WO2024131750A1 - T细胞抗原受体及其制备方法和应用 - Google Patents

T细胞抗原受体及其制备方法和应用 Download PDF

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WO2024131750A1
WO2024131750A1 PCT/CN2023/139719 CN2023139719W WO2024131750A1 WO 2024131750 A1 WO2024131750 A1 WO 2024131750A1 CN 2023139719 W CN2023139719 W CN 2023139719W WO 2024131750 A1 WO2024131750 A1 WO 2024131750A1
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seq
amino acid
chain
tcr
cells
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French (fr)
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孙娇钧
徐其龙
李雪姣
李晓丹
王歈
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北京永泰瑞科生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of biomedicine, and specifically relates to a T cell antigen receptor and a preparation method and application thereof.
  • T cell receptor is a molecule that T lymphocytes specifically recognize antigens and initiate immune responses. It is a heterodimeric cell surface protein of the immunoglobulin superfamily, which is associated with the non-variant protein of the CD3 complex involved in regulating signal transduction. TCR is the only receptor for specific antigen peptides presented on the major histocompatibility complex (MHC), and is crucial to the cellular immune function of the immune system. The binding of antigen-specific TCR to the MHC complex triggers direct physical contact and interaction between T cells and antigen-presenting cells, leading to a series of subsequent cell signaling and other physiological reactions, so that T cells with different antigen specificities can exert immune effects on their target cells.
  • MHC major histocompatibility complex
  • Cytomegalovirus is a DNA virus of the herpesvirus group. It is named cytomegalovirus because the infected cells will swell. CMV is one of the most common pathogens in humans and is widely prevalent in the population. Its infection rate in the adult population can be as high as 50%-100%. In individuals with normal immune systems, CMV infection usually does not cause any clinical symptoms, but after asymptomatic primary infection, the CMV virus is often not completely eliminated, and a persistent latent infection is formed in the patient's body. If the function of the immune system of an individual carrying CMV is suppressed, such as the onset of AIDS or immunosuppressive treatment after organ transplantation, the reactivation of latent CMV infection can lead to serious or even fatal diseases.
  • CMV infection may also be related to the onset and progression of some tumors.
  • CMV-pp65 antigen can be detected in half of glioma tissues. Therefore, the prevention and treatment of diseases related to CMV infection has also become one of the directions of drug development.
  • TCR-T cells for CMV infection using CMV-specific TCR.
  • patients with CMV reactivation can obtain effective immediate antiviral ability and eliminate the reactivated virus.
  • Patients who receive CMV-specific TCR-T infusion can also obtain lasting antiviral ability, which can significantly reduce the chance of subsequent reactivation.
  • Preliminary clinical studies have also shown that the CMV-pp65 vaccine can prolong the progression-free survival and overall survival rate of patients with brain glioma. Therefore, TCR targeting CMV-pp65 may also be used to treat CMV-related malignancies.
  • CN113881680A and CN102656188A both disclose TCRs that can recognize antigens of cytomegalovirus, but their screening methods do not pay special attention to the heterogeneity of TCR recognition, and the killing effect on target cells is not obvious.
  • CN106279404A discloses a soluble and stable heterodimeric TCR, which contains an artificial interchain disulfide bond between the ⁇ chain variable region and the ⁇ chain constant region. The TCR is soluble and stable, can be well renatured, refolded, purified, and can specifically bind to the original ligand. However, the patent does not disclose a specific TCR for cytomegalovirus.
  • TCR-T therapy can effectively control acute CMV disease and induce antiviral immune response; however, how to obtain effective T cells from cytotoxic T lymphocytes stimulated in the patient or in vitro, and quickly obtain their TCR sequences, so as to construct functional TCR-transduced specific T cells, is an obstacle to the application of TCR-T.
  • the present invention realizes the determination of TCR pairing sequences through high-throughput sequencing, and evaluates the expression level of important genes through immune cells to conduct specific T cell receptor research.
  • the antibody is amplified in vitro.
  • the original specific cytotoxic T lymphocytes are sequenced by single cells, and the TCR pairing information of the positive T cells after MHC tetramer sorting is obtained.
  • TCR is screened according to the clone sequence frequency, TCR-T is constructed, and in vitro functional verification is performed to evaluate the function of TCR-T, and finally the specific T cell receptor disclosed in the present invention is developed and obtained.
  • the HLA-A24-CMV-pp65 341-349 antigen complex disclosed in the present invention is composed of human leukocyte surface antigen HLA-A24 and CMV-pp65 341-349 short peptide (native sequence: QYDPVAALF (SEQ ID NO: 13)), and is expressed on the surface of target cells.
  • the first aspect of the present disclosure provides a TCR that recognizes human cytomegalovirus pp65 antigen, wherein the TCR has the characteristic of binding to the HLA-A24-CMV-pp65 341-349 antigen complex, and the TCR comprises at least one ⁇ chain variable region and/or one ⁇ chain variable region.
  • the T cell antigen receptor is an ⁇ heterodimer, and each comprises a TCR ⁇ chain variable region and a TCR ⁇ chain variable region.
  • the TCR ⁇ chain variable region comprises three complementary determining regions (CDRs) CDR1 ⁇ -CDR3 ⁇ , wherein CDR3 ⁇ comprises a sequence as shown in one of SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 31, or comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or at least 99% homology to one of SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 31.
  • the amino acid sequence of CDR3 of the TCR ⁇ chain variable region is as shown in one of SEQ ID NO: 3, SEQ ID NO: 22, and SEQ ID NO: 31.
  • CDR1 ⁇ of the TCR ⁇ chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 98% or at least 99% homologous to SEQ ID NO: 1;
  • CDR2 ⁇ comprises the amino acid sequence shown in SEQ ID NO: 2, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 98% or at least 99% homologous to SEQ ID NO: 2;
  • CDR1 ⁇ of the TCR ⁇ chain variable region comprises SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 D NO: 23, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 98% or at least 99% homologous to SEQ ID NO: 4, SEQ ID NO: 14,
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • CDR2 ⁇ MTLNGDE (SEQ ID NO: 2);
  • CDR3 ⁇ one of CARNTGKLIF (SEQ ID NO: 3), CAPSASKIIF (SEQ ID NO: 22), CAPQFNKFYF (SEQ ID NO: 31);
  • CDR1 ⁇ one of SQVTM (SEQ ID NO: 4), SGHVS (SEQ ID NO: 14), LNHDA (SEQ ID NO: 23);
  • CDR 3 ⁇ one of CSANPTGGGTEAFF (SEQ ID NO: 6), CASSLLTRTETQYF (SEQ ID NO: 16), CASSTTGLAGGPGNEQFF (SEQ ID NO: 25);
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • CDR 2 ⁇ ANQGSEA (SEQ ID NO: 5)
  • CDR 3 ⁇ CSANPTGGGTEAFF (SEQ ID NO: 6);
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • CDR3 ⁇ CASSTTGLAGGPGNEQFF(SEQ ID NO: 25);
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • variable region of the ⁇ chain of the TCR disclosed herein comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 32, or comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or more homologous to SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 32.
  • the ⁇ chain of the TCR disclosed herein comprises the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 34, or comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or more homologous to SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 34.
  • variable region of the ⁇ chain of the TCR disclosed herein comprises the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 33, or comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or more homologous to SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 33.
  • the ⁇ chain of the TCR disclosed herein comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 29, SEQ ID NO: 35, or comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or more homologous to SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 29, SEQ ID NO: 35.
  • the amino acids of the ⁇ chain and ⁇ chain of the TCR of the present disclosure are directly or indirectly connected, preferably indirectly connected, and more preferably connected using 2A peptides, which are short peptides (18-25 amino acids) derived from viruses. They are usually called "self-cleavage" peptides, which can produce multiple proteins from one transcription product.
  • 2A peptides used in the present disclosure include but are not limited to P2A, T2A, E2A, and F2A.
  • a 2A peptide such as P2A
  • P2A can also be linked to a furin cleavage site, a ser-gly linker (sgsg) and a p2a ribosomal skipping peptide to form a fp2A (furin-SGSG-p2A) linkage structure.
  • sgsg ser-gly linker
  • p2a ribosomal skipping peptide to form a fp2A (furin-SGSG-p2A) linkage structure.
  • the fp2A comprises the amino acid sequence shown in SEQ ID NO: 11.
  • connection order of the ⁇ chain and the ⁇ chain can be ⁇ chain, fp2A and ⁇ chain, or ⁇ chain, fp2A and ⁇ chain.
  • the TCR of the present disclosure comprises the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 30, SEQ ID NO: 36, or comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or more homologous to SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 30, SEQ ID NO: 36.
  • the CMV pp65 495-503 short peptide (native sequence: NLVPMVATV (SEQ ID NO: 46)) disclosed in the present invention is expressed on the surface of target cells.
  • the present disclosure provides a TCR that recognizes human cytomegalovirus pp65 antigen, wherein the TCR has the property of binding to CMV pp65 495-503 antigen complex, and the TCR comprises at least one ⁇ chain variable region and/or one ⁇ chain variable region.
  • the T cell antigen receptor is an ⁇ heterodimer, and each comprises a TCR ⁇ chain variable region and a TCR ⁇ chain variable region.
  • the TCR alpha chain variable region comprises three complementarity determining regions (CDRs) CDR1 ⁇ -CDR3 ⁇ , wherein CDR3 ⁇ comprises a sequence as shown in one of SEQ ID NO: 37, SEQ ID NO: 49, SEQ ID NO: 60, and SEQ ID NO: 69, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 98% or at least 99% homologous to one of SEQ ID NO: 37, SEQ ID NO: 49, SEQ ID NO: 60, and SEQ ID NO: 69; preferably, the amino acid sequence of CDR3 in the TCR alpha chain variable region is as shown in one of SEQ ID NO: 37, SEQ ID NO: 49, SEQ ID NO: 60, and SEQ ID NO: 69.
  • CDRs complementarity determining regions
  • the TCR ⁇ chain variable region comprises three CDR regions, CDR1 ⁇ -CDR3 ⁇ , wherein CDR3 comprises the sequence shown in SEQ ID NO: 40, SEQ ID NO: 52, SEQ ID NO: 63, and SEQ ID NO: 72, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 98% or at least 99% homologous to one of SEQ ID NO: 40, SEQ ID NO: 52, SEQ ID NO: 63, and SEQ ID NO: 72; preferably, the amino acid sequence of CDR3 in the TCR ⁇ chain variable region is as shown in one of SEQ ID NO: 40, SEQ ID NO: 52, SEQ ID NO: 63, and SEQ ID NO: 72.
  • the CDR1 ⁇ of the TCR ⁇ chain variable region comprises an amino acid sequence as shown in one of SEQ ID NO: 1, SEQ ID NO: 47, and SEQ ID NO: 58, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 98%, or at least An amino acid sequence with at least 99% homology;
  • CDR2 ⁇ comprises an amino acid sequence as shown in one of SEQ ID NO: 2, SEQ ID NO: 48, and SEQ ID NO: 59, or comprises an amino acid sequence with at least 85%, 90%, 95%, 98%, or at least 99% homology to one of SEQ ID NO: 2, SEQ ID NO: 48, and SEQ ID NO: 59;
  • the CDR1 ⁇ of the TCR ⁇ chain variable region comprises the amino acid sequence shown in one of SEQ ID NO: 38, SEQ ID NO: 50, SEQ ID NO: 61, and SEQ ID NO: 70, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 98% or at least 99% homologous to one of SEQ ID NO: 38, SEQ ID NO: 50, SEQ ID NO: 61, and SEQ ID NO: 70;
  • CDR2 ⁇ comprises the amino acid sequence shown in one of SEQ ID NO: 39, SEQ ID NO: 51, SEQ ID NO: 62, and SEQ ID NO: 71, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 98% or at least 99% homologous to one of SEQ ID NO: 39, SEQ ID NO: 51, SEQ ID NO: 62, and SEQ ID NO: 71.
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • CDR1 ⁇ one of SSNFYA (SEQ ID NO: 1), DSSSTY (SEQ ID NO: 47), TSGFNG (SEQ ID NO: 58)
  • CDR2 ⁇ one of MTLNGDE (SEQ ID NO: 2), IFSNMDM (SEQ ID NO: 48), NVLDGL (SEQ ID NO: 59)
  • CDR3 ⁇ one of CASINFNKFYF (SEQ ID NO: 37), CAEFTGTASKLTF (SEQ ID NO: 49), CAVTYNNARLMF (SEQ ID NO: 60), CARNYGQNFVF (SEQ ID NO: 69)
  • CDR1 ⁇ one of MDHEN (SEQ ID NO: 38), GTSNPN (SEQ ID NO: 50), MNHEY (SEQ ID NO: 61), DFQATT (SEQ ID NO: 70)
  • CDR 2 ⁇ One of SYDVKM (SEQ ID NO: 39), SVGIG (SEQ ID NO: 51), SMNVEV (SEQ ID NO: 62), SNEGSKA (SEQ ID NO: 71)
  • CDR 3 ⁇ One of CASSPLNGGATEAFF (SEQ ID NO: 40), CAWSDRAAFTDTQYF (SEQ ID NO: 52), CASSSVAGGRIEQFF (SEQ ID NO: 63), CSARDIKAQQWNIQYF (SEQ ID NO: 72).
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • CDR 3 ⁇ CASSPLNGGATEAFF (SEQ ID NO: 40)
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • CDR3 ⁇ CAEFTGTASKLTF (SEQ ID NO: 49)
  • CDR3 ⁇ CAWSDRAAFTDTQYF (SEQ ID NO: 52)
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • CDR3 ⁇ CAVTYNNARLMF (SEQ ID NO: 60)
  • CDR2 ⁇ SMNVEV (SEQ ID NO: 62)
  • CDR3 ⁇ CASSSVAGRIEQFF (SEQ ID NO: 63)
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • variable region of the ⁇ chain of the TCR disclosed herein comprises the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 53, SEQ ID NO: 64, SEQ ID NO: 73, or comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or more homologous to SEQ ID NO: 41, SEQ ID NO: 53, SEQ ID NO: 64, SEQ ID NO: 73.
  • the ⁇ chain of the TCR disclosed herein comprises the amino acid sequence of SEQ ID NO: 43, SEQ ID NO: 55, SEQ ID NO: 66, SEQ ID NO: 75, or comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or more homologous to SEQ ID NO: 43, SEQ ID NO: 55, SEQ ID NO: 66, SEQ ID NO: 75.
  • variable region of the ⁇ chain of the TCR disclosed herein comprises the amino acid sequence of SEQ ID NO: 42, SEQ ID NO: 54, SEQ ID NO: 65, SEQ ID NO: 74, or comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or more homologous to SEQ ID NO: 42, SEQ ID NO: 54, SEQ ID NO: 65, SEQ ID NO: 74.
  • the ⁇ chain of the TCR disclosed herein comprises the amino acid sequence of SEQ ID NO: 44, SEQ ID NO: 56, SEQ ID NO: 67, SEQ ID NO: 76, or comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or more homologous to SEQ ID NO: 44, SEQ ID NO: 56, SEQ ID NO: 67, SEQ ID NO: 76.
  • the amino acids of the ⁇ chain and ⁇ chain of the TCR of the present disclosure are directly or indirectly connected, preferably indirectly connected, and more preferably connected using 2A peptides, which are short peptides (18-25 amino acids) derived from viruses. They are usually called "self-cleavage" peptides, which can produce multiple proteins from one transcription product.
  • 2A peptides used in the present disclosure include but are not limited to P2A, T2A, E2A, and F2A.
  • a 2A peptide such as P2A
  • P2A can also be linked to a furin cleavage site, a ser-gly linker (sgsg) and a p2a ribosomal skipping peptide to form a fp2A (furin-SGSG-p2A) linkage structure.
  • sgsg ser-gly linker
  • p2a ribosomal skipping peptide to form a fp2A (furin-SGSG-p2A) linkage structure.
  • the fp2A comprises the amino acid sequence shown in SEQ ID NO: 11.
  • connection order of the ⁇ chain and the ⁇ chain can be ⁇ chain, fp2A and ⁇ chain, or ⁇ chain, fp2A and ⁇ chain.
  • the TCR of the present disclosure comprises the amino acid sequence shown in SEQ ID NO: 45, SEQ ID NO: 57, SEQ ID NO: 68, SEQ ID NO: 77 or comprises An amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or 99% homologous to SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:68, or SEQ ID NO:77.
  • the CMV pp65 501-509 short peptide (ATVQGQNLK (SEQ ID NO: 89)) disclosed in the present invention is expressed on the surface of target cells.
  • the present disclosure provides a TCR that recognizes human cytomegalovirus pp65 antigen, wherein the TCR has the property of binding to the CMV pp65 501-509 antigen complex, and the TCR comprises at least one ⁇ chain variable region and/or one ⁇ chain variable region.
  • the T cell antigen receptor is an ⁇ heterodimer, and each comprises a TCR ⁇ chain variable region and a TCR ⁇ chain variable region.
  • the TCR ⁇ chain variable region comprises three complementarity determining regions (CDRs) CDR1 ⁇ -CDR3 ⁇ , wherein CDR3 ⁇ comprises a sequence shown in one of CVITTSGTYKYIF (SEQ ID NO: 80), CAYRSFYTGANSKLTF (SEQ ID NO: 92), and CVVHSGGSYIPTF (SEQ ID NO: 103), or comprises an amino acid sequence having at least 85%, 90%, 95%, 98% or at least 99% homology with one of SEQ ID NO: 80, SEQ ID NO: 92, and SEQ ID NO: 103.
  • CDR3 ⁇ comprises a sequence shown in one of CVITTSGTYKYIF (SEQ ID NO: 80), CAYRSFYTGANSKLTF (SEQ ID NO: 92), and CVVHSGGSYIPTF (SEQ ID NO: 103)
  • SEQ ID NO: 80 comprises a sequence shown in one of CVITTSGTYKYIF (SEQ ID NO: 80), CAYRSF
  • the TCR ⁇ chain variable region comprises three CDR regions, CDR1 ⁇ -CDR3 ⁇ , wherein CDR3 ⁇ comprises a sequence shown in one of CASTINTYEQYF (SEQ ID NO: 83), CASSLYGGPGDQPQHF (SEQ ID NO: 95), and CASAQTIGAYNEQFF (SEQ ID NO: 106), or comprises an amino acid sequence having at least 85%, 90%, 95%, 98% or at least 99% homology with one of SEQ ID NO: 83, SEQ ID NO: 95, and SEQ ID NO: 106.
  • the CDR1 ⁇ of the TCR ⁇ chain variable region comprises the amino acid sequence shown in one of SEQ ID NO: 78, SEQ ID NO: 90, and SEQ ID NO: 101, or comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or at least 99% homology to one of SEQ ID NO: 78, SEQ ID NO: 90, and SEQ ID NO: 101;
  • the CDR2 ⁇ comprises the amino acid sequence shown in one of SEQ ID NO: 79, SEQ ID NO: 91, and SEQ ID NO: 102, or comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or at least 99% homology to one of SEQ ID NO: 79, SEQ ID NO: 91, and SEQ ID NO: 102;
  • the CDR1 ⁇ of the TCR ⁇ chain variable region comprises the amino acid sequence shown in one of SEQ ID NO: 81, SEQ ID NO: 93, and SEQ ID NO: 104, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 98% or at least 99% homologous to one of SEQ ID NO: 81, SEQ ID NO: 93, and SEQ ID NO: 104;
  • CDR2 ⁇ comprises the amino acid sequence shown in one of SEQ ID NO: 82, SEQ ID NO: 94, and SEQ ID NO: 105, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 98% or at least 99% homologous to one of SEQ ID NO: 82, SEQ ID NO: 94, and SEQ ID NO: 105.
  • CDR1 ⁇ one of VSGLRG (SEQ ID NO: 78), TSESDYY (SEQ ID NO: 90), NSASQS (SEQ ID NO: 101)
  • CDR2 ⁇ one of LYSAGEE (SEQ ID NO: 79), QEAYKQQN (SEQ ID NO: 91), VYSSGN (SEQ ID NO: 102)
  • CDR3 ⁇ One of CVITTSGTYKYIF (SEQ ID NO: 80), CAYRSFYTGANSKLTF (SEQ ID NO: 92), CVVHSGGSYIPTF (SEQ ID NO: 103)
  • CDR1 ⁇ one of MNHNS (SEQ ID NO: 81), SGHDT (SEQ ID NO: 93), MNHNY (SEQ ID NO: 104)
  • CDR 2 ⁇ one of SASEGT (SEQ ID NO: 82), YYEEEE (SEQ ID NO: 94), SVGAGI (SEQ ID NO: 105)
  • CDR 3 ⁇ one of CASTINTYEQYF (SEQ ID NO: 83), CASSLYGGPGDQPQHF (SEQ ID NO: 95), CASAQTIGAYNEQFF (SEQ ID NO: 106);
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • CDR3 ⁇ CAYRSFYTGANSKLTF (SEQ ID NO: 92)
  • CDR3 ⁇ CASSLYGGPGDQPQHF (SEQ ID NO: 95)
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • CDR3 ⁇ CVVHSGGSYIPTF (SEQ ID NO: 103)
  • CDR2 ⁇ SVGAGI (SEQ ID NO: 105)
  • CDR3 ⁇ CASAQTIGAYNEQFF (SEQ ID NO: 106).
  • variable region of the ⁇ chain of the TCR disclosed herein comprises an amino acid sequence of one of SEQ ID NO: 84, SEQ ID NO: 96, SEQ ID NO: 107, or comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or more homologous to one of SEQ ID NO: 84, SEQ ID NO: 96, SEQ ID NO: 107.
  • the ⁇ chain of the TCR disclosed herein comprises an amino acid sequence of one of SEQ ID NO: 86, SEQ ID NO: 98, SEQ ID NO: 109, or an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or more homologous to one of SEQ ID NO: 86, SEQ ID NO: 98, SEQ ID NO: 109.
  • variable region of the ⁇ chain of the TCR disclosed herein comprises an amino acid sequence of one of SEQ ID NO: 85, SEQ ID NO: 97, SEQ ID NO: 108, or comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or more homologous to one of SEQ ID NO: 85, SEQ ID NO: 97, SEQ ID NO: 108.
  • the ⁇ chain of the TCR disclosed herein comprises an amino acid sequence of one of SEQ ID NO: 87, SEQ ID NO: 99, SEQ ID NO: 110, or an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or more homologous to one of SEQ ID NO: 87, SEQ ID NO: 99, SEQ ID NO: 110.
  • the amino groups of the ⁇ chain and the ⁇ chain of the TCR of the present disclosure is directly or indirectly connected, preferably indirectly connected, and more preferably connected using 2A peptides.
  • 2A peptides are short peptides (18-25 amino acids) derived from viruses. They are usually called "self-cleaving" peptides, which can produce multiple proteins from one transcription product.
  • the 2A peptides used in the present disclosure include but are not limited to P2A, T2A, E2A, and F2A.
  • a 2A peptide such as P2A
  • P2A can also be linked to a furin cleavage site, a ser-gly linker (sgsg) and a p2a ribosomal skipping peptide to form a fp2A (furin-SGSG-p2A) linkage structure.
  • sgsg ser-gly linker
  • p2a ribosomal skipping peptide to form a fp2A (furin-SGSG-p2A) linkage structure.
  • the fp2A comprises the amino acid sequence shown in SEQ ID NO: 11.
  • connection order of the ⁇ chain and the ⁇ chain can be ⁇ chain, fp2A and ⁇ chain, or ⁇ chain, fp2A and ⁇ chain.
  • the TCR of the present disclosure comprises the amino acid sequence shown in one of SEQ ID NO: 88, SEQ ID NO: 100, SEQ ID NO: 111, or comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or more homologous to SEQ ID NO: 88, SEQ ID NO: 100, SEQ ID NO: 111.
  • the second aspect of the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the TCR described in the first aspect of the present disclosure or a complementary sequence thereof.
