WO2023179795A1 - 一种快速且简便地获得正确配对tcr的方法以及获得的tcr - Google Patents

一种快速且简便地获得正确配对tcr的方法以及获得的tcr Download PDF

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WO2023179795A1
WO2023179795A1 PCT/CN2023/084203 CN2023084203W WO2023179795A1 WO 2023179795 A1 WO2023179795 A1 WO 2023179795A1 CN 2023084203 W CN2023084203 W CN 2023084203W WO 2023179795 A1 WO2023179795 A1 WO 2023179795A1
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tcr
cancer
cells
cell
sequence
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PCT/CN2023/084203
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English (en)
French (fr)
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王文博
冯爱华
吉翔骏
王鹏
季翔
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立凌生物制药(苏州)有限公司
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Priority claimed from CN202210306873.XA external-priority patent/CN116836971A/zh
Priority claimed from CN202210562300.3A external-priority patent/CN115029341A/zh
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Publication of WO2023179795A1 publication Critical patent/WO2023179795A1/zh

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    • 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
    • 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
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

Definitions

  • the present invention relates to the field of biotechnology. More specifically, the present invention relates to a quick and easy method of obtaining correctly paired TCRs and to the TCRs obtained by this method.
  • T cell receptor is a receptor molecule specifically expressed on the surface of 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 the tumor antigen-specific TCR is transferred into ordinary T cells, it can give the T cells the ability to recognize tumor antigens. After being activated and proliferated in vitro, it 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-T cell therapy can be divided into the following steps: Step 1: Screening for appropriate tumor-specific antigens and the gene sequence of the TCR ⁇ chain that is highly specific for this antigen; Step 2: Isolating from the patient’s body out of T cells; the third step: load the above-mentioned TCR ⁇ chain gene sequence that can recognize tumor-specific antigens into the viral genome to construct a viral vector; the fourth step: transfect the virus into T cells and introduce the target TCR gene sequence into the T cells In the process, TCR-T cells that can specifically recognize tumor antigens are obtained; the fifth step: TCR-T cells are cultured in vitro and expanded in large quantities; the sixth step: a qualified number of TCR-T cells are infused into the patient for treatment.
  • TCR molecules are mainly composed of two chains, ⁇ and ⁇ .
  • the encoding genes V, (D)J, and C undergo germline rearrangement during T cell development, undergo positive selection and negative selection processes in the thymus, and finally have MHC recognition.
  • TCR ⁇ produced by mature T cells in the body constitutes a library of antigen recognition receptors (Repertoire) that can bind to tens of millions of antigens.
  • the successful acquisition of tumor antigen-specific TCR is an important prerequisite for TCR-T cell treatment of tumors.
  • the screening of tumor-specific TCR genes mainly involves obtaining T cells that specifically recognize tumor antigens, and then cloning their TCR genes.
  • tumor-specific TCR identification methods will greatly shorten the development cycle of TCR-T therapy and reduce costs.
  • single-cell sequencing technology can identify tumor-specific T cell clones at the single-cell level and help obtain high-affinity TCR sequences for tumors.
  • 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 purpose of the present invention is to provide a method for obtaining the correct paired TCR full-length gene quickly, simply and reliably.
  • Another object of the present invention is to provide TCR through the method of the present invention, so that it can be applied in clinical practice.
  • the present invention provides a method for obtaining correctly paired TCRs, which method includes the following steps:
  • the obtained cDNA and TCR variable region amplification products are subjected to library construction and NGS sequencing.
  • the paired TCR sequence information is determined by analyzing the NGS sequencing results of the TCR variable region, and then the matching TCR sequence information is found from the established TCR plasmid library. TCRs with the same sequence, thereby obtaining the paired TCR full-length gene sequence.
  • the "obtained activated T cells obtained cDNA of the TCR alpha variable region and TCR beta variable region of a single T cell with Barcode” in step 2) refers to labeling Barcode on a single T cell.
  • the cDNA of the TCR alpha variable region and TCR beta variable region of a single T cell with Barcode is then obtained by reverse transcription.
  • step 3 Barcode is labeled at the 3' end of the mRNA of the TCR alpha variable region and TCR beta variable region of a single T cell, then step 3) is:
  • the cDNA containing the Barcoded TCR alpha variable region and TCR beta variable region obtained in step 2) is first connected to the promoter sequence, and then recombined into the vector carrying the TCR alpha constant region and TCR beta constant region respectively, thereby amplifying Added TCR alpha variable region sequence and TCR beta variable region sequence;
  • step 3 is:
  • the cDNA containing the Barcoded TCR alpha variable region and TCR beta variable region obtained in step 2) is circularized for the second time, and then recombined into vectors carrying the TCR alpha constant region and TCR beta constant region respectively, thereby amplifying Add TCR alpha variable region sequence and TCR beta variable region sequence.
  • step 1) includes:
  • step 1-2 Use the tumor cells obtained in step 1-2 to stimulate T cells;
  • using the tumor cells obtained in step 1-2 to stimulate T cells includes combining the tumor cells obtained in step 1-2 Tumor cells and T cells were co-incubated.
  • the activation markers include but are not limited to CD137, OX40, CD40, ICOS, PD-1, CD39, CD103, CD69, CXCL13, GZMA; preferably CD137, CD39, CD103, CXCL13.
  • said steps 1-4 further include optionally detecting the expression of cytokines.
  • the cytokine is IFN- ⁇ .
  • said step 2) includes the following steps:
  • the circularization involves ligating the PCR product to a vector containing the TCR constant region.
  • the step 2-2 includes: obtaining the mRNA of 100-2000, preferably 100-800, most preferably 200-500 activated T cells; or
  • the mRNA of 100-50000, preferably 100-8000, most preferably 200-5000 activated T cells is obtained.
  • the vector carrying the TCR alpha constant region has a promoter, a TCR alpha constant region sequence and a PolyA site; the vector carrying the TCR beta constant region has a promoter, a TCR beta constant region Sequence and PolyA site.
  • step 4 sequence the TCR alpha variable region sequence and TCR beta variable region sequence obtained in step 3) and select the TCR alpha variable region sequence and TCR with the same cell Barcode beta variable region sequence to obtain correctly paired TCRs.
  • the sequencing is Sanger sequencing and/or NGS sequencing.
  • the method further includes the following steps:
  • the present invention provides a method for rapidly 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 transcription and construct full-length cDNA, and cDNA-specific PCR amplification to obtain the full-length cDNA transcriptome;
  • the cDNA full-length transcript is enriched, it is connected to the promoter sequence, circularized, and the TCR sequence is specifically amplified. After the amplification product is enriched, it is connected to an expression vector to obtain a full-length clone of the TCR, and the library sequence is compared to obtain it. Paired TCR sequences.
  • 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 cyclization method in the method is:
  • the promoter sequence is directly inserted before the TSO sequence;
  • the promoter is any genetic element that initiates mRNA transcription, including but not limited to CMV, EF1alpha, SV40, PGK1, CAG, T7, and Sp6;
  • the paired TCR sequences include TCR-alpha and TCR-beta sequences.
  • the expression vector is any plasmid with a replicon and a resistance gene element; the preferred expression vector is the linearized pMax-TRAC and pMax-TRBC vector;
  • the expression vector pMax has been artificially modified to remove the CMV promoter of the vector itself, add the TCR alpha constant region to obtain pMax-TRAC, and add the TCR beta constant region to obtain pMax-TRBC;
  • the purified TCR alpha-1 was cloned into pMax-TRAC through recombination, and the purified TCR beta-1 was cloned into pMax-TRBC to have a complete TCR expression cassette.
  • 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.
  • 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 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.
  • the paired TCR sequence of the present invention is obtained by the following method:
  • S102 Add 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
  • the promoter (Promoter) sequence is directly inserted before the TSO sequence.
  • the TSO sequence does not contain the ATG sequence, thus not affecting the expression of the TCR gene.
  • the constant region sequence and barcode sequence on the magnetic beads will be located before the promoter.
  • the nucleic acid sequence of CMV promoter sequence CMV-P is shown in Seq ID No.1.
  • TCR-alpha and TCR-beta After cloning the full length of TCR-alpha and TCR-beta according to the above steps, single clones are picked, plasmids are extracted and sequenced, and cell barcode comparison is performed. TCR-alpha and TCR-beta sequences with the same cell barcode are paired. TCR.
  • 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 tissue, extracting, and labeling the mRNA of the single cells through Barcode , reverse transcription and construct full-length cDNA, cDNA-specific PCR amplification to obtain the full-length cDNA transcriptome, first circularization after enrichment, specific amplification of the TCR sequence, second circularization after enrichment, and After enrichment again, the enriched product is ligated into an expression vector to obtain a full-length clone of TCR, and the Barcode sequence in the library 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) Perform specific amplification of TCR sequences to complete 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. After enrichment of TCR, 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 sequences.
  • 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.
  • a method for sorting individual tumor-reactive T cells is by using fluorescently labeled
  • the single cell suspension is stained and labeled with the labeled antibody and then sorted by a flow cytometer.
  • 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 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.
  • 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, which is prepared by using the method described in the first aspect.
  • the TCR is any one of TCR-1 to TCR-68.
  • the present invention provides the use of the TCR described in the second aspect in preparing genetically modified T cells.
  • the present invention provides a genetically modified T cell that expresses the TCR prepared by the method described in the first aspect or the TCR described in the second aspect.
  • 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 may refer to the individual's own T cells or allogeneic T cells.
  • the allogeneic T cells may be T cells from different individuals of the same species and/or T cells from a different species.
  • the allogeneic T cells may be T cells from different human individuals or T cells from other animal subjects.
  • the present invention provides the use of the genetically modified T cells described in the fourth aspect in a pharmaceutical composition for treating T cell-related diseases.
  • T cell-related diseases include cancer, infectious diseases, and autoimmune diseases.
  • the T cell-associated disease is tumor, or cancer.
  • the disease associated with T cells is an infectious disease.
  • the disease associated with T cells is an autoimmune disease.
  • the T cell-related disease is an organ transplant-related disease.
  • the TCR-T cells contained in the pharmaceutical composition are from the patient's own T cells.
  • the TCR sequence in the TCR-T cell is the TCR full-length sequence information obtained after obtaining mRNA from the patient's own T cells.
  • the genetically modified TCR-T cells provided by the present invention can be used to prepare pharmaceutical compositions for treating tumors.
  • the tumor that can be treated 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, Neuroblastoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumors, breast cancer, bronchial adenoma, Burkitt lymphoma, unknown primary carcinoma, central nervous system lymphoma, cerebellar astrocytes neoplasms, 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, epend
  • the genetically modified TCR-T cells provided by the present invention can be used to prepare pharmaceutical compositions for treating autoimmune diseases.
  • the autoimmune disease may be selected from the group consisting of arthritis, chronic obstructive pulmonary disease, ankylosing spondylitis, Crohn's disease, dermatomyositis, type I diabetes, endometriosis, Goodpasture's syndrome 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
  • the present invention provides a pharmaceutical composition comprising the genetically modified T cells described in the fourth aspect, and a pharmaceutically acceptable excipient.
  • 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 is used to treat tumors and autoimmune diseases;
  • the tumor is selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical cancer, AIDS-related cancer, AIDS Associated lymphoma, anal cancer, appendiceal cancer, astrocytoma, neuroblastoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial adenoma, Burkitt lymphoma, primary Unknown metastatic 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, connective tissue Proliferative small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's s
  • Said 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 cirrhosis, relapsing Polychondritis, rheumatoid arthritis,
  • the present invention provides a diagnostic and/or evaluation preparation.
  • the diagnostic and/or evaluation preparation provided by the invention contains the TCR-T cells obtained in the fourth aspect of the 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 diagnostic and/or evaluation preparations provided by the present invention can be used for biomarkers, antibody development, drug and vaccine evaluation, immune cell differentiation tracing, immune rejection and tolerance, minimal residual disease detection, food or other allergies Original 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 shows the use of flow cytometry in Example 1 to detect the proportion of CD8+ cells and tumor cells in bone marrow mononuclear cells
  • Figure 2 shows that after co-culturing the sorted CD8+ cells and tumor cells in Example 1, flow cytometry detects that tumor cells can activate CD8+ cells;
  • FIG. 3 shows the TCR alpha and TCR beta gene fragments obtained by electrophoresis detection PCR amplification in Example 2;
  • FIG. 4 shows the TCR-TRAC-pMax and TCR-TRBC-pMax maps of the TCR-expressing vectors in Example 3;
  • FIG. 5 shows the linearized gene fragments of the electrophoresis detection vector TCR-TRAC-pMax and TCR-TRBC-pMax in Example 3;
  • FIG. 6 shows the positive clones where the TCR alpha and TCR beta gene fragments were connected to the expression vector by electrophoresis detection in Example 3;
  • Figure 7 shows the flow cytometric detection of TCR-knocked out Jurkat cells in Example 4.
  • Figure 8 shows the expression of paired TCR plasmids electroporated into cells detected by flow cytometry in Example 4.
  • Figure 9 shows the detection of the function of the paired TCR screened in Example 4 after co-culture with tumor cells
  • Figure 10 shows an example diagram of the main band distribution of the cDNA library
  • Figure 11 shows an example of the main band distribution of the TCR V region library
  • Figure 12 shows the unscreened TCR clonotype distribution
  • Figure 13 shows the distribution of TCR clonotypes after screening
  • Figure 14 shows a schematic diagram of the circularization and amplification of TCR enriched products
  • Figure 15 shows the complete fragments of TCR alpha and TCR beta obtained by electrophoresis detection PCR amplification in Example 9;
  • Figure 16 is a schematic diagram of single cell labeling magnetic beads in Example 11.
  • FIG 17 is a flow chart of the TCR plasmid library construction technology in Example 11.
  • Figure 18 shows the TCR alpha and TCR beta gene fragments obtained by electrophoresis detection PCR amplification in Example 11;
  • Figure 19 shows the TCR alpha constant region and TCR beta constant region gene fragments obtained by electrophoresis detection PCR amplification in Example 11;
  • Figure 20 shows the TCR alpha full-length fragment and TCR beta full-length fragment obtained by electrophoresis detection PCR amplification in Example 11;
  • Figure 21 is the TCR-pMax map of the TCR-expressing vector in Example 11;
  • Figure 22 shows the positive clones in Example 11 where the TCR alpha and TCR beta full-length fragments were detected by electrophoresis and connected to the expression vector;
  • Figure 23 shows flow cytometric detection of TCR-knocked out Jurkat cells in Example 11.
  • Figure 24 shows the flow cytometric detection of the expression of paired TCR expression plasmids electroporated into cells in Example 11;
  • FIG. 25 is a flow chart of the TCR plasmid library construction technology in Example 12; wherein, TSO refers to template switch oligo, V region refers to the variable region of TCR, C region refers to the constant region of TCR, and Promoter is the promoter;
  • Figure 26 shows the TCR alpha and TCR beta gene fragments obtained by electrophoresis detection and PCR amplification in Example 12;
  • Figure 27 shows the linearized pMax-TRAC and pMax-TRBC vector maps in Example 12;
  • Figure 28 shows the positive clones where the TCR alpha and TCR beta gene fragments were connected to the expression vector through electrophoresis detection in Example 12.
  • the method uses DNA barcode to label the mRNA and cDNA of single-cell TCRs, adds labels to the paired TCRs, and then finds TCRs with the same labels through cloning screening for pairing, so that they can be paired in a reverse transcription system Realize paired TCR cloning of hundreds of cells to achieve fast, low-cost, and high-throughput acquisition of paired TCR. On this basis, the present invention was completed.
  • DNA Barcode is a DNA fragment with different bases, which is the label of the sample. Used to distinguish different samples, in this patent it refers to cell tag sequences. Its function is to specifically label individual cells so that each cell has a different barcode. If the gene sequences read by sequencing have the same barcode, these genes originate from the same cell.
  • method of cloning TCR gene and “method of obtaining paired TCR”, “method of the present invention” and “method of obtaining full-length TCR gene” have the same meaning, and they all refer to obtaining the complete form of an individual. TCR gene approach.
  • the present invention labels the mRNA and cDNA of single-cell TCRs through DNA barcode, adds tags to the paired TCRs, and then finds TCRs with the same tags through cloning screening for pairing, and performs reverse transcription in one step. Paired TCR cloning of hundreds of cells is achieved within the system, enabling fast, low-cost, and high-throughput acquisition of paired TCRs.
  • SMART switching mechanism at the 5'end of the RNA transcript
  • Takara Bio USA, Inc was cleverly applied to develop a unique high-throughput acquisition of paired TCR and screening of paired pairs in the downstream TCR technology. For the first time, the inventors achieved rapid and low-cost acquisition of correctly paired TCR genes without relying on gene synthesis.
  • SMART technology was first developed in 2001, which enables the construction of full-length cDNA libraries even if the starting material is RNA derived from a single cell.
  • the core of SMART technology is the use of a DNA-RNA composite oligo called TSO (Template switch oligo).
  • TSO can bind to the cDNA end generated by reverse transcription through the RNA part, and then the remaining part of TSO can be used as a constant template for cDNA amplification.
  • this project optimized the downstream function screening of paired TCRs.
  • DNA plasmids were used as vectors for high-throughput paired TCR screening, which greatly improved the screening efficiency.
  • the method provided by the present invention to obtain correctly paired TCRs includes the following steps:
  • tumor cell-activated T cells can be obtained through the following steps:
  • step 1-2 Use the tumor cells obtained in step 1-2 to stimulate T cells;
  • mononuclear cells can be derived from many sources, such as directly harvested from solid tumors.
  • step 2) can be implemented as follows:
  • the circularization involves ligating the PCR product to a vector containing the TCR constant region.
  • the inventors also discovered that when obtaining the mRNA of a single T cell, the number of cells can have a wide range.
  • the method of the present invention is applicable to both lower and higher cell numbers.
  • the method of the present invention can obtain mRNA from 100-2000, preferably 100-800, and most preferably 200-500 activated T cells.
  • the obtained cDNA and TCR variable region amplification products can be subjected to library construction and NGS sequencing, and the paired TCR sequence can be determined by analyzing the NGS sequencing results of the TCR variable region. information, and then search for TCRs with the same sequence as the NGS sequencing results from the established TCR plasmid library, thereby obtaining the paired TCR full-length gene sequence.
  • the method of the invention can obtain mRNA from 100-50000, preferably 100-8000, most preferably 200-5000 activated T cells.
  • the inventor further discovered that if Barcode is marked at the 3' end of the mRNA in the TCR alpha variable region and TCR beta variable region of a single T cell, then after obtaining the TCR expression vector, the barcode is located between the promoter and the gene, affecting gene expression.
  • the inventor further improved the present invention and obtained the TCR with Barcode
  • the cDNA of the alpha variable region and the TCR beta variable region are first connected to the promoter sequence, and then recombined into vectors carrying the TCR alpha constant region and the TCR beta constant region respectively, thereby amplifying the TCR alpha variable region sequence and TCR beta.
  • Variable region sequence alternatively, the obtained cDNA with Barcoded TCR alpha variable region and TCR beta variable region is circularized for the second time, and then recombined into vectors carrying TCR alpha constant region and TCR beta constant region respectively. , thereby amplifying the TCR alpha variable region sequence and the TCR beta variable region sequence.
  • this invention achieves rapid and low-cost acquisition of paired TCR genes without relying on gene synthesis
  • This invention uses DNA plasmids as carriers for the first time to conduct high-throughput paired TCR screening, which greatly improves the screening efficiency;
  • the present invention significantly reduces the time required for TCR screening
  • This invention lays a new foundation for the development of TCR drugs
  • the present invention can be applied to completely personalized TCR-T cell therapy.
  • Isolate mononuclear cells Use a tube containing anticoagulant to collect bone marrow samples from AML patients and store them at low temperature.
  • the bone marrow samples were poured into PBMC separation tubes (Dayou, Cat#7121012), centrifuged at 20°C and 800g for 15 minutes, and then the cell layer of mononuclear cells was aspirated.
  • the aspirated mononuclear cells were washed 1-2 times with RPMI1640 complete medium, and finally resuspended in RPMI1640 complete medium for later use.
  • T cell sorting Use Miltenyi anti-APC Micro Beads (130-090-855) and MS separation column (130-090-855) to sort out CD8+ T cells from PBMC.
  • Sorting of tumor cells Use Miltenyi magnetic beads and MS separation columns to sort out CD33+&CD34+ tumor cells from the CD8-negative cells obtained in the previous step.
  • Example 2 Using water-in-oil system to obtain single T cell TCR alpha and TCR beta variable region sequences
  • Cell preparation Flow cytometry detects T cells with upregulated CD137 expression. Use BD FACSjazz cell sorter (BD, 655489) to sort and collect CD137-positive T cells. The cell sorting process is performed according to the instructions of the instrument. Conduct activity measurement and cell counting on the sorted CD137-positive T cells to ensure that the cell viability rate is above 85%. Adjust the cell density to 1X10 6 /ml and prepare a single cell suspension.
