WO2021103617A1 - HLA-A11限制性乙型肝炎病毒HBc 141-151表位肽的T细胞受体及其应用 - Google Patents

HLA-A11限制性乙型肝炎病毒HBc 141-151表位肽的T细胞受体及其应用 Download PDF

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WO2021103617A1
WO2021103617A1 PCT/CN2020/105157 CN2020105157W WO2021103617A1 WO 2021103617 A1 WO2021103617 A1 WO 2021103617A1 CN 2020105157 W CN2020105157 W CN 2020105157W WO 2021103617 A1 WO2021103617 A1 WO 2021103617A1
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chain
cells
cell
seq
cell receptor
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周旭宇
魏训东
徐威
张建华
黄满
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中国科学院微生物研究所
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Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to the T cell receptor of the HLA-A11 restricted hepatitis B virus HBc 141-151 epitope peptide and its application.
  • Hepatocellular Carcinoma is abbreviated as liver cancer. It has hidden clinical manifestations and lacks typical symptoms in the early stage. Although the diagnosis and treatment of liver cancer have made some progress in recent years, its prognosis is still not ideal, and the five-year survival rate is extremely low. The reason is that, on the one hand, liver cancer is not sensitive to current chemotherapy drugs, and most patients with liver cancer lack effective treatment methods; on the other hand, liver cancer is often diagnosed in the late stage of the disease, which excludes local ablation, which can improve patients. Methods of illness. At present, surgical resection and liver transplantation are still the most effective methods for the treatment of liver cancer.
  • liver cancer develops to a certain stage, tumor cells may metastasize to other organs (such as lungs, bones, brain, etc.).
  • organs such as lungs, bones, brain, etc.
  • the current stage of the examination cannot be detected.
  • the tumor cells may be lurking in other organs Micro lesions may lead to recurrence of liver cancer, and the survival time of patients after surgery is not ideal. Therefore, there is an urgent need to find a new effective adjuvant treatment plan.
  • Immune cell therapy technology is the fastest growing sector in the field of biomedicine. In 2013, the journal Science Translational Medicine predicted that cell therapy will become "the third pillar of future medicine.” Since October 2017, the US FDA has successively approved the listing of CAR-T drugs from Novartis and KITE, creating a new era of immune cell therapy drugs and showing broad application prospects in clinical treatment.
  • the ideal target antigen is a tumor-specific antigen that is only expressed on the surface of tumor cells.
  • most antigens expressed by tumors are not tumor-specific, so most CAR-T and TCR-T target tumor-associated antigens, but this often leads to the possibility of "off-target”. At present, some tumor-related antigens are used more frequently, such as alpha-fetoprotein, NY-ESO, MAGE and other proteins belong to this state.
  • HBV Hepatitis B Virus
  • HCV Hepatitis C Virus
  • Chronic hepatitis B is an important risk factor for liver cirrhosis and liver cancer.
  • Chronic hepatitis B mainly involves antiviral immune response defects at various stages of the immune response, especially the decline in the number and function of HBV-specific CD8 + T cells, causing immune tolerance; at the same time, it is accompanied by immune-mediated inflammatory liver injury.
  • hepatitis B Currently, no drugs have been found to cure hepatitis B.
  • Clinical treatment for chronic hepatitis B is mainly antiviral-related drugs, including interferons (IFNs) and nucleoside and nucleotide drugs, which exert antiviral effects through immune regulation or interference with HBV replication.
  • IFNs interferons
  • nucleoside and nucleotide drugs which exert antiviral effects through immune regulation or interference with HBV replication.
  • these treatments cannot completely eliminate the virus, making patients prone to drug resistance, virus mutation, and repeated illnesses.
  • HBV-specific CD8 + T cells play a decisive role in the control of virus replication, virus clearance and clinical recovery of HBV infection.
  • the adoptive transfer of HBV-specific TCR gene-modified T cells (TCR-T) has been preliminarily proven to have very good antiviral activity.
  • HBV can also be used as a unique tumor-associated antigen.
  • the known HBV-HCC liver cancer cells do not express complete HBV antigens, in the natural history of chronic hepatitis B infection, the virus often integrates itself into the human genome, eventually forming HCC cells and carrying these HBV genomes.
  • Singaporean scholars found that although they do not express complete HBV antigens, HBV-HCC liver cancer cells have short fragments of HBV mRNAs.
  • HBV-specific T cell receptors TCRs
  • TCRs HBV-specific T cell receptors
  • liver cancer patients especially liver transplant patients
  • the HBV epitope on liver cancer cells as tumor-associated antigens for T cell adoptive immunotherapy may eliminate HBV-HCC liver cancer cells.
  • This liver-specific marker means that adverse reactions outside the tumor are largely predictable, with little or no involvement of other organs.
  • other teams have clinically studied the method of inducing TCR redirected T cells in vitro, using TCR-T cells that specifically recognize the restricted epitope of hepatitis B virus HLA-A2 HBs 183-191 for the treatment of liver cancer metastasis in liver transplant patients It proves that this redirected TCR-T cell has the activity of targeting specific liver cancer cells, showing great application potential and value.
  • HLA-A3 superfamily (including HLA-A11, HLA-A33, HLA-A68, HLA-A31, etc.) accounts for the largest proportion (about 52.7%) of the HLA classification of the Chinese population.
  • HLA-A3 superfamily members One of the distinguishing features of HLA-A3 superfamily members is that different members have common polypeptide binding characteristics, that is, they prefer to bind to polypeptide epitopes with basic amino acids at the C-terminal. Therefore, research on the treatment of liver cancer for the restricted population of HLA-A3 superfamily will have good popularity and versatility.
  • HLA-A11 is the most widely distributed in the A3 superfamily, and statistical analysis also shows that the HLA-A11 gene frequency is the highest among Chinese hepatitis B patients.
  • HLA transgenic animal models have played an important role in preclinical trials and basic experimental research.
  • HLA-A11 transgenic mice have been reported before.
  • various evidences indicate that HLA-A11 molecules prefer to bind to C-terminal basic epitope peptides, which are related to the antigen-associated transporter (TAP) in the mouse autoantigen presentation system
  • TAP antigen-associated transporter
  • HLA-A11/hTAP-LMP transgenic mice have a stronger ability to present HLA-A11 restricted CTL epitopes.
  • the purpose of the present invention is to prepare medicines for preventing and/or treating diseases caused by HBV infection.
  • the present invention first protects a T cell receptor that recognizes the HLA-A11-restricted hepatitis B virus HBc 141-151 epitope peptide, which can include an alpha chain and a beta chain.
  • the ⁇ chain may include three complementarity determining regions, and the amino acid sequence is as shown in the 48-53, 71-77 and 112-121 of SEQ ID No. 2; or these sequences have at most 3 , 2 or 1 amino acid change variants.
  • the ⁇ chain may include three complementarity determining regions, and the amino acid sequences are as shown in positions 46-50, 68-73, and 111-122 of SEQ ID No. 4, respectively; or these sequences have at most 3 , 2 or 1 amino acid change variants.
  • the amino acid sequence of the variable region of the ⁇ chain may be as shown in SEQ ID No. 2 at positions 22-112; or these sequences have at most 3, 2 or 1 amino acids Changed variants.
  • the amino acid sequence of the variable region of the ⁇ chain may be as shown in SEQ ID No. 4, positions 20-113; or variants of these sequences with at most 3, 2 or 1 amino acid changes.
  • amino acid sequence of the constant region of the ⁇ chain is shown at positions 133-268 of SEQ ID No.2.
  • amino acid sequence of the constant region of the ⁇ chain is shown at positions 133-305 of SEQ ID No. 4.
  • the amino acid sequence of the ⁇ chain may be as shown in SEQ ID No.2.
  • the amino acid sequence of the ⁇ chain may be as shown in SEQ ID No. 4.
  • the nucleic acid molecule encoding any of the aforementioned T cell receptors may include a nucleic acid molecule encoding the ⁇ chain of the T cell receptor and a nucleic acid molecule encoding the ⁇ chain of the T cell receptor.
  • the nucleotide sequences encoding the three complementarity determining regions in the ⁇ chain of the T cell receptor may be shown as the 142-159th, 211-231th and 334-363th positions of SEQ ID No. 1, respectively; Or a sequence that has more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% identity with these sequences and encodes the same amino acid residue.
  • the nucleotide sequences of the three complementarity determining regions in the ⁇ -strand encoding the T cell receptor can be as shown in SEQ ID No. 3, 136-150, 202-219, and 331-366, respectively; Or a sequence that has more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% identity with these sequences and encodes the same amino acid residue.
  • the nucleotide sequence encoding the variable region of the ⁇ chain may be as shown in SEQ ID No. 1 at positions 64-336; or with these sequences having more than 99%, more than 95%, more than 90%, or more than 85% Or a sequence that is more than 80% identical and encodes the same amino acid residue.
  • nucleotide sequence encoding the variable region of the ⁇ chain is shown in SEQ ID No. 3 at positions 58-339; or it has more than 99%, more than 95%, more than 90%, more than 85% with these sequences, or Sequences that are more than 80% identical and encode the same amino acid residues.
  • nucleotide sequence of the constant region of the ⁇ chain is shown at positions 397-807 of SEQ ID No. 1.
  • nucleotide sequence of the constant region of the ⁇ chain is shown at positions 397-918 of SEQ ID No. 3.
  • nucleotide sequence of the nucleic acid molecule encoding the alpha chain is shown in SEQ ID No. 1.
  • nucleotide sequence of the nucleic acid molecule encoding the ⁇ chain is shown in SEQ ID No. 3.
  • the cells can be Jurkat cells or T cells.
  • the T cell may specifically be a human T cell or a mouse T cell.
  • the vector can be a retroviral vector or a lentiviral vector.
  • the retroviral vector may specifically be a nucleic acid encoding the ⁇ chain of the T cell receptor inserted between the multiple cloning sites of the retroviral vector MSCV-IRES-GFP (such as restriction endonucleases XhoI and EcoRI) Molecule and a nucleic acid molecule encoding the beta chain of the T cell receptor, and the resulting recombinant plasmid.
  • MSCV-IRES-GFP such as restriction endonucleases XhoI and EcoRI
  • the retroviral vector may specifically be a recombinant plasmid obtained by inserting a DNA sequence between the multiple cloning sites of the retroviral vector MSCV-IRES-GFP (such as restriction endonucleases XhoI and EcoRI); the DNA sequence To connect the nucleic acid molecule encoding the ⁇ chain and the nucleic acid molecule encoding the ⁇ chain with the coding sequence of a linker peptide (such as a T2A self-cleaving polypeptide).
  • a linker peptide such as a T2A self-cleaving polypeptide
  • the retroviral vector may specifically be the replacement of the DNA fragment between the restriction enzymes XhoI and EcoRI of the retroviral vector MSCV-IRES-GFP with SEQ ID No.5
  • SEQ ID No.5 The DNA molecule shown, the resulting recombinant plasmid.
  • positions 1 to 804 are the complete coding genes of the ⁇ chain
  • positions 805 to 867 are the coding genes of the T2A self-splicing polypeptide
  • positions 868 to 1785 are the complete coding genes of the ⁇ chain.
  • MSCV-IRES-GFP replaces the small DNA fragments between the restriction enzymes XhoI and ClaI of the MO vector with the IRES nucleotide sequence (Genbank: MG550106.1) and fluorescent markers Protein GFP nucleotide sequence (Genbank: MH777595.1), the resulting recombinant plasmid.
  • the lentiviral vector can specifically be a multiple cloning site (such as restriction endonuclease EcoRI and BamHI) of the lentiviral packaging vector pCDH-MSCV-MCS-IRES-GFP (System Biosciences, code: CD731B-1) Insert the nucleic acid molecule encoding the ⁇ chain of the T cell receptor and the nucleic acid molecule encoding the ⁇ chain of the T cell receptor to obtain a recombinant plasmid.
  • pCDH-MSCV-MCS-IRES-GFP System Biosciences, code: CD731B-1
  • the lentiviral vector may specifically be a recombinant plasmid obtained by inserting a DNA sequence between the multiple cloning sites (such as restriction endonucleases EcoRI and BamHI) of the lentiviral packaging vector pCDH-MSCV-MCS-IRES-GFP;
  • the DNA sequence is formed by connecting the nucleic acid molecule encoding the ⁇ chain and the nucleic acid molecule encoding the ⁇ chain with the coding sequence of a connecting peptide (such as a T2A self-cleaving polypeptide).
  • the lentiviral vector may specifically be a DNA fragment between the restriction endonuclease EcoRI and BamHI of the lentiviral packaging vector pCDH-MSCV-MCS-IRES-GFP replaced with SEQ ID
  • the DNA molecule shown in No. 5 is a recombinant plasmid obtained.
  • positions 1 to 804 are the complete coding genes of the ⁇ chain
  • positions 805 to 867 are the coding genes of the T2A self-splicing polypeptide
  • positions 868 to 1785 are the complete coding genes of the ⁇ chain.
  • T cells having any of the above-mentioned T cell receptors also belong to the protection scope of the present invention.
  • a pharmaceutical composition containing any of the above-mentioned T cell receptors or "any of the above-mentioned expression cassettes, vectors or cells” also belongs to the protection scope of the present invention.
  • the pharmaceutical composition can be used to prevent and/or treat diseases caused by HBV infection.
  • the present invention also protects any of the above-mentioned T cell receptors, or, any of the above-mentioned nucleic acid molecules, or, any of the above-mentioned vectors or cells, or, any of the above-mentioned T cell receptors,
  • the application can be at least one of A1)-A4):
  • the target cell may be a spleen cell or a PBMC cell.
  • the spleen cells may specifically be spleen cells loaded with the polypeptide HBc 141-151 .
