T cell antigen receptor as well as preparation method and application thereof

Abstract

The invention relates to the technical field of T cell antigen receptors, in particular to a TCR (T cell receptor) capable of recognizing and combining a CMV-pp65495-503 antigen compound, nucleic acid containing a nucleotide sequence for coding the TCR, a carrier containing a nucleic acid molecule and a cell transduction the nucleic acid molecule or the carrier. The invention also relates to a pharmaceutical composition containing the TCR, the nucleic acid molecule, the carrier or the cell as active ingredients, and application of the TCR, the nucleic acid molecule, the carrier, the cell and the pharmaceutical composition in preparation of drugs for treating tumor or virus infection.

Description

Technical Field

[0001] 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. Background Art

[0002] T cell receptor (TCR) 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.

[0003] Cytomegalovirus (CMV) 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. In addition, CMV infection may also be related to the onset and progression of some tumors. For example, 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.

[0004] At present, a scheme for preparing TCR-T cells for CMV infection using CMV-specific TCR has been developed. After the TCR-T cells are infused back into the patient, 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. Summary of the invention

[0005] 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. Based on single-cell sequencing technology, 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.

[0006] The CMV pp65 described in the present disclosure 495-503 The short peptide (native sequence: NLVPMVATV (SEQ ID NO: 13)) is expressed on the surface of the target cells.

[0007] To achieve the above objectives, the first aspect of the present disclosure provides a TCR that recognizes human cytomegalovirus pp65 antigen, wherein the TCR has a binding affinity for CMV pp65. 495-503 The characteristics of the antigen complex, the TCR comprises at least one α chain variable region and / or one β chain variable region. Preferably, the T cell antigen receptor is an αβ heterodimer, and each comprises a TCR α chain variable region and a TCR β chain variable region.

[0008] In one embodiment, the TCRα chain variable region comprises three complementarity determining regions (CDRs) CDR1α-CDR3α, wherein CDR3α comprises the sequence shown in CASINFNKFYF (SEQ ID NO: 3) or one of SEQ ID NOs: 18-20, or comprises an amino acid sequence having at least 85%, 90%, 95%, 98% or at least 99% homology with one of SEQ ID NO: 3 or SEQ ID NOs: 18-20; preferably, the amino acid sequence of CDR3 of the TCRα chain variable region is as shown in SEQ ID NO: 3 or one of SEQ ID NOs: 18-20.

[0009] In one embodiment, the TCRβ chain variable region comprises three CDR regions, CDR1β-CDR3β, wherein CDR3 comprises the sequence shown in CASSPLNGGATEAFF (SEQ ID NO: 6), or comprises an amino acid sequence having at least 85%, 90%, 95%, 98% or at least 99% homology with one of SEQ ID NO: 6 and SEQ ID NO: 27-29; preferably, the amino acid sequence of CDR3 of the TCRβ chain variable region is as shown in one of SEQ ID NO: 6 and SEQ ID NO: 27-29.

[0010] In one embodiment, the CDR1α of the TCRα chain variable region comprises the amino acid sequence shown in one of SEQ ID NO:1 and SEQ ID NO:14-15, or comprises an amino acid sequence having at least 85%, 90%, 95%, 98% or at least 99% homology to one of SEQ ID NO:1 and SEQ ID NO:14-15; the CDR2α comprises the amino acid sequence shown in one of SEQ ID NO:2 and SEQ ID NO:16-17, or comprises an amino acid sequence having at least 85%, 90%, 95%, 98% or at least 99% homology to one of SEQ ID NO:2 and SEQ ID NO:16-17; the CDR1β of the TCRβ chain variable region comprises the amino acid sequence shown in one of SEQ ID NO:4 and SEQ ID NO:21-23, or comprises an amino acid sequence having at least 85%, 90%, 95%, 98% or at least 99% homology to one of SEQ ID NO:4 and SEQ ID NO:21-23; the CDR2β comprises the amino acid sequence shown in SEQ ID NO:2 NO:5, the amino acid sequence shown in one of SEQ ID NOs:24-26, or an amino acid sequence comprising at least 85%, 90%, 95%, 98% or at least 99% homologous to one of SEQ ID NO:5, SEQ ID NOs:24-26.

[0011] In a preferred embodiment, the amino acid sequences of CDR1α-CDR3α and CDR1β-CDR3β of the β chain of the TCR are as follows:

[0012] CDR1α: one of SSNFYA (SEQ ID NO: 1), DSSSTY (SEQ ID NO: 14), TSGFNG (SEQ ID NO: 15)

[0013] CDR2α: one of MTLNGDE (SEQ ID NO: 2), IFSNMDM (SEQ ID NO: 16), NVLDGL (SEQ ID NO: 17)

[0014] CDR3α: one of CASINFNKFYF (SEQ ID NO: 3), CAEFTGTASKLTF (SEQ ID NO: 18), CAVTYNNARLMF (SEQ ID NO: 19), CARNYGQNFVF (SEQ ID NO: 20)

[0015] CDR1β: one of MDHEN (SEQ ID NO: 4), GTSNPN (SEQ ID NO: 21), MNHEY (SEQ ID NO: 22), DFQATT (SEQ ID NO: 23)

[0016] CDR 2β: one of SYDVKM (SEQ ID NO: 5), SVGIG (SEQ ID NO: 24), SMNVEV (SEQ ID NO: 25), SNEGSKA (SEQ ID NO: 26)

[0017] CDR 3β: one of CASSPLNGGATEAFF (SEQ ID NO: 6), CAWSDRAAFTDTQYF (SEQ ID NO: 27), CASSSVAGGRIEQFF (SEQ ID NO: 28), CSARDIKAQQWNIQYF (SEQ ID NO: 29).

