発現するキメラ抗原受容体細胞が投与された患者から、生体試料を採取する工程、
（２）生体試料からＤＮＡを抽出する工程、及び
（３）抽出したＤＮＡを鋳型として、配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーと、配列番号９の塩基配列を有するプローブを組み合わせて、定量ＰＣＲを行い、生体試料中のキメラ抗原受容体細胞の量を測定する工程、
を含む、方法。
項１６Ｂ
項１５Ｂの評価方法による結果に基づいて、患者の治療方針を決定する方法。 Item 1B
A primer set comprising a primer having the base sequence of SEQ ID NO: 1 and a primer having the base sequence of SEQ ID NO: 2.
Item 2B
The primer set of Section 1B, wherein said primer set is used to detect a nucleic acid encoding a chimeric antigen receptor.
Item 3B
Item 2B, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence and the nucleotide sequence of SEQ ID NO: 2 or its complementary nucleotide sequence.
Item 4B
The primer set of paragraph 2B or 3B, wherein said chimeric antigen receptor comprises CD28 and CD3ζ.
Item 5B
Item 4B, wherein the CD28-encoding nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:5.
Item 6B
Item 4B or 5B, wherein the nucleotide sequence encoding CD3ζ comprises the nucleotide sequence of SEQ ID NO:6.
Item 7B
Claims 2B to 6B, wherein the detection is performed using quantitative PCR, a primer having the nucleotide sequence of SEQ ID NO: 1 is used as a forward primer, and a primer having the nucleotide sequence of SEQ ID NO: 2 is used as a reverse primer. A primer set according to any one of
Item 8B
The primer set of any of claims 2B-7B, wherein said chimeric antigen receptor is for an antigen against multiple myeloma.
Item 9B
The primer set according to any one of claims 2B to 8B, wherein said nucleic acid is contained in one or more cells selected from PBMCs, T cells, NK cells, and NK-T cells.
Item 10B
A kit for detecting a nucleic acid encoding a chimeric antigen receptor, comprising a primer set according to any one of claims 1B-9B.
Item 11B
The kit according to claim 10B, further comprising a probe having the base sequence of SEQ ID NO: 9 and used for detecting or quantifying a nucleic acid encoding a chimeric antigen receptor.
Item 12B
The primer set according to any of claims 2B-9B or the kit according to claims 10B or 11B, wherein said chimeric antigen receptor is present in a biological sample.
Item 13B
A method for the quantitative detection of chimeric antigen receptor cells expressing (simultaneously/together) CD28 and CD3ζ in a biological sample, comprising:
(1) collecting a biological sample from a patient to whom chimeric antigen receptor cells expressing CD28 and CD3ζ have been administered;
(2) a step of extracting DNA from a biological sample; and (3) using the extracted DNA as a template, a primer having the nucleotide sequence of SEQ ID NO: 1, a primer having the nucleotide sequence of SEQ ID NO: 2, and a nucleotide sequence of SEQ ID NO: 9. A step of performing quantitative PCR by combining probes having
A method for detecting chimeric antigen receptor cells, comprising:
Item 14B
13B. The method of claim 13B, wherein said patient is in need of treatment for multiple myeloma.
Item 15B
A method for evaluating efficacy of chimeric antigen receptor cell therapy in a patient in need of chimeric antigen receptor cell therapy, comprising:
(1) collecting a biological sample from a patient to whom chimeric antigen receptor cells expressing CD28 and CD3ζ have been administered;
(2) a step of extracting DNA from a biological sample; and (3) using the extracted DNA as a template, a primer having the nucleotide sequence of SEQ ID NO: 1, a primer having the nucleotide sequence of SEQ ID NO: 2, and a nucleotide sequence of SEQ ID NO: 9. and performing quantitative PCR to measure the amount of chimeric antigen receptor cells in the biological sample;
A method, including
Item 16B
A method of determining a treatment strategy for a patient based on the results of the evaluation method of Section 15B.
配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーを含む、プライマーセット。
項２Ｂ
前記プライマーセットが、キメラ抗原受容体をコードする核酸を検出するために用いられる、項１Ｂに記載のプライマーセット。
項３Ｂ
前記核酸が、配列番号１の塩基配列またはその相補的な塩基配列及び配列番号２の塩基配列またはその相補的な塩基配列からなる、項２Ｂに記載のプライマーセット。
項４Ｂ
前記キメラ抗原受容体がＣＤ２８及びＣＤ３ζを含む、項２Ｂ又は３Ｂに記載のプライマーセット。
項５Ｂ
前記ＣＤ２８をコードする塩基配列が配列番号５の塩基配列を含む、項４Ｂに記載のプライマーセット。
項６Ｂ
前記ＣＤ３ζをコードする塩基配列が配列番号６の塩基配列を含む、項４Ｂ又は５Ｂに記載のプライマーセット。
項７Ｂ
前記検出が定量ＰＣＲを用いて行われるものであって、配列番号１の塩基配列を有するプライマーがフォワードプライマー、配列番号２の塩基配列を有するプライマーがリバースプライマーとして使用される、請求項２Ｂ～６Ｂのいずれかに記載のプライマーセット。
項８Ｂ
前記キメラ抗原受容体が、多発性骨髄腫に対する抗原に対するキメラ抗原受容体である、請求項２Ｂ～７Ｂのいずれかに記載のプライマーセット。
項９Ｂ
前記核酸が、ＰＢＭＣ、Ｔ細胞、ＮＫ細胞、及びＮＫ－Ｔ細胞から選択される一種以上の細胞に含まれる、請求項２Ｂ～８Ｂのいずれかに記載のプライマーセット。
項１０Ｂ
請求項１Ｂ～９Ｂのいずれかに記載のプライマーセットを含む、キメラ抗原受容体をコードする核酸を検出するためのキット。
項１１Ｂ
更に、配列番号９の塩基配列を有するプローブを含有し、キメラ抗原受容体をコードする核酸を検出又は定量するために使用される、請求項１０Ｂに記載されるキット。
項１２Ｂ
前記キメラ抗原受容体が生体試料中に存在する、請求項２Ｂ～９Ｂのいずれかに記載のプライマーセット又は請求項１０Ｂ若しくは１１Ｂに記載のキット。
項１３Ｂ
生体試料中のＣＤ２８及びＣＤ３ζを（同時／一緒に）発現するキメラ抗原受容体細胞を定量的に検出するための方法であって、
（１）ＣＤ２８及びＣＤ３ζを発現するキメラ抗原受容体細胞が投与された患者から、生体試料を採取する工程、
（２）生体試料からＤＮＡを抽出する工程、及び
（３）抽出したＤＮＡを鋳型として、配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーと、配列番号９の塩基配列を有するプローブを組み合わせて、定量ＰＣＲを行う工程、
を含む、キメラ抗原受容体細胞の検出方法。
項１４Ｂ
前記患者が多発性骨髄腫の治療を必要とする、請求項１３Ｂに記載の方法。
項１５Ｂ
キメラ抗原受容体細胞による治療を必要とする患者における、キメラ抗原受容体細胞による治療奏功性を評価方法であって、
（１）ＣＤ２８及びＣＤ３ζを発現するキメラ抗原受容体細胞が投与された患者から、生体試料を採取する工程、
（２）生体試料からＤＮＡを抽出する工程、及び
（３）抽出したＤＮＡを鋳型として、配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーと、配列番号９の塩基配列を有するプローブを組み合わせて、定量ＰＣＲを行い、生体試料中のキメラ抗原受容体細胞の量を測定する工程、
を含む、方法。
項１６Ｂ
項１５Ｂの評価方法による結果に基づいて、患者の治療方針を決定する方法。 Item 1B
A primer set comprising a primer having the base sequence of SEQ ID NO: 1 and a primer having the base sequence of SEQ ID NO: 2.
Item 2B
The primer set of Section 1B, wherein said primer set is used to detect a nucleic acid encoding a chimeric antigen receptor.
Item 3B
Item 2B, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence and the nucleotide sequence of SEQ ID NO: 2 or its complementary nucleotide sequence.
Item 4B
The primer set of paragraph 2B or 3B, wherein said chimeric antigen receptor comprises CD28 and CD3ζ.
Item 5B
Item 4B, wherein the CD28-encoding nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:5.
Item 6B
Item 4B or 5B, wherein the nucleotide sequence encoding CD3ζ comprises the nucleotide sequence of SEQ ID NO:6.
Item 7B
Claims 2B to 6B, wherein the detection is performed using quantitative PCR, a primer having the nucleotide sequence of SEQ ID NO: 1 is used as a forward primer, and a primer having the nucleotide sequence of SEQ ID NO: 2 is used as a reverse primer. A primer set according to any one of
Item 8B
The primer set of any of claims 2B-7B, wherein said chimeric antigen receptor is for an antigen against multiple myeloma.
Item 9B
The primer set according to any one of claims 2B to 8B, wherein said nucleic acid is contained in one or more cells selected from PBMCs, T cells, NK cells, and NK-T cells.
Item 10B
A kit for detecting a nucleic acid encoding a chimeric antigen receptor, comprising a primer set according to any one of claims 1B-9B.
Item 11B
The kit according to claim 10B, further comprising a probe having the base sequence of SEQ ID NO: 9 and used for detecting or quantifying a nucleic acid encoding a chimeric antigen receptor.
Item 12B
The primer set according to any of claims 2B-9B or the kit according to claims 10B or 11B, wherein said chimeric antigen receptor is present in a biological sample.
Item 13B
A method for the quantitative detection of chimeric antigen receptor cells expressing (simultaneously/together) CD28 and CD3ζ in a biological sample, comprising:
(1) collecting a biological sample from a patient to whom chimeric antigen receptor cells expressing CD28 and CD3ζ have been administered;
(2) a step of extracting DNA from a biological sample; and (3) using the extracted DNA as a template, a primer having the nucleotide sequence of SEQ ID NO: 1, a primer having the nucleotide sequence of SEQ ID NO: 2, and a nucleotide sequence of SEQ ID NO: 9. A step of performing quantitative PCR by combining probes having
A method for detecting chimeric antigen receptor cells, comprising:
Item 14B
13B. The method of claim 13B, wherein said patient is in need of treatment for multiple myeloma.
Item 15B
A method for evaluating efficacy of chimeric antigen receptor cell therapy in a patient in need of chimeric antigen receptor cell therapy, comprising:
(1) collecting a biological sample from a patient to whom chimeric antigen receptor cells expressing CD28 and CD3ζ have been administered;
(2) a step of extracting DNA from a biological sample; and (3) using the extracted DNA as a template, a primer having the nucleotide sequence of SEQ ID NO: 1, a primer having the nucleotide sequence of SEQ ID NO: 2, and a nucleotide sequence of SEQ ID NO: 9. and performing quantitative PCR to measure the amount of chimeric antigen receptor cells in the biological sample;
A method, including
Item 16B
A method of determining a treatment strategy for a patient based on the results of the evaluation method of Section 15B.
目的核酸の検出が可能となる。一実施形態において目的核酸はキメラ抗原受容体をコードする核酸である。一実施形態において、目的核酸は、ＣＤ２８及びＣＤ３ζが直接的又は非直接的に連結されたポリペプチドをコードする核酸である。
It is possible to detect the target nucleic acid. In one embodiment, the nucleic acid of interest is a nucleic acid encoding a chimeric antigen receptor. In one embodiment, the nucleic acid of interest is a nucleic acid encoding a polypeptide in which CD28 and CD3ζ are directly or indirectly linked.
配列番号１の塩基配列は、CCACCCGCAAGCATTACCであり、共刺激分子ＣＤ２８をコードする塩基配列の一部と一致する。よって、配列番号１の塩基配列を有するプライマーは、ＣＤ２８をコードするＤＮＡ鎖の相補鎖に結合（ハイブリダイズ）可能である。配列番号２の塩基配列はCGCTCCTGCTGAACTTCACTCTであり、ＣＤ３ζをコードする塩基配列の一部に相補的である。よって、配列番号２の塩基配列を有するプライマーは、ＣＤ３ζをコードするＤＮＡ鎖に結合可能である。配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーを組み合わせて用いることにより、ＣＤ２８とＣＤ３ζが連結されたポリペプチドをコードするＤＮＡ鎖を検出することができる。一実施形態において、配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーを組み合わせ（プライマーセット）を用いたＰＣＲにより、ＣＤ２８とＣＤ３ζが連結されたポリペプチドをコードするＤＮＡ鎖を増幅させ、増副産物の存在を指標に当該ＤＮＡ鎖を検出できる。
The nucleotide sequence of SEQ ID NO: 1 is CCACCCGCAAGCATTACC, which matches part of the nucleotide sequence encoding the co-stimulatory molecule CD28. Therefore, the primer having the base sequence of SEQ ID NO: 1 can bind (hybridize) to the complementary strand of the DNA strand encoding CD28. The base sequence of SEQ ID NO: 2 is CGCTCCTGCTGAACTTCACTCT, which is complementary to part of the base sequence encoding CD3ζ. Therefore, the primer having the base sequence of SEQ ID NO: 2 can bind to the DNA strand encoding CD3ζ. By using a primer having the nucleotide sequence of SEQ ID NO: 1 and a primer having the nucleotide sequence of SEQ ID NO: 2 in combination, a DNA chain encoding a polypeptide in which CD28 and CD3ζ are linked can be detected. In one embodiment, a DNA chain encoding a polypeptide in which CD28 and CD3ζ are linked by PCR using a combination (primer set) of a primer having the nucleotide sequence of SEQ ID NO: 1 and a primer having the nucleotide sequence of SEQ ID NO: 2. can be amplified, and the DNA strand can be detected using the presence of the amplification product as an index.
配列番号１の塩基配列を有するプライマーを構成するヌクレオチドの数は任意である。一実施形態において、配列番号１の塩基配列を有するプライマーを構成するヌクレオチドの数は、１５個以上２５個以下であり、１６個以上２５個以下が好ましく、１８個以上２０個以下が好ましい。配列番号１の塩基配列を有するプライマーは、全体として、ＣＤ２８をコードするＤＮＡ鎖の相補鎖に対して相補的であることが好ましい。配列番号２の塩基配列を有するプライマーを構成するヌクレオチドの数は任意である。一実施形態において、配列番号２の塩基配列を有するプライマーを構成するヌクレオチドの数は、１５個以上２５個以下であり、１８個以上２５個以下が好ましい。配列番号２の塩基配列を有するプライマーは、全体として、ＣＤ３ζをコードするＤＮＡ鎖に対して相補的であることが好ましい。本プライマーセットにおいては、ＰＣＲのための調製を容易にするためにも、構成するヌクレオチドの数は、それぞれ１８個以上２５個以下の範囲で設定することが好ましい。上記の範囲を下回るヌクレオチド数では、十分な特異性が示されず、バックグラウンドの上昇を含めた精度の低下をもたらす。その一方で、ヌクレオチド数が上回る場合は、設計コストの増加を含めた利用の困難性を高めることから、実用面で望ましいものではない。
The number of nucleotides constituting the primer having the base sequence of SEQ ID NO: 1 is arbitrary. In one embodiment, the number of nucleotides constituting the primer having the base sequence of SEQ ID NO: 1 is 15 or more and 25 or less, preferably 16 or more and 25 or less, and preferably 18 or more and 20 or less. The primer having the base sequence of SEQ ID NO: 1 is preferably complementary to the complementary strand of the DNA strand encoding CD28 as a whole. The number of nucleotides constituting the primer having the base sequence of SEQ ID NO:2 is arbitrary. In one embodiment, the number of nucleotides constituting the primer having the base sequence of SEQ ID NO: 2 is 15 or more and 25 or less, preferably 18 or more and 25 or less. The primer having the base sequence of SEQ ID NO: 2 is preferably complementary to the DNA strand encoding CD3zeta as a whole. In this primer set, the number of constituent nucleotides is preferably set in the range of 18 or more and 25 or less for easy preparation for PCR. Nucleotide numbers below the above range do not exhibit sufficient specificity and result in decreased precision, including increased background. On the other hand, when the number of nucleotides exceeds, it is not desirable from a practical point of view because it increases the difficulty of utilization including an increase in design cost.
本発明において、構成するヌクレオチド長としては、ＣＤ２８をコードするプライマー長と、ＣＤ３ζをコードするプライマー長を比較した場合、１：１～１．５の範囲に設定されるものであり、好ましくは１：１．１～１．３、更に好ましくは１：１．１５～１．２５の範囲で設計される。本ヌクレオチド長は、ＣＤ２８とＣＤ３ζが逆に配置される場合にも、認識するＤＮＡ鎖に応じて設定されるものである。具体的には、配列番号１の塩基配列を有するプライマーを構成するヌクレオチドの数が１５個の場合、配列番号２の塩基配列を有するプライマーを構成するヌクレオチド数は１５～２３個から選択されるものであり、１８個の場合は１８～２５個から選択され、２０個の場合は２０～２５個、２５個の場合は２５個となる。ただし、相補的ではない配列（塩基）を修飾等の目的で５‘末または３’末に付加する場合においては、この限りではない。
In the present invention, the length of the constituent nucleotides is set in the range of 1:1 to 1.5, preferably 1, when comparing the length of the primer encoding CD28 and the length of the primer encoding CD3ζ. : 1.1 to 1.3, more preferably 1:1.15 to 1.25. This nucleotide length is set according to the DNA strand to be recognized even when CD28 and CD3ζ are arranged in reverse. Specifically, when the number of nucleotides constituting the primer having the nucleotide sequence of SEQ ID NO: 1 is 15, the number of nucleotides constituting the primer having the nucleotide sequence of SEQ ID NO: 2 is selected from 15 to 23. , and when there are 18, it is selected from 18 to 25, when it is 20, it is 20 to 25, and when it is 25, it is 25. However, this does not apply when a non-complementary sequence (nucleotide) is added to the 5'-end or 3'-end for the purpose of modification or the like.
配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーは、人工的に化学合成または単離することによって作成することができる。
A primer having the nucleotide sequence of SEQ ID NO: 1 and a primer having the nucleotide sequence of SEQ ID NO: 2 can be created by artificial chemical synthesis or isolation.
