WO2021223604A1 - 一种t细胞抗原受体、其多聚体复合物及其制备方法和应用 - Google Patents

一种t细胞抗原受体、其多聚体复合物及其制备方法和应用 Download PDF

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WO2021223604A1
WO2021223604A1 PCT/CN2021/089237 CN2021089237W WO2021223604A1 WO 2021223604 A1 WO2021223604 A1 WO 2021223604A1 CN 2021089237 W CN2021089237 W CN 2021089237W WO 2021223604 A1 WO2021223604 A1 WO 2021223604A1
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acid sequence
amino acid
cell
antigen
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PCT/CN2021/089237
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French (fr)
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贾乐梅
陈花
李文忠
王嘉盛
雷蕾
刘芳
赵学强
林欣
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华夏英泰（北京）生物技术有限公司
清华大学
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Priority to CA3182499A priority Critical patent/CA3182499A1/en
Priority to US17/923,522 priority patent/US20230181639A1/en
Priority to JP2022567894A priority patent/JP2023524602A/ja
Priority to EP21800580.9A priority patent/EP4148066A4/en
Priority to KR1020227042827A priority patent/KR20230061292A/ko
Publication of WO2021223604A1 publication Critical patent/WO2021223604A1/zh

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Definitions

  • the invention relates to the technical field of biomedicine, in particular to an antibody or an antigen binding fragment thereof, a T cell antigen receptor, a multimer complex, and a preparation method and application thereof.
  • Epstein-Barr virus is a gamma herpes virus isolated from Burkitt's lymphoma (BL) samples by Epstein and Barr in 1964. It is also the first recognized tumorigenicity. Virus.
  • the primary infection of EBV mainly occurs in the epithelial cells of the human oropharynx. Due to its remarkable B lymphocyte characteristics, it can infect B cells and remain latent in memory B cells for a long time, causing the infected person to carry it for life, so it is in adults. The positive rate of virus antibodies in the serum exceeds 90%.
  • EBV infection does not pose a big threat to most people with normal immune function, for people with congenital or acquired immune deficiency, EBV can cause a variety of life-threatening diseases.
  • EBV latent infection (6 nuclear proteins EBNA1, 2, 3A, 3B and 3C, EBNA-LP and 3 latent membrane proteins LMP1, LMP2A, LMP2B) can stimulate cell proliferation and transformation.
  • EBV-PTLD EBV-related post-transplant lymphoproliferative diseases
  • Burkitt EBV-related post-transplant lymphoproliferative diseases
  • Hodgkin Hodgkin's lymphoma, HL
  • NPC nasopharyngeal carcinoma
  • gastric cancer gastric cancer
  • the latent types of EBV are divided into stages I, II, and III.
  • antiviral therapy targeting Epstein-Barr virus has not been able to achieve effective control of a variety of EBV-related diseases due to drug resistance and serious side effects.
  • EBV-CTLs have been clinically applied in EBV-related lymphoma and nasopharyngeal carcinoma, and they are found to be safe in humans and have a certain therapeutic effect.
  • CTLs cultured in vitro also limit their clinical efficacy.
  • the number of tumor-associated EBV antigen-specific T cells in CTLs is small ( ⁇ 0.05%), so large doses (greater than 10 10 ) of multiple reinfusions (4-6 times) are generally required;
  • second, longer preparations The cycle leads to a long waiting time for treatment, and the accompanying changes in T cell differentiation subgroups further lead to weak in vivo functions; in addition, the prepared cells vary from person to person, and the efficacy is difficult to guarantee. Therefore, there is an urgent need to develop more specific, stronger tumor-killing ability in vivo, and longer-lasting cellular immunotherapy targeting EBV antigen for the treatment of EBV-related nasopharyngeal carcinoma or lymphoma.
  • T cells are an important part of the acquired immune system. They mediate the elimination of pathogens, autologous diseased cells and tumors, and are the protective force that maintains the body's homeostasis.
  • T cell receptor T cell receptor, TCR
  • MHC major histocompatibility complex
  • TCR-T cell therapy is to clone tumor/virus antigen peptides with high specificity and high affinity T cell receptors, and then introduce the patient's own T cells through gene transduction technology, so that the autologous T cells can specifically recognize the tumor/virus. Antigenic peptides, and then the technology to eliminate tumors or pathogens. Compared with CAR-T, TCR-T therapy can recognize a wider range of tumor antigens (intracellular proteins and membrane proteins), presents a stronger killing effect and is accompanied by a lower cytokine storm, and has greater treatment for solid tumors. potential.
  • TCR-T therapy has been observed in clinical trials of various solid tumors such as lung cancer, colon cancer, synovial cell sarcoma, etc., but because the main targets are mostly tumor-related antigens such as CEA, gp100, MART-1 , MAGA-A3 and NY-ESO-1, etc., found off-target effects and toxic side effects.
  • tumor-related antigens such as CEA, gp100, MART-1 , MAGA-A3 and NY-ESO-1, etc.
  • EBV antigen as a foreign antigen, has strong immunogenicity and is not prone to off-target effects, and is an ideal potential target.
  • the EBV latent membrane protein LMP2A can promote cell proliferation, survival and migration and assist in epithelial-mesenchymal transition. It is one of the main EBV genes expressed in all type II and type III diseases/malignancies. According to reports in the literature, the proportion of LMP2-specific CTL cells in the reinfused EBV-CTL is closely related to its clinical efficacy. These evidences indicate that LMP2 can be used as a target for the treatment of EBV infection-related type II/III diseases such as nasopharyngeal carcinoma.
  • Patent CN1526072A discloses a method for identifying the extracellular domain of EBV tumor-related latent membrane protein and a method for selecting antibody reagents that react with it, and specifically discloses the amino acid sequence of the extracellular domain of EBV LMP2.
  • Many EBV T cell CTL epitopes are disclosed in the patent CN1269804A.
  • Patent CN108289950A discloses a T cell receptor-like antibody medicament specific to human HLA-presented EBV LMP2, wherein the antigen peptide epitope recognized by the T cell receptor is CLGGLLTMV.
  • Patent WO2017085471 discloses a TCR sequence that specifically recognizes the antigen peptide as SSCSSCPLSK.
  • non-patent document Dual non-contiguous peptide occupancy of HLA class I evoke antiviral human CD8 T cell response and form neo-epitopes with self-antigens (Ziwei Xiao et al., Sci Rep, 2017) disclosed in HLA-A*1101 individual Among them, 7/8 individuals use the same TRBV4-1 fragment as the main recognition specificity of SSC.
  • Patent CN109306005A discloses an EB virus-specific T cell antigen receptor and its application. However, none of the prior art discloses the TCR described in the present invention.
  • the present invention found that the HLA-A*0201 restricted peptide FLYALALLL, HLA-A*1101 restricted peptide SSCSSCPLSK/SSCSSCPLTK, and the HLA-A*2402 restricted peptide PYLFWLAAI and TYGPVFMSL/TYGPVFMCL of the LMP2 protein are highly immunogenic epitopes. It can trigger the body to produce specific T lymphocytes and corresponding immune responses.
  • the present invention provides a variety of T cells that can specifically bind to the EBV latency protein LMP2 peptide (including the sequence FLYALALLL, PYLFWLAAI, TYGPVFMSL/TYGPVFMCL, SSSSCPLSK/SSCSSCPLTK) Antigen receptor and its application in preparing pharmaceutical composition for treating EBV-related diseases.
  • the TCR of the present invention can specifically recognize the corresponding pMHC complex, activate TCR T cells, and then produce high levels of cytokines IFN ⁇ , IL2, and TNF ⁇ , which can significantly kill target cells and prolong the lifespan of tumor-bearing mice.
  • the first aspect of the present invention provides a determinant complementary region (CDR) that binds to the EBV latency membrane protein LMP2, and the CDR is selected from one or two of SEQ ID NO: 35-117 A combination of the above.
  • CDR determinant complementary region
  • the CDRs include CDR1 ⁇ -CDR3 ⁇ and/or CDR1 ⁇ -CDR3 ⁇ .
  • the amino acid sequence of CDR1 ⁇ is as shown in SEQ ID NO: 35-44 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 35-44.
  • the amino acid sequence of CDR2 ⁇ is as SEQ ID NO: 35-44.
  • the amino acid sequence of CDR3 ⁇ is as shown in SEQ ID NO: 55-73 Any one of them has at least 80% homology with the amino acid sequence shown in any one of SEQ ID NO: 55-73
  • the amino acid sequence of CDR1 ⁇ is as shown in any one of SEQ ID NO: 74-84 or is similar to SEQ ID NO.
  • the amino acid sequence shown in any one of 74-84 has at least 80% homology
  • the amino acid sequence of CDR2 ⁇ is as shown in any one of SEQ ID NO: 85-96 or with any one of SEQ ID NO: 85-96
  • the amino acid sequence shown has at least 80% homology
  • the amino acid sequence of CDR3 ⁇ is at least 80% of the amino acid sequence shown in SEQ ID NO: 97-117 or any one of SEQ ID NO: 97-117 Homology.
  • the CDR is selected from any of the following groups:
  • the second aspect of the present invention provides an ⁇ -chain polypeptide that binds to the EBV latency membrane protein LMP2, and the ⁇ -chain polypeptide comprises CDR1 ⁇ , CDR2 ⁇ and/or CDR3 ⁇ .
  • the amino acid sequence of CDR1 ⁇ is as shown in SEQ ID NO: 35-44 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 35-44.
  • the amino acid sequence of CDR2 ⁇ is as SEQ ID NO: 35-44.
  • the amino acid sequence of CDR3 ⁇ is as shown in SEQ ID NO: 55-73 Any one of or has at least 80% homology with the amino acid sequence shown in any one of SEQ ID NOs: 55-73.
  • the ⁇ chain polypeptide comprises any of the following groups of CDR1 ⁇ -CDR3 ⁇ :
  • the third aspect of the present invention provides a ⁇ -chain polypeptide that binds to the EBV latency membrane protein LMP2, and the ⁇ -chain polypeptide comprises CDR1 ⁇ , CDR2 ⁇ and/or CDR3 ⁇ .
  • the amino acid sequence of CDR1 ⁇ is shown in SEQ ID NO: 74-84 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 74-84.
  • the amino acid sequence of CDR2 ⁇ is shown in SEQ ID NO: 74-84.
  • the amino acid sequence of CDR3 ⁇ is shown in SEQ ID NO: 97-117 Any one of or has at least 80% homology with the amino acid sequence shown in any one of SEQ ID NOs: 97-117.
  • the ⁇ -chain polypeptide comprises any of the following groups of CDR1 ⁇ -CDR3 ⁇ :
  • the fourth aspect of the present invention provides a T cell antigen receptor, which specifically binds to the EBV latency membrane protein LMP2.
  • the binding epitope includes any one or a combination of two or more of SEQ ID NO: 29-34.
  • the binding epitope includes any one or a combination of two or more of SEQ ID NO: 29, 30, 33, or 34.
  • the EBV latency membrane protein LMP2 includes the amino acid sequence shown in SEQ ID NO: 27 and/or SEQ ID NO: 28, or includes the amino acid sequence shown in SEQ ID NO: 27 and/or SEQ ID NO: 28 at least Amino acid sequence with 80% homology.
  • the T cell antigen receptor specifically binds to the LMP2 peptide from the EBV latent period membrane protein through the presentation of major histocompatibility complex molecules (MHC).
  • MHC major histocompatibility complex molecules
  • the T cell antigen receptor includes at least one ⁇ chain variable region and/or ⁇ chain variable region.
  • the T cell antigen receptor is an ⁇ heterodimer.
  • the T cell antigen receptor comprises CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and/or CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain.
  • the amino acid sequence of CDR1 ⁇ is as shown in SEQ ID NO: 35-44 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 35-44.
  • the amino acid sequence of CDR2 ⁇ is as SEQ ID NO: 35-44.
  • the amino acid sequence shown in any one of 74-84 has at least 80% homology
  • the amino acid sequence of CDR2 ⁇ is as shown in any one of SEQ ID NO: 85-96 or with any one of SEQ ID NO: 85-96
  • the amino acid sequence shown has at least 80% homology
  • the amino acid sequence of CDR3 ⁇ is at least 80% of the amino acid sequence shown in SEQ ID NO: 97-117 or any one of SEQ ID NO: 97-117 Homology.
  • the CDR1 ⁇ -CDR3 ⁇ and CDR1 ⁇ -CDR3 ⁇ may be SEQ ID NOs: 35, 45, 55, 74, 85, and 97, respectively; or SEQ ID NOs: 35, 45, 56, 74, 85, and 98; Or SEQ ID NO: 36, 46, 57, 75, 86, and 99; or SEQ ID NO: 37, 47, 58, 76, 87, and 100; or SEQ ID NO: 38, 48, 59, 74, 88 and 101; or SEQ ID NO: 39, 49, 60, 77, 89, and 102; or SEQ ID NO: 39, 49, 61, 77, 89, and 103; or SEQ ID NO: 40, 50, 62, 78, 90, and 104; or SEQ ID NO: 37, 47, 63, 79, 91, and 105; or SEQ ID NO: 37, 47, 64, 74, 88, and 106; or SEQ ID NO: 40,
  • the CDR1 ⁇ -CDR3 ⁇ and CDR1 ⁇ -CDR3 ⁇ are selected from any of the following groups:
  • the T cell antigen receptor amino acid sequence is selected from any one of SEQ ID NO: 5-26 or has the same amino acid sequence as any one of SEQ ID NO: 5-26. At least 80% homology.
  • the ⁇ chain and ⁇ chain of the T cell antigen receptor are connected by a linker sequence, and the linker sequence is preferably a furin-SGSG-p2A sequence (hereinafter abbreviated as fp2A).
  • the linker sequence is preferably a furin-SGSG-p2A sequence (hereinafter abbreviated as fp2A).
  • connection sequence may be ⁇ chain, fp2A, ⁇ chain, or ⁇ chain, fp2A, ⁇ chain.
  • connection sequence is ⁇ chain, fp2A, and ⁇ chain.
  • the T cell antigen receptor specifically binds to the EBV latency membrane protein LMP2 through the presentation of MHC or multimeric complexes.
  • the multimeric complex comprises an antigen peptide, and the antigen peptide comprises any one or a combination of two or more of SEQ ID NO: 29-34.
  • the multimeric complex further includes a monomer, a biotin molecule, and a streptavidin molecule or avidin molecule, wherein the monomer includes the extracellular region of the alpha chain of the MHC molecule and the beta 2m chain , The monomer is coupled with the biotin molecule, and the biotin molecule is combined with the streptavidin or avidin molecule.
  • the fifth aspect of the present invention provides an antibody or antigen-binding fragment thereof, which specifically binds to the EBV latency membrane protein LMP2.
  • the binding epitope of LMP2 includes any one or a combination of two or more of SEQ ID NO: 29-34.
  • the binding epitope of LMP2 includes any one or a combination of two or more of SEQ ID NO: 29, 30, 33 or 34.
  • the antibody or antigen-binding fragment thereof may also include Fab, Fab', Fab'-SH, Fv, scFv, (Fab')2, single domain antibody, diabody (dAb) or linear antibody. Fragment.
  • the antibody or antigen-binding fragment thereof is a single-domain antibody or single-chain antibody scFv.
  • the antibody or antigen-binding fragment thereof comprises CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain and/or CDR1 ⁇ -CDR3 ⁇ of the ⁇ chain.
  • the amino acid sequence of the antibody or antigen-binding fragment thereof is selected from any one of SEQ ID NO: 5-26 or is shown in any one of SEQ ID NO: 5-26 The amino acid sequence has at least 80% homology.
  • the sixth aspect of the present invention provides a nucleic acid that encodes the aforementioned antibody or antigen-binding fragment thereof, or the aforementioned T cell antigen receptor, or the aforementioned CDR, or the aforementioned ⁇ -chain polypeptide, or the aforementioned The ⁇ -chain polypeptide.
  • the nucleotide sequence encoding the antibody or antigen-binding fragment thereof, or T cell antigen receptor is selected from any one of SEQ ID NO: 122-143 or is combined with SEQ ID NO: 122-143.
  • the nucleotide sequence shown in NO: 122-143 has at least 80% homology.
  • the nucleotide sequence may be codon optimized.
  • the codon optimization includes changing a large number of rare codons used by viruses and the like into corresponding mammalian codons and/or removing mRNA unstable motifs and/or hidden splicing sites.
  • an expression vector is provided, and the expression vector comprises the above-mentioned nucleic acid.
  • the expression vector can be expressed in vivo or in vitro or in vitro. Further preferably, the expression vector is continuously expressed at a high level in cells in vivo.
  • the expression vector is a prokaryotic expression vector or a lentiviral expression vector.
  • the prokaryotic expression vector is Escherichia coli series.
  • the expression vector is pET-26b or pET28a+.
  • the expression vector is pHAGE-IRES-RFP.
  • sequence of connecting ⁇ chain, ⁇ chain and backbone vector in the expression vector is promoter, ⁇ chain, furin-p2A, ⁇ chain, IRES and RFP sequence.
  • the eighth aspect of the present invention provides a host cell, the host cell comprising the above-mentioned nucleic acid or the above-mentioned expression vector.