  • the nucleic acid sequence may be single-stranded or double-stranded, and may be DNA or RNA.
  • the nucleic acid sequence may be codon-optimized.
  • the codon optimization includes converting a large number of rare codons used by viruses, etc., into corresponding mammalian codons and/or removing mRNA unstable motifs and/or hidden splicing sites.
  • the third aspect of the present disclosure provides an expression vector, which comprises any nucleic acid described in the present disclosure.
  • the expression vector can be expressed in vivo, in vitro or in vitro. Further preferably, the expression vector is continuously expressed at a high level in cells in vivo.
  • the expression vector may be a prokaryotic expression vector or a retroviral vector.
  • the expression vector can be Rous sarcoma virus (RSV), lentivirus, human immunodeficiency virus (HIV), murine leukemia virus (MLV), equine infectious anemia virus (EIAV), mouse mammary cancer virus (MMTV), Fujinami sarcoma virus (FuSV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine leukemia virus (Mo-MLV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myeloproliferative virus 29 (MC29) or avian myeloerythroblastosis virus (AEV), etc. More preferably, the expression vector is a lentiviral expression vector.
  • a fourth aspect of the present disclosure provides a host cell, wherein the host cell comprises any nucleic acid or expression vector described in the present disclosure.
  • the host cell can be eukaryotic or prokaryotic. More preferably, the host cell is a eukaryotic cell, including but not limited to yeast cells, 293 cells, CHO cells, etc.
  • a fifth aspect of the present disclosure provides an immune cell, wherein the immune cell expresses the T cell antigen receptor described in the present disclosure.
  • the immune cells contain one or more nucleic acid sequences described in any one of the present disclosure.
  • the immune cells include but are not limited to stem cells and lymphocytes (including T cells and B cells). Further, the immune cells are B cells, and the B cells express the above-mentioned antibodies or antigen-binding fragments thereof.
  • the immune cells are T cells, and the T cell antigen receptor structure of the T cells is as described above. limited.
  • the T cells may be CD4 + T, CD8 + T, etc.
  • the immune cells are separated from T cells of the subject.
  • the immune cells are T cells from CMV seronegative donors.
  • a sixth aspect of the present disclosure provides a method for preparing immune cells, comprising transducing a nucleic acid sequence encoding the above-mentioned T cell antigen receptor into immune cells for expression.
  • the immune cells include but are not limited to stem cells, lymphocytes (including T cells and B cells). Further, the immune cells are B cells, and the B cells express the above-mentioned antibodies or antigen-binding fragments thereof.
  • the immune cells are T cells, and the T cell antigen receptor structure of the T cells is as defined above.
  • the preferred technical solution of the present disclosure also includes a step of knocking out the endogenous TCR of the cell.
  • the guide targeting the endogenous TCR can be constructed into a lentiviral vector and co-transfected into T cells with a packaging plasmid and a transfection reagent.
  • a seventh aspect of the present disclosure provides a method for preparing a recombinant T cell, comprising the following steps:
  • step 3 delivering the nucleic acid obtained in step 1) into the primary T cells described in step 2) to obtain recombinant T cells expressing any T cell antigen receptor described in the present disclosure.
  • the T cells are selected from hematopoietic stem cells or peripheral blood lymphocyte (PBL)-derived T cells.
  • PBL peripheral blood lymphocyte
  • An eighth aspect of the present disclosure provides a method for preparing a T cell antigen receptor, comprising the following steps:
  • step (3) transforming the expression vector obtained in step (2) into a host cell and then inducing its expression;
  • the positive T cells specifically bind to the cytomegalovirus (CMV) phosphoprotein pp65 antigen peptide presented by MHC.
  • CMV cytomegalovirus
  • the cytomegalovirus (CMV) phosphoprotein pp65 antigen peptide complex presented by MHC is a monomer or polymer complex.
  • the ninth aspect of the present disclosure provides the use of any T cell antigen receptor described in the first aspect, any nucleic acid described in the second aspect, the expression vector described in the third aspect, the host cell described in the fourth aspect, and the immune cell described in the fifth aspect in the preparation of products for diagnosing or treating tumors or CMV-related diseases.
  • the CMV-related disease is selected from neonatal CMV inclusion body disease, acute acquired CMV infection or diseases caused by CMV infection in immunocompromised persons.
  • the CMV-related disease is selected from liver, spleen or central nervous system diseases, disability, infectious mononucleosis, skeletal muscle pain, CMV retinitis, gastrointestinal CMV or encephalitis, etc.
  • the tenth aspect of the present disclosure provides the use of any T cell antigen receptor described in the present disclosure, any nucleic acid described in the present disclosure, the expression vector described in the present disclosure, the host cell described in the present disclosure, and the immune cell described in the present disclosure in T cell labeling, detection, cell sorting or activation.
  • the eleventh aspect of the present disclosure provides a pharmaceutical composition, which comprises any one of the following groups:
  • T cell antigen receptor described in the present disclosure
  • the pharmaceutical composition may further comprise pharmaceutically acceptable excipients.
  • the pharmaceutical composition can also be used together with other therapeutic agents.
  • the therapeutic agent can be an immunomodulator.
  • kit comprises any one of the following groups:
  • a method for detecting cytomegalovirus (CMV) phosphoprotein pp65 comprising contacting a sample to be detected with the T cell antigen receptor described in the present disclosure, and then detecting a complex formed by cytomegalovirus (CMV) phosphoprotein pp65 and the T cell antigen receptor.
  • CMV cytomegalovirus
  • the detection of cytomegalovirus (CMV) phosphoprotein pp65 is to detect the presence or content of cytomegalovirus (CMV) phosphoprotein pp65.
  • the presence refers to the presence or absence, and the content can be the expression level or protein concentration.
  • the antibody or antigen-binding fragment thereof or T cell antigen receptor comprises a detectable marker.
  • the marker may be His and/or HA.
  • the method for detecting cytomegalovirus (CMV) phosphoprotein pp65 is not a method for diagnosing a disease.
  • the sample to be detected is not an organism or its isolated tissue or cell.
  • cytomegalovirus (CMV) phosphoprotein pp65 exists in an organism or contains a certain concentration or expression level of cytomegalovirus (CMV) phosphoprotein pp65, it is not a definite disease, but only a possibility.
  • a method for treating and/or preventing CMV-related diseases comprising administering to an individual an effective amount of the T cell antigen receptor, the nucleic acid, the expression vector, the host cell, the immune cell or the pharmaceutical composition described in the present disclosure.
  • the method comprises the step of adoptively transferring T cells expressing the T cell antigen receptor disclosed herein to a subject.
  • the method comprises localizing the T cell antigen receptor described in the present disclosure in the vicinity of a CMV-associated disease (preferably a tumor or metastatic tumor) to enhance the efficacy of the toxin or immunostimulant.
  • a CMV-associated disease preferably a tumor or metastatic tumor
  • the invention is used for treating and/or preventing CMV reactivation after allogeneic hematopoietic stem cell transplantation.
  • CMV reactivation after organ transplantation (eg kidney, liver, pancreas, intestine, cornea), tissue transplantation, cell transplantation (pancreatic islet cells, limbal stem cells) or stem cell therapy.
  • organ transplantation eg kidney, liver, pancreas, intestine, cornea
  • tissue transplantation eg kidney, liver, pancreas, intestine, cornea
  • cell transplantation pancreatic islet cells, limbal stem cells
  • the T cells expressing the T cell antigen receptor disclosed herein are derived from a subject.
  • the T cells expressing the T cell antigen receptor disclosed herein and the hematopoietic stem cells, organs, tissues, cells or stem cells are derived from the same donor.
  • the fifteenth aspect of the present disclosure provides a method for diagnosing CMV-related diseases, the method comprising taking a sample, contacting the sample with the T cell antigen receptor described in the present disclosure, and then detecting the complex formed by CMV pp65 and the T cell antigen receptor.
  • the T cell antigen receptor comprises a detectable marker.
  • the TCR disclosed in the present invention can specifically recognize the corresponding CMV pp65 antigen peptide-MHC molecule complex, activate TCR T cells, and then produce high levels of cytokines IFN ⁇ , IL2, and TNF ⁇ , and significantly kill tumor cells both in vivo and in vitro experiments.
  • T cell antigen receptor is a molecule that can recognize peptides when presented by MHC molecules or their tetramers, and is usually present on the surface of T cells in the form of a complex with CD3 molecules.
  • the TCR of most T cells is composed of ⁇ and ⁇ peptide chains, and the TCR of a few T cells is composed of ⁇ and ⁇ peptide chains.
  • the “tumor” described in the present disclosure includes, but is not limited to, pancreatic cancer, liver cancer, colon cancer, rectal cancer, gastric cancer, lymphoma, basal cell carcinoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma.
  • the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia;
  • the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B cell lymphoma, T cell lymphoma, and Waldenstrom's macroglobulinemia;
  • the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma,
  • CMV-related diseases include diseases caused by CMV pp65 infection, including neonatal CMV inclusion disease, which can seriously affect the liver, spleen and central nervous system and cause disability. It also includes acutely acquired CMV infection, similar to infectious mononucleosis, including fever, skeletal muscle pain, etc. It also includes immunocompromised people, such as those who have undergone organ transplants, or those with HIV, where infection can lead to CMV retinitis, gastrointestinal CMV, and encephalitis, etc.
  • the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present disclosure.
  • prevention refers to all actions to suppress symptoms or delay the tension of specific symptoms by administering the product described in the present disclosure.
  • Diagnosis refers to finding out whether a patient has a disease or condition in the past, at the time of diagnosis, or in the future, or to finding out the progression or possible future progression of a disease, or to evaluating a patient's response to treatment.
  • Treatment means to slow down, interrupt, prevent, control, stop, alleviate, or reverse the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders, and refers to therapeutic intervention to improve the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.
  • an "effective amount” as described herein refers to an amount or dosage of a product described herein that provides the desired treatment or prevention after administration to a patient or an organ in a single or multiple doses.
  • the “product” described in the present disclosure includes, but is not limited to, the antibody or antigen-binding fragment thereof, the T cell antigen receptor, the nucleic acid, the expression vector, the host cell, the immune cell or the multimeric complex described in the present disclosure, and other reagents that assist or cooperate with the above-mentioned products.
  • the “product” described in the present disclosure may be a pharmaceutical composition such as a kit, a chip, an antibody conjugate or a multifunctional antibody.
  • the "subject" described in the present disclosure includes but is not limited to humans or non-human mammals.
  • the non-human mammals include but are not limited to mice, rats, monkeys, pigs or rabbits.
  • the "homology" described in the present disclosure refers to the fact that in terms of the use of amino acid sequences or nucleotide sequences, those skilled in the art can adjust the sequence according to actual work needs without changing the main structure or function of the original sequence, so that the used sequence has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% compared with the specific sequence described in the present disclosure.
  • the "having at least 80% homology with the amino acid sequence shown in any one of SEQ ID NO: 1-12, SEQ ID NO: 14-73" described in the present disclosure means that the amino acid sequence can be adjusted according to actual work needs, including one or more changes such as substitution, deletion and/or insertion of one or more amino acids, truncation, or lengthening of one or both ends, as long as it maintains more than 80% homology and retains the ability to bind to the pp65 epitope/MHC complex or the pp65 epitope/MHC molecule tetramer.
  • the change can be a substitution, addition or deletion.
  • the basic side chain includes but is not limited to lysine, arginine or histidine.
  • the acidic side chain includes but is not limited to aspartic acid or glutamic acid.
  • Uncharged amino acids include, but are not limited to, aspartyl, glutamine, serine, threonine or tyrosine.
  • Non-polar side chains include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan or cysteine.
  • at least 80% is, but is not limited to, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • the present invention has the following beneficial technical effects:
  • TCR that recognizes and binds to the CMV-pp65 antigen epitope polypeptide fragment, which can endow the transduced T cells with the ability to recognize the virus and its infected cells and initiate an immune response; it has the ability to specifically kill target cells.
  • the TCR obtained in the present disclosure has a new and specific CDR3 ⁇ / ⁇ structure, which is a naturally paired double-stranded structure; and the present disclosure adopts a single cell sequencing method to construct a library and sequence and compare the VDJ fragments of the cells multiple times to screen out the superior sequences.
  • the present disclosure also provides a nucleic acid encoding the TCR, a vector containing the nucleic acid molecule, a cell transducing the nucleic acid molecule or the vector, and a pharmaceutical composition comprising the TCR, nucleic acid molecule, vector or cell as an active ingredient, and uses of the TCR, nucleic acid molecule, vector, cell and pharmaceutical composition, which have drugability and industrial value.
  • FIG. 1 Schematic diagram of the component structure of the TCR sequence
  • FIG3 is the result of screening positive cells for TCR-T cell transduction in Example 2.
  • FIG4 is the result of screening positive cells for TCR-T cell transduction in Example 5.
  • FIG. 5 is the screening results of TCR-T cell transduction positive cells in Example 8.
  • the method of using a chemically synthesized short peptide C1 (amino acid sequence: QYDPVAALF (SEQ ID NO: 13)) to stimulate peripheral blood mononuclear cells (PBMC) from healthy donors in vitro (the HLA typing of healthy donors can be optional, and the HLA-A*2402 genotype in HLA-A*24 is used as an example in this embodiment) to induce and culture CD4-CD8+ positive CTL cells that can recognize the antigen peptide C1 and secrete IFN- ⁇ , specifically comprising the following steps:
  • Control group No peptide stimulation was added, and the donor PBMC cells were cultured in parallel under the same conditions as the experimental group as the control group;
  • both groups of cells were stimulated and incubated again with the addition of antigen peptide C1 to observe whether there were CD8+IFN- ⁇ + double-positive CTL cells (CD4-) that recognized the antigen peptide and secreted IFN- ⁇ ; in subsequent experiments, monoclonal cell lines were isolated and screened from the culture of CD4-CD8+ positive CTL cells that could recognize the antigen peptide and secrete IFN- ⁇ .
  • Lymphoblastoid cell line is an in vitro immortalized cell line obtained by transfecting human PBMC with EB virus.
  • LCL cell lines with different HLA restrictions can be prepared using PBMCs with different HLA restrictions.
  • PBMCs were isolated from peripheral blood and resuspended in 2 ml of RPMI1640 medium containing 10% FBS (referred to as RPMI/10% FBS, RPM1640 merchant: Thermo Fisher Scientific, catalog number: 22400-089, FBS merchant: Thermo Fisher Scientific, catalog number: 10099-141C).
  • the X-C1 cells of the experimental group of CD4-CD8+ positive CTL cells that can recognize antigenic peptides and secrete IFN- ⁇ in step 1 were diluted to 0.3 cell/2 ⁇ l and then spread on Terasaki plates (0-0.3 cells per well), and EBV-LCL cells (lymphoblastoid cell line (LCL cell line) is an in vitro immortalized cell line obtained after EB virus transfection of human PBMC) irradiated with 0.120J/ cm2 ultraviolet light were added as feeder cells to stimulate the growth of CTL positive T cells. After 5-7 days of static culture, the growth of monoclonal cells can be observed under the microscope.
  • EBV-LCL cells lymphoblastoid cell line (LCL cell line) is an in vitro immortalized cell line obtained after EB virus transfection of human PBMC) irradiated with 0.120J/ cm2 ultraviolet light
  • the monoclonal cell clusters were picked out and transferred to U-bottom 96-well plates, and further expanded and cultured in RPMI 1640 medium containing 30ng/ml OKT3 and 3000IU/ml IL-2; the cell culture was replenished/replaced/the culture container was replaced every 3-4 days, and the irradiated EBV-LCL feeder cells were added again every 30-40 days to maintain its specificity.
  • a T cell monoclonal cell line can be obtained, which has a cell number of the order of 10 7 and a viability of 80% to 90%.
  • a certain amount of cells is regularly taken for intracellular factor secretion detection to determine the presence of killer CTL cells.
  • TCR sequence sequencing From the monoclonal cell culture, samples were taken for TCR sequence sequencing, and the TCR sequences were obtained as follows:
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of TCR are as follows:
  • CDR2 ⁇ MTLNGDE (SEQ ID NO: 2);
  • CDR3 ⁇ one of CARNTGKLIF (SEQ ID NO: 3), CAPSASKIIF (SEQ ID NO: 22), CAPQFNKFYF (SEQ ID NO: 31);
  • CDR1 ⁇ one of SQVTM (SEQ ID NO: 4), SGHVS (SEQ ID NO: 14), LNHDA (SEQ ID NO: 23);
  • CDR 2 ⁇ One of ANQGSEA (SEQ ID NO: 5), FQNEAQ (SEQ ID NO: 15), SQIVND (SEQ ID NO: 24);
  • CDR 3 ⁇ one of CSANPTGGGTEAFF (SEQ ID NO: 6), CASSLLTRTETQYF (SEQ ID NO: 16), CASSTTGLAGGPGNEQFF (SEQ ID NO: 25);
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of one of the TCRs obtained are as follows:
  • CDR 2 ⁇ ANQGSEA (SEQ ID NO: 5)
  • CDR 3 ⁇ CSANPTGGGTEAFF (SEQ ID NO: 6);
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of another TCR obtained are as follows:
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of another TCR obtained are as follows:
  • CDR3 ⁇ CASSTTGLAGGPGNEQFF(SEQ ID NO: 25);
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of another TCR obtained are as follows:
  • the present invention further obtains paired TCR alpha sequences and TCR beta sequences, and the specific method is not limited to obtaining by sequencing, bioinformatics, etc.
  • TCR alpha sequence and TCR beta sequence obtained according to the first group of amino acid sequences in Table 1 are as follows:
  • TCR alpha sequence (amino acid sequence is SEQ ID NO: 9), TCR beta sequence (amino acid sequence is SEQ ID NO: 10); wherein the TCR alpha sequence includes the variable region V ⁇ (amino acid sequence is SEQ ID NO: 7),
  • the TCR beta sequence includes the variable region V ⁇ (amino acid sequence is SEQ ID NO: 8);
  • the variable region V ⁇ includes the following complementary determining regions: CDR1 ⁇ : SSNFYA; CDR2 ⁇ : MTLNGDE; CDR3 ⁇ : CARNTGKLIF (SEQ ID NO: 1-3), and the variable region V ⁇ includes the following complementary determining regions: CDR1 ⁇ : SQVTM; CDR2 ⁇ : ANQGSEA; CDR3 ⁇ : CSANPTGGGTEAFF (SEQ ID NO: 4-6).
  • TCR alpha sequence and TCR beta sequence can also be added before the above-mentioned TCR alpha sequence and TCR beta sequence, respectively, and after connecting them through the connecting sequence (SEQ ID NO: 11), the full-length sequence of TCR (SEQ ID NO: 12) can be obtained.
  • TCR alpha sequence and TCR beta sequence obtained according to the second group of amino acid sequences in Table 1 are as follows:
  • TCR alpha sequence (amino acid sequence is SEQ ID NO: 19), TCR beta sequence (amino acid sequence is SEQ ID NO: 20); wherein the variable region V ⁇ of the TCR alpha sequence includes the following complementary determining regions: CDR1 ⁇ : SSNFYA (SEQ ID NO: 1); CDR2 ⁇ : MTLNGDE (SEQ ID NO: 2); CDR3 ⁇ : CARNTGKLIF (SEQ ID NO: 3); the variable region V ⁇ includes the following complementary determining regions: CDR1 ⁇ : SGHVS (SEQ ID NO: 14); CDR2 ⁇ : FQNEAQ (SEQ ID NO: 15); CDR3 ⁇ : CASSLLTRTETQYF (SEQ ID NO: 16);
  • TCR alpha sequence and TCR beta sequence are connected via a connecting sequence (SEQ ID NO: 11) to obtain the full-length sequence of TCR (SEQ ID NO: 21).
  • TCR alpha sequence and TCR beta sequence obtained according to the third group of amino acid sequences in Table 1 are as follows:
  • TCR alpha sequence (amino acid sequence is SEQ ID NO: 28), TCR beta sequence (amino acid sequence is SEQ ID NO: 29); wherein the variable region V ⁇ of the TCR alpha sequence includes the following complementary determining regions: CDR1 ⁇ : SSNFYA (SEQ ID NO: 1); CDR2 ⁇ : MTLNGDE (SEQ ID NO: 2); CDR3 ⁇ : CAPSASKIIF (SEQ ID NO: 22); the variable region V ⁇ includes the following complementary determining regions: CDR1 ⁇ : LNHDA (SEQ ID NO: 23); CDR2 ⁇ : SQIVND (SEQ ID NO: 24); CDR3 ⁇ : CASSTTGLAGGPGNEQFF (SEQ ID NO: 25);
  • TCR alpha sequence and TCR beta sequence were connected via a connecting sequence (SEQ ID NO: 11) to obtain the full-length sequence of TCR (SEQ ID NO: 30).
  • TCR alpha sequence and TCR beta sequence obtained according to the fourth group of amino acid sequences in Table 1 are as follows:
  • TCR alpha sequence (amino acid sequence is SEQ ID NO: 35), TCR beta sequence (amino acid sequence is SEQ ID NO: 36); wherein the variable region V ⁇ of the TCR alpha sequence includes the following complementary determining regions: CDR1 ⁇ : SSNFYA (SEQ ID NO: 1); CDR2 ⁇ : MTLNGDE (SEQ ID NO: 2); CDR3 ⁇ : CAPQFNKFYF (SEQ ID NO: 31); the variable region V ⁇ includes the following complementary determining regions: CDR1 ⁇ : SGHVS (SEQ ID NO: 14); CDR2 ⁇ : FQNEAQ (SEQ ID NO: 15); CDR3 ⁇ : CASSLLTRTETQYF (SEQ ID NO: 16);
  • TCR alpha sequence and TCR beta sequence were connected via a connecting sequence (SEQ ID NO:11) to obtain the full-length sequence of TCR (SEQ ID NO:36).
  • Examples 2 and 3 both use the full-length sequence of TCR obtained based on the first group of amino acid sequences as an example for explanation.
  • the TCR gene sequence obtained in Example 1 was cloned into a lentiviral vector and transfected into the virus packaging line 293T cells. A virus solution is prepared and the virus solution is transduced into T cells to obtain T cells expressing the target TCR sequence.
  • the transfection procedure is as follows:
  • Lentiviral supernatant was produced by transfecting 293T cells with gag/pol packaging plasmid, VSV-G envelope plasmid and transfer construct containing lentiviral vector sequences as described below. That is, the DNA mixture was mixed in Opti-MEM (Life Technologies, Gaithersburg, MD, USA) and mixed with an equal volume of Opti-MEM containing Lipofectamine 3000 (Life Technologies). After incubation at room temperature for 15 minutes, the resulting mixture was applied to 293T cells. The medium containing lentivirus was collected 24 hours after transfection. After each collection, the supernatant was filtered through a 0.45 ⁇ m filter. The lentiviral harvests were combined and stored at 4°C and then ultracentrifuged at 20,000xg for 90 minutes. The lentiviral particles were resuspended in PBS.
  • the transduction procedure is as follows:
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • X-VIVO15 serum-free medium LONZA
  • T cells were transduced with lentiviral supernatant, followed by centrifugation at 1200 ⁇ g and 32°C for 2 hours. After 24 hours, the supernatant containing the viral vector was removed.
  • Non-transduced cells do not express specific TCRs.
  • T cells transduced with the lentiviral vectors containing the first group of TCRs, the second group of TCRs, the third group of TCRs, and the fourth group of TCRs in Example 1 respectively expressed specific TCRs (CD8 + Tetramer + data), that is, the transduction positive rate: the first group TCR transduction positive rate was 30.36%, the second group TCR transduction positive rate was 9.63%, the third group TCR transduction positive rate was 8.63%, and the fourth group TCR transduction positive rate was 3.28%, indicating that the transduction was successful.