  • the PBMC cells may specifically be PBMC cells carrying the polypeptide HBc 141-151 .
  • the spleen cells carrying the polypeptide HBc 141-151 may specifically be mice (such as HLA-A11 transgenic mice) carrying the polypeptide HBc 141-151 .
  • the polypeptide HBc 141-151 of load cell can be loaded PBMC polypeptide HBc 141-151 mice (e.g. HLA-A11 transgenic mice) or HLA-A11 + PBMC PBMC cells.
  • HLA-A11 + PBMC cells refer to PBMC cells of HLA-A11 positive healthy people.
  • the present invention also claims a method for preventing and/or treating diseases caused by HBV infection, which may include the following steps: the aforementioned T cell receptor, or, the nucleic acid molecule, or, the expression cassette , Vector or cell", or T cell containing any of the above-mentioned T cell receptors to prevent and/or treat diseases caused by HBV infection.
  • Any of the aforementioned diseases caused by HBV infection may be chronic hepatitis B or hepatocellular carcinoma.
  • TCR-T cells TCR transgene-positive CD8 cells
  • HBV polypeptide epitope-dependent activation and proliferation at the same time, the use of animal in vivo and in vitro killing target cell experiments proved that this pair of TCR has a good kill target cells (HLA-A11 transgenic mice loaded with polypeptide HBc 141-151 The activity of human spleen cells or PBMC cells); In vitro verification Human TCR-T also has the ability to specifically kill target cells ( HLA-A11 restricted human PBMC cells loaded with polypeptide HBc 141-151 ); in addition, animal experiments suggest this The TCR sequence may be one of the effective methods to eliminate HBV-infected cells.
  • Figure 1 is a schematic diagram of the process of screening HLA-A11 restricted HBV-specific TCR sequences.
  • Figure 2 shows the staining results of Jurkat cells expressing TCR.
  • FIG. 3 shows the identification of TCR transgenic mice.
  • FIG. 4 shows that CD8 + T cells (ie TCR-T cells) of TCR transgenic mice have HBV polypeptide-dependent activation and proliferation ability.
  • FIG. 5 shows that TCR-T cells have the function of killing target cells in vitro.
  • FIG. 6 shows that TCR-T cells have the function of killing target cells in vivo.
  • FIG. 7 shows that TCR-T cells can effectively eliminate chronic HBV infection in the body.
  • FIG. 8 shows that Human TCR-T cells have the function of killing target cells in vitro.
  • test materials used in the following examples are all purchased from conventional biochemical reagent stores.
  • HLA-A11/hTAP-LMP transgenic mice polypeptide HBc 123-157 , accessory polypeptide HBc 128-140 and polypeptide HBc 141-151 are described in the following documents: Man Huang, Wei Zhang, Jie Guo, Xundong Wei, Krung Phiwpan, Jianhua Zhang, Xuyu Zhou. Improved Transgenic Mouse Model for Studying HLA Class I Antigen Presentation. Scientific Report. 2016, doi:10.1038/srep33612.
  • tail vein hypertension injection method is described in the following documents: Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasma DNA. Gene Therapy. 1999, doi: 10.1038/sj. gt.3300947.
  • the pAAV/HBV1.2 plasmid is described in the following documents: Huang, LR, Wu, HL, Chen, PJ&Chen, DSAn immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection. Proc Natl Acad Sci USA 103.17862-17867, doi:10.1073/pnas.0608578103(2006)
  • 10 ⁇ PCR buffer, dNTPmix and Taq DNA polymerase are all products of TAKARA.
  • C57BL/6 mice and ICR mice are the products of Beijing Huafukang Biotechnology Co., Ltd.
  • B6D2F1 mice are products of Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • HLA-A11/hTAP-LMP transgenic mice will induce the primary CTL immune response against the HBc 141-151 epitope.
  • the pAAV/HBV1.2 plasmid was injected into the tail vein of HLA-A11/hTAP-LMP transgenic mice by tail vein high pressure injection, and each mouse was injected with 10 ⁇ g pAAV/HBV1.2 plasmid .
  • the pAAV/HBV1.2 plasmid contains 1.2 copies of the genome of the HBV virus.
  • the tail vein high pressure injection method can be used to transiently transfect mouse liver cells and express hepatitis B virus antigens and produce virus particles, so it can be used to simulate the early stage of HBV in mice infection.
  • the purpose of this step is to induce a secondary CTL immune response against HBc 141-151 after the early infection of HBV, and generate a large number of CD8 + T cells specific for the HBc 141-151 antigen.
  • step 2 On the 8th day after the completion of step 2, the HLA-A11/hTAP-LMP transgenic mice were sacrificed and the peripheral blood mononuclear cells were collected for Tetramer staining. After that, the HLA-A11/HBc 141-151 Tetramer (NIH Tetramer Facility) and CD8 double-positive T cells, using a mouth suction microinjection needle to manually suck the antigen-specific CTL under the microscope to obtain a single antigen-specific CTL cell.
  • step 3 After completing step 3, first prepare the reverse transcription system Mix1 (consisting of 0.5 ⁇ L Random primer and 4.5 ⁇ L DEPC-treated H 2 O) and the reverse transcription system Mix2 (consisting of 5 ⁇ L 2 ⁇ buffer and 0.5 ⁇ L RT enzyme); Then use a microinjection needle under the microscope to blow a single antigen-specific CTL cell into the reverse transcription system Mix1, ice bath at 70°C for 5 minutes, and ice bath for 2 minutes; then add the reverse transcription system Mix2, 25°C for 5 minutes, 42°C for 30 minutes , 85°C for 5min, synthesize single-cell cDNA.
  • the reverse transcription system Mix1 consisting of 0.5 ⁇ L Random primer and 4.5 ⁇ L DEPC-treated H 2 O
  • the reverse transcription system Mix2 consisting of 5 ⁇ L 2 ⁇ buffer and 0.5 ⁇ L RT enzyme
  • Random primer is a product of Shenggong Bioengineering (Shanghai) Co., Ltd.
  • 2 ⁇ buffer and RT enzyme are products of Beijing Quanshijin Biotechnology Co., Ltd.
  • step 5 After completing step 4, perform two rounds of TCR-specific degenerate primer PCR.
  • the first round of PCR TCR ⁇ -mix, 23 in the V area, 1 in the C area; TCR ⁇ -mix, 19 in the V area, and 1 in the C area.
  • the second round of PCR TCR ⁇ -mix(in), 23 in the V area, 1 in the C area; TCR ⁇ -mix(in), 19 in the V area, and 1 in the C area.
  • the nucleotide sequence of the primer is shown in Table 1.
  • the first round of PCR reaction system is 25 ⁇ L, see Table 2 for details.
  • the reaction procedure is: 95°C for 5 minutes; 95°C for 20s, 56°C for 20s, 72°C for 45s, 34 cycles; 72°C for 7 minutes.
  • the second round of PCR reaction system is 25 ⁇ L, see Table 3 for details.
  • the reaction procedure is: 95°C for 5 minutes; 95°C for 20s, 56°C for 20s, 72°C for 45s, 34 cycles; 72°C for 7 minutes.
  • a total of 40 antigen-specific CTLs were detected.
  • 5 antigen-specific CTLs can obtain ⁇ chain sequence and ⁇ chain sequence, that is, 5 pairs of TCR receptor sequences can be obtained.
  • the ⁇ chain and ⁇ chain of these 5 antigen-specific CTLs are exactly the same.
  • the coding gene of the ⁇ chain variable region is shown in SEQ ID No. 1 at positions 64-336; in SEQ ID No. 1, positions 142-159, 211-231, and 334-363, respectively It is the coding gene of the three CDRs.
  • the amino acid sequence of the variable region of the ⁇ chain is shown in SEQ ID No. 2 at positions 22-112; in SEQ ID No. 2, positions 48-53, 71-77, and 112-121, respectively. There are three complementary decision areas.
  • the coding genes of the ⁇ chain variable region are shown in SEQ ID No. 3 at positions 58-339; in SEQ ID No. 3, positions 136-150, 202-219, and 331-366, respectively It is the coding gene of the three CDRs.
  • amino acid sequence of the ⁇ chain variable region is shown in SEQ ID No. 4, positions 20-113; in SEQ ID No. 4, positions 46-50, 68-73, and 111-122, respectively. There are three complementary decision areas.
  • the TCR receptor composed of the ⁇ -chain sequence and ⁇ -chain sequence may have high affinity for the HLA-A11 restricted CTL epitope HBc 141-151 and come from the same T cell clone.
  • variable region of the ⁇ chain and the variable region of the ⁇ chain obtained in step 1 refer to the sequence of the constant region of the mouse genome ⁇ chain and ⁇ chain on NCBI to obtain and artificially synthesize HLA-A11 restricted hepatitis B virus HBc 141-151 specific TCR receptor alpha chain and beta chain complete coding gene.
  • SEQ ID No. 1 The complete coding gene of the ⁇ chain is shown in SEQ ID No. 1, which encodes the ⁇ chain shown in SEQ ID No. 2.
  • the complete coding gene of the ⁇ chain is shown in SEQ ID No. 3, which encodes the ⁇ chain shown in SEQ ID No. 4.
  • positions 1 to 804 are the complete coding genes of the ⁇ chain
  • positions 805 to 867 are the coding genes of the T2A self-splicing polypeptide
  • positions 868 to 1785 are the complete coding genes of the ⁇ chain.
  • the retroviral vector MSCV-IRES-GFP replaces the small DNA fragments between the restriction enzymes XhoI and ClaI of the MO vector with the IRES nucleotide sequence (Genbank: MG550106.1) and the fluorescent marker protein GFP nucleotide Sequence (Genbank: MH777595.1), the resulting recombinant plasmid.
  • the MO vector is recorded in the following documents: Tanyu Hu, Krung Phiwpan, Jitao Guo, et al. MicroRNA-142-3p Negatively Regulates Canonical Wnt Signaling Pathway. PLOS ONE. 2016, DOI: 10.1371/journal.pone.0158432.
  • the recombinant plasmid MSCV-TCR-GFP was replaced with the retroviral vector MSCV-NGFR-GFP, and the other steps were unchanged to obtain Jurkat cells expressing NGFR.
  • the retroviral vector MSCV-NGFR-GFP replaces the small DNA fragment between the restriction enzymes XhoI and EcoRI of the MO vector with the NGFR nucleotide sequence (sequence derived from addgene NGFR plasmid, Plasmid#27489), and then The small DNA fragment between restriction endonuclease EcoRI and ClaI was replaced with IRES nucleotide sequence (Genbank: MG550106.1) and fluorescent marker protein GFP nucleotide sequence (Genbank: MH777595.1) to obtain a recombinant plasmid.
  • TCR transgenic mice Construction and identification of HBc 141-151 specific TCR transgenic mice (hereinafter referred to as TCR transgenic mice)
  • the phCD2 plasmid is recorded in the following documents: Zhumabekov T, Corbella P, Tolani M. Kioussis D. Improved version of a human CD2 minigene based vector for T cell-specific expression in transgenic mice. Journal of Immunological Methods. 1995 (Sep 11; 185) ):133-40.
  • the p428 plasmid is recorded in the following documents: Sawada S, Scarborough JD, Killeen N, Littman DR. A lineage-specific transcriptional silencer regulations CD4 gene expression during T mphocyte development. Cell. 1994 Jun 17; 77(6): 917-29.
  • mice Three-week-old C57BL/6 female mice were intraperitoneally injected with PMSG (5U/mouse); 48h later, HCG (5U/mouse) was intraperitoneally injected; immediately after HCG injection, they were caged 1:1 with B6D2F1 male mice, and caged for 18 ⁇ 18. Check the vaginal plug after 24 hours, and the mice with the vaginal plug are put forward for use;
  • step (1) C57BL/6 female mice and B6D2F1 male mice were caged together. At the same time, ICR female mice and ligated ICR male mice were caged at a ratio of 2:1. After caged for 18-24 hours, check that there is negative Tethered mice (ie, pseudo-pregnant female mice) were put forward for use.
  • the C57BL/6 female mice with vaginal plug in step 2 (1) were sacrificed by dissection, and the mice were dissected, the upper part of the uterus was clamped with forceps, the fallopian tubes were separated into M2 medium, and the pots of the fallopian tubes were cut under a microscope Abdomen, let the eggs flow into the culture medium.
  • Anesthetize the pseudo-pregnant female mouse surgically remove the ovaries connected to the fallopian tubes and fix them with fat forceps, find the fallopian tube opening under the microscope.
  • the transplantation tube sucks the fertilized egg cultured after microinjection of DNA, inserts the transplantation tube port into the fallopian tube orifice, gently blows the liquid in the transplantation tube, puts the ovary and fallopian tube back into the abdominal cavity, and sutures the muscle and skin.
  • the first established mice were replaced with wild-type mice, and the other steps were unchanged, as a control.
  • CD8 + T cells ie TCR-T cells
  • TCR-T cells have HBV polypeptide-dependent activation and proliferation ability
  • the specific TCR transgenic positive T cells in TCR transgenic mice are TCR-T cells.
  • step 2 After completing step 1, add 2% FBSDMEM medium to make the volume reach 15 mL, centrifuge at 4°C and 2000 rpm for 2 min, and remove the supernatant to wash away unbound antibodies. Then every 3 ⁇ 10 7 cells was added 1mLGoatAnti-MouseIgG (QIAGEN, 310007) , 1mL GoatAnti-RatIgG (QIAGEN, NO: 310,107) and re-suspended magnetic beads (magnetic beads taken, containing 0.5% (v / v) BSA Washed with 2mM EDTA in 1 ⁇ PBS buffer twice, then resuspended in 1 ⁇ PBS buffer containing 0.5% (v/v) BSA and 2mM EDTA), and incubated at 4°C with rotation for 30min.