[0018] In a preferred embodiment, the amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of the TCR are as follows:

[0019] CDR1α: SSNFYA (SEQ ID NO: 1)

[0020] CDR2α: MTLNGDE (SEQ ID NO: 2)

[0021] CDR3α: CASINFNKFYF (SEQ ID NO: 3)

[0022] CDR1β: MDHEN (SEQ ID NO: 4)

[0023] CDR 2β: SYDVKM (SEQ ID NO: 5)

[0024] CDR 3β: CASSPLNGGATEAFF (SEQ ID NO: 6)

[0025] In a preferred embodiment, the amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of the TCR are as follows:

[0026] CDR1α: DSSSTY (SEQ ID NO: 14)

[0027] CDR2α: IFSNMDM (SEQ ID NO: 16)

[0028] CDR3α: CAEFTGTASKLTF (SEQ ID NO: 18)

[0029] CDR1β: GTSNPN (SEQ ID NO: 21)

[0030] CDR2β: SVGIG (SEQ ID NO: 24)

[0031] CDR3β: CAWSDRAAFTDTQYF (SEQ ID NO: 27)

[0032] In a preferred embodiment, the amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of the TCR are as follows:

[0033] CDR1α: TSGFNG (SEQ ID NO: 15)

[0034] CDR2α: NVLDGL (SEQ ID NO: 17)

[0035] CDR3α: CAVTYNNARLMF (SEQ ID NO: 19)

[0036] CDR1β: MNHEY (SEQ ID NO: 22)

[0037] CDR2β: SMNVEV (SEQ ID NO: 25)

[0038] CDR3β: CASSSVAGRIEQFF (SEQ ID NO: 28)

[0039] In a preferred embodiment, the amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of the TCR are as follows:

[0040] CDR1α: SSNFYA (SEQ ID NO: 1)

[0041] CDR2α: MTLNGDE (SEQ ID NO: 2)

[0042] CDR3α: CARNYGQNFVF (SEQ ID NO: 20)

[0043] CDR1β: DFQATT (SEQ ID NO: 23)

[0044] CDR2β: SNEGSKA (SEQ ID NO: 26)

[0045] CDR3β: CSARDIKAQQWNIQYF (SEQ ID NO: 29)

[0046] In one embodiment, the variable region of the α chain of the TCR of the present invention comprises the amino acid sequence of SEQ ID NO: 7, 39, 41, 43 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: 7, 39, 41, 43.

[0047] Preferably, the α chain of the TCR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 9, 30, 33, 36 or an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, 92%, 94%, 96%, 98% or 99% or more homologous to SEQ ID NO: 9, 30, 33, 36.

[0048] In one embodiment, the variable region of the β chain of the TCR of the present invention comprises the amino acid sequence of SEQ ID NO: 8, 40, 42, 44 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: 8, 40, 42, 44.

[0049] Preferably, the β chain of the TCR disclosed herein comprises the amino acid sequence of SEQ ID NO: 10, 31, 34, 37 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, 31, 34, 37.

[0050] In a specific embodiment of the present disclosure, preferably, the amino acids of the α chain and β 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. The 2A peptides used in the present disclosure include but are not limited to P2A, T2A, E2A, and F2A.

[0051] In some embodiments of the present disclosure, a 2A peptide, such as 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.

[0052] Further preferably, the fp2A comprises the amino acid sequence shown in SEQ ID NO:11.

[0053] In a specific embodiment of the present disclosure, the connection order of the α chain and the β chain can be α chain, fp2A and β chain, or β chain, fp2A and α chain.

[0054] In a specific embodiment of the present disclosure, the TCR of the present disclosure comprises the amino acid sequence shown in SEQ ID NO: 12, 32, 35, 38, 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: 12, 32, 35, 38.

[0055] 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.

[0056] In the technical solution of the present disclosure, the nucleic acid sequence may be single-stranded or double-stranded, and may be DNA or RNA.

[0057] In the preferred technical solution of the present disclosure, the nucleic acid sequence may be codon optimized. Further preferably, 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.

[0058] The third aspect of the present disclosure provides an expression vector, wherein the expression vector comprises any nucleic acid described in the present disclosure.

[0059] In the preferred technical solution of 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.

[0060] In the preferred technical solution of the present disclosure, the expression vector may be a prokaryotic expression vector or a retroviral vector.

[0061] In the preferred technical solutions disclosed herein, the expression vector may 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.

[0062] 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.

[0063] In the preferred technical solution of 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.

[0064] 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.

[0065] In a preferred technical solution of the present disclosure, the immune cells contain one or more nucleic acid sequences described in any one of the present disclosure.

[0066] In the preferred technical solutions of the present disclosure, 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.

[0067] In the preferred technical solution of the present disclosure, the T cells may be CD4 + T, CD8 + T et al.

[0068] In a preferred technical solution of the present disclosure, the immune cells are separated from T cells of the subject.

[0069] In a preferred technical solution of the present disclosure, the immune cells are T cells from CMV seronegative donors.

[0070] 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.

[0071] In the preferred technical solutions of the present disclosure, 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.

[0072] The preferred technical solution of the present disclosure also includes a step of knocking out the endogenous TCR of the cell. Specifically, 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.

[0073] A seventh aspect of the present disclosure provides a method for preparing a recombinant T cell, comprising the following steps:

[0074] 1) obtaining the nucleic acid described in any one of the second aspects of the present disclosure from a positive T cell clone;

[0075] 2) Isolation and culture of primary T cells;

[0076] 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.