  • BD FACSjazz cell sorter BD, 655489
  • Reverse transcription GEMs are collected, and the gel beads automatically dissolve in each oil droplet to release a large number of Barcode primer sequences, while the cells are lysed to release mRNA. Because the beads have been equipped with adapters, one of them is a poly dT sequence. Among the nucleic acids released by cell lysis, only mRNA has a polyA tail, so the poly dT on this bead can capture mRNA from numerous cleavage products.
  • Master Mix contains a reverse transcription reagent. When the mRNA is captured, a reverse transcription reaction occurs under the action of reverse transcriptase, producing a strand of cDNA with Barcode and UMI information for sequencing. Collect the mixed cleavage products and perform reverse transcription on a PCR machine: 53°C for 45 min; 85°C for 5 min; and store at 4°C.
  • the F-R1 primer sequence is:
  • the TCR alpha-R primer sequence is: AGTCTCTCAGCTGGTACACG (SEQ ID NO: 6)
  • the TCR beta-R primer sequence is: TCTGATGGCTCAAACACAGC (SEQ ID NO:7)
  • TCR-TRAV and TCR-TRBV a total of 2 products.
  • the 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.
  • the TCR enrichment product contains a part of the constant region sequence as the homologous sequence used for recombination.
  • TCR-pMax vector This vector carries the CMV promoter, TCR alpha and TCR beta constant region sequences and polyA sites.
  • the constant regions used were human TCR alpha and TCR beta constant regions.
  • Human TCR alpha constant region in vector It was named TRAC (see Seq ID No. 1 for the nucleic acid sequence and Seq ID No. 2 for the amino acid sequence).
  • the human TCR beta constant region in the vector is named TRBC (see Seq ID No. 3 for the nucleic acid sequence and Seq ID No. 4 for the amino acid sequence).
  • TCR-TRAC-pMax and TCR-TRBC-pMax vector maps are shown in Figure 4.
  • the TCR-TRAC-pMax and TCR-TRBC-pMax vectors also contain the constant region sequences of TCR alpha and TCR beta and the corresponding restriction sites, which is convenient for Example 2
  • the TCR products enriched in the vector were quickly cloned into this vector.
  • the purified TCR alpha enriched product is recombined into TCR-TRAC-pMax, and the purified TCR beta enriched product is recombined into TCR-TRBC-pMax vector.
  • the final TCR sequence has a complete constant region and TCR expression. box, which can efficiently express TCR subunits. The specific steps are:
  • the ll25-JJ-F1 primer sequence (bacteria detection primer) is: taggcacctattggtcttac (SEQ ID NO: 352)
  • the ll25-JJ-R1 primer sequence (bacterial detection primer) is: tcactgcattctagttgtgg (SEQ ID NO:353)
  • the initially identified positive clones were sent to the corresponding plasmid Sanger sequencing for verification.
  • a total of 192 clones of TCR alpha were sent for testing, and they were labeled according to TCR alpha1-192.
  • TCR beta sent 192 clones for testing, and they were labeled according to TCR beta 1-192.
  • the sequencing primer sequence is: taggcacctattggtcttac (SEQ ID NO: 354).
  • TCR alpha and TCR beta variable regions and the barcode sequence of single cells were obtained through Sanger sequencing.
  • the complete sequence is analyzed by DNA sequence analysis software to obtain the TCR-alpha and TCR-beta variable region sequences. It can be determined that TCR-alpha and TCR-beta clones with the same cell barcode are a pair of TCRs, that is, paired TCRs.
  • paired TCR sequences were obtained in all selected clones, and the paired TCR alpha and TCR beta plasmids were found. Out and marked.
  • Table 1 shows 24 pairs of paired TCRs with the same barcode.
  • the IMGT database https://www.imgt.org was further used to analyze the variable region types corresponding to different TCR-alpha and TCR-beta variable regions. The results are shown in Table 2 and Table 3.
  • the cell loading volume was selected to be 2500 cells.
  • a total of 300 clones were selected for TCR alpha and TCR beta for sequencing and sequence comparison.
  • sequence analysis it was found that there were only two pairs of paired TCRs among the selected clones, and the selected paired TCRs accounted for 0.67% of the total selected TCR clones.
  • the efficiency of obtaining paired TCRs was greatly reduced. Therefore, more clones need to be selected to obtain more paired TCRs, which greatly increases the workload and cost. Therefore, searching for paired TCRs with consistent barcodes by selecting single clones and Sanger sequencing requires strict restrictions on the number of cells loaded. Otherwise, the efficiency of obtaining paired TCRs will be greatly reduced, resulting in experimental failure.
  • Electroconversion conditions BTX-ECM830 1mm electric shock cup, voltage 250V, pulse time 1ms.
  • TCRab-1-24 24 paired TCR pairs with complete sequences among the 110 paired TCR plasmids were selected, named TCRab-1-24 respectively, and electroporated into Jurkat-KO-ER.
  • the electrical transfer process is:
  • the CD33/CD34 positive tumor cells obtained in Example 1 were plated into a 96-well flat-bottomed opaque white plate at a cell concentration of 1 ⁇ 10 6 /ml and 50ul/well.
  • Cells electroporated with Jurkat-KO-ER of different TCRs were added to the tumor cells according to the effect-to-target ratio of 1:1, that is, the cell concentration was 1 ⁇ 10 6 /ml, and 50ul/well was added to the tumor cells and mixed well. Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator.
  • TCR clones TCRab-2, TCRab-13 and TCRab-16 can specifically recognize tumor cells and can be further screened and evaluated.
  • TCRab 22, TCRab 33 and TCRab 66 primers were designed to amplify three TCR gene sequences respectively.
  • the PCR amplified bands were detected by agarose gel electrophoresis and then gel recovered and purified to obtain DNA fragments.
  • the three TCR gene sequences were recombined into the mRNA vector using a one-step rapid cloning kit (Yisheng Biotechnology Co., Ltd.: 10911ES20) to obtain recombinant products.
  • the three recombinant products were transferred into Escherichia coli DH5 ⁇ (Shanghai Weidi Biotechnology Co., Ltd.) strain.
  • T cell activation and mRNA electroporation Dilute human peripheral blood mononuclear cells (PBMCs, purchased from Shanghai Miaoshun Biotech) to 1 ⁇ 10 6 /ml, and use anti-human CD3 at a ratio of 1:3 between cells and magnetic beads. /CD28 magnetic beads (Thermo Fisher Scientific) activated T cells, and 300IU/ml IL-2 (stemcell: 78036) was added to the culture medium. Take a certain amount of activated T cells and centrifuge them at 1200 rpm for 5 minutes, then wash them twice with opti-MEM culture medium and set aside. Resuspend cells in 25ul of opti-MEM for every 1X10 ⁇ 6 cells and set aside.
  • PBMCs peripheral blood mononuclear cells
  • Fusion of mRNA on ice Add 1ug of mRNA per 1X10 ⁇ 6 cells and start electroporation. Electroconversion conditions: BTX-ECM830 1mm electric shock cup, voltage 250V, pulse time 1ms. After electroporation, quickly add 1 ml of T cell culture medium to the electroporation cup, and then transfer the cells to the culture wells for culture.
  • TCR-T was co-cultured with the patient's tumor cells, and T cells that were not electroporated were used as control cells.
  • Example 8 Rapid cloning of paired TCRs when the number of cells is large
  • T cells carrying tumor reactive T cell markers are obtained from tumors.
  • cDNA and TCR V region amplification products can be constructed and sequenced by NGS. Analyze the NGS sequencing results of the TCR V region to determine the paired TCR sequence information, and then search for TCRs with the same sequence as the NGS sequencing results from the TCR plasmid library established in Example 3, thereby obtaining the paired TCR full-length gene sequence without the need for gene synthesis. .
  • the basic steps are as follows:
  • Example 2 and Example 3 select a cell loading volume of 1,000-20,000 cells, and perform PCR After amplifying and connecting the TCR-pMax vector, the TCR alpha and TCR beta groups selected 100-10,000 clones respectively, extracted plasmids, established a plasmid library carrying the full-length TCR sequence, and then performed Sanger sequencing to determine the sequence information of each clone.
  • Use the IMGT website https://imgt.org/IMGT_vquest/input) to analyze the sanger sequencing results and obtain TCR V region type and CDR sequence information.
  • Dual Index Add Dual Index to the above magnetic bead purified product, perform PCR reaction, and then perform magnetic bead purification. Note: The function of Dual Index is to ensure that the data of each library can be correctly split after the sequencing is off the machine.
  • the cDNA and TCR V region library construction products were sequenced separately using a DNBSEQ-T7RS sequencer (Shenzhen MGI Technology Co., Ltd.). Perform paired-end sequencing based on the number of cells and the depth required for sequencing. Sequencing depth requirements: TCR V region library ⁇ 5000X, cDNA library ⁇ 20000X.
  • the Q30 quality value of the Fastq file of the sequencing data is not less than 85, and the data format is as shown in the figure:
  • the software Cell ranger was further used to analyze the cDNA sequencing results and obtain information on tumor response marker genes: PD-1, ENTPD-1, and CXCL13. Screen for TCRs that have the same barcode as tumor-responsive marker genes. These TCRs are likely to be tumor-responsive TCRs.
  • TCR clonotypes carrying the CXCL13 gene barcode the proportion of the top ten TCR clonotypes is shown in Figure 13.
  • the TCRs with the top ten clonotypes listed above were selected for downstream molecular cloning and functional studies.
  • TCRs with the same sequence were found in the TCR plasmid library sequenced by Sanger in Example 8.1. Make sure that the V region type of TCR is completely consistent with the CDR3 sequence information.
  • These paired plasmids carrying TCR genes are then selected and can be used for downstream vector construction and functional evaluation.
  • TCR cloning information and CDR3 sequence information are shown in Table 4. The above ten TCR sequences can be found in the TCR plasmid library sequenced by Sanger. It shows that the method provided by the present invention can efficiently clone the required paired TCR gene.
  • the cell loading volume was selected to be 21,000 cells.
  • a total of 600 single clones were selected for TCR alpha and TCR beta respectively for sequencing and sequence comparison to establish a TCR-containing Plasmid library of variable regions.
  • the top ten paired TCRs required for subsequent research were determined through NGS sequence analysis. Only one pair of the above ten TCR sequences can be found in the TCR plasmid library sequenced by Sanger, and the efficiency of obtaining paired TCRs required for research is greatly reduced. Therefore, more clones need to be selected to obtain more paired TCRs, which greatly Increased workload and cost. It shows that the cloning method in Example 8 provided by the present invention requires strict restrictions on the number of sampled cells, otherwise the efficiency of obtaining paired TCR will be greatly reduced, resulting in test failure.
  • the present invention redesigns the oligo sequence loaded on gel beads, adds the cyclization and amplification steps of the TCR enrichment product, and connects the 5' end element to the 3' end of the TCR gene fragment, thereby no longer affecting the cloned TCR Expression can be directly used for downstream applications (see Figure 14 for a schematic diagram). Since the cDNA amplification product only contains part of the TCR constant region, in the cyclization step, the present invention adds a gene fragment containing the remaining TCR constant region for recombination and cyclization. The basic steps are as follows:
  • Example 1 Place the amplified cDNA in a PCR tube, add another gene fragment: TCR constant region fragment (obtained by PCR), and add TE Buffer to 50ul. Place on a PCR machine and react at 95°C for 3 minutes. Immediately after the reaction, transfer the PCR tube to ice and keep in ice bath for 2 minutes.
  • TCR constant region fragment obtained by PCR
  • TCR-TRA and TCR-TRB a total of 2 products.
  • the two products were then connected to the empty pMax vector respectively.
  • the pMax vector itself has a CMV promoter and poly A site, so the full-length TCR expression cassette can be obtained directly.
  • Example 3 for identification of paired TCRs.
  • Examples 4-7 for downstream applications.
  • the 5' end of the TCR alpha and TCR beta full-length sequences obtained in this example does not have various barcode sequences and constant elements, and can Directly applicable to downstream screening and functional testing.
  • the inventors connected the obtained cDNA of the TCR alpha variable region and TCR beta variable region with Barcode to the promoter sequence; alternatively, the obtained TCR alpha with Barcode can be
  • the cDNA of the variable region and TCR beta variable region were circularized a second time in order to overcome the shortcomings that Barcode labeling may bring at the 3' end of the TCR alpha variable region and TCR beta variable region mRNA of a single T cell. at.
  • the inventors Using technical principles similar to those in the above embodiments, the inventors used a microplate system to obtain correctly paired TCRs.
  • 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 16).
  • 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 17.
  • 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.
  • Take 100 ⁇ l of resuspended cells approximately 300 to 500 cells, preferably 50 to 2000 cells
  • 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 in the 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.
  • cDNA amplification Prepare PCR Mix-2 on ice according to the following table, mix and centrifuge briefly
  • the PCR reaction procedure is:
  • Ampure XP purification magnetic beads are removed from 4°C 30 minutes in advance and returned to room temperature. Shake thoroughly before use.
  • 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.
  • the purified magnetic beads were removed from 4°C 30 minutes in advance and returned to room temperature.
  • the magnetic beads need to be mixed thoroughly before use.
  • the TCR R1 primer sequence GCGTCAGATGTGTATAAGAG (SEQ ID NO: 176);
  • TCR alpha-F-1 primer sequence AGTCTCTCAGCTGGTACACG (SEQ ID NO: 177);
  • TCR beta-F-1 primer sequence TCTGATGGCTCAAACACAGC (SEQ ID NO: 178).
  • the enriched products were detected by agarose gel electrophoresis, and the results are shown in Figure 18. 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.
  • the purified magnetic beads were removed from 4°C 30 minutes in advance and returned to room temperature. Shake thoroughly before use.
  • 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 amplification of part of the constant region Design PCR primers to amplify the remaining constant region sequence of TCR alpha-1 TCR alpha-2 from the TCR-TRAC-pMax vector, and add it to the 5' ends of the forward and reverse primers Homologous sequence of TCR alpha-1 fragment, constant region sequence TCR alpha-2.
  • the primer sequence is:
  • TCR alpha-2-F catatccagaaccctgaccc (SEQ ID NO: 179);
  • TCR alpha-2-R ctgtctcttatacacatctgacgcttagctggaccacagccgcagcg (SEQ ID NO: 180);
  • TCR beta-2-F gaggacctgaacaaggtgtt(SEQ ID NO:181);
  • TCR beta-2-R ctgtctcttatacacatctgacgcttagaaatcctttctcttgaccatg (SEQ ID NO: 182).
  • the PCR products were detected by agarose gel electrophoresis, and the results are shown in Figure 19. It can be seen from the electrophoresis results that the TCR alpha and TCR beta constant regions formed a single band after amplification, and the gene fragment was amplified successfully.
  • sol solution PE Add 3 times the volume of sol solution PE to the gel piece (if the gel weighs 0.1g, its volume can be regarded as 100 ⁇ l, then add 300 ⁇ l of sol solution PE). Solve at room temperature of 15-25°C for 5-10 minutes. During this period, gently turn the centrifuge tube up and down to ensure that the gel block is fully dissolved.
  • adsorption column CA5 Place the adsorption column CA5 back into the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove as much rinse liquid as possible. Place the adsorption column CA5 at room temperature for a few minutes and dry it thoroughly to prevent residual rinse solution from affecting the next experiment.
  • adsorption column CA5 Place the adsorption column CA5 into a clean centrifuge tube, add preheated ddH 2 O dropwise to the middle of the adsorption membrane, and leave it at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 2 minutes to collect the DNA solution.
  • the products after purification and recovery are TCR alpha-2 and TCR beta-2 respectively.
  • 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.
  • TCR alpha and TCR beta cyclization products were PCR amplified separately to enrich TCR.
  • the primer sequence is a
  • TCR-F ttgcctttctctccacaggggtacctggtatcaacgcagagtacttggg (SEQ ID NO: 183);
  • TCR-R cattctagttgtggtttgtccaaacctgcttggaacggtacatacttgct (SEQ ID NO: 184).
  • the PCR reaction procedure is:
  • the purified magnetic beads should be removed from 4°C and returned to room temperature 30 minutes in advance. Shake and mix thoroughly before use.
  • This vector carries the CMV promoter and polyA site.
  • the specific map is shown in Figure 21.
  • 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:
  • TCR-pMax was linearized and purified.
  • the volume of the transformation product should not exceed 1/10 of the competent volume.
  • the TCR-F-JJ primer sequence is: taggcacctattggtcttac (SEQ ID NO: 185);
  • the TCR-R-JJ primer sequence is: tcactgcattctagttgtgg (SEQ ID NO: 186).
  • the bacterial test results were detected by agarose gel electrophoresis, as shown in Figure 22. Bands with an inserted sequence length of approximately 1000bp are positive Cloning, and the identified positive clones were sent to the corresponding bacterial solution for Sanger sequencing verification.
  • the sequencing primers are:
  • TCR-seq-F acctattggtcttactga(SEQ ID NO:187);
  • TCR-seq-R:cattctagttgtggtttgtc (SEQ ID NO: 188).
  • 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 5 shows the variable region nucleotide sequences of 24 pairs of paired TCR sequences.
  • lentivirus carrying NFAT-luciferase to infect Jurkat cells (Cell Bank of the Chinese Academy of Sciences), and select monoclonal cultures to obtain Jurkat-NFAT-luciferase reporter cell lines (TCR reconstitution in Jurkat reporter cells facilitates the identification of novel tumor antigens by cDNA expression cloning.Int J Cancer. 2002 May 1;99(1):7-13).
  • CRISPR/Cas9 electroporation to simultaneously knock out the TCR gene, and select non-knocked-out cells as negative controls. The specific steps are:
  • Cas9 and sgRNA on ice add 10 ⁇ l Cas9 (500ng/ ⁇ l) protein (Nanjing GenScript), 2.5 ⁇ l TRAC-sgRNA and 2.5 ⁇ l TRBC-sgRNA) (400ng/ ⁇ l Nanjing GenScript) per 1 ⁇ 10 6 cells Rui), incubate the two at room temperature for 10 minutes, add the cells prepared in advance and start electroporation.
  • Electroconversion conditions BTX-ECM830 1mm electric shock cup, voltage 250V, pulse time 1ms.
  • TCR knockout 48 hours after electroporation knockout, a certain volume of cells was taken, anti-CD3-APC antibody (BD Pharmingen, 555335) was added to stain the cells, and incubated at 4°C for half an hour. After staining, cells were washed with PBS and resuspended, and flow cytometry was used to detect TCR knockdown. The results of TCR knockout in Jurkat cells are shown in Figure 23. From the flow cytometry results, it can be seen that the TCR in Jurkat cells has been completely knocked out. The effector cells prepared through this step are named: Jurkat-KO-ER.
  • 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:
  • Detect the expression of TCR in cells by flow cytometry 24 hours after electroporation of paired TCR plasmid into Jurkat-KO-ER cells, take a certain volume of cells, add anti-CD3-APC antibody (BD Pharmingen, 555335) to stain the cells, 4°C Incubate for half an hour. After staining, cells were washed with PBS and resuspended, and flow cytometry was used to detect the expression of TCR cells. Representative results of expression of electroporated TCR in Jurkat-KO-ER cells are shown in Figure 24. It can be seen from the figure that after 10 pairs of representative paired TCRs were electroporated into cells, CD3 expression resumed.
  • 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.
  • 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 16).
  • the DNA oligo includes: constant sequence 1, Barcode, constant sequence 2 and oligo dT sequence. Barcode is used to label single cells, Oligo dT is used to capture mRNA, and 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 25.
  • 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.
  • Take 100 ⁇ l of resuspended cells approximately 300 to 500 cells, preferably 50 to 2000 cells
  • 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 in the 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.
  • the primer sequence is:
  • cDNA-F TGGTATCAACGCAGAGTACTTGGG (SEQ ID NO: 261);
  • cDNA-R CCTACACGACGCTCTTCCGATC (SEQ ID NO: 262).
  • the PCR reaction procedure is:
  • Ampure XP purification magnetic beads are removed from 4°C 30 minutes in advance and returned to room temperature. Shake thoroughly before use.
  • the present invention directly inserts the promoter sequence before the TSO sequence during cyclization.
  • the TSO sequence does not contain the ATG sequence, thus not affecting the TCR gene. Express.
  • PCR amplification of CMV promoter Design PCR primers to amplify the CMV promoter sequence CMV-P from the pMax vector, and add cDNA homologous sequences to the 5' ends of the forward and reverse primers.
  • the primer sequence is:
  • sol solution PE Add 3 times the volume of sol solution PE to the gel piece (if the gel weighs 0.1g, its volume can be regarded as 100 ⁇ l, then add 300 ⁇ l of sol solution PE). Solve at room temperature of 15-25°C for 5-10 minutes. During this period, gently turn the centrifuge tube up and down to ensure that the gel block is fully dissolved.
  • adsorption column CA5 Place the adsorption column CA5 back into the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove as much rinse liquid as possible. Place the adsorption column CA5 at room temperature for a few minutes and dry it thoroughly to prevent residual rinse solution from affecting the next experiment.
  • adsorption column CA5 Place the adsorption column CA5 into a clean centrifuge tube, add preheated ddH 2 O dropwise to the middle of the adsorption membrane, and leave it at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 2 minutes to collect the DNA solution.
  • the purified product is used to prepare a reaction system according to the following table:
  • the TCR P1-F primer sequence GCGTCAGATGTGTATAAGAG (SEQ ID NO: 265)
  • TCR alpha-R primer sequence AGTCTCTCAGCTGGTACACG (SEQ ID NO: 266)
  • TCR beta-R primer sequence TCTGATGGCTCAAACACAGC (SEQ ID NO: 267)
  • TCR enriched products were detected by agarose gel electrophoresis, and the results are shown in Figure 26. It can be seen from the electrophoresis results that the TCR alpha variable region includes part of the constant region and promoter sequence, and the TCR beta variable region includes part of the constant region and promoter sequence, which form a single band after amplification.
  • the purified magnetic beads were removed from 4°C 30 minutes in advance and returned to room temperature. Be sure to mix thoroughly before use.
  • the purified product carries the 5' end barcode sequence and CMV promoter, the full length of the TCR variable region and part of the TCR constant region sequence.
  • one or more rounds of enrichment can be performed to ensure that there is enough product for downstream recombination reactions.
  • the expression vector pMax has been artificially modified to remove the CMV promoter of the vector itself, add the TCR alpha constant region to obtain pMax-TRAC, and add the TCR beta constant region to obtain pMax-TRBC.
  • the specific map is shown in Figure 27.
  • the purified TCR alpha-1 is cloned into pMax-TRAC through recombination, and the purified TCR beta-1 is cloned into pMax-TRBC to have a complete TCR expression cassette, which can efficiently express TCR subunits.
  • pMax-TRAC and pMax-TRBC were linearized and purified using BamHI (NEB: R3136S) and KpnI (NEB: R3142S).
  • the volume of the transformation product should not exceed 1/10 of the competent volume.
  • the TCR-F-JJ primer sequence is: taggcacctattggtcttac (SEQ ID NO: 268);
  • the TCR-R-JJ primer sequence is: tcactgcattctagttgtgg (SEQ ID NO: 269).
  • the bacterial test results were detected by agarose gel electrophoresis, as shown in Figure 28.
  • 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 tagaaactgggcttgtcgag (SEQ ID NO:270);
  • TCR-seq-R cattctagttgtggtttgtc (SEQ ID NO: 271).
  • 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 6 shows the variable region nucleotide sequences of 20 pairs of paired TCR sequences.