  • 1mLGoatAnti-MouseIgG QIAGEN, 310007
  • GoatAnti-RatIgG QIAGEN, NO: 310,107
  • step 2 use a magnet to adsorb the magnetic beads to remove CD4 + T cells and B cells. About 90% of the remaining cells in the supernatant are CD8 + T cells, and then label the resulting CD8 + cells with CFSE, CFSE
  • the basic process of labeling is: take the cells to be labeled, add CFSE diluent or CFSE stock solution as needed, mix well, incubate at 37°C and 5% CO 2 for 10 minutes; then add 10 times the volume of pre-warmed 1640 complete medium to stop Labeled and resuspended in PBS to obtain CFSE-labeled TCR-T cells.
  • mice Take the spleen cells of HLA-A11/hTAP-LMP transgenic mice, lyse red blood cells, count the cells, add fresh 1640 medium to dilute the cells to 1 ⁇ 10 7 cells/mL, and then transgene into HLA-A11/hTAP-LMP
  • the spleen cells of mice were added with polypeptide HBc 141-151 and the concentration in the system was 10 ⁇ g/mL, and cultured at 37° C. and 5% CO 2 for 1 hour.
  • step 5 wash twice with 1640 medium and count the cells.
  • step 6 Mix the cells obtained in step 5 and CFSE-labeled TCR-T cells 1:1, and incubate at 37°C and 5% CO 2 for 1 d, 2 d or 3 d.
  • step 6 After completing step 6, perform flow cytometry to analyze the proliferation and activation of TCR-T cells.
  • the polypeptide HBc 141-151 in the above method is replaced with a control polypeptide, and the other steps are unchanged, as a control.
  • the control polypeptide is specifically NP91 (NP91 is the 91-99 peptide fragment of PR8 influenza virus NP protein, synthesized by Beijing Xuheyuan Biotechnology Co., Ltd.).
  • polypeptide HBc 141-151 in the above method was replaced with Anti-CD3 antibody (clone number 145-2C11, Bioxcell), and the other steps were unchanged, as a positive control.
  • the detection result is shown in Figure 4 (the specific polypeptide is the polypeptide HBc 141-151 , and Anti-CD3 is the Anti-CD3 antibody).
  • the results show that HBc 141-151 can effectively activate TCR-T cells and can effectively stimulate the proliferation of TCR-T cells. It can be seen that TCR-T cells have the ability of HBV polypeptide-dependent activation and proliferation in vitro. When TCR-T cells are transfused back to HBV-positive animals or patients, they can effectively activate and expand the transfused TCR-T cells in vivo. .
  • TCR-T cells have the function of killing target cells in vitro
  • TCR transgenic mouse lymph nodes and spleen cells Take TCR transgenic mouse lymph nodes and spleen cells, treat the sample with magnetic beads that bind CD4 + T cells and B cells to remove CD4 + T cells and B cells, thereby enriching CD8 + T cells, and counting the cells.
  • HLA-A11/hTAP-LMP transgenic mice Take the spleen cells of HLA-A11/hTAP-LMP transgenic mice, lyse red blood cells, count the cells, add fresh 1640 medium to dilute the cells to 1 ⁇ 10 7 cells/mL, and then transfer to HLA-A11/hTAP-LMP
  • the spleen cells of transgenic mice were added with polypeptide HBc 141-151 and the concentration in the system was 10 ⁇ g/mL, and cultured at 37° C. and 5% CO 2 for 1 hour.
  • step (3) After completing step (2), wash twice with 1640 medium and count the cells.
  • step (3) Mix the cells obtained in step (3) with the CD8 + T cells of TCR transgenic mice, add 400U/mL IL- 2, and culture for 5 days at 37°C and 5% CO 2 to obtain in vitro activated TCR-T cells .
  • HLA-A11/hTAP-LMP transgenic mice Take the spleen cells of HLA-A11/hTAP-LMP transgenic mice, lyse red blood cells, count the cells, add fresh 1640 medium to dilute the cells to 1 ⁇ 10 7 cells/mL, and then transgene into HLA-A11/hTAP-LMP Add the polypeptide HBc 141-151 to the spleen cells of mice and make the concentration in the system 10 ⁇ g/mL, incubate at 37°C and 5% CO 2 for 1 hour, then wash twice with 1640 medium, count the cells, and obtain the loaded polypeptide HBc 141-151 target cells.
  • step 3 Add the target cells carrying the polypeptide HBc 141-151 obtained in step 2 into a 96-well plate, 1 ⁇ 10 4 cells/well; then add the in vitro activated TCR at a ratio of 10:1, 5:1 or 1:1 -T cells, cultured at 37°C and 5% CO 2 for 5 hours
  • Substrate, stop solution and cell lysate are all CytoTox Non-Radioactive Cytotoxicity Assay kit (Promega, catalog number G1780).
  • Step 3A) is: take a 96-well plate, add the same number of in vitro activated TCR-T cells as the experimental group, and incubate at 37° C. and 5% CO 2 for 5 hours.
  • Step 3B) is: take a 96-well plate, add the same number of target cells carrying the polypeptide HBc 141-151 as the experimental group, and incubate at 37° C. and 5% CO 2 for 5 hours.
  • Step 3C) is: take a 96-well plate, add the same number of target cells loaded with polypeptide HBc 141-151 and 10 ⁇ L of cell lysate as the experimental group, and incubate at 37° C. and 5% CO 2 for 5 hours.
  • Killing activity (experimental group value-T cell self-release value-target cell self-release value)/(target cell maximum release value-target cell self-release value)
  • polypeptide HBc 141-151 in the above method was replaced with NP91, and the other steps were unchanged, as a control.
  • T represents the ratio of TCR-T cells to target cells.
  • the results show that TCR-T cells can effectively kill target cells in vitro.
  • TCR-T cells have the function of killing target cells in vivo
  • TCR transgenic mouse lymph nodes and spleen cells Take TCR transgenic mouse lymph nodes and spleen cells, treat the sample with magnetic beads that bind CD4 + T cells and B cells to remove CD4 + T cells and B cells, thereby enriching CD8 + T cells, and counting the cells.
  • HLA-A11/hTAP-LMP transgenic mice Take the spleen cells of HLA-A11/hTAP-LMP transgenic mice, lyse red blood cells, count the cells, add fresh 1640 medium to dilute the cells to 1 ⁇ 10 7 cells/mL, and then transfer to HLA-A11/hTAP-LMP
  • the spleen cells of transgenic mice were added with polypeptide HBc 141-151 and the concentration in the system was 10 ⁇ g/mL, and cultured at 37° C. and 5% CO 2 for 1 hour.
  • step (3) After completing step (2), wash twice with 1640 medium and count the cells.
  • step (3) Mix the cells obtained in step (3) with the CD8+T cells of TCR transgenic mice, add 400U/mL IL- 2, and culture for 5 days at 37°C and 5% CO 2 to obtain in vitro activated TCR-T cell.
  • HLA-A11/hTAP-LMP transgenic mice Take the spleen cells of HLA-A11/hTAP-LMP transgenic mice, lyse the red blood cells, count the cells, add fresh 1640 medium to dilute the cells to 1 ⁇ 10 7 cells/mL, and then transfer to HLA-A11/hTAP-LMP
  • the spleen cells of transgenic mice were added with polypeptide HBc 141-151 and the concentration in the system was 10 ⁇ g/mL, cultured at 37° C. and 5% CO 2 for 1 hour, then washed twice with 1640 medium, and the cells were counted.
  • step (1) After completing step (1), resuspend the cells with PBS to obtain a spleen cell resuspension solution with a concentration of 5 ⁇ 10 7 /mL carrying the polypeptide HBc 141-151.
  • step (3) after completion of step (2), with lower concentrations of CFSE (CFSE low) marker polypeptide HBc load of splenocytes 141-151 (HBc polypeptide per mL of load must be added to the splenocytes 141-151 1 ⁇ L concentration of 0.5mM CFSE dilution; when CFSE labeling, the concentration of CFSE is 0.5 ⁇ M) to obtain spleen cells loaded with polypeptide HBc 141-151 and labeled with CFSE low.
  • CFSE CFSE low marker polypeptide HBc load of splenocytes 141-151
  • step (4) After completing step (4), wash twice with 1640 medium and count the cells.
  • step (5) After completing step (5), resuspend in PBS buffer to obtain a cell resuspension with a concentration of 5 ⁇ 10 7 /mL.
  • step (6) After completing step (6), use a higher concentration of CFSE (CFSE high ) to label the spleen cells that are not loaded with polypeptides (for each mL of spleen cells that are not loaded with peptides, add 1 ⁇ L of CFSE stock solution at a concentration of 5 mM; when performing CFSE labeling , The concentration of CFSE is 5 ⁇ M), and spleen cells labeled with CFSE high are obtained.
  • CFSE high concentration of CFSE
  • TCR-T cells have the function of killing target cells in vivo
  • each C57/B6J mouse was reinfused (tail vein infusion) of the in vitro activated TCR-T cells obtained in step 1 of 1 ⁇ 10 7 cells.
  • step (3) 24 hours after the completion of step (3), the mice were sacrificed, peripheral blood and spleen cells were harvested, red blood cells were lysed, and white blood cells were obtained by centrifugation to prepare a single cell suspension.
  • Flow cytometry detects the ratio of CFSE low to CFSE high in peripheral blood and spleen.
  • the killing function of TCR-T cells was quantitatively analyzed by the ratio of CFSE low and CFSE high.
  • step (3) is replaced with step K, and the other steps are unchanged, as a control for the reinfusion of PBS.
  • Step K is: 2h after completing step (2), each C57/B6J mouse is infused with the same volume of PBS buffer as the TCR-T cells activated in vitro in step (3).
  • test results are shown in Figure 6. Compared with the PBS control group, whether in the peripheral blood or the spleen, the cell population labeled with CFSE low decreased significantly compared with the cell population labeled with CFSE high. The results show that TCR-T cells can effectively kill target cells in vivo.
  • TCR-T cells can effectively eliminate chronic HBV infection in the body
  • RAAV/HBV1.3 virus (product of Beijing Wujiahe Institute of Molecular Medicine Co., Ltd.) was injected into the tail vein of HLA-A11/hTAP-LMP transgenic mice at a dose of 5 ⁇ 10 9 vg/mouse.
  • step (1) After completing step (1), observe the change curve of the virus gradient (hepatitis B virus s antigen, hepatitis B virus e antigen or viral DNA) under natural conditions, and predict the administration time of TCR-T cells based on the change curve.
  • virus gradient hepatitis B virus s antigen, hepatitis B virus e antigen or viral DNA
  • a mouse chronic HBV infection model is obtained.
  • the expression of HBsAg and HBeAg in the mouse chronic HBV infection model is regulated by the promoter of HBV virus DNA itself. Continuous expression of HBsAg and HBeAg can be observed in the liver and peripheral blood for 10 weeks.
  • HLA-A11/hTAP-LMP transgenic mice in the above method were replaced with C57BL/6 mice, and the other steps were unchanged to obtain chronic HBV infected C57BL/6 mice as a negative control.
  • TCR-T cells treat chronic HBV infection in HLA-A11/hTAP-LMP mice
  • TCR transgenic mouse lymph nodes and spleen cells Take TCR transgenic mouse lymph nodes and spleen cells, treat the sample with magnetic beads that bind CD4 + T cells and B cells to remove CD4 + T cells and B cells, thereby enriching CD8 + T cells, and counting the cells.
  • HLA-A11/hTAP-LMP transgenic mice Take the spleen cells of HLA-A11/hTAP-LMP transgenic mice, lyse red blood cells, count the cells, add fresh 1640 medium to dilute the cells to 1 ⁇ 10 7 cells/mL, and then transfer to HLA-A11/hTAP-LMP
  • the spleen cells of transgenic mice were added with polypeptide HBc 141-151 and the concentration in the system was 10 ⁇ g/mL, and cultured at 37° C. and 5% CO 2 for 1 hour.
  • step (3) After completing step (2), wash twice with 1640 medium, count the cells, then mix the cells and the CD8 + T cells of the TCR transgenic mice, and add 400U/mL IL-2, 37°C, 5% Cultured in CO 2 for 5 days, TCR-T cells activated in vitro were obtained.
  • step (4) After completing step (4), it is marked as day 0. Detect the OD 450nm value of HBsAg in serum every two days (HBsAg detection kit, Shanghai Kehua) and ALT concentration (ALT/GPT kit, automatic biochemical analyzer) MEDSOUL AMS-18) to determine the HBV infection in the body. A total of 8 days of testing.
  • step (4) On the 7th day after completing step (4), take the HLA-A11/hTAP-LMP transgenic mouse chronic HBV infection model, and inject 1 ⁇ 10 7 activated TCR-T cells in vitro for the second time.
  • step (6) Two days after completing step (6), detect the OD450nm value and ALT concentration of HBsAg in the serum.
  • Fig. 7 A The detection result of the OD 450nm value of HBsAg in the serum is shown in Fig. 7 A (WT 1#, 2#, 3#, 4# are C57BL/6 mice chronic HBV infection model, A11.hTAP 36#, A11.hTAP 37 #, A11.hTAP 39#, A11.hTAP 40# and A11.hTAP 43# are all HLA-A11/hTAP-LMP transgenic mice chronic HBV infection models).
  • the results of detection of ALT concentration in serum are shown in B in Figure 7 (WT 1#, 2#, 3#, 4# are C57BL/6 mice chronic HBV infection models, A11.hTAP 36#, A11.hTAP 37#, A11.
  • hTAP 39#, A11.hTAP 40# and A11.hTAP 43# are all HLA-A11/hTAP-LMP transgenic mice chronic HBV infection models). The results show that TCR-T cells can effectively eliminate HBV virus infection in the chronic HBV infection model of HLA-A11/hTAP-LMP transgenic mice.