上記プライマーセットが検出できる核酸（ＤＮＡ鎖）は、各プライマーが結合可能な領域を有する限り、任意である。一実施形態において、上記プライマーセットが検出できるＤＮＡ鎖は、配列番号１の塩基配列またはその相補的な塩基配列及び配列番号２の塩基配列またはその相補的な塩基配列を有することが好ましい。一実施形態において、そのようなＤＮＡ鎖は、ＣＤ２８及びＣＤ３ζを含むキメラ抗原受容体をコードするＤＮＡ鎖であり得る。キメラ抗原受容体は、ｓｃＦｖ、ＣＤ２８、及びＣＤ３ζを含むことが好ましく、これらがこの順で、必要に応じてリンカーを介して連結されていることが好ましく、ｓｃＦｖ－ＣＤ２８－ＣＤ３ζの順に構成されたＣＡＲコンストラクトを採用する場合、その検出には配列番号１の塩基配列及び配列番号２の塩基配列を有するプライマーをセットで用いることが好ましい。
The nucleic acids (DNA strands) that can be detected by the primer set are arbitrary as long as each primer has a binding region. In one embodiment, the DNA strand that can be detected by the primer set preferably has the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence and the nucleotide sequence of SEQ ID NO: 2 or its complementary nucleotide sequence. In one embodiment, such a DNA strand may be a DNA strand encoding a chimeric antigen receptor comprising CD28 and CD3ζ. The chimeric antigen receptor preferably comprises scFv, CD28, and CD3ζ, which are preferably linked in this order, optionally via a linker, and constructed in the order scFv-CD28-CD3ζ. When a CAR construct is employed, it is preferable to use a set of primers having the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2 for its detection.
上記ｓｃＦｖのアミノ酸配列及びそれをコードする塩基配列は任意である。一実施形態において、キメラ抗原受容体は、多発性骨髄腫に特異的であることが好ましい。そのようなキメラ抗原受容体は、多発性骨髄腫に特異的に結合するｓｃＦｖを有することが好ましい。このようなキメラ抗原受容体は、例えば、WO2017/026331に開示されている。一実施形態において、多発性骨髄腫に特異的に結合するｓｃＦｖを構成する軽鎖可変領域（ＶＬ）をコードするポリヌクレオチドは、下記の塩基配列を有することが好ましい。
CAAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAGGTTACATGCACTGGTTCCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGAGTCTGGGACCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTGACCCACCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG（配列番号３） The scFv amino acid sequence and the nucleotide sequence encoding it are arbitrary. In one embodiment, the chimeric antigen receptor is preferably specific for multiple myeloma. Such chimeric antigen receptors preferably have an scFv that specifically binds to multiple myeloma. Such chimeric antigen receptors are disclosed, for example, in WO2017/026331. In one embodiment, the polynucleotide encoding the light chain variable region (VL) that constitutes the scFv that specifically binds to multiple myeloma preferably has the following base sequence.
CAAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAGGTTACATGCACTGGTTCCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGAGTCTGGGACCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTGACCCACCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG（配列番号３）
CAAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAGGTTACATGCACTGGTTCCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGAGTCTGGGACCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTGACCCACCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG（配列番号３） The scFv amino acid sequence and the nucleotide sequence encoding it are arbitrary. In one embodiment, the chimeric antigen receptor is preferably specific for multiple myeloma. Such chimeric antigen receptors preferably have an scFv that specifically binds to multiple myeloma. Such chimeric antigen receptors are disclosed, for example, in WO2017/026331. In one embodiment, the polynucleotide encoding the light chain variable region (VL) that constitutes the scFv that specifically binds to multiple myeloma preferably has the following base sequence.
CAAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAGGTTACATGCACTGGTTCCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGAGTCTGGGACCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTGACCCACCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG（配列番号３）
一実施形態において、多発性骨髄腫に特異的に結合するｓｃＦｖを構成する重鎖可変領域（ＶＨ）をコードするポリヌクレオチドは、下記の塩基配列を有することが好ましい。
CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATGTTACCTGGAAGTGGTAGTTCTAACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGGGGGGATGGTAACTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA（配列番号４） In one embodiment, a polynucleotide encoding a heavy chain variable region (VH) that constitutes scFv that specifically binds to multiple myeloma preferably has the following base sequence.
CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATGTTACCTGGAAGTGGTAGTTCTAACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGGGGGGATGGTAACTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA（配列番号４）
CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATGTTACCTGGAAGTGGTAGTTCTAACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGGGGGGATGGTAACTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA（配列番号４） In one embodiment, a polynucleotide encoding a heavy chain variable region (VH) that constitutes scFv that specifically binds to multiple myeloma preferably has the following base sequence.
CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATGTTACCTGGAAGTGGTAGTTCTAACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGGGGGGATGGTAACTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA（配列番号４）
キメラ抗原受容体を構成するＣＤ２８のアミノ酸配列をコードする塩基配列は、配列番号１の塩基配列を含むことが好ましい。一実施形態において、ＣＤ２８のアミノ酸配列をコードする塩基配列は、下記の塩基配列を含むことが好ましい。
ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC（配列番号５）
上記配列における下線を引いた領域は配列番号１の塩基配列と同一である。 The nucleotide sequence encoding the amino acid sequence of CD28 that constitutes the chimeric antigen receptor preferably includes the nucleotide sequence of SEQ ID NO:1. In one embodiment, the nucleotide sequence encoding the CD28 amino acid sequence preferably includes the following nucleotide sequence.
ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC CCACCCGCAAGCATTACC AGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC（配列番号５）
The underlined region in the above sequence is identical to the base sequence of SEQ ID NO:1.
ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC（配列番号５）
上記配列における下線を引いた領域は配列番号１の塩基配列と同一である。 The nucleotide sequence encoding the amino acid sequence of CD28 that constitutes the chimeric antigen receptor preferably includes the nucleotide sequence of SEQ ID NO:1. In one embodiment, the nucleotide sequence encoding the CD28 amino acid sequence preferably includes the following nucleotide sequence.
ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC CCACCCGCAAGCATTACC AGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC（配列番号５）
The underlined region in the above sequence is identical to the base sequence of SEQ ID NO:1.
キメラ抗原受容体を構成するＣＤ３ζ（ＴＣＲζ、ＣＤ２４７）は、配列番号２の塩基配列に相補的な塩基配列によってコードされる領域を有することが好ましい。一実施形態において、ＣＤ３ζのアミノ酸配列をコードする塩基配列は、下記の塩基配列を含むことが好ましい。
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC（配列番号６）
上記配列における下線を引いた領域は配列番号２の塩基配列と相補的である。 CD3ζ (TCRζ, CD247) that constitutes the chimeric antigen receptor preferably has a region encoded by a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:2. In one embodiment, the nucleotide sequence encoding the CD3ζ amino acid sequence preferably includes the following nucleotide sequence.
AGAGTGAAGTTCAGCAGGAGCG CAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC（配列番号６）
The underlined region in the above sequence is complementary to the base sequence of SEQ ID NO:2.
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC（配列番号６）
上記配列における下線を引いた領域は配列番号２の塩基配列と相補的である。 CD3ζ (TCRζ, CD247) that constitutes the chimeric antigen receptor preferably has a region encoded by a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:2. In one embodiment, the nucleotide sequence encoding the CD3ζ amino acid sequence preferably includes the following nucleotide sequence.
AGAGTGAAGTTCAGCAGGAGCG CAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC（配列番号６）
The underlined region in the above sequence is complementary to the base sequence of SEQ ID NO:2.
ｓｃＦｖのＶＬとＶＨは任意のリンカーを介して連結し得る。一実施形態において、ＶＬとＶＨを連結するリンカーをコードする塩基配列はGGCTCCACTAGCGGTTCCGGCAAACCTGGCAGCGGAGAAGGCAGC（配列番号７）を含むことが好ましい。ＣＤ２８とＣＤ３ζは任意のリンカーを介して連結し得る。一実施形態において、ＣＤ２８とＣＤ３ζを連結するリンカーをコードする塩基配列はGCGGCCGCAを含むことが好ましい。
VL and VH of scFv can be linked via an arbitrary linker. In one embodiment, the nucleotide sequence encoding the linker connecting VL and VH preferably includes GGCTCCACTAGCGGTTCCGGCAAACCTGGCAGCGGAGAAGGCAGC (SEQ ID NO: 7). CD28 and CD3ζ can be linked via any linker. In one embodiment, the nucleotide sequence encoding the linker that connects CD28 and CD3ζ preferably contains GCGGGCCGCA.
キメラ抗原受容体をコードするポリヌクレオチドは、ｓｃＦｖをコードする領域よりもＮ末端側に更にシグナルペプチドをコードする領域を有していることが好ましい。シグナルペプチドをコードする塩基配列は任意であるが、ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCTTCAGTCATAATGTCCAGAGGA（配列番号８）を含むことが好ましい。
The polynucleotide encoding the chimeric antigen receptor preferably has a region encoding a signal peptide on the N-terminal side of the region encoding the scFv. The nucleotide sequence encoding the signal peptide is arbitrary, but preferably includes ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCTTCAGTCATAATGTCCAGAGGA (SEQ ID NO: 8).
一実施形態において、シグナルペプチド、ｓｃＦｖ、ＣＤ２８及びＣＤ３ζを含むキメラ抗原受容体をコードするポリヌクレオチドは、WO2017/026331または、Nature Medicine 23 12 2007 1436に記載されるものであり得る。キメラ抗原受容体、それをコードする核酸、及び当該核酸を含む（又は発現する）Ｔ細胞は、公知の手法で作成することができる。
In one embodiment, a polynucleotide encoding a chimeric antigen receptor comprising a signal peptide, scFv, CD28 and CD3ζ can be those described in WO2017/026331 or Nature Medicine 23 12 2007 1436. Chimeric antigen receptors, nucleic acids encoding them, and T cells containing (or expressing) the nucleic acids can be prepared by known techniques.
上記プライマーセットで検出する核酸は、一本鎖であっても二本鎖であってもよい。一実施形態において、核酸は二本鎖であることが好ましい。核酸が二本鎖である場合、二本鎖を構成する一本の鎖が配列番号１の塩基配列と相補的な塩基配列を有し、他方の鎖が配列番号２の塩基配列と相補的な塩基配列を有することが好ましい。上記プライマーセットで検出する核酸は、ＤＮＡでもＲＮＡでもよく、一実施形態において核酸はＤＮＡであることが好ましい。
The nucleic acids detected by the primer set may be either single-stranded or double-stranded. In one embodiment, it is preferred that the nucleic acid is double-stranded. When the nucleic acid is double-stranded, one strand constituting the double strand has a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, and the other strand has a complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO: 2. It preferably has a base sequence. The nucleic acid detected by the primer set may be DNA or RNA, and in one embodiment, the nucleic acid is preferably DNA.
上記プライマーセットを用いた目的核酸の検出は、ＰＣＲを用いて行うことが好ましい。ＰＣＲにおいて、配列番号１の塩基配列を有するプライマーは、フォワードプライマーとして使用されることが好ましく、配列番号２の塩基配列を有するプライマーはリバースプライマーとして使用されることが好ましい。一実施形態において、ＰＣＲは定性的に利用することも可能であるが、定量ＰＣＲであることが好ましい。定量ＰＣＲ法は、リアルタイムＰＣＲとも呼ばれ、一般的に、ＰＣＲにおいて増幅産物の生成過程を経時的にモニタリングする方法である。定量ＰＣＲを用いた検出では、通常、ＰＣＲ反応による増幅産物を光学的手段により検出する。増幅産物の検出は、ＰＣＲ反応終了時または終了後に行ってもよいし、ＰＣＲ反応工程と並行して行ってもよい。並行して行う場合、増幅産物の検出は、例えば、経時的に行うことができる。経時的な検出（モニタリング）は、例えば、連続的であっても非連続的（断続的）であってもよい。また、増幅産物が所定量となったＰＣＲサイクル数をカウントすることによって、試料中に含まれる標的核酸を定量することができる。更に、標準品から算出された検量線から試料中のコピー数を定量することもできる。
The detection of the target nucleic acid using the primer set is preferably performed using PCR. In PCR, the primer having the nucleotide sequence of SEQ ID NO: 1 is preferably used as a forward primer, and the primer having the nucleotide sequence of SEQ ID NO: 2 is preferably used as a reverse primer. In one embodiment, quantitative PCR is preferred, although PCR can be used qualitatively. Quantitative PCR, also called real-time PCR, is generally a method of temporally monitoring the process of generating amplified products in PCR. In the detection using quantitative PCR, the amplification product by PCR reaction is usually detected by optical means. The detection of the amplified product may be performed at or after the completion of the PCR reaction, or may be performed in parallel with the PCR reaction process. When performed in parallel, the detection of amplification products can be performed over time, for example. Detection (monitoring) over time can be, for example, continuous or discontinuous (intermittent). Also, by counting the number of PCR cycles at which the amplification product reaches a predetermined amount, the target nucleic acid contained in the sample can be quantified. Furthermore, it is also possible to quantify the copy number in a sample from a calibration curve calculated from standards.
一実施形態において、上記プライマーセットを用いて検出又は定量される核酸は、細胞内に存在するものであることが好ましい。核酸の形態は任意であり、例えば、プラスミドに組み込まれた状態であっても、ゲノムＤＮＡに組み込まれた状態であってもよい。当該核酸を有する細胞の種類は任意であるが、例えば、ＰＢＭＣ、Ｔ細胞、ＮＫ細胞、及びＮＫ－Ｔ細胞から選択される一種以上であることが好ましい。当該核酸又は当該核酸を有する細胞は、生体試料中に存在するものであることが好ましい。生体試料の種類は、任意であるが、血液（例えば、全血）であることが好ましい。
In one embodiment, the nucleic acid detected or quantified using the primer set is preferably present in cells. The form of the nucleic acid is arbitrary, and for example, it may be in a state of being incorporated into a plasmid or into a state of being incorporated into genomic DNA. Cells containing the nucleic acid may be of any type, but are preferably one or more selected from, for example, PBMCs, T cells, NK cells, and NK-T cells. The nucleic acid or cells having the nucleic acid are preferably present in a biological sample. The biological sample may be of any type, but is preferably blood (for example, whole blood).
一実施形態において、目的核酸の検出は、上記プライマーセットを用いたＰＣＲで増幅させた産物を特異的に認識する任意のプローブを用いて行うことが好ましい。そのようなプローブとしては、配列番号１の塩基配列を有するプライマーと、配列番号２の塩基配列を有するプライマーの間に存在する配列であれば、特に限定されるものではないが、例えば、CACCACGCGACTTCGCAGCCTAT（配列番号９）又はこれと相補的な塩基配列を有するオリゴヌクレオチドを上げることができる。プローブは、任意の手法（例えば、化学合成）で作成することができる。蛍光プローブに関しては、適切な市販のものを選択して利用することが可能である。例えば、蛍光プローブとしては、フルオレセイン（FAM）などを用いることができるが、これに限定されるものではない。その他、プローブには非蛍光クエンチャ―（NFQ）や、マイナーグルーブ結合（MGB）を付加することができる。
In one embodiment, the target nucleic acid is preferably detected using any probe that specifically recognizes the product amplified by PCR using the primer set. Such a probe is not particularly limited as long as it is a sequence present between the primer having the nucleotide sequence of SEQ ID NO: 1 and the primer having the nucleotide sequence of SEQ ID NO: 2. For example, CACCACGCGACTTCGCAGCCTAT ( SEQ ID NO: 9) or an oligonucleotide having a base sequence complementary thereto. Probes can be made by any technique (eg, chemical synthesis). Regarding the fluorescent probe, it is possible to select and use an appropriate commercially available one. For example, fluorescein (FAM) can be used as a fluorescent probe, but it is not limited to this. In addition, non-fluorescent quenchers (NFQ) and minor groove binding (MGB) can be added to the probe.
配列番号９の塩基配列を有するプローブを用いる場合、構成するヌクレオチドの数は任意である。一実施形態において、配列番号９の塩基配列を有するプローブを構成するヌクレオチドの数は、２０個～２５個以下であることが好ましい。本発明において、配列番号１の塩基配列を有するプライマーおよび配列番号２の塩基配列を有するプライマーと共に当該プローブを使用する場合、それぞれのプライマーとプローブとの間の塩基配列の距離は、５個～２５個のヌクレオチドの範囲で設定され、５個～１５個のヌクレオチド以下であることが好ましい。２５個を超す距離がある場合、反応時間の増加等を生じたり、その一方、５個より少ない場合には、プローブの蛍光の程度に影響を及ぼす可能性がある。
When using a probe having the nucleotide sequence of SEQ ID NO: 9, the number of constituent nucleotides is arbitrary. In one embodiment, the number of nucleotides constituting the probe having the base sequence of SEQ ID NO:9 is preferably 20 to 25 or less. In the present invention, when the probe is used together with a primer having the base sequence of SEQ ID NO: 1 and a primer having the base sequence of SEQ ID NO: 2, the base sequence distance between each primer and the probe is 5 to 25. It is set in the range of 1 nucleotide, preferably 5 to 15 nucleotides or less. More than 25 distances may result in increased reaction times, etc., while less than 5 may affect the degree of fluorescence of the probe.
キメラ抗原受容体をコードする核酸を検出するためのキットは、上記プライマーセットを含むことが好ましい。キットは、上記プライマーセットの他に任意の成分、試薬（例えば、ＰＣＲ試薬）、容器、機器等を含み得る。一実施形態において、キットは上記プローブを更に含むことが好ましい。ＰＣＲ試薬としては、例えば、ポリメラーゼ、dNTP（デオキシヌクレオシド三リン酸）、インターカレーター又は蛍光標識プローブ、バッファーなどを挙げることができる。
A kit for detecting a nucleic acid encoding a chimeric antigen receptor preferably contains the primer set. The kit may contain optional components, reagents (eg, PCR reagents), containers, equipment, etc., in addition to the primer set. In one embodiment, the kit preferably further comprises the probes described above. Examples of PCR reagents include polymerases, dNTPs (deoxynucleoside triphosphates), intercalators or fluorescence-labeled probes, and buffers.