  • the present invention can be used to evaluate the function of TCR-T cells in vitro by constructing target cells that overexpress the CMV-PP65 protein to which QYDPVAALF belongs.
  • the specific operation is as follows:
  • T cells expressing the target TCR sequence prepared in Example 2 were used as effector cells, among which the cells transduced with TCR lentivirus and expressed were the TCR group, and the cells not transduced were the control group (NC group);
  • the target cells in the present invention are tumor cells that overexpress the CMV-PP65 protein to which QYDPVAALF belongs, and are constructed by introducing the target antigen gene into the tumor cells:
  • Tumor cells were transduced with lentivirus containing the target antigen. After 1 day, the medium was changed to complete medium for 1-2 days, and then replaced with complete medium containing puromycin for continued culture. The expression of the target antigen in the tumor cells of the transduction group was detected.
  • the specific steps include:
  • the density of tumor cells was adjusted to 4-6 ⁇ 10 5 cells/ml using complete culture medium (complete culture medium used for OS-RC-2 human renal cancer cells: RPMI1640 culture medium (Gibco) containing 10% FBS), 1 ml/well was added to a 6-well plate, 8 ⁇ l Polybrene (1 mg/ml) was added to each well to a final concentration of 8 ⁇ g/ml, 10-30 ⁇ l of lentiviral vector containing the target antigen QYDPVAALF was added to each well, and a cell control well (non-transduced group) was set up, and the control well was added with the above 1 ml tumor cells and 8 ⁇ l Polybrene, mixed well, and incubated in a CO2 incubator (37°C, 5% CO2) for 1 day;
  • the cells were observed every 2 days and the complete medium containing 1 ⁇ g/ml puromycin was replaced once. The adherent cells were passaged when they were confluent;
  • Transduced and screened tumor cells that were transduced with the target antigen and cultured for more than 7 days were plated according to the limiting dilution method, 1 cell/well, 3 cells/well, cultured with complete medium containing puromycin and the cells in the cell wells were observed. Monoclonal wells were marked and cultured continuously until the cell number reached at least 6 ⁇ 10 7 cells and then frozen to obtain overexpressed target cells.
  • OS-RC-2-PP65 cells overexpressing PP65 protein prepared in step 2 were plated as target cells at 5 ⁇ 10 4 per well.
  • the effector cells of the TCR group and the effector cells of the NC group in step 1 were mixed with the target cells at effector-target ratios of 1:1, 5:1, 10:1, and 20:1, respectively, and incubated at 37°C.
  • the cells were analyzed using a real-time label-free cell function analyzer ( The killing efficiency was obtained by Real Time Cell Analyzer (RTCA, Agilent).
  • RTCA Real Time Cell Analyzer
  • Antigen-specific T cells are identical to Antigen-specific T cells.
  • the chemically synthesized antigen peptide C1 (amino acid sequence: NLVPMVATV (SEQ ID NO: 46)) is used to stimulate peripheral blood mononuclear cells (PBMC) from healthy donors in vitro (the HLA typing of healthy donors can be optional, and the HLA-A*0201 genotype in HLA-A*02 is used as an example in this embodiment), and CD4-CD8+ positive CTL cells that can recognize the antigen peptide C1 and secrete IFN- ⁇ are induced and cultured, which specifically includes the following steps:
  • Experimental group X-C1 cells cultured from donor PBMCs stimulated with antigen peptide C1 were used as the experimental group;
  • Control group No peptide stimulation was added, and the donor PBMC cells were cultured in parallel under the same conditions as the experimental group as the control group;
  • both groups of cells were stimulated and incubated again with the addition of antigen peptide C1 to observe whether there were CD8+IFN- ⁇ + double-positive CTL cells (CD4-) that recognized the antigen peptide and secreted IFN- ⁇ ; in subsequent experiments, monoclonal cell lines were isolated and screened from the culture of CD4-CD8+ positive CTL cells that could recognize the antigen peptide and secrete IFN- ⁇ .
  • Lymphoblastoid cell line is an in vitro immortalized cell line obtained by transfecting human PBMC with EB virus.
  • LCL cell lines with different HLA restrictions can be prepared using PBMCs with different HLA restrictions.
  • PBMCs were isolated from peripheral blood and resuspended in 2 ml of RPMI1640 medium containing 10% FBS (referred to as RPMI/10% FBS, RPM1640 merchant: Thermo Fisher Scientific, catalog number: 22400-089, FBS merchant: Thermo Fisher Scientific, catalog number: 10099-141C).
  • the X-C1 cells of the experimental group of CD4-CD8+ positive CTL cells that can recognize antigenic peptides and secrete IFN- ⁇ in step 1 were diluted to 0.3 cell/2 ⁇ l and then spread on Terasaki plates (0-0.3 cells per well), and EBV-LCL cells (lymphoblastoid cell line (LCL cell line) is an in vitro immortalized cell line obtained after EB virus transfection of human PBMC) irradiated with 0.120J/ cm2 ultraviolet light were added as feeder cells to stimulate the growth of CTL positive T cells. After 5-7 days of static culture, the growth of monoclonal cells can be observed under the microscope.
  • EBV-LCL cells lymphoblastoid cell line (LCL cell line) is an in vitro immortalized cell line obtained after EB virus transfection of human PBMC) irradiated with 0.120J/ cm2 ultraviolet light
  • the monoclonal cell clusters were picked out and transferred to U-bottom 96-well plates, and further expanded and cultured in RPMI 1640 medium containing 30ng/ml OKT3 and 3000IU/ml IL-2; the cell culture was replenished/replaced/the culture container was replaced every 3-4 days, and the irradiated EBV-LCL feeder cells were added again every 30-40 days to maintain its specificity.
  • a T cell monoclonal cell line can be obtained, which has a cell number of the order of 10 7 and a viability of 80% to 90%.
  • a certain amount of cells is regularly taken for intracellular factor secretion detection to determine the presence of killer CTL cells.
  • TCR sequence sequencing From the monoclonal cell culture, samples were taken for TCR sequence sequencing, and the TCR sequences were obtained as follows:
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of TCR are as follows:
  • CDR1 ⁇ one of SSNFYA (SEQ ID NO: 1), DSSSTY (SEQ ID NO: 47), TSGFNG (SEQ ID NO: 58)
  • CDR2 ⁇ one of MTLNGDE (SEQ ID NO: 2), IFSNMDM (SEQ ID NO: 48), NVLDGL (SEQ ID NO: 59)
  • CDR3 ⁇ one of CASINFNKFYF (SEQ ID NO: 37), CAEFTGTASKLTF (SEQ ID NO: 49), CAVTYNNARLMF (SEQ ID NO: 60), CARNYGQNFVF (SEQ ID NO: 69)
  • CDR1 ⁇ one of MDHEN (SEQ ID NO: 38), GTSNPN (SEQ ID NO: 50), MNHEY (SEQ ID NO: 61), DFQATT (SEQ ID NO: 70)
  • CDR 2 ⁇ One of SYDVKM (SEQ ID NO: 39), SVGIG (SEQ ID NO: 51), SMNVEV (SEQ ID NO: 62), SNEGSKA (SEQ ID NO: 71)
  • CDR 3 ⁇ One of CASSPLNGGATEAFF (SEQ ID NO: 40), CAWSDRAAFTDTQYF (SEQ ID NO: 52), CASSSVAGGRIEQFF (SEQ ID NO: 63), CSARDIKAQQWNIQYF (SEQ ID NO: 72).
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • CDR 3 ⁇ CASSPLNGGATEAFF (SEQ ID NO: 40)
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • CDR3 ⁇ CAEFTGTASKLTF (SEQ ID NO: 49)
  • CDR3 ⁇ CAWSDRAAFTDTQYF (SEQ ID NO: 52)
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • CDR3 ⁇ CAVTYNNARLMF (SEQ ID NO: 60)
  • CDR2 ⁇ SMNVEV (SEQ ID NO: 62)
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • the present invention further obtains paired TCR alpha sequences and TCR beta sequences, and the specific method is not limited to obtaining by sequencing, bioinformatics, etc.
  • TCR alpha sequence and TCR beta sequence obtained according to the fifth group of amino acid sequences in Table 3 are as follows:
  • the alpha sequence of TCR (amino acid sequence is SEQ ID NO:43), and the beta sequence of TCR (amino acid sequence is SEQ ID NO:44); wherein the TCR alpha sequence includes the variable region V ⁇ (amino acid sequence is SEQ ID NO:41), and the TCR beta sequence includes the variable region V ⁇ (amino acid sequence is SEQ ID NO:42); the variable region V ⁇ includes the following complementary determining regions: CDR1 ⁇ : SSNFYA; CDR2 ⁇ : MTLNGDE; CDR3 ⁇ : CASINFNKFYF (SEQ ID NO:1, 2, 37), and the variable region V ⁇ includes the following complementary determining regions: CDR1 ⁇ : MDHEN; CDR2 ⁇ : SYDVKM; CDR3 ⁇ : CASSPLNGGATEAFF (SEQ ID NO:38-40).
  • TCR alpha sequence and TCR beta sequence can also be added before the above-mentioned TCR alpha sequence and TCR beta sequence, respectively, and after connecting them through the connecting sequence (SEQ ID NO: 11), the full-length sequence of TCR (SEQ ID NO: 45) can be obtained.
  • TCR alpha sequence and TCR beta sequence obtained according to the sixth group of amino acid sequences in Table 3 are as follows:
  • TCR alpha sequence (amino acid sequence is SEQ ID NO: 55), TCR beta sequence (amino acid sequence is SEQ ID NO: 56); wherein the variable region V ⁇ of the TCR alpha sequence includes the following complementary determining regions: CDR1 ⁇ : DSSSTY (SEQ ID NO: 47); CDR2 ⁇ : IFSNMDM (SEQ ID NO: 48); CDR3 ⁇ : CAEFTGTASKLTF (SEQ ID NO: 49); the variable region V ⁇ includes the following complementary determining regions: CDR1 ⁇ : GTSNPN (SEQ ID NO: 50); CDR2 ⁇ : SVGIG (SEQ ID NO: 51); CDR3 ⁇ : CAWSDRAAFTDTQYF (SEQ ID NO: 52);
  • TCR alpha sequence and TCR beta sequence are connected via a connecting sequence (SEQ ID NO: 11) to obtain the full-length sequence of TCR (SEQ ID NO: 57).
  • TCR alpha sequence and TCR beta sequence obtained according to the seventh group of amino acid sequences in Table 3 are as follows:
  • TCR alpha sequence (amino acid sequence is SEQ ID NO: 66), TCR beta sequence (amino acid sequence is SEQ ID NO: 67); wherein the variable region V ⁇ of the TCR alpha sequence includes the following complementary determining regions: CDR1 ⁇ : TSGFNG (SEQ ID NO: 58); CDR2 ⁇ : NVLDGL (SEQ ID NO: 59); CDR3 ⁇ : CAVTYNNARLMF (SEQ ID NO: 60); the variable region V ⁇ includes the following complementary determining regions: CDR1 ⁇ : MNHEY (SEQ ID NO: 61); CDR2 ⁇ : SMNVEV (SEQ ID NO: 62); CDR3 ⁇ : CASSVAGGRIEQFF (SEQ ID NO: 63);
  • TCR alpha sequence and TCR beta sequence were connected via a connecting sequence (SEQ ID NO: 11) to obtain the full-length sequence of TCR (SEQ ID NO: 68).
  • TCR alpha sequence and TCR beta sequence obtained according to the eighth group of amino acid sequences in Table 3 are as follows:
  • TCR alpha sequence (amino acid sequence is SEQ ID NO: 75), TCR beta sequence (amino acid sequence is SEQ ID NO: 76); wherein the variable region V ⁇ of the TCR alpha sequence includes the following complementary determining regions: CDR1 ⁇ : SSNFYA (SEQ ID NO: 1); CDR2 ⁇ : MTLNGDE (SEQ ID NO: 2); CDR3 ⁇ : CARNYGQNFVF (SEQ ID NO: 69); the variable region V ⁇ includes the following complementary determining regions: CDR1 ⁇ : DFQATT (SEQ ID NO: 70); CDR2 ⁇ : SNEGSKA (SEQ ID NO: 71); CDR3 ⁇ : CSARDIKAQQWNIQYF (SEQ ID NO: 72);
  • TCR alpha sequence and TCR beta sequence were connected via a connecting sequence (SEQ ID NO: 11) to obtain the full-length sequence of TCR (SEQ ID NO: 77).
  • a full-length TCR containing a constant region is constructed and inserted into a lentiviral vector.
  • the element structure of the TCR sequence is shown in Figure 1.
  • Examples 5 and 6 are both illustrated with the full-length sequence of the TCR obtained based on the fifth group of amino acid sequences.
  • the TCR gene sequence obtained in Example 4 was cloned into a lentiviral vector, and the virus packaging system 293T cells were transfected to prepare a virus solution, and the virus solution was transduced into T cells to obtain T cells expressing the target TCR sequence.
  • the transfection procedure is as follows:
  • Lentiviral supernatant was produced by transfecting 293T cells with gag/pol packaging plasmid, VSV-G envelope plasmid and transfer construct containing lentiviral vector sequences as described below. That is, the DNA mixture was mixed in Opti-MEM (Life Technologies, Gaithersburg, MD, USA) and mixed with an equal volume of Opti-MEM containing Lipofectamine 3000 (Life Technologies). After incubation at room temperature for 15 minutes, the resulting mixture was applied to 293T cells. The medium containing lentivirus was collected 24 hours after transfection. After each collection, the supernatant was filtered through a 0.45 ⁇ m filter. The lentiviral harvests were combined and stored at 4°C and then ultracentrifuged at 20,000xg for 90 minutes. The lentiviral particles were resuspended in PBS. The structure of the transfer plasmid is shown in Figure 2.
  • the transduction procedure is as follows:
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • CD3/CD28 Dynabeads Gibco
  • X-VIVO15 serum-free medium LONZA
  • T cells were transduced with lentiviral supernatant, followed by centrifugation at 1200 ⁇ g and 32°C for 2 hours. After 24 hours, the supernatant containing the viral vector was removed.
  • the cells were suspended at 3 ⁇ 10 5 cells/ml in medium containing rhIL-2 (200 IU/mL), and X-VIVO15 serum-free medium supplemented with IL-2 (200 IU/mL) was supplemented every 2 to 3 days. TCR expression levels were detected by flow cytometry one day before harvest. That is, the transduction positive rate, and the results are shown in Figure 4.
  • the non-transduced cells (NC) do not express specific TCR.
  • T cells transduced with lentiviral vectors containing the fifth group of TCR, the sixth group of TCR, the seventh group of TCR, and the eighth group of TCR in Example 4 express specific TCR (CD8 + Tetramer + data), that is, the transduction positive rate: the fifth group TCR transduction positive rate is 25.75%, the sixth group TCR transduction positive rate is 23.77%, the seventh group TCR transduction positive rate is 17.51%, and the eighth group TCR transduction positive rate is 23.02%, indicating that the transduction is successful.
  • specific TCR CD8 + Tetramer + data
  • the present invention can be used to evaluate the function of TCR-T cells in vitro by constructing target cells that overexpress CMV-PP65 protein to which NLVPMVATV belongs.
  • the specific operation is as follows:
  • T cells expressing the target TCR sequence prepared in Example 5 are used as effector cells, among which the TCR group is transduced with TCR lentivirus and expressed, and the NC group is not transduced;
  • the target cells in the present invention are tumor cells that overexpress CMV-PP65 protein to which NLVPMVATV belongs, and are constructed by introducing target antigen genes into tumor cells:
  • Tumor cells were transduced with lentivirus containing the target antigen. After 1 day, the medium was changed to complete medium for 1-2 days, and then replaced with complete medium containing puromycin for continued culture. The expression of the target antigen in the tumor cells of the transduction group was detected.
  • the specific steps include:
  • the density of tumor cells was adjusted to 4-6 ⁇ 10 5 cells/ml using complete medium (the complete medium used for U-2OS human osteosarcoma cells was: McCoy's 5A medium (Gibco) containing 10% FBS), 1 ml/well was added to a 6-well plate, 8 ⁇ l Polybrene (1 mg/ml) was added to each well to a final concentration of 8 ⁇ g/ml, 10-30 ⁇ l of lentiviral vector containing the target antigen NLVPMVATV was added to each well, and a cell control well (non-transduced group) was set up, and the control well was added with the above 1 ml tumor cells and 8 ⁇ l Polybrene, mixed well, and incubated in a CO 2 incubator (37°C, 5% CO 2) for 1 day;
  • a CO 2 incubator 37°C, 5% CO 2
  • the cells were observed every 2 days and the complete medium containing 1 ⁇ g/ml puromycin was replaced once. The adherent cells were passaged when they were confluent;
  • Transduced and screened tumor cells that were transduced with the target antigen and cultured for more than 7 days were plated according to the limiting dilution method, 1 cell/well, 3 cells/well, cultured with complete medium containing puromycin and the cells in the cell wells were observed. Monoclonal wells were marked and cultured continuously until the cell number reached at least 6 ⁇ 10 7 cells and then frozen to obtain overexpressed target cells.
  • Real-time label-free dynamic cell analysis technology is a special process that integrates microelectronic cell sensor chips into The bottom of the cell detection plate is used to construct a cell impedance detection sensing system for real-time, dynamic, and quantitative tracking of changes in cell morphology, proliferation, and differentiation.
  • Antigen-specific T cells are identical to Antigen-specific T cells.
  • the chemically synthesized antigen peptide C1 (amino acid sequence: ATVQGQNLK (SEQ ID NO: 89)) was used to stimulate peripheral blood mononuclear cells (PBMC) from healthy donors in vitro (the HLA typing of healthy donors can be optional, and the HLA-A*1101 genotype in HLA-A*11 is used as an example in this embodiment to induce and culture CD4-CD8+ positive CTL cells that can recognize the antigen peptide C1 and secrete IFN- ⁇ , specifically including the following steps:
  • Experimental group X-C1 cells cultured from donor PBMCs stimulated with antigen peptide C1 were used as the experimental group;
  • Control group No peptide stimulation was added, and the donor PBMC cells were cultured in parallel under the same conditions as the experimental group as the control group;
  • both groups of cells were stimulated and incubated again with the addition of antigen peptide C1 to observe whether there were CD8+IFN- ⁇ + double-positive CTL cells (CD4-) that recognized the antigen peptide and secreted IFN- ⁇ ; in subsequent experiments, monoclonal cell lines were isolated and screened from the culture of CD4-CD8+ positive CTL cells that could recognize the antigen peptide and secrete IFN- ⁇ .
  • Lymphoblastoid cell line is an in vitro immortalized cell line obtained by transfecting human PBMC with EB virus.
  • LCL cell lines with different HLA restrictions can be prepared using PBMCs with different HLA restrictions.
  • PBMCs were isolated from peripheral blood and resuspended in 2 ml of RPMI1640 medium containing 10% FBS (referred to as RPMI/10% FBS, RPM1640 merchant: Thermo Fisher Scientific, catalog number: 22400-089, FBS merchant: Thermo Fisher Scientific, catalog number: 10099-141C).
  • the X-C1 cells of the experimental group of CD4-CD8+ positive CTL cells that can recognize antigenic peptides and secrete IFN- ⁇ in step 1 were diluted to 0.3 cell/2 ⁇ l and then spread on Terasaki plates (0-0.3 cells per well), and EBV-LCL cells (lymphoblastoid cell line (LCL cell line) is an in vitro immortalized cell line obtained after EB virus transfection of human PBMC) irradiated with 0.120J/ cm2 ultraviolet light were added as feeder cells to stimulate the growth of CTL positive T cells. After 5-7 days of static culture, the growth of monoclonal cells can be observed under the microscope.
  • EBV-LCL cells lymphoblastoid cell line (LCL cell line) is an in vitro immortalized cell line obtained after EB virus transfection of human PBMC) irradiated with 0.120J/ cm2 ultraviolet light
  • the monoclonal cell clusters were picked out and transferred to U-bottom 96-well plates, and further expanded and cultured in RPMI 1640 medium containing 30ng/ml OKT3 and 3000IU/ml IL-2; the cell culture was replenished/replaced/the culture container was replaced every 3-4 days, and the irradiated EBV-LCL feeder cells were added again every 30-40 days to maintain its specificity.
  • a T cell monoclonal cell line can be obtained, which has a cell number of the order of 10 7 and a viability of 80% to 90%.
  • a certain amount of cells is regularly taken for intracellular factor secretion detection to determine the presence of killer CTL cells.
  • TCR sequence sequencing From the monoclonal cell culture, samples were taken for TCR sequence sequencing, and the TCR sequences were obtained as follows:
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of TCR are as follows:
  • CDR1 ⁇ one of VSGLRG (SEQ ID NO: 78), TSESDYY (SEQ ID NO: 90), NSASQS (SEQ ID NO: 101)
  • CDR2 ⁇ one of LYSAGEE (SEQ ID NO: 79), QEAYKQQN (SEQ ID NO: 91), VYSSGN (SEQ ID NO: 102)
  • CDR3 ⁇ One of CVITTSGTYKYIF (SEQ ID NO: 80), CAYRSFYTGANSKLTF (SEQ ID NO: 92), CVVHSGGSYIPTF (SEQ ID NO: 103)
  • CDR1 ⁇ MNHNS (SEQ ID NO: 81), SGHDT (SEQ ID NO: 93), MNHNY (SEQ ID NO: 104)
  • CDR 2 ⁇ One of SASEGT (SEQ ID NO: 82), YYEEEE (SEQ ID NO: 94), SVGAGI (SEQ ID NO: 105)
  • CDR 3 ⁇ one of CASTINTYEQYF (SEQ ID NO: 83), CASSLYGGPGDQPQHF (SEQ ID NO: 95), CASAQTIGAYNEQFF (SEQ ID NO: 106);
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • CDR3 ⁇ CAYRSFYTGANSKLTF (SEQ ID NO: 92)
  • CDR3 ⁇ CASSLYGGPGDQPQHF (SEQ ID NO: 95)
  • amino acid sequences of CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain of the TCR are as follows:
  • CDR3 ⁇ CVVHSGGSYIPTF (SEQ ID NO: 103)
  • CDR2 ⁇ SVGAGI (SEQ ID NO: 105)
  • CDR3 ⁇ CASAQTIGAYNEQFF (SEQ ID NO: 106).
  • the present invention further obtains the paired TCR alpha sequence and TCR beta sequence.
  • the specific method is not limited to obtaining by sequencing, bioinformatics, etc.
  • the paired TCR alpha sequence and TCR beta sequence obtained according to the ninth group of amino acid sequences in Table 5 are as follows:
  • the alpha sequence of TCR (amino acid sequence is SEQ ID NO:86) and the beta sequence of TCR (amino acid sequence is SEQ ID NO:87); wherein the TCR alpha sequence includes the variable region V ⁇ (amino acid sequence is SEQ ID NO:84) and the TCR beta sequence includes the variable region V ⁇ (amino acid sequence is SEQ ID NO:85); the variable region V ⁇ includes the following complementary determining regions: CDR1 ⁇ : VSGLRG; CDR2 ⁇ : LYSAGEE; CDR3 ⁇ : CVITTSGTYKYIF (SEQ ID NO:78-80), and the variable region V ⁇ includes the following complementary determining regions: CDR1 ⁇ : MNHNS; CDR2 ⁇ : SASEGT; CDR3 ⁇ : CASTINTYEQYF (SEQ ID NO:81-83).
  • TCR alpha sequence and TCR beta sequence can also be added before the above-mentioned TCR alpha sequence and TCR beta sequence, respectively, and after connecting them through the connecting sequence (SEQ ID NO: 11), the full-length sequence of TCR (SEQ ID NO: 88) can be obtained.
