WO2023184616A1 - Procédé de détection d'une séquence de tcr clonée et son utilisation - Google Patents

Procédé de détection d'une séquence de tcr clonée et son utilisation Download PDF

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WO2023184616A1
WO2023184616A1 PCT/CN2022/087532 CN2022087532W WO2023184616A1 WO 2023184616 A1 WO2023184616 A1 WO 2023184616A1 CN 2022087532 W CN2022087532 W CN 2022087532W WO 2023184616 A1 WO2023184616 A1 WO 2023184616A1
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cancer
tcr
cell
cells
sequence
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王文博
冯爱华
王鹏
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立凌生物制药苏州有限公司
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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001111—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells

Definitions

  • the invention relates to the field of medical technology, and in particular to a detection method for cloning TCR sequences and its application.
  • T cell receptor is a characteristic marker on the surface of all T cells. It specifically recognizes the antigen peptide-MHC complex on antigen-presenting cells, thereby triggering a T cell immune response. Since TCR molecules determine the antigen recognition specificity of T cells, if tumor antigen-specific TCRs are transferred into ordinary T cells, the tumor antigen recognition ability of the T cells can be enhanced. After activation and proliferation in vitro, they can be injected into the patient's body. exert anti-tumor effect. Therefore, the method of introducing TCR genes can be used to easily obtain a large number of T cells that recognize specific antigens. T cells modified by TCR genes are called TCR-T.
  • TCR-T has become a research hotspot in tumor immunotherapy and is used clinically. Experiments have shown good therapeutic effects.
  • TCR-T cell therapy T Cell Receptor-Gene Engineered T Cells
  • TCR-T cell therapy by screening and identifying TCR sequences that can specifically bind to target antigens, and using genetic engineering methods to transfer them into T cells derived from the patient's peripheral blood (or Allogeneic T cells), and then the modified T cells are infused back into the patient's body so that they can specifically recognize and kill tumor cells expressing antigens, thereby achieving the purpose of treating tumors.
  • TCR is a heterodimer composed of two peptide chains, ⁇ and ⁇ . Each peptide chain is divided into variable region (V region), constant region (C region), transmembrane region and cytoplasmic region; Its cytoplasmic region is very short, and signal transmission is mainly carried out through CD3 molecules that are non-covalently bound to it.
  • TCR molecules belong to the immunoglobulin superfamily, and their antigen specificity exists in the V region; each V region has three hypervariable regions, CDR1, CDR2, and CDR3. Among them, CDR3 has the largest variation, which directly determines the antigen-binding specificity of the TCR. When the TCR recognizes the MHC-antigen peptide complex, CDR3 directly binds to the antigen peptide.
  • TCR screening technology is the core part of TCR-related drug development.
  • current TCR screening is costly, time-consuming, and low-efficiency.
  • acquisition of paired TCR full-length genes is the rate-limiting step in the entire process, which seriously affects the research and development of TCR-related drugs.
  • the third method is to perform single cell sorting on the T cell population, and then directly perform 5'RACE to amplify the full length of the TCR variable region at the single cell level, and then perform PCR amplification to obtain the full length of the TCR gene.
  • This method is relatively simple, it also requires a separate reaction system for each cell, consumes a lot of reagents, and the cost of the kit is very high, and it cannot amplify TCR genes in large quantities (An immunodominant NP105-113-B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease. Nat Immunol. 2022;01;23(1):50–61.).
  • the fourth method is to use single-cell DNA barcode to label each T cell in the T cell population, and then use high-throughput sequencing to obtain paired TCR full-length sequence information.
  • the paired TCRs determined by screening were gene synthesized to obtain full-length TCR genes.
  • this method can obtain paired TCR sequences in large quantities at low cost, it can only obtain sequence information and cannot directly obtain the full-length gene fragment of the TCR variable region. The cost of subsequent gene synthesis is high and it is impossible to synthesize TCRs in large quantities (Direct identification of neoantigenspecific TCRs from tumor specimens by high-throughput single-cell sequencing. Journal for ImmunoTherapy of Cancer 2021;9:e002595.).
  • Tumor-reactive T cells within tumors function through T cell receptors, but they are generally in an exhausted state and are difficult to use directly. How to quickly and cheaply clone the TCR sequences of these tumor-reactive T cells and then transduce them into primary T cells to prepare T cells in better condition is a technology expected to break through in the current field of tumor treatment.
  • the purpose of the present invention is to provide a fast and low-cost method for detecting the full-length sequence of cloned paired TCR and its application in the field of treatment, especially in the field of personalized treatment.