一実施形態において、上記プライマーセットを用いた定量ＰＣＲにて、ヒトの生体試料（例えば、血液）中のキメラ抗原受容体を検出し、その量を定量することができる。具体的には、（１）ＣＤ２８及びＣＤ３ζを発現するキメラ抗原受容体細胞が投与された患者から、生体試料を採取する工程、（２）生体試料からＤＮＡを抽出する工程、及び
（３）抽出したＤＮＡを鋳型として、配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーと、配列番号９の塩基配列を有するプローブを組み合わせて、定量ＰＣＲを行う工程、を含む方法によって、生体試料中のキメラ抗原受容体をコードする核酸を定量的に検出し、それに基づいて、生体試料中のキメラ抗原受容体細胞の量を測定することができる。よって、ＣＡＲ－Ｔ細胞療法において、ＣＡＲ－Ｔ細胞を投与後に被検体の生体試料を採取し、それを試料としてＣＡＲをコードする核酸の量を測定することにより、投与されたＣＡＲ－Ｔ細胞の動態（増減）を評価し、更にはＣＡＲ－Ｔ細胞療法の奏功性を評価することができる。また、評価結果を、薬効予測、薬剤投与の妥当性など、患者のその後の治療方針を決定に役立てることができる。 In one embodiment, a chimeric antigen receptor in a human biological sample (eg, blood) can be detected and its amount can be quantified by quantitative PCR using the primer set. Specifically, (1) a step of collecting a biological sample from a patient to whom chimeric antigen receptor cells expressing CD28 and CD3ζ were administered, (2) a step of extracting DNA from the biological sample, and (3) extraction. Using the obtained DNA as a template, a primer having the nucleotide sequence of SEQ ID NO: 1, a primer having the nucleotide sequence of SEQ ID NO: 2, and a probe having the nucleotide sequence of SEQ ID NO: 9 are combined to perform quantitative PCR. , the nucleic acid encoding the chimeric antigen receptor in the biological sample can be quantitatively detected, and based thereon, the amount of chimeric antigen receptor cells in the biological sample can be determined. Therefore, in CAR-T cell therapy, a biological sample is collected from a subject after administration of CAR-T cells, and is used as a sample to measure the amount of nucleic acid encoding CAR. The kinetics (increase or decrease) can be assessed and also the efficacy of CAR-T cell therapy can be assessed. In addition, the evaluation results can be used to determine subsequent treatment strategies for patients, such as prediction of drug efficacy and appropriateness of drug administration.
（３）抽出したＤＮＡを鋳型として、配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーと、配列番号９の塩基配列を有するプローブを組み合わせて、定量ＰＣＲを行う工程、を含む方法によって、生体試料中のキメラ抗原受容体をコードする核酸を定量的に検出し、それに基づいて、生体試料中のキメラ抗原受容体細胞の量を測定することができる。よって、ＣＡＲ－Ｔ細胞療法において、ＣＡＲ－Ｔ細胞を投与後に被検体の生体試料を採取し、それを試料としてＣＡＲをコードする核酸の量を測定することにより、投与されたＣＡＲ－Ｔ細胞の動態（増減）を評価し、更にはＣＡＲ－Ｔ細胞療法の奏功性を評価することができる。また、評価結果を、薬効予測、薬剤投与の妥当性など、患者のその後の治療方針を決定に役立てることができる。 In one embodiment, a chimeric antigen receptor in a human biological sample (eg, blood) can be detected and its amount can be quantified by quantitative PCR using the primer set. Specifically, (1) a step of collecting a biological sample from a patient to whom chimeric antigen receptor cells expressing CD28 and CD3ζ were administered, (2) a step of extracting DNA from the biological sample, and (3) extraction. Using the obtained DNA as a template, a primer having the nucleotide sequence of SEQ ID NO: 1, a primer having the nucleotide sequence of SEQ ID NO: 2, and a probe having the nucleotide sequence of SEQ ID NO: 9 are combined to perform quantitative PCR. , the nucleic acid encoding the chimeric antigen receptor in the biological sample can be quantitatively detected, and based thereon, the amount of chimeric antigen receptor cells in the biological sample can be determined. Therefore, in CAR-T cell therapy, a biological sample is collected from a subject after administration of CAR-T cells, and is used as a sample to measure the amount of nucleic acid encoding CAR. The kinetics (increase or decrease) can be assessed and also the efficacy of CAR-T cell therapy can be assessed. In addition, the evaluation results can be used to determine subsequent treatment strategies for patients, such as prediction of drug efficacy and appropriateness of drug administration.
ＣＡＲ－Ｔ細胞療法を必要とする患者は、例えば、癌を患う患者であり、癌は、例えば、血液癌又は固形癌であり得る。血液癌は、例えば、リンパ腫、白血病、又は骨髄腫であり得る。一実施形態において、ＣＡＲ－Ｔ細胞療法を必要とする患者は、多発性骨髄腫を患うヒトである。
A patient in need of CAR-T cell therapy is, for example, a patient with cancer, and the cancer can be, for example, blood cancer or solid cancer. A hematologic cancer can be, for example, lymphoma, leukemia, or myeloma. In one embodiment, the patient in need of CAR-T cell therapy is a human with multiple myeloma.
実施例１
１．ＣＡＲ－Ｔ細胞由来gDNA抽出
下記の手順でＣＡＲ－Ｔ細胞から検量線作成用のＣＡＲ－Ｔ細胞由来ゲノムＤＮＡを抽出する。この操作は、市販されるゲノムＤＮＡ抽出キット（例えば、DNeasy Blood & Tissue Kit）を用いて実施することができ、例えば下記の手順が例示される。なお、実施例で使用されるＣＡＲ－Ｔ細胞は、ａＩＴＧＢ７ ＣＡＲ－Ｔ細胞であり、対照物質は、非遺伝子導入Ｔ細胞（タカラバイオ株式会社）（以下、ＮＧＭＣとも称する）である。導入するａＩＴＧＢ７ ＣＡＲは、大阪大学より入手した。ａＩＴＧＢ７ ＣＡＲは、抗インテグリンβ７のｓｃＦＶ－ＣＤ２８－ＣＤ３ζを採用している。当該ａＩＴＧＢ７ ＣＡＲ－Ｔ細胞は、配列番号４～８の塩基配列を含む。
(1) ＣＡＲ－Ｔ細胞を37℃の恒温水槽内で解凍する。
(2) 1 × 107 cellsを分取する。
(3) 遠心分離（2000×g、5分、室温）後、上清を除去する。その後、タッピングを行い、PBS 400 μLを添加・混合し、Proteinase K 40 μL を添加する。
(4) 8 μLのRNase A（100 mg/mL）を加えて混和し、室温で約2分間インキュベートする。
(5) 400 μLのBuffer ALを添加・混合した後、56℃で10分間インキュベートする。
(6) インキュベート後、上記(5)の液を2 本に分ける（5 × 106 cells/424 μL）。
(7) 上記(6)の液それぞれに、200 μLのエタノールを添加・混合する。
(8) DNeasy mini spin column（以下、スピンカラム）にそれぞれの液を添加し、遠心分離（6000×g、1 分、室温）する。
(9) 遠心分離後、フロースルーを除去し、スピンカラムを新しいコレクションチューブにセットする。
(10) 500 μLのBuffer AW1をスピンカラムに添加し、遠心分離（6000×g、1分、室温）する。
(11) 遠心分離後、フロースルーを除去し、スピンカラムを新しいコレクションチューブにセットする。
(12) 500 μLのBuffer AW2をスピンカラムに添加し、遠心分離（20000×g、3分、室温）する。
(13) フロースルーを除去した後、再度遠心分離（20000×g、1分、室温）し、フロースルーを除去する。
(14) スピンカラムを新しいコレクションチューブにセットした後、50 μLのBuffer AEを添加する。
(15) 室温で約1分間静置した後、遠心分離（6000×g、1分、室温）し、溶出する。
(16) 溶出液を1つのチューブにまとめ、gDNA濃度をNanoDrop（サーモフィッシャー）等の分光光度計で測定する。
(17) ＣＡＲ－Ｔ細胞由来gDNA 濃度が50000 pg/μLになるようにBuffer AEで希釈した（検量線ストック溶液、S-1）後、分注し、超低温フリーザーで冷凍保存する（許容範囲：-90～-65℃）。 Example 1
1. Extraction of CAR-T Cell-Derived gDNA Genomic DNA derived from CAR-T cells for preparing a standard curve is extracted from CAR-T cells according to the following procedure. This operation can be performed using a commercially available genomic DNA extraction kit (eg, DNeasy Blood & Tissue Kit), and the following procedure is exemplified. The CAR-T cells used in the examples were aITGB7 CAR-T cells, and the control substance was non-transgenic T cells (Takara Bio Inc.) (hereinafter also referred to as NGMC). The aITGB7 CAR to be introduced was obtained from Osaka University. The aITGB7 CAR employs the anti-integrin β7 scFV-CD28-CD3ζ. The aITGB7 CAR-T cells contain the nucleotide sequences of SEQ ID NOS:4-8.
(1) Thaw CAR-T cells in a constant temperature water bath at 37°C.
(2) Collect 1×10 7 cells.
(3) Remove the supernatant after centrifugation (2000×g, 5 minutes, room temperature). Then, perform tapping, add and mix 400 μL of PBS, and add 40 μL of Proteinase K.
(4) Add 8 μL of RNase A (100 mg/mL), mix, and incubate at room temperature for about 2 minutes.
(5) Add 400 μL of Buffer AL, mix, and incubate at 56℃ for 10 minutes.
(6) After incubation, divide the solution of (5) above into two (5 × 10 6 cells/424 μL).
(7) Add 200 µL of ethanol to each of the solutions in (6) above and mix.
(8) Add each solution to a DNeasy mini spin column (hereafter referred to as spin column) and centrifuge (6000×g, 1 minute, room temperature).
(9) After centrifugation, remove the flow-through and set the spin column in a new collection tube.
(10) Add 500 μL of Buffer AW1 to the spin column and centrifuge (6000×g, 1 minute, room temperature).
(11) After centrifugation, remove the flow-through and set the spin column in a new collection tube.
(12) Add 500 μL of Buffer AW2 to the spin column and centrifuge (20000×g, 3 minutes, room temperature).
(13) After removing the flow-through, centrifuge again (20000×g, 1 minute, room temperature) to remove the flow-through.
(14) After setting the spin column in a new collection tube, add 50 μL of Buffer AE.
(15) After standing at room temperature for about 1 minute, centrifuge (6000×g, 1 minute, room temperature) to elute.
(16) Collect the eluate in one tube and measure the gDNA concentration with a spectrophotometer such as NanoDrop (Thermo Fisher).
(17) After diluting CAR-T cell-derived gDNA with Buffer AE so that the concentration is 50000 pg/μL (calibration curve stock solution, S-1), aliquot and store frozen in an ultra-low temperature freezer (allowable range: -90 to -65°C).
１．ＣＡＲ－Ｔ細胞由来gDNA抽出
下記の手順でＣＡＲ－Ｔ細胞から検量線作成用のＣＡＲ－Ｔ細胞由来ゲノムＤＮＡを抽出する。この操作は、市販されるゲノムＤＮＡ抽出キット（例えば、DNeasy Blood & Tissue Kit）を用いて実施することができ、例えば下記の手順が例示される。なお、実施例で使用されるＣＡＲ－Ｔ細胞は、ａＩＴＧＢ７ ＣＡＲ－Ｔ細胞であり、対照物質は、非遺伝子導入Ｔ細胞（タカラバイオ株式会社）（以下、ＮＧＭＣとも称する）である。導入するａＩＴＧＢ７ ＣＡＲは、大阪大学より入手した。ａＩＴＧＢ７ ＣＡＲは、抗インテグリンβ７のｓｃＦＶ－ＣＤ２８－ＣＤ３ζを採用している。当該ａＩＴＧＢ７ ＣＡＲ－Ｔ細胞は、配列番号４～８の塩基配列を含む。
(1) ＣＡＲ－Ｔ細胞を37℃の恒温水槽内で解凍する。
(2) 1 × 107 cellsを分取する。
(3) 遠心分離（2000×g、5分、室温）後、上清を除去する。その後、タッピングを行い、PBS 400 μLを添加・混合し、Proteinase K 40 μL を添加する。
(4) 8 μLのRNase A（100 mg/mL）を加えて混和し、室温で約2分間インキュベートする。
(5) 400 μLのBuffer ALを添加・混合した後、56℃で10分間インキュベートする。
(6) インキュベート後、上記(5)の液を2 本に分ける（5 × 106 cells/424 μL）。
(7) 上記(6)の液それぞれに、200 μLのエタノールを添加・混合する。
(8) DNeasy mini spin column（以下、スピンカラム）にそれぞれの液を添加し、遠心分離（6000×g、1 分、室温）する。
(9) 遠心分離後、フロースルーを除去し、スピンカラムを新しいコレクションチューブにセットする。
(10) 500 μLのBuffer AW1をスピンカラムに添加し、遠心分離（6000×g、1分、室温）する。
(11) 遠心分離後、フロースルーを除去し、スピンカラムを新しいコレクションチューブにセットする。
(12) 500 μLのBuffer AW2をスピンカラムに添加し、遠心分離（20000×g、3分、室温）する。
(13) フロースルーを除去した後、再度遠心分離（20000×g、1分、室温）し、フロースルーを除去する。
(14) スピンカラムを新しいコレクションチューブにセットした後、50 μLのBuffer AEを添加する。
(15) 室温で約1分間静置した後、遠心分離（6000×g、1分、室温）し、溶出する。
(16) 溶出液を1つのチューブにまとめ、gDNA濃度をNanoDrop（サーモフィッシャー）等の分光光度計で測定する。
(17) ＣＡＲ－Ｔ細胞由来gDNA 濃度が50000 pg/μLになるようにBuffer AEで希釈した（検量線ストック溶液、S-1）後、分注し、超低温フリーザーで冷凍保存する（許容範囲：-90～-65℃）。 Example 1
1. Extraction of CAR-T Cell-Derived gDNA Genomic DNA derived from CAR-T cells for preparing a standard curve is extracted from CAR-T cells according to the following procedure. This operation can be performed using a commercially available genomic DNA extraction kit (eg, DNeasy Blood & Tissue Kit), and the following procedure is exemplified. The CAR-T cells used in the examples were aITGB7 CAR-T cells, and the control substance was non-transgenic T cells (Takara Bio Inc.) (hereinafter also referred to as NGMC). The aITGB7 CAR to be introduced was obtained from Osaka University. The aITGB7 CAR employs the anti-integrin β7 scFV-CD28-CD3ζ. The aITGB7 CAR-T cells contain the nucleotide sequences of SEQ ID NOS:4-8.
(1) Thaw CAR-T cells in a constant temperature water bath at 37°C.
(2) Collect 1×10 7 cells.
(3) Remove the supernatant after centrifugation (2000×g, 5 minutes, room temperature). Then, perform tapping, add and mix 400 μL of PBS, and add 40 μL of Proteinase K.
(4) Add 8 μL of RNase A (100 mg/mL), mix, and incubate at room temperature for about 2 minutes.
(5) Add 400 μL of Buffer AL, mix, and incubate at 56℃ for 10 minutes.
(6) After incubation, divide the solution of (5) above into two (5 × 10 6 cells/424 μL).
(7) Add 200 µL of ethanol to each of the solutions in (6) above and mix.
(8) Add each solution to a DNeasy mini spin column (hereafter referred to as spin column) and centrifuge (6000×g, 1 minute, room temperature).
(9) After centrifugation, remove the flow-through and set the spin column in a new collection tube.
(10) Add 500 μL of Buffer AW1 to the spin column and centrifuge (6000×g, 1 minute, room temperature).
(11) After centrifugation, remove the flow-through and set the spin column in a new collection tube.
(12) Add 500 μL of Buffer AW2 to the spin column and centrifuge (20000×g, 3 minutes, room temperature).
(13) After removing the flow-through, centrifuge again (20000×g, 1 minute, room temperature) to remove the flow-through.
(14) After setting the spin column in a new collection tube, add 50 μL of Buffer AE.
(15) After standing at room temperature for about 1 minute, centrifuge (6000×g, 1 minute, room temperature) to elute.
(16) Collect the eluate in one tube and measure the gDNA concentration with a spectrophotometer such as NanoDrop (Thermo Fisher).
(17) After diluting CAR-T cell-derived gDNA with Buffer AE so that the concentration is 50000 pg/μL (calibration curve stock solution, S-1), aliquot and store frozen in an ultra-low temperature freezer (allowable range: -90 to -65°C).
２．血液試料からのgDNA抽出
下記の手順で血液試料からゲノムＤＮＡを抽出する。この操作は、市販されるゲノムＤＮＡ抽出キット（例えば、DNeasy Blood & Tissue Kit）を用いて実施することができ、例えば、下記の手順が例示される。
(1) 融解した抗凝固剤処理血液を100 μL分取する。これに20 μLのProteinase K を添加し、さらに100 μLのPBSを添加する。この液を1個体サンプルにつき、3本調製する（No.1～No.3）。
(2) 4 μLのRNase A（100 mg/mL）を加えて混和し、室温で約2分間インキュベートする。
(3) 200 μLのBuffer ALを添加・混和した後、56℃で10分間インキュベートする。インキュベート後、200 μLのエタノールを添加・混和する。
(4) スピンカラムに混和液を添加し、遠心分離（6000×g、1分、室温）する。目詰まりした場合、追加遠心操作をする。
(5) 遠心分離後、フロースルーを除去し、スピンカラムを新しいコレクションチューブにセットする。
(6) 500 μLのBuffer AW1をスピンカラムに添加し、遠心分離（6000×g、1分、室温）する。
(7) 遠心分離後、フロースルーを除去し、スピンカラムを新しいコレクションチューブにセットする。
(8) 500 μLのBuffer AW2をスピンカラムに添加し、遠心分離（20000×g、3分、室温）する。
(9) フロースルーを除去した後、再度遠心分離（20000×g、1分、室温）し、フロースルーを除去する。
(10) スピンカラムを新しいコレクションチューブにセットした後、No.1のサンプルに50 μLのBuffer AEを添加する。
(11) 室温で約1分間静置した後、遠心分離（20000×g、1分、室温）し、溶出する。
(12) No.1の溶出液を全量採取し、No.2のサンプルに添加する。
(13) 室温で約1分間静置した後、遠心分離（20000×g、1分、室温）し、溶出する。
(14) No.2の溶出液を全量採取し、No.3のサンプルに添加する。
(15) 室温で約1分間静置した後、遠心（20000×g、1分、室温）し、溶出する。
(16) 溶出液のgDNA濃度をNanoDrop等の分光光度計で測定する。
(17) マトリクス効果が認められないことを確認した1ウェルあたりのgDNA量（0.0696 μg）以下になるように、溶出液をBuffer AEで希釈する。溶出液のgDNA量が0.0696 μg未満となった場合は、最低希釈倍率（5倍）を適用する。
(18) 希釈後の溶出液を測定試料とし、分注後、超低温フリーザーにて冷凍保存（許容範囲：-90～-65℃）する。 2. Extraction of gDNA from Blood Samples Genomic DNA is extracted from blood samples according to the following procedure. This operation can be performed using a commercially available genomic DNA extraction kit (eg, DNeasy Blood & Tissue Kit), and the following procedure is exemplified, for example.