  • TCR alpha sequence and TCR beta sequence obtained according to the tenth group of amino acid sequences in Table 5 are as follows:
  • TCR alpha sequence (amino acid sequence is SEQ ID NO: 98), TCR beta sequence (amino acid sequence is SEQ ID NO: 99); wherein the variable region V ⁇ of the TCR alpha sequence includes the following complementary determining regions: CDR1 ⁇ : TSESDYY; CDR2 ⁇ : QEAYKQQN; CDR3 ⁇ : CAYRSFYTGANSKLTF (SEQ ID NO: 90-92); the variable region V ⁇ includes the following complementary determining regions: CDR1 ⁇ : SGHDT; CDR2 ⁇ : YYEEEE; CDR3 ⁇ : CASSLYGGPGDQPQHF (SEQ ID NO: 93-95);
  • TCR alpha sequence and TCR beta sequence are connected via a connecting sequence (SEQ ID NO: 11) to obtain the full-length sequence of TCR (SEQ ID NO: 100).
  • TCR alpha sequence and TCR beta sequence obtained according to the eleventh group of amino acid sequences in Table 5 are as follows:
  • TCR alpha sequence (amino acid sequence is SEQ ID NO: 109), TCR beta sequence (amino acid sequence is SEQ ID NO: 110); wherein the variable region V ⁇ of the TCR alpha sequence includes the following complementary determining regions: CDR1 ⁇ : NSASQS; CDR2 ⁇ : VYSSGN; CDR3 ⁇ : CVVHSGGSYIPTF (SEQ ID NO: 101-103); the variable region V ⁇ includes the following complementary determining regions: CDR1 ⁇ : MNHNY; CDR2 ⁇ : SVGAGI; CDR3 ⁇ : CASAQTIGAYNEQFF (SEQ ID NO: 104-106);
  • TCR alpha sequence and TCR beta sequence are connected via a connecting sequence (SEQ ID NO: 11) to obtain the full-length sequence of TCR (SEQ ID NO: 111).
  • Examples 8 and 9 both use the full-length sequence of TCR obtained based on the ninth group of amino acid sequences as an example.
  • the TCR gene sequence obtained in Example 7 was cloned into a lentiviral vector, and the virus packaging system 293T cells were transfected to prepare a virus solution, and the virus solution was transduced into T cells to obtain T cells expressing the target TCR sequence.
  • the transfection procedure is as follows:
  • Lentiviral supernatant was produced by transfecting 293T cells with gag/pol packaging plasmid, VSV-G envelope plasmid and transfer construct containing lentiviral vector sequences as described below. That is, the DNA mixture was mixed in Opti-MEM (Life Technologies, Gaithersburg, MD, USA) and mixed with an equal volume of Opti-MEM containing Lipofectamine 3000 (Life Technologies). After incubation at room temperature for 15 minutes, the resulting mixture was applied to 293T cells. The medium containing lentivirus was collected 24 hours after transfection. Each collection After that, the supernatant was filtered through a 0.45 ⁇ m filter.
  • the lentiviral harvests were combined and stored at 4°C, and then ultracentrifuged at 20,000 x g for 90 minutes.
  • the lentiviral particles were resuspended in PBS.
  • the structure of the transfer plasmid is shown in Figure 2.
  • the transduction procedure is as follows:
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • PBMCs peripheral blood cells
  • X-VIVO15 serum-free medium LONZA
  • T cells were transduced with lentiviral supernatant, followed by centrifugation at 1200 ⁇ g and 32°C for 2 hours. After 24 hours, the supernatant containing the viral vector was removed.
  • the cells were suspended in medium containing rhIL-2 (200 IU/mL) at 3 ⁇ 10 5 cells/ml, and X-VIVO15 serum-free medium supplemented with IL-2 (200 IU/mL) was supplemented every 2 to 3 days.
  • the TCR expression level i.e., the transduction positive rate, was detected by flow cytometry 1 day before harvest. The results are shown in Figure 5.
  • Non-transduced cells (NC) do not express specific TCRs.
  • T cells transduced with the lentiviral vectors containing the ninth group of TCR, the tenth group of TCR, and the eleventh group of TCR in Example 7 respectively expressed specific TCR (CD8 + Tetramer + data), that is, the transduction positive rate: the ninth group of TCR transduction positive rate was 59.09%, the tenth group of TCR transduction positive rate was 14.38%, and the eleventh group of TCR transduction positive rate was 91.67%, indicating that the transduction was successful.
  • the present invention can be used to evaluate the function of TCR-T cells in vitro by constructing target cells that overexpress the CMV-PP65 protein to which ATVQGQNLK belongs.
  • the specific operation is as follows:
  • T cells expressing the target TCR sequence prepared in Example 8 are used as effector cells, among which the TCR group is transduced with TCR lentivirus and expressed, and the NC group is not transduced;
  • the target cells in the present invention are tumor cells that overexpress the CMV-PP65 protein to which ATVQGQNLK belongs, and are constructed by introducing the target antigen gene into the tumor cells:
  • Tumor cells were transduced with lentivirus containing the target antigen. After 1 day, the medium was changed to complete medium for 1-2 days, and then replaced with complete medium containing puromycin for continued culture. The expression of the target antigen in the tumor cells of the transduction group was detected.
  • the specific steps include:
  • the density of tumor cells was adjusted to 4-6 ⁇ 10 5 cells/ml using complete medium (the complete medium used for Caki-2 renal clear cell carcinoma cells was: McCoy's 5A medium (Gibco) containing 10% FBS), 1 ml/well was added to a 6-well plate, 8 ⁇ l Polybrene (1 mg/ml) was added to each well to a final concentration of 8 ⁇ g/ml, 10-30 ⁇ l of lentiviral vector containing the target antigen ATVQGQNLK was added to each well, and a cell control well (non-transduced group) was set up, and the control well was added with the above 1 ml tumor cells and 8 ⁇ l Polybrene, mixed well, and incubated in a CO 2 incubator (37°C, 5% CO 2 ) for 1 day;
  • a CO 2 incubator 37°C, 5% CO 2
  • the cells were observed every 2 days and the complete medium containing 1 ⁇ g/ml puromycin was replaced once. The adherent cells were passaged when they were confluent;
  • Transduced and screened tumor cells that were transduced with the target antigen and cultured for more than 7 days were plated according to the limiting dilution method, 1 cell/well, 3 cells/well, cultured with complete medium containing puromycin and the cells in the cell wells were observed. Monoclonal wells were marked and cultured continuously until the cell number reached at least 6 ⁇ 10 7 cells and then frozen to obtain overexpressed target cells.
  • Caki-2-PP65 cells overexpressing PP65 protein prepared in step 2 were plated as target cells at 5 ⁇ 10 4 per well.
  • the effector cells of the TCR group and the effector cells of the NC group in step 1 were mixed with the target cells at the effector-target ratio of 1:1, 5:1, and 10:1, respectively, and incubated at 37°C.
  • the real-time label-free cell function analyzer Real Time Cell Analyzer, RTCA, Agilent
  • Real-time label-free dynamic cell analysis technology is to integrate the microelectronic cell sensor chip into the bottom of the cell detection plate through a special process to build a cell impedance detection sensor system that can track changes in cell morphology, proliferation and differentiation in real time, dynamically and quantitatively.

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Abstract

本发明涉及T细胞抗原受体技术领域，具体涉及能识别并结合CMVpp65抗原复合物的TCR，以及包含编码所述TCR的核苷酸序列的核酸，以及含有所述核酸分子的载体，以及转导所述核酸分子或所述载体的细胞，以及包含所述TCR、核酸分子、载体或细胞作为活性成分的药物组合物，以及所述TCR、核酸分子、载体、细胞、药物组合物分别用于制备治疗肿瘤或病毒感染的药物的用途。

Description

T细胞抗原受体及其制备方法和应用 技术领域
本公开属于生物医药领域，具体涉及一种T细胞抗原受体及其制备方法和应用。
背景技术
T细胞受体(T cell receptor,TCR)是T淋巴细胞特异性识别抗原、启动免疫应答的分子，为免疫球蛋白超家族的异二聚体细胞表面蛋白，其与参与调节信号转导的CD3复合物的无变异蛋白相关联。TCR是呈递在主要组织相容性复合体(MHC)上的特异性抗原肽的唯一受体，对免疫系统的细胞免疫功能至关重要，抗原特异性TCR与MHC复合物结合引发T细胞与抗原呈递细胞直接的物理接触，相互作用，导致一系列后续细胞信号传递和其他生理反应，从而使得不同抗原特异性的T细胞对其靶细胞发挥免疫效应。
巨细胞病毒(Cytomegalovirus，CMV)是一种疱疹病毒组DNA病毒，由于被感染的细胞会发生肿大，因此被命名为巨细胞病毒。CMV是人类最常见病原体之一，在人群中广泛流行，其在成人群体中的感染率可以高达50％-100％。在免疫系统正常的个体中，CMV的感染后通常不会出现任何临床症状，但是在无症状原发感染后，CMV病毒常常不能被完全清除，而在患者体内形成持续性地潜伏感染。如果携带CMV的个体免疫系统的功能被抑制，如艾滋病发病、器官移植后接受免疫抑制治疗，潜伏的CMV感染再度活化可导致严重甚至致死性疾病。此外CMV的感染还可能与部分肿瘤的发病和进展有关。例如，一半的胶质细胞瘤组织中，可以检测到CMV-pp65抗原，因此，预防和治疗与CMV感染相关的疾病也成为药物开发的方向之一。
目前，开发了利用CMV特异性TCR制备TCR-T细胞针对CMV感染的方案，将TCR-T细胞回输患者后，可让CMV再活化的患者获得有效的即时抗病毒能力，清除本次再活的病毒。而接受CMV特异性TCR-T回输的患者还能获得持久的抗病毒能力，能显著降低随后再活化的机率。初步的临床研究也显示CMV-pp65疫苗可延长脑胶质瘤患者的无进展生存期及总体生存率。因此，靶向CMV-pp65的TCR也有可能应用于治疗CMV相关的恶性肿瘤。
CN113881680A和CN102656188A均公开了能够识别巨细胞病毒的抗原的TCR，但其筛选方式没有特别关注TCR识别的异质性，对靶细胞的杀伤作用不明显。CN106279404A公开了一种可溶且稳定的异质二聚TCR，其在α链可变区与β链恒定区之间含有人工链间二硫键的异质二聚TCR，该TCR可溶且稳定，能够被很好地复性、重折叠、纯化同时能够与原配体特异性结合。但该专利未公开针对巨细胞病毒的特异性TCR。
发明内容
TCR-T疗法能够有效的控制急性CMV疾病，引起抗病毒免疫应答；但如何从病人体内或者体外刺激的细胞毒T淋巴细胞中获得有效的T细胞，快速的获得其TCR序列，从而构建有功能的TCR转导的特异性T细胞，是TCR-T应用的阻碍。本公开在单细胞测序技术的基础上，通过高通量测序实现TCR配对序列的测定，并通过免疫细胞对重要基因表达水平进行评估，进行特异性T细胞受体研究。
具体的，本公开的一种实现方式中，基于CMV pp65短肽抗原体外诱导扩增抗 原特异性的细胞毒T淋巴细胞，通过单细胞测序、MHC四聚体分选后阳性的T细胞的TCR配对信息，按照克隆序列频率筛选TCR，构建TCR-T，并进行体外的功能验证，评估TCR-T的功能，最终研发获得本公开的特异性T细胞受体。
本公开所述HLA-A24-CMV-pp65341-349抗原复合物由人白细胞表面抗原HLA-A24及CMV-pp65341-349短肽(天然序列：QYDPVAALF(SEQ ID NO：13))组成，表达于目标靶细胞表面。
为实现上述目的，本公开的第一方面，提供一种识别人巨细胞病毒pp65抗原的TCR，所述TCR具有结合HLA-A24-CMV-pp65341-349抗原复合物的特性，所述TCR至少包含一个α链可变区和/或β链可变区。优选的，所述的T细胞抗原受体是αβ异二聚体，且各自包含一个TCRα链可变区和一个TCRβ链可变区。
在其中一个实施方式中，所述TCRα链可变区包含CDR1α-CDR3α三个互补决定区(CDR)，其中CDR3α包含SEQ ID NO：3、SEQ ID NO：22、SEQ ID NO：31之一所示的序列，或包含与SEQ ID NO：3、SEQ ID NO：22、SEQ ID NO：31之一具有至少85％、90％、95％、98％或至少99％以上同源性的氨基酸序列。优选地，所述TCRα链可变区的CDR3的氨基酸序列如SEQ ID NO：3、SEQ ID NO：22、SEQ ID NO：31之一所示。
在其中一个实施方式中，TCRβ链可变区包含CDR1β-CDR3β三个CDR区，其中CDR3β包含SEQ ID NO：6、SEQ ID NO：16、SEQ ID NO：25所示的序列，或包含与SEQ ID NO：6、SEQ ID NO：16、SEQ ID NO：25之一具有至少85％、90％、95％、98％或至少99％以上同源性的氨基酸序列；优选地，所述TCRβ链可变区的CDR3β的氨基酸序列如SEQ ID NO：6、SEQ ID NO：16、SEQ ID NO：25之一所示。
在其中一个实施方式中，所述TCRα链可变区的CDR1α包含SEQ ID NO：1所示的氨基酸序列，或包含与SEQ ID NO：1具有至少85％、90％、95％、98％或至少99％以上同源性的氨基酸序列；CDR2α包含SEQ ID NO：2所示的氨基酸序列，或包含与SEQ ID NO：2具有至少85％、90％、95％、98％或至少99％以上同源性的氨基酸序列；所述TCRβ链可变区的CDR1β包含SEQ ID NO：4、SEQ ID NO：14、SEQ ID NO：23之一所示的氨基酸序列，或包含与SEQ ID NO：4、SEQ ID NO：14、SEQ ID NO：23具有至少85％、90％、95％、98％或至少99％以上同源性的氨基酸序列；CDR2β包含SEQ ID NO：5、SEQ ID NO：15、SEQ ID NO：24之一所示的氨基酸序列，或包含与SEQ ID NO：5、SEQ ID NO：15、SEQ ID NO：24之一具有至少85％、90％、95％、98％或至少99％同源性的氨基酸序列。
在优选地实施方式中，所述TCR的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)；
CDR2α：MTLNGDE(SEQ ID NO：2)；
CDR3α：CARNTGKLIF(SEQ ID NO：3)、CAPSASKIIF(SEQ ID NO：22)、CAPQFNKFYF(SEQ ID NO：31)之一；
CDR1β：SQVTM(SEQ ID NO：4)、SGHVS(SEQ ID NO：14)、LNHDA(SEQ ID NO：23)之一；
CDR 2β：ANQGSEA(SEQ ID NO：5)、FQNEAQ(SEQ ID NO：15)、SQIVND(SEQ ID NO：24)之一；
CDR 3β：CSANPTGGGTEAFF(SEQ ID NO：6)、CASSLLTRTETQYF(SEQ ID NO：16)、CASSTTGLAGGPGNEQFF(SEQ ID NO：25)之一；
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)
CDR2α：MTLNGDE(SEQ ID NO：2)
CDR3α：CARNTGKLIF(SEQ ID NO：3)
CDR1β：SQVTM(SEQ ID NO：4)
CDR 2β：ANQGSEA(SEQ ID NO：5)
CDR 3β：CSANPTGGGTEAFF(SEQ ID NO：6)；
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)
CDR2α：MTLNGDE(SEQ ID NO：2)
CDR3α：CARNTGKLIF(SEQ ID NO：3)
CDR1β：SGHVS(SEQ ID NO：14)
CDR2β：FQNEAQ(SEQ ID NO：15)
CDR3β：CASSLLTRTETQYF(SEQ ID NO：16)；
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)
CDR2α：MTLNGDE(SEQ ID NO：2)
CDR3α：CAPSASKIIF(SEQ ID NO：22)
CDR1β：LNHDA(SEQ ID NO：23)
CDR2β：SQIVND(SEQ ID NO：24)
CDR3β：CASSTTGLAGGPGNEQFF(SEQ ID NO：25)；
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)
CDR2α：MTLNGDE(SEQ ID NO：2)
CDR3α：CAPQFNKFYF(SEQ ID NO：31)
CDR1β：SGHVS(SEQ ID NO：14)
CDR2β：FQNEAQ(SEQ ID NO：15)
CDR3β：CASSLLTRTETQYF(SEQ ID NO：16)。