  • the present invention provides a method for cloning paired TCR sequences.
  • the method includes the following steps: sorting and capturing single tumor-reactive T cells in tumor tissues, extracting and labeling the mRNA of single cells, reverse transcribing and Construct full-length cDNA, cDNA-specific PCR amplification to obtain the full-length cDNA transcriptome, first circularization after enrichment, specific amplification of TCR sequence, second circularization after enrichment, and enrichment again.
  • the products are ligated into an expression vector to obtain a full-length clone of the TCR, and the library sequence is compared to obtain the paired TCR sequence.
  • TSO is added to the reverse transcription system during mRNA reverse transcription, and amplification completes the enrichment of the cDNA full-length transcriptome.
  • the enrichment of the full-length transcriptome is completed by amplifying the constant region sequence at the 5' end of the single-cell labeled magnetic beads and the TSO sequence added during the reverse transcription process.
  • the first circularization in the method is to circularize the obtained amplification product, using the constant sequence on the single-cell labeling magnetic beads and the TCR-specific primer (end of the constant region) ) performs specific amplification of TCR sequences and completes TCR enrichment.
  • the second circularization in the method is: after the obtained amplification product is subjected to a second circularization process, forward and reverse primers designed according to the constant sequence of single-cell labeling magnetic beads are used Enrich TCR. After enrichment, the cell barcode will be connected to the TCR constant region and located at the 3' end of the enriched product.
  • the paired TCR sequences include TCR-alpha and TCR-beta series.
  • the method for sorting single tumor-reactive T cells is as follows: tumor tissue is digested into single cells and prepared into a single cell suspension, and then injected into a microfluidic chip to obtain single tumor-reactive T cells.
  • the method for extracting and labeling the mRNA of a single cell is: after the single tumor-reactive T cell is separated, single-cell labeling magnetic beads are added to the microwell to complete the capture and labeling of the mRNA of the single cell. .
  • the surface of the single-cell labeling magnetic beads has single-stranded DNA oligo carrying two constant sequences, a specific barcode sequence and poly dT.
  • the single cell labeling magnetic beads carry different barcodes for labeling individual T cells, and poly dT is used to capture total mRNA and serve as a reverse transcription primer.
  • the library sequence comparison is: after cloning the full length of TCR-alpha and TCR-beta respectively, single clones are selected for cell barcode comparison, and TCR-alpha and TCR-beta with the same cell barcode are The TCR-beta sequence is a paired TCR.
  • a method for sorting single tumor-reactive T cells is to stain and label the single cell suspension with an antibody with a fluorescent label and then perform sorting using a flow cytometry sorter.
  • the method for sorting single tumor-reactive T cells is: digest the tumor tissue into single cells, use the antibody CXCL13-APC with a fluorescent label to stain and label the single cell suspension, and pass the single cells through The CXCL13-positive T cells in the tumor single cell suspension are sorted by a flow sorter and are tumor-reactive T cells.
  • the paired TCR sequence of the present invention is obtained by the following method:
  • single-cell labeling magnetic beads to the microwell, and use single-cell labeling magnetic beads to capture and label the mRNA of a single cell.
  • the surface of single-cell labeling magnetic beads carries two constant sequences, a specific barcode sequence and poly dT single-stranded DNA oligo. Each magnetic bead carries a different barcode for labeling individual T cells, and polydT is used to capture total mRNA and serve as a reverse transcription primer.
  • TSO Tempor switch oligo
  • TCR is enriched using forward and reverse primers designed based on the constant sequence of single-cell labeled magnetic beads. After enrichment, the cell barcode will be connected to the TCR constant region. Located at the 3' end of the enriched product. Finally, the enriched product of TCR was ligated into the expression vector to complete the full-length cloning of TCR.
  • TCR-alpha and TCR-beta sequences with the same cell barcode are paired TCRs.
  • the present invention provides a TCR-T cell.
  • the invention provides a TCR-T cell, which is obtained by injecting the paired TCR sequence obtained by the method of the invention into the corresponding T cell through bioengineering technology.
  • the corresponding T cells refer to the individual's own T cells or allogeneic T cells.
  • the allogeneic T cells are T cells of different individuals of the same species and/or T cells of different species.
  • the allogeneic T cells may be T cells from different human individuals or T cells from other animal bodies.
  • the present invention provides a pharmaceutical composition.
  • a pharmaceutical composition containing the TCR-T cells obtained in the second aspect of the present invention is obtained in the second aspect of the present invention.