(1) Take 100 μL of thawed anticoagulant-treated blood. Add 20 μL of Proteinase K to this, and then add 100 μL of PBS. Prepare 3 bottles of this liquid for each individual sample (No. 1 to No. 3).
(2) Add 4 μL of RNase A (100 mg/mL), mix, and incubate at room temperature for about 2 minutes.
(3) Add 200 μL of Buffer AL, mix, and incubate at 56℃ for 10 minutes. After incubation, add 200 µL of ethanol and mix.
(4) Add the mixture to the spin column and centrifuge (6000×g, 1 minute, room temperature). If clogged, perform additional centrifugation.
(5) After centrifugation, remove the flow-through and set the spin column in a new collection tube.
(6) Add 500 μL of Buffer AW1 to the spin column and centrifuge (6000×g, 1 minute, room temperature).
(7) After centrifugation, remove the flow-through and set the spin column in a new collection tube.
(8) Add 500 μL of Buffer AW2 to the spin column and centrifuge (20000×g, 3 minutes, room temperature).
(9) After removing the flow-through, centrifuge again (20000×g, 1 minute, room temperature) to remove the flow-through.
(10) After setting the spin column in a new collection tube, add 50 μL of Buffer AE to No.1 sample.
(11) After standing at room temperature for about 1 minute, centrifuge (20000×g, 1 minute, room temperature) to elute.
(12) Collect the entire volume of No.1 eluate and add to No.2 sample.
(13) After standing at room temperature for about 1 minute, centrifuge (20000×g, 1 minute, room temperature) to elute.
(14) Collect the entire volume of No.2 eluate and add to No.3 sample.
(15) After standing at room temperature for about 1 minute, centrifuge (20000×g, 1 minute, room temperature) to elute.
(16) Measure the gDNA concentration of the eluate with a spectrophotometer such as NanoDrop.
(17) Dilute the eluate with Buffer AE so that the amount of gDNA per well (0.0696 μg) or less was confirmed to have no matrix effect. If the amount of gDNA in the eluate is less than 0.0696 µg, apply the lowest dilution factor (5x).
(18) Use the diluted eluate as the measurement sample.
下記の手順で血液試料からゲノムＤＮＡを抽出する。この操作は、市販されるゲノムＤＮＡ抽出キット（例えば、DNeasy Blood & Tissue Kit）を用いて実施することができ、例えば、下記の手順が例示される。
(1) 融解した抗凝固剤処理血液を100 μL分取する。これに20 μLのProteinase K を添加し、さらに100 μLのPBSを添加する。この液を1個体サンプルにつき、3本調製する（No.1～No.3）。
(2) 4 μLのRNase A（100 mg/mL）を加えて混和し、室温で約2分間インキュベートする。
(3) 200 μLのBuffer ALを添加・混和した後、56℃で10分間インキュベートする。インキュベート後、200 μLのエタノールを添加・混和する。
(4) スピンカラムに混和液を添加し、遠心分離（6000×g、1分、室温）する。目詰まりした場合、追加遠心操作をする。
(5) 遠心分離後、フロースルーを除去し、スピンカラムを新しいコレクションチューブにセットする。
(6) 500 μLのBuffer AW1をスピンカラムに添加し、遠心分離（6000×g、1分、室温）する。
(7) 遠心分離後、フロースルーを除去し、スピンカラムを新しいコレクションチューブにセットする。
(8) 500 μLのBuffer AW2をスピンカラムに添加し、遠心分離（20000×g、3分、室温）する。
(9) フロースルーを除去した後、再度遠心分離（20000×g、1分、室温）し、フロースルーを除去する。
(10) スピンカラムを新しいコレクションチューブにセットした後、No.1のサンプルに50 μLのBuffer AEを添加する。
(11) 室温で約1分間静置した後、遠心分離（20000×g、1分、室温）し、溶出する。
(12) No.1の溶出液を全量採取し、No.2のサンプルに添加する。
(13) 室温で約1分間静置した後、遠心分離（20000×g、1分、室温）し、溶出する。
(14) No.2の溶出液を全量採取し、No.3のサンプルに添加する。
(15) 室温で約1分間静置した後、遠心（20000×g、1分、室温）し、溶出する。
(16) 溶出液のgDNA濃度をNanoDrop等の分光光度計で測定する。
(17) マトリクス効果が認められないことを確認した1ウェルあたりのgDNA量（0.0696 μg）以下になるように、溶出液をBuffer AEで希釈する。溶出液のgDNA量が0.0696 μg未満となった場合は、最低希釈倍率（5倍）を適用する。
(18) 希釈後の溶出液を測定試料とし、分注後、超低温フリーザーにて冷凍保存（許容範囲：-90～-65℃）する。 2. Extraction of gDNA from Blood Samples Genomic DNA is extracted from blood samples according to the following procedure. This operation can be performed using a commercially available genomic DNA extraction kit (eg, DNeasy Blood & Tissue Kit), and the following procedure is exemplified, for example.
(1) Take 100 μL of thawed anticoagulant-treated blood. Add 20 μL of Proteinase K to this, and then add 100 μL of PBS. Prepare 3 bottles of this liquid for each individual sample (No. 1 to No. 3).
(2) Add 4 μL of RNase A (100 mg/mL), mix, and incubate at room temperature for about 2 minutes.
(3) Add 200 μL of Buffer AL, mix, and incubate at 56℃ for 10 minutes. After incubation, add 200 µL of ethanol and mix.
(4) Add the mixture to the spin column and centrifuge (6000×g, 1 minute, room temperature). If clogged, perform additional centrifugation.
(5) After centrifugation, remove the flow-through and set the spin column in a new collection tube.
(6) Add 500 μL of Buffer AW1 to the spin column and centrifuge (6000×g, 1 minute, room temperature).
(7) After centrifugation, remove the flow-through and set the spin column in a new collection tube.
(8) Add 500 μL of Buffer AW2 to the spin column and centrifuge (20000×g, 3 minutes, room temperature).
(9) After removing the flow-through, centrifuge again (20000×g, 1 minute, room temperature) to remove the flow-through.
(10) After setting the spin column in a new collection tube, add 50 μL of Buffer AE to No.1 sample.
(11) After standing at room temperature for about 1 minute, centrifuge (20000×g, 1 minute, room temperature) to elute.
(12) Collect the entire volume of No.1 eluate and add to No.2 sample.
(13) After standing at room temperature for about 1 minute, centrifuge (20000×g, 1 minute, room temperature) to elute.
(14) Collect the entire volume of No.2 eluate and add to No.3 sample.
(15) After standing at room temperature for about 1 minute, centrifuge (20000×g, 1 minute, room temperature) to elute.
(16) Measure the gDNA concentration of the eluate with a spectrophotometer such as NanoDrop.
(17) Dilute the eluate with Buffer AE so that the amount of gDNA per well (0.0696 μg) or less was confirmed to have no matrix effect. If the amount of gDNA in the eluate is less than 0.0696 µg, apply the lowest dilution factor (5x).
(18) Use the diluted eluate as the measurement sample.
３．検量線用試料の調製
検量線ストック溶液（50000 pg/μL、S-1）を室温で融解後、下表１に従い、検量線用試料S-2～S-6を調製する。NTC試料（No template control）として、Buffer AEを使用する。検量線用試料は用時調製する。 3. Preparation of calibration curve samples After melting the calibration curve stock solution (50000 pg/μL, S-1) at room temperature, prepare calibration curve samples S-2 to S-6 according to Table 1 below. Buffer AE is used as an NTC sample (No template control). Prepare the samples for the calibration curve just before use.
検量線ストック溶液（50000 pg/μL、S-1）を室温で融解後、下表１に従い、検量線用試料S-2～S-6を調製する。NTC試料（No template control）として、Buffer AEを使用する。検量線用試料は用時調製する。 3. Preparation of calibration curve samples After melting the calibration curve stock solution (50000 pg/μL, S-1) at room temperature, prepare calibration curve samples S-2 to S-6 according to Table 1 below. Buffer AE is used as an NTC sample (No template control). Prepare the samples for the calibration curve just before use.
４．qPCR
市販される定量ＰＣＲ用試薬（例えば、LightCycler 480 Probe Master（ロシュ・ダイアグノスティックス株式会社製）を用いて定量ＰＣＲを行う。以下、LightCycler 480 Probe Masterを用いる場合を例に説明する。
（１）LightCycler 480 Probe Master付属のH2O（PCR-grade）を用い、下記の表２に示す組成のMaster Mix（1反応分）を用時調製する。 4. qPCR
Quantitative PCR is performed using commercially available quantitative PCR reagents (for example, LightCycler 480 Probe Master (manufactured by Roche Diagnostics Co., Ltd.). Hereinafter, the case of using LightCycler 480 Probe Master will be described as an example.
(1) Using H2O (PCR-grade) attached to the LightCycler 480 Probe Master, prepare a Master Mix (for one reaction) with the composition shown in Table 2 below before use.
市販される定量ＰＣＲ用試薬（例えば、LightCycler 480 Probe Master（ロシュ・ダイアグノスティックス株式会社製）を用いて定量ＰＣＲを行う。以下、LightCycler 480 Probe Masterを用いる場合を例に説明する。
（１）LightCycler 480 Probe Master付属のH2O（PCR-grade）を用い、下記の表２に示す組成のMaster Mix（1反応分）を用時調製する。 4. qPCR
Quantitative PCR is performed using commercially available quantitative PCR reagents (for example, LightCycler 480 Probe Master (manufactured by Roche Diagnostics Co., Ltd.). Hereinafter, the case of using LightCycler 480 Probe Master will be described as an example.
(1) Using H2O (PCR-grade) attached to the LightCycler 480 Probe Master, prepare a Master Mix (for one reaction) with the composition shown in Table 2 below before use.
定量ＰＣＲに用いるプライマー、及びプローブの配列を表３に示す。PCRには、StepOnePlus（商標）リアルタイムPCRシステム（サーモフィッシャーサイエンティフィック株式会社）を用いた。
Table 3 shows the sequences of the primers and probes used for quantitative PCR. A StepOnePlus (trademark) real-time PCR system (Thermo Fisher Scientific Co., Ltd.) was used for PCR.
（２）Master Mix（12 μL）及び8 μLの各試料を96ウェルホワイトプレートに添加する（計20 μL）。
（３）96ウェルホワイトプレートを遠心機でスピンダウンし、リアルタイムPCR装置にセットする。PCRの温度およびサイクル条件を例えば、下記表４のように設定する。 (2) Add Master Mix (12 µL) and 8 µL of each sample to a 96-well white plate (20 µL in total).
(3) Spin down the 96-well white plate with a centrifuge and set it in a real-time PCR device. The temperature and cycle conditions for PCR are set, for example, as shown in Table 4 below.
（３）96ウェルホワイトプレートを遠心機でスピンダウンし、リアルタイムPCR装置にセットする。PCRの温度およびサイクル条件を例えば、下記表４のように設定する。 (2) Add Master Mix (12 µL) and 8 µL of each sample to a 96-well white plate (20 µL in total).
(3) Spin down the 96-well white plate with a centrifuge and set it in a real-time PCR device. The temperature and cycle conditions for PCR are set, for example, as shown in Table 4 below.
５．データ解析
検量線用試料のCrossing point（Cp値、y軸）と理論濃度（pg/reaction）のlog concentration（x軸）から検量線を作成し、平均二乗誤差（MSE；Mean squared error）、Efficiencyを算出する。測定した各ウェルのCp値を得られた検量線の式に代入することにより検量線の逆回帰値および測定試料の定量値（pg/reaction）を算出する。これらの計算はいずれもリアルタイムPCR装置に付設の解析ソフト（LightCycler 480 Software、ロシュ・ダイアグノスティックス株式会社）を用いて実施できる。 5. Data analysis Create a calibration curve from the crossing point (Cp value, y-axis) of the sample for the calibration curve and the log concentration (x-axis) of the theoretical concentration (pg/reaction). Calculate By substituting the measured Cp value of each well into the formula of the obtained calibration curve, the inverse regression value of the calibration curve and the quantitative value (pg/reaction) of the measurement sample are calculated. All of these calculations can be performed using analysis software attached to the real-time PCR device (LightCycler 480 Software, Roche Diagnostics Co., Ltd.).
検量線用試料のCrossing point（Cp値、y軸）と理論濃度（pg/reaction）のlog concentration（x軸）から検量線を作成し、平均二乗誤差（MSE；Mean squared error）、Efficiencyを算出する。測定した各ウェルのCp値を得られた検量線の式に代入することにより検量線の逆回帰値および測定試料の定量値（pg/reaction）を算出する。これらの計算はいずれもリアルタイムPCR装置に付設の解析ソフト（LightCycler 480 Software、ロシュ・ダイアグノスティックス株式会社）を用いて実施できる。 5. Data analysis Create a calibration curve from the crossing point (Cp value, y-axis) of the sample for the calibration curve and the log concentration (x-axis) of the theoretical concentration (pg/reaction). Calculate By substituting the measured Cp value of each well into the formula of the obtained calibration curve, the inverse regression value of the calibration curve and the quantitative value (pg/reaction) of the measurement sample are calculated. All of these calculations can be performed using analysis software attached to the real-time PCR device (LightCycler 480 Software, Roche Diagnostics Co., Ltd.).
６．代表的な検量線データ
１～５の手順で得られた代表的な検量線データを下記表５に示す。
6. Representative Calibration Curve Data Representative calibration curve data obtained by procedures 1 to 5 are shown in Table 5 below.
１～５の手順で得られた代表的な検量線データを下記表５に示す。
実施例２
ヒト多発性骨髄腫細胞を尾静脈移植した重度免疫不全マウスにaITGB7 CAR-T細胞を投与した後、aITGB7 CAR-T細胞を特異的に認識するプライマーを用いてマウス血中でのaITGB7 CAR-T細胞量の変化を確認した。 Example 2
After administration of aITGB7 CAR-T cells to severely immunodeficient mice with tail vein transplantation of human multiple myeloma cells, aITGB7 CAR-T in mouse blood was detected using primers that specifically recognize aITGB7 CAR-T cells. Changes in cell amount were confirmed.
ヒト多発性骨髄腫細胞を尾静脈移植した重度免疫不全マウスにaITGB7 CAR-T細胞を投与した後、aITGB7 CAR-T細胞を特異的に認識するプライマーを用いてマウス血中でのaITGB7 CAR-T細胞量の変化を確認した。 Example 2
After administration of aITGB7 CAR-T cells to severely immunodeficient mice with tail vein transplantation of human multiple myeloma cells, aITGB7 CAR-T in mouse blood was detected using primers that specifically recognize aITGB7 CAR-T cells. Changes in cell amount were confirmed.
被験物質として、aITGB7 CAR-T細胞を用い、対照物質としては、NGMCを用いた。いずれのＴ細胞も遮光、－８０℃の条件で保管した。
AITGB7 CAR-T cells were used as the test substance, and NGMC was used as the control substance. All T cells were stored in the dark at -80°C.
投与液の調製
投与当日に、被験物質又は対照物質を細胞凍結保存液に必要量溶解し、投与濃度に調整した。調整後、遮光して氷中に静置した。 Preparation of Dosing Solution On the day of administration, the required amount of test substance or control substance was dissolved in a cell cryopreservation solution to adjust the administration concentration. After adjustment, it was shielded from light and allowed to stand in ice.
投与当日に、被験物質又は対照物質を細胞凍結保存液に必要量溶解し、投与濃度に調整した。調整後、遮光して氷中に静置した。 Preparation of Dosing Solution On the day of administration, the required amount of test substance or control substance was dissolved in a cell cryopreservation solution to adjust the administration concentration. After adjustment, it was shielded from light and allowed to stand in ice.
多発性骨髄腫細胞株は、ヒト多発性骨髄腫由来のMM.1Sを用いた。培養は、37℃、5%CO2の条件下、10%のウシ胎児血清、100 Units/mLのペニシリン及び100 μg/mLのストレプトマイシンを添加したRPMI 1640培地にて行った。ウシ胎児血清は、56℃に30分間加熱して非働化したものを使用した。
The multiple myeloma cell line used was MM.1S derived from human multiple myeloma. Cultivation was performed in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 Units/mL penicillin and 100 μg/mL streptomycin under conditions of 37° C., 5% CO 2 . Fetal bovine serum was inactivated by heating at 56°C for 30 minutes.
重度免疫不全マウスは、NOGマウスを用いた。5～6週齢の雌を20匹入手し、餌及び水（オートクレーブ滅菌した水道水）は自由摂取可能とした。
NOG mice were used as severely immunodeficient mice. Twenty 5- to 6-week-old females were obtained, and food and water (autoclave-sterilized tap water) were freely available.
試験方法
多発性骨髄腫細胞株MM.1Sを尾静脈移植10日後、1.33×105cells/bodyのaITGB7 CAR-Tを投与した。移植時には、血清不含RPMI 1640培地を用いた。NOGマウスでのPK試験を6タイムポイントで実施するため、各タイムポイントあたり3匹を割り付け、計18匹とした。採血のタイムポイントは、投与1時間後、24時間後、3日後、1週間後、2週間後、及び3週間後とした。残りの2匹は、検量線作成時の全血サンプル調製用として、aITGB7 CAR-Tを投与せずに採血した。Test method 10 days after tail vein transplantation of multiple myeloma cell line MM.1S, aITGB7 CAR-T was administered at 1.33×10 5 cells/body. Serum-free RPMI 1640 medium was used at the time of transplantation. Since the PK test in NOG mice was performed at 6 time points, 3 mice were assigned to each time point, for a total of 18 mice. Blood sampling time points were 1 hour, 24 hours, 3 days, 1 week, 2 weeks, and 3 weeks after administration. The remaining two animals were bled without administration of aITGB7 CAR-T for preparation of whole blood samples for standard curve preparation.