[0077] In a preferred technical solution of the present disclosure, the T cells are selected from hematopoietic stem cells or peripheral blood lymphocyte (PBL)-derived T cells.

[0078] An eighth aspect of the present disclosure provides a method for preparing a T cell antigen receptor, comprising the following steps:

[0079] (1) obtaining the nucleic acid described in any one of the second aspects of the present disclosure from a positive T cell clone;

[0080] (2) connecting the nucleic acid obtained in step (1) to a vector backbone to obtain an expression vector;

[0081] (3) transforming the expression vector obtained in step (2) into a host cell and then inducing its expression;

[0082] (4) Obtaining antibodies or antigen-binding fragments thereof or T cell antigen receptors.

[0083] In the preferred technical solution of the present disclosure, the positive T cells specifically bind to the cytomegalovirus (CMV) phosphoprotein pp65 antigen peptide presented by MHC. Further preferably, the cytomegalovirus (CMV) phosphoprotein pp65 antigen peptide complex presented by MHC is a monomer or polymer complex.

[0084] The ninth aspect of the present disclosure provides the use of any of the T cell antigen receptors described in the first aspect, any of the nucleic acids 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.

[0085] In the preferred technical scheme of the present disclosure, 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.

[0086] In the preferred technical scheme of the present invention, 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.

[0087] 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.

[0088] The eleventh aspect of the present disclosure provides a pharmaceutical composition, which comprises any one of the following groups:

[0089] 1) T cell antigen receptor described in the present disclosure;

[0090] 2) nucleic acids described in the present disclosure;

[0091] 3) the expression vector described in the present disclosure;

[0092] 4) the host cell described in the present disclosure;

[0093] 5) Immune cells as described in the present disclosure;

[0094] In the preferred technical solution of the present disclosure, the pharmaceutical composition may further comprise pharmaceutically acceptable excipients.

[0095] In the preferred technical solution of the present disclosure, the pharmaceutical composition can also be used together with other therapeutic agents. Further preferably, the therapeutic agent can be an immunomodulator.

[0096] In a twelfth aspect of the present disclosure, a kit is provided, wherein the kit comprises any one of the following:

[0097] 1) any T cell antigen receptor described in the present disclosure;

[0098] 2) any nucleic acid described in the present disclosure;

[0099] 3) any expression vector described in the present disclosure;

[0100] 4) any host cell described in the present disclosure;

[0101] 5) any immune cell described in the present disclosure; or

[0102] In the thirteenth aspect of the present disclosure, a method for detecting cytomegalovirus (CMV) phosphoprotein pp65 is provided, the method 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.

[0103] Preferably, 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.

[0104] Preferably, the antibody or antigen-binding fragment thereof or T cell antigen receptor comprises a detectable marker.

[0105] In a specific embodiment of the present disclosure, the marker may be His and / or HA.

[0106] In one embodiment, the method for detecting cytomegalovirus (CMV) phosphoprotein pp65 is not a method for diagnosing a disease. First, the sample to be detected is not an organism or its isolated tissue or cell, and second, even if 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.

[0107] In the fourteenth aspect of the present disclosure, a method for treating and / or preventing CMV-related diseases is provided, the method 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.

[0108] Preferably, the method comprises the step of adoptively transferring T cells expressing the T cell antigen receptor disclosed herein to a subject.

[0109] Preferably, 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 a toxin or immunostimulant.

[0110] Preferably, the invention is used for treating and / or preventing CMV reactivation after allogeneic hematopoietic stem cell transplantation.

[0111] Preferably, it is used for treating and / or preventing 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.

[0112] Preferably, the T cells expressing the T cell antigen receptor disclosed herein are derived from a subject.

[0113] Preferably, 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.

[0114] In a fifteenth aspect of the present disclosure, a method for diagnosing a CMV-related disease is provided, the method comprising taking a sample, contacting the sample with the T cell antigen receptor described in the present disclosure, and then detecting a complex formed by CMV pp65 and the T cell antigen receptor.

[0115] Preferably, the T cell antigen receptor comprises a detectable marker.

[0116] 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 IFNγ, IL2, and TNFα, and significantly kill tumor cells in both in vivo and in vitro experiments.

[0117] The "T cell antigen receptor" described in the present disclosure 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 α and β peptide chains, and the TCR of a few T cells is composed of γ and δ peptide chains.

[0118] 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. Wherein, 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, and chondrosarcoma. Preferably, the tumor is selected from pancreatic cancer, liver cancer, oral squamous cell carcinoma, colon cancer, ovarian cancer, gastric cancer. In a specific embodiment of the present disclosure, the tumor is a lymphoma.

[0119] The "CMV-related diseases" described in the present disclosure include diseases caused by CMV pp65 infection, including neonatal CMV inclusion disease, which can seriously affect the liver, spleen and central nervous system and 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 people who have undergone organ transplants, or people with HIV, and infection can lead to CMV retinitis, gastrointestinal CMV and encephalitis, etc.

[0120] When the term “comprising” described in the present disclosure is used to describe a protein or nucleic acid sequence, 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.

[0121] The term "prevention" as used in the present disclosure refers to all actions to suppress symptoms or delay the tension of specific symptoms by administering the product described in the present disclosure.

[0122] "Diagnosis" as used in this disclosure refers to finding out whether a patient has had 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.

[0123] "Treatment" as used in the present disclosure 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] The "subject" described in the present disclosure includes but is not limited to humans or non-human mammals. Preferably, the non-human mammals include but are not limited to mice, rats, monkeys, pigs or rabbits.