  • the pharmaceutical composition contains a pharmaceutically acceptable carrier.
  • the pharmaceutical composition contains a pharmaceutically acceptable carrier.
  • the carrier includes any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc. that are compatible with pharmaceutical administration.
  • Preferred examples of carriers or diluents involved in the dispersion medium include (but are not limited to) water, saline, Ringer's solution, dextrose solution and human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of these media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, its use is contemplated in the compositions. Supplementary active compounds can also be incorporated into the compositions.
  • the pharmaceutical composition is prepared into corresponding pharmaceutical preparations, which include but are not limited to intravenous, intradermal, subcutaneous, oral, transdermal, transmucosal and rectal administration and injection.
  • the pharmaceutical composition contains a second active agent different from TCR-T cells, and the second active agent includes drugs with anti-tumor effects, drugs that improve the patient's resistance, and/ Or drugs that increase patient tolerance, etc.
  • the pharmaceutical composition provided by the third aspect of the present invention is used to treat T cell-related diseases, including infectious diseases, tumors, autoimmune diseases, and organ transplantation.
  • the invention provides a diagnostic and/or assessment preparation.
  • a diagnostic and/or evaluation preparation containing the TCR-T cells obtained in the second aspect of the present invention is obtained in the second aspect of the present invention.
  • the preparation includes auxiliary materials, and the auxiliary materials include carriers or diluents;
  • the carrier or diluent is: any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents that are compatible with TCR-T cells.
  • the preparation is prepared into a preparation box.
  • the diagnostic and/or evaluation preparation is used to diagnose or evaluate a disease or event associated with T cells
  • the diseases or events include infectious diseases, tumors, autoimmune diseases, organ transplantation, etc.
  • the present invention further provides the use of the pharmaceutical composition of the third aspect of the present invention.
  • the pharmaceutical composition provided by the second aspect of the present invention is used to treat T cell-related diseases, including cancer, infectious diseases and autoimmune diseases.
  • the TCR-T cells contained in the pharmaceutical composition are from the patient's own T cells.
  • the TCR sequence in the TCR-T cells is the TCR full-length sequence information obtained after obtaining mRNA from the patient's own T cells.
  • the pharmaceutical composition is used for individual precise treatment of patients' own infectious diseases, tumors or immune diseases.
  • the cancer is selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical cancer, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, neurological Blastoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial adenoma, Burkitt lymphoma, unknown primary carcinoma, central nervous system lymphoma, cerebellar astrocytoma , cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative diseases, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma , esophageal cancer, Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer
  • the autoimmune disease is selected from the group consisting of arthritis, chronic obstructive pulmonary disease, ankylosing spondylitis, Crohn's disease, dermatomyositis, type I diabetes, endometriosis Goodpasture's syndrome, Graves' disease, Guillain-Balinese syndrome, Hashimoto's disease, hidradenitis suppurativa, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, interstitial cystitis, lupus erythematosus, Mixed connective tissue disease, morphea, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary disease cirrhosis, relapsing polychondritis, rheumatoid
  • the present invention further provides the use of the diagnostic and/or evaluation preparation provided in the fourth aspect of the present invention.
  • the diagnostic and/or evaluation preparations provided by the fourth aspect of the present invention are used for biomarkers, antibody development, drug and vaccine evaluation, immune cell differentiation tracing, immune rejection and tolerance, minimal residual disease detection, and food or other allergen detection.
  • the expression vector is a viral vector or a non-viral vector.
  • the vector contains a nucleic acid encoding TCR and a nucleic acid encoding CD8 ⁇ or CD8 ⁇ .
  • Figure 1 is a schematic diagram of single cell labeling magnetic beads in Example 2;
  • FIG. 2 is a flow chart of the TCR plasmid library construction technology in Example 2;
  • FIG. 3 shows the TCR alpha and TCR beta gene fragments obtained by electrophoresis detection and PCR amplification in Example 2;
  • FIG. 4 shows the TCR alpha constant region and TCR beta constant region gene fragments obtained by electrophoresis detection and PCR amplification in Example 2;
  • FIG. 5 shows the TCR alpha full-length fragment and the TCR beta full-length fragment obtained by PCR amplification by electrophoresis detection in Example 2;
  • FIG. 6 is a map of the TCR-expressing vector TCR-pMax in Example 2.
  • FIG. 7 shows the positive clones where the TCR alpha and TCR beta full-length fragments were connected to the expression vector by electrophoresis in Example 3;
  • Figure 8 is a flow cytometry analysis of TCR-knocked out Jurkat cells in Example 4.