多発性骨髄腫細胞株MM.1Sを尾静脈移植10日後、1.33×105cells/bodyのaITGB7 CAR-Tを投与した。移植時には、血清不含RPMI 1640培地を用いた。NOGマウスでのPK試験を6タイムポイントで実施するため、各タイムポイントあたり3匹を割り付け、計18匹とした。採血のタイムポイントは、投与1時間後、24時間後、3日後、1週間後、2週間後、及び3週間後とした。残りの2匹は、検量線作成時の全血サンプル調製用として、aITGB7 CAR-Tを投与せずに採血した。
MM.1S細胞尾静脈移植10日後に被験物質を尾静脈投与し、指定したタイムポイントでマウスから採血し、全血から単離したDNAに対して定量PCRを行うことで、血液中のCAR-T量を定量した。具体的な条件、手順は下記のとおりである。
10 days after MM.1S cell tail vein transplantation, the test substance was administered to the tail vein, blood was collected from the mice at the designated time points, and quantitative PCR was performed on the DNA isolated from the whole blood. The amount of T was quantified. Specific conditions and procedures are as follows.
移植用細胞懸濁液の調製
凍結保存された細胞を解凍後、3～6回継代培養した細胞を移植に使用した。5×105－2×106 cells/mLまで培養した細胞を遠心分離（240×g、設定温度4℃、5分間）し、上清を除去した。移植用血清不含培地を加えて細胞を懸濁させ、遠心分離（240×g、設定温度4℃、5分間）し、上清を除去した。この操作を更に2回繰り返した後、移植用血清不含培地を加えて細胞を懸濁させ、細胞懸濁液の一部をトリパンブルー溶液と混合し、血球計算盤を用いて生細胞をカウントし、下記表６に示す濃度の細胞懸濁液を調製した。調製後の細胞懸濁液は、移植時まで氷中に置いた。 Preparation of Cell Suspension for Transplantation After thawing the cryopreserved cells, cells subcultured 3 to 6 times were used for transplantation. The cells cultured to 5×10 5 -2×10 6 cells/mL were centrifuged (240×g, set temperature 4° C., 5 minutes), and the supernatant was removed. A serum-free medium for transplantation was added to suspend the cells, centrifuged (240×g, set temperature 4° C., 5 minutes), and the supernatant was removed. After repeating this operation two more times, serum-free medium for transplantation is added to suspend the cells, a portion of the cell suspension is mixed with trypan blue solution, and viable cells are counted using a hemocytometer. Then, cell suspensions having concentrations shown in Table 6 below were prepared. The prepared cell suspension was kept on ice until transplantation.
凍結保存された細胞を解凍後、3～6回継代培養した細胞を移植に使用した。5×105－2×106 cells/mLまで培養した細胞を遠心分離（240×g、設定温度4℃、5分間）し、上清を除去した。移植用血清不含培地を加えて細胞を懸濁させ、遠心分離（240×g、設定温度4℃、5分間）し、上清を除去した。この操作を更に2回繰り返した後、移植用血清不含培地を加えて細胞を懸濁させ、細胞懸濁液の一部をトリパンブルー溶液と混合し、血球計算盤を用いて生細胞をカウントし、下記表６に示す濃度の細胞懸濁液を調製した。調製後の細胞懸濁液は、移植時まで氷中に置いた。 Preparation of Cell Suspension for Transplantation After thawing the cryopreserved cells, cells subcultured 3 to 6 times were used for transplantation. The cells cultured to 5×10 5 -2×10 6 cells/mL were centrifuged (240×g, set temperature 4° C., 5 minutes), and the supernatant was removed. A serum-free medium for transplantation was added to suspend the cells, centrifuged (240×g, set temperature 4° C., 5 minutes), and the supernatant was removed. After repeating this operation two more times, serum-free medium for transplantation is added to suspend the cells, a portion of the cell suspension is mixed with trypan blue solution, and viable cells are counted using a hemocytometer. Then, cell suspensions having concentrations shown in Table 6 below were prepared. The prepared cell suspension was kept on ice until transplantation.
尾静脈移植に用いた動物の尾静脈内に、1 mL注射筒29G×1/2（テルモ株式会社）を用いて、で0.1mL（1×106－10×106 cells/body）の細胞懸濁液を移植した。
Inject 0.1 mL (1×10 6 -10×10 6 cells/body) of cells into the tail vein of the animal used for tail vein transplantation using a 1 mL syringe 29G×1/2 (Terumo Corporation). Suspension was implanted.
1 mL注射筒を用いて、0.1 mL（1.33×105 cells/body）の投与液（被験物質又は対照物質）を尾静脈内に移植した。
Using a 1 mL syringe, 0.1 mL (1.33×10 5 cells/body) of the administration solution (test substance or control substance) was implanted into the tail vein.
採血は、投与1時間後、24時間後、3日後、1週間後、2週間後、及び3週間後に、イソフルラン（アボット・ジャパン株式会社）吸入麻酔下で腹部後大静脈より行った。血液を採取後、直ちにヘパリン入りチューブに移し、転倒混和の後氷中に静置した。
Blood was collected from the retro-abdominal vena cava under isoflurane (Abbott Japan Co., Ltd.) inhalation anesthesia 1 hour, 24 hours, 3 days, 1 week, 2 weeks, and 3 weeks after administration. Immediately after the blood was collected, it was transferred to a heparin-filled tube, mixed by inversion, and allowed to stand on ice.
凍結融解したaITGB7 CAR-Tサンプル、NGMCサンプル、及びaITGB7 CAR-Tを投与した担がんマウス由来血液サンプルから、NucleoSpin（商標） Blood（MACHEREY-NAGEL）でゲノムDNAを抽出した。NanoDrop（商標）でDNA量を測定し、aITGB7 CARに対するプライマーセットを用いて、全ゲノム量に対する各サンプルのaITGB7 CARのDNA量を評価した。
Genomic DNA was extracted with NucleoSpin™ Blood (MACHEREY-NAGEL) from freeze-thawed aITGB7 CAR-T samples, NGMC samples, and blood samples derived from tumor-bearing mice administered with aITGB7 CAR-T. DNA content was measured with NanoDrop™ and the primer set for aITGB7 CAR was used to assess the DNA content of aITGB7 CAR in each sample relative to the total genome content.
検出系のaITGB7 CAR特異性解析用ゲノムDNA抽出
各サンプルのaITGB7 CARのDNA量を解析するため、凍結融解したaITGB7 CAR-T、及びNGMCから、ゲノムDNAを抽出した。 Genomic DNA Extraction for aITGB7 CAR Specificity Analysis of Detection System Genomic DNA was extracted from freeze-thawed aITGB7 CAR-T and NGMC in order to analyze the amount of aITGB7 CAR DNA in each sample.
各サンプルのaITGB7 CARのDNA量を解析するため、凍結融解したaITGB7 CAR-T、及びNGMCから、ゲノムDNAを抽出した。 Genomic DNA Extraction for aITGB7 CAR Specificity Analysis of Detection System Genomic DNA was extracted from freeze-thawed aITGB7 CAR-T and NGMC in order to analyze the amount of aITGB7 CAR DNA in each sample.
aITGB7 CAR特異性解析における検量線サンプルの調製
aITGB7 CARプラスミドを蒸留水で段階希釈し、1－1000 fg/mLの希釈液（10倍公比）を調製した。 Preparation of Standard Curve Samples for aITGB7 CAR Specificity Analysis The aITGB7 CAR plasmid was serially diluted with distilled water to prepare dilutions of 1-1000 fg/mL (10-fold common ratio).
aITGB7 CARプラスミドを蒸留水で段階希釈し、1－1000 fg/mLの希釈液（10倍公比）を調製した。 Preparation of Standard Curve Samples for aITGB7 CAR Specificity Analysis The aITGB7 CAR plasmid was serially diluted with distilled water to prepare dilutions of 1-1000 fg/mL (10-fold common ratio).
全血サンプルからのゲノムDNA抽出
各サンプルのaITGB7 CARのDNA量を解析するため、採血で得たaITGB7 CAR-Tを投与した担がんマウス由来血液サンプルから、ゲノムDNAを抽出した。 Genomic DNA Extraction from Whole Blood Samples In order to analyze the amount of aITGB7 CAR DNA in each sample, genomic DNA was extracted from blood samples derived from tumor-bearing mice to which aITGB7 CAR-T was administered.
各サンプルのaITGB7 CARのDNA量を解析するため、採血で得たaITGB7 CAR-Tを投与した担がんマウス由来血液サンプルから、ゲノムDNAを抽出した。 Genomic DNA Extraction from Whole Blood Samples In order to analyze the amount of aITGB7 CAR DNA in each sample, genomic DNA was extracted from blood samples derived from tumor-bearing mice to which aITGB7 CAR-T was administered.
全血サンプル用検量線サンプルの調製
aITGB7 CARプラスミドを蒸留水で段階希釈して0.01－100 pg/mLの希釈液を調製した。希釈したCARプラスミド又は蒸留水2 μLと採血で得た非投与マウス由来の全血198 μLを混合して、0及び0.1-1000 fg/mLの検量線用全血サンプルを調製した。これらから全血サンプルからのゲノムDNA抽出と同様にゲノムDNAを抽出し、検量線サンプルとした。 Preparation of Standard Curve Samples for Whole Blood Samples The aITGB7 CAR plasmid was serially diluted with distilled water to prepare 0.01-100 pg/mL dilutions. 2 μL of diluted CAR plasmid or distilled water and 198 μL of whole blood obtained by blood collection from untreated mice were mixed to prepare 0 and 0.1-1000 fg/mL whole blood samples for standard curves. Genomic DNA was extracted from these samples in the same manner as the genomic DNA extraction from whole blood samples, and used as a calibration curve sample.
aITGB7 CARプラスミドを蒸留水で段階希釈して0.01－100 pg/mLの希釈液を調製した。希釈したCARプラスミド又は蒸留水2 μLと採血で得た非投与マウス由来の全血198 μLを混合して、0及び0.1-1000 fg/mLの検量線用全血サンプルを調製した。これらから全血サンプルからのゲノムDNA抽出と同様にゲノムDNAを抽出し、検量線サンプルとした。 Preparation of Standard Curve Samples for Whole Blood Samples The aITGB7 CAR plasmid was serially diluted with distilled water to prepare 0.01-100 pg/mL dilutions. 2 μL of diluted CAR plasmid or distilled water and 198 μL of whole blood obtained by blood collection from untreated mice were mixed to prepare 0 and 0.1-1000 fg/mL whole blood samples for standard curves. Genomic DNA was extracted from these samples in the same manner as the genomic DNA extraction from whole blood samples, and used as a calibration curve sample.
ゲノムDNA濃度の測定
抽出したゲノムDNA溶液の濃度を測定した。抽出したゲノムDNA溶液はリアルタイムPCR反応に供するまで氷水上で保存した。「検出系のaITGB7 CAR特異性解析用ゲノムDNA抽出」で抽出したサンプルに関しては、DNA濃度測定後、蒸留水を用いて10倍公比で1000倍まで希釈した。 Measurement of Genomic DNA Concentration The concentration of the extracted genomic DNA solution was measured. The extracted genomic DNA solution was stored on ice water until it was subjected to real-time PCR reaction. With respect to the sample extracted in the "extraction of genomic DNA for aITGB7 CAR specificity analysis of detection system", after measuring the DNA concentration, it was diluted to 1000 times with distilled water at a common ratio of 10 times.
抽出したゲノムDNA溶液の濃度を測定した。抽出したゲノムDNA溶液はリアルタイムPCR反応に供するまで氷水上で保存した。「検出系のaITGB7 CAR特異性解析用ゲノムDNA抽出」で抽出したサンプルに関しては、DNA濃度測定後、蒸留水を用いて10倍公比で1000倍まで希釈した。 Measurement of Genomic DNA Concentration The concentration of the extracted genomic DNA solution was measured. The extracted genomic DNA solution was stored on ice water until it was subjected to real-time PCR reaction. With respect to the sample extracted in the "extraction of genomic DNA for aITGB7 CAR specificity analysis of detection system", after measuring the DNA concentration, it was diluted to 1000 times with distilled water at a common ratio of 10 times.
定量PCRに用いるプライマー、及びプローブは、表３に記載のものを用いた。
The primers and probes used for quantitative PCR were those listed in Table 3.
CAR遺伝子のPCR混和液は、１反応当たり、10 μLのPCR Master Mix (2X)、0.2 μLのForward primer、0.2 μLのReverse primer、0.5 μLのProbe、1.1 μLの蒸留水を混合して調製した。測定試料数に応じ必要な量を調製した。PCRプレートの各ウェルにPCR混和液を12 μLずつ分注し、対応するDNAサンプルを8 μL加えた。リアルタイムPCRの条件を表７に示す。
The CAR gene PCR mixture was prepared by mixing 10 μL of PCR Master Mix (2X), 0.2 μL of Forward primer, 0.2 μL of Reverse primer, 0.5 μL of Probe and 1.1 μL of distilled water per reaction. . A necessary amount was prepared according to the number of samples to be measured. 12 μL of the PCR mixture was dispensed into each well of the PCR plate, and 8 μL of the corresponding DNA sample was added. Table 7 shows the conditions for real-time PCR.
aITGB7 CARのDNA量の算出
リアルタイムPCR装置内蔵の解析ソフトを用いてCt値を算出し、ΔCt値を式１に従って算出し、定量PCRに用いたサンプル溶液中でのaITGB7 CAR DNAの相対値（RQ：Relative Quantification）は、式２に従って算出した。検量線サンプルのRQ値と検量線サンプルのプラスミド濃度をプロットして、近似直線の一次方程式を求め、各サンプルのaITGB7 CARのDNA濃度を算出した。全血サンプルに関しては、さらに単離した全ゲノム濃度で割ることで、一定量の血液中に存在するaITGB7 CARのDNA量を算出した。
ΔCt = Ct,x - Ct,r ----------（式 1）（ここで、Ct,x：サンプルのCt値の平均値、
Ct,r：検量線の内Ct値が算出できた最低濃度でのCt値の平均値）
RQ = 2^(-ΔCt) ----------------（式 2） Calculation of the amount of aITGB7 CAR DNA Calculate the Ct value using the analysis software built into the real-time PCR device, calculate the ΔCt value according to formula 1, and calculate the relative value of aITGB7 CAR DNA in the sample solution used for quantitative PCR (RQ : Relative Quantification) was calculated according to Equation 2. The RQ values of the calibration curve samples and the plasmid concentrations of the calibration curve samples were plotted to obtain a linear equation of an approximate straight line, and the aITGB7 CAR DNA concentration of each sample was calculated. For whole blood samples, the amount of aITGB7 CAR DNA present in a given volume of blood was calculated by further dividing by the total genome concentration isolated.
ΔCt = Ct,x - Ct,r ---------- (Equation 1) (where Ct,x is the average Ct value of the sample,
Ct,r: average value of Ct values at the lowest concentration for which Ct values could be calculated in the calibration curve)
RQ = 2^(-ΔCt) ---------------- (equation 2)
リアルタイムPCR装置内蔵の解析ソフトを用いてCt値を算出し、ΔCt値を式１に従って算出し、定量PCRに用いたサンプル溶液中でのaITGB7 CAR DNAの相対値（RQ：Relative Quantification）は、式２に従って算出した。検量線サンプルのRQ値と検量線サンプルのプラスミド濃度をプロットして、近似直線の一次方程式を求め、各サンプルのaITGB7 CARのDNA濃度を算出した。全血サンプルに関しては、さらに単離した全ゲノム濃度で割ることで、一定量の血液中に存在するaITGB7 CARのDNA量を算出した。
ΔCt = Ct,x - Ct,r ----------（式 1）（ここで、Ct,x：サンプルのCt値の平均値、
Ct,r：検量線の内Ct値が算出できた最低濃度でのCt値の平均値）
RQ = 2^(-ΔCt) ----------------（式 2） Calculation of the amount of aITGB7 CAR DNA Calculate the Ct value using the analysis software built into the real-time PCR device, calculate the ΔCt value according to formula 1, and calculate the relative value of aITGB7 CAR DNA in the sample solution used for quantitative PCR (RQ : Relative Quantification) was calculated according to Equation 2. The RQ values of the calibration curve samples and the plasmid concentrations of the calibration curve samples were plotted to obtain a linear equation of an approximate straight line, and the aITGB7 CAR DNA concentration of each sample was calculated. For whole blood samples, the amount of aITGB7 CAR DNA present in a given volume of blood was calculated by further dividing by the total genome concentration isolated.
ΔCt = Ct,x - Ct,r ---------- (Equation 1) (where Ct,x is the average Ct value of the sample,
Ct,r: average value of Ct values at the lowest concentration for which Ct values could be calculated in the calibration curve)
RQ = 2^(-ΔCt) ---------------- (equation 2)
（４）結果
定量PCRの結果を図１及び表８に示す。非遺伝子導入T細胞NGMCと比較して、aITGB7 CAR-Tに対してのみ、ゲノム量依存的に（希釈倍率に応じて）蛍光量の増加が認められ、aITGB7 CAR DNA量を特異的に評価可能であることが示された。 (4) Results The results of quantitative PCR are shown in FIG. 1 and Table 8. Compared to non-transgenic T-cell NGMC, only for aITGB7 CAR-T, a genomic amount-dependent increase in fluorescence was observed (according to the dilution ratio), and aITGB7 CAR DNA amount can be specifically evaluated. was shown to be
定量PCRの結果を図１及び表８に示す。非遺伝子導入T細胞NGMCと比較して、aITGB7 CAR-Tに対してのみ、ゲノム量依存的に（希釈倍率に応じて）蛍光量の増加が認められ、aITGB7 CAR DNA量を特異的に評価可能であることが示された。 (4) Results The results of quantitative PCR are shown in FIG. 1 and Table 8. Compared to non-transgenic T-cell NGMC, only for aITGB7 CAR-T, a genomic amount-dependent increase in fluorescence was observed (according to the dilution ratio), and aITGB7 CAR DNA amount can be specifically evaluated. was shown to be
全血中のaITGB7 CAR-T量の経時変化
各採血タイムポイント毎に測定した定量PCRの結果を図２に示す。1.33×105 cells/bodyでaITGB7 CAR-Tを投与後、1時間後からaITGB7 CARのDNAが全血中で検出され、投与2週間後に最大値を示すことが明らかとなった。 Time course of aITGB7 CAR-T amount in whole blood FIG. 2 shows the results of quantitative PCR measured at each blood sampling time point. After administration of aITGB7 CAR-T at 1.33×10 5 cells/body, DNA of aITGB7 CAR was detected in whole blood from 1 hour later, and showed a maximum level 2 weeks after administration.