[0128] The "homology" described in the present disclosure means 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) 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology compared with the specific sequence described in the present disclosure. For example, the "having at least 80% homology with the amino acid sequence shown in any one of SEQ ID NO: 1-12, SEQ ID NO: 14-44" 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. It can be a substitution between amino acids of the same polarity, a substitution between amino acids of the same charge, a substitution between uncharged amino acids, a substitution between aliphatic amino acids, a substitution between aromatic amino acids, a substitution between non-polar amino acids, or a substitution between amino acids in the side chain part with relevant properties. Among them, 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. Wherein, at least 80% includes, 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%.

[0129] Compared with the prior art, the present invention has the following beneficial technical effects:

[0130] 1. Obtained recognition and binding to CMV-pp65 495-503 TCR of antigen epitope polypeptide fragment can give transduced T cells the ability to recognize viruses and their infected cells and initiate immune responses; it has specific killing ability against target cells.

[0131] 2. The TCR obtained in the present disclosure has a new and specific CDR3α / β 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.

[0132] 3. 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 the use of the TCR, nucleic acid molecule, vector, cell and pharmaceutical composition, which have drugability and industrial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] Figure 1 Schematic diagram of the element structure of the TCR sequence;

[0134] Figure 2 TCR-T cell transduction positive cell screening results. DETAILED DESCRIPTION

[0135] Unless otherwise indicated or defined, all terms used have the usual meaning in the art, which will be understood by those skilled in the art. Reference is made to, for example, standard manuals, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual"; Lewin, "Genes VIII"; and Roitt et al., "Immunology" (8th edition), as well as the general prior art cited herein; in addition, unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can and have been performed in a manner known per se, which will be understood by those skilled in the art. Reference is also made to, for example, standard manuals, the above-mentioned general prior art and other references cited therein.

[0136] The present disclosure is described below with reference to examples, but the present disclosure is not limited to the examples.

[0137] Example 1 CMV-pp65 495-503 Cultivation and sequencing of specific T cells

[0138] 1. Antigen-specific T cells:

[0139] The peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated in vitro with chemically synthesized antigen peptide C1 (amino acid sequence: NLVPMVATV) (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 antigen peptide C1 and secrete IFN-γ were induced and cultured, which specifically includes the following steps:

[0140] 1) Experimental group: X-C1 cells cultured from donor PBMCs stimulated with antigen peptide C1 were used as the experimental group;

[0141] 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;

[0142] 2) When detecting the secretion of intracellular factors, both groups of cells were stimulated and incubated again with the addition of antigen peptide C1 to observe whether there were CD8+IFN-γ+ double-positive CTL cells (CD4-) that recognized the antigen peptide and secreted IFN-γ; 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-γ.

[0143] 2. Isolate and screen monoclonal cell lines

[0144] EBV-LCL cells: Lymphoblastoid cell line (LCL 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.

[0145] The specific process of constructing immortalized human B lymphocyte cell line LCLs transfected with EB virus is as follows:

[0146] 1) PBMCs were isolated from peripheral blood and resuspended in 2 ml of RPMI1640 medium containing 10% FBS (abbreviated as RPMI / 10% FBS, RPM1640 Merchant: Thermo Fisher Scientific

[0147] Catalog number: 22400-089, FBS merchant: Thermo Fisher Scientific, catalog number: 10099-141C).

[0148] 2) Pipette 10 μL of cell solution, add 90 μL RPMI / 10% FBS to dilute 10 times, and count the cells under a microscope.

[0149] 3) According to the counting results, calculate the required volume of B95-8 (purchased from the Chinese Academy of Sciences Cell Bank, catalog number GNO3) supernatant, where 1×10 6 Each PBMC cell corresponds to 500 μL of B95-8 supernatant.

[0150] 4) Two days in advance, culture 10 ml of B95-8 cells (ATCC) with an initial density of 1×10 6 After culturing in a 37°C, 5% CO2 incubator for 48 hours, the B95-8 cell supernatant was transferred to a centrifuge tube and centrifuged at 2000 rpm for 15 minutes.

[0151] 5) Use a 0.45 μm filter membrane to filter the B95-8 cell supernatant in the centrifuge tube for later use.

[0152] 6) Collect PBMC cells and centrifuge at 1000 rpm for 5 min, then discard the PBMC supernatant.

[0153] 7) According to the cell counting results, add an appropriate amount of B95-8 cell supernatant to resuspend the PBMC cells so that the PBMC cell concentration in the cell fluid is 2×10 6 / ml, then add 5ml of RPMI-10 (RPMI1640 medium with 10% FBS) containing 1μg / ml cyclosporin A, mix well, and transfer the cell suspension to a 25cm 2 The cells were cultured in culture flasks at 37°C in a 5% CO2 incubator for 3 weeks.

[0154] 8) At the end of 3 weeks of culture, the medium became acidic and the cells formed visible clumps. The cells increased in size, became clear, often hairy, and tended to form tight clumps of varying sizes, indicating that EBV immortalized B cells.

[0155] The X-C1 cells of the experimental group of CD4-CD8+ positive CTL cells that can recognize antigen peptides and secrete IFN-γ in step 1 were diluted to 0.3 cell / 2 μl and spread on Terasaki plates (0-0.3 cells per well), and 0.120 J / cm 2After ultraviolet irradiation, EBV-LCL cells (lymphoblastoid cell line (LCL cell line) is an in vitro immortalized cell line obtained by transfecting human PBMC with EB virus) are used 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. The monoclonal cell clusters are picked out and transferred to a U-bottom 96-well plate, and further expanded and cultured in RPMI 1640 medium containing 30ng / ml OKT3 and 3000IU / ml IL-2; the cell culture fluid is replenished / replaced / the culture container is replaced every 3-4 days, and the irradiated EBV-LCL feeder cells are added again every 30-40 days to maintain its specificity. In this way, a T cell monoclonal cell line can be obtained, of which 10 7 The cell quantity is of the same order of magnitude and the viability is maintained at 80% to 90%. During the expansion culture, a certain amount of cells is taken regularly to detect the secretion of intracellular factors to determine the presence of killer CTL cells.