  • the 293T cells were resuspended into single cells by pipetting, counted, and then adjusted with DMEM medium containing 10% (v/v) FBS to obtain a cell suspension with a concentration of 5 ⁇ 10 5 cells/mL. Take a petri dish (10 cm in size), spread 10 mL of cell suspension, and incubate overnight.
  • step (3) After completing step (2), transfection is performed when the confluence of 293T cells reaches 75%, and the medium is changed to DMEM medium 30 minutes before transfection.
  • step (4) After completing step (4), add 500 ⁇ LPEI mixture to 500 ⁇ L plasmid mixture, vortex to mix well, and incubate at room temperature for 20min; then gently add the incubated mixture to the 293T cells along the side wall of the culture dish, and gently shake the culture dish Mix well and incubate in a 37°C incubator. After 6-8h, the medium was replaced with 10 mL DMEM medium containing 10% (v/v) FBS.
  • step (5) collect the first virus supernatant, supplement with fresh DMEM medium, and store the virus supernatant at 4°C.
  • step (5) collect the second virus supernatant.
  • the first virus supernatant and the second virus supernatant were combined, centrifuged at 800 g for 5 min at room temperature, and the supernatant was collected.
  • step (7) After completing step (7), filter the supernatant through a 0.45 ⁇ m PES filter to remove cell debris, and collect the virus supernatant.
  • step (8) After completing step (8), transfer the virus supernatant to an ultracentrifuge tube. Centrifuge at 70,000g for 120 min at 4°C, carefully discard the supernatant, and collect the precipitate (white virus particles).
  • step (10) After completing step (9), take the precipitate, add 100 times the concentrated volume of 1640 medium to resuspend, and dissolve at 4°C overnight to obtain concentrated lentivirus. Store the concentrated lentivirus in an ultra-low temperature refrigerator at -80°C for later use.
  • step (2) After completing step (1), take a centrifuge tube (specification of 50 mL), add 5 mL of peripheral blood and 5 mL of PBS buffer, and mix thoroughly.
  • step (3) After completing step (2), add 5 mL of human peripheral blood lymphocyte separation solution (Tianjin Haoyang Biological Products Technology Co., Ltd., Item No. LTS1077) into the centrifuge tube, and use a disposable sterile dropper to take the diluted peripheral blood Superimpose carefully on the surface of the separation liquid along the wall of the tube, taking care to maintain a clear interface.
  • human peripheral blood lymphocyte separation solution Tetranjin Haoyang Biological Products Technology Co., Ltd., Item No. LTS1077
  • step (3) place the centrifuge tube in a centrifuge, adjust the speed up and speed down to the lowest, and centrifuge at 800g for 20 minutes.
  • the tube is divided into four layers after centrifugation.
  • the first layer is plasma and PBS
  • the second layer is a ring-shaped milky white lymphocyte layer
  • the third layer is a transparent separation layer
  • the fourth layer It is the layer of red blood cells and granulocytes.
  • Anti-CD3 antibody diluent Dilute anti-CD3 antibody (BioXcell, clone number: OKT3) with PBS buffer to a concentration of 3 ⁇ g/mL.
  • Anti-CD28 antibody diluent Dilute anti-CD28 antibody (BioXcell, clone number: CD28.2) with PBS buffer to a concentration of 1 ⁇ g/mL.
  • step (1) After completing step (1), take the 24-well plate, remove the liquid, and wash once with PBS buffer.
  • step (3) After completing step (2), take the 24-well plate, add 500 ⁇ L of human peripheral T lymphocyte diluent, incubate at 37°C for 48 hours; then centrifuge at 400 g for 5 minutes, collect the precipitate and resuspend it in 1640 medium to obtain Activated human peripheral T lymphocytes. Activated human peripheral T lymphocytes are used to infect lentivirus.
  • Human peripheral T lymphocyte diluent Dilute the human peripheral T lymphocytes obtained in step 2 to 4 ⁇ 10 6 cells/mL with 1640 medium.
  • step (1) After completing step (1), take the 24-well plate and centrifuge at 600g for 90 min at 32°C; then place the 24-well plate in a 37°C incubator for 24 hours.
  • step (3) After completing step (2), take the 24-well plate, carefully suck off 350 ⁇ L of medium in the infected well, and then add 1640 medium to make up to a volume of 2 mL, pipetting and mixing, and continue culturing in a 37°C incubator for 48 hours.
  • step (3) After completing step (3), first take an appropriate amount of infected T cells for flow cytometric detection of infection efficiency; then use anti-mouse CD8a antibody (Biolegend, clone number: 53-6.7) and anti-mouse TCRV ⁇ 10 antibody (BD Bioscience, Clone number: B21.5) Stain the infected T cells. After staining at 4°C for 30 minutes, perform the machine test.
  • the infection efficiency (TCRV ⁇ 10 positive rate) is greater than 15%, and the positive cells are the Human TCR-T cells successfully transferred to the TCR. , Used for follow-up killing experiments.
  • HumanTCR-T has the function of killing target cells in vitro
  • Substrate, stop solution and cell lysate are all CytoTox
  • the components in the Non-Radioactive Cytotoxicity Assay Kit (Promega, Catalog No. G1780).
  • step (1) After completing step (1), use 1640 medium to dilute HLA-A11 + PBMC cells to a concentration of 1 ⁇ 10 7 cells/mL, then add polypeptide HBc 141-151 and make the concentration in the system 10 ⁇ g /mL, 37°C, 5% CO 2 for 1 hour, then washed twice with 1640 medium, and counted cells to obtain target cells carrying the polypeptide HBc 141-151.
  • step (3) Add the target cells carrying the polypeptide HBc 141-151 obtained in step (2) into a 96-well plate, 2 ⁇ 10 4 cells/well; then add Human TCR- according to the effective target ratio of 2:1 or 1:1. T cells were cultured at 37°C and 5% CO 2 for 5 hours.
  • Step 3A) is: take a 96-well plate, add the same number of HumanTCR-T cells as the experimental group, and incubate at 37°C and 5% CO 2 for 5 hours.
  • Step 3B) is: take a 96-well plate, add the same number of target cells carrying the polypeptide HBc 141-151 as the experimental group, and incubate at 37° C. and 5% CO 2 for 5 hours.
  • Step 3C) is: take a 96-well plate, add the same number of target cells loaded with polypeptide HBc 141-151 and 10 ⁇ L of cell lysate as the experimental group, and incubate at 37° C. and 5% CO 2 for 5 hours.
  • Killing activity (experimental group value-T cell self-release value-target cell self-release value)/(target cell maximum release value-target cell self-release value)
  • polypeptide HBc 141-151 in the above method was replaced with NP91, and the other steps were unchanged, as a control.
  • FIG. 8 A is the acquisition of Human TCR-T cells; B is the in vitro killing target cell experiment of Human TCR-T cells, where E:T represents the ratio of Human TCR-T cells to target cells, and T+HBc 141- 151 means Human T cells kill the target cells incubated with HBc 141-151 polypeptide, TCR-T+NP 91 means Human TCR-T kills control cells incubated with NP 91 polypeptide, TCR-T+HBc 141-151 means Human TCR-T cells Kill the target cells incubated with HBc 141-151 polypeptide).
  • the results show that Human TCR-T cells can effectively kill target cells in vitro.
  • the inventors of the present invention isolated and identified a pair of HBV-specific TCR sequences, successfully constructed the pair of TCR transgenic mice, and verified in vitro that TCR transgene-positive CD8 cells (ie TCR-T Cell) has HBV polypeptide epitope-dependent activation and proliferation ability; using animal in vivo and in vitro killing target cell experiments, it is proved that this has a good killing target cell activity for TCR; in vitro verification Human TCR-T also has specificity The ability to kill HLA-A11 + target cells; in addition, animal experiments suggest that this pair of TCR sequences may be one of the effective methods to eliminate HBV-infected cells.