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Abstract

获得正确配对的TCR的方法，所述方法通过DNAbarcode对单细胞TCR的mRNA和cDNA进行标记，给配对的TCR加上标签，然后再通过克隆筛选找到具有相同标签的TCR进行配对，从而能够在一个反转录体系内实现数百个细胞的配对TCR克隆，实现快速，低成本，并且高通量的获取配对TCR。

Description

一种快速且简便地获得正确配对TCR的方法以及获得的TCR 技术领域
本发明涉及生物技术领域。更具体地说，本发明涉及快速且简便地获得正确配对TCR的方法以及通过这种方法获得的TCR。
背景技术
T细胞受体(T cell receptor，TCR)是特异性表达于T细胞表面的一种受体分子，它特异性识别抗原提呈细胞上的抗原肽-MHC复合物，进而触发T细胞免疫应答。由于TCR分子决定着T细胞的抗原识别特异性，如果将肿瘤抗原特异性的TCR转入普通T细胞中，能够赋予该T细胞肿瘤抗原的识别能力，经体外活化增殖后再输入患者体内，可以发挥抗肿瘤功效。因此利用TCR基因导入的方法可以方便地获得大量识别特定抗原的T细胞，经TCR基因修饰的T细胞被称为TCR-T，近年来TCR-T已经成为肿瘤免疫治疗中的研究热点，在临床实验中显示了良好的治疗效果。TCR-T细胞疗法(T Cell Receptor-Gene Engineered T Cells)，通过筛选和鉴定能够特异性结合靶点抗原的TCR序列，采用基因工程手段将其转入到患者外周血来源的T细胞中(或异源T细胞)，再将改造后的T细胞回输至患者体内，使其特异性识别和杀伤表达抗原的肿瘤细胞，从而达到治疗肿瘤的目的。TCR-T细胞疗法的技术流程可分为以下几步：第一步：筛选合适的肿瘤特异性抗原，以及对该抗原具有高度特异性的TCRαβ链的基因序列；第二步：从患者体内分离出T细胞；第三步：将上述能够识别肿瘤特异性抗原的TCR αβ链的基因序列装入病毒基因组，构建病毒载体；第四步：病毒转染T细胞，将目标TCR基因序列引入T细胞中，获得能够特异性识别肿瘤抗原的TCR-T细胞；第五步：TCR-T细胞体外培养，大量扩增；第六步：将数量合格的TCR-T细胞输入患者体内，进行治疗。
TCR分子主要由α和β两条链组成，其编码基因V、(D)J、C在T细胞发育过程中经过胚系重排，在胸腺中经过阳性选择和阴性选择过程，最终具有MHC识别限制性。机体成熟T细胞所产生的TCRαβ组成了一个能与成千万种抗原结合的抗原识别受体库(Repertoire)。成功获得肿瘤抗原特异性TCR是TCR-T细胞治疗肿瘤的重要前提，肿瘤特异性TCR基因的筛选主要通过获得肿瘤抗原特异性识别的T细胞，然后克隆其TCR基因。与此同时高通量肿瘤特异性TCR鉴定的方法，将大大缩短TCR-T疗法的开发周期，降低成本。目前单细胞测序技术能够从单细胞层面识别肿瘤特异性T细胞克隆，帮助获得肿瘤高亲和力的TCR序列。
TCR筛选技术是TCR相关药物研发最核心的部分。但是目前TCR筛选成本高，耗时长，效率低，尤其是配对TCR全长基因的获取，是整个过程的限速步骤，严重影响了TCR相关药物的研发。
因此，本领域急需能够快速、简便、可靠地获得正确配对TCR全长基因的方法。
发明内容
本发明的目的在于提供一种快速、简便、可靠地获得正确配对TCR全长基因的方法。
本发明还有一目的在于通过本发明的方法提供TCR，从而能够应用于临床。
在第一方面，本发明提供一种获得正确配对的TCR的方法，所述方法包括以下步骤：
1)获得肿瘤细胞激活的T细胞；
2)从步骤1)获得的激活的T细胞获得带有Barcode的单个T细胞的TCR alpha可变区和TCR beta可变区的cDNA；
3)将步骤2)获得的带有Barcode的TCR alpha可变区和TCR beta可变区的cDNA分别重组到携带TCR alpha恒定区和TCR beta恒定区的载体中，从而扩增TCR alpha可变区序列和TCR beta可变区序列；
4)从步骤3)获得的TCR alpha可变区序列和TCR beta可变区序列中挑选具有相同细胞Barcode的TCR alpha可变区序列和TCR beta可变区序列，从而获得正确配对的TCR；或者
将获得的cDNA和TCR可变区扩增产物进行建库和NGS测序，通过分析TCR可变区的NGS测序结果确定配对的TCR序列信息，然后从建立的TCR质粒库中寻找与NGS测序结果中序列相同的TCR，从而得到配对TCR全长基因序列。
在优选的实施方式中，步骤2)所述的“获得的激活的T细胞获得带有Barcode的单个T细胞的TCR alpha可变区和TCR beta可变区的cDNA”是指将Barcode标记在单个T细胞的TCR alpha可变区和TCR beta可变区的mRNA上，随后通过反转录获得带有Barcode的单个T细胞的TCR alpha可变区和TCR beta可变区的cDNA。
在优选的实施方式中，如果在步骤2)中，Barcode标记在单个T细胞的TCR alpha可变区和TCR beta可变区的mRNA的3’端，则步骤3)为：
将步骤2)获得的带有Barcode的TCR alpha可变区和TCR beta可变区的cDNA先与启动子序列连接，再分别重组到携带TCR alpha恒定区和TCR beta恒定区的载体中，从而扩增TCR alpha可变区序列和TCR beta可变区序列；
或者，步骤3)为：
将步骤2)获得的带有Barcode的TCR alpha可变区和TCR beta可变区的cDNA进行第两次环化，再分别重组到携带TCR alpha恒定区和TCR beta恒定区的载体中，从而扩增TCR alpha可变区序列和TCR beta可变区序列。
在具体的实施方式中，所述步骤1)包括：
1-1.从肿瘤患者样品中分离单个核细胞；
1-2.从步骤1-1得到的单个核细胞中分选出T细胞和肿瘤细胞；
1-3.利用步骤1-2得到的肿瘤细胞刺激T细胞；
1-4.任选检测激活标志物的表达，以供细胞分选；
或者
1-1.从肿瘤患者样品中分离单个核细胞；
1-2.任选检测激活标志物的表达，以供细胞分选。
在优选的实施方式中，利用步骤1-2得到的肿瘤细胞刺激T细胞包括将步骤1-2得到的 肿瘤细胞和T细胞共孵育。
在优选的实施方式中，所述激活标志物包括但不限于CD137、OX40、CD40、ICOS、PD-1、CD39、CD103、CD69、CXCL13、GZMA；优选CD137、CD39、CD103、CXCL13。
在优选的实施方式中，所述步骤1-4还包括任选检测细胞因子的表达。
在优选的实施方式中，所述的细胞因子是IFN-γ。
在具体的实施方式中，所述步骤2)包括以下步骤：
2-1.分选细胞活率在80％以上，优选在85％以上的激活的T细胞，并获得单细胞悬液；
2-2.获得细胞的mRNA；
2-3.利用Barcode引物序列与逆转录酶对获得的mRNA进行逆转录，从而得到带有Barcode的单个细胞的TCR alpha可变区和TCR beta可变区的cDNA；
2-4.对获得的cDNA进行纯化和PCR扩增；
2-5.将PCR产物进行环化和任选的线性化。
在优选的实施方式中，所述环化是将PCR产物连接含有TCR恒定区的载体。
在具体的实施方式中，所述步骤2-2包括：获得100-2000个，优选100-800个，最优选200-500个激活的T细胞的mRNA；或者
获得100-50000，优选100-8000个，最优选200-5000个激活的T细胞的mRNA。
在具体的实施方式中，所述步骤3)中，携带TCR alpha恒定区的载体具有启动子、TCR alpha恒定区序列和PolyA位点；携带TCR beta恒定区的载体具有启动子、TCR beta恒定区序列和PolyA位点。
在具体的实施方式中，所述步骤4)中，对步骤3)获得的TCR alpha可变区序列和TCR beta可变区序列进行测序并挑选具有相同细胞Barcode的TCR alpha可变区序列和TCR beta可变区序列，从而获得正确配对的TCR。
在优选的实施方式中，所述测序是进行Sanger测序和/或NGS测序。
在具体的实施方式中，所述方法还包括以下步骤：
5)任选对获得的TCR进行功能评价。
在另一实施方式中，本发明提供一种快速克隆配对TCR序列的方法，所述的方法包括以下步骤：肿瘤组织中分选并捕获单个肿瘤反应性T细胞，提取、标记单个细胞的mRNA，反转录并构建全长cDNA，cDNA特异性PCR扩增得到cDNA全长转录组；
cDNA全长转录组富集后，与启动子序列连接环化并进行TCR序列的特异性扩增，扩增产物富集后连接到表达载体中得到TCR的全长克隆，进行文库序列比对获得配对的TCR序列。
在优选的实施方式中，所述的方法中，mRNA反转录时在反转录体系中加入TSO，扩增完成cDNA全长转录组的富集。
在优选的实施方式中，所述的方法中，通过单细胞标记磁珠5’端的恒定区序列及反转录过程中添加上的TSO序列，扩增完成全长转录组的富集。
在优选的实施方式中，所述的方法中环化方法为：
环化时直接将启动子序列插入到TSO序列之前；所述的启动子是任一启动mRNA转录的基因元件，包括但不限于CMV、EF1alpha、SV40、PGK1、CAG、T7、Sp6；
设计PCR引物扩增CMV启动子序列CMV-P，并在正向和反向引物的5’端添加cDNA同源序列后进行TCR序列的特异性扩增，完成TCR的扩增。
在优选的实施方式中，所述的配对TCR序列包含TCR-alpha和TCR-beta序列。
在优选的实施方式中，所述的表达载体为任一具有复制子和抗性基因元件的质粒；优选的表达载体为线性化pMax-TRAC和pMax-TRBC载体；
表达载体pMax经过人工改造去掉载体自身的CMV启动子，添加TCR alpha恒定区得到pMax-TRAC，添加TCR beta恒定区得到pMax-TRBC；
通过重组的方法将纯化后的TCR alpha-1克隆到pMax-TRAC，将纯化后的TCR beta-1克隆到pMax-TRBC后具有完整的TCR表达框。
在优选的实施方式中，分选单个肿瘤反应性T细胞的方法为：肿瘤组织消化为单细胞并制备成单细胞悬液后注入微流控芯片获得单个肿瘤反应性T细胞。
在一种具体实施方式中，分选单个肿瘤反应性T细胞的方法为：通过使用带有荧光标签的抗体对单细胞悬液进行染色标记后进行流式分选仪进行分选。
在一种具体实施方式中，分选单个肿瘤反应性T细胞的方法为：将肿瘤组织消化为单细胞，使用带有荧光标签的抗体CXCL13-APC对单细胞悬液进行染色标记，单细胞通过流式分选仪分选肿瘤单细胞悬液中CXCL13阳性的T细胞即为肿瘤反应性T细胞。
在一种具体实施方式中，所述的提取、标记单个细胞mRNA的方法为：单个肿瘤反应性T细胞分离后，在微孔中加入单细胞标记磁珠，完成单个细胞的mRNA的捕获和标记。
在一种具体实施方式中，所述的单细胞标记磁珠表面有携带2个恒定的序列，特异barcode序列和poly dT的单链DNA oligo。
在一种具体实施方式中，所述的单细胞标记磁珠携带不同的barcode，用于标记单个T细胞，poly dT用于捕获总mRNA并作为反转录引物。
在一种具体实施方式中，所述的文库序列比对为：分别进行TCR-alpha和TCR-beta全长克隆后，挑取单克隆进行细胞barcode比对，具有相同细胞barcode的TCR-alpha和TCR-beta序列为配对TCR。
在一个具体实施方式中，本发明的配对TCR序列是通过如下方法获得：
S101、将一定数量组织细胞注入到定制的微流控芯片，芯片表面有可以容纳单个细胞的微孔，根据“泊松分布”的原理完成单个细胞的分离。
S102、在微孔中加入单细胞标记磁珠，利用单细胞标记磁珠完成单个细胞的mRNA的捕获和标记。单细胞标记磁珠表面有携带2个恒定的序列，特异barcode序列和poly dT的单链DNA oligo。每一个磁珠携带不同的barcode，用于标记单个T细胞，polydT用于捕获总mRNA并作为反转录引物。
S103、单个T细胞进行标记后，开始mRNA的反转录，获取cDNA。
S104、在反转录体系中加入TSO(Template switch oligo)，作为后续PCR的引物结合区。通过单细胞标记磁珠5’端的恒定区序列及反转录过程中添加上的TSO序列，扩增完成全长转录组的富集。
S105、将获得的扩增产物与扩增的启动子序列进行重组环化处理，然后使用单细胞标记磁珠上的恒定区序列和TCR恒定区特异性引物进行TCR序列的特异性扩增，完成TCR的富集。
在环化时直接将启动子(Promoter)序列插入到TSO序列之前，TSO序列中不包含ATG序列，从而不影响TCR基因的表达。磁珠上的恒定区序列和barcode序列将位于启动子之前。设计PCR引物从pMax载体上扩增CMV启动子序列CMV-P，并在正向和反向引物的5’端添加cDNA同源序列，PCR扩增CMV启动子。CMV启动子序列CMV-P的核酸序列如见Seq ID No.1所示。
S106、将获得的TCR富集产物与具有TCR恒定区的表达载体重组环化相连，然后转化大肠杆菌进行质粒扩增。
S107、按照上述步骤分别进行TCR-alpha和TCR-beta全长克隆后，挑取单克隆，提取质粒并测序后进行细胞barcode比对，具有相同细胞barcode的TCR-alpha和TCR-beta序列为配对TCR。
在另一实施方式中，本发明提供一种克隆配对TCR序列的方法，所述的方法包括以下步骤：肿瘤组织中分选并捕获单个肿瘤反应性T细胞，提取、通过Barcode标记单个细胞的mRNA，反转录并构建全长cDNA，cDNA特异性PCR扩增得到cDNA全长转录组，富集后第一次环化，进行TCR序列的特异性扩增、富集后第二次环化并再次富集，富集产物连接到表达载体中得到TCR的全长克隆，进行文库中Barcode序列比对获得配对的TCR序列。
在优选的实施方式中，所述的方法中，mRNA反转录时在反转录体系中加入TSO，扩增完成cDNA全长转录组的富集。
在优选的实施方式中，所述的方法中，通过单细胞标记磁珠5’端的恒定区序列及反转录过程中添加上的TSO序列，扩增完成全长转录组的富集。
在优选的实施方式中，所述的方法中第一次环化为，将获得的扩增产物进行环化处理，使用单细胞标记磁珠上的恒定序列和TCR特异性引物(恒定区末端)进行TCR序列的特异性扩增，完成TCR的富集。
在优选的实施方式中，所述的方法中第二次环化为，将获得的扩增产物进行第二次环化处理后，使用根据单细胞标记磁珠恒定序列设计的正反向引物进行TCR的富集，富集后细胞barcode将与TCR恒定区相连，位于富集产物3’端。
在优选的实施方式中，所述的配对TCR序列包含TCR-alpha和TCR-beta序列。
在优选的实施方式中，分选单个肿瘤反应性T细胞的方法为：肿瘤组织消化为单细胞并制备成单细胞悬液后注入微流控芯片获得单个肿瘤反应性T细胞。
在一种具体实施方式中，分选单个肿瘤反应性T细胞的方法为：通过使用带有荧光标 签的抗体对单细胞悬液进行染色标记后进行流式分选仪进行分选。
在一种具体实施方式中，分选单个肿瘤反应性T细胞的方法为：将肿瘤组织消化为单细胞，使用带有荧光标签的抗体CXCL13-APC对单细胞悬液进行染色标记，单细胞通过流式分选仪分选肿瘤单细胞悬液中CXCL13阳性的T细胞即为肿瘤反应性T细胞。
在一种具体实施方式中，所述的提取、标记单个细胞mRNA的方法为：单个肿瘤反应性T细胞分离后，在微孔中加入单细胞标记磁珠，完成单个细胞的mRNA的捕获和标记。
在一种具体实施方式中，所述的单细胞标记磁珠表面有携带2个恒定的序列，特异barcode序列和poly dT的单链DNA oligo。
在一种具体实施方式中，所述的单细胞标记磁珠携带不同的barcode，用于标记单个T细胞，poly dT用于捕获总mRNA并作为反转录引物。
在一种具体实施方式中，所述的文库序列比对为：分别进行TCR-alpha和TCR-beta全长克隆后，挑取单克隆进行细胞barcode比对，具有相同细胞barcode的TCR-alpha和TCR-beta序列为配对TCR。
进一步，在一个具体实施方式中，本发明的配对TCR序列是通过如下方法获得：
S01、将一定数量组织细胞注入到定制的微流控芯片，芯片表面有可以容纳单个细胞的微孔，根据“泊松分布”的原理完成单个细胞的分离。
S02、在微孔中加入单细胞标记磁珠，利用单细胞标记磁珠完成单个细胞的mRNA的捕获和标记。单细胞标记磁珠表面有携带2个恒定的序列，特异barcode序列和poly dT的单链DNA oligo。每一个磁珠携带不同的barcode，用于标记单个T细胞，polydT用于捕获总mRNA并作为反转录引物。
S03、单个T细胞进行标记后，开始mRNA的反转录，获取cDNA。
S04、在反转录体系中加入TSO(Template switch oligo)，作为后续PCR的引物结合区。通过单细胞标记磁珠5’端的恒定区序列及反转录过程中添加上的TSO序列，扩增完成全长转录组的富集。
S05、将获得的扩增产物进行环化处理，使用单细胞标记磁珠上的恒定序列和TCR特异性引物(恒定区末端)进行TCR序列的特异性扩增，完成TCR的富集。
S06、将获得的扩增产物进行第二次环化处理后，使用根据单细胞标记磁珠恒定序列设计的正反向引物进行TCR的富集，富集后细胞barcode将与TCR恒定区相连，位于富集产物3’端。最后将TCR的富集产物连接到表达载体中，完成TCR的全长克隆。
S07、按照上述步骤分别进行TCR-alpha和TCR-beta全长克隆后，挑取单克隆进行细胞barcode比对，具有相同细胞barcode的TCR-alpha和TCR-beta序列为配对TCR。
在第二方面，本发明提供一种TCR，所述TCR采用第一方面所述方法制备得到。
在优选的实施方式中，所述TCR是TCR-1到TCR-68中的任一种。
在第三方面，本发明提供第二方面所述的TCR在制备遗传改造的T细胞中的用途。
在第四方面，本发明提供一种遗传改造的T细胞，所述T细胞表达采用第一方面所述方法制备得到的TCR或第二方面所述的TCR。
在一些实施例中，本发明提供的一种TCR-T细胞，所述TCR-T细胞是通过生物工程技术将本发明方法获得的配对TCR序列注入相应的T细胞后获得。
在这些方法中，所述相应的T细胞可以指个体自身T细胞或异源T细胞。
在这些方法中，所述异源T细胞可以是同类物种的不同个体的T细胞和/或不同物种的T细胞。
在这些方法中，所述异源T细胞可以是来自人类不同个体的T细胞或来自其它动物体的T细胞。
在第五方面，本发明提供第四方面所述的遗传改造的T细胞在用于治疗与T细胞相关的疾病的药物组合物的用途。
与T细胞相关的疾病包括癌症、感染性疾病和自身免疫病。在一些实施例中，与T细胞相关的疾病是肿瘤，或者癌症。一些实施例中，与T细胞相关的疾病是感染性疾病。一些实施例中，与T细胞相关的疾病是自体免疫性疾病。在一些实施例中，与T细胞相关的疾病是器官移植相关疾病。
在一些实施例中，所述药物组合物中所含有的TCR-T细胞是来自患者自身的T细胞。
在一些实施例中，所述的TCR-T细胞中的TCR序列是从患者自身T细胞获取mRNA后获得的TCR全长序列信息。
在一些实施方式中，本发明提供的遗传改造的TCR-T细胞可用于制备治疗肿瘤的药物组合物。在一些实施例中，可被治疗的肿瘤选自下组：急性淋巴细胞白血病、急性髓性白血病、肾上腺皮质癌、AIDS相关癌症、AIDS相关淋巴瘤、肛门癌、阑尾癌、星形细胞瘤、神经母细胞瘤、基底细胞癌、胆管癌、膀胱癌、骨癌、脑肿瘤、乳腺癌、支气管腺瘤、伯基特淋巴瘤、原发性未知癌、中枢神经系统淋巴瘤、小脑星形细胞瘤、宫颈癌、儿童癌症、慢性淋巴细胞白血病、慢性骨髓性白血病、慢性骨髓增生性疾病、结肠癌、皮肤T细胞淋巴瘤、结缔组织增生性小圆细胞瘤、子宫内膜癌、室管膜瘤、食道癌、尤因氏肉瘤、生殖细胞肿瘤、胆囊癌、胃癌、胃肠道类癌肿瘤、胃肠道间质瘤、胶质瘤、毛细胞白血病、头颈癌、心脏癌、肝细胞癌、霍奇金淋巴瘤、咽下癌、眼内黑色素瘤、胰岛细胞癌、卡波西肉瘤、肾癌、喉癌、唇癌和口腔癌、脂肪肉瘤、肝癌、肺癌、淋巴瘤、白血病、巨球蛋白血症、骨恶性纤维组织细胞瘤/骨肉瘤、成神经管细胞瘤、黑色素瘤、间皮瘤、原发灶隐匿的转移性鳞状颈癌、口腔癌、多发性内分泌肿瘤综合症、骨髓增生异常综合症、骨髓性白血病、鼻腔和副鼻窦癌、鼻咽癌、神经母细胞瘤、非霍奇金淋巴瘤、非小细胞肺癌、口腔癌、口咽癌、骨肉瘤/骨恶性纤维组织细胞瘤、卵巢癌、卵巢上皮癌、卵巢生殖细胞肿瘤、胰腺癌、胰腺癌胰岛细胞、副鼻窦和鼻腔癌、甲状旁腺癌、阴茎癌、咽癌、嗜铬细胞瘤、松果体星形细胞瘤、松果体生殖细胞瘤、垂体腺瘤、胸膜肺母细胞瘤、浆细胞瘤、原发性中枢神经系统淋巴瘤、前列腺癌、直 肠癌、肾细胞癌、肾盂和输尿管移行细胞癌、视网膜母细胞瘤、横纹肌肉瘤、唾液腺癌、肉瘤、皮肤癌、默克尔细胞皮肤癌、小肠癌、软组织肉瘤、鳞状细胞癌、胃癌、T细胞淋巴瘤、咽喉癌、胸腺瘤、胸腺癌、甲状腺癌、滋养细胞肿瘤、原发部位未知癌、尿道癌、子宫肉瘤、阴道癌、外阴癌、瓦尔登斯特伦巨球蛋白血症和威尔姆斯瘤。
在一些实施例中，本发明提供的遗传改造的TCR-T细胞可用于制备治疗自体免疫性疾病的药用组合物。所述的自体免疫性疾病可以选自下组：关节炎、慢性阻塞性肺疾病、强直性脊柱炎、克罗恩病、皮肌炎、I型糖尿病、子宫内膜异位症、Goodpasture氏综合症、Graves氏病、格林-巴厘综合症、桥本氏病、化脓性汗腺炎、川崎病、IgA肾病、原发性血小板减少性紫癜、间质性膀胱炎、红斑狼疮、混合性结缔组织病、硬斑病、重症肌无力、嗜睡症、神经性肌强直、寻常型天疱疮、恶性贫血、银屑病、银屑病关节炎、多发性肌炎、原发性胆汁性肝硬化、复发性多软骨炎、类风湿关节炎、精神分裂症、硬皮病、干燥综合症、僵人综合症、颞动脉炎、溃疡性结肠炎、脉管炎、白斑病以及韦格纳肉芽肿。
在第六方面，本发明提供一种药物组合物，所述药物组合物包含第四方面所述的遗传改造的T细胞，以及药学上可接受的赋形剂。
在另一项具体实施方式中，所述的药物组合物含有不同于TCR-T细胞的第二活性制剂，所述第二活性制剂包括具有抗肿瘤作用的药物、提高患者抵抗力的药物和/或增加患者耐受性的药物等。