[0156] 3. Sequencing and construction of TCR

[0157] From the monoclonal cell culture, samples were taken for TCR sequence sequencing, and the TCR sequences were obtained as follows:

[0158] The amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of TCR are as follows:

[0159] CDR1α: one of SSNFYA (SEQ ID NO: 1), DSSSTY (SEQ ID NO: 14), TSGFNG (SEQ ID NO: 15)

[0160] CDR2α: one of MTLNGDE (SEQ ID NO: 2), IFSNMDM (SEQ ID NO: 16), NVLDGL (SEQ ID NO: 17)

[0161] CDR3α: one of CASINFNKFYF (SEQ ID NO: 3), CAEFTGTASKLTF (SEQ ID NO: 18), CAVTYNNARLMF (SEQ ID NO: 19), CARNYGQNFVF (SEQ ID NO: 20)

[0162] CDR1β: one of MDHEN (SEQ ID NO: 4), GTSNPN (SEQ ID NO: 21), MNHEY (SEQ ID NO: 22), DFQATT (SEQ ID NO: 23)

[0163] CDR 2β: one of SYDVKM (SEQ ID NO: 5), SVGIG (SEQ ID NO: 24), SMNVEV (SEQ ID NO: 25), SNEGSKA (SEQ ID NO: 26)

[0164] CDR 3β: one of CASSPLNGGATEAFF (SEQ ID NO: 6), CAWSDRAAFTDTQYF (SEQ ID NO: 27), CASSSVAGGRIEQFF (SEQ ID NO: 28), CSARDIKAQQWNIQYF (SEQ ID NO: 29).

[0165] In a preferred embodiment, the amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of the TCR are as follows:

[0166] CDR1α: SSNFYA (SEQ ID NO: 1)

[0167] CDR2α: MTLNGDE (SEQ ID NO: 2)

[0168] CDR3α: CASINFNKFYF (SEQ ID NO: 3)

[0169] CDR1β: MDHEN (SEQ ID NO: 4)

[0170] CDR 2β: SYDVKM (SEQ ID NO: 5)

[0171] CDR 3β: CASSPLNGGATEAFF (SEQ ID NO: 6)

[0172] In a preferred embodiment, the amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of the TCR are as follows:

[0173] CDR1α: DSSSTY (SEQ ID NO: 14)

[0174] CDR2α: IFSNMDM (SEQ ID NO: 16)

[0175] CDR3α: CAEFTGTASKLTF (SEQ ID NO: 18)

[0176] CDR1β: GTSNPN (SEQ ID NO: 21)

[0177] CDR2β: SVGIG (SEQ ID NO: 24)

[0178] CDR3β: CAWSDRAAFTDTQYF (SEQ ID NO: 27)

[0179] In a preferred embodiment, the amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of the TCR are as follows:

[0180] CDR1α: TSGFNG (SEQ ID NO: 15)

[0181] CDR2α: NVLDGL (SEQ ID NO: 17)

[0182] CDR3α: CAVTYNNARLMF (SEQ ID NO: 19)

[0183] CDR1β: MNHEY (SEQ ID NO: 22)

[0184] CDR2β: SMNVEV (SEQ ID NO: 25)

[0185] CDR3β: CASSSVAGRIEQFF (SEQ ID NO: 28)

[0186] In a preferred embodiment, the amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of the TCR are as follows:

[0187] CDR1α: SSNFYA (SEQ ID NO: 1)

[0188] CDR2α: MTLNGDE (SEQ ID NO: 2)

[0189] CDR3α: CARNYGQNFVF (SEQ ID NO: 20)

[0190] CDR1β: DFQATT (SEQ ID NO: 23)

[0191] CDR2β: SNEGSKA (SEQ ID NO: 26)

[0192] CDR3β: CSARDIKAQQWNIQYF (SEQ ID NO: 29)

[0193] The amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of the above-mentioned TCR are specifically shown in Table 1 below:

[0194] Table 1 Amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of TCR

[0195]

[0196] Based on obtaining CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of the first to fourth groups of TCRs in Table 1 above, 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.

[0197] For example, the paired TCR alpha sequence and TCR beta sequence obtained according to the first group of amino acid sequences in Table 1 are as follows:

[0198] 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 Vα (amino acid sequence is SEQ ID NO:7), and the TCR beta sequence includes the variable region Vβ (amino acid sequence is SEQ ID NO:8); the variable region Vα includes the following complementary determining regions: CDR1α: SSNFYA; CDR2α: MTLNGDE; CDR3α: CASINFNKFYF (SEQ ID NO:1-3), and the variable region Vβ includes the following complementary determining regions: CDR1β: MDHEN; CDR2β: SYDVKM; CDR3β: CASSPLNGGATEAFF (SEQ ID NO:4-6).

[0199] A guide sequence can be added before the above-mentioned TCR alpha sequence and TCR beta sequence, respectively, and after being connected via a connecting sequence (SEQ ID NO: 11), a full-length sequence of TCR (SEQ ID NO: 12) can be obtained.