在其中一个实施方式中，本公开的TCR的α链的可变区包含SEQ ID NO：7、SEQ ID NO：17、SEQ ID NO：26、SEQ ID NO：32的氨基酸序列或包含与SEQ ID NO：7、SEQ ID NO：17、SEQ ID NO：26、SEQ ID NO：32至少80％、或至少85％、或至少90％、92％、94％、96％、98％或99％以上同源性的氨基酸序列。
优选地，本公开的TCR的α链包含SEQ ID NO：9、SEQ ID NO：19、SEQ ID NO：28、SEQ ID NO：34的氨基酸序列或包含与SEQ ID NO：9、SEQ ID NO：19、SEQ ID NO：28、SEQ ID NO：34至少80％、或至少85％、或至少90％、92％、94％、96％、98％或99％以上同源性的氨基酸序列。
在其中一个实施方式中，本公开的TCR的β链的可变区包含SEQ ID NO：8、SEQ ID NO：18、SEQ ID NO：27、SEQ ID NO：33的氨基酸序列或包含与SEQ ID NO：8、SEQ ID NO：18、SEQ ID NO：27、SEQ ID NO：33至少80％、或至少85％、或至少90％、92％、94％、96％、98％或99％以上同源性的氨基酸序列。
优选地，本公开的TCR的β链包含SEQ ID NO：10、SEQ ID NO：20、SEQ ID NO：29、SEQ ID NO：35的氨基酸序列或包含与SEQ ID NO：10、SEQ ID NO：20、SEQ ID NO：29、SEQ ID NO：35至少80％、或至少85％、或至少90％、92％、94％、96％、98％或99％以上同源性的氨基酸序列。
在本公开的一个具体实施方式中，优选的，本公开的TCR的α链与β链的氨基酸直接连接或间接连接，优选为间接连接，更优选使用2A肽连接，2A肽是来源于病毒的短肽(18-25个氨基酸)，它们通常被称为“自我剪切”肽，能使一条转录产物产生多种蛋白。本公开使用的2A肽包括但不限于P2A、T2A、E2A、F2A。
在本公开的一些实施方式中，2A肽，如P2A还可以与furin切割位点、ser-gly连接子(sgsg)和p2a核糖体跳肽连接，形成fp2A(furin-SGSG-p2A)连接结构。
进一步优选的，所述fp2A包含SEQ ID NO：11所示的氨基酸序列。
在本公开的一个具体实施方式中，所述的α链与β链的连接顺序，可以为α链、fp2A与β链，或者，β链、fp2A与α链。
在本公开的一个具体实施方式中，本公开的TCR包含SEQ ID NO：12、SEQ ID NO：21、SEQ ID NO：30、SEQ ID NO：36所示的氨基酸序列或包含与SEQ ID NO：12、SEQ ID NO：21、SEQ ID NO：30、SEQ ID NO：36至少80％、或至少85％、或至少90％、92％、94％、96％、98％或99％以上同源性的氨基酸序列。
本公开所述CMV pp65495-503短肽(天然序列：NLVPMVATV(SEQ ID NO:46))表达于目标靶细胞表面。
为实现上述目的，本公开提供一种识别人巨细胞病毒pp65抗原的TCR，所述TCR具有结合CMV pp65495-503抗原复合物的特性，所述TCR至少包含一个α链可变区和/或β链可变区。优选的，所述的T细胞抗原受体是αβ异二聚体，且各自包含一个TCRα链可变区和一个TCRβ链可变区。
在其中一个实施方式中，所述TCRα链可变区包含CDR1α-CDR3α三个互补决定区(CDR)，其中CDR3α包含SEQ ID NO：37、SEQ ID NO：49、SEQ ID NO：60、SEQ ID NO：69之一所示的序列，或包含与SEQ ID NO：37、SEQ ID NO：49、SEQ ID NO：60、SEQ ID NO：69之一具有至少85％、90％、95％、98％或至少99％以上同源性的氨基酸序列；优选地，所述TCRα链可变区的CDR3的氨基酸序列如SEQ ID NO：37、SEQ ID NO：49、SEQ ID NO：60、SEQ ID NO：69之一所示。
在其中一个实施方式中，TCRβ链可变区包含CDR1β-CDR3β三个CDR区，其中CDR3包含SEQ ID NO：40、SEQ ID NO：52、SEQ ID NO：63、SEQ ID NO：72所示的序列，或包含与SEQ ID NO：40、SEQ ID NO：52、SEQ ID NO：63、SEQ ID NO：72之一具有至少85％、90％、95％、98％或至少99％以上同源性的氨基酸序列；优选地，所述TCRβ链可变区的CDR3的氨基酸序列如SEQ ID NO：40、SEQ ID NO：52、SEQ ID NO：63、SEQ ID NO：72之一所示。
在其中一个实施方式中，所述TCRα链可变区的CDR1α包含SEQ ID NO：1、SEQ ID NO：47、SEQ ID NO：58之一所示的氨基酸序列，或包含与SEQ ID NO：1、SEQ ID NO：47、SEQ ID NO：58之一具有至少85％、90％、95％、98％或至少 99％以上同源性的氨基酸序列；CDR2α包含SEQ ID NO：2、SEQ ID NO：48、SEQ ID NO：59之一所示的氨基酸序列，或包含与SEQ ID NO：2、SEQ ID NO：48、SEQ ID NO：59之一具有至少85％、90％、95％、98％或至少99％以上同源性的氨基酸序列；
所述TCRβ链可变区的CDR1β包含SEQ ID NO：38、SEQ ID NO：50、SEQ ID NO：61、SEQ ID NO：70之一所示的氨基酸序列，或包含与SEQ ID NO：38、SEQ ID NO：50、SEQ ID NO：61、SEQ ID NO：70之一具有至少85％、90％、95％、98％或至少99％以上同源性的氨基酸序列；CDR2β包含SEQ ID NO：39、SEQ ID NO：51、SEQ ID NO：62、SEQ ID NO：71之一所示的氨基酸序列，或包含与SEQ ID NO：39、SEQ ID NO：51、SEQ ID NO：62、SEQ ID NO：71之一具有至少85％、90％、95％、98％或至少99％同源性的氨基酸序列。
在优选地实施方式中，所述TCR的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)、DSSSTY(SEQ ID NO：47)、TSGFNG(SEQ ID NO：58)之一
CDR2α：MTLNGDE(SEQ ID NO：2)、IFSNMDM(SEQ ID NO：48)、NVLDGL(SEQ ID NO：59)之一
CDR3α：CASINFNKFYF(SEQ ID NO：37)、CAEFTGTASKLTF(SEQ ID NO：49)、CAVTYNNARLMF(SEQ ID NO：60)、CARNYGQNFVF(SEQ ID NO：69)之一
CDR1β：MDHEN(SEQ ID NO：38)、GTSNPN(SEQ ID NO：50)、MNHEY(SEQ ID NO：61)、DFQATT(SEQ ID NO：70)之一
CDR 2β：SYDVKM(SEQ ID NO：39)、SVGIG(SEQ ID NO：51)、SMNVEV(SEQ ID NO：62)、SNEGSKA(SEQ ID NO：71)之一
CDR 3β：CASSPLNGGATEAFF(SEQ ID NO：40)、CAWSDRAAFTDTQYF(SEQ ID NO：52)、CASSSVAGGRIEQFF(SEQ ID NO：63)、CSARDIKAQQWNIQYF(SEQ ID NO：72)之一。
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)
CDR2α：MTLNGDE(SEQ ID NO：2)
CDR3α：CASINFNKFYF(SEQ ID NO：37)
CDR1β：MDHEN(SEQ ID NO：38)
CDR 2β：SYDVKM(SEQ ID NO：39)
CDR 3β：CASSPLNGGATEAFF(SEQ ID NO：40)
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：DSSSTY(SEQ ID NO：47)
CDR2α：IFSNMDM(SEQ ID NO：48)
CDR3α：CAEFTGTASKLTF(SEQ ID NO：49)
CDR1β：GTSNPN(SEQ ID NO：50)
CDR2β：SVGIG(SEQ ID NO：51)
CDR3β：CAWSDRAAFTDTQYF(SEQ ID NO：52)
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：TSGFNG(SEQ ID NO：58)
CDR2α：NVLDGL(SEQ ID NO：59)
CDR3α：CAVTYNNARLMF(SEQ ID NO：60)
CDR1β：MNHEY(SEQ ID NO：61)
CDR2β：SMNVEV(SEQ ID NO：62)
CDR3β：CASSSVAGGRIEQFF(SEQ ID NO：63)
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)
CDR2α：MTLNGDE(SEQ ID NO：2)
CDR3α：CARNYGQNFVF(SEQ ID NO：69)
CDR1β：DFQATT(SEQ ID NO：70)
CDR2β：SNEGSKA(SEQ ID NO：71)
CDR3β：CSARDIKAQQWNIQYF(SEQ ID NO：72)
在其中一个实施方式中，本公开的TCR的α链的可变区包含SEQ ID NO：41、SEQ ID NO：53、SEQ ID NO：64、SEQ ID NO：73的氨基酸序列或包含与SEQ ID NO：41、SEQ ID NO：53、SEQ ID NO：64、SEQ ID NO：73至少80％、或至少85％、或至少90％、92％、94％、96％、98％或99％以上同源性的氨基酸序列。
优选地，本公开的TCR的α链包含SEQ ID NO：43、SEQ ID NO：55、SEQ ID NO：66、SEQ ID NO：75的氨基酸序列或包含与SEQ ID NO：43、SEQ ID NO：55、SEQ ID NO：66、SEQ ID NO：75至少80％、或至少85％、或至少90％、92％、94％、96％、98％或99％以上同源性的氨基酸序列。
在其中一个实施方式中，本公开的TCR的β链的可变区包含SEQ ID NO：42、SEQ ID NO：54、SEQ ID NO：65、SEQ ID NO：74的氨基酸序列或包含与SEQ ID NO：42、SEQ ID NO：54、SEQ ID NO：65、SEQ ID NO：74至少80％、或至少85％、或至少90％、92％、94％、96％、98％或99％以上同源性的氨基酸序列。
优选地，本公开的TCR的β链包含SEQ ID NO：44、SEQ ID NO：56、SEQ ID NO：67、SEQ ID NO：76的氨基酸序列或包含与SEQ ID NO：44、SEQ ID NO：56、SEQ ID NO：67、SEQ ID NO：76至少80％、或至少85％、或至少90％、92％、94％、96％、98％或99％以上同源性的氨基酸序列。
在本公开的一个具体实施方式中，优选的，本公开的TCR的α链与β链的氨基酸直接连接或间接连接，优选为间接连接，更优选使用2A肽连接，2A肽是来源于病毒的短肽(18-25个氨基酸)，它们通常被称为“自我剪切”肽，能使一条转录产物产生多种蛋白。本公开使用的2A肽包括但不限于P2A、T2A、E2A、F2A。
在本公开的一些实施方式中，2A肽，如P2A还可以与furin切割位点、ser-gly连接子(sgsg)和p2a核糖体跳肽连接，形成fp2A(furin-SGSG-p2A)连接结构。
进一步优选的，所述fp2A包含SEQ ID NO：11所示的氨基酸序列。
在本公开的一个具体实施方式中，所述的α链与β链的连接顺序，可以为α链、fp2A与β链，或者，β链、fp2A与α链。
在本公开的一个具体实施方式中，本公开的TCR包含SEQ ID NO：45、SEQ ID NO：57、SEQ ID NO：68、SEQ ID NO：77所示的氨基酸序列或包含与 SEQ ID NO：45、SEQ ID NO：57、SEQ ID NO：68、SEQ ID NO：77至少80％、或至少85％、或至少90％、92％、94％、96％、98％或99％以上同源性的氨基酸序列。
本公开所述CMV pp65501-509短肽(ATVQGQNLK(SEQ ID NO:89))表达于目标靶细胞表面。
为实现上述目的，本公开提供一种识别人巨细胞病毒pp65抗原的TCR，所述TCR具有结合CMV pp65501-509抗原复合物的特性，所述TCR至少包含一个α链可变区和/或β链可变区。优选的，所述的T细胞抗原受体是αβ异二聚体，且各自包含一个TCRα链可变区和一个TCRβ链可变区。
在其中一个实施方式中，所述TCRα链可变区包含CDR1α-CDR3α三个互补决定区(CDR)，其中CDR3α包含CVITTSGTYKYIF(SEQ ID NO：80)、CAYRSFYTGANSKLTF(SEQ ID NO：92)、CVVHSGGSYIPTF(SEQ ID NO：103)之一所示的序列，或包含与SEQ ID NO：80、SEQ ID NO：92、SEQ ID NO：103之一具有至少85％、90％、95％、98％或至少99％以上同源性的氨基酸序列。
在其中一个实施方式中，TCRβ链可变区包含CDR1β-CDR3β三个CDR区，其中CDR3β包含CASTINTYEQYF(SEQ ID NO：83)、CASSLYGGPGDQPQHF(SEQ ID NO：95)、CASAQTIGAYNEQFF(SEQ ID NO：106)之一所示的序列，或包含与SEQ ID NO：83、SEQ ID NO：95、SEQ ID NO：106之一具有至少85％、90％、95％、98％或至少99％以上同源性的氨基酸序列。
在其中一个实施方式中，所述TCRα链可变区的CDR1α包含SEQ ID NO：78、SEQ ID NO：90、SEQ ID NO：101之一所示的氨基酸序列，或包含与SEQ ID NO：78、SEQ ID NO：90、SEQ ID NO：101之一具有至少85％、90％、95％、98％或至少99％以上同源性的氨基酸序列；CDR2α包含SEQ ID NO：79、SEQ ID NO：91、SEQ ID NO：102之一所示的氨基酸序列，或包含与SEQ ID NO：79、SEQ ID NO：91、SEQ ID NO：102之一具有至少85％、90％、95％、98％或至少99％以上同源性的氨基酸序列；
所述TCRβ链可变区的CDR1β包含SEQ ID NO：81、SEQ ID NO：93、SEQ ID NO：104之一所示的氨基酸序列，或包含与SEQ ID NO：81、SEQ ID NO：93、SEQ ID NO：104之一具有至少85％、90％、95％、98％或至少99％以上同源性的氨基酸序列；CDR2β包含SEQ ID NO：82、SEQ ID NO：94、SEQ ID NO：105之一所示的氨基酸序列，或包含与SEQ ID NO：82、SEQ ID NO：94、SEQ ID NO：105之一具有至少85％、90％、95％、98％或至少99％同源性的氨基酸序列。
CDR1α：VSGLRG(SEQ ID NO：78)、TSESDYY(SEQ ID NO：90)、NSASQS(SEQ ID NO：101)之一
CDR2α：LYSAGEE(SEQ ID NO：79)、QEAYKQQN(SEQ ID NO：91)、VYSSGN(SEQ ID NO：102)之一
CDR3α：CVITTSGTYKYIF(SEQ ID NO：80)、CAYRSFYTGANSKLTF(SEQ ID NO：92)、CVVHSGGSYIPTF(SEQ ID NO：103)之一
CDR1β：MNHNS(SEQ ID NO：81)、SGHDT(SEQ ID NO：93)、MNHNY(SEQ ID NO：104)之一
CDR 2β：SASEGT(SEQ ID NO：82)、YYEEEE(SEQ ID NO：94)、SVGAGI(SEQ ID NO：105)之一
CDR 3β：CASTINTYEQYF(SEQ ID NO：83)、CASSLYGGPGDQPQHF(SEQ ID NO：95)、CASAQTIGAYNEQFF(SEQ ID NO：106)之一；
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：VSGLRG(SEQ ID NO：78)
CDR2α：LYSAGEE(SEQ ID NO：79)
CDR3α：CVITTSGTYKYIF(SEQ ID NO：80)
CDR1β：MNHNS(SEQ ID NO：81)
CDR 2β：SASEGT(SEQ ID NO：82)
CDR 3β：CASTINTYEQYF(SEQ ID NO：83)
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：TSESDYY(SEQ ID NO：90)
CDR2α：QEAYKQQN(SEQ ID NO：91)
CDR3α：CAYRSFYTGANSKLTF(SEQ ID NO：92)
CDR1β：SGHDT(SEQ ID NO：93)
CDR2β：YYEEEE(SEQ ID NO：94)
CDR3β：CASSLYGGPGDQPQHF(SEQ ID NO：95)
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：NSASQS(SEQ ID NO：101)
CDR2α：VYSSGN(SEQ ID NO：102)
CDR3α：CVVHSGGSYIPTF(SEQ ID NO：103)
CDR1β：MNHNY(SEQ ID NO：104)
CDR2β：SVGAGI(SEQ ID NO：105)
CDR3β：CASAQTIGAYNEQFF(SEQ ID NO：106)。
在其中一个实施方式中，本公开的TCR的α链的可变区包含SEQ ID NO：84、SEQ ID NO：96、SEQ ID NO：107之一的氨基酸序列或包含与SEQ ID NO：84、SEQ ID NO：96、SEQ ID NO：107之一至少80％、或至少85％、或至少90％、92％、94％、96％、98％或99％以上同源性的氨基酸序列。
优选地，本公开的TCR的α链包含SEQ ID NO：86、SEQ ID NO：98、SEQ ID NO：109之一的氨基酸序列或包含与SEQ ID NO：86、SEQ ID NO：98、SEQ ID NO：109之一至少80％、或至少85％、或至少90％、92％、94％、96％、98％或99％以上同源性的氨基酸序列。
在其中一个实施方式中，本公开的TCR的β链的可变区包含SEQ ID NO：85、SEQ ID NO：97、SEQ ID NO：108之一的氨基酸序列或包含与SEQ ID NO：85、SEQ ID NO：97、SEQ ID NO：108之一至少80％、或至少85％、或至少90％、92％、94％、96％、98％或99％以上同源性的氨基酸序列。
优选地，本公开的TCR的β链包含SEQ ID NO：87、SEQ ID NO：99、SEQ ID NO：110之一的氨基酸序列或包含与SEQ ID NO：87、SEQ ID NO：99、SEQ ID NO：110之一至少80％、或至少85％、或至少90％、92％、94％、96％、98％或99％以上同源性的氨基酸序列。
在本公开的一个具体实施方式中，优选的，本公开的TCR的α链与β链的氨基 酸直接连接或间接连接，优选为间接连接，更优选使用2A肽连接，2A肽是来源于病毒的短肽(18-25个氨基酸)，它们通常被称为“自我剪切”肽，能使一条转录产物产生多种蛋白。本公开使用的2A肽包括但不限于P2A、T2A、E2A、F2A。
在本公开的一些实施方式中，2A肽，如P2A还可以与furin切割位点、ser-gly连接子(sgsg)和p2a核糖体跳肽连接，形成fp2A(furin-SGSG-p2A)连接结构。
进一步优选的，所述fp2A包含SEQ ID NO：11所示的氨基酸序列。
在本公开的一个具体实施方式中，所述的α链与β链的连接顺序，可以为α链、fp2A与β链，或者，β链、fp2A与α链。
在本公开的一个具体实施方式中，本公开的TCR包含SEQ ID NO：88、SEQ ID NO：100、SEQ ID NO：111之一所示的氨基酸序列或包含与SEQ ID NO：88、SEQ ID NO：100、SEQ ID NO：111至少80％、或至少85％、或至少90％、92％、94％、96％、98％或99％以上同源性的氨基酸序列。
本公开的第二方面提供一种核酸，所述核酸包含编码本公开第一方面所述TCR的核苷酸序列或其互补序列。
本公开的技术方案中，所述的核酸序列可以是单链或双链，可以是DNA也可以是RNA。
本公开优选的技术方案中，所述的核酸序列可以是经过密码子优化的。进一步优选的，所述的密码子优化包括将病毒等使用的大量稀有密码子变为对应的哺乳动物密码子和/或移除mRNA不稳定基序和/或隐藏的剪接位点。
本公开的第三方面提供了一种表达载体，所述的表达载体包含本公开任一所述的核酸。
本公开优选的技术方案中，所述的表达载体能够在体内或体外或离体条件下表达。进一步优选的，所述的表达载体在体内细胞中持续高水平表达。
本公开优选的技术方案中，所述的表达载体可以是原核表达载体或逆转录病毒载体。
本公开优选的技术方案中，所述的表达载体可以是劳氏肉瘤病毒(RSV)、慢病毒、人免疫缺陷病毒(HIV)、鼠科白血病病毒(MLV)、马传染性贫血病毒(EIAV)、小鼠乳腺癌病毒(MMTV)、Fujinami肉瘤病毒(FuSV)、FBR鼠骨肉瘤病毒(FBR MSV)、莫洛尼氏鼠白血病病毒(Mo‐MLV)、莫洛尼氏鼠肉瘤病毒(Mo‐MSV)、Abelson鼠白血病病毒(A‐MLV)、禽髓细胞增生病毒29(MC29)或禽骨髓成红细胞增多症病毒(AEV)等等。更进一步优选的，所述的表达载体是慢病毒表达载体。
本公开的第四方面提供了一种宿主细胞，所述的宿主细胞包含本公开任一所述的核酸或表达载体。
本公开优选的技术方案中，所述的宿主细胞可以是真核的或原核的。更优选的，所述的宿主细胞为真核细胞，包括但不限于酵母细胞、293细胞、CHO细胞等。
本公开的第五方面提供了一种免疫细胞，所述的免疫细胞表达本公开所述的T细胞抗原受体。
本公开优选的技术方案中，所述的免疫细胞包含一个或多个本公开任一所述的核酸序列。
本公开优选的技术方案中，所述的免疫细胞包括但不限于干细胞、淋巴细胞(包括T细胞、B细胞)。进一步，所述免疫细胞是B细胞，所述B细胞表达上述的抗体或其抗原结合片段。所述免疫细胞是T细胞，所述T细胞的T细胞抗原受体结构如上所 限定。
本公开优选的技术方案中，所述的T细胞可以是CD4+T、CD8+T等。
本公开优选的技术方案中，所述的免疫细胞分离自受试者的T细胞。
本公开优选的技术方案中，所述的免疫细胞是来自CMV血清阴性供体的T细胞。
本公开的第六方面提供一种免疫细胞的制备方法，包括将编码上述T细胞抗原受体的核酸序列转导至免疫细胞中表达获得。
本公开优选的技术方案中，所述的免疫细胞包括但不限于干细胞、淋巴细胞(包括T细胞、B细胞)。进一步，所述免疫细胞是B细胞，所述B细胞表达上述的抗体或其抗原结合片段。所述免疫细胞是T细胞，所述T细胞的T细胞抗原受体结构如上所限定。
本公开优选的技术方案中，还包括敲除细胞内源性TCR的步骤。具体的，可以为将靶向内源TCR的guide构建至慢病毒载体，与包装质粒、转染试剂共转至T细胞。
本公开第七方面提供了一种重组T细胞的制备方法，包括如下步骤：
1)从阳性T细胞克隆得到本公开第二方面任一所述的核酸；
2)分离、培养原代T细胞；
3)将步骤1)得到的核酸递送至步骤2)所述的原代T细胞中，获得表达本公开任一所述T细胞抗原受体的重组T细胞。
本公开优选的技术方案中，所述的T细胞选自造血干细胞或外周血淋巴细胞(PBL)源T细胞。
本公开第八方面提供了一种T细胞抗原受体的制备方法，包括如下步骤：
(1)从阳性T细胞克隆得到本公开第二方面任一所述的核酸；
(2)将步骤(1)得到的核酸连接至载体骨架，获得表达载体；
(3)将步骤(2)获得的表达载体转化至宿主细胞，然后诱导其表达；
(4)获得抗体或其抗原结合片段或者T细胞抗原受体。
本公开优选的技术方案中，所述的阳性T细胞为与MHC呈递的巨细胞病毒(CMV)磷蛋白pp65抗原肽特异性结合。进一步优选的，所述的MHC呈递的巨细胞病毒(CMV)磷蛋白pp65抗原肽复合物为单体或多聚体复合物。
本公开第九方面提供了第一方面任一所述的T细胞抗原受体、第二方面任一所述的核酸、第三方面所述的表达载体、第四方面所述的宿主细胞、第五方面所述的免疫细胞在制备诊断或治疗肿瘤或与CMV相关疾病的产品中的应用。
本公开优选的技术方案中，所述的CMV相关疾病选自新生儿CMV包涵体病、急性获得性CMV感染或免疫缺陷人员CMV感染导致的疾病。
本公开优选的技术方案中，所述的CMV相关疾病选自肝、脾或中枢神经系统疾病，残疾，传染性单核细胞增多症，骨骼肌疼痛，CMV视网膜炎，胃肠CMV或脑炎等等。
本公开第十方面提供了本公开任一所述的T细胞抗原受体、本公开任一所述的核酸、本公开所述的表达载体、本公开所述的宿主细胞、本公开所述的免疫细胞在T细胞标记、检测、细胞分选或活化中的应用。
本公开的十一方面提供了一种药物组合物，所述的药物组合物包含下列任一组：
1)本公开所述的T细胞抗原受体；
2)本公开所述的核酸；
3)本公开所述的表达载体；
4)本公开所述的宿主细胞；
5)本公开所述的免疫细胞；
本公开优选的技术方案中，所述的药物组合物还可以包含药学上可接受的辅料。
本公开优选的技术方案中，所述的药物组合物还可以与其他治疗剂共同使用。进一步优选的，所述的治疗剂可以为免疫调节剂。
本公开的第十二方面，提供了一种试剂盒，所述的试剂盒包含下列任一组：
1)本公开任一所述的T细胞抗原受体；
2)本公开任一所述的核酸；
3)本公开任一所述的表达载体；
4)本公开任一所述的宿主细胞；
5)本公开任一所述的免疫细胞；或
本公开的第十三方面，提供了一种检测巨细胞病毒(CMV)磷蛋白pp65的方法，所述的方法包括将待检测样品与本公开所述的T细胞抗原受体接触，然后检测巨细胞病毒(CMV)磷蛋白pp65与T细胞抗原受体形成的复合物。
优选的，所述检测巨细胞病毒(CMV)磷蛋白pp65为检测巨细胞病毒(CMV)磷蛋白pp65的存在或含量。其中，所述的存在表示有无，所述的含量可以为表达量或蛋白浓度等。