各採血タイムポイント毎に測定した定量PCRの結果を図２に示す。1.33×105 cells/bodyでaITGB7 CAR-Tを投与後、1時間後からaITGB7 CARのDNAが全血中で検出され、投与2週間後に最大値を示すことが明らかとなった。 Time course of aITGB7 CAR-T amount in whole blood FIG. 2 shows the results of quantitative PCR measured at each blood sampling time point. After administration of aITGB7 CAR-T at 1.33×10 5 cells/body, DNA of aITGB7 CAR was detected in whole blood from 1 hour later, and showed a maximum level 2 weeks after administration.
実施例３
１．ゲノムＤＮＡの抽出
ａＩＴＧＢ７ ＣＡＲ－Ｔ細胞を投与したヒトドナーから取得したＰＢＭＣから、ＱＩＡａｍｐ ＤＮＡ Ｍｉｎｉ Ｋｉｔ（ＱＩＡＧＥＮ製）に付属のプロトコルに従って、ｇＤＮＡを抽出した。ｇＤＮＡサンプルは、必要に応じて－２０℃で保管した。
（１）１０ミリリットルのＰＢＳを１５ｍＬチューブに移した。
（２）ＰＢＭＣを３７℃の水浴内で融解し、ＰＢＭＣを１５ｍＬチューブに移した。
（３）チューブを遠心分離（３００×ｇ、１０分間、室温）した後、上清を除去した。
（４）タッピング後に１ｍＬのＰＢＳを１５ｍＬチューブに移し、ＰＢＭＣを懸濁した。
（５）２０マイクロリットルの細胞懸濁液を２０μＬのＶｉａＳｔａｉｎ ＡＯＰＩ Ｓｔａｉｎｉｎｇ Ｓｏｌｕｔｉｏｎ（Ｎｅｘｃｅｌｏｍ Ｂｉｏｓｃｉｅｎｃｅ製）と混合し、Ｃｅｌｌｏｍｅｔｅｒ Ａｕｔｏ ２０００（Ｎｅｘｃｅｌｏｍ Ｂｉｏｓｃｉｅｎｃｅ製）を用いて細胞数を計数した。
（６）ピペットによって懸濁した後、懸濁液（５×１０６細胞／チューブ以下）を１．５ｍＬチューブ（三角底）に移した。
（７）チューブを遠心分離（２０００×ｇ、５分間、室温）し、上清を除去した。
（８）タッピング後に２００μＬのＰＢＳを添加し、ＰＢＭＣを懸濁した（ＰＢＭＣ懸濁液）。
（９）２０マイクロリットルのプロテイナーゼＫを添加し、十分に混合した。
（１０）４マイクロリットルのＲＮａｓｅ Ａ（１００ｍｇ／ｍＬ）を添加し、混合し、室温で２分間インキュベートした。
（１１）２００マイクロリットルのＢｕｆｆｅｒ ＡＬを添加し、１５秒間ボルテックスした。チューブを短時間遠心分離した。
（１２）チューブを５６℃で１０分間インキュベートした。チューブを短時間遠心分離した。
（１３）２００マイクロリットルのエタノール（９９．５％）を添加し、１５秒間ボルテックスした。チューブを短時間遠心分離した。
（１４）ステップ（１３）の混合物をＱＩＡａｍｐ Ｍｉｎｉ Ｓｐｉｎ Ｃｏｌｕｍｎ（ＱＩＡＧＥＮ製）にアプライした。
（１５）チューブを室温にて６０００×ｇで１分間遠心分離した。
（１６）カラムを新たなコレクションチューブ（２ｍＬ）にセットし、５００μＬのＢｕｆｆｅｒ ＡＷ１（ＱＩＡＧＥＮ製）を添加した。
（１７）チューブを室温にて６０００×ｇで１分間遠心分離した。
（１８）カラムを新たなコレクションチューブ（２ｍＬ）にセットし、５００μＬのＢｕｆｆｅｒ ＡＷ２（ＱＩＡＧＥＮ製）を添加した。
（１９）チューブを室温にて２００００×ｇで３分間遠心分離した。
（２０）カラムを新たなコレクションチューブ（２ｍＬ）にセットした。
（２１）チューブを室温にて２００００×ｇで１分間遠心分離した。
（２２）カラムを新たなコレクションチューブ（１．５ｍＬ）にセットし、２００μＬのＢｕｆｆｅｒ ＡＥを添加した。
（２３）チューブを室温で５分間インキュベートした。
（２４）チューブを室温にて６０００×ｇで１分間遠心分離した。
（２５）溶出液を各ドナーにつき１本のチューブに合わせ、分光光度計を用いてｇＤＮＡ濃度を測定した。Ｂｕｆｆｅｒ ＡＥをブランクとして用いた。
分光光度計は、ＮａｎｏＤｒｏｐ ２０００（サーモフィッシャー）を用い、以下の条件で測定した。
モード：核酸
サンプルタイプ：ＤＮＡ
エクスポートデータ：核酸濃度、Ａ２６０ｎｍ、Ａ２８０ｎｍ、Ａ２６０ｎｍ／Ａ２８０ｎｍ、Ａ２６０ｎｍ／Ａ２３０ｎｍ Example 3
1. Extraction of Genomic DNA From PBMCs obtained from human donors to whom aITGB7 CAR-T cells were administered, gDNA was extracted according to the protocol attached to QIAamp DNA Mini Kit (manufactured by QIAGEN). gDNA samples were stored at -20°C as needed.
(1) 10 milliliters of PBS was transferred to a 15 mL tube.
(2) PBMC were thawed in a 37° C. water bath and transferred to a 15 mL tube.
(3) After centrifuging the tube (300×g, 10 minutes, room temperature), the supernatant was removed.
(4) After tapping, 1 mL of PBS was transferred to a 15 mL tube to suspend PBMCs.
(5) 20 microliters of cell suspension was mixed with 20 μL of ViaStain AOPI Staining Solution (manufactured by Nexcelom Bioscience), and the number of cells was counted using Cellometer Auto 2000 (manufactured by Nexcelom Bioscience).
(6) After suspending with a pipette, the suspension (5×10 6 cells/tube or less) was transferred to a 1.5 mL tube (triangular bottom).
(7) The tube was centrifuged (2000×g, 5 minutes, room temperature) and the supernatant was removed.
(8) After tapping, 200 μL of PBS was added to suspend PBMC (PBMC suspension).
(9) 20 microliters of Proteinase K was added and mixed well.
(10) 4 microliters of RNase A (100 mg/mL) was added, mixed and incubated for 2 minutes at room temperature.
(11) Added 200 microliters of Buffer AL and vortexed for 15 seconds. The tube was centrifuged briefly.
(12) The tubes were incubated at 56°C for 10 minutes. The tube was centrifuged briefly.
(13) Added 200 microliters of ethanol (99.5%) and vortexed for 15 seconds. The tube was centrifuged briefly.
(14) The mixture of step (13) was applied to QIAamp Mini Spin Column (manufactured by QIAGEN).
(15) The tube was centrifuged at 6000 xg for 1 minute at room temperature.
(16) The column was set in a new collection tube (2 mL), and 500 μL of Buffer AW1 (manufactured by QIAGEN) was added.
(17) The tube was centrifuged at 6000 xg for 1 minute at room temperature.
(18) The column was set in a new collection tube (2 mL), and 500 μL of Buffer AW2 (manufactured by QIAGEN) was added.
(19) The tube was centrifuged at 20000 xg for 3 minutes at room temperature.
(20) The column was set in a new collection tube (2 mL).
(21) The tube was centrifuged at 20000 xg for 1 minute at room temperature.
(22) The column was set in a new collection tube (1.5 mL), and 200 µL of Buffer AE was added.
(23) The tubes were incubated at room temperature for 5 minutes.
(24) The tube was centrifuged at 6000 xg for 1 minute at room temperature.
(25) The eluate was combined into one tube for each donor and the gDNA concentration was measured using a spectrophotometer. Buffer AE was used as a blank.
A NanoDrop 2000 (Thermo Fisher) was used as a spectrophotometer, and measurements were made under the following conditions.
Mode: Nucleic Acid Sample Type: DNA
Export data: nucleic acid concentration, A260nm, A280nm, A260nm/A280nm, A260nm/A230nm
１．ゲノムＤＮＡの抽出
ａＩＴＧＢ７ ＣＡＲ－Ｔ細胞を投与したヒトドナーから取得したＰＢＭＣから、ＱＩＡａｍｐ ＤＮＡ Ｍｉｎｉ Ｋｉｔ（ＱＩＡＧＥＮ製）に付属のプロトコルに従って、ｇＤＮＡを抽出した。ｇＤＮＡサンプルは、必要に応じて－２０℃で保管した。
（１）１０ミリリットルのＰＢＳを１５ｍＬチューブに移した。
（２）ＰＢＭＣを３７℃の水浴内で融解し、ＰＢＭＣを１５ｍＬチューブに移した。
（３）チューブを遠心分離（３００×ｇ、１０分間、室温）した後、上清を除去した。
（４）タッピング後に１ｍＬのＰＢＳを１５ｍＬチューブに移し、ＰＢＭＣを懸濁した。
（５）２０マイクロリットルの細胞懸濁液を２０μＬのＶｉａＳｔａｉｎ ＡＯＰＩ Ｓｔａｉｎｉｎｇ Ｓｏｌｕｔｉｏｎ（Ｎｅｘｃｅｌｏｍ Ｂｉｏｓｃｉｅｎｃｅ製）と混合し、Ｃｅｌｌｏｍｅｔｅｒ Ａｕｔｏ ２０００（Ｎｅｘｃｅｌｏｍ Ｂｉｏｓｃｉｅｎｃｅ製）を用いて細胞数を計数した。
（６）ピペットによって懸濁した後、懸濁液（５×１０６細胞／チューブ以下）を１．５ｍＬチューブ（三角底）に移した。
（７）チューブを遠心分離（２０００×ｇ、５分間、室温）し、上清を除去した。
（８）タッピング後に２００μＬのＰＢＳを添加し、ＰＢＭＣを懸濁した（ＰＢＭＣ懸濁液）。
（９）２０マイクロリットルのプロテイナーゼＫを添加し、十分に混合した。
（１０）４マイクロリットルのＲＮａｓｅ Ａ（１００ｍｇ／ｍＬ）を添加し、混合し、室温で２分間インキュベートした。
（１１）２００マイクロリットルのＢｕｆｆｅｒ ＡＬを添加し、１５秒間ボルテックスした。チューブを短時間遠心分離した。
（１２）チューブを５６℃で１０分間インキュベートした。チューブを短時間遠心分離した。
（１３）２００マイクロリットルのエタノール（９９．５％）を添加し、１５秒間ボルテックスした。チューブを短時間遠心分離した。
（１４）ステップ（１３）の混合物をＱＩＡａｍｐ Ｍｉｎｉ Ｓｐｉｎ Ｃｏｌｕｍｎ（ＱＩＡＧＥＮ製）にアプライした。
（１５）チューブを室温にて６０００×ｇで１分間遠心分離した。
（１６）カラムを新たなコレクションチューブ（２ｍＬ）にセットし、５００μＬのＢｕｆｆｅｒ ＡＷ１（ＱＩＡＧＥＮ製）を添加した。
（１７）チューブを室温にて６０００×ｇで１分間遠心分離した。
（１８）カラムを新たなコレクションチューブ（２ｍＬ）にセットし、５００μＬのＢｕｆｆｅｒ ＡＷ２（ＱＩＡＧＥＮ製）を添加した。
（１９）チューブを室温にて２００００×ｇで３分間遠心分離した。
（２０）カラムを新たなコレクションチューブ（２ｍＬ）にセットした。
（２１）チューブを室温にて２００００×ｇで１分間遠心分離した。
（２２）カラムを新たなコレクションチューブ（１．５ｍＬ）にセットし、２００μＬのＢｕｆｆｅｒ ＡＥを添加した。
（２３）チューブを室温で５分間インキュベートした。
（２４）チューブを室温にて６０００×ｇで１分間遠心分離した。
（２５）溶出液を各ドナーにつき１本のチューブに合わせ、分光光度計を用いてｇＤＮＡ濃度を測定した。Ｂｕｆｆｅｒ ＡＥをブランクとして用いた。
分光光度計は、ＮａｎｏＤｒｏｐ ２０００（サーモフィッシャー）を用い、以下の条件で測定した。
モード：核酸
サンプルタイプ：ＤＮＡ
エクスポートデータ：核酸濃度、Ａ２６０ｎｍ、Ａ２８０ｎｍ、Ａ２６０ｎｍ／Ａ２８０ｎｍ、Ａ２６０ｎｍ／Ａ２３０ｎｍ Example 3
1. Extraction of Genomic DNA From PBMCs obtained from human donors to whom aITGB7 CAR-T cells were administered, gDNA was extracted according to the protocol attached to QIAamp DNA Mini Kit (manufactured by QIAGEN). gDNA samples were stored at -20°C as needed.
(1) 10 milliliters of PBS was transferred to a 15 mL tube.
(2) PBMC were thawed in a 37° C. water bath and transferred to a 15 mL tube.
(3) After centrifuging the tube (300×g, 10 minutes, room temperature), the supernatant was removed.
(4) After tapping, 1 mL of PBS was transferred to a 15 mL tube to suspend PBMCs.
(5) 20 microliters of cell suspension was mixed with 20 μL of ViaStain AOPI Staining Solution (manufactured by Nexcelom Bioscience), and the number of cells was counted using Cellometer Auto 2000 (manufactured by Nexcelom Bioscience).
(6) After suspending with a pipette, the suspension (5×10 6 cells/tube or less) was transferred to a 1.5 mL tube (triangular bottom).
(7) The tube was centrifuged (2000×g, 5 minutes, room temperature) and the supernatant was removed.
(8) After tapping, 200 μL of PBS was added to suspend PBMC (PBMC suspension).
(9) 20 microliters of Proteinase K was added and mixed well.
(10) 4 microliters of RNase A (100 mg/mL) was added, mixed and incubated for 2 minutes at room temperature.
(11) Added 200 microliters of Buffer AL and vortexed for 15 seconds. The tube was centrifuged briefly.
(12) The tubes were incubated at 56°C for 10 minutes. The tube was centrifuged briefly.
(13) Added 200 microliters of ethanol (99.5%) and vortexed for 15 seconds. The tube was centrifuged briefly.
(14) The mixture of step (13) was applied to QIAamp Mini Spin Column (manufactured by QIAGEN).
(15) The tube was centrifuged at 6000 xg for 1 minute at room temperature.
(16) The column was set in a new collection tube (2 mL), and 500 μL of Buffer AW1 (manufactured by QIAGEN) was added.
(17) The tube was centrifuged at 6000 xg for 1 minute at room temperature.
(18) The column was set in a new collection tube (2 mL), and 500 μL of Buffer AW2 (manufactured by QIAGEN) was added.
(19) The tube was centrifuged at 20000 xg for 3 minutes at room temperature.
(20) The column was set in a new collection tube (2 mL).
(21) The tube was centrifuged at 20000 xg for 1 minute at room temperature.
(22) The column was set in a new collection tube (1.5 mL), and 200 µL of Buffer AE was added.
(23) The tubes were incubated at room temperature for 5 minutes.
(24) The tube was centrifuged at 6000 xg for 1 minute at room temperature.
(25) The eluate was combined into one tube for each donor and the gDNA concentration was measured using a spectrophotometer. Buffer AE was used as a blank.
A NanoDrop 2000 (Thermo Fisher) was used as a spectrophotometer, and measurements were made under the following conditions.
Mode: Nucleic Acid Sample Type: DNA
Export data: nucleic acid concentration, A260nm, A280nm, A260nm/A280nm, A260nm/A230nm
２．標準用液の調製
標準プラスミドストック溶液（１．００×１０８コピー／μＬ、ＳＳ）を室温で融解し、下記表９に従って標準溶液Ｃ１～Ｃ１０を調製した。Ｂｕｆｆｅｒ ＡＥをＮＴＣ（鋳型なしのコントロール）サンプルとして用いた。標準プラスミドは、ａＩＴＧＢ７ ＣＡＲ－Ｔ細胞が有するＤＮＡの塩基配列の部分配列を有する直鎖プラスミドである。 2. Preparation of Standard Solution A standard plasmid stock solution (1.00×10 8 copies/μL, SS) was thawed at room temperature, and standard solutions C1 to C10 were prepared according to Table 9 below. Buffer AE was used as the NTC (no template control) sample. The standard plasmid is a linear plasmid having a partial DNA base sequence of aITGB7 CAR-T cells.
標準プラスミドストック溶液（１．００×１０８コピー／μＬ、ＳＳ）を室温で融解し、下記表９に従って標準溶液Ｃ１～Ｃ１０を調製した。Ｂｕｆｆｅｒ ＡＥをＮＴＣ（鋳型なしのコントロール）サンプルとして用いた。標準プラスミドは、ａＩＴＧＢ７ ＣＡＲ－Ｔ細胞が有するＤＮＡの塩基配列の部分配列を有する直鎖プラスミドである。 2. Preparation of Standard Solution A standard plasmid stock solution (1.00×10 8 copies/μL, SS) was thawed at room temperature, and standard solutions C1 to C10 were prepared according to Table 9 below. Buffer AE was used as the NTC (no template control) sample. The standard plasmid is a linear plasmid having a partial DNA base sequence of aITGB7 CAR-T cells.