在优选的实施方式中，所述药物组合物用于治疗肿瘤和自体免疫性疾病；所述的肿瘤选自下组：急性淋巴细胞白血病、急性髓性白血病、肾上腺皮质癌、AIDS相关癌症、AIDS相关淋巴瘤、肛门癌、阑尾癌、星形细胞瘤、神经母细胞瘤、基底细胞癌、胆管癌、膀胱癌、骨癌、脑肿瘤、乳腺癌、支气管腺瘤、伯基特淋巴瘤、原发性未知癌、中枢神经系统淋巴瘤、小脑星形细胞瘤、宫颈癌、儿童癌症、慢性淋巴细胞白血病、慢性骨髓性白血病、慢性骨髓增生性疾病、结肠癌、皮肤T细胞淋巴瘤、结缔组织增生性小圆细胞瘤、子宫内膜癌、室管膜瘤、食道癌、尤因氏肉瘤、生殖细胞肿瘤、胆囊癌、胃癌、胃肠道类癌肿瘤、胃肠道间质瘤、胶质瘤、毛细胞白血病、头颈癌、心脏癌、肝细胞癌、霍奇金淋巴瘤、咽下癌、眼内黑色素瘤、胰岛细胞癌、卡波西肉瘤、肾癌、喉癌、唇癌和口腔癌、脂肪肉瘤、肝癌、肺癌、淋巴瘤、白血病、巨球蛋白血症、骨恶性纤维组织细胞瘤/骨肉瘤、成神经管细胞瘤、黑色素瘤、间皮瘤、原发灶隐匿的转移性鳞状颈癌、口腔癌、多发性内分泌肿瘤综合症、骨髓增生异常综合症、骨髓性白血病、鼻腔和副鼻窦癌、鼻咽癌、神经母细胞瘤、非霍奇金淋巴瘤、非小细胞肺癌、口腔癌、口咽癌、骨肉瘤/骨恶性纤维组织细胞瘤、卵巢癌、卵巢上皮癌、卵巢生殖细胞肿瘤、胰腺癌、胰腺癌胰岛细胞、副鼻窦和鼻腔癌、甲状旁腺癌、阴茎癌、咽癌、嗜铬细胞瘤、松果体星形细胞瘤、松果体生殖细胞瘤、垂体腺瘤、胸膜肺母细胞瘤、浆细胞瘤、原发性中枢神经系统淋巴瘤、前列腺癌、直肠癌、肾细胞癌、肾盂和输尿管移行细胞癌、视网膜母细胞瘤、横纹肌肉瘤、唾液腺癌、肉瘤、皮肤癌、默克尔细胞皮肤癌、小肠癌、软组织肉瘤、鳞状细胞癌、胃癌、T细胞淋巴瘤、咽喉癌、胸腺瘤、胸腺癌、甲状腺癌、滋养 细胞肿瘤、原发部位未知癌、尿道癌、子宫肉瘤、阴道癌、外阴癌、瓦尔登斯特伦巨球蛋白血症和威尔姆斯瘤；
所述的自体免疫性疾病选自下组：关节炎、慢性阻塞性肺疾病、强直性脊柱炎、克罗恩病、皮肌炎、I型糖尿病、子宫内膜异位症、Goodpasture氏综合症、Graves氏病、格林-巴厘综合症、桥本氏病、化脓性汗腺炎、川崎病、IgA肾病、原发性血小板减少性紫癜、间质性膀胱炎、红斑狼疮、混合性结缔组织病、硬斑病、重症肌无力、嗜睡症、神经性肌强直、寻常型天疱疮、恶性贫血、银屑病、银屑病关节炎、多发性肌炎、原发性胆汁性肝硬化、复发性多软骨炎、类风湿关节炎、精神分裂症、硬皮病、干燥综合症、僵人综合症、颞动脉炎、溃疡性结肠炎、脉管炎、白斑病以及韦格纳肉芽肿。
第七方面，本发明提供一种诊断和/或评估制剂。
在一些实施例中，本发明提供的诊断和/或评估制剂含有本发明第四方面获得的TCR-T细胞。
进一步的，所述的制剂包括辅料，所述辅料包括载体或稀释剂；
所述的载体或稀释剂为：与TCR-T细胞相容的任何和所有溶剂、分散介质、包衣、等渗和吸收延迟剂。
进一步的，所述的制剂制备为制剂盒。
在另一项具体实施方式中，所述的诊断和/或评估制剂用于诊断或评估与T细胞相关的疾病或事件；
所述疾病或事件包括感染性疾病、肿瘤、自身免疫疾病和器官移植等。
在一些实施例中，本发明提供的诊断和/或评估制剂可用于生物标志物、抗体开发、用药和疫苗评估、免疫细胞分化溯源、免疫排斥和耐受、微小残留病检测、食品或其它过敏原检测。
在另一项具体实施方式中，所述的表达载体为病毒载体或非病毒载体。
在另一项具体实施方式中，所述的载体包含编码TCR的核酸和编码CD8αβ或CD8α的核酸。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了实施例1中利用流式细胞仪检测骨髓单个核细胞中CD8+细胞和肿瘤细胞的比例；
图2显示了实施例1分选的CD8+细胞与肿瘤细胞共培养后流式检测肿瘤细胞可以激活CD8+细胞；
图3显示了实施例2电泳检测PCR扩增得到的TCR alpha和TCR beta基因片段；
图4显示了实施例3表达TCR的载体TCR-TRAC-pMax和TCR-TRBC-pMax图谱；
图5显示了实施例3电泳检测载体TCR-TRAC-pMax和TCR-TRBC-pMax线性化基因片段；
图6显示了实施例3电泳检测TCR alpha和TCR beta基因片段连接到表达载体上的阳性克隆；
图7显示了实施例4流式检测敲除TCR的Jurkat细胞；
图8显示了实施例4流式检测配对的TCR质粒电转到细胞中表达情况；
图9显示了实施例4筛选的配对TCR与肿瘤细胞共培养后检测其功能；
图10显示了cDNA文库主条带分布示例图；
图11显示了TCR V区文库主条带分布示例图；
图12显示了未筛选TCR克隆型分布；
图13显示了筛选后TCR克隆型分布；
图14显示了TCR富集产物的环化和扩增示意图；
图15显示了实施例9电泳检测PCR扩增得到的TCR alpha和TCR beta完整片段；
图16为实施例11的单细胞标记磁珠示意图；
图17为实施例11的TCR质粒文库构建技术流程图；
图18为实施例11的电泳检测PCR扩增得到的TCR alpha和TCR beta基因片段；
图19为实施例11的电泳检测PCR扩增得到的TCR alpha恒定区和TCR beta恒定区基因片段；
图20为实施例11的电泳检测PCR扩增得到的TCR alpha全长片段和TCR beta全长片段；
图21为实施例11中表达TCR的载体TCR-pMax图谱；
图22为实施例11中电泳检测TCR alpha和TCR beta全长片段连接到表达载体上的阳性克隆；
图23为实施例11中，流式检测敲除TCR的Jurkat细胞；
图24为实施例11中，流式检测配对的TCR表达质粒电转到细胞中表达情况；
图25为实施例12中TCR质粒文库构建技术流程图；其中，TSO指template switch oligo，V region指TCR的可变区、C region指TCR的恒定区、Promoter为启动子；
图26为实施例12中电泳检测PCR扩增得到的TCR alpha和TCR beta基因片段；
图27为实施例12中，线性化pMax-TRAC和pMax-TRBC载体图谱；
图28为实施例12中，电泳检测TCR alpha和TCR beta基因片段连接到表达载体上的阳性克隆。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现了一种全新的获得正确配对的TCR的 方法，所述方法通过DNA barcode对单细胞TCR的mRNA和cDNA进行标记，给配对的TCR加上标签，然后再通过克隆筛选找到具有相同标签的TCR进行配对，从而能够在一个反转录体系内实现数百个细胞的配对TCR克隆，实现快速，低成本，并且高通量的获取配对TCR。在此基础上完成了本发明。
DNA Barcode
DNA Barcode是带有不同碱基的DNA片段，是样品的标签。用于区分不同的样品，在该专利中指细胞标签序列。它的作用是特异性的标记单个细胞，使得每个细胞具有不同的barcode。如果测序读取的基因序列具有同样的barcode，则这些基因来源于同一个细胞。
本发明的克隆TCR基因的方法
在本发明中，“克隆TCR基因的方法”与“获得配对TCR的方法”、“本发明的方法”以及“获取全长TCR基因的方法”等具有相同的意义，均是指获取个体的完整TCR基因的方法。
在本领域中，目前主要的克隆TCR基因的方法有以下几种：
1.对T细胞群进行单细胞分选，然后对单细胞进行裂解和反转录，最后使用靶向TCR可变区的多重兼并引物以及靶向TCR恒定区的引物进行单细胞水平PCR，扩增TCR可变区。这是一种快速低成本获取TCR可变区的方法，但是这种方法由于使用的兼并引物在TCR可变区内部，因此扩增得到的区域只是部分区域，不是TCR全长，无法直接应用于下游分子构建和蛋白表达。一般需要进行数据库序列比对，从数据库拿到全长序列后进行基因合成，才能获得全长TCR序列(Rapid Identification and Evaluation of Neoantigen-reactive T-Cell Receptors From Single Cells.J Immunother.2021Jan；44(1):1-8)。
2.对T细胞群进行单细胞分选，然后使用激活剂激活扩增单个T细胞，一般需要扩增两周左右的时间，让单个T细胞变成一个T细胞克隆群，细胞数目达到几百个或者上千个。然后使用5’RACE(rapid amplification of cDNA ends)方法进行TCR可变区全长扩增，扩增后可以得到TCR全长基因，后续直接扩增得到TCR全长基因。但是这种方法耗时很长，很多单细胞不能成功形成多细胞克隆，试剂盒成本很高无法大量扩增TCR基因(Development of a CD8co-receptor independent T-cell receptor specific for tumor-associated antigen MAGE-A4for next generation T-cell-based immunotherapy.J Immunother Cancer.2021；9(3))。
3.与第二种方法类似，对T细胞群进行单细胞分选，然后直接在单细胞水平进行5‘RACE扩增TCR可变区全长，后续再进行PCR扩增得到TCR全长基因。这种方法虽然比较简单，但是同样因为每个细胞需要一个单独的反应体系，试剂消耗大，试剂盒成本很高，无法大量扩增TCR基因(An immunodominant NP105-113-B*07:02cytotoxic T cell response controls viral replication and is associated with less severe COVID-19disease.Nat Immunol.2022；01；23(1):50-61.)。
4.使用单细胞DNA barcode对T细胞群中的每一个T细胞进行标记，然后使用高通量测序的方法获得配对TCR全长序列信息。对筛选确定的配对TCR进行基因合成得到全长 TCR基因。这种方法虽然可以大量低成本的获得配对TCR序列，但是只能得到序列信息，无法直接获得TCR可变区全长基因片段，后续基因合成成本高，无法大量进行TCR的合成(Direct identification of neoantigenspecific TCRs from tumor specimens by high-throughput single-cell sequencing.Journal for ImmunoTherapy of Cancer 2021；9:e002595.)。
与现有技术相比，本发明通过DNA barcode对单细胞TCR的mRNA和cDNA进行标记，给配对的TCR加上标签，然后再通过克隆筛选找到具有相同标签的TCR进行配对，在一个反转录体系内实现数百个细胞的配对TCR克隆，实现快速，低成本，并且高通量的获取配对TCR。在本发明的开发中，巧妙的应用了SMART(switching mechanism at the 5'end of the RNA transcript)技术(Takara Bio USA,Inc)，在下游开发出了独特的高通量获取配对TCR和筛选配对TCR的技术。本发明人首次实现不依赖基因合成的，快速、低成本的获取正确配对的TCR基因。
SMART技术最早在2001年被开发出来，该技术可以实现全长cDNA文库的构建，即使起始材料是来源于单个细胞的RNA。SMART技术的核心是使用叫做TSO(Template switch oligo)的DNA-RNA复合oligo，TSO通过RNA部分可以结合反转录产生的cDNA末端，然后TSO其余部分可以作为cDNA扩增的恒定模版。
另外，本项目优化了配对TCR的下游功能筛选。首次使用DNA质粒为载体进行高通量配对TCR筛选，大大提高了筛选效率。
通过上述两方面对TCR筛选的优化，使常规需要2-3个月才能完成的流程缩短为3到4周，成本降低为原来的1/5-1/10，解决了TCR序列筛选成本高，耗时长，效率低等问题，为TCR类药物的发展奠定基础，也使得个体化TCR-T作为一种细胞疗法成为可能。
在具体的实施方式中，本发明提供的获得正确配对的TCR的方法包括以下步骤：
1)获得肿瘤细胞激活的T细胞；
2)从步骤1)获得的激活的T细胞获得带有Barcode的单个T细胞的TCR alpha可变区和TCR beta可变区的cDNA；
3)将步骤2)获得的带有Barcode的TCR alpha可变区和TCR beta可变区的cDNA分别重组到携带TCR alpha恒定区和TCR beta恒定区的载体中，从而扩增TCR alpha可变区序列和TCR beta可变区序列；
4)从步骤3)获得的TCR alpha可变区序列和TCR beta可变区序列中挑选具有相同细胞Barcode的TCR alpha可变区序列和TCR beta可变区序列，从而获得正确配对的TCR。
本领域技术人员知晓如何获得肿瘤细胞激活的T细胞。例如，可以通过以下步骤获得肿瘤细胞激活的T细胞：
1-1.从肿瘤患者样品中分离单个核细胞；
1-2.从步骤1-1得到的单个核细胞中分选出T细胞和肿瘤细胞；
1-3.利用步骤1-2得到的肿瘤细胞刺激T细胞；
1-4.任选检测激活标志物的表达，以供细胞分选。
本领域技术人员知晓，单个核细胞的来源可以有许多种，例如直接从实体肿瘤内部分 选表达肿瘤反应性T细胞特异性标志物基因的T细胞群。因此，肿瘤细胞与T细胞的共培养并非在任何情况下均是必需的。
在具体的实施方式中，上述步骤2)可以如下所述实现：
2-1.分选细胞活率在80％以上，优选在85％以上的激活的T细胞，并获得单细胞悬液；
2-2.获得细胞的mRNA；
2-3.利用Barcode引物序列与逆转录酶对获得的mRNA进行逆转录，从而得到带有Barcode的单个细胞的TCR alpha可变区和TCR beta可变区的cDNA；
2-4.对获得的cDNA进行纯化和PCR扩增；
2-5.将PCR产物进行环化和任选的线性化。
在优选的实施方式中，所述环化是将PCR产物连接含有TCR恒定区的载体。
进一步地，本发明人还发现获取单个T细胞的mRNA时，细胞的数量可以有较宽的范围。换言之，本发明的方法既适用于较低的细胞数，也适用于较高的细胞数。在具体的实施方式中，本发明的方法可以从100-2000个，优选100-800个，最优选200-500个激活的T细胞中获取mRNA。
当将本发明的方法应用于较高数量的细胞时，可以将获得的cDNA和TCR可变区扩增产物进行建库和NGS测序，通过分析TCR可变区的NGS测序结果确定配对的TCR序列信息，然后从建立的TCR质粒库中寻找与NGS测序结果中序列相同的TCR，从而得到配对TCR全长基因序列。在这种情况下，本发明的方法可以从100-50000，优选100-8000个，最优选200-5000个激活的T细胞中获取mRNA。
本发明人进一步发现，如果Barcode标记在单个T细胞的TCR alpha可变区和TCR beta可变区的mRNA的3’端，则在获得TCR表达载体后，barcode位于启动子和基因之间，影响基因的表达。因此，为克服Barcode标记在单个T细胞的TCR alpha可变区和TCR beta可变区的mRNA的3’端带来的缺陷，本发明人进一步改进了本发明，将获得的带有Barcode的TCR alpha可变区和TCR beta可变区的cDNA先与启动子序列连接，再分别重组到携带TCR alpha恒定区和TCR beta恒定区的载体中，从而扩增TCR alpha可变区序列和TCR beta可变区序列；或者，将获得的带有Barcode的TCR alpha可变区和TCR beta可变区的cDNA进行第两次环化，再分别重组到携带TCR alpha恒定区和TCR beta恒定区的载体中，从而扩增TCR alpha可变区序列和TCR beta可变区序列。
本发明的优点：
1.本发明首次实现不依赖基因合成的，快速、低成本地获取配对的TCR基因；
2.本发明首次使用DNA质粒为载体进行高通量配对TCR筛选，大大提高了筛选效率；
3.本发明显著降低TCR筛选所需的时间；
4.本发筛选TCR的成本显著降低；
5.本发明为TCR类药物的发展奠定了新的基础；
6.本发明可以应用于完全个体化的TCR-T细胞疗法。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例：
实施例1.骨髓来源T细胞激活
1.分离单个核细胞：使用含有抗凝剂的管子采集AML病人骨髓样本，低温保存。骨髓样本倒入PBMC分离管(达优，Cat#7121012)，20℃，800g离心15min后吸取单个核细胞的细胞层。吸取的单个核细胞用RPMI1640完全培养基清洗1-2次，最后用RPMI1640完全培养基重悬细胞备用。取少量分离得到的骨髓单个核细胞细胞加入anti-CD8-PE(Biolegend，344706)、anti-CD33-PE(Biolegend，366606)和anti-CD34-PE(Biolegend，343608)对细胞进行染色，4℃孵育半小时。染色完成后，用PBS清洗细胞后重悬，使用流式细胞仪检测T细胞和肿瘤细胞(CD33或CD34阳性细胞)比例。流式细胞仪检测骨髓单个核细胞中CD8细胞和肿瘤细胞的比例，结果见图1A和1B。
2.T细胞分选：利用美天旎anti-APC Micro Beads(130-090-855)和MS分离柱(130-090-855)从PBMC中先分选出CD8+T细胞。
肿瘤细胞的分选：上一步骤中分选得到的CD8阴性的细胞，用美天旎磁珠和MS分离柱从中分选出CD33+&CD34+肿瘤细胞。
3.激活标志物CD137检测：
3.1细胞共培养：分选得到的CD33+&CD34+肿瘤细胞计数后，按照细胞浓度2×105/ml，100ul/孔铺至96孔平底板中。根据效靶比2:1，将CD8+T细胞的密度调整至2×105/ml，按照100ul每孔接种到96孔板中与肿瘤细胞混合共培养。
3.2检测CD137表达：共培养24h后，吸弃上清。剩余的细胞取60ul，加入anti-CD8-APC(BD Pharmingen，555369)、anti-CD137-PE(BD Pharmingen，555956)抗体对细胞进行染色，4℃孵育半小时。染色完成后，用PBS清洗细胞后重悬，使用流式细胞仪检测CD137的表达。共培养后的细胞流式检测结果见图2，从流式结果可以看出分选的CD8+T细胞与肿瘤细胞共培养后能检测到CD137表达上调。
实施例2.使用油包水体系获取单个T细胞TCR alpha和TCR beta可变区序列
1.细胞制备：流式检测到CD137表达上调的T细胞，使用BD FACSjazz cell sorter(BD，655489)分选收集CD137阳性的T细胞，细胞分选流程按照仪器的说明书操作。对分选得到CD137阳性的T细胞进行活性测定和细胞计数，保证细胞活率在85％以上，根据计数将细 胞密度调整到1X106/ml，制备成单细胞悬液。
2.单细胞分选：选择300-600个(100-2000个细胞范围内)活性状态良好的T细胞上样，将样品命名为ST。将Master Mix(10x Genomics，Chromium Next GEM Single Cell 5'Library&Gel Bead Kit v1.1，PN-1000165)和细胞悬液共70ul、含有barcode序列的凝胶珠50ul和45ul的油滴分别加入到Chromium Next GEM Chip G的不同小室并且不要产生气泡，经由微流体“双十字”交叉系统形成油滴包裹的凝胶珠(Gel Bead-In-EMulsions，GEMs)。
3.反转录：收集GEMs，凝胶珠在每个油滴内自动溶解释放大量Barcode引物序列，同时细胞裂解释放出mRNA。因为beads已带上接头，其中有一段是poly dT序列，在细胞裂解释放的核酸中，只有mRNA带有polyA的尾巴，于是这段beads上面poly dT就可以从众多的裂解产物里捕获到mRNA。同时Master Mix中带有反转录试剂，当mRNA被捕获后，在逆转录酶的作用下发生逆转录反应，产生用于测序的带有Barcode和UMI信息的cDNA一链。收集混合裂解后的产物在PCR仪上进行反转录：53℃45min；85℃5min；4℃保存。
4.反转录产物纯化
4.1将纯化磁珠取出后上下颠倒8-10次并且多次震荡，直到所有磁珠都充分悬浮。
4.2在反转录产物中加入200ul的磁珠，并在室温下孵育5min。放置在磁性分离器2min，吸出上清液将EP管继续留在磁性分离器上。
4.3每管加入300ul 80％的乙醇清洗30s，无需震荡并保持EP管在磁性分离器上30s。吸出上清液。重复该步骤一次。
4.4最后一次尽可能吸尽上清液室温下风干2min，过干可能会降低产量。
4.5将EP管从磁性分离器上取下来，加入35.5ul洗脱溶液，室温下静置2min。
4.6将EP管放在磁性分离器上1-2min直到磁珠被磁铁吸附，吸取35ul上清到新的EP管中，尽量不要吸到磁珠。
5.cDNA扩增和纯化
5.1在冰上预先配制以下试剂：
将配制好的65ul的cDNA扩增mix全部加入到上一步骤纯化的35ul产物中。
5.2PCR扩增条件：
5.3cDNA产物纯化：
5.3.1将纯化磁珠从冰箱取出后上下颠倒8-10次并且多次震荡，直到所有磁珠都充分悬 浮。
5.3.2在cDNA产物中加入0.6X的磁珠60ul，并在室温下孵育5min。放置在磁性分离器上2min，吸出上清液，将EP管继续留在磁性分离器上。
5.3.3每管加入200ul 80％的乙醇清洗30s，无需震荡并保持EP管在磁性分离器上。吸出上清液，重复此步骤两次，最后一次尽可能吸尽上清液。
5.3.4室温下风干2min，过干可能会降低产量。
5.3.5加入45.5ul洗脱溶液混匀，室温下静置5min，将EP管放在磁性分离器上1-2min直到磁珠被磁铁吸附，吸取45ul上清到新的EP管中，尽量不要吸到磁珠。
6.TCR富集
6.1取纯化后的cDNA 2ul加入到33ul的ddH2O,得到总体积为35ul样品备用。
在冰上预先配制以下试剂：
将以上配制好的65ul扩增mix全部加入到备用的35ul样品中。
6.2 PCR扩增条件：
6.3 TCR富集产物纯化：
6.3.1将纯化磁珠从冰箱取出后上下颠倒8-10次并且多次震荡，直到所有磁珠都充分悬浮。
6.3.2在一轮TCR产物中加入0.8X的磁珠80ul，并在室温下孵育5min。放置在磁性分离器上2min，吸出上清液，将EP管继续留在磁性分离器上。
6.3.3每管加入200ul 80％的乙醇清洗30s，无需震荡并保持EP管在磁性分离器上。吸出上清液，重复此步骤两次，最后一次尽可能吸尽上清液。
6.3.4室温下风干2min，过干可能会降低产量。
6.3.5加入35.5ul洗脱溶液混匀，室温下静置5min，将EP管放在磁性分离器上1-2min直到磁珠被磁铁吸附，吸取45ul上清到新的EP管中，尽量不要吸到磁珠。
7.PCR分别扩增TCR alpha和TCR beta可变区
7.1对富集后的TCR产物分别扩增TCR alpha和TCR beta可变区
PCR扩增体系：50ul