  • Figure 9 shows the expression status of the paired TCR expression plasmids electroporated into cells detected by flow cytometry in Example 4.
  • tumor tissue is removed through surgery, and then the tumor tissue is digested into single cells.
  • the single cell suspension is stained and labeled using antibodies with fluorescent tags: CD3-FITC, CD45-PE and CXCL13-APC.
  • the single cells were sorted by a flow cytometer (Sony; SH800S) to sort CXCL13-positive T cells in the tumor single cell suspension.
  • This part of T cells carrying CXCL13 may be tumor-reactive T cells.
  • Other tumor-reactive T cell signatures include: CD39 (ENTPD-1) and CD200.
  • the magnetic beads carry DNA oligo ( Figure 1).
  • the DNA oligo includes: constant sequence 1, Barcode, constant sequence 2 and oligo dT sequence. Barcode is used to label individual cells. Oligo dT is used to capture mRNA. Constant sequence 1 and constant sequence 2 are used for cyclization and PCR processes. The overall technical flow of the present invention is shown in Figure 2.
  • the surface of the customized microfluidic chip has 20,000 micropores for accommodating single cells (preferably 1,000 to 150,000 pores).
  • Place the microfluidic chip on a clean petri dish use a 200 ⁇ l pipette to draw 200 ⁇ l of 100% absolute ethanol from the inlet and inject it into the chip. You can use the pipette to pump 100% absolute ethanol back and forth in the chip. Until bubbles no longer appear in the chip, remove the liquid from the sample outlet in time. Repeat flushing 2 to 3 times, remove the liquid at the sample outlet, and then draw 200 ⁇ l of 0.02% PBST (PBS contains 0.02% Tween-20) and inject it into the chip from the inlet. Control the time to within 10 seconds and remove the liquid at the sample outlet promptly. . Keep a small amount of liquid at the sample outlet, cover the petri dish, and let it stand at room temperature for later use.
  • the recovered single-cell labeling magnetic beads can be placed on a magnetic stand, and the supernatant can be aspirated to increase the single-cell labeling. After the density of cell labeling magnetic beads is injected again into the vacancy, let it sit for 10 seconds and then rinse. In the same way, if there are many vacancies for single-cell labeling magnetic beads at the outlet end of the chip, the recovered single-cell labeling magnetic beads can be injected into the outlet slot, and a pipette can be used to suck the single-cell labeling magnetic beads into the vacancies from the inlet end and let stand. Rinse again after 10 seconds.
  • step 4.8 Repeat step 4.8 once.
  • the thermal cover of the PCR instrument is 85°C: react at 50°C for 1 hour; react at 75°C for 10 minutes; store at 4°C.
  • the product is not purified and enzyme is added for enzymatic digestion. Place the PCR tube on ice and prepare the enzyme digestion system according to the following table.
  • TCR R1 primer 1.5 ⁇ l
  • Amplification Enzyme 1ul cyclization product about 20ng ddH2O variable Total To 50 ⁇ l
  • TCR R1 primer sequence GCGTCAGATGTGTATAAGAG;
  • TCR alpha-F-1 primer sequence AGTCTCTCAGCTGGTACACG;
  • TCR beta-F-1 primer sequence TCTGATGGCTCAAACACAGC.
  • the enriched products were detected by agarose gel electrophoresis, and the results are shown in Figure 3. It can be seen from the electrophoresis results that the TCR alpha and TCR beta variable regions, including part of the constant region, form a single band after amplification.
  • step 3 for a total of 2 rinses.
  • the purified product carries the 5' end barcode sequence and other elements, the full length of the TCR variable region and part of the TCR constant region sequence.
  • PCR primers to amplify the remaining constant region sequence TCR alpha-2 of TCR alpha-1 from the TCR-TRAC-pMax vector, and add TCR alpha to the 5' ends of the forward and reverse primers -1 fragment homologous sequence, constant region sequence TCR alpha-2 (see Seq ID No.1 for the nucleic acid sequence and Seq ID No.2 for the amino acid sequence).
  • Design PCR primers to amplify the remaining constant region sequence TCR beta-2 of TCR beta-1 from the TCR-TRBC-pMax vector, and add the TCR beta-1 fragment to the 5' ends of the forward and reverse primers.
  • Homologous sequence, constant region sequence TCR beta-2 see Seq ID No.3 for nucleic acid sequence and Seq ID No.4 for amino acid sequence).