  • the host cell may be eukaryotic or prokaryotic. More preferably, the host cells are yeast cells, 293 cells, CHO cells, Escherichia coli and the like.
  • the host cell is Stbl3, BL21 or transetta.
  • the ninth aspect of the present invention provides an immune cell that expresses the above-mentioned CDR, the above-mentioned ⁇ -chain polypeptide, the above-mentioned ⁇ -chain polypeptide, the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned T cell Antigen receptor.
  • the immune cells include but are not limited to lymphocytes (including T cells and B cells). Further, the immune cell is a B cell, and the B cell expresses the above-mentioned antibody or antigen-binding fragment thereof.
  • the immune cell is a T cell, and the T cell antigen receptor structure of the T cell is as defined above.
  • the tenth aspect of the present invention provides a method for preparing immune cells, comprising encoding the above-mentioned CDR, the above-mentioned ⁇ -chain polypeptide, the above-mentioned ⁇ -chain polypeptide, the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned T cell
  • the nucleic acid sequence of the antigen receptor is transfected into immune cells to be expressed.
  • the immune cells include but are not limited to lymphocytes (including T cells and B cells). Further, the immune cell is a B cell, and the B cell expresses the aforementioned antibody or antigen-binding fragment thereof.
  • the immune cell is a T cell, and the T cell antigen receptor structure of the T cell is as defined above.
  • the guide targeting the endogenous TCR can be constructed into a lentiviral vector, and it can be co-transfected to T cells with the packaging plasmid and transfection reagent.
  • the eleventh aspect of the present invention provides a method for preparing recombinant T cells, which includes the following steps:
  • step 1) The nucleic acid sequence obtained in step 1) is delivered to the primary T cell described in step 2) to obtain a recombinant T cell expressing the CDR, the ⁇ chain polypeptide, the ⁇ chain polypeptide, or the T cell antigen receptor.
  • the T cells are selected from hematopoietic stem cells or peripheral blood lymphocytes (PBL)-derived T cells.
  • PBL peripheral blood lymphocytes
  • the twelfth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof or a T cell antigen receptor, which includes the following steps:
  • step (2) Connect the nucleic acid sequence obtained in step (1) to the vector backbone to obtain an expression vector;
  • the positive T cells specifically bind to the EBV latency membrane protein LMP2 peptide presented by MHC.
  • the EBV latency membrane protein LMP2 peptide presented by MHC is a monomer or a multimeric complex.
  • the thirteenth aspect of the present invention provides a multimeric complex comprising any one of the above-mentioned T cell antigen receptors.
  • it also includes a monomer, a biotin molecule, and a fluorescently-labeled streptavidin molecule or avidin molecule, wherein the monomer includes an antigen peptide, an extracellular region of the alpha chain of an MHC molecule, and a beta 2m chain , The monomer is coupled with the biotin molecule, and the biotin molecule is combined with the streptavidin or avidin molecule.
  • the C-terminus of the extracellular region of the alpha chain of the MHC molecule is connected with an avi-tag sequence.
  • the extracellular region of the alpha chain of the MHC molecule does not contain a signal peptide sequence. And add M amino acids in front of the mature peptide sequence.
  • the ⁇ 2m chain does not contain a signal peptide sequence. And add two amino acids M and A in front of the mature peptide sequence.
  • the ⁇ 2m chain does not contain a signal peptide and two amino acids are added before the mature peptide sequence, preferably M and A.
  • the antigen peptide comprises any one or a combination of two or more of SEQ ID NO: 29-34.
  • the antigen peptide comprises any one or a combination of two or more of SEQ ID NO: 29, 30, 33 or 34.
  • the multimeric complex comprises:
  • T cell antigen receptor preferably, it is any one of SEQ ID NO: 5-26 or has at least 80% homology with the amino acid sequence shown in any one of SEQ ID NO: 5-26.
  • the monomers include antigenic peptides, the extracellular region of the MHC molecule ⁇ chain with the avi-tag sequence connected to the C-terminus, and the ⁇ 2m chain without signal peptide; the antigenic peptides are selected from SEQ ID NO: Any one of 29-34 or a combination of two or more;
  • a streptavidin molecule or avidin molecule wherein the monomer is coupled to the biotin molecule, and the biotin molecule is combined with the streptavidin or avidin.
  • the MHC molecules are MHC class I molecules or MHC class II molecules. More preferably, the MHC molecules are MHC class I molecules.
  • the MHC molecule is selected from HLA-A*0201, HLA-A*2402 and HLA-A*1101.
  • the amino acid sequence of the alpha chain of the MHC molecule is as shown in any one of SEQ ID NO: 1-3 or any one of SEQ ID NO: 1-3 Have at least 80% homology.
  • the ⁇ 2m chain of the MHC molecule is at least 80% homologous to the amino acid sequence shown in any one of SEQ ID NO: 4 or any one of SEQ ID NO: 4 sex.
  • the monomer In order to improve the binding specificity of the antigen peptide-MHC tetramer and the T cell antigen receptor, the monomer also includes chemical modification, mutation, insertion and/or deletion of at least one amino acid.
  • the extracellular region of the alpha chain of the MHC molecule and the beta 2m chain are non-covalently bonded.
  • the multimeric complex contains at least one monomer.
  • each monomer is coupled with at least one biotin molecule.
  • the fourteenth aspect of the present invention provides a method for preparing the above-mentioned polymer complex, which includes the following steps:
  • step II Refolding the antigen peptide and the MHC molecule ⁇ chain extracellular region and ⁇ 2m chain connected to the avi-tag sequence at the C-terminus obtained in step I) to prepare a monomer;
  • step IV Reacting the biotinylated monomer obtained in step III) with fluorescently labeled streptavidin or avidin to prepare an antigen peptide-MHC tetramer.
  • step IV Co-incubate the antigen peptide-MHC molecule tetramer obtained in step IV) with T cells to form a T cell antigen receptor and antigen peptide-MHC molecule tetramer complex, and catch specific T cell antigen receptors.
  • the step I) includes separately cloning the nucleotide sequence of the ⁇ chain extracellular region of the MHC molecule encoding the C-terminal avi-tag sequence and the nucleotide sequence of the ⁇ 2m chain of the MHC molecule, after being connected to the vector, Transform into expressing bacteria and cultivate, add inducer, and extract inclusion bodies.
  • the expression bacteria are cultured to an OD 600 value between 0.2 and 0.4.
  • the final molar concentration of the inducer after adding is 0.5-1 mM.
  • the expression is induced for 4-6 hours.
  • the step II) includes the refolding of the ⁇ 2m chain, that is, the antigen peptide, the ⁇ 2m chain of the MHC molecule, and the extracellular region of the MHC molecule ⁇ chain with the C-terminus connected to the avi-tag sequence are sequentially added to the dilution buffer in a light-proof water bath,
  • the refolding of the ⁇ 2m chain includes denaturation of inclusion bodies, adding protease inhibitors, and then dialysis.
  • the molar ratio of the antigen peptide, the ⁇ 2m chain without signal peptide and the ⁇ chain with the avi-tag sequence connected to the C terminal is (30-50):(2-2.5):1. More preferably, it is 40:2:1.
  • the step II) further includes a step of purifying the monomer.
  • the biotinylation in step III) is to combine the monomer with BiomixA and BiomixB under the catalysis of BirA enzyme.
  • the step III) further includes a step of purifying the biotinylated monomer.
  • the molar ratio of the monomer and streptavidin in the step IV) is (4-7):1.
  • the fifteenth aspect of the present invention provides an application of the above-mentioned multimeric complex in the preparation, screening or detection of the antibody or its antigen-binding fragment or T cell antigen receptor of the present invention.
  • the sixteenth aspect of the present invention provides the above-mentioned CDR, the above-mentioned ⁇ -chain polypeptide, the above-mentioned ⁇ -chain polypeptide, the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned T cell antigen receptor, the above-mentioned nucleic acid, and the above-mentioned expression
  • the EBV-related diseases are selected from infectious mononucleosis, linked lymphocytic proliferation syndrome, viral hematopoietic syndrome, oral hairy mucosal leukoplakia, viral meningitis, peripheral neuritis, Viral pneumonia, viral myocarditis, nasopharyngeal carcinoma, Hodgkin’s lymphoma, Burkitt’s lymphoma, gastric cancer, hepatocellular carcinoma, lymphoepithelioid sarcoma, salivary gland tumors, breast cancer, thymoma, primary exudative Lymphoma or B/T/NK cell lymphoma.
  • the seventeenth aspect of the present invention provides the aforementioned CDR, the aforementioned ⁇ -chain polypeptide, the aforementioned ⁇ -chain polypeptide, the aforementioned antibody or antigen-binding fragment thereof, the aforementioned T cell antigen receptor, the aforementioned nucleic acid, and the aforementioned expression
  • the eighteenth aspect of the present invention provides a pharmaceutical composition comprising any one of the following groups:
  • the pharmaceutical composition may also include pharmaceutically acceptable excipients.
  • the pharmaceutical composition can also be used together with other therapeutic agents.
  • the therapeutic agent may be an immunomodulator.
  • the nineteenth aspect of the present invention provides a kit, said kit comprising any one of the following groups:
  • the twentieth aspect of the present invention relates to a method for detecting EBV LMP2.
  • the method includes contacting a sample to be tested with the antibody or antigen-binding fragment thereof or T cell antigen receptor of the present invention, and then detecting EBV LMP2 Complexes formed with antibodies or antigen-binding fragments or T cell antigen receptors.
  • the detecting EBV LMP2 is detecting the presence or content of EBV LMP2.
  • the presence indicates presence or absence, and the content may be expression level or protein concentration.
  • the antibody or antigen-binding fragment thereof or T cell antigen receptor includes a detectable label.
  • the marker may be His and/or HA.
  • the method for detecting EBV LMP2 in the present invention is not a method for diagnosis of diseases.
  • the sample to be tested is not an organism or its isolated tissues or cells.
  • EBV LMP2 is present in the organism or contains a certain concentration or expression level of EBV LMP2, it is not certain that it is a disease, but a possibility.
  • the twenty-first aspect of the present invention relates to a method for treating EBV-related diseases, said method comprising administering to an individual an effective amount of 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 pharmaceutical composition.
  • the method for treating EBV-related diseases includes positioning the T cell antigen receptor of the present invention in the vicinity of EBV-related diseases (preferably tumors or metastatic tumors) to increase toxins or immunostimulants The effectiveness of.
  • EBV-related diseases preferably tumors or metastatic tumors
  • the EBV-related diseases are selected from infectious mononucleosis, linked lymphocytic proliferation syndrome, viral hematopoietic syndrome, oral hairy mucosal leukoplakia, viral meningitis, peripheral neuritis, viral pneumonia , Viral myocarditis, nasopharyngeal carcinoma, Hodgkin’s lymphoma, Burkitt’s lymphoma, gastric cancer, hepatocellular carcinoma, lymphoepithelioid sarcoma, salivary gland tumor, breast cancer, thymoma, primary exudative lymphoma or B/T/NK cell lymphoma.
  • the twenty-second aspect of the present invention relates to a method for diagnosing EBV-related diseases.
  • the method includes taking a sample, contacting the sample with the antibody or antigen-binding fragment thereof or T cell antigen receptor of the present invention, and then Detect the complex formed by EBV LMP2 and antibody or its antigen-binding fragment or T cell antigen receptor.
  • the antibody or antigen-binding fragment thereof or T cell antigen receptor includes a detectable label.
  • the TCR of the present invention can specifically recognize the corresponding EBV LMP2 antigen peptide or pMHC complex, activate TCR T cells, and then produce high levels of cytokines IFN ⁇ , IL2, and TNF ⁇ , which can significantly kill target cells and prolong tumor-bearing The life span of mice.
  • the "antigen-binding fragments" of the present invention include but are not limited to: Fab fragments, which have VL, CL, VH and CH1 domains; Fab' fragments, which have one or more cysteine at the C-terminus of the CH1 domain Fab fragments of residues; Fd fragments, which have VH and CH1 domains; Fd' fragments, which have VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; Fv fragments, which have antibodies The single-arm VL and VH domains; dAb fragments, which consist of VH or VL domains; isolated CDR regions; F(ab')2 fragments, which contain two Fabs connected by a disulfide bridge at the hinge region A bivalent fragment of a fragment; a single-chain antibody molecule (such as a single-chain Fv; scFv); a "diabody" with two antigen-binding sites, which contains the same polypeptide
  • the "CDR” in the present invention is a short fragment of immunoglobulin (Ig) or T cell antigen receptor (TCR), which binds to an epitope alone or in combination with other CDRs.
  • the immunoglobulin can be an antibody, and the CDR corresponds to the complementarity determining region in the variable sequence of the antibody. For each variable region, there are three CDRs in each variable region of the heavy chain and the light chain, which are called CDR1, CDR2, and CDR3 of the heavy chain or light chain, respectively.
  • the CDRs are present in the ⁇ chain or the ⁇ chain, and there are three CDRs in the ⁇ chain or the ⁇ chain, which are called CDR1, CDR2, and CDR2 of the ⁇ chain or ⁇ chain, respectively.
  • CDR3 The exact boundaries of these CDRs are defined differently according to different systems.
  • the system described by Kabat et al. Kabat et al. (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides clear residue numbers for antibody variable regions
  • the system also provides residue boundaries defining the three CDRs. These CDRs can be called Kabat CDRs.
  • Each complementarity determining region can contain amino acid residues from the "complementarity determining region" as defined by Kabat. Chothia et al. ( Chothia & Lesk, J. Mol. Biol, 196: 901-917 (1987) and Chothia et al., Nature 342: 877-883 (-1989)) found that some sub-parts in the Kabat CDR adopt almost the same peptide skeleton image Although there is great diversity at the amino acid sequence level. These sub-parts are called L1, L2 and L3 or H1, H2 and H3, respectively, where "L” and “H” represent light chain and heavy chain regions, respectively. These regions It can be called Chothia CDR, which has a boundary that overlaps with Kabat CDR.
  • CDR CDR boundary definitions that may not strictly follow one of the above systems, but will still overlap with Kabat CDR.
  • the method used herein can use the definitions based on any of these systems.
  • CDR although the preferred embodiment uses the CDR defined by Kabat or Chothia.
  • the residue boundaries of the CDR in the TCR are the same as those described above.
  • the "antibody variable region” refers to the light chain and the heavy chain of the antibody molecule.
  • Complementarity determining regions (CDRs, CDR1, CDR2, and CDR3) and framework regions (FR) are part of the amino acid sequence.
  • VH refers to the variable domain of the heavy chain.
  • VL refers to the variable domain of the light chain.
  • LMP2 in the present invention includes LMP2A and LMP2B.
  • LMP2A and LPM2B differ in that there is an additional N-terminal 119 amino acid cytoplasmic domain in LMP2A, except that the structures of LMP2A and LPM2B are the same.
  • both LMP2A or LMP2B contain the antigen peptide region bound by the T cell antigen receptor or antibody of the present invention.
  • the "homology" in the present invention means that in terms of using amino acid sequences or nucleotide sequences, those skilled in the art can adjust the sequence according to actual work requirements without changing the main structure or function of the original sequence.
  • 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%, 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%
  • the “having at least 80% homology with the amino acid sequence shown in SEQ ID NO: 35-44” in the present invention means that it retains binding to the EBV latency membrane protein LMP2 peptide epitope: MHC complex
  • SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44 can be adjusted according to actual work needs, such as substitution, deletion and/or insertion of one or more amino acids Wait.
  • said at least 80% includes 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%.
  • the "individual” in the present invention 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 "effective amount” in the present invention refers to the amount or dose of the product of the present invention that provides the desired treatment or prevention after being administered to a patient or organ in a single or multiple doses.
  • diagnosis in the present invention 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 finding out the progression of the disease or possible future progression, or assessing the patient's response to treatment.
  • Treatment in the present invention means slowing down, interrupting, preventing, controlling, stopping, reducing, or reversing the progress or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve all disease-related signs, The complete elimination of symptoms, disorders, or disorders, and refers to therapeutic intervention to improve the signs, symptoms, etc. of the disease or pathological state after the disease has begun to develop.
  • the "product" of the present invention includes, but is not limited to, the antibody or antigen-binding fragment thereof of the present invention, the T cell antigen receptor, the nucleic acid, the expression vector, the host cell, The immune cells or the multimeric complexes, and other reagents that assist or cooperate with the above-mentioned products.
  • the “products” of the present invention can be pharmaceutical compositions such as kits, chips, antibody conjugates or multifunctional antibodies.
  • TRBV means T cell receptor ⁇ variable region
  • TRBC means T cell receptor ⁇ constant region
  • Figure 1 SDS PAGE detection results of monomers, where M is the protein Marker, and bands 1 and 2 are monomers;
  • Figure 2 The results of tetramer staining.
  • Figure 2A shows the results of A0201-FLYALALLL-tetramer and A1101-SSCSSCPLSK-tetramer staining.
  • Figure 2B shows the results of A2402-TYGPVFMSL-tetramer staining with commercial Contrast of tetramers;
  • Figure 3 Tetramer staining results, where Figure 3A shows the staining results of A2402-TYGPVFMCL-tetramer and A2402-TYGPVFMSL-tetramer; Figure 3B shows A1101-SSCSSCPLSK-tetramer, namely A1101-SSCSSCPLTK-tetramer Staining results; Figure 3C shows the staining results of A2402-PYLFWLAAI-tetramer.