  • the present invention has isolated and identified a pair of HBV-specific TCR sequences, successfully constructed this pair of TCR transgenic mice, and verified in vitro that TCR transgene-positive CD8 cells (ie TCR-T cells) have HBV polypeptide epitope dependence Sexual activation and proliferation; using animal in vivo and in vitro killing target cell experiments at the same time, it proves that this pair of TCR has a good kill target cell (HLA-A11 transgenic mice loaded with polypeptide HBc 141-151 spleen cells or PBMC cells) In vitro verification Human TCR-T also has the ability to specifically kill target cells ( HLA-A11 restricted human PBMC cells loaded with the polypeptide HBc 141-151 ); in addition, animal experiments suggest that this pair of TCR sequences may be the elimination of HBV One of the effective ways to infect cells.
  • the HLA-A11 restricted HBc 141-151 epitope peptide T cell receptor provided by the present invention has important application value.

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Abstract

本发明公开了HLA-A11限制性乙型肝炎病毒HBc 141-151表位肽的T细胞受体及其应用。该T细胞受体包含α链和β链；α链包含三个互补决定区，氨基酸序列分别如SEQ ID No.2的第48-53位、第71-77位和第112-121位所示；β链包含三个互补决定区，氨基酸序列分别如SEQ ID No.4的第46-50位、第68-73位和第111-122位所示。实验证明，该T细胞受体不仅具有HBV多肽表位依赖性的活化和增殖能力，而且体内和体外均具有良好的杀伤靶细胞的活性，还能够有效清除体内HBV的慢性感染。本发明具有很重要的应用价值。

Description

HLA-A11限制性乙型肝炎病毒HBc 141-151表位肽的T细胞受体及其应用 技术领域
本发明属于生物医学领域，具体涉及HLA-A11限制性乙型肝炎病毒HBc 141-151表位肽的T细胞受体及其应用。
背景技术
肝细胞癌(Hepatocellular Carcinoma，HCC)简称肝癌，其临床表现隐匿，早期缺乏典型症状，尽管近年来肝癌的诊断和治疗有了一定的进展，其预后仍然不理想，五年生存率极低。究其原因，一方面是肝癌对目前化疗药物不敏感，大多数肝癌患者缺乏有效的治疗方法；另一方面，肝癌往往在疾病的晚期才被诊断，这就排除了局部消融这些原本可以改善病人病情的方法。目前，手术切除与肝移植仍然是治疗肝癌最有效的方法。但是肝癌发展到一定阶段，肿瘤细胞可能转移到其它器官(如肺部、骨骼、脑等)，行肝移植前，现阶段的检查检测不到，术后因免疫抑制状态，潜伏在其它器官的微病灶可能导致肝癌复发，患者术后生存期不理想。因此，亟需找寻新的有效辅助治疗方案。
免疫细胞治疗技术是生物医药领域中发展最为迅速的板块。2013年，科学转化医学(Science Translational Medicine)杂志预测，细胞治疗将成为“未来医学第三大支柱”。2017年10月以来，美国FDA相继批准诺华与KITE公司的CAR-T药物上市，开创免疫细胞治疗药物的新时代，在临床治疗中展现出广阔的应用前景。在实体肿瘤免疫细胞治疗领域，理想的目标抗原是仅在肿瘤细胞表面表达的肿瘤特异性抗原。不幸的是肿瘤表达的大多数抗原不具备肿瘤特异，因此大多数的CAR-T、TCR-T都以肿瘤相关性抗原作为靶点，但这往往会导致“脱靶”的可能性。目前使用较多的一些肿瘤相关抗原，甲胎蛋白、NY-ESO、MAGE等蛋白都属于这一状态。
在诸多肝癌相关环境危险因素中，乙型肝炎病毒(Hepatitis B Virus，HBV)或丙型肝炎病毒(Hepatitis C Virus，HCV)感染与肝癌的发生直接相关。高达80％的肝癌归因于HBV或HCV感染，在我国主要是HBV感染为主，90％肝癌患者HBsAg呈阳性。HBV感染人体后可刺激机体产生一系列体液免疫和细胞免疫反应，通常可以清除感染的病毒而痊愈；但如果机体免疫反应低下，不足以清除病毒，则病毒可持续存在，可发展成为慢性乙肝。慢性乙肝是肝硬化和肝癌发生的重要危险因素。慢性乙肝主要涉及免疫反应各个阶段的抗病毒免疫反应缺陷，尤其是HBV特异性CD8 +T细胞的数量下降、功能降低，引起免疫耐受；同时伴随免疫介导的炎症性肝损伤。
目前，尚未发现能够治愈乙肝的药物。临床上针对慢性乙肝的治疗手段主要为抗病毒相关药物的治疗，包括干扰素类(IFNs)及核苷和核苷酸类药物等，通过免疫调节或干扰HBV复制发挥抗病毒作用。然而，这些治疗手段不能彻底清除病毒，使得患者容易出现耐药、病毒变异以及病情反复等情况。
HBV特异性CD8 +T细胞在控制病毒的复制、清除病毒和HBV感染的临床恢复上起着决定性作用。HBV特异性TCR基因修饰的T细胞(TCR-T)过继转移，已初步证明有非常好的抗病毒活性。在肝癌领域，HBV也可作为一种独特的肿瘤相关性抗原。虽然已知的HBV-HCC肝癌细胞不表达完整的HBV抗原，但是在慢性乙型肝炎的自然史感染中，病毒往往将自身整合到人类基因组中，最终形成HCC细胞并携带这些HBV基因组。新加坡学者研究发现，尽管不表达完整HBV抗原，HBV-HCC肝癌细胞却带有短片段的HBV mRNAs，这些mRNAs可以编码能够被HBV特异性T细胞识别并活化的表位多肽，他们入组了2例肝移植患者肝癌复发和肺部转移瘤，根据肿瘤中HBV mRNA表达选择HBV特异性T细胞受体(TCRs)，利用基因工程手段让自体T细胞表达这些TCRs，并过继转移到病人体内。这些TCR-T细胞不影响肝功能，在1例患者中，5/6肺转移瘤在1年内体积减小。该结果提示在肝癌病人，特别是肝移植患者治疗中，将肝癌细胞上HBV抗原表位当做肿瘤相关抗原进行T细胞过继免疫治疗有可能将HBV-HCC肝癌细胞清除掉。这种肝特异性标记意味着肿瘤外的不良反应在很大程度上是可以预测的，很少或不涉及其它器官。此外，也有其它团队临床研究通过体外诱导TCR重定向的T细胞的方法，将特异识别乙肝病毒HLA-A2限制性表位HBs 183-191的TCR-T细胞用于治疗肝移植患者中的肝癌转移，证明了这种重定向TCR-T细胞具有靶向特定肝癌细胞的活性，展现出巨大的应用潜力和价值。
现有报道的治疗性T细胞的研究主要局限在HLA-A2人群的研究，而针对其它HLA人群的关注却明显不足。HLA-A3超家族(包含HLA-A11、HLA-A33、HLA-A68、HLA-A31等)在中国人群HLA分型中占着最大比例(约52.7％)。HLA-A3超家族成员的显著特征之一是不同的成员具有共同的多肽结合特性，即偏好结合C末端带碱性氨基酸的多肽表位。因此，针对HLA-A3超家族限制性的人群进行肝癌的治疗研究将具有很好的大众性和通用性。其中，HLA-A11是A3超家族中分布最广的，统计分析也表明中国乙肝患者中HLA-A11基因频率最高。
HLA转基因动物模型在临床前试验和基础实验研究中发挥了重要作用。HLA-A11转基因小鼠之前已有报道，然而多方面的证据表明，HLA-A11分子偏好结合C末端碱性的表位多肽，与小鼠自身抗原提呈系统中抗原相关转运蛋白(TAP)的多肽结合偏好性不符，因而HLA-A11转基因小鼠在递呈HLA-A11限制性表位上存在明显缺陷。
将含人源TAP-LMP基因簇的BAC转基因小鼠(hTAP-LMP转基因小鼠)与HLA-A11转基因小鼠杂交，得到优化型的HLA-A11/hTAP-LMP转基因小鼠。与HLA-A11转基因小鼠相比，HLA-A11/hTAP-LMP转基因小鼠具有更强的提呈HLA-A11限制性CTL表位的能力。
综上所述，免疫细胞技术治疗乙肝和肝癌进入到临床试验阶段并取得了进展，显示出广阔的应用前景。但是在中国人群为主的HLA-A11限制性肝癌治疗亟需进行深入的研究。
发明公开
本发明的目的是制备用于预防和/或治疗由HBV感染所致的疾病的药物。
本发明首先保护一种识别HLA-A11限制性乙型肝炎病毒HBc 141-151表位肽的T细胞受体，可包含α链和β链。所述α链可包含三个互补决定区，氨基酸序列分别如SEQ ID No.2的第48-53位、第71-77位和第112-121位所示；或这些序列的具有至多3个、2个或1个氨基酸改变的变体。所述β链可包含三个互补决定区，氨基酸序列分别如SEQ ID No.4的第46-50位、第68-73位和第111-122位所示；或这些序列的具有至多3个、2个或1个氨基酸改变的变体。
所述T细胞受体中，所述α链的可变区的氨基酸序列可如SEQ ID No.2的第22-112位所示；或这些序列的具有至多3个、2个或1个氨基酸改变的变体。所述β链的可变区的氨基酸序列可如SEQ ID No.4的第20-113位所示；或这些序列的具有至多3个、2个或1个氨基酸改变的变体。
所述α链的恒定区的氨基酸序列为SEQ ID No.2的第133-268位所示。
所述β链的恒定区的氨基酸序列为SEQ ID No.4的第133-305位所示。
所述T细胞受体中，所述α链的氨基酸序列可如SEQ ID No.2所示。所述β链的氨基酸序列可如SEQ ID No.4所示。
编码上述任一所述T细胞受体的核酸分子也属于本发明的保护范围。
编码上述任一所述T细胞受体的核酸分子可包含编码所述T细胞受体的α链的核酸分子和编码所述T细胞受体的β链的核酸分子。
编码所述T细胞受体的α链中三个互补决定区的核苷酸序列分别可如SEQ ID No.1的第142-159位、第211-231位和第334-363位所示；或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码相同氨基酸残基的序列。
编码所述T细胞受体的β链中三个互补决定区的核苷酸序列分别可如SEQ ID No.3的第136-150位、第202-219位和第331-366位所示；或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码相同氨基酸残基的序列。
编码所述α链的可变区的核苷酸序列可如SEQ ID No.1的第64-336位所示；或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码相同氨基酸残基的序列。
编码所述β链的可变区的核苷酸序列如SEQ ID No.3的第58-339位所示；或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码相同氨基酸残基的序列。
所述α链的恒定区的核苷酸序列为SEQ ID No.1的第397-807位所示。
所述β链的恒定区的核苷酸序列为SEQ ID No.3的第397-918位所示。
编码所述α链的核酸分子的核苷酸序列如SEQ ID No.1所示。
编码所述β链的核酸分子的核苷酸序列如SEQ ID No.3所示。
含有上述任一所述核酸分子的表达盒、载体或细胞也属于本发明的保护范围。
所述细胞可为Jurkat细胞或T细胞。所述T细胞具体可为人T细胞或小鼠T细胞。
所述载体可为逆转录病毒载体或慢病毒载体。
所述逆转录病毒载体具体可为向逆转录病毒载体MSCV-IRES-GFP的多克隆位点(如限制性内切酶XhoI和EcoRI)之间插入编码所述T细胞受体的α链的核酸分子和编码所述T细胞受体的β链的核酸分子，得到的重组质粒。
所述逆转录病毒载体具体可为向逆转录病毒载体MSCV-IRES-GFP的多克隆位点(如限制性内切酶XhoI和EcoRI)之间插入DNA序列，得到的重组质粒；所述DNA序列为将编码所述α链的核酸分子和编码所述β链的核酸分子用连接肽(如T2A自剪切多肽)的编码序列连接而成。
在本发明的一个实施例中，所述逆转录病毒载体具体可为将逆转录病毒载体MSCV-IRES-GFP的限制性内切酶XhoI和EcoRI之间的DNA小片段替换为SEQ ID No.5所示的DNA分子，得到的重组质粒。SEQ ID No.5中，第1至804位为α链的完整编码基因，第805至867位为T2A自剪切多肽的编码基因，第868至1785位为β链的完整编码基因。
上述任一所述逆转录病毒载体MSCV-IRES-GFP是将MO载体的限制性内切酶XhoI和ClaI之间的DNA小片段替换为IRES核苷酸序列(Genbank：MG550106.1)和荧光标记蛋白GFP核苷酸序列(Genbank：MH777595.1)，得到的重组质粒。
所述慢病毒载体具体可为向慢病毒包装载体pCDH-MSCV-MCS-IRES-GFP(System Biosciences，编号：CD731B-1)的多克隆位点(如限制性内切酶EcoRI和BamHI)之间插入编码所述T细胞受体的α链的核酸分子和编码所述T细胞受体的β链的核酸分子，得到的重组质粒。
所述慢病毒载体具体可为向慢病毒包装载体pCDH-MSCV-MCS-IRES-GFP的多克隆位点(如限制性内切酶EcoRI和BamHI)之间插入DNA序列，得到的重组质粒；所述DNA序列为将编码所述α链的核酸分子和编码所述β链的核酸分子用连接肽(如T2A自剪切多肽)的编码序列连接而成。
在本发明的一个实施例中，所述慢病毒载体具体可为将慢病毒包装载体pCDH-MSCV-MCS-IRES-GFP的限制性内切酶EcoRI和BamHI之间的DNA小片段替换为SEQ ID No.5所示的DNA分子，得到的重组质粒。SEQ ID No.5中，第1至804位为α链的完整编码基因，第805至867位为T2A自剪切多肽的编码基因，第868至1785位为β链的完整编码基因。
具有上述任一所述T细胞受体的T细胞也属于本发明的保护范围。
含有上述任一所述T细胞受体的T细胞或“上述任一所述表达盒、载体或细胞”的药物组合物也属于本发明的保护范围。所述药物组合物可用于预防和/或治疗由HBV感染所致的疾病。
本发明还保护上述任一所述T细胞受体、或、上述任一所述核酸分子、或、上述任一所述载体或细胞、或、上述任一所述T细胞受体的T细胞，的应用，可为A1)-A4)中的至少一种：
A1)制备预防和/或治疗由HBV感染所致的疾病的药物；
A2)预防和/或治疗由HBV感染所致的疾病；
A3)体内或体外杀伤靶细胞；
A4)清除HBV的慢性感染。
上述应用中，所述靶细胞可为脾细胞或PBMC细胞。所述脾细胞具体可为负载多肽HBc 141-151的脾细胞。所述PBMC细胞具体可为负载多肽HBc 141-151的PBMC细胞。所述负载多肽HBc 141-151的脾细胞具体可为小鼠(如HLA-A11转基因小鼠)负载多肽HBc 141-151的脾细胞。所述负载多肽HBc 141-151的PBMC细胞可为负载多肽HBc 141-151的小鼠(如HLA-A11转基因小鼠)PBMC或HLA-A11 +PBMC细胞。HLA-A11 +PBMC细胞是指HLA-A11阳性健康人的PBMC细胞。
本发明还要求保护一种预防和/或治疗由HBV感染所致疾病的方法，可包括如下步骤：以前文所述的T细胞受体、或、所述核酸分子、或、“所述表达盒、载体或细胞”、或、含有上述任一所述T细胞受体的T细胞来预防和/或治疗由HBV感染所致疾病。
上述任一所述由HBV感染所致的疾病可为慢性乙型肝炎或肝细胞癌。
本发明的发明人经过大量实验，分离并鉴定了一对HBV特异性的TCR序列，成功构建了这对TCR的转基因小鼠，并且在体外验证了TCR转基因阳性的CD8细胞(即TCR-T细胞)具有HBV多肽表位依赖性的活化和增殖能力；同时利用动物体内和体外杀伤靶细胞实验，证明了这对TCR具有很好的杀伤靶细胞(HLA-A11转基因小鼠负载多肽HBc 141-151的脾细胞或PBMC细胞)的活性；体外验证Human TCR-T也具有特异性的杀伤靶细胞(负载多肽HBc 141-151的HLA-A11限制性人PBMC细胞)的能力；此外，动物实验提示这对TCR序列可能是清除HBV感染细胞的有效方法之一。本发明提供的HLA-A11限制性HBc 141-151表位肽的T细胞受体具有重要的应用价值。
附图说明
图1为筛选HLA-A11限制性HBV特异性TCR序列的流程示意图。
图2为表达TCR的Jurkat细胞的染色结果。
图3为TCR转基因小鼠的鉴定。
图4为TCR转基因小鼠的CD8 +T细胞(即TCR-T细胞)具有HBV多肽依赖的活化增殖能力。
图5为TCR-T细胞具有体外杀伤靶细胞的功能。
图6为TCR-T细胞具有体内杀伤靶细胞的功能。
图7为TCR-T细胞可以有效的清除体内HBV的慢性感染。
图8为Human TCR-T细胞具有体外杀伤靶细胞的功能。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
HLA-A11/hTAP-LMP转基因小鼠、多肽HBc 123-157、辅助多肽HBc 128-140和多肽HBc 141-151记载于如下文献中：Man Huang，Wei Zhang，Jie Guo，Xundong Wei，Krung Phiwpan，Jianhua Zhang，Xuyu Zhou.Improved Transgenic Mouse Model for Studying HLA Class I Antigen Presentation.Scientific Report.2016，doi:10.1038/srep33612.