其中F-R1引物序列为：
TCR alpha-R引物序列为：AGTCTCTCAGCTGGTACACG(SEQ ID NO:6)
TCR beta-R引物序列为：TCTGATGGCTCAAACACAGC(SEQ ID NO:7)
PCR扩增条件：
琼脂糖凝胶电泳检测PCR产物，结果见图3。从电泳结果可以看出，TCR alpha和TCR beta可变区扩增后形成单一的条带。
7.2 PCR产物纯化，按照试剂盒提供的标准操作流程进行(天根生化科技有限公司，DP219-03)操作。
7.2.1将单一的DNA条带从琼脂糖凝胶中切下(尽量切除多余部分)放入干净的离心管中，称取重量。
7.2.2向凝胶块中加入3倍体积溶胶液PE(如果凝胶重0.1g，其体积可视为100ul，则加入300ul溶胶液PE)。室温15℃-25℃溶胶5-10min，期间不断温和地上下翻转离心管，以确保胶块充分溶解。
7.2.3将上一步所得溶液加入一个吸附柱CA5中(吸附柱放入收集管中)，室温放置2min，12000rpm离心30-60s，倒掉收集管中的废液，将吸附柱CA5放入收集管中。
7.2.4向吸附柱CA5中加入600ul漂洗液PW，12000rpm离心30-60s，倒掉收集管中的废液，将吸附柱CA5放入收集管中。
7.2.5重复操作步骤7.2.4。
7.2.6将吸附柱CA5放回收集管中，12000rpm离心2min，尽量除尽漂洗液。将吸附柱CA5置于室温放置数分钟，彻底地晾干，以防止残留的漂洗液影响下一步实验。
7.2.7将吸附柱CA5放到一个干净的离心管中，向吸附膜中间位置悬空滴加适量洗脱缓冲液TB或提前预热的ddH2O，室温放置2min。12000rpm离心2min收集DNA溶液。纯化回收后的产物分别标记为：TCR-TRAV和TCR-TRBV，共2个产物。该产物携带5‘端barcode序列和其他元件，TCR可变区全长以及部分的TCR恒定区序列。
注：TCR富集产物包含一部分恒定区序列，作为重组所用的同源序列。
实施例3.配对全长TCR序列获得
1.连接TCR-pMax载体：该载体携带CMV启动子，TCR alpha和TCR beta恒定区序列和polyA位点。恒定区使用人TCR alpha和TCR beta恒定区。载体中人TCR alpha恒定区 命名为TRAC(核酸序列见Seq ID No.1，氨基酸序列见Seq ID No.2)。载体中人的TCR beta恒定区命名为TRBC(核酸序列见Seq ID No.3，氨基酸序列见Seq ID No.4)，其中TCR-TRAC-pMax和TCR-TRBC-pMax载体图谱见图4，从图谱可以看出TCR-TRAC-pMax和TCR-TRBC-pMax载体中除了包含基因表达所需要的启动子、还包含TCR alpha和TCR beta的恒定区序列和相应的酶切位点，便于实施例2中富集的TCR产物快速克隆到该载体中。
人的TCR alpha恒定区核酸序列Seq ID No.1
人的TCR alpha恒定区氨基酸序列Seq ID No.2
人的TCR beta恒定区核酸序列Seq ID No.3
人的TCR beta恒定区氨基酸序列Seq ID No.4
通过重组的方法将纯化后的TCR alpha富集产物重组到TCR-TRAC-pMax、纯化后的TCR beta富集产物重组到TCR-TRBC-pMax载体，最终的TCR序列具有完整的恒定区和TCR表达框，可以高效表达TCR亚基。具体步骤为：
1.1制备线性化载体：利用BamHI(NEB:R3136S)和KpnI(NEB:R3142S)将TCR-TRAC-pMax、TCR-TRBC-pMax线性化并纯化。TCR-TRAC-pMax、TCR-TRBC-pMax线性化后进行琼脂糖凝胶电泳检测，结果见图5。
1.2重组反应：
50℃反应20min
2.转化大肠杆菌
2.1在冰上解冻感受态细胞DH5α(上海唯地生物科技有限公司)，一般转化产物的体积不超过感受态体积的1/10。
2.2取10ul重组产物，加入到100ul感受态细胞中，轻弹管壁数下混匀，在冰上放置20min。
2.3 42℃热激90s，冰上放置2min，加600ul无双抗LB培养基(上海生工)，37℃摇床220rpm复苏1h，4500rpm离心5min。吸去600ul上清，剩余的菌液全部涂含Amp平板，37℃微生物培养箱培养过夜。
3.菌检
第二天从转化TCR alpha和TCR beta基因片段的平板上分别挑选300个单菌落于96孔深孔板中37℃摇床摇2h，深孔板每孔放入1ml含Amp抗性LB培养基。每个取2ul菌液PCR初步鉴定阳性克隆。
PCR菌检体系：20ul
其中ll25-JJ-F1引物序列(菌检引物)为：taggcacctattggtcttac(SEQ ID NO:352)
ll25-JJ-R1引物序列(菌检引物)为：tcactgcattctagttgtgg(SEQ ID NO:353)
PCR菌检条件：
琼脂糖凝胶电泳检测菌检结果见图6，从电泳结果可以看出插入序列长度在750bp的条带为阳性克隆，筛选得到的阳性克隆的剩余菌液使用无内毒素质粒小提中量试剂盒(天根生化科技有限公司，DP118-02)抽提质粒。
4.Sanger测序
对初步鉴定出来的阳性克隆送相应的质粒sanger测序验证。TCR alpha共送测192个克隆，分别按照TCR alpha1-192进行标记。TCR beta送测192个克隆，分别按照TCR beta 1-192进行标记。其中测序引物序列为：taggcacctattggtcttac(SEQ ID NO:354)。
5.序列比对，挑选配对TCR序列
通过sanger测序得到TCR alpha和TCR beta可变区的完整序列以及单个细胞的barcode序列。完整序列通过DNA序列分析软件分析得到TCR-alpha和TCR-beta可变区序列，可以判定具有同一个细胞barcode的TCR-alpha和TCR-beta克隆为一对TCR即配对TCR。通过这种方法在所有挑选的克隆中获得配对的TCR序列，将配对TCR alpha和TCR beta质粒找 出并标记。共筛选到110对配对的TCR质粒，然后将配对的TCR alpha和TCR beta质粒混合到一起重新按照TCRab1-110进行标记。其中表1展示了其中的24对具有相同barcode的配对TCR。进一步使用IMGT数据库(https://www.imgt.org)分别分析不同TCR-alpha和TCR-beta可变区对应的可变区类型，结果见表2和表3。
表1.24对具有相同barcode的配对TCR序列