  • Figure 4 Schematic diagram of the connection of TCR ⁇ chain and ⁇ chain in pHAGE vector, where the sequence of connection is promoter, ⁇ chain, furin-p2A, ⁇ chain, IRES and RFP sequence in sequence;
  • Figure 5 Flow cytometric detection of HLA-A*A0201 FLYALALLL specific TCR (E23, E240,), HLA-A*A2402 TYGPVFM S L/TYGPVFM C L specific TCR (E44) and PYLFWLAAI specific TCR (E29, E180) -1,) the surface expression of the membrane, of which, one of the BV421 series of fluoresceins, the full name of BV is Brilliant Violet. ;
  • FIG. 6 Flow cytometric detection of HLA-A*A0201 FLYALALLL specific TCR (E23, E240,), HLA-A*A2402 TYGPVFM S L/TYGPVFM C L specific TCR (E44) and PYLFWLAAI specific TCR (E29, E180) -1,) the binding affinity of the EBV LMP2 tetramer probe;
  • Figure 7 Flow cytometric detection of the binding affinity of the HLA-A*1101 epitope SSCSSCPLSK (LSK) and SSCSSCPLTK (LTK) specific TCR to the tetramer probe;
  • Figure 8 IL-2 production of JC5-TCR cells stimulated by different concentrations of LSK peptides
  • Figure 9 EC50 statistical results of LSK epitope-specific TCR, where public TCR represents a TCR with a conserved CDR3 motif, and private TCR represents a TCR with no conserved sequence in the CDR3 region;
  • FIG. 10 IL-2 production of JC5-TCR cells stimulated by different concentrations of LTK peptides
  • Figure 11 EC50 statistical results of LTK epitope-specific TCR, where public TCR represents a TCR with a conserved CDR3 motif, and private TCR represents a TCR with no conserved sequence of the CDR3 region;
  • Figure 12 IL2 release levels of LSK-specific TCR T cells after incubation with EBV-LCL cells;
  • FIG. 13 IFN ⁇ release levels of LSK-specific TCR T cells after incubation with EBV-LCL cells
  • FIG. 14 Proliferation results of TCR T cells under long-term stimulation of excessive target cells in vitro
  • Figure 15 Detection diagram of the killing ability of TCR T cells under long-term stimulation of excessive target cells in vitro
  • FIG. 16 E23-TCRT, E240-TCRT cytokine IL2, TNF ⁇ and IFN ⁇ release levels and target cell luciferase level detection results, where E23, E240 represent the TCR prepared in Example 2, and NE represents the blank control group , NT stands for T cell group only, Ctrl stands for Raji cells not transfected with LMP2, 0.5:1, 1:1, 2:1 are effective target ratios;
  • FIG. 17 Detection results of the release levels of E29-TCRT, E180-1-TCRT cytokines IL2, TNF ⁇ and IFN ⁇ , where E29, E180-1 represent the TCR prepared in Example 2, and 1G4 represents the antigen EY-ESO- 1 is the control TCR, NE stands for the blank control group, and NT stands for the T cell group only;
  • FIG. 18 Detection results of the release levels of E44-TCRT cytokines IL2, TNF ⁇ and IFN ⁇ , where E44 represents the TCR prepared in Example 2, E9 represents the positive control TCR capable of recognizing the antigen LMP2, RFP represents the negative control group, and NE represents Blank control group, NT stands for T cell group only;
  • FIG. 19 BFP method to determine the structural affinity of public TCR results
  • FIG. 20 Alanine Scanning method analyzes the contribution of each amino acid in the E141-TCR CDR3 region to recognize antigens and kill target cells;
  • FIG. 21 Alanine Scanning method analyzes the contribution of each amino acid in the E141-TCR CDR3 region to recognize antigens and activate T cells;
  • FIG. 22 Evaluation of E23-TCR in an animal model of lymphoma, E240-TCR inhibits tumor growth in mice;
  • FIG. 23 Evaluation of E29-TCR and E44-TCR inhibiting tumor growth in mice in an animal model of lymphoma
  • Figure 25 Evaluation of E141-TCR's inhibition of tumor growth in mice in a solid tumor animal model
  • FIG. 26 Solid tumor model, statistical results of tumor growth in mice without T cell injection group (PBS), control TCR-T cell injection group (TCR-1G4), and EBV TCR-T injection group (E141-TCR);
  • FIG. 27 Solid tumor model, specific proliferation of TCR T cells in mice without T cell injection group (PBS), control TCR-T cell injection group (TCR-1G4), and EBV TCR-T injection group (E141-TCR) statistical results.
  • HLA-A*0201 with optimized expression sequence (its amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 118), HLA-A*2402 (its amino acid sequence is shown in SEQ ID NO: 2, the nucleotide sequence is shown in SEQ ID NO: 119) and HLA-A*1101 (the amino acid sequence is shown in SEQ ID NO: 3, and the nucleotide sequence is shown in SEQ ID NO: 120)
  • the ⁇ chain and ⁇ 2m chain of) (the amino acid sequence is shown in SEQ ID NO: 4, and the nucleotide sequence is shown in SEQ ID NO: 121).
  • the structure of the ⁇ chain is that the extracellular region sequence of the corresponding HLA-type ⁇ chain is linked with the Avi-tag sequence, separated by BamHI restriction sites to provide biotinylation sites.
  • the signal peptide sequence is removed from the ⁇ 2m chain, and two amino acids (M and A) are added in front of the mature peptide sequence.
  • the expression vector is PET28a+, and the expression strain is transetta or BL21.
  • the concentration of IPTG was 0.5mM, and the expression was induced for 4h. Extract ⁇ chain and ⁇ 2m chain protein inclusion bodies.
  • HLA-A*0201 type corresponds to the epitope FLYALALLL (SEQ ID NO: 29)
  • HLA-A*2402 type corresponds to the epitope PYLFWLAAI (SEQ ID NO: 30)
  • TYGPVFMSL SEQ ID NO: 31
  • TYGPVFMCL SEQ ID NO: 32
  • HLA-A*1101 type corresponds to the epitope SSCSSCPLSK (SEQ ID NO: 33), SSCSSCPLTK (SEQ ID NO: 34).
  • step 2) add the antigen peptide in step 2), the corresponding step 1) the ⁇ 2m chain renaturation protein and the ⁇ chain protein in a molar ratio of 40:2:1 in order to add to the reduction system
  • the folding reaction was 72h.
  • the obtained product was purified by Superdex75 10/300GL column. The purified products were collected and biotinylated with avidity kit, and the biotinylated monomer was purified again, and the purity of the monomer was detected by gel electrophoresis.
  • step 4) Combine the biotinylated monomer described in step 3) with APC-labeled streptavidin to obtain the corresponding tetramers, named A0201–FLYALALLL-tetramer, A2402-PYLFWLAAI-tetramer, respectively , A2402-TYGPVFMSL-tetramer, A2402-TYGPVFMCL-tetramer, A1101-SSCSSCPLSK-tetramer and A1101-SSCSSCPLTK-tetramer.
  • PBMC peripheral blood mononuclear cells
  • the SDS PAGE detection result of the monomer is shown in Figure 1.
  • the obtained monomer clearly shows that the heavy chain (the C-terminus is connected to the extracellular region of the ⁇ chain of the Avi-tag sequence) corresponds to the light chain (the ⁇ 2m chain of the signal peptide region is removed)
  • the size of the protein, and the purity is high.
  • the constructed tetramers were respectively incubated with cells infected with the corresponding HLA-type TCR.
  • Exemplary, constructed A0201-FLYALALLL-tetramer, A2402-TYGPVFMSL-tetramer, A2402-TYGPVFMCL-tetramer, A2402-PYLFWLAAI-tetramer, A1101-SSCSSCPLTK-tetramer, A1101-SSCSSCPLSK- Tetramers were respectively incubated with cells infected with the corresponding HLA-type TCR (LLL tetramer corresponds to TCR E23; AAI tetramer corresponds to TCR E29; MSL/MCL tetramer corresponds to TCR E44; LSK/LTK tetramer corresponds to TCR E141), compared with MBL’s commercial tetramer, the percentage of positive cells detected by A0201-FLYALALLL-tetramer is 70.5%, which is much higher than the commercial
  • the TCR captured by the tetramer constructed by the present invention can simultaneously recognize the wild-type epitope and the mutant epitope, and prevent immune escape.
  • both A2402 HLA-type wild-type epitope (MCL) and mutant epitope (MSL) tetramers can be recognized by TCR E44 fished by MSL tetramer.
  • both A1101 HLA-type wild-type epitope (LSK) and mutant epitope (LTK) tetramers can be recognized by TCR E141 fished by LSK tetramer.
  • Tetramers with different epitopes of the same HLA type catch the corresponding specific TCRs.
  • the AAI tetramers can detect cells that are positive for TCR and E29.
  • the reverse transcription primer is: TRBC1-TCAGGCAGTATCTGGAGTCATTG (SEQ ID NO: 144)
  • the PCR amplification primers are:
  • Upstream primer 1 TRBV_F1 (see SEQ ID NO: 147-185 and 366)
  • Upstream primer 2 TRBV_F2 (see SEQ ID NO: 186-225)
  • Downstream primer 1 TRBC2-GCACCTCCTTCCCATTCACC (SEQ ID NO: 145)
  • Downstream primer 2 TRBC3-GCTTCTGATGGCTCAAACACAG (SEQ ID NO: 146)
  • a PCR system is 20 ⁇ L, the annealing temperature is 60° C., and the reaction is 30 cycles. Take 1 ⁇ L of the product of the first round of PCR as a template for the second round of PCR.
  • the second round of PCR system is 30 ⁇ L, the annealing temperature is 60° C., and the reaction is carried out for 30 cycles. Then the second round of PCR products were run on agarose gel, and the corresponding size bands were cut and recovered (Tiangen Gum Recovery Kit), and sent for sequencing.
  • the sequencing primer was downstream primer 2.
  • TCR ⁇ gene sequence specific E23, E240, E29, E180-1, E44, E141, E149, E168, E170, E244, E245, E254, E301, E304, E305, E307, E314, E315, E316, E317, E318
  • the TCR ⁇ gene sequence of E320 is shown in the "double underlined" nucleotide sequence of SEQ ID NO: 122-143, respectively.
  • the reverse transcription primer is: TRAC1-CGACCAGCTTGACATCACAG (SEQ ID NO: 226)
  • the PCR amplification primers are:
  • Upstream primer 3 TRAV_F1 (see SEQ ID NO: 229-273)
  • Upstream primer 4 TRAV_F2 (see SEQ ID NO: 274-315)
  • Downstream primer 3 TRAC2-GTTGCTCTTGAAGTCCATAGACCTC (SEQ ID NO: 227)
  • Downstream primer 4 TRAC3-CAGGGTCAGGGTTCTGGATA (SEQ ID NO: 228)
  • a PCR system is 20 ⁇ L, the annealing temperature is 60° C., and the reaction is 30 cycles. Take 1 ⁇ L of the product of the first round of PCR as a template for the second round of PCR.
  • the second round of PCR system is 30 ⁇ L, the annealing temperature is 60° C., and the reaction is carried out for 30 cycles.
  • the second round of PCR products were run on agarose gel, and the corresponding size bands were cut and recovered (Tiangen Gum Recovery Kit) and sent for sequencing.
  • the sequencing primer was downstream primer 4.
  • TCR ⁇ gene sequence specific E23, E240, E29, E180-1, E44, E141, E149, E168, E170, E244, E245, E254, E301, E304, E305, E307, E314, E315, E316, E317, E318
  • the TCR ⁇ gene sequence of E320 is shown in the nucleotide sequence of the "wave underlined" in SEQ ID NO: 122-143, respectively.
  • TCR ⁇ , fp2A, TCR ⁇ were amplified by long primers (including fp2A sequence) by overlap-PCR (KOD-Plus-Neo, TOYOBO) to obtain TCR ⁇ -fp2A-TCR ⁇ fragments, named E23, E240, E29, E180-1, E44 , E141, E149, E168, E170, E244, E245, E254, E301, E304, E305, E307, E314, E315, E316, E317, E318, E320 pHAGE-TCR plasmids.
  • the primers for amplification are:
  • Downstream primer 6 agggatcctctagactcgagctagcTCAGCTGGACCACAGCCGCA (SEQ ID NO: 361)
  • the PCR system is 50 ⁇ L
  • the annealing temperature is 60° C.
  • the reaction is carried out for 30 cycles.
  • Recover the PCR product by running gel (Tiangen Gum Recycling Kit), take 1 ⁇ L of the recovered product as a template, and use the upstream primer 5 and downstream primer 6 to perform overlap PCR.
  • the PCR system is 50 ⁇ L
  • the annealing temperature is 60°C
  • the reaction is 30 cycles.
  • Run the agarose gel to obtain a band of about 1800 bp, which is cut and recovered.
  • the lentiviral vector pHAGE-IRES-RFP was double digested with NotI and NheI.
  • the digestion system is 40 ⁇ L, which contains 1.5 ⁇ L of NotI and NheI respectively.
  • the plasmid is 2-3 ⁇ g, digested at 37°C for 6h, and then 1 ⁇ L of alkaline phosphatase (NEB) is added to the system and treated for 1h to reduce the self-ligation of the plasmid.
  • the cut plasmid is recovered by running gel, and the concentration is measured with nanodrop, which is used as a backbone for plasmid construction.
  • the sequencing primers are the primers seq-pHAGE-F and seq-pHAGE-R on the pHAGE carrier, and the downstream primer 4.
  • Example 3 pMHC tetramer staining method to detect TCR membrane expression and affinity
  • the guide sequence (TRA_oligo1-CACCGTCTCTCAGCTGGTACACGGC (SEQ ID NO: 362), TRA_oligo2-AAACGCCGTGTACCAGCTGAGAGAC (SEQ IDNO: 363), TRB_oligo1-CACCGGGCTCAAATRCAB_GColigo1-CACCGGGCTCAAATRCAB_GColigo1-CACCGGGCTCAAATRCAB_GC:CC364) was designed in the constant regions of the ⁇ chain and ⁇ chain. AAACGAGGTCGCTGTGTTTGAGCCC) (SEQ ID NO: 365).
  • the guide sequences of the synthetic ⁇ chain and ⁇ chain were constructed into sgRNA-LentiCRISPR-puro and sgRNA-LentiCRISPR-BSD lentiviral vectors, respectively, and co-transfected 293T with the packaging plasmids psPAX2, pMD2.G and PEI transfection reagent in a certain ratio
  • Cells were collected for 48h and 72h cell culture supernatants, and the two concentrated viruses simultaneously infect the human JurkatT cell line. After 48 hours of infection, they were killed with appropriate concentrations of puromycin and blasticidin until all control cells of the two drugs died. The surviving cells were cultured in 96-well plates by flow sorting single cells. For the obtained monoclonal cell line, its expression was identified with TCR ⁇ chain and ⁇ chain antibodies respectively.
  • the cell line with defects in both chains is the obtained Jurkat T cell with endogenous TCR knockout, named JC5.
  • pHAGE-TCR plasmids such as E23 and E240 constructed in Example 2 were mixed with the packaging plasmids psPAX2, pMD2.G and the transfection reagent PEI according to a certain ratio to transfect 293T cells.
  • Example 3 In view of the fact that the pMHC tetramer in Example 3 detects the structural affinity of TCR, and the tetramer is tetravalently bound to the TCR on the surface of JC5.
  • TCR we stably integrated HLA-A*1101 molecules in T2 cells and constructed T2-HLA-A*1101 cell line to quantify the half-maximal effect antigen concentration (EC50) of TCR. Functional activity.
  • the synthesized LMP2 epitope was diluted with DMSO solvent to a storage concentration of 4 mg/mL. Then the epitope peptides were serially diluted with complete medium to obtain 2 ⁇ 10 -8 -2 ⁇ 10 -4 M LSK and LTK peptide solutions, which were added to 1 ⁇ 10 6 cells in a volume of 1:100. In mL of T2-HLA-A*1101 cell suspension, mix well and spread the cells in a 96-well plate with 100 ⁇ L per well. At the same time, add 100 ⁇ L of JC5-TCR cells at a concentration of 1 ⁇ 10 6 cells/mL, and mix well. Then is the T2 incubation system with the peptide concentration of 1 ⁇ 10 -10 -1 ⁇ 10 -6 M.
  • FIG. 8 and Figure 10 respectively represent the IL2 production of JC5-TCR cells stimulated by different concentrations of LSK and LTK peptides.
  • the corresponding EC50 value can be calculated by prism.
  • the EC50 value of three repetitions is shown in Fig. 9 and Fig. 11. It can be seen from Figures 8-11 that for the synthesized LSK epitope, TCR E149, E304, E170, and E315 have excellent functional activities, while for the LTK epitope, E149, E254, E170, E316, E317, E318 all show better Good functional activity.
  • the results of E244 and E307 are consistent with the tetramer staining, and both have weaker recognition ability for the SSCSSCPL S K/SSCSSCPL T K epitope.