优选的，所述的抗体或其抗原结合片段或者T细胞抗原受体包括可检测的标记物。
在本公开的一个具体实施方式中，所述的标记物可以是His和/或HA。
在其中一个实施方案中，所述的检测巨细胞病毒(CMV)磷蛋白pp65的方法，不是疾病的诊断方法。首先，待检测样品并非生物体或其离体组织或细胞，其次，即便生物体中存在巨细胞病毒(CMV)磷蛋白pp65或者包含一定浓度或表达水平的巨细胞病毒(CMV)磷蛋白pp65也并非确定是疾病，只是一种可能性。
本公开的第十四方面，提供了一种治疗和/或预防与CMV相关疾病的方法，所述的方法包括给予个体有效量的本公开所述的T细胞抗原受体、所述的核酸、所述的表达载体、所述的宿主细胞、所述的免疫细胞或所述的药物组合物。
优选的，所述的方法包括将表达本公开所述T细胞抗原受体的T细胞过继性转移至受试者的步骤。
优选的，所述的方法包括将本公开所述的T细胞抗原受体定位在与CMV相关疾病(优选为肿瘤或转移性肿瘤)的附近，以提高毒素或免疫刺激剂的效力。
优选的，用于治疗和/或预防同种异体造血干细胞移植后的CMV再活化。
优选的，用于治疗和/或预防器官移植(例如肾、肝、胰、肠、角膜)、组织移植、细胞移植(胰岛细胞、角膜缘干细胞)或干细胞治疗后的CMV再活化。
优选的，所述的表达本公开所述T细胞抗原受体的T细胞衍生自受试者。
优选的，所述的表达本公开所述T细胞抗原受体的T细胞与所述造血干细胞、器官、组织、细胞或干细胞衍生自相同的供体。
本公开的第十五方面，提供了一种诊断与CMV相关疾病的方法，所述的方法包括取样，将样品与本公开所述的T细胞抗原受体接触，然后检测CMV pp65与T细胞抗原受体形成的复合物。
优选的，所述的T细胞抗原受体包括可检测的标记物。
本公开所述的TCR能够特异性地识别对应的CMV pp65抗原肽-MHC分子复合物，激活TCR T细胞，进而产生高水平的细胞因子IFNγ、IL2、TNFα，体内体外试验均显著杀伤肿瘤细胞。
本公开所述的“T细胞抗原受体”是能够识别当由MHC分子或其四聚体递呈时的肽的分子，通常与CD3分子呈复合物形式存在于T细胞表面。大多数T细胞的TCR由α和β肽链组成，少数T细胞的TCR由γ和δ肽链组成。
本公开所述的“肿瘤”包括但不限于胰腺癌、肝癌、结肠癌、直肠癌、胃癌、淋巴瘤、基底细胞癌、非小细胞肺癌、白血病、卵巢癌、鼻咽癌、乳腺癌、子宫内膜癌、、膀胱癌、肺癌、支气管癌、骨癌、前列腺癌、、胆管癌、食管癌、肾癌、甲状腺癌、头颈部癌、睾丸癌、胶质母细胞瘤、星形细胞瘤、黑色素瘤、骨髓增生异常综合征、以及肉瘤。其中，所述的白血病选自急性淋巴细胞性(成淋巴细胞性)白血病、急性骨髓性白血病、髓性白血病、慢性淋巴细胞性白血病、多发性骨髓瘤、浆细胞白血病、以及慢性骨髓性白血病；所述淋巴瘤选自霍奇金淋巴瘤和非霍奇金淋巴瘤，包括B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、边缘区B细胞淋巴瘤、T细胞淋巴瘤、和瓦尔登斯特伦巨球蛋白血症；所述肉瘤选自骨肉瘤、尤文肉瘤、平滑肌肉瘤、滑膜肉瘤、软组织肉瘤、血管肉瘤、脂肪肉瘤、纤维肉瘤、横纹肌肉瘤、以及软骨肉瘤。优选的，所述的肿瘤选自胰腺癌、肝癌、口腔鳞状上皮癌、结肠癌、卵巢癌、胃癌。在本公开的一个具体实施方式中，所述的肿瘤为淋巴瘤。
本公开所述的“与CMV相关疾病”包含CMV pp65感染所导致的疾病，包括新生儿CMV包涵体病，严重可影响肝、脾和中枢神经系统以及残疾。还包括急性获得性CMV感染，类似于传染性单核细胞增多症，包括发烧、骨骼肌疼痛等等。还包括免疫缺陷人员，例如移植过器官的人，或患有HIV的人，感染可导致CMV视网膜炎、胃肠CMV和脑炎等等。
本公开所述的“包含”在本公开中用于描述蛋白质或核酸的序列时，所述蛋白质或核酸可以是由所述序列组成，或者在所述蛋白质或核酸的一端或两端可以具有额外的氨基酸或核苷酸，但仍然具有本公开所述的活性。
本公开所述的“预防”是指通过施用本公开所述的产品来抑制症状或者延缓特定症状紧张的所有行为。
本公开所述的“诊断”是指以查明患者过去、诊断时或将来是否患有疾病或病症，或者是查明疾病的进展或将来可能的进展，或者是评估患者对治疗的反应。
本公开所述的“治疗”表示减缓、中断、阻止、控制、停止、减轻、或逆转一种体征、症状、失调、病症、或疾病的进展或严重性，但不一定涉及所有疾病相关体征、症状、病症、或失调的完全消除，且是指在疾病已开始发展后改善疾病或病理状态的体征、症状等等的治疗干预。
本公开所述的“有效量”是指在以单个或多个剂量给予至患者或器官之后提供所希望的治疗或预防的本公开所述的产品的量或剂量。
本公开所述的“产品”包括但不限于本公开所述的抗体或其抗原结合片段、所述的T细胞抗原受体、所述的核酸、所述的表达载体、所述的宿主细胞、所述的免疫细胞或者所述的多聚体复合物，以及其他辅助或与上述上述产品协同的试剂。
本公开所述的“产品”可以为试剂盒、芯片、抗体偶联物或多功能抗体等药物组合物。
本公开所述的“受试者”包括但不限于人或非人哺乳动物。优选的，所述的非人哺乳动物包括但不限于小鼠、大鼠、猴子、猪或兔子等。
本公开所述的“同源性”是指在使用氨基酸序列或核苷酸序列的方面，本领域技术人员可以在不改变原序列主要结构或功能的前提下，根据实际工作需要对序列进行调整，使使用序列与本公开所述的具体序列相比，具有(包括但不限于)1％，2％，3％，4％，5％，6％，7％，8％，9％，10％，11％，12％，13％，14％，15％，16％，17％，18％，19％，20％，21％，22％，23％，24％，25％，26％，27％，28％，29％，30％，31％，32％，33％，34％，35％，36％，37％，38％，39％，40％，41％，42％，43％，44％，45％，46％，47％，48％，49％，50％，51％，52％，53％，54％，55％，56％，57％，58％，59％，60％，70％，80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的同源性。例如，本公开所述的“与SEQ ID NO：1-12、SEQ ID NO：14-73中任一种所示氨基酸序列具有至少80％同源性”即为在保留与巨细胞病毒(CMV)磷蛋白pp65肽表位：MHC复合物结合功能的前提下，可以根据实际工作需要对氨基酸序列进行调整，包含一个或多个改变例如取代、缺失和/或插入一个或多个氨基酸等，截短，或者一端或两端的加长，只要其保持80％以上同源性并且保留与pp65表位/MHC复合物或pp65表位/MHC分子四聚体结合的能力。所述的改变可以是取代、添加或缺失。可以是相同极性的氨基酸之间的取代、相同电荷的氨基酸之间的取代、不带电荷氨基酸之间的取代、脂肪族氨基酸之间的取代、芳香族氨基酸之间的取代、无极性氨基酸之间的取代或者具有相关性质的侧链部分的氨基酸之间的取代。其中，碱性侧链包括但不限于赖氨酸、精氨酸或组氨酸。酸性侧链包括但不限于天冬氨酸或谷氨酸。不带电荷的氨基酸包括但不限于天冬氨酰、谷氨酰胺、丝氨酸、苏氨酸或酪氨酸。非极性侧链包括但不限于甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸或半胱氨酸。其中，所述的至少80％包括但不限于80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％。
与现有技术相比，本公开具有下述有益技术效果：
1、获得了识别并结合CMV-pp65抗原表位多肽片段的TCR，其可赋予转导后的T细胞以识别病毒及其感染细胞、并启动免疫应答的能力；对靶细胞具有特异性杀伤能力。
2、本公开得到的TCR具有全新的、特定的CDR3α/β结构，为自然配对的双链结构；且本公开采用单个细胞测序方法，对细胞的VDJ片段进行了建库和多次测序并比对，筛选出优效序列。
3、本公开还提供包含编码所述TCR的核酸、以及含有所述核酸分子的载体、转导所述核酸分子或所述载体的细胞，以及包含所述TCR、核酸分子、载体或细胞作为活性成分的药物组合物，以及所述TCR、核酸分子、载体、细胞、药物组合物的用途，具有成药性和产业化价值。
附图说明
图1 TCR序列的元件结构示意图；
图2实施例2转移质粒的结构；
图3实施例2中TCR-T细胞转导阳性细胞筛选结果；
图4实施例5中TCR-T细胞转导阳性细胞筛选结果；
图5实施例8中TCR-T细胞转导阳性细胞筛选结果。
具体实施方式
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。参考例如标准手册，如Sambrook et al.,“MolecularCloning:ALaboratory Manual”；Lewin,“Genes VIII”；及Roitt et al.,“Immunology”(第8版)，以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
以下参照实施例说明本公开。但本公开不局限于实施例。
实施例1 CMV-pp65341-349特异性T细胞的培养、检测及测序
利用化学合成的短肽C1(氨基酸序列为：QYDPVAALF(SEQ ID NO：13))体外刺激来源于健康人供者的外周血单个核细胞(PBMC)(健康人供者的HLA分型可以为任选，本实施例中采用HLA-A*24中的HLA-A*2402基因型做为示例)，诱导并培养出可识别抗原肽C1并分泌IFN-γ的CD4-CD8+阳性CTL细胞，具体包括以下步骤：
1)实验组：经抗原肽C1刺激供者PBMC培养得到的X-C1细胞培养，作为实验组；
对照组：不加入多肽刺激，其他与实验组相同条件并行培养的供者PBMC细胞培养，作为对照组；
2)胞内因子分泌检测时，两组细胞均加入抗原肽C1再次刺激孵育，观察是否存在CD8+IFN-γ+双阳性CTL细胞(CD4-)识别抗原肽并分泌IFN-γ；后续实验从可识别抗原肽并分泌IFN-γ的CD4-CD8+阳性CTL细胞的培养中分离筛选出单克隆化的细胞株。
1.分离筛选出单克隆化的细胞株
EBV-LCL细胞：淋巴母细胞系(LCL细胞系)是经EB病毒转染人源PBMC后获得的体外永生化细胞系，利用不同HLA限制性的PBMC可制备不同HLA限制性的LCL细胞系。
构建EB病毒转染的永生化人源B淋巴细胞系LCLs具体过程如下：
1)从外周血分离得到PBMC，重悬于2ml含10％FBS的RPMI1640培养基(简称RPMI/10％FBS，RPM1640商家：Thermo Fisher Scientific货号：22400-089，FBS商家：Thermo Fisher Scientific，货号：10099-141C)中。
2)吸取10μL细胞液，加入90μL RPMI/10％FBS稀释10倍，在显微镜下进行细胞计数。
3)根据计数结果，计算所需B95-8(购自中国科学院细胞库，货号为GNO3)上清液的体积，其中每1×106个PBMC细胞对应500μL B95-8上清液。
4)提前两天培养10ml B95-8细胞(ATCC)，初始密度为1×106个/ml，置于37℃，5％CO2培养箱培养48h后，吸取B95-8细胞上清液转移至离心管中，2000rpm离心15min。
5)使用0.45μm滤膜对离心管内的B95-8细胞上清液过滤待用。
6)收集PBMC细胞。1000rpm离心5min，弃去PBMC上清液。
7)根据细胞计数结果，加入适量B95-8细胞上清液重悬PBMC细胞，使细胞液中PBMC细胞浓度为2×106/ml，然后添加5ml包含1μg/ml环孢素A的RPMI-10(10％FBS的RPMI1640培养基)，混匀后，将上述细胞悬浮液转移至25cm2的培养瓶内，在37℃，5％CO2的培养箱中培养3周。
8)在3周的培养结束时，培养基变为酸性，细胞形成可见的团块，细胞体积增大，细胞清晰，通常多毛，并倾向于形成不同大小的紧密团块，表明EBV发生了B细胞的永生。
将步骤1中可识别抗原肽并分泌IFN-γ的CD4-CD8+阳性CTL细胞的实验组的X-C1细胞稀释至0.3cell/2μl后铺Terasaki板(每孔0-0.3个细胞)，并加入0.120J/cm2紫外线照射后EBV-LCL细胞(淋巴母细胞系(LCL细胞系)是经EB病毒转染人源PBMC后获得的体外永生化细胞系)作为饲养细胞刺激CTL阳性T细胞生长，经5～7天静置培养后，镜下可观察到单克隆化细胞的生长。将单克隆细胞团挑出并转移到U型底96孔板中，在含30ng/ml OKT3和3000IU/ml IL-2的RPMI 1640培养基中进一步扩增培养；每3～4天给细胞培养补液/换液/更换培养容器，每30～40天再次加入照射后EBV-LCL饲养细胞刺激以保持其特异性。如此，可得到T细胞单克隆化的细胞株，其有107数量级细胞量且活率保持在80％～90％。在扩增培养中，定期取一定量细胞进行胞内因子分泌检测以确定杀伤CTL细胞的存在。
2.测序，并构建TCR
从单克隆细胞培养中，取样进行TCR序列的测序，获得TCR序列如下：
TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)；
CDR2α：MTLNGDE(SEQ ID NO：2)；
CDR3α：CARNTGKLIF(SEQ ID NO：3)、CAPSASKIIF(SEQ ID NO：22)、CAPQFNKFYF(SEQ ID NO：31)之一；
CDR1β：SQVTM(SEQ ID NO：4)、SGHVS(SEQ ID NO：14)、LNHDA(SEQ ID NO：23)之一；
CDR 2β：ANQGSEA(SEQ ID NO：5)、FQNEAQ(SEQ ID NO：15)、SQIVND(SEQ ID NO：24)之一；
CDR 3β：CSANPTGGGTEAFF(SEQ ID NO：6)、CASSLLTRTETQYF(SEQ ID NO：16)、CASSTTGLAGGPGNEQFF(SEQ ID NO：25)之一；
获得的其中一条TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)
CDR2α：MTLNGDE(SEQ ID NO：2)
CDR3α：CARNTGKLIF(SEQ ID NO：3)
CDR1β：SQVTM(SEQ ID NO：4)
CDR 2β：ANQGSEA(SEQ ID NO：5)
CDR 3β：CSANPTGGGTEAFF(SEQ ID NO：6)；
获得的另外一条TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)
CDR2α：MTLNGDE(SEQ ID NO：2)
CDR3α：CARNTGKLIF(SEQ ID NO：3)
CDR1β：SGHVS(SEQ ID NO：14)
CDR2β：FQNEAQ(SEQ ID NO：15)
CDR3β：CASSLLTRTETQYF(SEQ ID NO：16)；
获得的另外一条TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)
CDR2α：MTLNGDE(SEQ ID NO：2)
CDR3α：CAPSASKIIF(SEQ ID NO：22)
CDR1β：LNHDA(SEQ ID NO：23)
CDR2β：SQIVND(SEQ ID NO：24)
CDR3β：CASSTTGLAGGPGNEQFF(SEQ ID NO：25)；
获得的另外一条TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)
CDR2α：MTLNGDE(SEQ ID NO：2)
CDR3α：CAPQFNKFYF(SEQ ID NO：31)
CDR1β：SGHVS(SEQ ID NO：14)
CDR2β：FQNEAQ(SEQ ID NO：15)
CDR3β：CASSLLTRTETQYF(SEQ ID NO：16)。
上述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列具体如下表1所示：
表1
在获得上述表1种第一组至第四组的TCR的α链的CDR1α-CDR3α，β链的CDR1β-CDR3β的基础上，本发明进一步获得配对的TCR的alpha序列和TCR beta序列，具体方式不限于如通过测序、生物信息学等方式获得。
如根据表1中第一组氨基酸序列获得的配对的TCR的alpha序列和TCR的beta序列如下：
TCR的alpha序列(氨基酸序列为SEQ ID NO:9)，TCR beta序列(氨基酸序列为SEQ ID NO:10)；其中TCR alpha序列包括可变区Vα(氨基酸序列为SEQ ID NO:7)， TCR beta序列包括可变区Vβ(氨基酸序列为SEQ ID NO:8)；可变区Vα包括如下互补决定区：CDR1α：SSNFYA；CDR2α：MTLNGDE；CDR3α：CARNTGKLIF(SEQ ID NO:1-3)，可变区Vβ包括如下互补决定区：CDR1β：SQVTM；CDR2β：ANQGSEA；CDR3β：CSANPTGGGTEAFF(SEQ ID NO:4-6)。
在上述TCR alpha序列和TCR beta序列前还可以分别添加引导序列，经由连接序列(SEQ ID NO:11)连接后，得到TCR的全长序列(SEQ ID NO:12)。
如根据表1中第二组氨基酸序列获得的配对的TCR的alpha序列和TCR的beta序列如下：
TCR的alpha序列(氨基酸序列为SEQ ID NO:19)，TCR beta序列(氨基酸序列为SEQ ID NO:20)；其中TCR alpha序列的可变区Vα包括如下互补决定区：CDR1α：SSNFYA(SEQ ID NO：1)；CDR2α：MTLNGDE(SEQ ID NO：2)；CDR3α：CARNTGKLIF(SEQ ID NO：3)；可变区Vβ包括如下互补决定区：CDR1β：SGHVS(SEQ ID NO：14)；CDR2β：FQNEAQ(SEQ ID NO：15)；CDR3β：CASSLLTRTETQYF(SEQ ID NO：16)；
上述TCR alpha序列和TCR beta序列，经由连接序列(SEQ ID NO:11)连接后，得到TCR的全长序列(SEQ ID NO:21)。
如根据表1中第三组氨基酸序列获得的配对的TCR的alpha序列和TCR的beta序列如下：
TCR的alpha序列(氨基酸序列为SEQ ID NO:28)，TCR beta序列(氨基酸序列为SEQ ID NO:29)；其中TCR alpha序列的可变区Vα包括如下互补决定区：CDR1α：SSNFYA(SEQ ID NO：1)；CDR2α：MTLNGDE(SEQ ID NO：2)；CDR3α：CAPSASKIIF(SEQ ID NO：22)；可变区Vβ包括如下互补决定区：CDR1β：LNHDA(SEQ ID NO：23)；CDR2β：SQIVND(SEQ ID NO：24)；CDR3β：CASSTTGLAGGPGNEQFF(SEQ ID NO：25)；
上述TCR alpha序列和TCR beta序列，经由连接序列(SEQ ID NO:11)连接后，得到TCR的全长序列(SEQ ID NO:30)。
如根据表1中第四组氨基酸序列获得的配对的TCR的alpha序列和TCR的beta序列如下：
TCR的alpha序列(氨基酸序列为SEQ ID NO:35)，TCR beta序列(氨基酸序列为SEQ ID NO:36)；其中TCR alpha序列的可变区Vα包括如下互补决定区：CDR1α：SSNFYA(SEQ ID NO：1)；CDR2α：MTLNGDE(SEQ ID NO：2)；CDR3α：CAPQFNKFYF(SEQ ID NO：31)；可变区Vβ包括如下互补决定区：CDR1β：SGHVS(SEQ ID NO：14)；CDR2β：FQNEAQ(SEQ ID NO：15)；CDR3β：CASSLLTRTETQYF(SEQ ID NO：16)；
上述TCR alpha序列和TCR beta序列，经由连接序列(SEQ ID NO:11)连接后，得到TCR的全长序列(SEQ ID NO:36)。
之后构建包含恒定区的全长TCR并插入慢病毒载体中。TCR序列的元件结构如图1所示。
为了便于说明，实施例2和实施例3均以第一组氨基酸序列的基础上获得的TCR的全长序列做为示例说明。
实施例2制备CMV-pp65341-349特异性TCR基因修饰的T细胞
将实施例1获得的TCR基因序列克隆至慢病毒载体，转染病毒包装系293T细胞， 制备成病毒液，将病毒液转导T细胞，获得表达目的TCR序列的T细胞。
转染操作如下：
通过用gag/pol包装质粒、VSV-G包膜质粒和包含如下所述慢病毒载体序列的转移构建体转染293T细胞产生慢病毒上清液。也就是，将DNA混合物在Opti-MEM(LifeTechnologies，Gaithersburg，MD，USA)混合，并与等体积的含有Lipofectamine 3000(LifeTechnologies)的Opti-MEM混合。在室温下孵育15分钟后，将所得混合物施加到293T细胞。在转染后24小时收集含有慢病毒的培养基。每次收集后，将上清液通过0.45μm滤膜过滤。合并慢病毒收获物，并储存于4℃下，然后在20,000xg下超速离心90分钟。将慢病毒颗粒重悬于PBS中即得。
转移质粒的结构如图2所示。
转导操作如下：
第0天，采集供者材料血，分离PBMC。PBMC经CD3/CD28 Dynabeads(Gibco)磁珠活化2天(磁珠：细胞＝3：1)，以1×106个细胞/ml重悬于含200IU/mL IL-2的X-VIVO15无血清培养基(LONZA)中。第2天使用慢病毒上清液转导T细胞，之后在1200×g、32℃条件下离心2小时。24小时后，除去含有病毒载体的上清液。将细胞以3×105个细胞/ml悬浮于含rhIL-2(200IU/mL)的培养基，每2至3天补充有IL-2(200IU/mL)的X-VIVO15无血清培养基。收获前1天流式检测TCR表达水平，即转导阳性率，结果如图3所示，未经转导的细胞(NC)不表达特异性TCR。分别使用包含实施例1第一组TCR、第二组TCR、第三组TCR、第四组TCR的慢病毒载体转导的T细胞表达特异性TCR(CD8+Tetramer+的数据)，也即是转导阳性率：第一组TCR转导阳性率为30.36％，第二组TCR转导阳性率为9.63％，第三组TCR转导阳性率为8.63％，第四组TCR转导阳性率为3.28％，表明转导成功。
实施例3 CMV-pp65341-349特异性TCR基因修饰的T细胞的体外功能验证
本发明通过构建过表达QYDPVAALF所属的CMV-PP65蛋白的靶细胞，可以用于体外评价TCR-T细胞的功能。具体操作如下：
1、制备效应细胞：实施例2中制备的表达目的TCR序列的T细胞做为效应细胞，其中转导TCR慢病毒并表达的是TCR组，不转导的是对照组(NC组)；
2、制备过表达靶细胞：
本发明中的靶细胞为过表达QYDPVAALF所属的CMV-PP65蛋白的肿瘤细胞，通过在肿瘤细胞中引入目的抗原基因的方式构建：
使用含有目的抗原的慢病毒转导肿瘤细胞，1天后换液使用完全培养基培养1-2天，更换成含嘌呤霉素的完全培养基继续培养，并对转导组肿瘤细胞目的抗原的表达情况进行检测。
具体包括以下步骤：
1)慢病毒转导目的抗原基因
使用完全培养基(OS-RC-2人肾癌细胞所用的完全培养基为：含10％FBS的RPMI1640培养基(Gibco))调整肿瘤细胞的密度至4～6×105cells/ml，1ml/孔加入6孔板，每孔添加8μl Polybrene(1mg/ml)至终浓度为8μg/ml，每孔添加10-30μl含目的抗原QYDPVAALF的慢病毒载体，设置细胞对照孔(未转导组)，对照孔添加组分为上述的1ml肿瘤细胞及8μl Polybrene，混匀，置于CO2培养箱(37℃，5％CO2)内孵育1天；
弃上清，每孔添加2ml完全培养基，置于CO2培养箱内孵育2天；
2)转导肿瘤细胞嘌呤霉素筛选
弃上清，每孔添加2ml含有1μg/ml嘌呤霉素(Solarbio)的完全培养基，置于CO2培养箱内孵育；
每2天观察一次细胞，并更换一次含有1μg/ml嘌呤霉素的完全培养基，贴壁细胞长满时传代；
使用含有1μg/ml嘌呤霉素的完全培养基筛选培养7天，若对照组仍有活细胞，需要提高嘌呤霉素浓度继续培养直至对照组细胞全部死亡；
3)含目的抗原的细胞单克隆化培养
转导并筛选培养7天以上的转导目的抗原的肿瘤细胞按照有限稀释法进行铺板，1个细胞/孔、3个细胞/孔，使用含有嘌呤霉素的完全培养基进行培养并观察细胞孔中细胞的情况，标记出单克隆孔，持续进行培养至细胞数量达到至少6×107cells以上时冻存，既获得过表达靶细胞。
3、体外杀伤实验：步骤2制备的过表达PP65蛋白的OS-RC-2-PP65做为靶细胞按照每孔5×104个铺板，步骤1中TCR组的效应细胞和NC组的效应细胞，分别按照效靶比1:1、5:1、10:1、20:1与靶细胞混合，37℃共孵育，采用实时无标记细胞功能分析仪(Real Time Cell Analyzer，RTCA，Agilent)获得杀伤效率。
采集效应细胞铺板后4小时数据进行杀伤效率评价；具体杀伤结果如表2所示。
表2
实施例4 CMV-pp65495-503特异性T细胞的培养及测序
1、抗原特异性T细胞：
利用化学合成的抗原肽C1(氨基酸序列为：NLVPMVATV(SEQ ID NO：46))体外刺激来源于健康人供者的外周血单个核细胞(PBMC)(健康人供者的HLA分型可以为任选，本实施例中采用HLA-A*02中的HLA-A*0201基因型做为示例)，诱导并培养出可识别抗原肽C1并分泌IFN-γ的CD4-CD8+阳性CTL细胞，具体包括以下步骤：
3)实验组：经抗原肽C1刺激供者PBMC培养得到的X-C1细胞培养，作为实验组；
对照组：不加入多肽刺激，其他与实验组相同条件并行培养的供者PBMC细胞培养，作为对照组；
4)胞内因子分泌检测时，两组细胞均加入抗原肽C1再次刺激孵育，观察是否存在CD8+IFN-γ+双阳性CTL细胞(CD4-)识别抗原肽并分泌IFN-γ；后续实验从可识别抗原肽并分泌IFN-γ的CD4-CD8+阳性CTL细胞的培养中分离筛选出单克隆化的细胞株。
3.分离筛选出单克隆化的细胞株
EBV-LCL细胞：淋巴母细胞系(LCL细胞系)是经EB病毒转染人源PBMC后获得的体外永生化细胞系，利用不同HLA限制性的PBMC可制备不同HLA限制性的LCL细胞系。
构建EB病毒转染的永生化人源B淋巴细胞系LCLs具体过程如下：
9)从外周血分离得到PBMC，重悬于2ml含10％FBS的RPMI1640培养基(简称RPMI/10％FBS，RPM1640商家：Thermo Fisher Scientific 货号：22400-089，FBS商家：Thermo Fisher Scientific，货号：10099-141C)中。