下述实施例中，尾静脉高压注射法记载于如下文献中：Liu F，Song Y，Liu D.Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA.Gene Therapy.1999，doi:10.1038/sj.gt.3300947。
下述实施例中，pAAV/HBV1.2质粒记载于如下文献中：Huang，L.R.，Wu，H.L.，Chen，P.J.&Chen，D.S.An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection.Proc Natl Acad Sci USA 103.17862–17867，doi:10.1073/pnas.0608578103(2006)
10×PCR buffer、dNTPmix和Taq DNA polymerase均为TAKARA公司的产品。C57BL/6小鼠和ICR小鼠均为北京华阜康生物科技股份有限公司的产品。B6D2F1小鼠为北京维通利华实验动物技术有限公司的产品。
实施例1、TCR-T细胞的获得及其应用
一、应用HLA-A11/hTAP-LMP转基因小鼠筛选获得A11限制性HBV特异性TCR序列
1、将100μL含100μg多肽HBc 123-157和100μg辅助多肽HBc 128-140的PBS缓冲液和100μL IFA混合，乳化；然后皮下多点注射至HLA-A11/hTAP-LMP转基因小鼠。HLA-A11/hTAP-LMP转基因小鼠体内将诱导出针对HBc 141-151表位的初次CTL免疫应答。
2、完成步骤1后第14天，采用尾静脉高压注射法向HLA-A11/hTAP-LMP转基因小鼠的尾静脉注射pAAV/HBV1.2质粒，每只小鼠注射10μg pAAV/HBV1.2质粒。
pAAV/HBV1.2质粒包含HBV病毒1.2个拷贝的基因组，采用尾静脉高压注射法可瞬时转染小鼠肝细胞并表达乙肝病毒抗原以及产生病毒颗粒，因而可用于 在小鼠体内模拟HBV的早期感染。进行该步骤的目的是HBV的早期感染后，诱导针对HBc 141-151的二次CTL免疫应答，产生大量HBc 141-151抗原特异性的CD8 +T细胞。
3、完成步骤2后第8天，处死HLA-A11/hTAP-LMP转基因小鼠并收集外周血单核细胞，进行Tetramer染色，之后流式分选出HLA-A11/HBc 141-151Tetramer(NIH Tetramer Facility)和CD8双阳性的T细胞，使用口吸式显微注射针将抗原特异性CTL在显微镜下手动吸取单个细胞，即获得单个抗原特异性CTL细胞。
4、完成步骤3后，首先配制反转录体系Mix1(由0.5μL Random引物和4.5μLDEPC处理的H 2O组成)和反转录体系Mix2(由5μL 2×buffer和0.5μL RT酶组成)；然后在显微镜下使用显微注射针将单个抗原特异性CTL细胞吹入反转录体系Mix1中，70℃冰浴5min，冰浴2min；之后加入反转录体系Mix2，25℃5min，42℃30min，85℃5min，合成单细胞cDNA。
Random引物为生工生物工程(上海)股份有限公司的产品。
2×buffer和RT酶为北京全式金生物技术有限公司的产品。
5、完成步骤4后，进行两轮TCR特异性兼并引物PCR。
第一轮PCR：TCRα-mix，V区23条，C区1条；TCRβ-mix，V区19条，C区1条。
第二轮PCR：TCRα-mix(in)，V区23条，C区1条；TCRβ-mix(in)，V区19条，C区1条。
引物的核苷酸序列具体见表1。
表1
Figure PCTCN2020105157-appb-000001
Figure PCTCN2020105157-appb-000002
第一轮PCR反应体系为25μL，具体见表2。反应程序为：95℃5min；95℃20s，56℃20s，72℃45s，34个循环；72℃7min。
表2
Figure PCTCN2020105157-appb-000003
Figure PCTCN2020105157-appb-000004
第二轮PCR反应体系为25μL，具体见表3。反应程序为：95℃5min；95℃20s，56℃20s，72℃45s，34个循环；72℃7min。
表3
Figure PCTCN2020105157-appb-000005
6、完成步骤5后，两轮PCR结束后得到配对的TCR-α和TCR-β可变区序列。切胶回收PCR扩增产物，测序。将测序结果在IMGT网站(网址为：http://www.imgt.org/IMGT_vquest/vquest？livret＝0&Option＝mouseTcR)上进行分析，得到单个细胞的α链序列和β链序列。
共检测40个抗原特异性CTL。其中5个抗原特异性CTL可以获得α链序列和β链序列，即得到5对TCR受体序列。经过分析，这5个抗原特异性CTL的α链和β链完全相同。
α链可变区的编码基因如SEQ ID No.1的第64-336位所示；其中，SEQ ID No.1中，第142-159位、第211-231位和第334-363位分别为三个CDR的编码基因。
α链可变区的氨基酸序列如SEQ ID No.2的第22-112位所示；其中，SEQ ID No.2中，第48-53位、第71-77位和第112-121位分别为三个互补决定区。
β链可变区的编码基因如SEQ ID No.3的第58-339位所示；其中，SEQ ID No.3中，第136-150位、第202-219位和第331-366位分别为三个CDR的编码基因。
β链可变区的氨基酸序列如SEQ ID No.4的第20-113位所示；其中，SEQ ID No.4中，第46-50位、第68-73位和第111-122位分别为三个互补决定区。
这对α链序列和β链序列组成的TCR受体可能对HLA-A11限制性CTL表位HBc 141-151具有高亲和力，并来自同一个T细胞克隆。
步骤3-6的实验流程图见图1。
二、表达TCR的Jurkat细胞的获得及鉴定
1、根据步骤一得到的α链可变区和β链可变区的序列，参考NCBI上小鼠基因组α链和α链的恒定区序列，获得并人工合成HLA-A11限制性乙型肝炎病毒 HBc 141-151特异性TCR受体α链和β链的完整编码基因。
α链的完整编码基因如SEQ ID No.1所示，编码SEQ ID No.2所示的α链。
β链的完整编码基因如SEQ ID No.3所示，编码SEQ ID No.4所示的β链。
2、将逆转录病毒载体MSCV-IRES-GFP的限制性内切酶XhoI和EcoRI之间的DNA小片段替换为SEQ ID No.5所示的DNA分子，其它序列均不变，得到重组质粒MSCV-TCR-IRES-GFP(即重组质粒MSCV-TCR-GFP)。SEQ ID No.5中，第1至804位为α链的完整编码基因，第805至867位为T2A自剪切多肽的编码基因，第868至1785位为β链的完整编码基因。
逆转录病毒载体MSCV-IRES-GFP是将MO载体的限制性内切酶XhoI和ClaI之间的DNA小片段替换为IRES核苷酸序列(Genbank：MG550106.1)和荧光标记蛋白GFP核苷酸序列(Genbank：MH777595.1)，得到的重组质粒。
MO载体记载于如下文献：Tanyu Hu，Krung Phiwpan，Jitao Guo，et al.MicroRNA-142-3p Negatively Regulates Canonical Wnt Signaling Pathway.PLOS ONE.2016，DOI:10.1371/journal.pone.0158432.
3、培养Jurkat细胞至数量2×10 7以上，收集细胞，用不含抗生素的1640培养基清洗2遍，最后一遍洗后用不含抗生素1640培养基重悬至5×10 7/mL，将上述细胞分为400μL/份加至电转杯(BIO-RAD，货号：165-2088)，同时加入40μg重组质粒MSCV-TCR-GFP，混匀，将电转杯放入电转仪(BIO-RAD，Gene Pulser Xcell TM)中，电压250V，电容950μF电转，将重组质粒MSCV-TCR-GFP导入Jurkat细胞，得到表达TCR的Jurkat细胞。
按照上述方法，将重组质粒MSCV-TCR-GFP替换为逆转录病毒载体MSCV-NGFR-GFP，其它步骤均不变，得到表达NGFR的Jurkat细胞。
逆转录病毒载体MSCV-NGFR-GFP是将MO载体的限制性内切酶XhoI和EcoRI之间的DNA小片段替换为NGFR核苷酸序列(序列来源于addgene NGFR质粒,Plasmid#27489)，再将限制性内切酶EcoRI和ClaI之间的DNA小片段替换为IRES核苷酸序列(Genbank：MG550106.1)和荧光标记蛋白GFP核苷酸序列(Genbank：MH777595.1)，得到的重组质粒。
4、利用Tetramer(HLA-A11/HBc 141-151)对步骤3得到的表达TCR的Jurkat细胞或表达NGFR的Jurkat细胞进行染色。
染色结果见图2。结果表明，TCR对HLA-A11限制性表位具有较强的亲和力。
三、HBc 141-151特异性TCR转基因小鼠(以下简称TCR转基因小鼠)的构建和鉴定
1、重组质粒phCD2-TCR-α和重组质粒p428-TCR-β的获得
(1)向phCD2质粒的限制性内切酶EcoRI的识别位点处插入SEQ ID No.1所示的TCR-α基因序列，其它序列均不变，得到重组质粒phCD2-TCR-α。
phCD2质粒记载于如下文献：Zhumabekov T,Corbella P,Tolaini M.Kioussis D.Improved version of a human CD2 minigene based vector for T cell-specific  expression in transgenic mice.Journal of Immunological Methods.1995 Sep 11；185(1):133-40.
(2)向p428质粒的限制性内切酶SalI的识别位点处插入SEQ ID No.3所示的TCR-β基因序列，其它序列均不变，得到重组质粒p428-TCR-β。
p428质粒记载于如下文献：Sawada S,Scarborough JD,Killeen N,Littman DR.A lineage-specific transcriptional silencer regulates CD4gene expression during T ly mphocyte development.Cell.1994 Jun 17；77(6):917-29.
2、超排以及假孕母鼠的准备：
(1)超排
向3周龄的C57BL/6雌性小鼠腹腔注射PMSG(5U/只)；48h以后，腹腔注射HCG(5U/只)；注射HCG后立即与B6D2F1雄鼠1:1合笼，合笼18～24h后检查阴栓，有阴栓的小鼠提出待用；
(2)假孕母鼠的准备
在步骤(1)中C57BL/6雌性小鼠与B6D2F1雄鼠合笼的同时，将ICR雌鼠与结扎的ICR雄鼠以2:1的比例合笼，合笼18～24h后检查，有阴栓的小鼠(即假孕母鼠)提出待用。
3、取卵
将步骤2中(1)有阴栓的C57BL/6雌性小鼠脱颈处死，并解剖小鼠，用镊子夹住子宫上部，将输卵管分离至M2培养基中，在显微镜下划开输卵管的壶腹部，使卵流到培养液中。向培养液里加入1mg/mL的透明质酸酶，以去除受精卵周围的颗粒细胞。选择状态较好的受精卵，并转移至塑料皿(直径35mm)里由矿物油覆盖着的M2培养基液滴之中，在二氧化碳孵箱(37℃，5％二氧化碳，95％空气)中培养直到受精卵适合注射为止。
4、向受精卵原核注入DNA溶液
首先，将1mL注射器的活塞切两段长度为0.5cm的小柱，用砂轮切割一片宽度为0.5cm的盖玻片，75％(v/v)乙醇水溶液消毒。用凡士林把切下来的两段小柱按盖玻片的长度黏在载玻片上，利用1mL的注射器在两小柱中央滴两小滴M2溶液，同时在盖玻片上滴一滴，然后将盖玻片反转盖在两小柱上压紧，将注射器针头插入盖玻片的M2液滴里缓慢推入M2溶液，使围成的小室充满M2溶液。随后将完成的注射室放到显微操作仪的载物台上，用胚胎移植管将一组受精卵(约100枚)移到注射室中。将固定针和注射针装上并调整到视野中央，调整各自的X、Y和Z轴，使固定针、注射针和合子处于同一水平面上。推动注射针透过透明质带，进入原核，利用压力泵的持续压力(约150hPa)向原核注入DNA(浓度为3-5ng/μL的重组质粒phCD2-TCR-α和浓度为3-5ng/μL的重组质粒p428-TCR-β按体积比1:1混合而成)至原核稍微膨大，然后将注射针迅速撤出。调节固定针为正压，使注射过的合子脱落，再调节为负压吸附另一个合子进行注射。合子注射完毕后，立即移回M16培养基中，至于37℃的培养箱培养(约12h)。
5、受精卵移植
将假孕母鼠麻醉，手术取出卵巢连接输卵管并用脂肪镊固定，在显微镜下找到输卵管开口。移植管吸取经显微注射DNA后培养成活的受精卵，将移植管口插入输卵管口后轻轻将移植管内的液体吹入，将卵巢连同输卵管放回腹腔，缝合肌肉和皮肤。
6、TCR转基因小鼠的鉴定
取步骤5处理后假孕母鼠分娩的首建小鼠进行基因型鉴定，具体步骤如下：取首建小鼠的尾静脉血，使用ACK红细胞裂解液裂解红细胞，离心，收集白细胞。使用anti-mouse CD8a(Biolegend，克隆号：53-6.7)以及anti-mouse TCRVβ10(BDBioscience，克隆号：B21.5)的抗体对获得的白细胞进行染色。
按照上述方法，将首建小鼠替换为野生型小鼠，其它步骤均不变，作为对照。
染色结果见图3。结果表明，TCR转基因小鼠的体内可以检测到CD8细胞高表达特异性TCRVβ。
四、TCR转基因小鼠的CD8 +T细胞(即TCR-T细胞)具有HBV多肽依赖的活化增殖能力
TCR转基因小鼠中特异性TCR转基因阳性T细胞即为TCR-T细胞。
1、取TCR转基因小鼠淋巴结以及脾脏细胞，计数并将细胞浓度稀释至3×10 7/mL。每3×10 7个细胞加入1μganti-CD4抗体(BioXcell，克隆号：GK1.5)，4℃旋转孵育30min。
2、完成步骤1后，加入2％FBSDMEM培养基使体积达到15mL，4℃、2000rpm离心2min，去除上清，以洗去未结合的抗体。然后每3×10 7个细胞加入1mLGoatAnti-MouseIgG(QIAGEN，310007)、1mL GoatAnti-RatIgG(QIAGEN，货号：310107)和磁珠重悬液(取磁珠，用含0.5％(v/v)BSA和2mM EDTA的1×PBS缓冲液洗涤2次，然后用含0.5％(v/v)BSA和2mM EDTA的1×PBS缓冲液重悬获得)，4℃旋转孵育30min。