  • Example 5 Construction of human primary TCR T cells and in vitro functional testing
  • PBMC mononuclear cells
  • the infection method was centrifugation at 1500 rpm at 32°C for 2 hours. Change the medium to terminate the infection, and continue to place it in a 37°C cell incubator for culture. Three days after infection, TCR-positive cells can be sorted out by flow cytometry to obtain TCRT cells (including the above-mentioned E23, E240, E29, E180-1, E44, E141, E149, E168, E170, E244, E245, E254, E301 , E304, E305, E307, E314, E315, E316, E317, E318, E320).
  • the virus particles loaded with LMP2-RFP, HLA-A*0201-BSD/HLA-A*2402-BSD/HLA-A*1101-BSD and Luciferase-GFP were used to infect Raji cells in logarithmic growth phase with a lentiviral system. .
  • Raji cells stably expressing both LMP2, HLA-A molecules and Luciferas-GFP were obtained, named Raji-HLA-A*A0201/2402/1101-LMP2-luciferase.
  • virus particles of HLA-A*0201-BSD/HLA-A*2402-BSD/HLA-A*1101-BSD were infected with logarithmic growth phase EBV-LCL cells.
  • EBV-LCL cells stably expressing HLA-A molecules were obtained, named EBV-LCL-HLA-A*0201, EBV-LCL-HLA-A*2402, EBV-LCL-HLA-A*1101 cells, respectively .
  • EBV-LCL is the immortalized human B cell after being infected by the Epstein-Barr virus, which more realistically mimics the antigen level in tumor cells in the body. Therefore, the TCR T cells and EBV-LCL-HLA-A*1101 cells that recognize the SSCSSCPL S K/SSCSSCPL T K epitope will be 8:1, 4:1, 2:1, 1:1, 0.5 according to the effective target ratio. :1, 0.25:1 for co-cultivation, in which the target cells are fixed to 1 ⁇ 10 5 cells. After a total of 24 hours of incubation, the supernatant was collected to detect the secreted cytokines IL2 ( Figure 12) and IFN- ⁇ ( Figure 13).
  • TCR E141, E170, E254 and E315 combined with endogenous levels of LSK can significantly activate T cells.
  • E315TCR T cells not only exhibited the best IFN- ⁇ levels at various target concentration ratios, but also had higher IL2 values. The results show that the TCR prepared by the present invention can effectively mediate the recognition of endogenous tumor antigens.
  • TCR T cells and Raji-HLA-A*1101-LMP2-luciferase cells, which recognize the epitope of SSCSSCPL S K/SSCSSCPL T K were co-cultured according to the effective target ratio of 1:3.
  • Cells were collected for flow analysis on day 1, 3, and 5.
  • the medium used was 1640 complete medium without IL2, and the initial TCR T cells were 1 ⁇ 10 5 cells.
  • the samples at each time point were incubated independently, and the remaining co-culture was performed on 2 and 4 days. Incubate the sample for half of the medium and add the target cells.
  • the cells used for flow cytometry are first stained with anti-human CD3 antibody, and the cells of a specified volume are collected and recorded on the machine, and the number of T cells in the system can be obtained by conversion (see Figure 14). It can be seen from the proliferation curve of the absolute number of T cells that E315-TCR T cells have the best activation and proliferation after recognizing two epitopes. In addition, further analysis of the effective target ratio in the system (see Figure 15) showed that the same as the proliferation results, E315-TCRT cells showed the strongest tumor clearance ability.
  • E23-TCRT and E240-TCRT cells constructed in the examples of the present invention can be specifically activated by EBV LMP2 antigen peptide presenting cells, and can significantly kill target cells.
  • the Raji-HLA-A*2402-antigenic peptides, Raji cells without antigenic peptides, T2-HLA2402 cells without antigen peptides, and E29-TCRT, E180-1-TCRT cells, and 1G4T cells were combined with E29-TCRT, E180-1-TCRT cells, and 1G4T cells respectively. After co-cultivation at a ratio of 1:3, the cells and supernatant were collected after 24 hours of co-cultivation, and the activation of E29-TCR T and 180-1-TCRT cells was preliminarily detected.
  • TCR positive cells can be sorted out by flow cytometry.
  • the sorted TCR T cells and Raji-LMP2-luciferase target cells were co-cultured according to an effective target ratio of 1:1, and the T cells were 1 ⁇ 10 5 cells. After a total of 24 hours of incubation, the cells and the supernatant were collected.
  • Example 7 Animal model construction and EBV TCRT in vivo function test
  • Epstein-Barr virus mainly infects nasopharyngeal epithelial cells and B cells, and is closely related to the occurrence and development of nasopharyngeal carcinoma and a variety of B-cell lymphomas.
  • a B-cell lymphoma mouse model and a nasopharyngeal carcinoma solid tumor model were constructed respectively to verify the in vivo function of the identified TCR. 1. Lymphoma model and TCRT in vivo function test
  • mice 3 ⁇ 10 5 Raji-HLA-A*1101/0201/2402-LMP2-luciferase tumor cells were inoculated into NOD/Scid IL-2R ⁇ null (NCG) female mice for 5-6 weeks through the tail vein to construct a lymphoma model (see Figure 22, 23, 24), marked as the first day, on the fifth day, the mice were divided into 3 groups, respectively A: PBS injection group (injection of equal volume of PBS); B: control TCRT cell injection group (TCR -1G4 T cells); C: EBV TCRT injection group (E141-TCRT cells), mice in group B/C were injected with 5 ⁇ 10 6 TCR T cells through the tail vein, group A was injected with an equal volume of 200 ⁇ L PBS.
  • A PBS injection group (injection of equal volume of PBS)
  • B control TCRT cell injection group (TCR -1G4 T cells)
  • C EBV TCRT injection group (E141-TCRT cells) mice in group B/C were
  • the second injection was performed on the 8th day, and the specific operation was the same as that on the 5th day.
  • the specific reinfusion volume and reinfusion time of other TCR T cells are shown in Figure 22 and 23.
  • the EBV-specific E141-TCRT cells constructed in the examples of the present invention can significantly kill tumor cells in mice and improve the survival rate of mice.
  • the EBV-specific E23-TCRT, E240-TCRT, E29-TCRT, and E44-TCRT cells constructed in the examples of the present invention can also significantly kill tumor cells in mice. , Improve the survival rate of mice.
  • mice 1 ⁇ 10 6 C666-1-HLA-A*1101-LMP2-luciferase tumor cells were subcutaneously inoculated into NCG female mice aged 5-6 weeks to construct a solid tumor model of nasopharyngeal carcinoma (see Figure 25), which was marked as 0 Day and 7 days later, the mice were divided into 3 groups, namely A: PBS injection group (injected equal volume of PBS); B: control TCR-T cell injection group (TCR-1G4T cells); C: EBV TCR-T In the injection group (E141-TCRT cells), mice in the B/C group were injected with 3 ⁇ 10 6 T cells through the tail vein, and the mice in the A group were injected with an equal volume of 200 ⁇ L PBS.
  • A PBS injection group (injected equal volume of PBS)
  • B control TCR-T cell injection group (TCR-1G4T cells)
  • C EBV TCR-T In the injection group (E141-TCRT cells)
  • mice in the B/C group were
  • the EBV-specific E141-TCRT cells constructed in the examples of the present invention can significantly kill tumor cells in mice ( Figure 25, Figure 26), and reinfused The TCR T cells have undergone specific proliferation in vivo ( Figure 27).

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Abstract

提供了一种抗体或其抗原结合片段、T细胞抗原受体、表达T细胞抗原受体(TCR)的免疫细胞，及其制备方法和应用。所述的TCR能够特异性地识别对应的pMHC复合物，激活TCR T细胞，进而产生高水平的细胞因子IFNγ、IL2、TNFα，显著地杀伤靶细胞并延长负荷肿瘤的小鼠的寿命。

Description

一种T细胞抗原受体、其多聚体复合物及其制备方法和应用 技术领域
本发明涉及生物医药技术领域，具体涉及一种抗体或其抗原结合片段、T细胞抗原受体、多聚体复合物，及其制备方法和应用。
背景技术
EB病毒(Epstein-Barr virus，EBV)是Epstein和Barr于1964年从伯基特氏淋巴瘤(Burkitt’s lymphoma，BL)的样本中分离的一种γ疱疹病毒，也是第一个被公认的致瘤病毒。EBV原发感染主要发生在人类口咽部的上皮细胞，后因其显著的嗜B淋巴细胞特性，能感染B细胞并在记忆B细胞中长期潜伏存在，导致被感染者终身携带，故而在成人血清中病毒抗体的阳性率超过90％。尽管EBV感染对绝大多数免疫功能正常的人并不会构成太大的威胁，但对于患有先天性或获得性免疫缺陷的人群，EBV会导致多种危及生命的疾病。此外，EBV潜伏感染表达的蛋白(6个核蛋白EBNA1、2、3A、3B和3C，EBNA-LP以及3个潜伏期膜蛋白LMP1、LMP2A、LMP2B)能刺激细胞增生和转化，已被证实与多种恶性肿瘤的发生、发展、临床预后直接相关，包括EBV相关的移植后淋巴细胞增生性疾病(EBV related post-transplant lymphoproliferative diseases，EBV-PTLD)、伯基特氏淋巴瘤、霍奇金淋巴瘤(Hodgkin’s lymphoma，HL)、鼻咽癌(Nasopharyngeal carcinoma，NPC)、胃癌(Gastric cancer)等。根据潜伏蛋白在这些疾病中的差异表达，将EBV潜伏型分为I、II、III期。目前，靶向EB病毒的抗病毒治疗由于耐药性以及严重副作用等问题，尚不能实现对多种EBV相关疾病的有效控制。