10)吸取10μL细胞液，加入90μL RPMI/10％FBS稀释10倍，在显微镜下进行细胞计数。
11)根据计数结果，计算所需B95-8(购自中国科学院细胞库，货号为GNO3)上清液的体积，其中每1×106个PBMC细胞对应500μL B95-8上清液。
12)提前两天培养10ml B95-8细胞(ATCC)，初始密度为1×106个/ml，置于37℃，5％CO2培养箱培养48h后，吸取B95-8细胞上清液转移至离心管中，2000rpm离心15min。
13)使用0.45μm滤膜对离心管内的B95-8细胞上清液过滤待用。
14)收集PBMC细胞。1000rpm离心5min，弃去PBMC上清液。
15)根据细胞计数结果，加入适量B95-8细胞上清液重悬PBMC细胞，使细胞液中PBMC细胞浓度为2×106/ml，然后添加5ml包含1μg/ml环孢素A的RPMI-10(10％FBS的RPMI1640培养基)，混匀后，将上述细胞悬浮液转移至25cm2的培养瓶内，在37℃，5％CO2的培养箱中培养3周。
16)在3周的培养结束时，培养基变为酸性，细胞形成可见的团块，细胞体积增大，细胞清晰，通常多毛，并倾向于形成不同大小的紧密团块，表明EBV发生了B细胞的永生。
将步骤1中可识别抗原肽并分泌IFN-γ的CD4-CD8+阳性CTL细胞的实验组的X-C1细胞稀释至0.3cell/2μl后铺Terasaki板(每孔0-0.3个细胞)，并加入0.120J/cm2紫外线照射后EBV-LCL细胞(淋巴母细胞系(LCL细胞系)是经EB病毒转染人源PBMC后获得的体外永生化细胞系)作为饲养细胞刺激CTL阳性T细胞生长，经5～7天静置培养后，镜下可观察到单克隆化细胞的生长。将单克隆细胞团挑出并转移到U型底96孔板中，在含30ng/ml OKT3和3000IU/ml IL-2的RPMI 1640培养基中进一步扩增培养；每3～4天给细胞培养补液/换液/更换培养容器，每30～40天再次加入照射后EBV-LCL饲养细胞刺激以保持其特异性。如此，可得到T细胞单克隆化的细胞株，其有107数量级细胞量且活率保持在80％～90％。在扩增培养中，定期取一定量细胞进行胞内因子分泌检测以确定杀伤CTL细胞的存在。
4.测序，并构建TCR
从单克隆细胞培养中，取样进行TCR序列的测序，获得TCR序列如下：
TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)、DSSSTY(SEQ ID NO：47)、TSGFNG(SEQ ID NO：58)之一
CDR2α：MTLNGDE(SEQ ID NO：2)、IFSNMDM(SEQ ID NO：48)、NVLDGL(SEQ ID NO：59)之一
CDR3α：CASINFNKFYF(SEQ ID NO：37)、CAEFTGTASKLTF(SEQ ID NO：49)、CAVTYNNARLMF(SEQ ID NO：60)、CARNYGQNFVF(SEQ ID NO：69)之一
CDR1β：MDHEN(SEQ ID NO：38)、GTSNPN(SEQ ID NO：50)、MNHEY(SEQ ID NO：61)、DFQATT(SEQ ID NO：70)之一
CDR 2β：SYDVKM(SEQ ID NO：39)、SVGIG(SEQ ID NO：51)、SMNVEV(SEQ ID NO：62)、SNEGSKA(SEQ ID NO：71)之一
CDR 3β：CASSPLNGGATEAFF(SEQ ID NO：40)、CAWSDRAAFTDTQYF(SEQ ID NO：52)、CASSSVAGGRIEQFF(SEQ ID NO：63)、CSARDIKAQQWNIQYF(SEQ ID NO：72)之一。
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)
CDR2α：MTLNGDE(SEQ ID NO：2)
CDR3α：CASINFNKFYF(SEQ ID NO：37)
CDR1β：MDHEN(SEQ ID NO：38)
CDR 2β：SYDVKM(SEQ ID NO：39)
CDR 3β：CASSPLNGGATEAFF(SEQ ID NO：40)
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：DSSSTY(SEQ ID NO：47)
CDR2α：IFSNMDM(SEQ ID NO：48)
CDR3α：CAEFTGTASKLTF(SEQ ID NO：49)
CDR1β：GTSNPN(SEQ ID NO：50)
CDR2β：SVGIG(SEQ ID NO：51)
CDR3β：CAWSDRAAFTDTQYF(SEQ ID NO：52)
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：TSGFNG(SEQ ID NO：58)
CDR2α：NVLDGL(SEQ ID NO：59)
CDR3α：CAVTYNNARLMF(SEQ ID NO：60)
CDR1β：MNHEY(SEQ ID NO：61)
CDR2β：SMNVEV(SEQ ID NO：62)
CDR3β：CASSSVAGGRIEQFF(SEQ ID NO：63)
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：SSNFYA(SEQ ID NO：1)
CDR2α：MTLNGDE(SEQ ID NO：2)
CDR3α：CARNYGQNFVF(SEQ ID NO：69)
CDR1β：DFQATT(SEQ ID NO：70)
CDR2β：SNEGSKA(SEQ ID NO：71)
CDR3β：CSARDIKAQQWNIQYF(SEQ ID NO：72)
上述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列具体如下表3所示：
表3
在获得上述表3中第五组至第八组的TCR的α链的CDR1α-CDR3α，β链的CDR1β-CDR3β的基础上，本发明进一步获得配对的TCR的alpha序列和TCR beta序列，具体方式不限于如通过测序、生物信息学等方式获得。
如根据表3中第五组氨基酸序列获得的配对的TCR的alpha序列和TCR的beta序列如下：
TCR的alpha序列(氨基酸序列为SEQ ID NO:43)，TCR beta序列(氨基酸序列为SEQ ID NO:44)；其中TCR alpha序列包括可变区Vα(氨基酸序列为SEQ ID NO:41)，TCR beta序列包括可变区Vβ(氨基酸序列为SEQ ID NO:42)；可变区Vα包括如下互补决定区：CDR1α：SSNFYA；CDR2α：MTLNGDE；CDR3α：CASINFNKFYF(SEQ ID NO:1、2、37)，可变区Vβ包括如下互补决定区：CDR1β：MDHEN；CDR2β：SYDVKM；CDR3β：CASSPLNGGATEAFF(SEQ ID NO:38-40)。
在上述TCR alpha序列和TCR beta序列前还可以分别添加引导序列，经由连接序列(SEQ ID NO:11)连接后，得到TCR的全长序列(SEQ ID NO:45)。
如根据表3中第六组氨基酸序列获得的配对的TCR的alpha序列和TCR的beta序列如下：
TCR的alpha序列(氨基酸序列为SEQ ID NO:55)，TCR beta序列(氨基酸序列为SEQ ID NO:56)；其中TCR alpha序列的可变区Vα包括如下互补决定区：CDR1α：DSSSTY(SEQ ID NO：47)；CDR2α：IFSNMDM(SEQ ID NO：48)；CDR3α：CAEFTGTASKLTF(SEQ ID NO：49)；可变区Vβ包括如下互补决定区：CDR1β：GTSNPN(SEQ ID NO：50)；CDR2β：SVGIG(SEQ ID NO：51)；CDR3β：CAWSDRAAFTDTQYF(SEQ ID NO：52)；
上述TCR alpha序列和TCR beta序列，经由连接序列(SEQ ID NO:11)连接后，得到TCR的全长序列(SEQ ID NO:57)。
如根据表3中第七组氨基酸序列获得的配对的TCR的alpha序列和TCR的beta序列如下：
TCR的alpha序列(氨基酸序列为SEQ ID NO:66)，TCR beta序列(氨基酸序列为SEQ ID NO:67)；其中TCR alpha序列的可变区Vα包括如下互补决定区：CDR1α：TSGFNG(SEQ ID NO：58)；CDR2α：NVLDGL(SEQ ID NO：59)；CDR3α：CAVTYNNARLMF(SEQ ID NO：60)；可变区Vβ包括如下互补决定区：CDR1β：MNHEY(SEQ ID NO：61)；CDR2β：SMNVEV(SEQ ID NO：62)；CDR3β：CASSSVAGGRIEQFF(SEQ ID NO：63)；
上述TCR alpha序列和TCR beta序列，经由连接序列(SEQ ID NO:11)连接后，得到TCR的全长序列(SEQ ID NO:68)。
如根据表3中第八组氨基酸序列获得的配对的TCR的alpha序列和TCR的beta序列如下：
TCR的alpha序列(氨基酸序列为SEQ ID NO:75)，TCR beta序列(氨基酸序列为SEQ ID NO:76)；其中TCR alpha序列的可变区Vα包括如下互补决定区：CDR1α：SSNFYA(SEQ ID NO：1)；CDR2α：MTLNGDE(SEQ ID NO：2)；CDR3α：CARNYGQNFVF(SEQ ID NO：69)；可变区Vβ包括如下互补决定区：CDR1β：DFQATT(SEQ ID NO：70)；CDR2β：SNEGSKA(SEQ ID NO：71)；CDR3β：CSARDIKAQQWNIQYF(SEQ ID NO：72)；
上述TCR alpha序列和TCR beta序列，经由连接序列(SEQ ID NO:11)连接后，得到TCR的全长序列(SEQ ID NO:77)。
之后构建包含恒定区的全长TCR并插入慢病毒载体中。TCR序列的元件结构如图1所示。为了便于说明，实施例5和实施例6均以第五组氨基酸序列的基础上获得的TCR的全长序列做为示例说明。
实施例5制备CMV-pp65495-503特异性TCR基因修饰的T细胞
将实施例4获得的TCR基因序列克隆至慢病毒载体，转染病毒包装系293T细胞，制备成病毒液，将病毒液转导T细胞，获得表达目的TCR序列的T细胞。
转染操作如下：
通过用gag/pol包装质粒、VSV-G包膜质粒和包含如下所述慢病毒载体序列的转移构建体转染293T细胞产生慢病毒上清液。也就是，将DNA混合物在Opti-MEM(LifeTechnologies，Gaithersburg，MD，USA)混合，并与等体积的含有Lipofectamine 3000(LifeTechnologies)的Opti-MEM混合。在室温下孵育15分钟后，将所得混合物施加到293T细胞。在转染后24小时收集含有慢病毒的培养基。每次收集后，将上清液通过0.45μm滤膜过滤。合并慢病毒收获物，并储存于4℃下，然后在20,000xg下超速离心90分钟。将慢病毒颗粒重悬于PBS中即得。转移质粒的结构如图2所示。
转导操作如下：
第0天，采集供者材料血，分离PBMC。PBMC经CD3/CD28 Dynabeads(Gibco)磁珠活化2天(磁珠：细胞＝3：1)，以1×106个细胞/ml重悬于含200IU/mL IL-2的X-VIVO15无血清培养基(LONZA)中。第2天使用慢病毒上清液转导T细胞，之后在1200×g、32℃条件下离心2小时。24小时后，除去含有病毒载体的上清液。将细胞以3×105个细胞/ml悬浮于含rhIL-2(200IU/mL)的培养基，每2至3天补充有IL-2(200IU/mL)的X-VIVO15无血清培养基。收获前1天流式检测TCR表达水平， 即转导阳性率，结果如图4所示，未经转导的细胞(NC)不表达特异性TCR。分别使用包含实施例4第五组TCR、第六组TCR、第七组TCR、第八组TCR的慢病毒载体转导的T细胞表达特异性TCR(CD8+Tetramer+的数据)，也即是转导阳性率：第五组TCR转导阳性率为25.75％，第六组TCR转导阳性率为23.77％，第七组TCR转导阳性率为17.51％，第八组TCR转导阳性率为23.02％，表明转导成功。
实施例6 CMV-pp65495-503特异性TCR基因修饰的T细胞的体外功能验证
本发明通过构建过表达NLVPMVATV所属的CMV-PP65蛋白的靶细胞，可以用于体外评价TCR-T细胞的功能。具体操作如下：
1、制备效应细胞：实施例5中制备的表达目的TCR序列的T细胞做为效应细胞，其中转导TCR慢病毒并表达的是TCR组，不转导的是NC组；
2、制备过表达靶细胞：
本发明中的靶细胞为过表达NLVPMVATV所属的CMV-PP65蛋白的肿瘤细胞，通过在肿瘤细胞中引入目的抗原基因的方式构建：
使用含有目的抗原的慢病毒转导肿瘤细胞，1天后换液使用完全培养基培养1-2天，更换成含嘌呤霉素的完全培养基继续培养，并对转导组肿瘤细胞目的抗原的表达情况进行检测。
具体包括以下步骤：
1)慢病毒转导目的抗原基因
使用完全培养基(U-2OS人骨肉瘤细胞所用的完全培养基为：含10％FBS的McCoy's 5A培养基(Gibco))调整肿瘤细胞的密度至4～6×105cells/ml，1ml/孔加入6孔板，每孔添加8μl Polybrene(1mg/ml)至终浓度为8μg/ml，每孔添加10-30μl含目的抗原NLVPMVATV的慢病毒载体，设置细胞对照孔(未转导组)，对照孔添加组分为上述的1ml肿瘤细胞及8μl Polybrene，混匀，置于CO2培养箱(37℃，5％CO2)内孵育1天；
弃上清，每孔添加2ml完全培养基，置于CO2培养箱内孵育2天；
2)转导肿瘤细胞嘌呤霉素筛选
弃上清，每孔添加2ml含有1μg/ml嘌呤霉素(Solarbio)的完全培养基，置于CO2培养箱内孵育；
每2天观察一次细胞，并更换一次含有1μg/ml嘌呤霉素的完全培养基，贴壁细胞长满时传代；
使用含有1μg/ml嘌呤霉素的完全培养基筛选培养7天，若对照组仍有活细胞，需要提高嘌呤霉素浓度继续培养直至对照组细胞全部死亡；
3)含目的抗原的细胞单克隆化培养
转导并筛选培养7天以上的转导目的抗原的肿瘤细胞按照有限稀释法进行铺板，1个细胞/孔、3个细胞/孔，使用含有嘌呤霉素的完全培养基进行培养并观察细胞孔中细胞的情况，标记出单克隆孔，持续进行培养至细胞数量达到至少6×107cells以上时冻存，既获得过表达靶细胞。
3、体外杀伤实验：步骤2制备的过表达PP65蛋白的U-2OS-PP65做为靶细胞按照每孔5×104个铺板，步骤1中TCR组的效应细胞和NC组的效应细胞，分别按照效靶比1:1、5:1、10:1、20:1与靶细胞混合，37℃共孵育，采用实时无标记细胞功能分析仪(Real Time Cell Analyzer，RTCA，Agilent)获得杀伤效率。(实时无标记动态细胞分析技术是通过特殊工艺，将微电子细胞传感器芯片整合到 细胞检测板的底部，用以构建实时、动态、定量跟踪细胞形态和增殖分化等改变的细胞阻抗检测传感系统。
当贴壁生长在微电极表面的细胞引起贴壁电极界面阻抗的改变时，这种改变与细胞的实时功能状态改变呈相关性，通过实时动态的电极阻抗检测可以获得细胞生理功能相关的生物信息，包括细胞生长、伸展、形态变化、死亡和贴壁等)。采集效应细胞铺板后4小时数据进行杀伤效率评价，具体杀伤结果如表4所示。
表4
实施例7 CMV-pp65501-509特异性T细胞的培养及测序
1、抗原特异性T细胞：
利用化学合成的抗原肽C1(氨基酸序列为：ATVQGQNLK(SEQ ID NO：89))体外刺激来源于健康人供者的外周血单个核细胞(PBMC)(健康人供者的HLA分型可以为任选，本实施例中采用HLA-A*11中的HLA-A*1101基因型做为示例，诱导并培养出可识别抗原肽C1并分泌IFN-γ的CD4-CD8+阳性CTL细胞，具体包括以下步骤：
5)实验组：经抗原肽C1刺激供者PBMC培养得到的X-C1细胞培养，作为实验组；
对照组：不加入多肽刺激，其他与实验组相同条件并行培养的供者PBMC细胞培养，作为对照组；
6)胞内因子分泌检测时，两组细胞均加入抗原肽C1再次刺激孵育，观察是否存在CD8+IFN-γ+双阳性CTL细胞(CD4-)识别抗原肽并分泌IFN-γ；后续实验从可识别抗原肽并分泌IFN-γ的CD4-CD8+阳性CTL细胞的培养中分离筛选出单克隆化的细胞株。
2、分离筛选出单克隆化的细胞株
EBV-LCL细胞：淋巴母细胞系(LCL细胞系)是经EB病毒转染人源PBMC后获得的体外永生化细胞系，利用不同HLA限制性的PBMC可制备不同HLA限制性的LCL细胞系。
构建EB病毒转染的永生化人源B淋巴细胞系LCLs具体过程如下：
17)从外周血分离得到PBMC，重悬于2ml含10％FBS的RPMI1640培养基(简称RPMI/10％FBS，RPM1640商家：Thermo Fisher Scientific 货号：22400-089，FBS商家：Thermo Fisher Scientific，货号：10099-141C)中。
18)吸取10μL细胞液，加入90μL RPMI/10％FBS稀释10倍，在显微镜下进行细胞计数。
19)根据计数结果，计算所需B95-8(购自中国科学院细胞库，货号为GNO3)上清液的体积，其中每1×106个PBMC细胞对应500μL B95-8上清液。
20)提前两天培养10ml B95-8细胞(ATCC)，初始密度为1×106个/ml，置于37℃，5％CO2培养箱培养48h后，吸取B95-8细胞上清液转移至离心管中，2000rpm离心15min。
21)使用0.45μm滤膜对离心管内的B95-8细胞上清液过滤待用。
22)收集PBMC细胞。1000rpm离心5min，弃去PBMC上清液。
23)根据细胞计数结果，加入适量B95-8细胞上清液重悬PBMC细胞，使细胞液中PBMC细胞浓度为2×106/ml，然后添加5ml包含1μg/ml环孢素A的RPMI-10(10％FBS的RPMI1640培养基)，混匀后，将上述细胞悬浮液转移至25cm2的培养瓶内，在37℃，5％CO2的培养箱中培养3周。
24)在3周的培养结束时，培养基变为酸性，细胞形成可见的团块，细胞体积增大，细胞清晰，通常多毛，并倾向于形成不同大小的紧密团块，表明EBV发生了B细胞的永生。
将步骤1中可识别抗原肽并分泌IFN-γ的CD4-CD8+阳性CTL细胞的实验组的X-C1细胞稀释至0.3cell/2μl后铺Terasaki板(每孔0-0.3个细胞)，并加入0.120J/cm2紫外线照射后EBV-LCL细胞(淋巴母细胞系(LCL细胞系)是经EB病毒转染人源PBMC后获得的体外永生化细胞系)作为饲养细胞刺激CTL阳性T细胞生长，经5～7天静置培养后，镜下可观察到单克隆化细胞的生长。将单克隆细胞团挑出并转移到U型底96孔板中，在含30ng/ml OKT3和3000IU/ml IL-2的RPMI 1640培养基中进一步扩增培养；每3～4天给细胞培养补液/换液/更换培养容器，每30～40天再次加入照射后EBV-LCL饲养细胞刺激以保持其特异性。如此，可得到T细胞单克隆化的细胞株，其有107数量级细胞量且活率保持在80％～90％。在扩增培养中，定期取一定量细胞进行胞内因子分泌检测以确定杀伤CTL细胞的存在。
5.测序，并构建TCR
从单克隆细胞培养中，取样进行TCR序列的测序，获得TCR序列如下：
TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：VSGLRG(SEQ ID NO：78)、TSESDYY(SEQ ID NO：90)、NSASQS(SEQ ID NO：101)之一
CDR2α：LYSAGEE(SEQ ID NO：79)、QEAYKQQN(SEQ ID NO：91)、VYSSGN(SEQ ID NO：102)之一
CDR3α：CVITTSGTYKYIF(SEQ ID NO：80)、CAYRSFYTGANSKLTF(SEQ ID NO：92)、CVVHSGGSYIPTF(SEQ ID NO：103)之一
CDR1β：MNHNS(SEQ ID NO：81)、SGHDT(SEQ ID NO：93)、MNHNY (SEQ ID NO：104)之一
CDR 2β：SASEGT(SEQ ID NO：82)、YYEEEE(SEQ ID NO：94)、SVGAGI(SEQ ID NO：105)之一
CDR 3β：CASTINTYEQYF(SEQ ID NO：83)、CASSLYGGPGDQPQHF(SEQ ID NO：95)、CASAQTIGAYNEQFF(SEQ ID NO：106)之一；
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：VSGLRG(SEQ ID NO：78)
CDR2α：LYSAGEE(SEQ ID NO：79)
CDR3α：CVITTSGTYKYIF(SEQ ID NO：80)
CDR1β：MNHNS(SEQ ID NO：81)
CDR 2β：SASEGT(SEQ ID NO：82)
CDR 3β：CASTINTYEQYF(SEQ ID NO：83)
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：TSESDYY(SEQ ID NO：90)
CDR2α：QEAYKQQN(SEQ ID NO：91)
CDR3α：CAYRSFYTGANSKLTF(SEQ ID NO：92)
CDR1β：SGHDT(SEQ ID NO：93)
CDR2β：YYEEEE(SEQ ID NO：94)
CDR3β：CASSLYGGPGDQPQHF(SEQ ID NO：95)
在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
CDR1α：NSASQS(SEQ ID NO：101)
CDR2α：VYSSGN(SEQ ID NO：102)
CDR3α：CVVHSGGSYIPTF(SEQ ID NO：103)
CDR1β：MNHNY(SEQ ID NO：104)
CDR2β：SVGAGI(SEQ ID NO：105)
CDR3β：CASAQTIGAYNEQFF(SEQ ID NO：106)。
上述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列具体如下表5所示：
表5
在获得上述表5中第九组至十一组的TCR的α链的CDR1α-CDR3α，β链的CDR1β-CDR3β的基础上，本发明进一步获得配对的TCR的alpha序列和TCR beta序 列，具体方式不限于如通过测序、生物信息学等方式获得。
如根据表5中第九组氨基酸序列获得的配对的TCR的alpha序列和TCR的beta序列如下：
TCR的alpha序列(氨基酸序列为SEQ ID NO:86)，TCR beta序列(氨基酸序列为SEQ ID NO:87)；其中TCR alpha序列包括可变区Vα(氨基酸序列为SEQ ID NO:84)，TCR beta序列包括可变区Vβ(氨基酸序列为SEQ ID NO:85)；可变区Vα包括如下互补决定区：CDR1α：VSGLRG；CDR2α：LYSAGEE；CDR3α：CVITTSGTYKYIF(SEQ ID NO:78-80)，可变区Vβ包括如下互补决定区：CDR1β：MNHNS；CDR2β：SASEGT；CDR3β：CASTINTYEQYF(SEQ ID NO:81-83)。
在上述TCR alpha序列和TCR beta序列前还可以分别添加引导序列，经由连接序列(SEQ ID NO:11)连接后，得到TCR的全长序列(SEQ ID NO:88)。
如根据表5中第十组氨基酸序列获得的配对的TCR的alpha序列和TCR的beta序列如下：
TCR的alpha序列(氨基酸序列为SEQ ID NO:98)，TCR beta序列(氨基酸序列为SEQ ID NO:99)；其中TCR alpha序列的可变区Vα包括如下互补决定区：CDR1α：TSESDYY；CDR2α：QEAYKQQN；CDR3α：CAYRSFYTGANSKLTF(SEQ ID NO：90-92)；可变区Vβ包括如下互补决定区：CDR1β：SGHDT；CDR2β：YYEEEE；CDR3β：CASSLYGGPGDQPQHF(SEQ ID NO：93-95)；
上述TCR alpha序列和TCR beta序列，经由连接序列(SEQ ID NO:11)连接后，得到TCR的全长序列(SEQ ID NO:100)。
如根据表5中第十一组氨基酸序列获得的配对的TCR的alpha序列和TCR的beta序列如下：
TCR的alpha序列(氨基酸序列为SEQ ID NO:109)，TCR beta序列(氨基酸序列为SEQ ID NO:110)；其中TCR alpha序列的可变区Vα包括如下互补决定区：CDR1α：NSASQS；CDR2α：VYSSGN；CDR3α：CVVHSGGSYIPTF(SEQ ID NO：101-103)；可变区Vβ包括如下互补决定区：CDR1β：MNHNY；CDR2β：SVGAGI；CDR3β：CASAQTIGAYNEQFF(SEQ ID NO：104-106)；
上述TCR alpha序列和TCR beta序列，经由连接序列(SEQ ID NO:11)连接后，得到TCR的全长序列(SEQ ID NO:111)。
之后构建包含恒定区的全长TCR并插入慢病毒载体中。TCR序列的元件结构如图1所示。
为了便于说明，实施例8和实施例9均以第九组氨基酸序列的基础上获得的TCR的全长序列作为示例说明。
实施例8制备CMV-pp65501-509特异性TCR基因修饰的T细胞
将实施例7获得的TCR基因序列克隆至慢病毒载体，转染病毒包装系293T细胞，制备成病毒液，将病毒液转导T细胞，获得表达目的TCR序列的T细胞。
转染操作如下：
通过用gag/pol包装质粒、VSV-G包膜质粒和包含如下所述慢病毒载体序列的转移构建体转染293T细胞产生慢病毒上清液。也就是，将DNA混合物在Opti-MEM(LifeTechnologies，Gaithersburg，MD，USA)混合，并与等体积的含有Lipofectamine 3000(LifeTechnologies)的Opti-MEM混合。在室温下孵育15分钟后，将所得混合物施加到293T细胞。在转染后24小时收集含有慢病毒的培养基。每次收集 后，将上清液通过0.45μm滤膜过滤。合并慢病毒收获物，并储存于4℃下，然后在20,000xg下超速离心90分钟。将慢病毒颗粒重悬于PBS中即得。转移质粒的结构如图2所示。