3、完成步骤2后，用磁铁吸附磁珠，除去CD4 +T细胞和B细胞，上清液中剩余细胞大约有90％为CD8 +T细胞，随后对得到的CD8 +细胞进行CFSE标记，CFSE标记的基本过程为：取准备标记的细胞，根据需要加入CFSE稀释液或CFSE原液，充分混匀，37℃、5％CO 2培养10min；之后加入10倍体积预热的1640完全培养基以停止标记，并用PBS重悬，得到CFSE标记的TCR-T细胞。
4、取HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞，裂解红细胞，细胞计数，加入新鲜1640培养基将细胞稀释至1×10 7个/mL，然后向HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞中加入多肽HBc 141-151并使其在体系中的浓度为10μg/mL，37℃、5％CO 2培养1h。
5、完成步骤4后，用1640培养基洗涤两次，细胞计数。
6、将步骤5得到的细胞和CFSE标记的TCR-T细胞1:1混合，37℃、5％CO 2培 养1d、2d或3d。
7、完成步骤6后，进行流式检测，分析TCR-T细胞的增殖和活化情况。
将上述方法中的多肽HBc 141-151替换为对照多肽，其它步骤均不变，作为对照。对照多肽具体为NP91(NP91为PR8流感病毒NP蛋白91-99肽段，由北京旭和源生物科技有限公司合成)。
将上述方法中的多肽HBc 141-151替换为Anti-CD3抗体(克隆号145-2C11，Bioxcell)，其它步骤均不变，作为阳性对照。
检测结果见图4(特异性多肽为多肽HBc 141-151，Anti-CD3为Anti-CD3抗体)。结果表明，HBc 141-151可以有效的活化TCR-T细胞，并且可以有效的刺激TCR-T细胞的增殖。由此可见，TCR-T细胞具有HBV多肽依赖的体外活化增殖能力，当把TCR-T细胞回输给HBV阳性的动物或者患者时，可以有效的体内激活和扩增回输的TCR-T细胞。
五、TCR-T细胞具有体外杀伤靶细胞的功能
为了体外验证TCR具有特异性的杀伤靶细胞的能力，进行体外杀伤实验。具体步骤如下：
1、TCR-T细胞的活化
(1)取TCR转基因小鼠淋巴结以及脾脏细胞，用结合CD4 +T细胞以及B细胞的磁珠处理样品，去除CD4 +T细胞以及B细胞，从而富集CD8 +T细胞，细胞计数。
(2)取HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞，裂解红细胞，细胞计数，加入新鲜1640培养基将细胞稀释至1×10 7个/mL，然后向HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞中加入多肽HBc 141-151并使其在体系中的浓度为10μg/mL，37℃、5％CO 2培养1h。
(3)完成步骤(2)后，用1640培养基洗涤两次，细胞计数。
(4)将步骤(3)得到的细胞和TCR转基因小鼠的CD8 +T细胞等数量混合，加入400U/mLIL-2，37℃、5％CO 2培养5d，得到体外活化的TCR-T细胞。
2、取HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞，裂解红细胞，细胞计数，加入新鲜1640培养基将细胞稀释至1×10 7个/mL，然后向HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞中加入多肽HBc 141-151并使其在体系中的浓度为10μg/mL，37℃、5％CO 2培养1h，之后1640培养基洗涤两次，细胞计数，得到负载多肽HBc 141-151的靶细胞。
3、将步骤2得到的负载多肽HBc 141-151的靶细胞加入96孔板中，1×10 4个/孔；随后按10:1、5:1或1:1的比例加入体外活化的TCR-T细胞，37℃、5％CO 2培养5h。
4、取96孔板，250g离心4min；然后将50μL上清转移至新的96孔板，每孔加入底物50μL，室温、避光孵育30min；之后每孔加入50μL终止液，使用酶标仪(波长490nm)读取数值，即实验组数值。
底物、终止液和细胞裂解液均CytoTox
Figure PCTCN2020105157-appb-000006
Non-Radioactive Cytotoxicity Assay试剂盒(Promega公司，货号G1780)。
5、杀伤实验本底数值检测
(1)按照上述步骤1-4，将步骤3替换为步骤3A)，其它步骤均不变，得到T细胞自释放值。步骤3A)为：取96孔板，加入与实验组同等数量的体外活化的TCR-T细胞，37℃、5％CO 2培养5h。
(2)按照上述步骤1-4，将步骤3替换为步骤3B)，其它步骤均不变，得到靶细胞自释放值。步骤3B)为：取96孔板，加入与实验组同等数量的负载多肽HBc 141-151的靶细胞，37℃、5％CO 2培养5h。
(3)按照上述步骤1-4，将步骤3替换为步骤3C)，其它步骤均不变，得到靶细胞最大释放值。步骤3C)为：取96孔板，加入与实验组同等数量的负载多肽HBc 141-151的靶细胞和10μL细胞裂解液，37℃、5％CO 2培养5h。
6、计算杀伤活性
杀伤活性＝(实验组数值-T细胞自释放值-靶细胞自释放值)/(靶细胞最大释放值-靶细胞自释放值)
将上述方法中的多肽HBc 141-151替换为NP91，其它步骤均不变，作为对照。
检测结果见图5(E：T表示TCR-T细胞和靶细胞的比例)。结果表明，TCR-T细胞在体外可以有效的杀伤靶细胞。
六、TCR-T细胞具有体内杀伤靶细胞的功能
为了体内验证TCR具有特异性的杀伤靶细胞的能力，进行体内杀伤实验。将负载多肽HBc 141-151的HLA-A11/hTAP-LMP细胞用较低浓度CFSE(CFSE low)标记，未负载多肽的HLA-A11/hTAP-LMP细胞作为对照用较高浓度CFSE(CFSE high)标记，将两种细胞等量混合后回输至C57/B6J小鼠体内，随后回输预先活化的TCR-T细胞，使用流式细胞仪检测C57/B6J小鼠体内CFSE low标记的细胞群是否会被TCR-T细胞杀伤。具体步骤如下：
1、TCR-T细胞的活化
(1)取TCR转基因小鼠淋巴结以及脾脏细胞，用结合CD4 +T细胞以及B细胞的磁珠处理样品，去除CD4 +T细胞以及B细胞，从而富集CD8 +T细胞，细胞计数。
(2)取HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞，裂解红细胞，细胞计数，加入新鲜1640培养基将细胞稀释至1×10 7个/mL，然后向HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞中加入多肽HBc 141-151并使其在体系中的浓度为10μg/mL，37℃、5％CO 2培养1h。
(3)完成步骤(2)后，用1640培养基洗涤两次，细胞计数。
(4)将步骤(3)得到的细胞和TCR转基因小鼠的CD8+T细胞等数量混合，加入400U/mL IL-2，37℃、5％CO 2培养5d，得到体外活化的TCR-T细胞。
2、靶细胞的制备
(1)取HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞，裂解红细胞，细胞计数，加入新鲜1640培养基将细胞稀释至1×10 7个/mL，然后向 HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞中加入多肽HBc 141-151并使其在体系中的浓度为10μg/mL，37℃、5％CO 2培养1h，之后1640培养基洗涤两次，细胞计数。
(2)完成步骤(1)后，用PBS重悬细胞，得到浓度为5×10 7/mL的负载多肽HBc 141-151的脾细胞重悬液。
(3)完成步骤(2)后，用较低浓度的CFSE(CFSE low)标记负载多肽HBc 141-151的脾细胞(每mL负载多肽HBc 141-151的脾细胞需加入1μL浓度为0.5mM的CFSE稀释液；进行CFSE标记时，CFSE的浓度为0.5μM)，得到负载多肽HBc 141-151且CFSE low标记的脾细胞。
(4)取HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞，裂解红细胞，细胞计数；然后37℃、5％CO 2培养1h(培养基为1640培养基)。
(5)完成步骤(4)后，用1640培养基洗涤两次，细胞计数。
(6)完成步骤(5)后，用PBS缓冲液重悬，得到浓度为5×10 7/mL的细胞重悬液。
(7)完成步骤(6)后，用较高浓度的CFSE(CFSE high)标记未负载多肽的脾细胞(每mL未负载多肽的脾细胞需加入1μL浓度为5mM的CFSE原液；进行CFSE标记时，CFSE的浓度为5μM)，得到CFSE high标记的脾细胞。
3、TCR-T细胞具有体内杀伤靶细胞的功能
(1)将负载多肽HBc 141-151且CFSE low标记的脾细胞和CFSE high标记的脾细胞等数量混合，得到混合细胞。
(2)取C57/B6J小鼠，每只C57/B6J小鼠回输(尾静脉输入)混合细胞2×10 7个。
(3)完成步骤(2)的2h后，每只C57/B6J小鼠回输(尾静脉输入)步骤1获得的体外活化的TCR-T细胞1×10 7个。
(4)完成步骤(3)的24h后，处死小鼠，收获外周血和脾细胞，裂解红细胞，离心，获得白细胞，制成单细胞悬液。流式细胞仪检测外周血以及脾脏中CFSE low与CFSE high的比例。通过CFSE low与CFSE high的比例定量分析TCR-T细胞的杀伤功能。
按照上述步骤(1)-(4)，将步骤(3)替换为步骤K，其它步骤均不变，作为回输PBS对照。步骤K为：完成步骤(2)的2h后，每只C57/B6J小鼠回输与步骤(3)中体外活化的TCR-T细胞等体积的PBS缓冲液。
检测结果见图6。与回输PBS对照组相比，不论在外周血或是脾脏中，CFSE low标记的细胞群相较CFSE high的细胞群均有明显的下降。结果表明，TCR-T细胞在体内可以有效的杀伤靶细胞。
七、TCR-T细胞可以有效的清除体内HBV的慢性感染
1、小鼠慢性HBV感染模型的建立
小鼠慢性HBV感染模型的建立的方法记载于如下文献中：董小岩，尉迟捷， 王刚，等.高嗜肝性8型重组腺相关病毒体内转导法制备乙型肝炎病毒持续感染小鼠模型.病毒学报.2010，26(6):425-431。具体步骤如下：
(1)向HLA-A11/hTAP-LMP转基因小鼠尾静脉注射rAAV/HBV1.3病毒(北京五加和分子医学研究所有限公司的产品)，注射剂量为5×10 9vg/只。
完成步骤(1)后，观察自然情况下病毒梯度(乙肝病毒s抗原、乙肝病毒e抗原或病毒DNA)变化曲线，并根据变化曲线预测TCR-T细胞的给药时间。
(2)完成步骤(1)后1个月，得到小鼠慢性HBV感染模型。小鼠慢性HBV感染模型中HBsAg和HBeAg的表达受HBV病毒DNA自身的启动子等元件调控，在肝脏和外周血中可以连续10周观察到持续表达的HBsAg和HBeAg。
将上述方法中的HLA-A11/hTAP-LMP转基因小鼠替换为C57BL/6小鼠，其它步骤均不变，获得慢性HBV感染的C57BL/6小鼠，作为阴性对照。
2、TCR-T细胞治疗HLA-A11/hTAP-LMP小鼠的慢性HBV感染
(1)取TCR转基因小鼠淋巴结以及脾脏细胞，用结合CD4 +T细胞以及B细胞的磁珠处理样品，去除CD4 +T细胞以及B细胞，从而富集CD8 +T细胞，细胞计数。
(2)取HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞，裂解红细胞，细胞计数，加入新鲜1640培养基将细胞稀释至1×10 7个/mL，然后向HLA-A11/hTAP-LMP转基因小鼠的脾脏细胞中加入多肽HBc 141-151并使其在体系中的浓度为10μg/mL，37℃、5％CO 2培养1h。
(3)完成步骤(2)后，用1640培养基洗涤两次，细胞计数，然后将细胞和TCR转基因小鼠的CD8 +T细胞等数量混合，加入400U/mLIL-2，37℃、5％CO 2培养5d，得到体外活化的TCR-T细胞。
(4)取HLA-A11/hTAP-LMP转基因小鼠慢性HBV感染模型，每只小鼠回输(尾静脉输入)体外活化的TCR-T细胞1×10 7个。
(5)完成步骤(4)后记为第0天，每隔两天检测血清中HBsAg的OD 450nm值(HBsAg检测试剂盒，上海科华)和ALT浓度(ALT/GPT kit，全自动生化分析仪MEDSOUL AMS-18)，从而判断体内HBV病毒的感染情况。共检测8天。
(6)完成步骤(4)后第7天，取HLA-A11/hTAP-LMP转基因小鼠慢性HBV感染模型，二次回输体外活化的TCR-T细胞1×10 7个。
(7)完成步骤(6)后2天，检测血清中HBsAg的OD450nm值和ALT浓度。
(8)将步骤(4)-(7)中的HLA-A11/hTAP-LMP转基因小鼠慢性HBV感染模型替换为C57BL/6小鼠慢性HBV感染模型，其它步骤均不变，作为对照。
血清中HBsAg的OD 450nm值的检测结果见图7中A(WT 1#，2#，3#，4#均为C57BL/6小鼠慢性HBV感染模型，A11.hTAP 36#、A11.hTAP 37#、A11.hTAP 39#、A11.hTAP 40#和A11.hTAP 43#均为HLA-A11/hTAP-LMP转基因小鼠慢性HBV感染模型)。血清中ALT浓度检测结果见图7中B(WT 1#，2#，3#，4#均为C57BL/6小鼠慢性HBV感染模型，A11.hTAP 36#、A11.hTAP 37#、A11.hTAP 39#、A11.hTAP 40#和A11.hTAP 43#均为HLA-A11/hTAP-LMP转基因小鼠慢性 HBV感染模型)。结果表明，TCR-T细胞可以有效的清除HLA-A11/hTAP-LMP转基因小鼠慢性HBV感染模型体内的HBV病毒感染。
八、Human TCR-T细胞具有体外杀伤靶细胞的功能
为了体外验证Human TCR-T具有特异性的杀伤靶细胞的能力，进行体外杀伤实验。具体步骤如下：
1、慢病毒的包装与浓缩
(1)将慢病毒包装载体pCDH-MSCV-MCS-IRES-GFP(SystemBiosciences，编号：CD731B-1)的限制性内切酶EcoRI与BamHI之间的DNA小片段替换为TCRDNA片段(SEQ ID No.5所示)，得到pCDH-MSCV-TCR-GFP质粒。