Papadopounds等最先发现，输注EBV健康携带者的淋巴细胞可以治愈PTLD患者，随后临床发展出自体EBV-CTLs细胞疗法。目前EBV-CTLs已在EBV相关淋巴瘤和鼻咽癌中开展了临床应用，发现其人体内安全性良好并有一定的治疗效果，但体外培养的CTLs的诸多不足也限制了其临床疗效。首先，CTLs中肿瘤相关EBV抗原的特异性T细胞数量较少(<0.05％)，因此一般需进行大剂量(大于10 10)的多次回输(4-6次)；其次，较长的制备周期导致等待治疗时间长，伴随着的T细胞分化亚群的变化进一步导致其体内功能弱；此外，制备的细胞因人而异，疗效难以保证。因此，亟待开发更特异性、体内杀伤肿瘤能力更强、更持久的靶向EBV抗原的细胞免疫疗法，用于EBV相关鼻咽癌或淋巴瘤的治疗。
T细胞是获得性免疫系统的重要组成部分，介导了病原体、自身病变细胞和肿瘤的清除，是维持机体稳态的防护力量。T细胞受体(T cell receptor，TCR)是T细胞的身份标识，TCR通过对靶细胞主要组织相容性复合体(MHC)及所提呈的抗原复合体的识别，引发T细胞的激活和一系列后续细胞 信号传递和其他生理反应，从而使得抗原特异性的T细胞对其靶细胞发挥免疫效应。TCR-T细胞疗法是将肿瘤/病毒抗原肽高特异性、高亲和力的T细胞受体克隆后，通过基因转导技术导入患者自体的T细胞，使自体T细胞可以特异的识别肿瘤/病毒的抗原多肽，进而清除肿瘤或病原体的技术。与CAR-T相比，TCR-T疗法可识别更广泛的肿瘤抗原(胞内蛋白和膜蛋白)，呈现更加强的杀伤作用的同时伴随较低细胞因子风暴，对实体瘤的治疗有更大的潜力。目前TCR-T疗法已在肺癌、结肠癌、滑膜细胞肉瘤等多种实体肿瘤的临床试验中观察到显著疗效，但因为主要针对的靶点多为肿瘤相关抗原如CEA、gp100、MART-1、MAGA-A3和NY-ESO-1等，发现了脱靶效应和毒副作用。对于EBV感染相关的恶性疾病，EBV抗原作为外来抗原，有较强的免疫原性并且不易发生脱靶效应，是理想的潜在靶标。
EBV潜伏膜蛋白LMP2A能促进细胞的增殖、存活和迁移并协助上皮间质转化，是所有II型和III型疾病/恶性肿瘤中表达的EBV主要基因之一。据文献报道，在回输的EBV-CTL中，LMP2特异性的CTL细胞比例与其临床疗效密切相关，这些证据表明LMP2可以作为EBV感染相关的II/III型疾病如鼻咽癌治疗的靶点。例如：专利CN1526072A公开了EBV肿瘤相关潜伏膜蛋白的胞外结构域的鉴定方法和与之反应的抗体试剂的选择方法，并具体公开了EBV LMP2胞外域的氨基酸序列。专利CN1269804A中公开的众多EBV的T细胞CTL表位。专利CN108289950A公开了一种特异于人HLA呈递的EBV LMP2的T细胞受体样抗体药剂，其中所述的T细胞受体识别的抗原肽表位为CLGGLLTMV。专利WO2017085471公开了一种特异识别抗原肽为SSCSSCPLSK的TCR序列。此外，非专利文献Dual non-contiguous peptide occupancy of HLA class I evoke antiviral human CD8 T cell response and form neo-epitopes with self-antigens(Ziwei Xiao等，Sci Rep，2017)公开了在HLA-A*1101个体中，有7/8的个体利用相同的TRBV4-1片段作为SSC的主要识别特异性。专利CN109306005A公开了一种EB病毒特异性T细胞抗原受体及其应用。但是，现有技术中均未公开本发明所述的TCR。
发明内容
本发明发现LMP2蛋白的HLA-A*0201限制肽FLYALALLL，HLA-A*1101限制肽SSCSSCPLSK/SSCSSCPLTK，以及HLA-A*2402限制肽PYLFWLAAI和TYGPVFMSL/TYGPVFMCL是免疫原性较强的抗原表位，可以引发机体产生特异的T淋巴细胞和相应的免疫反应，因此，本发明提供了多种可以特异性结合EBV潜伏期蛋白LMP2肽(包含序列FLYALALLL、PYLFWLAAI、TYGPVFMSL/TYGPVFMCL、SSCSSCPLSK/SSCSSCPLTK)的T细胞抗原受体及其在制备治疗与EBV相关疾病的药物组合物中的应用。本发明所述的TCR能够特异性地识别对应的pMHC复合物，激活TCR T细胞，进而产生高水平的细胞因子IFNγ、IL2、TNFα，显著地杀伤靶细胞并延长负荷肿瘤的小鼠的寿命。
具体的，本发明的第一方面，提供了一种与EBV潜伏期膜蛋白LMP2结合的决定簇互补区(CDR)，所述的CDR选自SEQ ID NO：35-117中的一种或两种以上的组合。
优选的，所述的CDR包括CDR1α-CDR3α和/或CDR1β-CDR3β。
其中，CDR1α的氨基酸序列如SEQ ID NO：35-44中的任一种或与SEQ ID NO：35-44中任一种所示氨基酸序列具有至少80％同源性，CDR2α的氨基酸序列如SEQ ID NO：45-54中的任一种或与SEQ ID NO：45-54中任一种所示氨基酸序列具有至少80％同源性，CDR3α的氨基酸序列如SEQ ID NO：55-73中的任一种或与SEQ ID NO：55-73中任一种所示氨基酸序列具有至少80％同源性，CDR1β的氨基酸序列如SEQ ID NO：74-84中的任一种或与SEQ ID NO：74-84中任一种所示氨基酸序列具有至少80％同源性，CDR2β的氨基酸序列如SEQ ID NO：85-96中的任一种或与SEQ ID NO：85-96中任一种所示氨基酸序列具有至少80％同源性，CDR3β的氨基酸序列如SEQ ID NO：97-117中的任一种或与SEQ ID NO：97-117中任一种所示氨基酸序列具有至少80％同源性。
在本发明的一个具体实施方式中，所述的CDR选自下列任一组：
Figure PCTCN2021089237-appb-000001
Figure PCTCN2021089237-appb-000002
Figure PCTCN2021089237-appb-000003
本发明的第二方面，提供了一种与EBV潜伏期膜蛋白LMP2结合的α链多肽，所述的α链多肽包含CDR1α、CDR2α和/或CDR3α。
其中，CDR1α的氨基酸序列如SEQ ID NO：35-44中的任一种或与SEQ ID NO：35-44中任一种所示氨基酸序列具有至少80％同源性，CDR2α的氨基酸序列如SEQ ID NO：45-54中的任一种或与SEQ ID NO：45-54中任一种所示氨基酸序列具有至少80％同源性，CDR3α的氨基酸序列如SEQ ID NO：55-73中的任一种或与SEQ ID NO：55-73中任一种所示氨基酸序列具有至少80％同源性。
优选的，所述的α链多肽包含下列任一组的CDR1α-CDR3α：
Figure PCTCN2021089237-appb-000004
Figure PCTCN2021089237-appb-000005
本发明的第三方面，提供了一种与EBV潜伏期膜蛋白LMP2结合的β链多肽，所述的β链多肽包含CDR1β、CDR2β和/或CDR3β。
其中，CDR1β的氨基酸序列如SEQ ID NO：74-84中的任一种或与SEQ ID NO：74-84中任 一种所示氨基酸序列具有至少80％同源性，CDR2β的氨基酸序列如SEQ ID NO：85-96中的任一种或与SEQ ID NO：85-96中任一种所示氨基酸序列具有至少80％同源性，CDR3β的氨基酸序列如SEQ ID NO：97-117中的任一种或与SEQ ID NO：97-117中任一种所示氨基酸序列具有至少80％同源性。
在本发明的一个具体实施方式中，所述的β链多肽包含下列任一组的CDR1β-CDR3β：
Figure PCTCN2021089237-appb-000006
Figure PCTCN2021089237-appb-000007
本发明的第四方面，提供了一种T细胞抗原受体，所述的T细胞抗原受体特异性结合EBV潜伏期膜蛋白LMP2。
优选的，结合表位包含SEQ ID NO：29-34中的任一种或两种以上的组合。
进一步优选的，结合表位包含SEQ ID NO：29、30、33或34中的任一种或两种以上的组合。
进一步优选的，所述的EBV潜伏期膜蛋白LMP2包含SEQ ID NO：27和/或SEQ ID NO：28所示的氨基酸序列，或者包含与SEQ ID NO：27和/或SEQ ID NO：28具有至少80％同源性的氨基酸序列。
优选的，所述的T细胞抗原受体通过主要组织相容性复合物分子(MHC)的呈递，与来自EBV潜伏期膜蛋白LMP2肽特异性结合。
优选的，所述的T细胞抗原受体至少包含一个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。
优选的，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，CDR1α的氨基酸序列如SEQ ID NO：35-44中的任一种或与SEQ ID NO：35-44中任一种所示氨基酸序列具有至少80％同源性，CDR2α的氨基酸序列如SEQ ID NO：45-54中的任一种或与SEQ ID NO：45-54中任一种所示氨基酸序列具有至少80％同源性，CDR3α的氨基酸序列如SEQ ID NO：55-73中的任一种或与SEQ ID NO：55-73中任一种所示氨基酸序列具有至少80％同源性，CDR1β的氨基酸序列如SEQ ID NO：74-84中的任一种或与SEQ ID NO：74-84中任一种所示氨基酸序列具有至少80％同源性，CDR2β的氨基酸序列如SEQ ID NO：85-96中的任一种或与SEQ ID NO：85-96中任一种所示氨基酸序列具有至少80％同源性，CDR3β的氨基酸序列如SEQ ID NO：97-117中的任一种或与SEQ ID NO：97-117中任一种所示氨基酸序列具有至少80％同源性。
优选的，所述的CDR1α-CDR3α及CDR1β-CDR3β可以分别为SEQ ID NO：35、45、55、74、85及97；或者为SEQ ID NO：35、45、56、74、85及98；或者为SEQ ID NO：36、46、57、75、86及99；或者为SEQ ID NO：37、47、58、76、87及100；或者为SEQ ID NO：38、48、59、74、88及101；或者为SEQ ID NO：39、49、60、77、89及102；或者为SEQ ID NO：39、49、61、77、89及103；或者为SEQ ID NO：40、50、62、78、90及104；或者为SEQ ID NO：37、47、63、79、91及105；或者为SEQ ID NO：37、47、64、74、88及106；或者为SEQ ID NO：40、50、65、78、90及107；或者为SEQ ID NO：39、49、60、80、92及108；或者为SEQ ID NO：39、49、60、77、89及109；或者为SEQ ID NO：39、49、61、77、89及110；或者为SEQ ID NO：40、50、66、81、93及111；或者为SEQ ID NO：41、51、67、82、94及112；或者为SEQ ID NO：39、49、68、77、89及102；或者为SEQ ID NO：42、52、69、83、95及113；或者为SEQ ID NO：43、53、70、76、87及114；或者为SEQ ID NO：44、54、71、74、88及115；或者为SEQ ID NO：37、47、72、84、96及116；或者为SEQ ID NO：40、50、73、82、94及117。
在本发明的一个具体实施方式中，所述的CDR1α-CDR3α及CDR1β-CDR3β选自下列任一组：
Figure PCTCN2021089237-appb-000008
Figure PCTCN2021089237-appb-000009
Figure PCTCN2021089237-appb-000010
Figure PCTCN2021089237-appb-000011
在本发明的一个具体实施方式中，所述T细胞抗原受体氨基酸序列选自SEQ ID NO：5-26中的任一种或与SEQ ID NO：5-26任一种所示氨基酸序列具有至少80％同源性。
优选的，所述T细胞抗原受体的α链和β链通过linker序列连接，linker序列优选为furin-SGSG-p2A序列(以下简写fp2A)。
优选的，所述的连接顺序可以为α链、fp2A、β链，或者β链、fp2A、α链。
在本发明的一个具体实施方式中，所述的连接顺序为β链、fp2A、α链。
优选的，所述的T细胞抗原受体通过MHC或多聚体复合物的呈递，与EBV潜伏期膜蛋白LMP2特异性结合。其中，所述的多聚体复合物包含抗原肽，所述的抗原肽包含SEQ ID NO：29-34中的任一种或两种以上的组合。优选的，所述的多聚体复合物还包括单体，生物素分子，以及链霉亲和素分子或亲和素分子，其中，所述单体包括MHC分子α链胞外区和β2m链，所述单体与所述生物素分子偶联，所述生物素分子与所述链霉亲和素或亲和素分子结合。
本发明的第五方面，提供了一种抗体或其抗原结合片段，所述的抗体或其抗原结合片段特异性结合EBV潜伏期膜蛋白LMP2。
优选的，所述LMP2的结合表位包含SEQ ID NO：29-34中的任一种或两种以上的组合。
进一步优选的，所述LMP2的结合表位包含SEQ ID NO：29、30、33或34中的任一种或两种以上的组合。
优选的，所述的抗体或其抗原结合片段还可以包含如Fab、Fab’、Fab’-SH、Fv、scFv、(Fab’)2、单结构域抗体、双抗体(dAb)或线性抗体的片段。
优选的，所述的抗体或其抗原结合片段为单域抗体或单链抗体scFv。
优选的，所述抗体或其抗原结合片段包含α链的CDR1α-CDR3α和/或β链的CDR1β-CDR3β。在本发明的一个具体实施方式中，所述的抗体或其抗原结合片段的氨基酸序列选自SEQ ID NO：5-26中的任一种或与SEQ ID NO：5-26任一种所示氨基酸序列具有至少80％同源性。
本发明的第六方面，提供了一种核酸，所述的核酸编码上述的抗体或其抗原结合片段，或者上述的T细胞抗原受体，或者上述的CDR，或者上述的α链多肽，或者上述的β链多肽。
在本发明的一个具体实施方式中，所述的编码抗体或其抗原结合片段，或者T细胞抗原受体的核苷酸序列选自SEQ ID NO：122-143中的任一种或与SEQ ID NO：122-143任一种所示核苷酸序列具有至少80％同源性。
优选的，所述的核苷酸序列可以是经密码子优化的。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。
本发明的第七方面，提供了一种表达载体，所述的表达载体包含上述的核酸。
优选的，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。
优选的，所述的表达载体为原核表达载体或慢病毒表达载体。
进一步优选的，所述的原核表达载体为大肠杆菌系列。在本发明的一个具体实施方式中，所述的表达载体为pET-26b或pET28a+。
在本发明的一个具体实施方式中，所述的表达载体为pHAGE-IRES-RFP。
进一步优选的，所述的表达载体中β链、α链与骨架载体的连接顺序为启动子、β链、furin-p2A、α链、IRES和RFP序列。
本发明的第八方面，提供了一种宿主细胞，所述的宿主细胞包含上述的核酸或上述的表达载体。
优选的，所述的宿主细胞可以是真核的或原核的。更优选的，所述的宿主细胞为酵母细胞、293细胞、CHO细胞、大肠杆菌等。
在本发明的一个具体实施方式中，所述的宿主细胞为Stbl3、BL21或transetta。
本发明的第九方面，提供了一种免疫细胞，所述的免疫细胞表达上述的CDR、上述的α链多肽、上述的β链多肽、上述的抗体或其抗原结合片段，或者上述的T细胞抗原受体。
优选的，所述的免疫细胞包括但不限于淋巴细胞(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。
本发明的第十方面，提供了一种免疫细胞的制备方法，包括将编码上述CDR、上述的α链多肽、上述的β链多肽、上述的抗体或其抗原结合片段，或者所述的T细胞抗原受体的核酸序列转染至免疫细胞中表达获得。
优选的，所述的免疫细胞包括但不限于淋巴细胞(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。
优选的，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。
本发明的第十一方面，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细胞克隆得到编码上述的T细胞抗原受体的核酸序列；
2)分离、培养原代T细胞；
3)将步骤1)得到的核酸序列递送至步骤2)所述的原代T细胞中，获得表达上述CDR、上述α链多肽、上述β链多肽或者T细胞抗原受体的重组T细胞。
优选的，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。
本发明的第十二方面，提供了一种抗体或其抗原结合片段或T细胞抗原受体的制备方法，包括如下步骤：
(1)从阳性T细胞克隆得到编码上述抗体或其抗原结合片段，或上述的T细胞抗原受体的核酸序列；
(2)将步骤(1)得到的核酸序列连接至载体骨架，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。
优选的，所述的阳性T细胞为与MHC呈递的EBV潜伏期膜蛋白LMP2肽特异性结合。进一步优选的，所述的MHC呈递的EBV潜伏期膜蛋白LMP2肽为单体或多聚体复合物。
本发明的第十三方面，提供了一种多聚体复合物，其包含上述任一所述的T细胞抗原受体。
优选的，其还包括单体，生物素分子，以及带荧光标记的链霉亲和素分子或亲和素分子，其中，所述单体包括抗原肽、MHC分子α链胞外区和β2m链，所述单体与所述生物素分子偶联，所述生物素分子与所述链霉亲和素或亲和素分子结合。
优选的，所述的MHC分子α链胞外区的C端连接avi-tag序列。
优选的，所述的MHC分子α链胞外区不含信号肽序列。并在成熟肽序列前面添加M氨基酸。
优选的，所述的β2m链不含有信号肽序列。且在成熟肽序列前面添加M和A两个氨基酸。
在本发明的一个具体实施方式中，所述的β2m链为不含信号肽且在成熟肽序列前加了两个氨基酸，优选为M、A。
优选的，所述的抗原肽包含SEQ ID NO：29-34中的任一种或两种以上的组合。
进一步优选的，所述的抗原肽包含SEQ ID NO：29、30、33或34中的任一种或两种以上的组合。
在本发明的一个具体实施方式中，所述的多聚体复合物包含：
(1)T细胞抗原受体；优选为SEQ ID NO：5-26中的任一种或与SEQ ID NO：5-26任一种所示氨基酸序列具有至少80％同源性。
(3)单体，所述的单体包括抗原肽、C端连接avi-tag序列的MHC分子α链胞外区和不含信号肽的β2m链；所述的抗原肽选自SEQ ID NO：29-34中的任一种或两种以上的组合；
(4)生物素分子；以及
(5)链霉亲和素分子或亲和素分子；其中，所述单体与所述生物素分子偶联，所述生物素分子与所述链霉亲和素或亲和素结合。
优选的，所述的MHC分子为MHC I类分子或MHC II类分子。更优选的，所述的MHC分子为MHC I类分子。
优选的，所述的MHC分子选自HLA-A*0201、HLA-A*2402和HLA-A*1101。
在本发明的一个具体实施方式中，所述的MHC分子α链的氨基酸序列如SEQ ID NO：1-3中的任一种或SEQ ID NO：1-3中的任一种所示氨基酸序列具有至少80％同源性。
在本发明的一个具体实施方式中，所述的MHC分子的β2m链如SEQ ID NO：4中的任一种或SEQ ID NO：4中的任一种所示氨基酸序列具有至少80％同源性。