表2.TCR-alpha可变区对应的可变区类型






表3.TCR-beta可变区对应的可变区类型






实施例4.
参考实施例2和实施例3，选择细胞上样量为2500个细胞，经过PCR扩增和连接TCR-pMax载体后，TCR alpha和TCR beta分别挑选了共300个克隆进行测序和序列比对。经过序列分析发现，在挑选的克隆中只有两对配对的TCR，筛选到的配对的TCR占挑取的TCR总克隆的0.67％，获得配对TCR的效率大大降低。因此需要挑选更多的克隆才可能获得更多的配对TCR，大大增加了工作量和成本。因此通过挑取单克隆和sanger测序寻找barcode一致的，配对的TCR需要对上样细胞数进行严格的限制，否则获得配对TCR的效率大大降低，导致试验失败。
实施例5.高通量TCR功能评价
1.效应细胞构建和测试
使用带有NFAT-luciferase的慢病毒感染Jurkat细胞(中国科学院细胞库)，并挑取单克隆培养得到Jurkat-NFAT-luciferase的reporter细胞系(TCR reconstitution in Jurkat reporter cells facilitates the identification of novel tumor antigens by cDNA expression cloning.Int J Cancer.2002 May 1；99(1):7-13)。使用CRISPR/Cas9电转同时敲除TCR基因，选择未敲除的细胞做阴性对照。具体步骤为：
1.1取一定量的Jurkat-NFAT-luciferase细胞，1200rpm离心5min后用培养基opti-MEM洗2次备用。每1X106细胞用25ul的opti-MEM重悬细胞后备用。
1.2冰上准备Cas9和sgRNA：每1X106细胞加入10ul Cas9(500ng/ul)蛋白(南京金斯瑞提供)、2.5ul的TRAC-sgRNA和2.5ul的TRBC-sgRNA)(400ng/ul南京金斯瑞提供)，两者常温下孵育10min，加入提前准备好的细胞开始电转。
1.3电转条件:BTX-ECM830的1mm电击杯，电压为250V，脉冲时间1ms。
1.4电转结束迅速在电击杯里加入1ml培养基，然后将细胞转入培养孔里培养。
1.5电转敲除48h后，取一定体积的细胞，加入anti-CD3-APC抗体(BD Pharmingen，555335)对细胞进行染色，4℃孵育半小时。染色完成后，用PBS清洗细胞后重悬，使用流式细胞仪检测TCR敲除的情况。Jurkat细胞中敲除TCR结果见图7，从流式结果可以看出Jurkat细胞中的TCR已全部敲除，CD3不再表达。通过该步骤制备得到的效应细胞命名为：Jurkat-KO-ER。
2.配对的TCR质粒电转进效应细胞
根据电转最优条件，选取110对配对的TCR质粒中序列完整的24对配对的TCR，分别命名为TCRab-1-24，电转进Jurkat-KO-ER。电转流程为：
2.1取Jurkat-KO-ER细胞，1200rpm离心5min后用培养基opti-MEM洗2次备用。
2.2每1X106的Jurkat-KO-ER细胞加入配对的TCR质粒4ug(TCR alpha和TCR beta质粒各2ug)，补足培养基opti-MEM至100ul。
2.3将质粒和细胞混合物100ul加入到96孔电转板(BTX，45-0450)，电压为260V，脉冲时间1ms。电转后将细胞转移到新的96孔板中，将细胞继续培养。
3.筛选有功能的TCR
3.1流式检测细胞中TCR的表达：配对TCR质粒电转进Jurkat-KO-ER的细胞24h后，取一定体积的细胞，加入anti-CD3-APC抗体(BD Pharmingen，555335)对细胞进行染色，4℃孵育半小时。染色完成后，用PBS清洗细胞后重悬，使用流式细胞仪检测TCR细胞的表达情况。电转的TCR在Jurkat-KO-ER细胞的表达的代表性结果见图8。从图8可以看出，4对代表性的配对的TCR电转到细胞内以后，CD3恢复表达。
3.2细胞铺板：将实施例1中获取的CD33/CD34阳性肿瘤细胞按照细胞浓度1×106/ml，50ul/孔铺至96孔平底不透明白板中。将电转了不同TCR的Jurkat-KO-ER的细胞按照效靶比1:1即细胞浓度1×106/ml，50ul/孔加入到肿瘤细胞中混匀。将96孔板放置于37℃，5％CO2细胞培养箱中培养。
3.3酶标仪检测：细胞共培养24h后测定细胞中荧光素酶活性(相对光单位，RLU)，来检测每个TCR的功能。具体步骤为：每孔加入2X的D-luciferin底物(Thermo Fisher Scientific：88293)100ul混匀避光显色5min，在酶标仪用化学发光模式检测荧光强度。由图9可见，TCR克隆：TCRab-2，TCRab-13和TCRab-16能够特异性的识别肿瘤细胞，可以做进一步筛选评价。
实施例6.TCR alpha和beta克隆至mRNA载体
以TCRab 22，TCRab 33和TCRab 66为模板设计引物分别扩增3个TCR基因序列，PCR扩增条带经琼脂糖凝胶电泳检测后分别进行胶回收纯化得到DNA片段。利用一步法快速克隆试剂盒(翌圣生物科技有限公司：10911ES20)分别将3个TCR基因序列重组进mRNA载体得到重组产物。将3个重组产物分别转入大肠杆菌DH5α(上海唯地生物科技有限公司)菌株，第二天挑取中等大小的白色菌落在含Amp抗生素的液体LB培养基中摇过夜，第二天抽提质粒，酶切鉴定正确后送苏州金唯智生物科技有限公司测序验证，测序验证3个载体均构建正确。得到载体分别为TCRab-22-m、TCRab-33-m和TCRab-66-m。
实施例7.mRNA TCR-T制备和功能评价
1.T细胞激活和mRNA电转：将人外周血单个核细胞(PBMCs，购自上海妙顺生物)稀释到1×106/ml，按照细胞与磁珠1:3的比例使用抗人的CD3/CD28磁珠(Thermo Fisher Scientific)激活T细胞，同时培养基中添加300IU/ml的IL-2(stemcell：78036)。取一定量激活的T细胞1200rpm离心5min后用培养基opti-MEM洗2次备用。每1X10^6细胞用25ul的opti-MEM重悬细胞后备用。冰上融合mRNA：每1X10^6细胞加入1ug的mRNA，开始电转。电转条件:BTX-ECM830的1mm电击杯，电压为250V，脉冲时间1ms。电转结束迅速在电击杯里加入1ml的T细胞培养基，然后将细胞转入培养孔里培养。
2.电转24h后，取一定体积的细胞，加入抗小鼠TCR恒定区的抗体对细胞进行染色，4℃孵育半小时。染色完成后，用PBS清洗细胞后重悬，使用流式细胞仪检测TCR表达的情况。通过该步骤制备得到了mRNA TCR-T。
3.体外功能评价
TCR-T与病人的肿瘤细胞共培养，未电转的T细胞作为对照细胞。
实施例8.细胞数目较多时对配对TCR进行快速克隆
从肿瘤中获取携带肿瘤反应性T细胞标志物的T细胞，在细胞数量比较多的情况下(1000-50000个细胞)，可以将cDNA和TCR V区扩增产物进行建库和NGS测序，通过分析TCR V区的NGS测序结果确定配对的TCR序列信息，然后从实施例3建立的TCR质粒库中寻找与NGS测序结果中序列相同的TCR，从而得到配对TCR全长基因序列，不需要基因合成。基本步骤如下：
1.参考实施例2和实施例3技术流程，选择细胞上样量为1000-20000个细胞，经过PCR 扩增和连接TCR-pMax载体后，TCR alpha和TCR beta组分别挑选100-10000个克隆，提取质粒，建立携带全长TCR序列的质粒库，然后进行sanger测序，确定每一个克隆的序列信息。使用IMGT网站(https://imgt.org/IMGT_vquest/input)分析sanger测序结果，获得TCR V区类型和CDR序列信息。
2.将实施例2中获得的细胞cDNA和TCR V区产物进行建库。基本步骤如下：
1)DNA片段化、末端修复和A-tailing。投入起始量50ng目的产物建库，在PCR反应体系中加入试剂Fragmentation Enzyme、Fragmentation Buffer进行段化、末端修复和添加A尾系列反应。
2)Adaptor连接。向上述PCR产物加入Adaptor Oligos，在DNA连接酶的作用下，进行PCR反应，从而添加adaptor序列。
3)产物纯化。对连接反应后的PCR产物使用0.6X SPRIselect Reagent(货号：19723500)按照试剂盒提供的实验步骤进行纯化。
4)添加Library Index。向上述磁珠纯化后产物中添加Dual Index，进行PCR反应，之后再进行磁珠纯化。注：Dual Index作用是用于每个文库在测序下机后数据可以正确拆分。
5)质控。使用Qubit 3.0Fluorometer(Thermo Fisher Scientific公司)或Qsep1(Bioptic公司)对cDNA和TCR V区建库产物进行质控。TCR V文库和cDNA文库质量要求：浓度≥10ng/ul，总量要求不低于80ng，主条带集中分布在400bp–700bp，300bp以下片段占比要小于20％，达到标准后送NGS测序。cDNA建库质控图见图10。TCR建库质控图见图11。
3.NGS测序和分析
建库完成后，使用DNBSEQ-T7RS测序仪(深圳华大智造科技股份有限公司)，对cDNA和TCR V区建库产物分别测序。根据细胞数量的多少和测序所需要的深度进行双端测序。测序深度要求：TCR V区文库≥5000X，cDNA文库≥20000X。测序下机数据Fastq文件Q30质量值不低于85，数据格式如图所示：
然后使用软件Cell ranger分析TCR的NGS测序结果。获得不同配对TCR克隆型的全长序列以及克隆数信息以下结果：在所有测到的TCR克隆型中，前十位TCR克隆型所占比例见图12。进一步使用软件Cell ranger分析cDNA的测序结果，获得肿瘤反应性标志物基因：PD-1、ENTPD-1和CXCL13等的信息。筛选具有和肿瘤反应性标志物基因相同barcode的TCR，这些TCR很可能是肿瘤反应性TCR。在携带CXCL13基因barcode的TCR克隆型中，前十位TCR克隆型所占比例见图13。挑选上述克隆型数量在前十位的TCR进行下游分子克隆和功能研究。
4.配对TCR挑选
根据实施例8.3中通过NGS方法筛选的配对TCR序列信息(图13)，在实施例8.1中经sanger测序的TCR质粒库中找相同序列的TCR。确定TCR的V区类型和CDR3序列信息完全一致。然后将这些配对的携带TCR基因的质粒挑选出来，可用于下游载体构建和功能评价。TCR克隆信息和CDR3序列信息见表4。上述十种TCR序列在经sanger测序的TCR质粒库中都能够找到。说明本发明提供的方法可以高效的克隆到需要的配对TCR基因。
表4TCR克隆信息和CDR3序列信息。
5.配对TCR克隆
根据配对TCR序列信息分别设计上下游引物，然后使用Gibson重组法，通过三片段重组将配对TCR alpha和TCR beta全长序列克隆到目的质粒。TCR alpha和TCR beta全长序列之间以2A序列连接。这些同时携带TCR alpha和TCR beta全长序列的目的质粒可用于下游的功能验证。
实施例9.
参考实施例8，选择细胞上样量为21000个细胞，经过PCR扩增和连接TCR-pMax载体后，TCR alpha和TCR beta分别挑选了共600个单克隆进行测序和序列比对，建立含有TCR可变区的质粒库。参考实施例8，经过NGS序列分析确定后续研究需要的前十位配对TCR。上述十种TCR序列在经sanger测序的TCR质粒库中只能够找到1对，获得研究需要的配对TCR的效率大大降低。因此需要挑选更多的克隆才可能获得更多的配对TCR，大大 增加了工作量和成本。说明本发明提供的实施例8中克隆方法需要对上样细胞数进行严格的限制，否则获得配对TCR的效率大大降低，导致试验失败。
实施例10.获取TCR全长
由于上述实施例获得的TCR alpha和TCR beta全长序列5‘端包含各种barcode序列和一些恒定元件，可能会影响后续TCR的表达。因此本发明重新设计了凝胶珠负载的oligo序列，增加了TCR富集产物的环化和扩增步骤，将5’端的元件连接到TCR基因片段的3‘端，从而不再影响所克隆TCR的表达，可以直接进行下游应用(示意图见图14)。由于cDNA扩增产物只包含部分的TCR恒定区，因此在环化步骤本发明添加了包含其余TCR恒定区的基因片段进行重组环化。基本步骤如下：
1.扩增产物环化
1.1参考实施例1步骤，将扩增的cDNA放于PCR管中，再添加另一个基因片段：TCR恒定区片段(通过PCR获得)，用TE Buffer补充至50ul。置于PCR仪上95℃反应3min。反应结束后立即将PCR管转移至冰上，冰浴2min。
1.2按照下表配制反应体系：
将上述反应液加入步骤1.1反应后的PCR管中混匀后，瞬时离心将反应液收集至管底。37℃反应15min。反应结束后取出PCR管立即进入下步反应。
1.3酶切消化，取出Digestion Enzyme置于冰上自然融解，混合均匀，瞬时离心。取2ul的Digestion Enzyme加入步骤1.2反应后的PCR管中混匀后，涡旋震荡瞬时离心将反应液收集至管底。37℃反应15min。反应结束后，向每个反应中加入7ul Digestion Stop Buffer混匀后，使用移液器轻轻吹打混合均匀，瞬时离心使全部反应液置于PCR管底部。
1.4环化产物纯化
1.4.1取出DNA Clean Beads充分震荡混匀，室温恢复半个小时再使用。
1.4.2吸取170ul DNA Clean Beads至步骤1.3的产物中，用移液器轻轻吹打混匀，室温孵育10min。
1.4.3孵育结束后瞬时离心，将1.5mLEP管置于磁力架，静置5min至液体澄清，用移液器小心吸弃上清。
1.4.4保持1.5ml EP管置于磁力架上，沿离心管侧壁加入500ul新鲜配制的80％乙醇漂洗磁珠及管壁，小心吸取并丢弃上清。重复该步骤一次。
1.4.5将离心管瞬时离心后放置于磁力架上，使用10ul吸头移去少量残留乙醇，注意勿吸到磁珠。
1.4.6打开离心管管盖，并于室温静置约5min，至乙醇挥发完全。
1.4.7移出离心管，加入22ul TE Buffer进行DNA洗脱，用移液器轻轻吹打至混匀，室 温孵育5min。
1.4.8瞬时离心，将1.5ml EP管置于磁力架上静置2min至液体完全澄清，使用移液器小心转移20ul上清液转移到新的1.5ml EP管中，既得到纯化的产物。
2.全长TCR富集
2.1对富集后的TCR产物分别扩增TCR-alpha和TCR-beta全长。扩增结果见图11。PCR的正向和反向引物在TSO序列内部(见图15)。
PCR扩增体系：50ul
PCR扩增条件：
2.2TCR全长纯化，按照试剂盒提供的标准操作流程进行(天根生化科技有限公司，DP219-03)操作。
2.2.1将单一的DNA条带从琼脂糖凝胶中切下(尽量切除多余部分)放入干净的离心管中，称取重量。
2.2.2向凝胶块中加入3倍体积溶胶液PE(如果凝胶重0.1g，其体积可视为100ul，则加入300ul溶胶液PE)。室温15℃-25℃溶胶5-10min，期间不断温和地上下翻转离心管，以确保胶块充分溶解。
2.2.3将上一步所得溶液加入一个吸附柱CA5中(吸附柱放入收集管中)，室温放置2min，12000rpm离心30-60s，倒掉收集管中的废液，将吸附柱CA5放入收集管中。
2.2.4向吸附柱CA5中加入600ul漂洗液PW，12000rpm离心30-60s，倒掉收集管中的废液，将吸附柱CA5放入收集管中。
2.2.5重复操作步骤2.2.4。
2.2.6将吸附柱CA5放回收集管中，12000rpm离心2min，尽量除尽漂洗液。将吸附柱CA5置于室温放置数分钟，彻底地晾干，以防止残留的漂洗液影响下一步实验。
2.2.7将吸附柱CA5放到一个干净的离心管中，向吸附膜中间位置悬空滴加适量洗脱缓冲液TB或提前预热的ddH2O，室温放置2min。12000rpm离心2min收集DNA溶液。纯化回收后的产物分别标记为：TCR-TRA和TCR-TRB，共2个产物。然后将两个产物分别连接到空的pMax载体，pMax载体本身具有CMV启动子和poly A位点，因此可以直接获得TCR全长表达框。参考实施例3进行配对TCR的鉴定。参考实施例4-7进行下游应用。本实施例获得的TCR alpha和TCR beta全长序列5‘端没有各种barcode序列和恒定元件，可以 直接应用于下游筛选和功能测试。
在以下实施例11和12中，本发明人将获得的带有Barcode的TCR alpha可变区和TCR beta可变区的cDNA与启动子序列连接；或者，将获得的带有Barcode的TCR alpha可变区和TCR beta可变区的cDNA进行第两次环化，以便为克服Barcode标记在单个T细胞的TCR alpha可变区和TCR beta可变区的mRNA的3’端可能带来的不足之处。
实施例11
采用与上述实施例相类似的技术原理，本发明人利用微孔板体系获得正确配对TCR。
1.肿瘤反应性T细胞的富集
首先通过外科手术取肿瘤组织，然后将肿瘤组织消化为单细胞。将单细胞进行处理后，使用带有荧光标签的抗体对单细胞悬液进行染色标记：CD3-FITC，CD45-PE和CXCL13-APC。将单细胞通过流式分选仪(Sony；SH800S)分选肿瘤单细胞悬液中CXCL13阳性的T细胞。携带CXCL13的这部分T细胞可能为肿瘤反应性T细胞。其他肿瘤反应性T细胞的标签包括：CD39(ENTPD-1)和CD200等。
2.使用磁珠标记配对TCR alpha和TCR beta链并构建TCR质粒文库。磁珠上携带DNA oligo(图16)，DNA oligo包括：恒定序列1，Barcode，恒定序列2和oligo dT序列。Barcode用于标记单个细胞。Oligo dT用于捕获mRNA。恒定序列1和恒定序列2用于环化和PCR过程。本发明整体技术流程见图17。
2.1单细胞分选
(1)将分选的细胞进行活性测定和细胞计数，保证细胞活率在85％以上，根据计数用PBS将细胞密度调整到2×105/ml～1×106/ml，制备成单细胞悬液。
(2)微流控芯片的处理
定制的微流控芯片表面有容纳单个细胞的微孔，数量为2万个(优选1千～15万个孔)。将微流控芯片置于干净的培养皿上，用200μl的移液器吸取200μl的100％无水乙醇从进样口注入芯片，可使用移液器在芯片中来回抽吸100％无水乙醇直至芯片中不再出现气泡，及时移除出样口的液体。重复冲洗2～3次，移除出样口处液体后吸取200μl的0.02％PBST(PBS中包含0.02％Tween-20)从进口处注入芯片，时间控制在10s以内及时移除出样口处液体。出样口处保留少量液体，最后盖上培养皿盖，室温静置备用。
(3)注入细胞
移除进出样口多余液体，加200μl的PBS润洗芯片，然后移除出样口及进样口多余液体，重复润洗1次。吸取100μl重悬好的细胞(大约300～500个细胞，优选50～2000个细胞范围)，缓慢匀速注入芯片，立即移除出样口多余液体。静置5min使细胞落入微孔内，静置期 间可在显微镜下观察细胞落入微孔情况。待细胞落入微孔内，吸取200μl的PBS缓慢匀速注入芯片冲洗掉多余细胞，立即移除进出样口液体。用PBS重复1次，冲洗掉留在表面未落入微孔内的细胞。
(4)注入单细胞标记磁珠
吸取60μl重悬好的单细胞标记磁珠，缓慢匀速加入芯片进样口。多次吸取100μl的PBS，缓慢匀速加入进样口，使单细胞标记磁珠缓慢流动，并及时吸取出样口单细胞标记磁珠，直至达到芯片的另一端，在此期间收集进出样口的多余单细胞标记磁珠。吸取200μl的PBS缓慢匀速注入芯片，吸去进出样口多余液体。用PBS重复1次至冲洗掉多余的单细胞标记磁珠。显微镜下观察单细胞标记磁珠掉入孔中的情况，若芯片进口端单细胞标记磁珠空缺较多，可将回收的单细胞标记磁珠置于磁力架上，吸除上清液提高单细胞标记磁珠密度后再次注入到空缺处静置10s后再冲洗。同理，若芯片出口端单细胞标记磁珠空缺较多，可将回收的单细胞标记磁珠注入到出口槽处，用移液器从进口端将单细胞标记磁珠吸入空缺处，静置10s后再冲洗。
2.2细胞裂解和mRNA捕获
(1)注入Lysis Buffer
吸取100μl的Lysis Buffer从进样口缓慢注入芯片时间约15s，立即移除进出样口多余液体。室温静置20min用于裂解细胞并释放mRNA，此步骤可以让单细胞标记磁珠捕获mRNA。
(2)取出单细胞标记磁珠
取1.5mL离心管，标记后置于1.5mL规格磁力架上。
保持磁力架置于芯片底部，用200μl的Wash Buffer加入到出样口凹槽，快速润洗出样口凹面，润洗完毕后立即移除液体重复润洗3次。
将200μl的wash buffer加入出样口，将磁力架转移置于芯片顶部，静置1min，保持磁力架在芯片顶部，将200μl移液器吸头插入进样口，吸取200μl液体，收集到含有单细胞标记磁珠的液体转移至预冷的1.5mL离心管内。重复该步骤1次，收集捕获到mRNA的全部单细胞标记磁珠。
2.3反转录和扩增
将装有单细胞标记磁珠的离心管短暂离心后置于1.5ml规格磁力架上，待溶液澄清后，小心吸除上清液。从磁力架上取下离心管，加入1mL的Wash Buffer，用移液器轻轻吹吸混匀后短暂离心置于磁力架上，待溶液澄清后小心移除上清。
从磁力架上取下离心管，加入500μl的1×Wash Buffer，用移液器轻轻吹吸混匀后短暂离心置于磁力架上，待溶液澄清后小心移除上清。
取下离心管，短暂离心后再置于磁力架上，用20μl的移液器吸取残余的液体。只留下 离心管底部的单细胞标记磁珠。
在冰上按照如下表格配制RT Mix-1，混匀并短暂离心
将配制好的200μl RT Mix加入到以上步骤中的TCR Barcode Beads，并用移液器吹吸混匀。置于提前设置好的金属浴中，42℃，转速1300rpm，反应90min(提前预热)。
cDNA扩增:在冰上按照如下表格配制PCR Mix-2，混匀并短暂离心
将上一步骤的反转录产物离心，放置于1.5ml规格的磁力架上，待溶液澄清后小心移除上清。
将离心管从磁力架上取下，向管中加入配制好的400μl PCR Mix，一边吹打混匀，一边分装到8联排管中，每管分装50μl。
盖好8联排管盖，置于PCR仪中扩增。PCR反应程序为：
2.4 cDNA产物纯化
Ampure XP纯化磁珠提前30min从4℃中取出恢复室温。使用前要充分振荡混匀。
将PCR扩增产物收集到1.5ml离心管中短暂离心。加入0.6×纯化磁珠涡旋混匀后，室 温孵育5min短暂离心，置于1.5mL规格磁力架上静置5min至液体透明澄清，小心吸除上清液至新的1.5mL离心管中。
保持离心管始终处于磁力架上，加入800μl新配制的80％乙醇漂洗磁珠。室温孵育30s小心移除上清。
重复上述步骤一次。
取下离心管短暂离心，再次置于磁力架上，吸去多余酒精，开盖晾干约2min。
取下离心管，加入100μl Buffer EB，用移液器吹打混匀，室温孵育5min，短暂离心后静置于磁力架上至液体透明澄清。
吸取上清并转移至新的EP管中，加入80μl磁珠(0.8×产物体积)，吹打混匀后室温孵育5min短暂离心，置于磁力架上静置5min至液体透明澄清，小心移除上清至新的1.5mL离心管中。
保持PCR管始终处于磁力架上，加入800μl新配制的80％乙醇漂洗磁珠。室温孵育30s小心移除上清。
重复上述步骤一次。
取下离心管短暂离心，再次置于磁力架上，吸去多余酒精，开盖晾干约2min。
取下离心管，加入20μl Buffer EB，用移液器吹打混匀，室温孵育5min。
短暂离心后静置于磁力架上至液体透明澄清。
吸取上清并转移至新的EP管中即为纯化产物。
2.5第一次环化
(1)纯化cDNA产物按照下表配制反应体系
使用移液器轻柔充分混匀，短暂离心后将反应管置于PCR仪中。PCR仪热盖85℃：50℃反应1小时；75℃反应10min；4℃保存。
(2)酶切
环化后产物不纯化，加入酶进行酶切。将PCR管置于冰上按照如下表格配制酶切体系。
使用移液器轻柔充分混匀，短暂离心后将反应管置于PCR仪中：37℃反应30min；4℃保存。
(3)环化产物纯化
纯化磁珠提前30min从4℃中取出，恢复室温。使用前磁珠需充分混匀。
将PCR管中的液体瞬离后计算体积。加入1.3×产物体积的磁珠，吹打混匀后，室温孵育5min，短暂离心置于磁力架上静置5min至液体透明澄清，小心移除上清至新的PCR管中。
保持PCR管始终处于磁力架上，加入200μl新配制的80％乙醇漂洗磁珠。室温孵育30s，小心移除上清。重复改步骤两次。
取下PCR管短暂离心，再次置于磁力架上吸去多余酒精，晾干。
取下PCR管，加入20μl Nuclease-free Water，吹吸混匀磁珠，室温孵育5min，短暂离心后静置于磁力架上至液体透明澄清。
吸取上清并转移至新EP管中，即为纯化的环化产物。
2.6 TCR第一轮富集
(1)将PCR管置于冰上按照如下表格配制富集PCR Mix-3