[0200] For example, the paired TCR alpha sequence and TCR beta sequence obtained according to the second group of amino acid sequences in Table 1 are as follows:

[0201] TCR alpha sequence (amino acid sequence of SEQ ID NO: 30), TCR beta sequence (amino acid sequence of SEQ ID NO: 31); wherein the TCR alpha sequence includes a variable region Vα (amino acid sequence of SEQ ID NO: 39), and the TCR beta sequence includes a variable region Vβ (amino acid sequence of SEQ ID NO: 40); wherein the variable region Vα of the TCR alpha sequence includes the following complementary determining regions: CDR1α: DSSSTY (SEQ ID NO: 14); CDR2α: IFSNMDM (SEQ ID NO: 16); CDR3α: CAEFTGTASKLTF (SEQ ID NO: 18); the variable region Vβ includes the following complementary determining regions: CDR1β: GTSNPN (SEQ ID NO: 21); CDR2β: SVGIG (SEQ ID NO: 24); CDR3β: CAWSDRAAFTDTQYF (SEQ ID NO: 27);

[0202] The above-mentioned 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: 32).

[0203] For example, the paired TCR alpha sequence and TCR beta sequence obtained according to the third group of amino acid sequences in Table 1 are as follows:

[0204] TCR alpha sequence (amino acid sequence of SEQ ID NO: 33), TCR beta sequence (amino acid sequence of SEQ ID NO: 34); wherein the TCR alpha sequence includes a variable region Vα (amino acid sequence of SEQ ID NO: 41), and the TCR beta sequence includes a variable region Vβ (amino acid sequence of SEQ ID NO: 42); wherein the variable region Vα of the TCR alpha sequence includes the following complementary determining regions: CDR1α: TSGFNG (SEQ ID NO: 15); CDR2α: NVLDGL (SEQ ID NO: 17); CDR3α: CAVTYNNARLMF (SEQ ID NO: 19); the variable region Vβ includes the following complementary determining regions: CDR1β: MNHEY (SEQ ID NO: 22); CDR2β: SMNVEV (SEQ ID NO: 25); CDR3β: CASSSVAGGRIEQFF (SEQ ID NO: 28);

[0205] The above-mentioned 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: 35).

[0206] For example, the paired TCR alpha sequence and TCR beta sequence obtained according to the fourth group of amino acid sequences in Table 1 are as follows:

[0207] TCR alpha sequence (amino acid sequence of SEQ ID NO: 36), TCR beta sequence (amino acid sequence of SEQ ID NO: 37); wherein the TCR alpha sequence includes a variable region Vα (amino acid sequence of SEQ ID NO: 43), and the TCR beta sequence includes a variable region Vβ (amino acid sequence of SEQ ID NO: 44); wherein the variable region Vα of the TCR alpha sequence includes the following complementary determining regions: CDR1α: SSNFYA (SEQ ID NO: 1); CDR2α: MTLNGDE (SEQ ID NO: 2); CDR3α: CARNYGQNFVF (SEQ ID NO: 20); the variable region Vβ includes the following complementary determining regions: CDR1β: DFQATT (SEQ ID NO: 23); CDR2β: SNEGSKA (SEQ ID NO: 26); CDR3β: CSARDIKAQQWNIQYF (SEQ ID NO: 29);

[0208] The above-mentioned 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: 38).

[0209] The full-length TCR including the constant region is then constructed and inserted into a lentiviral vector. The element structure of the TCR sequence is as follows Figure 1 shown.

[0210] For ease of explanation, 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.

[0211] Example 2 Preparation of CMV-pp65 495-503 T cells modified with specific TCR genes

[0212] The TCR gene sequence obtained in Example 1 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.

[0213] The transfection procedure is as follows:

[0214] 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 culture medium containing lentivirus was collected 24 hours after transfection. After each collection, the supernatant was filtered through a 0.45 μm filter membrane. The lentiviral harvest was combined and stored at 4 ° C, and then ultracentrifuged at 20,000 x g for 90 minutes. The lentiviral particles were resuspended in PBS.

[0215] The structure of the transfer plasmid is as follows:

[0216]

[0217] The transduction procedure is as follows:

[0218] On day 0, donor blood was collected and PBMCs were isolated. PBMCs were activated with CD3 / CD28 Dynabeads (Gibco) magnetic beads for 2 days (beads: cells = 3:1) and cultured at 1×10 6 The cells were resuspended in X-VIVO15 serum-free medium (LONZA) containing 200 IU / mL IL-2 at 100 cells / ml. On the second day, the T cells were transduced with the lentiviral supernatant and then centrifuged at 1200×g and 32°C for 2 hours. After 24 hours, the supernatant containing the viral vector was removed. The cells were plated at 3×10 5 10 cells / ml were suspended in medium containing rhIL-2 (200 IU / mL), and X-VIVO15 serum-free medium supplemented with IL-2 (200 IU / mL) was added 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 were as follows Figure 2 As shown, the non-transduced cells (NC) do not express specific TCR. T cells transduced with the lentiviral vectors containing the first group of TCR, the second group of TCR, the third group of TCR, and the fourth group of TCR in Example 1 express specific TCR (CD8 + Tetramer + The data of the first group (P < 0.05) show that the TCR transduction positive rate was 25.75%, the TCR transduction positive rate was 23.77%, the TCR transduction positive rate was 17.51%, and the TCR transduction positive rate was 23.02%, indicating that the transduction was successful.

[0219] Example 3 CMV-pp65 495-503 In vitro functional validation of T cells modified with specific TCR genes

[0220] 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:

[0221] 1. Preparation of effector cells: The T cells expressing the target TCR sequence prepared in Example 2 are used as effector cells, among which the TCR group is transduced with TCR lentivirus and expressed, and the NC group is not transduced;

[0222] 2. Preparation of overexpression target cells:

[0223] 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:

[0224] 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.