为提高抗原肽-MHC四聚体与T细胞抗原受体结合的特异性，所述的单体还包括至少一个氨基酸的化学修饰、突变、插入和/或缺失。
优选的，所述的MHC分子α链胞外区和β2m链为非共价结合。
优选的，所述的多聚体复合物至少包含一个单体。
优选的，每个单体至少偶联一个生物素分子。
本发明的第十四方面，提供了一种上述的多聚体复合物的制备方法，包括如下步骤：
I)表达和纯化在C端连接avi-tag序列的MHC分子α链胞外区和β2m链；
II)将抗原肽、步骤I)获得的在C端连接avi-tag序列的MHC分子α链胞外区和β2m链复性折叠，制备单体；
III)将步骤II)制备的单体生物素化，获得生物素化的单体；
IV)将步骤III)获得的生物素化的单体与带荧光标记的链霉亲和素或亲和素反应，制备抗原肽-MHC四聚体。
V)将步骤IV)得到的抗原肽-MHC分子四聚体与T细胞共孵育，形成T细胞抗原受体与抗原肽-MHC分子四聚体复合物，钓取特异性T细胞抗原受体。
优选的，所述步骤I)中包括分别克隆在编码C端连接avi-tag序列的MHC分子α链胞外区的核苷酸序列和MHC分子β2m链的核苷酸序列，连接至载体后，转化到表达菌中培养，加入诱导剂，提取包涵体。
进一步优选的，所述表达菌培养至OD 600值在0.2-0.4之间。
进一步优选的，所述诱导剂加入后的最终摩尔浓度为0.5-1mM。
优选的，诱导表达4-6h。
优选的，所述步骤II)包括β2m链的复性折叠，即将抗原肽、MHC分子β2m链、C端连接avi-tag序列的MHC分子α链胞外区依次加入稀释缓冲液中避光水浴，其中，所述的β2m链的复性折叠包括包涵体变性、加入蛋白酶抑制剂，而后透析。
优选的，所述的抗原肽、不含信号肽的β2m链与C端连接avi-tag序列的α链的摩尔比为(30-50)：(2-2.5)：1。进一步优选为40:2:1。
优选的，所述步骤II)中还包含纯化单体的步骤。
优选的，所述步骤III)中的生物素化是在BirA酶的催化下，将单体与BiomixA、BiomixB结合。
优选的，所述步骤III)中还包括纯化生物素化单体的步骤。
优选的，所述步骤IV)中的单体与链霉亲和素反应的摩尔比为(4-7)：1。
本发明的第十五方面，提供了一种上述多聚体复合物在制备、筛选或检测本发明所述抗体或其抗原结合片段或T细胞抗原受体中的应用。
本发明的第十六方面，提供了上述的CDR、上述的α链多肽、上述的β链多肽、上述的抗体或其抗原结合片段、上述的T细胞抗原受体、上述的核酸、上述的表达载体、上述的宿主细胞、上述的免疫细胞或者上述的多聚体复合物在制备诊断或治疗与EBV相关疾病的产品中的应用。
优选的，所述的与EBV相关疾病选自传染性单核细胞增多症、连锁淋巴细胞增生综合症、 病毒性嗜血细胞综合症、口腔毛状黏膜白斑病、病毒性脑膜炎、周围神经炎、病毒性肺炎、病毒性心肌炎、鼻咽癌、霍奇金淋巴瘤、伯基特淋巴瘤、胃癌、肝细胞癌、淋巴上皮样肉瘤、唾液腺肿瘤、乳腺癌、胸腺瘤、原发性渗出性淋巴瘤或B/T/NK细胞淋巴瘤。
本发明的第十七方面，提供了上述的CDR、上述的α链多肽、上述的β链多肽、上述的抗体或其抗原结合片段、上述的T细胞抗原受体、上述的核酸、上述的表达载体、上述的宿主细胞、上述的免疫细胞或者上述的多聚体复合物在T细胞标记、检测、细胞分选或活化中的应用。
本发明的第十八方面，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：
i)本发明所述的CDR；
ii)本发明所述的α链多肽；
iii)本发明所述的β链多肽；
iv)本发明所述的抗体或其抗原结合片段；
v)本发明所述的T细胞抗原受体；
vi)本发明所述的核酸；
vii)本发明所述的表达载体；
viii)本发明所述的宿主细胞；
ix)本发明所述的免疫细胞；或
x)本发明所述的多聚体复合物。
优选的，所述的药物组合物还可以包含药学上可接受的辅料。
优选的，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。
本发明的第十九方面，提供了一种试剂盒，所述的试剂盒包含下列任一组：
i)本发明所述的CDR；
ii)本发明所述的α链多肽；
iii)本发明所述的β链多肽；
iv)本发明所述的抗体或其抗原结合片段；
v)本发明所述的T细胞抗原受体；
vi)本发明所述的核酸；
vii)本发明所述的表达载体；
viii)本发明所述的宿主细胞；
ix)本发明所述的免疫细胞；或
x)本发明所述的多聚体复合物。
本发明的第二十方面，涉及一种检测EBV LMP2的方法，所述的方法包括将待检测样品与本发明所述的抗体或其抗原结合片段或者T细胞抗原受体接触，然后检测EBV LMP2与抗体或其抗原结合片段或者T细胞抗原受体形成的复合物。
优选的，所述检测EBV LMP2为检测EBV LMP2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。
在本发明的一个具体实施方式中，所述的标记物可以是His和/或HA。
本发明所述的检测EBV LMP2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EBV LMP2或者包含一定浓度或表达水平的EBV LMP2也并非确定是疾病，只是一种可能性。
本发明的第二十一方面，涉及一种治疗与EBV相关疾病的方法，所述的方法包括给予个体有效量的本发明所述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、所述的免疫细胞或所述的药物组合物。
优选的，所述的治疗与EBV相关疾病的方法包括将本发明所述的T细胞抗原受体定位在与EBV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。
进一步，所述EBV相关疾病选自传染性单核细胞增多症、连锁淋巴细胞增生综合症、病毒性嗜血细胞综合症、口腔毛状黏膜白斑病、病毒性脑膜炎、周围神经炎、病毒性肺炎、病毒性心肌炎、鼻咽癌、霍奇金淋巴瘤、伯基特淋巴瘤、胃癌、肝细胞癌、淋巴上皮样肉瘤、唾液腺肿瘤、乳腺癌、胸腺瘤、原发性渗出性淋巴瘤或B/T/NK细胞淋巴瘤。
本发明的第二十二方面，涉及一种诊断与EBV相关疾病的方法，所述的方法包括取样，将样品与本发明所述的抗体或其抗原结合片段或T细胞抗原受体接触，然后检测EBV LMP2与抗体或其抗原结合片段或T细胞抗原受体形成的复合物。优选的，所述的抗体或其抗原结合片段或T细胞抗原受体包括可检测的标记物。
本发明所述的TCR能够特异性地识别对应的EBV LMP2抗原肽或pMHC复合物，激活TCR T细胞，进而产生高水平的细胞因子IFNγ、IL2、TNFα，显著地杀伤靶细胞并延长负荷肿瘤的小鼠的寿命。
本发明所述的“抗原结合片段”包括但不限于：Fab片段，其具有VL、CL、VH和CH1域；Fab'片段，其是在CH1域的C端具有一个或多个半胱氨酸残基的Fab片段；Fd片段，其具有VH和CH1域；Fd'片段，其具有VH和CH1域和在CH1域的C端的一个或多个半胱氨酸残基；Fv片段，其具有抗体的单一臂的VL和VH域；dAb片段，其由VH域或VL域组成；分离的CDR区；F(ab')2片段，其是包含由铰链区处的二硫桥连接的两个Fab'片段的二价片段；单链抗体分子(例如单链Fv；scFv)；具有两个抗原结合位点的"双抗体"，其包含同一多肽链中与轻链可变域(VL)连接的重链可变域(VH)；"线性抗体"，其包含一对串联Fd区段(VH-CH1-VH-CH1)，该区段与互补的轻链多肽一起形成一对抗原结合区；和任何前述物质的修饰的形式，其保留了抗原结合活性。
本发明所述的“CDR”是免疫球蛋白(Ig)或T细胞抗原受体(TCR)的短片段，其单独或者与其他CDR组合结合抗原表位。所述的免疫球蛋白可以为抗体，则CDR对应抗体可变序列内的互补决定区。对于每个可变区，在重链和轻链的每个可变区中有三个CDR，其分别称为重链或轻链的CDR1、CDR2和CDR3。在所述的T细胞抗原受体(TCR)中CDR存在于α链或β链中，在α链或β链中各存在三个 CDR，其分别称为α链或β链的CDR1、CDR2和CDR3。这些CDR的确切边界根据不同的系统而不同定义。Kabat等人(Kabat et al,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987)和(1991))描述的系统不仅提供了适用于抗体可变区的明确的残基编号系统，而且还提供了限定三个CDR的残基边界。这些CDR可以称为Kabat CDR。每个互补决定区可以包含来自如由Kabat定义的“互补决定区”的氨基酸残基。Chothia等人(Chothia&Lesk,J.Mol.Biol,196:901-917(1987)和Chothia et al.,Nature 342:877-883(-1989))发现，Kabat CDR内的某些子部分采用几乎相同的肽骨架构象，尽管在氨基酸序列水平上具有大的多样性。这些子部分分别称为L1、L2和L3或H1、H2和H3，其中“L”和“H”分别表示轻链和重链区。这些区域可以称为Chothia CDR，其具有与Kabat CDR重叠的边界。还有其它CDR边界定义可以不严格遵循上述系统之一，但是仍将与Kabat CDR重叠，本文使用的方法可以利用根据任何这些系统定义的CDR，尽管优选实施方案使用Kabat或Chothia定义的CDR。所述的CDR在TCR中的残基边界同上所述。所述的“抗体可变区”是指抗体分子的轻链和重链中包括互补决定区(CDR，即CDR1、CDR2和CDR3)和框架区(FR)的氨基酸序列的部分。VH是指重链的可变域。VL是指轻链的可变域。
本发明所述的“LMP2”包含LMP2A和LMP2B。LMP2A和LPM2B的区别在于LMP2A中存在一个额外的N-末端119个氨基酸的胞质结构域，除此之外LMP2A和LPM2B的结构均相同。同时，无论是LMP2A或LMP2B均包含本发明所述的T细胞抗原受体或者抗体结合的抗原肽区域。
本发明所述的“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，使使用序列与本发明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，本发明所述的“与SEQ ID NO：35-44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EBV潜伏期膜蛋白LMP2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SEQ ID NO：35、36、37、38、39、40、41、42、43或44进行调整，例如取代、缺失和/或插入一个或多个氨基酸等。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％。
本发明所述的“个体”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。
本发明所述的“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发明所述的产品的量或剂量。
本发明所述的“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。
本发明所述的“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。
本发明所述的“产品”包括但不限于本发明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。
本发明所述的“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。
本发明所述的“和/或”包括择一列出的项目以及任何数量的项目组合。
本发明所述的“包括”是开放式的描述，含有所描述的指定成分或步骤，以及不会实质上影响技术效果的其他指定成分或步骤。
本发明所述的“TRBV”表示是T细胞受体β可变区，“TRBC”表示是T细胞受体β恒定区。
附图说明
以下，结合附图来详细说明本发明的实施例，其中：
图1：单体的SDS PAGE检测结果，其中，M为蛋白Marker，条带1、2为单体；
图2：四聚体染色结果，其中，图2A为A0201-FLYALALLL-四聚体、A1101-SSCSSCPLSK-四聚体染色结果，图2B为A2402-TYGPVFMSL-四聚体在体积梯度检测实验中与商业化四聚体的对比；
图3：四聚体染色结果，其中，图3A为A2402-TYGPVFMCL-四聚体及A2402-TYGPVFMSL-四聚体染色结果；图3B为A1101-SSCSSCPLSK-四聚体即A1101-SSCSSCPLTK-四聚体染色结果；图3C为A2402-PYLFWLAAI-四聚体染色结果。
图4：TCRβ链和α链在pHAGE载体中连接示意图，其中连接顺序依次为启动子、β链、furin-p2A、α链、IRES和RFP序列；
图5：流式细胞检测HLA-A*A0201 FLYALALLL特异性TCR(E23，E240,)、HLA-A*A2402 TYGPVFM SL/TYGPVFM CL特异性TCR(E44)及PYLFWLAAI特异性TCR(E29,E180-1,)的膜表面表达情况，其中，BV421系列荧光素中的一种，BV的全称是Brilliant Violet。；
图6：流式细胞检测HLA-A*A0201 FLYALALLL特异性TCR(E23，E240,)、HLA-A*A2402  TYGPVFM SL/TYGPVFM CL特异性TCR(E44)及PYLFWLAAI特异性TCR(E29,E180-1,)与EBV LMP2四聚体探针结合的亲和力；
图7：流式细胞检测HLA-A*1101表位SSCSSCPLSK(LSK)和SSCSSCPLTK(LTK)特异性TCR与四聚体探针结合的亲和力；
图8：不同浓度的LSK肽段刺激下JC5-TCR细胞的IL-2产量；
图9：LSK表位特异性TCR的EC50统计结果，其中public TCR代表CDR3基序保守的TCR，private TCR代表不具有CDR3区保守序列的TCR；
图10：不同浓度的LTK肽段刺激下JC5-TCR细胞的IL-2产量；
图11：LTK表位特异性TCR的EC50统计结果，其中public TCR代表CDR3基序保守的TCR，private TCR代表不具有CDR3区保守序列的TCR；
图12：LSK特异TCR T细胞与EBV-LCL细胞孵育后的IL2释放水平；
图13：LSK特异TCR T细胞与EBV-LCL细胞孵育后的IFNγ释放水平；
图14：体外过量靶细胞长期刺激下TCR T细胞的增殖结果图；
图15：体外过量靶细胞长期刺激下TCR T细胞的杀伤能力检测图；
图16：E23-TCRT、E240-TCRT细胞因子IL2、TNFα及IFNγ的释放水平及靶细胞荧光素酶水平的检测结果图，其中，E23,E240代表实施例2制备的TCR，NE代表空白对照组，NT代表只有T细胞组，Ctrl为未转LMP2的Raji细胞，0.5：1、1：1、2:1为效靶比；
图17：E29-TCRT、E180-1-TCRT细胞因子IL2、TNFα及IFNγ的释放水平的检测结果，其中，E29,E180-1代表实施例2制备的TCR，1G4代表能够识别抗原EY-ESO-1的对照TCR，NE代表空白对照组，NT代表只有T细胞组；
图18：E44-TCRT细胞因子IL2、TNFα及IFNγ的释放水平的检测结果，其中，E44代表实施例2制备的TCR，E9代表能够识别抗原LMP2的阳性对照TCR，RFP代表阴性对照组，NE代表空白对照组，NT代表只有T细胞组；
图19：BFP法测定public TCR的结构亲和力结果图；
图20：Alanine Scanning法分析E141-TCR CDR3区域各个氨基酸识别抗原并杀伤靶细胞能力的贡献；
图21：Alanine Scanning法分析E141-TCR CDR3区域各个氨基酸识别抗原并激活T细胞能力的贡献；
图22：淋巴瘤动物模型中评价E23-TCR,E240-TCR在小鼠体内抑制肿瘤生长情况；
图23：淋巴瘤动物模型中评价E29-TCR、E44-TCR在小鼠体内抑制肿瘤生长情况；
图24：淋巴瘤动物模型中评价E141-TCR在小鼠体内抑制肿瘤生长情况；
图25：实体瘤动物模型中评价E141-TCR在小鼠体内抑制肿瘤生长情况；
图26：实体瘤模型，无T细胞注射组(PBS)、对照TCR-T细胞注射组(TCR-1G4)、及EBV TCR-T注射组(E141-TCR)的小鼠肿瘤生长情况统计结果；
图27：实体瘤模型，无T细胞注射组(PBS)、对照TCR-T细胞注射组(TCR-1G4)、及EBV TCR-T注射组(E141-TCR)的小鼠体内TCR T细胞特异增殖统计结果。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：EBV抗原表位四聚体的构建和效果检测
一、EBV抗原表位四聚体的构建
1)表达序列优化的HLA-A*0201(其氨基酸序列如SEQ ID NO：1所示，核苷酸序列如SEQ ID NO：118所示)、HLA-A*2402(其氨基酸序列如SEQ ID NO：2所示，核苷酸序列如SEQ ID NO：119所示)和HLA-A*1101(其氨基酸序列如SEQ ID NO：3所示，核苷酸序列如SEQ ID NO：120所示)的α链和β2m链(其氨基酸序列如SEQ ID NO：4所示，核苷酸序列如SEQ ID NO：121所示)。其中，α链的结构为相应HLA型α链的胞外区序列链接了Avi-tag序列，以BamHI酶切位点间隔，以提供生物素化的位点。β2m链去掉了信号肽序列，在成熟肽序列前面加了两个氨基酸(M和A)。表达载体为PET28a+，表达菌为transetta或BL21。IPTG浓度为0.5mM，诱导表达4h。提取α链和β2m链蛋白包涵体。
2)EBV抗原表位(抗原肽)的选择：HLA-A*0201型对应抗原表位FLYALALLL(SEQ ID NO：29)，HLA-A*2402型对应抗原表位PYLFWLAAI(SEQ ID NO：30)、TYGPVFMSL(SEQ ID NO：31)、TYGPVFMCL(SEQ ID NO：32)，HLA-A*1101型对应抗原表位SSCSSCPLSK(SEQ ID NO：33)、SSCSSCPLTK(SEQ ID NO：34)。
3)pMHC I单体折叠及纯化：将步骤2)所述抗原肽、相应的步骤1)所述β2m链复性蛋白和α链蛋白以摩尔比40:2:1的比例按顺序加入还原体系中，折叠反应72h。将得到的产物过Superdex75 10/300GL柱子纯化。收集纯化产物用avidity试剂盒生物素化，再次纯化得到生物素化的单体，凝胶电泳检测单体纯度。
4)将步骤3)所述生物素化的单体与APC标记的链霉亲和素结合反应，得到对应的四聚体，分别命名A0201–FLYALALLL-四聚体、A2402-PYLFWLAAI-四聚体、A2402-TYGPVFMSL-四聚体、A2402-TYGPVFMCL-四聚体、A1101–SSCSSCPLSK-四聚体及A1101-SSCSSCPLTK-四聚体。
二、EBV抗原表位四聚体效果检测
1.分离人外周血单核细胞(PBMC)，制备细胞悬液，细胞密度为1×10 6细胞/mL。
2.将细胞离心，3000rpm，5min。去除上清，用50μl含有1％血清的PBS重悬。
3.加入2μl四聚体室温孵育30min。
4.加入2μl CD8抗体，冰上孵育20min。
5.加入1ml PBS，3000rpm，5min。