其中TCR R1引物序列：GCGTCAGATGTGTATAAGAG(SEQ ID NO:176)；
TCR alpha-F-1引物序列：AGTCTCTCAGCTGGTACACG(SEQ ID NO:177)；
TCR beta-F-1引物序列：TCTGATGGCTCAAACACAGC(SEQ ID NO:178)。
(2)配制的PCR mix轻柔充分混匀，将反应管置于PCR仪中，PCR反应程序为：
琼脂糖凝胶电泳检测富集产物，结果见图18。从电泳结果可以看出，TCR alpha和TCR beta可变区包括部分恒定区扩增后形成单一的条带。
(3)第一轮富集产物纯化
纯化磁珠提前30min从4℃中取出恢复室温。使用前要充分震荡混匀。
将PCR管中的液体瞬离，计算体积。加入30μl磁珠(0.6×产物体积)，吹打混匀后室温孵育5min，短暂离心后置于磁力架上静置5min至液体透明澄清，小心移除上清至新的PCR管中。
保持PCR管始终处于磁力架上，加200μl新配制的80％乙醇漂洗磁珠。室温孵育30s，小心移除上清。
重复以上步骤，共计漂洗2次。
取下PCR管短暂离心，再次置于磁力架上，吸去多余酒精晾干。
取下PCR管加入20μl的Buffer EB，吹吸混匀磁珠室温孵育5min，短暂离心后静置于磁力架上至液体透明澄清。
吸取上清并转移至新的EP管中即为纯化产物，标记为TCR alpha-1和TCR beta-1。该纯化产物携带5’端barcode序列和其他元件，TCR可变区全长以及部分的TCR恒定区序列。
2.7第二次环化
(1)PCR扩增部分恒定区：设计PCR引物从TCR-TRAC-pMax载体上扩增TCR alpha-1剩余的恒定区序列TCR alpha-2，并在正向和反向引物的5’端添加TCR alpha-1片段的同源序列，恒定区序列TCR alpha-2。按照同样的方法设计PCR引物从TCR-TRBC-pMax载体上扩增TCR beta-1剩余的恒定区序列TCR beta-2，并在正向和反向引物的5’端添加TCR beta-1片段的同源序列，恒定区序列TCR beta-2。
PCR扩增体系：
其中引物序列为：
TCR alpha-2-F：catatccagaaccctgaccc(SEQ ID NO:179)；
TCR alpha-2-R：ctgtctcttatacacatctgacgcttagctggaccacagccgcagcg(SEQ ID NO:180)；
TCR beta-2-F：gaggacctgaacaaggtgtt(SEQ ID NO:181)；
TCR beta-2-R：ctgtctcttatacacatctgacgcttagaaatcctttctcttgaccatg(SEQ ID NO:182)。
PCR扩增条件：
琼脂糖凝胶电泳检测PCR产物，结果图19。从电泳结果可以看出，TCR alpha和TCR beta恒定区扩增后形成单一的条带，基因片段扩增成功。
(2)PCR产物纯化，按照试剂盒提供的标准操作流程进行(天根生化科技有限公司，DP219-03)操作。
将单一的DNA条带从琼脂糖凝胶中切下(尽量切除多余部分)放入干净的离心管中，称取重量。
向凝胶块中加入3倍体积溶胶液PE(如果凝胶重0.1g，其体积可视为100μl，则加入300μl溶胶液PE)。室温15～25℃溶胶5～10min，期间不断温和地上下翻转离心管，以确保胶块充分溶解。
将上一步所得溶液加入一个吸附柱CA5中(吸附柱放入收集管中)，室温放置2min，12000rpm离心30～60s，倒掉收集管中的废液，将吸附柱CA5放入收集管中。
向吸附柱CA5中加入600μl漂洗液PW，12000rpm离心30～60s，倒掉收集管中的废液， 将吸附柱CA5放入收集管中。
重复上述操作步骤。
将吸附柱CA5放回收集管中，12000rpm离心2min，尽量除尽漂洗液。将吸附柱CA5置于室温放置数分钟，彻底地晾干，以防止残留的漂洗液影响下一步实验。
将吸附柱CA5放到一个干净的离心管中，向吸附膜中间位置悬空滴加提前预热的ddH2O，室温放置2min。12000rpm离心2min收集DNA溶液。纯化回收后的产物分别为TCR alpha-2和TCR beta-2。
(3)第二次环化
纯化的产物按照下表配制反应体系：
50℃反应1小时进行环化反应。
(4)环化产物纯化
提前半小时取出DNA Clean Beads置于室温，使用前充分震荡混匀。
吸取DNA Clean Beads(0.5×产物体积)至以上步骤的产物中，用移液器轻轻吹打混匀，室温孵育10min。
孵育结束后瞬时离心，将1.5mLEP管置于磁力架，静置5min至液体澄清，用移液器小心吸取并丢弃上清。
保持1.5ml EP管置于磁力架上，加入500μl新鲜配制的80％乙醇漂洗磁珠及管壁，小心吸取并丢弃上清。重复该步骤一次。
保持1.5ml EP管固定于磁力架上，打开1.5ml EP管管盖，室温干燥。
将1.5ml EP管从磁力架上取下，加入22μl的TE Buffer进行DNA洗脱，用移液器轻轻吹打至混匀，室温静置10min。
瞬时离心，将1.5ml的EP管置于磁力架上，静置5min至液体澄清，转移20μl上清液转移到新的1.5ml EP管中。
2.8 TCR第二轮富集
(1)TCR alpha和TCR beta环化产物分别PCR扩增进行TCR的富集。
PCR扩增体系：

其中引物序列为
TCR-F：ttgcctttctctccacaggggtacctggtatcaacgcagagtacttggg(SEQ ID NO:183)；
TCR-R：cattctagttgtggtttgtccaaacctgcttggaacggtacatacttgct(SEQ ID NO:184)。
PCR反应程序为：
第二轮富集的产物琼脂糖凝胶电泳检测为图20。从电泳结果可以看出，二次环化后TCR alpha和TCR beta全长片段扩增成功。
(2)第二轮富集产物纯化
纯化磁珠提前30min从4℃中取出恢复室温，使用前要充分振荡混匀。
将PCR管中的液体瞬离计算体积。加入30μl磁珠(0.5×产物体积)，吹打混匀后室温孵育5min，短暂离心后置于磁力架上静置5min至液体透明澄清，小心移除上清至新的PCR管中。
保持PCR管始终处于磁力架上，加入200μl新配制的80％乙醇漂洗磁珠。室温孵育30s，小心移除上清。
重复上述步骤3，共计漂洗2次。
取下PCR管短暂离心，再次置于磁力架上，吸去多余酒精，晾干。
取下PCR管，加入20μl的Buffer EB，吹吸混匀磁珠室温孵育5min，短暂离心后静置于磁力架上至液体透明澄清。
吸取上清并转移至新的EP管中，即为纯化产物。
2.9连接TCR表达载体
该载体携带CMV启动子和polyA位点，具体图谱见图21。通过重组的方法将纯化后的TCR-alpha和TCR-beta全长分别克隆到TCR-pMax载体后具有完整的TCR表达框，可以高效表达TCR亚基。具体步骤为：
(1)制备线性化TCR-pMax载体：利用XhoI(NEB:R0146S)和KpnI(NEB:R3142S)
将TCR-pMax线性化并纯化。
(2)重组反应：
50℃反应20min.
2.10转化大肠杆菌
在冰上解冻感受态细胞DH5α(上海唯地生物科技有限公司)，一般转化产物体积不能超过感受态体积的1/10。
取10μl重组产物，加入到100μl感受态细胞中，轻柔混匀，在冰上放置20min。
42℃热激90s，冰上放置2min，加700μl无双抗LB培养基(上海生工)，37℃摇床220rpm复苏40min，5000rpm离心3min。吸去700μl上清，剩余的液体移液枪混匀后全部涂含Amp平板，37℃微生物培养箱培养过夜。
3.TCR文库序列比对，获取配对TCR序列
第二天从转化了TCR-alpha和TCR-beta基因片段的平板上分别挑选192个单菌落于200μl的Amp抗性LB培养基37℃摇床摇2h，取2μl菌液PCR初步鉴定阳性克隆。
PCR菌检体系：20μl
其中TCR-F-JJ引物序列为：taggcacctattggtcttac(SEQ ID NO:185)；
TCR-R-JJ引物序列为：tcactgcattctagttgtgg(SEQ ID NO:186)。
PCR菌检条件：
菌检结果用琼脂糖凝胶电泳检测见图22。插入序列长度大约在1000bp的条带为阳性 克隆，鉴定出来的阳性克隆送相应菌液sanger测序验证。其中测序引物为：
TCR-seq-F：acctattggtcttactga(SEQ ID NO:187)；
TCR-seq-R:cattctagttgtggtttgtc(SEQ ID NO:188)。
序列比对和TCR配对
通过sanger测序得到的TCR alpha和TCR beta完整序列和单个细胞barcode，得到的完整序列通过DNA序列分析软件分析得到TCR alpha和TCR beta全长序列，同时判定具有相同barcode序列的TCR alpha和TCR beta克隆为一对TCR即为配对TCR。通过这种方法在挑选的克隆中共找出86对配对的TCR。即获取可以配对TCR的全长序列。
其中表5展示了其中24对配对TCR序列的可变区核苷酸序列。







4.高通量TCR表达检测
4.1效应细胞构建和测试
使用带有NFAT-luciferase的慢病毒感染Jurkat细胞(中国科学院细胞库)，并挑取单克隆培养得到Jurkat-NFAT-luciferase的reporter细胞系(TCR reconstitution in Jurkat reporter cells facilitates the identification of novel tumor antigens by cDNA expression cloning.Int J Cancer. 2002 May 1；99(1):7-13)。使用CRISPR/Cas9电转同时敲除TCR基因，选择未敲除的细胞做阴性对照。具体步骤为：
取一定量的Jurkat-NFAT-luciferase细胞，1200rpm离心5min后用培养基opti-MEM洗2次备用。每1×10 6细胞用25μl的opti-MEM重悬细胞后备用。
冰上准备Cas9和sgRNA：每1×106细胞加入10μl Cas9(500ng/μl)蛋白(南京金斯瑞)、2.5μl的TRAC-sgRNA和2.5μl的TRBC-sgRNA)(400ng/μl南京金斯瑞)，两者常温下孵育10min，加入提前准备好的细胞开始电转。
电转条件：BTX-ECM830的1mm电击杯，电压为250V，脉冲时间1ms。
电转结束迅速在电击杯里加入1ml培养基，然后将细胞转入培养孔里培养。
电转敲除48h后，取一定体积的细胞，加入anti-CD3-APC抗体(BD Pharmingen，555335)对细胞进行染色，4℃孵育半小时。染色完成后，用PBS清洗细胞后重悬，使用流式细胞仪检测TCR敲除的情况。Jurkat细胞中敲除TCR结果见图23，从流式结果可以看出Jurkat细胞中的TCR已全部敲除。通过该步骤制备得到的效应细胞命名为：Jurkat-KO-ER。
4.2配对的TCR质粒电转进效应细胞
根据电转最优条件，选24对配对的TCR质粒中序列完整的10对配对的TCR，分别命名为TCRab-1-10，电转进Jurkat-KO-ER。电转流程为：
取Jurkat-KO-ER细胞，1200rpm离心5min后用培养基opti-MEM洗2次备用。
每1×106的Jurkat-KO-ER细胞加入配对的TCR质粒4ug(TCRalpha和TCRbeta质粒各2ug)，补足培养基opti-MEM至100μl。
将质粒和细胞混合物100μl加入96孔电转板(BTX，45-0450)，电压为260V，脉冲时间1ms。电转后将细胞转移到新的96孔板中，将细胞继续培养。
4.3筛选有功能的TCR
流式检测细胞中TCR的表达：配对TCR质粒电转进Jurkat-KO-ER的细胞24h后，取一定体积的细胞，加入anti-CD3-APC抗体(BD Pharmingen，555335)对细胞进行染色，4℃孵育半小时。染色完成后，用PBS清洗细胞后重悬，使用流式细胞仪检测TCR细胞的表达情况。电转的TCR在Jurkat-KO-ER细胞的表达的代表性结果见图24。从图可以看出，10对代表性的配对的TCR电转到细胞内以后，CD3恢复表达。
5.将配对的TCR克隆至转座子载体
使用常规的分子克隆方法将TCR alpha和TCR beta全长序列克隆至转座子载体中。
6.个体化TCR-T细胞治疗
将携带活性TCR序列的转座子通过电转递送到患者T细胞中，制备个体化的TCR-T细胞。这些细胞可以用来肿瘤患者的治疗。
实施例12
1.肿瘤反应性T细胞的富集
首先通过外科手术取肿瘤组织，然后将肿瘤组织消化为单细胞。将单细胞进行处理后，使用带有荧光标签的抗体对单细胞悬液进行染色标记：CD3-FITC，CD45-PE和CXCL13-APC。将单细胞通过流式分选仪(Sony；SH800S)分选肿瘤单细胞悬液中CXCL13阳性的T细胞。携带CXCL13的这部分T细胞可能为肿瘤反应性T细胞。其他肿瘤反应性T细胞的标签包括：CD39(ENTPD-1)和CD200等。
2.使用磁珠标记配对TCR alpha和TCR beta链并构建TCR质粒文库。磁珠上携带DNA oligo(图16)，DNA oligo包括：恒定序列1，Barcode，恒定序列2和oligo dT序列。Barcode用于标记单个细胞，Oligo dT用于捕获mRNA，恒定序列1和恒定序列2用于环化和PCR过程。本发明整体技术流程见图25。
2.1单细胞分选
(1)将分选的细胞进行活性测定和细胞计数，保证细胞活率在85％以上，根据计数用PBS将细胞密度调整到2×105/ml～1×106/ml，制备成单细胞悬液。
(2)微流控芯片的处理
定制的微流控芯片表面有容纳单个细胞的微孔，数量为2万个(优选1千～15万个孔)。将微流控芯片置于干净的培养皿上，用200μl的移液器吸取200μl的100％无水乙醇从进样口注入芯片，可使用移液器在芯片中来回抽吸100％无水乙醇直至芯片中不再出现气泡，及时移除出样口的液体。重复冲洗2～3次，移除出样口处液体后吸取200μl的0.02％PBST(PBS中包含0.02％Tween-20)从进口处注入芯片，时间控制在10s以内及时移除出样口处液体。出样口处保留少量液体，最后盖上培养皿盖，室温静置备用。
(3)注入细胞
移除进出样口多余液体，加200μl的PBS润洗芯片，然后移除出样口及进样口多余液体，重复润洗1次。吸取100μl重悬好的细胞(大约300～500个细胞，优选50～2000个细胞范围)，缓慢匀速注入芯片，立即移除出样口多余液体。静置5min使细胞落入微孔内，静置期间可在显微镜下观察细胞落入微孔情况。待细胞落入微孔内，吸取200μl的PBS缓慢匀速注入芯片冲洗掉多余细胞，立即移除进出样口液体。用PBS重复1次，冲洗掉留在表面未落入微孔内的细胞。
(4)注入单细胞标记磁珠
吸取60μl重悬好的单细胞标记磁珠，缓慢匀速加入芯片进样口。多次吸取100μl的PBS，缓慢匀速加入进样口，使单细胞标记磁珠缓慢流动，并及时吸取出样口单细胞标记磁珠，直至达到芯片的另一端，在此期间收集进出样口的多余单细胞标记磁珠。吸取200μl的PBS缓慢匀速注入芯片，吸去进出样口多余液体。用PBS重复1次至冲洗掉多余的单细 胞标记磁珠。显微镜下观察单细胞标记磁珠掉入孔中的情况，若芯片进口端单细胞标记磁珠空缺较多，可将回收的单细胞标记磁珠置于磁力架上，吸除上清液提高单细胞标记磁珠密度后再次注入到空缺处静置10s后再冲洗。同理，若芯片出口端单细胞标记磁珠空缺较多，可将回收的单细胞标记磁珠注入到出口槽处，用移液器从进口端将单细胞标记磁珠吸入空缺处，静置10s后再冲洗。
2.2细胞裂解和mRNA捕获
(1)注入Lysis Buffer
吸取100μl的Lysis Buffer从进样口缓慢注入芯片时间约15s，立即移除进出样口多余液体。室温静置20min用于裂解细胞并释放mRNA，此步骤可以让单细胞标记磁珠捕获mRNA。
(2)取出单细胞标记磁珠
取1.5mL离心管，标记后置于1.5mL规格磁力架上。
保持磁力架置于芯片底部，用200μl的Wash Buffer加入到出样口凹槽，快速润洗出样口凹面，润洗完毕后立即移除液体重复润洗3次。
将200μl的wash buffer加入出样口，将磁力架转移置于芯片顶部，静置1min，保持磁力架在芯片顶部，将200μl移液器吸头插入进样口，吸取200μl液体，收集到含有单细胞标记磁珠的液体转移至预冷的1.5mL离心管内。重复该步骤1次，收集捕获到mRNA的全部单细胞标记磁珠。
2.3反转录和扩增
将装有单细胞标记磁珠的离心管短暂离心后置于1.5ml规格磁力架上，待溶液澄清后，小心吸除上清液。从磁力架上取下离心管，加入1mL的Wash Buffer，用移液器轻轻吹吸混匀后短暂离心置于磁力架上，待溶液澄清后小心移除上清。
从磁力架上取下离心管，加入500μl的1×Wash Buffer，用移液器轻轻吹吸混匀后短暂离心置于磁力架上，待溶液澄清后小心移除上清。
取下离心管，短暂离心后再置于磁力架上，用20μl的移液器吸取残余的液体。只留下离心管底部的单细胞标记磁珠。
在冰上按照如下表格配制RT Mix-1，混匀并短暂离心。

将配制好的200μl RT Mix-1加入到以上步骤中的TCR Barcode Beads，并用移液器吹吸混匀。置于提前设置好的金属浴中，42℃，转速1300rpm，反应90min(提前预热)。
cDNA扩增
在冰上按照如下表格配制PCR Mix-2，混匀并短暂离心。
其中引物序列为：
cDNA-F：TGGTATCAACGCAGAGTACTTGGG(SEQ ID NO:261)；
cDNA-R：CCTACACGACGCTCTTCCGATC(SEQ ID NO:262)。
将上一步骤的反转录产物离心，放置于1.5ml规格的磁力架上，待溶液澄清后小心移除上清。
将离心管从磁力架上取下，向管中加入配制好的400μl PCR Mix-2，一边吹打混匀，一边分装到8联排管中，每管分装50μl。
盖好8联排管盖，置于PCR仪中扩增。PCR反应程序为：
2.4 cDNA产物纯化
Ampure XP纯化磁珠提前30min从4℃中取出恢复室温。使用前要充分振荡混匀。
将PCR扩增产物收集到1.5m离心管中短暂离心。加入0.6×纯化磁珠涡旋混匀后，室温孵育5min短暂离心，置于1.5mL规格磁力架上静置5min至液体透明澄清，小心吸除上清液至新的1.5mL离心管中。
保持离心管始终处于磁力架上，加入800μl新配制的80％乙醇漂洗磁珠。室温孵育30s小心移除上清。
重复上述步骤一次。
取下离心管短暂离心，再次置于磁力架上，吸去多余酒精，开盖晾干约2min。
取下离心管，加入20μl ddH2O，用移液器吹打混匀，室温孵育5min短暂离心后静置于磁力架上至液体透明澄清。
吸取上清并转移至新的EP管中即为纯化产物。
2.5环化
(1)为了防止barcode和恒定区序列对后续表达的影响，本发明在环化时直接将启动子(Promoter)序列插入到TSO序列之前，TSO序列中不包含ATG序列，从而不影响TCR基因的表达。
PCR扩增CMV启动子：设计PCR引物从pMax载体上扩增CMV启动子序列CMV-P，并在正向和反向引物的5’端添加cDNA同源序列。
PCR扩增体系：
其中引物序列为：
CMV-P-F序列：
CMV-P-F序列：
PCR扩增条件：