转导操作如下：
第0天，采集供者材料血，分离PBMC。PBMC经CD3/CD28Dynabeads(Gibco)磁珠活化2天(磁珠：细胞＝3：1)，以1×106个细胞/ml重悬于含200IU/mL IL-2的X-VIVO15无血清培养基(LONZA)中。第2天使用慢病毒上清液转导T细胞，之后在1200×g、32℃条件下离心2小时。24小时后，除去含有病毒载体的上清液。将细胞以3×105个细胞/ml悬浮于含rhIL-2(200IU/mL)的培养基，每2至3天补充有IL-2(200IU/mL)的X-VIVO15无血清培养基。收获前1天流式检测TCR表达水平，即转导阳性率，结果如图5所示，未经转导的细胞(NC)不表达特异性TCR。分别使用包含实施例7第九组TCR、第十组TCR、第十一组TCR的慢病毒载体转导的T细胞表达特异性TCR(CD8+Tetramer+的数据)，也即是转导阳性率：第九组TCR转导阳性率为59.09％，第十组TCR转导阳性率为14.38％，第十一组TCR转导阳性率为91.67％。表明转导成功。
实施例9 CMV-pp65501-509特异性TCR基因修饰的T细胞的体外功能验证
本发明通过构建过表达ATVQGQNLK所属的CMV-PP65蛋白的靶细胞，可以用于体外评价TCR-T细胞的功能。具体操作如下：
1、制备效应细胞：实施例8中制备的表达目的TCR序列的T细胞做为效应细胞，其中转导TCR慢病毒并表达的是TCR组，不转导的是NC组；
2、制备过表达靶细胞：
本发明中的靶细胞为过表达ATVQGQNLK所属的CMV-PP65蛋白的肿瘤细胞，通过在肿瘤细胞中引入目的抗原基因的方式构建：
使用含有目的抗原的慢病毒转导肿瘤细胞，1天后换液使用完全培养基培养1-2天，更换成含嘌呤霉素的完全培养基继续培养，并对转导组肿瘤细胞目的抗原的表达情况进行检测。
具体包括以下步骤：
1)慢病毒转导目的抗原基因
使用完全培养基(Caki-2肾透明细胞癌细胞所用的完全培养基为：含10％FBS的McCoy's 5A培养基(Gibco))调整肿瘤细胞的密度至4～6×105cells/ml，1ml/孔加入6孔板，每孔添加8μl Polybrene(1mg/ml)至终浓度为8μg/ml，每孔添加10-30μl含目的抗原ATVQGQNLK的慢病毒载体，设置细胞对照孔(未转导组)，对照孔添加组分为上述的1ml肿瘤细胞及8μl Polybrene，混匀，置于CO2培养箱(37℃，5％CO2)内孵育1天；
弃上清，每孔添加2ml完全培养基，置于CO2培养箱内孵育2天；
2)转导肿瘤细胞嘌呤霉素筛选
弃上清，每孔添加2ml含有1μg/ml嘌呤霉素(Solarbio)的完全培养基，置于CO2培养箱内孵育；
每2天观察一次细胞，并更换一次含有1μg/ml嘌呤霉素的完全培养基，贴壁细胞长满时传代；
使用含有1μg/ml嘌呤霉素的完全培养基筛选培养7天，若对照组仍有活细胞，需要提高嘌呤霉素浓度继续培养直至对照组细胞全部死亡；
3)含目的抗原的细胞单克隆化培养
转导并筛选培养7天以上的转导目的抗原的肿瘤细胞按照有限稀释法进行铺板，1个细胞/孔、3个细胞/孔，使用含有嘌呤霉素的完全培养基进行培养并观察细胞孔中细胞的情况，标记出单克隆孔，持续进行培养至细胞数量达到至少6×107cells以上时冻存，既获得过表达靶细胞。
3、体外杀伤实验：步骤2制备的过表达PP65蛋白的Caki-2-PP65做为靶细胞按照每孔5×104个铺板，步骤1中TCR组的效应细胞和NC组的效应细胞，分别按照效靶比1:1、5:1、10:1与靶细胞混合，37℃共孵育，采用实时无标记细胞功能分析仪(Real Time Cell Analyzer，RTCA，Agilent)获得杀伤效率。(实时无标记动态细胞分析技术是通过特殊工艺，将微电子细胞传感器芯片整合到细胞检测板的底部，用以构建实时、动态、定量跟踪细胞形态和增殖分化等改变的细胞阻抗检测传感系统。
当贴壁生长在微电极表面的细胞引起贴壁电极界面阻抗的改变时，这种改变与细胞的实时功能状态改变呈相关性，通过实时动态的电极阻抗检测可以获得细胞生理功能相关的生物信息，包括细胞生长、伸展、形态变化、死亡和贴壁等。)采集效应细胞铺板后4小时数据进行杀伤效率评价，具体杀伤结果如表6所示。
表6
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明权利要求保护的范围。

Claims (15)

  1. 一种T细胞抗原受体，其特征在于，其中，所述的T细胞抗原受体包含α链的CDR1α-CDR3α和/或β链的CDR1β-CDR3β，其中，CDR3α包含SEQ ID NO：3、SEQ ID NO：22、SEQ ID NO：31、SEQ ID NO：37、SEQ ID NO：49、SEQ ID NO：60、SEQ ID NO：69、SEQ ID NO：80、SEQ ID NO：92、SEQ ID NO：103之一所示的氨基酸序列，或包含与SEQ ID NO：3、SEQ ID NO：22、SEQ ID NO：31、SEQ ID NO：37、SEQ ID NO：49、SEQ ID NO：60、SEQ ID NO：69、SEQ ID NO：80、SEQ ID NO：92、SEQ ID NO：103之一具有至少85％以上同源性的氨基酸序列；和/或其中，CDR3β包含SEQ ID NO：6、SEQ ID NO：16、SEQ ID NO：25、SEQ ID NO：40、SEQ ID NO：52、SEQ ID NO：63、SEQ ID NO：72、SEQ ID NO：83、SEQ ID NO：95、SEQ ID NO：106之一所示的氨基酸序列，或包含与SEQ ID NO：6、SEQ ID NO：16、SEQ ID NO：25、SEQ ID NO：40、SEQ ID NO：52、SEQ ID NO：63、SEQ ID NO：72、SEQ ID NO：83、SEQ ID NO：95、SEQ ID NO：106之一具有至少85％以上同源性的氨基酸序列，
    优选地，CDR1α包含SEQ ID NO：1、SEQ ID NO：47、SEQ ID NO：58、SEQ ID NO：78、SEQ ID NO：90、SEQ ID NO：101之一所示的氨基酸序列，或包含与SEQ ID NO：1、SEQ ID NO：47、SEQ ID NO：58、SEQ ID NO：78、SEQ ID NO：90、SEQ ID NO：101之一具有至少85％以上同源性的氨基酸序列；CDR2α包含SEQ ID NO：2、SEQ ID NO：48、SEQ ID NO：59、SEQ ID NO：79、SEQ ID NO：91、SEQ ID NO：102之一所示的氨基酸序列，或包含与SEQ ID NO：2、SEQ ID NO：48、SEQ ID NO：59、SEQ ID NO：79、SEQ ID NO：91、SEQ ID NO：102之一具有至少85％以上同源性的氨基酸序列；CDR1β包含SEQ ID NO：4、SEQ ID NO：14、SEQ ID NO：23、SEQ ID NO：38、SEQ ID NO：50、SEQ ID NO：61、SEQ ID NO：70、SEQ ID NO：81、SEQ ID NO：93、SEQ ID NO：104之一所示的氨基酸序列，或包含与SEQ ID NO：4、SEQ ID NO：14、SEQ ID NO：23、SEQ ID NO：38、SEQ ID NO：50、SEQ ID NO：61、SEQ ID NO：70、SEQ ID NO：81、SEQ ID NO：93、SEQ ID NO：104之一具有至少85％以上同源性的氨基酸序列；CDR2β包含SEQ ID NO：5、SEQ ID NO：15、SEQ ID NO：24、SEQ ID NO：39、SEQ ID NO：51、SEQ ID NO：62、SEQ ID NO：71、SEQ ID NO：82、SEQ ID NO：94、SEQ ID NO：105之一所示的氨基酸序列，或包含与SEQ ID NO：5、SEQ ID NO：15、SEQ ID NO：24、SEQ ID NO：39、SEQ ID NO：51、SEQ ID NO：62、SEQ ID NO：71、SEQ ID NO：82、SEQ ID NO：94、SEQ ID NO：105之一具有至少85％以上同源性的氨基酸序列。
  2. 根据权利要求1所述的T细胞抗原受体，其特征在于，所述的T细胞抗原受体特异性结合CMV pp65。
  3. 根据权利要求1所述的T细胞抗原受体，其特征在于，所述的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
    CDR1α：SSNFYA(SEQ ID NO：1)、DSSSTY(SEQ ID NO：47)、TSGFNG(SEQ ID NO：58)、VSGLRG(SEQ ID NO：78)、TSESDYY(SEQ ID NO：90)、NSASQS(SEQ ID NO：101)之一；
    CDR2α：MTLNGDE(SEQ ID NO：2)、IFSNMDM(SEQ ID NO：48)、NVLDGL(SEQ ID NO：59)、LYSAGEE(SEQ ID NO：79)、QEAYKQQN(SEQ ID NO：91)、VYSSGN(SEQ ID NO：102)之一；
    CDR3α：CARNTGKLIF(SEQ ID NO：3)、CAPSASKIIF(SEQ ID NO：22)、CAPQFNKFYF(SEQ ID NO：31)、CASINFNKFYF(SEQ ID NO：37)、CAEFTGTASKLTF(SEQ ID NO：49)、CAVTYNNARLMF(SEQ ID NO：60)、CARNYGQNFVF(SEQ ID NO：69)、CVITTSGTYKYIF(SEQ ID NO：80)、CAYRSFYTGANSKLTF(SEQ ID NO：92)、CVVHSGGSYIPTF(SEQ ID NO：103)之一；
    CDR1β：SQVTM(SEQ ID NO：4)、SGHVS(SEQ ID NO：14)、LNHDA(SEQ ID NO：23)、MDHEN(SEQ ID NO：38)、GTSNPN(SEQ ID NO：50)、MNHEY(SEQ ID NO：61)、DFQATT(SEQ ID NO：70)、MNHNS(SEQ ID NO：81)、SGHDT(SEQ ID NO：93)、MNHNY(SEQ ID NO：104)之一；
    CDR 2β：ANQGSEA(SEQ ID NO：5)、FQNEAQ(SEQ ID NO：15)、SQIVND(SEQ ID NO：24)、SYDVKM(SEQ ID NO：39)、SVGIG(SEQ ID NO：51)、SMNVEV(SEQ ID NO：62)、SNEGSKA(SEQ ID NO：71)、SASEGT(SEQ ID NO：82)、YYEEEE(SEQ ID NO：94)、SVGAGI(SEQ ID NO：105)之一；
    CDR 3β：CSANPTGGGTEAFF(SEQ ID NO：6)、CASSLLTRTETQYF(SEQ ID NO：16)、CASSTTGLAGGPGNEQFF(SEQ ID NO：25)、CASSPLNGGATEAFF(SEQ ID NO：40)、CAWSDRAAFTDTQYF(SEQ ID NO：52)、CASSSVAGGRIEQFF(SEQ ID NO：63)、CSARDIKAQQWNIQYF(SEQ ID NO：72)、CASTINTYEQYF(SEQ ID NO：83)、CASSLYGGPGDQPQHF(SEQ ID NO：95)、CASAQTIGAYNEQFF(SEQ ID NO：106)之一；
    在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
    CDR1α：SSNFYA(SEQ ID NO：1)
    CDR2α：MTLNGDE(SEQ ID NO：2)
    CDR3α：CARNTGKLIF(SEQ ID NO：3)
    CDR1β：SQVTM(SEQ ID NO：4)
    CDR 2β：ANQGSEA(SEQ ID NO：5)
    CDR 3β：CSANPTGGGTEAFF(SEQ ID NO：6)；
    在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
    CDR1α：SSNFYA(SEQ ID NO：1)
    CDR2α：MTLNGDE(SEQ ID NO：2)
    CDR3α：CARNTGKLIF(SEQ ID NO：3)
    CDR1β：SGHVS(SEQ ID NO：14)
    CDR2β：FQNEAQ(SEQ ID NO：15)
    CDR3β：CASSLLTRTETQYF(SEQ ID NO：16)；
    在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
    CDR1α：SSNFYA(SEQ ID NO：1)
    CDR2α：MTLNGDE(SEQ ID NO：2)
    CDR3α：CAPSASKIIF(SEQ ID NO：22)
    CDR1β：LNHDA(SEQ ID NO：23)
    CDR2β：SQIVND(SEQ ID NO：24)
    CDR3β：CASSTTGLAGGPGNEQFF(SEQ ID NO：25)；
    在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
    CDR1α：SSNFYA(SEQ ID NO：1)
    CDR2α：MTLNGDE(SEQ ID NO：2)
    CDR3α：CAPQFNKFYF(SEQ ID NO：31)
    CDR1β：SGHVS(SEQ ID NO：14)
    CDR2β：FQNEAQ(SEQ ID NO：15)
    CDR3β：CASSLLTRTETQYF(SEQ ID NO：16)；
    在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
    CDR1α：SSNFYA(SEQ ID NO：1)
    CDR2α：MTLNGDE(SEQ ID NO：2)
    CDR3α：CASINFNKFYF(SEQ ID NO：37)
    CDR1β：MDHEN(SEQ ID NO：38)
    CDR 2β：SYDVKM(SEQ ID NO：39)
    CDR 3β：CASSPLNGGATEAFF(SEQ ID NO：40)；
    在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
    CDR1α：DSSSTY(SEQ ID NO：47)
    CDR2α：IFSNMDM(SEQ ID NO：48)
    CDR3α：CAEFTGTASKLTF(SEQ ID NO：49)
    CDR1β：GTSNPN(SEQ ID NO：50)
    CDR2β：SVGIG(SEQ ID NO：51)
    CDR3β：CAWSDRAAFTDTQYF(SEQ ID NO：52)；
    在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
    CDR1α：TSGFNG(SEQ ID NO：58)
    CDR2α：NVLDGL(SEQ ID NO：59)
    CDR3α：CAVTYNNARLMF(SEQ ID NO：60)
    CDR1β：MNHEY(SEQ ID NO：61)
    CDR2β：SMNVEV(SEQ ID NO：62)
    CDR3β：CASSSVAGGRIEQFF(SEQ ID NO：63)；
    在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
    CDR1α：SSNFYA(SEQ ID NO：1)
    CDR2α：MTLNGDE(SEQ ID NO：2)
    CDR3α：CARNYGQNFVF(SEQ ID NO：69)
    CDR1β：DFQATT(SEQ ID NO：70)
    CDR2β：SNEGSKA(SEQ ID NO：71)
    CDR3β：CSARDIKAQQWNIQYF(SEQ ID NO：72)；
    在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
    CDR1α：VSGLRG(SEQ ID NO：78)
    CDR2α：LYSAGEE(SEQ ID NO：79)
    CDR3α：CVITTSGTYKYIF(SEQ ID NO：80)
    CDR1β：MNHNS(SEQ ID NO：81)
    CDR 2β：SASEGT(SEQ ID NO：82)
    CDR 3β：CASTINTYEQYF(SEQ ID NO：83)；
    在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
    CDR1α：TSESDYY(SEQ ID NO：90)
    CDR2α：QEAYKQQN(SEQ ID NO：91)
    CDR3α：CAYRSFYTGANSKLTF(SEQ ID NO：92)
    CDR1β：SGHDT(SEQ ID NO：93)
    CDR2β：YYEEEE(SEQ ID NO：94)
    CDR3β：CASSLYGGPGDQPQHF(SEQ ID NO：95)；
    在优选地实施方式中，所述TCR的α链的CDR1α-CDR3α和β链的CDR1β-CDR3β的氨基酸序列如下所示：
    CDR1α：NSASQS(SEQ ID NO：101)
    CDR2α：VYSSGN(SEQ ID NO：102)
    CDR3α：CVVHSGGSYIPTF(SEQ ID NO：103)
    CDR1β：MNHNY(SEQ ID NO：104)
    CDR2β：SVGAGI(SEQ ID NO：105)
    CDR3β：CASAQTIGAYNEQFF(SEQ ID NO：106)。
  4. 根据权利要求1-3任一所述的T细胞抗原受体，其α链的可变区包含SEQ ID NO：7、SEQ ID NO：17、SEQ ID NO：26、SEQ ID NO：32、SEQ ID NO：41、SEQ ID NO：53、SEQ ID NO：64、SEQ ID NO：73、SEQ ID NO：84、SEQ ID NO：96、SEQ ID NO：107所示的氨基酸序列或包含与SEQ ID NO：7、SEQ ID NO：17、SEQ ID NO：26、SEQ ID NO：32、SEQ ID NO：41、SEQ ID NO：53、SEQ ID NO：64、SEQ ID NO：73、SEQ ID NO：84、SEQ ID NO：96、SEQ ID NO：107具有至少85％以上同源性的氨基酸序列；
    优选地，其β链的可变区包含SEQ ID NO：8、SEQ ID NO：18、SEQ ID NO：27、SEQ ID NO：33、SEQ ID NO：42、SEQ ID NO：54、SEQ ID NO：65、SEQ ID NO：74、SEQ ID NO：85、SEQ ID NO：97、SEQ ID NO：108所示的氨基酸序列或包含与SEQ ID NO：8、SEQ ID NO：18、SEQ ID NO：27、SEQ ID NO：33、SEQ ID NO：42、SEQ ID NO：54、SEQ ID NO：65、SEQ ID NO：74、SEQ ID NO：85、SEQ ID NO：97、SEQ ID NO：108具有至少85％以上同源性的氨基酸序列；
    优选地，其α链包含SEQ ID NO：9、SEQ ID NO：19、SEQ ID NO：28、SEQ ID NO：34、SEQ ID NO：43、SEQ ID NO：55、SEQ ID NO：66、SEQ ID NO：75、SEQ ID NO：86、SEQ ID NO：98、SEQ ID NO：109所示的氨基酸序列或包含与SEQ ID NO：9、SEQ ID NO：19、SEQ ID NO：28、SEQ ID NO：34、SEQ ID NO：43、SEQ ID NO：55、SEQ ID NO：66、SEQ ID NO：75、SEQ ID NO：86、SEQ ID NO：98、SEQ ID NO：109具有至少85％以上同源性的氨基酸序列；
    优选地，其β链包含SEQ ID NO：10、SEQ ID NO：20、SEQ ID NO：29、SEQ ID NO：35、SEQ ID NO：44、SEQ ID NO：56、SEQ ID NO：67、SEQ ID NO：76、SEQ ID NO： 87、SEQ ID NO：99、SEQ ID NO：110所示的氨基酸序列或包含与SEQ ID NO：10、SEQ ID NO：20、SEQ ID NO：29、SEQ ID NO：35、SEQ ID NO：44、SEQ ID NO：56、SEQ ID NO：67、SEQ ID NO：76、SEQ ID NO：87、SEQ ID NO：99、SEQ ID NO：110具有至少85％以上同源性的氨基酸序列。
  5. 根据权利要求1-4任一所述的T细胞抗原受体，其特征在于，其α链与β链的氨基酸直接连接或间接连接，优选为间接连接，更优选通过fp2A连接，优选地，所述fp2A的氨基酸序列如SEQ ID NO：11所示；
    优选地，其包含SEQ ID NO：12、SEQ ID NO：21、SEQ ID NO：30、SEQ ID NO：36、SEQ ID NO：45、SEQ ID NO：57、SEQ ID NO：68、SEQ ID NO：77、SEQ ID NO：88、SEQ ID NO：100、SEQ ID NO：111所示的氨基酸序列，或包含与SEQ ID NO：12、SEQ ID NO：21、SEQ ID NO：30、SEQ ID NO：36、SEQ ID NO：45、SEQ ID NO：57、SEQ ID NO：68、SEQ ID NO：77、SEQ ID NO：88、SEQ ID NO：100、SEQ ID NO：111具有至少85％以上同源性的氨基酸序列。
  6. 一种编码权利要求1-5任一所述的T细胞抗原受体的核酸。
  7. 一种表达载体，其特征在于，所述的表达载体包含权利要求6所述的核酸。
  8. 一种宿主细胞，其特征在于，所述的宿主细胞包含权利要求6所述的核酸或权利要求7所述的表达载体。
  9. 一种免疫细胞，其特征在于，所述的免疫细胞表达权利要求1-5任一所述的T细胞抗原受体。
  10. 一种免疫细胞的制备方法，其特征在于，所述的制备方法包括将编码权利要求6所述的核酸序列转导至免疫细胞中表达获得。
  11. 一种重组T细胞的制备方法，其特征在于，包括如下步骤：
    1)从CMV阳性T细胞克隆得到权利要求6所述的核酸；
    2)分离、培养原代T细胞；
    3)将步骤1)的核酸递送至步骤2)所述的原代T细胞中，获得表达权利要求1-5任一所述的T细胞抗原受体的重组T细胞。
  12. 一种T细胞抗原受体的制备方法，其特征在于，包括如下步骤：
    (1)从阳性T细胞克隆得到权利要求6所述的核酸；
    (2)将步骤(1)得到的核酸连接至载体，获得表达载体；
    (3)将步骤(2)获得的表达载体转化至宿主细胞，然后诱导其表达；
    (4)获得T细胞抗原受体。
  13. 权利要求1-5任一所述的T细胞抗原受体、权利要求6所述的核酸、权利要求7所述的表达载体、权利要求8所述的宿主细胞、权利要求9任一所述的免疫细胞在制备诊断、预防或治疗与CMV相关疾病的产品中的应用，或在T细胞标记、检测、细胞分选或活化中的应用。
  14. 一种药物组合物，所述的药物组合物包含下列任一组：
    1)权利要求1-5任一所述的T细胞抗原受体；
    2)权利要求6所述的核酸；
    3)权利要求7所述的表达载体；
    4)权利要求8所述的宿主细胞；或
    5)权利要求9所述的免疫细胞。
  15. 一种试剂盒，所述的试剂盒包含下列任一组：
    1)权利要求1-5任一所述的T细胞抗原受体；
    2)权利要求6所述的核酸；
    3)权利要求7所述的表达载体；
    4)权利要求8所述的宿主细胞；
    5)权利要求9所述的免疫细胞。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102656188A (zh) * 2009-09-29 2012-09-05 Ucl商务股份有限公司 能够识别来自巨细胞病毒的抗原的t细胞受体
CN110357953A (zh) * 2019-07-17 2019-10-22 深圳市因诺转化医学研究院 识别人巨细胞病毒pp65抗原的TCR
CN113880937A (zh) * 2020-07-01 2022-01-04 华夏英泰（北京）生物技术有限公司 T细胞抗原受体、其多聚体复合物及其制备方法和应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102656188A (zh) * 2009-09-29 2012-09-05 Ucl商务股份有限公司 能够识别来自巨细胞病毒的抗原的t细胞受体
CN110357953A (zh) * 2019-07-17 2019-10-22 深圳市因诺转化医学研究院 识别人巨细胞病毒pp65抗原的TCR
CN113880937A (zh) * 2020-07-01 2022-01-04 华夏英泰（北京）生物技术有限公司 T细胞抗原受体、其多聚体复合物及其制备方法和应用
CN113880953A (zh) * 2020-07-01 2022-01-04 华夏英泰（北京）生物技术有限公司 T细胞抗原受体、其多聚体复合物及其制备方法和应用
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