(2)将293T细胞吹打重悬成单细胞，计数，然后用含10％(v/v)FBS的DMEM培养基调节，得到浓度为5×10 5个/mL的细胞悬浮液。取培养皿(规格为10cm)，铺10mL细胞悬浮液，培养过夜。
(3)完成步骤(2)后，待293T细胞汇合度为75％时进行转染，转染前30min更换培养基为DMEM培养基。
(4)转染预混物准备
向500μLDMEM培养基中加入12μg pCDH-MSCV-TCR-GFP质粒、9μg psPAX2和6μg pMG2.D，涡旋充分混匀，得到质粒混合物。
psPAX2和pMG2.D均为北京天恩泽基因科技有限公司的产品。
向500μLDMEM培养基中加入27μgPEI，涡旋混匀，静置5min，得到PEI混合物。
(5)完成步骤(4)后，向500μL质粒混合物中加入500μLPEI混合物，涡旋充分混匀，室温孵育20min；然后将孵育完成的混合物轻柔沿培养皿侧壁加入293T细胞中，轻晃培养皿混匀，37℃培养箱培养。6-8h后将培养基更换为10mL含10％(v/v)FBS的DMEM培养基。
(6)完成步骤(5)后第48h，收集第一次病毒上清，补充新鲜DMEM培养基，病毒上清4℃保存。
(7)完成步骤(5)后第72h，收集第二次病毒上清。将第一次病毒上清和第二次病毒上清合并，800g、室温离心5min，收集上清。
(8)完成步骤(7)后，将所述上清使用0.45μmPES滤膜过滤除去细胞碎片，收集病毒上清。
(9)完成步骤(8)后，将所述病毒上清转移至超速离心管。4℃、70000g离心120min，小心弃上清，收集沉淀(白色病毒颗粒)。
(10)完成步骤(9)后，取所述沉淀，加入100倍浓缩体积的1640培养基重悬，4℃溶解过夜，得到浓缩慢病毒。将浓缩慢病毒分装保存于-80℃超低温冰箱，备用。
2、人外周淋巴细胞的分离
(1)抽取人外周静脉血5mL。
(2)完成步骤(1)后，取离心管(规格为50mL)，加入5mL外周血和5mLPBS缓冲液，充分混匀。
(3)完成步骤(2)后，向离心管中加入5mL人外周血淋巴细胞分离液(天津市灏洋生物制品科技有限公司，货号LTS1077)，使用一次性无菌滴管取稀释后外周血沿管壁小心叠加于分离液液面之上，注意保持清楚界面。
(4)完成步骤(3)后，将离心管置于离心机中，调节升速和降速为最低，800g离心20min。
(5)完成步骤(4)后，离心结束后管内分为四层，第一层为血浆和PBS，第二层为环状乳白色淋巴细胞层，第三层为透明分离液层，第四层为红细胞和粒细胞层。用移液器小心吸取第二层环状乳白色淋巴细胞层至无菌离心管(规格为50mL)，向离心管中加入40mL PBS缓冲液，混匀细胞，800g离心5min，弃上清，之后使用1640培养基重悬细胞，计数备用。
3、人外周T淋巴细胞的活化
(1)取24孔板，每孔加入500μL anti-CD3抗体稀释液和500μL anti-CD28抗体稀释液，4℃包被过夜。
anti-CD3抗体稀释液：用PBS缓冲液稀释anti-CD3抗体(BioXcell，克隆号：OKT3)至浓度为3μg/mL获得。
anti-CD28抗体稀释液：用PBS缓冲液稀释anti-CD28抗体(BioXcell，克隆号：CD28.2)至浓度为1μg/mL获得。
(2)完成步骤(1)后，取所述24孔板，移除液体，并用PBS缓冲液清洗一次。
(3)完成步骤(2)后，取所述24孔板，加入500μL人外周T淋巴细胞稀释液，37℃培养箱培养48h；之后400g离心5min，收集沉淀并使用1640培养基重悬，得到活化的人外周T淋巴细胞。活化的人外周T淋巴细胞用于感染慢病毒。
人外周T淋巴细胞稀释液：用1640培养基将步骤2获得的人外周T淋巴细胞稀释至4×10 6个/mL获得。
4、慢病毒感染人外周T淋巴细胞
(1)取24孔板，每孔加入5×10 5个活化的人外周T淋巴细胞和200μL浓缩慢病毒，然后用1640培养基补体积至500μL；最后加入polybrene和IL2，并使polybrene和IL2在体系中的浓度分别为8μg/mL和40U/mL。
(2)完成步骤(1)后，取24孔板，600g、32℃离心90min；然后将24孔板放入37℃培养箱感染24h。
(3)完成步骤(2)后，取所述24孔板，小心吸掉感染孔中350μL培养基，然后加入1640培养基补足至体积2mL并吹打混匀，37℃培养箱继续培养48h。
(4)完成步骤(3)后，先取适量感染后T细胞进行流式检测感染效率；然后使用anti-mouse CD8a抗体(Biolegend，克隆号：53-6.7)和anti-mouse TCRVβ10抗体(BD Bioscience，克隆号：B21.5)对感染后T细胞进行染色，4℃染色30min 后进行上机检测，感染效率(TCRVβ10阳性率)大于15％，阳性细胞即为成功转入TCR的Human TCR-T细胞，用于后续杀伤实验。
5、HumanTCR-T体外具有杀伤靶细胞的功能
底物、终止液和细胞裂解液均为CytoTox
Figure PCTCN2020105157-appb-000007
Non-Radioactive Cytotoxicity Assay试剂盒(Promega公司，货号G1780)中的组件。
(1)抽取HLA-A11阳性健康人外周静脉血5mL，然后按照步骤2的方法分离获得淋巴细胞，即HLA-A11 +PBMC细胞。
(2)完成步骤(1)后，用1640培养基将HLA-A11 +PBMC细胞稀释至浓度为1×10 7个/mL，然后加入多肽HBc 141-151并使其在体系中的浓度为10μg/mL，37℃、5％CO 2培养1h，之后1640培养基洗涤两次，细胞计数，得到负载多肽HBc 141-151的靶细胞。
(3)将步骤(2)得到的负载多肽HBc 141-151的靶细胞加入96孔板中，2×10 4个/孔；随后按效靶比例为2:1或1:1加入Human TCR-T细胞，37℃、5％CO 2培养5h。
(4)取96孔板，250g离心4min；然后将50μL上清转移至新的96孔板，每孔加入底物50μL，室温、避光孵育30min；之后每孔加入50μL终止液，使用酶标仪(波长490nm)读取数值，即实验组数值。
(5)杀伤实验本底数值检测
①按照上述步骤(1)-(4)，将步骤(3)替换为步骤3A)，其它步骤均不变，得到T细胞自释放值。步骤3A)为：取96孔板，加入与实验组同等数量的HumanTCR-T细胞，37℃、5％CO 2培养5h。
②按照上述步骤(1)-(4)，将步骤(3)替换为步骤3B)，其它步骤均不变，得到靶细胞自释放值。步骤3B)为：取96孔板，加入与实验组同等数量的负载多肽HBc 141-151的靶细胞，37℃、5％CO 2培养5h。
③按照上述步骤(1)-(4)，将步骤(3)替换为步骤3C)，其它步骤均不变，得到靶细胞最大释放值。步骤3C)为：取96孔板，加入与实验组同等数量的负载多肽HBc 141-151的靶细胞和10μL细胞裂解液，37℃、5％CO 2培养5h。
6、计算杀伤活性
杀伤活性＝(实验组数值-T细胞自释放值-靶细胞自释放值)/(靶细胞最大释放值-靶细胞自释放值)
将上述方法中的多肽HBc 141-151替换为NP91，其它步骤均不变，作为对照。
将上述方法中的HumanTCR-T替换为未转染TCR的Human T细胞，其它步骤均不变，作为对照。
检测结果见图8(A为Human TCR-T细胞的获得；B为Human TCR-T细胞体外杀伤靶细胞实验，其中E：T表示Human TCR-T细胞和靶细胞的比例，T+HBc 141-151为Human T细胞杀伤孵育HBc 141-151多肽的靶细胞，TCR-T+NP 91为Human TCR-T杀伤孵育NP 91多肽的对照细胞，TCR-T+HBc 141-151为Human TCR-T细胞杀伤孵育HBc 141-151多肽的靶细胞)。结果表明，Human TCR-T细胞在体外 可以有效的杀伤靶细胞。
综上所述，本发明的发明人分离并鉴定了一对HBV特异性的TCR序列，成功构建了这对TCR的转基因小鼠，并且在体外验证了TCR转基因阳性的CD8细胞(即TCR-T细胞)具有HBV多肽表位依赖性的活化和增殖能力；利用动物体内和体外杀伤靶细胞实验，证明了这对TCR具有很好的杀伤靶细胞的活性；体外验证Human TCR-T也具有特异性的杀伤HLA-A11 +靶细胞的能力；此外，动物实验提示这对TCR序列可能是清除HBV感染细胞的有效方法之一。
工业应用
本发明分离并鉴定了一对HBV特异性的TCR序列，成功构建了这对TCR的转基因小鼠，并且在体外验证了TCR转基因阳性的CD8细胞(即TCR-T细胞)具有HBV多肽表位依赖性的活化和增殖能力；同时利用动物体内和体外杀伤靶细胞实验，证明了这对TCR具有很好的杀伤靶细胞(HLA-A11转基因小鼠负载多肽HBc 141-151的脾细胞或PBMC细胞)的活性；体外验证Human TCR-T也具有特异性的杀伤靶细胞(负载多肽HBc 141-151的HLA-A11限制性人PBMC细胞)的能力；此外，动物实验提示这对TCR序列可能是清除HBV感染细胞的有效方法之一。本发明提供的HLA-A11限制性HBc 141-151表位肽的T细胞受体具有重要的应用价值。

Claims (17)

  1. 一种识别HLA-A11限制性HBc 141-151表位肽的T细胞受体，包含α链和β链；
    所述α链包含三个互补决定区，氨基酸序列分别如SEQ ID No.2的第48-53位、第71-77位和第112-121位所示；或这些序列的具有至多3个、2个或1个氨基酸改变的变体；
    所述β链包含三个互补决定区，氨基酸序列分别如SEQ ID No.4的第46-50位、第68-73位和第111-122位所示；或这些序列的具有至多3个、2个或1个氨基酸改变的变体。
  2. 如权利要求1所述T细胞受体，其特征在于：
    所述α链的可变区的氨基酸序列如SEQ ID No.2的第22-112位所示；；或这些序列的具有至多3个、2个或1个氨基酸改变的变体；
    所述β链的可变区的氨基酸序列如SEQ ID No.4的第20-113位所示；或这些序列的具有至多3个、2个或1个氨基酸改变的变体。
  3. 如权利要求1所述T细胞受体，其特征在于：
    所述α链的恒定区的氨基酸序列为SEQ ID No.2的第133-268位所示；
    所述β链的恒定区的氨基酸序列为SEQ ID No.4的第133-305位所示。
  4. 如权利要求1所述T细胞受体，其特征在于：
    所述α链的氨基酸序列如SEQ ID No.2所示；
    所述β链的氨基酸序列如SEQ ID No.4所示。
  5. 编码权利要求1至4任一所述T细胞受体的核酸分子。
  6. 如权利要求5所述的核酸分子，其特征在于：编码所述T细胞受体的核酸分子包含编码所述T细胞受体的α链的核酸分子和编码所述T细胞受体的β链的核酸分子；
    编码所述T细胞受体的α链中三个互补决定区的核苷酸序列分别如SEQ ID No.1的第142-159位、第211-231位和第334-363位所示；或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码相同氨基酸残基的序列；
    编码所述T细胞受体的β链中三个互补决定区的核苷酸序列分别如SEQ ID No.3的第136-150位、第202-219位和第331-366位所示；或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码相同氨基酸残基的序列。
  7. 如权利要求5所述的核酸分子，其特征在于：
    编码所述α链的可变区的核苷酸序列如SEQ ID No.1的第64-336位所示；或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码相同氨基酸残基的序列；
    编码所述β链的可变区的核苷酸序列如SEQ ID No.3的第58-339位所示； 或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码相同氨基酸残基的序列。
  8. 如权利要求5所述的核酸分子，其特征在于：
    所述α链的恒定区的核苷酸序列为SEQ ID No.1的第397-807位所示；
    所述β链的恒定区的核苷酸序列为SEQ ID No.3的第397-918位所示。
  9. 如权利要求5所述的核酸分子，其特征在于：
    编码所述α链的核酸分子的核苷酸序列如SEQ ID No.1所示；
    编码所述β链的核酸分子的核苷酸序列如SEQ ID No.3所示。
  10. 含有权利要求5至9任一所述核酸分子的表达盒、载体或细胞。
  11. 如权利要求10所述的载体，其特征在于：所述载体为逆转录病毒载体或慢病毒载体；
    所述逆转录病毒载体为向逆转录病毒载体MSCV-IRES-GFP的多克隆位点之间插入编码所述T细胞受体的α链的核酸分子和编码所述T细胞受体的β链的核酸分子，得到的重组质粒；
    所述慢病毒载体为向慢病毒包装载体pCDH-MSCV-MCS-IRES-GFP的多克隆位点之间插入编码所述T细胞受体的α链的核酸分子和编码所述T细胞受体的β链的核酸分子，得到的重组质粒。
  12. 如权利要求10所述的细胞，其特征在于：所述细胞为T细胞或Jurkat细胞。
  13. 具有权利要求1至4任一所述T细胞受体的T细胞。
  14. 含有权利要求10所述载体或细胞、或、含有权利要求13所述T细胞的药物组合物。
  15. 权利要求1至4任一所述T细胞受体、或、权利要求5至9任一所述的核酸分子、或、权利要求10至12任一所述表达盒、载体或细胞、或、权利要求13所述T细胞受体的T细胞，的应用，为A1)-A4)中的至少一种：
    A1)制备预防和/或治疗由HBV感染所致的疾病的药物；
    A2)预防和/或治疗由HBV感染所致的疾病；
    A3)体内或体外杀伤靶细胞；
    A4)清除HBV的慢性感染。
  16. 一种预防和/或治疗由HBV感染所致疾病的方法，包括如下步骤：以权利要求1至4任一所述T细胞受体、或、权利要求5至9任一所述的核酸分子、或、权利要求10至12任一所述表达盒、载体或细胞、或、含有权利要求13所述T细胞受体的T细胞来预防和/或治疗由HBV感染所致疾病。
  17. 如权利要求15所述的应用或权利要求16所述的方法，其特征在于：所述由HBV感染所致的疾病为慢性乙型肝炎或肝细胞癌。
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