[0225] The specific steps include:

[0226] 1) Lentiviral transduction of target antigen gene

[0227] The density of tumor cells was adjusted to 4-6×10 using complete culture medium (the complete culture medium used for U-2OS human osteosarcoma cells was McCoy's 5A medium (Gibco) containing 10% FBS). 5 cells / ml, 1 ml / well was added to a 6-well plate, 8 μl Polybrene (1 mg / ml) was added to each well to a final concentration of 8 μg / ml, 10-30 μl of lentiviral vector containing the target antigen NLVPMVATV was added to each well, and a cell control well (untransduced group) was set up, and the control well was added with the above 1 ml tumor cells and 8 μl Polybrene, mixed, and placed in a CO2 incubator (37°C, 5% CO2) for 1 day;

[0228] Discard the supernatant, add 2 ml of complete medium to each well, and incubate in a CO2 incubator for 2 days;

[0229] 2) Puromycin selection of transduced tumor cells

[0230] The supernatant was discarded, and 2 ml of complete medium containing 1 μg / ml puromycin (Solarbio) was added to each well and incubated in a CO incubator;

[0231] The cells were observed every 2 days and the complete medium containing 1 μg / ml puromycin was replaced once. The adherent cells were passaged when they were confluent;

[0232] Use complete medium containing 1 μg / ml puromycin for screening and culture for 7 days. If there are still living cells in the control group, increase the concentration of puromycin and continue culturing until all cells in the control group die;

[0233] 3) Monoclonal culture of cells containing target antigen

[0234] Transduction and screening of tumor cells transduced with target antigens that have been cultured for more than 7 days were plated according to the limiting dilution method, 1 cell / well, 3 cells / well, cultured in complete medium containing puromycin, and the cells in the cell wells were observed. The monoclonal wells were marked and cultured continuously until the number of cells reached at least 6×10 7 When the cells are above 100, the target cells with overexpression can be obtained.

[0235] 3. In vitro killing experiment: U-2OS-PP65 cells overexpressing PP65 protein prepared in step 2 were used as target cells at 5×10 4 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, 10:1, and 20:1, respectively, and incubated at 37°C. The cells were analyzed using a real-time label-free cell function analyzer ( Real Time Cell Analyzer, RTCA, Agilent) to obtain the killing efficiency. (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.

[0236] When cells attached to the microelectrode surface cause changes in the impedance of the attached electrode interface, this change is correlated with the real-time functional state changes of the cells. Through real-time dynamic electrode impedance detection, biological information related to cell physiological functions can be obtained, including cell growth, extension, morphological changes, death and adhesion. ) The data were collected 4 hours after the effector cells were plated to evaluate the killing efficiency. The specific killing results are shown in Table 2.

[0237] Table 2

[0238]

[0239] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A T cell antigen receptor, characterized in that The T cell antigen receptor comprises CDR1α-CDR3α of the α chain and / or CDR1β-CDR3β of the β chain, wherein CDR3α comprises the amino acid sequence shown in SEQ ID NO: 3, one of SEQ ID NOs: 18-20, or comprises an amino acid sequence having at least 85% homology with one of SEQ ID NO: 3, SEQ ID NOs: 18-20; and / or wherein CDR3β comprises the amino acid sequence shown in SEQ ID NO: 6, one of SEQ ID NOs: 27-29, or comprises an amino acid sequence having at least 85% homology with one of SEQ ID NO: 6, SEQ ID NOs: 27-29, Preferably, CDR1α comprises an amino acid sequence as shown in one of SEQ ID NO: 1 and SEQ ID NO: 14-15, or comprises an amino acid sequence having at least 85% homology to one of SEQ ID NO: 1 and SEQ ID NO: 14-15; CDR2α comprises an amino acid sequence as shown in one of SEQ ID NO: 2 and SEQ ID NO: 16-17, or comprises an amino acid sequence having at least 85% homology to one of SEQ ID NO: 2 and SEQ ID NO: 16-17; CDR1β comprises an amino acid sequence as shown in one of SEQ ID NO: 4 and SEQ ID NO: 21-23, or comprises an amino acid sequence having at least 85% homology to one of SEQ ID NO: 4 and SEQ ID NO: 21-23; CDR2β comprises SEQ ID NO: 5 and SEQ ID NO: The amino acid sequence shown in any one of SEQ ID NO: 24-26, or an amino acid sequence having at least 85% homology with any one of SEQ ID NO: 5 and SEQ ID NO: 24-26.

2. The T cell antigen receptor according to claim 1, characterized in that The T cell antigen receptor specifically binds to CMV pp65.