6.去除上清，加入1ml PBS，3000rpm，5min。
7.去除上清，用500μl的4％多聚甲醛重悬细胞，用滤膜过滤细胞悬液。
8.用流式细胞仪检测阳性细胞。
三、实验结果
单体的SDS PAGE检测结果见图1。如图所示，经复性折叠和HPLC纯化，得到的单体清晰地显示重链(C端连接Avi-tag序列的α链胞外区)和轻链(去除信号肽区的β2m链)对应大小的蛋白，且纯度较高。
将构建好的四聚体分别与感染了对应HLA型TCR的细胞共孵育。示例性的，构建的A0201-FLYALALLL-四聚体、A2402-TYGPVFMSL-四聚体、A2402-TYGPVFMCL-四聚体、A2402-PYLFWLAAI-四聚体、A1101-SSCSSCPLTK-四聚体、A1101-SSCSSCPLSK-四聚体分别与感染了对应HLA型TCR的细胞共孵育(LLL四聚体对应TCR E23；AAI四聚体对应TCR E29；MSL/MCL四聚体对应TCR E44；LSK/LTK四聚体对应TCR E141)，与MBL公司商业化四聚体相比，A0201-FLYALALLL-四聚体检测阳性细胞百分比为70.5％，远高于商业化的FLYALALLL四聚体(59.7％)，A1101-SSCSSCPLSK-四聚体检测阳性细胞百分比为20.3％，明显高于商业化的SSCSSCPLSK-四聚体(18.1％)，如图2A所示。充分表明本发明的四聚体在检测细胞阳性率方面表现了高度的特异性和染色效果。在进一步的体积梯度检测实验中发现，自主研发的A2402-TYGPVFMSL四聚体特异性结合效果远高于商业化四聚体(参见图2B)。
通过本发明构建的四聚体所钓取的TCR能够同时识别野生型抗原表位和突变型抗原表位，防止免疫逃逸。如图3-A，A2402 HLA型野生型抗原表位(MCL)和突变型抗原表位(MSL)四聚体均能被由MSL四聚体钓取的TCR E44所识别。图3-B所示，A1101 HLA型野生型抗原表位(LSK)和突变 型抗原表位(LTK)四聚体均能被由LSK四聚体钓取的TCR E141所识别。同一HLA型不同抗原表位四聚体钓取对应特异性的TCR，如图3-C所示，AAI四聚体能检测到对应TCR E29阳性的细胞。
实施例2：pHAGE-TCR-RFP载体的构建
一、获取EBV LMP2表位特异的TCR的β和α基因片段
1)取实施例1中制备的A0201–FLYALALLL-四聚体、A2402-PYLFWLAAI-四聚体、A2402-TYGPVFMSL-四聚体、A2402-TYGPVFMCL-四聚体、A1101–SSCSSCPLSK-四聚体及A1101-SSCSSCPLTK-四聚体，与外周血染色，将四聚体染色阳性的T细胞进行流式单细胞分选，反转录获得cDNA(
Figure PCTCN2021089237-appb-000012
IV Reverse Transcriptase,Invitrogen)。根据多重PCR(Multiplex PCR)原理，通过两轮PCR(KOD-Plus-Neo，TOYOBO)扩增出TCRβ基因的可变区片段。
反转录引物为：TRBC1-TCAGGCAGTATCTGGAGTCATTG(SEQ ID NO：144)
PCR扩增引物为：
上游引物1：TRBV_F1(见SEQ ID NO：147-185和366)
上游引物2：TRBV_F2(见SEQ ID NO：186-225)
下游引物1：TRBC2-GCACCTCCTTCCCATTCACC(SEQ ID NO：145)
下游引物2：TRBC3-GCTTCTGATGGCTCAAACACAG(SEQ ID NO：146)
具体的，根据PCR聚合酶KOD-Plus-Neo产品说明书，一轮PCR体系为20μL，退火温度为60℃，反应30个循环。取第一轮PCR反应的产物1μL，作为二轮PCR的模板，二轮PCR体系为30μL，退火温度为60℃，反应30个循环。然后将第二轮PCR产物跑琼脂糖胶，将对应大小的条带进行切胶回收(天根胶回收试剂盒)，送测序，测序引物为下游引物2。得到TCRβ基因序列，具体的E23、E240、E29、E180-1、E44、E141、E149、E168、E170、E244、E245、E254、E301、E304、E305、E307、E314、E315、E316、E317、E318、E320的TCRβ基因序列分别如SEQ ID NO：122-143的“双下划线”所示核苷酸序列。
2)如上，对四聚体染色阳性T细胞的进行反转录获得cDNA(
Figure PCTCN2021089237-appb-000013
IV Reverse Transcriptase,Invitrogen)。根据产品说明书通过两轮PCR(KOD-Plus-Neo,TOYOBO)扩增出TCRα基因片段。
反转录引物为：TRAC1-CGACCAGCTTGACATCACAG(SEQ ID NO：226)
PCR扩增引物为：
上游引物3：TRAV_F1(见SEQ ID NO：229-273)
上游引物4：TRAV_F2(见SEQ ID NO：274-315)
下游引物3：TRAC2-GTTGCTCTTGAAGTCCATAGACCTC(SEQ ID NO：227)下游引物4： TRAC3-CAGGGTCAGGGTTCTGGATA(SEQ ID NO：228)
具体的，根据PCR聚合酶KOD-Plus-Neo产品说明书，一轮PCR体系为20μL，退火温度为60℃，反应30个循环。取第一轮PCR反应的产物1μL，作为二轮PCR的模板，二轮PCR体系为30μL，退火温度为60℃，反应30个循环。然后将第二轮PCR产物跑琼脂糖胶，将对应大小的条带进行切胶回收(天根胶回收试剂盒)，送测序，测序引物为下游引物4。得到TCRα基因序列，具体的E23、E240、E29、E180-1、E44、E141、E149、E168、E170、E244、E245、E254、E301、E304、E305、E307、E314、E315、E316、E317、E318、E320的TCRα基因序列分别如SEQ ID NO：122-143的“波浪下划线”所示核苷酸序列。
二、pHAGE-TCR载体的构建
将TCRβ，fp2A，TCRα经长引物(包含fp2A序列)overlap-PCR扩增(KOD-Plus-Neo,TOYOBO)得到TCRβ-fp2A-TCRα片段，分别命名为E23,E240,E29,E180-1,E44,E141，E149,E168,E170,E244,E245,E254,E301,E304,E305,E307,E314,E315,E316,E317,E318,E320的pHAGE-TCR质粒。
扩增的引物为：
上游引物5见表1。
表1
Figure PCTCN2021089237-appb-000014
Figure PCTCN2021089237-appb-000015
Figure PCTCN2021089237-appb-000016
下游引物5：
TCTCCAGCCTGCTTCAGCAGGCTGAAGTTAGTAGCTCCGCTTCCGCTccgtttccgccgGAAATCCTTTCTCTTGACCATG(SEQ ID NO：338)
上游引物6见表2。
表2
Figure PCTCN2021089237-appb-000017
Figure PCTCN2021089237-appb-000018
下游引物6：agggatcctctagactcgagctagcTCAGCTGGACCACAGCCGCA(SEQ ID NO：361)
具体的，先用引物5和引物6分别将TCRβ和TCRα扩增出来，PCR体系为50μL，退火温度为60℃，反应30个循环。将PCR产物跑胶回收(天根胶回收试剂盒)，分别取回收产物1μL作为模板，用上游引物5和下游引物6进行overlap PCR，PCR体系为50μL，退火温度为60℃，反应30个循环。跑琼脂糖胶得到约1800bp的条带，进行切胶回收。
将慢病毒载体pHAGE-IRES-RFP用NotI和NheI进行双酶切。酶切体系为40μL，其中含NotI和NheI分别1.5μL，质粒2-3μg，37℃酶切6h，然后在体系里加入1μL碱性磷酸酶(NEB)，处理1h以减少质粒自连，将酶切后的质粒跑胶回收，用nanodrop测浓度，作为骨架用于构建质粒。
根据Clone Express II One Step Cloning kit产品说明书，将TCR通过overlap与酶切后线性化的pHAGE-IRES-RFP载体进行连接(见图4)，转化至Stbl3菌株，在含氨苄的LB平板上培养12-16h，挑取单克隆隆菌进行测序，测序引物选用pHAGE载体上的引物seq-pHAGE-F和seq-pHAGE-R，以及下游引物4。获得相应TCR，简称分别为E23,E240,E29,E180-1,E44,E141，E149,E168,E170,E244,E245,E254,E301,E304,E305,E307,E314,E315,E316,E317,E318,E320。
实施例3：pMHC四聚体染色法检测TCR的膜表达和亲和力
1、构建内源性TCR敲除的JurkatT细胞系
基于Jurkat细胞TCR的序列特征，在α链和β链的恒定区设计guide序列(TRA_oligo1-CACCGTCTCTCAGCTGGTACACGGC(SEQIDNO：362),TRA_oligo2-AAACGCCGTGTACCAGCTGAGAGAC(SEQIDNO：363),TRB_oligo1-CACCGGGCTCAAACACAGCGACCTC(SEQIDNO：364),TRB_oligo2-AAACGAGGTCGCTGTGTTTGAGCCC)(SEQIDNO：365).
将合成的α链和β链的guide序列分别构建到sgRNA-LentiCRISPR-puro和sgRNA-LentiCRISPR-BSD慢病毒载体中，与包装质粒psPAX2，pMD2.G和PEI转染试剂按照一定的比例共转293T细胞，收取48h和72h的细胞培养上清，浓缩后的两个病毒同时感染人类JurkatT细胞系。感染48h后用合适浓度的嘌呤霉素和杀稻瘟菌素进行药杀，直至两个药物各自的对照组细胞全部死亡。存活的细胞用流式分选单细胞到96孔板中进行培养。对于获得的单克隆细胞系，用TCRα链和β链的抗体分别对其表达进行鉴定，两条链都缺陷的细胞株即为获得的内源性TCR敲除的Jurkat T细胞，命名为JC5。
2、构建稳定整合EBV TCR的JC5细胞系
将实施例2构建的上述E23、E240等pHAGE-TCR质粒分别与包装质粒psPAX2，pMD2.G以及转染试剂PEI按照一定的比例混合，转染293T细胞。收取48h和72h的细胞培养上清，浓缩后感染 对数生长期的JC5细胞(MOI＝0.3)。感染3天后，用抗人CD3和抗人TCRαβ流式抗体对细胞进行染色，将相同TCR表达水平的细胞分选后进行培养，即为JC5-TCR细胞系。
3、TCR上膜情况及亲和力检测
取1×10 6JC5-TCR细胞，用BrilliantViolet 421 TM anti-humanTCRαβ(Biolegend)以及相应的EBV LMP2 pMHC四聚体-APC(Tetramer-APC)染色，之后进行流式分析。
由图5，6可知，所制备的针对EBV LMP2 HLA-A*A0201 FLYALALLL的特异E23-TCR、E240-TCR针对EBV LMP2HLA-A*A2402 PYLFWLAAI的特异E29-TCR、E180-1-TCR及针对EBV LMP2HLA-A*A2402 TYGPVFM SL/TYGPVFM CL的特异E44-TCR均可以正确表达并且展示在细胞膜外，且与对应四聚体探针具有一定的亲和力。
由图7可知，所制备的针对EBV LMP2HLA-A*A1101 SSCSSCPL SK/SSCSSCPL TK的特异TCR中，除了E244-TCR和E307-TCR外，其余构建的TCR均对SSCSSCPL SK/SSCSSCPL TK表位有较好的结合。这一结果不仅表明了自制四聚体能成功的用于鉴定特异结合的T细胞，并且表明所获得的TCR具有良好的亲和力。
实施例4：TCR功能活性和EC50
鉴于实施例3中pMHC四聚体检测的是TCR的结构亲和能力，并且四聚体与JC5表面的TCR是四价结合。为进一步鉴定TCR的活性，我们在T2细胞中稳定整合了HLA-A*1101分子，构建了T2-HLA-A*1101细胞系用于定量TCR的半最大效应抗原浓度(EC50)，比较TCR的功能活性。
1、构建稳定整合HLA-A*1101的T2细胞系
克隆HLA-A*1101分子和β2m分子(来源于人)，用fp2A连接后构建到pHAGE-BSD载体中，与包装质粒psPAX2，pMD2.G和PEI转染试剂按照一定的比例共转293T细胞进行包毒，用于感染T2细胞系。感染48h的T2细胞用合适浓度的杀稻瘟菌素进行药杀，直至对照组细胞全部死亡，获得T2-HLA-A*1101细胞系。
2、TCR功能活性和EC50的测定
将合成的LMP2抗原表位用DMSO溶剂稀释到4mg/mL的储存浓度。然后用完全培养基对表位肽段进行连续梯度稀释，获得2×10 -8-2×10 -4M的LSK和LTK肽段溶液，以1：100的体积添加到1×10 6细胞/mL的T2-HLA-A*1101细胞悬液中，混匀后将细胞铺在96孔板中，每孔100μL，同时加入100μL浓度为1×10 6细胞/mL的JC5-TCR细胞，混匀后即为肽段浓度是1×10 -10-1×10 -6M的T2孵育体系。共培养24h后，收集培养基上清，用ELISA试剂盒检测IL2产量。实验重复三次。图8和图10分别代表了不同浓度的LSK和LTK肽段刺激下JC5-TCR细胞的IL2产量。相应的EC50值可以通过prism 计算获得，三次重复的EC50值具体如图9和图11。由图8-11可知，对于合成的LSK抗原表位，TCR E149,E304，E170,E315具有优异的功能活性，而对于LTK表位，E149,E254，E170,E316,E317,E318都展现了较好的功能活性。E244和E307的结果与四聚体染色一致，均对SSCSSCPL SK/SSCSSCPL TK表位的识别能力较弱。
实施例5：人原代TCR T细胞的构建及体外功能检测
1、人类原代T细胞的分离、培养和慢病毒感染
为了进一步验证所筛选到的TCR对EBV LMP2抗原的识别和杀伤功能，采用淋巴细胞分离液Ficoll从志愿者的外周血中分离得到单个核细胞(PBMC)，然后根据EasySep Human T cell isolation kit(stem cell technologies)产品说明书，从PBMC中通过阴选的方法获得T细胞，用含有100U/mL IL2的1640完全培养基将细胞重悬至1×10 6细胞/mL，培养在抗CD3/CD28抗体包被的培养皿中进行激活。激活48h后，用慢病毒体系将装载有TCR的病毒颗粒(由实施例3制备)感染T细胞，感染方法为32℃下1500rpm离心2h，取出后放在37℃细胞培养箱培养10h，然后通过添换培养基终止感染，并继续放在37℃细胞培养箱进行培养。感染三天后可用流式细胞仪将TCR阳性的细胞分选出来，获得TCRT细胞(包括上述E23,E240,E29,E180-1,E44,E141，E149,E168,E170,E244,E245,E254,E301,E304,E305,E307,E314,E315,E316,E317,E318,E320)。
2、靶细胞的构建
用慢病毒体系将分别装载有LMP2-RFP、HLA-A*0201-BSD/HLA-A*2402-BSD/HLA-A*1101-BSD以及Luciferase-GFP的病毒颗粒感染对数生长期的Raji细胞。通过药物筛选及流式分选，得到同时稳定表达LMP2、HLA-A分子以及Luciferas-GFP的Raji细胞，命名为Raji-HLA-A*A0201/2402/1101-LMP2-luciferase。此外，将HLA-A*0201-BSD/HLA-A*2402-BSD/HLA-A*1101-BSD的病毒颗粒感染对数生长期的EBV-LCL细胞。通过药物筛选，得到稳定表达HLA-A分子的EBV-LCL细胞，分别命名为EBV-LCL-HLA-A*0201、EBV-LCL-HLA-A*2402、EBV-LCL-HLA-A*1101细胞。
3、TCR在人原代T细胞中的体外功能验证
1)验证TCR对内源水平抗原表位的识别能力
EBV-LCL即为被EB病毒侵染后永生化的人B细胞，较为真实的模拟了体内肿瘤细胞中的抗原水平。因此，将识别SSCSSCPL SK/SSCSSCPL TK表位的TCR T细胞和EBV-LCL-HLA-A*1101细胞按照效靶比为8:1、4:1、2:1、1:1、0.5:1、0.25:1进行共培养，其中靶细胞固定为1×10 5细胞。共孵育24h后，收集上清用于检测分泌的细胞因子IL2(图12)和IFN-γ(图13)。从细胞因子释放水平来看，TCR： E141，E170，E254以及E315结合内源水平的LSK后可以显著地激活T细胞。尤其是E315TCR T细胞，不仅在各个效靶比浓度下展现了最好的IFN-γ水平，相应的IL2值也较高，结果表明本发明制备的TCR能有效介导肿瘤内源抗原的识别。
2)验证TCR T细胞对肿瘤细胞的长期杀伤能力
将识别SSCSSCPL SK/SSCSSCPL TK表位的TCR T细胞和Raji-HLA-A*1101-LMP2-luciferase细胞按照效靶比为1:3进行起始共培养，记为0天，然后分别在1天，3天，5天收集细胞进行流式分析。其中，所用培养基为不含IL2的1640完全培养基，起始的TCR T细胞为1×10 5细胞，各个时间点的样品独立孵育，并分别在2天，4天的时候对剩余的共孵育样品进行半换液，补加靶细胞。用于流式分析的细胞先用抗人CD3抗体进行染色，上机时收集记录指定体积的细胞，通过换算得知体系里T细胞的数量(见图14)。由绝对T细胞数量的增殖曲线可见，E315-TCR T细胞在识别两个抗原表位后进行了最好的激活和增殖。此外，进一步分析体系里的效靶比例(见图15)，与增殖结果相同，E315-TCRT细胞展现了最强的肿瘤清除能力。
3)TCR在人原代T细胞中的体外功能验证
将Raji-HLA-A*0201-LMP2、未转LMP2的Raji细胞与E23-TCRT，E240-TCRT细胞及1G4 T细胞按照1：0.5、1：1、1：2的数量比例进行共培养后，共培养24h后，分别收集细胞和上清，初步检测了E23-TCRT和E240-TCRT细胞的激活和靶细胞的死亡情况。从胞外细胞因子TNFα、IL2和IFNγ(见图16)的释放水平来看，E240-TCRT及E23-TCRT同靶细胞共孵育后，与对照组1G4 T相比，可以显著地引起T细胞的激活。且从靶细胞裂解后的释放的荧光素酶量可反映出靶细胞死亡情况(见图16)。实验结果证明，本发明实施例构建的E23-TCRT及E240-TCRT细胞可以特异性地被EBV LMP2抗原肽提呈细胞激活，且能够显著地杀伤靶细胞。
将Raji-HLA-A*2402-抗原肽、未转抗原肽的Raji细胞，加抗原肽，及不加抗原肽的T2-HLA2402细胞分别与E29-TCRT、E180-1-TCRT细胞、1G4T细胞按照1：3的数量比例进行共培养后，共培养24h后，分别收集细胞和上清，初步检测了E29-TCR T及180-1-TCRT细胞的激活情况。从胞外细胞因子IL2和IFNγ(见图17)的释放水平来看，E29-TCRT及180-1-TCRT细胞同靶细胞共孵育后，与对照组1G4T相比，可以显著地引起T细胞的激活。此外从胞外细胞因子(见图18)的释放水平来看，E44-TCRT细胞同靶细胞共孵育后，与对照组RFP T相比，也可以显著地引起T细胞的激活。实验结果证明，本发明实施例构建的E29-TCRT、E180-1-TCRT及E44-TCRT细胞可以特异性地被EBV LMP2抗原肽提呈细胞激活，且能够显著地杀伤靶细胞。