３．試験（ＱＣ）サンプルの調製
標準プラスミドストック溶液（１．００×１０８コピー／μＬ、ＳＳ）を室温で融解した後、下記の表１０及び１１に従って試験サンプルＨ、Ｍ、Ｌ、ＬＬ及びＢＬを調製した。上記１で抽出した各ｇＤＮＡを室温で融解し、Ｂｕｆｆｅｒ ＡＥで希釈して、５０ｎｇ／μＬのｇＤＮＡを調製した。 3. Preparation of Test (QC) Samples After thawing a standard plasmid stock solution (1.00×10 8 copies/μL, SS) at room temperature, test samples H, M, L, LL and BL were prepared according to Tables 10 and 11 below. prepared. Each gDNA extracted in 1 above was melted at room temperature and diluted with Buffer AE to prepare 50 ng/μL of gDNA.
標準プラスミドストック溶液（１．００×１０８コピー／μＬ、ＳＳ）を室温で融解した後、下記の表１０及び１１に従って試験サンプルＨ、Ｍ、Ｌ、ＬＬ及びＢＬを調製した。上記１で抽出した各ｇＤＮＡを室温で融解し、Ｂｕｆｆｅｒ ＡＥで希釈して、５０ｎｇ／μＬのｇＤＮＡを調製した。 3. Preparation of Test (QC) Samples After thawing a standard plasmid stock solution (1.00×10 8 copies/μL, SS) at room temperature, test samples H, M, L, LL and BL were prepared according to Tables 10 and 11 below. prepared. Each gDNA extracted in 1 above was melted at room temperature and diluted with Buffer AE to prepare 50 ng/μL of gDNA.
Ｈ、Ｍ、Ｌ、ＬＬ及びＢＬをｑＰＣＲに使用。ドナー数ｎに応じてＨ－１～Ｈ－ｎ、Ｍ－１～Ｍ－ｎ、Ｌ－１～Ｌ－ｎ、ＬＬ－１～ＬＬ－ｎ、ＢＬ－１～ＢＬ－ｎを調製した。
H, M, L, LL and BL used for qPCR. H-1 to Hn, M-1 to Mn, L-1 to Ln, LL-1 to LL-n, and BL-1 to BL-n were prepared according to the number of donors n.
４．リアルタイムＰＣＲ
（１）十分な量のＰＣＲ Ｍａｓｔｅｒ Ｍｉｘを使用当日に調製した。１回の反応の内容を下に記載する。ＬｉｇｈｔＣｙｃｌｅｒ ４８０ Ｐｒｏｂｅ Ｍａｓｔｅｒに同梱のＨ２Ｏ（ＰＣＲグレード）を使用した。
LightCycler 480 Probe Master １０．０μＬ
20×Pre Mixed Primer and Probe １．０μＬ
Ｈ ２ Ｏ（ＰＣＲグレード） １．０μＬ
合計 １２．０μＬ
上記「Pre Mixed Primer and Probe」は、配列番号１の塩基配列からなるフォワードプライマー、配列番号２の塩基配列からなるリバースプライマー、及び配列番号９の塩基配列を有するプローブ（5’- FAM-CACCACGCGACTTCGCAGCCTATC-NFQ-MGB- 3’）を含む（FAM: Fluorescein, NFQ: Non-Fluorescent Quencher, MGB: Minor Groove Binder）。 4. Real-time PCR
(1) A sufficient amount of PCR Master Mix was prepared on the day of use. The contents of one reaction are described below. The H2O (PCR grade) supplied with the LightCycler 480 Probe Master was used.
LightCycler 480 Probe Master 10.0 μL
20×Pre Mixed Primer and Probe 1.0 μL
H2O ( PCR grade) 1.0 μL
Total 12.0 μL
The above "Pre Mixed Primer and Probe" includes a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1, a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2, and a probe having the nucleotide sequence of SEQ ID NO: 9 (5'-FAM-CACCACGCGACTTCGCAGCCTATC- NFQ-MGB-3') (FAM: Fluorescein, NFQ: Non-Fluorescent Quencher, MGB: Minor Groove Binder).
（１）十分な量のＰＣＲ Ｍａｓｔｅｒ Ｍｉｘを使用当日に調製した。１回の反応の内容を下に記載する。ＬｉｇｈｔＣｙｃｌｅｒ ４８０ Ｐｒｏｂｅ Ｍａｓｔｅｒに同梱のＨ２Ｏ（ＰＣＲグレード）を使用した。
LightCycler 480 Probe Master １０．０μＬ
20×Pre Mixed Primer and Probe １．０μＬ
Ｈ ２ Ｏ（ＰＣＲグレード） １．０μＬ
合計 １２．０μＬ
上記「Pre Mixed Primer and Probe」は、配列番号１の塩基配列からなるフォワードプライマー、配列番号２の塩基配列からなるリバースプライマー、及び配列番号９の塩基配列を有するプローブ（5’- FAM-CACCACGCGACTTCGCAGCCTATC-NFQ-MGB- 3’）を含む（FAM: Fluorescein, NFQ: Non-Fluorescent Quencher, MGB: Minor Groove Binder）。 4. Real-time PCR
(1) A sufficient amount of PCR Master Mix was prepared on the day of use. The contents of one reaction are described below. The H2O (PCR grade) supplied with the LightCycler 480 Probe Master was used.
LightCycler 480 Probe Master 10.0 μL
20×Pre Mixed Primer and Probe 1.0 μL
H2O ( PCR grade) 1.0 μL
Total 12.0 μL
The above "Pre Mixed Primer and Probe" includes a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1, a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2, and a probe having the nucleotide sequence of SEQ ID NO: 9 (5'-FAM-CACCACGCGACTTCGCAGCCTATC- NFQ-MGB-3') (FAM: Fluorescein, NFQ: Non-Fluorescent Quencher, MGB: Minor Groove Binder).
（２）ＰＣＲ Ｍａｓｔｅｒ Ｍｉｘ（１２μＬ）を３８４ウェルプレートの各ウェルに移し、鋳型（標準溶液及びＱＣサンプル、８μＬ）を各ウェルに添加し、混合した（合計２０μＬ）。試験は３ウェルで実施した（ｎ＝３）。
(2) PCR Master Mix (12 μL) was transferred to each well of a 384-well plate, template (standard solution and QC sample, 8 μL) was added to each well and mixed (20 μL in total). Tests were performed in triplicate wells (n=3).
（３）３８４ウェルプレートを密閉し、短時間遠心分離した。ｒｅａｌ－ｔｉｍｅ ＰＣＲ Ｓｙｓｔｅｍ ＬｉｇｈｔＣｙｃｌｅｒ（商標） ４８０ＩＩを用いて下記の条件でリアルタイムＰＣＲを行った。サイクル２のステップ３の終了時に蛍光強度を測定した。
(3) The 384-well plate was sealed and centrifuged briefly. Using real-time PCR System LightCycler (trademark) 480II, real-time PCR was performed under the following conditions. Fluorescence intensity was measured at the end of step 3 of cycle 2.
５．データ分析
Ｃｐ値をＡｂｓ Ｑｕａｎｔ／２ｎｄ Ｄｅｒｉｖａｔｉｖｅ Ｍａｘ法（交点）によって算出した。各標準溶液の濃度及びＣｐ値を用いて標準曲線をプロットした後、平均二乗誤差（ＭＳＥ）を算出した。標準曲線は、非線形（多項式）回帰直線である。標準溶液のＣｐ値及び標準曲線を用いて逆回帰値を算出した。サンプルのＣｐ値及び標準曲線を用いてサンプル濃度を算出した。逆回帰値の平均、ＣＶ及びサンプル濃度を算出した。逆回帰値の平均及び理論値を用いて、次の式によりＲＥ（相対誤差）（％）を算出した。
ＲＥ（％）＝（｛逆回帰値の平均（コピー／μＬ）／理論値（コピー／μＬ）｝－１）×１００ＣＶ（％）＝（ＳＤ／平均）×１００ 5. Data Analysis Cp values were calculated by the Abs Quant/2nd Derivative Max method (intersection point). After plotting the standard curve using the concentrations and Cp values of each standard solution, the mean squared error (MSE) was calculated. The standard curve is a non-linear (polynomial) regression line. Reverse regression values were calculated using the Cp values of the standard solutions and the standard curve. Sample concentrations were calculated using the Cp values of the samples and the standard curve. Mean of inverse regression values, CV and sample concentration were calculated. RE (relative error) (%) was calculated by the following formula using the average of the inverse regression values and the theoretical value.
RE (%) = ({mean of inverse regression values (copies/μL)/theoretical value (copies/μL)}−1)×100 CV (%)=(SD/mean)×100
Ｃｐ値をＡｂｓ Ｑｕａｎｔ／２ｎｄ Ｄｅｒｉｖａｔｉｖｅ Ｍａｘ法（交点）によって算出した。各標準溶液の濃度及びＣｐ値を用いて標準曲線をプロットした後、平均二乗誤差（ＭＳＥ）を算出した。標準曲線は、非線形（多項式）回帰直線である。標準溶液のＣｐ値及び標準曲線を用いて逆回帰値を算出した。サンプルのＣｐ値及び標準曲線を用いてサンプル濃度を算出した。逆回帰値の平均、ＣＶ及びサンプル濃度を算出した。逆回帰値の平均及び理論値を用いて、次の式によりＲＥ（相対誤差）（％）を算出した。
ＲＥ（％）＝（｛逆回帰値の平均（コピー／μＬ）／理論値（コピー／μＬ）｝－１）×１００ＣＶ（％）＝（ＳＤ／平均）×１００ 5. Data Analysis Cp values were calculated by the Abs Quant/2nd Derivative Max method (intersection point). After plotting the standard curve using the concentrations and Cp values of each standard solution, the mean squared error (MSE) was calculated. The standard curve is a non-linear (polynomial) regression line. Reverse regression values were calculated using the Cp values of the standard solutions and the standard curve. Sample concentrations were calculated using the Cp values of the samples and the standard curve. Mean of inverse regression values, CV and sample concentration were calculated. RE (relative error) (%) was calculated by the following formula using the average of the inverse regression values and the theoretical value.
RE (%) = ({mean of inverse regression values (copies/μL)/theoretical value (copies/μL)}−1)×100 CV (%)=(SD/mean)×100
Ｃｐ値、ＭＳＥ、逆回帰値及びサンプル濃度は、リアルタイムＰＣＲシステム用の分析ソフトウェア（ＬｉｇｈｔＣｙｃｌｅｒ（商標） ４８０ソフトウェア、Roche Diagnostics）を用いて算出した。
Cp values, MSE, inverse regression values and sample concentrations were calculated using analysis software for real-time PCR systems (LightCycler™ 480 software, Roche Diagnostics).
６． 検証項目
ｑＰＣＲは、１回の反復試験当たり３ウェルで実施した。 6. Validation Items qPCR was performed in 3 wells per replicate.
ｑＰＣＲは、１回の反復試験当たり３ウェルで実施した。 6. Validation Items qPCR was performed in 3 wells per replicate.
６－１ 較正曲線
ＴａｑＭａｎ ｑＰＣＲアッセイの直線性を決定するために、Ｃ１～Ｃ１０を用いて標準曲線を作成した。
較正標準の許容基準は、以下の通りであった：
（１）ＭＳＥが０．２未満。
（２）逆回帰値のＣＶが１０点中少なくとも９点で３０％未満（Ｃ１及びＣ１０が３０％未満であるものとする）。
（３）逆回帰値のＲＥが１０点中少なくとも９点で±３０％以内（Ｃ１及びＣ１０が±３０％以内であるものとする）。
（４）ＮＴＣのＣｐ値が４５．００であるか、又はＣ１０のＣｐ値よりも明らかに大きいものとする。 6-1 Calibration Curve To determine the linearity of the TaqMan qPCR assay, C1-C10 were used to generate a standard curve.
Acceptance criteria for calibration standards were as follows:
(1) MSE less than 0.2.
(2) The CV of the inverse regression value is less than 30% on at least 9 points out of 10 (C1 and C10 shall be less than 30%).
(3) RE of the inverse regression value is within ±30% at least 9 points out of 10 (C1 and C10 shall be within ±30%).
(4) The Cp value of NTC is 45.00 or clearly greater than the Cp value of C10.
ＴａｑＭａｎ ｑＰＣＲアッセイの直線性を決定するために、Ｃ１～Ｃ１０を用いて標準曲線を作成した。
較正標準の許容基準は、以下の通りであった：
（１）ＭＳＥが０．２未満。
（２）逆回帰値のＣＶが１０点中少なくとも９点で３０％未満（Ｃ１及びＣ１０が３０％未満であるものとする）。
（３）逆回帰値のＲＥが１０点中少なくとも９点で±３０％以内（Ｃ１及びＣ１０が±３０％以内であるものとする）。
（４）ＮＴＣのＣｐ値が４５．００であるか、又はＣ１０のＣｐ値よりも明らかに大きいものとする。 6-1 Calibration Curve To determine the linearity of the TaqMan qPCR assay, C1-C10 were used to generate a standard curve.
Acceptance criteria for calibration standards were as follows:
(1) MSE less than 0.2.
(2) The CV of the inverse regression value is less than 30% on at least 9 points out of 10 (C1 and C10 shall be less than 30%).
(3) RE of the inverse regression value is within ±30% at least 9 points out of 10 (C1 and C10 shall be within ±30%).
(4) The Cp value of NTC is 45.00 or clearly greater than the Cp value of C10.
６－２ バッチ内再現性
Ｈ、Ｍ、Ｌ及びＬＬを１つのレベルにつき３回の反復試験で分析することによってＣＶ及びＲＥを評価した。
ＣＶ及びＲＥの許容基準は、以下の通りであった：
ＱＣサンプルのＣＶが３０％未満。
ＱＣサンプル値のＲＥが±３０％以内。 6-2 Intra-batch reproducibility CV and RE were evaluated by analyzing H, M, L and LL with three replicates per level.
Acceptance criteria for CV and RE were as follows:
CV of QC sample is less than 30%.
RE of QC sample value within ±30%.
Ｈ、Ｍ、Ｌ及びＬＬを１つのレベルにつき３回の反復試験で分析することによってＣＶ及びＲＥを評価した。
ＣＶ及びＲＥの許容基準は、以下の通りであった：
ＱＣサンプルのＣＶが３０％未満。
ＱＣサンプル値のＲＥが±３０％以内。 6-2 Intra-batch reproducibility CV and RE were evaluated by analyzing H, M, L and LL with three replicates per level.
Acceptance criteria for CV and RE were as follows:
CV of QC sample is less than 30%.
RE of QC sample value within ±30%.
６－３ バッチ間再現性
Ｈ、Ｍ、Ｌ及びＬＬを１つのレベルにつき３回の反復試験で分析することによってＣＶ及びＲＥを評価した。
バッチ間再現性は、３つの異なるバッチからの４つの濃度のＱＣサンプルによって決定した。バッチ間再現性結果の使用が許容可能であった。
ＣＶ及びＲＥの許容基準は、以下の通りであった：
ＱＣサンプルのＣＶが３０％未満。
ＱＣサンプル値のＲＥが±３０％以内。 6-3 Batch-to-Batch Reproducibility CV and RE were evaluated by analyzing H, M, L and LL in triplicate per level.
Batch-to-batch reproducibility was determined with four concentrations of QC samples from three different batches. The use of batch-to-batch reproducibility results was acceptable.
Acceptance criteria for CV and RE were as follows:
CV of QC sample is less than 30%.
RE of QC sample value within ±30%.
Ｈ、Ｍ、Ｌ及びＬＬを１つのレベルにつき３回の反復試験で分析することによってＣＶ及びＲＥを評価した。
バッチ間再現性は、３つの異なるバッチからの４つの濃度のＱＣサンプルによって決定した。バッチ間再現性結果の使用が許容可能であった。
ＣＶ及びＲＥの許容基準は、以下の通りであった：
ＱＣサンプルのＣＶが３０％未満。
ＱＣサンプル値のＲＥが±３０％以内。 6-3 Batch-to-Batch Reproducibility CV and RE were evaluated by analyzing H, M, L and LL in triplicate per level.
Batch-to-batch reproducibility was determined with four concentrations of QC samples from three different batches. The use of batch-to-batch reproducibility results was acceptable.
Acceptance criteria for CV and RE were as follows:
CV of QC sample is less than 30%.
RE of QC sample value within ±30%.
６－４ マトリックス効果
Ｈ、Ｍ、Ｌ及びＬＬを１つのレベルにつき１回の反復試験で分析することによってＣＶ及びＲＥを評価した。
マトリックス効果は、６人の異なるドナーからの４つの濃度のＱＣサンプルによって決定した。
ＣＶ及びＲＥの許容基準は、以下の通りであった：
ＱＣサンプルのＣＶが３０％未満。
ＱＣサンプル値のＲＥが±３０％以内。 6-4 Matrix Effects CV and RE were assessed by analyzing H, M, L and LL with one replicate per level.
Matrix effects were determined with 4 concentrations of QC samples from 6 different donors.
Acceptance criteria for CV and RE were as follows:
CV of QC sample is less than 30%.
RE of QC sample value within ±30%.
Ｈ、Ｍ、Ｌ及びＬＬを１つのレベルにつき１回の反復試験で分析することによってＣＶ及びＲＥを評価した。
マトリックス効果は、６人の異なるドナーからの４つの濃度のＱＣサンプルによって決定した。
ＣＶ及びＲＥの許容基準は、以下の通りであった：
ＱＣサンプルのＣＶが３０％未満。
ＱＣサンプル値のＲＥが±３０％以内。 6-4 Matrix Effects CV and RE were assessed by analyzing H, M, L and LL with one replicate per level.
Matrix effects were determined with 4 concentrations of QC samples from 6 different donors.
Acceptance criteria for CV and RE were as follows:
CV of QC sample is less than 30%.
RE of QC sample value within ±30%.
６－５ 選択性
６人のドナーから１回の反復試験でＢＬのＣｐ値を決定することにより、選択性についてｑＰＣＲを行った。
選択性の許容基準は、以下の通りであった：
Ｃｐ値が決定されないか、又はＣ１０のＣｐ値よりも明らかに大きい。 6-5 Selectivity qPCR was performed for selectivity by determining BL Cp values in one replicate from 6 donors.
Selectivity acceptance criteria were as follows:
The Cp value is either not determined or is clearly greater than that of C10.
６人のドナーから１回の反復試験でＢＬのＣｐ値を決定することにより、選択性についてｑＰＣＲを行った。
選択性の許容基準は、以下の通りであった：
Ｃｐ値が決定されないか、又はＣ１０のＣｐ値よりも明らかに大きい。 6-5 Selectivity qPCR was performed for selectivity by determining BL Cp values in one replicate from 6 donors.