3. The T cell antigen receptor according to claim 1, characterized in that The amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain are as follows: CDR1α: one of SSNFYA (SEQ ID NO: 1), DSSSTY (SEQ ID NO: 14), TSGFNG (SEQ ID NO: 15) CDR2α: one of MTLNGDE (SEQ ID NO: 2), IFSNMDM (SEQ ID NO: 16), NVLDGL (SEQ ID NO: 17) CDR3α: one of CASINFNKFYF (SEQ ID NO: 3), CAEFTGTASKLTF (SEQ ID NO: 18), CAVTYNNARLMF (SEQ ID NO: 19), CARNYGQNFVF (SEQ ID NO: 20) CDR1β: one of MDHEN (SEQ ID NO: 4), GTSNPN (SEQ ID NO: 21), MNHEY (SEQ ID NO: 22), DFQATT (SEQ ID NO: 23) CDR 2β: one of SYDVKM (SEQ ID NO: 5), SVGIG (SEQ ID NO: 24), SMNVEV (SEQ ID NO: 25), SNEGSKA (SEQ ID NO: 26) CDR 3β: one of CASSPLNGGATEAFF (SEQ ID NO: 6), CAWSDRAAFTDTQYF (SEQ ID NO: 27), CASSSVAGGRIEQFF (SEQ ID NO: 28), CSARDIKAQQWNIQYF (SEQ ID NO: 29). In a preferred embodiment, the amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of the TCR are as follows: CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α: CASINFNKFYF (SEQ ID NO: 3) CDR1β: MDHEN (SEQ ID NO: 4) CDR 2β: SYDVKM (SEQ ID NO: 5) CDR 3β: CASSPLNGGATEAFF (SEQ ID NO: 6) In a preferred embodiment, the amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of the TCR are as follows: CDR1α: DSSSTY (SEQ ID NO: 14) CDR2α: IFSNMDM (SEQ ID NO: 16) CDR3α: CAEFTGTASKLTF (SEQ ID NO: 18) CDR1β: GTSNPN (SEQ ID NO: 21) CDR2β: SVGIG (SEQ ID NO: 24) CDR3β: CAWSDRAAFTDTQYF (SEQ ID NO: 27) In a preferred embodiment, the amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of the TCR are as follows: CDR1α: TSGFNG (SEQ ID NO: 15) CDR2α: NVLDGL (SEQ ID NO: 17) CDR3α: CAVTYNNARLMF (SEQ ID NO: 19) CDR1β: MNHEY (SEQ ID NO: 22) CDR2β: SMNVEV (SEQ ID NO: 25) CDR3β: CASSSVAGRIEQFF (SEQ ID NO: 28) In a preferred embodiment, the amino acid sequences of CDR1α-CDR3α of the α chain and CDR1β-CDR3β of the β chain of the TCR are as follows: CDR1α: SSNFYA (SEQ ID NO: 1) CDR2α: MTLNGDE (SEQ ID NO: 2) CDR3α: CARNYGQNFVF (SEQ ID NO: 20) CDR1β: DFQATT (SEQ ID NO: 23) CDR2β: SNEGSKA (SEQ ID NO: 26) CDR3β: CSARDIKAQQWNIQYF (SEQ ID NO: 29).

4. The T cell antigen receptor according to any one of claims 1 to 3, wherein the variable region of the α chain comprises the amino acid sequence shown in SEQ ID NO: 7, 39, 41, 43 or comprises an amino acid sequence having at least 85% homology with SEQ ID NO: 7, 39, 41, 43; Preferably, the variable region of the β chain comprises the amino acid sequence shown in SEQ ID NO: 8, 40, 42, 44 or comprises an amino acid sequence having at least 85% homology with SEQ ID NO: 8, 40, 42, 44; Preferably, the α chain comprises the amino acid sequence shown in SEQ ID NO: 9, 30, 33, 36 or comprises an amino acid sequence having at least 85% homology with SEQ ID NO: 9, 30, 33, 36; Preferably, the β chain comprises the amino acid sequence shown in SEQ ID NO: 10, 31, 34, 37 or comprises an amino acid sequence having at least 85% homology with SEQ ID NO: 10, 31, 34, 37.

5. The T cell antigen receptor according to any one of claims 1 to 4, characterized in that The amino acids of the α chain and the β chain are directly or indirectly connected, preferably indirectly connected, more preferably connected through fp2A, and preferably, the amino acid sequence of fp2A is as shown in SEQ ID NO: 11; Preferably, it comprises the amino acid sequence shown in SEQ ID NO: 12, 32, 35, 38, or comprises an amino acid sequence having at least 85% homology with SEQ ID NO: 12, 32, 35, 38.

6. A nucleic acid encoding the T cell antigen receptor according to any one of claims 1 to 5.

7. An expression vector, characterized in that: The expression vector comprises the nucleic acid according to claim 6.

8. A host cell, characterized in that The host cell comprises the nucleic acid according to claim 6 or the expression vector according to claim 7.

9. An immune cell, characterized in that The immune cells express the T cell antigen receptor according to any one of claims 1-5.

10. A method for preparing immune cells, characterized in that: The preparation method comprises transducing the nucleic acid sequence encoding claim 6 into immune cells for expression.

11. A method for preparing a recombinant T cell, characterized in that: The steps include: 1) Obtaining the nucleic acid of claim 6 from CMV-positive T cell clones; 2) Isolation and culture of primary T cells; 3) delivering the nucleic acid of step 1) into the primary T cells of step 2) to obtain recombinant T cells expressing the T cell antigen receptor of any one of claims 1-5.

12. A method for preparing a T cell antigen receptor, characterized in that: The steps include: (1) obtaining the nucleic acid of claim 6 from a positive T cell clone; (2) connecting the nucleic acid obtained in step (1) to a vector to obtain an expression vector; (3) transforming the expression vector obtained in step (2) into a host cell and then inducing its expression; (4) Acquire T cell antigen receptors.

13. Use of the T cell antigen receptor according to any one of claims 1 to 5, the nucleic acid according to claim 6, the expression vector according to claim 7, the host cell according to claim 8, or the immune cell according to any one of claim 9 in the preparation of products for diagnosing, preventing or treating CMV-related diseases, or in T cell labeling, detection, cell sorting or activation.

14. A pharmaceutical composition comprising any one of the following: 1) The T cell antigen receptor according to any one of claims 1 to 5; 2) The nucleic acid of claim 6; 3) The expression vector of claim 7; 4) The host cell of claim 8; or 5) The immune cell according to claim 9.

15. A kit comprising any one of the following: 1) The T cell antigen receptor according to any one of claims 1 to 5; 2) The nucleic acid of claim 6; 3) The expression vector of claim 7; 4) The host cell according to claim 8; 5) The immune cell according to claim 9.

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