实施例6：共享CDR3基序的TCR功能比较
由于鉴定到的识别SSCSSCPLSK/SSCSSCPLTK表位的E141,E149,E254,E301,E304以及E314在TCRα链和β链的CDR3高变区均非常保守，序列高度相似至相差一个氨基酸但是功能上相去甚远(图9和图11)。因为将具有这些保守CDR3基序的TCR定义为public TCR，将不具有这些保守序列的TCR定义为private TCR。并在此实施例中对public TCR的结构亲和力和功能进行分析。
1、BFP法测定public TCR的结构亲和力
将红细胞固定在一侧的微量吸液管上，然后将特异包埋有pMHC分子的珠子吸附在红细胞表面，形成超敏的生物膜力学探针(BFP)，同时将JCR-TCR细胞固定在另一侧的微量吸液管上，通过压电式传感器控制两者的接触，每个循环接触时外加压力为10pN，接触时间为0.1s，然后以1000pN/s的速度分开，进行下一个循环重复。整个过程用显微镜记录红细胞-珠子表面的形变，判断是否有键形成以及持续时间。由图19的成键持续时间可知，public TCR之间对LSK表位的结构亲和力参差不齐，其中E304具有较好的功能，与T2的结果吻合(图9)。
2、Alanine Scanning法分析保守的CDR3基序的功能
将TCR E141α链和β链CDR3区域首位后的氨基酸依次突变成丙氨酸，命名为a2-a9以及b2-b11，构建到pHAGE慢病毒载体中，包毒后感染人原代T细胞(MOI＝10)，感染三天后可用流式细胞仪将TCR阳性的细胞分选出来。分选后的TCR T细胞和Raji-LMP2-luciferase靶细胞按照效靶比为1:1进行共培养，其中T细胞为1×10 5细胞。共孵育24h后，分别收集细胞和上清，细胞沉淀裂解后用于检测尚存活靶细胞释放的荧光素酶量(图20)，上清用于检测TCR T细胞分泌的细胞因子IL2(图21)。根据图20和图21可知，与HLA unmatched-Raji细胞、或未添加TCR-T细胞以及添加了未转染TCR的pan-T细胞的HLA matched-Raji这些对照组的本底值相比，未突变的E141遇到HLA matched-Raji后能有效的清除靶细胞，并且特异的产生大量的细胞因子IL2。但是，在a3、a5、a6以及b6、b7、b8这些位点，单个氨基酸的突变足以让TCR T细胞彻底失活。这些实验结果共同表明，尽管这些public TCR共享同一个保守的基序，相互之间的功能可能会因为一个氨基酸的差异而相去甚远。
实施例7：动物模型构建及EBV TCRT体内功能检测
EB病毒主要感染鼻咽上皮细胞和B细胞，与鼻咽癌和多种B细胞淋巴瘤的发生发展密切相关。本实施例分别构建了B细胞淋巴瘤小鼠模型和鼻咽癌实体瘤模型，以验证所鉴定的TCR的体内功能。1、淋巴瘤模型及TCRT体内功能检测
将3×10 5Raji-HLA-A*1101/0201/2402-LMP2-luciferase肿瘤细胞通过尾静脉接种给5-6周的NOD/Scid IL-2Rγnull(NCG)雌鼠，构建淋巴瘤模型(见图22，23，24)，记为第1天，第5天时，将小鼠分为3个小组，分别为A：PBS注射组(注射等体积PBS)；B：对照TCRT细胞注射组(TCR-1G4  T细胞)；C：EBV TCRT注射组(E141-TCRT细胞)，B/C组小鼠尾静脉注射5×10 6TCR T细胞，A组注射等体积200μL PBS。第8天时进行二次注射，具体操作与第5天一致。其他TCR T细胞具体回输量及回输时间见图22，23。在接下来的几周监测小鼠体内肿瘤细胞生长情况，T细胞体内增殖情况以及小鼠存活情况。如图24所示，与对照组比较，本发明实施例构建的EBV特异性E141-TCRT细胞能够显著地杀伤小鼠体内肿瘤细胞，提高小鼠的存活率。此外，如图22，23所示，与对照组比较，本发明实施例构建的EBV特异性E23-TCRT、E240-TCRT、E29-TCRT、E44-TCRT细胞也能够显著地杀伤小鼠体内肿瘤细胞，提高小鼠的存活率。
2、实体瘤模型
将1×10 6C666-1-HLA-A*1101-LMP2-luciferase肿瘤细胞通过皮下接种给5-6周的NCG雌鼠，构建鼻咽癌实体瘤模型(见图25)，记为第0天，7天后，将小鼠分为3个小组，分别为A：PBS注射组(注射等体积PBS)；B：对照TCR-T细胞注射组(TCR-1G4T细胞)；C：EBV TCR-T注射组(E141-TCRT细胞)，B/C组小鼠尾静脉注射3×10 6T细胞，A组注射等体积200μL PBS。在接下来的几周监测小鼠体内肿瘤细胞生长情况，T细胞体内增殖情况以及小鼠存活情况。如图25，图26以及图27所示，与对照组比较，本发明实施例构建的EBV特异性E141-TCRT细胞能够显著地杀伤小鼠体内肿瘤细胞(图25，图26)，并且回输的TCR T细胞在体内进行了特异的增殖(图27)。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。

Claims (32)

  1. 一种与EBV潜伏期膜蛋白LMP2结合的决定簇互补区(CDR)，其特征在于，所述的CDR选自SEQ ID NO：35-117中的一种或两种以上的组合。
  2. 根据权利要求1所述的CDR，其特征在于，所述的CDR包括CDR1α-CDR3α和/或CDR1β-CDR3β，其中，CDR1α的氨基酸序列如SEQ ID NO：35-44中的任一种或与SEQ ID NO：35-44中任一种所示氨基酸序列具有至少80％同源性，CDR2α的氨基酸序列如SEQ ID NO：45-54中的任一种或与SEQ ID NO：45-54中任一种所示氨基酸序列具有至少80％同源性，CDR3α的氨基酸序列如SEQ ID NO：55-73中的任一种或与SEQ ID NO：55-73中任一种所示氨基酸序列具有至少80％同源性，CDR1β的氨基酸序列如SEQ ID NO：74-84中的任一种或与SEQ ID NO：74-84中任一种所示氨基酸序列具有至少80％同源性，CDR2β的氨基酸序列如SEQ ID NO：85-96中的任一种或与SEQ ID NO：85-96中任一种所示氨基酸序列具有至少80％同源性，CDR3β的氨基酸序列如SEQ ID NO：97-117中的任一种或与SEQ ID NO：97-117中任一种所示氨基酸序列具有至少80％同源性。
  3. 根据权利要求1或2所述的CDR，其特征在于，所述的CDR选自下列任一组：
    Figure PCTCN2021089237-appb-100001
    Figure PCTCN2021089237-appb-100002
    Figure PCTCN2021089237-appb-100003
  4. 一种与EBV潜伏期膜蛋白LMP2结合的α链多肽，其特征在于，所述的α链多肽包含CDR1α、CDR2α和/或CDR3α，其中，CDR1α的氨基酸序列如SEQ ID NO：35-44中的任一种或与SEQ ID NO：35-44中任一种所示氨基酸序列具有至少80％同源性，CDR2α的氨基酸序列如SEQ ID NO：45-54中的任一种或与SEQ ID NO：45-54中任一种所示氨基酸序列具有至少80％同源性，CDR3α的氨基酸序列如SEQ ID NO：55-73中的任一种或与SEQ ID NO：55-73中任一种所示氨基酸序列具有至少80％同源性。
  5. 根据权利要求4所述的α链多肽，其特征在于，所述的α链多肽包含下列任一组的CDR1α-CDR3α：
    Figure PCTCN2021089237-appb-100004
    Figure PCTCN2021089237-appb-100005
  6. 一种与EBV潜伏期膜蛋白LMP2结合的β链多肽，其特征在于，所述的β链多肽包含CDR1β、CDR2β和/或CDR3β，其中，CDR1β的氨基酸序列如SEQ ID NO：74-84中的任一种或与SEQ ID NO：74-84中任一种所示氨基酸序列具有至少80％同源性，CDR2β的氨基酸序列如SEQ ID NO：85-96 中的任一种或与SEQ ID NO：85-96中任一种所示氨基酸序列具有至少80％同源性，CDR3β的氨基酸序列如SEQ ID NO：97-117中的任一种或与SEQ ID NO：97-117中任一种所示氨基酸序列具有至少80％同源性。
  7. 根据权利要求6所述的β链多肽，其特征在于，所述的β链多肽包含下列任一组的CDR1β-CDR3β：
    Figure PCTCN2021089237-appb-100006
    Figure PCTCN2021089237-appb-100007
  8. 一种抗体或其抗原结合片段，其特征在于，所述的抗体或其抗原结合片段特异性结合EBV潜伏期膜蛋白LMP2，所述的抗体或其抗原结合片段包含α链的CDR1α-CDR3α和/或β链的CDR1β-CDR3β，其中，CDR1α的氨基酸序列如SEQ ID NO：35-44中的任一种或与SEQ ID NO：35-44中任一种所示氨基酸序列具有至少80％同源性，CDR2α的氨基酸序列如SEQ ID NO：45-54中的任一种或与SEQ ID NO：45-54中任一种所示氨基酸序列具有至少80％同源性，CDR3α的氨基酸序列如SEQ ID NO：55-73中的任一种或与SEQ ID NO：55-73中任一种所示氨基酸序列具有至少80％同源性，CDR1β的氨基酸序列如SEQ ID NO：74-84中的任一种或与SEQ ID NO：74-84中任一种所示氨基酸序列具有至少80％同源性，CDR2β的氨基酸序列如SEQ ID NO：85-96中的任一种或与SEQ ID NO：85-96中任一种所示氨基酸序列具有至少80％同源性，CDR3β的氨基酸序列如SEQ ID NO：97-117中的任一种或与SEQ ID NO：97-117中任一种所示氨基酸序列具有至少80％同源性。
  9. 根据权利要求8所述的抗体或其抗原结合片段，其特征在于，所述LMP2的结合表位包含SEQ ID NO：29-34中的任一种或两种以上的组合。
  10. 根据权利要求8或9所述的抗体或其抗原结合片段，其特征在于，所述的CDR1α-CDR3α及CDR1β-CDR3β选自下列任一组：
    Figure PCTCN2021089237-appb-100008
    Figure PCTCN2021089237-appb-100009
    Figure PCTCN2021089237-appb-100010
    Figure PCTCN2021089237-appb-100011
  11. 根据权利要求8-10任一所述的抗体或其抗原结合片段，其特征在于，所述的抗体或其抗原结合片段为单域抗体或单链抗体scFv。
  12. 根据权利要求8-11任一所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段的氨基酸序列选自SEQ ID NO：5-26中的任一种或与SEQ ID NO：5-26任一种所示氨基酸序列具有至少80％同源性。
  13. 一种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EBV潜伏期膜蛋白LMP2，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，CDR1α的氨基酸序列如SEQ ID NO：35-44中的任一种或与SEQ ID NO：35-44中任一种所示氨基酸序列具有至少80％同源性，CDR2α的氨基酸序列如SEQ ID NO：45-54中的任一种或与SEQ ID NO：45-54中任一种所示氨基酸序列具有至少80％同源性，CDR3α的氨基酸序列如SEQ ID NO：55-73中的任一种或与SEQ ID NO：55-73中任一种所示氨基酸序列具有至少80％同源性，CDR1β的氨基酸序列如SEQ ID NO：74-84中的任一种或与SEQ ID NO：74-84中任一种所示氨基酸序列具有至少80％同源性，CDR2β的氨基酸序列如SEQ ID NO：85-96中的任一种或与SEQ ID NO：85-96中任一种所示氨基酸序列具有至少80％同源性，CDR3β的氨基酸序列如SEQ ID NO：97-117中的任一种或与SEQ ID NO：97-117中任一种所示氨基酸序列具有至少80％同源性。
  14. 根据权利要求13所述的T细胞抗原受体，其特征在于，所述LMP2的结合表位包含SEQ ID NO：29-34中的任一种或两种以上的组合。
  15. 根据权利要求13或14所述的T细胞抗原受体，其特征在于，所述的CDR1α-CDR3α及CDR1β-CDR3β选自下列任一组：
    Figure PCTCN2021089237-appb-100012
    Figure PCTCN2021089237-appb-100013
    Figure PCTCN2021089237-appb-100014
    Figure PCTCN2021089237-appb-100015
  16. 根据权利要求13-15任一所述的T细胞抗原受体，其特征在于，其氨基酸序列选自SEQ ID NO：5-26中的任一种或与SEQ ID NO：5-26任一种所示氨基酸序列具有至少80％同源性。
  17. 一种核酸，其特征在于，所述的核酸编码权利要求1-3任一所述的CDR、权利要求4-5任一所述的α链多肽、权利要求6-7任一所述的β链多肽、权利要求8-12任一所述的抗体或其抗原结合片段或者权利要求13-16任一所述的T细胞抗原受体。
  18. 一种表达载体，其特征在于，所述的表达载体包含权利要求17所述的核酸。
  19. 一种宿主细胞，其特征在于，所述的宿主细胞包含权利要求17所述的核酸或权利要求18所述的表达载体。
  20. 一种免疫细胞，其特征在于，所述的免疫细胞表达权利要求1-3任一所述的CDR、权利要求4-5任一所述的α链多肽、权利要求6-7任一所述的β链多肽、权利要求8-12任一所述的抗体或其抗原结合片段或者权利要求13-16任一所述的T细胞抗原受体。
  21. 一种重组T细胞的制备方法，其特征在于，包括如下步骤：
    1)从阳性T细胞克隆得到权利要求17的核酸序列；
    2)分离、培养原代T细胞；
    3)将步骤1)得到的核酸序列递送至步骤2)所述的原代T细胞中，获得表达权利要求1-3任一所述的CDR、权利要求4-5任一所述的α链多肽、权利要求6-7任一所述的β链多肽或者权利要求13-16任一所述T细胞抗原受体的重组T细胞。
  22. 一种抗体或其抗原结合片段或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17的核酸序列；
    (2)将步骤(1)得到的核酸序列连接至载体骨架，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。
  23. 一种多聚体复合物，其特征在于，所述多聚体复合物包含权利要求13-16任一所述的T细胞抗原受体。
  24. 根据权利要求23所述的多聚体复合物，其特征在于，所述多聚体复合物还包括单体，生 物素分子，以及链霉亲和素分子或亲和素分子，其中，所述单体包括MHC分子α链胞外区和β2m链及抗原肽，所述单体与所述生物素分子偶联，所述生物素分子与所述链霉亲和素或亲和素分子结合。
  25. 根据权利要求24所述的多聚体复合物，其特征在于，所述的抗原肽包含SEQ ID NO：29-34中的任一种或两种以上的组合。
  26. 根据权利要求24或25所述的多聚体复合物，其特征在于，所述的MHC分子选自HLA-A*0201、HLA-A*2402和HLA-A*1101。
  27. 一种权利要求23-26任一所述的多聚体复合物的制备方法，其特征在于，包括如下步骤：
    I)表达和纯化在C端连接avi-tag序列的MHC分子α链胞外区和β2m链；
    II)将抗原肽、步骤I)获得的在C端连接avi-tag序列的MHC分子α链胞外区和β2m链复性折叠，制备单体；
    III)将步骤II)制备的单体生物素化，获得生物素化的单体；
    IV)将步骤III)获得的生物素化的单体与带荧光标记的链霉亲和素或亲和素反应，制备抗原肽-MHC分子四聚体。
    V)将步骤IV)得到的抗原肽-MHC分子四聚体与T细胞共孵育，形成T细胞抗原受体与抗原肽-MHC分子四聚体复合物，钓取特异性T细胞抗原受体。
  28. 权利要求1-3任一所述的CDR、权利要求4-5任一所述的α链多肽、权利要求6-7任一所述的β链多肽、权利要求8-12任一所述的抗体或其抗原结合片段或者权利要求13-16任一所述的T细胞抗原受体、权利要求17所述的核酸、权利要求18所述的表达载体、权利要求19所述的宿主细胞、权利要求20所述的免疫细胞或者权利要求23-26任一所述的多聚体复合物在制备诊断或治疗与EBV相关疾病的产品中的应用。
  29. 根据权利要求28所述的应用，其特征在于，所述的与EBV相关疾病选自传染性单核细胞增多症、连锁淋巴细胞增生综合症、病毒性嗜血细胞综合症、口腔毛状黏膜白斑病、病毒性脑膜炎、周围神经炎、病毒性肺炎、病毒性心肌炎、鼻咽癌、霍奇金淋巴瘤、伯基特淋巴瘤、胃癌、肝细胞癌、淋巴上皮样肉瘤、唾液腺肿瘤、乳腺癌、胸腺瘤、原发性渗出性淋巴瘤或B/T/NK细胞淋巴瘤。
  30. 权利要求1-3任一所述的CDR、权利要求4-5任一所述的α链多肽、权利要求6-7任一所述的β链多肽、权利要求8-12任一所述的抗体或其抗原结合片段或者权利要求13-16任一所述的T细胞抗原受体、权利要求17所述的核酸、权利要求18所述的表达载体、权利要求19所述的宿主细胞、权利要求20所述的免疫细胞或者权利要求23-26任一所述的多聚体复合物在T细胞标记、检测、细胞分选或活化中的应用。
  31. 一种药物组合物，所述的药物组合物包含下列任一组：
    i)权利要求1-3任一所述的CDR；
    ii)权利要求4-5任一所述的α链多肽；
    iii)权利要求6-7任一所述的β链多肽；
    iv)权利要求8-12任一所述的抗体或其抗原结合片段；
    v)权利要求13-16任一所述的T细胞抗原受体；
    vi)权利要求17所述的核酸；
    vii)权利要求18所述的表达载体；
    viii)权利要求19所述的宿主细胞；
    ix)权利要求20所述的免疫细胞；或
    x)权利要求23-26任一所述的多聚体复合物。
  32. 一种试剂盒，所述的试剂盒包含下列任一组：
    i)权利要求1-3任一所述的CDR；
    ii)权利要求4-5任一所述的α链多肽；
    iii)权利要求6-7任一所述的β链多肽；
    iv)权利要求8-12任一所述的抗体或其抗原结合片段；
    v)权利要求13-16任一所述的T细胞抗原受体；
    vi)权利要求17所述的核酸；
    vii)权利要求18所述的表达载体；
    viii)权利要求19所述的宿主细胞；
    ix)权利要求20所述的免疫细胞；或
    x)权利要求23-26任一所述的多聚体复合物。
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