Selectivity acceptance criteria were as follows:
The Cp value is either not determined or is clearly greater than that of C10.
６－６ 凍結融解安定性
Ｈ及びＬを１つのレベルにつき１回の反復試験で分析することによってＣＶ及びＲＥを評価した。ＱＣサンプルを－２０℃で０回、１回及び３回凍結融解した後、ｑＰＣＲを行って凍結融解安定性を確認した。融解操作は、ＱＣサンプルをＲＴで静置することによって行った。
凍結融解安定性の許容基準は、以下の通りであった：
ＱＣサンプルのＣＶが３０％未満である。
ＱＣサンプル値のＲＥが±３０％以内である。 6-6 Freeze-Thaw Stability CV and RE were assessed by analyzing H and L with one replicate per level. QC samples were freeze-thawed 0, 1 and 3 times at −20° C. before performing qPCR to confirm freeze-thaw stability. The melting operation was performed by standing the QC samples at RT.
Acceptance criteria for freeze-thaw stability were as follows:
QC samples have a CV less than 30%.
The RE of the QC sample values is within ±30%.
Ｈ及びＬを１つのレベルにつき１回の反復試験で分析することによってＣＶ及びＲＥを評価した。ＱＣサンプルを－２０℃で０回、１回及び３回凍結融解した後、ｑＰＣＲを行って凍結融解安定性を確認した。融解操作は、ＱＣサンプルをＲＴで静置することによって行った。
凍結融解安定性の許容基準は、以下の通りであった：
ＱＣサンプルのＣＶが３０％未満である。
ＱＣサンプル値のＲＥが±３０％以内である。 6-6 Freeze-Thaw Stability CV and RE were assessed by analyzing H and L with one replicate per level. QC samples were freeze-thawed 0, 1 and 3 times at −20° C. before performing qPCR to confirm freeze-thaw stability. The melting operation was performed by standing the QC samples at RT.
Acceptance criteria for freeze-thaw stability were as follows:
QC samples have a CV less than 30%.
The RE of the QC sample values is within ±30%.
６－７ 長期貯蔵安定性
Ｈ及びＬを１つのレベルにつき１回の反復試験で分析することによってＣＶ及びＲＥを評価した。ｑＰＣＲを行い、初期濃度（０ヶ月）を確認した。ＱＣサンプルを－２０℃で１ヶ月、３ヶ月、６ヶ月及び１２ヶ月（±１週間）保持した後、ｑＰＣＲを行って長期貯蔵安定性を確認した。
長期貯蔵安定性の許容基準は、以下の通りである：
ＱＣサンプルのＣＶが３０％未満である。
ＱＣサンプル値のＲＥが±３０％以内である。 6-7 Long Term Storage Stability CV and RE were assessed by analyzing H and L with one replicate per level. qPCR was performed to confirm the initial concentration (0 months). QC samples were kept at −20° C. for 1 month, 3 months, 6 months and 12 months (±1 week) before performing qPCR to confirm long-term storage stability.
Acceptance criteria for long-term storage stability are as follows:
QC samples have a CV less than 30%.
The RE of the QC sample values is within ±30%.
Ｈ及びＬを１つのレベルにつき１回の反復試験で分析することによってＣＶ及びＲＥを評価した。ｑＰＣＲを行い、初期濃度（０ヶ月）を確認した。ＱＣサンプルを－２０℃で１ヶ月、３ヶ月、６ヶ月及び１２ヶ月（±１週間）保持した後、ｑＰＣＲを行って長期貯蔵安定性を確認した。
長期貯蔵安定性の許容基準は、以下の通りである：
ＱＣサンプルのＣＶが３０％未満である。
ＱＣサンプル値のＲＥが±３０％以内である。 6-7 Long Term Storage Stability CV and RE were assessed by analyzing H and L with one replicate per level. qPCR was performed to confirm the initial concentration (0 months). QC samples were kept at −20° C. for 1 month, 3 months, 6 months and 12 months (±1 week) before performing qPCR to confirm long-term storage stability.
Acceptance criteria for long-term storage stability are as follows:
QC samples have a CV less than 30%.
The RE of the QC sample values is within ±30%.
７．測定結果
７－１ 較正曲線
較正曲線の各濃度（Ｃ１～Ｃ１０）について、ＣＶは０％～１７．７％の範囲となり、ＲＥは－１０．３％～９．７％の範囲であった。平均二乗誤差は、０．００４１４～０．００８２０であった。ＮＴＣは４５．００サイクルまで増幅されなかった。
較正曲線は全ての基準を満たしたため、較正曲線は許容可能である。 7. Measurement Results 7-1 Calibration Curve For each concentration (C1 to C10) of the calibration curve, the CV ranged from 0% to 17.7% and the RE ranged from -10.3% to 9.7%. The mean squared error ranged from 0.00414 to 0.00820. NTC was not amplified until 45.00 cycles.
The calibration curve is acceptable because it met all criteria.
７－１ 較正曲線
較正曲線の各濃度（Ｃ１～Ｃ１０）について、ＣＶは０％～１７．７％の範囲となり、ＲＥは－１０．３％～９．７％の範囲であった。平均二乗誤差は、０．００４１４～０．００８２０であった。ＮＴＣは４５．００サイクルまで増幅されなかった。
較正曲線は全ての基準を満たしたため、較正曲線は許容可能である。 7. Measurement Results 7-1 Calibration Curve For each concentration (C1 to C10) of the calibration curve, the CV ranged from 0% to 17.7% and the RE ranged from -10.3% to 9.7%. The mean squared error ranged from 0.00414 to 0.00820. NTC was not amplified until 45.00 cycles.
The calibration curve is acceptable because it met all criteria.
７－２ バッチ内再現性
各ＱＣサンプル（Ｈ、Ｍ、Ｌ及びＬＬ）について、ＣＶは０．４％～２０．０％の範囲であり、ＲＥは－６．５％～１６．０％の範囲であった。ＱＣサンプルが全ての許容基準を満たしたため、バッチ内再現性は許容可能である。 7-2 Intra-batch reproducibility For each QC sample (H, M, L and LL), the CV ranged from 0.4% to 20.0% and the RE from -6.5% to 16.0%. was in the range. The intra-batch reproducibility is acceptable as the QC samples met all acceptance criteria.
各ＱＣサンプル（Ｈ、Ｍ、Ｌ及びＬＬ）について、ＣＶは０．４％～２０．０％の範囲であり、ＲＥは－６．５％～１６．０％の範囲であった。ＱＣサンプルが全ての許容基準を満たしたため、バッチ内再現性は許容可能である。 7-2 Intra-batch reproducibility For each QC sample (H, M, L and LL), the CV ranged from 0.4% to 20.0% and the RE from -6.5% to 16.0%. was in the range. The intra-batch reproducibility is acceptable as the QC samples met all acceptance criteria.
７－３ バッチ間再現性
各ＱＣサンプル（Ｈ、Ｍ、Ｌ及びＬＬ）について、ＣＶは５．２％～９．２％の範囲であり、ＲＥは－２．９％～５．２％の範囲であった。ＱＣサンプルが全ての許容基準を満たしたため、バッチ間再現性は許容可能である。 7-3 Batch-to-batch reproducibility For each QC sample (H, M, L and LL), the CV ranged from 5.2% to 9.2% and the RE from -2.9% to 5.2%. was in the range. Batch-to-batch reproducibility is acceptable as the QC samples met all acceptance criteria.
各ＱＣサンプル（Ｈ、Ｍ、Ｌ及びＬＬ）について、ＣＶは５．２％～９．２％の範囲であり、ＲＥは－２．９％～５．２％の範囲であった。ＱＣサンプルが全ての許容基準を満たしたため、バッチ間再現性は許容可能である。 7-3 Batch-to-batch reproducibility For each QC sample (H, M, L and LL), the CV ranged from 5.2% to 9.2% and the RE from -2.9% to 5.2%. was in the range. Batch-to-batch reproducibility is acceptable as the QC samples met all acceptance criteria.
７－４ マトリックス効果
各ＱＣサンプル（Ｈ、Ｍ、Ｌ及びＬＬ）について、ＣＶは０．５％～１３．２％の範囲であり、ＲＥは－１２．５％～５．０％の範囲であった。ＱＣサンプルが全ての許容基準を満たしたため、マトリックス効果は許容可能である。 7-4 Matrix Effects For each QC sample (H, M, L and LL), CV ranged from 0.5% to 13.2% and RE ranged from -12.5% to 5.0%. there were. Matrix effects are acceptable as the QC samples met all acceptance criteria.
各ＱＣサンプル（Ｈ、Ｍ、Ｌ及びＬＬ）について、ＣＶは０．５％～１３．２％の範囲であり、ＲＥは－１２．５％～５．０％の範囲であった。ＱＣサンプルが全ての許容基準を満たしたため、マトリックス効果は許容可能である。 7-4 Matrix Effects For each QC sample (H, M, L and LL), CV ranged from 0.5% to 13.2% and RE ranged from -12.5% to 5.0%. there were. Matrix effects are acceptable as the QC samples met all acceptance criteria.
７－５ 選択性
６人のドナーからの各ＢＬは増幅しなかった。ＢＬが許容基準を満たしたため、選択性は許容可能である。 7-5 Selectivity Each BL from 6 donors did not amplify. The selectivity is acceptable because the BL met the acceptance criteria.
６人のドナーからの各ＢＬは増幅しなかった。ＢＬが許容基準を満たしたため、選択性は許容可能である。 7-5 Selectivity Each BL from 6 donors did not amplify. The selectivity is acceptable because the BL met the acceptance criteria.
７－６ 凍結融解安定性
各ＱＣサンプル（Ｈ及びＬ）について、ＣＶは０．９％～３．０％の範囲であり、ＲＥは－４．１％～１８．０％の範囲であった。ＱＣサンプルが基準を満たしたため、凍結融解安定性は、－２０℃／室温で３回まで許容可能であると決定された。 7-6 Freeze-Thaw Stability For each QC sample (H and L), CV ranged from 0.9% to 3.0% and RE ranged from -4.1% to 18.0%. . Freeze-thaw stability was determined to be acceptable up to 3 times at −20° C./room temperature as the QC samples met the criteria.
各ＱＣサンプル（Ｈ及びＬ）について、ＣＶは０．９％～３．０％の範囲であり、ＲＥは－４．１％～１８．０％の範囲であった。ＱＣサンプルが基準を満たしたため、凍結融解安定性は、－２０℃／室温で３回まで許容可能であると決定された。 7-6 Freeze-Thaw Stability For each QC sample (H and L), CV ranged from 0.9% to 3.0% and RE ranged from -4.1% to 18.0%. . Freeze-thaw stability was determined to be acceptable up to 3 times at −20° C./room temperature as the QC samples met the criteria.
７－７ 長期貯蔵安定性
ＱＣサンプル（ＱＣＨ及びＱＣＬ）の初期濃度のＣＶ及びＲＥは、許容基準を満たした。 7-7 Long Term Storage Stability The initial concentration CV and RE of the QC samples (QCH and QCL) met the acceptance criteria.
ＱＣサンプル（ＱＣＨ及びＱＣＬ）の初期濃度のＣＶ及びＲＥは、許容基準を満たした。 7-7 Long Term Storage Stability The initial concentration CV and RE of the QC samples (QCH and QCL) met the acceptance criteria.
８． 結論
以上の結果から、上記１～４に示すアッセイ法が、ＣＤ２８及びＣＤ３ζを含むＣＡＲ－Ｔ細胞の測定に適していることが確認された。 8. Conclusion From the above results, it was confirmed that the assay methods shown in 1 to 4 above are suitable for measuring CAR-T cells containing CD28 and CD3ζ.
以上の結果から、上記１～４に示すアッセイ法が、ＣＤ２８及びＣＤ３ζを含むＣＡＲ－Ｔ細胞の測定に適していることが確認された。 8. Conclusion From the above results, it was confirmed that the assay methods shown in 1 to 4 above are suitable for measuring CAR-T cells containing CD28 and CD3ζ.
以上の結果は、臨床を含めた生体内においてaITGB7 scFV－CD28－CD3ζを構成するaITGB7（MMG49）CAR-Tを投与した場合に、組織サンプル中のCAR-T量を測定できることを示している。
The above results show that the amount of CAR-T in tissue samples can be measured when aITGB7 (MMG49) CAR-T, which constitutes aITGB7 scFV-CD28-CD3ζ, is administered in vivo, including clinically.
本発明は、以上の実施例の内容に制限されるものではない。
The present invention is not limited to the contents of the above examples.
Claims (12)
- 配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーを含む、プライマーセット。 A primer set comprising a primer having the base sequence of SEQ ID NO: 1 and a primer having the base sequence of SEQ ID NO: 2.
- 前記プライマーセットが、キメラ抗原受容体をコードする核酸を検出するために用いられる、請求項１に記載のプライマーセット。 2. The primer set of claim 1, wherein said primer set is used to detect nucleic acids encoding chimeric antigen receptors.
- 前記核酸が、配列番号１の塩基配列またはその相補的な塩基配列及び配列番号２の塩基配列またはその相補的な塩基配列からなる、請求項２に記載のプライマーセット。 3. The primer set according to claim 2, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence and the nucleotide sequence of SEQ ID NO: 2 or its complementary nucleotide sequence.
- 前記キメラ抗原受容体がＣＤ２８及びＣＤ３ζを含む、請求項２又は３に記載のプライマーセット。 4. The primer set of claim 2 or 3, wherein said chimeric antigen receptor comprises CD28 and CD3zeta.
- 前記検出が定量ＰＣＲを用いて行われるものであって、配列番号１の塩基配列を有するプライマーがフォワードプライマー、配列番号２の塩基配列を有するプライマーがリバースプライマーとして使用される、請求項２～４のいずれかに記載のプライマーセット。 Claims 2 to 4, wherein the detection is performed using quantitative PCR, the primer having the nucleotide sequence of SEQ ID NO: 1 is used as a forward primer, and the primer having the nucleotide sequence of SEQ ID NO: 2 is used as a reverse primer. A primer set according to any one of
- 前記キメラ抗原受容体が、多発性骨髄腫に対するキメラ抗原受容体である、請求項２～５のいずれかに記載のプライマーセット。 The primer set according to any one of claims 2 to 5, wherein said chimeric antigen receptor is a chimeric antigen receptor for multiple myeloma.
- 前記核酸が、ＰＢＭＣ、Ｔ細胞、ＮＫ細胞、及びＮＫ－Ｔ細胞から選択される一種以上の細胞に含まれる、請求項２～６のいずれかに記載のプライマーセット。 The primer set according to any one of claims 2 to 6, wherein the nucleic acid is contained in one or more cells selected from PBMCs, T cells, NK cells, and NK-T cells.
- 請求項１～７のいずれかに記載のプラマーセットを含む、キメラ抗原受容体をコードする核酸を検出するためのキット。 A kit for detecting a nucleic acid encoding a chimeric antigen receptor, comprising a primer set according to any one of claims 1-7.
- 更に、配列番号９の塩基配列を有するプローブを含有し、キメラ抗原受容体をコードする核酸を検出又は定量するために使用される、請求項８に記載されるキット。 9. The kit according to claim 8, further comprising a probe having the base sequence of SEQ ID NO: 9 and used for detecting or quantifying a nucleic acid encoding a chimeric antigen receptor.
- 前記キメラ抗原受容体が生体試料中に存在する、請求項２～７のいずれかに記載のプライマーセット又は請求項８若しくは９に記載のキット。 The primer set according to any one of claims 2 to 7 or the kit according to claim 8 or 9, wherein said chimeric antigen receptor is present in a biological sample.
- 生体試料中のＣＤ２８及びＣＤ３ζを発現するキメラ抗原受容体細胞を検出するための方法であって、
（１）ＣＤ２８及びＣＤ３ζを発現するキメラ抗原受容体細胞が投与された患者から取得した生体試料からＤＮＡを抽出する工程、及び
（２）抽出したＤＮＡを鋳型として、配列番号１の塩基配列を有するプライマー及び配列番号２の塩基配列を有するプライマーと、配列番号９の塩基配列を有するプローブを組み合わせて、定量ＰＣＲを行う工程、
を含む、キメラ抗原受容体細胞の検出方法。 A method for detecting chimeric antigen receptor cells expressing CD28 and CD3ζ in a biological sample, comprising:
(1) a step of extracting DNA from a biological sample obtained from a patient to whom chimeric antigen receptor cells expressing CD28 and CD3ζ were administered; and (2) using the extracted DNA as a template, having the nucleotide sequence of SEQ ID NO: 1 A step of performing quantitative PCR by combining a primer and a primer having the nucleotide sequence of SEQ ID NO: 2 with a probe having the nucleotide sequence of SEQ ID NO: 9;
A method for detecting chimeric antigen receptor cells, comprising: - 前記患者が多発性骨髄腫の治療を必要とする、請求項１１に記載の方法。 The method of claim 11, wherein said patient is in need of treatment for multiple myeloma.
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| WO2017026331A1 (en) * | 2015-08-11 | 2017-02-16 | 国立大学法人大阪大学 | Antibody |
| CN109971835A (en) * | 2017-12-28 | 2019-07-05 | 上海细胞治疗研究院 | A kind of probe that lock nucleic acid is modified and a kind of method for measuring CAR copy number |
| CN109971836A (en) * | 2017-12-28 | 2019-07-05 | 上海细胞治疗研究院 | The method and kit of double fluorescent quantitative PCR measurement CAR copy number |
| US20200085871A1 (en) * | 2017-03-17 | 2020-03-19 | University Of Tennessee Research Foundation | Methods of using cytotoxic t cells for treatment of autoimmune diseases |
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| WO2017026331A1 (en) * | 2015-08-11 | 2017-02-16 | 国立大学法人大阪大学 | Antibody |
| US20200085871A1 (en) * | 2017-03-17 | 2020-03-19 | University Of Tennessee Research Foundation | Methods of using cytotoxic t cells for treatment of autoimmune diseases |
| CN109971835A (en) * | 2017-12-28 | 2019-07-05 | 上海细胞治疗研究院 | A kind of probe that lock nucleic acid is modified and a kind of method for measuring CAR copy number |
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