CMV启动子扩增后形成大约600bp单一的条带，基因片段扩增成功。
(2)PCR产物纯化，按照试剂盒提供的标准操作流程进行(天根生化科技有限公司，DP219-03)操作。
将单一的DNA条带从琼脂糖凝胶中切下(尽量切除多余部分)放入干净的离心管中，称取重量。
向凝胶块中加入3倍体积溶胶液PE(如果凝胶重0.1g，其体积可视为100μl，则加入300μl溶胶液PE)。室温15～25℃溶胶5～10min，期间不断温和地上下翻转离心管，以确保胶块充分溶解。
将上一步所得溶液加入一个吸附柱CA5中(吸附柱放入收集管中)，室温放置2min，12000rpm离心30～60s，倒掉收集管中的废液，将吸附柱CA5放入收集管中。
向吸附柱CA5中加入600μl漂洗液PW，12000rpm离心30～60s，倒掉收集管中的废液，将吸附柱CA5放入收集管中。
重复上述操作步骤。
将吸附柱CA5放回收集管中，12000rpm离心2min，尽量除尽漂洗液。将吸附柱CA5置于室温放置数分钟，彻底地晾干，以防止残留的漂洗液影响下一步实验。
将吸附柱CA5放到一个干净的离心管中，向吸附膜中间位置悬空滴加提前预热的ddH2O，室温放置2min。12000rpm离心2min收集DNA溶液。
(3)纯化的cDNA和CMV启动子环化
纯化的产物按照下表配制反应体系：
使用移液器轻柔充分混匀，短暂离心后将反应管置于PCR仪中，50℃反应25min进行环化反应。
(4)环化产物纯化
提前半小时取出DNA Clean Beads置于室温，使用前充分震荡混匀。
加入01×DNA Clean Beads涡旋混匀后加入前一步骤的产物中，用移液器轻轻吹打混匀，室温孵育10min。
孵育结束后瞬时离心，将1.5mL EP管置于磁力架，静置5min至液体澄清，用移液器小心吸取并丢弃上清。
保持1.5ml EP管置于磁力架上，加入500μl新鲜配制的80％乙醇漂洗磁珠及管壁，小 心吸取并丢弃上清。重复该步骤一次。
保持1.5ml EP管固定于磁力架上，打开1.5ml EP管管盖，室温干燥。
将1.5ml EP管从磁力架上取下，加入22μl的ddH2O进行DNA洗脱，用移液器轻轻吹打至混匀，室温静置10min。
瞬时离心，将1.5ml的EP管置于磁力架上，静置5min至液体澄清，转移20μl上清液转移到新的1.5ml EP管中。
2.6TCR的富集
(1)将PCR管置于冰上按照如下表格配制富集mix-3
其中TCR P1-F引物序列：GCGTCAGATGTGTATAAGAG(SEQ ID NO:265)
TCR alpha-R引物序列：AGTCTCTCAGCTGGTACACG(SEQ ID NO:266)
TCR beta-R引物序列：TCTGATGGCTCAAACACAGC(SEQ ID NO:267)
(2)配制的PCR mix-3轻柔充分混匀，将反应管置于PCR仪中，PCR反应程序为：
琼脂糖凝胶电泳检测TCR富集产物，结果见图26。从电泳结果可以看出，TCR alpha可变区包括部分恒定区以及启动子序列，TCR beta可变区包括部分恒定区以及启动子序列扩增后都形成单一的条带。
(3)TCR富集产物纯化
纯化磁珠提前30min从4℃中取出恢复室温。使用前一定要充分混匀。
将PCR管中的液体瞬离，计算体积。加入25μl磁珠(0.5×产物体积)，吹打混匀后室温孵育5min，短暂离心后置于磁力架上静置5min至液体透明澄清，小心移除上清至新的PCR管中。
保持PCR管始终处于磁力架上，加入200μl新配制的80％乙醇漂洗磁珠。室温孵育30s， 小心移除上清。
重复以上步骤，共计漂洗2次。
取下PCR管短暂离心，再次置于磁力架上，吸去多余酒精晾干。
取下PCR管加入20μl的ddH2O，吹吸混匀磁珠室温孵育5min，短暂离心后静置于磁力架上至液体透明澄清。
吸取上清并转移至新的EP管中即为纯化产物，标记为TCR alpha-1和TCR beta-1。该纯化产物携带5’端barcode序列和CMV启动子，TCR可变区全长以及部分的TCR恒定区序列。根据产物的多少可以进行一轮或者多轮富集，保证有足够的产物进行下游的重组反应。
2.7连接TCR表达载体
该表达载体pMax经过人工改造去掉载体自身的CMV启动子，添加TCR alpha恒定区得到pMax-TRAC，添加TCR beta恒定区得到pMax-TRBC，具体图谱见图27。通过重组的方法将纯化后的TCR alpha-1克隆到pMax-TRAC，将纯化后的TCR beta-1克隆到pMax-TRBC后具有完整的TCR表达框，可以高效表达TCR亚基。
具体步骤为：
制备线性化pMax-TRAC和pMax-TRBC载体：利用BamHI(NEB:R3136S)和KpnI(NEB:R3142S)将pMax-TRAC和pMax-TRBC线性化并纯化。
重组反应：
50℃反应20min。
转化大肠杆菌
在冰上解冻感受态细胞DH5α(上海唯地生物科技有限公司)，一般转化产物体积不能超过感受态体积的1/10。
取10μl重组产物，加入到100μl感受态细胞中，轻柔混匀，在冰上放置20min。
42℃热激90s，冰上放置2min，加700μl无双抗LB培养基(上海生工)，37℃摇床220rpm复苏40min，5000rpm离心3min。吸去700μl上清，剩余的液体移液枪混匀后全部涂含Amp平板，37℃微生物培养箱培养过夜。
3.TCR文库序列比对，获取配对TCR序列
第二天从转化了TCR-alpha和TCR-beta基因片段的平板上分别挑选192个单菌落于200μl的Amp抗性LB培养基37℃摇床摇2h，取2μl菌液PCR初步鉴定阳性克隆。
PCR菌检体系：20μl
其中TCR-F-JJ引物序列为：taggcacctattggtcttac(SEQ ID NO:268)；
TCR-R-JJ引物序列为：tcactgcattctagttgtgg(SEQ ID NO:269)。
PCR菌检条件：
菌检结果用琼脂糖凝胶电泳检测见图28。插入序列长度大约在1000bp的条带为阳性克隆，鉴定出来的阳性克隆送相应菌液sanger测序验证。其中测序引物为：
TCR-seq-F：tagaaactgggcttgtcgag(SEQ ID NO:270)；
TCR-seq-R:cattctagttgtggtttgtc(SEQ ID NO:271)。
序列比对和TCR配对
通过sanger测序得到的TCR alpha和TCR beta完整序列和单个细胞barcode，得到的完整序列通过DNA序列分析软件分析得到TCR alpha和TCR beta全长序列，同时判定具有相同barcode序列的TCR alpha和TCR beta克隆为一对TCR即为配对TCR。通过这种方法在挑选的克隆中共找出86对配对的TCR。即获取可以配对TCR的全长序列。
其中表6展示了其中20对配对TCR序列的可变区核苷酸序列。
表6. 20对配对TCR序列的可变区核苷酸序列





在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (36)

  1. 一种获得正确配对的TCR的方法，所述方法包括以下步骤：
    1)获得肿瘤细胞激活的T细胞；
    2)从步骤1)获得的激活的T细胞获得带有Barcode的单个T细胞的TCR alpha可变区和TCR beta可变区的cDNA；
    3)将步骤2)获得的带有Barcode的TCR alpha可变区和TCR beta可变区的cDNA分别重组到携带TCR alpha恒定区和TCR beta恒定区的载体中，从而扩增TCR alpha可变区序列和TCR beta可变区序列；
    4)从步骤3)获得的TCR alpha可变区序列和TCR beta可变区序列中挑选具有相同细胞Barcode的TCR alpha可变区序列和TCR beta可变区序列，从而获得正确配对的TCR。
  2. 如权利要求1所述的方法，其特征在于，所述步骤1)包括：
    1-1.从肿瘤患者样品中分离单个核细胞；
    1-2.从步骤1-1得到的单个核细胞中分选出T细胞和肿瘤细胞；
    1-3.利用步骤1-2得到的肿瘤细胞刺激T细胞；
    1-4.任选检测激活标志物的表达，以供细胞分选；
    或者
    1-1.从肿瘤患者样品中分离单个核细胞；
    1-2.任选检测激活标志物的表达，以供细胞分选。
  3. 如权利要求1所述的方法，其特征在于，所述步骤2)包括以下步骤：
    2-1.分选细胞活率在80％以上，优选在85％以上的激活的T细胞，并获得单细胞悬液；
    2-2.获得细胞的mRNA；
    2-3.利用Barcode引物序列与逆转录酶对获得的mRNA进行逆转录，从而得到带有Barcode的单个细胞的TCR alpha可变区和TCR beta可变区的cDNA；
    2-4.对获得的cDNA进行纯化和PCR扩增；
    2-5.将PCR产物进行环化和任选的线性化。
  4. 如权利要求3所述的方法其特征在于，所述步骤2-2包括：获得100-2000个，优选100-800个，最优选200-500个激活的T细胞的mRNA；或者
    获得100-50000，优选100-8000个，最优选200-5000个激活的T细胞的mRNA。
  5. 如权利要求1所述的方法，其特征在于，所述步骤3)中，携带TCR alpha恒定区的载体具有启动子、TCR alpha恒定区序列和PolyA位点；携带TCR beta恒定区的载体具有启动子、TCR beta恒定区序列和PolyA位点。
  6. 如权利要求1所述的方法，其特征在于，如果在步骤2)中，Barcode标记在单个T细胞的TCR alpha可变区和TCR beta可变区的mRNA的3’端，则步骤3)为：
    将步骤2)获得的带有Barcode的TCR alpha可变区和TCR beta可变区的cDNA先与启动子序列连接，再分别重组到携带TCR alpha恒定区和TCR beta恒定区的载体中，从而扩增 TCR alpha可变区序列和TCR beta可变区序列；
    或者，步骤3)为：
    将步骤2)获得的带有Barcode的TCR alpha可变区和TCR beta可变区的cDNA进行第两次环化，再分别重组到携带TCR alpha恒定区和TCR beta恒定区的载体中，从而扩增TCR alpha可变区序列和TCR beta可变区序列。
  7. 如权利要求1所述的方法，其特征在于，所述步骤4)中，对步骤3)获得的TCR alpha可变区序列和TCR beta可变区序列进行测序并挑选具有相同细胞Barcode的TCR alpha可变区序列和TCR beta可变区序列，从而获得正确配对的TCR。
  8. 如权利要求1所述的方法，其特征在于，所述方法还包括以下步骤：
    5)任选对获得的TCR进行功能评价。
  9. 一种克隆配对TCR序列的方法，其特征在于，所述的方法包括以下步骤：肿瘤组织中分选并捕获单个肿瘤反应性T细胞，提取、通过Barcode标记单个细胞的mRNA，反转录并构建全长cDNA，cDNA特异性PCR扩增得到cDNA全长转录组，富集后第一次环化，进行TCR序列的特异性扩增、富集后第二次环化并再次富集，富集产物连接到表达载体中得到TCR的全长克隆，进行文库中Barcode序列比对获得配对的TCR序列。
  10. 根据权利要求9所述的一种克隆配对TCR序列的方法，其特征在于：所述的方法中，mRNA反转录时在反转录体系中加入TSO，扩增完成cDNA全长转录组的富集。
  11. 根据权利要求10所述的一种克隆配对TCR序列的方法，其特征在于：所述的方法中，通过单细胞标记磁珠5’端的恒定区序列及反转录过程中添加上的TSO序列，扩增完成全长转录组的富集。
  12. 根据权利要求9所述的一种克隆配对TCR序列的方法，其特征在于：所述的方法中第一次环化为，将获得的扩增产物进行环化处理，使用单细胞标记磁珠上的恒定序列和TCR特异性引物(恒定区末端)进行TCR序列的特异性扩增，完成TCR的富集。
  13. 根据权利要求9所述的一种克隆配对TCR序列的方法，其特征在于：所述的方法中第二次环化为，将获得的扩增产物进行第二次环化处理后，使用根据单细胞标记磁珠恒定序列设计的正反向引物进行TCR的富集，富集后细胞barcode将与TCR恒定区相连，位于富集产物3’端。
  14. 根据权利要求9所述的一种克隆配对TCR序列的方法，其特征在于：所述的配对TCR序列包含TCR-alpha和TCR-beta序列。
  15. 根据权利要求9所述的一种快速克隆配对TCR序列的方法，其特征在于，分选单个肿瘤反应性T细胞的方法为：肿瘤组织消化为单细胞并制备成单细胞悬液后注入微流控芯片获得单个肿瘤反应性T细胞。
  16. 一种快速克隆配对TCR序列的方法，所述的方法包括以下步骤：肿瘤组织中分选并捕获单个肿瘤反应性T细胞，提取、标记单个细胞的mRNA，反转录并构建全长cDNA，cDNA特异性PCR扩增得到cDNA全长转录组；
    其特征在于：cDNA全长转录组富集后，与启动子序列连接环化并进行TCR序列的特 异性扩增，扩增产物富集后连接到表达载体中得到TCR的全长克隆，进行文库序列比对获得配对的TCR序列。
  17. 根据权利要求16所述的一种快速克隆配对TCR序列的方法，其特征在于：所述的方法中，mRNA反转录时在反转录体系中加入TSO，扩增完成cDNA全长转录组的富集。
  18. 根据权利要求17所述的一种快速克隆配对TCR序列的方法，其特征在于：所述的方法中，通过单细胞标记磁珠5’端的恒定区序列及反转录过程中添加上的TSO序列，扩增完成全长转录组的富集。
  19. 根据权利要求16所述的一种快速克隆配对TCR序列的方法，其特征在于，所述的方法中环化方法为：
    环化时直接将启动子序列插入到TSO序列之前；所述的启动子是任一启动mRNA转录的基因元件，包括但不限于CMV、EF1alpha、SV40、PGK1、CAG、T7、Sp6；
    设计PCR引物扩增CMV启动子序列CMV-P，并在正向和反向引物的5’端添加cDNA同源序列后进行TCR序列的特异性扩增，完成TCR的扩增；
    CMV启动子序列CMV-P的核酸序列如Seq ID No.1所示。
  20. 根据权利要求19所述的一种快速克隆配对TCR序列的方法，其特征在于：所述的PCR引物包括CMV-P-F和CMV-P-F；
    CMV-P-F序列：
    AGATCGGAAGAGCGTCGTGTAGGGGCATTGATTATTGACTAGTTAT；
    CMV-P-F序列：
    CCCCAAGTACTCTGCGTTGATACCAACCTCTGCTTATATAGACCTCC。
  21. 根据权利要求16所述的一种快速克隆配对TCR序列的方法，其特征在于：所述的配对TCR序列包含TCR-alpha和TCR-beta系列。
  22. 根据权利要求16所述的一种快速克隆配对TCR序列的方法，其特征在于：所述的表达载体为任一具有复制子和抗性基因元件的质粒；优选的表达载体为线性化pMax-TRAC和pMax-TRBC载体；
    表达载体pMax经过人工改造去掉载体自身的CMV启动子，添加TCR alpha恒定区得到pMax-TRAC，添加TCR beta恒定区得到pMax-TRBC；
    通过重组的方法将纯化后的TCR alpha-1克隆到pMax-TRAC，将纯化后的TCR beta-1克隆到pMax-TRBC后具有完整的TCR表达框。
  23. 根据权利要求16所述的一种快速快速克隆配对TCR序列的方法，其特征在于，分选单个肿瘤反应性T细胞的方法为：肿瘤组织消化为单细胞并制备成单细胞悬液后注入微流控芯片获得单个肿瘤反应性T细胞。
  24. 一种TCR，所述TCR采用权利要求1-23中任一项所述方法制备得到。
  25. 如权利要求24所述的TCR，其特征在于，所述TCR是选自TCR01-TCR68中的任一种。
  26. 权利要求24或25所述的TCR在制备遗传改造的T细胞中的用途。
  27. 一种遗传改造的TCR-T细胞，所述T细胞表达采用权利要求1-23中任一项所述方法制备得到的TCR或权利要求24或25所述的TCR。
  28. 根据权利要求27所述的一种TCR-T细胞，其特征在于：所述相应的T细胞是指个体自身T细胞或异源T细胞。
  29. 权利要求27或28所述的遗传改造的T细胞在制备治疗与T细胞相关的疾病的药物组合物中的用途。
  30. 一种药物组合物，所述药物组合物包含权利要求27或28所述的遗传改造的T细胞以及药学上可接受的赋形剂。
  31. 根据权利要求30所述的药物组合物用于制备治疗与T细胞相关的疾病的药物的用途，所述疾病包括感染性疾病、肿瘤、自身免疫疾病和器官移植。
  32. 根据权利要求31所述的药物组合物的用途，其特征在于：所述T细胞相关的疾病为癌症，选自由急性淋巴细胞白血病、急性髓性白血病、肾上腺皮质癌、AIDS相关癌症、AIDS相关淋巴瘤、肛门癌、阑尾癌、星形细胞瘤、神经母细胞瘤、基底细胞癌、胆管癌、膀胱癌、骨癌、脑肿瘤、乳腺癌、支气管腺瘤、伯基特淋巴瘤、原发性未知癌、中枢神经系统淋巴瘤、小脑星形细胞瘤、宫颈癌、儿童癌症、慢性淋巴细胞白血病、慢性骨髓性白血病、慢性骨髓增生性疾病、结肠癌、皮肤T细胞淋巴瘤、结缔组织增生性小圆细胞瘤、子宫内膜癌、室管膜瘤、食道癌、尤因氏肉瘤、生殖细胞肿瘤、胆囊癌、胃癌、胃肠道类癌肿瘤、胃肠道间质瘤、胶质瘤、毛细胞白血病、头颈癌、心脏癌、肝细胞癌、霍奇金淋巴瘤、咽下癌、眼内黑色素瘤、胰岛细胞癌、卡波西肉瘤、肾癌、喉癌、唇癌和口腔癌、脂肪肉瘤、肝癌、肺癌、淋巴瘤、白血病、巨球蛋白血症、骨恶性纤维组织细胞瘤/骨肉瘤、成神经管细胞瘤、黑色素瘤、间皮瘤、原发灶隐匿的转移性鳞状颈癌、口腔癌、多发性内分泌肿瘤综合症、骨髓增生异常综合症、骨髓性白血病、鼻腔和副鼻窦癌、鼻咽癌、神经母细胞瘤、非霍奇金淋巴瘤、非小细胞肺癌、口腔癌、口咽癌、骨肉瘤/骨恶性纤维组织细胞瘤、卵巢癌、卵巢上皮癌、卵巢生殖细胞肿瘤、胰腺癌、胰腺癌胰岛细胞、副鼻窦和鼻腔癌、甲状旁腺癌、阴茎癌、咽癌、嗜铬细胞瘤、松果体星形细胞瘤、松果体生殖细胞瘤、垂体腺瘤、胸膜肺母细胞瘤、浆细胞瘤、原发性中枢神经系统淋巴瘤、前列腺癌、直肠癌、肾细胞癌、肾盂和输尿管移行细胞癌、视网膜母细胞瘤、横纹肌肉瘤、唾液腺癌、肉瘤、皮肤癌、默克尔细胞皮肤癌、小肠癌、软组织肉瘤、鳞状细胞癌、胃癌、T细胞淋巴瘤、咽喉癌、胸腺瘤、胸腺癌、甲状腺癌、滋养细胞肿瘤、原发部位未知癌、尿道癌、子宫肉瘤、阴道癌、外阴癌、瓦尔登斯特伦巨球蛋白血症和威尔姆斯瘤组成的组，所述的自体免疫性疾病选自由关节炎、慢性阻塞性肺疾病、强直性脊柱炎、克罗恩病、皮肌炎、I型糖尿病、子宫内膜异位症、Goodpasture氏综合症、Graves氏病、格林-巴厘综合症、桥本氏病、化脓性汗腺炎、川崎病、IgA肾病、原发性血小板减少性紫癜、间质性膀胱炎、红斑狼疮、混合性结缔组织病、硬斑病、重症肌无力、嗜睡症、神经性肌强直、寻常型天疱疮、恶性贫血、银屑病、银屑病关节炎、多发性肌炎、原发性胆汁性肝硬化、复发性多软骨炎、类风湿关节炎、精神分裂症、硬皮病、干燥综合症、僵人综合症、颞动脉炎、溃疡性结肠炎、脉管炎、白斑病以及韦格纳肉芽肿组成的组。
  33. 一种诊断和/或评估制剂，所述诊断和/或评估制剂中含有权利要求27或28所述的TCR-T细胞。
  34. 根据权利要求33所述的诊断和/或评估制剂，其特征在于：所述的制剂制备为制剂盒。
  35. 根据权利要求33或34所述的诊断和/或评估制剂用于制备诊断或评估与T细胞相关的疾病或事件的试剂盒的用途；
    所述疾病或事件包括感染性疾病、肿瘤、自身免疫疾病和器官移植。
  36. 根据权利要求35所述的制剂的用途，进一步用于生物标志物、抗体开发、用药和疫苗评估、免疫细胞分化溯源、免疫排斥和耐受、微小残留病检测、食品或其它过敏原检测。
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