  • the primer sequence is:
  • TCR alpha-2-F catatccagaaccctgaccc
  • TCR alpha-2-R ctgtctcttatacacatctgacgcttagctggaccacagccgcagcg;
  • TCR beta-2-F gaggacctgaacaaggtgtt
  • TCR beta-2-R ctgtctcttatacacatctgacgcttagaaatcctttctcttgaccatg.
  • the purified product is used to prepare a reaction system according to the following table:
  • the cyclization reaction was carried out at 50°C for 1 hour.
  • step 7.3 Add DNA Clean Beads (0.5 ⁇ product volume) to the product of step 7.3, mix gently by pipetting, and incubate at room temperature for 10 minutes.
  • TCR alpha and TCR beta cyclization products are PCR amplified respectively to enrich TCR.
  • the primer sequence is a
  • TCR-F ttgcctttctctccacaggggtacctggtatcaacgcagagtacttggg;
  • TCR-R cattctagttgtggtttgtccaaacctgcttggaacggtacatacttgct.
  • the PCR reaction procedure is:
  • step 3 for a total of 2 rinses.
  • This vector carries the CMV promoter and polyA site.
  • the specific map is shown in Figure 6.
  • the purified full-length TCR-alpha and TCR-beta are cloned into the TCR-pMax vector through recombination, and then have a complete TCR expression cassette, which can efficiently express TCR subunits.
  • the specific steps are:
  • the TCR-F-JJ primer sequence is: taggcacctattggtcttac;
  • the TCR-R-JJ primer sequence is: tcactgcattctagttgtgg.
  • the bacterial test results were detected by agarose gel electrophoresis, as shown in Figure 7.
  • the band with an inserted sequence length of approximately 1000 bp is a positive clone, and the identified positive clones are sent to the corresponding bacterial solution for Sanger sequencing verification.
  • the sequencing primers are:
  • TCR-seq-F acctattggtcttactga
  • TCR alpha and TCR beta and the barcode of a single cell obtained through Sanger sequencing.
  • the complete sequence obtained was analyzed by DNA sequence analysis software to obtain the full-length sequence of TCR alpha and TCR beta.
  • TCR alpha and TCR beta clones with the same barcode sequence were determined.
  • a pair of TCRs is a paired TCR.
  • a total of 86 paired TCR pairs were found in the selected clones by this method. That is, the full-length sequence that can be paired with TCR is obtained.
  • Table 1 shows the variable region nucleotide sequences of 24 pairs of paired TCR sequences.
  • Electroconversion conditions BTX-ECM830 1mm electric shock cup, voltage 250V, pulse time 1ms.
  • TCRab-1-10 10 paired TCR pairs with complete sequences among the 24 paired TCR plasmids were selected, named TCRab-1-10 respectively, and electroporated into Jurkat-KO-ER.
  • the electrical transfer process is:
  • TCR alpha and TCR beta were cloned into transposon vectors using conventional molecular cloning methods.
  • the transposon carrying the active TCR sequence is delivered into the patient's T cells through electroporation to prepare personalized TCR-T cells. These cells can be used to treat cancer patients.
  • any combination of various embodiments of the present invention can also be carried out. As long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.

Abstract

Procédé de clonage et d'appariement rapides d'une séquence de TCR et son utilisation. Le procédé permettant de cloner et d'appairer rapidement la séquence de TCR comprend les étapes suivantes : tri et capture d'un seul lymphocyte T réactif à la tumeur dans un tissu tumoral ; extraction et marquage de l'ARNm du lymphocyte unique, réalisation d'une transcription inverse et construction d'un ADNc de pleine longueur ; réalisation d'une amplification PCR spécifique sur l'ADNc pour donner un transcriptome de pleine longueur d'ADNc ; réalisation d'une première cyclisation après enrichissement pour l'amplification spécifique de la séquence de TCR ; réalisation d'une seconde cyclisation après l'enrichissement et réalisation d'un nouvel enrichissement ; connexion d'un produit d'enrichissement à un vecteur d'expression pour obtenir un clone pleine longueur de TCR ; et réalisation d'un alignement de séquences de la banque pour obtenir une séquence de TCR appariée. Le clonage de TCR appariés de centaines de cellules est réalisé dans un système de transcription inverse, afin de permettre une acquisition rapide, peu coûteuse et à haut débit de TCR appariés.
PCT/CN2022/087532 2022-03-31 2022-04-19 Procédé de détection d'une séquence de tcr clonée et son utilisation WO2023184616A1 (fr)

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